Encyclopedia of Virology, Volume 2: Viruses as Infectious Agents: Human and Animal Viruses 0128234067, 9780128234068

Citation preview

ENCYCLOPEDIA OF VIROLOGY FOURTH EDITION

Volume 2

ENCYCLOPEDIA OF VIROLOGY FOURTH EDITION EDITORS IN CHIEF

Dennis H. Bamford Molecular and Integrative Biosciences Research Programme Faculty of Biological and Environmental Sciences University of Helsinki, Helsinki, Finland

Mark Zuckerman South London Specialist Virology Centre King’s College Hospital NHS Foundation Trust London, United Kingdom and Department of Infectious Diseases School of Immunology and Microbial Sciences, King’s College London Medical School London, United Kingdom

Volume 2

AMSTERDAM  BOSTON  HEIDELBERG  LONDON  NEW YORK  OXFORD PARIS  SAN DIEGO  SAN FRANCISCO  SINGAPORE  SYDNEY  TOKYO Academic Press is an imprint of Elsevier

ACADEMIC PRESS

Academic Press is an imprint of Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

50 Hampshire Street, 5th Floor, Cambridge MA 02139, United States Copyright r 2021 Elsevier Ltd. unless otherwise stated. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers may always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-814515-9

For information on all publications visit our website at http://store.elsevier.com

Publisher: Oliver Walter Acquisitions Editor: Priscilla Braglia

Content Project Manager: Katarzyna Miklaszewska Associate Content Project Manager: Gayathri S. Designer: Matthew Limbert .

EDITORS IN CHIEF

Dennis H. Bamford, PhD, is Professor Emeritus of Virology at the Faculty of Biological and Environmental Sciences, University of Helsinki, Finland. He obtained his PhD in 1980 from the Department of Genetics, University of Helsinki. During 1981–1982 he was an EMBO postdoctoral fellow at the Public Health Research Institute of the City of New York, United States, and during 1983–1992 he worked as a Senior Scientist at the Academy of Finland. In 1993 he was appointed Professor of General Microbiology at the University of Helsinki. He was awarded the esteemed Academy Professorship twice, in 2002–2007 and 2012–2016, and he also served twice as the Director of the Finnish Center of Excellence (in Structural Virology, 2000–2005, and in Virus Research, 2006–2011). Prof. Bamford has had continuous external research funding (e.g., from several European Union, Academy of Finland, TEKES and Jusélius Foundation funds, as well as the Human Frontier Science Program). He is an EMBO member and has held several positions of trust in scientific and administrative organizations. Prof. Bamford has published approx. 400 articles in international peer-reviewed journals in virology, microbiology, biochemistry, and molecular biology (36 of them in high impact journals). About half of the primary articles have been published with international collaborators showing high international integration. He has also been invited to give 56 keynote and plenary presentations in major international meetings. Prof. Bamford has supervised over 35 Master’s and over 40 PhD theses. Seven of his graduate students or post docs have obtained a professorship and a similar number have a principal investigator status. Prof. Bamford has studied virus evolution from a structure-centered perspective, showing that seemingly unrelated viruses, such as bacteriophage PRD1 and human adenovirus have similar virion architecture. When the corona virion architecture was gradually revealed, it was observed that its structural elements were close to those seen in RNA bacteriophage phi6 so that phi6 has been actively used as surrogate for pathogenic viruses - quite a surprise!

Dr. Mark Zuckerman is Head of Virology, Consultant Medical Virologist, and Honorary Senior Lecturer at South London Specialist Virology Centre, King’s College Hospital NHS Foundation Trust and King’s College London Medical School, Department of Infectious Diseases, School of Immunology and Microbial Sciences in London, United Kingdom. His interests include the clinical interface between developing molecular diagnostic tests relevant to the local population of patients, respiratory virus infections, herpesvirus infections in immunocompromised patients and blood-borne virus transmission incidents in the healthcare setting. He has chaired the UK Clinical Virology Network, Royal College of Pathologists Virology Specialty Advisory Committee and Virology Examiners Panel and is a member of the Specialty Advisory Committee on Transfusion Transmitted Viruses. He is a co-author on four editions of the “Mims’ Medical Microbiology” textbook, has written chapters in a number of other textbooks and has over 100 publications in international peer-reviewed journals and is an associate editor for two journals.

v

EDITORIAL BOARD Editors in Chief Dennis H. Bamford Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Mark Zuckerman South London Specialist Virology Centre, King’s College Hospital NHS Foundation Trust, London, United Kingdom and Department of Infectious Diseases, School of Immunology and Microbial Sciences, King’s College London Medical School, London, United Kingdom

Section Editors Claude M. Fauquet St Louis, MO, United States Michael Feiss Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, United States Elizabeth E. Fry Department of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Said A. Ghabrial† Department of Plant Pathology, University of Kentucky, Lexington, KY, United States Eric Hunter Department of Pathology and Laboratory Medicine, Emory University School of Medicine and Emory Vaccine Center, Emory University, Atlanta, GA, United States Ilkka Julkunen Institute of Biomedicine, University of Turku, Turku, Finland Peter J. Krell Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada Mart Krupovic Archaeal Virology Unit, Institut Pasteur, Paris, France Maija Lappalainen HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Hubert G.M. Niesters Department of Medical Microbiology and Infection Prevention, Division of Clinical Virology, University Medical Center Groningen, Groningen, The Netherlands Massimo Palmarini MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom David Prangishvili Institut Pasteur, Paris, France and Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia David I. Stuart Department of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom and Diamond Light Source, Didcot, United Kingdom Nobuhiro Suzuki Institute of Plant Stress and Resources (IPSR), Okayama University, Kurashiki, Japan

†

Deceased.

vii

SECTION EDITORS Claude Fauquet received his PhD in biochemistry from University Louis Pasteur in Strasburg, France in 1974. Dr. Fauquet joined the Institut de Recherche pour le Dévelopement (IRD) and worked there as a plant virologist for 28 years, and served in Ivory Coast, West Africa for 14 years. In 1991, he founded the International Laboratory for Tropical Agricultural Biotechnology (ILTAB) at The Scripps Research Institute, CA, United States. ILTAB was then hosted by the Donald Danforth Plant Science Center, St. Louis, MO, from 1999 to 2012. In 2003, he co-founded the Global Cassava Partnership for the 21st Century (GCP21), which he directed until 2019 and which goal is to improve the cassava crop worldwide. Dr. Fauquet is an international leader in plant virology including taxonomy, epidemiology, molecular virology, and in gene-silencing as an antiviral strategy. He was Secretary of the International Committee on Taxonomy of Viruses (ICTV) for 18 years and the editor of several ICTV Reports including the VIIIth ICTV Report in 2005. He has published more than 300 research papers in reviewed journals and books. He is a fellow of the American Association for the Advancement of Science, of the American Phytopathological Society and a member of the St. Louis Academy of Sciences. In 2007, Dr. Fauquet was knighted “Chevalier de l’Ordre des Palmes Académiques” by the French Minister of High Education and Research.

Dr. Michael Feiss is Professor Emeritus in the Department of Microbiology and Immunology of the Carver College of Medicine at the University of Iowa, IA, United States. Dr. Feiss received his PhD in Genetics at the University of Washington followed by a postdoctoral traineeship in the laboratory of Dr. Allan Campbell at Stanford. Dr. Feiss is a microbial geneticist who studies virus assembly with an emphasis on how a DNA virus, bacteriophage lambda, packages viral DNA into the empty prohead shell. The lab investigates how sites in the viral DNA orchestrate the initiation and termination of the DNA packaging process. This work includes comprehensive examination of the DNA recognition sites. A related interest is study of terminase, the viral DNA packaging enzyme, including the functional domains for protein–DNA and protein–protein interactions. A second focus has been the roles of the bacterial host’s IHF and DnaJ proteins in the lytic life cycle of the virus. More recent work has involved a genetic dissection of the role of terminase’s ATPase center that powers translocation of viral DNA into the prohead. This interest in the ATP hydrolysis-driven packaging motor involves a multidisciplinary collaboration examining the kinetics of DNA packaging during individual packaging events. Finally, recent studies have also looked at how the packaging process has diverged among several lambda-like phages, including phages 21, N15, and Gifsy-1.

Elizabeth E. Fry is a senior postdoctoral scientist in structural biology at the University of Oxford, Oxford, United Kingdom, where she received her DPhil. for studies relating to the structure determination of Foot-and-Mouth Disease Virus. Specializing in structural virology, Dr. Fry has studied many virus/viral protein structures but her primary focus is on picornavirus structure and function, in particular receptor interactions and virus uncoating. She is particularly interested in rationally designing virus-like-particles as next generation vaccines to reduce the inherent risks in handling live viruses.

ix

x

Section Editors Said A. Ghabrial† received his BSc in 1959 from Cairo University, Cairo, Egypt, and his PhD from Louisiana State University, Baton Rouge, LA, United States, in 1965. Dr. Ghabrial did postdoctoral research at the University of California, Davis, CA, United States, before returning to Cairo, where he served as a plant virologist in the Ministry of Agriculture. He returned to the United States in 1970 to do postdoctoral research at Purdue University, West Lafayette, IN. In 1972, he joined the Plant Pathology Department at the University of Kentucky, Lexington, KY, United States, where he rose to the rank of professor in 1986 and worked until 2013. Dr. Ghabrial has served as an associate and senior editor of Phytopathology. He served on the editorial boards of the Encyclopedia of Virology, 3rd edition and Encyclopedia of Plant Pathology, and edited a thematic issue of Advances in Virus Research on “Mycoviruses”. He was a member of the American Phytopathological Society (APS) and the American Society for Virology (ASV); in July 2002 he was elected as a Fellow of the American Phytopathological Society. He also acted as Chair of the ICTV Subcommittee on Fungal Viruses in 1987–1993 and 2011–2014. His long professional career allowed him to make many scientific achievements in phytopathology and virology. Among them are molecular dissection of a legume-infecting RNA virus, bean pod mottle virus (BPMV), development of BPMV-based vectors, discovery of a transmissible debilitation disease of the phytopathogenic ascomycete, Helminthosporium victoriae (Cochliobolus victoriae), establishment of a viral etiology of the H. victoriae disease, and advancement of structural biology of diverse fungal viruses.

Eric Hunter, PhD, is Professor of Pathology and Laboratory Medicine at Emory University, Atlanta, GA, United States. He serves as Co-Director of the Emory Center for AIDS Research and is a Georgia Research Alliance Eminent Scholar. Dr. Hunter’s research focus has been the molecular virology and pathogenesis of retroviruses, including human immunodeficiency virus. He has made significant contributions to the understanding of the role of retroviral glycoprotein structural features during viral entry and providing unique insights into the assembly and replication of this virus family. In recent years the emphasis of his research has been on HIV transmission and pathogenesis, defining the extreme genetic bottleneck and selection of viruses with unique traits during HIV heterosexual transmission. He has described the selection of fitter viruses at the target mucosa, a gender difference in the extent of selection bias, and a role for genital inflammation in reducing selection. His research has defined the impact of HIV adaptation to the cellular immune response on immune recognition and control of HIV after transmission, as well as on virus replicative fitness in vitro and in vivo. Recent work highlights the roles that virus replicative fitness and sex of the host play in defining disease progression in a newly infected individual. His bibliography includes over 300 peer-reviewed articles, reviews, and book chapters. He has also been the recipient of four NIH merit awards for his work on retrovirus and HIV molecular biology. Dr. Hunter served as the Editor in Chief of the journal AIDS Research and Human Retroviruses for 10 years. He was Chair of the AIDS Vaccine Research Subcommittee which is charged with providing advice and consultation on AIDS vaccine research to the National Institute of Allergy and Infectious Diseases and continues to serve on editorial boards for several academic journals and on external advisory committees for several government, academic, and commercial institutions.

Ilkka Julkunen graduated as an MD/PhD in 1984 from the Department of Virology, University of Helsinki, Helsinki, Finland. He worked as a postdoctoral research fellow at Memorial SloanKettering Cancer Center in New York, United States, in 1986–1989, followed by positions as a senior scientist, group leader and research professor at Finnish Institute for Health and Welfare in 1989–2013. In 2013 he became a Professor of Virology at the University of Turku, Turku, Finland. The research interests of Dr. Julkunen have concentrated on innate and adaptive humoral immunity in viral and microbial infections. He has studied intracellular signaling and RIG-I and TLR-mediated activation of interferon system in human macrophages and dendritic cells and stable cell lines in response to human and avian influenza, Sendai, Zika and coronavirus infections. In addition, he has analyzed the downregulation of innate immunity by viral regulatory proteins from influenza, HCV, flavi-, filo- and coronaviruses. He has expertise in vaccinology, biotechnology and development of methods to analyze antiviral immunity, he has also been actively involved in research training and collaborations with biotechnological industry.

†

Deceased.

Section Editors

xi

Peter Krell started his career in virology early as a summer high school student working for the Canadian Forestry Service studying the resistance of nuclear polyhedrosis viruses (now called baculoviruses) to environmental exposure with Dr. Fred T. Bird at the Insect Pathology Research Institute in Sault Ste. Marie, ON, Canada. He received his BSc and MSc in biology from Carleton University studying the iridovirus Tipula Iridescent Virus with Dr Peter Lee, in Ottawa, the Canadian capital. For his PhD he headed east to Dalhousie University in Halifax, Nova Scotia on the Atlantic coast. In addition to enjoying the salt sea air, fresh cod, lobster and mussels, he studied the molecular biology of polydnaviruses under the guidance of Dr Don Stoltz. Heading south to Texas A&M University in College Station, TX, United States, as a Postdoctoral Fellow he worked with Dr. Max Summer, of baculovirus fame, and Dr. Brad Vinson continuing to study polydnaviruses, but also became steeped in the early days of molecular baculovirology. He then accepted a faculty position in the Department of Microbiology and Immunology at the University of Guelph in Guelph, ON, Canada. There he switched to baculovirus research, which was more tractable, due in part to available cell cultures and focused on viral DNA replication and functional genomics, particularly on chitinase, cathepsin and ME53. In collaboration with Dr. Eva Nagy he studied molecular biology of different animal viruses, notably Fowl Avian adenoviruses and their development as vaccine vectors, but also on the birnavirus infectious pancreatic necrosis virus, the coronavirus porcine endemic diarrhea virus, fowlpox virus and the paramyxovirus Newcastle disease virus. He has been involved extensively with virus taxonomy, being active in the International Committee on Taxonomy of Viruses (ICTV) as member of the Polydnaviridae and Baculoviridae study groups, national representative of Canada on the ICTV, member of the Executive Committee for the ICTV and Chair of the ICTV Invertebrate Virus Subcommittee. In terms of governance, Peter Krell was President of the Canadian Society of Microbiology, Secretary and later President of the Society for Invertebrate Pathology, as well as being on the Editorial Boards of the Canadian Journal of Microbiology and the ASM Journal of Virology. While at the University of Guelph, he rose through the ranks to Professor and is currently University Professor Emeritus.

Mart Krupovic is the Head of the Archaeal Virology Unit in the Department of Microbiology at the Institut Pasteur of Paris, France. He received his MSc in Biochemistry in 2005 from the Vilnius University, Vilnius, Lithuania and PhD in 2010 in general microbiology from the University of Helsinki, Helsinki, Finland. His current research focuses on the diversity, origin, and evolution of viruses, as well as molecular mechanisms of virus–host interactions in archaea. He has published over 170 journal articles and serves as an editor or on the editorial boards of Biology Direct, Research in Microbiology, Scientific Reports, Virology, and Virus Evolution. He is also a member of the Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) and chairs the Archaeal Viruses Subcommittee of the ICTV.

Maija Lappalainen, MD, PhD, Associate Professor of Clinical Microbiology, is the Head of Clinical Microbiology in the HUS Diagnostic Center, HUSLAB, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. In her thesis during the years 1987–1992 she studied the incidence and diagnostics of congenital toxoplasmosis. After PhD, her research interest has been in diagnostic clinical virology, viral hepatitis, respiratory infections, viral infections in the immunocompromised patients and viral infections during pregnancy.

xii

Section Editors

Hubert G.M. Niesters (1958) studied biology and chemistry in Nijmegen, the Netherlands. After obtaining his PhD in Utrecht (Prof. dr. M. Horzinek and Prof. dr. B. van der Zeijst, 1987) on the molecular epidemiology of infectious bronchitis virus, he worked as a post-doctoral fellow with Prof. dr. Jim Strauss at the California Institute of Technology (Pasadena, United States) on the replication of Alphaviruses. He received a Niels Stensen fellowship (The Netherlands) and an E.S. Gosney fellowship (Caltech) during this period. After returning to the Netherlands (1989), he became a research associate in medical microbiology at the Diagnostic Medical Center (Delft) but moved back to clinical virology as a senior research associate in 1991 at the Erasmus University Medical Center Rotterdam (Head Prof. dr. Ab Osterhaus). From 1993 to 2007, he was responsible for the molecular diagnostics unit. During this period, he was involved in the discovery and characterization of several new viruses and variants. In 2007, he became full professor and director of the Laboratory of Clinical Virology within the Department of Medical Microbiology at the University Medical Center Groningen and University of Groningen. He has been actively involved in the implementation and development of new technologies like real-time amplification and automation within clinical virology. He has been focusing on molecular diagnostics and its use and the clinical value in a transplant setting, as well as in monitoring treatment of hepatitis viruses. Recently, his interest focuses on rapid regional epidemiology, automation including MiddleWare solutions for molecular diagnostics, as well as the cost–benefit of rapid point-ofimpact molecular testing. Special interest is focused on raising awareness for the detection of enteroviruses (enterovirus D68) and its relationship with acute flaccid myelitis (AFM). Since 2017, he is the Chair of the executive board of QCMD (Quality Control of Molecular Diagnostics, Glasgow). He is an auditor and team leader for the Dutch Council of Accreditation and Co-Editor in Chief of the Journal of Clinical Virology. He is an (co)-author of more than 250 peer-reviewed papers, chapters and reviews including emerging viruses, such as enterovirus D68 and hepatitis E virus (H-index 80). For his entire work, he received in 2016 the “Ed Nowakowski Senior Memorial Clinical Virology Award” from the Pan American Society for Clinical Virology.

Massimo Palmarini is the Director of the MRC-University of Glasgow Centre for Virus Research and Chair of Virology at the University of Glasgow, Glasgow, United Kingdom. A veterinarian by training, his research programs focus on the biology, evolution and pathogenesis of arboviruses and the mechanisms of virus cross-species transmission. His work is funded by the MRC and the Wellcome Trust. Massimo Palmarini has been elected Fellow of the Academy of Medical Sciences, of the Royal Society of Edinburgh and of the Royal Society of Biology and he was a Wolfson-Royal Society Research Merit Awardee. He is a Wellcome Trust Investigator.

David Prangishvili, PhD, Honorary Professor at the Institut Pasteur, Paris, France, and Professor at Tbilisi State University, Tbilisi, Georgia, is one of the pioneers in studies on the biology of Archaea and their viruses. His scientific career spans ex-USSR (Institute of Molecular Biology, Moscow; 1970–1976), Georgia (Georgian National Academy of Sciences, Tbilisi; 1976–1991), Germany (Max-Planck Institute for Biochemistry, Munich; University of Regensburg; 1991–2004) and France (Institut Pasteur, Paris, 2004–2020). In the research groups headed by him, several dozens of new species and eight new families of archaeal viruses have been discovered and characterized, which display remarkable diversity of unique morphotypes and exceptional genome contents. The results of his research contribute to the knowledge on viral diversity on our planet and change the field of prokaryotic virology, leading to the notion that viruses of hyperthermophilic Archaea form a particular group in the viral world, distinctive from viruses of Bacteria and Eukarya, and to the recognition of the virosphere of Archaea as one of the distinct features of this Domain of Life. David Prangishvili is a member of the Academia Europaea, the European Academy of Microbiology, and the Georgian National Academy of Sciences.

Section Editors

xiii

David I. Stuart is MRC Professor of Structural Biology in the Nuffield Department of Medicine, Oxford University, Oxford, United Kingdom, Life Science Director at Diamond Light Source and Director of Instruct-ERIC (pan-European organisation providing shared access to infrastructure and methods for structural biology). He has diverse interests in structural virology from picornaviruses, double-stranded RNA viruses and enveloped RNA viruses. His drive to develop structural techniques led to the determination of the structure of Bluetongue virus (1995) and then the first membrane containing virus, PRD1. More recently, he has been at the fore-front of bringing Cryo-EM technology to bear on virus structure determination and its future role in visualizing virus function in cellulo. In addition to basic science he has a strong commitment to structural vaccinology and the development of antiviral drugs.

Dr. Nobuhiro Suzuki, PhD, received his MSc (1985) in phytopathology and PhD (1989) in virology from Tohoku University in Sendai, Japan. Dr. Suzuki currently serves as a full Professor of the Institute of Plant Stress and Resources, formerly Institute of Plant Sciences and Bioresouces at Okayama University and as an Editor of Virus Research, Frontiers in Virology, Journal of General Plant Pathology, Virology Journal, and Biology. He has also been Guest Editor to PLoS Pathogens, PNAS, and mBio, and an Editorial Board member of Virology and Journal of Virology. Suzuki Laboratory focuses on characterization of diverse viruses infecting phytopathogenic fungi and exploration of their interplays. Recent achievements include the discovery of a neo-virus lifestyle exhibited by a (+)ssRNA virus and an unrelated dsRNA virus in a plant pathogenic fungus and of multilayer antiviral defense in fungi involving Dicer. Prior to coming to Kurashiki, Okayama Prefecture, he was a visiting fellow of the Center for Agricultural Biotechnology at the University of Maryland Biotechnology Institute (UMBI), College Park, MA, United States, for 4 years (1997–2001) to study molecular biology of hypoviruses in the laboratory of Professor Donald L. Nuss. Before visiting UMBI, he served as an assistant professor and a lecturer of the Biotechnology Institute at the Akita Prefectural College of Agriculture, Japan, for 11 years (1988–1998) where he was engaged in a project on molecular characterization of rice dwarf phytoreovirus, a member of the family Reoviridae. He received awards from the Japanese Phytopathological Society of Japan and Japanese Society for Virology for his outstanding achievements in plant and fungal virology.

FOREWORD I am delighted to write the foreword to this wonderful Fourth Edition of the Encyclopedia of Virology. The Third Edition was published in 2008, how the world has changed in the intervening years. The release of the updated fourth edition could not be more timely or more prescient. It is superb and a huge tribute to the authors, Elsevier the publisher, and to the brilliant editors, Dennis Bamford and Mark Zuckerman. SARS-CoV-2 has dominated the world since it emerged in 2019 and affected every continent and every aspect of life. A reminder, if it were needed, of the impact of infectious diseases, the importance of virology and the vulnerability and interconnectivity of our world. There is no doubt that with rapidly changing ecology, urbanization, climate change, increased travel, and fragile public health systems, epidemics and pandemics will become more frequent, more complex and harder to prevent and contain. Most of these epidemics will be caused by viruses, those we know about and maybe able to predict and some we do not know of that will emerge from animals, plants or the environment. Our changing climate will change the epidemiology of viruses, their vectors and the infections they cause, hence the critical importance of this totally revised Fourth Edition of the Encyclopedia of Virology which brings together research and an understanding of viruses in animals, plants, bacteria and fungi, the environment, and among humans. Never has a holistic, one-health understanding been more important. That starts with an understanding of the fundamentals of virology, a field of science that has been transformed in the years since the Third Edition. An understanding transformed by embracing traditional fields of molecular and structural biology, genomics, and influenced by immunology, genetics, pharmacology and increasingly by epidemiology and mathematics. Events of 2020 and 2021 also show why it is so important to integrate within traditional virology an understanding of the animal and human health and behavior, of climate change and its impact on the ecology of viruses, plant sciences and vectors. And why we must understand the viruses we think we know well, and those viruses less extensively studied. Research is critical to this, research that pushes the boundaries of what we know, has the humility to seek answers to things we do not understand and shares that knowledge with the widest possible community. That research will be most exciting at the interface between disciplines, most impactful when dynamic, open, inclusive, global, and collaborative. This is what the Fourth Edition of the Encyclopedia of Virology, the largest reference source of research in virology sets out to achieve. It is a wonderful contribution to a critical field of knowledge. It contains new chapters, every chapter revised and updated by a dedicated global community who have come together to provide what is a brilliant and inspiring reference. It is an honor to contribute in a very small way to the timely release of the Fourth Edition of the Encyclopedia of Virology. Jeremy Farrar

xv

PREFACE The fourth edition of the Encyclopedia of Virology is encyclopedic, but we wanted to move away from an alphabetical list, apart from where it was more logical, to a vision that encompassed a different structure. Articles describing novel trends as well as original discoveries in specific subfields of virology have been distributed into a set of five volumes, namely Fundamentals of Virology, Human and Animal Viruses, Plant Viruses, Bacterial, Archaeal, Fungal, Algal and Invertebrate Viruses, and Diagnosis, Treatment and Prevention of Virus Infections. We had hoped that the new edition would ‘go viral’ but it was ironic that the time to publication 12 years after the previous edition had been made a bit longer due to a virus infection. The world encountered a devastating global pandemic, COVID-19, caused by a new type of a coronavirus, SARS-CoV-2. Scientists in many disciplines all over the world started immediate efforts to discover solutions as to how to mitigate and stop the spread of the pandemic. Virology moved from being a highly specialized subject to one in which everyone became a virologist, proving just how significant the different aspects of virology are in terms of understanding the nature of viral infection. Since the previous edition, the growth in the field of general virology has been enormous, including huge advances in basic science, identification of novel viruses, diagnostic methods, treatment and prevention. Taking this into account, the introduction of the articles within the Encyclopedia are very timely and crucial for providing a wealth of knowledge of the latest findings in the field of virology to a vast range of people, whether school students, undergraduates, postgraduates, teachers, scientists, researchers, journalists and others interested in infections and the conflict between the host and the pathogen. Pandemic viruses have become a serious public concern in the changing world. We can ask ourselves whether we have reached the point in which nature can no longer cope with the consequences of increased population density and human activities that are harmful to the environment. Although several pandemics have threatened mankind before, this COVID-19 pandemic has highlighted the massive adverse economic consequences towards the wellbeing of society and the importance of research in virology. We aimed to produce a Major Reference Work that differs in approach to others and binds all the virology disciplines together. Chapters have been included on origin, evolution and emergence of viruses, environmental virology and ecology, epidemiology, techniques for studying viruses, viral life cycles, structure, entry, genome and replication, assembly and packaging and taxonomy and viral–host interactions. Information has been included on all known species of viruses infecting bacteria, fungi, plants, vertebrates and invertebrates. Additional topics include antiviral classification and examples of their use in management of infection, diagnostic assays and vaccines, as well as the economic importance of viral diseases of crops and their control. This edition used viral classification according to the 9th Report of the International Committee on Taxonomy of Viruses published in 2012. Updating it to the 10th Report in 2020 was affected by the pandemic and can be found online at http://ictv.global/report/. We wish to acknowledge the hard work, interest, flexibility and patience, during such difficult times both socially and professionally, of everybody involved in the process of writing this edition of the Encyclopedia of Virology, especially Katarzyna Miklaszewska, Priscilla Braglia, Sam Crowe and colleagues at Elsevier. We sincerely thank all the authors and section editors for their excellent contributions to this edition.

Book Cover Image: Viruses are obligate parasites and all cells have their own viruses increasing the total number of viruses to the estimated astronomical number of 1031 that extends the number of stars in the universe. The viral string illustrates how pandemic viruses surround the globe. The original picture was created by Dr. Nina Atanasova (Finnish Meteorological Institute and University of Helsinki) and amended by Matthew Limbert at Elsevier. Dennis H. Bamford Mark Zuckerman

xvii

HOW TO USE THE ENCYCLOPEDIA Structure of the Encyclopedia All articles in the encyclopedia are arranged thematically as a series of entries within subjects/sections, apart from volume 2 where there it was more logical to have articles arranged alphabetically. There are three features to help you easily find the topic you are interested in: a thematic contents list, a full subject index, and contributors. 1. Thematic contents list: The alphabetical contents list, which appears at the front of each volume, lists the entries in the order that they appear in the encyclopedia. 2. Index: The index appears at the end of volume 5 and includes page numbers for quick reference to the information you are looking for. The index entries differentiate between references to a whole entry, a part of an entry, and a table or figure. 3. Contributors: At the start of each volume there is a list of the authors who contributed to all volumes.

xix

LIST OF CONTRIBUTORS Stephen T. Abedon The Ohio State University, Mansfield, OH, United States Peter Abrahamian Agricultural Research Service, US Department of Agriculture, Beltsville, MD, United States Jônatas S. Abrahão Federal University of Minas Gerais, Belo Horizonte, Brazil Florence Abravanel Toulouse University Hospital, Toulouse, France and Toulouse University Paul Sabatier, Toulouse, France Nicola G.A. Abrescia Center for Cooperative Research in Biosciences, Basque Research and Technology Alliance, Derio, Spain; Ikerbasque, Basque Foundation for Science, Bilbao, Spain; and Center for Biomedical Research in the Liver and Digestive Diseases Network, Carlos III Health Institute, Madrid, Spain Gian Paolo Accotto Institute for Sustainable Plant Protection, National Research Council of Italy, Torino, Italy

Aleksandra Alimova The City University of New York (CUNY), School of Medicine, The City College of New York, New York, NY, United States Juan C. Alonso National Biotechnology Center–Spanish National Research Council, Madrid, Spain Imran Amin National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Stephanie E. Ander University of Colorado School of Medicine, Aurora, CO, United States Danielle E. Anderson Duke-NUS Medical School, Singapore, Singapore Ida Bagus Andika Qingdao Agricultural University, Qingdao, China Ana C.d.S.P. Andrade Federal University of Minas Gerais, Belo Horizonte, Brazil Juana Angel Pontifical Javeriana University, Bogota, Colombia

Elisabeth Adderson St. Jude Children’s Research Hospital, Memphis, TN, United States and University of Tennessee Health Sciences Center, Memphis, TN, United States

Vanesa Anton-Vazquez King’s College Hospital, London, United Kingdom

Mustafa Adhab University of Baghdad, Baghdad, Iraq

Guido Antonelli Sapienza University of Rome, Rome, Italy

Alexey A. Agranovsky Lomonosov Moscow State University, Moscow, Russia Nasim Ahmed National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Maher Al Rwahnih University of California, Davis, CA, United States Olufemi J. Alabi Texas A& M AgriLife Research and Extension Center, Weslaco, TX, United States Aurélie A. Albertini Institute for Integrative Biology of the Cell (I2BC), French Alternative Energies and Atomic Energy Commission, French National Center for Scientific Research, Paris-Sud University, University of Paris-Saclay, Gif-sur-Yvette, France

Josefa Antón University of Alicante, Alicante, Spain Nanako Aoki Tokyo University of Agriculture and Technology, Fuchu, Japan Timothy D. Appleby King’s College Hospital, London, United Kingdom Miguel Arenas Department of Biochemistry, Genetics and Immunology, University of Vigo, Vigo, Spain and CINBIO (Biomedical Research Center), University of Vigo, Vigo, Spain Basil Arif Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste Marie, ON, Canada

xxi

xxii

List of Contributors

Vicente Arnau Institute for Integrative Systems Biology (I2SysBio), University of Valencia–Spanish National Research Council, Valencia, Spain Gaurav Arya Duke University, Durham, NC, United States Leyla Asadi University of Alberta, Edmonton, AB, Canada Sassan Asgari The University of Queensland, Brisbane, QLD, Australia Nina S. Atanasova Finnish Meteorological Institute, Helsinki, Finland and University of Helsinki, Helsinki, Finland Houssam Attoui UMR1161 Virologie, INRAE – French National Research Institute for Agriculture, Food and Environment, ANSES, Ecole Nationale Vétérinaire d’Alfort, University of Paris-Est, Maisons-Alfort, France Silvia Ayora National Biotechnology Center–Spanish National Research Council, Madrid, Spain

Xiaoyong Bao The University of Texas Medical Branch, Galveston, TX, United States Yiming Bao Beijing Institute of Genomics, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Beijing, China Alan D.T. Barrett The University of Texas Medical Branch, Galveston, TX, United States Diana P. Baquero Archaeal Virology Unit, Institut Pasteur, Paris, France and Sorbonne University, Paris, France Moshe Bar-Joseph Agricultural Research Organization, Volcani Center, Bet Dagan, Israel Rachael S. Barr Bristol Royal Hospital for Children, Bristol, United Kingdom Ralf Bartenschlager Heidelberg University, Heidelberg, Germany

Walid Azab Free University of Berlin, Berlin, Germany

David L.V. Bauer Francis Crick Institute, London, United Kingdom

Sasha R. Azar The University of Texas Medical Branch, Galveston, TX, United States

Oliver W. Bayfield University of York, York, United Kingdom

Fengwei Bai The University of Southern Mississippi, Hattiesburg, MS, United States Dalan Bailey The Pirbright Institute, Pirbright, United Kingdom S.C. Baker Loyola University of Chicago, Maywood, IL, United States Fausto Baldanti University of Pavia, Pavia, Italy and Scientific Institute for Research, Hospitalization and Healthcare, San Matteo Polyclinic Foundation, Pavia, Italy Logan Banadyga Public Health Agency of Canada, Winnipeg, MB, Canada Ashley C. Banyard Animal and Plant Health Agency, Addlestone, United Kingdom; University of West Sussex, Falmer, United Kingdom; and St. George's Medical School, University of London, London, United Kingdom

Sally A. Baylis Paul-Ehrlich-Institute, Langen, Germany Philippa M. Beard The Pirbright Institute, Pirbright, United Kingdom and The Roslin Institute, University of Edinburgh, United Kingdom Paul Becher University of Veterinary Medicine, Hannover, Germany Björn Becker Saarland University, Saarbrücken, Germany Karen L. Beemon Johns Hopkins University, Baltimore, MD, United States Martin Beer Friedrich-Loeffler-Institute, Insel Riems, Germany Jose Miguel Benito Health Research Institute of the Jiménez Díaz Foundation, Autonomous University of Madrid and Rey Juan Carlos University Hospital, Móstoles, Spain Mária Benko ˝ Institute for Veterinary Medical Research, Center for Agricultural Research, Budapest, Hungary

List of Contributors

Max Bergoin National Institute of Scientific Research – ArmandFrappier Health Research Centre, Laval, QC, Canada Sabrina Bertin Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, Rome, Italy Shweta Bhatt University of Copenhagen, Copenhagen, Denmark Dennis K. Bideshi California Baptist University, Riverside, CA, United States and University of California, Riverside, CA, United States Yves Bigot INRAE – French National Research Institute for Agriculture, Food and Environment, Nouzilly, France Richard J. Bingham University of York, York, United Kingdom

Maxime Boutier University of Liège, Liège, Belgium P.R. Bowser Cornell University, Ithaca, NY, United States Daniel Bradshaw Public Health England, London, United Kingdom Claude Bragard University of Louvain, Louvain-la-Neuve, Belgium Aaron C. Brault Centers for Disease Control and Prevention, Fort Collins, CO, United States Nicolas Bravo-Vasquez St. Jude Children’s Research Hospital, Memphis, TN, United States Rob W. Briddon University of Agriculture, Faisalabad, Pakistan Thomas Briese Columbia University, New York, NY, United States

Vera Bischoff Institute for Chemistry and Biology of the Marine Environment, Oldenburg, Germany

Paul Britton The Pirbright Institute, Pirbright, United Kingdom

Kate N. Bishop Francis Crick Institute, London, United Kingdom

Thomas J. Brouwers Athena Institute, VU Amsterdam, Amsterdam, The Netherlands

Lindsay W. Black The University of Maryland School of Medicine, Baltimore, MD, United States Romain Blanc-Mathieu Institute for Chemical Research, Kyoto University, Kyoto, Japan Soile Blomqvist National Institute for Health and Welfare, Helsinki, Finland Bryony C. Bonning University of Florida, Gainesville, FL, United States Lisa M. Bono Rutgers, The State University of New Jersey, New Brunswick, NJ, United States Alexia Bordigoni Aix-Marseille University, CNRS, IRD, Mediterranean Institute of Oceanography, Marseille, France and Aix-Marseille University, IRD257, Assistance-Publique des Hôpitauxde Marseille, UMR Microbes, Evolution, Phylogeny and Infections (MEPHI), IHU Méditerranée Infection, Marseille, France Mihnea Bostina University of Otago, Dunedin, New Zealand

xxiii

Kevin E. Brown Frimley Park Hospital, Frimley, United Kingdom and Immunisation and Countermeasures Division, Public Health England, London, United Kingdom Corina P.D. Brussaard NIOZ Royal Netherlands Institute for Sea Research, Den Burg, Texel, The Netherlands and Utrecht University, Utrecht, The Netherlands Harald Brüssow Laboratory of Gene Technology, Department of Biosystems, KU Leuven, Leuven, Belgium Joachim J. Bugert Bundeswehr Institute of Microbiology, Munich, Germany Jozef J. Bujarski Northern Illinois University, DeKalb, IL, United States and Polish Academy of Sciences, Poznan, Poland Laura Burga University of Otago, Dunedin, New Zealand Sara H. Burkhard University Hospital of Zurich, Zurich, Switzerland Cara C. Burns Centers for Disease Control and Prevention, Atlanta, GA, United States

xxiv

List of Contributors

Felicity Burt University of the Free State, Bloemfontein, South Africa Kerry S. Burton Leamington Spa, United Kingdom Sarah J. Butcher University of Helsinki, Helsinki, Finland Mathias Büttner Leipzig University, Leipzig, Germany Jesse Cahill Sandia National Labs, Albuquerque, NM, United States Marianna Calabretto Sapienza University of Rome, Rome, Italy Thierry Candresse The National Research Institute for Agriculture, Food and the Environment, University of Bordeaux, Villenave d′Ornon, France Alan J. Cann University of Leicester, Leicester, United Kingdom Lorenzo Capucci The Lombardy and Emilia Romagna Experimental Zootechnic Institute, Brescia, Italy Irene Carlon-Andres University of Oxford, Oxford, United Kingdom José M. Casasnovas National Center for Biotechnology, Spanish National Research Council (CSIC), Madrid, Spain J.W. Casey Cornell University, Ithaca, NY, United States R.N. Casey Cornell University, Ithaca, NY, United States Sherwood R. Casjens University of Utah, Salt Lake City, UT, United States Antonella Casola The University of Texas Medical Branch, Galveston, TX, United States José R. Castón National Center for Biotechnology, Spanish National Research Council, Madrid, Spain

Patrizia Cavadini The Lombardy and Emilia Romagna Experimental Zootechnic Institute, Brescia, Italy Supranee Chaiwatpongsakorn Nationwide Children’s Hospital, Columbus, OH, United States Supriya Chakraborty Jawaharlal Nehru University, New Delhi, India Yu-Chan Chao Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Tyler P. Chavers Centers for Disease Control and Prevention, Atlanta, GA, United States Keping Chen Jiangsu University, Zhenjiang, China Xiaorui Chen Genomics Research Center, Academia Sinica, Taipei, Taiwan Yanping Chen Bee Research Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD, United States Dayna Cheng National Cheng Kung University, Tainan, Taiwan Quentin Chesnais University of Strasbourg, Colmar, France Sotaro Chiba Nagoya University, Nagoya, Japan Wah Chiu Stanford University, Stanford, CA, United States David Chmielewski Stanford University, Stanford, CA, United States Irma E. Cisneros The University of Texas Medical Branch, Galveston, TX, United States Lark L. Coffey University of California, Davis, CA, United states Alanna B. Cohen Rutgers University, New Brunswick, NJ, United States

Carlos E. Catalano University of Colorado Anschutz Medical Campus, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, United States

Jeffrey I. Cohen Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States

Roberto Cattaneo Mayo Clinic, Rochester, MN, United States

Seth Coleman Rice University, Houston, TX, United States

List of Contributors

Miquel Coll Institute for Research in Biomedicine, Barcelona, Spain and Institute for Molecular Biology of Barcelona, Barcelona, Spain John Collinge UCL Institute of Prion Diseases, London, United Kingdom Carina Conceicao The Pirbright Institute, Pirbright, United Kingdom Gabriela N. Condezo National Center for Biotechnology, Spanish National Research Council, Madrid, Spain

xxv

Amy Davis St Jude Children’s Research Hospital, Memphis, TN, United States William O. Dawson Citrus Research and Education Center, Lake Alfred, FL, United States and University of Florida, Lake Alfred, FL, United States Erik De Clercq Rega Institute for Medical Research, KU Leuven, Leuven, Belgium Raoul J. de Groot Utrecht University, Utrecht, The Netherlands

Michaela J. Conley MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom

Juan C. de la Torre The Scripps Research Institute, La Jolla, CA, United States

Charles A Coomer University of Oxford, Oxford, United Kingdom

Marcelo De las Heras University of Zaragoza, Zaragoza, Spain

Anne K. Cordes Hannover Medical School, Institute of Virology, Hannover, Germany

Juliana Gabriela Silva de Lima Federal University of Rio Grande do Norte, Natal, Brazil

Mauricio Cortes Jr. Department of Chemistry, College of Arts and Sciences, Fort Wayne, IN, United States Robert H.A. Coutts University of Hertfordshire, Hatfield, United Kingdom Jeff A. Cowley CSIRO Livestock Industries, Brisbane, QLD, Australia Robert W. Cross The University of Texas Medical Branch, Galveston, TX, United States Henryk Czosnek The Hebrew University of Jerusalem, Rehovot, Israel Håkon Dahle Department of Biological Sciences, University of Bergen, Bergen, Norway Janet M. Daly University of Nottingham, Sutton Bonington, United Kingdom Subha Das Okayama University, Kurashiki, Japan

Athos S. de Oliveira University of Brasília, Brasília, Brazil Nicole T. de Stefano University of Florida, Gainesville, FL, United States Greg Deakin NIAB-EMR, East Malling, United Kingdom Philippe Delfosse University of Luxembourg, Esch-sur-Alzette, Luxembourg Natacha Delrez University of Liège, Liège, Belgium Tatiana A. Demina Molecular and Integrative Biosciences Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Ismail Demir Department of Biology, Karadeniz Technical University, Trabzon, Turkey Zihni Demirbağ Department of Biology, Karadeniz Technical University, Trabzon, Turkey

Indranil Dasgupta University of Delhi, New Delhi, India

X. Deng Loyola University of Chicago, Maywood, IL, United States

Sibnarayan Datta Defence Research Laboratory, Defence Research and Development Organisation (DRDO), Tezpur, Assam, India

Cécile Desbiez Plant Pathology Unit, INRAE – French National Research Institute for Agriculture, Food and Environment, Montfavet, France

xxvi

List of Contributors

Christelle Desnues Aix-Marseille University, CNRS, IRD, Mediterranean Institute of Oceanography, Marseille, France and Aix-Marseille University, IRD 257, Assistance-Publique des Hôpitaux de Marseille, UMR Microbes, Evolution, Phylogeny and Infections (MEPHI), IHU Méditerranée Infection, Marseille, France

Lucy Dorrell University of Oxford, Oxford, United Kingdom

Samantha J. DeWerff University of Illinois at Urbana-Champaign, Urbana, IL, United States

Andreas Dotzauer University of Bremen, Bremen, Germany

Daniele Di Carlo Sapienza University of Rome, Rome, Italy Arturo Diaz La Sierra University, Riverside, CA, United States Alfredo Diaz-Lara University of California, Davis, CA, United States Ralf G. Dietzgen The University of Queensland, St. Lucia, QLD, Australia Michele Digiaro International Center for Advanced Mediterranean Agronomic Studies (CIHEAM), Mediterranean Agronomic Institute of Bari, Valenzano, Italy Michael Dills Montana State University, Bozeman, MT, United States Wayne Dimech National Serology Reference Laboratory, Fitzroy, VIC, Australia Savithramma P. Dinesh-Kumar University of California, Davis, CA, United States Linda K. Dixon The Pirbright Institute, Pirbright, United Kingdom Valerian V. Dolja Oregon State University, Corvallis, OR, United States Aušra Domanska University of Helsinki, Helsinki, Finland Leslie L. Domier Agricultural Research Service, US Department of Agriculture, Urbana, IL, United States Pilar Domingo-Calap Institute for Integrative Systems Biology (I2SysBio), University of Valencia-CSIC, Valencia, Spain Tatiana Domitrovic Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Sarah M. Doore Michigan State University, East Lansing, MI, United States

Rosemary A. Dorrington Rhodes University, Grahamstown, South Africa Andor Doszpoly Hungarian Academy of Sciences, Budapest, Hungary

Simon B. Drysdale St George’s University Hospitals NHS Foundation Trust, London, United Kingdom and St George’s, University of London, London, United Kingdom Robert L. Duda University of Pittsburgh, Pittsburgh, PA, United States Carol Duffy University of Alabama, Tuscaloosa, AL, United States Siobain Duffy Rutgers, The State University of New Jersey, New Brunswick, NJ, United States David D. Dunigan University of Nebraska–Lincoln, Lincoln, NE, United States Stéphane Duquerroy University of Paris-Saclay, Orsay, France and Institut Pasteur, Paris, France Bas E. Dutilh Utrecht University, Utrecht, The Netherlands and Radboud University Medical Center, Nijmegen, The Netherlands Michael Edelstein Faculty of Medicine, Bar Ilan University, Ramat Gan, Israel Herman K. Edskes National Institutes of Health, Bethesda, MD, United States Rosina Ehmann Bundeswehr Institute of Microbiology, Munich, Germany Toufic Elbeaino International Center for Advanced Mediterranean Agronomic Studies (CIHEAM), Mediterranean Agronomic Institute of Bari, Valenzano, Italy Joanne B. Emerson University of California, Davis, CA, United States Ann Emery University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

List of Contributors

xxvii

Christine E. Engeland University Hospital Heidelberg and German Cancer Research Center, Heidelberg, Germany and Witten/Herdecke University, Witten, Germany

Elvira Fiallo-Olivé Institute for Mediterranean and Subtropical Horticulture “La Mayora”–Spanish National Research Council–University of Malaga, Algarrobo-Costa, Málaga, Spain

Luis Enjuanes National Center for Biotechnology – Spanish National Research Council (CNB-CSIC), Madrid, Spain

Andrew E. Firth University of Cambridge, Cambridge, United Kingdom

Katri Eskelin University of Helsinki, Helsinki, Finland Rosa Esteban Institute of Biology and Functional Genomics, CSIC/University of Salamanca, Salamanca, Spain Mary K. Estes Baylor College of Medicine, Houston, TX, United States Cassia F. Estofolete São José do Rio Preto School of Medicine, São José do Rio Preto, Brazil Alyssa B. Evans National Institutes of Health, Hamilton, MT, United States Øystein Evensen Norwegian University of Life Sciences, Oslo, Norway Alex Evilevitch Department of Experimental Medical Science, Lund University, Lund, Sweden Montserrat Fàbrega-Ferrer Institute for Research in Biomedicine, Barcelona, Spain and Institute for Molecular Biology of Barcelona, Barcelona, Spain Francesco Faggioli Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, Rome, Italy Bentley A. Fane University of Arizona, Tucson, AZ, United States Brian A. Federici University of California, Riverside, CA, United States F. Fenner Australian National University, Canberra, ACT, Australia Isabel Fernández de Castro Cell Structure Laboratory, National Center for Biotechnology – Spanish National Research Council (CNB-CSIC), Madrid, Spain Giovanni Ferrara University of Alberta, Edmonton, AB, Canada

Roland A. Fleck King’s College London, London, United Kingdom Ricardo Flores Polytechnic University of Valencia, Higher Council of Scientific Research, Valencia, Spain Ervin Fodor University of Oxford, Oxford, United Kingdom Anthony R. Fooks Animal and Plant Health Agency, Addlestone, United Kingdom; University of Liverpool, Liverpool, United Kingdom; and St. George's Medical School, University of London, London, United Kingdom Patrick Forterre Archeal Virology Unit, Institut Pasteur, Paris, France and French National Center for Scientific Research, Institute of Integrative Biology of the Cell, University of Paris-Saclay, Gif sur Yvette, France Rennos Fragkoudis University of Nottingham, Sutton Bonington, United Kingdom and University of Edinburgh, Edinburgh, United Kingdom Manuel A. Franco Pontifical Javeriana University, Bogota, Colombia Giovanni Franzo Department of Animal Medicine, Production and Health (MAPS), Padua University, Padua, Italy Graham L. Freimanis The Pirbright Institute, Pirbright, United Kingdom Juliana Freitas-Astúa Brazilian Agricultural Research Corporation (Embrapa) Cassava and Fruits, Cruz das Almas, Brazil Elizabeth E. Fry Department of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Marc Fuchs Cornell University, Geneva, NY, United States Tsutomu Fujimura Institute of Biology and Functional Genomics, CSIC/University of Salamanca, Salamanca, Spain

xxviii

List of Contributors

Kuko Fuke Tokyo University of Agriculture and Technology, Fuchu, Japan

Said A. Ghabrial† Department of Plant Pathology, University of Kentucky, Lexington, KY, United States

Toshiyuki Fukuhara Tokyo University of Agriculture and Technology, Fuchu, Japan

Clément Gilbert Evolution, Genomes, Behavior and Ecology Laboratory, CNRS University of Paris-Sud UMR 9191, IRD UMR 247, Gif-sur-Yvette, France

To S. Fung South China Agricultural University, Guangzhou, China Yahya Z.A. Gaafar Julius Kuehn Institute – Federal Research Center for Cultivated Plants, Braunschweig, Germany Toni Gabaldon Barcelona Supercomputing Center-National Center for Supercomputing, Institute of Research in Biomedicine, and Catalan Institution for Research and Advanced Studies, Barcelona, Spain Morgan Gaïa University of Paris-Saclay, Evry, France José Gallardo National Center for Biotechnology, Spanish National Research Council, Madrid, Spain Hernan Garcia-Ruiz University of Nebraska–Lincoln, Lincoln, NE, United States Juan A. García National Center for Biotechnology-Spanish National Research Council, Madrid, Spain Matteo P. Garofalo The University of Texas Medical Branch, Galveston, TX, United States Yves Gaudin Institute for Integrative Biology of the Cell (I2BC), French Alternative Energies and Atomic Energy Commission, French National Center for Scientific Research, Paris-Sud University, University of Paris-Saclay, Gif-sur-Yvette, France Andrew D.W. Geering The University of Queensland, St. Lucia, QLD, Australia Thomas W. Geisbert The University of Texas Medical Branch, Galveston, TX, United States Andrea Gentili Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, Rome, Italy Volker Gerdts University of Saskatchewan, Saskatoon, SK, Canada

Robert L. Gilbertson University of California, Davis, CA, United States Efstathios S. Giotis Imperial College London, London, United Kingdom and University of Essex, Colchester, United Kingdom Laurent Glais French Federation of Seed Potato Growers/Research, Development, Promotion of Seed Potato, Paris, France and Institute for Genetics, Environment and Plant Protection, Agrocampus West, French National Institute for Agriculture, Food and Environment, University of Rennes 1, Le Rheu, France Miroslav Glasa Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia Ido Golding University of Illinois at Urbana-Champaign, Urbana, IL, United States Esperanza Gomez-Lucia Complutense University of Madrid, Madrid, Spain Zheng Gong Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China Andrea González-González University of Florida, Gainesville, FL, United States Michael M. Goodin University of Kentucky, Lexington, KY, United States Alexander E. Gorbalenya Leiden University Medical Center, Leiden, The Netherlands Paul Gottlieb The City University of New York (CUNY), School of Medicine, The City College of New York, New York, NY, United States M.-A. Grandbastien INRAE – French National Research Institute for Agriculture, Food and Environment, Versailles, France †

Deceased.

List of Contributors

Meritxell Granell National Center for Biotechnology, Madrid, Spain and Institute of Chemical Research of Catalonia (ICIQ), Tarragona, Spain

Sébastien Halary National Museum of Natural History, UMR 7245 CNRS/MNHN Molécule de Communication et Adaptation des Micro-organismes, Paris, France

Patrick L. Green The Ohio State University, Columbus, OH, United States

Aron J. Hall Centers for Disease Control and Prevention, Atlanta, GA, United States

Sandra J. Greive University of York, York, United Kingdom

John Hammond Floral and Nursery Plants Research, Agricultural Research Service, US Department of Agriculture, Beltsville, MD, United States

Diane E. Griffin Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States Jonathan M. Grimes University of Oxford, Oxford, United Kingdom Nigel Grimsley Integrative Biology of Marine Organisms Laboratory, Banuyls-sur-Mer, France and Sorbonne University, Banuyls-sur-Mer, France Bruno Gronenborn Institute for Integrative Biology of the Cell, CNRS, University of Paris-Sud, CEA, Gif sur Yvette, France Julianne H. Grose Brigham Young University, Provo, UT, United States Scott Grytdal Centers for Disease Control and Prevention, Atlanta, GA, United States

Rosemarie W. Hammond Agricultural Research Service, US Department of Agriculture, Beltsville, MD, United States Virginia Hargest St Jude Children’s Research Hospital, Memphis, TN, United States and University of Tennessee Health Science Center, Memphis, TN, United States Scott J. Harper Washington State University, Prosser, WA, United States Balázs Harrach Institute for Veterinary Medical Research, Center for Agricultural Research, Budapest, Hungary Masayoshi Hashimoto The University of Tokyo, Tokyo, Japan Muhammad Hassan University of Agriculture, Faisalabad, Pakistan

Duane J. Gubler Duke-NUS Medical School, Singapore, Singapore

Asma Hatoum-Aslan University of Alabama, Tuscaloosa, AL, United States

Peixuan Guo College of Pharmacy, The Ohio State University, Columbus, OH, United States

Philippa C. Hawes The Pirbright Institute, Pirbright, United Kingdom

Tongkun Guo Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China Anne-Lise Haenni Institut Jacques Monod, French National Center for Scientific Research, Paris Diderot University, Paris, France Susan L. Hafenstein Pennsylvania State University, Hershey, PA, United States Ahmed Hafez Biotechvana, Valencia, Spain; Pompeu Fabra University, Barcelona, Spain; and Minia University, Minya, Egypt Marie Hagbom Linköping University, Linköping, Sweden

xxix

Janelle A. Hayes University of Massachusetts Medical School, Worcester, MA, United States Guijuan He Virginia Tech, Blacksburg, VA, United States Klaus Hedman University of Helsinki, Helsinki, Finland and Helsinki University Hospital, Helsinki, Finland Albert Heim Hannover Medical School, Hanover, Germany Gary L. Hein University of Nebraska–Lincoln, Lincoln, NE, United States Manfred Heinlein IBMP-CNRS, University of Strasbourg, Strasbourg, France

xxx

List of Contributors

Mercedes Hernando-Pérez National Center for Biotechnology, Spanish National Research Council, Madrid, Spain Carmen Hernández Institute for Plant Molecular and Cell Biology (Spanish National Research Council–Polytechnic University of Valencia), Valencia, Spain Etienne Herrbach University of Strasbourg, Colmar, France Stephen Higgs Biosecurity Research Institute, Kansas State University, Manhattan, KS, United States Bradley I. Hillman Rutgers University, New Brunswick, NJ, United States Deborah M. Hinton National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States Judith Hirsch Plant Pathology Unit, INRAE – French National Research Institute for Agriculture, Food and Environment, Montfavet, France Jody Hobson-Peters Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia

Elisabeth Huguet Research Institute on Insect Biology, French National Center for Scientific Research, University of Tours, Tours, France Roger Hull John Innes Centre, Norwich, United Kingdom Kiwamu Hyodo Okayama University, Kurashiki, Japan Eugénie Hébrard Interactions Plantes Microorganismes Environnement, Institut de Recherche pour le Développement, Centre de coopération internationale en recherche agronomique pour le développement, University of Montpellier, Montpellier, France Martin Hölzer University of Jena, Jena, Germany Tetsuro Ikegami The University of Texas Medical Branch at Galveston, Galveston, TX, United States Niina Ikonen Finnish Institute for Health and Welfare, Helsinki, Finland Cihan I˙nan Department of Molecular Biology and Genetics, Karadeniz Technical University, Trabzon, Turkey

Natalie M. Holste University of Nebraska–Lincoln, Lincoln, NE, United States

I˙kbal Agah I˙nce Department of Medical Microbiology, Acıbadem University School of Medicine, Istanbul, Turkey

Jin S. Hong Kangwon National University, Chunchon, South Korea

Katsuaki Inoue Diamond Light Source, Didcot, United Kingdom

Margaret J. Hosie MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom

Toru Iwanami Tokyo University of Agriculture, Tokyo, Japan

Olivia G. Howell University of Alabama, Tuscaloosa, AL, United States

Jacques Izopet Toulouse University Hospital, Toulouse, France and Toulouse University Paul Sabatier, Toulouse, France

Liya Hu Baylor College of Medicine, Houston, TX, United States Zhaoyang Hu Jiangsu University, Zhenjiang, China Kuan-Ying A. Huang Chang Gung Memorial Hospital, Taoyuan, Taiwan Yu Huang Peking University, Beijing, China Natalia B. Hubbs Hanover College, Hanover, IN, United States

Fauziah Mohd Jaafar UMR1161 Virologie, INRAE – French National Research Institute for Agriculture, Food and Environment, ANSES, Ecole Nationale Vétérinaire d’Alfort, University of Paris-Est, Maisons-Alfort, France Andrew O. Jackson China Agricultural University, Beijing, China Daral J. Jackwood The Ohio State University/OARDC, Wooster, OH, United States

List of Contributors

Jean-Rock Jacques Cellular and Molecular Epigenetics (GIGA), Liège, Belgium and Molecular Biology (TERRA), Gembloux, Belgium Tiffany Jenkins Nationwide Children’s Hospital, Columbus, OH, United States and The Ohio State University, Columbus, OH, United States Jeffrey D. Jensen Arizona State University, Tempe, AZ, United States Daohong Jiang Huazhong Agricultural University, Wuhan, China Zhihao Jiang China Agricultural University, Beijing, China

xxxi

Laura Kakkola University of Turku, Turku, Finland Hannimari Kallio-Kokko University of Helsinki and Helsinki University Hospital, Helsinki, Finland Nassim Kamar Toulouse University Hospital, Toulouse, France and Toulouse University Paul Sabatier, Toulouse, France Phyllis J. Kanki Harvard T.H. Chan School of Public Health, Boston, MA, United States Peter Karayiannis University of Nicosia, Nicosia, Cyprus

Allison R. Jilbert The University of Adelaide, Adelaide, SA, Australia

Henry M. Kariithi Kenya Agricultural and Livestock Research Organization, Nairobi, Kenya

Peng Jing Department of Chemistry, College of Arts and Sciences, Fort Wayne, IN, United States

Brian A. Kelch University of Massachusetts Medical School, Worcester, MA, United States

Xixi Jing Central South University, Changsha, China

Karen E. Keller Horticultural Crops Research Unit, Agricultural Research Service, US Department of Agriculture, Corvallis, OR, United States

Meesbah Jiwaji Rhodes University, Grahamstown, South Africa Kyle L. Johnson The University of Texas at El Paso, El Paso, TX, United States and CQuentia, Memphis, TN, United States Welkin E. Johnson Boston College, Chestnut Hill, MA, United States Ian M. Jones University of Reading, Reading, United Kingdom and London School of Hygiene and Tropical Medicine, London, United Kingdom Ramon Jordan Agricultural Research Service, US Department of Agriculture, Beltsville, MD, United States Thomas Joris Cellular and Molecular Epigenetics (GIGA), Liège, Belgium and Molecular Biology (TERRA), Gembloux, Belgium Ilkka Julkunen Institute of Biomedicine, University of Turku, Turku, Finland Sandra Junglen Charité - University Medicine Berlin, Berlin, Germany Masanori Kaido Kyoto University, Kyoto, Japan

Japhette E. Kembou-Ringert University of Tel Aviv, Tel Aviv, Israel Peter J. Kerr University of Sydney, Sydney, NSW, Australia and CSIRO Health and Biosecurity, Black Mountain Laboratories, Canberra, ACT, Australia Tiffany King Nationwide Children’s Hospital, Columbus, OH, United States and The Ohio State University College of Medicine, Columbus, OH, United States Andrea Kirmaier Boston College, Chestnut Hill, MA, United States Thomas Klose Purdue University, West Lafayette, IN, United States Barbara G. Klupp Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany David M. Knipe Harvard Medical School, Boston, MA, United States Nick J. Knowles The Pirbright Institute, Pirbright, United Kingdom Guus Koch Wageningen Bioveterinary Research, Lelystad, The Netherlands

xxxii

List of Contributors

Renate Koenig Julius Kühn Institute – Federal Research Center for Cultivated Plants, Braunschweig, Germany Susanne E. Kohalmi The University of Western Ontario, London, ON, Canada Hideki Kondo Okayama University, Kurashiki, Japan Jennifer L. Konopka-Anstadt Centers for Disease Control and Prevention, Atlanta, GA, United States Eugene V. Koonin National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, United States and National Institutes of Health, Bethesda, MD, United States Marion P.G. Koopmans Erasmus Medical Center, Rotterdam, The Netherlands Richard Kormelink Wageningen University and Research, Wageningen, The Netherlands Ioly Kotta-Loizou Imperial College London, London, United Kingdom Peter J. Krell Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada Mart Krupovic Archaeal Virology Unit, Institut Pasteur, Paris, France

Manish Kumar Jawaharlal Nehru University, New Delhi, India Gael Kurath US Geological Survey, Western Fisheries Research Center, Seattle, WA, United States Satu Kurkela University of Helsinki and Helsinki University Hospital, Helsinki, Finland Wan-Chun Lai Chang Gung Memorial Hospital, Taoyuan, Taiwan Kevin Lamkiewicz University of Jena, Jena, Germany Rebecca K. Lane University of Texas Health Science Center at San Antonio, San Antonio, TX, United States Andrew S. Lang Memorial University of Newfoundland, St. John’s, NL, Canada Daniel Carlos Ferreira Lanza Federal University of Rio Grande do Norte, Natal, Brazil Maija Lappalainen HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Katherine LaTourrette University of Nebraska–Lincoln, Lincoln, NE, United States

Andreas Kuhn University of Hohenheim, Stuttgart, Germany

Chris Lauber TWINCORE – Center for Experimental and Clinical Infection Research, Hannover, Germany

Jens H. Kuhn National Institutes of Health, Frederick, MD, United States

Antonio Lavazza The Lombardy and Emilia Romagna Experimental Zootechnic Institute, Brescia, Italy

Richard J. Kuhn Purdue University, West Lafayette, IN, United States

C. Martin Lawrence Montana State University, Bozeman, MT, United States

Suvi Kuivanen University of Helsinki, Helsinki, Finland

Hervé Lecoq Plant Pathology Unit, INRAE – French National Research Institute for Agriculture, Food and Environment, Montfavet, France

Ranjababu Kulasegaram Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom Raghavendran Kulasegaran-Shylini Department of Pathogen Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom Gaurav Kumar University of Delhi, New Delhi, India

Young-Min Lee Utah State University, Logan, UT, United States Kristen N. LeGault University of California, Berkeley, CA, United States James Legg International Institute of Tropical Agriculture, Dar es Salaam, Tanzania

List of Contributors

xxxiii

Anne Legreve University of Louvain, Louvain-la-Neuve, Belgium

Walter Ian Lipkin Columbia University, New York, NY, United States

Petr G. Leiman The University of Texas Medical Branch, Galveston, TX, United States

Jan G. Lisby Copenhagen University Hospital Hvidovre, Hvidovre, Denmark

Stanley M. Lemon Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, NC, United States and Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, NC, United States

Ding X. Liu South China Agricultural University, Guangzhou, China

Sebastian Leptihn Zhejiang University-Edinburgh University Institute, Zhejiang University, Haining, China Dennis J. Lewandowski University of Florida, Lake Alfred, FL, United States Sébastien Lhomme Toulouse University Hospital, Toulouse, France and Toulouse University Paul Sabatier, Toulouse, France Dawei Li China Agricultural University, Beijing, China Guangdi Li Central South University, Changsha, China Guoqing Li Huazhong Agricultural University, Wuhan, China Yi Li Peking University, Beijing, China Zhefeng Li College of Pharmacy, The Ohio State University, Columbus, OH, United States Zhenghe Li Zhejang University, Hangzhou, China

Qiang Liu University of Saskatchewan, Saskatoon, SK, Canada Sijun Liu Iowa State University, Ames, IA, United States Carlos Llorens Biotechvana, Scientific Park University of Valencia, Valencia, Spain L. Sue Loesch-Fries Purdue University, West Lafayette, IN, United States George P. Lomonossoff John Innes Centre, Norwich, United Kingdom L. Letti Lopez The University of Texas at Austin, Austin, TX, United States Alan T. Loynachan University of Kentucky, Lexington, KY, United States Garry A. Luke University of St. Andrews, St. Andrews, United Kingdom M. Luo University of Alabama at Birmingham, Birmingham, AL, United States Juan J. López-Moya Center for Research in Agricultural Genomics and Spanish National Research Council, Barcelona, Spain

Jia Q. Liang South China Agricultural University, Guangzhou, China

Che Ma Genomics Research Center, Academia Sinica, Taipei, Taiwan

Sebastian Liebe Institute of Sugar Beet Research, Göttingen, Germany

Stuart A. MacFarlane The James Hutton Institute, Invergowrie, United Kingdom

João Paulo Matos Santos Lima Federal University of Rio Grande do Norte, Natal, Brazil

Saichetana Macherla J. Craig Venter Institute, La Jolla, CA, United States

Bruno Lina HCL Department of Virology, National Reference Center for Respiratory Viruses, Institute of Infectious Agents, Croix-Rousse Hospital, Lyon, France and Virpath Laboratory, International Center of Research in Infectiology (CIRI), INSERM U1111, CNRS—UMR 5308, École Normale Supérieure de Lyon, University Claude Bernard Lyon, Lyon University, Lyon, France

Kensaku Maejima The University of Tokyo, Tokyo, Japan Fabrizio Maggi University of Pisa, Pisa, Italy and University of Insubria, Varese, Italy Melissa S. Maginnis The University of Maine, Orono, ME, United States

xxxiv

List of Contributors

Edgar Maiss Leibniz University Hannover, Hannover, Germany

Chikara Masuta Hokkaido University, Sapporo, Japan

Kira S. Makarova National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, United States

Carlos P. Mata University of Leeds, Leeds, United Kingdom

Ariana Manglli Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, Rome, Italy

Jelle Matthijnssens Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

Annette Mankertz Robert Koch-Institute, Berlin, Germany

Claire P. Mattison Centers for Disease Control and Prevention, Atlanta, GA, United States and Cherokee Nation Assurance, Arlington, VA, United States

Pilar Manrique The Ohio State University, Wexner Medical Center, Columbus, OH, United States

William McAllister Rowan University School of Osteopathic Medicine, Stratford, NJ, United States

Shahid Mansoor National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan

Alison A. McBride National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States

Marco Marklewitz Institute of Virology, Charité – University Medicine Berlin, Berlin, Germany Giovanni P. Martelli† University of Bari Aldo Moro, Bari, Italy Darren P. Martin University of Cape Town, Cape Town, South Africa Robert R. Martin Horticultural Crops Research Unit, Agricultural Research Service, US Department of Agriculture, Corvallis, OR, United States Manuel Martinez-Garcia University of Alicante, Alicante, Spain Francisco Martinez-Hernandez University of Alicante, Alicante, Spain Natalia Martín-González Autonomous University of Madrid, Madrid, Spain Joaquín Martínez Martínez Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, United States Manja Marz University of Jena, Jena, Germany Andrea Marzi National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States Hema Masarapu Sri Venkateswara University, Tirupati, India †

Deceased.

Michael McChesney University of California, Davis, CA, United States Elaine McCulloch Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom Andrew J. McMichael University of Oxford, Oxford, United Kingdom Alexander McPherson University of California, Irvine, CA, United States Irene K. Meki French National Center for Scientific Research, Montpellier, France Ulrich Melcher Oklaoma State University, Stillwater, OK, United States Tomas A Melgarejo University of California, Davis, CA, United States Michael J. Melzer Department of Plant and Environmental Protection Sciences, University of Hawaii, Honolulu, HI, United States Luiza Mendonça University of Oxford, Oxford, United Kingdom Xiang-Jin Meng Virginia Polytechnic Institute and State University, Blacksburg, VA, United States Peter P.C. Mertens University of Nottingham, Sutton Bonington, United Kingdom

List of Contributors

Thomas C. Mettenleiter Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany Philip D. Minor St Albans, United Kingdom Ali Mirazimi National Veterinary Institute, Uppsala, Sweden and Karolinska Hospital University, Huddinge, Sweden Nischay Mishra Columbia University, New York, NY, United States Edward S. Mocarski Emory University School of Medicine, Atlanta, GA, United States Florian Mock University of Jena, Jena, Germany Volker Moennig University of Veterinary Medicine, Hannover, Germany Ian J. Molineux The University of Texas at Austin, Austin, TX, United States Aderito L. Monjane Norwegian Veterinary Institute, Oslo, Finland Jacen S. Moore University of Tennessee Health Science Center, Memphis, TN, United States Marc C. Morais The University of Texas Medical Branch, Galveston, TX, United States Cristina Moraru Institute for Chemistry and Biology of the Marine Environment, Oldenburg, Germany Hiromitsu Moriyama Tokyo University of Agriculture and Technology, Tokyo, Japan Sergey Y. Morozov Lomonosov Moscow State University, Moscow, Russia Thomas E. Morrison University of Colorado School of Medicine, Aurora, CO, United States Léa Morvan University of Liège, Liège, Belgium Benoît Moury Plant Pathology Unit, INRAE – French National Institute for Agriculture, Food and Environment, Montfavet, France

xxxv

Muhammad Mubin University of Agriculture, Faisalabad, Pakistan Nicolas J. Mueller University Hospital of Zurich, Zurich, Switzerland Emmanuelle Muller The French Agricultural Research Center for International Development, Joint Research Units–Biology and Genetics of Plant-Pathogen Interactions, Montpellier, France and Biology and Genetics of PlantPathogen Interactions, University of Montpellier, The French Agricultural Research Center for International Development, French National Institute for Agricultural Research, Montpellier SupAgro, Montpellier, France John S. Munday Massey University, Palmerston North, New Zealand Jacob H. Munson-McGee Montana State University, Bozeman, MT, United States and Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, United States Hacer Muratoğlu Department of Molecular Biology and Genetics, Karadeniz Technical University, Trabzon, Turkey Kenan C. Murphy University of Massachusetts Medical School, Worcester, MA, United States Ugrappa Nagalakshmi University of California, Davis, CA, United States Keizo Nagasaki Kochi University, Nankoku, Japan Nazia Nahid GC University, Faisalabad, Pakistan and University of Agriculture, Faisalabad, Pakistan Venugopal Nair The Pirbright Institute, Pirbright, United Kingdom Remziye Nalçacıoğlu Department of Molecular Biology and Genetics, Karadeniz Technical University, Trabzon, Turkey Shigetou Namba The University of Tokyo, Tokyo, Japan Rubab Z. Naqvi National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Rachel Nash The Pirbright Institute, Surrey, United Kingdom C.K. Navaratnarajah Purdue University, West Lafayette, IN, United States

xxxvi

List of Contributors

Maria A. Navarrete-Muñoz Biotechvana, Madrid, Spain; Institute of Health Research-Jiménez Díaz Foundation, Autonomous University of Madrid; and Rey Juan Carlos University Hospital, Móstoles, Spain Jesús Navas-Castillo Institute for Mediterranean and Subtropical Horticulture “La Mayora”–Spanish National Research Council– University of Malaga, Algarrobo-Costa, Málaga, Spain Muhammad S. Nawaz-ul-Rehman University of Agriculture, Faisalabad, Pakistan Christopher L. Netherton The Pirbright Institute, Pirbright, United Kingdom Thu V.P. Nguyen Baylor College of Medicine, Houston, TX, United States Annette Niehl Julius Kühn Institute – Federal Research Center for Cultivated Plants, Braunschweig, Germany Hubert G.M. Niesters Department of Medical Microbiology and Infection Prevention, Division of Clinical Virology, University Medical Center Groningen, Groningen, The Netherlands Jozef I. Nissimov University of Waterloo, Waterloo, ON, Canada Norman Noah London School of Hygiene and Tropical Medicine, London, United Kingdom Mauricio L. Nogueira São José do Rio Preto School of Medicine, São José do Rio Preto, São Paulo, Brazil Johan Nordgren Linköping University, Linköping, Sweden C. Micha Nübling Paul-Ehrlich-Institute, Langen, Germany Visa Nurmi University of Helsinki, Helsinki, Finland

Hanna M. Oksanen Molecular and Integrative Biosciences Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Graziele P. Oliveira Federal University of Minas Gerais, Belo Horizonte, Brazil Francesco Origgi University of Bern, Bern, Switzerland Nikolaus Osterrieder Free University of Berlin, Berlin, Germany Robert A. Owens Beltsville Agricultural Research Center, Beltsville, MD 20705, United States Emine Özsahin University of Guelph, Guelph, ON, Canada Sergi Padilla-Parra University of Oxford, Oxford, United Kingdom; Department of Infectious Diseases, Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom; and Randall Division of Cell and Molecular Biophysics, King’s College London, London, United Kingdom Joshua Pajak Duke University, Durham, NC, United States Massimo Palmarini MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom Amanda R. Panfil The Ohio State University, Columbus, OH, United States Marcus Panning Institute of Virology, Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Donald L. Nuss University of Maryland, Rockville, MD, United States

Vitantonio Pantaleo National Research Council, Research Unit of Bari, Bari, Italy

M. Steven Oberste Centers for Disease Control and Prevention, Atlanta, GA, United States

Anna Papa Aristotle University of Thessaloniki, Thessaloniki, Greece

Hiroyuki Ogata Institute for Chemical Research, Kyoto University, Kyoto, Japan Ane Ogbe University of Oxford, Oxford, United Kingdom

Nikolaos Pappas Utrecht University, Utrecht, The Netherlands Hanu R. Pappu Washington State University, Pullman, WA, United States

List of Contributors

xxxvii

Kristin N. Parent Michigan State University, East Lansing, MI, United States

Jean-Marie Peron Toulouse University Hospital, Toulouse, France and Toulouse University Paul Sabatier, Toulouse, France

Colin R. Parrish Cornell University, Ithaca, NY, United States

Karin E. Peterson National Institutes of Health, Hamilton, MT, United States

A. Lorena Passarelli Kansas State University, Manhattan, KS, United States

Karel Petrzik Biology Center CAS, Institute of Plant Molecular Biology, České Budějovice, Czech Republic

Basavaprabhu L. Patil ICAR–Indian Institute of Horticultural Research, Bengaluru, India Jade Pattyn University of Antwerp, Antwerp, Belgium T.A. Paul Cornell University, Ithaca, NY, United States Lillian Pavlik Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste Marie, ON, Canada Susan L. Payne Texas A& M University, College Station, TX, United States Michael N. Pearson The University of Auckland, Auckland, New Zealand Mark E. Peeples Nationwide Children’s Hospital, Columbus, OH, United States and The Ohio State University College of Medicine, Columbus, OH, United States Ben Peeters Wageningen Bioveterinary Research, Lelystad, The Netherlands Joseph S.M. Peiris The University of Hong Kong, Pok Fu Lam, Hong Kong Malik Peiris The University of Hong Kong, Pok Fu Lam, Hong Kong Judit J. Pénzes National Institute of Scientific Research – ArmandFrappier Health Research Centre, Laval, QC, Canada Miryam Pérez-Cañamás Institute for Plant Molecular and Cell Biology (Spanish National Research Council–Polytechnic University of Valencia), Valencia, Spain

Mahtab Peyambari Pennsylvania State University, State College, PA, United States Sujal Phadke J. Craig Venter Institute, La Jolla, CA, United States Hanh T. Pham National Institute of Scientific Research – ArmandFrappier Health Research Centre, Laval, QC, Canada Mauro Pistello University of Pisa, Pisa, Italy Daniel Ponndorf John Innes Centre, Norwich, United Kingdom Leo L.M. Poon The University of Hong Kong, Pok Fu Lam, Hong Kong Welkin H. Pope University of Pittsburgh, Pittsburgh, PA, United States Minna M. Poranen University of Helsinki, Helsinki, Finland Claudine Porta The Pirbright Institute, Pirbright, United Kingdom and University of Oxford, Oxford, United Kingdom Samuel S. Porter National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States and University of Maryland, College Park, MD, United States Frank A. Post King's College Hospital NHS Foundation Trust, London, United Kingdom

Marta Pérez-Illana National Center for Biotechnology, Spanish National Research Council, Madrid, Spain

Nils Poulicard Interactions Plantes Microorganismes Environnement, Institut de Recherche pour le Développement, Centre de coopération internationale en recherche agronomique pour le développement, University of Montpellier, Montpellier, France

Jaume Pérez-Sánchez Institute of Aquaculture Torre de la Sal, Spanish National Research Council, Castellon, Spain

David Prangishvili Institut Pasteur, Paris, France and Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia

xxxviii

List of Contributors

B. V. Venkataram Prasad Baylor College of Medicine, Houston, TX, United States Lalita Priyamvada Centers for Disease Control and Prevention, Atlanta, GA, United States Simone Prospero Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland

Chris M. Rands University of Geneva Medical School and Swiss Institute of Bioinformatics, Geneva, Switzerland Venigalla B. Rao The Catholic University of America, Washington, DC, United States Janne J. Ravantti University of Helsinki, Helsinki, Finland

Elisabeth Puchhammer-Stöckl Medical University of Vienna, Vienna, Austria

Mandy Ravensbergen Wageningen University and Research, Wageningen, The Netherlands

Jianming Qiu University of Kansas Medical Center, Kansas City, KS, United States

Georget Y. Reaiche-Miller The University of Adelaide, Adelaide, SA, Australia

S.L. Quackenbush Colorado State University, Fort Collins, CO, United States

D.V.R. Reddy International Crops Research Institute for the Semi-Arid Tropics, Hyderabad, India

Killian J. Quinn King’s College Hospital, London, United Kingdom

Vishwanatha R.A.P. Reddy The Pirbright Institute, Pirbright, United Kingdom

Diego F. Quito-Avila Department of Life Sciences, ESPOL Polytechnic University, Guayaquil, Ecuador

Juan Reguera Aix-Marseille University, French National Center for Scientific Research, Marseille, France and French National Institute of Health and Medical Research, Marseille, France

Frank Rabenstein Julius Kühn Institute, Quedlinburg, Germany Sheli R. Radoshitzky United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States Saleem U. Rahman National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Mbolarinosy Rakotomalala FOFIFA, Antananarivo, Madagascar

William K. Reisen University of California, Davis, CA, United states Jingshan Ren University of Oxford, Oxford, United Kingdom Renato O. Resende University of Brasilia, Brasilia, Brazil Peter A. Revill The Peter Doherty Institute of Infection and Immunity, Royal Melbourne Hospital, Melbourne, VIC, Australia

Norma Rallon Institute of Health Research-Jiménez Díaz Foundation, Autonomous University of Madrid and Rey Juan Carlos University Hospital, Móstoles, Spain

Félix A. Rey Institut Pasteur, Paris, France

Robert P. Rambo Diamond Light Source, Didcot, United Kingdom

Simone G. Ribeiro Embrapa Genetic Resources and Biotechnology, Brasília, Brazil

Bertha Cecilia Ramirez The Institute for Integrative Biology of the Cell, The French Alternative Energies and Atomic Energy Commission, French National Center for Scientific Research, University of Paris-Sud, University of Paris-Saclay, Gif-sur-Yvette, France María D. Ramos-Barbero University of Alicante, Alicante, Spain

Lara Rheinemann University of Utah, Salt Lake City, UT, United States

Daniel Rigling Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland Cristina Risco Cell Structure Laboratory, National Center for Biotechnology – Spanish National Research Council (CNB-CSIC), Madrid, Spain

List of Contributors

Efraín E. Rivera-Serrano Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, NC, United States and Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, NC, United States Cécile Robin INRAE – French National Research Institute for Agriculture, Food and Environment, UMR BIOGECO, Cestas, France Rodrigo A.L. Rodrigues Federal University of Minas Gerais, Belo Horizonte, Brazil Elina Roine University of Helsinki, Helsinki, Finland Maria R. Rojas University of California, Davis, CA, United States Marilyn J. Roossinck Pennsylvania State University, State College, PA, United States Vera I.D. Ros Wageningen University and Research, Wageningen, The Netherlands Cristina Rosa Pennsylvania State University, University Park, PA, United States Hanna Rose Leibniz University Hannover, Hannover, Germany David A. Rosenbaum University of Florida, Gainesville, FL, United States Shannan L. Rossi The University of Texas Medical Branch, Galveston, TX, United States Michael G. Rossmann† Purdue University, West Lafayette, IN, United States

xxxix

Polly Roy Department of Pathogen Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom and University of Reading, Reading, United Kingdom Aaron P. Roznowski The University of Texas at Austin, Austin, TX, United States and University of Arizona, Tucson, AZ, United States Luisa Rubino Institute for Sustainable Plant Protection, National Research Council, Bari, Italy Olli Ruuskanen Turku University Hospital, Turku, Finland Eugene V. Ryabov USDA, Agricultural Research Service, Beltsville, MD, United States Martin D. Ryan University of St. Andrews, St. Andrews, United Kingdom Ki H. Ryu Seoul Women’s University, Seoul, South Korea Hanns-Joachim Rziha Eberhard Karls University of Tübingen, Tübingen, Germany Sead Sabanadzovic Mississippi State University, Starkville, MS, United States Roghaiyeh Safari Cellular and Molecular Epigenetics (GIGA), Liège, Belgium and Molecular Biology (TERRA), Gembloux, Belgium Azeez Sait Sahul Hameed C. Abdul Hakeem College, Melvisharam, India

L. Roux University of Geneva Medical School, Geneva, Switzerland

Nicole Samies University of Alabama at Birmingham, Birmingham, AL, United States

Simon Roux US Department of Energy Joint Genome Institute, Walnut Creek, CA, United States

Carmen San Martín National Center for Biotechnology, Spanish National Research Council, Madrid, Spain

J. Rovnak Colorado State University, Fort Collins, CO, United States David J. Rowlands University of Leeds, Leeds, United Kingdom †

Deceased.

Ruth-Anne Sandaa Department of Biological Sciences, University of Bergen, Bergen, Norway Hélène Sanfaçon Agriculture and Agri-Food Canada, Summerland, BC, Canada

xl

List of Contributors

Rafael Sanjuán Institute for Integrative Systems Biology (I2SysBio), University of Valencia-CSIC, Valencia, Spain

Declan C. Schroeder University of Reading, Reading, United Kingdom and University of Minnesota, St. Paul, MN, United States

Neeraja Sankaran Utrecht University, Utrecht, The Netherlands

Stacey Schultz-Cherry St. Jude Children’s Research Hospital, Memphis, TN, United States

Fernando Santos University of Alicante, Alicante, Spain Cecilia Sarmiento Tallinn University of Technology, Tallinn, Estonia Takahide Sasaya National Agriculture and Food Research Organization, Fukuyama, Japan Preethi Sathanantham Virginia Tech, Blacksburg, VA, United States Panayampalli S. Satheshkumar Centers for Disease Control and Prevention, Atlanta, GA, United States Yukiyo Sato Okayama University, Kurashiki, Japan Andreas Sauerbrei Jena University Hospital, Jena, Germany Eugene I. Savenkov Swedish University of Agricultural Sciences, Uppsala, Sweden and Linnean Center for Plant Biology, Uppsala, Sweden Carita Savolainen-Kopra National Institute for Health and Welfare, Helsinki, Finland Kay Scheets Oklahoma State University, Stillwater, OK, United States Uffe V. Schenider Copenhagen University Hospital Hvidovre, Hvidovre, Denmark Richard H. Scheuermann J. Craig Venter Institute, La Jolla, CA, United States; University of California, San Diego, CA, United States; La Jolla Institute for Immunology, La Jolla, CA, United States; and Global Virus Network, Baltimore, MD, United States Manfred J. Schmitt Saarland University, Saarbrücken, Germany James E. Schoelz University of Missouri, Columbia, MO, United States Jason R. Schrad Michigan State University, East Lansing, MI, United States

Thomas F. Schulz Hannover Medical School, Institute of Virology, Hannover, Germany and German Center for Infection Research, Hannover-Braunschweig Site, Braunschweig, Germany Catherine A. Scougall The University of Adelaide, Adelaide, SA, Australia Kimberley D. Seed University of California, Berkeley, CA, United States Joaquim Segalés Departament of Animal Health and Anatomy, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain; Animal Health Research Center (CReSA) – Institute of Agrifood Research and Technology (IRTA), Campus UAB, Barcelona, Spain; and OIE Collaborating Center for the Research and Control of Emerging and Re-emerging Swine Diseases in Europe (IRTA-CReSA), Barcelona, Spain Mateo Seoane-Blanco National Center for Biotechnology, Madrid, Spain Madhumati Sevvana Purdue University, West Lafayette, IN, United States Kazım Sezen Department of Biology, Karadeniz Technical University, Trabzon, Turkey Arvind Sharma Institut Pasteur, Paris, France Sumit Sharma Linköping University, Linköping, Sweden James M. Sharp University of Zaragoza, Zaragoza, Spain and Edinburgh, United Kingdom Qunxin She Shandong University, Qingdao, China Keith E. Shearwin The University of Adelaide, Adelaide, SA, Australia Hanako Shimura Hokkaido University, Sapporo, Japan Reina S. Sikkema Erasmus Medical Center, Rotterdam, The Netherlands

List of Contributors

Aaron Simkovich Agriculture and Agri-Food Canada, London, ON, Canada and The University of Western Ontario, London, ON, Canada Peter Simmonds University of Oxford, Oxford, United Kingdom Tarja Sironen University of Helsinki, Helsinki, Finland Susanna Sissonen Finnish Institute for Health and Welfare, Helsinki, Finland Michael A. Skinner Imperial College London, London, United Kingdom Douglas E. Smith University of California, San Diego, La Jolla, CA, United States Melvyn Smith Viapath Analytics, Specialist Virology Centre, King’s College NHS Foundation Trust, London, United Kingdom Thomas J. Smith The University of Texas Medical Branch, Galveston, TX, United States Teemu Smura Helsinki University Hospital and University of Helsinki, Helsinki, Finland Eric J. Snijder Leiden University Medical Center, Leiden, The Netherlands Gisela Soboll Hussey Michigan State University, East Lansing, MI, United States Maria Söderlund-Venermo University of Helsinki, Helsinki, Finland Merike Sõmera Tallinn University of Technology, Tallinn, Estonia Eun G. Song Seoul Women’s University, Seoul, South Korea Milan J. Sonneveld Erasmus University Medical Center, Rotterdam, The Netherlands Beatriz Soriano Biotechvana, Scientific Park University of Valencia and Institute for Integrative Systems Biology (I2SysBio), University of Valencia–Spanish National Research Council, Valencia, Spain

xli

Thomas E. Spencer University of Missouri, Columbia, MO, United States Pothur Sreenivasulu Sri Venkateswara University, Tirupati, India Ashley L. St. John Duke-NUS Medical School, Singapore, Singapore David K. Stammers University of Oxford, Oxford, United Kingdom John Stanley John Innes Centre, Colney, United Kingdom Glyn Stanway University of Essex, Colchester, United Kingdom Thilo Stehle University of Tuebingen, Tuebingen, Germany and Vanderbilt University School of Medicine, Nashville, TN, United States Gregory W. Stevenson Iowa State University, Ames, IA, United States Lucy Rae Stewart Agricultural Research Service, US Department of Agriculture, Wooster, OH, United States C.C.M.M. Stijger Wageningen University and Research Center, Bleiswijk, The Netherlands Peter G. Stockley University of Leeds, Leeds, United Kingdom David Stone Weymouth Laboratory, Centre for Environment, Fisheries and Aquaculture Science, Weymouth, United Kingdom Ashley E Strother The University of Texas Medical Branch, Galveston, TX, United States Sundharraman Subramanian Michigan State University, East Lansing, MI, United States William C. Summers Yale University, New Haven, CT, United States Liying Sun Northwest A& F University, Yangling, China Wesley I. Sundquist University of Utah, Salt Lake City, UT, United States Petri Susi University of Turku, Turku, Finland Curtis A. Suttle University of British Columbia, Vancouver, BC, Canada

xlii

List of Contributors

Nobuhiro Suzuki Institute of Plant Stress and Resources (IPSR), Okayama University, Kurashiki, Japan Lennart Svensson Linköping University, Linköping, Sweden and Karolinska Institute, Stockholm, Sweden Ronald Swanstrom University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Nicholas M.I. Taylor University of Copenhagen, Copenhagen, Denmark Xu Tengzhi University of California, Davis, CA, United States Raquel Tenorio Cell Structure Laboratory, National Center for Biotechnology – Spanish National Research Council (CNB-CSIC), Madrid, Spain

Daniele M. Swetnam University of California, Davis, CA, United states

Robert B. Tesh The University of Texas Medical Branch, Galveston, TX, United States

Moriah L. Szpara Pennsylvania State University, University Park, PA, United States

Vaskar Thapa Pennsylvania State University, State College, PA, United States

Keisuke Tabata Heidelberg University, Heidelberg, Germany

John E. Thomas The University of Queensland, Brisbane, QLD, Australia

Anna Taglienti Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, Rome, Italy

Julie A. Thomas Rochester Institute of Technology, Rochester, NY, United States

Naoki Takeshita Tokyo University of Agriculture and Technology, Fuchu, Japan Kana Takeshita Urayama Tokyo University of Agriculture and Technology, Fuchu, Japan Michael E. Taliansky The James Hutton Institute, Dundee, United Kingdom Pan Tao The Catholic University of America, Washington, DC, United States

Lynn C. Thomason Frederick National Laboratory for Cancer Research, Frederick, MD, United States Elizabeth Ashley Thompson The University of Southern Mississippi, Hattiesburg, MS, United States Jeremy R. Thompson Cornell University, Ithaca, NY, United States Antonio Tiberini Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, Rome, Italy

Jacqueline E. Tate Centers for Disease Control and Prevention, Atlanta, GA, United States

Peter Tijssen National Institute of Scientific Research – ArmandFrappier Health Research Centre, Microbiology and Immunology, Laval, QC, Canada

Satyanarayana Tatineni Agricultural Research Service, US Department of Agriculture, Lincoln, NE, United States and University of Nebraska–Lincoln, Lincoln, NE, United States

Yuji Tomaru Japan Fisheries Research and Education Agency, Kanagawa, Japan

Sisko Tauriainen University of Turku, Turku, Finland Norbert Tautz University of Luebeck, Luebeck, Germany Paulo Tavares Institute for Integrative Biology of the Cell, CEA, CNRS, University of Paris-Sud, University of Paris-Saclay, Gif-sur-Yvette, France

Laura Tomassoli Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, Rome, Italy Ruben Torres National Biotechnology Center–Spanish National Research Council, Madrid, Spain Jia Q. Truong The University of Adelaide, Adelaide, SA, Australia

List of Contributors

Erkki Truve† Tallinn University of Technology, Tallinn, Estonia Chih-Hsuan Tsai Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Roman Tuma University of Leeds, Leeds, United Kingdom and University of South Bohemia, České Budějovice, Czech Republic Topi Turunen Infectious Disease Unit, Espoo, Finland and Finnish Institute for Health and Welfare, Helsinki, Finland Reidun Twarock University of York, York, United Kingdom Ioannis E. Tzanetakis University of Arkansas, Fayetteville, United States Antti Vaheri University of Helsinki, Helsinki, Finland Eeva J. Vainio Natural Resources Institute Finland (Luke), Helsinki, Finland Anna M. Vaira Institute for Sustainable Plant Protection, National Research Council of Italy, Torino, Italy Steven M. Valles Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, US Department of Agriculture, Gainesville, FL, United States Adrián Valli National Center for Biotechnology-Spanish National Research Council, Madrid, Spain Rodrigo A. Valverde Louisiana State University Agricultural Center, Baton Rouge, United States Pierre Van Damme University of Antwerp, Antwerp, Belgium Rene A.A. van der Vlugt Wageningen University and Research Center, Wageningen, The Netherlands Bernard A.M. Van der Zeijst Leiden University Medical Center, Leiden, The Netherlands Koenraad Van Doorslaer University of Arizona, Tucson, AZ, United States †

Deceased.

xliii

James L. Van Etten University of Nebraska–Lincoln, Lincoln, NE, United States Suzanne van Meer University Medical Center Utrecht, Utrecht, The Netherlands Monique M. van Oers Wageningen University and Research, Wageningen, The Netherlands Mark J. van Raaij National Center for Biotechnology, Madrid, Spain Marc H.V. Van Regenmortel University of Strasbourg, Strasbourg, France Piet A. van Rijn Wageningen Bioveterinary Research, Lelystad, The Netherlands and North-West University, Potchefstroom, South Africa Alain Vanderplasschen University of Liège, Liège, Belgium Dana L. Vanlandingham College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States Olli Vapalahti Helsinki University Hospital and University of Helsinki, Helsinki, Finland Mark Varrelmann Institute of Sugar Beet Research, Göttingen, Germany Nikos Vasilakis The University of Texas Medical Branch, Galveston, TX, United States Michael Veit Free University of Berlin, Berlin, Germany Česlovas Venclovas Vilnius University, Vilnius, Lithuania H. Josef Vetten Julius Kühn Institute, Braunschweig, Germany Marli Vlok University of British Columbia, Vancouver, BC, Canada Anne-Nathalie Volkoff Diversity, Genomes and Insects-Microorganisms Interactions, National Institute of Agricultural Research, University of Montpellier, Montpellier, France Ian E.H. Voorhees Cornell University, Ithaca, NY, United States Alex Vorsters University of Antwerp, Antwerp, Belgium

xliv

List of Contributors

Jonathan D.F. Wadsworth UCL Institute of Prion Diseases, London, United Kingdom

Kerstin Wernike Friedrich-Loeffler-Institute, Insel Riems, Germany

Peter J. Walker The University of Queensland, St. Lucia, QLD, Australia

Rachel J. Whitaker University of Illinois at Urbana-Champaign, Urbana, IL, United States

Paul Wallace Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom

K. Andrew White York University, Toronto, ON, Canada

Aiming Wang Agriculture and Agri-Food Canada, London, ON, Canada

Anna E. Whitfield North Carolina State University, Raleigh, NC, United States

Jen-Ren Wang National Cheng Kung University, Tainan, Taiwan

Richard Whitley University of Alabama at Birmingham, Birmingham, AL, United States

Lin-Fa Wang Duke-NUS Medical School, Singapore, Singapore Nan Wang Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Xiangxi Wang Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Xiaofeng Wang Virginia Tech, Blacksburg, VA, United States Katherine N. Ward University College London, London, United Kingdom Matti Waris University of Turku, Turku, Finland Ranjit Warrier Purdue University, West Lafayette, IN, United States Daniel Watterson Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia Marta L. Wayne University of Florida, Gainesville, FL, United States

Reed B. Wickner National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States Luc Willems Cellular and Molecular Epigenetics (GIGA), Liège, Belgium and Molecular Biology (TERRA), Gembloux, Belgium Brian J. Willett MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kindom Alexis Williams The University of Texas Medical Branch, Galveston, TX, United States Stephen A. Winchester Frimley Park Hospital, Frimley, United Kingdom and Immunisation and Countermeasures Division, Public Health England, London, United Kingdom Clayton W. Winkler National Institutes of Health, Hamilton, MT, United States

Friedemann Weber FB 10 – Institute for Virology, Justus Liebig University Giessen, Giessen, Germany

Stephan Winter Leibniz Institute – DSMZ – German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany

Sung-Chan Wei Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

William M. Wintermantel Agricultural Research Service, US Department of Agriculture, Salinas, CA, United States

Robin A. Weiss University College London, London, United Kingdom

Jennifer Wirth Montana State University, Bozeman, MT, United States

Tao Weitao Southwest Baptist University, Bolivar, MO, United States

Yuri I. Wolf National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, United States

List of Contributors

xlv

Thorsten Wolff Robert Koch Institute, Berlin, Germany

Lawrence S. Young University of Warwick, Coventry, United Kingdom

Blaide Woodburn University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Mark J. Young Montana State University, Bozeman, MT, United States

Michael E. Woodson The University of Texas Medical Branch, Galveston, TX, United States Courtney Woolsey The University of Texas Medical Branch, Galveston, TX, United States Chien-Fu Wu Tokyo University of Agriculture and Technology, Fuchu, Japan Mingde Wu Huazhong Agricultural University, Wuhan, China Songsong Wu Huazhong Agricultural University, Wuhan, China Yan Xiang University of Texas Health Science Center at San Antonio, San Antonio, TX, United States Jiatao Xie Huazhong Agricultural University, Wuhan, China Zhuang Xiong Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China Hajime. Yaegashi Institute of Fruit Tree and Tea Science, NARO, Morioka, Japan Mehtap Yakupoğlu Trabzon University, Trabzon, Turkey Yasuyuki Yamaji The University of Tokyo, Tokyo, Japan Meng Yang China Agricultural University, Beijing, China Teng-Chieh Yang Scarsdale, NY, United States Qin Yao Jiangsu University, Zhenjiang, China Tianyou Yao Baylor College of Medicine, Houston, TX, United States Nobuyuki Yoshikawa Iwate University, Morioka, Japan George R. Young Francis Crick Institute, London, United Kingdom

Ry Young Texas A& M University, College Station, TX, United States Isaac T. Younker University of Alabama, Tuscaloosa, AL, United States Qian Yu School of Life Sciences, Jiangsu University, Zhenjiang, China Sang-Im Yun Utah State University, Logan, UT, United States Fauzia Zarreen University of Delhi, New Delhi, India Francisco M. Zerbini Federal University of Viçosa, Viçosa, Brazil Dong-Xiu Zhang University of Maryland, Rockville, MD, United States Jianqiang Zhang Iowa State University, Ames, IA, United States Junjie Zhang Texas A& M University, College Station, TX, United States Long Zhang College of Pharmacy, The Ohio State University, Columbus, OH, United States Pan Zhang Central South University, Changsha, China Peijun Zhang University of Oxford, Oxford, United Kingdom and Electron Bio-Imaging Centre, Diamond Light Source, Didcot, United Kingdom Tao Zhang Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China Yongliang Zhang China Agricultural University, Beijing, China Zhenlu Zhang Shandong Agricultural University, Tai’an, China Lixia Zhou College of Pharmacy, The Ohio State University, Columbus, OH, United States

xlvi

List of Contributors

Ling Zhu Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Heiko Ziebell Julius Kühn Institute – Federal Research Center for Cultivated Plants, Braunschweig, Germany John Ziebuhr The Queen's University of Belfast, Belfast, United Kingdom

Jeffrey J. Zimmerman Iowa State University, Ames, IA, United States Falk Zucker Institute for Chemistry and Biology of the Marine Environment, Oldenburg, Germany

CONTENT OF ALL VOLUMES Editors in Chief

v

Editorial Board

vii

Section Editors

ix

Foreword

xv

Preface

xvii

Guide to Use

xix

List of Contributors

xxi

VOLUME 1 The Virus as a Concept – Fundamentals of Virology A Brief History of Virology David J Rowlands

3

The Origin of Viruses Patrick Forterre and Morgan Gaïa

14

The Virocell Concept Patrick Forterre

23

Virus Taxonomy Jens H Kuhn

28

The Greater Virus World and Its Evolution Eugene V Koonin and Valerian V Dolja

38

The Virus Species Concept Peter Simmonds

47

Genetic Diversity and Evolution of Viral Populations Rafael Sanjuán and Pilar Domingo-Calap

53

Mechanisms of RNA Virus Evolution Lisa M Bono and Siobain Duffy

62

Mechanisms of DNA Virus Evolution Moriah L Szpara and Koenraad Van Doorslaer

71

Paleovirology Clément Gilbert

79

Evolution Steered by Structure Nicola GA Abrescia

87

xlvii

xlviii

Content of all Volumes

Pairwise Sequence Comparison in Virology Tao Zhang, Zheng Gong, Tongkun Guo, Zhuang Xiong, and Yiming Bao

100

Computational Analysis of Recombination in Viral Nucleotide Sequences Miguel Arenas

108

Phylogeny of Viruses Alexander E Gorbalenya and Chris Lauber

116

Virus Bioinformatics Nikolaos Pappas, Simon Roux, Martin Hölzer, Kevin Lamkiewicz, Florian Mock, Manja Marz, and Bas E Dutilh

124

Metagenomics in Virology Simon Roux, Jelle Matthijnssens, and Bas E Dutilh

133

Database and Analytical Resources for Viral Research Community Sujal Phadke, Saichetana Macherla, and Richard H Scheuermann

141

Classification of the Viral World Based on Atomic Level Structures Janne J Ravantti and Nicola GA Abrescia

153

Isolating, Culturing, and Purifying Viruses With a Focus on Bacterial and Archaeal Viruses Katri Eskelin and Hanna M Oksanen

162

High Throughput Sequencing and Virology Graham L Freimanis and Nick J Knowles

175

Single-Virus Genomics: Studying Uncultured Viruses, One at a Time Manuel Martinez-Garcia, Francisco Martinez-Hernandez, and Joaquín Martínez Martínez

184

Biophysical Characterizations in the Solution State Robert P Rambo and Katsuaki Inoue

191

Virus Crystallography Jonathan M Grimes

199

Advanced Light and Correlative Microscopy in Virology Sergi Padilla-Parra, Charles A Coomer, and Irene Carlon-Andres

208

Atomic Force Microscopy (AFM) Investigation of Viruses Alexander McPherson

218

Cryo-Electron Microscopy (CEM) Structures of Viruses David Chmielewski and Wah Chiu

233

Analysis of Viruses in the Cellular Context by Electron Tomography Peijun Zhang and Luiza Mendonça

242

Mathematical Modeling of Virus Architecture Reidun Twarock

248

Principles of Virus Structure Madhumati Sevvana, Thomas Klose, and Michael G Rossmann†

257

Structures of Small Icosahedral Viruses Elizabeth E Fry, Jingshan Ren, and Claudine Porta

278

Structural Principles of the Flavivirus Particle Organization and of Its Conformational Changes Stéphane Duquerroy, Arvind Sharma, and Félix A Rey

290

Reoviruses (Reoviridae) and Their Structural Relatives Liya Hu, Mary K Estes, and B V Venkataram Prasad

303

†

Deceased.

Content of all Volumes

xlix

Structures of Tailed Phages and Herpesviruses (Herpesviridae) Montserrat Fàbrega-Ferrer and Miquel Coll

318

Adenoviruses (Adenoviridae) and Their Structural Relatives Gabriela N Condezo, Natalia Martín-González, Marta Pérez-Illana, Mercedes Hernando-Pérez, José Gallardo, and Carmen San Martín

329

Negative Single-Stranded RNA Viruses (Mononegavirales): A Structural View Juan Reguera

345

Structure of Retrovirus Particles (Retroviridae) David K Stammers and Jingshan Ren

352

Structure of Helical Viruses C Martin Lawrence

362

Giant Viruses and Their Virophage Parasites Rodrigo AL Rodrigues, Ana CdSP Andrade, Graziele P Oliveira, and Jônatas S Abrahão

372

Viral Replication Cycle AJ Cann

382

Viral Receptors José M Casasnovas and Thilo Stehle

388

Bacterial and Archeal Virus Entry Minna M Poranen and Aušra Domanska

402

Nonenveloped Eukaryotic Virus Entry Ian M Jones and Polly Roy

409

Enveloped Virus Membrane Fusion Aurélie A Albertini and Yves Gaudin

417

Genome Replication of Bacterial and Archaeal Viruses Česlovas Venclovas

429

Viral Transcription David LV Bauer and Ervin Fodor

439

Translation of Viral Proteins Martin D Ryan and Garry A Luke

444

Recombination Jozef J Bujarski

460

Assembly of Viruses: Enveloped Particles CK Navaratnarajah, R Warrier, and RJ Kuhn

468

Assembly of Viruses: Nonenveloped Particles M Luo

475

Virion Assembly: From Small Picornaviruses (Picornaviridae) to Large Herpesviruses (Herpesviridae) Ling Zhu, Nan Wang, and Xiangxi Wang

480

Genome Packaging Richard J Bingham, Reidun Twarock, Carlos P Mata, and Peter G Stockley

488

Virus Factories Isabel Fernández de Castro, Raquel Tenorio, and Cristina Risco

495

Release of Phages From Prokaryotic Cells Jesse Cahill and Ry Young

501

Virus Budding Lara Rheinemann and Wesley I Sundquist

519

l

Content of all Volumes

Vesicle-Mediated Transcytosis and Export of Viruses Efraín E Rivera-Serrano and Stanley M Lemon

529

Vector Transmission of Animal Viruses Houssam Attoui, Fauziah Mohd Jaafar, Rennos Fragkoudis, and Peter PC Mertens

542

The Human Virome Alexia Bordigoni, Sébastien Halary, and Christelle Desnues

552

Epidemiology of Human and Animal Viral Diseases Michael Edelstein

559

Zoonosis, Emerging and Re-Emerging Viral Diseases Janet M Daly

569

Antiviral Innate Immunity: Introduction Friedemann Weber

577

Humoral and T Cell-Mediated Immunity to Viruses Ane Ogbe and Lucy Dorrell

584

Antigenicity and Antigenic Variation Kuan-Ying A Huang, Xiaorui Chen, Che Ma, Dayna Cheng, Jen-Ren Wang, and Wan-Chun Lai

597

Antigen Presentation Andrew J McMichael

601

Defense Against Viruses and Other Genetic Parasites in Prokaryotes Kira S Makarova, Yuri I Wolf, and Eugene V Koonin

606

Defective-Interfering Viruses L Roux

617

Ecology and Global Impacts of Viruses Joanne B Emerson

621

The Role of Retroviruses in Cellular Evolution Andrea Kirmaier and Welkin E Johnson

627

The Role of Bacteriophages in Bacterial Evolution Chris M Rands and Harald Brüssow

633

Viruses and Their Potential for Bioterrorism Dana L Vanlandingham and Stephen Higgs

644

The Use of Viral Promoters in Expression Vectors Ian M Jones

652

Oncolytic Viruses Laura Burga and Mihnea Bostina

658

Biotechnology Approaches to Modern Vaccine Design George P Lomonossoff and Daniel Ponndorf

662

Viruses: Impact on Science and Society Neeraja Sankaran and Robin A Weiss

671

VOLUME 2 Viruses as Infectious Agents: Human and Animal Viruses Adenoviruses (Adenoviridae) Balázs Harrach and Mária Benkő

3

Content of all Volumes

li

African Horse Sickness Virus (Reoviridae) Piet A van Rijn

17

African Swine Fever Virus (Asfarviridae) Linda K Dixon, Rachel Nash, Philippa C Hawes, and Christopher L Netherton

22

Akabane Virus and Schmallenberg Virus (Peribunyaviridae) Martin Beer and Kerstin Wernike

34

Alphaviruses Causing Encephalitis (Togaviridae) Diane E Griffin

40

Anelloviruses (Anelloviridae) Fabrizio Maggi and Mauro Pistello

48

Animal Lentiviruses (Retroviridae) Esperanza Gomez-Lucia

56

Animal Morbilliviruses (Paramyxoviridae) Carina Conceicao and Dalan Bailey

68

Animal Papillomaviruses (Papillomaviridae) John S Munday

79

Astroviruses (Astroviridae) Virginia Hargest, Amy Davis, and Stacey Schultz-Cherry

92

Avian Hepadnaviruses (Hepadnaviridae) Allison R Jilbert, Georget Y Reaiche-Miller, and Catherine A Scougall

100

Avian Herpesviruses (Herpesviridae) Vishwanatha RAP Reddy and Venugopal Nair

112

Avian Influenza Viruses (Orthomyxoviridae) Nicolas Bravo-Vasquez and Stacey Schultz-Cherry

117

Avian Leukosis and Sarcoma Viruses (Retroviridae) Karen L Beemon

122

Bluetongue Virus (Reoviridae) Raghavendran Kulasegaran-Shylini and Polly Roy

127

Borna Disease Virus and Related Bornaviruses (Bornaviridae) Susan L Payne

137

Bovine Leukemia Virus (Retroviridae) Thomas Joris, Roghaiyeh Safari, Jean-Rock Jacques, and Luc Willems

144

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae) Paul Becher, Volker Moennig, and Norbert Tautz

153

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae) Philippa M Beard

165

Chikungunya Virus (Togaviridae) Thomas E Morrison and Stephanie E Ander

173

Circoviruses (Circoviridae) Giovanni Franzo and Joaquim Segalés

182

Coronaviruses: General Features (Coronaviridae) Paul Britton

193

Coronaviruses: Molecular Biology (Coronaviridae) X Deng and SC Baker

198

lii

Content of all Volumes

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae) Ali Mirazimi, Felicity Burt, and Anna Papa

208

Dengue Viruses (Flaviviridae) Ashley L St. John and Duane J Gubler

218

Ebola Virus (Filoviridae) Andrea Marzi and Logan Banadyga

232

Enteroviruses (Picornaviridae) Carita Savolainen-Kopra, Soile Blomqvist, and Petri Susi

245

Enveloped, Positive-Strand RNA Viruses (Nidovirales) L Enjuanes, AE Gorbalenya, RJ de Groot, JA Cowley, J Ziebuhr, and EJ Snijder

256

Epstein–Barr Virus (Herpesviridae) Lawrence S Young

267

Equine Herpesviruses (Herpesviridae) Gisela Soboll Hussey, Nikolaus Osterrieder, and Walid Azab

278

Equine, Canine, and Swine Influenza (Orthomyxoviridae) Janet M Daly and Japhette E Kembou-Ringert

287

Feline Calicivirus (Caliciviridae) Margaret J Hosie and Michaela J Conley

294

Feline Leukemia and Sarcoma Viruses (Retroviridae) Brian J Willett and Margaret J Hosie

300

Fish and Amphibian Alloherpesviruses (Herpesviridae) Maxime Boutier, Léa Morvan, Natacha Delrez, Francesco Origgi, Andor Doszpoly, and Alain Vanderplasschen

306

Fish Retroviruses (Retroviridae) TA Paul, RN Casey, PR Bowser, JW Casey, J Rovnak, and SL Quackenbush

316

Fish Rhabdoviruses (Rhabdoviridae) Gael Kurath and David Stone

324

Foot-and-Mouth Disease Viruses (Picornaviridae) David J Rowlands

332

Fowlpox Virus and Other Avipoxviruses (Poxviridae) Efstathios S Giotis and Michael A Skinner

343

Hantaviruses (Hantaviridae) Tarja Sironen and Antti Vaheri

349

Henipaviruses (Paramyxoviridae) Lin-Fa Wang and Danielle E Anderson

355

Hepatitis A Virus (Picornaviridae) Andreas Dotzauer

362

Hepatitis B Virus (Hepadnaviridae) Peter Karayiannis

373

Hepatitis C Virus (Flaviviridae) Ralf Bartenschlager and Keisuke Tabata

386

Hepeviruses (Hepeviridae) Xiang-Jin Meng

397

Herpes Simplex Virus 1 and 2 (Herpesviridae) David M Knipe and Richard Whitley

404

Content of all Volumes

liii

History of Virology: Vertebrate Viruses F Fenner

414

Human Boca- and Protoparvoviruses (Parvoviridae) Maria Söderlund-Venermo and Jianming Qiu

419

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae) Ding X Liu, Jia Q Liang, and To S Fung

428

Human Cytomegalovirus (Herpesviridae) Edward S Mocarski

441

Human Immunodeficiency Virus (Retroviridae) Blaide Woodburn, Ann Emery, and Ronald Swanstrom

460

Human Metapneumovirus (Pneumoviridae) Antonella Casola, Matteo P Garofalo, and Xiaoyong Bao

475

Human Norovirus and Sapovirus (Caliciviridae) Sumit Sharma, Marie Hagbom, Lennart Svensson, and Johan Nordgren

483

Human Papillomaviruses (Papillomaviridae) Alison A McBride and Samuel S Porter

493

Human Parainfluenza Viruses (Paramyxoviridae) Elisabeth Adderson

502

Human Pathogenic Arenaviruses (Arenaviridae) Sheli R Radoshitzky and Juan C de la Torre

507

Human Polyomaviruses (Papillomaviridae) Melissa S Maginnis

518

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae) Amanda R Panfil and Patrick L Green

528

Infectious Bursal Disease Virus (Birnaviridae) Daral J Jackwood

540

Infectious Pancreatic Necrosis Virus (Birnaviridae) Øystein Evensen

544

Influenza A Viruses (Orthomyxoviridae) Laura Kakkola, Niina Ikonen, and Ilkka Julkunen

551

Influenza B, C and D Viruses (Orthomyxoviridae) Thorsten Wolff and Michael Veit

561

Jaagsiekte Sheep Retrovirus (Retroviridae) James M Sharp, Marcelo De las Heras, Massimo Palmarini, and Thomas E Spencer

575

Japanese Encephalitis Virus (Flaviviridae) Sang-Im Yun and Young-Min Lee

583

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae) Anne K Cordes and Thomas F Schulz

598

Marburg and Ravn Viruses (Filoviridae) Courtney Woolsey, Thomas W Geisbert, and Robert W Cross

608

Measles Virus (Paramyxoviridae) Roberto Cattaneo and Michael McChesney

619

Molluscum Contagiosum Virus (Poxviridae) Joachim J Bugert and Rosina Ehmann

629

liv

Content of all Volumes

Mumps Virus (Paramyxoviridae) Stephen A Winchester and Kevin E Brown

634

Murine Leukemia and Sarcoma Viruses (Retroviridae) George R Young and Kate N Bishop

643

Newcastle Disease Virus (Paramyxoviridae) Ben Peeters and Guus Koch

648

Orthobunyaviruses (Peribunyaviridae) Alyssa B Evans, Clayton W Winkler, and Karin E Peterson

654

Parapoxviruses (Poxviridae) Hanns-Joachim Rziha and Mathias Büttner

666

Parechoviruses (Picornaviridae) Sisko Tauriainen and Glyn Stanway

675

Parvoviruses of Carnivores, and the Emergence of Canine Parvovirus (Parvoviridae) Colin R Parrish, Ian EH Voorhees, and Susan L Hafenstein

683

Polioviruses (Picornaviridae) Philip D Minor

688

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae) Jianqiang Zhang, Alan T Loynachan, Gregory W Stevenson, and Jeffrey J Zimmerman

697

Prions of Vertebrates Jonathan DF Wadsworth and John Collinge

707

Pseudorabies Virus (Herpesviridae) Thomas C Mettenleiter and Barbara G Klupp

714

Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus (Caliciviridae) Lorenzo Capucci, Patrizia Cavadini, and Antonio Lavazza

724

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae) Peter J Kerr

730

Rabies and Other Lyssaviruses (Rhabdoviridae) Ashley C Banyard and Anthony R Fooks

738

Respiratory Syncytial Virus (Pneumoviridae) Tiffany King, Tiffany Jenkins, Supranee Chaiwatpongsakorn, and Mark E Peeples

747

Rhinoviruses (Picornaviridae) Matti Waris and Olli Ruuskanen

757

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae) Tetsuro Ikegami

765

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae) Katherine N Ward

778

Rotaviruses (Reoviridae) Juana Angel and Manuel A Franco

789

Rubella Virus (Picornaviridae) Annette Mankertz

797

Saint Louis Encephalitis Virus (Flaviviridae) William K Reisen, Lark L Coffey, Daniele M Swetnam, and Aaron C Brault

805

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae) Joseph SM Peiris and Leo LM Poon

814

Content of all Volumes

lv

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Coronaviridae) Malik Peiris

825

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae) Phyllis J Kanki

827

Sindbis Virus (Togaviridae) Satu Kurkela

837

Tick-Borne Encephalitis Virus (Flaviviridae) Teemu Smura, Suvi Kuivanen, and Olli Vapalahti

843

Transmissible Gastroenteritis Virus of Pigs and Porcine Epidemic Diarrhea Virus (Coronaviridae) Qiang Liu and Volker Gerdts

850

Vaccinia Virus (Poxviridae) Yan Xiang and Rebecca K Lane

854

Varicella-Zoster Virus (Herpesviridae) Jeffrey I Cohen

860

Variola and Monkeypox Viruses (Poxviridae) Lalita Priyamvada and Panayampalli S Satheshkumar

868

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae) Peter J Walker and Robert B Tesh

875

West Nile Virus (Flaviviridae) Fengwei Bai and Elizabeth Ashley Thompson

884

Yellow Fever Virus (Flaviviridae) Ashley E Strother and Alan DT Barrett

891

Zika Virus (Flaviviridae) Nikos Vasilakis, Shannan L Rossi, Sasha R Azar, Irma E Cisneros, Cassia F Estofolete, and Mauricio L Nogueira

899

VOLUME 3 Viruses as Infectious Agents: Plant Viruses An Introduction to Plant Viruses Roger Hull

3

Emerging and Re-Emerging Plant Viruses Sabrina Bertin, Francesco Faggioli, Andrea Gentili, Ariana Manglli, Anna Taglienti, Antonio Tiberini, and Laura Tomassoli

8

Emerging Geminiviruses (Geminiviridae) Muhammad S Nawaz-ul-Rehman, Nazia Nahid, and Muhammad Mubin

21

Movement of Viruses in Plants Manfred Heinlein

32

Plant Antiviral Defense: Gene-Silencing Pathways Vitantonio Pantaleo, Chikara Masuta, and Hanako Shimura

43

Plant Resistance to Viruses: Engineered Resistance Marc Fuchs

52

Plant Resistance to Viruses: Natural Resistance Associated With Dominant Genes Mandy Ravensbergen and Richard Kormelink

60

lvi

Content of all Volumes

Plant Resistance to Viruses: Natural Resistance Associated With Recessive Genes Masayoshi Hashimoto, Kensaku Maejima, Yasuyuki Yamaji, and Shigetou Namba

69

Plant Viral Diseases: Economic Implications Basavaprabhu L Patil

81

Retrotransposons of Plants M-A Grandbastien

98

Vector Transmission of Plant Viruses Etienne Herrbach and Quentin Chesnais

106

Viral Suppressors of Gene Silencing Hernan Garcia-Ruiz

116

Virus-Induced Gene Silencing (VIGS) Xu Tengzhi, Ugrappa Nagalakshmi, and Savithramma P Dinesh-Kumar

123

Alfalfa Mosaic Virus (Bromoviridae) L Sue Loesch-Fries

132

Alphaflexiviruses (Alphaflexiviridae) Sergey Y Morozov and Alexey A Agranovsky

140

Alphasatellites (Alphasatellitidae) Rob W Briddon and Muhammad S Nawaz-ul-Rehman

149

Amalgaviruses (Amalgaviridae) Ioannis E Tzanetakis, Sead Sabanadzovic, and Rodrigo A Valverde

154

Badnaviruses (Caulimoviridae) Andrew DW Geering

158

Banana Bunchy Top Virus (Nanoviridae) John E Thomas

169

Barley Yellow Dwarf Viruses (Luteoviridae) Leslie L Domier

176

Bean Common Mosaic Virus and Bean Common Mosaic Necrosis Virus (Potyviridae) Ramon Jordan and John Hammond

184

Bean Golden Mosaic Virus and Bean Golden Yellow Mosaic Virus (Geminiviridae) Francisco M Zerbini and Simone G Ribeiro

192

Beet Curly Top Virus (Geminiviridae) Robert L Gilbertson, Tomas A Melgarejo, Maria R Rojas, William M Wintermantel, and John Stanley

200

Beet Necrotic Yellow Vein Virus (Benyviridae) Sebastian Liebe, Annette Niehl, Renate Koenig, and Mark Varrelmann

213

Benyviruses (Benyviridae) Annette Niehl, Sebastian Liebe, Mark Varrelmann, and Renate Koenig

219

Betaflexiviruses (Betaflexiviridae) Nobuyuki Yoshikawa and Hajime Yaegashi

229

Betasatellites and Deltasatelliles (Tolecusatellitidae) Muhammad S Nawaz-ul-Rehman, Nazia Nahid, Muhammad Hassan, and Muhammad Mubin

239

Bluner-, Cile-, and Higreviruses (Kitaviridae) Diego F Quito-Avila, Juliana Freitas-Astúa, and Michael J Melzer

247

Brome Mosaic Virus (Bromoviridae) Guijuan He, Zhenlu Zhang, Preethi Sathanantham, Arturo Diaz, and Xiaofeng Wang

252

Content of all Volumes

lvii

Bromoviruses (Bromoviridae) Jozef J Bujarski

260

Bymoviruses (Potyviridae) Annette Niehl and Frank Rabenstein

268

Cacao Swollen Shoot Virus (Caulimoviridae) Emmanuelle Muller

274

Carmo-Like Viruses (Tombusviridae) Miryam Pérez-Cañamás and Carmen Hernández

285

Cassava Brown Streak Viruses (Potyviridae) Basavaprabhu L Patil

293

Cassava Mosaic Viruses (Geminiviridae) James Legg and Stephan Winter

301

Caulimoviruses (Caulimoviridae) James E Schoelz and Mustafa Adhab

313

Cheraviruses, Sadwaviruses and Torradoviruses (Secoviridae) Toru Iwanami and René AA van der Vlugt

322

Citrus Tristeza Virus (Closteroviridae) Moshe Bar-Joseph, Scott J Harper, and William O Dawson

327

Closteroviruses (Closteroviridae) Marc Fuchs

336

Comoviruses and Fabaviruses (Secoviridae) George P Lomonossoff

348

Cotton Leaf Curl Disease (Geminiviridae) Nasim Ahmed, Imran Amin, and Shahid Mansoor

355

Cowpea Mosaic Virus (Secoviridae) George P Lomonossoff

364

Cucumber Mosaic Virus (Bromoviridae) Judith Hirsch and Benoît Moury

371

Dianthovirus (Tombusviridae) Kiwamu Hyodo and Masanori Kaido

383

Endornaviruses (Endornaviridae) Toshiyuki Fukuhara

388

Fimoviruses (Fimoviridae) Toufic Elbeaino and Michele Digiaro

396

Furoviruses (Virgaviridae) Annette Niehl and Renate Koenig

405

Geminiviruses (Geminiviridae) Jesús Navas-Castillo and Elvira Fiallo-Olivé

411

Hordeiviruses (Virgaviridae) Zhihao Jiang, Meng Yang, Yongliang Zhang, Andrew O Jackson, and Dawei Li

420

Idaeoviruses (Mayoviridae) Robert R Martin and Karen E Keller

430

Ilarviruses (Bromoviridae) Aaron Simkovich, Susanne E Kohalmi, and Aiming Wang

439

lviii

Content of all Volumes

Luteoviruses (Luteoviridae) Leslie L Domier

447

Machlomovirus and Panicoviruses (Tombusviridae) Kay Scheets

456

Maize Streak Virus (Geminiviridae) Darren P Martin and Aderito L Monjane

461

Nanoviruses (Nanoviridae) Bruno Gronenborn and H Josef Vetten

470

Necro-Like Viruses (Tombusviridae) Luisa Rubino and Giovanni P Martelli†

481

Nepoviruses (Secoviridae) Hélène Sanfaçon

486

Ophioviruses (Aspiviridae) Anna M Vaira and John Hammond

495

Orthotospoviruses (Tospoviridae) Renato O Resende and Hanu R Pappu

507

Ourmiaviruses (Botourmiaviridae) Gian Paolo Accotto and Cristina Rosa

516

Papaya Ringspot Virus (Potyviridae) Cécile Desbiez and Hervé Lecoq

520

Pecluviruses (Virgaviridae) Hema Masarapu, Pothur Sreenivasulu, Philippe Delfosse, Claude Bragard, Anne Legreve, and DVR Reddy

528

Pepino Mosaic Virus (Alphaflexiviridae) Rene AA van der Vlugt and CCMM Stijger

539

Plant Reoviruses (Reoviridae) Yu Huang and Yi Li

545

Plant Resistance to Geminiviruses Basavaprabhu L Patil, Supriya Chakraborty, Henryk Czosnek, Elvira Fiallo-Olivé, Robert L Gilbertson, James Legg, Shahid Mansoor, Jesús Navas-Castillo, Rubab Z Naqvi, Saleem U Rahman, and Francisco M Zerbini

554

Plant Rhabdoviruses (Rhabdoviridae) Ralf G Dietzgen, Michael M Goodin, and Zhenghe Li

567

Plant Satellite Viruses (Albetovirus, Aumaivirus, Papanivirus, Virtovirus) Mart Krupovic

581

Plum Pox Virus (Potyviridae) Miroslav Glasa and Thierry Candresse

586

Poleroviruses (Luteoviridae) Hernan Garcia-Ruiz, Natalie M Holste, and Katherine LaTourrette

594

Pomoviruses (Virgaviridae) Eugene I Savenkov

603

Potato Virus Y (Potyviridae) Laurent Glais and Benoît Moury

612

Potexviruses (Alphaflexiviridae) Ki H Ryu, Eun G Song, and Jin S Hong

623

†

Deceased.

Content of all Volumes

lix

Potyviruses (Potyviridae) Adrián Valli, Juan A García, and Juan J López-Moya

631

Quinviruses (Betaflexiviridae) Ki H Ryu and Eun G Song

642

Reverse-Transcribing Viruses (Belpaoviridae, Metaviridae, and Pseudoviridae) Carlos Llorens, Beatriz Soriano, Maria A Navarrete-Muñoz, Ahmed Hafez, Vicente Arnau, Jose Miguel Benito, Toni Gabaldon, Norma Rallon, Jaume Pérez-Sánchez, and Mart Krupovic

653

Rice Tungro Disease (Secoviridae, Caulimoviridae) Gaurav Kumar, Fauzia Zarreen, and Indranil Dasgupta

667

Rice Yellow Mottle Virus (Solemoviridae) Eugénie Hébrard, Nils Poulicard, and Mbolarinosy Rakotomalala

675

Satellite Nucleic Acids and Viruses Olufemi J Alabi, Alfredo Diaz-Lara, and Maher Al Rwahnih

681

Secoviruses (Secoviridae) Jeremy R Thompson

692

Sequiviruses and Waikaviruses (Secoviridae) Lucy Rae Stewart

703

Solemoviruses (Solemoviridae) Cecilia Sarmiento, Merike Sõmera, and Erkki Truve†

712

Tenuiviruses (Phenuiviridae) Bertha Cecilia Ramirez and Anne-Lise Haenni

719

Tobacco Mosaic Virus (Virgaviridae) Marc HV Van Regenmortel

727

Tobamoviruses (Virgaviridae) Ulrich Melcher, Dennis J Lewandowski, and William O Dawson

734

Tobraviruses (Virgaviridae) Stuart A MacFarlane

743

Tomato Leaf Curl New Delhi Virus (Geminiviridae) Supriya Chakraborty and Manish Kumar

749

Tomato Spotted Wilt Virus (Tospoviridae) Hanu R Pappu, Anna E Whitfield, and Athos S de Oliveira

761

Tomato Yellow Leaf Curl Viruses (Geminiviridae) Henryk Czosnek

768

Tombusvirus-Like Viruses (Tombusviridae) K Andrew White

778

Tombusviruses (Tombusviridae) Luisa Rubino and Kay Scheets

788

Tritimoviruses and Rymoviruses (Potyviridae) Satyanarayana Tatineni and Gary L Hein

797

Triviruses (Betaflexiviridae) Yahya ZA Gaafar and Heiko Ziebell

805

Tymoviruses (Tymoviridae) Rosemarie W Hammond and Peter Abrahamian

818

†

Deceased.

lx

Content of all Volumes

Umbraviruses (Tombusviridae) Eugene V Ryabov and Michael E Taliansky

827

Varicosaviruses (Rhabdoviridae) Takahide Sasaya

833

Virgaviruses (Virgaviridae) Eugene I Savenkov

839

Viroids (Pospiviroidae and Avsunviroidae) Ricardo Flores and Robert A Owens

852

Watermelon Mosaic Virus and Zucchini Yellow Mosaic Virus (Potyviridae) Cécile Desbiez and Hervé Lecoq

862

VOLUME 4 Viruses as Infectious Agents: Bacterial, Archaeal, Fungal, Algal, and Invertebrate Viruses Bacterial Viruses History of Virology: Bacteriophages William C Summers

3

Icosahedral Phages – Single-Stranded DNA (φX174) Bentley A Fane and Aaron P Roznowski

10

Single-Stranded RNA Bacterial Viruses Peter G Stockley and Junjie Zhang

21

Enveloped Icosahedral Phages – Double-Stranded RNA (φ6) Paul Gottlieb and Aleksandra Alimova

26

Membrane-Containing Icosahedral DNA Bacteriophages Roman Tuma, Sarah J Butcher, and Hanna M Oksanen

36

Tailed Double-Stranded DNA Phages Robert L Duda

45

Helical and Filamentous Phages Andreas Kuhn and Sebastian Leptihn

53

Replication of Bacillus Double-Stranded DNA Bacteriophages Silvia Ayora, Paulo Tavares, Ruben Torres, and Juan C Alonso

61

Lytic Transcription William McAllister and Deborah M Hinton

69

Lysogeny Keith E Shearwin and Jia Q Truong

77

Decision Making by Temperate Phages Ido Golding, Seth Coleman, Thu VP Nguyen, and Tianyou Yao

88

Mobilization of Phage Satellites Kristen N LeGault and Kimberley D Seed

98

Portal Vertex Peng Jing and Mauricio Cortes Jr.

105

Content of all Volumes

lxi

Prohead, the Head Shell Pre-Cursor Marc C Morais and Michael E Woodson

115

Enzymology of Viral DNA Packaging Machines Carlos E Catalano

124

DNA Packaging: DNA Recognition Sandra J Greive and Oliver W Bayfield

136

DNA Packaging: The Translocation Motor Janelle A Hayes and Brian A Kelch

148

Biophysics of DNA Packaging Joshua Pajak, Gaurav Arya, and Douglas E Smith

160

Energetics of the DNA-Filled Head Alex Evilevitch

167

Bacteriophage Receptor Proteins of Gram-Negative Bacteria Sarah M Doore, Kristin N Parent, Sundharraman Subramanian, Jason R Schrad, and Natalia B Hubbs

175

Tail Structure and Dynamics Shweta Bhatt, Petr G Leiman, and Nicholas MI Taylor

186

Bacteriophage Tail Fibres, Tailspikes, and Bacterial Receptor Interaction Mateo Seoane-Blanco, Mark J van Raaij, and Meritxell Granell

194

Phage Genome and Protein Ejection In Vivo Ian J Molineux, L Letti Lopez, and Aaron P Roznowski

206

Dealing With the Whole Head: Diversity and Function of Capsid Ejection Proteins in Tailed Phages Lindsay W Black and Julie A Thomas

219

Jumbo Phages Isaac T Younker and Carol Duffy

229

CRISPR-Cas Systems and Anti-CRISPR Proteins: Adaptive Defense and Counter-Defense in Prokaryotes and Their Viruses Asma Hatoum-Aslan and Olivia G Howell

242

Bacteriophage: Therapeutics and Diagnostics Development Teng-Chieh Yang

252

Bacteriophage Vaccines Pan Tao and Venigalla B Rao

259

Bacteriophage Diversity Julianne H Grose and Sherwood R Casjens

265

Genetic Mosaicism in the Tailed Double-Stranded DNA Phages Welkin H Pope

276

Bacteriophages of the Human Microbiome Pilar Manrique, Michael Dills, and Mark J Young

283

Bacteriophage: Red Recombination System and the Development of Recombineering Technologies Lynn C Thomason and Kenan C Murphy

291

Nanotechnology Application of Bacteriophage DNA Packaging Nanomotors Tao Weitao, Lixia Zhou, Zhefeng Li, Long Zhang, and Peixuan Guo

302

General Ecology of Bacteriophages Stephen T Abedon

314

Marine Bacteriophages Vera Bischoff, Falk Zucker, and Cristina Moraru

322

lxii

Content of all Volumes

Ecology of Phages in Extreme Environments Tatiana A Demina and Nina S Atanasova

342

Archaeal Viruses Diversity of Hyperthermophilic Archaeal Viruses David Prangishvili, Mart Krupovic, and Diana P Baquero

359

Euryarchaeal Viruses Tatiana A Demina and Hanna M Oksanen

368

Vesicle-Like Archaeal Viruses Elina Roine and Nina S Atanasova

380

Virus–Host Interactions in Archaea Diana P Baquero, David Prangishvili, and Mart Krupovic

387

Antiviral Defense Mechanisms in Archaea Qunxin She

400

Discovery of Archaeal Viruses in Hot Spring Environments Using Viral Metagenomics Jennifer Wirth, Jacob H Munson-McGee, and Mark J Young

407

Metagenomes of Archaeal Viruses in Hypersaline Environments Fernando Santos, María D Ramos-Barbero, and Josefa Antón

414

Extreme Environments as a Model System to Study How Virus–Host Interactions Evolve Along the Symbiosis Continuum Samantha J DeWerff and Rachel J Whitaker

419

Fungal Viruses An Introduction to Fungal Viruses Nobuhiro Suzuki

431

Cross-Kingdom Virus Infection Liying Sun, Hideki Kondo, and Ida Bagus Andika

443

Diversity of Mycoviruses in Aspergilli Ioly Kotta-Loizou

450

Evolution of Mycoviruses Mahtab Peyambari, Vaskar Thapa, and Marilyn J Roossinck

457

Mixed Infections of Mycoviruses in Phytopathogenic Fungus Sclerotinia sclerotiorum Jiatao Xie and Daohong Jiang

461

Mycovirus-Mediated Biological Control Daniel Rigling, Cécile Robin, and Simone Prospero

468

Mycoviruses With Filamentous Particles Michael N Pearson

478

Prions of Yeast and Fungi Reed B Wickner and Herman K Edskes

487

Single-Stranded DNA Mycoviruses Daohong Jiang

493

Structure of Double-Stranded RNA Mycoviruses José R Castón, Nobuhiro Suzuki, and Said A Ghabrial†

504

†

Deceased.

Content of all Volumes

lxiii

Ustilago maydis Viruses and Their Killer Toxins Alexis Williams and Thomas J Smith

513

Vegetative Incompatibility in Filamentous Fungi Songsong Wu, Daohong Jiang, and Jiatao Xie

520

Viral Diseases of Agaricus bisporus, the Button Mushroom Kerry S Burton and Greg Deakin

528

Viral Killer Toxins Manfred J Schmitt and Björn Becker

534

Alternaviruses (Unassigned) Hiromitsu Moriyama, Nanako Aoki, Kuko Fuke, Kana Takeshita Urayama, Naoki Takeshita, and Chien-Fu Wu

544

Barnaviruses (Barnaviridae) Peter A Revill

549

Botybirnaviruses (Botybirnavirus) Mingde Wu, Guoqing Li, Daohong Jiang, and Jiatao Xie

552

Chrysoviruses (Chrysoviridae) - General Features and Chrysovirus-Related Viruses Ioly Kotta-Loizou, Robert HA Coutts, José R Castón, Hiromitsu Moriyama, and Said A Ghabrial†

557

Fungal Partitiviruses (Partitiviridae) Eeva J Vainio

568

Fusariviruses (Unassigned) Sotaro Chiba

577

Giardiavirus (Totiviridae) Juliana Gabriela Silva de Lima, João Paulo Matos Santos Lima, and Daniel Carlos Ferreira Lanza

582

Hypoviruses (Hypoviridae) Dong-Xiu Zhang and Donald L Nuss

589

Megabirnaviruses (Megabirnaviridae) Yukiyo Sato and Nobuhiro Suzuki

594

Mitoviruses (Mitoviridae) Bradley I Hillman and Alanna B Cohen

601

Mycoreoviruses (Reoviridae) Bradley I Hillman and Alanna B Cohen

607

Mymonaviruses (Mymonaviridae) Daohong Jiang

615

Narnaviruses (Narnaviridae) Rosa Esteban and Tsutomu Fujimura

621

Phlegiviruses (Unassigned) Karel Petrzik

627

Plant and Protozoal Partitiviruses (Partitiviridae) Hanna Rose and Edgar Maiss

632

Quadriviruses (Quadriviridae) Hideki Kondo, José R Castón, and Nobuhiro Suzuki

642

Totiviruses (Totiviridae) Bradley I Hillman and Alanna B Cohen

648

†

Deceased.

lxiv

Content of all Volumes

Yado-kari Virus 1 and Yado-nushi Virus 1 (Unassigned) Subha Das and Nobuhiro Suzuki

658

Yeast L-A Virus (Totiviridae) Reed B Wickner, Tsutomu Fujimura, and Rosa Esteban

664

Algal Viruses Algal Marnaviruses (Marnaviridae) Marli Vlok, Curtis A Suttle, and Andrew S Lang

671

Algal Mimiviruses (Mimiviridae) Ruth-Anne Sandaa, Håkon Dahle, Corina PD Brussaard, Hiroyuki Ogata, and Romain Blanc-Mathieu

677

Miscellaneous Algal Viruses (Alvernaviridae, Bacilladnaviridae, Dinodnavirus, Reoviridae) Keizo Nagasaki, Yuji Tomaru, and Corina PD Brussaard

684

Phycodnaviruses (Phycodnaviridae) James L Van Etten, David D Dunigan, Keizo Nagasaki, Declan C Schroeder, Nigel Grimsley, Corina PD Brussaard, and Jozef I Nissimov

687

Invertebrate Viruses An Introduction to Viruses of Invertebrates Peter Krell

699

Ascoviruses (Ascoviridae) Sassan Asgari, Dennis K Bideshi, Yves Bigot, and Brian A Federici

724

Baculovirus–Host Interactions: Repurposing Host-Acquired Genes (Baculoviridae) A Lorena Passarelli

732

Baculoviruses: General Features (Baculoviridae) Vera ID Ros

739

Baculoviruses: Molecular Biology and Replication (Baculoviridae) Monique M van Oers

747

Bidensoviruses (Bidnaviridae) Qin Yao, Zhaoyang Hu, and Keping Chen

759

Bunyaviruses of Arthropods (Mypoviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phunuiviridae, Wupedeviridae) Sandra Junglen

764

Dicistroviruses (Dicistroviridae) Yanping Chen and Steven M Valles

768

Entomobirnaviruses (Birnaviridae) Marco Marklewitz

776

Hytrosaviruses (Hytrosaviridae) Henry M Kariithi and Irene K Meki

780

Iflaviruses (Iflaviridae) Bryony C Bonning and Sijun Liu

792

Iridoviruses of Invertebrates (Iridoviridae) İkbal Agah İnce

797

Mesoniviruses (Mesoniviridae) Jody Hobson-Peters and Daniel Watterson

804

Content of all Volumes

lxv

Nimaviruses (Nimaviridae) Peter Krell and Emine Ozsahin

808

Nodaviruses of Invertebrates and Fish (Nodaviridae) Kyle L Johnson and Jacen S Moore

819

Nudiviruses (Nudiviridae) Yu-Chan Chao, Chih-Hsuan Tsai, and Sung-Chan Wei

827

Parvoviruses of Invertebrates (Parvoviridae) Judit J Pénzes, Hanh T Pham, Qian Yu, Max Bergoin, and Peter Tijssen

835

Polydnaviruses (Polydnaviridae) Anne-Nathalie Volkoff and Elisabeth Huguet

849

Poxviruses of Insects (Poxviridae) Basil Arif, Lillian Pavlik, Remziye Nalçacıoğlu, Hacer Muratoğlu, Cihan İnan, Mehtap Yakupoğlu, Emine Özsahin, Ismail Demir, Kazım Sezen, and Zihni Demirbağ

858

Reoviruses of Invertebrates (Reoviridae) Peter Krell

867

Rhabdoviruses of Insects (Rhabdoviridae) Andrea González-González, Nicole T de Stefano, David A Rosenbaum, and Marta L Wayne

883

Sarthroviruses (Sarthroviridae) Azeez Sait Sahul Hameed

888

Solinviviruses (Solinviviridae) Steven M Valles and Andrew E Firth

892

Tetraviruses (Alphatetraviridae, Carmotetraviridae, Permutotetraviridae) Rosemary A Dorrington, Tatiana Domitrovic, and Meesbah Jiwaji

897

VOLUME 5 Diagnosis, Treatment and Prevention of Virus Infections Diagnosis Introduction to Virus Diagnosis and Treatment Maija Lappalainen and Hubert GM Niesters

3

Electron Microscopy for Viral Diagnosis Roland A Fleck

5

Serological Approaches for Viral Diagnosis Klaus Hedman and Visa Nurmi

15

A Brief History of the Development of Diagnostic Molecular-Based Assays Hubert GM Niesters

22

Sequencing Strategies Sibnarayan Datta

27

Validating Real-Time Polymerase Chain Reaction (PCR) Assays Melvyn Smith

35

Rapid Point-of-Care Assays Jan G Lisby and Uffe V Schenider

45

lxvi

Content of all Volumes

Standardization of Diagnostic Assays Sally A Baylis, C Micha Nübling, and Wayne Dimech

52

Quality Assurance in the Clinical Virology Laboratory Paul Wallace and Elaine McCulloch

64

Biosafety and Biosecurity in Diagnostic Laboratories Hannimari Kallio-Kokko and Susanna Sissonen

82

Screening for Viral Infections Walter Ian Lipkin, Nischay Mishra, and Thomas Briese

91

Clinical Diagnostic Virology Marcus Panning

98

Virus Diagnosis in Immunosuppressed Individuals Elisabeth Puchhammer-Stöckl and Fausto Baldanti

105

Diagnosis; Future Prospects on Direct Diagnosis Marianna Calabretto, Daniele Di Carlo, Fabrizio Maggi, and Guido Antonelli

112

Treatment Antiviral Classification Guangdi Li, Xixi Jing, Pan Zhang, and Erik De Clercq

121

Antiretroviral Therapy – Nucleoside/Nucleotide and Non-Nucleoside Reverse Transcriptase Inhibitors Timothy D Appleby and Killian J Quinn

131

Protease Inhibitors Vanesa Anton-Vazquez and Frank A Post

139

HIV Integrase Inhibitors and Entry Inhibitors Daniel Bradshaw and Ranjababu Kulasegaram

145

Management of Respiratory Syncytial Virus Infections (Pneumoviridae) Rachael S Barr and Simon B Drysdale

155

Management of Influenza Virus Infections (Orthomyxoviridae) Bruno Lina

160

Management of Herpes Simplex Virus Infections (Herpesviridae) Nicole Samies and Richard Whitley

175

Management of Varicella-Zoster Virus Infections (Herpesviridae) Andreas Sauerbrei

181

Treatment and Prevention of Herpesvirus Infections in the Immunocompromised Host Sara H Burkhard and Nicolas J Mueller

190

Management of Adenovirus Infections (Adenoviridae) Albert Heim

197

Management of Hepatitis A and E Virus Infection Sébastien Lhomme, Florence Abravanel, Jean-Marie Peron, Nassim Kamar, and Jacques Izopet

206

Management of Patients With Chronic Hepatitis B (Hepadnaviridae) and Chronic Hepatitis D Infection (Deltavirus) Milan J Sonneveld and Suzanne van Meer

217

Studying Population Genetic Processes in Viruses: From Drug-Resistance Evolution to Patient Infection Dynamics Jeffrey D Jensen

227

Content of all Volumes

Virus-Based Cancer Therapeutics Roberto Cattaneo and Christine E Engeland

lxvii

233

Prevention Surveillance of Infectious Diseases Norman Noah

247

Preparing for Emerging Zoonotic Viruses Reina S Sikkema and Marion PG Koopmans

256

Use of Immunoglobulins in the Prevention of Viral Infections Leyla Asadi and Giovanni Ferrara

267

Vaccine Production, Safety, and Efficacy Thomas J Brouwers and Bernard AM Van der Zeijst

281

Vaccines Against Viral Gastroenteritis Scott Grytdal, Tyler P Chavers, Claire P Mattison, Jacqueline E Tate, and Aron J Hall

289

Human Papillomavirus (HPV) Vaccines and Their Impact Jade Pattyn, Pierre Van Damme, and Alex Vorsters

295

Influenza Vaccination Topi Turunen

300

Polio Eradication M Steven Oberste, Cara C Burns, and Jennifer L Konopka-Anstadt

310

Subject Index

315

HUMAN AND ANIMAL VIRUSES

Adenoviruses (Adenoviridae) Balázs Harrach and Mária Benko˝, Institute for Veterinary Medical Research, Center for Agricultural Research, Budapest, Hungary r 2021 Elsevier Ltd. All rights reserved.

Nomenclature

HAdV Human adenovirus kbp Kilo base pair, 1000 bp SAdV Simian adenovirus

AdV Adenovirus bp Base pair

Glossary CAR A cellular receptor characterized by its property that Coxsackie virus and many adenovirus types attach to it. Co-evolution The continuous parallel evolution of a host and its virus, consequently showing similar phylogeny (evolutionary tree topology). Fiber Antenna like projections from the adenovirus virions, which is used to attach the virus to the cells.

PCR Polymerase chain reaction is a molecular biological method to make many copies of a specific DNA segment. Phylogenetics The study of the evolutionary history and relationships among groups of organisms. Squamata The largest order of reptiles, comprising lizards, snakes and amphisbaenians (worm lizards) collectively called squamates or scaled reptiles.

Adenoviruses (AdVs) are middle-sized, nonenveloped, icosahedral, double-stranded DNA viruses of vertebrates. The prefix adeno comes from the Greek word άdήn (gland), reflecting the first isolation of a virus of this type from human adenoid tissue 67 years ago. AdVs have since been isolated from many hosts, including representatives of every major vertebrate class from fish to mammals. Thanks to the polymerase chain reaction (PCR), a rapidly increasing number of novel AdVs is being detected in wild life, but isolation and in vitro propagation of such viruses are usually hampered by the lack of appropriate permissive cell cultures. Certain AdVs can cause disease or even death in humans or animals; nonetheless they are mostly harmless in non-immuno-compromised, healthy individuals. AdVs have been used as model organisms in molecular biology, and important findings of general relevance have emerged from such studies, including the phenomenon of splicing in eukaryotes. AdVs have become one of the most popular vector systems for virus-based gene therapy and vaccination and have potential as antitumor tools. Wide prevalence in diverse host species and a substantially conserved genome organization make AdVs an ideal model for studying virus evolution.

Classification Adenoviruses (AdVs) belong to the family Adenoviridae. Very recently, higher taxonomical levels have been introduced: realm Varidnaviria, kingdom Bamfordvirae, phylum Preplasmiviricota, class Tectiliviricetes, order Rowavirales. This is based on similarities to other viruses and consequently on their anticipated evolutionary history. Thus certain bacterial viruses, namely the 'bacteriophages' in Tectiviridae, and also a virus of archaea living in hot springs (Sulfolobus turreted icosahedral virus, Turriviridae), and the green alga virus Paramecium bursaria Chlorella virus 1 (Phycodnaviridae) were described as having common evolutionary roots with AdVs. Adenoviridae, Tectiviridae, Turriviridae with bacteriophages classified into Corticoviridae are grouped into a common class, and with Phycodnaviridae and additional families into a common kingdom based mainly on their double jelly-roll major capsid protein. There are five official genera and one pending genus in the family. Four genera (Mastadenovirus, Aviadenovirus, Ichtadenovirus, and Testadenovirus) comprise AdVs that are hypothesized to have likely co-evolved with the host, namely mammals, birds, fish, and turtles (testudines), respectively. The remaining two genera (Atadenovirus and Siadenovirus) have broader host range. Atadenoviruses were named after the bias (toward high A þ T) in the genome of their initial representatives, found in various ruminant and avian hosts, as well as in a marsupial-associated sample. However, every known AdV detected in squamate hosts (lizards, snakes, and worm lizards) also belongs to this genus. The base composition of the DNA of the squamate-reptilian AdVs is equilibrated. Siadenoviruses were isolated from, or detected by PCR in, birds, a frog, and a few tortoise species. This genus was named after the presence of a gene encoding a putative sialidase; however novel siadenoviruses, detected in penguins recently, were found devoid of this gene. Within each genus, the viruses are grouped into species, which are labeled with the name of the host and the genus, supplemented with letters of the alphabet, e.g., Human mastadenovirus D (Table 1). Host origin is only one of several criteria that are used to demarcate the species. Phylogenetic distance is the most significant criterion, with species defined as being separated by more than 10%–15% amino acid sequence divergence of the DNA polymerase by maximum likelihood analysis. Genome organization characteristics is another important criterion, yet additional traits such as the G þ C content, crossneutralization values, biological characteristics including pathogenicity, hemagglutination properties and the ability to recombine, are considered, too. Ideally, all these ancillary data are consistent with the results of the phylogenetic calculations.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00057-6

3

4

Adenoviruses (Adenoviridae)

Table 1

The taxonomy of family Adenoviridaea

Mastadenovirus Species

Type (abbreviated name)

Strain

Full genomea

Bat mastadenovirus A Bat mastadenovirus B Bat mastadenovirus C Bat mastadenovirus D Bat mastadenovirus E Bat mastadenovirus F Bat mastadenovirus G Bat mastadenovirus H Bat mastadenovirus I Bat mastadenovirus J unassigned bat AdVs Bovine mastadenovirus Bovine mastadenovirus Bovine mastadenovirus Canine mastadenovirus

BtAdV  3 BtAdV  2 BtAdV  4 BtAdV  7 BtAdV  8 BtAdV  9 BtAdV  11 straw-colored fruit bat AdV Egyptian fruit bat AdV Asian particolored bat AdV dozens of bat AdVs BAdV  1 BAdV  3 BAdV  10 CAdV  1, 2

TJM PPV/1 WIV9 WIV12 WIV13 WIV17 250-A 06–106 3085 Vs9

þ þ þ þ þ þ þ þ þ þ – þ þ – þ

A B C A

WBR  1 Ma268 RI261, Toronto A26/61 1319 M1 Tt11018 M1 385/75.9

Odocoileus AdV  2 (OdAdV  2) bottlenose AdV  2 (BDAdV  2) bottlenose AdV  1 (BDAdV  1) EAdV  1 EAdV  2 HAdV  12, 18, 31, chimpanzee strain ch1 HAdV  3, 7, 11, 14, 16, 21, 34, 35, 50, simian AdV  21 (SAdV  21ch), 27–28ch/go, 29ch, 32–33ch, 35bo/ch, 41go, 46–47go Human mastadenovirus C BAdV  9, HAdV  1, 2, 5, 6, SAdV  31ch, 34ch, 40ch, 42bo, 43go, 44bo, 45go Human mastadenovirus D HAdV  8–10, 13, 15, 17, 19, 20, 22–30, 32, 33, 36–39, 42–49, 51, þ 23 genotypes (mainly recombinants) Human mastadenovirus E HAdV  4, SAdV  22–26ch, 30ch, 36ch, 37bo/ch, 38–39ch Human mastadenovirus F HAdV  40, 41 Human mastadenovirus G HAdV  52, SAdV  1cy, 2rh, 7rh, 11–12rh, 15rh, 51–53rh Murine mastadenovirus A MAdV  1 Murine mastadenovirus B MAdV  2 K87 Murine mastadenovirus C MAdV  3 Ovine mastadenovirus A BAdV  2, OAdV  2–5 Ovine mastadenovirus B goat AdV  2 (GAdV  2), OAdV  1 Ovine mastadenovirus C OAdV  6, 8 8: 7508 ECC  2011 Platyrrhini mastadenovirus A titi monkey AdV  1 (TMAdV  1) unassigned New World monkey AdVs squirrel monkey AdV  1, 419 NWM AdV strains

Deer mastadenovirus B Dolphin mastadenovirus A Dolphin mastadenovirus B Equine mastadenovirus A Equine mastadenovirus B Human mastadenovirus A Human mastadenovirus B

Polar bear mastadenovirus A Porcine mastadenovirus A Porcine mastadenovirus B Porcine mastadenovirus C Sea lion mastadenovirus A Simian mastadenovirus A Simian mastadenovirus B

PBAdV  1 PAdV  1–3 PAdV  4 PAdV  5 California sea lion AdV  1 (CSLAdV  1) SAdV  3–4rh, 6rh, 9–10 m, 14rh, 48cy SAdV  5rh, 8cy, 49–50cy, þ 10 untyped rh/cy strains, baboon AdV  1 (BaAdV  1)

Simian mastadenovirus C Simian mastadenovirus D Simian mastadenovirus E Simian mastadenovirus F Simian mastadenovirus G Simian mastadenovirus H Simian mastadenovirus I unassigned simian AdVs Skunk mastadenovirus A

baboon AdV  2, 3 (olive baboon, BaAdV), SAdV  19yb SAdV  13m SAdV  16gr SAdV  17–18gr SAdV  20gr SAdV  54rh SAdV  55gs 414 Old World monkey AdVs SkAdV  1

BK35

Zc11–030

P9 C8 17: B  105, ATCC VR  541 23336 WIV19 PB1

þ þ þ þ þ þ þ

all HAdVs/ SAdVs þ þ þ 52, SAdV  1, 2, 7, 11, 51–53 þ þ þ BAdV  2 – 8 þ squirrel monkey  1 þ 3 þ þ þ 3, 6, 48 8, 49, 50, þ 10 strains, BaAdV  1 þ þ þ þ þ þ þ – þ

Adenoviruses (Adenoviridae)

Table 1

5

Continued

Mastadenovirus Species

Type (abbreviated name)

Strain

Full genomea

Squirrel mastadenovirus A

SqAdV  1

DE/2013/Sciurus vulgaris/ 2013Pa405–00252

þ

Tree shrew mastadenovirus A Guinea pig mastadenovirus A (pending) unassigned prosimian AdVs unassigned mastadenoviruses

TSAdV  1 GPAdV  1

Aviadenovirus Duck aviadenovirus B Falcon aviadenovirus A Fowl aviadenovirus A Fowl aviadenovirus B Fowl aviadenovirus C Fowl aviadenovirus D Fowl aviadenovirus E Goose aviadenovirus A Pigeon aviadenovirus A Pigeon aviadenovirus B Psittacine aviadenovirus B Psittacine aviadenovirus C Turkey aviadenovirus B Turkey aviadenovirus C Turkey aviadenovirus D unassigned turkey adenovirus unassigned aviadenoviruses

411 prosimian AdV strains from alpaca, Asian house shrew, cat, Chinese striped hamster, Daurian ground squirrel, deer mouse, harbor porpoise, rabbit, rat, seal, South West China vole DAdV  2 FaAdV  1 FAdV  1 FAdV  5 FAdV  4, 10 FAdV  2, 3, 9, 11 FAdV  6, 7, 8a, 8b GoAdV  1–4 PiAdV  1 PiAdV  2 PsAdV  4 (red-bellied parrot) PsAdV  1 (Senegal parrot) TAdV  1 TAdV  4 TAdV  5 TAdV  2 from cranes, goldfinch, European greenfinch, great tit, gull, Meyer’s parrot, neotropic cormorant, Pacific black duck, smooth-billed ani, tropical screech owl, vitelline masked weaver, white-eyed parakeet

Atadenovirus Bovine atadenovirus D Bovine atadenovirus E (pending) Bovine atadenovirus F (candidate) Deer atadenovirus A Duck atadenovirus A Lizard atadenovirus A

BAdV  4, 5, 8 BAdV  6 BAdV  7 Odocoileus AdV  1 (deer AdV  1, OdAdV  1) DAdV  1 LiAdV  1, 2

Lizard atadenovirus B (pending) Ovine atadenovirus D Possum atadenovirus A Psittacine atadenovirus A Snake atadenovirus A unassigned snake AdVs

bearded dragon AdV  1 (BDAdV  1) goat AdV  1 (GAdV  1), OAdV  7 PoAdV  1 PsAdV  3 (southern mealy amazon) SnAdV  1 SnAdV  2, SnAdV  3

unassigned avian atadenoviruses

chimney swift, common chaffinch, eastern spinebill, Eurasian bullfinch, Eurasian siskin, European greenfinch, European robin, long-billed corella, song thrush, tropical screech owl, vitelline masked weaver, white-eyed parakeet, white-plumed honeyeater from anole, blue-tongued skink, chameleon, East African spiny-tailed lizard, emerald monitor, fat-tailed gecko, Greek tortoise, Japalura tree dragon, long-tailed grass lizard, tokay gecko, tree dragon, white-throated monitor, worm lizard

unassigned reptilian atadenoviruses

Siadenovirus Frog siadenovirus A Great tit siadenovirus A Raptor siadenovirus A Skua siadenovirus A

AUS96

FrAdV  1 GTAdV  1 RAdV  1 (Harris hawk, eagle owls) South Polar skua AdV  1 (SPSAdV  1)

þ þ – –

GR CELO 340 ON1, C  2B P7-A, 75, A2-A, 380 CR119, YR36, TR59, 764 P29 IDA YPDS-Y-V1. A19.11–2013 CS15–4016 18VIR149_ITA_2018 D90/2 CS15–4016 1277BT, D1648 TAV  2

þ – þ þ þ þ þ 4 þ þ þ þ þ þ þ – –

4: THT/62

4 þ – CA_AdV_Ohc 98–6943 þ þ gila monster, Mexican beaded 2 lizard BD5H2 þ 7 – HKU/Parrot19 þ corn snake, python þ California kingsnake, asp – viper, gopher snake eastern eastern spinebill (‘passerine spinebill  1 AdV-1’): AU2787

–

þ – þ þ (Continued )

6

Adenoviruses (Adenoviridae)

Table 1

Continued

Mastadenovirus Species

Type (abbreviated name)

Turkey siadenovirus A Penguin siadenovirus A Psittacine siadenovirus D (pending)

TAdV  3 chinstrap penguin AdV  2, gentoo penguin AdV  4 PsAdV  5, 6

Psittacine siadenovirus E (pending) unassigned psittacine siadenoviruses

PsAdV  7 PsAdV  2 (plum headed parakeet, cockatoo, eastern rosella, scarlet chested parrot, orange-bellied parrot, etc.) from double-barred finch, Eurasian blackcap, Gouldian finch, pigeon, Sulawesi tortoise, zebra finch

unassigned siadenoviruses Ichtadenovirus Sturgeon ichtadenovirus A Testadenovirus (pending) Pond slider testadenovirus A (pending) Box turtle testadenovirus A (cand) Pancake tortoise testadenovirus A (cand) unassigned testadenovirus

Strain

Pacific parrotlet, budgerigar strain BrdKdnyDNA little corella

Full genomea þ þ þ þ – –

white sturgeon AdV  1 (WSAdV  1)

WSAdV1/1996

þ

red-eared slider AdV  1 (RESAdV  1)

2010Z01

–

box turtle AdV  1, eastern box turtle AdV  1 (EBTAdV  1) pancake tortoise AdV  1

EBTAdV  1: R08–207

–

R08–227

–

red-footed tortoise AdV  1

R13–677

–

Unassigned Viruses in the Family crocodile adenovirus

–

Available full genome sequences are noted by þ or by the type number of the sequenced serotype(s) if those listed are not all sequenced and available. In case of the common name of ‘simian AdV’, two letter abbreviations show the original hosts: bo, bonobo; ch, chimpanzee; cy, cynomolgus monkey (crab-eating macaque); go, gorilla; gr, grivet; gs, golden snubnosed monkey; m, macaque sp.; rh, rhesus macaque; yb, yellow baboon. a

Along these rules, e.g., chimpanzee AdVs are classified into different human AdV species. Human AdVs have been studied the most extensively and are grouped into seven species (Human mastadenovirus A to Human mastadenovirus G; abbreviated informally to HAdV–A to HAdV–G). The species are further subdivided to numbered (sero)types, abbreviated in a hyphenated form such as e.g., HAdV–5. The AdV taxonomy, i.e., clustering similar AdVs together, while separating those that are 'adequately' different, and especially correct naming of the taxa, is prone to human errors and discussions partly because of the lack of knowledge available at a certain time point. As we discover novel AdVs, we find that several AdV types are capable of crossing species barriers of different levels. Thus, some names referring to hosts will not be correct or the best description in the future. Nonetheless, to avoid confusion, it is advisable to keep the historical names based on the first findings. Type classification had been based on serology. In the past decade, however, new type numbers are being assigned to novel AdVs, the full genome sequence of which seems to warrant this. Consequently, HAdVs with type numbers above 52 do not necessarily represent distinct serotypes. The emerging large quantity of data causes problems also by clearly demonstrating that we will find a very large number of instances of homologous recombination, especially in the case of the best studied human (or in general primate) AdVs. These recombinants have obviously a mixture of biological and genetic characteristics of the 'original' types. The six clusters of AdVs, defined by gene organization and/or phylogenetic calculations, correspond to the five approved (Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus, Ichtadenovirus) and the pending sixth genus (Testadenovirus) (Fig. 1). The evolutionary distances among the AdVs seem to be generally proportional to those among the main lineages of their vertebrate hosts (in evolutionary order: fishes, turtles, squamates, birds, mammals) supporting the theory on the co-evolution of virus and host. However, there are a few exceptions when very distantly related viruses infect the same host, for example, bovine mastadenoviruses and atadenoviruses infect cattle, whereas the Sulawesi tortoise siadenovirus and testadenoviruses infect testudines (turtles). Obviously, besides co-evolution, several host switches also occurred. According to this hypothesis, the mastadenoviruses, aviadenoviruses, and testadenoviruses represent continuous co-evolving lineages, while squamate AdVs (atadenoviruses) have supposedly switched to ruminants, birds, and common tortoise. The host origin of the lineage of siadenoviruses is presently unknown. Representatives of this genus were found to infect birds, tortoises, and a frog. Similarly, the provenance of the white sturgeon AdV, the single member of the genus Ichtadenovirus is not clear either, as no other fish AdV was ever confirmed by targeted examinations or during the aquatic environmental metagenomics studies to date. By phylogenetic analyses, more recent host switches can also be recognized within the genera, for example from Old World monkeys and apes to humans, or from bats to dog-like carnivores, horse, and skunk.

Adenoviruses (Adenoviridae)

7

8

Adenoviruses (Adenoviridae)

Virion Structure Adenovirus virions are non-enveloped, icosahedral particles 90 nm in diameter. The capsid consists of 240 nonvertex capsomers (called hexons), each 8–10 nm in diameter, and 12 vertex capsomers (penton bases), each with one or more (2  3) protruding antenna-like structures called fiber(s) 9–77.5 nm in length. The members of genus Aviadenovirus usually have two fibers per vertex. The number of fiber structures is not directly connected to the number of fiber genes. Fowl adenovirus 1 (FAdV–1), FAdV–4, and FAdV–10 even have two, tandem fiber genes of different lengths, resulting in two fibers of different sizes at each vertex. Members of species Human mastadenovirus F also have two fiber genes of different lengths, but the fiber structures are displayed separately on alternate vertices. Lizard adenovirus 2 (from Mexican beaded lizard) also has two fiber genes of different size. Three long fibers are displayed in some vertices while a single short fiber in the others. The major capsomers (hexons) are formed by the interaction of three identical polypeptides, designated as polypeptide II (the Roman numeral label is related to the relative mobility of structural proteins under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each hexon has two characteristic parts: a triangular top with three ‘towers’, and a pseudohexagonal base with a central cavity. Hexons, or more exactly their basal part, are packed tightly to form a protein shell that protects the inner components of the virion. In members of the genus Mastadenovirus, polypeptide IX is located on the outer part of the capsid, keeping together the hexons in each facet (12 copies of protein IX per facet) (Fig. 2). In HAdVs, polypeptide IX also links two facets together across the icosahedral edge. However, polypeptide IX is not present in the members of any other genus. Two monomers of IIIa are located underneath the vertex region, and multiple copies of polypeptide VI are underneath the hexons. Penton bases are formed at the vertices by the interaction of five copies of polypeptide III, and are tightly associated with one, two or three fibers. Fibers are homotrimers of polypeptide IV, and have three structural domains, tail, shaft and the distal knob. There are six monomers of polypeptide VIII per facet: three of them wedged between polypeptide IIIa and the peripentonal hexons, and the other three around the icosahedral 3-fold symmetry axis, contributing with polypeptide IX to the stabilization of the facet. The core consists of the DNA genome complexed with four polypeptides: V, VII, X, and a virus-coded terminal protein (TP). Polypeptide V is found only in mastadenoviruses. Atadenoviruses have two unique structural proteins: p32K and LH3. The major capsid protein, LH3 forms prominent ‘knobs’ on the surface of the atadenoviruses and is located in the same relative position among the hexon subunits as protein IX in the mastadenovirus but sits on top of the central hexon trimers (probably holding the outer capsid together). A virus-coded protease is also packaged bound to the genome. Adenoviruses are stable on storage in the frozen state. They are insensitive to lipid solvents. Heat sensitivity varies among the genera.

Genome The adenovirus genome is a single linear molecule of double-stranded DNA (24,630–48,395 bp) containing inverted terminal repeats (ITR) of 26–721 bp at its termini. The 50 ends of each DNA strand are linked covalently to the TP. The range of nucleotide composition is 33.24%–66.92% G þ C. The genetic organization of the central part of the genome is conserved throughout the family, whereas the distal parts show large variations in length and gene content (Fig. 3). Splicing was first discovered in AdVs, and is a common means of expressing mRNAs in this virus family. In the conserved region, most of late genes are expressed by splicing from the rightward-oriented major late promoter, located in overlap with the DNA-dependent DNA polymerase (pol) gene. The early genes encoding pol, the precursor of TP (pTP), and the DNA-binding protein (DBP) are expressed by splicing from leftward-oriented promoters.

Life Cycle Replication of various human adenoviruses has been studied in detail, in particular with HAdV–2. Virus entry takes place via interactions of the fiber knob with specific receptors (most often the coxsackievirus and adenovirus receptor or CAR) on the surface of a susceptible cell followed by internalization via interactions between the penton base and cellular av integrins. Protein VI mediates the release of virions from the endosomes, allowing dynein-mediated transport on microtubules to nuclear pores. After uncoating, the virus core is delivered to the nucleus, i.e., the site of virus transcription, DNA replication and assembly. Virus infection mediates the shutdown of host DNA synthesis and later RNA and protein synthesis. Transcription of the AdV genome by host RNA polymerase II involves both DNA strands of the genome and initiates (in HAdV–2) from five early (E1A, E1B, E2, E3, and E4), two intermediate (IX and IVa2), the major late (L) and the U exon protein (UXP) promoters. All primary transcripts are capped and polyadenylated, with complex splicing patterns producing families of mRNAs. In primate adenoviruses, there are one Fig. 1 Phylogenetic tree of adenoviruses based on maximum likelihood analysis of DNA-dependent DNA polymerase (pol) amino acid sequences from adenoviruses one from each species and pending species, or distinct strain with full pol sequence known. Multiple alignment: MultAlin; model selection: ProtTest 2.4; maximum likelihood calculation: PhyML 3.1 with model LG þ I þ G and Shimodaira-Hasegawa-like branch test for statistical test for branch support on the platform of Galaxy/Pasteur. Unrooted calculation; white sturgeon AdV–1 and slider AdV–1 are selected as outgroup for visualization. From the name of the adenovirus types the word 0 adenovirus/AdV0 was removed for clarity. In case of the common names of ‘psittacine AdV’ and ‘simian AdV‘,the original hosts are shown in brackets. Adenovirus species are indicated. The bar indicates 20% difference between two neighboring sequences. SH (Shimodaira-Hasegawa) branch support values shown at the nodes.

Adenoviruses (Adenoviridae)

9

Fig. 2 Stylized section of a mastadenovirus particle. Capsid proteins (II, III, IIIa, IV, VI, VIII and IX) and core proteins (V, VII, X and terminal protein /TP/) are labeled by Roman numbers related to the relative mobility of structural proteins under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Protein V and IX (in red squares) occure only in mastadenoviruses. Instead of protein IX, a structural protein called LH3 can be found in almost all atadenoviruses. As the structure of the nucleoprotein core has not been established, the polypeptides associated with the DNA are shown in hypothetical locations. Adapted from Harrach, B., Benko˝, M., Both, G.W., et al., 2011. Family Adenoviridae, In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Elsevier, pp. 125–141.

or two virus-associated (VA) RNA genes. These are transcribed by cellular RNA polymerase III and facilitate translation of late mRNAs and block the cellular interferon response. In non-primate mastadenoviruses, no VA RNA genes could be identified. A nonhomologous VA RNA gene has been mapped in some aviadenoviruses near the right end of the genome. The replication of aviadenoviruses has been shown to involve significantly different pathways from those characterized in HAdVs. This is not unexpected, given the considerable differences in gene layout between the non-conserved regions of the genome. About 40 different polypeptides (the largest number being in aviadenoviruses and ichtadenovirus, the smallest in siadenoviruses) are produced. Almost a third of these compose the virion, including a virus-encoded cysteine protease, which processes a number of precursor proteins (these are prefixed with p). With the exception of polypeptides V and IX, the other structural proteins found in mastadenoviruses are conserved in every genus. Products of the four early regions of mastadenoviruses modulate the host cell's transcriptional machinery (E1 and E4), assemble the virus DNA replication complex (E2), and subvert host defense mechanisms (E3). The E2 region (encoding DBP, pol, and pTP) is well conserved throughout the family, while the E3 and E4 regions show great variation in length and gene content even among the mastadenoviruses. E1A, E1B 19K, and the E3 and E4 regions (with the exception of 34K, called ORF6 in HAdVs) occur only in mastadenoviruses. Genes encoding proteins related to 34K are also present in atadenoviruses, usually in duplicate. Intermediate (IX, only in mastadenoviruses, and IVa2) and late gene products (in many mastadenoviruses expressed from five transcription units, L1–L5) are concerned with assembly and maturation of the virion. Late proteins include 52K (scaffolding protein) and pIIIa (L1); III (penton base), pVII (major core protein), V (minor core protein, only in mastadenoviruses) and pX (L2); pVI, hexon (II), and protease (L3); 100K, 33K (and its unspliced version, 22K), and pVIII (L4); and fiber (IV) (L5). Seemingly, there are no lipids in adenovirus particles. However, the fiber proteins and some of the nonstructural proteins are glycosylated.

Epidemiology Adenoviruses are abundant and widespread and theoretically every reptilian, avian and mammalian species could host at least one AdV type. Endemic or enzootic infections that are frequently unapparent seem to be very common. In contrast, epidemic or epizootic occurrence is relatively rare but normally has much more significant consequences. The non-enveloped virions, shed by infected individuals, are rather stable in the environment and can preserve infectivity for long periods. The virions are not only able to tolerate adverse environmental effects, such as drought and moderate temperature or pH changes, but are also resistant to lipid solvents and simple disinfectants. There is evidence that HAdVs can persist and perhaps even retain infectivity in natural or communal water sources. Moreover, freshwater and marine bivalves have also been found to accumulate AdVs. Because of their

10

Adenoviruses (Adenoviridae)

Fig. 3 Schematic illustration of the different genome organizations found in representative members of the four thoroughly analyzed adenovirus genera. Black arrows depict genes conserved in every genus, gray arrows show genes present in more than one genus, and colored arrows show genusspecific genes. Adapted from Harrach, B., Benko˝, M., Both, G.W., et al., 2011. Family Adenoviridae, In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Elsevier, pp. 125–141. with permission.

stability, AdVs have become a major indicator of viral pollution in the environment. Vertical transmission has also been documented in several host species, including birds and cattle. Although different AdV types have different affinities for various organs and tissues, primary virus shedding is likely through the intestines. AdVs are often transmitted by person-to-person contact, particularly among young children where fecal–oral spread is also common. Aerosol transmission is probably not rare in crowded populations. Adenovirus DNA may be found in tonsillar tissue, peripheral blood lymphocytes, and lung epithelial cells long after clinical disease has abated. Swimming-pool-related outbreaks, particularly by strains causing keratoconjunctivitis or pharyngitis, are not uncommon. In poultry and other farm animals, transportation, crowding, and mixing of different populations can lead to mass infections accompanied by disease outbreaks.

Adenoviruses (Adenoviridae)

11

Clinical Features The tropism and pathogenicity of HAdVs are largely species- and type-dependent. Respiratory pathogens are common, although infections of a large variety of different organs have been described. The fiber protein mediates primary tissue tropism, and some of the specific determinants, epitopes, and receptors have been mapped. The most common clinical manifestations connected to members of each human AdV species are listed below. Members of Human mastadenovirus B are divided into two groups informally termed 'subspecies' HAdV–B1 and HAdV–B2 since they represent two phylogenetic lineages (Fig. 1).

HAdV–A HAdV–12, HAdV–18, and HAdV–31 can replicate efficiently in the intestines, and, based on serological surveys, are common in the population, especially in children with gastrointestinal disease. However, their role in the etiology of infant diarrhea is yet to be determined. Serotypes belonging to this species are generally difficult to isolate and culture.

HAdV–B1 Respiratory pathogens HAdV–3, HAdV–7, HAdV–16, and HAdV–21 belong here (and HAdV–50). Seasonal outbreaks of febrile respiratory disease, mainly during winter, can be caused in infants by HAdV–7 in most parts of the world. Similar diseases, usually with a less severe outcome, can be seen among school children. In some countries, HAdV–3 is the dominant serotype, whereas HAdV–7 seems to have a number of different genotypes that shift occasionally in certain geographic areas. In addition to HAdV–7 and HAdV–21, HAdV–4 (species HAdV–E), and HAdV–14 (HAdV–B2) are most often implicated in the etiology of acute respiratory outbreaks among freshly enlisted military recruits in the USA.

HAdV–B2 HAdV–11, HAdV–34, and HAdV–35 cause persistent interstitial infection in the kidney and hemorrhagic cystitis. These HAdV types, along with HAdV–50, are most often shed in the feces or urine of acquired immune deficiency syndrome (AIDS) patients and organ or tissue transplantation recipients. Re-emergence of a genomic variant of HAdV–14 causing severe, sometimes fatal respiratory infections was reported first from the USA in 2006, later also detected in Europe and China.

HAdV–C The low serotype designations (HAdV–1, HAdV–2, HAdV–5, and HAdV–6) of viruses in this species reflect the relative ubiquity of these viruses. These HAdVs can be isolated easily and, indeed, comprise approximately half of all HAdV serotypes reported to the World Health Organization. HAdV–1, HAdV–2, and HAdV–5 are known to maintain endemic infections, and most teenagers will have had infections with more than one serotype. The site of persistence is lymphoid tissue, and shedding can last for a couple of years after primary infection.

HAdV–D This species contains 32 serotypes, and therefore encompasses well over one half of all known HAdVs. HAdV–8, HAdV–19, and HAdV–37 cause epidemic keratoconjunctivitis, especially in dry climates or densely populated areas. Other serotypes are rarely isolated except from immunocompromised patients. Virus shedding by asymptomatic healthy individuals was found to be common in Africa. Recently, by whole genome analysis, a large number of double or multiple HAdV–D intertypic recombinants, involved in epidemic keratoconjunctivitis, have been recognized. Several recombinant strains were assigned to novel type number (genotypes) without testing or confirming their serological distinctness.

HAdV–E This species contains a single human serotype, namely HAdV–4, although different variants ('genotypes') of this virus have been described. HAdV–4 strains have been most often implicated in respiratory diseases among military recruits in the USA. The species encompass a number of novel isolates originating from chimpanzees and bonobos. In Japan, HAdV–4 is the second most important cause of adenovirus-associated eye disease after HAdV–8.

HAdV–F The so-called enteric or fastidious HAdV–40 and HAdV–41 are classified here. The genetic distance between these two serotypes comes close to meriting separation into different species, but, for practical reasons, including indistinguishable pathology, they remain in a single species. These viruses are a major cause of infantile diarrhea all over the world. In Europe, a shift in dominance

12

Adenoviruses (Adenoviridae)

from HAdV–40 in the 1970s to HAdV–41 after 1992 has been observed. Because of a deletion in the E1 region near the left end of the genome, HAdV–40 and HAdV–41 can be isolated and cultured only in complementing, transformed cell lines such as 293 or PER.C6.

HAdV–G The most recently described HAdV serotype, HAdV–52, was recovered from a patient with diarrhea. No further cases or serologic evidence on the circulation of this virus have been reported yet. Interestingly, this species encompasses numerous monkey AdVs besides HAdV–52.

Epizootiology Simian Adenoviruses A series of simian viruses (termed SVs) were isolated from various tissue cultures prepared from apes and monkeys. These were numbered serially, irrespective of the virus family to which they belong, since identification and allocation to a family were performed later only. A considerable number of the SV isolates have been identified as adenoviruses. In many cases, unfortunately, the original SV numbers have been retained as the numbers of the AdV type, and thus a somewhat confusing system of SAdV numbering can still be encountered in publications, as well as in the records of the American Type Culture Collection. To date, there are 455 simian adenovirus types (SAdV–1 through SAdV–55) and dozens of detected but not well characterized SAdVs. SAdV–1 to SAdV–20 originated from Old World monkeys, whereas SAdV–21 to SAdV–47, and further strains were isolated from apes (chimpanzees, bonobos, and gorillas). SAdV–48 to SAdV–55, and further variants are again from Old World monkeys. In the past years, novel adenoviruses have been described from New World monkeys. In one case, the possible transmission of the virus from diseased animals to the caretakers was also observed. Very recently, a dozen different prosimian AdVs were detected by PCR screening of samples directly from Madagascar and from zoos. Data concerning prevalence or pathogenicity of AdVs in their natural hosts are increasing continuously. An interesting mixing and grouping can be observed among the primate AdVs, suggesting that they can sometimes be less host specific and supposedly able to infect individuals of closely related host species.

Canine and Other Carnivoran Adenoviruses For a long period, only canine AdVs had been known to infect carnivores. CAdV–1 is the causative agent of infectious canine hepatitis (Rubarth’s disease), a life-threatening disease of puppies. Regular vaccination worldwide has decreased the number of clinical cases. Viruses, serologically indistinguishable from CAdV–1, have been found to cause encephalitis in foxes as well as infections in numerous other carnivorous animals including wolfs, coyotes, jackals, otters, raccoons, and even bears. The disease caused by a genetically closely related virus, CAdV–2, is called kennel cough and is common among dog breeder stocks. There exist a couple of publications reporting cases of inclusion-body-hepatitis-like condition in felids. Yet, focused attempts to find a distinct feline adenovirus yielded only a single case of cat AdV amplification and partial sequencing from paraffin embedded sample. It is likely, however, that cats living in close proximity with humans can harbor HAdV–1 or HAdV–5 from species HAdV–C. More recently, specific adenoviruses have been described from hosts, namely seals and several sea lions, belonging to the superfamily of pinnipeds. Similarly, a distinct AdV had been found in polar bears died in zoos. Phylogeny inference and genome analyses imply that the AdVs isolated from, or detected in, some carnivorous animals (CAdV–1, CAdV–2, skunk AdV–1) have a close common ancestor with the AdVs discovered in different bats recently. This would explain the elevated pathogenicity of these AdVs in the newly invaded hosts. Indeed, skunk AdV was discovered from independent deadly diseases of pet African hedgehogs in Japan and the USA, in a marmoset in a Hungarian zoo and in North American porcupines.

Porcine Adenoviruses At least five porcine adenovirus types (PAdV–1 to PAdV–5) are recognized. These represent three different species. Species Porcine mastadenovirus A contains three serotypes (PAdV–1 to PAdV–3) that are fairly similar to each other and cause no specific diseases. PAdV–4 (in species PAdV–B) has been described as associated with neurological disease, whereas PAdV–5 (in species PAdV–C) is more distantly related to PAdV–A. The pathogenic roles of porcine adenoviruses need further investigation.

Equine Adenoviruses Two equine adenovirus serotypes (EAdV–1 and EAdV–2) have been isolated to date. EAdV–1 seems to be more prevalent and causes clinical respiratory diseases. It seems to be genetically close to certain bat AdVs so that a host switch has been suspected. On

Adenoviruses (Adenoviridae)

13

the other hand, EAdV–2 appears to be the AdV co-evolved with horses. It usually causes gastrointestinal infection with mild or selflimiting disease in foals. Certain Arabian horse lineages carry a genetic defect that, when present in the homozygous state, results in severe combined immunodeficiency disease. Affected animals are incapable of mounting an immune response, and foals often die of pneumonia due to equine adenovirus infection, which would be generally harmless in immunocompetent individuals.

Bovine, Ovine, Caprine, and Other Ruminant Adenoviruses Ruminant animals can harbor rather diverse AdVs. Several isolates originating from cattle, sheep, goat and deer species belong to various species in the genus Mastadenovirus. Unusual BAdVs, substantially differing from mastadenoviruses, as judged for example by the absence of the genus-specific antigen, were described four decades ago. These BAdV serotypes are now classified in a separate genus, Atadenovirus. One ovine (OAdV–7), one goat (GAdV–1), and one deer adenovirus (Odocoileus AdV–1) also belong here. Bovine mastadenoviruses were recognized early on and were found occasionally to cause enzootic bronchopneumonia or calf pneumo-enteritis. However, experimental infection of young calves with the isolated adenovirus seldom, if at all, resulted in reproduction of disease. The early isolates of BAdV–10 originated from diseased or dead animals. In phylogeny reconstructions, BAdV–10 represents an independent lineage. It also has a very simple E3 region and a variable fiber gene. Therefore, it has been proposed to be in the process of switching from one host to another. Crossing the host barrier has also been suggested for BAdV–2, with two subtypes, each infecting cattle and sheep, respectively. Based on evolutionary relationships, BAdV–2 is presently classified into species Ovine mastadenovirus A together with serotypes OAdV–2 to OAdV–5. The newest isolated OAdV type is mastadenovirus OAdV–8. The atadenovirus Odocoileus AdV–1 caused hemorrhagic epizooty killing thousands of mule deer in California.

Avian Adenoviruses Birds are known to harbor AdVs from three different genera. In addition to the host origin, members of the genus Aviadenovirus can be distinguished from mastadenoviruses by the lack of the genus-common complement-fixing antigen. Twelve aviadenovirus serotypes have been described from chicken. A specific condition, referred to as hydropericardium syndrome, has been clearly associated with fowl adenovirus 4 (FAdV–4) from species Fowl aviadenovirus C. It occurs mainly in Asia causing great economical losses. Another specific disease the so-called gizzard erosion is caused by FAdV–1 (species FAdV–A). Infection with at least ten additional FAdV types may cause inclusion body hepatitis in chicken. Novel aviadenoviruses have been isolated from, or detected by PCR and sequencing in, numerous host species including falcons, finches, gulls, parrots, pigeon, neotropic cormorant, Pacific black duck, and in unpublished cases from many other wild birds (Table 1). The genus Atadenovirus contains duck adenovirus 1 (DAdV–1), the causative agent of the so-called egg drop syndrome (EDS) of chickens. The disease, causing dramatic decrease in the egg production of laying flocks, was first experienced in Europe in 1976 then soon became known worldwide. Retrospective serological studies confirmed the presence of hemagglutination inhibitory antibodies to EDS virus in archive serum samples originating from a large number of wild and domestic bird species, with predominance in waterfowl, which are now considered as the main reservoir. The presence of novel atadenoviruses has been reported in parrots, songbirds, chimney swift, finches, robin and tropical screech owl (Table 1). Additional yet unpublished atadenoviruses have been detected in various wild birds. Another poultry pathogen, namely turkey adenovirus 3 (TAdV–3), is classified into the genus Siadenovirus. The distinctness of this virus, also known as turkey hemorrhagic enteritis virus, has been recognized early based on the lack of common cross-reacting antigen with aviadenoviruses or DAdV–1. Serologically indistinguishable strains have been associated with various pathological entities in turkey, pheasant, and chicken. Besides TAdV–3, the genus Siadenovirus encompasses the single known adenovirus from an amphibian host, frog adenovirus 1 (FrAdV–1). Recently, numerous novel siadenoviruses have been demonstrated by PCR and sequencing in different diseased or dead birds, including finches, parrots, pigeons, owls and a hawk, as well as penguins and skuas from the Antarctica. Psittacine AdV–2 seems to have high pathogenicity and is capable of crossing the species barrier. It has been detected in representatives of more than seven different species belonging to psittaciformes.

Reptilian Adenoviruses Adenovirus infections in various snake and lizard species are reported frequently, but the number of virus isolates is still limited. In Germany, captive boid snakes, from different collections and breeders, seem to be infected by the same type of AdV, and also by a parvovirus (snake adeno-associated virus). Bearded dragons, a reptile pet of growing popularity, have a specific adenoviral infection and disease often manifesting in neurological signs, the so-called ‘star-gazing’. The AdVs found in squamate reptiles, belong to genus Atadenovirus, and at least 25 distinct atadenovirus types, detected by PCR and sequencing, have been reported. An unusual virion morphology with triple fiber projection per vertices was discovered during the characterization of an AdV isolate

14

Adenoviruses (Adenoviridae)

from Mexican beaded lizard. This virus was also found to have two fiber genes, a trait that usually broadens the host-cell range during infection. In a dead specimen of the common (Greek) tortoise an atadenovirus was detected supposedly as a result of host switching. Most recently, novel AdVs representing a thus far unknown lineage, proposed as an additional genus (Testadenovirus), have been reported from testudines, including red-eared slider, yellow-bellied slider, box turtle, eastern box turtle, pancake tortoise, and red-footed tortoise. An interesting case of siadenovirus infection in confiscated Sulawesi tortoises has been described. Significant fatalities and transmission of this virus to other turtles has also been reported. The pathogenic role of the adenoviral infections in reptiles needs further studies nonetheless the introduction of the practice of quarantining is highly recommended.

Frog and Fish Adenoviruses Data concerning adenovirus infection of aquatic vertebrates are scarce. In fact, only a single AdV has been isolated from each of the two host classes, amphibians and fish. Interestingly, the frog AdV was isolated from a renal tumor of a leopard frog on a reptilian cell line (TH1) prepared from turtle heart tissue. FrAdV–1 has a genome organization similar to that of TAdV–3, and the common evolutionary origin of these two viruses in genus Siadenovirus is supported by phylogenetic calculations. The only adenovirus isolate available from fish was obtained from a healthy adult specimen of an ancient Chondrostei species, the white sturgeon. Experimental transmission of the virus failed to produce any clinical signs. Genomic characterization of the sturgeon adenovirus implied the need for creating a new genus, Ichtadenovirus. Nuclear inclusion bodies typical of adenoviruses and adenovirus-like particles have been observed by light and electron microscopy, respectively, in damaged tissues of a couple of sea fish species, but molecular confirmation of AdV infection has not been successful. Some of these suspected viruses turned out to have genes similar to those in polyomavirus, but having larger and different genomes thus being candidate for members of a new family, still to be proposed officially (Adomaviridae).

Pathogenesis Adenovirus infections of man generally occur in childhood, and the outcome varies in severity from asymptomatic to explosive outbreaks of upper or lower respiratory tract manifestations. Less commonly, AdVs cause gastrointestinal, ophthalmic, urinary, and neurological diseases. The vast majority of adenovirus-caused diseases are self-limiting. However, immunocompromised patients, above all organ transplant recipients, individuals infected with human immunodeficiency virus and developing AIDS, and those receiving radiation and chemotherapy against tumors, represent special populations that are prone to experience grave, frequently fatal consequences of AdV infection. In numerous cases, the organ to be transplanted itself proves to be the source of invasive AdVs. Sporadic fatal infections may occasionally occur in healthy, immunocompetent individuals. In such cases, the presence of certain predisposing immunogenetic factors cannot be excluded. Cellular immune responses are also important for the recovery from acute adenovirus infection. Peripheral blood mononuclear cells have been found to exhibit proliferative responses to HAdV–2 antigen. This function is mediated by CD4 þ T cells, which seem to recognize conserved antigens among different HAdV serotypes. The incubation period from infection to clinical signs is estimated to be 1–7 days and may be dose dependent. Clinical symptoms during the initial viremia are dominated by fever and general malaise. Recovery from infection is associated with the development of serotype-specific neutralizing antibodies that protect against disease or reinfection with the same serotype. Pathology by AdVs is partially the consequence of viral replication and cell lysis. Correspondingly, in various tissues and organs, such as bronchial epithelium, liver, kidney, and spleen, disseminated necrotic foci can be observed upon necropsy and histopathological examination. Characteristic intranuclear inclusion bodies and so-called smudge cells contain large amounts of adenovirus capsid proteins. Besides the lesions caused by virus replication, direct toxic effects of high doses of structural proteins, as well as the host’s inflammatory respond, may contribute to the aggravation of pathology. Experimental infection of animals with various AdV types seldom results in pathology similar to that experienced with the same virus under natural circumstances. One of the few exceptions is turkey hemorrhagic enteritis, the pathogenesis of which has been studied in detail. TAdV–3 causes intestinal hemorrhages and immunosuppression. By in situ DNA hybridization and PCR, the presence of TAdV–3-specific DNA as evidence for virus replication has been demonstrated in the immunoglobulin M-bearing B lymphocytes and macrophage-like cells, but not in CD4 þ or CD8 þ T lymphocytes. Interestingly, fewer virions were present in the intestines, which are the principal site of pathology, than in the neighboring lymphoid organs including spleen and cecal tonsils. This finding strongly suggests that the intestinal lesions induced by TAdV–3 are mediated by the immune system. Systemic or intestinal hemorrhagic disease of ruminants seems to be related to virus replication in endothelial cells.

Diagnosis With the development of modern techniques, especially PCR and direct DNA sequencing, the number of AdVs detected in various organ samples of human or animal origin has increased rapidly. However, there is no official agreement or convention on the

Adenoviruses (Adenoviridae)

15

criteria that would be prerequisites for the approval of new AdV types. The conventional methods, including virus isolation in tissue culture, raising antisera, and performing a large set of cross-neutralization tests, are cumbersome, and the majority of medical and veterinary diagnostic laboratories do not possess appropriate prototype strain and serum collections. Full genomic sequences should validate new types even in the absence of isolated virus strains. However, the value of short sequences from PCR fragments is still a topic of debate. In principle, PCR and sequencing should be able to replace serotyping if appropriate targets are identified. Human medical laboratories use commercially available tests, such as complement fixation and enzyme immunoassay, to detect adenovirus-specific antibodies that cross-react with all serotypes. Nearly all adults have serologic evidence of past infection with one or more AdV types. For the detection of viral DNA by PCR, the most common target was initially the gene encoding the major capsid protein, the hexon. With subsequent restriction enzyme digestion, typing systems for HAdVs and FAdVs have also been elaborated. Recently, a nested PCR method, targeting the most conserved part of the adenovirus-coded DNA-dependent DNA polymerase gene, became very popular. The highly degenerate consensus primers seem to be capable of facilitating amplification of DNA from every adenovirus, irrespective of its genus affiliation.

Treatment No specific anti-adenovirus therapy has yet been established. Recent advances in understanding the pathophysiology of fulminant adenovirus diseases in immunocompromised patients have prompted the consideration of applying donor lymphocyte infusions after transplantation. Cidofovir is a monophosphate nucleotide analog that, after undergoing cellular phosphorylation, competitively inhibits incorporation of dCTP into virus DNA by the viral DNA polymerase. Incorporation of the compound disrupts further chain elongation. Efforts to improve its effect are in progress. There are a growing number of positive experiences with using this drug to combat adenovirus infections. Successful disinfection of poultry houses and stables can be performed with the use of chlorine-releasing agents, iodophors or quaternary ammonium compounds.

Prevention In the USA, orally administrable, live, enteric-coated vaccines against HAdV–4, HAdV–7, and HAdV–21 were used in military units for a couple of decades. The vaccine went out of production in 1996 and the supplies were exhausted by 1999. Soon, re-emergence of HAdV–7 and especially HAdV–4 was verified and about 12% of all recruits were affected by adenovirus disease. The vaccination program, restored at the military in October 2011, caused a prompt 75% reduction in the incidence of febrile respiratory illness among the new trainees. In the veterinary practice, dog vaccination schedules all over the world invariably include a live or killed CAdV–2 component against infectious canine hepatitis. Inactivated vaccine for horses against equine adenoviruses has been prepared in Australia. In farm animals, inactivated bivalent vaccines (containing one mastadenovirus and one atadenovirus BAdV type) have been in use in several countries for controlling enzootic calf pneumonia or pneumo-enteritis. In poultry practice, commercially available or experimental vaccines for the prevention of EDS or turkey hemorrhagic enteritis are applied occasionally. There are several attempts ongoing for the production of recombinant subunit vaccines, which should be safer than vaccines derived from tissue culture or the organs of infected birds.

Adenoviruses as Vectors HAdV–5 has been engineered and used most extensively as a gene delivery vector with a view to applications in gene therapy, immunization and antitumor medicine. Other human adenoviruses such as HAdV–3,  4,  26,  35,  41,  48,  49, etc. (either replication-competent or replication-deficient) or even nonhuman serotypes (OAdV–7, CAdV–2, chimpanzee and rhesus monkey AdVs), are being tested as a means of overcoming the problem posed by preexisting neutralizing antibodies in the human population, and also to achieve targeting to specific organs and tissues. Bovine, porcine, canine, and fowl AdV types have also been tested as novel antigen delivery vectors for immunizing animals of the respective species.

Further Reading Alonso-Padilla, J., Papp, T., Kaján, G.L., et al., 2016. Development of novel adenoviral vectors to overcome challenges observed with HAdV–5 based constructs. Molecular Therapy 24, 6–16. Condezo, G.N., San Martín, C., 2017. Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center. PLoS Pathogens 13, e1006320.

16

Adenoviruses (Adenoviridae)

Crenshaw, B.J., Jones, L.B., Bell, C.R., Kumar, S., Matthews, Q.L., 2019. Perspective on adenoviruses: epidemiology, pathogenicity, and gene therapy. Biomedicines 7, e61. Doszpoly, A., Harrach, B., LaPatra, S., Benko˝, M., 2019. Unconventional gene arrangement and content revealed by full genome analysis of the white sturgeon adenovirus, the single member of the genus Ichtadenovirus. Infection, Genetics and Evolution 75, 103976. Gilson, T., Blanchette, P., Ballmann, M.Z., et al., 2016. Using the E4orf6-based E3 ubiquitin ligase as a tool to analyze the evolution of adenoviruses. Journal of Virology 90, 7350–7367. Greber, U.F., Flatt, J.W., 2019. Adenovirus entry: From infection to immunity. Annual Review of Virology 6, 177–197. Hiwarkar, P., Kosulin, K., Cesaro, S., et al., 2018. Management of adenovirus infection in patients after haematopoietic stem cell transplantation: state-of-the-art and real-life current approach. A position statement on behalf of the infectious diseases working party of the European Society of Blood and Marrow Transplantation. Reviews in Medical Virology 28, e1980. Hoeben, R.C., Uil, T.G., 2013. Adenovirus DNA replication. Cold Spring Harbor Perspectives in Biology 5, a013003. Lenman, A., Liaci, A.M., Liu, Y., et al., 2018. Polysialic acid is a cellular receptor for human adenovirus 52. Proceedings of the National Academy of Sciences of the United States of America 115, E4264–E4273. Mennechet, F.J.D., Paris, O., Ouoba, A.R., et al., 2019. A review of 65 years of human adenovirus seroprevalence. Expert Review of Vaccines 18, 597–613. Needle, D.B., Selig, M.K., Jackson, K.A., et al., 2019. Fatal bronchopneumonia caused by skunk adenovirus 1 in an African pygmy hedgehog. Journal of Veterinary Diagnostic Investigation 31, 103–106. Pénzes, J.J., Menéndez-Conejero, R., Condezo, G.N., et al., 2014. Molecular characterization of a lizard adenovirus reveals the first atadenovirus with two fiber genes and the first adenovirus with either one short or three long fibers per penton. Journal of Virology 88, 11304–11314. Podgorski, I.I., Pantó, L., Földes, K., et al., 2018. Adenoviruses of the most ancient primate lineages support the theory on virus  host co-evolution. Acta Veterinaria Hungarica 66, 474–487. San Martín, C., 2012. Latest insights on adenovirus structure and assembly. Viruses 4, 847–877. Singh, A.K., Nguyen, T.H., Vidovszky, M.Z., et al., 2017. Structure and N-acetyl glucosamine binding of the distal domain of murine adenovirus 2 fibre. Journal of General Virology 99, 1494–1508.

Relevant Websites https://sites.google.com/site/adenoseq Adenovirus sequences. Google Sites. https://www.visual-science.com/projects/adenovirus Human adenovirus. Scientific illustration. Visual Science. https://talk.ictvonline.org/files/proposals/animal_dna_viruses_and_retroviruses Pending Proposals. International Committee on Taxonomy of Viruses (ICTV). https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10508 Adenoviridae. NCBI. NIH. https://talk.ictvonline.org/taxonomy Virus Taxonomy. International Committee on Taxonomy of Viruses (ICTV).

African Horse Sickness Virus (Reoviridae) Piet A van Rijn, Wageningen Bioveterinary Research, Lelystad, The Netherlands and North-West University, Potchefstroom, South Africa r 2021 Elsevier Ltd. All rights reserved.

Classification African horse sickness virus (AHSV) is a virus species within the genus Orbivirus belonging to the subfamily Sedoreovirinae within the Reoviridae family. Other orbivirus species are equine encephalosis virus, also infecting horses, and bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) both infecting instead ruminants. AHSV causes African Horse Sickness (AHS), which is a notifiable disease according to the World Organization of Animal Health (OIE). BTV is the prototype orbivirus and shares morphology and many molecular and virological properties with AHSV. Nine serotypes of AHSV have been recognized.

Virion Structure and Genome AHSV is a non-enveloped virus of 70–80 nm in diameter with an icosahedral structure. The viral genome of double-stranded (ds) RNA is about 19 kb in total and fragmented in ten genome segments (Seg-1 to Seg-10) varying in length from 0.8 to 3.9 kb. The segmented genome encodes seven structural proteins VP1-VP7 and at least four non-structural proteins NS1, NS2, NS3/NS3a, and NS4. Structural proteins VP2, 3, 5 and 7 form a three-layered virus particle harboring enzymatic proteins VP1, VP4 and VP6 in addition to one copy of each dsRNA genome segment. The inner protein layer consists of 120 VP3 molecules. VP3 displays two conformations in dimers and are clustered around a five-fold axis and form a replication complex together with the enzymatic proteins while VP3 decamers forms a subcore particle in an orbicular configuration. The subcore forms the core particle by a layer of 780 copies of VP7. A third protein layer, which is the outer shell, consists of VP2 and VP5 and determines the serotype of the infectious virus particle. Serotypes have been recognized by specific neutralizing antibodies and reflect genetic differences observed in Seg-2, which encode serotype dominant VP2. Serotypes 1 and 2, 3 and 7, and 5 and 8 induce some cross-neutralizing antibodies and display a higher percentage of genetic homology between each other compared to other serotypes. Non-structural proteins are not part of the virus particle but are expressed in the infected cells and support virus replication.

Life Cycle AHSV infects cells of the insect vector as well as of the mammalian host. After entering the cell, the outer shell is removed and the uncoated virus, the core particle, is released in the cytoplasm. Messenger RNAs (mRNAs) are synthesized inside the transcriptionally active core particle from all ten dsRNA genome segments and secreted into the cytoplasm. In this way, the viral genome is not exposed to cellular defense mechanisms triggered by dsRNA. Translation of viral mRNAs is specifically enhanced by the NS1 protein. Later after infection, mRNAs are recruited from the cytoplasm into replication complexes in virus inclusion bodies (VIBs) formed by the NS2 protein. Pre-subcore particles are formed within VIB, and initiate the replication of recruited single-stranded RNAs into dsRNA genome segments. The mechanism by which exactly one copy of each RNA segment is recruited into one virus particle is not completely understood but likely occurs according to a “follow-the-leader” process initiated by specific intersegmental RNA-RNA interactions and macromolecule interactions. After formation of core particles and assembly of the outer shell in a late stage, the progeny virus is released from the infected cell through an NS3/NS3a mediated process using cellular transport mechanisms and/or by budding on the cell membrane causing a cytopathogenic effect (CPE). Virus release from culicoides cells is dependent on NS3/NS3a protein and indeed infected culicoides cells show no CPE. In order to complete its life cycle, replication and transmission of AHSV between equines and midges are crucial. Midges are infected by feeding on viremic equines. Saliva, gut components, viral proteins, virus dose, and likely the gut microbiome influence AHSV infection of gut cells. Progeny virus is released into the hemolymph and, after dissemination throughout the midge, several organs are infected. Infection of salivary glands leads to virus release into saliva and transmission of AHSV to susceptible equines by every next blood meal. The interplay between virus and midge is very specific, since only a very few culicoides species are competent midges able to transmit AHSV to susceptible animals. Saliva, as well as trauma by bites, presumably enhance the infection of the equine host. In conclusion, the life cycle of AHSV requires replication in both the competent midge and susceptible equine hosts and at least two subsequent blood feedings by competent midges on equines.

Epidemiology AHS is an arthropod-borne noncontagious disease of Equidae. AHS is endemic in tropical sub-Saharan Africa where one or more serotypes at any given time are circulating asymptomatically in zebra and the African donkey. In subtropical regions,

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20937-0

17

18

African Horse Sickness Virus (Reoviridae)

AHS displays a seasonal occurrence with the first cases appearing in late summer while the disease declines or even disappears in the autumn due to lower temperatures and the reduced midge population. Occasionally, the affected areas can expand by changes of climate and environmental conditions. In the past, AHS outbreaks have also been reported outside Africa in the South–East Asia and the Iberian Peninsula. Local spread of AHS is always associated with the presence of competent infected biting midges spreading by wind and active flight. The most relevant competent midges for AHSV in Africa are Culicoides imicola and Culicoides bolitinos. Usually, midges become persistently infected and are lifelong virus source for onward transmission. In contrast, susceptible equine hosts are thought to be infectious only temporarily. African wildlife is an important AHSV reservoir. For example, infected zebra can be viremic for up to 40 days as opposed to domestic horses, if they survive the infection, are usually viremic for 2–3 weeks. Movement of viremic equines over long distances can lead to new epidemics but these depend on the presence of local competent midges for onward transmission. For instance, the last recorded European AHS epidemic occurred in Spain (in 1987–90) and was caused by the transmission of AHSV to local horses by indigenous Culicoides imicola which fed on imported viremic zebra. In the 21st century, the distribution of several midge-borne orbiviruses and their vectors has been dramatically expanding both to the south and north in different parts of the world. Presumably, climate change and globalization will further expand habitats of potential competent midges. Culicoides sonorensis in the Americas and Culicoides brevitarsis in Australia are known BTV transmitters but their vector competence for AHSV is unknown. The largest recorded Bluetongue outbreak in Europe in 2006–2009 was unexpectedly spread by indigenous midge species belonging to the Culicoides obsoletus group. Accordingly, the epidemiology of AHS could drastically change in the future and might include introduction and expansion of the disease to historically AHS-free regions with a moderate climate.

Clinical Features The incubation period in horses after natural AHSV infection is 5–9 days, whereas after experimental infection the incubation period is in general 3 days but depends on the dose and route of virus infection. The prognosis for infected horses is extremely poor as the mortality rate is 70%–95%. The fatality rate for African donkeys and zebra is very low (5% to 10%), whereas approximately 50% of infected mules will survive AHSV infection. There are four classical clinical forms of AHS in equines. The peracute, pulmonary form occurs in fully susceptible animals and has a short course. Infected horses with this form will rarely recover. The rise in body temperature starts after 3–5 days and can reach 40–41°C. This disease form is marked by rapidly progressive respiratory failure leading to a higher breathing frequency and further supported by abdominal heave lines. The horse appears exhausted and can stand with the forelimbs spread with an extended head and neck, profuse sweating, and dilated nostrils. Uncontrollable coughing with frothy, serofibrinous fluid exuding from the nostrils is common in the terminal stage of the disease. The respiratory distress usually starts suddenly a few hours before death. The subacute, cardiac, edematous form starts with rising in body temperature to 39–41°C about 7 days after infection and lasts in general for a couple of days. The fatality rate of this form is about 50%. The head may show severe swelling, including eyelids, lips, cheeks, tongue, intermandibular space, and laryngeal region. A characteristic symptom of this form is the swelling of the supraorbital fossae that may include conjunctival swelling with petechiae. The swellings may extend to the neck down to the chest. Remarkably, ventral edema and edema of the lower limbs are not seen. Paralysis of the esophagus may result in aspiration pneumonia and sublingual hemorrhages. The horse becomes very depressed and may lie down frequently for short periods. In the end, the horse dies by heart failure in about two weeks after onset of clinical signs. Horses that recover show gradually reducing swellings. The most commonly observed mixed, acute form displays signs of both the pulmonary and edematous forms with mortality rates around 70%. Mostly, signs are observed at post-mortem examination of fatal cases. Mild pulmonary signs seem to be not progressive, whereas edematous swellings and fluid in the body cavities seem to result in heart failure and death. Still, other typical signs of the pulmonary form are also observed in cases of the mixed form. The fourth form is horse sickness fever, which is the mildest form of the disease and is often missed. The incubation period is long (9 days) and the rise in body temperature can slowly reach 40°C. Clinical signs are not obvious. This mild form is usually seen in resistant or partially protected equines such as zebra, donkeys, or previously vaccinated horses. Horses that recover from natural AHSV infection develop lifelong immunity for the respective serotype and partial protection against closest related serotypes. Foals of immune mares show passive immunity for 3–6 months depending on the amount of AHSV directed antibodies received by uptake of colostrum.

Pathology The pulmonary form shows the most characteristic post-mortem changes such as edema of the lungs resulting in foam production in the air pipe and bronchia and hydrothorax (Fig. 1). Extensive alveolar edema and particolored hyperemia of the lungs are seen in peracute cases, whereas extensive interstitial edema is observed for more prolonged courses. The lungs may appear reasonably normal, but hydrothorax is common and the thoracic cavity may contain several liters of fluid. Less frequent are periaortic and peritracheal edematous infiltration. Cardiac lesions are not obvious, but epicardial and endocardial petechial hemorrhages are sometimes observed. Particularly, the lymph nodes in the thoracic and abdominal cavities are edematous and enlarged. Regarding the gastrointestinal tract, hyperemia of the

African Horse Sickness Virus (Reoviridae)

19

Fig. 1 Severe characteristic post-mortem observations in a horse which died as a result of AHSV infection. (A). Hydrothorax. (B). Formation of foam in the air pipe and bronchia.

glandular fundus of the stomach, hyperemia and petechial hemorrhages in the mucosa and serosa of the small and large intestines, subcapsular hemorrhages in the spleen, and congestion in the renal cortex are reported. The cardiac form typically shows yellow, gelatinous infiltration in the subcutaneous and intermuscular fascia, primarily of the head, neck, and shoulders. Infrequently, lesions may be observed in the chest, ventral abdomen, and rump. Hydropericardium is common, with extensive petechial and ecchymotic hemorrhages on the epicardium and endocardium. In contrast to the pulmonary form, lung edema is not present or very mild, and hydrothorax is rare. In addition to those found for the pulmonary form, submucosal edema of the cecum, large colon, and rectum is more pronounced. The mixed, acute form of the disease shows a combination of those found in the pulmonary and cardiac forms.

Diagnosis Clinical diagnosis is not very specific especially in the early phase of AHS. However, additional factors support a presumptive diagnosis, such as other AHS suspicions or lethal confirmed AHS cases in the vicinity of the suspected horse. In a later phase of the disease, once characteristic clinical signs typical for AHS develop, differentiation from other equine diseases also improves clinical diagnosis. Specific findings by post-mortem examination further increase the differential diagnosis. AHS can spread very rapidly and causes huge economic and enormous socio-emotional impact by its severity. Therefore, AHS is a notifiable disease according to the World Organization of Animal Health (OIE). Any suspicion of AHS must be promptly reported to national veterinary authorities. The suspected horses should be correctly diagnosed with laboratory diagnostics as soon as possible in order to quickly install effective, feasible, and acceptable control measures. Further, the suspected horse must be protected against bites of midges to prevent further spread of disease by housing and use of horse blankets, repellents, and/or insecticides. The veterinary authority

20

African Horse Sickness Virus (Reoviridae)

will advise in this and further arrange and submit blood samples to an authorized laboratory, often the national reference laboratory for AHS. Preferably, all sick and healthy equines on the premises should be blood sampled. In the case of deceased horses, samples of spleen, lung lymph nodes and samples of any other organs with obvious pathology should be collected too, refrigerated and transported on ice for diagnostic purposes. Many highly sensitive and specific laboratory diagnostic tools for AHS have been developed and are operational in authorized laboratories to quickly confirm or exclude AHSV infection. Real-time reverse transcriptase polymerase chain reaction (PCR) assays for AHSV have been developed and are operational in diagnostic laboratories as first-line diagnostic tool. Several genome segments have been targeted which also opens the possibility for confirmatory PCR assays. Confirmation could be also performed with Seg-2 based PCR assays in order to identify the virus serotype. The latter can be supportive to find the original virus source, and is important in case vaccination against the spreading of a specific AHSV serotype is considered. In addition, virus isolation is performed to further characterize and investigate the causing virus, including serotyping with virus neutralization tests using reference sera against each of the nine serotypes. However, virus isolation is not routinely used for diagnostic purposes because this method is less sensitive, laborious and time-consuming. At one week after AHSV infection or later, antibodies against AHSV are detected by ELISA specific for VP7 antibodies and confirms AHSV infection. High throughput ELISAs are routinely used and are highly sensitive and specific detecting all nine AHSV serotypes. Neutralizing antibodies are detected later after infection with serum neutralization tests using nine reference strains of AHSV serotypes. Neutralizing antibodies are in majority serotype specific and directed to VP2.

Treatment, Control, and Prevention There is no adequate treatment for AHS, and appropriate supportive therapy can be provided based on the observed clinical signs. AHSV infections must be detected and controlled as soon as possible. Importantly, control measures should prevent the spread of AHS to disease-free areas, countries or horse populations. Enforcing quarantine periods, mandatory protocols for clinical inspections and test procedures in combination with insect control measures prior to allowing import of equines are described for international equine transports over long distances. However, local expansion of an affected area is hard to stop due to local transport, movement of animals and, more importantly, spread by competent midges. National authorities in AHS endemic, as well as in AHS-free countries, together with national reference laboratories, horse industries, owner organizations, and veterinary services have developed contingency plans describing control of AHS according to recommendations of OIE and WHO. In general terms, the suspected horse is temporarily contained with respect to access of midges, and official authorities must be informed. After confirmation by laboratory diagnosis, control measures as described in contingency plans are instantly implemented. Follow-up control measures regarding confirmed cases and (suspected) horses on the same premise are under debate and vary between national contingency plans. For example, infected and/or suspected horses are euthanized or receive supportive therapy while insect control strategies are undertaken. All contingency plans describe the installation of different zones: the affected zone around the affected holding (3 km diameter), surrounded by the protective zone (100 km diameter) and the surveillance zone of an additional 50 km radius around the protective zone. Actions are different for these zones and vary in different national contingency plans. Movement of equines from the affected zone is prohibited and is restricted or allowed in combination with negative test results, quarantine period and vector control measures for the other zones. The temporary or permanent housing of equines, removal of midge breeding places, and use of repellents, insecticides, and horse blankets are also described in contingency plans. Clinical inspections and surveillance are intensified in all zones. These control measures are adopted and specified to the local or national situation with regard to feasibility, such as the capacity of accommodation/stables, possibilities for insect control, the behavior of competent midges (if known), and the number of equine wildlife and domestic horses. Furthermore, contingency plans describe the possibility of vaccination in order to prevent disease and eventually eradicate AHS. In most cases, the decision by authorities to start vaccination campaigns in the affected area or country depends on the success of already undertaken actions, the feasibility, and of course, the availability of suitable AHS vaccine. Vaccination is the most effective way to protect horses and to eradicate AHS outbreaks. Still, success by vaccination strongly depends on the profile (efficacy, safety, costs, acceptance) of the used vaccine. Currently, conventionally live-attenuated vaccines (LAV) are available for AHS in several African countries. Subsequent vaccinations with affordable cocktail vaccines for multiple serotypes protect horses against all nine serotypes. LAVs can, however, induce viremia and can spread to, and by, midges. Indeed, virulent variants (partially) derived from LAVs by reassortment events and/or by reversion to virulence have been reported. The Spanish AHS outbreak has been eradicated, first by use of LAV, and later by inactivated AHS vaccine which is more expensive but safer. Both vaccine types do not enable DIVA (differentiating infected from vaccinated individuals) and complicate eradication programs, and safe trade of equines is strongly hampered by this shortcoming. Experimental vector- and subunit vaccines are completely safe and DIVA vaccines in combination with VP7 based ELISAs. Protection is mainly based on immune responses against outer shell proteins. Further, several virus-based vaccine platforms have been developed by genetic modification and applied for all nine serotypes by exchange of outer shell proteins. Consequently, virus-based vaccines induce full-blown immune responses against almost all viral proteins. Disabled infectious single cycle/cell (DISC) vaccine lacks essential protein VP6 and non-essential NS4. In effect, DISC vaccine is produced by in trans complementation on cells constitutively expressing VP6 protein. DISC vaccination leads to an abortive infection, since no progeny infectious virus can be produced in equines. AHSV without non-essential NS4 or NS3/NS3a are attenuated, protective and similarly produced as LAVs. AHSV without NS3/NS3a does not cause viremia and does not propagate in midges and is therefore named disabled

African Horse Sickness Virus (Reoviridae)

21

infectious single animal (DISA) vaccine. Furthermore, the DISA vaccine platform is DIVA compatible with the experimental NS3/NS3a based ELISA and a DIVA PCR test. All these experimental (vector) vaccine platforms, including easy-to-produce deletion mutants of AHSV, are very promising but are classified as genetically modified organisms and regulatory hurdles hamper their use at the moment. In conclusion, there is a desperate need for improved AHS vaccines in order to reduce the economic and socioemotional impact caused by this devastating viral disease of horses.

Further Reading Alberca, B., Bachanek-Bankowska, K., Cabana, M., et al., 2014. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge. Vaccine 32, 3670–3674. Erasmus, B.J., 1973. The pathogenesis of African Horsesickness. In: Proceedings of the 3rd International Conference on Equine Infectious Diseases. Basel, Paris: Karger. pp. 1–11. Erasmus, B.J., 1976. A new approach to polyvalent immunisation against African horse sickness. In: Proceedings of the Fourth International Conference on Equine Infectious Diseases, Lyon, France, September 1976. Princeton, N.J.: Veterinary Publications, pp. 401–403. Guthrie, A.J., Quan, M., 2009. African horse sickness. In: Mair, T.S., Hutchinson, R.E. (Eds.), Infectious Diseases of the Horse. Fordham: Equine Veterinary Journal Ltd, pp. 72–82. Lulla, V., Losada, A., Lecollinet, S., et al., 2017. Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge. Vaccine 35, 4262–4269. Lulla, V., Lulla, A., Wernike, K., et al., 2016. Assembly of replication-incompetent African horse sickness virus particles: Rational design of vaccines for all serotypes. Journal of Virology 90, 7405–7414. Mertens, P.P.C., Maan, S., Samuel, A., Attoui, H., 2005. Orbiviruses, Reoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. London: Elsevier/Academic Press, pp. 466–483. OIE (World Organisation for Animal Health), 2010. African horse sickness. In: Fernandez, P.J., White, W.R. (Eds.), Atlas of Transboundary Animal Diseases. Paris: OIE, pp. 12–18. Sanchez-Vizcaíno, J.M., 2004. Control and eradication of African horse sickness with vaccine. In: Schudel, A., Lombard, M. (Eds.), Control of Infectious Diseases by Vaccination, Developments in biologicals, vol. 119, Basel: S. Karger AG, pp. 255–258. van de Water, S.G., van Gennip, R.G., Potgieter, C.A., Wright, I.M., van Rijn, P.A., 2015. VP2 exchange and NS3/NS3a deletion in African horse sickness virus (AHSV) in development of Disabled Infectious Single Animal vaccine candidates for AHSV. Journal of Virology 89, 8764–8772. van Rijn, P.A., Maris-Veldhuis, M.A., Boonstra, J., van Gennip, R.G.P., 2018a. Diagnostic DIVA tests accompanying the Disabled Infectious Single Animal (DISA) vaccine platform for African horse sickness. Vaccine 36, 3584–3592. van Rijn, P.A., Maris-Veldhuis, M.A., Potgieter, C.A., van Gennip, R.G.P., 2018b. African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform. Vaccine 36, 1925–1933. Weyer, C.T., Grewar, J.D., Burger, P., et al., 2016. African horse sickness caused by genome reassortment and reversion to virulence of live, attenuated vaccine viruses, South Africa, 2004–2014. Emerging Infectious Diseases 22, 2087–2096.

African Swine Fever Virus (Asfarviridae) Linda K Dixon, Rachel Nash, Philippa C Hawes, and Christopher L Netherton, The Pirbright Institute, Pirbright, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Nomenclature ASFV African swine fever virus ATF4 Activating transcription factor 4 BER Base excision repair Bcl-2 B-cell lymphoma 2 CHOP C/EBP homologous protein or GADD153 CSF Classical swine fever CSFV Classical swine fever virus dpi days post-infection dTTP deoxy thymidine triphosphate ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum EU European Union GADD34 Growth arrest and DNA damage-inducible protein 34 g/cm3 grams per cubic centimetre IAP Inhibitor of apoptosis protein ICP34.5 Infected cell protein 34.5 IFN Interferon IL1 Interleukin 1

Glossary Apoptosis A highly regulated form of programmed cell death. Capsomers Capsomers are subunits of the capsid, an outer covering of protein that protects the virus genetic material. Clathrin-mediated endocytosis A vesicular transport event that facilitates the internalization of molecules including receptors. This is involved in various processes including signal transduction, receptor recycling, virus entry. The process involves clathrin, a tri-skeleton shaped scaffold protein. C-type lectin A type of carbohydrate-binding protein domain known as a lectin. The C-type lectin require calcium for binding. C-type lectin have diverse functions including regulation of cell adhesion, immune responses and apoptosis. Entry fusion complex The entry fusion complex is a multicomponent protein complex required for fusion of the virus envelope with cellular membranes to enable virus entry. ERCC4-like nuclease An enzyme involved in DNA recombination or repair. Genotypes This term is used to describe genetic variants of a given microoganism, in this case African swine fever virus Filopodia Slender cytoplasmic projections that contain actin filaments cross-linked into bundles by actin-binding proteins.

kbp kilobase pairs kDa kilodaltons MGF Multigene family ml millilitre NF-kB Nuclear factor kappa B NFAT Nuclear factor of activated-T cells mg milligram nm nanometre NTPase Nucleoside triphosphatase OIE World Organisation for Animal Health ORF Open reading frame PCR Polymerase chain reaction RNAP RNA polymerase ROS Reactive oxygen species SUMO Small Ubiquitin-like Modifier TBP Tata box binding protein TFIIB Transcription factor IIB TFS Transcription factor S VLTF Vaccinia virus late transcription factor VETFL Vaccinia virus early transcription factor

Guanyl transferase Three enzymes (RNA triphosphatase, guanyl transferase and methyl transferase) which are involved in the addition of the methylated 5’ cap to mRNA. Immunogold electron microscopy Immunogold labelling is a technique which involves binding of antibodies attached to gold particles to proteins. These complexes can then be detected by electron microscopy to localise specific proteins. Macropinocytosis Is a form of uptake of materials including solutes from the extracellular fluid. The uptake mechanism is non-specific and taken advantage of by many pathogens. Once inside cells macropinosomes mature through endosomes Multigene families Multigene families are groups of genes from the same organism that encode proteins with similar sequences over all or part of the sequence. Myristoylation Involves covalent attachment of a myristoyl group by an amide bond to an alpha amino group of an N-terminal glycine residue. Commonly this can act as a membrane anchor for proteins. Sylvatic cycle The sylvatic cycle is the fraction of the pathogen population’s lifespan spent cycling between wild animals and vectors.

African Swine Fever Virus Classification The Asfarviridae family consists of a single genus Asfivirus which contains one member: African swine fever virus (ASFV). The virus contains a double-stranded DNA genome varying in length between 170 and 193 kbp with terminal cross-links and inverted terminal

22

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20925-4

African Swine Fever Virus (Asfarviridae)

23

repeats. The predominantly cytoplasmic replication cycle and genome structure resembles that of the Poxviridae although the icosahedral capsid structure differs. African swine fever virus encodes enzymes and factors required for replication and transcription in the cytoplasm, in common with other large DNA virus families that have a replication stage in the cytoplasm. Genome sequencing has shown that ASFV is only distantly related to other viruses but shares the highest number of conserved genes with several viruses that infect amoeba, including Faustovirus, kaumoebavirus and Pacmanvirus. These giant viruses have genome sizes of greater than 400 kbp and share approximately 30 genes with ASFV including genes involved in replication, transcription and virus morphogenesis.

Virion Structure ASFV has a multi-layered structure consisting of a nucleoprotein core, 70–100 nm in diameter, inside a protein core shell which is surrounded by an internal lipid layer (Fig. 1). The icosahedral capsid is assembled on this internal envelope. These intracellular mature particles are 170–190 nm in diameter and gain an additional membrane as they bud from the plasma membrane of the infected cell to form the extracellular enveloped virus that is 175–215 nm in diameter. Both the intracellular mature and extracellular enveloped forms of the virus are infectious. The capsid show icosahedral symmetry (T- 189–217) corresponding to 1892–2172 capsomers each observed as a hexagonal prism with a central hole. The distance between capsomers is 7.4–8.1 nm. Mass spectrometry analysis of purified virus particles has identified 68 virus encoded proteins of which more than 20 have no known function. About 20 additional proteins in the virus particle were identified as host proteins. ASFV encodes for two polyproteins, pp220 (CP2475L) and pp62 (CP530R) which are proteolytically cleaved, by a virus-encoded cysteine protease (S273R). Mature peptide products of molecular weight 150, 37, 34, 14, and 5 kDa from pp20 and 35, 15, and 8 kDa from pp62 are incorporated into the core shell. The main virus capsid protein, p72 (B646L), represents about 10% of the mass of the virus particle. The p49 (B438L) protein is required for forming vertices of the capsid and is also present in the virus particle. Proteins present in the nucleoprotein core include a histone-like DNA binding protein A104R, components of the multi-subunit RNA polymerase and other enzymes and factors required for early transcription for example the mRNA capping and polyadenylation enzymes, early transcription factors and three helicases. Enzymes involved in the virus encoded DNA repair system are also packaged in the virus particle presumably in the core. Only one viral protein has conclusively been demonstrated to be in the external envelope, the type I membrane protein encoded by EP402R which contains an extracellular domain similar to host CD2

Fig. 1 African swine fever virus structure. Electron micrograph showing the Ba71v isolate of ASFV budding from a Vero cell with a cartoon highlighting the different structural elements. Known viral contents of the nucleoid, core shell, internal envelope, capsid and external envelope are indicated. The scale bar is 100 nm.

24

African Swine Fever Virus (Asfarviridae)

protein. The virus CD2 like protein (CD2v) is required for the binding of red blood cells to virus infected cells and extracellular virions. A number of membrane proteins are found on the internal virus envelope including p54 (pE183L), p22 (pKP177R), p17 (pD117L) and p12 (pO61R). The latter was previously shown to be involved in virus attachment. Several ASFV proteins have similarities to components of the vaccinia virus entry fusion complex and it is possible that a similar ASFV complex is localised to the internal envelope. Virus particles band at 1.095 g/cm−3 in Percoll and 1.19–1.24 g/cm−3 in CsCl. The S20W is about 35,005. Virus infectivity is stable over a wide pH range between 4 and 13 but is inactivated by treatment at 60°C for 30 min or by UV irradiation. Chemical treatment with some disinfectants, for example 1% formaldehyde, 2% NaOH or paraphenylphenolic compounds effectively inactivate virus.

Genome Organisation The ASFV genome consists of linear double stranded DNA varying between 170 and 193 kbp depending on the isolate. The genome termini are covalently closed and consist of two flip-flop forms that are inverted and complementary to each other. Adjacent to the termini are tandem repeat arrays. Genome sequences present in sequence databases generally do not include all of the terminal repeat sequences or terminal crosslinks. The ASFV genome consists of about 150–167 open reading frames (ORFs) which are closely spaced and read from both DNA strands as shown on Fig. 2. In some genome regions a number of consecutive ORFs are on the same DNA strand. Some of these genes are part of multigene families (MGFs) that have evolved on the virus genome by a process of gene duplication and in some examples transposition to the other end of the genome. The number of ORFs is based on the definition that the minimum size for an encoded protein is 60 amino acids and the ORFs do not overlap extensively with other major ORFs. Thus, some smaller ORFs may encode functional proteins but may have been missed from the current sequence annotations of the ASFV genome. Information from the transcript map of the ASFV genome may help in identification of any additional expressed small ORFs. The

Fig. 2 Genome of African swine fever virus. Schematic representation of the African swine fever virus Georgia 2007/1 isolate. Open reading frames are indicated by arrows indicating the direction that these are read.

African Swine Fever Virus (Asfarviridae)

25

intergenic region between ORFs is generally short and contains a short promoter sequence immediately upstream of the ORF. The virus encoded RNA polymerase and transcription factors bind to initiate transcription from the downstream ORF. These promoter sequences are generally AT rich and consensus promoter sequences for replication stage specific transcripts, (immediate early, early, intermediate, late) have not been identified. Partial sequencing of the gene for the major capsid protein, p72/B646L, has been used to assign ASFV isolates to 24 different genotypes falling in three main clades. These were all found in East Africa, where the ancient sylvatic cycle is present. Genotypes that have spread more widely in domestic pigs include the genotype I in central and West Africa, genotype IX in Kenya and Uganda and genotype II in the Caucasus, Russian Federation, Europe and China. Gain or loss of members of multigene families (MGF) is the main mechanism for genome variability. Loss of some members of MGFs 360 and 505/530 (see below) have been associated with reduction in virulence for domestic pigs. In addition frame shift mutations have been identified in some genes, notably the genes coding for pEP402R/CD2v and the C-type lectin p153R in some low virulence virus isolates. The expression of pEP402R/ CD2v protein is required to cause the binding of red blood cells to infected cells, a process referred to as haemadsorbtion. Thus virus isolates that do not express this protein do not cause haemadsorbtion. The different functional classes of ASFV encoded proteins are indicated on the genome map in Fig. 2 and in Table 1 and summarised below.

Multigene families A striking feature of the ASFV genome is the large number of genes that belong to one of several multigene families, named MGF 100, MGF 110, MGF 300, MGF 360, MGF 505/530 based on the average number of amino acids encoded by family members. The numbers of copies of these genes varies between virus isolates. This varies between 5 and 13 for MGF 110, 3–4 for MGF 300, 11–16 for MGF 360 and 8–10 for MGF 530/505. The MGF 100 contains up to 5 members. In addition the gene for the p22/ KP177R virion transmembrane protein has one or two related copies at the opposite genome end in most of the field isolates although these are missing from the genome of the tissue-culture adapted isolate BA71V. These gene families have evolved on the ASFV genome by a process of gene duplication and transposition from one genome end to the other. The functions are largely unknown although some members of MGF 360 and MGF 505/530 play a role in suppressing the type I interferon (IFN) response. MGF110 encode proteins with signal peptides and conserved cysteine motifs and some members contain the amino acid sequence KDEL which are involved in retrieval of proteins from the endoplasmic reticulum (ER).

Polymerases ASFV transcription does not require the functional host RNA polymerase II since it proceeds in the presence of inhibitors of the host enzyme. From sequence comparisons with eukaryotic RNA polymerase II, ASFV encoded subunits of a multisubunit DNAdependent RNA polymerase (RNAP) have been identified. These include subunits 1, 2, 3, 5, 6, 7, 10. This is fewer than the 12 subunits coded for by the eukaryotic RNAP suggesting some mechanistic differences. The RNAP 3 subunit has a C-terminal extension dissimilar to the eukaryotic homologue which may represent a fusion with RNAP subunit 11. In addition ASFV codes for homologues of the eukaryotic transcription factors TFIIB and TFS and a putative TATA Box Binding protein (TBP). In eukaryotes TBP binds to specific promoter sequences, initiator factor TFIIB binds TBP and recruits the RNAP. The elongation factor TFS helps the polymerase move past arrest sites. ASFV codes for proteins with sequence similarity to vaccinia virus encoded transcription factors including the early factor VETFL and late factors VLTF2 and VLTF3. Transcript modification enzymes have also been identified including a poly A polymerase and 5’ capping enzyme with guanylyltransferase capability. Functional characterisation of the ASFV transcription machinery has however not yet been completed.

DNA replication Sequence comparisons have identified a processive DNA polymerase family B, involved in genome replication and a putative DNA primase involved in initiating DNA replication. Other proteins involved in DNA replication include a DNA topoisomerase class II involved in unwinding of the duplex DNA during replication and a PCNA like protein, potentially involved in clamping the DNA polymerase to the genome. Other proteins potentially involved in replication include those with motifs of helicases and nucleoside triphosphatases (NTPases) and proteins with similarity to ERCC4-like nucleases and a lambda like exonuclease which may be involved in resolution of head to head concatameric replicating genomes into single units. The proteins involved in DNA replication are not packaged in the virus particle but are expressed early during infection.

Helicases Six helicases are encoded and presumed to have roles in unwinding/winding DNA or RNA to allow access to the virus transcription or replication apparatus. Of these helicases, three are packaged in the virus particle and likely to have roles in enabling early gene transcription.

Nucleotide metabolism ASFV encodes genes for several enzymes involved in nucleotide metabolism in order to modulate the pool of dNTPs available for DNA replication. These include ribonucleotide reductase, thymidine kinase, thymidylate kinase, deoxyuridine triphosphatase (dUTPase). There is essentially no synthesis of thymidylate in macrophages and both the thymidine kinase and dUTPase genes are required for replication in porcine macrophages, but not the Vero cell culture line that supports replication of some isolates of

26

African Swine Fever Virus (Asfarviridae)

Table 1 ASFV genes and their functions. List of ASFV genes and their functions, as well as uncharacterised proteins that have been identified in purified virions by mass spectrometry. Genes are ordered by function and genes which do not code for virion components and no specific function has been described (such as the multigene family proteins) are not included Gene Name

Function

Virion Component

Nucleotide metabolism F778R F334L K196R A240L

Ribonucleotide reductase large subunit Ribonucleotide reductase small subunit Thymidine kinasea Thymidylate kinase

N N N N

Transcription EP424R NP868R C475L B263R NP1450L EP1242L H359L D205R C147L D339L CP80R C315R I243L B175L B385R G1340L

FtsJ-like RNA methyltransferase mRNA-capping enzymea Poly A polymerase large subunit Putative TATA binding protein RNA polymerase subunit 1 RNA polymerase subunit 2 RNA polymerase subunits 3 & 11 RNA polymerase subunit 5 RNA polymerase subunit 6 RNA polymerase subunit 7 RNA polymerase subunit 10 TFIIB like transcription factor Transcription factor SII (TFIIS) Vaccinia A1-like transcription factor Vaccinia A2-like transcription factor Vaccinia A7-like transcription factor

Y Y Y N Y Y Y Y Y Y N N N N N Y

Helicases F1055L QP509L Q706L A859L D1133L B962L

Helicase Helicase Helicase Helicase Helicase Helicase

N N Y N Y Y

DNA replication G1211R P1192R EP364R D345L C962R E301R

DNA polymerase (Group of α-like) DNA topoisomerase IIa ERCC4 nuclease domain Lambda-like exonuclease Putative DNA primase Similar to proliferating cell nuclear

N N N N N N

DNA repair E296R NP419L O174L E165R

Apurinic/apyrimidinic endonuclease class II DNA ligasea DNA polymerase Xa dUTPasea

Y Y Y Y

superfamily superfamily superfamily superfamily superfamily superfamily

II II. II. II. II. II.

Vaccine virus D11 like Vaccinia A18R orthologue Vaccinia D6-like Vaccinia I8 like

Structural proteins and those involved in morphogenesis CP2475L 220 kDa polyprotein precursor of structural proteins p5, p150, p37, p14 and p34a CP530R 62 kDa polyprotein precursor of structural proteins p35, p15 and p8a E120R Capsid protein p14.5. Required for virion egressa B438L Capsid protein p49. Required for vertice formation EP402R CD2 homologuea O61R Inner envelope protein p12. Involved in attachmenta D117L Inner envelope protein p17a KP177R Inner envelope protein p22a E183L Inner envelope protein p54. Binds to LC8 chain of dyneina H108R Inner envelope proteina E248R Inner envelope protein. Involved in post-entry fusiona B646L Major capsid protein p72a K78R Nucleocapsid protein p10a A104R Nucleocapsid protein pA104Ra

Y Y Y Y Y Y Y Y Y Y Y Y Y Y

African Swine Fever Virus (Asfarviridae)

Table 1

27

Continued

Gene Name

Function

Virion Component

CP204L E199L S273R A137R

P32/P30 early phosphoprotein Structural protein, transmembrane regiona SUMO-1 proteasea Structural protein p11.5. Protected pigs have antibody response to this protein.

Y Y Y Y

Uncharacterised virion components K145R Uncharacterised C129R Uncharacterised C717R Uncharacterised E184L Uncharacterised E423R Uncharacterised F317L Uncharacterised H124R Uncharacterised H171R Uncharacterised H240R Uncharacterised I73R Uncharacterised K421R Uncharacterised M1249L Uncharacterised QP383R Uncharacterised M448R Uncharacterised CP312R Uncharacterised C105R Uncharacterised B117L Uncharacterised B169L Uncharacterised C257L Uncharacterised CP123L Uncharacterised E146L Uncharacterised EP84R Uncharacterised I177L Uncharacterised

late protein protein protein protein protein protein protein protein protein early protein protein protein protein protein. Possible RNA ligase protein. Protected pigs have antibody response to this protein protein. Was C122R in original Ba71v sequence transmembrane protein transmembrane protein transmembrane protein transmembrane protein transmembrane protein transmembrane protein transmembrane protein

Other enzymes and host cell modulation A179L Bcl-2 family apoptosis inhibitora L83L Binds IL-1a EP153R C-type lectin-like A224L IAP family apoptosis inhibitora DP148R Inhibits interferon signallinga DP96R Inhibits TBK1 and IKKβa EP152R Interacts with BAG6. Essentiala A238L IκB-like protein. Inhibits host gene transcriptiona DP71L MyD116, GADD34, ICP34.5 homologue. Translation modulator D250R Nudix hydrolase/decapping mRNAa H339R α-NAC binding proteina R298L Serine/threonine protein kinasea B119L Similar to S.cerevisiae ERV1 and vaccinia E10Ra I329L TLR3 agonist. Inhibits IFNa B318L Trans-geranylgeranyl-diphosphate synthasea I215L Ubiquitin-conjugating enzymea

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N Y N N Y N N N Y Y Y N N N

a

Indicates a protein for which there is experimental evidence for its function.

ASFV. It is likely that early expression of these genes increase the dTTP/dUTP ratio to facilitate efficient replication in the macrophage natural target cells for ASFV. These enzymes are not packaged into virus particles.

DNA repair ASFV codes for components of a base excision DNA repair (BER) system which could be an adaptation required for replication in the highly oxidising environment of the macrophage cytoplasm. The high concentrations of reactive oxygen species (ROS) in macrophages can result in DNA lesions which could either introduce mutations into the viral genome or interrupt reading of the genome by the viral DNA and RNA polymerases. Sequence analysis has identified a DNA polymerase X, an ATP-dependent DNA ligase and a class II apurinic/apyrimidinic endonuclease that could constitute a BER system. A DNA glycosylase is required to initiate the repair process and the apparent lack of an ASFV gene for this enzyme suggests either that the enzyme is a very distant homologue or that the virus recruits a host DNA glycosylase. However, the known components of the BER system are packaged into the virion which implies that the as yet unidentified DNA glycosylase enzyme is also likely to be virally encoded. The BER

28

African Swine Fever Virus (Asfarviridae)

pathway is required for replication in the macrophage as deletion mutants lacking the gene for the class II endonuclease are viable in Vero cells, but not macrophages.

Virion morphogenesis and structural proteins The genes identified as coding for virus structural proteins and those involved in other aspects of virus assembly are shown on Table 1 and on Fig. 1. Initial studies identified virus structural proteins biochemically and mapped the genes for some of these to the virus genome. More recently a proteomic approach using mass spectrometry was used to more fully characterise the proteins present in purified virus particles. The results from these studies generally agree well and are complemented by immunogold electron microscopy to localise proteins in the virus particle. The recent proteomic analysis identified 68 virus proteins in the purified extracellular virions. From these 44 had not previously been characterised and many of these are of unknown function. The major structural proteins include the VP72 (B646L) major capsid protein, the products of polyproteins pp220 and pp62 which are cleaved by a virus encoded SUMO-like cysteine protease into products of molecular weights 150, 37, 34, 14 and 5 kDa or 35, 15 and 8 kDa respectively. These protein products are incorporated into the shell of the nucleoprotein core. Fifteen of the 26 virion proteins containing transmembrane domains were identified in the virus particle and all except one presumed to be incorporated into the inner envelope of the virus particle since only the virus encoded CD2v/EP402R protein has conclusively been shown to be in the outer envelope. Proteins in the inner envelope include several with similarity to the vaccinia virus entry fusion complex and others that have been implicated in virus binding and entry to cells. The vaccinia virus entry fusion complex has 11 proteins each containing a transmembrane domain at either the N or C-terminus but no predicted glycosylation sites or signal peptides. These are features shared with a number of the ASFV virion proteins (Table 1). Enzymes and factors packaged in the virus also include the RNA polymerase and other components of the early transcription machinery, components of the virus-encoded DNA repair system, others such as two DNA binding proteins, a serine protein kinase and inhibitor of apoptosis protein (see Table 1).

Proteins that inhibit host defences By comparison with vaccinia virus, which shares a similar replication cycle to ASFV, it is estimated that more than one third of ASFV encoded proteins may be non-essential for replication in cells but have roles including evasion of host defences or modulation of host cell function.

Inhibitors of apoptosis ASFV encodes several inhibitors of apoptosis targeting different pathways. These include a member of the Bcl-2 protein family, A179L that binds to a broad range of pro-apoptotic Bcl-2 family members to inhibit their activity. This protein also binds to Beclin-1 and may also inhibit autophagy. The A224L protein belongs to the IAP inhibitor of apoptosis family and has been shown to bind directly to caspase 3 inhibiting its activity. The DP71L protein shares a conserved domain with host protein GADD34 and the Herpes simplex virus-encoded ICP34.5 protein. These proteins up regulate global protein synthesis by targeting host protein phosphatase 1 to dephosphorylate translation initiation factor eIF-2alpha. This activity also prevents translation of a class of mRNAs with short upstream open reading frames that are normally only translated under conditions of cellular stress. These include mRNAs for transcription factors ATF4 and CHOP which activate transcription of genes involved in apoptosis induction. Thus expression of the DP71L protein can this inhibit stress-induced induction of apoptosis. Additional ASFV proteins that inhibit this pathway are predicted since in cells infected with deletion mutants of the DP71L gene stress-induced phosphorylation of eIF-2 alpha is also inhibited.

Inhibitors of type I interferon and inflammatory responses ASFV encodes a number of inhibitors of the type I interferon response, the main early host response to inhibit virus replication. These were identified either by ectopic expression of ASFV genes identified by sequence comparison or by random screening of ASFV expressed proteins. Alternatively, comparison of type I IFN induction in cells infected with virulent compared to gene deleted attenuated viruses showed that deletion of some genes belonging to MGFs 360 or 505/530 deletion resulted in increased type I IFN induction indicating they function to suppress IFN induction. Other IFN inhibitory proteins include the I329L protein which inhibits the Toll-like receptor 3 activated induction of type I IFN. Other proteins including DP96R inhibit the type I IFN response. Deletion of DP96R, DP148R and MGF 360 and 505/530 genes results in virus attenuation. The A238L protein inhibits transcriptional activation induced by several factors including NF-kB and NFAT mediated by the p300 transcriptional co-activator and its deletion from the virus genome increases several pro-inflammatory responses including TNF-alpha expression, inducible nitric oxide synthesis. The L83L protein has been shown to bind to IL-1 but deletion of the gene did not reduce the virulence of the highly virulent Georgia 2007/1 strain.

Other enzymes The virus encoded protein kinase, ubiquitin conjugating enzyme and geranyl pyrophosphate synthase may modulate host protein function although their roles are unknown. The protein kinase is packaged in virus particles suggesting it has a role early during infection.

African Swine Fever Virus (Asfarviridae)

29

Life Cycle A diagram of the replication cycle is shown in Fig. 3.

Entry ASFV enters swine macrophages by clathrin-mediated endocytosis as well as macropinocytosis. ASFV can also enter tick midgut phagocytic digestive cells attached to red blood cells. The receptor or receptors on the surface of swine macrophages that ASFV binds to are presently unknown, however it is unlikely to be CD163 as gene-edited pigs lacking this gene are fully susceptible to

Fig. 3 Replication cycle of African swine fever virus. (1) African swine fever virions enter by receptor mediated endocytosis and enter the endosomal pathway. Virions lose their external envelope and the capsid disassembles in a pH dependent manner in multivesicular bodies. (2) The internal envelope fuses with the endosomal membrane and the core enters the cytoplasm. (3) Expression of early genes begins and (4) DNA replication initiates, possibly involving a nuclear step. (5) Virus factories begin to coalesce in a perinuclear regions of the cytoplasm and exclude host proteins except those required for replication, such as the translational apparatus. Viral proteins and DNA accumulate in factories and striking hexagonal assembly intermediates are seen forming on viral membranes. (6) Fully formed intracellular virions egress along microtubules from the factory to the plasma membrane, from which they bud (7) into the extracellular space gaining an external envelope. Some virions are seen on the tips of cellular projections which label for actin.

30

African Swine Fever Virus (Asfarviridae)

ASFV. After entry, the outer envelope and capsid is lost in a pH dependent process within endosomal compartments before the inner envelope fuses with late endosomal membranes delivering the core to cytoplasm. Both the intracellular mature form and extracellular enveloped forms of ASFV are infectious. The precise role of individual viral proteins in entry is unclear. P12/pO63R has long been considered an attachment protein, however this protein has now been shown to localise to the inner envelope rather than the outer and therefore may play a role at a later stage of the entry process. Likewise, antibodies that recognise p54/pE184L, p30/pCP204L and p72/pB646L are capable of neutralising infectivity, but these are components of the inner envelope or capsid. Infection of cells with an ASFV conditional lethal mutant with the E248R gene under control of an inducible promoter showed that expression of the pE248R protein is required for fusion of the virion inner envelope with endosomal membranes. This suggests that this protein may be part of an entry fusion complex similar to that identified in vaccinia virus. Bioinformatics analysis showed similarity of some components of the vaccinia virus entry fusion complex with ASFV virion proteins (see Table 1). The CD2v/pEP402R protein is the only virus protein that has been localised to the outer envelope of virions although it is not essential for virus replication. This suggests that interaction of the virus outer envelope with cells may involve a non-specific interaction although alternative explanations are possible.

Transcription ASFV gene expression is tightly regulated with immediate early, early, intermediate and late classes of transcripts described. Early gene transcription begins immediately following entry of virus cores into the cytoplasm. RNA polymerase and other factors packaged in the virus core carry out early gene transcription in partial uncoated cores immediately following entry into the cytoplasm. Shut off of immediate early transcripts is dependent on protein synthesis, whereas initiation of intermediate and late gene expression, as well as the shut off of some early transcripts is dependent on viral DNA synthesis. Whole genome transcription data is currently lacking and therefore the temporal expression patterns are restricted to a few known genes. Late genes are predominately structural proteins involved in the assembly and morphogenesis of progeny virions, whereas early genes tend to be involved in modulating the host immune response and replication, although this is not an absolute rule.

DNA replication DNA replication takes place predominantly in perinuclear cytoplasmic factory areas at a later stage of replication. The similarity between the genome termini of ASFV and poxviruses suggests they share similarities in DNA replication. This is proposed to involve introduction of single strand nicks in the duplex DNA close to genome termini, providing a template for DNA polymerase to synthesise a strand complimentary to some of the terminal inverted repeats and terminal loop structure. Subsequently, these strands fold back such that replication can continue along the genome creating duplexes as predominantly head to head concatemers. Resolution of concatameric full length genomes is proposed to involve extrusion or Holliday- like junctions and resolution by virus encoded enzymes. In support of this model head to head genomic fragments have been detected in ASFV infected cells as well as concatameric genomes of double unit length. An early phase of DNA replication takes place in the nucleus of infected cells and ASFV DNA has been observed juxtaposed to the inner nuclear envelope. Low molecular weight fragments of DNA are synthesised in the nucleus and cytoplasm early during the replication cycle with larger intermediate and full length fragments appearing exclusively in the cytoplasm. It is tempting to speculate that the sub-genomic fragments synthesised in the nucleus are transported to the cytoplasm where they act to prime full length genome synthesis. However evidence for this model is currently lacking since radioactive label incorporated into the shorter fragments could not be chased into the full length genome.

Assembly and morphogenesis Progeny virions are assembled in discrete perinuclear regions of the cytoplasm called virus factories. Formation of these replication sites coincides with the initiation of cytoplasmic DNA synthesis, however the morphology of virus factories change as replication progresses and serve to concentrate and organise translation, transcription, DNA synthesis and assembly within one cellular compartment. Virions are assembled on the inner envelope with the capsid assembled on the convex surface and the core on the concave. Intermediate forms resembling half formed hexagons are readily visible within virus factories. The inner envelope consists of a single lipid bilayer which by comparison to vaccinia is likely stabilised in part by the major capsid protein, p72. The endoplasmic reticulum has been suggested as the source of the membrane used for the inner envelope, however this was in part based on the detection of viral and cellular endoplasmic reticulum proteins in purified virions by immunoblot which were not present when virions were analysed by mass spectrometry. Membrane targeting of virus proteins is crucial for ASFV morphogenesis. Assembly of the p72 on the convex surface is dependent on a virally encoded chaperone pB602L and correct processing of pp220 into its mature proteins is dependent on myristoylation, as well as the viral protease pS273R mentioned above. Formation of the nucleocapsid and DNA packaging is poorly understood, however is dependent on the prior formation of the core as deletion mutants lacking pp220 and pp62 form ‘empty’ particles that lack the electron dense nucleocapsid. Once complete, pE120R mediates the transport of virions along microtubules by kinesin motor proteins to the plasma membrane where they gain their external envelope as they bud out of the cell. ASFV can also induce filopodia like actin projections at the cell surface which may aid cell-to-cell spread.

African Swine Fever Virus (Asfarviridae)

31

Epidemiology African swine fever virus (ASFV) was first identified in Kenya in the early 20th century as the cause of a highly lethal disease of domestic pigs that was initially called East African swine fever and later African swine fever (ASF). Wild African suids, in particular the warthog were considered the most likely source of infection. Warthogs did not show clinical signs but spread disease to domestic pigs which they came into contact with. Subsequently, other African wild suids, including the bushpig, red river hog and giant forest hog were also shown to be infected without clinical signs. Soft ticks of the Ornithodoros species which inhabit warthog burrows were later shown to be biological vectors of ASFV. The virus can persistently infect ticks over long periods and be transmitted between them transtadially and transexually. Transovarial transmission has been demonstrated for O. moubata, the tick vector present in warthog burrows. The virus is very well adapted to replication in hosts in the ancient sylvatic cycle and presumed to have been present over a very long time period. This is supported by the higher genetic diversity of isolates from the regions where this cycle is present. ASF was subsequently identified in some other East African countries where the ancient sylvatic cycle is present. Since then the virus spread through central and West Africa and is now endemic or causes sporadic outbreaks in almost all sub-Saharan Africa. However, the ancient sylvatic cycle has not been described apart from in East Africa. The virus was contained in sub-Saharan Africa until the late 1950s when there were outbreaks in Spain and Portugal, this was followed with further outbreaks across Western Europe, the Caribbean and Brazil until the 1990s when disease was finally eradicated, although ASFV remains endemic in Sardinia. In 2007 ASFV spread into the Caucasus and from there to the Russian Federation. From 2013 further spread to Eastern Europe was observed. More recently there has been an increase in outbreaks across Europe including: Moldova in September 2016, Czech Republic in June 2017, then Romania in July 2017, followed by Hungary in April 2018, and Bulgaria in August 2018. ASFV was also identified in wild boar in Belgium in September 2018. In addition to European outbreaks ASFV has also been identified for the first time in China in August 2018, with the initial report in the Liaoning region in North East China and has since been reported throughout the country, including Beijing. The virus also appeared in Mongolia in January 2019. The current spread of ASFV presents an enormous threat to the global pig industry. ASFV can be transmitted through multiple routes including direct contact with infected animals, tick bites, fomites, and contaminated pig products. ASFV is transmitted by direct contact with the virus and in sub-Saharan Africa it is also transmitted by the soft tick Ornithodoros spp, acting as a viral vector. ASFV can be transmitted through direct contact with infected blood which contains up to 109 infectious units of virus per ml, or saliva, urine or faeces each of which can contain up to 105 infectious units per ml/mg. ASFV has also been shown to be spread by fomites such as feed, equipment and vehicles. Contaminated pig products are another source of infection, with ASFV persisting for months in pork products. This also highlights the risks associated with swill feeding in transmission of ASFV. There is also potential of secretions from infected wild boar being a risk to transmission, by contaminating the environment that is potentially shared with domestic pigs. Wild boar pose another route of transmission to domestic pigs, as in Europe wild boar as equally susceptible to ASFV, and there has been observations in recent outbreaks of dead wild boar from ASFV in the vicinity of ASF-affected farms, as well close to national borders, with some reports suggesting that pig and wild boar interactions with carcass sites play a role in ASFV transmission.

Clinical Features The clinical signs caused by ASFV infection vary depending on the virus isolate and host. The virus is very well adapted to its ancient wildlife hosts in eastern and southern Africa. These include several wild suid species such as warthogs, bushpigs and red river hogs and soft ticks of the Ornithodoros species. In these hosts few clinical signs are observed but virus can persist in tissues, particularly spleen and lymph nodes. In warthogs and bushpigs experimental infection with highly virulent ASFV was observed to cause a transient low fever coincident with a peak of replication in blood. In contrast ASFV infection in domestic pigs and wild boar generally results in an acute haemorrhagic fever with a very high case fatality approaching 100%. Different courses of ASF disease have been described in the field and in experimental infections. Highly virulent isolates can cause a peracute disease which may result in death within 4 days post-infection (dpi) without evident lesions and an acute form of disease which can result in death of almost all infected pigs within 4–21 dpi. In subacute disease forms, caused by moderately virulent isolates, incubation periods are longer, clinical signs tend to be less marked and mortality rates are lower varying between 30% and 70%. A typical disease course results in early clinical signs of fever, reduced eating or loss of appetite and increasing inactivity and lethargy or morbidity. External signs of haemorrhage including cyanosis or skin reddening around the tips of ears, and other extremities, conjunctivitis, bloody diarrhoea can be observed but animals may die without these signs. Clinical signs may typically be observed starting from 3 to 5 dpi but this may vary depending on the dose, route of infection or virus isolate involved. Lower virulence isolates can cause chronic forms of disease characterised by low mortality but signs such as delayed growth, emaciation, joint swelling, skin ulcers and lesions. Some pigs may remain persistently infected with virus in lymph tissues over a prolonged period. Infections of wild boar are clinically very similar to that in domestic pigs and result in similar fatality rates.

Pathogenesis The main target cells for ASFV replication are those of the myeloid lineage. Monocytes and macrophages at an intermediate to late stage of differentiation are most susceptible to productive infection. Cell markers determining susceptibility to infection have not

32

African Swine Fever Virus (Asfarviridae)

been clearly defined. There are reports indicating that dendritic cells may also be susceptible to infection. Vascular endothelial cells have been shown to be infected in cell culture. At later stages of acute disease in animals several other cell types, including lymphocytes and platelets, have been observed to contain virus particles or proteins although virus replication does not occur. The oral-nasal route is the main route for ASFV infection in suids. In areas where the Ornithodoros spp soft tick plays a role infection by tick bites may occur. Following infection the virus replicates mainly in mononuclear phagocytic cells in the tonsils, sub-mandibular or other lymph nodes and spreads through lymph and blood to secondary organs of replication. The virus encodes a number of proteins that inhibit apoptosis but at later stages of the replication cycle apoptosis of the infected cells is observed. Spread of virus in apoptotic bodies may provide a mechanism for virus spread avoiding inflammatory responses induced by necrotic cell death. Virus replication in macrophages has been reported to result in activation of both infected and bystander uninfected macrophages as detected by immunohistochemistry and measured by increased phagocytosis and cytokine secretion. A feature of acute ASF disease is the massive apoptosis observed in both T and B lymphocytes in infected tissues and blood. This apoptosis is observed in uninfected bystander lymphocytes in infected tissues, particularly surrounding infected macrophages. This suggests that factors on the surface of or secreted from infected macrophages are involved in inducing the apoptosis of lymphocytes. Little is known of the mechanism by which apoptosis is induced although TNF-alpha has been identified as a possible contributing factor. In acute and sub-acute forms of disease, ASFV replicates rapidly at primary and secondary sites in lymph tissues including spleen and spreads systemically in blood. This indicates a failure of the host’s early innate responses to control virus replication, presumed mainly to result from the many and diverse mechanisms the virus has to inhibit these responses. High levels of virus replication in blood result in typical signs of haemorrhagic pathology including destruction of vascular endothelial cells, leakiness of blood vessels, thrombo and lymphocytopenia and induction of disseminated intravascular coagulation. Typically very high levels of virus replication are observed in blood, up to 108 or 109 per ml. In some infected animals with moderately virulent isolates, levels of virus replication in blood are lower (up to 106 to 107 per ml) and some individuals may recover from clinical signs. In pigs infected with low virulence isolates transient low levels of virus in blood may be observed and low levels of virus replication in tissues are detected. Animals that recover may be persistently infected over a period of at least several months. In experimental studies virus was shown to be cleared from persistently infected animals.

Immune Responses African swine fever virus typically causes a lethal haemorrhagic fever in affected swine, however survivors discovered in the field were found to have robust immunity against homologous strains of the virus. Isolates of reduced virulence obtained from both the field and through experimental passage through tissue culture permitted a more thorough analysis of the immune response to African swine fever and mechanisms that contribute to protection. Pigs infected with virulent strains of the virus typically exhibit titres of 107–109 infectious units of virus per millilitre of blood. This in combination with high serum levels of pro-inflammatory cytokines such as interferon, tumour necrosis factor alpha and interleukins -1β, -6 and -8 likely contribute to the lymphopenia and other pathological signs seen in animals suffering acute forms of the disease. A combination of rapid viral replication, disease progression and immunosuppression are likely responsible for the failure to generate ASFV-specific adaptive responses in naïve animals infected with virulent strains of the virus. Animals infected with low virulence strains typically do not display elevated serum levels of cytokines, but develop both humoral and cellular immune responses that protect animals from acute disease induced by related virulent strains of the virus. Antibodies of the IgM class specific to ASFV first appear between 7 and 11 days post infection and those of the IgG class appear from 14 days onwards. Serum from recovered animals can neutralize ASFV infectivity in vitro as well as mediate lysis of infected cells. The importance of the antibody response in protecting animals from acute disease caused by virulent ASFV strains has been demonstrated by experiments where serum was passively transferred to naïve pigs. Proliferation of ASF specific lymphocytes can be seen from 10 days post infection with attenuated strains of the virus. Pigs recovered from attenuated ASF infection have circulating T-cells that secrete interleukin-2 and interferon gamma after re-exposure to virus, as well as T-cells that have ASFV-specific cytolytic activity. Antibody depletion studies with anti-CD8 monoclonals have shown that the cellular response is essential for the protection afforded by low virulence strains of ASFV. Current research is focussed on identifying precise correlates of protection and identifying which of the 150 or more open reading frames encode for protective antigens.

Diagnosis and Control Diagnosis of ASF requires laboratory confirmation since clinical signs are similar to several other diseases including classical swine fever (CSF). Detection of virus genome by polymerase chain reaction (PCR) is the usual method used for diagnosis. Detection of infectious virus by replication in primary porcine macrophage cultures is also used, in order to confirm the presence of, and characterise, infectious virus. The haemadsorbtion of red blood cells to infected monocytes/macrophages or immunofluorescence using antibodies against one of the virion proteins is used to detect infection in cell culture. Kits are commercially available to detect virus antigen by ELISA or using lateral flow devices but the sensitivity of these tests is lower than PCR or virus isolation and they should not be used as the only detection method.

African Swine Fever Virus (Asfarviridae)

33

Detection of ASFV specific antibodies using ELISA assay can be used in some situations to identify animals that have been infected. Pigs or wild boar infected with highly virulent isolates usually die before an antibody response is detected. Therefore this method alone is not recommended as the only diagnostic test to detect infection. Detection of ASFV specific antibodies can indicate that isolates of reduced virulence are circulating. To obtain a more complete assessment of the epidemiological situation both ASFV antigen and specific antibodies is recommended. African swine fever is a notifiable disease to the OIE (World Organisation for Animal Health) as well as national and other international veterinary services. Following confirmation of an ASF outbreak in pigs, veterinary authorities in most countries will cull animals on infected premises and disinfect the premises. In addition movement of pigs is banned from a surrounding protected zone and surveillance for ASF carried out in a wider zone. Infection results in a ban of export trade from the infected area. Varying strategies are applied in different countries to allow continued trade internal to the country or region (for example the EU). If infected wild boar are detected control is more difficult to implement. Following a single point of introduction of ASF to wild boar in the Czech Republic in 2017, disease was eventually eradicated by fencing an extended infected zone and shooting all wild boar in this zone. Reducing contact between wild suids and domestic pigs can also help control ASFV spread particularly if combined with improved on farm biosecurity. In S. Africa double fencing of farms in areas where ASFV infected wildlife are present has proven effective in preventing outbreaks on farms. The availability of a vaccine would increase options for control. It was established in the 1950s and 1960s that pigs that recover from infection with less virulent strains of ASFV are protected against challenge with related virulent viruses, indicating that vaccine development should be successful. However inactivated virus does not induce protection against challenge even when delivered with recent adjuvants. Moreover it has proven difficult to develop a subunit vaccine since the complexity of the genome makes it difficult to identify protective antigens. Live attenuated vaccines are the most promising approach for vaccine development in the shorter term since high levels of protection can be achieved in the absence of significant replication of the challenge virus. Live attenuated strains isolated from the field have provided excellent models for understanding mechanism of protective immunity but safety issues have precluded their commercial development. Rational attenuation of virus by gene deletion has provided a number of very promising candidates for further evaluation. Deletion of genes that control innate immune responses, in particular the type I interferon response, has been an effective mechanism to attenuate virulent ASFV and induce a protective response. Delivery of live attenuated vaccines in baits to wild boar was successfully used in the eradication of CSFV from Europe and should also be effective for ASFV.

Further Reading Afonso, C.L., Piccone, M.E., Zaffuto, K.M., et al., 2004. African swine fever virus multigene family 360 and 530 genes affect host interferon response. Journal of Virology 78, 1858–1864. Alejo, A., Matamoros, T., Guerra, M., Andres, G., 2018. A proteomic atlas of the African swine fever virus particle. Journal of Virology 92, e01293. (18). Alonso, C., Borca, M., Dixon, L., et al., 2018. ICTV virus taxonomy profile: Asfarviridae. Journal of General Virology 99, 613–614. Andreani, J., Khalil, J.Y.B., Sevvana, M., et al., 2017. Pacmanvirus, a new giant icosahedral virus at the crossroads between asfarviridae and faustoviruses. Journal of Virology 91. Blome, S., Gabriel, C., Beer, M., 2013. Pathogenesis of African swine fever in domestic pigs and European wild boar. Virus Research 173, 122–130. Dixon, L.K., Chapman, D.A.G., Netherton, C.L., Upton, C., 2013. African swine fever virus replication and genomics. Virus Research 173, 3–14. Dixon, L.K., Sanchez-Cordon, P.J., Galindo, I., Alonso, C., 2017. Investigations of pro- and anti-apoptotic factors affecting African swine fever virus replication and pathogenesis. Viruses 9. Gonzalez, A., Talavera, A., Almendral, J.M., Vinuela, E., 1986. Hairpin loop structure of African swine fever virus-DNA. Nucleic Acids Research 14, 6835–6844. Granja, A.G., Perkins, N.D., Revilla, Y., 2008. A238L inhibits NF-ATc2, NF-kappa B, and c-Jun activation through a novel mechanism involving protein kinase C-theta-mediated up-regulation of the amino-terminal transactivation domain of p300. Journal of Immunology 180, 2429–2442. Hawes, P.C., Netherton, C.L., Wileman, T.E., Monaghan, P., 2008. The envelope of intracellular African swine fever virus is composed of a single lipid bilayer. Journal of Virology 82, 7905–7912. Hernaez, B., Guerra, M., Salas, M.L., Andres, G., 2016. African Swine fever virus undergoes outer envelope disruption, capsid disassembly and inner envelope fusion before core release from multivesicular endosomes. Plos Pathogens 12. Jori, F., Vial, L., Penrith, M.L., et al., 2013. Review of the sylvatic cycle of African swine fever in sub-Saharan Africa and the Indian ocean. Virus Research 173, 212–227. McCullough, K.C., Basta, S., Knotig, S., et al., 1999. Intermediate stages in monocyte-macrophage differentiation modulate phenotype and susceptibility to virus infection. Immunology 98, 203–212. Netherton, C.L., Wileman, T.E., 2013. African swine fever virus organelle rearrangements. Virus Research 173, 76–86. Penrith, M.-L., 2013. History of ‘swine fever’ in Southern Africa. Journal of the South African Veterinary Association-Tydskrif Van Die Suid-Afrikaanse Veterinere Vereniging. 84. Rodriguez, J.M., Salas, M.L., 2013. African swine fever virus transcription. Virus Research 173, 15–28. Salas, M.L., Andres, G., 2013. African swine fever virus morphogenesis. Virus Research 173, 29–41. Sanchez-Cordon, P.J., Montoya, M., Reis, A.L., Dixon, L.K., 2018. African swine fever: A re-emerging viral disease threatening the global pig industry. Veterinary Journal 233, 41–48. Sanchez-Vizcaino, J.M., Mur, L., Gomez-Villamandos, J.C., Carrasco, L., 2015. An update on the epidemiology and pathology of African swine fever. Journal of Comparative Pathology 152, 9–21. Takamatsu, H.-H., Denyer, M.S., Lacasta, A., et al., 2013. Cellular immunity in ASFV responses. Virus Research 173, 110–121.

Akabane Virus and Schmallenberg Virus (Peribunyaviridae) Martin Beer and Kerstin Wernike, Friedrich-Loeffler-Institute, Insel Riems, Germany © 2021 Published by Elsevier Ltd.

Glossary Ankyloses Abnormal stiffening and immobility of a joint due to fusion of the bones. Arthrogryposis Rigid fixation of the joints. Brachygnathia Shortness or recession of the mandible. Dystocia Abnormal labor or parturition. Encephalomyelitis Inflammation of the brain and the spinal cord. Hydranencephaly Replacement of the brain’s cerebral hemispheres by sacs filled with cerebrospinal fluid. Kyphosis Abnormally excessive convex curvature of the spine.

Lordosis Excessive inward curvature of the spine. Micromyelia Abnormal smallness or shortness of the spinal cord. Nystagmus Involuntary, rhythmic oscillating motions of the eyes. Porencephaly Small cavities in the brain substance. Scoliosis Lateral deviation of the spine. Strabismus Condition in which the eyes do not point in the same direction. Torticollis Twisting of the neck causing the head to rotate or tilt.

Introduction The genus Orthobunyavirus consists of more than 170 viruses and comprise several members of public health or veterinary importance. Among the viruses that cause diseases in animals are Akabane virus and Schmallenberg virus, which infect predominantly ruminants and are able to cross the placental barrier and induce fetal malformation. Both Akabane virus and Schmallenberg virus, are transmitted between their mammalian hosts by blood-sucking insect vectors, specifically biting midges of the genus Culicoides. While Akabane virus was initially described in the 1950th, Schmallenberg virus has been discovered for the first time in 2011. Both viruses are named according to the place of origin of the samples, in which the viruses were detected for the first time. Akabane virus was initially detected in mosquitoes caught in a village in Japan, and Schmallenberg virus was isolated from the blood of acutely infected cattle kept near the German city of Schmallenberg.

Classification Akabane virus and Schmallenberg virus are members of the genus Orthobunyavirus in the family Peribunyaviridae (order Bunyavirales). Orthobunyaviruses are grouped into 18 serogroups on the basis of serological relatedness and both, Akabane virus and Schmallenberg virus, belong to the Simbu serogroup. Further Simbu serogroup viruses are for example the eponymous Simbu virus, Aino virus, Douglas virus, Peaton virus, Sathuperi virus, Shamonda virus, and Shuni virus. All of them infect animals. The only known zoonotic member of this serogroup is Oropouche virus, which is present in South America.

Virion Structure Akabane and Schmallenberg virions are spherical, about 100 nm in diameter and relatively simple in their composition, possessing only four structural proteins: the nucleocapsid protein, the viral RNA-dependent RNA polymerase and the surface glycoproteins Gn and Gc. The nucleocapsid protein encapsidates the three RNA segments to form ribonucleoprotein complexes, which associate with the viral RNA-dependent RNA polymerase and are contained within the envelope of the particle (Fig. 1). The viral envelope consists of a lipid layer, which is derived from the Golgi complex of the host cell. On the surface of the virions, locally ordered lattice of spikes protrude from the membrane. These spikes consist of the glycoproteins Gn and Gc, which form heterodimers via disulfide bonds and are anchored in the lipid envelope (Fig. 1).

Genome The genome of Akabane virus and Schmallenberg virus consists of three segments of negative-stranded RNA, which are named according to their size as large (L), medium (M), and small (S) segment. The L-segment encodes the viral RNA-dependent RNA polymerase. The M-segment encodes the surface glycoproteins Gn and Gc, as well as a non-structural protein (NSm) in the coding

34

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20939-4

Akabane Virus and Schmallenberg Virus (Peribunyaviridae)

35

Fig. 1 Virion morphology and genome organization of Schmallenberg virus. The genome consists of three segments of negative-stranded RNA, which are referred to as S, M, and L. The genomic segments have complementary terminal sequences that may base-pair and form circular or panhandle structures. The three RNA segments are encapsidated by the nucleocapsid protein (N) to form ribonucleoprotein complexes, which associate with the viral RNA-dependent RNA polymerase and are contained within the lipid envelope of the particle. The glycoproteins Gn and Gc are inserted in the membrane. Reprinted from Wernike, K., et al., 2014. Schmallenberg virus-two years of experiences. Preventive Veterinary Medicine 116 (4), 423-434, Copyright (2014), with permission from Elsevier.

order Gn-NSm-Gc. The S-segment encodes the nucleocapsid protein and the non-structural protein NSs in alternative overlapping reading frames. The three RNA segments have complementary terminal sequences and, as a consequence, the RNAs may base-pair and form circular or panhandle structures (Fig. 1).

Viral Replication The basic features of the replication cycle are similar to those of other Bunyaviruses, although insect-transmitted viruses such as Akabane virus and Schmallenberg virus have to multiply in two different hosts, i.e., the insect vectors and the mammalian hosts. Cell entry, which occurs by clathrin-mediated endocytosis, involves interactions between the viral glycoproteins Gn and/or Gc and cell surface receptors, which have not been yet identified. Uncoating of the viral particles likely occurs when the endosomes become acidified, thereby initiating fusion of the viral membrane and endosomal membrane. After the release of the ribonucleoprotein complexes into the cytoplasm, the genomic RNA is transcribed into messenger RNAs (mRNAs) by the virion-associated RNA polymerase. This process is termed primary transcription. The viral RNA polymerase exhibits, in addition to its polymerase activity, an endonuclease activity and this is used to recruit primers for RNA transcription from the host cell mRNA (so-called capsnatching mechanism). During translation of the mRNA, the virion-associated RNA polymerase switches from primary transcription to replication. Following translation of the primary transcripts into viral proteins, the genome is replicated via a complementary full-length positive-strand RNA, the so-called antigenome, and then further mRNA synthesis ensues, which is termed secondary transcription. Maturation occurs at the membranes of the Golgi complex. The ribonucleoproteins are transported to the Golgi apparatus that has been modified by the insertion of the viral glycoproteins Gn and Gc. After budding into Golgi membrane-derived vesicles, the viral particles are trafficked to the cell surface via the exocytic pathway involving actin filaments. Subsequently, the fusion of the vesicular membranes with the plasma membrane leads to the release of infectious virions.

Epidemiology Akabane virus, Schmallenberg virus and other orthobunyaviruses persist in nature by alternate cycling of the virus between mammalian hosts and midges after biting of the mammalian host by the insects. The geographical distribution of the viruses depends on the distribution, seasonal activity, and abundance of the insect vectors responsible for their transmission. Most of Africa, the Middle East, Asia, and Australia may be regarded as being endemic for Akabane virus. Schmallenberg virus initially emerged in the German/Dutch border region and, thereafter, spread throughout the European continent reaching the Scandinavian countries and the British Isles at the North, the Mediterranean region at the South and Eastern European countries such as Poland, Lithuania and Russia. Within endemically infected areas, Akabane virus and Schmallenberg virus established patterns of cyclic circulation, meaning that for one or perhaps two seasons the viruses are detected only sporadically, while in the intermediate years the viruses re-appear to a greater extent. The differences in the level of virus circulation are presumably related to the overall herd immunity rate in the ruminant population and to the abundance of the vector insects, which is influenced by climate.

36

Akabane Virus and Schmallenberg Virus (Peribunyaviridae)

The principal vectors of Akabane virus, Schmallenberg virus and further Simbu serogroup members are small biting midges of the genus Culicoides. Multiple Culicoides species are often present in the same place at the same time. From an epidemiological perspective, important considerations are the infection rates of Culicoides with Akabane or Schmallenberg virus and the vector competence, i.e., the physiological ability of a vector organism to acquire, maintain and transmit an infectious agent. The Simbu serogroup viruses are transmitted from the insect vectors to the mammalian hosts with a high level of efficiency, partly because of the relatively high virus infection rates of the Culicoides migdes and the large number of individual Culicoides that may attack an animal. Akabane virus has been detected in the Culicoides species C. brevitarsis, C. oxystoma, C. imicola, C. milnei and C. wadai, while the following Culicoides species were implicated in the transmission of Schmallenberg virus: C. obsoletus sensu stricto, C. scoticus, C. chiopterus, C. dewulfi, and C. nubeculosus. Experimental infection studies with laboratory reared lines of Culicoides provided evidence of successful dissemination in C. sonorensis. Although Akabane virus has been initially isolated from mosquitoes, they are not considered to be true competent vectors of neither this virus nor of Schmallenberg virus. In regions with a temperate climate, there is a distinct seasonal pattern of virus transmission, which coincides with warm, moist summer and autumn months and is a consequence of the abundance of the insect vectors. Insect numbers start to increase in the late spring and early summer months, usually peak in early autumn and virus transmission ceases with the onset of very low temperatures and the first frosts. Even in subtropical and tropical regions, there is a tendency towards seasonal transmission, with the highest infection rates in summer and declining transmission rates in the periods of lower rainfall.

Clinical Features Akabane virus and Schmallenberg virus infect predominantly ruminants. Clinical symptoms have been observed only in domestic animals so far. Direct and indirect detections in association with clinical signs have occurred in adult cattle, sheep, and goats or in their fetuses or newborn offspring. In addition, Schmallenberg virus genome has been detected in several wild and exotic ruminant species such as alpacas, llama, mouflon, water buffalo or elk. Furthermore, antibodies against Akabane virus have been found in horses, pigs, camel, deer, and a wide selection of African wildlife, while no infections have been detected in marsupials in Australia. Antibodies against Schmallenberg virus have been detected in various domestic and wild ruminants, free-ranging wild boar, and a range of ungulates kept in European zoos, but clinical signs related to Akabane or Schmallenberg virus infections have not been described in any wild or exotic species. An infection of adult domestic ruminants with Akabane virus or Schmallenberg virus is either asymptomatic or associated with mild, short-lasting, non-specific clinical signs such as fever, decreased milk production, or diarrhea. Some strains of Akabane virus may induce encephalitis in newborn calves in rare cases and the Iriki strain of Akabane virus, which is present in Japan and Korea, has been occasionally associated with cases of encephalitis in adult cattle. In contrast to the mild symptoms observed in adult animals, both viruses have significant impact on developing fetuses. When naïve dams are infected during a critical phase of gestation, the viruses can cause stillbirth or premature birth, the birth of mummified fetuses or severe congenital malformations known as “arthrogryposis-hydranencephaly syndrome” (AHS). The range of defects and the percentage of affected newborns varies depending on the herd management and the time period of virus transmission. In herds with a year-round mating system, the full range of congenital defects may be seen, while in herds with a very restricted, seasonal mating period, only a few types of abnormality may be noticed. An infection early in pregnancy may be associated with embryonic or fetal death and/or abortion, given that an increased number of repeated estrus cycles, which suggest early embryonic or fetal loss, and an increasing rate of abortions have been reported from affected flocks. When fetuses are infected in early to mid-gestation, the viruses may induce congenital lesions. The most common lesions affect the skeletal muscle, the central nervous system and/or the axial skeleton; lesions can affect either one system or can occur in combination. Affected animals may show arthrogryposis (Fig. 2) associated with histological evidence of muscular hypoplasia, characterized by a reduction in number and diameter of the myofibrils, which may be associated with a loss of cross-striation in myofibrils and fatty replacement. In the central nervous system, the most common lesions are hydranencephaly, porencephaly, cerebellar hypoplasia and micromyelia. These lesions are sometimes associated with nonsuppurative encephalomyelitis and neuronal degeneration and necrosis. The animals are either stillborn or, in cases of less severe malformation, born alive. The following clinical signs or behavioral abnormalities suggestive for lesions in the central nervous system have been described in alive newborns: hypertonicity, hyperreflexia, depression, blindness, ventrolateral strabismus, nystagmus, deafness, dullness, inability to stand, inability to suckle, or aimless wandering. The animals may also display vertebral malformations, including lordosis, kyphosis, scoliosis, torticollis, ankyloses or brachygnathia inferior. These lesions can also appear in isolation with no other clinical sign. In sheep and goats infected with Akabane virus, there may also be pulmonary hypoplasia, which has not been observed after Schmallenberg virus infections. The described defects are usually life-threatening and most affected animals die after birth or are euthanized. The nature of the musculoskeletal and nervous lesions is similar in cattle and sheep. However, only a proportion of newborns whose mothers were infected during gestation show clinical signs or pathomorphological alterations. Even the birth of one malformed and one healthy offspring within the same litter has been described. In cows or ewes delivering severely malformed offspring, dystocia is common and, as a consequence, many of those animals require embryotomy or Caesarian section.

Akabane Virus and Schmallenberg Virus (Peribunyaviridae)

37

Fig. 2 Genesis of fetal malformations in calves and lambs after infection of their mothers with Akabane virus or Schmallenberg virus during a critical period of pregnancy. When naïve dams are infected, they show a short-lived viremia during which the virus may be transmitted to the developing fetus. Dependent on the stage of pregnancy, fetal infections can led to stillbirth, premature birth, dystocia or varying degrees of malformation. Modified from Höper, D., et al., 2012. Schmallenberg-Virus, ein neues Virus in Europa. Deutsches Tierärzteblatt 4 (2012), 500-505, with permission.

Pathogenesis The onset of the two to six days lasting viremia usually occurs one to six days after infection and, in most cases, is not accompanied by clinical signs. The viruses are not associated to any blood cell population, but are circulating freely in the serum, which might assist in their timely elimination from the blood stream. Similarly to other orthobunyaviruses, the NSs protein of Akabane virus and Schmallenberg virus represents an important virulence factor. In infected mammalian cells, the NSs protein counteracts the shutoff of host cell protein synthesis and the induction of interferon. In addition, NSs is involved in other functions like the regulation of translation, apoptosis, and viral polymerase activity. Viruses, in which the NSs protein has been deleted by reverse genetics, do not induce detectable virus replication in interferon-competent hosts. As mentioned above, when naïve pregnant animals are infected, the viruses may replicate in the placenta and cross the placental barrier resulting in either abortion, premature birth, still birth or varying degrees of fetal malformation depending on the time of gestation when infection occurred. The susceptibility of the growing embryo or fetus to an infection depends on the maturity of the placentomes and fetal target organs and on the development of the fetal immune system. If the infection of naïve pregnant female takes place before the first placentomes are established (between days 30 and 45 after conception in ruminants), the embryo may be protected from viral invasion. The second key point is related to the maturation of the fetal immune system, since the critical timeframe for the induction of malformations is most probably limited to the time before the fetus becomes immunocompetent. Bovine fetuses develop first antibodies between days 60 and 150 of gestation while in lambs, immunocompetence develops from 65 to 70 days of pregnancy. Consequently, the timeframe during which an infection might lead to malformation ranges from about 30 to 150 days after conception in cattle and from about 30 to 60 days in small ruminants (Fig. 2). Target cells of the viruses within the developing fetuses are neurons in the cerebral cortex, brainstem and spinal cord. An infection of these cells during ontogenesis leads to malformation of the central nervous system such as hydranencephaly, porencephaly, cerebellar hypoplasia or micromyelia. The effects on skeletal muscle are mainly secondary, although a primary viral myositis sometimes occurs. Arthrogrypotic newborns show restrictions to movements of the limb joints, which is a result of changes of the soft tissues. The joint surfaces are usually normally developed and the bones are unaffected, while the muscles are

38

Akabane Virus and Schmallenberg Virus (Peribunyaviridae)

usually reduced in size and paler in color than normal, which is a result of both, neurogenic muscular atrophy and primary infection of the muscles. Most of the births of dead and/or malformed lambs and calves induced by Akabane virus or Schmallenberg virus infections of their mothers were reported after full-term pregnancy, leading to a time gap of several months between infection and birth (Fig. 2). However, not every infection during the critical period of pregnancy leads to the transplacental transmission of the virus to the fetus. Furthermore, the crossing of the placental barrier does not inevitably result in abortion or fetal malformation. The incidence of affected fetuses or newborns varies depending on the virus, and in the case of Akabane virus, on the strain, but it is usually low. For Schmallenberg virus, an average malformation rate of about 0.5%–3% was estimated for cattle, while on average of 8% of lambs born in infected herds showed typical congenital malformation. However, in some sheep holdings, more than 30% of the pregnant ewes of a lambing season aborted or gave birth to malformed offspring. In cattle naturally infected by Akabane virus, the incidence of defects may be, dependent on the virus strain, as high as 25%–50%, if the animals are infected at the most critical stages of gestation with a very virulent strain. In sheep infected at the most susceptible stages of gestation, the incidence of fetal infection can range from 15% to 80%. When fetuses are infected once they are immunocompetent or become able to develop specific antibodies during an ongoing infection, the antibodies produced by the fetus itself might assist in the clearance of the virus from the fetus. Due to the occasional elimination of the virus between infection of the fetus and birth, some newborns suspected of Akabane virus or Schmallenberg virus infection test negative for virus or viral RNA. However, the presence of antibodies in the blood of the newborns before the intake of the colostrum of their mothers might be used for diagnostic purposes. In adult animals, first neutralizing antibodies are commonly detectable between two and three weeks after infection and they prevent from reinfection with the same virus type. Akabane virus-specific antibodies are measurable for at least two years and antibodies against Schmallenberg virus usually persist for many years, if not even lifelong. Maternally-derived antibodies, which are transferred from seropositive dams to their offspring by colostrum ingestion during the first hours of life, decay over time and are not measurable any more after five to six months.

Diagnosis The most widely used method for the direct virus detection is the analysis of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) or real-time RT-PCR. Different RT-PCR assays targeting either the L-, M-, or S-segment of either Akabane virus or Schmallenberg virus have been developed. Furthermore, group-specific real-time RT-PCR assays, which allow for the detection of all members of the Simbu serogroup, have been established. In addition to the detection of viral RNA, the viruses can be isolated on insect cell lines (e.g., Culicoides variipennis larvae (KC) cells) or mammalian cell lines (e.g., baby hamster kidney (BHK) or African green monkey (Vero) cells). Test systems for antibody detection include micro-neutralization, indirect immunofluorescence tests and various in-house or commercially available ELISAs. The choice of the sample material is critical for both, virus or viral genome and antibody detection. In adult animals, the preferred material for detecting acute infections and for antibodies is serum, with the detection of virus or viral genome being time-restricted by the short viremia of only a few days. Suitable sample materials for virus or viral genome detection in malformed fetuses or newborns include different parts of the brain, placenta, meconium, and hair swabs (¼amniotic fluid collected by a swab from the hair of the fetus or newborn). However, not all calves or lambs by clinical presentation suspected of Akabane virus or Schmallenberg virus infection test positive by RT-PCR. The most accepted explanation for this phenomenon is that the virus, which has caused the initial lesions weeks or months before abortion, stillbirth or birth, is no longer present in the newborn. In addition to the direct virus detection, the detection of antibodies in fetuses or newborns is also a valuable tool to confirm a congenital infection, when serum samples are taken prior to colostrum intake or when fetal heart blood is used as sample matrix from aborted or stillborn fetuses. For differential diagnosis in cases of newborns showing congenital defects, other teratogenic pathogens should be taken into consideration. Examples include further Simbu serogroup viruses such as Shuni virus or Aino virus, other bunyaviruses (e.g., Cache Valley virus, Rift Valley fever virus) or further viral agents including bovine viral diarrhea, border disease, bluetongue, Chuzan or Wesselsbron viruses. Furthermore, a range of non-infectious causes of fetal malformation such as maternal intoxication (e.g., by ingestion of toxic plants) or an inherited defect should also be considered.

Prevention Treatment options are not available, but there are two main approaches for prophylaxis: management strategies and vaccination. As Akabane virus and Schmallenberg virus are transmitted by insect vectors, insect-proof nets or mesh to shield animal housing or the use of insecticides or repellents could be taken into consideration to prevent potentially infected vectors from biting susceptible animals. However, case-control studies provided either no or only little evidence for protective effects of those treatments. An option to prevent from transplacental transmission of the virus from the dam to the developing fetus could be an intelligent management system. At least in regions with temperate climate, the mating period could be adjusted in order to avoid that susceptible animals are in the critical phase of gestation during the season of the highest activity of the insect vectors, i.e., in

Akabane Virus and Schmallenberg Virus (Peribunyaviridae)

39

summer and autumn. When grazing is used, another option is to adapt the management system and keep young animals outside during the major vector season. In this way, young animals are exposed to insect vectors, which might potentially lead to an infection. The immunity acquired in the course of an infection early in life may prevent fetal infection during a later pregnancy. However, this management concept requires a high number of infected insect organisms every year and a very high transmission rate of the virus from the vector to the animal host to ensure that every young animal is bitten and infected. Therefore, a much more reliable method of prophylaxis is vaccination. Akabane vaccines have been produced in Japan and Australia. In both countries, inactivated vaccines have been developed either as monovalent Akabane vaccine or as multivalent vaccines to prevent infections with Akabane virus, Aino virus and the likewise teratogenic reovirus Chuzan virus. The vaccination scheme involves two immunizations administered four weeks apart from each other and annual revaccination is recommended. In experimental infection trials, the vaccines were safe when used in pregnant animals. In addition to inactivated vaccines, a live attenuated vaccine has been produced in Japan by serial passage of Akabane virus in cell culture. This vaccine likewise prevents from infection. However, in pregnant ewes this vaccine may cause intrauterine infection of the fetus and, therefore, it is not recommended for use in sheep. Immunization with inactivated Akabane vaccines do not lead to an in vivo cross-protection against Schmallenberg virus infections. Against infections with Schmallenberg virus, virus-specific monovalent inactivated vaccines have been licensed for the European market. These vaccines confer a protective immunity in cattle after two immunizations administered three weeks apart, while sheep have to be vaccinated only once. Annual revaccination is recommended. Apart from inactivated vaccines, liveattenuated, subunit, DNA-mediated and live-vectored vaccines have been developed, but none of them is currently commercially available.

Further Reading Elliott, R.M., 2014. Orthobunyaviruses: Recent genetic and structural insights. Nature Reviews Microbiology 12, 673–685. European Commission: Report on the outcome of Technical and Scientific Studies as described in Annex I of Commission Implementing Decision 2012/349. Plyusnin, A., Elliott, R.M., 2011. The Bunyaviridae: Molecular and Cellular Biology. Norfolk: Caister Academic Press, (VI, ISBN: 978-1-904455-90-5). Saeed, M.F., Li, L., Wang, H., Weaver, S.C., Barrett, A.D., 2001. Phylogeny of the Simbu serogroup of the genus Bunyavirus. Journal of General Virology 82, 2173–2181. World Organisation for Animal Health, 2018. Chapter 2.9.1. Bunyaviral diseases of animals (excluding Rift Valley fever and Crimean-Congo haemorrhagic fever). In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organisation for Animal Health. Zientara, S., Verwoerd, D., Pastoret, P.-P., 2015a. New developments in major vector-borne diseases. Part I: An overview. Scientific and Technical Review 34 (1). Zientara, S., Verwoerd, D., Pastoret, P.-P., 2015b. New developments in major vector-borne diseases. Part II: Important diseases for veterinarians. Scientific and Technical Review 34 (2).

Relevant Websites https://talk.ictvonline.org/ictv-reports/ictv_9th_report/negative-sense-rna-viruses-2011/w/negrna_viruses/205/bunyaviridae Bunyaviridae. https://talk.ictvonline.org/ictv-reports/ictv_9th_report/negative-sense-rna-viruses-2011/w/negrna_viruses/206/bunyaviridae-figures Bunyaviridae-Figures. https://www.fli.de/en/institutes/institute-of-diagnostic-virology-ivd/reference-laboratories/nrl-for-sbv/ NRL for SBV: Friedrich-Loeffler-Institut. http://www.oie.int/en/scientific-expertise/specific-information-and-recommendations/schmallenberg-virus/ Schmallenberg virus: OIE-World Organisation for Animal Health.

Alphaviruses Causing Encephalitis (Togaviridae) Diane E Griffin, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Arbovirus Virus maintained in an infection cycle involving hematophagous arthropods and vertebrate hosts.

Encephalitis Inflammation of the brain associated with cognitive changes.

History Nomenclature of the encephalitic alphaviruses recognizes the characteristic geographic distribution of each and the initial identification of the viruses as causes of encephalitis in horses. The first clear record of epidemic equine encephalitis is from 1831, when an outbreak in Massachusetts, USA, resulted in the deaths of 75 horses. Over the next 100 years several outbreaks of encephalitis in horses occurred along the Atlantic seaboard. The virus causing eastern equine encephalitis (EEEV) was first isolated from the brain of an affected horse in 1933 by Tenbroek and Merrill. However, two years earlier Meyer, Haring and Howitt had isolated a virus from the central nervous system (CNS) tissues of two horses involved in an epidemic of equine encephalitis in the San Joaquin Valley of California. EEEV and the Western equine encephalitis virus (WEEV) were antigenically distinct. Both diseases occurred in summertime epidemics, and in 1933, Kelsor showed that mosquitoes could transmit WEEV and shortly thereafter mosquito transmission of EEEV was demonstrated. In 1938 both EEEV and WEEV were isolated from humans with encephalitis occurring in the same regions as the equine cases. In 1936 equine encephalitis occurred in the Goajira region of Venezuela and the virus causing this epizootic, isolated independently by Beck and Wyckoff and Kubes and Rios, was not neutralized by sera from animals immunized with EEEV or WEEV and was designated Venezuelan equine encephalitis virus (VEEV).

Taxonomy and Classification In 1954 three serologic groups (A, B and C) of arthropod-borne viruses were distinguished by Casals and Brown on the basis of cross-reactivity in hemagglutination inhibition (HI) tests. EEEV, WEEV and VEEV constituted the original group A arboviruses. A second cross-reacting set, including dengue, St. Louis encephalitis and yellow fever viruses, constituted the group B arboviruses and the other viruses, mostly from Brazil, were designated group C viruses. As viruses became classified on the fundamental properties of the virion and genome, the group A viruses became the alphaviruses (genus Alphavirus) within the Togaviridae family of enveloped plus-strand RNA viruses. HI classifies alphaviruses into six broad antigenic complexes and EEEV, WEEV, and VEEV form three of these complexes. Viruses within each complex have been subtyped using reactivity with monoclonal antibodies, kinetic HI or neutralization assays. The EEE complex includes Madariaga virus (MADV) as well as EEEV. The WEE complex includes Aura, Fort Morgan, Highlands J, Sindbis, Trocara and Whataroa viruses in addition to WEEV. The VEE complex includes Cabassou, Everglades, Mosso das Pedras, Mucambo, Pixuna, Rio Negro and Tonate in addition to VEEV. WEEV and VEEV can also be subdivided into epizootic and enzootic strains. Epizootic strains of VEEV are equine-virulent and are further subdivided antigenically into subtypes IAB and IC (epizootic strains) and ID, IE, and IF (enzootic strains). Sequence information, has confirmed the validity of most of these serologic distinctions.

Virion and Genome Structure Properties of the Virion Alphavirus virions are 60–65 nm in diameter. The RNA is enclosed in a capsid formed by a single protein arranged as an icosahedron. The nucleocapsid is enclosed in a lipid envelope derived from the host cell plasma membrane that contains the viralencoded glycoproteins, E1 and E2. These proteins form heterodimers that are grouped as trimers to form knobs on the virion surface arranged with a T ¼ 4 symmetry. Glycoproteins are arranged with 240 copies of each protein interacting with 240 copies of capsid protein (Fig. 1). The virion is sensitive to ether and detergent. Infectivity can be inactivated by heat or acid. The viruses are stable at  701C for long periods.

Properties of the Genome The 49S genome is a nonsegmented, capped and polyadenylated message-sense RNA that is infectious. Complete sequence information is available for representative members of all antigenic complexes. The genomes contain approximately 11,700

40

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00027-8

Alphaviruses Causing Encephalitis (Togaviridae)

41

Fig. 1 Cryo-EM structure of eastern equine encephalitis virus showing radially colored surface representation. The right half shows a section of the map with the lipid bilayer indicated between the black dotted lines. From Hasan, S.S., Sun, C., Kim, A.S., et al., 2018. Cryo-EM structures of eastern equine encephalitis virus reveal mechanisms of virus disassembly and antibody neutralization. Cell Reports 25, 3136–3147.

nucleotides and have two open reading frames. The nonstructural proteins (nsPs) are encoded at the 50 -end and the structural proteins at the 30 -end. The 30 untranslated region (UTR) is important for negative-strand synthesis, the 50 UTR for genome synthesis and 21 nucleotides at the junction between the nonstructural and structural genes, for synthesis of the subgenomic RNA. Analysis of codon usage shows an underutilization of the dinucleotide CpG.

Properties of the Viral Proteins Nonstructural proteins The 50 end of the genome encodes four nsPs that function in replication of viral RNA and in production of the genomic and subgenomic RNAs. The nsPs are translated as a polyprotein from the genomic RNA and sequentially processed to individual proteins by the papain-like protease in the C-terminal portion of nsP2. nsP1 has methyl transferase and guanylyl transferase activities, is palmitoylated and binds the replication complex to membranes. The N-terminal domain of nsP2 has helicase, ATPase, GTPase, methyl transferase and 50 -triphosphatase activity. nsP3 is a phosphoprotein that induces membrane remodeling for the formation of cytoplasmic vacuoles and regulates RNA synthesis in a cell-type-dependent manner. nsP3 has a highly conserved N-terminal macrodomain, a middle zinc-binding region and a poorly conserved, unstructured, acidic and highly phosphorylated C-terminal hypervariable domain that interacts with many cellular proteins to regulate viral replication and assembly of stress granules. nsP4 is the RNA-dependent-RNA polymerase (RdRp).

Structural proteins Five structural proteins (C, E3, E2, 6K and E1) are translated as a polyprotein from the subgenomic RNA and a sixth small transframe (TF) protein is produced as the result of ribosomal frameshifting. The capsid protein is 259 (EEEV, WEEV) to 275 (VEEV) amino acids long. The N-terminal portion is conserved, basic and binds the genomic RNA and the C-terminal portion interacts with the cytoplasmic tail of E2 and with other capsid proteins to form the nucleocapsid. Capsid also inhibits host gene expression. The E3 protein is 59 (VEEV), 60 (WEEV) or 63 (EEEV) amino acids long, serves as a signal sequence for E2 and, in vitro, is shed into the supernatant fluid after cleavage by a furin-like protease in the trans Golgi network. The E2 glycoprotein is a transmembrane protein that is 420 (EEEV) to 423 (WEEV, VEEV) amino acids long and has two (EEEV) or three (WEEV, VEEV) N-linked glycosylation sites. The intracytoplasmic portion has a second stretch of hydrophobic amino acids. The 6K protein is 55 (WEEV, VEEV) or 56 (EEEV) amino acids long, serves as a signal peptide for E1, can form ion channels, remains with interior membranes, interacts with viral glycoproteins and facilitates virus budding. The E1 protein is 439 (WEEV), 441 (EEEV) or 442 (VEEV) amino acids long and has one or two N-linked glycosylation sites. E1 has a short (one or two residue) intracytoplasmic tail and a positionally conserved internal hydrophobic stretch of amino acids in the N-terminal portion of the protein that serves as

42

Alphaviruses Causing Encephalitis (Togaviridae)

nsP1, nsP2, nsP3, nsP4

Plus-strand replicase

Continued proteolytic processing by nsP2 in trans P123, nsP4

FXR

Minus strand replicase

Translation of nonstructural proteins and initial processing by nsP2 in cis 5'cap

nsP1

nsP2

nsP3

nsP4

Capsid

PE2

6K

E1

(A)n3'

Transcription of full-length minus-strand RNA 5'

3' Transcription of full-length plus-strand genomic RNA 5'cap

Transcription of subgenomic RNA (A)n3'

5'cap

Capsid

(A)n3' 6K E1 Translation of structural proteins and autoproteolytic cleavage by C

PE2

Capsid, PE2-6K-E1 Processing in ER PE2, 6K, E1 Processing in trans Golgi E3, E2

Fig. 2 Replication of new world alphaviruses. Genome is message from which nsPs are translated followed by synthesis of full-length negative sense genomic RNA by P123 þ nsP4 (early replicase) and formation of spherules with dsRNA, a step dependent on host FXR. P123 processed to individual nsPs forms the stable plus-strand late replicase and synthesis of genomic and subgenomic RNA. Structural proteins are translated from subgenomic RNA.

the fusion peptide for virion entry into the cell. TF is 70 (VEEV), 72 (WEEV) or 92 (EEEV) amino acids long, palmitoylated, trafficks to the plasma membrane and small amounts are incorporated into virions. Ribosomal frameshifting that leads to TF protein synthesis (o30% of the time) eliminates E1 synthesis.

Replication Initial attachment of virus to the cell likely involves interaction of the E1 glycoprotein, which has hemagglutination activity, with phospholipids on the cell surface. Efficient subsequent entry involves binding of E2 to a variety of cellular proteins followed by receptor-mediated endocytosis. Within the acidified endosomal compartment the glycoprotein spikes undergo a conformational change that results in fusion with the endosomal membrane and release of the nucleocapsid into the cytoplasm. Acidified endosomes may not be essential for infection of mosquito cells. Once released from the nucleocapsid by interaction with ribosomes, the virion genomic RNA serves as mRNA and translation of the nsPs as 2 polyproteins (P123 and P1234) is initiated (Fig. 2). Sequential polyprotein processing regulates the synthesis of minus strand full-length template RNA and plus strand genomic and subgenomic RNAs. nsP4 is first cleaved from P1234 and the resulting P123 polyprotein and nsP4 initiate formation of short-lived early replication complexes within spherules at the plasma membrane that synthesize a genome-length minus-strand template viral RNA. Formation of replication complexes requires interaction of the hypervariable domain of nsP3 with host proteins including members of the fragile X syndrome family of RNA-binding proteins. As P123 is processed to the individual nsPs, these complexes convert to stable replication complexes that synthesize plus-strand 49S genomic and 26S subgenomic RNAs within cytopathic vacuoles. The 26S RNA represents the 30 portion of the genome from which the structural proteins are translated as a polyprotein despite cessation of host protein synthesis. After translation of the capsid protein is complete, it is autocatalytically cleaved from the nascent chain. pE2 (precursor: E3 plus E2) and E1 are transported into the endoplasmic reticulum, cleaved by cellular signal peptidases and processed through the Golgi with a final processing of pE2 into E2 and E3 by furin. At the plasma membrane nucleocapsids align with regions containing the E1-E2 heterodimers and bud from the cell surface. The process may be different in mosquito cells, where intracytoplasmic compartments containing mature virions are seen in vitro. However, these structures have not been seen during ultrastructural studies of alphavirus infection in mosquitoes.

Geographic and Seasonal Distribution The encephalitic alphaviruses are transmitted primarily by mosquitoes during the warm months of the year and geographically restricted. EEEV is endemic in eastern Canada and from New Hampshire along the Atlantic seaboard and Gulf Coast to Texas in the

Alphaviruses Causing Encephalitis (Togaviridae)

43

United States and in the Caribbean. Inland foci exist around the Great Lakes. Recently EEEV has been detected as far north as Nova Scotia suggesting a northward expansion. In the northern part of the range, cases occur between July and October, while in the southern region cases can occur throughout the year. It is likely that EEEV is periodically reintroduced into northern regions from areas of year-round transmission by migratory birds or wind-borne mosquitoes. Three antigenic subtypes of MADV (formerly South American EEEV) are enzootic in the Amazon River basin, the coasts of South and Central America and Brazil with the cycle maintained in moist forests. Viruses of the WEE complex are widely distributed throughout the Americas as three closely related but antigenically distinct viruses (WEEV, HJV and Fort Morgan virus). HJV is endemic on the East Coast of the United States in the same areas as EEEV. WEEV is widely distributed in the western plains and valleys of the United States and Canada and is found in South America. Enzootic VEEVs are perennially active in subtropical and tropical areas of the Americas (subtype II in Florida; ID and IE in Central America; IF and III-VI in South America). Epizootics have appeared in Venezuela, Colombia, Peru and Ecuador at approximately 10-year intervals.

Host Range and Virus Propagation The life cycle of the encephalitic alphaviruses involves replication in invertebrate vectors, primarily mosquitoes, and in reservoir vertebrate hosts, primarily birds (EEEV and WEEV) or small mammals (VEEV). In epidemic periods additional hosts, such as horses and humans, are infected. EEEV causes encephalitis in humans, horses, emus, pigeons and pheasants. Other birds are susceptible to infection but remain asymptomatic, despite prolonged viremia. The amplifying species for EEEV in North America are wading birds and migratory songbirds. Infection has also been reported in turtles and snakes. In South America, forest-dwelling rodents and marsupials are likely reservoirs for MADV. In invertebrates, EEEV is most consistently recovered from the Culiseta melanura in North America and Culex taeniopus in the Caribbean with MADV recovered from Cx. (Melanconion) spp. in South America. Outbreaks of EEE are initiated when the virus spreads from the enzootic cycle involving ornithophilic mosquitoes (e.g., Cs. melanura) into mosquito populations that feed on a wider variety of hosts. Mosquitoes of many species can serve as bridge vectors to humans and equids. WEEV causes encephalitis in horses and humans, but disease is now rare. The enzootic cycle in North America involves domestic and wild birds and Cx. tarsalis for WEEV in the western United States and songbirds and Culiseta melanura for HJV in the eastern United States. Serosurveys and virus isolations have demonstrated evidence of natural infection in chickens and other domestic birds, passerine birds, pheasants, rodents, rabbits, ungulates and snakes. Epizootic strains of VEEV can cause disease in horses and in humans. Infection of horses with enzootic strains of VEEV is asymptomatic and may immunize horses for protection against epizootic strains. Enzootic strains produce mild disease in humans. A variety of wild birds are susceptible to infection, but small mammals serve as an important reservoir, with efficient transmission of infection by Cx. (Melanconion) mosquitoes. In addition to the native hosts, a number of laboratory animals are susceptible to infection. All three viruses cause encephalitis in monkeys, mice, rats, guinea pigs and hamsters. Disease is generally age-dependent, so that young animals more often develop fatal infections than do adult animals. Because of this susceptibility primary isolates of these viruses have often been made in newborn mice. In vitro, the viruses are routinely propagated in cultures of chick embryo fibroblasts, BHK-21 cells or Vero cells. Most strains will form plaques in these cells and plaque assays provide the usual basis for virus quantification. Mosquito cell lines support replication, often without cytopathic effect. Many other cell lines (e.g., L cells, HeLa cells) support replication as well.

Genetics Alphaviruses show genetic changes by accumulation of point mutations in the genomic RNA, but this occurs at a rate that is slower than that predicted for other RNA viruses. Recombination is infrequent, but can be demonstrated in vitro and occurs at least occasionally in nature because WEEV is the result of recombination between EEEV and Sindbis-like viruses.

Evolution The alphaviruses derive from a single unknown protoalphavirus and viruses in the alphavirus superfamily have a similar genetic organization and include many RNA plant viruses. In general, amino acids important in secondary structure (e.g., cysteine) have been conserved for the structural glycoproteins E1 and E2 consistent with the similar three-dimensional structure of all alphaviruses. Sequence analyzes suggest that the encephalitic alphaviruses evolve slowly in nature. The capsid protein and E1 glycoprotein are the most conserved of the structural proteins, whereas the E2 glycoprotein is more divergent. EEEV has evolved independently in North and South America over the last 1000 years and there is currently one group in North America and the Caribbean and three groups in South America. Based on sequence divergence, as well as ecologic and pathogenesis distinctions the three South American lineages were recently classified as a different alphavirus species MADV. North

44

Alphaviruses Causing Encephalitis (Togaviridae)

Fig. 3 Numbers of human cases of Eastern equine encephalitis reported in the United States from 1964 to 2019. Data are from the US Centers for Disease Control and Prevention.

American EEEV isolates are highly conserved, varying by less than 3% over 100 years consistent with birds as the reservoir hosts. MADV isolates are evolving more rapidly, consistent with small mammals as the reservoir hosts. Rates of divergence of WEEV and HJV of 0.1%–0.2% per year have been estimated. It is hypothesized that short transmission seasons and limited host mobility constrain genetic diversity in a geographic region.

Epidemiology EEEV causes localized outbreaks of equine, emu, pheasant and human encephalitis in the summers. Cases of equine encephalitis are usually the first indicators of an outbreak. In North America the primary enzootic cycle is maintained in shaded freshwater swamps where the vector is the ornithophilic swamp mosquito Culiseta melanura and the reservoir hosts are migratory passerine songbirds. Young birds are probably most important for virus amplification because they are more susceptible to infection, have a prolonged viremia and are less defensive towards mosquitoes. Human and equine cases usually occur within 5 miles of the swamp, with virus being transmitted by epizootic vectors such as Coquillettidia perturbans, Aedes sollicitans, and, potentially, Aedes albopictus. The enzootic vector in the Caribbean is probably Culex taeniopus and for MADV in South America is probably Culex (Melanoconion) spp. Epizootics appear approximately every 5–10 years and are usually associated with heavy rainfall that increases the populations of enzootic and epizootic mosquito vectors. There is no evidence for overwintering in mosquitoes so the mechanism for maintenance in northern areas is not known and may require reintroduction from southern areas of transmission. Human infections are unusual, with an average of 5–10 cases per year in the United States (Fig. 3). Serological surveys in the northeastern United States suggest that there are approximately 23 inapparent infections for every case of encephalitis, but children are more susceptible and this declines to only 8:1 for children under 4 years of age. WEEV is maintained in an endemic cycle involving domestic and passerine birds and Culex tarsalis, a mosquito particularly adapted to irrigated agricultural areas. Interseasonal persistence occurs in saltwater marshes, where vertical transmission of WEEV in Cx. tarsalis has been demonstrated. Occasional isolations have been made from Cx. stigmatosoma, Aedes melanimon and Ae. dorsalis, also competent vectors. Transmission from this enzootic cycle has resulted in small numbers of cases of encephalitis in humans. In the past, there have been widespread summer epizootics of equine encephalitis in North America with significant extension into humans. The estimated case to infection ratio is 1:58 in children under five years and 1:1150 in adults. Recently, for reasons that are not completely clear, cases of equine and human encephalitis have declined with the last human case in 1998 (Fig. 4). In the eastern United States, HJV has been isolated from ornithophilic mosquitoes (Culiseta melanura, the enzootic vector for EEEV) in freshwater swamp habitats along the Atlantic coast, as well as from birds. HJV can occasionally cause encephalitis in horses and is a recognized pathogen for turkeys, pheasants, emus and whooping cranes. The lack of significant human disease during equine outbreaks of WEE in South America may be related to the feeding habits of the vector or to a difference in virulence for humans of the South American strains. VEEV is maintained in enzootic cycles by Culex (Melanoconion) spp. mosquitoes that live in tropical and subtropical swamps throughout the Americas and breed near aquatic plants. They feed at dawn and dusk on a wide variety of rodents, birds and other vertebrates. Humans living in these areas have a high prevalence of antibody, but little recognized disease. Epizootic strains of VEEV arise by mutation from enzootic strains and are isolated only during outbreaks. Epizootics have occurred primarily in Latin America in cattle ranching areas during the rainy season. Formalin-inactivated vaccines containing residual live virus are suspected to be responsible for initiating the 1969–72 outbreak in Central America that extended to Texas. During epizootics equids are important amplifying species. Virus has been isolated from several species of mosquitoes including Aedes taeniorhynchus, Ae.

Alphaviruses Causing Encephalitis (Togaviridae)

45

Fig. 4 Numbers of human cases of Western equine encephalitis reported in the United States from 1964 to 2019. Data are from the US Centers for Disease Control and Prevention.

aegypti, Mansonia dubitans and Psorophora confinnis. The incidence of encephalitis in clinically ill humans is generally less than 5% and the overall mortality less than 1%.

Transmission and Tissue Tropism The primary mode of alphavirus transmission to birds and mammals is through the bite of an infected mosquito that inoculates virus extravascularly. Mosquitoes become infected by feeding on a viremic host, are able to transmit the virus 4–10 days later (extrinsic incubation) and remain persistently infected. Maintenance of this cycle requires an amplifying host that develops a viremia of sufficient magnitude to infect mosquitoes feeding on it. Other modes of transmission are of occasional importance. EEEV persists in the feather follicles of infected pheasants and secondary transmission among penned pheasants can occur through feather picking and cannibalism. VEEV can be transmitted by the respiratory route between infected horses and to humans in the laboratory. WEEV and VEEV can be transmitted transplacentally. In mammals, EEEV replicates primarily in muscle and in neurons and glial cells of the CNS, with occasional involvement of liver and lymphatic tissue. Skeletal and myocardial muscle and the CNS of infected birds contain virus. In mosquitoes there is infection of the midgut, muscle and salivary glands without involving the nervous system. WEEV replicates primarily in skeletal and cardiac muscle, brown fat and the choroid plexus, ependyma and neurons in the CNS of mammals. Little is known of the tissue tropism of WEEV in birds. Epizootic strains of VEEV infect the upper respiratory tract, lymphatic and myeloid tissue, pancreas, liver and CNS to varying degrees in different mammals. Mosquito infection is initiated in the midgut epithelial cells and spreads through the hemolymph to the salivary glands and flight muscles.

Pathogenicity EEEV is the most virulent of the alphaviruses, causing severe encephalitis in humans, horses, dogs, pigs, pigeons, emus, quail and pheasants. The case-fatality rate in humans is 30%–50%, up to 90% in horses and 50%–70% in pheasants. Laboratory studies indicate a similar neurovirulence of EEEV for monkeys, mice and hamsters. Hamsters also develop hepatitis and lymphatic organ infection. At 3–4 weeks of age mice become relatively resistant to peripheral, but not intracranial, inoculation. Birds vary in their susceptibility, with birds of some species developing disease, while birds of many other species show no morbidity or mortality, despite a prolonged viremia. Pheasant deaths are caused by encephalitis while young chickens develop myocarditis. MADV is much less virulent, but infection with lineage III viruses has been associated with cases of fever and encephalitis. Mosquitoes develop persistent infection of the fat body and salivary glands. WEEV in the western United States caused epidemics of encephalitis in humans, horses and emus, but the case-fatality rate of 10% for humans, 20%–40% for horses and 10% for emus is lower than for EEEV in the eastern U.S. HJV has been rarely reported to cause fatal disease in horses and turkeys. South American strains of WEEV cause fatal encephalitis in horses, but little disease in humans. Epizootic strains are neurovirulent and neuroinvasive in adult mice, whereas enzootic strains are not. HJV is intermediate in virulence between North and South American strains of WEEV. With increasing age mice become relatively resistant to fatal HJV infection, whereas hamsters remain susceptible. Infection of horses with epizootic strains of VEEV frequently causes fatal disease associated with leukopenia and a high-titered, prolonged viremia. In contrast to EEE and WEE, encephalitis is not always apparent and virus is shed in nasal, eye and mouth

46

Alphaviruses Causing Encephalitis (Togaviridae)

secretions as well as in urine and milk. Experimental infection of laboratory animals produces a variety of disease patterns. In guinea pigs and rabbits, VEEV causes necrosis in lymph nodes, spleen, thymus, intestinal and conjunctival lymphoid tissue, liver and bone marrow. Hamsters develop encephalitis and pancreatitis in addition to widespread infection of myeloid and lymphoid tissues. In rats and mice there is more limited destruction of lymphoid and myeloid tissue and death is usually due to encephalitis. Virus enters the CNS through the olfactory tract and causes neuronal infection and apoptosis. Neonatal mice show extensive replication in many tissues, including brain, myocardium and pancreas. Comparative studies of the virulent Trinidad donkey and avirulent TC-83 strains of IA serotype VEEV and construction of recombinant viruses have led to identification of the 50 noncoding region and the E2 envelope glycoprotein as important determinants of virulence for mice, whereas changes in nsP3 and E2 determine virulence for horses.

Clinical Features of Infection EEEV is the most virulent of the encephalitic alphaviruses, causing a high mortality due to encephalitis. Prodromal symptoms of fever, headache and myalgias are common. The onset of encephalitis tends to be fulminant and is associated with continued fever, increased headache, meningismus, obtundation and seizures. The overall case-fatality rate is 30% in recent studies, with higher rates in children and the elderly. Recovery is more likely in individuals that have a long (5–7 day) prodrome and do not develop coma. Sequelae are common, with more than 80% of survivors having significant neurological residua, including paralysis, seizures and cognitive deficits. The diagnosis is usually made by detection of antibody in serum or cerebrospinal fluid. WEEV can cause encephalitis with signs and symptoms similar to those caused by EEEV. There is a 3–5 day prodrome of fever and headache that may progress to irritability, nuchal rigidity, photophobia and altered mental status. Severe disease, seizures, fatal encephalitis and significant sequelae are more likely to occur in infants and in young children. Infection with epizootic strains of VEEV usually causes relatively mild disease in humans. Illness in adults typically is manifested by fever, headache, myalgias and pharyngitis 2–5 days after exposure. Severe disease, including fulminant reticuloendothelial infection and encephalitis may occur in young children. Children recovering from encephalitis may be left with neurological deficits. Fetal abnormalities and spontaneous abortions may occur with infection during pregnancy. The cerebrospinal fluid during alphavirus encephalitis usually shows a moderate (up to 4000 cells/mm3) pleocytosis with predominance of either polymorphonuclear or mononuclear cells. Protein is usually elevated and the glucose is normal. The electroencephalogram and magnetic resonance imaging scans are often abnormal, but computed tomographic scans may be normal or indicative only of edema.

Pathology and Histopathology Initial CNS infection with EEEV in experimental animals is of the capillary endothelial or choroid plexus epithelial cells and spread within the CNS can be cell to cell or through cerebrospinal fluid. The targeted cell within the CNS is the neuron and damage to this cell may be severe and irreversible. Histopathology demonstrates a diffuse meningoencephalitis with widespread neuronal destruction, neuronophagia, gliosis and perivascular inflammation, with polymorphonuclear and mononuclear leukocytes early, and vasculitis and vessel occlusion late. Hamsters also exhibit necrosis of hepatocytes and lymphatic tissue accompanied by local infiltration of mononuclear leukocytes. Initial infection of mosquitoes is of midgut epithelial cells. Infection is facilitated when virus in the serum is concentrated next to these cells as the infected blood meal clots. Infected midgut epithelial cells subsequently degenerate and slough and this process may facilitate penetration of the virus into the hemocoel and rapid dissemination of the infection. Pathological examination of brains from fatal human cases of WEE demonstrates early perivascular extravasation of blood followed by endothelial hyperplasia, perivascular mononuclear and polymorphonuclear inflammation and parenchymal necrosis. Areas of neuronal degeneration, glial nodules and demyelination are found. Neonatal mice develop acute inflammation and necrosis in skeletal and smooth muscle, cartilage and bone marrow. In animals with encephalitis, the brain shows multifocal areas of necrosis and widespread lymphocytic infiltration of the leptomeninges and perivascular regions of the brain parenchyma. The heart shows a necrotizing, inflammatory myocarditis. Infiltration of mononuclear leukocytes into areas of lung, liver and brown fat also occurs. The pathology of VEE in horses includes cellular depletion of bone marrow, spleen and lymph nodes, pancreatic necrosis and, in cases with encephalitis, swelling of vascular endothelial cells, edema and mononuclear cell cuffing of cerebral vessels and meningitis followed by the later appearance of demyelinating lesions. Small mammals with widespread involvement of the reticuloendothelial system may develop ileal necrosis. Both innate and adaptive immune responses are induced by infection. Interferon a/b is produced quickly and virus-specific IgM is often detectable by enzyme immunoassay or neutralization assay very early after onset of the disease. IgM in either serum or cerebrospinal fluid provides a means for rapid diagnosis of infection. IgG antibody appears 10–14 days after disease onset and can be measured by enzyme immunoassay, HI or neutralization. Many lines of evidence suggest that recovery from infection is primarily dependent on the antibody response. Extensive experimental studies to define the antibody specificity and the mechanisms of recovery and protection have been done using infection of mice. Neutralizing and nonneutralizing antibodies against multiple epitopes on the E1, E2 and E3 glycoproteins can protect against challenge and promote recovery.

Alphaviruses Causing Encephalitis (Togaviridae)

47

Cellular immunity has received more limited study, but virus-specific lymphoproliferative and cytotoxic responses have been documented and a mononuclear inflammatory response is common. The importance of cellular immune responses for recovery or for contribution to fatal disease has not been established but interferon-g can contribute to virus clearance from the CNS. Prior infection with VEEV increases subsequent antibody responses to unrelated antigens. Anti-thymocyte globulin extends time to death in mice infected with VEEV, suggesting a T cell-mediated immunopathogenic component to fatal disease.

Prevention, Treatment and Control Prevention of infection relies on efforts to control mosquito populations by spraying and reduction of breeding places. Individual use of protective measures such as mosquito repellents and protective clothing are important. Vaccines against EEEV, WEEV and VEEV are available for horses and against EEEV for birds. Experimental human vaccines against EEEV, WEEV and VEEV also are available for laboratory workers exposed to these agents. Most of these vaccines consist of formalin-inactivated virus, but TC-83 is a successful live attenuated vaccine against VEEV for use in horses. TC-83 is also used as a vaccine for humans, although side effects are common. No antiviral agents are of proven usefulness, but supportive treatment of these infections is important.

Future Construction of full-length cDNA alphavirus clones that can be transcribed into infectious RNA has provided important tools for understanding the functions of various genes and their importance for replication and virulence in the multiple hosts necessary for maintenance of these viruses in their natural cycles. The structures of each of the viral proteins both as individual proteins and polyproteins are needed for interpretation of much of the sequence and virulence data that has been acquired. Further information on the roles of host cell proteins and cell-type dependent differences in replication are likely to provide the next level of understanding of virus-host relationships. There is an ongoing need to develop effective vaccines and therapies.

Further Reading Carossino, M., Thiry, E., de la Grandiere, A., Barrandeguy, M.E., 2014. Novel vaccination approaches against equine alphavirus encephalitides. Vaccine 32, 311–319. Forrester, N.L., Coffey, L.L., Weaver, S.C., 2014. Arboviral bottlenecks and challenges to maintaining diversity and fitness during mosquito transmission. Viruses 6, 3991–4004. Griffin, D.E., Weaver, S.C., 2019. Alphaviruses. In: Knipe, D.M., Howley, P.M., Whelan, S. (Eds.), Field’s Virology, seventh ed. Wolters Kluwer. Kumar, B., Manuja, A., Gulati, B.R., Virmani, N., Tripathi, B.N., 2018. Zoonotic viral diseases of equines and their impact on human and animal health. The Open Virology Journal 12, 80–98. Lundberg, L., Carey, B., Kehn-Hall, K., 2017. Venezuelan equine encephalitis virus capsid-the clever caper. Viruses 9, 279. Ronca, S.E., Dineley, K.T., Paessler, S., 2016. Neurological sequelae resulting from encephalitic alphavirus infection. Frontiers in Microbiology 7, 959. Sharma, A., Knollmann-Ritschel, B., 2019. Current understanding of the molecular basis of Venezuelan equine encephalitis virus pathogenesis and vaccine development. Viruses 11, 164.

Anelloviruses (Anelloviridae) Fabrizio Maggi, University of Pisa, Pisa, Italy and University of Insubria, Varese, Italy Mauro Pistello, University of Pisa, Pisa, Italy r 2021 Elsevier Ltd. All rights reserved. This is an update of P. Biagini, P. de Micco, Anellovirus, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00694-4.

Glossary Human virome The total collection of viral nucleic acids, both RNA and DNA, that make up the viral community in and on the human body. Immunosuppression Partial or complete suppression of the immune response of an individual. MicroRNA A small non-coding RNA molecule that regulates gene expression by sequestering the messenger RNA (mRNA) and interfering with translation of the mRNA into proteins.

Representational difference analysis Molecular technique used to identify and characterize differences between two complex genomes. Viral species A monophyletic group of viruses the properties of which are unique and can be distinguished from those of other species by multiple criteria. Viremia The presence of viruses in the blood.

Introduction Anelloviruses (AV) are a large group of agents discovered in recent years that infect humans and various animal species. The first indication of their existence was in 1997 when a Japanese team applied representational difference analysis to search for novel hepatotropic viruses in the blood of patients with cryptogenetic post-transfusion hepatitis. They found a novel virus with a particularly small genome formed by a circular single-stranded DNA of negative polarity. This new virus was initially believed a novel hepatitis virus and named TT virus (TTV) after the initials of the first patient in whom it was identified. The significance of TTV was then changed in 2004 when it became the acronym of Torquetenovirus (from the Latin words torques and tenuis, meaning necklace and thin, respectively) to comply with the International Committee on Taxonomy of Viruses (ICTV) rule stating that no official virus designation shall be derived from people’s names. The discovery of TTV also opened a Pandora’s box of new and novel roles for viruses. Soon after its identification, it became clear that TTV was neither associated to hepatitis nor to any known disease, as the virus was found to be widespread in healthy and sick people. In the 2000s, the discovery of TTV was followed by identification of numerous related, previously unrecognized viruses, detected in diseased and healthy individuals and with genome properties resembling, yet sometime quite divergent from, those of TTV. Furthermore, polymerase chain reaction (PCR) testing of some blood donors yielded amplicons much shorter than expected for a bona fide TTV. Further analyses demonstrated the existence of additional viruses clearly related to but with a smaller genome than TTV. Those groups of viruses were named Torquetenominivirus (TTMV), and Torquetenomidivirus (TTMVD). Since 2009, all of these viruses are classified in the newly established family Anelloviridae (from anellus, Latin for ring, to indicate the circular genome) (Fig. 1). Although knowledge on TTV and related AV has accumulated rapidly, many fundamental aspects of their infection remain unresolved. AV have been found to be amazingly widespread, with abundant viral DNA detectable in the plasma of 80% or more of worldwide general population, but their significance for human health is still unknown. It has been proposed that AV should be considered completely nonpathogenic and recent evidence showing that they represent the most abundant members of the human virome speak in favor of this hypothesis. However, at this time, it seems wiser to consider AV a “disease orphan”, similar to other viruses that were found to produce significant pathologies many years after their discovery.

Classification Initially, AV were classified within the Circoviridae family. This grouping was based on the high genome relatedness between AV and the chicken anemia virus (CAV), a prototype circovirus that causes severe damage to the poultry industry, and from the assumption that all known vertebrate viruses with small circular, single-stranded DNA genomes were circoviruses. Subsequent deeper sequence analysis of AV genomes showed that, although they share many genetic features with the circoviruses, they differed considerably from other viruses of this family (e.g., porcine circovirus, beak and feather disease virus of parrots) and had no major sequence homology. Recognition of these differences and other unique attributes have led to group AV in the Anelloviridae, a family coined ad hoc by the ICTV. The progressive discovery of AV human and animal strains exhibiting genomes highly divergent and with size ranging from B2 kb to B4 kb, makes reliable phylogenetic and taxonomic analyses of full-length sequences complicated. From this standpoint, nucleotide sequencing and genetic analysis of the entire open reading frame-1 (ORF1) is the most convenient approach for

48

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20996-5

Anelloviruses (Anelloviridae)

49

Fig. 1 Phylogenetic tree based on full-length nucleotide sequences of viral species of Anelloviridae.

classifying AV, which are grouped in genera, species and isolates to cover their wide range of genetic variability. Based on the currently available data, AV are divided into 12 genera, each of which is designated by a letter of the Greek alphabet with the only exception of the genus Gyrovirus, that has been recently moved from Circoviridae to Anelloviridae (Table 1). Within a given genus, the ORF1 DNA genomes of all members have more than 56% divergence with AV of other genera. A species is designated for those AV within a given genus that share 45%–80% identity, while a viral isolate within a species has more than 80% identity with other isolates within the species. By using these criteria, human AV are clustered among 3 of the genera: Alphatorquevirus (identifying the TTV sequences) Betatorquevirus (TTMV sequences), and Gammatorquevirus (TTMDV sequences); the remaining 9 genera include animal AV only. Human TTV sequences are genetically classified in at least 29 major species, each of which consists of numerous isolates. TTMV and TTMDV has been investigated in less detail, and existing data indicate that they may be more homogeneous compared to TTV, but the recognition of much more diversity is expected as the number of available sequences increases.

Virion Structure and Genome Knowledge of physical-chemical properties of AV is incomplete and has been obtained by studying plasma and, less commonly, fecal extracts of TTV infected individuals. As determined by PCR analysis of infected serum passed through polycarbonate filters of decreasing pore size, the TTV particle is approximately spherical and has a diameter of between 30 and 32 nm (below 30 nm for TTMV). Isopycnic density was found to be 1.32–1.33 g/ml in cesium chloride (1.27–1.28 g/cm3 for TTMV) and 1.26 g/ml in sucrose gradients. Because

50

Anelloviruses (Anelloviridae)

Table 1

List of genera and species in the family of Anelloviridae

Family

Genus

No. of species included

Representative species

Anelloviridae

Alphatorquevirus Betatorquevirus Deltatorquevirus Epsilontorquevirus Etatorquevirus Gammatorquevirus Gyrovirus Iotatorquevirus Kappatorquevirus Lambdatorquevirus Thetatorquevirus Zetatorquevirus Unclassified

29 12 1 1 2 15 1 2 2 1 1 1 At least 28

Human torquetenovirus Human torquetenominivirus Torque teno tupaia virus Torque teno tamarin virus Torque teno felis virus Human torquetenomidivirus Chicken anemia virus Torque teno sus virus 1 Torque teno sus virus k2 Torque teno zalophus virus Torque teno canis virus Torque teno douroucouli virus Torque teno rodent virus

density does not change following Tween 80 and is poorly affected by solvent/detergent treatments, TTV has been deduced lack an external lipid envelope and to be as stable to inactivation by chemical and physical agents as parvoviruses and circoviruses. The component of AV currently best understood is the genome. Early characterization showed that nucleic acids extracted from TTV were sensitive to DNase I and mung bean nuclease but resistant to RNase A and specific restriction enzymes, indicating that the viral genome is single-stranded DNA. Initially, when the prototype Japanese isolate TA278 was sequenced to about 90%, it was proposed that the genome was linear. When sequencing of TA278 genome was further extended, it was then recognized that the two extremities were connected by a guanine and cytosine (GC)-rich stretch of about 100 nucleotides (nt), that was hard to amplify and sequence in earlier studies and that completes the covalently closed circular molecule. Hybridization/nuclease protection assays also showed that virions encapsidate the minus strand. The genomes of many of the human and animal AV have been sequenced in their entirety, and show similar genomic organization. However, although all consist of a circular molecule of single-stranded DNA, AV genomes differ in size; TTV is 3.5–3.9 kilobases (Kb), TTMDV around 3.2 Kb, and TTMV 2.7–2.9 Kb. All are negative sense, contain an untranslated region (UTR) of about 1.2 kb, and a potential coding region of approximately 2.6 kb. The UTR appears to control virus replication and expression through a number of important regulatory sequences and secondary structures (those formed by the 100 nt high GC region are particularly important), and it possess a short nucleotide sequence highly conserved among all AV isolates. The coding region consists of two major potential protein-coding genes, named ORF1 and ORF2. These ORFs are of different size, are present in the plus strand complementary to the genomic DNA, partially overlap and are in different reading frames. In the prototype TA278 sequence, ORF1 spans nt 589–2898 and ORF2 spans nt 107–712, which correspond to a coding capacity of 770 and 150 amino acid (aa), respectively. The length of the two ORFs may vary in individual isolates. ORF1 is believed to encode the putative capsid protein, which also contains specific motifs: (1) an arginine-reach hydrophobic region at the N-terminus that is believed to mediate binding of the genome to the capsid and its transportation to the cell nucleus; (2) a hypervariable region (HVR) located centrally that encodes potential glycosylation sites that vary in number and location in individual AV strains and that might affect several biological properties of the resulting protein, including antigenic specificity; (3) short aa motifs typical of replication-associated proteins (Rep) of viral DNAs replicating using the rolling circle mechanism. The smaller ORF2 encodes for a putative nonstructural protein of approximately 100–120 aa that is thought to possess phosphatase activity and be involved in viral replication. Sequence analysis of AV isolates predict additional putative ORFs that might encode for proteins which function is poorly characterized. Among these, there are two phosphorylated proteins, one resembles the gene NS5A of hepatitis C virus, the other, named TTV-derived apoptosis-inducing protein (TAIP), has been shown to induce p53-independent apoptosis in various human hepato-cellular carcinoma cell lines at levels comparable with that of CAV apoptin.

Life Cycle Although the discovery of TTV was over twenty years ago, the AV life cycle is poorly defined. Lack of in vitro systems to efficiently cultivate and propagate viral isolates has greatly limited studies of this family of viruses. This includes the screening of cell types supporting replication of AV, the identification of the receptor(s) on the cell surface used for viral entry, the dissection of the biosynthetic machinery exploited to replicate the virus, identification of intracellular signaling pathways perturbed, and visualization of cytopathic effects on infected cells. Our current understanding of AV replication mechanisms is thus mostly based on inference from what is known about other single stranded DNA viruses, even though these have not been studied in great detail either. What is certain about is that AV are found in many organs and infect different cell types. It is presumed, therefore, that the cellular receptor(s) used for viral entry is/are widespread in tissues and exposed by a wide array of cell types. A precise definition of

Anelloviruses (Anelloviridae)

51

cell types supporting virus replication is not yet available. Since related single-stranded DNA viruses require actively multiplying cells for productive replication, it is conceivable that replication of most, if not all, AV depends on cellular proteins expressed during the S-phase of cell cycle. It has also been shown that the same cell can be infected with more than one AV, thus explaining why recombination is a frequent occurrence among these viruses. Transcription analysis of prototype TTV strains have led to the identification of at least three RNA transcripts of different lengths (3.0, 1.2, and 1.0 Kb). Such transcripts share common 50 and 30 termini and are encoded by the positive DNA strand through alternative splicing. However, the transcriptional strategies of different AV vary considerably. The mechanism of DNA replication is not known with certainty but there are several indications pointing to a rolling circle mechanism. Such a mechanism, particularly well-suited for single-stranded DNA viruses, is indicated by the presence in ORF1 of conserved Rep protein motifs typical of the many viruses that replicate in this manner. The absence of motifs typical of known DNA polymerases in the encoded products suggests that the genome is replicated by cellular enzymes. Due to the great genetic diversity of AV, it has also been hypothesized that the viral DNA is replicated by polymerases with poor or no proof-reading activity. Indeed, rates of nucleotide change found in the HVR region of TTV are at least ten-fold higher compared other DNA viruses and are similar to that observed for RNA viruses. Using a combined computational and synthetic approach, microRNA-coding regions have been identified in diverse human TTVs and likely produce TTV microRNA in vivo. To this aim, TTV utilizes the host microRNA biogenesis machinery to produce biologically active microRNAs. Although their role in viral replication is unclear, it has been hypothesized that they promote viral persistence and immune evasion. Modes of virion assembly and release from producer cells are not known. It is likely that virus particles are assembled in the cytoplasm and, in analogy with what observed with other nonenveloped viruses, released by cell lysis. However, recent findings demonstrating that TTV particles are sequestered within circulating extracellular vesicles suggests other routes of egress of progeny virions. Available data do not support the idea that TTV can persist as an episome or is integrated into host cell DNA.

Epidemiology AV are among the most successful viruses of humans. Indeed, the best documented aspect of AV epidemiology is the remarkably high diffusion of infection in general population and throughout the world. Epidemiological surveys employing PCR assays of high sensitivity and searching UTR or the most conserved segments of encoding regions in the blood, have shown that TTV infects nearly two third of the general population and that prevalence is not related to geographical, socio-economical, and age- and gender-related factors. Interestingly, prevalence is high in geographically segregated populations that have limited contact with the rest of the world. TTMV and TTMDV have a prevalence of active infections similar to TTV, thus individuals who carry multiple AV in their blood and/or other tissues are the rule rather than an exception. Metagenomic analyses provided evidence for the presence of as many as 47 different species of AV in the gut of one infant at 12 months of age. Systematic studies performed using standardized and consistent protocols will allow the proportion of individuals carrying multiple species of TTV to be defined and meaningful comparisons of the study cohorts to be performed. Although in some surveys viremia rates increase with age and peak in adulthood, AV infection is frequent already in the early months of life. This implies that AV infection is highly contagious and uses various routes of spread. That AV can be transmitted via blood and blood products has been repeatedly documented. Most screenings performed early after TTV discovery found the highest detection rates amongst polytransfused, thalassemic, and long-term hemodialysis patients, as well as hemophiliacs, and intravenous drug abusers, clearly indicating that TTV is a blood-borne virus. These observations, again to comply with ITCV rules, prompted a change of the original TTV acronym from Japanese patient initials to “Transfusion Transmitted Virus”. As mentioned above, this denomination was replaced by the current definition as soon as it became evident that TTV viremia rates in general population were far too high to be explained by blood-borne transmission. Indeed, TTV was later found in feces, respiratory secretions, and various biological fluids. The common occurrence of TTV in feces of viremic patients suggests that oral-fecal route is a significant mode of transmission. This finding combined with the high prevalence of virus carriers and a high resistance to physicochemical agents, leading to the presence of the virus in food, wastewater, drinking water, and river water, likely contributes to a particularly high dissemination of TTV in human environment. Attempts are underway to evaluate whether TTV might be useful as a marker of anthropic pollution instead of or in addition to enteric bacteria. Mother-to-fetus transmission of TTV is also very common. TTV DNA sequences similar to those in the corresponding maternal blood were detected in newborns and cord bloods in amounts as high or higher than the respective peripheral blood. Alternative routes of AV spread have been poorly investigated, although TTV and related AV have been detected in nasopharyngeal and genital secretions, suggesting that, at least in some instances, the virus can be transmitted via respiratory and sexual routes. AV DNA sequences are also found in saliva, tears, skin, and hairs implying that household contact transmission is also possible. Whether non-human AV or AV-like viruses, reported to be widespread in chickens, pigs, cats, dogs and other farm and companion animals, contribute to AV epidemiology in humans is unclear. More detailed analysis of these viruses is needed to establish whether animal AV add to the already enormous reservoir of AV genomes existing in nature and contribute to human AV evolution and diversification. Few studies have investigated the geographical distributions of TTV species. Some species have been found to be very common worldwide, others seem to be less frequent or clustered in specific geographic regions. It is also uncertain whether these data reflect

52

Anelloviruses (Anelloviridae)

real differences in species distribution or are biased by preferential amplification of selected species by the molecular assay used. Whether species frequency varies with age of infected subjects and/or route of transmission also remains to be determined. Extensive investigations with next generation sequencing (NGS) or other standardized tools are warranted to obtain a wide and precise epidemiological picture.

Clinical Features To date, the medical significance of TTV and AV in general is unclear. Undoubtedly, the circumstances in which TTV was discovered greatly influenced initial studies in the attempts to link the virus to a clinical disease. The temporal association between TTV viremia and the elevation of transaminases observed in transfused patients in whom TTV was demonstrated, as well as the apparent ability of TTV to replicate in the liver, led to the suggestion that the virus was a possible cause of acute and chronic liver disease, particularly those that have a putative viral cause but are unrelated to known viruses. Later findings disputed this correlation and, to date, although it cannot be excluded that TTV infection could be occasionally associated with occasional liver injury of varying severity, the virus is no longer considered an important cause of clinically overt liver disease. Attempts to evaluate the possible role of TTV and other AV in the genesis of extrahepatic illnesses of unknown origin have, therefore, been explored. Given the chronic nature of most if not all TTV infections, most studies have been focused on chronic diseases such as diabetes, cryoglobulinemia, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Kawasaki syndrome, multiple sclerosis and other neurological diseases for which a viral etiology has long been suspected. In all cases, a direct role of TTV in disease etiology has been excluded or proved inconclusive. Because TTV infection is acquired early in life, other studies were performed to correlate TTV infection with acute pathologies of presumable viral origin occurring in infants. Here, it has been shown that TTV might behave as a respiratory virus and be involved in inducing or aggravating some acute pediatric respiratory diseases in which no other etiological agents were detected. Additional diseases for which a role of TTV has been sought in children are asthma and bronchiectasis. In fact, a correlation between TTV load in nasal fluid and severity of the associated respiratory deficit was observed in both these diseases. Other studies also implicated TTV or TTMV in a few renal ailments and in certain forms of anemia, neutropenia, and thrombocytopenia. However, until final proof of these or other findings, TTV and other AV should be considered “viruses waiting for a disease” or “ disease orphans” in the same way that other viruses have been linked to a disease years after their discovery (e.g., echoviruses, reoviruses, Epstein-Barr virus, etc.). So far, AV behave exactly as formerly orphan viruses and we are still seeking occasional infections sufficiently aggressive to trigger significant clinical disease. Additionally, it cannot be excluded that only some types of AV are pathogenic but their virulence is blurred by the plethora of non-pathogenic types. An unresolved question is whether AV modulate the host’s adaptive immune response. It has been demonstrated that TTV interacts systematically with many cellular sensors, including those modulating immune and inflammatory responses. Because of the small number of focused studies performed and the limitations of in vitro methods, detailed information on the mechanisms involved in AV-cell interactions and pathways involved is lacking. A few studies have showed that TTV ORF2 protein suppressed NFκB activity, which is crucial for expression of many genes connected to inflammation. It has also been shown that the TTV genome, as well as its replicative intermediate in infected cells, may influence the synthesis of pro-inflammatory cytokines evoked following stimulation of Toll-like receptors. Again, different TTV species have been shown to encode microRNAs, which are often sequestered within plasma exosomes of infected individuals. Interestingly, at least one TTV species encodes a microRNA in vivo that targets IFN signaling and, therefore, possibly modulates immune evasion by antagonizing the host antiviral response. Overall, these findings support the idea that TTV influences various biological processes and this leads to an elevated inflammatory status that is either generalized or in specific body sites. Further, perturbation of biological processes may be proportional to the type(s) and amount of TTV(s) circulating in the infected host. From another perspective, put forward following discovery of TTV and its surprisingly high prevalence, the fact that no disease is associated with active infection and that the virus undergoes sustained and continuous replication in healthy individuals has led to the hypothesis that AV is devoid of pathogenic potential. This idea strengthened since initial observations to the point that TTV is now considered integral part of the normal human microflora. Further, TTV can be considered a stepping stone for reconsidering some key aspects of virology. Once thought to be only present in the host during disease, viruses have recently been demonstrated to be numerous in various body sites of healthy subjects, and the term virome has been coined to describe the collection of viral species present in a human organ. This sort of viral “flora” is an ensemble of “innocuous” entities such as bacteriophages, endogenous retroviruses, eukaryotic viruses not associated with diseases, and noxious viruses causing acute, chronic or latent illnesses. Thanks to NGS technology, the human virome has been studied in various compartments, such as respiratory tract, gut, and skin in healthy and sick conditions. To date it is known that some components of human virome are present only in a few areas and few individuals, others are disseminated in almost all body sites of a very high percentage of people. AV, and particularly TTV, are the paradigmatic example of the latter. They are the most representative and abundant viruses of the human virome and their load in plasma is proposed as a simple and general measure of immune system function of infected hosts. In keeping with this notion, it has been shown that TTV viremia increases after autologous hematopoietic stem cell transplantation (HSCT) but returns to pre-HSCT levels once the immune system regains its functionality. Thus, monitoring TTV viremia might prove useful in estimating the time interval between the chemotherapy courses required to minimize the occurrence of opportunistic infections. Again, the immunological status of solid organ transplant recipients and the shape and composition of the virome are closely connected. In transplanted patients, marked expansion of viral species of Anelloviridae family and high titer of virus indicate

Anelloviruses (Anelloviridae)

53

Fig. 2 Factors impacting the size of TTV viremia. P, proven; EA, some direct evidence available; H, hypothetical.

Table 2

Dynamic of chronic TTV viremia, relative to other viruses

Virus

Mean virion half-life in plasma

New virions entering plasma per day

Hepatitis B Hepatitis C HIV-1 TTV

28.8 h 2.7 h o1h 4.3 h

41.7  1013 41.3  1012 49.8  109 42.0  1010

strong immunosuppression and risks of opportunistic infection. Conversely, a lower-than-average burden of TTV is indicative of insufficient immunosuppression and risk of graft rejection.

Pathogenesis A well-known feature of AV is the ability to establish, in many if not all exposed individuals, chronic productive infections characterized by continuous and abundant presence of the virus in the bloodstream. The course of infection is mostly unknown and the small amount of information available almost exclusively concerns TTV. Body sites and cell types where AV undergoes primary amplification following body entry as well as tissues sustaining its continuous replication and shedding in blood circulation are unknown. In children hospitalized for acute respiratory infections, higher TTV loads were measured in the nasal secretions compared to blood and, more interestingly and in some individuals, the same viral isolate was detected earlier in nares than bloodstream, suggesting that the respiratory tract is a site of primary TTV amplification. After its entry, TTV becomes detectable in the peripheral blood within one or a few weeks, indicating that dissemination of infection throughout the body is rapid. The most frequent outcome of TTV infection is a prolonged, likely lifelong TTV viremia sustained by genuine or, in a minority of cases, by a mix of viruses acquired through multiple exposures. Cases of self-limited infection have been also reported in which viremia spontaneously disappears a few weeks to several years post-exposure. It is unclear whether this is the result of an eradication of infection, decreased virus shedding into blood and/or increased virus clearance, or an infection entered in a latent inactive state. In chronically infected persons, the levels of plasma TTV vary extensively, ranging between 101 to over 109 DNA copies per ml. In many individuals, TTV viremia remains essentially stable over time, in others it undergoes wide fluctuations over time under the influence of various factors that may directly or indirectly impact on viremia size (Fig. 2). During replication, TTV is produced in large amounts: in kinetic studies of TTV viremia in patients treated with interferon for concomitant hepatitis C, it has been calculated that 1010 TTV virions are generated per day with nearly all virus entirely cleared and replenished in plasma on a daily basis (Table 2). Remaining aspects of TTV interactions with the infected host are far less well characterized. For instance, the target tissues and organs from which TTV is continuously released into the bloodstream and the fate of cells sustaining viral replication are unknown. Almost all anatomical body sites have been found to harbor variably abundant TTV. These include liver, lung, pancreas,

54

Anelloviruses (Anelloviridae)

spleen, kidney, lymph nodes, skeletal muscles, thyroid gland, bone marrow and circulating leukocytes. Only the cerebrospinal fluid is usually virus negative provided that the blood-brain barrier is intact. The presence of double-stranded viral DNA and viral mRNA, i.e., two important cellular markers of TTV replication, has been reported in liver, bone marrow, and circulating peripheral blood mononuclear cells, but the methods to detecting such replicative intermediates have many pitfalls, thus leaving ample room for questioning the findings. Most findings support the idea that proliferating hematopoietic cells are an important source of circulating TTV. The idea is strengthened by the fact that baseline TTV viremia decreased to undetectable levels in patients under myelosuppressive treatments (e.g., cyclophosphamide and total body irradiation) for bone marrow transplantation. Since neither viral receptor nor in vivo tropism are known, it cannot be ruled out that other cells sustain TTV replication and that different species of TTV and AV replicate in different cell types, tissues and body compartments. How rapidly and effective the host immune system restraints an invading AV is also unknown. While nothing is known about anti-TTV cell mediated immune responses, current knowledge about humoral responses suggests that TTV elicits antiviral antibodies that fail to eradicate the virus, at least in the great majority of cases. The frequent identification of people infected by multiple and heterologous TTV species highlights the inability of anti-TTV specific antibodies to clear the virus or protect against subsequent infections. Sequential examination of patients accidentally infected through contaminated blood and of chimpanzees experimentally infected with human AV has shown that the antibody response mounts rather slowly. TTV-specific IgM become detectable in serum 10–21 weeks post-infection, 2–7 weeks after the first detection of TTV DNA in the blood. IgM declined in titer later on to disappear within 5–11 weeks, although longer persistence was reported. IgG antibodies generally developed with a lag of several weeks, increased in titer concomitant with IgM decline, and last with little variation for a prolonged period of time or even indefinitely. Most if not all the TTV detected by molecular methods in the serum of chronically infected subjects is immunoprecipitated with anti-human IgG sera, indicating that TTV circulate in the blood bound to Ig. Conversely, in individuals with putative acute infections, free TTV particles in blood are abundant and often overwhelm the IgG complexed ones.

Diagnosis Laboratory diagnosis of AV infection is still at infancy. This is due to the following reasons: (1) there are no reliable and sensitive tissue culture systems for AV isolation and propagation; (2) there are no easy-to-use immunological methods to detect and titrate AV induced antibodies, and this in part accounts for their unclear significance in the course of viral infection; (3) detection of viral antigens in plasma is also unavailable. Diagnosis of infections relies, therefore, on the detection of TTV genome in blood or other specimens. Here too there are some caveats: most PCR assays are incapable of detecting the entire spectrum of AV species, standardization is far from being achieved because each laboratory sets up its own test independently, there have been few interlaboratory exchanges of methods and molecular constructs and there are no international standards. As a matter of fact, validated, in-vitro diagnostic labeled assays to detect and quantitate TTV DNA are badly needed but uncertainties about the clinical relevance of AV infection has discouraged most commercial companies from investing in this field. Because of great genetic variability of AV, identification of the viral DNA segment to be targeted for amplification dictates the sensitivity and spectrum of viral species detection. This is true in general for all viruses and crucial for AV, where variability is an order of magnitude higher compared to other viruses. Historically, the first PCR protocols were designed to target the TTV ORF1 gene. Soon after their development, however, it became clear that they greatly underestimated the prevalence rates of the infection since PCR primers annealed to poorly conserved sequences. As reported above, sequence homology of the UTR is higher than ORF1 across the different TTV isolates, particularly for a segment that is shorter than 100 nucleotides, making it suitable for developing PCR assays capable of detecting all or nearly all the 29 species currently identified. Indeed, when compared to ORF1-PCRs, UTR-PCRs greatly increased the rates of samples scored positive for TTV DNA. It has however been pointed out that even UTR-PCR assays can be biased towards some species, unless primers are carefully designed. UTR-PCR assays capable of simultaneous detection of all TTMV and TTMDV species have not been developed at the moment. It is evident that correlation of AV to a specific disease and/or to use the viruses as potential biomarkers of immune function is difficult without precise quantification of the virus. Recently developed real-time PCRs and droplet digital PCRs targeting TTV UTR and measuring the viral content in plasma and other biological samples hold promises to reduce the gap. TTV viremia as determined with UTR-PCRs is 10- to 100-fold higher compared to ORF1-PCRs, reflecting the higher sensitivity of former assays as well as detection of TTV species poorly or not amplified by latter. The lower limits of detection of quantitative PCR assays are on average 10–100 DNA copies per ml of blood, a threshold that is likely sufficient, based on available information and because of the high levels of viremia established for most diagnostic purposes. TTV genotyping exploits genomic sequences informative for classification and, for this purpose, ORF1 gene is an apt target as it permits grouping of all AV by either sequencing of the whole gene or using genotype-specific primers in standard PCR assays. The concomitant presence of multiple AV, a frequent occurrence in most subjects, is a confounding factor that impairs reliability and applicability of genotyping assays. NGS technology is a valid alternative for precise genotyping. In the quest for establishing routes of transmission and disease association it is crucial to extend examination to other clinical specimens. TTV DNA has been detected in feces, saliva, nose swabs, throat swabs, and breast milk of viremic subjects. Of note, some biological fluids scored positive also in persons with no detectable viremia.

Anelloviruses (Anelloviridae)

55

Treatment and Prevention As long as their clinical implications remain undefined, there will be little attention paid to the prevention and control of AV infection. It is also clear that, based on knowledge accrued so far, there is little doubt that prevention of infection is extremely difficult in the absence of specific polyvalent vaccines. There are no drugs of proven efficacy for AV infection, and the current lack of information on AV pathogenicity does not justify specific clinical trials. In patients treated with IFNa alone or in association with ribavirin for HBV or HCV infections TTV viremia did not change or decreased only transiently. Antiretroviral drugs have no anti-TTV activity. Specific antivirals are likely to be developed should one day AV infection be considered clinically relevant and worth treating.

Further Reading De Vlaminck, I., Khush, K.K., Strehl, C., et al., 2013. Temporal response of the human virome to immunosuppression and antiviral therapy. Cell 155, 1178–1187. Fernández-Ruiz, M., Albert, E., Giménez, E., et al., 2019. Monitoring of alphatorquevirus DNA levels for the prediction of immunosuppression – Related complications after kidney transplantation. American Journal of Transplantation 19, 1139–1149. Jaksch, P., Kundi, M., Görzer, I., et al., 2018. Torque teno virus as a novel biomarker targeting the efficacy of immunosuppression after lung transplantation. Journal of Infectious Diseases 218, 1922–1928. Lim, E.S., Zhou, Y., Zhao, G., et al., 2015. Early life dynamics of the human gut virome and bacterial microbiome in infants. Nature Medicine 21, 1228–1234. Maggi, F., Bendinelli, M., 2009. Immunobiology of the torque teno viruses and other anelloviruses. Current Topics of Microbiology and Immunology 331, 65–90. Maggi, F., Focosi, D., Statzu, M., et al., 2018. Early post-transplant torquetenovirus viremia predicts cytomegalovirus reactivations in solid organ transplant recipients. Scientific Reports 8, 15490. Ninomiya, M., Nishizawa, T., Takahashi, M., et al., 2007. Identification and genomic characterization of a novel human torque teno virus of 3.2 kb. Journal of General Virology 88, 1939–1944. Nishizawa, T., Okamoto, H., Konishi, K., et al., 1997. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochemical and Biophysical Research Communications 241, 92–97. Okamoto, H., Nishizawa, T., Kato, N., et al., 1998. Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown etiology. Hepatology Research 10, 1–16. Spandole, S., Cimponeriu, D., Berca, L.M., Mihăescu, G., 2015. Human anelloviruses: An update of molecular, epidemiological and clinical aspects. Archives of Virology 160, 893–908. Strassl, R., Schiemann, M., Doberer, K., et al., 2018. Quantification of torque teno virus viremia as a prospective biomarker for infectious disease in kidney allograft recipients. Journal of Infectious Diseases 218, 1191–1199. Takahashi, K., Iwasa, Y., Hijikata, M., Mishiro, S., 2000. Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus. Archivies of Virology 145, 979–993.

Animal Lentiviruses (Retroviridae) Esperanza Gomez-Lucia, Complutense University of Madrid, Madrid, Spain r 2021 Elsevier Ltd. All rights reserved.

Glossary CAP Methylated guanosine linked to the first nucleotide of the RNA, essential to initiate protein synthesis, protect RNA from the exonuclease cleavage, and recruit protein factors for several functions. CD4 þ T-lymphocyte (or T-cell) T-cell identified by the CD4 marker associated to the TCR (T-cell receptor for the antigen), which enables the cell to recognize antigen processed by antigen processing cells (macrophages, monocytes, dendritic cells). After recognition, the T-cell becomes activated and can collaborate with other cells of the immune system, such as B-cells (for antibody production) and other T-cells (for cell-mediated immune responses). Chemokine Chemotactic molecule which stimulates inflammation. Chemokines are usually produced by the injured tissues, and attract neutrophils, monocytes, macrophages, lymphocytes, dendritic cells, eosinophils, and basophils. Endogenous lentivirus Lentivirus which infected sexual cells (sperm or ovum) and became part of the germline, being passed from generation to generation throughout thousands or millions of years. Usually endogenous lentiviruses have acquired mutations which avoid them from productive replication (replication deficient).

Enhancer Sequences in the 50 non-coding region of the genome which are recognized by cellular factors and trigger RNA transcription. Exogenous lentivirus Lentivirus which is transmitted in a contagious manner, either after budding and being recognized by receptors, or by cell-to-cell contact. Usually it is replication competent. Immunodeficiency State in which the immune system is unable completely or in part to fight infectious disease and cancer. Lipid rafts Microdomains in the plasma membrane less fluid than the surrounding lipid bilayer, enriched with cholesterol, saturated phospholipids and membrane proteins. Macrophage Cell derived from blood monocytes, with high phagocytic ability, which resides in tissues. It participates both in the innate and adaptive immunities. Promoter Region at the 50 end of genes which triggers RNA transcription for protein synthesis. Provirus DNA genome of retroviruses, after the viral single-stranded RNA is reverse transcribed into doublestranded DNA and integrates in a host chromosome.

Introduction Lentiviruses are grouped in a genus within the family Retroviridae. The name derives from the Latin word lenti which means slow, alluding to the long incubation period of the disease, which in most cases lasts years. During this period the virus seems to be controlled by the immune system but steadily replicates and increases in numbers. When the viral load is too high and the damage to the different systems, mainly the immune system, grows unendurable, the disease becomes apparent and the infection turns fatal. The discovery that acquired immune deficiency syndrome (AIDS) was caused by a lentivirus, human immune deficiency virus (HIV), prompted the study of lentiviruses in other species. Up-to-date lentiviruses have been identified to infect humans and other primates, cats, cattle, sheep, goats and horses. Recently lentiviruses have been shown in rabbits and ferrets. Most animal lentiviral infections are notifiable diseases to the World Organization of Animal Health (OIE). All lentiviruses have similar biochemical, morphological and antigenic properties. They share common features regarding virion particle structure, genetics, infection kinetics, tropism, immune response elicited, transmission, etc. In general, lentiviruses infect immune cells (mainly macrophages and T-lymphocytes) and induce several types of immune dysfunctions (macrophage subset differentiation, T-cell anergy, etc.), inflammation, and organ/tissue impairments. As in other retroviral infections, infected cells carry the retroviral genome (provirus) throughout their life and pass the infection to their progeny. Unlike other viruses, lentiviruses may efficiently infect non-dividing cells.

Classification Genus Lentivirus is one of the six genera of the subfamily Orthoretrovirinae within the family Retroviridae. The genus, that traditionally contained seven members, has recently expanded to 10 after the addition in the 2018 release of the International Committee on Taxonomy of Viruses (ICTV) of Puma lentivirus (PLV), Jembrana disease virus (JDV), and HIV-2. All lentiviruses are genetically related and must have derived from a single ancestor, which has co-evolved with the hosts that originated from the initial one. Thus, lentiviruses may be classified according to the hosts they infect. Primate lentiviruses include human immune deficiency virus (HIV) type 1 (HIV-1) and type 2 (HIV-2), as well as simian immune deficiency virus (SIV), which has been identified in many different non-human primates with genomic differences between them. Small ruminant lentiviruses (SRLV) include visna-maedi virus (VMV) of sheep and

56

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00005-9

Animal Lentiviruses (Retroviridae)

57

Fig. 1 Phylogenetic tree of the genus Lentivirus after alignment. Names of the viruses are shown in Table 1. HIV-1 and HIV-2 are in blue to more easily locate their phylogenetic relationships. RELIK is an endogenous lentivirus found in the rabbit genome.

caprine arthritis encephalitis virus (CAEV) of goats. Cattle lentiviruses had a single representative, bovine immunodeficiency virus (BIV), but as mentioned above, now also the closely related JDV is considered. As of now, lentiviruses of carnivores have been shown to infect felids exclusively and include feline immunodeficiency virus (FIV) and PLV. Lastly, the lentivirus of horses is named equine infectious anemia virus (EIAV) (Fig. 1). Within the primate lentiviruses, for example, HIV-1 and HIV-2 differ in that the sequences only present no more than 50% homology between them, and that HIV-1 has vpx while HIV-2, vpu. This reflects that HIV-1 and HIV-2 originated from different ancestors, HIV-1 from chimpanzees and gorillas, and HIV-2 from sooty mangabey. As lentiviruses are genetically related, they have certain cross-reactivity between the Gag proteins, but cross-reactivity is non-existent between proteins codified by the gene env. For a long time lentiviruses had been considered to be only exogenous or contagious, but recently the germlines of leporids (rabbits and hares) and ferrets have been shown to carry endogenous lentiviruses, remnants of ancient infections which have lost the ability to produce infectious virions (see below).

Virion Structure As in the rest of the retroviruses, lentiviral particles are rounded. The size is slightly larger than in other retroviruses, 100–130 nm in diameter; SIV particles can reach even 150 nm in diameter. They are enveloped, with short projections distributed evenly on the envelope, composed of two proteins: surface proteins (SU), the outermost, and transmembrane (TM), which anchors SU to the envelope. Inside the virion electron micrographs reveal a core shaped like a truncated cone or rod, composed of capsid proteins (CA). The matrix proteins (MA) fill the space between the envelope and the core to give consistency to the viral particle. The capsid contains two identical helical nucleocapsids, each consisting of a molecule of RNA, surrounded by the nucleocapsid proteins (NC) and associated with the proteins that are involved in the early stages of viral replication: the protease (PR), reverse transcriptase (RT) and integrase (IN), as well as Nef, Vif and Vpr in the lentiviruses that have them (Fig. 2).

Genome Both molecules of single-stranded RNA are identical. They are 7700–11,000 nucleotides (nt) long (Table 1) and are linked by hydrogen bonds. They have a CAP sequence at the 50 end and a polyadenylated tail around 200 nt long in the 30 end. Regardless of these characteristics, they do not act as mRNA. As in other retroviruses, lentiviruses possess three main genes, gag (which encodes the inner proteins), pol (which encodes the proteins for replication, protease, reverse transcriptase and integrase) and env (which

58

Animal Lentiviruses (Retroviridae)

Fig. 2 Schematic representation of the HIV-1 genome and a particle of lentivirus.

Table 1 Species of lentiviruses recognized by the International Committee on Taxonomy of Viruses (ICTV) in the 2018 release (https://talk. ictvonline.org/ictv-reports/ictv_9th_report/reverse-transcribing-dna-and-rna-viruses-2011/w/rt_viruses/161/retroviridae). GenBank Accession Numbers of the prototype strains or that corresponds to the complete sequence of the virus, total length of the genome (expressed in nucleotides, nt) and accessory genes present in each species are shown Virus

Complete name

Abreviation

GenBank

nt

Accessory genes

Carnivores

Feline immunodeficiency virus Puma lentivirus

FIV PLV

EU117992 NC_038669

9891 9100

rev response element, vif, orfA vif

Cattle

Bovine immunodeficiency virus Jembrana disease virus

BIV JDV

M32690 NC_001654

8482 7732

tat, vif, rev, vpw, vpy, tmx tat, rev, vif, tmx, Jdvgp5

Small ruminants

Visna-Maedi Virus Caprine arthritis and encephalitis virus

VMV CAEV

NC_001452 NC_001463

9202 9189

vif, rev, vpr-like vif, rev, vpr-like

Primates

Human immunodeficiency virus type 1 Human immunodeficiency virus type 2 Simian immunodeficiency virus Equine infectious anemia virus

HIV-1 HIV-2 SIV EIAV

K02007 NC_001722 KU892415 M16575

9737 10,359 10,276 8407

tat, tat, tat, tat,

Horses

rev, rev, rev, rev,

vif, vpr, vpu, nef vif, vpr, vpx, nef vif, vpr, vpx, nef S2

encodes the external glycoproteins). Besides these genes, lentiviruses have genes the products of which regulate the synthesis and processing of the viral RNA (tat and rev), and other accessory genes (vif, vpr, vpu, nef, tmx, etc.) that play an important role in the viral replication, in counteracting the restriction factors of the host and in pathogenesis (Fig. 3). HIV and SIV are the lentiviruses with the highest number of accessory genes, followed by BIV (Table 1). Several of these accessory genes may have similar functions to other genes with different names in other lentiviral species, so the list may be misleadingly long. For example, BIV has tat, rev, vif, vpw, vpy, and tmx genes, the last three with functions similar to HIV-1 vpr, vpu and nef, but with limited sequence similarity to the human virus counterparts. The role in pathogenesis of several of these genes has not been ascertained unequivocally or they may have several functions carried out by single genes in other lentiviruses. This is the case of FIV orfA, which acts by increasing the net translation of viral gene products similar to HIV Tat, but it does not increase transcription or use a Tar element in the same way as Tat. This gene also shares similar functions to vpu, vpr and nef of HIV-1. Animal lentiviruses, except for HIV and primate lentiviruses, also encode a protein involved in regulating cellular dNTP ratios, the viral dUTPase, although BIV encodes a dUTPaserelated gene without enzymatic activity. At each end of the viral RNA there are the same B100 nucleotides, a sequence known as repeat region (R). In addition, at the 50 end there is a unique region about 70 nucleotide long (U5), and at the 30 one of about 450 nucleotides long (U3). These regions are essential to start reverse transcription, integration and post-integration transcription. During the process of reverse transcription these unique regions are duplicated, forming two identical structures that are known as long terminal repeats (LTR), with a structure U3-RU5 (Fig. 4). In the stage of provirus synthesis (when the lentiviral genome is double-stranded DNA) LTRs flank the genome at both ends. The 50 end of the R region in the 50 LTR is where transcription begins ( þ 1 transcription). The U3 region of the 50 LTR is extremely important because it effectively controls transcription. In this region there are two main areas: a promoter area and an

Animal Lentiviruses (Retroviridae)

59

Fig. 3 Proviral genomic structure of most common animal lentiviruses, showing genes gag, pol, and env and accessory genes. Several accessory genes are encoded in non-consecutive sequences and are expressed following splicing of the RNA.

enhancer area. The promoter area is closer to the beginning of transcription, and exhibits a TATA box 20–50 nt upstream of the cap site, þ 1 of transcription. The TATA box is present in the promoter region of many eukaryotic genes. Some lentiviruses also have a CCAAT box, another promoter sequence. The enhancer region is further upstream from the beginning of transcription and contains numerous sequences that are recognized by cell signaling factors. Upon activation of these enhancers, the promoter area triggers the beginning of the synthesis of RNA and DNA (Fig. 4). These sequences which bind cell signaling factors are collectively known as transcription factor binding sites or TBS, and include AP-1, AP-4, ATF, and many others. In some cases there is a TBS for NF-κB or for LBP-1. In different lentiviruses, a variety of other sites exist, these include a gamma-activated site or GAS (FIV, HIV and CAEV), an interferon stimulated response element (ISRE, in FIV or HIV), interferon response factor-1 (IRF-1, CAEV), and hormone response element (HRE, FIV and HIV). The significance of these TBS is that viruses are silent in non-activated cells, but upon activation, the signaling process culminates in the entrance of molecules to the nucleus that can stimulate lentiviral replication and production of viruses. The other two regions of the 50 LTR, R and U5, play a primary role during the reverse transcription process. The LTRs have different sizes depending on the lentiviral species, ranging from about 311 nt (PLV) to 855 nt (HIV-2). The region between the 50 LTR and the beginning of gag is defined as a non-coding region. It contains the primer binding site (PBS, 18 nt long complementary to the tRNA that functions as primer in the reverse transcription process), the packaging signal (c), and the leader region (L), which precedes the first coding sequence. This non-coding region serves as a donor for splicing processes that give rise to different subgenomic mRNAs. All retroviruses contain a sequence rich in purine, or polypurine tract (PPT) immediately before the U3 region of the 30 end. The PPT contains the site for initiating the positive strand of the viral DNA synthesis.

Encoded Proteins Proteins encoded by essential genes are similar in all lentiviruses. External or surface proteins are usually glycosylated (at least SU), while inner proteins usually are not. Both external and inner proteins are very immunogenic, but the antibodies formed are not

60

Animal Lentiviruses (Retroviridae)

Fig. 4 Schematic representation of viral RNA, proviral DNA and LTRs. The viral RNA contains identical R sequences at both ends, and a unique U5 in the 50 end, and U3 in the 30 end. This U5 and U3 become duplicated during the reverse transcription to give the LTRs, formed by identical U3-R-U5 at both ends. This viral DNA, known as provirus, integrates in the cell genome. There, it remains silent till cell signals cross the nuclear membrane and react with specific sequences in the U3 of the 50 LTR known as transcription factor binding sites or TBS (some of them are shown). This activates the promoter region of the LTR, leading to transcription from the 50 end of R or cap site.

able to eliminate the infection. The inner proteins are relatively conserved and can exhibit cross-reactions between different lentiviruses. They are named according to their weight (Table 2). Mature proteins are cleaved from polyprotein precursors. Accessory proteins and their functions are listed in Table 3.

Replication Cycle The replication of retroviruses took time to be elucidated in part because the concept that RNA could be transcribed into DNA went against the accepted dogma that genetic information flows from the DNA to RNA, and from there to proteins. Unlike other retroviruses, which replicate in dividing cells, lentiviruses are preferentially expressed in differentiated cells. Some receptors and coreceptors have been identified, but many other receptors have not been identified yet. For example, despite the similar pathogenesis of HIV and FIV, unlike HIV the latter does not employ CD4 as a receptor, but CD134, a member of the family of receptors for TNF-a and nerve growth factors. In the case of SRLV it is likely that mannosylated residues in the Env protein are sensed by the mannose receptor (CD206), a C-type lectin endocytic receptor expressed on macrophages and dendritic cells. While the receptors for BIV or JDV have not yet been identified, there is evidence that BIV may use the C-C chemokine receptor 5 (CCR5), the main coreceptor used by the majority of strains of HIV-1 for infection of cells. After the recognition between SU protein and its receptor, there are conformational changes that motivate the exposure of a hidden domain that in the case of HIV allows interaction with the co-receptors, CCR5 or CXCR4. This causes further changes that expose the fusion domain of TM, with the resulting fusion of the virus envelope with the cell membrane or of the endosome, allowing the entry of the viral particle (Fig. 5). Another entry mechanism can be in cells with receptors for the Fc domain of immunoglobulins (FcR) that trap antibody-coated particles (e.g., VMV). The penetration process concludes with the release of the core into the cytoplasm. The host cell has mechanisms to prevent this process, including TRIM5a, which the virus has to circumvent. Once in the cytoplasm, the RT, likely in the context of a partially disrupted capsid, begins to transcribe the retroviral genome. First, thanks to the RNA dependent DNA polymerase activity (RdDp), it transcribes a single-stranded (minus sense) DNA molecule. As the RNA-DNA hybrid moves through the RT its RNase H activity degrades almost simultaneously the original viral RNA which served as template and through the DNA dependent DNA polymerase (DdDp) activity, an additional molecule of RT transcribes the complementary strand of nascent DNA, synthesizing a molecule of double-stranded DNA. In a complex process that involves duplication of regions from the 50 and 30 ends of the viral genome, LTRs that flank the provirus (DNA retroviral genome) are generated. Vif and Nef are important at this early stage, a reason for being present in the viral particle. Nef increases the effectiveness of RT, so that larger amounts of reverse transcribed DNA are produced, and Vif blocks the cell cytidine deaminase APOBEC3 that would increase the rate of error of the RT.

Animal Lentiviruses (Retroviridae)

61

Table 2 Gene products of the lentiviruses, some of their functions and presence in some lentiviral species. In lentiviruses, the number that accompanies the name of the proteins represents its approximate weight in kDa. -, not present. Blank cells, no specific name. Names of the viruses are shown in Table 1 Gene

Protein

Function

HIV-1

FIV

BIV

VMV

EIAV

gag-pol gag

Gag-Pol Gag MA

Precursor of the viral structural proteins and enzymes Precursor of the structural proteins of the virus Matrix. Inner membrane layer. Confers rigidity and stability to the virion. Myristilated Capsid. Encapsulates the nucleocapsids. Intervenes in packaging. Synthesized in large quantities and its detection is used for diagnosis in some lentiviral infections Nucleocapsid. Surrounds and protects RNA Protein for viral encapsidation and budding of assembled particle

Pr160 Pr55 p17

Pr50 p15

Pr170 Pr53 p16

Pr175 Pr55 p16

p15

p24

p24

p26

p25

p26

p7 p6

p10 –

p7 –

p14 –

p11 p9

p12

p14

p10

p66/p51

p65

p68

– p32

p14 p31

gp160 gp120

gp41

CA

NC P6 pol

PR

Protease: Proteolytic cleavage of Gag and Gag-Pol to produce structural proteins and mature virion Reverse transcriptase. Triple function: RNA dependent DNA polymerase (RdDp), DNA dependent DNA polymerase (DdDp) and RNAse H to give the end result of double-stranded DNA from single-stranded RNA

RT

dUTPase IN env

PrEnv SU

TM

Integrase. Integration of proviral DNA in the host genome Precursor of the viral envelope proteins Surface protein on the viral envelope located in the outside of the virion. Interaction with receptors. Main domain for receptor binding. Attachment of virus to target cell. Determinant of tropism. Immunogenic, stimulates the synthesis of specific antibodies. It interacts with the extracellular domain of the TM. Always glycosylated Transmembrane protein. With a cytoplasmic domain and a hydrophobic domain within the cell membrane. The interaction of TM with the lipid bilayer allows the anchoring of the SU-TM complex to the viral envelope. Mediates fusion between viral envelope and cell membrane. Induces syncytia. Immunosuppressant. Frequently glycosylated

–

p14 p35

gp95

gpp145 gp100

pr185 gp135

gp90

gp40

gp45

gp46

gp4

yes

Table 3 Regulatory and accessory genes, proteins encoded, their function and viruses in which they are present. CD4, molecule on surface of T-helper cells. MHC-I, major histocompatibility complex type I. Full names of the viruses are shown in Table 1 Gene

Protein Function

Virus

tat

Tat

BIV, CAEV, EIAV, HIV-1, HIV-2, JDV, SIV, VMV

rev

Rev

nef

Nef

vif

Vif

vpr vpu vpx vpw vpy tmx orfA

Vpr Vpu

Jdvgp5 S2

Tmx

Potent transactivator that may greatly stimulate viral expression. Phosphoprotein located in the nucleus and nucleolus of the infected cells. Elongation of mRNA and transcription Regulates transport, processing and translation of viral mRNAs. Phosphoprotein located in the nucleus and nucleolus of infected cells. Endocytosis of CD4 and MHC-I at the cell membrane. Increase in the efficacy of RT, generating higher amounts or retrotranscribed DNA. Increase in the viral infectivity. Cell activation. Induction of apoptosis. Downregulation of E2 ubiquitin conjugating enzyme Enhancement of viral infectivity. Stabilization of the pre-integration complex. Blockage of the cell restriction APOBEC3G (cytidine deaminase) which increases the error rate of RT Arrest of the cell cycle at G2. Transport of the pre-integration complex to the cell nucleus Enhancement of virion release. Degradation of CD4 and MHC-I

BIV, CAEV, EIAV, HIV-1, HIV-2, JDV, VMV HIV-1, HIV-2, SIV

BIV, CAEV, FIV, HIV-1, HIV-2, JDV, SIV, VMV

HIV-1, HIV-2, SIV HIV-1 HIV-2, SIV Similar function to Vpr BIV Similar function to Vpu BIV Similar function to Nef BIV, JDV Similar functions to Tat, Vpu, Vpr and Nef. Arrest of the cell cycle at G2. Downregulation FIV of E2 ubiquitin conjugating enzymes Unknown BIV Similar function to Nef EIAV

62

Animal Lentiviruses (Retroviridae)

Fig. 5 Schematic representation of the replication cycle of lentiviruses. Reverse transcriptase (RT) is shown in the shape of a star.

Proviral DNA, in the form of a pre-integration complex (PIC), crosses the nuclear membrane and, using the viral enzyme integrase present in the PIC, it is integrated into a host cell's chromosome. Vpr (also present in the virus particle) plays an important role in this process, directing the nuclear localization of the pre-integration complex and interfering with the cell cycle, stopping it in the G2 phase to facilitate integrase-mediated integration of the proviral DNA into the cell genome. Once integrated, proviral DNA behaves as a Mendelian gene: when the cell divides, the progeny cells will contain the provirus. For this reason, lentiviral infections are lifelong, especially taking into account the cells that they infect, most of them indispensable for the immune response. Although integration is random, HIV-1 prefers to integrate within genes, while other retroviruses prefer exons or non-coding sequences. Integration appears to be a prerequisite for the infectivity, and although there is extra-chromosomal proviral DNA, it is not expressed. The integrated provirus can remain without being expressed for months or years, until the appropriate signaling molecules cross the nuclear membrane. They recognize specific short sequences of the U3 region of the 50 LTR, the TBS mentioned above, triggering the transcription of the lentiviral genome by the cellular RNA polymerase II, as if it were a common cellular gene. Therefore, lentiviruses are not expressed in resting cells and have to wait till these are activated, remaining latent till this occurs. This allows them to wait for adequate conditions to continue their replication cycle. Integrated DNA serves as a template for the cellular RNA polymerase II to transcribe both spliced subgenomic mRNA, which will be translated to the characteristic polyproteins, and full-lenth viral RNA, which will be packaged into the viral particle. Accessory genes are expressed first, especially tat, rev, and nef. When the products of these genes reach appropriate levels, Rev facilitates the export of unspliced and partially spliced transcripts to the cytoplasm and translated in the form of polyproteins. Of the proteins encoded by env, at least SU (and usually also TM) undergoes N-linked glycosylation. Both SU and TM are inserted into discrete microdomains in the plasma membrane related to lipid rafts. The internal proteins of the virus undergo limited modification (fatty acylation at N-terminus of MA) and accumulate at the plasma membrane and associate with the proteins SU and TM, in order to initiate the process of budding and assembly. Once concluded, the immature particles are released by budding. The PR cleaves the polyproteins inside the virions, giving rise to the mature virus particles. During these final stages, proteins such as cell MHC-I or CD4 are redirected to the degradation route, so they are not expressed on the cell surface, where they could interfere with virus release.

Reverse Transcription The process of retrotranscription copies the information of the viral genome, contained in the molecule of single-stranded RNA, into a molecule of double-stranded DNA. During this process, the RT performs three enzymatic functions: DNA-dependent RNA-polymerase (RdDp), Ribonuclease H (RNase H), and DNA-dependent DNA-polymerase (DdDp). The RT activity lacks exonuclease activity (repairing) and, in addition, it has a high frequency of error when incorporating nucleotides, which makes that the final error rate of the RT ranges between 3  10–3 and 3  10–5, depending on the retrovirus. The error rate of RT is higher with RNA templates than with DNA templates and varies according to the context of the reading. A characteristic of the activity of the RT is that throughout the process it switches from one strand of nucleic acid to the other, and may make mistakes at this time, allowing recombination. Reverse transcription begins with the hybridization of the tRNA, which acts as the primer, with the PBS close to the 50 end of the viral genome (Fig. 6). From here, it extends the strand in the direction 50 until it reaches the end of R. RNA that forms part of the newly formed RNA-DNA hybrid is digested by the RNase H activity of the RT. The 30 end of the newly synthesized DNA and, now as a single strand, hybridizes with the R region of the 30 end of the viral RNA. This process is called the first strand transfer. RdDp

Animal Lentiviruses (Retroviridae)

63

Fig. 6 Process of reverse transcription. The segments delimited by an orange outline represent RNA.

activity continues to extend the DNA molecule all the way towards the 50 end. RNase H activity degrades viral RNA 50 - 30 , but is unable to degrade the polypurine tract (PPT) adjacent to the U3 region of the template RNA. This PPT functions as the primer to start DNA synthesis using DNA as template, in a 30 direction This synthesis is extended by the primer, by copying a new PBS. RNase H activity removes the last vestiges of RNA, both in the area of PPT as in the primer. This creates two complementary PBS areas in each chain of DNA, allowing the second strand transfer to occur. As a new site is formed by where the DNA can be transcribed, RT, through its DdDp activity, synthesizes a strand of DNA complementary to the already synthesized. DNA synthesis continues until RT completes the complementary DNA, concluding the synthesis of the 50 LTR.

Biological Properties Variability Lentiviruses can achieve diversity both within individual hosts and within populations through spontaneous mutation rates ranging from 103–106 substitutions per site per year due to low polymerase fidelity and lack of proofreading capacity of RT. In addition to this remarkable mutation rate, lentiviruses can also create diversity by recombination, because RT needs to switch template from one strand of nucleic acid molecule to the other during the process of transcription (see above). These switches can match the nascent strand with non-corresponding sequences, causing recombination. This occurs in all lentiviruses, but the frequency of crossovers and factors promoting recombination can be variable. From the perspective of the lentivirus, variability has several advantages. Both mutations and recombination increase viral genetic diversity in fluctuating environments; the most

64

Animal Lentiviruses (Retroviridae)

obvious implication of this is the development of escape mutants that allow the viral population to avoid the selective pressure applied by the host immune response or antivirals. Recombination is a mechanism to prevent the accumulation of deleterious mutations in the population, which would decrease its fitness. The extraordinary variability of some of the lentiviruses poses a major obstacle to vaccine development and drug usage. This, together with the persistent presence of lentiviruses in the infected cell, makes it very difficult to fight against these viruses.

Natural Hosts As mentioned above, lentiviruses derive from a common ancestor that infected its host millions of years ago, and co-evolved with it as the progeny of this host diverged genetically. For this reason, related viruses may infect closely related species. Thus, it is possible that sheep are infected by CAEV and VMV by goats. It has also been shown that HIV-1 and HIV-2 come from SIV. SIV underwent several inter-species jumps; in the case of HIV-1, from chimpanzees and gorillas to humans, and in the case of HIV-2, from the sooty mangabey to humans. However, the mechanisms by which viruses from other animal species are able to infect a new one after these inter-species jumps remain unknown. In the case of FIV, although it usually is not passed between cat species due to the elusive and territorial nature of these animals, variants of the virus in different wild species have been isolated, including pumas, Asian leopard cats, ocelots, bobcats, jaguarundis, Pallas cats, cheetahs, leopards, lions, snow leopards and spotted hyenas. VMV can also infect wild ruminant species, such as mouflons. SIV infects a very high number of simian species, and it has been determined that up to 40 of the 69 species of African non-human primates act as natural hosts of SIV, including African green monkeys and mandrills.

Transmission As other retroviruses, the lentiviral virions are sensitive to many physical and chemical agents including desiccation, temperature, acidic or basic pH values, etc. For these reasons they are usually transmitted in “short distances” by direct methods. Lentiviruses are usually exogenous viruses. This means that they are transmitted horizontally or in a contagious way, through cell-to-cell contact, as well as to the progeny of the infected cell. However, in the rare occasions in which a germ cell, a spermatozoid or an ovum, is infected, the provirus becomes integrated into the genome and is transmitted vertically from generation to generation of the animal, according to the patterns of Mendelian inheritance. These retroviruses that remain in the genome over millions of years are called endogenous retroviruses. Till recently, it was thought that lentiviruses had no capacity for germline integration. However, endogenous lentivirus-like viruses have been described recently in rabbits and lemurs, called RELIK and PSIV, respectively. This supports the concept that lentiviruses are more widespread than previously thought, can be incorporated into the host germline, and are ancient, existing prior to 12 million years ago. The phylogenetic relationships of RELIK (which is also found in hares) are shown in Fig. 1. For some lentiviruses, the most common horizontal transmission routes are the sexual route, the direct inoculation in tissues or bloodstream, through syringes with contaminated blood and bites (FIV and SIV), or during childbirth when the virus enters the body of the neonate through mucosal surfaces (HIV). In some cases lentiviruses can be transmitted by colostrum or milk, although not in SIV, possibly due to the low levels of CCR5 in the newborn. Some lentiviruses (i.e., BIV, HIV) can cross the placenta and infect in utero. EIAV is exceptional amongst not only lentiviruses but also retroviruses in that the transmission is performed by arthropod vectors (horseflies and deerflies). In JDV arthropods are also partly responsible for transmission.

Tropism In many cases, target cells are those involved in the immune response. Some of the lentiviral species are lympho-tropic (infecting lymphocytes), others are macrophage-tropic (infecting cells of the monocyte/macrophage lineage, including in some cases, microglia, such as VMV, CAEV or EIAV), while others have a mixed tropism (infecting both lymphocytes and macrophages, such as SIV, HIV and FIV). Within the lympho-tropic species and strains, some infect T-helper cells (CD4 þ ), cytotoxic T-cells (CD8 þ ), Tgδ cells (common in cattle), null cells or B lymphocytes (e.g., BIV and JDV). Infected T-lymphocytes present an abnormal pattern of cytokine secretion, with increased expression of IFN and certain interleukins, such as IL-12 and IL-18. Some lentiviruses can also infect dendritic cells, which are key in immune responses. CAEV and VMV infect monocytes, but are not expressed until the cells mature to macrophages in the target organs.

Pathogenic Processes Lentiviral infections usually follow a similar course, with an initial acute transitory stage, a latency stage in which the immune system is able to control the infection, and a final stage consisting of a new phase of fast replication, which determines the typical syndrome. However, many lentiviral infections pass unnoticed in animals because the virus does not produce any clinical sign. Asymptomatic infected animals can transmit the infection (carriers); the infection and the disease become apparent when the virus infects a different population. Typically, the acute stage, which develops 4–6 weeks after infection, is characterized by fever, lymphadenopathy, leukopenia (due frequently to neutropenia) and weight loss. This acute stage can last for a few weeks up to several months. This is followed by an asymptomatic stage, in which the animal appears to be healthy or with minimal clinical signs, which may last several years, during which the virus replicates at low rate, and the immune response is able to control it, but

Animal Lentiviruses (Retroviridae)

65

producing a progressive impoverishment of the affected cell population. Finally, the immune response is unable to control the virus and the disease becomes apparent, with a significant increase of the viral load, an important deterioration of the immune system, immune deficiency with presence of respiratory, digestive, and possibly neurological clinical signs, as well as weight loss and hyperthermia, which lead to the death of the animal.

Immune Deficiency Immune deficiency is one of the most common pathologies produced by lentiviruses. Several species include in their name this term, such as HIV-1, HIV-2, SIV, FIV and BIV, although in the latter the direct role of the virus in the clinical disease in infected cattle under natural conditions has not been shown clearly, and its true economic impact is hypothetical. Immune deficiency develops because many lentiviruses infect lymphocytes, especially T-cells. In the course of infection, CD4 þ T-cells (T-helper cells) decrease, either by the direct action of the virus or due to the antiviral defense mechanisms. This decrease in CD4 þ T-cells is exploited by opportunistic infections that become established and impair the clinical status of the animal, being responsible for its death in the majority of cases. Immune deficiency is often accompanied by hematological abnormalities, mainly anemia, characteristic of FIV and EIAV infections. It is produced because viral surface glycoproteins coat red blood cells or erythrocytes, triggering complement-mediated destruction of these cells through a mechanism of type II hypersensitivity. Another consequence of immune deficiency is the development of tumors, which can be produced by an uncontrolled growth of cells (lymphoma). Some of the alterations that appear as a consequence of immune deficiency are located on the skin and oral cavity (gingivitis), respiratory and gastro-intestinal tracts. Frequently the liver, kidneys and central nervous system are impaired.

Inflammation VMV and CAEV produce mainly inflammation; processes associated with BIV and JDV also seem to be more related to inflammation than with immune deficiency. This could be due to the earlier sacrifice of cattle than of the other animal species mentioned in the paragraph of immune deficiency. The inflammation directly attributed to infection by CAEV or VMV is located in the mammary gland, joints, lungs or central nervous system (meningoencephalitis), prevailing one over the other depending on the infecting virus. As in the other lentiviruses, CAEV and VMV can remain latent for many months with little or no expression. The myriad of mechanisms that regulate viral replication and lead to increased viral loads and more serious outcomes of disease remain unclear. However, it is recognized that specific cell transcription factors activate viral gene expression when interacting with specific proviral DNA sequences included in the LTR, the transcription factor binding sites (TBS) mentioned above. SRLV constant replication in tissue and the subsequent immune response produce chronic inflammation causing pathological changes observed in the target organs of animals infected by the virus. As a result, the multisystem inflammatory disease that characterizes SRLV infection has an immunopathogenic origin. The main change in the affected tissues is infiltration by mononuclear cells (lymphocytes, macrophages and plasma cells). Cells accumulate and organize in lymphoid follicles. The final phase of the pathogenesis begins when the clinical disease appears. Depending on the clinical form, the animal can die rapidly or remain chronically infected. The inflammatory processes triggered by lentiviruses are associated with the production of cytokines, which can regulate viral transcription through the cellular signaling pathways mediated by the TBS in the proviral LTR as explained above. For example, cytokines IFN-g and TNF-a, present in arthritic joints of animals infected by CAEV, have shown to activate viral transcription via recognition of the corresponding gamma activation site (GAS) and TNF activation site (TAS) present in the U3 of CAEV.

Cell Defenses and Viral Mechanisms to Evade Them When attacked by viruses, cells display innate response mechanisms to avoid their productive infection. Some of the cellular mechanisms to fight against lentiviruses are TRIM5a, APOBEC3 or tetherin. TRIM5a induces a premature uncoating of the viral core, by recognizing the incoming viral capsid, inducing its proteasome-dependent degradation. This impairs integration and significantly reduces proviral load and viral production. APOBEC3 is a cytidine-deaminase that causes uracil accumulation in negative-strand nascent DNA, leading to detrimental G-to-A mutations in the proviral DNA. This molecule may be incorporated into progeny virions in producer cells (passenger APOBEC3) and inhibit lentiviral replication in the following replication cycle in target cells. Lastly, tetherin is able to trap virions on the surface of infected cells, preventing virus release, and therefore, virus transmission between cells. These mechanisms are very specific against particular lentiviruses, and this innate immune response is responsible for preserving the species barrier. In response, lentiviruses have developed mechanisms to counteract these restriction factors. For example, the high genetic heterogeneity found within the capsid region is likely a way to escape TRIM5a. In HIV, FIV, BIV, SRLV and EIAV infections, the viral protein Vif has the main function of avoiding APOBEC3 incorporation into viral particles, restricting the probability of incorporating wrong nucleotides. Vif also triggers the degradation of most APOBEC3 proteins. Vpu is particularly dedicated to bind tetherin and degrade it via proteasome. This so-called “evolutionary arms race” has led to the selection of somatic genes encoding virus-interacting proteins. So far, these innate mechanisms have been partially unveiled in the simian or human counterparts, and equine, ovine, bovine, and feline lentiviruses are being actively studied to decipher the important restriction mechanisms in these species.

66

Animal Lentiviruses (Retroviridae)

Animal Lentiviral Infections Feline Immunodeficiency and Puma Lentiviral Infections Feline immunodeficiency virus was initially isolated from cats (Felis catus) in Petaluma (CA, USA) in 1986. As mentioned above, besides cats, other felids, such as cheetah, lions, leopards or hyenas may be infected by the virus. The most recent release of the ICTV (2018) recognizes two different viral species, highly related to FIV, identified in mountain lions (Puma concolor) and bobcats (Lynx rufus), which are known as puma lentivirus A (PLVA) and B (PLVB). However, the list of FIV-related lentiviruses in wild felids is increasing quickly. FIV is considered a good animal model for the study of HIV, as its biochemical and morphological properties are very similar, though antigenically they are distinct. Also, though both HIV and FIV infect CD4 þ T-cells, the latter enters cells through CD134, a member of the TNF-a and growth factor receptors. CD134 is highly expressed in active lymphocytes, monocytes/macrophages and dendritic cells. FIV is mostly transmitted through direct inoculation of the virus through bites, the reason why it is most prevalent in adult male cats. After an initial period of 4–6 weeks with no signs, cats develop an acute phase with hyperthermia, lymphadenopathy, leukopenia (mostly neutropenia), and weight loss. This is followed by a long asymptomatic phase which may last many years. In fact, 12% of the healthy cat populations are considered to be unapparent carriers. Infected cats may die in old age of etiologies unrelated to FIV-infection. However, some develop clinical immune deficiency with increased viral loads due to the immune impairment, accompanied with respiratory and intestinal clinical signs, gingiva-stomatitis, hyperthermia, etc., which constitute the so called feline acquired immune deficiency syndrome or FAIDS. FIV, rather than opportunistic bacteria, may be directly responsible for the intestinal dysfunction, as it may cause blunting of the villi and dilation of the crypts. In contrast to FIV, lentiviruses of wild felids have not historically been associated with significant pathogenicity. However, several captive African lions have manifested clinical signs or hematologic abnormalities associated with lentiviral infection. A commercial vaccine is available to prevent FIV infection in cats, but its use remains controversial, including whether or not the vaccine effectively induces cross-protective immunity against the various naturally circulating virus strains.

Bovine Immunodeficiency (BI) and Jembrana Disease (JD) BIV was first isolated in 1969 in the USA from a progressively deteriorating 8-year old pregnant Holstein dairy cow (Bos taurus), identified as R-29, with wasting syndrome, lymphadenopathy, leukocytosis, lymphocytosis, fatigue, diminished milk yield and neurologic disorders. Serology has shown that BIV is present worldwide; however, the direct role of BIV in clinical disease among cattle infected under natural conditions is not clearly demonstrated and the true economic impact of BIV-infection remains conjectural. However, an acute disease in Bali cattle (Bos javanicus), Jembrana disease, endemic to parts of Indonesia, was described in the 1990s and is thought to have arisen from BIV via an unknown transmission event. The clinical signs observed in BIVinfection are more related to the inflammatory events characteristic of VM or CAE (see below) than immune deficiency itself. BIV targets monocytes and macrophages, inducing some immune dysfunction such as decreased superoxide anion production, phagocytic activity and chemotaxis. On the other hand, JDV has a high morbidity and mortality of up to 20%. JD is characterized by an acute stage 5–12 days post infection, with fever, lymphadenomegaly, lethargy, anorexia, panleukopenia, splenomegaly, hemorrhages and very high viral loads. There is no recurrence of disease in recovered animals but viral loads remain high during at least 2 years. JDV is often described as an atypical lentivirus, because of the low level of viral variation in vivo, the delayed antibody response, and the ensuing immunological control which prevents subsequent heterologous infection. There is an inactivated tissue-derived vaccine against JDV to control the disease in Indonesia.

Visna-maedi (VM) and Caprine Arthritis and Encephalitis (CAE) VM was first described in Iceland in the 1930s. VMV was isolated from cases of interstitial pneumonitis (maedi ¼ shortness of breath in Icelandic) and demyelinating leukoencephalomyelitis (visna ¼ wasting in Icelandic). CAEV was first isolated in 1980. They are collectively called Small Ruminant Lentiviruses (SRLV). Transmission is related to body fluids, mainly respiratory exudates and milk or colostrum. Both VM and CAE are prevalent worldwide, although the prevalence is much higher in developed countries, which seems to be related to the custom of feeding lambs or kids with milk pooled from all the mothers, which favors transmission, and to the intensive rearing system. Both viruses can infect sheep and goats and cross-infections have been observed both experimentally and naturally. Nevertheless, the infection by VMV is more frequent in sheep and the infection by CAEV in goats. SRLV have in vivo tropism for monocytes, macrophages and dendritic cells, but may also infect cells of the central nervous system and others. As with other lentiviral infections, there is an initial short phase of viremia. Infected monocytes and macrophages spread the infection. The replication in monocytes and macrophages does not take place until these cells mature in the target organs. The infection remains latent for a period of time which depends on the viral strain and mainly on the individual susceptibility, and the animal can die in a short time or remain chronically infected. The continual replication of SRLV in tissues and the ensuing immune response causes a chronic inflammation that originates the pathological changes observed in target organs of SRLV-infected animals. Consequently, as in other lentiviral infections, the multisystem inflammatory disease that characterizes SRLV infections is immune-pathogenic in nature. The final stage of the pathogenesis begins when the clinical disease starts. Clinical forms of both VM and CAE are respiratory, nervous, mammary and joint disease. Although normally the process is

Animal Lentiviruses (Retroviridae)

67

subclinical, a small percentage of the animals can display one or more of these forms or even all four of them. Sheep usually develop respiratory and mammary clinical signs, while goats display nervous and joint forms.

Equine Infectious Anemia (EIA) EIA is considered a worldwide disease that occurs only in members of the family Equidae. It was first identified as an infectious disease of horses by veterinarians in France in 1843, and shown to be produced by a filterable agent in 1904. The disease is different from other retroviral diseases in that it is transmitted mostly by insect vectors (especially horseflies and deerflies), but also by hypodermic needles and other veterinary instruments. Sexual transmission of EIAV has not been demonstrated to date. The cell target during persistent EIAV infections appears to be exclusively cells of the monocyte/macrophage lineage, especially macrophages resident in tissues, such as liver, kidney, spleen. Viremia observed during episodes of chronic EIA result primarily from the production and release of EIAV from infected tissue macrophages, rather than an extensive infection of blood monocytes. Field isolates and laboratory strains of EIAV differ markedly in their pathogenicity; processes can be asymptomatic to lethal. Characteristically, the course of the infection is initially acute (first month), followed by chronic disease (first year) to progress into an asymptomatic lifelong stage thereafter. Horses suffer acute and chronic disease, while donkeys and mules typically experience asymptomatic infections. Acute disease is often associated with the first exposure to the virus, with fever, thrombocytopenia and hemorrhages evident from 7 to 30 days after exposure, though anemia or edema is not seen at this point. After this first stage, horses become clinically healthy but recurring episodes of the disease appear at irregular intervals, ranging from weeks to several months. Stress or immunosuppression trigger these viremia and reactivation episodes. Typically, horses will develop weight loss, anemia, diarrhea, and edema. Most of these signs are thought to be the combined effect of immune-mediated lysis of virusinfected cells and an immune complex-mediated inflammatory response. During the first year after infection, clinical episodes in horses with chronic EIA are more severe and frequent, and they usually disappear thereafter, and the horse becomes a lifelong inapparent carrier. Currently there is no effective vaccine for the prevention of EIAV infection and disease, mostly due to the large antigenic diversity of the virus.

Further Reading Bhatia, S., Patil, S.S., Sood, R., 2013. Bovine immunodeficiency virus: A lentiviral infection. Indian Journal of Virology 24, 332–341. doi:10.1007/s13337-013-0165-9. Burrell, C.J., Howard, C.R., Murphy, F.A., 2017. Retroviruses. In: Fenner and White’s Medical Virology. Elsevier, pp. 317–344. doi:10.1016/B978-0-12-375156-0.00023-0. Hosie, M.J., Addie, D., Belák, S., et al., 2009. Feline immunodeficiency. ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 575–584. doi:10.1016/j.jfms.2009.05.006. ICTV, 2018. International Committee on Taxonomy of Viruses, Available at: https://talk.ictvonline.org/taxonomy/. Murphy, B., 2017. Retroviridae. In: Fenner’s Veterinary Virology. Elsevier, 269–297. doi:10.1016/B978-0-12-800946-8.00014-3. Payne, S., 2017. Family retroviridae. Viruses. 287–301. doi:10.1016/B978-0-12-803109-4.00036-2. Ryu, W.-S., 2016. Molecular Virology of Human Pathogenic Viruses, first ed. Elsevier Science. Available at: https://www.elsevier.com/books/molecular-virology-of-humanpathogenic-viruses/ryu/978-0-12-800838-6 (accessed 8.03.19). Stover, M.G., Watson, R.R., 2015. Animal lentiviruses. In: Health of HIV Infected People. Elsevier, pp. 349–365. doi:10.1016/B978-0-12-800767-9.00020-0.

Animal Morbilliviruses (Paramyxoviridae) Carina Conceicao and Dalan Bailey, The Pirbright Institute, Pirbright, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Classification The Morbillivirus genus belongs to the Paramyxoviridae family in the order Mononegavirales. Within the genus there are now seven recognized species (summarised in Table 1). For the purpose of clarity and concordance with the published literature, we will refer to individual viruses using their commonly used abbreviations (Table 1). The morbilliviruses are genetically closely related, with the most distant, formally classified, virus being feline morbillivirus (FmoPV) (Table 1 and Fig. 1). At the genomic level nucleotide identities vary between 53% (FmoPV and measles virus, MeV) and 76% (canine distemper virus, CDV and phocine distemper virus, PDV). Current theories on the evolution of morbilliviruses are summarised excellently in Nambulli et al. (2016) (see “Further Reading”). Of particular interest, rinderpest virus, RPV (or its ancestor) is thought to have emerged following the domestication of cattle. This ancestral virus is also thought to be the source of MeV, following zoonotic transmission to humans. It is well known that MeV spread to the North and South Americas in the 1500s causing devastating epidemics in indigenous populations; however, it is less widely appreciated that CDV is thought to have originated in the Americas, and then spread to Europe following colonisation. PDV may in turn have originated from spillover of CDV into aquatic mammals, perhaps as recently as the twentieth century. The recent identification of a wealth of morbillivirus sequences in bats and rodents now indicates that the established morbilliviruses may have actually emerged from related small mammal viruses. The close molecular relationship of morbilliviruses is also evident in their related antigenicity, with various viruses being able to provide cross-protective immunity.

Virion Structure Pleomorphism is a feature of morbilliviruses, which translates to the production of particles ranging from 150 to 500 nm in diameter. These viruses are surrounded by a lipid bilayer derived from the host cell decorated with the viral glycoproteins haemagglutinin (H) and fusion protein (F), which can be seen as extruding spikes on the surface of morbillivirus virions (Fig. 2(A)). Inside the viral envelope, the matrix protein M forms a highly ordered lattice that interacts with the lipid bilayer, supporting the virion and packaging the viral genome within. The morbillivirus genome consists of a non-segmented, single-stranded, negative sense RNA molecule encapsidated by the nucleoprotein N in a helical nucleocapsid (Fig. 2(A)). The RNA-dependent RNA polymerase (RdRp) termed the large (L) protein and its co-factor, the phosphoprotein (P), associate with the N-RNA complex, forming the ribonucleoprotein complex (RNP). These RNP complexes assemble into a helical Table 1

Host range, epidemiology and phylogeny of morbillivirus species identified to date

Former species Species (2018-onwards) name (pre 2018)

Common Typical host abbreviation (For isolates)

Canine morbillivirus

Canine distemper CDV virus

Cetacean morbillivirus

CMV

Feline morbillivirus Measles morbillivirus Phocine morbillivirus Rinderpest morbillivirus

FmoPV/ FeMV MeV/MV

Small ruminant morbillivirus

Measles virus

Sub-species classification

Domestic 10 lineages dogs/ (see Section Epidemiology Carnivores and Host Range) Previously defined as two Cetaceans species: Porpoise and (whales, dolphin morbilliviruses dolphins) Domestic cats Not defined to date Humans

Phocine PDV distemper virus Rinderpest virus RPV

Pinnipeds (seals) Cattle

Peste-des-petits- PPRV ruminants virus

Sheep and goats

Geographic location

Genome length; % identity to MeV

Example sequence accession number

Global

15,690 nt; 61%

NC_001921.1

Uncharacterised (likely global)

15,702 nt; 64%

NC_005283.1

16,050 nt; 53%

JQ411014.1

15,894 nt

NC_001498.1

15,696 nt; 60%

NC_028249.1

15,882 nt; 69%

NC_006296.2

15,948 nt; 65%

NC_006383.2

Uncharacterised (likely global) 19 genotypes identified to date, Global only 6 currently circulatinga Not defined to dateb Uncharacterised (likely global) Extinct Lineages 1–3 (see Section Epidemiology and Host Range) Northern and Lineages 1–4 (see sub-Saharan Africa, Section Epidemiology Middle East, Asia and Host Range)

a

Circulating genotypes are B3, D4, D8, D9, G3 and H1 (CDC, https://www.cdc.gov/measles/lab-tools/genetic-analysis.html). The 1998 and 2002 outbreaks may form two distinct lineages.

b

68

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20938-2

Animal Morbilliviruses (Paramyxoviridae)

69

Fig. 1 Genetic diversity and ecology of the morbilliviruses. Rooted phylogenetic tree of the morbilliviruses, constructed using a neighbour-joining algorithm from full-length H sequences with at least 4 representative sequences of each species. Each branch corresponds to the diversity found within the sequences analysed with the genetic distance between branches indicated (0.2). The host silhouettes indicate the naturally infected hosts where disease has been recorded.

Fig. 2 Virion structure and genome composition of the morbilliviruses. (A) Schematic representation of morbillivirus virions illustrating the virally-encoded glycoproteins embedded in the host-derived envelope. Inside the M protein supports the viral envelope and interacts with the ribonucleoprotein complex (RNP), composed of the RNA genome encapsidated by the N protein, the viral polymerase L and its co-factor, the P protein. (B) Morbillivirus genomes strictly adhere to the “rule of six”, with one monomer of N protein encapsidating 6 nucleotides (nt) of RNA. (C) The morbillivirus genome comprises six genes that encode for six structural proteins (3′ - N, P, M, F, H and L - 5′) as well as two nonstructural proteins V and C, expressed from the P gene, by gene editing and through use of an alternative open reading frame (ORF), respectively. The ORFs and intergenic regions are drawn to scale. (D) The RNA genome of morbilliviruses is composed of seven cis-acting elements that define the gene start (GS), IG and gene end (GE) for each of the six genes. At the 3′ and 5′ ends are conserved and complementary sequences that constitute the genome promoter (GP) for the production of viral mRNA and the antigenome promoter (AGP) for the production of full-length RNA genome, respectively.

configuration forming a characteristic herringbone-like structure, visible by electron microscopy. The RNP is the minimum required unit to initiate transcription and replication of viral RNA in vitro. Infectious morbillivirus virions possess at least one RNP; however, it has been shown for MeV that more than one RNP can be incorporated without affecting viral fitness.

Genome Morbillivirus show variable genome lengths ranging from 15.5 to just over 16 kb (Table 1). Genome length conforms to the ‘rule of six’, since each N monomer encapsidates six nucleotides of RNA. The rule of six ensures the whole viral genome is encapsidated by the nucleoprotein, as this would otherwise negatively affect transcription and replication activity by the RdRp (Fig. 2(B)). Interestingly, PPRV was shown to not strictly conform to this ‘rule of six’ as two nucleotide insertions, or one nucleotide deletion at the 5′ end of a minigenome, did not severely affect the RdRp activity. However, the hexameric genome length of morbillivirus is prominently evident in nature, as demonstrated by a novel variant of PPRV that possessed a 6-nucleotide insertion in its genome,

70

Animal Morbilliviruses (Paramyxoviridae)

when compared with other strains of PRRV. All morbillivirus genomes are composed of six genes (3′ N, P, M, F, H and L 5′) which encode for eight proteins (Fig. 2(C)). The two non-structural proteins C and V are encoded within the P open reading frame (ORF), by alternative translation initiation and RNA editing, respectively. All genes are separated by untranslated regions (UTRs), which contain in the following order, gene-stop signals (including polyadenylation signals) at the end of the upstream gene, a nontranscribed trinucleotide-intergenic region (IG) and lastly, gene-start signals for the expression of downstream genes (Fig. 2(D)). In morbilliviruses the IG is always 3 nt; however, the whole UTR region between genes can be up to 200 nucleotides in length, except for the UTR between the M and F ORFs, which can be over 1000 nucleotides. This long UTR is unique to morbilliviruses and is thought to play a role in mRNA translation, glycoprotein expression and virulence, as reduction in its size resulted in CDV attenuation in vivo. There are also conserved and complementary sequences at the 3′ and 5′ end of the genome, used as promoters by the RdRp (Fig. 2(D)). Preceding the N ORF at the 3′ end is a conserved 52-nucleotide long leader sequence, followed by an IG and the 3′ UTR of the N gene. This 109-nucleotide long region constitutes the genome promoter (GP) that directs the production of viral messenger RNA (mRNA) or full-length antigenome (positive sense RNA), which subsequently serves as the template for the production of nascent full-length genomic RNA (negative sense). Similarly, at the 5′ end of the genome there is a conserved 37-nucleotides long trailer sequence that together with the 5′ UTR of the L gene comprises the antigenome promoter (AGP), which directs production of full-length genomic RNA from the antigenome. Of note, the recently identified FmoPV possesses a 400nucleotide long trailer sequence, giving it the largest morbillivirus genome identified to date (16,050 nucleotides). Although relatively well conserved, these GP and AGP sequences are thought to be species-specific; however, weak interactions can occur between the RNP of one virus and the GP and AGP of another, as was shown for RPV and PPRV using surrogate minigenome reporter assays. Even minor changes in sequence can have significant consequences on viral replication in vivo, e.g., exchanging the GP and AGP between a vaccine and virulent strain of RPV significantly altered pathogenesis in infected cattle.

Proteins The nucleoprotein is the most abundant structural protein in morbillivirus particles, since significant amounts are required to encapsidate the full length RNA genome during the formation of the RNP. PPRV and RPV encode a 525-amino acid (aa) long protein whereas the N protein of CDV and aquatic morbilliviruses (cetacean morbillivirus, CeMV and PDV) is 523-aa long. All paramyxovirus N proteins can be divided into two main regions: the Ncore located at the N-terminus and the Ntail located at the C-terminus. The Ncore is a conserved structural domain containing the regions for self-assembly, RNA binding and P binding, while the disordered Ntail is involved in mediating RdRp activity via interactions with the Ncore and the RdRp co-factor P. The importance of Ntail was illustrated by reports that truncated forms reduced the pathogenesis of recombinant CDV viruses in vivo. The predisposition of this protein to create nucleocapsid-like structures can also be observed when the protein is expressed in isolation in different systems. A closer look at these structures revealed that Ncore is the main domain responsible for formation of nucleocapsid structures, as mutations impaired N-N assembly and RNA packaging. The functional RNP complex is also composed of the phosphoprotein P, which both acts as a chaperone for the N protein by interacting with Ncore to maintain solubility in the cytoplasm but also as cofactor for the RdRp L polymerase. While PPRV encodes a 509-aa long P protein, the respective RPV and PDV proteins are 507-aa long, with CeMV encoding the smallest P protein (506 aa). The P gene also encodes two other gene products: the non-structural proteins C and V. The C protein is 186-aa long and produced by leaky scanning from an alternative start codon downstream of the P protein ATG. By contrast, the non-structural V protein is produced by viral RdRp-mediated RNA editing. At a specific motif of three G residues within the P gene sequence a G residue is inserted during transcription, resulting in a subset of V viral mRNAs that share the same N-terminal sequence as P but have a frame-shifted C termini. The C protein has been shown to regulate transcription and replication of genomic RNA in RPV and inhibit type I interferon (IFN) responses in PPRV. Although the C protein is broadly thought to act as an IFN antagonist, Messling et al. suggested that this protein is mostly involved in viral spread within the host, while the V protein is the main functional effector of IFN inhibition. In fact, the V proteins of RPV, PPRV and CDV have all been shown to inhibit type I and type II IFN responses. Mechanistically, V protein blocks type II IFN responses through interactions with STAT1, preventing translocation to the nucleus and activation of interferon-stimulated genes (ISGs). The M protein plays a pivotal role in viral structure and assembly of progeny virions, and is the minimum requirement for the release of virus-like particles (VLPs). During morbillivirus infection, the M protein associates with the RNP via interactions with the N-terminus of the N protein, not only mediating viral RNA encapsidation into progeny virions, but also particle synthesis. In addition, the matrix protein is involved in recruiting the glycoproteins to the inner surface of the cell membrane to enable virus budding. The majority of the M protein is found within the cytoplasm; however, it can also be found in the nucleus of infected cells, where for MeV it was reported to inhibit host transcriptional machinery. The fusion protein F is one of two viral glycoproteins present on the surface of the virion, orchestrating virus-cell and cell-cell fusion during morbillivirus infection. F is produced as an inactive precursor F0, which is proteolytically cleaved and folded into the metastable prefusion state consisting of F1 and F2 subunits, linked by a disulfide bond. The F1 subunit is known to contain an N-terminal hydrophobic fusion peptide, two heptad regions (HR1 at the N-terminus and HR2 at the C-terminus), a transmembrane domain and a C-terminal cytoplasmic tail. A third heptad repeat termed HR3 is located in the F2 subunit. Together, these heptad regions mediate membrane fusion upon F triggering via conformational changes that enable the fusion peptide (located in HR1) to dock with the target membrane. For the functionally trimeric F protein to be activated and promote virus-cell membrane

Animal Morbilliviruses (Paramyxoviridae)

71

fusion, the viral particle first needs to bind its cellular receptor, an interaction mediated by the viral attachment protein H. The tetrameric H protein interacts with the F trimer primarily through an extended stalk domain. Each H monomer is composed of an N-terminal cytoplasmic tail, a transmembrane domain, stalk and a β-propeller with six β-sheets arranged as a globular head at the C-terminus. Structural studies of the H protein bound to its cellular receptors have revealed that β4, β5 and β6 constitute the receptor binding domain (RBD). The H protein is the main target of the host’s humoral immune response and many of neutralizing antibodies (nAbs) raised upon morbillivirus infection recognize the RBD, presumably serving to block attachment. Since this protein binds to two distinct cellular receptors, the ability to alter of the RBD sequence to drift away from nAb recognition is limited, which may explain the mono-serotypic nature of morbillivirus. Lastly, the largest protein encoded within the morbillivirus genome is the RdRp polymerase. This protein is responsible for transcription of genomic RNA into mRNA as well as replication of nascent full length RNA genomes via a full length positive-sense intermediate, the antigenome. Since the entire morbillivirus replication cycle occurs in the cytoplasm of the cell, the L protein also carries out capping, methylation and polyadenylation of nascent viral mRNAs to allow efficient translation by the host cell machinery.

Life Cycle Morbillivirus infection begins with the attachment of virions to the surface of the host cell via a proteinaceous cellular receptor. CD46 was the first receptor identified, however it was later shown that this is only recognized by laboratory-adapted MeV strains and not by wild type (WT) strains. Later, the signalling lymphocyte activation molecule (SLAMF1) was identified as the immune cell receptor for both WT and vaccine strains. This receptor is mainly expressed on activated B and T cells, macrophages and dendritic cells. In addition to being lymphotropic, morbilliviruses are also epitheliotropic; with the cellular adhesion molecule Nectin-4 being the receptor on epithelial cells. Experiments for many of the established morbilliviruses indicate that SLAMF1 and Nectin-4 are universal morbillivirus receptors. Genome entry occurs following F protein-mediated fusion of viral and cellular membranes; however, recently it was shown that SLAMF1 can also mediate endocytosis of the virions in a macropinocytosis-like manner. The H and F proteins exist as a hetero-oligomeric complex on the surface of the virion. Binding of H to its cellular receptor brings the viral and cellular membranes into close proximity to enable fusion. To date, five models of morbillivirus fusion triggering have been described. The generally accepted model, summarised in Fig. 3, is that morbillivirus fusion is triggered following receptor attachment by H, which results in conformational changes in the head domain of the H protein and partial unfolding of the central stalk region. Unfolding of this region, which is known to interact with F, causes dissociation of the highly energetic and unstable prefusion form of this trimer. Receptor binding by H lowers the energy barrier required for F triggering and this, coupled with the release of the prefusion form, enables the protein to undergo a series of irreversible conformational changes until it stabilizes in its post-fusion state. This includes structural rearrangement of the heptad regions, resulting in docking of the fusion peptide in the target membrane and ‘pulling’ of the viral and cellular membranes into close proximity. Although fusion with the cellular membrane typically occurs at neutral pH, this is a highly energetic process, thus more than one F trimer might be triggered to overcome this energy barrier, as seen with other paramyxovirus. Finally, an F-mediated membrane pore is formed between the viral and cellular membranes, which expands in an actin cytoskeleton-dependent manner to enable the release of the viral RNP into the cytoplasm of the host cell. Following F and H-mediated entry, the RNP is released into the cytoplasm of the host cell. Here, the incoming RdRp initiates the process of transcription by first binding to the GP sequence at the 3′ end of the genomic RNA. For morbilliviruses, transcription continues via a ‘stop-start’ mechanism, with the RdRp only moving to the next gene once the mRNA from the previous gene has been synthesized and released. Importantly, during this gene-transition process at the IG the RdRp may fall from the genomic

Fig. 3 Step-wise model for membrane fusion in the morbilliviruses. The H protein binds to the SLAM receptor (1), resulting in conformational changes in H (2). These changes promote detachment of the H and F proteins and activation of the latter, triggering structural changes in F (3). The heptad repeats of F form a three-helix bundle wherein the fusion peptide docks with the plasma membrane (4), pulling the two membranes into close proximity and promoting merging of the viral and cellular membranes (5). Lastly, F in its stable post-fusion state enables pore formation and expansion with consequent release of the RNP into the cytoplasm (6).

72

Animal Morbilliviruses (Paramyxoviridae)

Fig. 4 Transcription and replication of the morbillivirus RNA genome. The RNA genome is transcribed by the viral RdRp, which can only commence transcriptional activity at the 3′ end of this molecule. Following transcription of each gene, beginning with N, the RdRp either falls off the template at the intergenic region or continues transcription of the next gene, e.g., P. This results in a transcriptional gradient whereby the genes closest to the GP are transcribed to higher levels than the promoter distal genes, i.e., L is transcribed with the least frequency. The RdRp also carries out genome replication from the antigenome promoter (AGP at the 5′ end of the genome), a process which occurs following the production of an encapsidated, full-length intermediate called the antigenome. This serves as the template for production of full-length, N-encapsidated RNA genomes which can be incorporated into progeny virions.

template. Since re-attachment of the polymerase and further transcription can only commence from the 3′ GP, a transcriptional gradient results, whereby the genes closest to the GP are transcribed at higher levels than promoter distal genes (Fig. 4). This gene expression strategy reflects the relative requirement for individual proteins in viral replication, with the order of the genes being conserved in all morbilliviruses. Of note, the F and H glycoproteins are synthesized in the ribosomes of the rough endoplasmic reticulum and trafficked through the Golgi complex where post-translational modifications occur, including proteolytic cleavage of F0 and glycosylation of both proteins. The RdRp also carries out replication of nascent full-length RNA genomes, initially through production of a positive-sense intermediate; the antigenome, which is also N-encapsidated (Fig. 4). Once this positive-sense intermediate is produced, the RdRp binds to the AGP at the 3′ end of the antigenome and synthesizes nascent, full-length, negative-sense RNA genomes, also N-encapsidated. The switch from transcription to replication is poorly understood; however, the most widely accepted model is that increasing levels of soluble N protein mediate the interchange from transcription to replication. Anderson et al. showed that deletion of the proximal region of the 3′ UTR between the M and F ORFs of CDV influenced replication initiation but the exact mechanism for this remains poorly characterised. It has also been suggested that the presence of two variable-state RdRps, specific for replication or transcription, mediate this switch. Finally, the synthesized viral components assemble at the plasma membrane to be incorporated into progeny virions that are released from the host cell. The exact mechanism by which morbilliviruses assemble and bud remains largely uncharacterized, therefore we will couple studies on other paramyxoviruses to address this step of the life cycle. It is widely accepted that the M protein plays a major role in assembly of the viral proteins and RNPs as well as budding of nascent virions. Budding of morbilliviruses from polarised epithelial cells occurs at the apical surface, requiring directional movement of the viral components to this surface. Apical budding is largely dependent on specific sorting of the glycoproteins, as otherwise these proteins accumulate at the basolateral membrane. This sorting is accomplished via interactions between the M protein and the cytoplasmic tails of the glycoproteins, as demonstrated for MeV, Sendai virus and para-influenza virus 5. Furthermore, budding at the apical surface also requires direct trafficking of the RNP complex to this site. Nakatsu et al. recently showed that apical budding of MeV in polarised epithelial cells is dependent on trafficking of the RNP complex to the cell membrane via the recycling endosome pathway (REP), employing Rab11A-positive vesicles. In contrast, budding from nonpolarized cells occurs in a REP-independent manner. Another cellular component required for efficient release of morbilliviruses is the actin cytoskeleton, as its specific disruption with pharmacological inhibitors severely impairs the trafficking of the M protein and RNPs to the surface of the cell. It has been suggested that once the viral proteins reach the cell surface they accumulate at lipid raft domains; CDV virions were reported to contain cholesterol while MeV RNPs and M proteins were shown to localize to lipid rafts. In contrast to viral assembly, particle budding requires actin cytoskeleton destabilization, allowing the mature bud to form. Bringolf et al. used a structure-guided mutagenesis approach to show that M protein oligomerization plays a key role in promoting virus budding, identifying that accumulation of M dimers at the cell surface induces membrane curvature and formation of the viral bud leading to virus egress, which Salditt et al. showed to be ESCRT-independent. Once the viral bud is formed, nascent virions ‘pinch off’ from the surface of the cell via a mechanism that remains largely uncharacterised. Lastly, it has been shown that morbilliviruses downregulate their cellular receptors on the surface of infected cells, a process which could facilitate release of cell-free virions. The life cycle of morbilliviruses is summarised in Fig. 5.

Animal Morbilliviruses (Paramyxoviridae)

73

Fig. 5 Morbillivirus life cycle in the cell. A schematic overview of the morbillivirus replication cycle with viral proteins coloured, as in Fig. 2. (1) The virion attaches to the cell receptor through its H protein, which activates the F protein and triggers fusion of the viral and cellular membranes, with release of the RNP into the cytoplasm of the host cell; (2) the viral genome is transcribed, capped and polyadenylated by the RdRp to be translated by the host cell ribosomes (3); (4) Unlike the other viral proteins, the F and H proteins are translated by ribosomes in the rough endoplasmic reticulum and further processed in the Golgi complex; (5) At a later stage, the RdRp switches from genome transcription to genome replication to produce nascent viral RNA that will be incorporated into progeny virions; (6) The RNP as well as the M, F and H proteins are sorted to the surface of the cell membrane where nascent particles are generated by budding.

Epidemiology and Host Range RPV Historically recognized as significant diseases of humans and animals, RPV and MeV were properly described during the 16th and 17th centuries; however, the first evidence for rinderpest disease dates back to the 4th century AD. RPV is thought to have originated in Asia, followed by emergence in Europe in the 18th century, where the virus was described as the causative agent of cattle plague. By the end of the 19th century, RPV had reached the African continent, spreading to Australia and South America by the middle of the 20th century. Discovery of the virus, coupled with sequencing technologies, allowed RPV isolates to be classified into three lineages based on sequence variability (up to 11%) in the F gene. These lineages accurately represented the geographic distribution of RPV during the 20th century: lineage I included East, West and North African isolates, lineage II represented isolates from East and West African countries, while lineage III included Southern Asian isolates of RPV. The high prevalence of RPV across three continents and its enormous economic impact on agriculture, especially in twentieth century Africa, resulted in the implementation of a coordinated mass vaccination campaign to eradicate the disease. Following years of effort RPV was declared eradicated in 2011 (see below).

PPRV In the middle of the 20th century, PPRV was identified as the causative agent of ‘peste des petits ruminants’ (plague of small ruminants) in West Africa. Although initially thought to be a variant of RPV the origin of this genetically distinct virus remains uncertain. In fact PPRV is thought to have circulated in sheep and goats for a long time before being officially identified.

74

Animal Morbilliviruses (Paramyxoviridae)

Nevertheless PPRV spread throughout Africa and reached Asia in the second half of the 20th century. Currently, PPRV circulates in over 70 African and Asian countries and poses a major threat to over 80% of the global sheep and goat populations, a distribution that has made it also a target for eradication. To date, four lineages of PPRV have been classified, based on sequencing of the C-terminal domain of the N gene. As with RPV, this reflects the geographical distribution of this virus across Africa, the Middle East and Asian countries. Lineages I, II and III are endemic in West and East African countries, respectively, while lineage IV is endemic in the Middle East and South Asian countries, although its distribution is increasing. Lineages II and III are thought to have originated independently and at the same time, while lineage IV is endemic in Asia. Worryingly, PPRV has shown evidence of a broadening host range, with lineage IV PPRV recently being detected in camels and saiga antelope, and lineage II in deer. Additionally, evidence of PPRV infection has also been detected in cattle and gazelles.

CDV CDV is believed to have originated in the Americas before spreading into Europe, Africa and Asia following colonisation. It was initially thought that CDV was restricted to the Canidae; however, in the 1990s, CDV outbreaks caused fatalities amongst wildlife species including lions, tigers and leopards. A sudden CDV outbreak in the Serengeti National Park in Tanzania, suspected to have originated in African wild dogs, caused unexpectedly high morbidity and mortality in the African lion and spotted hyena populations. Indeed, CDV outbreaks pose a great threat to endangered species. For example a severe outbreak in the black-footed ferret population, with mortality rates of more than 70%, rendered this species extinct in the wild in the 1980s. More recently, CDV was reported in endangered Ethiopian wolves and it also now poses a major threat to Nicaragua’s jaguar population, following transmission from CDV-infected hunting dogs. To date, CDV has been reported to naturally or experimentally infect almost all families of the order Carnivora. Significantly, CDV outbreaks have also been reported in monkeys in China and Japan, raising concerns that this virus can cross-over the species barrier to infect primates. Experimental adaptation in vitro has shown that CDV can rapidly adapt to use both epithelial and immune human cell receptors. The worldwide prevalence of CDV has resulted in isolates being categorized into various phylogenetic lineages. CDV lineages are categorized based on H protein variability, with more than 5% amino acid diversity being the cut-off for delineation. Over the last 40 years, 10 lineages of CDV have been described based on this criterion, with the lineages also reflecting geographical distribution: two North American, three European, two South American, two Asian and one South African lineage(s). North America lineage 1 includes the vaccine strains of CDV while North America 2 viruses are known to be currently circulating in the USA; however, recently a third lineage has also been reported in domestic dogs. In South America, Europe lineage 1 strains were predominant until the identification of CDV strains in wildlife in Argentina, which led to the classification of South America lineage 2. The same authors suggested renaming Europe 1 to Europe/South America 1. Furthermore, Espinal et al. have also reported the presence of a third lineage in South America in Colombian domestic dogs. In Europe, CDV strains from Europe lineage 3 are currently circulating in Italy, affecting wolf populations in particular, and contributing to a spill-over event in badgers. The first CDV infection in anteaters has recently been reported in Brazil, with these strains clustering in the Europe/South America 1 lineage. Lastly, phylogenetic analysis of Serengeti isolates has revealed that these constitute a different lineage from the currently circulating South African lineage.

Aquatic Mammal Morbilliviruses PDV was one of the first aquatic morbilliviruses to be identified and, to date, has caused two large outbreaks in European harbor seals. These outbreaks were originally thought to be CDV-related, as an epizootic event was taking place at the same time following contact of infected terrestrial mammals with seals; however, the harbor seals were later shown to be infected with a genetically and antigenically distinct virus that showed similar clinical manifestations. The second aquatic morbillivirus, CeMV was first isolated in 1991 with a host range largely restricted to dolphins, whales and porpoises. To date the exact origin of CeMV remains unknown, however, phylogenetic analysis suggests that CeMV shares a common ancestor with RPV and PPRV, suggesting that its origin might be a spill-over event. CeMV is presumed to be endemic in cetacean populations with an outbreak in pilot whales recently reported in the Canary Islands.

Emerging Morbilliviruses Recently, a morbillivirus was detected in domestic house cats, firstly in China, with later reports from Japan, the USA and South Africa, suggesting the presence of a previously unidentified morbillivirus, largely restricted to these animals. Phylogenetic analysis based on the six structural proteins revealed that FmoPV clusters together with other morbilliviruses; however, FmoPV is somewhat distinct and less closely related to the other recognized members of the Morbillivirus genus. Interestingly, a recent study by Drexler et al. showed that bats and rodents may serve as a reservoir for a large number of previously uncharacterized morbilliviruses. The authors isolated multiple viral sequences which upon analysis were closely related to morbilliviruses, yet distinct enough to create their own phylogenetic clusters. This study also identified two bat isolates that were closely related to CDV and PDV, raising concerns of possible zoonotic transmission.

Animal Morbilliviruses (Paramyxoviridae)

75

Clinical Features The following is a summary of the classical clinical signs and symptoms seen in animal morbillivirus infections. A typical acute infection in animals can be classified into five distinct stages: Incubation, Prodromal, Mucosal, Diarrhoeic and Convalescent – a categorisation that corresponds well with the ‘in-tissue’ spread of infection described in the Pathogenesis section below. For RPV infections in cattle, which were often severe, the development of clinical disease was often summarised more succinctly as diarrhoea, dehydration and death. The initial incubation period is followed by a transient pyrexia. Around this period oculo-nasal discharges (associated with virus excretion and consistent with conjunctivitis and rhinitis) begin to develop which can become mucopurulent over time. Similarly, mucosal necrosis develops with erosions and ulcerations in the oral cavity, digestive and/or respiratory tracts. This worsens over time as shedding of necrotic material leads to severe erosions, bloody diarrhoea and a general worsening condition in the animal. Emaciation, dehydration and an associated drop in temperature to sub-normal levels can follow, all contributing to lethality. Other more non-specific signs of infection include restlessness, depression and anorexia as well as shallow and rapid breathing. In pregnant animals infection of the foetus and/or frequent abortion can occur; however, there is little evidence for teratogenic effects or the development of persistently infected ‘carrier states’ in new-borns. The mortality rate in morbillivirus infections can vary greatly, dependent on a number of factors. In immune-naïve herds mortality rates of 100% have been recorded, especially for the ruminant morbilliviruses. However, for other morbilliviruses a significant percentage of infections may be sub-clinical in nature, e.g., CDV infections in domestic dogs. It is known that disease severity varies based on the strain of host as well as its immune status (vaccinated, immunosuppressed or waning maternally-derived immunity). Other complex factors, such as herd immunity levels and geographical endemicity may also contribute to severity in the wild. Variations within the viral sequence strain might also contribute to the development of disease, perhaps best exemplified by the low virulence RPV strains that were circulating in East Africa prior to eradication. In animals that survive an acute infection, one of the hallmarks of morbillivirus infection is a prolonged period of immunosuppression, frequently associated with secondary bacterial infections or indeed viral co-infections. Antigen and viral RNA can persist long after the resolution of acute infection; however, the significance of this to both viral epidemiology and immunosuppression is not well understood. The progression of disease in morbillivirus-infected animals can also vary between hosts and there are a range of specific clinical signs that appear more specific to one virus than others; however, whether these are the result of virus- or host-specific differences is not well understood. Noticeable differences include a more frequent neural pathology in certain hosts. For CDV and CeMV encephalitis can be prolonged and chronic, with lesions forming in the CNS and brain. This is associated with abnormal behaviour, poor coordination, seizures and possible paralysis and can result in extensive demyelination. CDVinfected dogs can develop old dog encephalitis (ODE) which is reminiscent of the chronic MeV infection in humans, subsclerosing pan-encephalitis (SSPE) and many of these neurological symptoms develop long after the resolution of acute infection. Hyperkeratosis is most dominantly seen in CDV-infected domestic dogs; however, keratosis of the flippers, head, trunk, and tail have been reported for aquatic mammal morbilliviruses. In morbillivirus-infected ruminants, digestive pathology is more severe with the Peyer’s patches being particularly badly affected. In the large intestines of ruminants submucosal capillaries running along the crests of the mucosal folds become congested giving rise to zebra-stripes – a pathological hallmark of disease. There are, however, specific differences between the ruminant viruses as well, e.g., PPRV is more likely to cause a primary viral pneumonia with frequent secondary microbial infections involving Pasteurella spp. and Mycoplasma spp. Significant respiratory infections are also evident in aquatic mammals, where bronchopneumonia is evident as well as necrosis of various tissues accompanied by atelectasis, congestion, edema and emphysema; the presence of parasitic and/or mycotic secondary infections in these animals has also been identified. FeMV infections perhaps represent the greatest outlier and currently there is no clear evidence that the virus utilises the biphasic life cycle described for other morbilliviruses. FeMV infections appear chronic and possibly sub-clinical in domestic cats with the kidneys and urine being associated with virus persistence and transmission, respectively.

Pathogenesis As discussed previously, morbilliviruses are immuno and epithelio-tropic viruses; tropisms defined by their use of specific proteinaceous receptors. Accordingly, infection of cell-types expressing the two established receptors, SLAMF1 (aka CD150) and Nectin-4, determines the route and progression of disease within the mammalian host. SLAMF1, the immune cell receptor, is found on activated lymphocytes (B and T cells) as well as macrophages and dendritic cell subsets. The physiological role of SLAMF1 is predominately immune cell signalling; however, a role in the innate immune response to Gram-negative bacteria was also recently uncovered. Nectin-4, in contrast, is found on epithelial cells, mainly at mucosal surfaces in the respiratory and digestive tract. Physiologically, Nectin-4 is critical for cellular adhesion, forming an essential component of the adherens junction between polarised epithelial cells. Morbilliviruses are highly infectious, and despite their enveloped particle, are thought to be capable of persisting in aerosolised droplets or on contaminated fomites or bedding for extended periods of time. For the majority of animal morbilliviruses the primary source of infection is thought to be inhalation of contaminated respiratory droplets. The prevalent model for virus dissemination through the host (summarised in Fig. 6) is built on various pathogenesis and receptor studies, including animal models of MeV infection. Initially, resident SLAMF1-expressing cells, such as dendritic cells or macrophages in the respiratory tract

76

Animal Morbilliviruses (Paramyxoviridae)

Fig. 6 Model of animal morbillivirus spread in an infected host. Virus particles enter via the respiratory route (1), with the virus first infecting SLAMF1-positive immune cells in tissues in the upper respiratory tract, perhaps the tonsil (2). Infected immune cells drain to local lymph nodes seeding subsequent rounds of immune cell infection and enabling the virus to reach more remote lymphatic tissues and immune-related organs such as the spleen (3). Immune cell-associated viraemia subsequently allows viral dissemination to other organs, such as the intestine, lungs and trachea (4). In these tissue infection spreads into the epithelial cells of these organs, via Nectin-4 mediated entry; a receptor found on the basolateral surface of polarised cells. This second stage of the infection plays a key role in the development of many clinical signs observed in animal morbillivirus infections. Lastly, nascent virions are released into the lumen of the digestive or respiratory tract, allowing spread to naïve animals (5). Of note, the neurological infections seen in some CDV-infected dogs are not illustrated here.

are infected. However, despite high levels of Nectin-4 expression in the oral cavity and upper respiratory tract this receptor is not immediately accessible because adherens junctions are buried on the basolateral surface of polarised cells. Indeed, infected immune cells first migrate to local lymph nodes, leading to a burst of viral replication in resident B and T cells. Subsequently, infected cells migrate from lymphatic tissues into the blood generating a cell-associated viraemia. It is only then, upon contact between morbillivirus-infected immune cells and the basolateral surface of mucosal epithelia that the virus spreads to Nectin-4positive cells. Replication within epithelial tissues leads to preferential release of nascent virus into the lumen of the respiratory or digestive tract favouring transmission to new hosts. This biphasic life cycle has led to SLAMF1 and Nectin-4 being referred to as the entry and exit receptors, respectively. This widely accepted model for morbillivirus infection is supported by pathological observations following autopsies of naturally or experimentally infected animals. Morbilliviruses can be detected in the bronchus-associated lymphoid tissue (BALT) and tracheabronchial lymph nodes within a few days of infection. In addition, a significant incubation period is observed (without epithelial pathology), associated with an acute leucopoenia – indicative of localised replication in lymphatic tissues. Marked necrosis and depletion of lymphocytes can be seen in a range of immune tissues such as the spleen and thymus as well as lymph nodes surrounding both the respiratory and digestive tract, where germinal centre B cells are frequently found to be infected. Subsequent virus spread to the epithelium is associated with immune cell infiltration into the lamina propria and degeneration of epithelial cells. Fused epithelial cell syncytia can often be observed as well as intracytoplasmic and intranuclear inclusion bodies.

Diagnosis and Prevention The control and prevention of morbillivirus disease is of global relevance to human health, agriculture and wildlife. In fact, in many ways, morbillivirus diseases primed many of the modern approaches to veterinary vaccination and epidemic control (see Rinderpest case study below). The varied approaches are summarised in this section.

Animal Morbilliviruses (Paramyxoviridae)

77

Certain animal morbilliviruses, such as RPV or PPRV, are notifiable diseases – enabling a co-ordinated response to infection, especially in non-endemic settings. For these viruses in particular there are a multitude of available tests and tools for diagnosing viral infection, as well as advisory information on which samples and tissues to harvest (managed by the World Organisation for Animal Health, OIE). This advice is likely applicable to all morbillivirus infections; however, the availability of validated diagnostic assays may vary. The recommended sites for sampling are swabs from the eye, nose and any obvious mucosal erosions or lesions, as well as uncoagulated blood. As with many infections the aim is to sample and isolate live virus, viral RNA, viral antigen and/or host-specific antibodies. Virus isolation can be made on a broad range of cell-lines, e.g., Veros, where the efficiency of recovery can be boosted by over-expression of one of the cognate receptors, SLAMF1 or Nectin-4. There are various established RTPCR or RT-qPCR tests to detect viral RNA, whilst viral antigen can be detected by agar gel immunodiffusion, immunocapture ELISA or penside test assays. The isolation of virus-specific antibodies is important for serological and epidemiological monitoring, as well as the assessment of vaccination responses. Of note, all morbilliviruses appear to be mono-serotypic in nature enabling sero-surveillance. Various established tests are available including virus neutralisation tests (VNTs), indirect ELISAs and competitive ELISAs, all of which target immune responses to the F, H and/or N protein. Sequencing of viral isolates, often targeting the N, F or H genes, has proved beneficial for phylogenetic characterisation and tracking of outbreaks. Basic approaches to limit the spread of disease are effective at controlling morbillivirus outbreaks. In non-endemic settings the segregation of animals and culling of infected and contact individuals has proven effective, along with adequate disposal of infected carcasses. Often this is not practical in non-endemic or wildlife settings, where vaccination is the only realistic option. The best animal vaccines must have the following properties: They must be efficacious and safe in all animals and breeds, immunity must be easily testable in vitro, they must not be immunosuppressive, and finally they should provide lifelong or near-lifelong immunity. Fortunately, many of the licenced morbillivirus vaccines share these properties, the most widely used of which are live attenuated variants. The two commonly used CDV vaccine strains were derived from adaptation to ferrets, chicken embryos and later chicken cell lines (Onderstepoort) or canine kidney cell lines (Rockburn). Whilst these are widely, and successfully, used in dogs their efficacy in wildlife is less clear, with examples of vaccine-associated virulent disease. The difficulties associated with testing and using vaccines in wildlife also means that few vaccine trials have been performed in aquatic mammals, although there is evidence of CDV vaccines providing cross-protective immunity. The other widely used morbillivirus vaccines are the live attenuated PPRV vaccines, generated following serial passage of field strains in tissue culture (Veros). Recently, researchers have demonstrated that the two most widely used vaccines are efficacious against all lineages of circulating PPRV, supporting their use in future control and eradication campaigns. Despite the availability of efficacious vaccines there is a requirement for next generation tools. For example, a vaccine that would allow epidemiologists and veterinarians to distinguish infected from vaccinated animals (DIVA) would prove beneficial in a post-outbreak or eradication setting. Researchers have investigated this by modifying viral epitopes, using alternative viral vectors or recombinant protein platforms to express immunogens or indeed making recombinant morbilliviruses and there is great promise that one such vaccine will soon come to the market, likely for PPRV. Lastly, it is worth noting that recombinant morbilliviruses are also used as vectors themselves to deliver and express antigens from other important viruses, e.g., a CDV expressing Rabies G protein.

Treatment In general supportive care revolves around maintaining adequate hydration and the avoidance of secondary bacterial infections. Although there are no licenced antivirals for treating sick animals, there are a number of candidate antiviral compounds that have shown efficacy in the lab either by blocking fusion or inhibiting the polymerase.

Rinderpest Eradication: A Success Story Although RPV was declared eradicated by the FAO and OIE in 2011 the history of control of this disease goes back hundreds of years. Historically, outbreaks of RPV in Europe and Asia are thought to have originated in the Caspian basin (presumably brought in by or with marauding armies). To an extent, the severity and frequency of these outbreaks is thought to have precluded the development of sustainable bovine agriculture. However, in 1711, following RPV disease in the papal herds, Pope Clement XI ordered a physician, Dr. Lancisi, to investigate the issue. He implemented some of the first detailed epidemic control strategies in animals including movement restriction, slaughter and burial of carcasses in lime. The success of this strategy was quickly adopted by other countries in Europe to control RPV infections; many of these approaches are still in use today. Training in the implementation of these strategies led to the development of the first veterinary school (Lyon, France 1761) as well as state veterinary services, with other countries in Europe soon following suit. Rinderpest was also instrumental in establishment of the OIE, while RPV was one of the first issues tackled by FAO in inaugural meetings. The impact of RPV on wildlife should also not be overlooked, as this virus caused devastating epidemics in wild ungulates, especially in Africa. Interestingly, wildlife were initially thought to be the source of infections; however, following the successful removal of RPV from cattle populations in the Serengeti ecosystem, the buffalo and wildebeest populations soared. The history of RPV vaccines tracks a shift from treatment to prevention and finally eradication. Initial vaccinations involved immunization with sera from convalescent animals. This was replaced by killed vaccines and then ultimately live attenuated viruses adapted to animals: goats, rabbits and eggs but later tissue culture

78

Animal Morbilliviruses (Paramyxoviridae)

– primary calf kidney cultures. The history and politics of RPV eradication is a fascinating area summarised excellently elsewhere (see further reading); however, the key factors that led to these live attenuated vaccines being successfully used to eradicate RPV are as follows: There was a limited geographical distribution of the disease and no latency or persistence. In addition, infected animals had a short infectious period and transmission was reliant on direct or close indirect contact. This, combined with a safe universal vaccine, usable in all animals and providing lifelong immunity contributed to the success of a global approach led by international organisations with a clear mandate and unified strategy. Many of these criteria apply to PPRV and the FAO and OIE have recently launched a global strategy for PPRV eradiation. Whilst eradication is also a meaningful target for the human morbillivirus MeV it seems unlikely that CDV or any of the aquatic mammal morbilliviruses are realistic targets, given their global distribution and more complicated host-range.

Further Reading Beineke, A., Baumgartner, W., Wohlsein, P., 2015. Cross-species transmission of canine distemper virus-an update. One Health 1, 49–59. Chang, A., Dutch, R.E., 2012. Paramyxovirus fusion and entry: Multiple paths to a common end. Viruses 4 (4), 613–636. de Vries, R.D., Ludlow, M., de Jong, A., et al., 2017. Delineating morbillivirus entry, dissemination and airborne transmission by studying in vivo competition of multicolor canine distemper viruses in ferrets. PLOS Pathogens 13 (5), e1006371. Drexler, J.F., Corman, V.M., Muller, M.A., et al., 2012. Bats host major mammalian paramyxoviruses. Nature Communications 3, 796. Duignan, P.J., Van Bressem, M.F., Baker, J.D., et al., 2014. Phocine distemper virus: Current knowledge and future directions. Viruses 6 (12), 5093–5134. Nambulli, S., Sharp, C.R., Acciardo, A.S., Drexler, J.F., Duprex, W.P., 2016. Mapping the evolutionary trajectories of morbilliviruses: What, where and whither. Current Opinion in Virology 16, 95–105. Pastoret, P.-P., Yamanouchi, K., Mueller-Doblies, U., et al., 2006. Chapter 5: Rinderpest – An old and worldwide story: History to c. 1902. In: Barrett, T., Pastoret, P.-P., Taylor, W.P. (Eds.), Rinderpest and Peste des Petits Ruminants. Oxford: Academic Press. (86-VI). Van Bressem, M.F., Duignan, P.J., Banyard, A., et al., 2014. Cetacean morbillivirus: Current knowledge and future directions. Viruses 6 (12), 5145–5181.

Animal Papillomaviruses (Papillomaviridae) John S Munday, Massey University, Palmerston North, New Zealand r 2021 Elsevier Ltd. All rights reserved.

Glossary Cell mediated immunity The part of the immune response that is directly mediated by cells such as cytotoxic T-lymphocytes. Humoral immunity The part of the immune response that is mediated by antibodies. Immunohistochemistry Using antibodies to identify proteins within histological sections. In situ hybridization Using antisense probes to identify DNA or RNA within histological sections. Oncoproteins Proteins that promote cell growth and division.

Open reading frame A section of nucleotide sequences between a start and a stop codon. Papilloma While it is acknowledged that papillomas can either be papillomavirus-induced hyperplastic lesions or non-papillomavirus-induced benign neoplasms of the epithelium, in the text the term ‘papilloma’ is used to indicate an area of focal epithelial thickening caused by papillomavirus infection. The term papilloma is therefore used to describe a ‘wart’ in human medicine. Retinoblastoma protein A protein that controls cell division. Loss of retinoblastoma protein will favor cell division. Viral capsid The protein shell of a virus.

Classification The Papillomaviridae family includes two subfamilies, the Firstpapillomavirinae and the Secondpapillomavirinae. The Firstpapillomavirinae currently contains 51 genera while only one genus is currently classified within the Secondpapillomavirinae subfamily. Genera in the Firstpapillomavirinae are named using the Greek alphabet. As there are now more papillomavirus genera than letters in the Greek alphabet, the prefixes dyo- and treis- have been used. For example the deltapapillomavirus genus was the fourth genus established and this was prior to the dyodeltapapillomavirus genus which was established prior to the treisdeltapapillomavirus genus. Within the Secondpapillomavirinae subfamily, genera are named according to the Semitic abjads (an abjad is similar to an alphabet, except only consonants are represented). Alefpapillomavirus is only genus currently within the Secondpapillomavirinae subfamily. Knowledge of the genera of papillomaviruses is useful as papillomaviruses within each genus often have closely related hosts. Additionally papillomaviruses within the same genera often have similar biological properties and therefore usually cause similar diseases. For example papillomaviruses in the Lambdapapillomavirus genus all infect carnivores and cause proliferative papillomas, most frequently in the oral cavity. Within genera, the papillomaviruses are further subdivided into numerically-named species. Currently there are 133 papillomavirus species within the 51 papillomavirus genera. Papillomaviruses within the different species can be expected to have the same, or closely related, hosts and similar biological behaviors. For example, within the deltapapillomaviruses, the species 4 deltapapillomaviruses all cause fibropapillomas in domestic cattle while the species 1 lambdapapillomaviruses all cause oral papillomas in felids including domestic cats, snow leopards, bobcats, and lions. Within the species, papillomaviruses are subclassified into individual papillomavirus types. Each papillomavirus type is named sequentially using the scientific name of the host species. For example, the sixth fully classified papillomavirus of domestic cattle is Bos taurus papillomavirus type 6, which is a species 1 xipapillomavirus within the xipapillomavirus genus. With the notable exception of the bovine species 4 deltapapillomaviruses, papillomaviruses are highly species-specific. Around 500 individual papillomavirus types have been fully classified, including around 175 from the non-human species. Papillomaviruses have been identified in virtually every species that has been intensively studied including cattle, horses, dogs, cats, sheep, deer, rabbits, bears, sea lions, dolphins, primates, birds, a variety of laboratory and free-ranging rodents, bats, snakes, and turtles. Most species are likely to be infected by multiple papillomavirus types from multiple papillomavirus genera and further research will undoubtedly identify many more papillomavirus types in animals. Classification of papillomaviruses has traditionally been using the genetic sequence of the L1 open reading frame (ORF) (Fig. 1(a)). Viruses of different subfamilies have less than 45% similarity in their L1 ORF while papillomaviruses in different genera have less than 60% similarity. Papillomaviruses with less than 70% similarity are considered to be in different species and less than 90% similarity is present between papillomaviruses of different types. While this scheme is simple, many viruses will show overlap between multiple different genera and species, making classification difficult. To overcome these areas of overlap, papillomaviruses are additionally classified based on their host species and the type of lesions that they cause. As papillomaviruses tend to infect either mucosal or cutaneous epithelium, human papillomaviruses are also classified into mucosal or cutaneous types. While the same is likely to be true in animals, this classification is not widely used in veterinary medicine. In humans, papillomaviruses are also classified based on the likelihood that they will cause cancer. Therefore the ‘lowrisk’ alphapapillomaviruses cause warts that spontaneously resolve and rarely, if ever, progress to cancer. In contrast, the ‘high-risk’

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20923-0

79

80

Animal Papillomaviruses (Papillomaviridae)

Fig. 1 (a). Schematic representation of select papillomavirus genera (courtesy Dr. Neroli Thomson, Massey University, New Zealand). (b). Genetic organization of Felis catus papillomavirus type 2 and human papillomavirus type 16 showing open reading frames within the viral genome and the long control region (LCR; courtesy of Dr. Neroli Thomson, Massey University, New Zealand).

Animal Papillomaviruses (Papillomaviridae)

81

Table 1 Summary of papillomaviruses that have been detected in five domestic animal species. It should be noted that not all papillomavirus types within a genus will cause all the associated lesions listed. For example, Canis familiaris papillomavirus (CPV)-17 is the only canine Taupapillomavirus type associated with oral SCCs and only CPV-2 and -7 have been associated with cutaneous papillomas. Additionally, while papillomaviruses are listed as being associated with lesions, for many the role of the papillomavirus in lesion development still remains unproven. SCC, squamous cell carcinoma; BISC, Bowenoid in situ carcinoma; BPV, Bos taurus papillomavirus; OaPV, Ovis aries papillomavirus; EcPV, Equus caballus papillomavirus; CPV, Canis familiaris papillomavirus; FcaPV, Felis catus papillomavirus Species

Papillomavirus genus

Papillomavirus types

Predominant associated lesions

Cattle

Delta

BPV-1, -2, -13, -14

Xi

BPV-3, -4, -6, -9, -10, -11, -12, -15, -17, -20, -23, -24

Epsilon Dyoxi Dyokappa Unclassified

BPV-5, -8 BPV-7 BPV-16, -18, -22 BPV-19, -21

Cutaneous and esophageal fibropapillomas Bladder neoplasia Cutaneous and upper alimentary papillomas Upper alimentary SCC Cutaneous papilloma Cutaneous papilloma Cutaneous papilloma Cutaneous papilloma

Sheep

Delta Dyolambda

OaPV-1, -2, -4 OaPV-3

Cutaneous fibropapilloma Cutaneous SCC

Horses

Zeta Dyoiota

EcPV-1 EcPV-2, -4, -5

Dyorho Unclassified Delta

EcPV-3, 6, 7 EcPV-8 BPV-1, -2, 13

Cutaneous papilloma Penile papilloma Penile SCC Aural plaque Aural plaque Cutaneous papilloma Equine sarcoid

Lambda

CPV-1, -6

Tau

CPV-2, -7, -13, -17, -19

Chi

CPV-3, -4, -5, -8, -9, -10, -11, -12, -14, -15, -16, -18, -20

Lambda Dyotheta

FcaPV-1 FcaPV-2

Unclassified Delta

FcaPV-3, -4, -5 BPV-14

Dogs

Cats

Oral papillomas Cutaneous papillomas Cutaneous papillomas Oral SCC Viral pigmented plaques Cutaneous SCC Oral papillomas Viral plaques/BISC Cutaneous SCC Viral plaques/BISC Feline sarcoid

alphapapillomaviruses cause around 5% of all human cancers. While papillomaviruses are not commonly subdivided into ‘low’ and ‘high’ risk categories in veterinary medicine, there is increasing evidence that a subset of papillomavirus types cause cancer in animals. The papillomavirus types that are currently recognized from the major domestic species are contained in Table 1.

Virion Structure Papillomavirus virions are non-enveloped and around 55 nm in diameter. The viral capsid consists of 360 copies of the L1 protein which are arranged in 72 pentamers (five-sided arrays) and around 12 copies of the L2 protein. Each capsid contains one copy of the papillomavirus DNA although both “empty” and “full” virus particles can be seen by electron microscopy. Papillomaviruses are resistant to diverse environmental insults and infectivity survives lipid solvents and detergents, low pH, and high temperatures.

Genome The genome consists of a single molecule of circular double-stranded DNA that is typically around 7500 bp in length although the genome of individual papillomavirus types ranges from 5748 bp to 8607 bp. Several different translational strategies are utilized to enhance the limited coding capacity of the papillomavirus genome. The genome encodes 6–9 proteins depending on the individual papillomavirus type. These proteins are subdivided into the early (E) and late (L) proteins (Fig. 1(b)). The E proteins are non-structural proteins and have important regulatory and replicative

82

Animal Papillomaviruses (Papillomaviridae)

Fig. 2 Schematic representation of papillomaviral replication with stratified epithelium. Arrows indicate where the predominant layers of the epithelium at which each papillomaviral protein is produced and where papillomaviral DNA replication occurs (courtesy Dr. Neroli Thomson, Massey University, New Zealand).

functions. The E1 protein is a viral DNA helicase and is responsible for unwinding the double-stranded DNA. The main function of the E2 protein it to control the papillomavirus replication cycle. As will be discussed, replication of papillomaviruses is closely coordinated with epithelial cell replication and differentiation and the E5, E6 and E7 proteins alter the normal regulation of epithelial cells to maximize papillomavirus replication. As these proteins can promote cell division and therefore potentially cause neoplastic transformation of a cell, they are often referred to as ‘viral oncoproteins’. In the majority of papillomavirus types, E7 is considered to be the protein that most significantly influences epithelial cell regulation. The E7 protein influences cell division by binding, and degrading, the retinoblastoma protein, an important cell cycle checkpoint. The E7 of most papillomavirus types has a retinoblastoma binding site (LXCXE) although binding to retinoblastoma protein can occur even without this site, possibly due to poorly-defined alternative pRb binding sites within the E7 carboxyl-terminal domain. In contrast, promotion of cell regulation by bovine deltapapillomaviruses appears to be primarily through interactions between the E5 protein and the platelet-derived growth factor-β receptor. The two L proteins form the viral capsid and these are only expressed close to the completion of the viral replication cycle.

Life Cycle Infection by most papillomavirus types is currently believed to be restricted to stratified squamous epithelium. This is because papillomavirus replication is dependent on, and intimately linked to, the growth and differentiation of these epithelial cells (Fig. 2). Infection by a papillomavirus occurs when micro-trauma allows the papillomavirus to interact with the basement membrane, which then facilitates viral entry into a basal cell. The papillomavirus genome and some viral E proteins then enter the nucleus resulting in the production of 10–200 episomal copies of viral genomic DNA. As basal cells replicate, the episomal DNA spreads through the basal cell population and infection is maintained indefinitely within the infected epithelium. During this phase of replication, the papillomavirus does not significantly alter normal epithelial cell regulation and such infections are asymptomatic. While the infection is maintained within the host, viral replication does not occur and the infection at this stage is not infective. For a papillomavirus to complete its life cycle, it has to be present within a basal cell that undergoes terminal differentiation. However, once a basal cell has terminally differentiated, it loses the ability to divide, resulting in degradation of the nucleus. As replication of the papillomavirus DNA is dependent on the host cell nuclear machinery, a key feature of papillomaviruses is their ability to prevent cells from leaving the S-phase of the mitotic cycle. By ensuring the infected keratinocytes keep dividing, not only does the papillomavirus retain the nuclear machinery within the cell, but also amplifies the infection by ensuring that each dividing keratinocyte contains replicating papillomavirus. As the infected keratinocytes approach the surface of the epithelium viral capsids are produced and virions are assembled. An infected cell may produce 10,000–100,000 new virus particles. Papillomaviruses do not cause cell lysis and virions are only released after the epithelial cell has been sloughed from the epithelial surface and degraded. Replication of the papillomaviral DNA begins when an initiation complex binds to a single unique origin of replication resulting in unwinding of the DNA. A single DNA strand is synthesized continuously in the direction of unwinding with an additional DNA strand synthesized discontinuously in the opposite direction. As replication proceeds, the torsional strain created by the unwinding of the parental strands of DNA is released by the E1 protein. Bidirectional replication proceeds around the full genomic DNA circle resulting in the formation of two new strands of circular DNA. Infection by papillomaviruses can result in a variety of clinical manifestations that include asymptomatic infection, papillomas (warts), viral plaques, and neoplasia. The manifestation of infection is largely dependent on whether or not the papillomavirus is replicating and the ability of the viral oncoproteins to promote epithelial cell division. Most papillomavirus infections are asymptomatic. Such asymptomatic infections may be due to a papillomavirus infection remaining latent and not replicating.

Animal Papillomaviruses (Papillomaviridae)

83

Alternatively, papillomavirus infections may remain asymptomatic when replication by the papillomavirus occurs only at a slow rate. Slow viral replication only mildly increases epithelial cell proliferation and visible changes in the epithelium will not develop. Papillomavirus infections that are characterized by slow viral replication generally stimulate no, or a weak, immune reaction. Therefore such infections may persist, resulting in the production of small numbers of infectious virions throughout the life of the host. In contrast, if the papillomavirus is able to stimulate marked epithelial proliferation, the thickened epithelium will become folded resulting in a visible papilloma. By stimulating such marked epithelial proliferation large numbers of infective virions are produced. However, the changes to the epithelium will be detected by the host resulting in an immune response, regression of the papilloma, and prevention of further viral replication. Viral plaques tend to be caused by papillomaviruses types that are normally asymptomatic, but an inability of the host to limit the rate of replication enables them to stimulate greater epithelial proliferation. Papillomaviruses can also predispose to neoplasia. The likelihood that a papillomavirus will cause neoplasia is not dependent on the degree of epithelial proliferation that is able to be induced by the papillomavirus. Indeed the human papillomaviruses that cause warts rarely cause cancer.

Epidemiology There are currently few studies investigating the epidemiology of papillomavirus infections in animals. However, it appears likely that there are significant differences in the epidemiology of infection between papillomaviruses that are generally asymptomatic and papillomaviruses that cause papillomas. The overwhelming majority of papillomaviruses are well adapted to their hosts and infections by these papillomavirus types typically remain asymptomatic. These papillomaviruses infect a high proportion of individuals with infection being from the dam, either during the birth process or due to the close contact between the mother and newborn shortly after birth. While direct contact is the most accepted mechanism of transfer, there is some evidence that transplacental transfer of papillomaviruses could be possible. Examples of papillomaviruses that are transmitted this way include the human betapapillomaviruses that ubiquitously infect the skin of people and Felis catus papillomavirus type 2 which can be detected in almost all cats from very early in life. Papillomaviruses that cause papillomas appear to have a different epidemiology as evidence suggests that papilloma development occurs when an animal is first infected by the papillomavirus. This infection could be the result of direct contact or indirect contact from a fomite. The initial infection results in a period of rapid virus replication that results in the development of a visible papilloma. Due to the large number of virions produced in a papilloma, an animal is likely to be highly contagious at this time. However, after a variable period of time, the body mounts a cell-mediated immune response against the papilloma, inhibiting viral replication and causing papilloma regression. Due to the ability of the infection to be maintained within the basal cells, it is likely that the infection is never completely resolved although it appears that few, if any, infectious virions are produced. This method of infection and disease development is illustrated by the ‘outbreaks’ of papillomas that have been rarely reported in animals.

Clinical Features Infection by papillomaviruses can result in four distinct clinical manifestations that will be described separately. Due to differences between the species, these manifestations of papillomaviral disease will also be described separately for ruminants, horses, dogs, and cats. It should be noted, many other non-human species develop lesions due to papillomavirus infection and the list of species herein is not intended to be comprehensive.

Papillomas Papillomas are the result of papillomavirus-induced thickening and folding of the epithelium. They mostly develop in young animals, presumably at the time of first infection by the causative papillomavirus type. Humoral antibodies are formed that protect the animal from additional infection by the papillomavirus type and, after a variable amount of time, the body mounts a cell-mediated immune response that causes spontaneous regression of the papilloma. The infection is probably not completely cleared and later immunosuppression may allow the papillomavirus to cause further papillomas. As papillomas typically regress and do not contain mutations within the cell DNA, they are not considered neoplasms. Papillomavirus-induced papillomas are commonly referred to as ‘warts’ in humans.

Ruminants Papillomas in cattle are common with most animals developing these at some time in their lives. Papillomaviruses can be spread by direct contact or by fomites such as contaminated milking equipment, rubbing posts or wire fences. Sexual transmission of papillomavirus-induced genital papillomas is likely.

84

Animal Papillomaviruses (Papillomaviridae)

Papillomas in cattle can be subdivided into squamous papillomas that consist solely of proliferating epithelium and fibropapillomas in which there is proliferation of both the overlying epithelium and the underlying dermal fibroblasts. Cattle develop mucocutaneous papillomas and papillomas of the upper alimentary tract. Mucocutaneous papillomas of cattle are most common around the head, but also commonly develop on the penis, vulva, and teats (Fig. 3(a)). As recent studies have revealed that most papillomas contain numerous BPV types, whether or not papillomas in particular locations are more likely to be caused by a specific BPV type is unknown. However, it appears likely that fibropapillomas are caused by infection with the deltapapillomavirus types, predominantly BPV-1 and -2. Rarely florid persistent mucocutaneous papillomas can be present and these can cause morbidity or mortality by interfering with vision or predisposing to secondary infection. It is currently unknown why some cattle appear unable to make an effective cell-mediated immune response against the papillomas. As these animals are invariably slaughtered, it is also unknown whether these warts would eventually have spontaneously resolved or would have progressed to neoplasia. Upper alimentary papillomas develop from the caudal aspect of the oral cavity to the rumen. They are subdivided into squamous papillomas and fibropapillomas that are thought be to be caused by BPV-4 and BPV-2 respectively. These papillomas typically remain small and self-resolve and clinical disease due to these papillomas is extremely rare. However, in animals that are exposed to the immunosuppressive and carcinogenic chemicals in bracken fern, upper alimentary squamous papillomas can become persistent and these papillomas can undergo neoplastic transformation. Sheep develop cutaneous squamous papillomas and fibropapillomas most commonly around the muzzle, feet, and mammary gland. As with other species, the papillomas spontaneously resolve. Sheep are infected by three deltapapillomavirus types, although it is currently unknown which of these cause fibropapillomas. Fibropapillomas of the mammary glands have also been reported in goats.

Horses Equine papillomas can be subdivided into those of the genitals and those that develop in other mucocutaneous areas. All are squamous papillomas and fibropapillomas are not recognized in horses. Genital papillomas in horses are thought to be caused by Equus caballus papillomavirus type 2 (EcPV-2). Papillomas are more common in males and usually develop on the free part of the penis (Fig. 3(b)). Unlike papillomas in other species, equine genital papillomas most often develop in middle-aged or older animals. It is currently unknown how EcPV-2 is spread between horses,

Fig. 3 (a). Teat papillomas on an Angus cow. Multiple filiform squamous papillomas are visible. (b). Penile papillomas on a horse. Numerous papillomas are present and these have extended to involve the majority of the free part of the penis (courtesy Dr. Richard Malik, Centre for Veterinary Education, University of Sydney, Australia). (c). Oral and cutaneous papillomas on a dog. Despite their large size and number, such papillomas rarely cause discomfort to the dog. (courtesy Dr. Stephen White, University of California at Davis, California, USA). (d). Pigmented plaques on a dog. Multiple sessile darkly pigmented plaques are visible scattered over the legs of this dog (courtesy Dr. Mark Turnwald, Belmont Veterinary Clinic, North Shore City, New Zealand).

Animal Papillomaviruses (Papillomaviridae)

85

but as papillomas develop in castrated horses, sexual transmission is unlikely. The biological behavior of equine genital papillomas is currently unresolved with some spontaneously resolving, others becoming numerous and persistent, and a proportion progressing to penile squamous cell carcinoma (SCC). Non-genital warts in horses are common and are thought to be predominantly caused by EcPV-1. They are most common in younger horses on the muzzle, lips, distal limbs, and eyelids. Papillomas are typically multiple and individual papillomas can coalesce into a single large mass. Non-genital papillomas in horses typically regress within 3 months and persistent infections or progression to neoplasia appears to be extremely uncommon.

Dogs Papillomas are common in dogs and are subdivided into those of the oral cavity and those of the skin. The majority of oral papillomas are believed to be caused by Canis familiaris papillomavirus type 1 (CPV-1, formerly canine oral papillomavirus, COPV). Oral papillomas typically develop in younger dogs and the exophytic vegetative lesions can be multiple and extensive (Fig. 3(c)). However, despite the spectacular clinical appearance, it is rare for papillomas to interfere with eating or breathing by the animal. Most cases will spontaneously resolve within 3 months, although around 30% of papillomas will persist for longer and complete resolution can take up to a year in some cases. There are rare reports of persistent canine oral papillomas and these probably develop due to an ineffective papillomavirus-specific cell-mediated immune response. Such papillomas may be predisposed to progression to oral SCC, although this appears to be an extremely rare event. Canine cutaneous papillomas have been associated with CPV-1, -2, -6, and -7. They are most common in young dogs and affected dogs often develop multiple papillomas. Papillomas develop most frequently in areas that are more likely to have skin abrasions such as the face, ears, and extremities. Canine cutaneous papillomas are further subdivided into exophytic papillomas in which the folded epidermis protrudes above the surface of the skin and inverted papillomas in which the folded epithelium is contained within a central depressed cup-shaped structure. Both exophytic and inverted papillomas are expected to spontaneously regress and it is currently uncertain whether these two lesions are caused by different papillomavirus types. Progression of a cutaneous papilloma to a SCC appears to be extremely rare in dogs.

Cats Domestic cats are unusual because both cutaneous and oral papillomas are extremely rarely reported. Feline oral papillomas appear as clusters of small pale flattened plaques on the underside of the tongue. These papillomas are thought to be caused by FcaPV-1. They are not associated with clinical signs of disease and probably spontaneously regress.

Viral Plaques Viral plaques are much less common than papillomas. They probably develop when the host is unable to suppress infection by a normally asymptomatic papillomavirus type. The failure to inhibit papillomavirus replication results in moderate epithelial thickening and the development of a plaque. As the lesions do not necessarily develop when the animal is first infected, plaques tend to develop on older animals than papillomas which typically develop in younger animals. While spontaneous resolution of plaques can occur, they can persist over a long period of time and some may progress to SCCs.

Horses Horses develop precancerous plaques of the penis and aural plaques within the concave surface of the ear pinna. Penile plaques are thought to be caused by EcPV-2 and probably represent an intermediate lesion between a papilloma and a SCC. Aural plaques have been associated with EcPV-3, -4, -5 and -6 with the papillomaviruses thought to be spread between horses by species of black flies (Simulium spp.). The prevalence of aural plaques is not known and it is possible many horses have these lesions without their owners seeking veterinary attention. Aural plaques can develop on horses of any age and consist of multiple discrete grey-white, roughened papules that can coalesce to form plaques up to several cm in diameter. Aural plaques may be unilateral or bilateral and are typically only a cosmetic concern. Aural plaques do not regress spontaneously, but have not been reported to progress to SCC.

Dogs Canine pigmented plaques are thought to be caused by a number of closely-related Chipapillomavirus types. Some breeds of dogs, in particular Pugs and Vislas appear to be predisposed to pigmented plaques suggesting a breed-specific failure in normal suppression of replication by these papillomavirus types. Dogs receiving immunosuppressive therapy are also at increased risk for plaque development, although plaques often develop on dogs with no identifiable immunosuppression. Plaques are typically dark, multiple, and 1–10mm in diameter. They are most common on the ventrum and medial aspects of the limbs (Fig. 3(d)). Canine pigmented plaques are considered cosmetically undesirable, but usually do not cause significant morbidity. There are rare reports of progression to SCC although it is currently unknown whether or not plaques caused by specific papillomavirus types are more likely to progress to neoplasia.

86

Animal Papillomaviruses (Papillomaviridae)

Fig. 4 (a). Pigmented viral plaques/Bowenoid in situ carcinomas on the face of a domestic short haired cat. Two lesions are visible on the face of this cat (arrows; courtesy Dr. Richard Malik, Centre for Veterinary Education, University of Sydney, Australia). (b). Papillomas and squamous cell carcinomas of the penis of a horse. The penis of this horse is covered by papillomatous growths, some of which have progressed to a squamous cell carcinoma. (c). Viral plaques/Bowenoid in situ carcinomas and squamous cell carcinomas on a Devon Rex cat. Histology was consistent with the squamous carcinomas developing from pre-existing viral plaque/Bowenoid in situ carcinomas. Some lesions were covered by a thick layer of keratin (arrow; from Munday, J.S., Benfell, M.W., French, A., Orbell, G.M., Thomson, N., 2016. Bowenoid in situ carcinomas in two Devon Rex cats: Evidence of unusually aggressive neoplasm behavior in this breed and detection of papillomaviral gene expression in primary and metastatic lesions. Veterinary Dermatology 27, 215–e55, with permission). (d). Equine sarcoid. This sarcoid developed close to the eye of this 3-year-old horse (arrow; courtesy Dr. Luca Panizzi, Massey University, New Zealand).

Cats Papillomavirus-induced cutaneous plaques in cats have historically been subdivided into viral plaques and Bowenoid in situ carcinomas (BISCs). However, as transitional lesions between the two lesions have been reported, they are best considered different severities of the same disease process. Feline viral plaques/BISCs are most often caused by FcaPV-2. Most cats are infected with FcaPV-2 shortly after birth and disease development appears to be primarily determined by, as yet poorly understood, host factors. Viral plaques/BISCs are uncommon in cats and usually develop in middle-aged or older animals. Lesions are most common on the head and neck of cats and can be single or multiple (Fig. 4(a)). Lesions are typically pigmented, mildly raised, and hairless. More advanced lesions can be ulcerated or covered by thick scaling. Lesions can spontaneously resolve, be persistent without progressing, or slowly increase in size and number. In addition, viral plaques/BISCs are pre-neoplastic and all viral plaques/BISCs should be carefully monitored for progression to a SCC. Viral plaques/BISCs tend to develop in Devon Rex and Sphinx cats at an earlier age and these lesions are predisposed to rapid progression to highly metastatic SCCs in these breeds.

Squamous Cell Carcinomas In humans, papillomaviruses cause around 5% of all cancers including almost all SCCs of the cervix and 20%–30% of oral SCCs. The papillomaviruses that cause these cancers are a select group of sexually-transmitted ‘high-risk’ alpha papillomavirus types. A key feature of these papillomaviruses is the ability of their E6 proteins to prevent accurate replication of epithelial cell DNA and to inhibit cell apoptosis. This predisposes the proliferating epithelial cells to developing genetic mutations which can result in the development of neoplasia. While papillomaviruses in the domestic species have been associated with neoplasia development, the role of the papillomavirus in cancer development and the mechanisms by which the papillomaviruses cause neoplastic transformation are much less well understood.

Ruminants Vulval papillomas have been reported to progress to SCCs in cattle that have been exposed to high levels of ultraviolet (UV) light. However, it is currently unknown whether or not the vulval papillomas are caused by papillomavirus infection and there is currently little evidence that papillomaviruses cause skin cancer in cattle.

Animal Papillomaviruses (Papillomaviridae)

87

Upper alimentary tract SCCs are extremely common in cattle that ingest bracken fern. As these SCCs can develop within a preexisting papilloma, it is possible that BPV-4 could influence the development of these cancers. However, the precise role of the papillomavirus is currently uncertain and PV DNA is not detectible in the SCCs. Furthermore, bracken fern has directly carcinogenic properties suggesting that ingestion of bracken fern could be a more significant factor in the development of cancer than the presence of the papillomavirus. The critical role of bracken fern is supported by the rarity of upper alimentary tract SCCs in cattle that are not exposed to bracken fern. Cutaneous squamous cell carcinomas in sheep were first proposed to be influenced by papillomavirus infection in 1982 when it was observed that some SCCs developed as a progression from a papilloma and that some SCCs contained PV particles. More recently, Ovis aires papillomavirus type 3 (OaPV-3) DNA and gene expression was found more frequently in a series of ovine cutaneous SCCs than in non-neoplastic samples of skin. Current evidence suggests that OaPV-3 could be a significant co-factor with UV light in the development of cutaneous SCCs in sheep. However, as SCCs rarely develop in sheep in the absence of high levels of solar radiation, it is difficult to determine the relative importance of papillomavirus infection and UV light in the development of these lesions. It is also impossible to exclude that higher rates of OaPV-3 are present within the SCCs simply because these cancers provide a more permissive environment for papillomavirus infection.

Horses Penile SCCs are associated with EcPV-2 infection in horses. Currently the mechanisms by which EcPV-2 causes neoplasia are poorly understood. However, it is hypothesized that a high proportion of penile SCCs develop due to progression of a virallyinduced papilloma to a precancerous plaque and subsequently to an invasive cancer (Fig. 4(b)). The degree to which EcPV-2 promotes the progression from papilloma to cancer is uncertain and it is possible that additional cofactors are required for neoplasm development. While both smegma and exposure to sunlight have been suggested as potential co-factors, there is currently little evidence supporting either as significant in the development of penile cancer in horses. In addition to penile SCCs, there is some evidence that EcPV-2 may also cause a proportion of vulval and oropharyngeal cancers in horses, although currently too few cases have been evaluated to draw conclusions about the role of the papillomavirus in these neoplasms.

Dogs Cutaneous viral plaques and papillomas have occasionally been reported to progress to SCCs in dogs, although the overwhelming majority of cutaneous SCCs in this species do not appear to be caused by papillomavirus infection. There are just two reports of oral SCCs being influenced by papillomavirus infection in dogs. In one case, CPV-17 was found within multiple oral plaques and SCCs in a dog. These lesions contained histological evidence of viral replication suggesting that the papillomavirus was the cause of the lesions. Interestingly, CPV-17 has not subsequently been detected in any canine lesion. In the second report a dog had oral papillomas for over 18 months before one progressed to an invasive SCC. Overall, as with cutaneous SCCs, evidence currently suggests that papillomaviruses are not a significant cause of oral SCCs in dogs.

Cats Papillomaviruses were first associated with feline cutaneous SCCs in 2008 when FcaPV-2 DNA was amplified significantly more frequently from SCCs than from non-neoplastic skin samples. Subsequent evidence has suggested that papillomaviruses could influence the development of 33%–45% of feline cutaneous SCCs. A papillomavirus etiology of a SCC may be most likely for a cancer that develops in skin that is protected from sunlight and it is possible that these SCCs could develop from a pre-existing viral plaque/BISC (Fig. 4(c)). The role of papillomaviruses in SCCs that develop in sun exposed skin is less clear. In people there is good evidence that papillomaviruses and sunlight act as co-factors in the development of SCCs and the same could be true in cats. However, as papillomaviruses commonly asymptomatically infect skin, it is currently not possible to definitively exclude the papillomaviruses being present in the SCCs simply because they provide a more permissive environment. While PV DNA is detectable in a small proportion of feline oral SCCs, there is no evidence that PVs are a significant cause of these neoplasms.

Rabbits Infection of cottontail rabbits with Sylvilagus floridanus papillomavirus type 1 (formerly Shope’s papillomavirus, cottontail rabbit papillomavirus, CRPV) causes multiple papillomas which progress to SCCs in around a quarter of rabbits. The consistent development of papillomas and cancer due to papillomavirus inoculation enabled rabbits to be used as an animal model during the development of HPV vaccines.

Sarcoids Sarcoids are proliferations of fibroblasts that are induced by cross-species infection by bovine deltapapillomaviruses. Sarcoids do not metastasize, but tend to be invasive making complete excision difficult.

88

Animal Papillomaviruses (Papillomaviridae)

Horses Sarcoids have been reported in horses, donkeys, mules and zebras and are the most common skin tumor of equids worldwide. Sarcoids can be single or multiple and may occur anywhere on horses but are more common on the head, neck, and limbs (Fig. 4(d)). They often develop at sites of previous surgery or trauma. Sarcoids are most common in young to middle-aged horses and breed predispositions in Quarter horses, Appaloosas and Arabians have been described. Sarcoids are thought to be caused by infection with BPV-1, -2 or -13, although sarcoids cannot be induced experimentally by inoculation with these papillomaviruses. It is currently not clear whether BPVs are transmitted from infected cattle, from infected horses, or from both. Horses have historically been considered dead-end hosts of BPV infection, with early studies detecting only BPV DNA in dermal fibroblasts, but never infectious BPV virions. However, expression of the BPV L1 gene has been detected in sarcoids, suggesting the possibility of viral replication within the lesion. Additionally, full-length BPV genomes complexed with L1 capsid proteins, possibly representing virions, have been detected in two equine sarcoids.

Cats Feline sarcoids appear to be caused by BPV-14. As cattle are the definitive host of the presumptive causative papillomavirus, it is unsurprisingly that feline sarcoids are restricted to cats that have contact with cattle and are only seen regularly in ‘barn cats’ in the Midwest of the United States of America. Feline sarcoids most frequently develop on the nasal philtrum, although they can develop anywhere on the body or in the mouth. The mechanism of spread is poorly understood, but the lesion distribution suggests that biting flies or cat-fight wounds may be important for BPV-14 infection. BPV-14 is a deltapapillomavirus that is closely related to the BPVs that cause equine sarcoids. However, it is interesting that BPV-14 has not been detected in an equine sarcoid and neither BPV-1 nor -2 have been detected in a feline sarcoid. This suggests that, although the deltapapillomaviruses can cause cross-species infection, the range of species able to be infected may be limited.

Other Neoplasms Deltapapillomavirus DNA and viral proteins, including those of types -2, -13, and -14, have been found more frequently in bladder neoplasms of cattle and water buffalo than in non-neoplastic bladder samples. While this suggests a possible carcinogenic role of the BPVs, the development of bladder neoplasia in cattle is dependent on the ingestion of bracken fern. Therefore, it cannot be excluded that the high rates of BPV detection in the bladder tumors is due to the immunosuppressive action of bracken fern, rather than because the papillomaviruses directly contribute to cancer development. It is interesting to note that deltapapillomavirus infections of the bladder of cattle are productive and this appears to be a rare example of papillomaviruses being able to replicate in a location other than squamous epithelium. How bladder infection occurs is currently unknown. However, as papillomavirus DNA is detectible in the blood, hematogenous infection of the bladder mucosa may be most likely. Interestingly a wide range of bladder tumors are associated with papillomavirus infection including urothelial and mesenchymal types.

Pathogenesis Papillomaviruses cause disease by stimulating increased epithelial cell replication. If there is a marked increase in replication then the infection can result in a papilloma while a more moderate increase in replication could result in a viral plaque or an asymptomatic infection. Due to their ability to interfere with the normal regulation of cell division, some papillomaviruses may influence the development of neoplasia. Because papillomaviruses do not cause cell lysis and only produce viral antigens in the superficial layers of the epidermis, infection may elicit no immune response. If an immune response is made, it consists of the production of antibodies that protect against subsequent infection by this papillomavirus type and a cell-mediated response against cells that are infected by the papillomavirus. The initiation of a cell-mediated response is responsible for the resolution of a developed papilloma.

Diagnosis A papillomavirus etiology of a lesion can be diagnosed by clinical examination, histology of the lesion, immunohistochemistry, or molecular techniques. Not all methods of diagnosis are appropriate for each of the types of papillomavirus-induced lesion. Histology of a papillomavirus-induced lesion generally reveals thickened epithelium (Fig. 5(a)). If the papillomavirus is replicating within the lesion, papillomavirus-induced cell changes may be visible. These changes include the presence of enlarged cells with increased quantities of blue to grey slightly granular cytoplasm, cells that have shrunken nuclei that are surrounded by a clear halo (koilocytes), cells with basophilic or eosinophilic cytoplasmic bodies, cells with enlarged vesicular nuclei, or cells with eosinophilic intranuclear viral inclusion bodies (Fig. 5(b)). Clumping of keratohyalin granules can also be visible within the superficial layers of the epidermis.

Animal Papillomaviruses (Papillomaviridae)

89

Fig. 5 (a). Canine papilloma. The papilloma consists of marked epithelial proliferation. The papilloma is clearly demarcated from underlying dermis and the thickened epidermis is covered by increased keratin. This papilloma was caused by Canis familiaris papillomavirus type 1. (b). Canine papilloma. Higher magnification of the papilloma reveals papillomavirus-induced cell changes. These consist of enlarged cells with clear cytoplasm, cells with vesicular nuclei, and prominent clumping of keratohyalin granules.

Fig. 6 Papillomavirus-associated cutaneous squamous cell carcinoma from a cat. Note the intense cytoplasmic and nuclear immunostaining using antibodies against p16CDKN2A protein (p16). This immunostaining pattern suggests that the behavior of the neoplastic cells was altered by papillomavirus infection.

There are two strategies that can be used to detect a papillomavirus etiology of a lesion using immunohistochemistry. Firstly, anti-papillomaviral antibodies can be used to detect the papillomavirus L1 protein. A significant disadvantage of this strategy is that L1 protein (and therefore immunostaining) will only be present in lesions that contain active viral replication and it is rare for immunostaining to be present in a lesion that does not contain papillomavirus-induced cell changes. In addition, as there are no commercially-available antibodies to most papillomavirus types of domestic species, the cross-reactivity of the antibodies is often unknown. The second strategy involves using immunohistochemistry to detect p16CDKN2A protein (p16). Papillomaviruses consistently alter cell regulation by mechanisms that increase cell p16. Therefore the detection of increased p16 in a cell is consistent with the behavior of that cell having been altered by papillomavirus infection (Fig. 6). An advantage of this strategy is that p16 immunostaining will be increased regardless of the replication status of the papillomavirus in the lesion. Additionally, a commercially available anti-p16 antibody (clone G175–405) has been shown to cross react with the canine and feline p16 protein, although this antibody does not appear to cross react with the equine p16 protein. As the bovine deltapapillomaviruses cause cell proliferation by alternative pathways, increased p16 is less likely to be present in lesions caused by these papillomaviruses. Molecular techniques available include PCR and in situ hybridization. PCR has the advantage of being extremely sensitive and, unlike immunohistochemistry, able to determine the papillomavirus type present within the lesion. However, as papillomaviruses often incidentally infect the skin of animals, simply detecting papillomavirus DNA within a lesion is not conclusive evidence that the papillomavirus caused the lesion. As in situ hybridization is able to localize the DNA within a lesion, this technique provides greater evidence that the papillomavirus was the cause of the lesion. However, as with PCR, the possibility of incidental infection cannot be excluded. The sensitivity of in situ hybridization is lower than PCR.

90

Animal Papillomaviruses (Papillomaviridae)

Papillomas Papillomas are usually diagnosed by clinical examination with the presence of multiple exophytic lesions in the mouth or over the skin of a younger animal consistent with this diagnosis. Histology is rarely needed but reveals thickened folded epithelium. As papillomas are formed due to florid viral replication, papillomas are expected to contain prominent papillomavirus-induced cell changes and immunostaining using anti-papillomaviral antibodies.

Viral Plaques Viral plaques can be suspected on clinical examination due to their appearance and location. However, canine pigmented plaques and feline viral plaques/BISCs will often require histology for definitive diagnosis. Histology of a viral plaque reveals a welldemarcated focus of mild to moderate epidermal hyperplasia that often has a scalloped appearance. The hyperplasia tends to remain superficial with little involvement of adjacent hair follicles. Cell atypia and crowding can be present, especially in more advanced feline viral plaques/BISCs. Feline and canine plaques can contain increased melanin while hypomelanosis is described as a feature of equine aural plaques. Papillomavirus-induced cell changes and immunostaining using anti-papillomavirus antibodies are variably present. Feline viral plaques/BISCs contain intense immunostaining with anti-p16 antibodies. It is currently unknown whether similar immunohistochemical changes are visible in canine or equine viral plaques.

Squamous Cell Carcinomas and Other Neoplasms Squamous cell carcinomas are characterized by the presence of invasion of dysplastic cells through the basement membrane of the epithelium. Significant viral replication is not typically present within either SCCs or bovine bladder neoplasms and papillomavirus-associated neoplasms are not expected to contain papillomavirus-induced cell changes or immunostaining using antipapillomavirus antibodies. There is good correlation between the presence of papillomavirus DNA and p16 immunostaining in feline cutaneous squamous cell carcinomas (SCCs), suggesting that, like human oral SCCs, p16 immunostaining may be a good indicator of a papillomaviral etiology of a feline cutaneous SCC. Additionally, p16 immunostaining was observed in a canine oral SCC that was associated with papillomavirus infection. The papillomavirus-associated cancers are expected to contain viral nucleic acids and molecular tests can be used to localize these within the lesions.

Sarcoids Histology reveals a proliferation of well-differentiated dermal fibroblast-like cells underlying a thickened epidermis that has characteristic broad interlacing rete pegs. As these lesions support little viral replication, neither papillomavirus-induced cell changes nor immunostaining using anti-papillomavirus antibodies are present. Molecular tests can detect papillomavirus DNA, mainly within proliferating fibroblasts. In cats, as BPV-14 does not appear to infect the skin of cats asymptomatically, the detection of BPV-14 from a skin tumor provides strong evidence to support a diagnosis of feline sarcoid. While the detection of BPV DNA from an equine skin mass is supportive of a diagnosis of equine sarcoid, caution has to be used as BPV DNA has been detected in skin from horses without sarcoids.

Treatment Papillomas Papillomas in all species typically cause little or no morbidity and are expected to spontaneously regress after a variable length of time. For the overwhelming majority of cases, benign neglect is the treatment of choice. Surgical excision, laser treatment, or cryotherapy can be used for lesions that develop in locations that cause pain or interfere with function. Papillomas can recur after surgical excision, although such warts are expected to subsequently regress once the body mounts an appropriate cell-mediated immune response. Numerous medical therapies have been proposed to hasten the resolution of papillomas. However, none has sufficient evidence of efficacy from controlled studies to be confident of any benefit. Crushing of papillomas or injecting excised papillomas back into animals (autologous vaccination) have been historically used, but is not supported by controlled studies. Likewise the use of papillomavirus vaccines has not been shown to influence papilloma regression in either humans or animals.

Viral Plaques Viral plaques appear to only rarely spontaneously regress. As plaques are mainly a cosmetic concern in dogs and horses, they can often be left without further treatment. In cats, viral plaques/BISCs should be carefully observed due to the potential of these lesions to progress to a SCC. Treatment options for viral plaques include surgical excision, laser therapy, or cryotherapy. Imiquimod cream has been used to treat plaques in cats and horses. Imiquimod is not specific for papillomaviruses, but works by stimulating a local immune response that can cause the resolution of epidermal lesions.

Animal Papillomaviruses (Papillomaviridae)

91

Squamous Cell Carcinomas and Sarcoids There are numerous techniques for removing these neoplasms that are out of the scope of this discussion. In cats it is possible to subdivide cutaneous SCCs into those that are associated with papillomavirus infection and those that are not. Evidence suggests that papillomavirus-associated SCCs may have a more favorable prognosis than those not caused by papillomavirus infection.

Prevention Vaccines that contain the L1 capsid protein prevent papillomavirus infection and subsequent papillomavirus-induced neoplasia in people. This suggests that vaccination could also be used to prevent papillomavirus-induced disease in animals. However, there are some key considerations regarding the use of papillomavirus vaccines in animals. Firstly, there appears to be little cross-protection between papillomavirus types. Therefore, vaccines would have to be created that protect against each individual papillomavirus type. Secondly, a vaccination has to be given prior to first infection. All the papillomavirus types that are included in the human vaccines are sexually transmitted. This is critical as it can be guaranteed that people will not be infected by these papillomaviruses prior to vaccination. In the domestic species, the papillomaviruses that cause papillomas appear to most frequently infect their hosts as young adults possibly providing an opportunity to prevent infection by vaccination. In contrast, FcaPV-2, EcPV-2, and the viruses that cause canine pigmented plaques probably infect their hosts soon after birth. If so, novel strategies to allow vaccination prior to first infection will have to be developed. Additional research is required to determine when horses are infected by the BPVs that cause sarcoids. Lastly, vaccines are likely to be expensive and it seems unlikely that a vaccine against self-regressing papillomas will be a commercial success. If papillomaviruses are found to be a key factor in the development of esophageal and bladder cancer in cattle and water buffalo then vaccination against these economically-important diseases could be viable. However, if bracken fern is found to be the major factor causing these cancers, preventing papillomavirus infection may not result in significant protection against cancer.

Further Reading Akgul, B., Cooke, J.C., Storey, A., 2006. HPV-associated skin disease. Journal of Pathology 208, 165–175. Bergvall, M., Melendy, T., Archambault, J., 2013. The E1 proteins. Virology 445, 35–56. Borzacchiello, G., Roperto, F., 2008. Bovine papillomaviruses, papillomas and cancer in cattle. Veterinary Research 39, 45. Doorbar, J., Quint, W., Banks, L., et al., 2012. The biology and life-cycle of human papillomaviruses. Vaccine 30 (S5), F55–F70. Egawa, N., Doorbar, J., 2017. The low-risk papillomaviruses. Virus Research 231, 119–127. Gil da Costa, R.M., Peleteiro, M.C., Pires, M.A., DiMaio, D., 2017. An update on canine, feline and bovine papillomaviruses. Transboundary and Emerging Diseases 64, 1371–1379. Lange, C.E., Favrot, C., 2011. Canine papillomaviruses. The Veterinary Clinics of North America Small Animal Practice 41, 1183–1195. Munday, J.S., 2014a. Bovine and human papillomaviruses: A comparative review. Veterinary Pathology 51, 1063–1075. Munday, J.S., 2014b. Papillomaviruses in felids. Veterinary Journal 199, 340–347. Munday, J.S., Kiupel, M., 2010. Papillomavirus-associated cutaneous neoplasia in mammals. Veterinary Pathology 47, 254–264. Munday, J.S., Thomson, N.A., Luff, J.A., 2017. Papillomaviruses in dogs and cats. Veterinary Journal 225, 23–31. Van Doorslaer, K., Chen, Z., Bernard, H.U., et al., 2018. ICTV virus taxonomy profile: Papillomaviridae. The Journal of General Virology 99, 989–990. Vande Pol, S.B., Klingelhutz, A.J., 2013. Papillomavirus E6 oncoproteins. Virology 445, 115–137. zur Hausen, H., 2009. Papillomaviruses in the causation of human cancers – A brief historical account. Virology 384, 260–265.

Relevant Websites https://talk.ictvonline.org/ictv-reports/ictv_online_report/dsdna-viruses/w/papillomaviridae Papillomaviridae – Papillomaviridae – dsDNA Viruses – International. https://pave.niaid.nih.gov/ PaVE: Papilloma virus genome database.

Astroviruses (Astroviridae) Virginia Hargest, St. Jude Children’s Research Hospital, Memphis, TN, United States and University of Tennessee Health Science Center, Memphis, TN, United States Amy Davis and Stacey Schultz-Cherry, St. Jude Children’s Research Hospital, Memphis, TN, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Capsid The protein coat that surrounds the genetic material of a virus. Fomite An object that can harbor pathogenic organisms and therefore may be involved in the route of transmission of the pathogen. Subgenomic RNA (sgRNA) Viral RNA which can be directly translated into the desired viral proteins without needing to translate the entire genome. Usually translated in excess compared to genomic RNA.

T ¼ 3 Icosahedron In relation to the symmetry of virion structure, a virus with a triangulation number of 3 is composed of 12 pentameric and 20 hexameric capsid subunits for a total of 180 capsid proteins. Transepithelial Electrical Resistance (TER) Method used to assess the barrier function of epithelial cells by measuring current passing through the cells on both transcellular and paracellular paths. Uncoating A condition when the protein capsid of the virus is dismantled revealing the viral genome inside the host organism.

Classification Astrovirus, a non-enveloped positive-sense single-stranded RNA virus, was first discovered in 1975 in the stool of infants suffering from gastroenteritis. The virus obtained its name after Madeley and Cosgrove noted a star-like morphology of a portion of the virions under electron microscopy. In 1993, complete sequencing of the viral genome prompted its classification to a new family to be called Astroviridae. The Astroviridae family has since been divided into two genera: Avastrovirus, those viruses that infect avian species, and Mamastrovirus, those viruses that infect mammalian species (Fig. 1). Avastrovirus and Mamastrovirus are also split into two genogroups based on of their genetic relatedness within the hypervariable capsid protein.

Fig. 1 Phylogenetic classification of Astroviridae. Tree generated using capsid protein amino acid sequences. Based on Ninth Report of the International Committee on Taxonomy of Viruses, 2012.

92

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20997-7

Astroviruses (Astroviridae)

93

Prior to 2008, humans were thought to be infected by only eight closely related genotypes of astrovirus known as human astrovirus type 1 through 8 (HAstV1–8). These eight genotypes are now known as the classical HAstV genotypes. With the advent of unbiased fullgenome sequencing and new pathogen discovery techniques, eight more novel HAstV genotypes were identified, receiving names after the location of their discovery: Melbourne (MLB 1–3) and Virginia (VA 1–5). In the same year the VA genotypes were discovered, the same viruses were discovered in samples from Nigeria, Pakistan, and Nepal. These viruses are sometimes termed HAstV-HMO due to their relatedness to human, mink and ovine astroviruses, however, for the purposes of this review will be termed HAstV-VA. Phylogenetic analysis revealed that the newly identified MLB and VA genotypes are more closely related to other mammalian astroviruses than the classical genotypes creating three genetically distinct clades of HAstV within Mamastrovirus.

Virion Structure The star-like appearance of astrovirus noted by Madeley and Cosgrove is due to spikes studded on the virion surface. Within the past decade, extensive progress has been made in our knowledge of the capsid’s structure using methods including electron and cryo-electron microscopy, X-ray crystallography, and density ultracentrifugation. The crystal structures for the HAstV8 spike, HAstV1 full capsid, HAstV2 spike, and turkey astrovirus type 2 spike have all been described. These studies demonstrate that astrovirus particles are approximately 43 nm in diameter with T ¼ 3 icosahedral symmetry (Fig. 2A) and closely resemble the structure of the human hepatitis E virus virion. The capsid protein is translated as an inactive VP90 precursor that undergoes cleavage before assembly into an infectious virion. First, intracellular caspases cleave VP90 to generate VP70, of which 180 copies are needed to form an immature virion. Two separate studies reported that this caspase cleavage is necessary for viral particles to be released from the cell. Once released from the cell, extracellular proteases further cleave VP70 of the immature virions into VP34, VP27, and VP25 to form mature virions. VP34 makes up the continuous inner core while VP27/VP25 form the spike domains (Fig. 2A). The immature virion has an estimated 90 spikes which is reduced to just 30 spikes on an infectious particle following this final cleavage. Through antigenic studies, these spikes are thought to contain the binding site to the yet to be identified host cell receptor. The astrovirus capsid appears to have other functional roles although its full role in the replication cycle remains an area of active research. The capsid has been reported to bind both complement 1q and mannose-binding lectin to inhibit the classical and lectin pathways of complement. This could be a clue as to how astrovirus is able to infect without causing noticeable histological changes. The astrovirus capsid has also been shown to act as a novel enterotoxin in vitro and in vivo.

Genome The astrovirus genome is a single-stranded positive-sense RNA segment of approximately 6.2–7.7 kb. The genetic material contains both 50 and 30 untranslated regions and three open reading frames (ORFs) flanked by 50 viral genome-linked protein (VPg) and a 30

Fig. 2 Astrovirus Genome and Virion Structure. (A) T ¼ 3 icosahedral structure of the astrovirus capsid composed by an inner core of VP34 and VP27/VP25 in the spike domains. (B) The three open reading frames (ORF) of astrovirus flanked by a 50 viral genome linked protein (VPg) and a 30 poly(A) tail with known protein coding regions designated by inset boxes. Darker regions of ORF2 indicate hypervariable regions. Modified from SIB Swiss Institute of Bioinformatics, ViralZone Astroviridae.

94

Astroviruses (Astroviridae)

poly(A) tail (Fig. 2B). ORF1a and ORF1b, located at the 50 end of the genome, both encode the astrovirus nonstructural proteins. While astrovirus could have as many as five nonstructural proteins, the three characterized nonstructural proteins include a serine protease, the VPg, and an RNA-dependent RNA polymerase (RdRp). A ribosomal frameshift signal is present between ORF1a and ORF1b and is critical for the correct translation of the RdRp. ORF2, at the 50 end of the genome, encodes the astrovirus structural protein and is expressed from a subgenomic RNA (sgRNA). This sgRNA was confirmed when two species of viral RNA were detected in astrovirus infected cells, both full-length genomic RNA and a roughly 2.8 kb sgRNA. This allows for an excess of astrovirus capsid protein to be translated and ready for assembly during the viral replication cycle.

Life Cycle Binding and Entry Despite dedicated work, much of the astrovirus replication cycle remains unknown. Our understanding comes mainly from studying a few of the classical HAstV genotypes, and inference from the replication cycles of closely related viral families like Togaviridae and Caliciviridae. The cellular receptor or receptors for astroviruses are still unknown, and given that different cell types have different susceptibilities for various HAstV genotypes it is likely a variety of binding factors may be needed for cell entry. For example, while human colorectal adenocarcinoma (Caco-2) cells can support the replication of all HAstV1–8 genotypes, baby hamster kidney (BHK-21) cells can only be infected by HAstV2, and human colorectal carcinoma epithelial (HTC-15) cells can support only HAstV1 replication. Crystallization of the capsid and spike proteins revealed conserved polysaccharide binding motifs meaning these could be involved in viral binding. Nevertheless, pretreatment of Caco-2 cells with heparinase was unable to inhibit viral binding, while pretreatment with chondroitinase was only able to reduce infectivity, showing further research is needed to understand how these sites could be involved in binding. After attachment of the virus to the host cell, entry appears to be facilitated by clathrin-mediated endocytosis. Electron microscopy has displayed astrovirus particles localized in coated pits and vesicles and blocking clathrin assembly has been shown to inhibit HAstV8 replication. Once endocytosed, vesicles containing astrovirus particles must reach late endosomes. Blocking Rab7 was shown to block replication of HAstV8 suggesting the acidic environment found in late endosomes is required for proper uncoating and release of the viral genome.

Uncoating and Replication While the exact mechanism for viral uncoating is unknown, studies show the process is membrane associated and mediated by an endoplasmic reticulum signal motif located in the C-terminus of VP90. Analysis of recombinant viral-like particles (VLPs) showed that divalent cations in the capsid protein provide stability to the viral particles. Therefore, the acidic environment of late endosomes and typically low cation concentration in the cytoplasm should both promote virus uncoating. Once the viral genome has been released, the first proteins translated after viral uncoating are the nonstructural proteins encoded by ORF1a and ORF1b. The nonstructural polyprotein, nsp1a, is cleaved by the viral serine protease, conceivably along with unidentified cellular proteases into smaller proteins some whose function is still unknown. One of the nsp1a proteins, nsp1a/4, is a VPg that has been shown to localize with viral RNA and is thought to facilitate viral replication and possibly transcription from the negative-sense RNA template. Although direct interaction between the VPg and viral genome has not been confirmed, pretreatment of viral RNA with proteinase K before transfection into permissive cells significantly reduced the infectious virus produced, indicating a protein moiety is required for successful replication. The production of the capsid protein involves both genomic and sgRNA, with an excess of sgRNA at 12 h post-infection (hpi) coinciding with higher levels of capsid protein production. This is characteristic common among many positive-sense single-stranded RNA viruses as a means of evading repression of translation by the host. While caliciviruses possess a VPg on their sgRNA segments to initiate its translation, it is unclear if astroviruses utilize a similar mechanism. The precise role host proteins play during astrovirus replication has yet to be fully addressed, although one study has implicated host proteins involved in fatty acid and cholesterol synthesis, phosphatidylinositol and inositol metabolism, and RNA helicase activity, all being necessary viral replication. Our lab has demonstrated that the increase in phosphorylated ERK1/2 protein that occurs within 15 min after viral binding is required for successful viral replication. Recently, others have reported the ubiquitin proteasome system is also needed replication of the viral genome and sgRNA.

Assembly and Release The location for viral assembly appears to be genotype dependent with HAstV4 and HAstV8 accumulating in double membrane vacuoles near the nucleus, while ovine astrovirus has been found in lysosomes and autophagic vesicles. In these membraned sites, viral capsids can self-assemble into full virions or VPLs, and viral release occurs via an unknown, nonlytic mechanism without significant cell death. It has been hypothesized that astrovirus release could occur through similar nonlytic mechanisms used by rotavirus or poliovirus, or through a form of cell membrane destabilization. Overall, many questions remain involving the replication cycle of astrovirus and more research is needed to elucidate key aspects of every step in the cycle including what

Astroviruses (Astroviridae)

95

receptors and binding factors are used, the mechanism of viral release without inducing cell death, and how cellular factors are involved in this process.

Epidemiology Astrovirus is spread via the fecal-oral route and can be transmitted through contaminated food and water, human-to-human, or through nosocomial infection. The fecal-oral route of transmission was confirmed through two volunteer studies, where after ingesting astrovirus subjects developed symptoms and detectable serological responses.

Classical HAstV Classical HAstV infections are predominantly reported in pediatric population, but the elderly and immunocompromised are also susceptible to infection. Although less common, healthy adults have been reported with HAstV infections. The age range of typical infection is newborn to 5 years, with up to 90% of children ages 5 years and up having detectable serum antibodies. Although there tends to be a seasonal pattern with higher incidence in winter, possibly due to enhanced stability in colder temperatures, astrovirus circulates year-round with infections reported in summer months as well. Classical HAstVs are thought to be responsible for between 2% and 9% of all acute, non-bacterial diarrhea in children. Reported in 2013, the mean incidence of HAstV infection was 11% worldwide, with 7% incidence in rural areas and 23% in urban areas. The incidence of infection tends to be higher in developing countries, most likely due to poor hygiene and water treatment practices. Astrovirus infection statistics are greatly hindered by underreporting and asymptomatic infections. However, the development of more sensitive real-time RT-PCR have enabled significantly higher rates of HAstV infections in asymptomatic children to be detected. With these new screening methods, the rate of HAstV positive samples from asymptomatic children is thought to be roughly 4%. Of the classical genotypes, HAstV1 is the most frequently isolated worldwide, with the exception of Mexico where HAstV2 dominates. The incidence of HAstV infection has been decreasing over the past three decades from 22% in the 1980s to just 5% in the 2000s. This could be due to novel HAstVs outcompeting the classical genotypes, since reports have shown their prevalence is rising.

Novel HAstV The association between the MLB and VA clades of HAstV and clinical disease is still somewhat ambiguous compared to the classical genotypes, which makes accurate reporting difficult. Published reports have exhibited this in the large variation in prevalence from one geographic location to another. One review of novel HAstV prevalence, describes an overall positivity rate from stool samples 1.5% lower than that observed for classic HAstVs. However, one study from Japan in 2016 found more MLB-HAstV positive samples, 10.6%, than classic HAstVs, 5.1%. Although most case reports exhibit a generally low prevalence of novel HAstVs, studies have revealed the seroprevalence of HAstV-MLB1 to be 86% and HAstV-VA1 to be 65% indicating a large portion of the population have seen these viruses previously. In a study of immunocompromised pediatric oncology patients, half of the samples were positive for HAstV1, however HAstV-VA2 and HAstV-MLB1 were also present in 21% and 13% of samples, respectively. The authors of this study did comment that the PCR screening method used was unable to detect other members of the HAstV-VA and HAstV-MLB clades, so these genotypes may have been more prevalent than reported.

Clinical Features Human Disease The typical symptom of HAstV infection is watery diarrhea that lasts 2–4 days and can be less commonly accompanied by fever, headaches, abdominal pain and anorexia. Not all HAstV infections present with symptoms, since many infections in healthy individuals tend to be asymptomatic, which could be due to previous exposure to the virus conferring immunity. In the immunocompromised population, HAstV infections are of increased interest due to recent reports of severe symptoms and extra-gastrointestinal dissemination. First reported in 2010, a case report involving a 15-year old boy with X-linked agammaglobulinaemia suggested HAstV to be the causative agent in encephalitis and meningitis. While virus specific primers were unable to detect the virus, next-generation sequencing (NGS) identified the presence of HAstV in the patient’s biopsy samples. Since then, eight additional cases have linked encephalitis and meningitis to HAstV infection in predominately immunocompromised patients, though one case was in an otherwise healthy individual. In eight out of the nine cases, a non-classical genotype was identified in patient samples. These studies suggest that astroviruses have the potential to cause systemic infection, highlighting the need to research astrovirus pathogenesis with both classical and non-classical genotypes. Especially when epidemiological studies have shown infections caused by non-canonical viruses are becoming less rare.

96

Astroviruses (Astroviridae)

Animal Disease As in humans, diarrhea is the main symptom of astrovirus infection in many animal species including turkeys, chicken, cattle, lambs, piglets, and deer, among others, although cases of asymptomatic infections in animals have been reported. Chicken astrovirus infection has been associated with two different conditions in chicken hatchlings, runting-stunting syndrome and “white chick” syndrome. These syndromes are characterized by decreased hatch-rate, poor weight gain, increased chick mortality and weakness. It can lead to pale plumage in chickens where “white chick” syndrome gets its name. Likewise, astrovirus infections in turkeys also causes stunted growth associated with poult enteritis mortality syndrome (PEMS). PEMS affects newly hatched turkey poults and is characterized by diarrhea, dehydration, and increased mortality. Along with diarrhea associated disease in animals, there have been cases of astrovirus-associated encephalitis and meningitis in mink and cows. Recently, porcine astrovirus type 3 was detected in central nervous system tissues from pigs suffering a neurologic disorder characterized by hind limb weakness to quadriplegia and occasionally convulsions. With improved screening, detection and sequencing techniques, we are now able to detect the variety of symptoms associated with astrovirus infection, however further research is still needed to discover how the virus is causing disease.

Pathogenesis Obstacles Several obstacles must be overcome in order to uncover the mechanisms of astrovirus pathogenesis. To date, researchers make do without proper cell culture methods for all astrovirus genotypes or a well-characterized or clinically relevant small animal model. Caco-2 cells are the most common cell culture model for in vitro studies on classical HAstV genotypes. However, this is an immortalized cancerous cell line that may not completely mimic the same mechanisms occurring in vivo. Furthermore, cell culture systems for the MLB and VA genotypes are limited or non-existent, which greatly limits our ability to study these novel genotypes. Modeling astrovirus disease in vivo has also been challenging. The turkey poult model has historically been the most widely used animal model in astrovirus research. Although poults develop clinical symptoms upon infection with turkey astrovirus type-2 (TAstV2), this virus is an Avastrovirus and genetically distinct from Mamastroviruses. It is still unclear if characteristics of disease resulting from Avastrovirus infection translate to classical or non-classical HAstV infection. Studies using turkey poults are also burdened by the lack of a cell culture system in which to grow virus; researchers are restricted to generating the virus in embyronated turkey eggs. Furthermore, planning experiments is difficult due to a low hatch rate, approximately 50%, decreasing overall sample sizes, and the highly transmissible nature of astrovirus requiring uninfected controls to be housed separately to avoid cross-contamination. Most importantly, turkey-specific antibodies and reagents are rarely commercially available, which greatly reduces which technical methods can be utilized. In 2012, four murine astroviruses (MuAstV, STL1 – STL4) were identified in mice housed at Washington University in St. Louis, MO. This discovery offered the potential to develop a traditional small animal model to study astrovirus. Unlike TAstV-infected turkey poults, MuAstV-infected mice do not exhibit diarrhea yet exhibit low level persistence and viral shedding for long periods of time. MuAstV has also been identified in mice purchased from multiple vendors, making obtaining mice without underlying astrovirus infection challenging. The characterization of the MuAstV infection is ongoing, but researchers are hopeful this model holds the key many future discoveries. Nevertheless, using the resources available, researchers are expanding the body of knowledge on astrovirus pathogenesis (summarized in Fig. 3).

Mechanism of Disease To date, most pathogenesis studies have focused on understanding the mechanism by which astrovirus induces diarrhea. Reports have shown astrovirus infection in turkey poults results in relocalization of the sodium transport proteins NHE3 and SGLT-1 from the cell membrane into the cytoplasm of intestinal epithelial cells, supporting a hypothesis that astrovirus disrupts ionic transport causing sodium malabsorption and osmotic diarrhea. Other intestinal pathogens disrupt the intestinal epithelium by causing inflammation and cell death. However, astrovirus-induced diarrhea occurs without triggering cell death or inflammation, although there is one report of HAstV8 inducing apoptosis in Caco-2 cells. Instead HAstV causes barrier permeability by disrupting intestinal cellular junctions. Tight junctions are highly regulated cell-cell associations that maintain cell polarity and regulate the passage of nutrients, other solutes and microorganisms from the intestinal lumen across the epithelium. Cellular junctions are comprised of the tight junction complex, which includes proteins such as occludin, claudin, ZO-1, and cadherins. Loss of tight junctions can result in increasing fluid in the lumen of the intestine and diarrhea. Multiple studies report that AstV infection alters permeability of the intestinal barrier caused by redistribution of junctional proteins. In vitro, HAstV infection caused a drop in transepithelial resistance (TER) in Caco-2 monolayers and allowed for greater flux of FITC–dextran across the cell monolayer. The decrease in TER and increase in flux began between 16 and 20 hpi, with maximum permeability achieved by 36 and 48 hpi. HAstV1 has been shown to increase intestinal barrier permeability in vitro, possibly due to a reduction in actin fibers, reduction of occludin from the junctional complex and loss of E-cadherin by 24 hpi. Studies in the turkey poult yielded comparable results, where infected poults had increased lumenal-to-serosal flux and disruption

Astroviruses (Astroviridae)

97

Fig. 3 Astrovirus increases barrier permeability in the gut epithelium. (A) Normal intestinal epithelial cells maintain tightly regulated barrier permeability. (B) In infected cells, intestinal epithelial cells upregulate iNOS inducing macrophages to secrete NO, and TGF-b within the epithelial cells is activated. IFN-b is induced in an effort to control infection. Tight junction proteins and the sodium transporters are relocalized, leading to the breakdown of barrier permeability allowing exchange of ions and solutes across the intestinal epithelium.

of F-actin. There is evidence that MuAstV infection also increased intestinal permeability, as indicated by orally administered FITC-dextran crossing the intestinal epithelium reaching the blood. Remarkably, astrovirus-induced disruption of barrier integrity was found to be independent of active replication. Reports by Moser et al. found both UV-inactivated HAstV and recombinant capsid protein could decrease barrier permeability in Caco-2 cells as well as activate ERK1/2. In vivo, administration of the recombinant TAstV2 capsid protein induced acute diarrhea, and capsid inoculated animals experienced relocalization of SGLT-1 from the plasma membrane, comparable to those infected with virus.

Immune Response The immune response to astrovirus is not well-characterized, but studies suggest that infection does not produce overt inflammation. It is assumed that the innate immune system plays a vital role in controlling the virus because the course of infection with astrovirus is relatively short. One essential arm of the innate immune system is the interferon response. Astrovirus replication was found to be impacted by the type I interferon (IFN) response. Two reports have shown IFN-b levels increase after HAstV infection and the addition of exogenous IFN-b can protect epithelial barrier integrity. Contrasting the induction of barrier permeability, productive replication is required to induce IFN-b in vitro. In vivo, IFNAR knockout mice, which lack the IFN-a receptor making them unable to respond to type I IFNs, could not clear an astrovirus infection unlike wild-type mice. Along with inhibiting complement factors, astrovirus may be modulating the immune response by activating the immunosuppressive cytokine, TGF-b. Infected turkey poults exhibited increased levels of active TGF-b to up 12 days after infection. Astrovirus has been shown to not only infect intestinal epithelial cells but also intestinal macrophages, although infection in macrophages led to abortive replication. While astrovirus infection results in decreased macrophage function and viability, infected macrophages produce more nitrogen oxide (NO) because of increased inducible NO synthase (iNOS) produced by infected epithelial cells. The NO produced by macrophages has been reported to then inhibit astrovirus replication in turkey poults. The innate immune response controls the initial spread of astrovirus and enables the host to mount an effective humoral and cell-mediated adaptive response. Nearly 70% of healthy adults have detectable antibodies against astroviruses. Studies in human volunteers have shown a direct link between having anti-astrovirus antibodies and disease, where subjects without antibodies to the virus had more severe symptoms. Further reports have shown individuals can develop anti-astrovirus antibodies without the presence of symptoms as is the case with poultry abattoir workers with antibodies against TAstV. This finding was contrary to the widely held belief that astrovirus infections were species-specific, and provided evidence supporting the possibility for zoonotic transmission. However, further work is needed in this area of investigation.

98

Astroviruses (Astroviridae)

Recently, antibodies that neutralized viral infectivity have been discovered. Bogdanoff et al. have mapped the neutralizing epitopes of HAstV2 demonstrating only antibodies to spike protein, not the core, can prevent binding to Caco-2 cells. Additional analysis of several genotype specific neutralizing antibodies determined a quaternary epitope on each side of the dimeric capsid spike necessary for neutralization. These studies could help determine components for future vaccines to induce the production of cross-protective neutralizing antibodies against astrovirus. In terms of cell-mediated adaptive response, astrovirus specific T cells are also thought to play a significant role in viral clearance. A challenge of biopsy specimens from the small intestine of healthy adults with inactivated HAstV demonstrated HAstVspecific CD4 þ and CD8 þ T cells reside in these tissues and could prevent reinfection. Recent work in the mouse model has shown Rag1 knockout mice, which lack B and T cells, shed more virus in their feces than wild-type mice. This study also detected higher viral replication in intestinal tissues, mesenteric lymph nodes, spleen, liver, and kidneys of Rag1 knockout mice, indicating B and T cells are necessary in preventing systemic spread of astrovirus.

Diagnosis Initially, HAstV was detected via electron microscopy. However, using electron microscopy is time consuming and is impractical for high-throughput screening purposes. In 1995, the first conventional RT-PCR and enzyme immunoassays were developed to determine the antigenic groups of HAstV. Several real-time RT-PCR methods have now been developed for quick screening of HAstV, including many that are multiplexed to detect additional enteric viruses. However, these methods do not detect non-classical HAstV VA or MLB genotypes. To date, there is only one real-time RT-PCR multiplex method accurately detects classical HAstV1–8, MLB1, and VA2. Developing more comprehensive real-time RT-PCR methods to detect the 16 known genotypes has been hindered by limited sequence information available through public databases. Adding to the sequences available and validating new detection methods is essential for researchers to better delineate astrovirus epidemiology and relationship to disease.

Treatment Currently, there are no vaccine or medical treatment options for HAstV. This is in part due to the short duration and relatively mild symptoms that HAstV infection causes. The low interest in developing a HAstV vaccine could be because of its believed low clinical impact or the need for a multivalent vaccine to cover multiple genotypes. Although most infections resolve without intervention, if treatment is necessary, it generally involves alleviating symptoms. HAstV-induced diarrhea has the potential to cause dehydration in infected individuals and therefore may require oral or intravenous fluid replacement. Since immunocompromised individuals could be at risk of astrovirus spreading systemically, it has been suggested intravenous IgG be used as treatment, although more studies are needed to determine the efficacy of such treatments.

Prevention The best form of prevention is to stop the spread of the virus. In order to fully disinfect surfaces and contaminated fomites most healthcare facilities regularly use 90% ethanol, however this treatment was shown to be ineffective against astrovirus. Astrovirus is a remarkably hearty virus that can remain stable at low pH, is resistant to many detergents, and even lipid and chlorine solvents are incapable of inactivating the virus. However, reports have shown that formaldehyde and peroxymonosulfate can fully inactivate the virus. Temperature modulation also has very little effect since astrovirus is also very stable at low temperatures and can remain infectious for up to ten years. Astrovirus has been detected in environmental water samples and shown to persist in drinking water, proving the importance of water treatment in preventing the spread of the virus. Typical water treatment involves chlorine disinfection with residual free chlorine in the range of 0.2–1 mg/L, and a maximum residual disinfectant level of 4mg/L allowed by the Environmental Protection Agency. Astrovirus infectivity has been shown to be decreased by 3 logs at the height of the typical range, 1mg/L free chlorine, for two hours. Most studies to date on disinfection and inactivation have focused on classical HAstV genotypes. While bleach use is recommended for the inactivation of novel HAstV genotypes, more research is necessary to determine exact protocols for their inactivation.

Further Reading Arias, C.F., DuBois, R.M., 2017. The astrovirus capsid: A review. Viruses 9 (1), 15. Bogdanoff, W.A., Perez, E.L., López, T., Arias, C.F., DuBois, R.M., 2017. Structural basis for escape of human astrovirus from antibody neutralization: Broad implications for rational vaccine design. Journal of Virology 92 (1). Bosch, A., Pintó, R.M., Guix, S., 2014. Human astroviruses. Clinical Microbiology Reviews 27 (4), 1048–1074.

Astroviruses (Astroviridae)

99

Cortez, V., Freiden, P., Gu, Z., et al., 2017. Persistent infections with diverse co-circulating astroviruses in pediatric oncology patients, Memphis, Tennessee, USA. Emerging Infectious Diseases 23 (2), 288–290. Cortez, V., Meliopoulos, V.A., Karlsson, E.A., et al., 2017. Astrovirus biology and pathogenesis. Annual Review of Virology 4 (1), 327–348. Donato, C., Vijaykrishna, D., 2017. The broad host range and genetic diversity of mammalian and avian astroviruses. Viruses 9 (5). Espinosa, R., López, T., Bogdanoff, W.A., et al., 2019. Isolation of neutralizing monoclonal antibodies to human astrovirus and characterization of virus variants that escape neutralization. Journal of Virology 93 (2), doi:10.1128/JVI.01465-18. Gu, Z., Zhu, H., Rodriguez, A., et al., 2015. Comparative evaluation of broad-panel PCR assays for the detection of gastrointestinal pathogens in pediatric oncology patients. The Journal of Molecular Diagnostics 17 (6), 715–721. Johnson, C., Hargest, V., Cortez, V., Meliopoulos, V.A., Schultz-Cherry, S., 2017. Astrovirus pathogenesis. Viruses 9 (1), 22. Karlsson, E.A., Small, C.T., Freiden, P., et al., 2015. Non-human primates harbor diverse mammalian and avian astroviruses including those associated with human infections. PLOS Pathogens 11 (11), e1005225. Meliopoulos, V.A., Marvin, S.A., Freiden, P., et al., 2016. Oral administration of astrovirus capsid protein is sufficient to induce acute diarrhea in vivo. mBio 7 (6). Mendez, E., Arias, C., 2013. Astrovirus. In: Fields, B., Knipe, D., Howley, P. (Eds.), Fields Virology, sixth ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins. Moser, L.A., Carter, M., Schultz-Cherry, S., 2007. Astrovirus increases epithelial barrier permeability independently of viral replication. Journal of Virology 81 (21), 11937–11945. Schultz-Cherry, S., 2013. Astrovirus Research. New York: Springer. Vu, D.L., Bosch, A., Pintó, R.M., Guix, S., 2017. Epidemiology of classic and novel human astrovirus: Gastroenteritis and beyond. Viruses 9 (2).

Avian Hepadnaviruses (Hepadnaviridae) Allison R Jilbert, Georget Y Reaiche-Miller, and Catherine A Scougall, The University of Adelaide, Adelaide, SA, Australia r 2021 Elsevier Ltd. All rights reserved. This is an update of A.R. Jilbert, W.S. Mason, Hepadnaviruses of Birds, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00414-3.

Nomenclature nt

nucleotide(s)

Glossary anti-DHBc antibodies Antibodies to DHBcAg anti-DHBs antibodies Antibodies to DHBsAg cccDNA Covalently closed circular DNA CTL Cytotoxic T lymphocyte(s) DHBcAg Duck hepatitis B virus core Ag DHBeAg Duck hepatitis B virus e Ag DHBsAg Duck hepatitis B virus surface Ag

DHBV Duck hepatitis B virus dslDNA Double stranded-linear hepadnavirus DNA HBV Human hepatitis B virus ORF Open reading frame rcDNA Relaxed circular hepadnavirus DNA RT Reverse transcriptase TP Terminal protein TR Terminal redundancy

Classification Avian hepadnaviruses belong to the avihepadnavirus genus of the hepadnaviridae family and are related phylogenetically to human hepatitis B virus (HBV) and other viruses of the orthohepadnavirus genus, which infect mammals. Avian hepadnaviruses are related phylogenetically to each other through similarities in genome sequence and organization of open reading frames (ORF). Only two species have been assigned within the avihepadnavirus genus: duck hepatitis B virus (DHBV), first isolated from Pekin ducks (Anas domesticus), and heron hepatitis B virus (HHBV), isolated from grey herons (Ardea cinerae). HHBV was assigned as a species based both on genome divergence and a host range difference from DHBV. DHBV isolates have been found not only in domesticated ducks, but also in the puna teal (Anas puna), the Chiloe wigeon (Anas sibilatrix) and, in the wild, the mallard, the species from which Pekin ducks and most other domesticated ducks (except Muscovy) were derived. Avian hepadnaviruses less closely related to DHBV but currently unassigned as distinct species have also been isolated. Viruses isolated from geese include the Ross’ goose hepatitis virus (RGHV) from Ross’ geese (Anser rossii) and Mandarin ducks (Aix galericulata), the snow goose hepatitis B virus (SGHBV) from snow geese (Anser caerulescens) and the sheldgoose hepatitis B virus from the ashy-headed sheldgoose (ASHBV) (Chloephaga poliocephala) and Orinoco sheldgoose (OSHBV) (Neochen jubata). The crane hepatitis B virus (CCHBV) has been isolated from demoiselle cranes (Anthropoides virgo) and grey crowned cranes (Balearica regulorum). Parrot hepatitis B virus (PHBV) has been isolated from ring-necked parakeets (Psittacula krameri), an African grey parrot (Psittacus erithacus), an Alexandrine Parakeet (Psittacula eupatria) and a crimson rosella (Platycercus elegans). The stork hepatitis B virus (STHBV) has been isolated from white storks (Ciconia ciconia) and the elegant-crested tinamou hepatitis B virus (ETHBV) has been isolated from elegant-crested tinamou (Eudromia elegans) (Fig. 1(A); Table 1A).

Virus Structure Avian hepadnaviruses are enveloped viruses possessing an icosahedral nucleocapsid and B3000 nt relaxed circular double-stranded DNA (rcDNA) genome (Fig. 2). DHBV, the prototypic avian hepadnavirus, has a diameter of 40–45 nm. The envelope of DHBV is composed of a lipid bilayer containing 2 transmembrane DHBV surface antigen (DHBsAg) proteins, PreS/S and S (36 and 17 kDa respectively). These two proteins are synthesized from the same ORF, with initiation of PreS/S occurring upstream of S and share a common carboxy-terminus. An additional B28 kDa DHBsAg protein has been detected in DHBV-infected liver but it is unclear if this is a degradation product of PreS/S or is translated from an AUG codon mapping between the start sites of PreS/S and S. The envelope proteins, PreS/S and S, participate in virion formation and also self-assemble into non-infectious DHBsAg particles. DHBsAg assembles in the endoplasmic reticulum and is released from infected hepatocytes in a 500–1000-fold excess over infectious virions. DHBsAg particles do not contain a virus nucleocapsid or viral nucleic acids and are pleomorphic and spherical, with a diameter of B35–60 nm. Many are almost indistinguishable from DHBV virions by electron microscopy (EM). The nucleocapsid of DHBV is icosahedral, as observed by cryo-EM, has a diameter of B38 nm and is composed of 90 or 120 subunits arranged in T ¼ 3 or T ¼ 4 symmetry. The subunits are dimers of the 30 kDa non-glycosylated DHBV core antigen (DHBcAg) protein, or C protein. The rcDNA genome, virus encoded polymerase and cellular chaperones are contained in the nucleocapsid. The DHBV polymerase is a 90 kDa protein that functions as an RNA-dependent DNA polymerase and a DNA-

100

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20980-1

Avian Hepadnaviruses (Hepadnaviridae)

101

Fig. 1 Phylogenetic relationships of exogenous avian hepadnaviruses and endogenous avian hepadnavirus elements. (A). Phylogenetic analysis of full-length exogenous avian hepadnavirus genomes (Table 1A) was performed using ClustalW sequence alignment with the ClustalW cost matrix, the Neighbor-Joining method with the HKY nucleotide model and 1000 bootstraps and is shown as an unrooted tree. Scale bar ¼ 0.09 nucleotide substitutions per site. (B). Phylogenetic analysis of partial polymerase amino acid (AA) sequences of the endogenous avian hepadnavirus elements, eZHBV_C (HAS) (Hypothetical Ancestral Sequence described by Suh, A., Brosius, J., Schmitz, J., Kriegs, J.O., 2013. The genome of a mesozoic paleovirus reveals the evolution of hepatitis B viruses. Nature Communications 4, 1791; 778 AA), eBHBV-1-BK008520.1 (679 AA), eBHBV-2BK008521.1 (592 AA) and eJHBV-HQ116565.1 (195 AA) (Table 1B), compared to full-length polymerase AA sequences (785–793 AA) of selected exogenous avian hepadnaviruses (Table 1A). Analysis was performed using ClustalW sequence alignment with the BLOSUM cost matrix, NeighborJoining method with the Jukes-Cantor model and 1000 bootstrap replicates and is shown as an unrooted tree. Scale bar ¼ 0.09 AA substitutions per site. The percentage of bootstrap replicates that supported each branch is shown in both A and B.

dependent DNA polymerase. The polymerase protein has 4 separate domains listed from the N-terminal end, including a terminal protein (TP), ‘spacer’, reverse transcriptase (RT) and RNase H domain. Also released from hepatocytes and circulating in the bloodstream of infected ducks is DHBV e antigen (DHBeAg), which includes proteolytically processed forms of the PreC/C protein ranging in size from a non-glycosylated 27 kDa to B30–33 kDa glycosylated forms. DHBeAg is thought to be similar in function to the better described HBV e antigen: based upon studies in HBV transgenic mice, HBV e antigen appears to suppress the host immune response to HBV. Thus, it is possible that DHBeAg performs a similar role, facilitating the development of chronic DHBV infections.

Genome The avian hepadnaviruses have rcDNA genomes with similar genome organization to the orthohepadnaviruses, including the presence of overlapping ORFs all present on the negative strand (Fig. 2). The genomes of DHBV isolates range in size from 3021 to 3027 nt (Table 1A). The negative strand of rcDNA is nicked, with a 9 nt terminal redundancy (r) and with the virus polymerase covalently attached to its 50 end. Similarly, the positive strand of the DHBV genome has an 18 nt RNA primer attached to its 50 end and is incomplete in rcDNA genomes, with a minimum gap of 12 nt (Fig. 2); as illustrated below (Fig. 4), this gap is likely due to the inability of the polymerase to displace the positive strand RNA primer from DR2. The negative strand of rcDNA contains a P ORF, PreS/S ORF and PreC/C ORF, encoding the polymerase, PreS/S and S proteins, and DHBcAg and DHBeAg proteins, respectively. The P ORF, read in a þ 1 frame, completely overlaps the PreS/S ORF, read in a

102

Avian Hepadnaviruses (Hepadnaviridae)

Table 1A

Exogenous Avian Hepadnaviruses

Clone name

Accessiona

‘Chinese’ DHBV: AusDHBV DHBVS31cg DHBVS5cg DHBVQCA34 DHBVS18-B DHBV22 DHBV26

AJ006350.1 M32991.1 M32990.1 X60213.1 M21953.1 X58568.1 X58569.1

‘Western country’ DHBV: DHBV1 X58567.1 DHBV3 DQ195079.1 DHBV P2–3 M60677.1 DHBV16 K01834.1 DHBV47045 AF047045.1 DHBV F1–6 X12798.1 DHBVCG X74623.1 SGHBV1–13 SGHBV1–15 SGHBV1–19 SGHBV1–7 SGHBV1–9 PTHBV CWHBV OSHBV ASHBV CCHBV1 CCHBV2 CCHBV3 RGHBV-MDa RGHBV-MDb RGHBV STHBV-1 STHBV-7 STHBV-16 STHBV-21 HHBV-4 ETHBV ETHBV PHBV-A PHBV-A PHBV-B PHBV-D PHBV-G PHBV-G

AF110996.1 AF110997.1 AF110998.1 AF110999.1 AF111000.1 AY494851.1 AY494850.1 AY494852.1 AY494853.1 AJ441111.1 AJ441112.1 AJ441113.1 AY494848.1 AY494849.1 M95589.1 AJ251934.1 AJ251935.1 AJ251936.1 AJ251937.1 M22056.1 KY977506.1 KY977507.1 JN565944.1 JX274026.1 JX274024.1 JX274022.1 JX274018.1 JX274019.1

Genome Size (nt)

%c

Host

3027 3027 3027 3027 3024 3024 3024

100.0 95.9 94.8 94.7 93.7 91.9 91.4

Duck, Duck, Duck, Duck, Duck, Duck, Duck,

IDHBV

3021 3021 3021 3021 3021 3021 3021

90.0 90.6 89.7 89.5 89.5 89.4 89.0

MDHBVa MDHBVb HPUGENM SHE251934 SHE251935 SHE251936 SHE251937 HPUCG 160014 160050 PL P337 P410 P830 P902 P1233

3024 3024 3024 3024 3024 3024 3024 3018 3051 3021 3018 3018 3021 3012 3018 3033 3033 3033 3033 3027 3024 3024 3048 3042 3042 3039 3036 3036

88.9 89.0 88.9 88.9 89.0 88.9 88.9 86.0 85.1 83.3 83.3 83.4 81.8 82.7 81.7 76.6 76.6 76.5 76.6 76.3 75.6 75.6 74.5 74.9 75.3 74.5 73.3 73.3

Alt. Nameb

HPUS31CG HPUS5CG DHVBCG HPUGA

HPUCGE HPUCGD

Species

Locationd

Anas Anas Anas Anas Anas Anas Anas

domesticus domesticus domesticus domesticus domesticus domesticus domesticus

Australia Shanghai, China Shanghai, China Shanghai, China Shanghai, China Chi-tung County, China Shanghai, China

Goose, domestic Duck, Pekin Duck, Pekin Duck, Pekin Duck Duck Duck

Anas Anas Anas Anas Anas Anas Anas

domesticus domesticus domesticus domesticus domesticus domesticus domesticus

Germany Germany USA USA Canada Germany India

Snow Goose Snow Goose Snow Goose Snow Goose Snow Goose Puna Teal Chiloe Wigeon Orinco Sheldgoose Ashy-Headed Sheldgoose Grey Crowned Crane Grey Crowned Crane Grey Crowned Crane Mandarin Duck Mandarin Duck Ross’ Goose White Stork White Stork White Stork White Stork Grey Heron Elegant-Crested Tinamou Elegant-Crested Tinamou Ring-necked Parakeet Ring-necked Parakeet African Grey Parrot Alexandrine Parakeet Crimson Rosella Ring-necked Parakeet

Anser caerulescens Anser caerulescens Anser caerulescens Anser caerulescens Anser caerulescens Anas puna Anas sibilatrix Neochen jubata Chloephaga poliocephala Balearica regulorum Balearica regulorum Balearica regulorum Aix galericulata Aix galericulata Anser rossii Ciconia ciconia Ciconia ciconia Ciconia ciconia Ciconia ciconia Ardea cinerae Eudromia elegans Eudromia elegans Psittacula krameri Psittacula krameri Psittacula erithacus Psittacula eupatria Platycercus elegans Psittacula krameri

Pekin white brown domestic domestic domestic domestic

Germany Germany Germany Germany Germany USA USA USA USA Germany Germany Germany USA USA USA Germany Germany Germany Germany Germany Germany Germany Poland Poland Poland Poland Poland Poland

a

GenBank accession number. Alternate name. c Percentage sequence identity to AusDHBV-AJ006350.1. d Location of the bird/collection where the virus is found. b

þ 2 frame, leading to out of frame overlap between the ‘spacer’ and RT domains of the polymerase and the PreS and S regions of the surface proteins respectively. The P ORF also overlaps out of frame with the downstream region of the C ORF, read in a þ 3 frame, leading to overlap between the TP domain of the polymerase and the C-terminal domain of DHBcAg (Fig. 2). These overlaps place evolutionary constraints on the genome with single mutations potentially leading to changes in more than one ORF. The positive strand of rcDNA does not encode any functional ORFs. The DHBV genome was originally thought to contain only 3 ORFs, and to lack an ORF encoding a protein analogous to the orthohepadnavirus X protein. This is surprising because the HBV X protein is required for transcription of HBV DNA, which is otherwise suppressed by a host-complex targeting episomal DNAs. It was subsequently discovered that DHBV has a fourth ORF, the X ORF, which lacks a conventional start codon but can be read in þ 2 frame to direct synthesis of a candidate DHBV X protein. The significance of this

Avian Hepadnaviruses (Hepadnaviridae)

103

Fig. 2 DHBV genome organization. The rcDNA genome of AusDHBV (GenBank AJ006350) is shown at the top, with the virus polymerase covalently attached to the 50 end of the negative strand and an 18 nt RNA primer attached to the 50 end of the positive strand. The genome is numbered clockwise from a conserved EcoR1 site at nt 3027/1 and contains 2 direct repeat (DR) sequences (DR1 and DR2, nt 2541–2552 and 2483–2494 respectively). The location of the P, PreS/S, PreC/C and X ORFs, pregenomic, L and S mRNAs and common polyadenylation site (nt 2778–2783) are also shown. None of the major mRNAs are spliced. Thus, each mRNA has its own promoter. Pregenomic RNA has a terminal redundancy (TR) of 243 nt and is the mRNA for both the DHBcAg and polymerase proteins from the C ORF and P ORF respectively. Pregenomic RNA lacks the AUG of the PreC/C ORF, and DHBeAg is synthesised instead from an mRNA a few nt longer at the 50 end than pregenomic RNA (not shown). The L and S mRNAs encode the DHBsAg proteins, PreS/S and S, respectively. The X ORF, which lacks a conventional start codon, is used to produce X mRNA (not shown) allowing synthesis of a candidate X protein (as described in the text). The more rarely synthesized double stranded-linear DNA (dslDNA) is shown at the bottom.

observation is still unclear. Knockout of the X gene of DHBV did not alter the time course of DHBV infection: the X gene deletion mutant spread rapidly through the liver of newly-hatched ducks and caused persistent infection. Interestingly, other avian hepadnaviruses including HHBV, STHBV, RGHBV, ASHBV and OSHBV, all have an AUG codon near the beginning of the X ORF. The difference in size between the genomes of the avian (e.g., DHBV, 3021–3027 nt) and the orthohepadnaviruses (e.g., HBV, 3200–3300 nt), is due in part to the absence of a 150 nt stretch in the S ORF of the avian virus. This region in the HBV genome encodes the ‘a’ determinant, a highly immuno-dominant region present in HBV surface antigen. Most neutralizing anti-HBV surface antigen antibodies are directed towards the ‘a’ determinant. Indeed, HBV strains with mutations in the ‘a’ determinant can establish infection and replicate in vaccinated hosts as vaccine escape mutants. The effect of the absence of an ‘a’ determinant in DHBV is unknown but both DHBV PreS/S and S proteins can induce high-titer neutralizing antibodies.

Life Cycle Although the avian and orthohepadnaviruses share only 40% genome sequence homology, their life cycles and replication strategies are similar. In brief, their rcDNA genome is converted in the nucleus to covalently closed circular DNA (cccDNA), which is used as the transcriptional template for all the viral mRNAs, including the pregenome (Fig. 2). Pregenomic RNA, in addition to functioning as the mRNA for DHBcAg and polymerase, is encapsidated by DHBcAg and reverse transcribed, in the cytoplasm, into rcDNA and double stranded-linear DNA (dslDNA) (Fig. 3).

104

Avian Hepadnaviruses (Hepadnaviridae)

cccDNA Integration

~90% rcDNA

~10% dslDNA

Fig. 3 DHBV infection of hepatocytes. Virions, shown at the top may have either an rcDNA or, more rarely, a dslDNA genome. Upon infection, virus nucleocapsids lose their envelope and move to the nucleus. Virus DNA is released from nucleocapsids and probably enters the nucleus at nuclear pores. Both rcDNA and dslDNA give rise to cccDNA, the virus transcriptional template. cccDNA is used by host cell RNA polymerase II to make all viral mRNAs including the pregenomic RNA that is reverse transcribed into rcDNA or dslDNA. The dslDNA form also has a propensity to integrate into host cell DNA. Early in infection, cccDNA copy number increases to 5–50 copies/cell by intracellular amplification, in which newly made rcDNA and dslDNA migrate to the nucleus rather than being exported as progeny virus; later, nucleocapsids containing rcDNA and dslDNA bud into the ER and are released as progeny virus.

Hepatocytes, the major parenchymal cell of the liver, are the primary site of DHBV infection. Hepatocyte specificity is determined by a cell surface receptor for virus binding and entry and by a viral preference for liver-enriched transcription factors. Receptor binding occurs via the PreS region of DHBsAg and is followed by receptor-mediated endocytosis. The cellular protein carboxypeptidase D (180 kDa) binds DHBsAg particles and DHBV with high affinity and is found on both internal and external membranes of the cell. An additional DHBV binding protein, glycine decarboxylase (120 kDa), has also been identified in liver, kidney and pancreas, all sites of DHBV infection. These 2 proteins are potential components of a DHBV receptor complex but definitive proof that they mediate virus infection is still lacking. In contrast, the receptor used by HBV was recently identified as the human sodium taurocholate co-transporting polypeptide (hNTCP). hNTCP, a multiple transmembrane transporter predominantly expressed in the liver, binds the N-terminal region of the HBV large surface protein, leading to viral entry via receptormediated endocytosis. The immediate sequel to DHBV uptake is transport of rcDNA to the nucleus (Fig. 3). Following transport, the covalently linked polymerase protein, the RNA primer and the 9 nt terminal redundancy in the negative strand are removed, the positive strand is completed, and ligation of the ends of each DNA strand takes place to form cccDNA. Once formed, cccDNA is transcribed by host RNA polymerase II to produce pregenomic RNA which is translated allowing assembly of nucleocapsids from DHBcAg and packaging of pregenomic RNA and polymerase. Pregenomic RNA is greater than genomic length due to a terminal redundancy (TR) of 243 nt (Figs. 2 and 4). Its packaging into nucleocapsids is facilitated by the presence of an encapsidation signal, epsilon (S), located near its 50 end (at nt 2566–2622) and again at the same position (2566-2622) within the 30 TR (Fig. 4(A)). Pregenomic RNA also contains DR1, a 12 nt sequence located 6 nt from the 50 end of the pregenome at nt 2541–2552 and at the same position (nt 2541–2552) in the 30 TR, where it is referred to as DR1*. The same 12 nt sequence is located upstream of DR1 at nt 2483–2494, where it is named DR2.

Avian Hepadnaviruses (Hepadnaviridae)

105

Fig. 4 DHBV DNA synthesis. Pregenomic RNA, which has a terminal redundancy (TR) of 243 nt plus a poly-A tail, has 2 potential encapsidation signals, epsilon (S), located at the 50 and 30 ends, but only the 50 copy is functional for pregenome encapsidation. Pregenomic RNA is packaged into nucleocapsids following binding of virus polymerase to the 50 S sequence. DNA synthesis then begins with reverse transcription of 4 nt of the bulge in the stem loop structure S, 50 UUAC 30 and leads to synthesis of the first 4 nt of negative strand DNA, 50 GTAA 30 as shown in B. Polymerase with the nascent transcript then translocates to the right hand copy of DR1, DR1*, and reverse transcription of the negative strand and degradation of the pregenomic RNA template by RNase H proceeds (C, D). Following completion of the negative strand, the 50 18 nt of the pregenome, including the 50 cap and DR1, are typically translocated to DR2 and positive strand synthesis then initiates from this RNA primer. In this case, rcDNA formation is of necessity an early step in positive strand elongation (E, F). About 10% of the time, positive strand synthesis initiates without translocation of the primer (G), leading to the formation of dslDNA (H).

Once the polymerase is translated, it may bind to its own message, pregenomic RNA, via the 50 S sequence, blocking its further translation, and facilitating packaging into nucleocapsids. DNA synthesis begins with reverse transcription of 4 nt (50 UUAC 30 ) in the bulge in the stem loop structure of S leading to synthesis of 4 nt of DNA (50 GTAA 30 ) (Fig. 4(B)). A tyrosine residue in the TP domain of polymerase serves as the primer of reverse transcription and the polymerase remains covalently attached to the 50 end of the newly formed 4 nt DNA sequence. Following synthesis of the first 4 nt, the DNA-polymerase complex is translocated to DR1* (Fig. 4(C)), where the 4 nt can base pair due to sequence homology. Reverse transcription re-initiates and continues to the 50 end of the pregenome, to produce a full-length negative strand DNA with a 9 nt terminal redundancy. Most of the pregenomic RNA is degraded during negative strand elongation by the RNase H activity of polymerase (Fig. 4(D)). However, the 50 B18 nt of the RNA pregenome, including the cap and all of the 50 copy of DR1, escapes RNase H degradation and serves as the primer for synthesis of the positive strand. To facilitate positive strand synthesis, the B18 nt RNA primer is first translocated to DR2, near the 50 end of the negative strand, where it hybridizes due to the sequence identity of DR1 and DR2 (Fig. 4(E)). Positive strand synthesis then occurs in a 50 to 30 direction to the 50 end of the negative strand template. Circularization then occurs to allow continuation of positive strand synthesis to produce mature rcDNA (Fig. 4(F)). This circularization is facilitated by the 9 nt TR on the negative strand template and the covalent attachment of the TP domain of the polymerase to the 50 end of the negative strand.

106

Avian Hepadnaviruses (Hepadnaviridae)

In B10% of cases, translocation of the B18 nt RNA primer from DR1 to DR2 does not occur and positive strand synthesis occurs by in situ priming, to produce dslDNA (Fig. 3, Fig. 4(G, H)). The dslDNA molecule has the 18 nt RNA primer at the 50 end of the positive strand and the polymerase covalently attached to the 50 end of the negative strand and is slightly longer than genome length. Interestingly, the virus polymerase and the B18 nt RNA primer remain associated with virus rcDNA and dslDNA genomes throughout virus assembly. Each nucleocapsid contains only a single copy of polymerase, supporting the model that a single protein molecule is simultaneously the TP primer of DNA synthesis and the polymerase for both RNA- and DNA-dependent DNA synthesis. Nucleocapsids containing newly made rcDNA and dslDNA are enveloped by budding into the endoplasmic reticulum and exported as progeny virus via the Golgi, or are transported to the nucleus to make additional copies of cccDNA, typically found at 5–50 copies/hepatocyte (Fig. 3). DHBV cccDNA molecules exist within the hepatocyte nucleus as a population of virus minichromosomes that bind up to 20 nucleosomes. cccDNA does not undergo semi-conservative replication. New copies are therefore formed solely from rcDNA and dslDNA synthesized in the cytoplasm. However, formation of cccDNA from dslDNA involves illegitimate recombination and typically involves loss of sequences, so that the cccDNA formed from dslDNA is generally defective. Nuclear transport of nucleocapsids containing rcDNA and dslDNA is negatively regulated by the virus envelope proteins, which direct nucleocapsids into the pathway of virus assembly. Negative regulation is essential because excessive accumulation of cccDNA will kill the host cell. Negative regulation may also be important because of the stability of cccDNA, and new synthesis of cccDNA may only be necessary to restore cccDNA levels in progeny cells following mitosis. Negative regulation of cccDNA synthesis also occurs during HBV infection, but it is not known if the HBV envelope proteins have an essential role in this process. Nuclear transport of nucleocapsids containing dslDNA and, to a lesser extent, rcDNA, can also result in the integration of viral DNA at random sites in the cell chromosome via illegitimate recombination to create unique virus-cell DNA junctions (Fig. 3). Integrated forms of rcDNA and dslDNA are replication incompetent as they cannot be used to transcribe pregenomic RNA, which is greater than genomic length, and because virus sequences may be lost during the integration process, particularly from the ends of the dslDNA molecule. Integrated DNA has been detected in B0.01%–0.1% of hepatocytes during transient HBV infections and higher levels accumulate during chronic HBV infections. Because integration occurs at random sites in host DNA, virus-cell DNA junctions can be used as molecular markers to identify and track the fate of individual hepatocyte lineages and possibly to gain insights into the development of primary hepatocellular carcinoma (HCC).

Epidemiology DHBV is naturally transmitted in ovo from an infected female duck to the egg, with virus replication occurring in the yolk sac and liver of the developing embryo, leading to immune tolerance and congenital DHBV infection. Congenital infection is likely further facilitated because DHBV infection is not cytopathic. DHBV infections are therefore maintained within flocks and it is not unusual in some commercial flocks to find the presence of DHBV infection in 100% of birds. DHBV infection can also be transmitted horizontally through parenteral exposure to infected blood and, since infection of adult ducks usually results in transient infection, duck flocks may contain a mixture of congenitally DHBV-infected and DHBV immune ducks. Female ducks that are immune to DHBV will have anti-DHBs and anti-DHBc antibodies circulating in their bloodstream as well as in the egg yolk which they can pass to their ducklings providing the newly hatched ducks with temporary resistance to DHBV infection.

Pathogenesis and Clinical Features One major difference between DHBV and HBV are the clinical features of infection that result from differences in their mode of transmission and maintenance. DHBV is maintained primarily by in ovo transmission to the developing embryo, while HBV is maintained by horizontal transmission via infected body fluids from mother to child at birth or, during the first year of life, by transmission to the child from infected siblings or adults. Thus, both viruses cause persistent infection, but the degree of immune tolerance to DHBV is much greater following in ovo transmission than following horizontal transmission of HBV, which may be why immune responses to HBV-infected cells can lead to severe clinical outcomes of chronic HBV infection. Humans with chronic HBV infection, if exposed to the virus at less than a year of age, often develop a “healthy carrier” state with high levels of virus replication and little liver disease. This healthy carrier state can last for 10–20 years but can progress to flares of immune-mediated liver disease with fluctuating levels of HBV DNA in the liver and bloodstream and chronic liver disease. If HBV infection is acquired in adulthood and chronic infection develops the accompanying immune-mediated liver disease is similar and can include inflammation, liver failure, fibrosis, cirrhosis, or HCC. Following in ovo transmission congenitally DHBV infected ducks support virus replication in 495% of hepatocytes and have high levels of virus DNA and DHBsAg circulating in the bloodstream but this widespread infection generally results in an absence of clinical features and only mild, if any, hepatitis, similar to the “healthy carrier” state in humans. Interestingly, although 495% of hepatocytes remain infected, levels of DHBV DNA and DHBsAg in the bloodstream gradually decrease over 800 days. Whilst the cause of this gradual decline is unknown, anti-DHBc antibodies, which are usually detected within 7–14 days in experimentally infected adult ducks, are not detected until B90 days post-hatch, consistent with a high degree of immune tolerance and the delayed activation of immune responses.

Avian Hepadnaviruses (Hepadnaviridae)

107

In summary, despite a high and persistent level of DHBV infection and replication, congenitally DHBV-infected ducks are immune tolerant to DHBV and progression to immune-mediated liver disease, cirrhosis and HCC have not been reported. Although HCC was detected in ducks in a Chinese province, aflatoxin exposure may have been common and the key contributory factor in the development of HCC. Amyloid disease of the liver is common in domestic ducks but does not appear to be linked to DHBV infection. DHBV can also be transmitted experimentally to result in persistent or transient infection with clinical outcomes determined by the age of the ducks at the time of infection and the dose of virus inoculated. Experimental inoculation of newly-hatched ducks with DHBV invariably results in persistent infection with an absence of immune responses capable of clearing infection or producing chronic liver disease. In experiments where DHBV was titrated in newly-hatched (1-3-day-old) ducks, persistent infection was established by intravenous inoculation of pooled DHBV-positive serum containing the equivalent of B1 virus genome. Virus spread throughout the liver to infect 495% hepatocytes within 28 days, showing not only that newly-hatched ducks are highly susceptible to DHBV infection but that the DHBV inoculum, which was derived from pooled sera from congenitally DHBV-infected ducks, had a high specific infectivity with a one-to-one ratio of infectious virus particles to DNA genomes. In related experiments in 3-day-old ducks inoculated with 1500 infectious doses of DHBV, infection was first detected in widelyscattered individual hepatocytes, then in pairs and small groups of cells and resulted in infection of 495% hepatocytes within 13–15 days. Spread of virus occurred both to adjacent hepatocytes presumably by spread within the space of Disse (cell-to-cell spread) and via the blood by release of virus into the liver sinusoids. Susceptibility to persistent DHBV infection declines with age. As ducks increase in age, larger doses are required to establish persistent infection, consistent with the idea that larger doses of virus are required to overwhelm the developing immune system: inoculation of 2-week-old ducks with 4  104 infectious doses of DHBV resulted in infection of randomly-scattered hepatocytes with subsequent virus clearance. Increasing the dose to 1  106 resulted in virus spread to 495% of hepatocytes and persistent infection. As the age of the duck further increases, larger doses are required to establish persistent infection: inoculation of 6-weekold ducks with 1.5–4.5  1010 infectious doses of DHBV resulted in transient infection with successful clearance of infected hepatocytes and accompanying increases in activated Kupffer cells and apoptotic hepatocytes in 11 out of 16 ducks, while the remaining 5 ducks developed persistent infection. Persistent infection was accompanied by mild mononuclear cell infiltration of portal tracts, but no evidence of lobular hepatitis or extensive liver damage was seen within a 1-month timeframe. While showing that susceptibility to persistent infection decreases with age, these studies also raised the question of how the immune system can clear an infection in which virtually all hepatocytes are infected. This is a problem because under most circumstances, including DHBV clearance, the hepatocyte population is mostly self-renewing. This has been partially resolved by subsequent experiments in DHBV-infected ducks, woodchuck hepatitis virus-infected woodchucks and HBV-infected chimpanzees. Current understanding is that the “clearance phase” of transient hepadnavirus infection involves 3 steps: (1). A strong CD8-positive CTL response that kills a large fraction of infected hepatocytes, with direct loss of replicative intermediates and cccDNA; (2). A concurrent CTL-induced interferon-gamma response that eliminates nucleocapsids containing replicating DNA from surviving hepatocytes; and (3). Since hepatocytes are a largely self-renewing population, proliferation of surviving hepatocytes to restore liver cell mass. In this way hepatocytes that survive give rise to the uninfected hepatocytes that populate the liver at recovery. What has remained unclear is the fate during this “clearance phase” of the 5–50 copies of cccDNA present in the nucleus of infected hepatocytes; in particular, those hepatocytes not killed by anti-viral CTL. On one hand there is evidence to suggest that cccDNA elimination requires the turnover of the infected hepatocytes (modeling studies have shown that if cytokines eliminate nucleocapsids containing replicative DNA, and cccDNA survives mitosis, cccDNA-free hepatocytes will be generated by dilution of cccDNA through 2-3 turnovers of the entire hepatocyte population, while if cccDNA does not survive mitosis, generation of cccDNA-free hepatocytes would require o1 turnover). On the other hand, published evidence also suggests that cytokine-mediated effects might lead to loss of cccDNA; whether this is a significant effect in vivo and sufficient to explain cccDNA loss during virus clearance remains unclear. Although hepatocytes are the main cell type infected by DHBV, infection also occurs in extrahepatic tissues. In ducks infected in ovo, with transmission of DHBV from the bloodstream of the female duck to the egg, virus replication first occurs in the endodermal yolk sac, then in the developing liver and pancreas of the embryo (Fig. 5(A–C)). In the liver, hepatocytes and bile duct cells are infected with DHBV and express high levels of DHBcAg and DHBsAg. Virus replication also occurs in scattered acinar cells and in alpha and beta endocrine islet cells in the pancreas, and in glomeruli and tubular epithelial cells in the kidney of developing embryos. Similar patterns of extrahepatic DHBV infection also occur following experimental transmission in ducklings (Fig. 5(C–E)). Viral antigen accumulation in kidney glomeruli many represent bound immune complexes. Cells located in the red pulp and germinal centers of the spleen have been shown to contain DHBV DNA. Mononuclear cells in the red pulp support DHBV replication early after infection while germinal centers contain non-replicating DHBV DNA associated with follicular dendritic cells. Overall, the significance of viral replication in extrahepatic tissues to the pathogenesis of DHBV infection remains unclear.

Diagnosis In ducks with congenital and persistent DHBV infection, where 495% of hepatocytes are infected, DHBV virions are released into the bloodstream resulting in titers of up to 1  1010 virions/ml. 35–60 nm DHBsAg particles that lack a virus nucleocapsid are also released from infected hepatocytes, in 500–1000-fold excess over virions, resulting in B5–50 ug/ml of DHBsAg. Serum samples can be tested for DHBV DNA by PCR and for DHBsAg using enzyme-linked immunosorbent assay (ELISA). DHBV

108

Avian Hepadnaviruses (Hepadnaviridae)

A

B

C

D

E

F

50 uM

Fig. 5 Hepatic and extrahepatic DHBV infection. Immunoperoxidase staining of DHBcAg in yolk sac (A), liver (B, E), pancreas (C, F) and kidney (D). DHBV is transmitted in ovo from the bloodstream of a DHBV-infected female duck to the egg resulting in DHBV infection in (A) yolk sac cells, (B) liver hepatocytes and bile duct cells (arrow) and (C) pancreatic acinar cells in a 14-day fertilized duck embryo. DHBV infection was also detected in (D) kidney glomerular cells (arrow), (E) liver hepatocytes and bile duct cells (arrow) and (F) pancreatic acinar and islets cells (arrow) in a 2-week-old congenitally DHBV-infected duck. Ethanol:acetic acid-fixed tissues were immunostained with polyclonal anti-DHBc antibodies and counterstained with haematoxylin. Magnification  226. Bar ¼ 50 uM.

infection can also be detected by immunoperoxidase or immunofluorescence staining of ethanol:acetic acid-fixed and frozen liver tissue using monoclonal or polyclonal anti-DHBs or anti-DHBc antibodies or by using PCR techniques to detect DHBV DNA in extracts from liver and extrahepatic tissues. Detection of cccDNA is performed by selective extraction of protein-free DNA followed by detection by Southern blot hybridization or by a specific PCR assay for cccDNA with primers spanning the incomplete region of the rcDNA genome to reduce detection of rcDNA and replicative intermediates. DHBsAg and DHBcAg proteins are highly immunogenic. Anti-DHBc antibodies can be detected by ELISA in the serum of ducks during transient and persistent DHBV infections. Anti-DHBs antibodies are produced during the resolution phase of transient infection and are generally not detected in ducks with persistent infection except in complexes with circulating DHBsAg. AntiDHBs antibodies bind virus particles and are able to block DHBV infection of cells. For this reason, ducks that have recovered from transient infection are immune to challenge with DHBV. Cellular immune responses that occur during DHBV infections are more difficult to study due to a lack of reagents for studying the immune system in the duck; however, in vitro stimulation assays for detection of viral antigen specific T-cell responses have been developed and anti-duck CD4 and CD8 monoclonal antibodies are available for further studies. The recent cloning of cDNA for the duck interferon-alpha and –gamma, duck T-cell markers including CD3, CD4, CD8, MHC I and II and duck TLRs now increase the feasibility of studies of the immune response during DHBV infection.

Treatment Because of its reproducible kinetics and predictable outcomes of infection, the DHBV model has been widely used for the evaluation of anti-viral therapies. Similarities in the replication strategy and structure and activity of the HBV and DHBV polymerase enzymes have allowed anti-viral drugs designed for chronic HBV to be evaluated, either as monotherapy or in combination, in DHBV-infected ducks. A range of nucleoside and nucleotide analogs (NA), that inhibit replication by acting as chain terminators, have been tested including ganciclovir, penciclovir, adefovir, entecavir (ETV), tenofovir disproxil fumarate (TDF), emtricitabine and clevudine. Treatment of DHBV-infected ducks with NA blocks reverse transcription and inhibits the synthesis of rcDNA and dslDNA in the cytoplasm of infected cells. However, cccDNA, which is present at 5–50 copies in the nucleus of each infected cell at the outset of treatment, is often reduced (presumably by hepatocyte turnover) but not eliminated, and since cccDNA is the template for transcription of viral RNA, viral antigen expression also persists, and virus replication can rebound if treatment is discontinued. A further issue is the high error rate of the viral polymerase, which can introduce mutations into the polymerase RT domain, allowing the emergence of drug-resistant DHBV strains. Some NA (e.g., ETV and TDF) can be used in monotherapy in treatment naïve patients, but long-term anti-viral therapy for chronic HBV infection more commonly uses combinations of different NA to lower the risk of emergence of multidrug-resistant HBV strains. Newer direct-acting anti-viral agents as well as immunomodulators are also in development for chronic HBV infection. Studies of anti-viral therapy in the DHBV model have demonstrated the stability and long in vivo half-life of cccDNA. It is not surprising therefore that cccDNA persists in trace amounts following the resolution of transient HBV infection and that infection

Avian Hepadnaviruses (Hepadnaviridae)

109

can rebound in situations of immune depletion; for example, during immune suppression following transplantation with donor liver containing residual HBV cccDNA. Studies in the DHBV model have shown that levels of cccDNA present in the liver following the resolution of transient infection are unaffected by treatment with ETV, indicating that residual cccDNA is highly stable and is present in a cell population with a low rate of turnover as compared to typical hepatocytes. The stability and persistence of cccDNA has also been studied in the liver of newly-hatched ducks in which high levels of hepatocyte mitosis occur due to liver growth. Although these results suggested that cccDNA could survive hepatocyte mitosis, there was large duck-to-duck variation in cccDNA levels suggesting that despite therapy with ETV, cccDNA may be maintained to some extent by low levels of viral DNA replication in the growing liver. DHBV-infected ducks have also provided a model system for anti-viral drug therapy in combination with DNA vaccines and have been used to assess nucleic acid polymers developed by REPLICor Inc (which function as broad spectrum anti-viral agents by inhibiting the release of HBsAg from hepatocytes) to protect against and as therapy for persistent DHBV infection. The identification and cloning of the cellular receptor used by HBV, hNTCP, and the ability to express the hNTCP in mammalian cells, has conferred susceptibility to HBV infection on a whole range of transformed cell lines and will enable studies of anti-viral drugs, virus replication and cccDNA to be performed in vitro in cell lines infected with HBV. These systems are likely to decrease reliance on the DHBV model, but are unlikely to replace the model, which allows studies of the complex interplay between host and virus factors during in vivo infection.

Endogenous Avian Hepadnavirus Elements Recent advances in computational genomics have led to the discovery of endogenous avian hepadnavirus elements in the germline of many bird species, representing most of the major bird orders. Endogenous avian hepadnavirus elements were first identified in the zebra finch (Taeniopygia guttata, family Estrildidae) and designated as endogenous zebra finch HBV (eZHBV). eZHBVs have been detected on 10 different chromosomes as site-specific integrated viral fragments that cover B70% of the avian hepadnavirus genome. eZHBVs have also been found in three other estrildid finches: the black throated finch (Poephila cincta), the scaly breasted munia (Lonchura punctulata) and the Gouldian finch (Erythrura Gouldiae) and in the rough-legged hawk (Buteo lagopus) and the black-headed gull (Chroicocephalus ridibundus). Endogenous avian hepadnavirus elements have also been found in the dark-eyed junco (Junco hyemalis, family Emberizidae) and designated as endogenous Junco HBV (eJHBV). The first full-length endogenous avian hepadnavirus elements were found in the genome of the budgerigar (Melopsittacus undulatus, family Psittaculidae) and, along with additional partial genome fragments, were named endogenous budgerigar HBV (eBHBV) (Table 1B). Partial and full-length eBHBVs do not share site-specific orthologous integration points with the identified eZHBVs and, like the individual eZHBVs, represent separate integration events. All of the eZHBV, eJHBV and eBHBV sequences described to date have lost their AUG start codons or contain stop codons or missense mutations that would prevent them from encoding functional proteins. However, analysis of the putative polymerase AA sequences of eZHBV, eJHBV and eBHBV shows that they are related to, but distinct from the exogenous avian hepadnaviruses, AusDHBV, SGHBV1-7, CCHBV1, RGHBV, ETHBV, PHBV-A, STHBV-1 and HHBV-4 (Fig. 1(B)). Phylogenetic analysis suggests that endogenous avian hepadnavirus elements were created through multiple integrations of rcDNA or dslDNA into germline DNA earlier in evolutionary history when the species, or an ancestor, supported exogenous avian hepadnavirus infection. Integration of hepadnavirus DNA into the host genome occurs at a random site and, once fixed in the host germline, the site-specific integration is passed on to progeny by long-term vertical inheritance and can be used as a genetic marker of ancestry. For example, eZHBVs, which were the first endogenous viral elements to be described from a reverse transcribing DNA virus, revealed a deep timescale of the association between hepadnaviruses and birds. The detection of eZHBV sequences in Table 1B

Endogenous Avian Hepadnavirus Elements

Clone name

Accessiona

Length (nt)b

Host

Species

Locationc

eZHBV eZHBV eZHBV eZHBV eZHBV_C eZHBV_C eBHBV-1 eBHBV-2 eJHBV

HQ116569.1 HQ116568.1 HQ116566.1 HQ116567.1 KC750096.1d KC750099.1e BK008520.1/JQ978775.1 BK008521.1/JQ978784.1 HQ116565.1/HQ116576.1

1076 1044 803 1034 4295 4291 4865/4859 3856/3856 1096/325

Zebra Finch Black Throated Finch Scaly Breasted Munia Gouldian Finch Rough-legged Hawk Black-headed Gull Budgerigar Budgerigar Dark-Eyed Junco

Taeniopygia guttata Poephila cincta Lonchura punctulata Erythrura gouldiae Buteo lagopus Chroicocephalus ridibundus Melopsittacus undulatus Melopsittacus undulatus Junco hyemalis

Fitzroy Crossing, Australia Chillagoe, QLD, Australia Pasir Ris, Singapore Seattle, USA Germany Germany Shanghai, China Shanghai, China Arlington, TX, USA

a

GenBank accession number. Length of GenBank sequence containing partial or full-length endogenous avian hepadnavirus elements þ /  fragments of germline DNA. c Location of the bird/collection where the virus is found. d Shares 93.8% identity with eZHBV_C (HAS) described by Suh et al., 2013. e Shares 91.8% identity with eZHBV_C (HAS) described by Suh et al., 2013. b

110

Avian Hepadnaviruses (Hepadnaviridae)

Fig. 6 Evolution of endogenous avian hepadnavirus elements. (A). Endogenous avian hepadnavirus elements reveal a long association between hepadnaviruses and birds. Presence/absence analyzes of eZHBV insertion loci revealed eZHBV endogenization events in the ancestor of Neoaves, during early passeroid evolution and during recent ploceid evolution. The youngest eZHBV (eZHBV_M) was found in the zebra finch but not in the Gouldian finch, weaver, pipit, rosefinch, dunnock, leafbird or sunbird, placing its endogenization after the speciation of the zebra finch in the Miocene, 12.1 million years ago (MYA). Five distinct site-specific orthologous eZHBVs integrants (eZHBV_O1-eZHBV_O5) have been identified during the Oligocene (26.6–34.2 MYA) based on their presence in the finch, but not in the leafbird or sunbird, and one (eZHBV_E) during the Eocene (34.2–35 MYA) which was found in finches and the leafbird but not in the sunbird. The oldest eZHBV integrant (eZHBV_C) is believed to have integrated into a zebra finch ancestor between 82 and 94 MYA, during the Cretaceous Period. The avian tree of life is shown as a simplified chronogram based on molecular dates of interordinal avian relationships, as well as relationships within Passeriformes and Ploceidae. Nucleotide substitution rates (substitutions/site/year) based on HAS (hypothetical ancestral sequences) were calculated for the respective parts of eZHBV evolution and denoted above the tree. Endogenization events are depicted as icosahedrons and temporal ranges of insertion events are shown as colored rectangles. Colored scale at the bottom bar represents millions of years. Reproduced from Suh, A., Brosius, J., Schmitz, J., Kriegs, J.O., 2013. The genome of a mesozoic paleovirus reveals the evolution of hepatitis B viruses. Nature Communications 4, 1791, Fig. 1, with permission. (B). Distribution of endogenous avian hepadnavirus elements across the avian phylogeny. Chromosome and whole DNA genome shotgun assemblies of 48 avian species were screened in silico using tBLASTn with a library of hepadnavirus protein sequences. 46 out of 48 host species generated high-identity matches to hepadnavirus proteins and are marked in red on the species tree, while 2 DNA samples were negative and are marked in white. The phylogeny is based on the results of a phylogenomics consortium whole DNA genome analyzes across all the species shown. Reproduced with modifications from Cui, J., Zhao, W., Huang, Z., et al., 2014. Low frequency of paleoviral infiltration across the avian phylogeny. Genome Biology 15 (12), 539, Fig. 1, with permission.

different avian species is illustrated in Fig. 6(A) (reproduced with permission Suh et al., 2013). The youngest eZHBV integrant to be described (eZHBV_M; corresponding to 15% of the avian hepadnavirus genome) is thought to have integrated into the germline genome of a zebra finch ancestor in the Miocene, 12.1 MYA. Five additional integration events have been identified for eZHBVs (eZHBV_O1-eZHBV_O5; 3.3%–9.0% of the avian hepadnavirus genome) during the Oligocene (26.6–34.2 MYA), and one (eZHBV_E; 14.8% of the avian hepadnavirus genome) during the Eocene (34.2–35 MYA). The oldest eZHBV integrant to be dated so far (eZHBV_C; 99% of the avian hepadnavirus genome) is believed to have integrated between 82 and 94 MYA, during the Cretaceous Period. The detection of eZHBV_C in all major taxa of Neoaves (which includes most modern birds) and its absence in non-Neoaves (e.g., the chicken and the ostrich) places the time of integration after the separation of Neoaves and non-Neoaves but before the diversification of Neoaves. Mining of whole genome sequence data recently made available for 48 bird species (representing thirty-two of thirty-five proposed bird orders and including all Neoavian orders) has revealed the widespread presence of endogenous avian hepadnavirus

Avian Hepadnaviruses (Hepadnaviridae)

111

elements. As can be seen in Fig. 6(B) (adapted and reproduced from Cui et al., 2014), endogenous avian hepadnavirus elements were detected in 46 out of 48 species including the zebra finch and budgerigar as previously discussed, but also detected in species that support exogenous avian hepadnavirus infection including the Grey crowned crane and the mallard, the species from which most domesticated ducks are derived. The diversity of avian species containing endogenous avian hepadnavirus elements suggests that multiple integrations have occurred over the evolutionary history of birds.

Acknowledgments The authors wish to acknowledge Dr William Mason for critical reading of the manuscript and helpful discussions, and Ms Tahlia Perry and Prof Frank Grutzner for assistance with the phylogenetic analysis.

References Cui, J., Zhao, W., Huang, Z., et al., 2014. Low frequency of paleoviral infiltration across the avian phylogeny. Genome Biology 15 (12), 539. Suh, A., Brosius, J., Schmitz, J., Kriegs, J.O., 2013. The genome of a mesozoic paleovirus reveals the evolution of hepatitis B viruses. Nature Communications 4, 1791.

Further Reading Cui, J., Holmes, E.C., 2012. Endogenous hepadnaviruses in the genome of the budgerigar (Melopsittacus undulatus) and the evolution of avian hepadnaviruses. Journal of Virology 86 (14), 7688–7691. Foster, W.K., Miller, D.S., Scougall, C.A., et al., 2005. Effect of antiviral treatment with entecavir on age- and dose-related outcomes of duck hepatitis B virus infection. Journal of Virology 79 (9), 5819–5832. Gilbert, C., Feschotte, C., 2010. Genomic fossils calibrate the long-term evolution of hepadnaviruses. PLOS Biology 8 (9). Guo, H., Mason, W.S., Aldrich, C.E., et al., 2005. Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity. Journal of Virology 79 (5), 2729–2742. Jilbert, A.R., Miller, D.S., Scougall, C.A., Turnbull, H., Burrell, C.J., 1996. Kinetics of duck hepatitis B virus infection following low dose virus inoculation: One virus DNA genome is infectious in neonatal ducks. Virology 226 (2), 338–345. Jilbert, A.R., Wu, T.T., England, J.M., et al., 1992. Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement. Journal of Virology 66 (3), 1377–1388. Lauber, C., Seitz, S., Mattei, S., et al., 2017. Deciphering the origin and evolution of hepatitis B viruses by means of a family of non-enveloped fish viruses. Cell Host Microbe 22 (3), 387–399. Le Mire, M.F., Miller, D.S., Foster, W.K., Burrell, C.J., Jilbert, A.R., 2005. Covalently closed circular DNA is the predominant form of duck hepatitis B virus DNA that persists following transient infection. Journal of Virology 79 (19), 12242–12252. Mason, W.S., 2015. Animal models and the molecular biology of hepadnavirus infection. Cold Spring Harbor Perspectives in Medicine 5 (4). Miller, D.S., Bertram, E.M., Scougall, C.A., Kotlarski, I., Jilbert, A.R., 2004. Studying host immune responses against duck hepatitis B virus infection. Methods in Molecular Medicine 96, 3–25. Piasecki, T., Harkins, G.W., Chrzastek, K., et al., 2013. Avihepadnavirus diversity in parrots is comparable to that found amongst all other avian species. Virology 438 (2), 98–105. Reaiche-Miller, G.Y., Thorpe, M., Low, H.C., et al., 2013. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver. Virology 446 (1–2), 357–364. Summers, J., Jilbert, A.R., Yang, W., et al., 2003. Hepatocyte turnover during resolution of a transient hepadnaviral infection. Proceedings of the National Academy of Sciences of the United States of America 100 (20), 11652–11659.

Avian Herpesviruses (Herpesviridae) Vishwanatha RAP Reddy and Venugopal Nair, The Pirbright Institute, Pirbright, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Classification Avian herpesviruses (AHV) are widespread among most variety of domestic, wild and captive bird species. Similar to mammalian herpesviruses, most AHV have narrow host range in their natural hosts, although some of these viruses infect different bird species belonging to the same or to unrelated families or even orders. Based on the International Committee on Taxonomy of Viruses (ICTV), AHV are classified as alphaherpesviruses, under subfamily Alphaherpesvirinae, family Herpesviridae and order Herpesvirales. These viruses include Gallid herpesvirus type 1 (GaHV1) and Psittacid herpesvirus type 1 (PsHV1) that belong to the genus Iltovirus, and Gallid herpesvirus type 2 and 3 (GaHV2 and 3), Meleagrid herpesvirus 1 (MeHV1), Anatid herpesvirus 1 (AnHV1) and Columbid herpesvirus 1 (CoHV1) that belong to the genus Mardivirus. Several taxonomically unassigned AHV were also identified in a wide variety of birds, such as falcons (Falconid herpesvirus 1, FaHV1), cranes (Gruid herpesvirus 1, GrHV1), cormorant (Phalacrocoracid herpesvirus 1, PhHV1), eagles (Acciptrid herpesvirus 1, AcHV1), storks (Ciconiid herpesvirus 1, CiHV1), owls (Strigid herpesvirus 1, StHV1), quails (Perdicid herpesvirus 1, PdHV1), penguins (Sphenicid herpesvirus 1, SpHV1) and loons (Gaviid herpesvirus 1, GavHV1) (Fig. 1). Additional AHV infections have also been reported in several wild- and free-range bird species such as the prairie falcon, red-headed falcon, little pied cormorant, demoiselle cranes, Sudan crowned cranes, paradise cranes, Japanese cranes, hooded cranes, black storks, American white storks, black-footed penguin, satyr tragopan, toucans, and many passerine birds.

Fig. 1. Genetic relationship of avian herpesviruses with other alphaherpesviruses based on 44 592 amino acid sequences of 52 core genes of the viruses is shown. Gallid herpesvirus 1 (GaHV1 ) and Psittacid herpesvirus 1 (PsHV1) are closely clustered under Iltovirus genus. Gallid herpesvirus 2 and 3 (GaHV2 and 3), Meleagrid herpesvirus 1 (MeHV1), Anatid herpesvirus 1 (AnHV1) and (Falconid herpesvirus 1, FaHV1) are closely grouped under Mardivirus genus. Above figure adapted from Pfaff, F., Schulze, C., König, P., et al., 2017. A novel alphaherpesvirus associated with fatal diseases in banded Penguins. Journal of General Virology 98, 89–95. doi:10.1099/jgv.0.000698.

112

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20930-8

Avian Herpesviruses (Herpesviridae)

113

Most AHV of wild or free-range birds have been named according to the species of the avian hosts. However, a number of others in wild- and free-range birds still remain as nameless orphan AHV. In some cases, AHV are named after the pathological characteristics associated with the diseases or in honor of scientists who first reported it. Examples include infectious laryngotracheitis virus (ILTV or GaHV1), duck enteritis virus (DEV or AnHV1), hepatosplenitis infectiosa strigum virus (HSiS or StHV1), inclusion body disease virus (FaHV1, GrHV1 and AcHV1), herpesvirus of turkeys (HVT or MeHV1), Pacheco’s parrot disease virus (PPDV or PsHV1), Marek’s disease virus (MDV or GaHV2), and Smadel’s disease virus (CoHV1). This article mainly will focus on ILTV, PPDV, MDV and DEV. For information on other AHV, please refer to further reading details. ILTV is a highly contagious respiratory virus of chickens that is transmitted by inhalation, direct bird-to-bird contact and indirectly through fomites and people. ILTV actively replicates in the epithelium of larynx, trachea and conjunctiva, leading to severe epithelial damage, hemorrhages and laryngotracheitis (hence the term ‘infectious laryngotracheitis’). After an acute phase of infection, characterized by clinical signs such as nasal discharge, conjunctivitis and drop in egg production, ILTV establishes a lifelong latency in the trigeminal ganglion. During severe forms of the disease, additional signs such as gasping, expectoration of bloody mucus, dyspnea and death due to asphyxia are also observed. Major economic impact of ILT comes from production losses, high morbidity and mortality and from the costs of vaccination. Live attenuated and recombinant viral vectored vaccines are available to protect against ILT. Most outbreaks are associated with reversions of the live vaccines strains to a virulent strains. Pacheco’s parrot disease virus (PPDV) or Psittacine herpesvirus 1 (PsHV1) is a highly contagious, and potentially fatal respiratory virus of psittacine birds, characterized by acute necrotizing lesions of the crop, intestines, liver and pancreas. Pacheco’s parrot disease (PPD) has been reported in psittacine birds in most geographical locations of the world, and is a major concern in the captive parrots of exotic collections. Amazon parrots, cockatiels, Roselle, Galah and macaws are some of the worldwide affected bird species of the PPDV. PPDV outbreaks commonly occur in the quarantine stations and markets, where parrots are held in close proximity. Thus, worldwide exotic pet breeders and companion bird markets are of great concern about the PPD. Other types of psittacine herpesviruses distinct from PsHV1 such as psittacid herpesvirus 2 and 3 have been identified, serotyped and phylogenetically characterized. Marek’s disease virus (MDV-1) or Gallid herpesvirus 2 (GaHV2) is a contagious oncogenic virus, causes neoplastic and neuropathic disease in chickens, commonly known as Marek’s disease (MD). Only, MDV-1 causes clinical oncogenic disease in chickens. Gallid herpesvirus 3 (GaHV3) and herpesvirus of turkeys [HVT or Meleagrid herpesvirus 1 (MeHV1)] are non-oncogenic apathogenic herpesviruses of chickens and turkeys, respectively. GaHV3 and HVT are usually used as the live vaccines against pathogenic MD. Duck enteritis virus (DEV) or Anatid herpesvirus 1 (AnHV1) is a highly contagious virus of waterfowl species such as ducks, geese and swans, which causes acute disease called duck viral enteritis (DVE), sometimes also referred to as duck plague. Waterfowls of all ages are susceptible to DEV and the disease is characterized by vascular damage, tissue hemorrhages, lymphoid organ lesions, digestive mucosal eruptions, degenerative changes in parenchymatous organs and death. DVE causes significant economic losses in domestic and wild water fowls due to mortality, reduced egg production and meat condemnations. Total morbidity and mortality may range from 5% to 100%.

Host Range Many AHV appear to be group-specific in a given bird species and exhibit a very narrow host range. Chickens are the primary natural hosts of ILTV, although pheasants, pheasant-chicken crosses and peafowls are also infected with this virus. ILTV is commonly isolated and propagated in the embryonated chicken eggs, where it induces opaque plaques on the chorioallantoic membrane (CAM) resulting from necrosis and proliferation of the infected cells. Plaques on CAM can appear as early as two days post-inoculation (PI) and embryo deaths occur between 2 and 8 days PI. ILTV can also be propagated in a variety of primary avian cell cultures such as chicken embryo kidney (CEK), chicken embryo liver (CEL), chicken embryo lung and chicken kidney cells (CKC), as well as in the LMH (leghorn male hepatoma) cell line. PsHV1 grows in primary chicken embryo fibroblasts (CEF) cultures where it readily shows syncytial plaques. MDV-1 is routinely propagated in CEF, embryonic CKC, and/or duck embryo fibroblasts (DEF). Several lymphoblastoid cell lines that grow continuously as suspension cultures have been developed from MD lymphomas. These cell lines are routinely used to understand the pathobiology of MD lymphomas.

Virion Structure Broadly AHV have hexagonal nucleocapsids with icosahedral symmetry, with typical morphological features of herpesvirus particles. The 80–125 nm in diameter icosahedral nucleocapsids is made of elongated hollow 162 capsomers-150 hexamers and 12 pentamers. Overall diameter of the enveloped virus can vary. ILTV with irregular envelope has a diameter of 195–250 nm, while the enveloped MDV particles are around 150–160 nm. In general, the morphologies of members of the Mardivirus are similar, although HVT capsids appear to show a unique crossed appearance.

Genome AHV isolates have linear double stranded DNA genomes of approximately 150–200 kilobase pairs (kbp). Based on the genome organization, herpesviruses are classified in to six different groups A to F including class D (ILTV and PsHV1) or E (MDV-1, GaHV3

114

Avian Herpesviruses (Herpesviridae)

and HVT) genomes. Class D herpesvirus genomes consist of long and short unique regions (UL and US) interspersed with inverted repeat sequences, such as internal repeat (IR) and terminal repeat (TR) sequences. Class E herpesvirus genomes, consist of unique long (UL) and unique short sequence (US), flanked by a sets of inverted repeat sequences: the terminal repeat long (TRL), internal repeat long (IRL), internal repeat short (IRS), and terminal repeat short (TRS), respectively. The 153-kbp ILTV genome, predicted to have 79 open reading frames (ORFs), has a 47.9% G+C content, while the 163-kbp PPDV genome, with 73 predicted ORFs, has a 60.9% G+C content. The majority of ILTV and PPDV ORFs have homologous ORFs in other alphaherpesviruses, such as HSV1. However, many unique ORFs also do exist in the ILTV and PPDV genomes. For example, ORFs A, B, C, D, E, F, UL0 and UL1 are largely specific of ILTV and PPDV. A large internal inversion of UL22 to UL44 genes within the UL segment has been also reported to be specific to ILTV and PPDV genomes. UL47, a conserved homolog among alphaherpesvirues, is absent at the corresponding positions of ILTV and PPDV genomes, but a homolog with weak identity (18%) is found to be inserted between the US3 and US4 genes of US region. ILTV and PPDV genomes also have differences in gene organization, for example, a UL16 homolog that is conserved throughout all herpesviruses, is absent in ILTV but present in PPDV. In contrast, ILTV contains UL48, UL0 ORFs and US9, neither of which are present in PPDV. Further, the ILTV inverted and terminal repeats are 2535 bp (18.5%) shorter than the PPDV, and each repeat consists of three genes: ICP4, US10 and a homolog of the MDV sORF4/3. Conversely, only ICP4 is present in inverted and terminal repeats of PPDV, US10 and sORF4/3 are present in US region. The full genome sequences of MDV, GaHV3 and HVT are very similar of approximately 160–180 kb with a varying G+C content of ~43.9%, ~53.6% and ~47.6%, respectively. A total of 103 ORFs in MDV genome, 102 in GaHV3 and 99 in HVT were clearly identified, in addition to a number of microRNAs. The majority of UL and US genes of Maridiviruses are homologous to the genes encoded by the alphaherpesviruses. Many virus-specific genes have been identified, and are located predominantly in the IRL and TRL regions. Meq (MDV EcoRI Q), vIL8 (viral interleukin 8), vLIP (viral lipase), pp38/pp24 and vTR (RNA telomerase subunit) genes have been identified that are unique for MDV. The Meq gene encodes a 339-amino acid oncoprotein, which contains a basic leucine zipper (bZIP) transcriptional regulator domain at the N terminal and the proline rich repeat transactivator domain at the C terminal. The Meq protein is expressed consistently in the nucleus of lymphoma cells and tumor cell lines. The vIL8 gene is located in the long repeat region, and originally was identified as a spliced Meq variant. The vIL8 functions as a chemoattractant for chicken mononuclear cells, and may be important for the switch of infection from B to T lymphocytes. The vLIP gene, required for efficient lytic replication, encodes a soluble N glycosylated protein similar to α/β beta fold of pancreatic lipases. MDV-1 genome encodes the pp38/pp24 complex within the TRL/IRL and the UL region. MDV-encoded vTR gene, with nearly 88% sequence identity to the chicken telomerase RNA (cTR) indicating its transduction from the host genome, is present in the IRL/TRL region of the MDV genome. HVT encodes a two copies of vNr13 protein, a Bcl2 homolog, not seen in the other alphaherpesviruses. The vNr13 is strikingly similar to chicken Nr13 protein (63.7% amino acid identity), an antiapoptotic member of the Bcl2 like proteins. Most DEV strains have approximately of 160-kbp long genome with a G+C content of 44.89%. With around 78 ORFs, many are homologs seen in other alphaherpesviruses. Five DEV genes were unique among the AHV, while three appear to be specific to DEV.

Replication Cycle The replication cycle of ILTV is similar to other alphaherpesviruses, which usually starts with the initial attachment to cell surface, which is enhanced by viral glycoproteins gB, gD and gH-L complex. After initial attachment, the nucleocapsid is released into the cytoplasm and transported to the nucleus. Upon injection of the viral genome in the nucleus via the nucleopore, viral DNA replication and transcription occur. In general, the alphaherpesvirus genome transcription strictly follows a regulated cascade at different time points grouped into immediate early (IE), early (E) and late (L) genes. Commonly, IE and E genes, and their products regulate transcription of L genes. First several enzymes and DNA binding proteins are expressed, which are required for DNA replication, e.g., DNA polymerase and thymidine kinase. This is followed by the expression of viral structural proteins such as the capsid, tegument and envelope glycoproteins. The newly synthesized DNA concatemers cleaves into monomeric units and assemble with capsids to form nucleocapsids in the nucleus. The assembled nucleocapsids exploit an envelopment/de-envelopment pathway for transport to the cytosol. The nucleocapsids acquire an envelope by budding through the inner leaflet of the nuclear membrane. The primary enveloped nucleocapsids, undergo a fusion with the outer nuclear membrane, leading to the release of de-enveloped/naked nucleocapsids into the cytoplasm. In the cytoplasm, main tegument proteins are added to the nucleocapsids, before obtaining their final envelope by budding into the Golgi-derived vesicles. Enveloped mature virions released into the extracellular space by exocytosis/cell lysis and membrane vacuolar fusion. The replication cycle of Mardiviruses are similar to other cell-associated herpesviruses such as varicella zoster virus (VZV). The precise virus entry, attachment and replication pathways of MDV remains still unsolved because of strict cell-associated nature of the virus, and only few steps of the replication cycle are available. US3-kinase has been reported to be involved in the morphogenesis as well as cell-to-cell spread of virus through the effect on stress fiber breakdown and polymerization of actin. The glycoproteins gE, gI and gM play a role in the transfer of virus from infected to uninfected cells.

Epidemiology Majority of AHV are highly contagious especially in intensive production system where the viruses are transmitted very efficiently between birds. Many AHV appear to be group-specific in a given bird species and exhibit a very narrow host range, suggesting their co-

Avian Herpesviruses (Herpesviridae)

115

evolution over millions of years. Chickens are the primary natural hosts of ILTV, although pheasants, pheasant-chicken crosses and peafowls are also infected with this virus. Phylogenetic analysis have suggested that AHV species such as ILTV and PsHV-1 have separated from mammalian alphaherpesviruses around 200 million years ago, and have been co-evolving since then with their specific hosts. PsHV1-infected birds shed the virus from the oral or respiratory secretions and droppings. As in the case of ILTV infection, PsHV1 is transmitted by direct bird to bird contact or indirectly by contact with contaminated fomites or environment. Latent virus persists in the flock over generations, and act as source of new infection following reactivation during stress with factors such as the onset of egg laying and mixing of chickens. ILTV has been also reported in extra respiratory tissues like liver, spleen, kidneys, heart, intestines, bursa Fabricius and Harderian glands. Epidemiology of ILTV is also affected by their ability for potential recombination. Water appears to be the natural means of DEV transmission among infected water fowls. DEV establishes latency in the TG and lymphoid tissues including peripheral blood lymphocytes, and recovered waterfowls become carriers and shed the virus intermittently. Viruses such as MDV mainly infects chickens, although quails, turkeys and pheasants are also susceptible to the disease. Because of their long-term survival in the infected environment in the poultry house dust, coupled with continuous shedding from dander and dust in the latently-infected hosts, diseases such as MD are difficult to be eradicated from the infected flocks. While vaccination still continues to be the most effective approach for control of MD, the leakiness of the current vaccines due to their inability to prevent virus shedding has been thought to be one factor driving virulence of the MDV strains.

Clinical Features Clinical features of AHV infections vary between viruses and tissues targeted by different viruses. ILTV-associated diseases range from mild to severe forms. The mild form of the disease is characterized by nasal discharge, mild conjunctivitis, mild respiratory rales, reduced egg production and no mortality, while the severe form is characterized by open mouth breathing with expectoration of bloody mucoid material, severe conjunctivitis, high morbidity, and moderate to severe mortality. The clinical signs are reported in chickens of all ages but the most characteristic are observed in adult birds. The important gross pathological changes of ILT include hemorrhages and diphtheritic changes in larynx and trachea, with mucoid plugs/casts in the trachea and presence of eosinophilic inclusion bodies in mucosal cells. Clinical signs of PPD include anorexia, depression, nasal discharge, diarrhea, tremors and instability. The majority of infected birds die very rapidly before the onset of clinical signs. Gross pathological lesions include abnormal changes in the respiratory, muscular and circulatory system, bone marrow, thyroid and adrenal glands, liver, spleen, kidneys, the urogenital and gastrointestinal tract, and the nervous system. Histopathological changes include necrotizing lesions in many organs, hemorrhages and congestion of the liver, spleen and kidneys, and the presence of intranuclear inclusion bodies. Clinical manifestations of MD vary according to the specific syndromes. In general, the classical form of the disease is characterized by unilateral paralysis of the legs and/or wings. In acute forms of disease with visceral lymphomas, affected birds may appear clinically normal but have extensive neoplastic involvement visible on post-mortem examination. Non-specific signs such as weight loss, paleness, anorexia, and diarrhea may also be seen in the chickens affected with lymphomas. The incidence of MD is quite variable in commercial flocks and in general are low since the introduction of vaccination. Severely affected peripheral nerves may show loss of cross-striations, yellow discoloration and edematous appearance. Lymphomas may occur in a variety of visceral organs. Lymphomas can be found in the lung, spleen, bursa, thymus, heart, adrenal gland, kidney, liver, mesentery, proventriculus, intestine, iris, skeletal muscle, skin and gonads. Microscopically two main types of lymphoproliferative lesions are recognized in peripheral nerves. Type A lesions are considered as neoplastic mainly consisting of CD30+ CD4+ T cells. Demyelination and Schwann cell proliferation of peripheral nerves are also observed in type A lesions. Type B lesions are mostly inflammatory, characterized by diffuse, light to moderate infiltration of lymphocytes and plasma cells. MD lymphomatous lesions in MD are more proliferative than those in nerves. Appearance is similar to type A lesions, mainly consist of T lymphocytes and lymphoblasts, B cells, macrophages and natural killer cells (NK cells). Majority of transformed T cells express CD4 and MHC II. In mature breeder duck flocks acutely infected with DEV, sudden, high and persistent mortality with drop in egg production have been reported. As infection progresses, other signs such as loss of appetite, ataxia, photophobia, associated pasted eye-lids, droopiness, ruffled feathers, nasal discharge and watery diarrhea are observed. Characteristic lesions of DVE are intravascular coagulopathy and necrotic degenerative changes in mucosa and submucosa of gastrointestinal tract in lymphoid and parenchymatous organs. Petechial or larger extravasations of blood may be found or in the myocardium and other visceral organs. Liver, pancreas, intestine, lungs, and kidney surfaces may be covered with petechial hemorrhages. Mucosal lesions are observed in the oral cavity, esophagus, ceca and cloaca. Microscopically, initial lesions occur in the walls of blood vessels. The endothelial lining is disrupted and connective tissue of the wall becomes less compact. Eosinophilic intranuclear and cytoplasmic inclusions are observed in epithelial cells of digestive tract.

Pathogenesis Pathogenesis of AHV can differ based on their tissue tropism and diseases. ILTV enters its host through upper respiratory and ocular routes, and then primarily replicates in the epithelium of larynx, tracheal and conjunctival mucosae, respiratory sinuses, air sacs and lungs. During the first 7 days of infection, ILTV replication in the larynx and trachea, leads to epithelial damage and hemorrhages. Several authors have reported that active replication of ILTV slows down after 6 days post inoculation (dpi), and virus may remain at very low levels up to 10 dpi. ILTV infection spreads to trigeminal ganglion (TG) in 4–7 days, where the virus establishes latency.

116

Avian Herpesviruses (Herpesviridae)

Transmission of MDV occurs through the inhalation of infectious dander via the respiratory route, where the virus replication is thought to be initiated in macrophages. After 24 h of infection, cytolytic replication takes place in the lymphoid organs such as spleen, bursa and thymus, where productive cytolytic infection is initiated in the B cells, followed by the T cells. After the initial cytolytic infection associated with immunosuppression, MDV enters a latency phase in T-cells with potential integration in the telomeric regions of the host chromosomes. Finally, some of the latently-infected CD4+ T cells are transformed leading to the development of T-cell lymphomas in multiple organs. MDV cytolytic replication continues in the feather follicle epithelium (FFE), the only known site from where cell-free infectious virus is assembled and released into the environment.

Diagnosis Monitoring and surveillance of AHV infections are important to recognize the distribution and occurrence conditions in the flocks. Diagnosis of AHV infections in poultry flocks are made using pathological, virological and molecular methods. Pathological diagnosis using gross pathological lesions or histopathological changes are often sufficient if the clinical symptoms and lesions are typical of the diseases associated with the AHV infections. Virological methods of diagnosis include virus isolation using cell culture, chicken embryos and animal models, as well as by the detection of viral antigens in the suspected samples. While virus isolation may not be suitable for all AHV isolates, especially as some of the virus isolates may not easily replicate in the culture systems as further adaptation is required. Serological detection of viral antigen-specific antibodies is also valuable for diagnosis of AHV infections, particularly for detection of infections at the flock levels. PCR-based molecular methods of detection of AHVspecific nucleic acids are increasingly used in the diagnosis due to their inherent speed, sensitivity, reproducibility, and because of the potential to be used on clinical samples. Real-time PCR capable of multiplexing for simultaneous detection of multiple AHV has been developed, which give quantitative data on the copy numbers of AHV isolates in clinical samples. While all of these diagnostic tools are valuable in detecting AHV in the samples, it has to be borne in mind that simple detection of viruses, nucleic acids or proteins alone in the samples does not conclusively show that the clinical disease is indeed caused by these pathogens, especially AHV species such as MDV are ubiquitous present even in clinically normal birds.

Prevention Control strategies for diseases caused by different AHV can vary based on epidemiological features and disease characteristics although some of the basic principles are common. ILTV is controlled by coordinated efforts of rapid diagnosis, vaccination protocol, and biosecurity measures to prevent further spread. Currently, ILT control is achieved through the use of commercially available live attenuated or genetically-engineered recombinant herpesvirus of turkeys (HVT) or Fowl pox virus (FPV) vectored vaccines. Live attenuated vaccines such as the chicken embryo origin (CEO) vaccines are effective in inducing strong immunity but possess considerable residual virulence, and can cause disease, particularly when birds are stressed. Such viruses can then spread by bird-to-bird passages to initiate new outbreaks as well as causing emergence of virulent viruses. Thus, the use of attenuated vaccines has been restricted to ILTV-enzootic areas. Genetically engineered recombinant vaccines are effective to limit signs of disease, but have only limited effect on virus shedding. Thus further innovations in the design and development of new vaccines are required for more effective control of future ILTV outbreaks. Inactivated vaccines are available against PSHV1, and are developed by using common inactivating agents. There are three main serotypes of the PsHV1 and infection of one serotype does not protect against with another serotype, suggesting the potential value of autogenous vaccines. However, development of autogenous vaccines requires time for virus isolation and vaccine production. Strict quarantine measures along with diagnostic approaches are also critical, especially for exotic pet bird breeders and companion bird keepers. For MD, vaccination is the principal strategy for the prevention and control. MD vaccines are administered to 18-day old embryos in ovo or 1-day old chicks at hatch. Currently used MD vaccines include the GaHV2 strain CVI988 (Rispens), and naturally apathogenic GaHV3 strain SB-1 and HVT strain Fc126. HVT-based vaccines are also extensively used as recombinant viral vectors for protecting against multiple avian diseases. Recent studies have suggested that MD vaccines such as HVT have the potential to drive virulence because of their limited effects on MDV replication and shedding. Inactivated and attenuated vaccines available against DEV are effective in control especially when combined with quarantine measures during import and export of domestic and wild waterfowl species.

Further Reading Garcia, M., Spatz, S., Guy, J.S., 2013. Laryngotracheitis. In: Swayne, D.E., Glison, J.R., McDougald, L.R., et al. (Eds.), Diseases of Poultry, thirteen ed. pp. 161–179. Kaleta, E.F., Docherty, D.E., 2007. Avian herpesviruses. In: Thomas, N.J., Hunter, D.B., Atkinson, C.T., et al. (Eds.), Infectious Disease of Wild Birds. Ames: Blackwell Publishing, pp. 63–86. Metwally, S.A., 2013. Duck virus enteritis (duck plague). In: Swayne, D.E., Glison, J.R., McDougald, L.R., et al. (Eds.), Diseases of Poultry, thirteen ed. pp. 431–440. Schat, K.A., Nair, V., 2013. Marek’s disease. In: Swayne, D.E., Glison, J.R., McDougald, L.R., et al. (Eds.), Diseases of Poultry, thirteen ed. pp. 161–179. Thureen, D.R., Keeler Jr., C.L., 2006. Psittacid herpesvirus 1 and infectious laryngotracheitis virus: Comparative genome sequence analysis of two avian alphaherpesviruses. Journal of Virology 80, 7863–7872.

Avian Influenza Viruses (Orthomyxoviridae) Nicolas Bravo-Vasquez and Stacey Schultz-Cherry, St. Jude Children’s Research Hospital, Memphis, TN, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary “DIVA" (differentiating infected from vaccinated animals) Method used to differentiate influenza virus vaccinated from infected animals. Genetic clade A taxonomic group that comprises a single common ancestor and all descendants of that ancestor. Highly pathogenic avian influenza virus (HPAI) Designation given to select H5 and H7 avian influenza viruses that kill chickens. Intravenous pathogenicity index (IVPI) Means of testing the level of pathogenicity of an influenza virus isolate by

observing clinical signs in infected birds over a ten-day period. Strains are considered highly pathogenic if they cause more than 75% mortality within 8 days. Low pathogenic avian influenza virus (LPAI) Designation given to avian influenza viruses that do not kill chickens. Reservoir A reservoir host or carrier that harbors pathogenic organisms, without injury to itself and serves as a source from which other individuals can be infected. Spillover Term describing the spread of influenza virus into another host. Can refer to spread from wild birds into poultry.

Introduction Avian influenza viruses (AIV) are an ongoing risk to domestic, pet, exotic (zoo) and wild birds worldwide. Disease outbreaks can threaten biodiversity by causing high morbidity and high mortality in endangered species and can also have a significant impact on a country’s economy by halting trade among countries, forcing market closures and halting exportations of avian products and sub products from affected countries. While members of the class Aves are most susceptible to disease, a broad variety of animal species including humans can be infected with AIV leading to significant public health concerns. Indeed, at least three human influenza pandemics of the 20th century (1918, 1957 and 1968) were caused by avian-human reassorted viruses. More recently, AIV subtypes H5, H7, H9, H10, and H6 have been associated with zoonotic infections in humans resulting in a call for increased global AIV surveillance activities by the World Health Organization (WHO), Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (OIE).

Taxonomy AIV belongs to the Orthomyxoviridae family, which is comprised of the following genera: Alphainfluenzavirus (including influenza A virus), Betainfluenzavirus (influenza B virus), Gammainfluenzavirus (influenza C virus), Deltainfluenza virus (influenza D virus in cattle and swine), Thogotovirus (a tick-borne virus), Isavirus and Quaranjavirus. As mentioned above, AIV are solely found in the Alphainfluenzavirus. Like all influenza viruses, they can be further classified into subtypes based on the genetic and antigenic properties of their surface glycoproteins, HA and neuraminidase (NA). Combinations of all 16 HA subtypes (H1-H16) and 9 subtypes of NA (N1-N9) have been isolated from birds. The endemic circulation of H5 viruses in many parts of the world has led to further subgrouping of the HA into unique genetic clades. A clade is a taxonomic group that comprises a single common ancestor and all descendants of that ancestor. There are geographical differences in the circulation of some HPAI H5 clades.

Virion Structure and Genome AIV virion is pleomorphic with a size that may vary between 80 and 120 nm and may be either spherical or filamentous. The genome is composed of eight single strand negative-sense RNA segments that code for over 10 proteins including the nucleoprotein (NP), nuclear export protein (NEP) and nonstructural protein (NS), a polymerase heterotrimeric complex (polymerase basic protein 1 (PB1); polymerase basic protein 2 (PB2) and polymerase acid protein (PA)), HA, NA and matrix protein 2 (M2, ion transport channel), and the matrix protein 1 (M1) that maintains the virion shape. The virus is surrounded by a lipid envelope derived from the host plasma membrane. The trimeric HA protein has receptor (cell surface expressed sialic acid) binding activity mediating cell entry. It is the predominant surface glycoprotein being up to 80% of the total proteins present in the viral envelope. The second most predominant surface glycoprotein, the tetrameric NA, has neuraminidase activity that cleaves sialic acid molecules allowing viral egress. HA and NA must work in concert to support productive viral replication. More recently, additional accessory proteins have been identified including PB1-F2, PB2-F2, PB1-N40, PA-X and PA-N155 and PA-N182 that play crucial roles in the viral life cycle.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20932-1

117

118

Avian Influenza Viruses (Orthomyxoviridae)

Life Cycle Avian influenza viruses follow the same replication cycle as mammalian influenza viruses; attachment, entry, fusion, vRNA transport into the nucleus, transcription, replication, translation, assembly and export. If the life cycle is the same, why don’t avian viruses replicate in mammalian cells and vice versa? There are several species-specific restriction factors throughout the replication cycle that block this from occurring. First, HA on the virion attaches to host cell surface expressed sialic acids (SA). The avian influenza viruses have tropism for cellular receptors with terminal α2,3 sialic acid (SAα2,3Gal), which can be found throughout the respiratory and intestinal tract of birds and the respiratory tracts of mammals including swine, horses and humans (deep lung). Mammalian viruses bind preferentially to α2-6 linked SA. Second, after attachment, AIV virions enters the cells via receptor-mediated endocytosis where the pH change within the endosome induces a conformation change in HA exposing the HA2 protein resulting in membrane fusion. While the precise pH required for the HA conformational change differs by viral strain and host species, overall HA stability has been linked to pandemic potential and ability to cross-species. Third, after fusion, the viral ribonucleoproteins (vRNPs) are released into the cytoplasm where they co-opt host factors for transportation into the nucleus for replication initiation. Genome replication represents one of the major host range barriers between avian and mammalian influenza viruses. There are several species-specific host restriction factors that block AIV replication in mammalian cells and vice versa. These include cytoplasmic pattern recognition factors and interferon-stimulated genes (MxA, IFITM3, DDX21 as examples) and specific importin-α nuclear import proteins. Once the vRNPs have entered the nucleus, the genome is transcribed followed by replication. The AIV polymerases perform genome replication poorly in mammalian cells. This is likely due to a combination of species-specific host factors like ANP32A and DDX17 in addition to specific mutations in all three viral polymerase genes as well as in NP, NEP with PB2 E627K being the best understood. Difference in temperature at which polymerase enzymatic activity is regulated also contributes to species-specific blocks. Finally, host responses that limit virus replication may differ for avian and influenza viruses including evasion of autophagy, disruption of MAVS signaling and regulating interferon induction. Understanding the cellular and host mechanisms underlying cross-species infections will increase our ability to predicate viruses with pandemic potential. AIV are typically propagated in the allantoic cavity of embryonated chicken eggs or in tissue culture cells including MadinDarby canine kidney (MDCK) cells or primary chicken embryo fibroblasts (CEF) but embryonated duck and turkey eggs can also be used. While embryonated chicken eggs are the preferred option since they can support the growth of a broad range of AIV strains, many field isolates may not rapidly grow, can be contaminated with Newcastle disease virus, and HPAI AIV may be difficult to propagate to high titers in embryonated eggs given the rapid death of the embryo.

Epidemiology AIV is highly contagious. It is spread by respiratory aerosols, bodily fluids (feces) and feathers from infected animals, contaminated fomites and environment including water. Birds play a fundamental role in the epidemiology of influenza virus. Wild bird, especially wild waterfowl (including Anseriformes) and shorebirds (including Charadriiformes), are considered the natural reservoirs for influenza virus. More than 80 different HA and NA combinations have been identified in wild waterfowl to date; much greater diversity than any other host species. These birds have the potential to spread virus between countries and even continents during migration. AIV spillover events from wild-birds to poultry can result in large outbreaks if not properly contained. According to the FAO database, ~18,924 confirmed AIV outbreaks were reported during the current decade. The majority occurring in Asia (63%), followed by Africa (19%), Europe (15%) and the Americas (3%) (Fig. 1), primarily due to the endemic spread of HPAI H5 viruses. Since 2004, more than 18,800 HPAI H5 outbreaks have been reported in 61 countries. According to the sanitary code for terrestrial animals of the OIE, H5 and H7 AIV, regardless of pathogenicity, requires immediate notification and control measures. In many areas of the world, live bird markets (LBM) are commonplace and part of the culture. These are also main risk factors for the spread of AIV within the market, to other areas through the movement of poultry, and for human infections. Most of the birds coming into the market are maintained in subsistence-oriented small-scale production systems commonly known as backyard production systems (BPS). BPS systems are the most common form of animal production worldwide, with domestic birds being the species that are preferably bred in this type of production system, while the production of pigs can be considered as a complementary activity. BPS animals and their products are typically consumed by their owners, transported and sold in local markets or used as gifts. Sick birds may be slaughtered, sold and consumed. Most BPS do not apply basic hygiene and biosecurity measures and the birds are often in contact with wild birds, swine and other mammals including humans. Live bird market closures have been shown to be an effective way to stop human AIV infections.

Clinical Features and Pathogenesis Unlike humans and other mammals, influenza disease in birds is caused solely by the influenza virus A genera of the Orthomyxoviridae family. Disease ranges from asymptomatic to lethal depending on the viral strain and bird species. AIV may be designated as low or highly pathogenic. Historically, this was based solely on the ability of the virus to kill experimentally infected domestic chickens (intravenous pathogenicity index, IVPI). Today, this designation can be initially based on specific genetic

Avian Influenza Viruses (Orthomyxoviridae)

119

Fig. 1 Global avian influenza virus outbreaks reported to the FAO between 2010 and October 2018. Blue symbol ¼ AIV outbreaks in captive animals; black circles ¼ AIV outbreaks in domestic animals; green squares ¼ AIV outbreaks in wildbirds; and red triangle ¼ AIV outbreaks in humans.

signatures within the hemagglutinin (HA) protein. It is important to note that low and high pathogenicity does not refer to the disease symptoms or infectivity of the virus in humans, mammals or other species of birds. The majority of AIV infections in wild birds are asymptomatic with the virus replicating primarily in the intestines and can be associated with shedding of high viral concentrations in the feces. Viral replication can also be found in the respiratory tract. In domestic birds and poultry, disease can also range from a mild drop in egg production to highly pathogenic. Most avian influenza viruses cause a low pathogenic avian influenza virus infection (LPAI), where it is possible to notice respiratory symptoms (coughing, sneezing, sinus swelling and respiratory discharge), sinusitis, and respiratory tract lesions. While less severe than highly pathogenic infections (HPAI), LPAI viral infections can cause considerable losses, especially in turkeys, due to anorexia, depression and decreased egg production. In contrast, highly pathogenic avian influenza (HPAI) virus infections are associated with severe systemic disease with variable clinical presentations, including respiratory signs, ocular and nasal discharge, cough and dyspnea, swelling of the breasts and head and necrotic combs (Fig. 2). Mortality can reach 100%. Should a bird survive a peracute infection, they may experience central nervous system involvement resulting in lack coordination, wing droop or even paralysis. Microscopic lesions may be present, though their location and severity will vary. HPAI viruses have hitherto been restricted to select H5 and H7 AIV subtypes; however, the majority of H5 and H7 AIVs are low pathogenic. HPAI variants can emerge from LPAI H5 and H7 viruses circulating in domestic poultry populations. It was thought that the primary difference between a HPAI and LPAI virus was solely based on the presence of a polybasic cleavage site in the HA allowing proteolytic processing by ubiquitous host proteases. More recent studies have shown that pathogenicity may be more complicated and involve multiple genetic changes beyond the HA. In humans, the first example of AIV causing severe and even fatal infections in humans occurred in 1997 when 18 (6 fatal) H5N1 infections were reported in Hong Kong. AIV infections in humans can range from mild to severe and include conjunctivitis, influenza-like illness (fever, cough, sore throat, muscle aches) that can be accompanied by nausea, abdominal pain, diarrhea, and vomiting. Severe respiratory illness including shortness of breath, difficulty breathing, pneumonia, acute respiratory distress, and respiratory failure, neurologic changes and the involvement of other organ systems have also been reported. While the Asian lineage H7N9 and HPAI Asian lineage H5N1 viruses have been responsible for most human illness worldwide to date, including the most serious illnesses and highest mortality, there have been reports of humans infected with H5Nx, H9, H10, and H6 AIV subtypes. In most cases, the infected people had direct contact with infected poultry or objects contaminated by their feces and human-to-human transmission has been inefficient. The possibility that the virus could mutate or reassort and acquire the ability to transmit easily between humans is a continual concern.

Prevention HPAI outbreaks represent a main threat to the poultry industry. As detailed in the OIE Terrestrial Animal Health Code, the OIE must be notified of all cases of HPAI found in any domestic or wild bird by a country’s veterinary authorities. LPAI H5 and H7 AIV strains in poultry are also notifiable given their potential to mutate into highly pathogenic viruses or to infect other species.

120

Avian Influenza Viruses (Orthomyxoviridae)

Fig. 2 Gross lesions in chickens, ducks and geese following experimental infection with HPAI viruses. Reproduced from the OIE Scientific and Technical Review (www.oie.int) – Pantin-Jackwood, M.J., Swayne, D.E., 2009. Pathogenesis and pathobiology of avian influenza virus infection in birds. In: Mettenleiter, T., (Ed.), Avian influenza, Revue scientifique et technique (International Office of Epizootics) 28 (1), 113–136. doi:10.20506/rst.28.1.1869. (a) Oedema of comb, wattles, and periorbital tissues, A/chicken/Puebla/1994 H5N2 HPAI virus; (b) Severe subcutaneous haemorrhage and oedema of feet and leg shanks, A/Hong Kong/156/97 (H5N1); (c) Severe oedema, necrosis and haemorrhage of comb and wattles, highly pathogenic embryo derivative A/chicken/NJ/12508/86 (H5N2); (d) Thickened dermis from oedema of distal leg, A/chicken/Queretaro/14589-660/94 (H5N2); (e) Severe pulmonary oedema and haemorrhage in the lung, A/Hong Kong/156/97 (H5N1); (f) Petechial haemorrhages in epicardial fat, A/chicken/NJ/12508/86 (H5N2); (g) Submucosal haemorrhage surrounding ducts of glands in proventriculus, A/chicken/Hong Kong/156/97 (H5N1); (h) Multifocal haemorrhage in the fascial plane of the gastrocnemius muscle (pars intermedia, A/chicken/Hong Kong/220/1997 (H5N1) virus; (i) Haemorrhage in lymphoid tissue of Peyer’s patches and Meckel’s diverticulum of the jejunum, A/Hong Kong/220/97 (H5N1); (j) Haemorrhage in caecal tonsils and rectum, A/Hong Kong/483/97; (k) Bile-stained loose droppings from a 2-week-old Pekin duck, A/Egret/HK/757.2/02 (H5N1); (l) Two-week-old Embden goose with torticollis, A/chicken/Hong Kong/220/1997 (H5N1); (m) Mottling of the pancreas in a 2-week-old Embden goose, A/chicken/Hong Kong/220/1997 (H5N1).

Veterinary authorities within countries must establish programs to ensure early detection of AIV, response capacities and ongoing implementation of biosecurity measures to protect farms and commercial poultry. If infection is detected, a policy of culling infected and contact animals is typically followed to rapidly contain, control and eradicate the disease. The requirements for containment include:

• • • • • •

Humane destruction of all infected and exposed animals (following OIE animal welfare standards); Appropriate disposal of carcasses, litter and all animal products; Surveillance and tracing of potentially infected or exposed poultry; Strict quarantine and controls on movement of poultry and any potentially contaminated vehicles and personnel; Thorough cleaning and decontamination of infected premises. Due to the biochemicals characteristics of AIV they are susceptible to a broad variety of disinfectants, among them sodium hypochlorite, quaternary ammonias, 60%–95% ethanol, povidone and other disinfectants. They also can be easily inactivated by medium temperatures (56–60°C for a minimum of 60 min), ionizing radiation and extreme pH either acid or basic (pH 1-3 or pH 10-14); A period of at least 21 days before restocking.

Avian Influenza Viruses (Orthomyxoviridae)

121

This may be applied at the level of the infected farm only or include farms within a short radius of the infected premise. All of this is done in conjunction with active surveillance. Vaccination can be used to protect susceptible populations from infection. Vaccine strategies can be an effective emergency measure in an outbreak or as a routine measure in endemic areas. A decision to use vaccination must include an exit strategy detailing the conditions that must be met for vaccination to be stopped. The goal of an AIV vaccine for poultry is to reduce virus excretion if they are subsequently infected thus preventing virus circulation. There are a variety of vaccine types available for poultry including monovalent and bivalent inactivated vaccines, subunit killed vaccines, live recombinant vaccines, and DNAbased vaccines as examples. Consideration must be given to routes of administration and timing of administration, differences in host species immune responses, cost, and methods to differentiate vaccinated from infected birds. "DIVA" (differentiating infected from vaccinated animals) was one system that successfully enabled detection of field exposure in a vaccinated population resulting in eradication of LPAI H7 viruses in Italy. The basis of DIVA was use of a vaccine that contained a virus possessing the same HA, but a different NA, as the field virus allowing detection of antibodies to the NA of the field virus. For example, a vaccine containing an H7N3 virus can be used against an H7N1 field virus.

Further Reading van Dijk, J.G., Verhagen, J.H., Wille, M., Waldenström, J., 2018. Host and virus ecology as determinants of influenza A virus transmission in wild birds. Current Opinion in Virology 28, 26–36. (PMID: 29121508). Forument, M., Holmes, E.C., 2015. Avian influenza virus exhibits distinct evolutionary dynamics in wild birds and poultry. BMC Evolutionary Biology 15, 120. (PMCID: PMC4481119). Global Consortium for H5N8 and Related Influenza Viruses, 2016. Role for migratory wild birds in the global spread of avian influenza H5N8. Science 354 (6309), 213–217. (PMCID: PMC5972003). Long, J.S., Mistry, B., Haslam, S.M., Barclay, W.S., 2018. Host and viral determinants of influenza A virus species specificity. Nature Reviews Microbiology 28.(PMID: 30487536). Pantin-Jackwood, M., Swayne, D.E., Smith, D., Shepherd, E., 2013. Effect of species, breed and route of virus inoculation on the pathogenicity of H5N1 highly pathogenic influenza (HPAI) viruses in domestic ducks. Veterinary Research 22 (44), 62. (PMID: 23876184). Pantin-Jackwood, M.J., Swayne, D.E., 2009. Pathogenesis and pathobiology of avian influenza virus infection in birds. In: Mettenleiter, T., (Ed.), Avian influenza, Revue scientifique et technique (International Office of Epizootics) 28 (1), 113–136. doi:10.20506/rst.28.1.1869. Ramey, A.M., DeLiberto, T.J., Berhane, Y., Swayne, D.E., Stallknecht, D.E., 2018. Lessons learned from research and surveillance directed at highly pathogenic influenza A viruses in wild birds inhabiting North America. Virology 518, 55–63. (PMID: 29453059). Russell, C.J., Hu, M., Okda, F.A., 2018. Influenza hemagglutinin protein stability, activation, and pandemic risk. Trends in Microbiology 26 (10), 841–853. (PMID: 29681430). Shaw, M.L., Palese, P., 2013. Orthomyxoviridae. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology., sixth ed. Philadelphia, PA: Lippincott Williams & Wilkins, pp. 1151–1186. Swayne, D.E., Spackman, E., Pantin-Jackwood, M., 2014. Success factors for avian influenza vaccine use in poultry and potential impact at the wild bird-agricultural interface. Ecohealth 11 (1), 94–108. (PMID: 24026475). Wright, P.F., Neumann, G., Kawaoka, Y., 2013. Orthomyxoviruses. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology., sixth ed. Philadelphia, PA: Lippincott Williams & Wilkins, pp. 1187–1244. Yoon, S.W., Webby, R.J., Webster, R.G., 2014. Evolution and ecology of influenza A viruses. Current Topics in Microbiology and Immunology 385, 359–375. (PMID: 24990620).

Relevant Websites http://www.offlu.net/fileadmin/home/en/publications/pdf/OFFLUsurveillance.pdf OFFLU. Strategy document for surveillance and monitoring of influenzas in animals. http://www.oie.int/en/animal-health-in-the-world/avian-influenza-portal/press-releases/ OIE. Avian Influenza Poratal. http://www.oie.int/en/standard-setting/terrestrial-code/access-online/ OIE. Terrestrial Animal Health Code. http://www.fao.org/avianflu/en/strategies.html Strategy and Policy – FAO. http://www.who.int/influenza/about/en/ WHO. About the Global Influenza Programme.

Avian Leukosis and Sarcoma Viruses (Retroviridae) Karen L Beemon, Johns Hopkins University, Baltimore, MD, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Avian leukosis virus (ALV) First avian retrovirus, identified in 1908 by Ellerman and Bang, causes lymphomas by insertional activation of host proto-oncogenes. Avian sarcoma/leukosis viruses (ASLV) Includes ALV and RSV and other avian retroviruses. Bic Precursor to microRNA 155, discovered by ALV insertional mutagenesis in 1989; over-expressed in many human tumors. Endogenous retroviruses (ERVs) Retroviruses that have infected germ cells and are transmitted genetically. Insertional mutagenesis Mechanism of oncogenesis by ALV, which activates a nearby host oncogene after integration into the host genome. Myc expression is commonly induced.

LTR Long terminal repeat formed at both ends of the provirus during reverse transcription and contains U3, R, and U5 sequences. MC29 Defective ALV containing the myc oncogene. Myc A transcription factor that is oncogenic when its expression is dysregulated by ALV transduction or insertional mutagenesis. Oncogene A gene that causes cancer. Rous sarcoma virus (RSV) First transforming retrovirus, discovered in 1911 by Peyton Rous, causes rapid sarcomas. RSV carries an oncogene src, - see below. Src The first oncogene to be discovered, as part of Rous Sarcoma Virus, and also the first protein tyrosine kinase to be identified.

History Retroviruses of birds have been studied for over 110 years. Avian leukosis virus (ALV) was discovered in 1908 by Ellermann and Bang in Denmark, who transmitted erythroleukemia between chickens by cell-free filtrates. Rous sarcoma virus (RSV) was isolated by Peyton Rous in 1911 in New York and shown to transmit sarcomas through cell-free extracts from a chicken sarcoma. Rous was awarded the Nobel Prize in 1966 for showing that cancer can be transmitted by a virus; this was the beginning of tumor virology. Numerous isolates of ALV and of related transforming viruses, which cause sarcomas and a variety of hematopoietic neoplasms, were reported in the decades that followed. Progress in understanding their nature was slow until the development of cell culture assays in the late 1950s and the use of genetic and cell biological approaches to study replication and transformation. The discovery of reverse transcriptase (RT) in RSV in 1970 by Howard Temin and of the origin and mechanism of action of viral oncogenes in the decade following led to an explosion of research activity. In addition to the genetic information of ALV needed for replication, RSV has a transduced oncogene, src, that enables it to induce sarcomas rapidly in vivo and to transform cells in culture. Src was the first retroviral oncogene to be characterized and the first tyrosine kinase to be identified. Bishop and Varmus received the Nobel Prize in 1989 for showing src is derived from its host. ALV-J was isolated in 1988 in England and is currently an economic problem in chicken flocks, especially in Asia.

Classification The avian leukosis viruses comprise a single genus Alpharetrovirus, of the family Retroviridae and the subfamily Orthoretrovirinae. Although they share many structural and biological characteristics with the mammalian C-type gammaretroviruses (such as murine leukemia virus), these two groups are not closely related. All ALVs are closely related to one another, sharing considerable sequence and antigenic identity. Isolates are differentiated by subgroup (i.e., based on receptor utilization and determined by the env gene), as well as the presence or absence of oncogenes. Subgroups A–E and the more recently isolated subgroups J and K infect chickens. ALV-J was derived by recombination, acquiring a new env gene from an endogenous retroelement, EAV-HP. Subgroups F–I refer to endogenous viruses found in ring-necked pheasant (subgroup F), golden pheasant (subgroup G), Hungarian partridge (subgroup H), and Gambel’s quail (subgroup I). ALVs are endemic in flocks of domestic chickens (Gallus gallus) worldwide and will replicate efficiently in species closely related to the chicken such as quail, turkeys, and pheasants. Some RSV subgroups can transform mammalian cells and induce tumors in mammals, but with greatly reduced efficiency. The restriction of the virus to avian species is due to a lack of suitable receptors for most subgroups as well as to blocks to viral gene expression. The rare transformants that arise in RSV-infected mammalian cells often display rearrangements in proviral DNA that relieve this block. A variety of cell types from gallinaceous birds (including chickens, turkeys, and quail) can be used to propagate avian retroviruses. Primary and secondary fibroblast cultures or cell lines (DF-1) are most commonly used, as well as lines derived from quail tumors (QT6). To avoid problems associated with frequent recombination, it is advisable to use cells that do not contain

122

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21510-0

Avian Leukosis and Sarcoma Viruses (Retroviridae)

123

related endogenous proviruses, such as cells from species other than chickens or from chickens bred to lack endogenous proviruses closely related to ALVs.

Virion Structure Like all retroviruses, ALVs are transmitted as enveloped virions of about 100 nm diameter, derived by budding from the host cell membrane. Lipids derived from the host plasma membrane are present in the viral envelope and make up 35% of the virion weight. Within the retrovirus family, they are defined as having a C-type morphology. Small, dispersed spikes project from the surface of the virion; these are trimers of the two env-encoded proteins, SU (surface) and TM (transmembrane). The internal core of the virion appears in electron micrographs as a centrally located, roughly spherical structure about 30 nm in diameter. Immature virions seen during or shortly after budding have a more open, spherical core structure, substantially larger in diameter than the mature one. The core comprises about 1500 copies each of the five gag-encoded proteins (MA, p10, CA, NC, and protease) and about 100 copies each of pol-encoded reverse transcriptase and integrase. The avian retroviruses are unique in encoding the viral aspartyl protease as part of the gag gene; in other types of retroviruses, it is part of pol. Virions of ALV have an equilibrium density in sucrose solutions of about 1.16–1.18 g/ml and a sedimentation coefficient of about 600S. They are quite labile and are readily inactivated by extremes of pH, as well as by heat or mild detergent treatments. They are somewhat radiation resistant, perhaps reflecting the recombinational repair capability provided by the dimeric genome.

Genome The ALV genome consists of a homodimer of positive-sense, single-stranded (ss) RNA about 7500 nt in length. The genomic complexity of retroviruses was first determined for RSV by oligonucleotide fingerprinting and found to be the size of one 35S RNA subunit. Subsequently, all retroviruses were found to be diploid. The RNA of RSV was found to be about 2 kb larger than that of ALV due to the presence of the src oncogene. Transforming viruses, in which an oncogene has been inserted, have genomes varying in length from about 3200 nt (for UR2 virus) to about 9300 nt (for nondefective RSV). In most of these viruses, the oncogene has replaced some of the viral genes, leading to a genetically defective virus, which requires co-infection with helper ALV for replication. Oncogenes transduced by ALV from chickens include src, myc, myb, erbA, erbB/EGFR, fps, ros, and jun. The avian retroviral genome is modified and processed by cell machinery. It contains a 50 m7GpppGm cap and a 30 poly (A) sequence, as well as internal m6A residues. The gene order of retroviruses was first established with the avian retroviruses. ALV: 50 -gag-pol-env-30 RSV: 50 -gag-pol-env-src-30 The RSV primary transcript is incompletely spliced to yield an unspliced mRNA (which also serves as the viral genomic RNA) and spliced mRNAs for env and src. The negative regulator of splicing (NRS) within the gag gene, as well as inefficient 30 splice sites, inhibit complete splicing. Important noncoding regions found near the end of the genome are necessary to provide signals for virus replication. These include an 18–21 base sequence (R) repeated at each end as well as unique sequences U3 (c. 250 nt) near the 30 end and U5 (c. 80 nt) near the 50 end which are duplicated in the long terminal repeat (LTR) during reverse transcription. The LTR contains sequences controlling transcription initiation (enhancers and promoters) and polyadenylation. Adjacent to these are the sites for initiation of reverse transcription: the primer binding (PB) sequence next to U5. Between PB and the beginning of gag is an approximately 300 nt leader region, which contains signals important for the dimerization and packaging of the genome into virions. There are also three short open reading frames in this region upstream of gag. The direct repeat (DR) sequences flanking the src gene in RSV are necessary for cytoplasmic accumulation of full-length viral RNA. ALV has one copy of the DR sequence, which is involved in export of the RNA from the nucleus to the cytoplasm, with the help of cellular proteins NXF1 and Dbp5. Further, an RNA stability element (RSE) downstream of the Gag termination codon inhibits nonsense-mediated decay of the ALV genome. The RSE binds PTBP1 and inhibits binding of UPF1 to the terminating ribosome complex. The ALV genome encodes nine proteins, the products of three genes. The Gag proteins constitute the major structural components and are sufficient to form virus-like particles. The Gag-Pro precursor is processed during release of virus into four Gag proteins: MA (matrix, 19 kDa) which interacts with the cell membrane; p10, a 10 kDa protein; CA (capsid, 27 kDa) which forms the core shell structure; and NC (nucleocapsid, 12 kDa), an RNA-binding protein necessary for specific encapsidation of genomic RNA. The Gag-Pro precursor also contains the 15 kDa PR (protease) peptide necessary for processing all internal virion proteins. The Pol reading frame is expressed as a fusion protein with Gag and Pro, processed to yield RT (usually present as a heterodimer of 98 and 66 kDa reflecting partial processing by PR), and integrase (IN, about 32 kDa); these are the two enzymatic components necessary for synthesis and integration of the DNA provirus. The env gene encodes the Env precursor (Pr95) which is processed as a membrane protein and cleaved by host cell proteases to yield the SU and TM glycoproteins. SU has an apparent molecular weight of about 85 kDa, about half of which is due to the presence of approximately 14 N-linked carbohydrate side chains. The TM

124

Avian Leukosis and Sarcoma Viruses (Retroviridae)

glycoprotein has an apparent molecular weight of about 37 kDa. The SU and TM products remain as a disulfide-bonded heterodimer with SU containing the activity necessary for receptor binding and TM mediating fusion with the cellular membrane.

Life Cycle Replication of avian retroviruses requires reverse transcription of the RNA genome to DNA and its integration into the host genome. This group of viruses provided some of the important early models for studying these processes. Howard Temin proposed and demonstrated reverse transcription of RSV RNA by a virus-encoded enzyme and received the Nobel Prize in 1975 for this work. The viral integrase protein is necessary for cleavage of the host genome and insertion of the provirus. The cellular FACT protein complex facilitates this process for the avian viruses. The ALV proviruses show fairly random integration sites with a slight preference for transcription start sites in tissue culture. However, in tumors there is a strong selection for integration near oncogenes, and the ALV regulatory sequences drive expression of the cellular oncogenes. Entry of the virion into the cell follows interaction with a specific receptor on the cell surface. Viral subgroups (A–J) have been identified on the basis of distinct receptor recognition. The presence of receptors for specific subgroups is polymorphic among birds. Four unlinked genetic loci (Tv-a, Tv-b, Tv-c, and TV-j) for ALV receptors have been genetically identified in chickens. The dominance of susceptibility over resistance alleles at each of these loci implies that they encode the receptor directly. The Tv-b locus has several alleles, controlling susceptibility to subgroups B, D, and E. Receptors for ASLV subgroups A, B, C, D, E, and J have been identified. The Tv-a receptor resembles a portion of the receptor for low-density lipoprotein and is unrelated to other known retroviral receptors. The receptors for B, D, and E are all in the tumor necrosis factor receptor family. The ALV-C receptor is in the butyrophilin family and consists of two immunoglobulin-like domains. ALV-J uses the Na þ /H þ exchanger 1 as its receptor. The newly isolated ALV-K shares the Tv-a receptor with subgroup A viruses. Entry of the virion core into the cell is by fusion of viral and cellular membranes, which is mediated by the viral glycoproteins. Once within the cytoplasm of the infected cell, reverse transcription within the permeabilized core structure copies the ssRNA genome into double-stranded DNA. This process – which varies little from that of other retroviruses – includes a series of ‘jumps’ from one end of the template to the other. The DNA product is longer than the genomic RNA template and has generated a long terminal repeat (LTR) at both ends. The LTR contains sequences necessary for DNA integration and for synthesis and processing of viral RNA. Like other retroviruses, ALVs exhibit very high rates of homologous recombination – a consequence of the diploid genome and the ‘jumping’ mechanism of reverse transcription. The latter also permits relatively high rates of nonhomologous recombination, leading to frequent rearrangements of the genome as well as the occasional acquisition of foreign sequences such as oncogenes. Integration of viral DNA into fairly random sites in the cell genome is accomplished by the IN protein which has entered the cell with the virion and remains associated with the DNA. The cellular FACT protein complex interacts with ALV IN and promotes integration.The process of integration leads to the insertion of the linear viral DNA into cell DNA. Integrated ALV DNA is characterized by the loss of two bases from each end of the viral LTR sequence and the duplication of six bases of the cell DNA target sequence at the integration site. RNA transcription of the provirus is mediated by cellular RNA polymerase II, directed to the correct initiation site by promoter and enhancer sequences in the LTR. The strength of the enhancer elements is a major factor distinguishing pathogenic from nonpathogenic (RAV-0) ALV isolates. Unlike the more complex retroviruses, there is no apparent role of virus-encoded proteins in regulating the transcription process. Processing of the viral transcripts includes addition of poly(A) following a canonical signal (AAUAAA) in the RNA derived from the 30 LTR and splicing of the fraction of the transcripts destined to become mRNA for the env and src genes. Splicing removes most of the gag, pro, and pol sequences. Importantly, a fraction of the primary unspliced RNA transcript serves as genomic RNA and as the mRNA for Gag and Pol polyproteins. Translation of the full-length mRNA leads to two products: The Gag precursor of 76 kDa and the Gag-Pol precursor of 180 kDa. Synthesis of the latter polyprotein is made possible by a  1 translational frameshift about 5% of the time, reading through the termination codon at the end of Gag. Assembly of the precursors appears to be at the cell surface and is coincident with budding. Release of the immature particle (characterized by a hollow, symmetrical core which almost fills the virion) is rapidly followed by cleavage of the Gag and Gag-Pol precursors. This cleavage is accompanied by condensation of the core into its mature form. Since the PR protein embedded in the Gag precursor contains only one-half of the active site, dimerization of this domain is necessary for cleavage to occur. This requirement probably helps to delay cleavage until the appropriate time after budding. Once infected, the host cell is usually not killed by virus replication. A strong superinfection resistance due to blockage or loss of viral receptors develops soon after infection and prevents accumulation of proviruses by reinfection. In some cases, weak or slow development of superinfection resistance is associated with a cytopathic interaction of the virus with its host cell.

Epidemiology ALV’s have been a major cause of death in poultry, mainly by induction of leukoses in hematopoietic cells. ALV spreads by both vertical and horizontal transmission and is controlled mainly by eradication of breeding stock containing ALV. ALV’s subgroup A and B were the predominant subgroups in the 20th century and shown to cause B cell lymphomas, originating in the bursa of Fabricius and metastasizing to other organs. They were largely brought under control in the late 20th century.

Avian Leukosis and Sarcoma Viruses (Retroviridae)

125

However, a new subgroup of ALV (subgroup J), causing myeloid tumors, was isolated from meat-type (broiler) chickens in 1988 in England. Similar viruses were isolated from broiler chickens in the US and China. By 2004 ALV-J infection had spread to layer chickens in China, causing hemangiomas and other diseases, as well as reduced egg laying. It has since become a major economic problem in Asia. Their pathogenicity is evolving rapidly with a change in host range and tumor types. The layer chicken isolates have incurred genetic changes in the 30 UTR and env gene. ALVs are endemic in flocks of domestic chickens (Gallus gallus) worldwide and will replicate efficiently in species closely related to the chicken such as quail, turkeys, and pheasants. Some RSV subgroups can transform mammalian cells and induce tumors in mammals, but with greatly reduced efficiency. The restriction of the virus to avian species is due to a lack of suitable receptors for most subgroups as well as to blocks to viral gene expression. The rare transformants that arise in RSV-infected mammalian cells often display rearrangements in proviral DNA that relieve this block. A variety of cell types from gallinaceous birds (including chickens, turkeys, and quail) can be used to propagate avian retroviruses. Primary and secondary fibroblast cultures or cell lines (DF-1) are most commonly used, as well as lines derived from quail tumors (QT6). To avoid problems associated with frequent recombination, it is advisable to use cells that do not contain related endogenous proviruses, such as cells from species other than chickens or from chickens bred to lack endogenous proviruses closely related to ALVs. Transmission of virus is principally vertical by infection of the offspring through virus secreted into the egg. Indeed, high titers of ALV are often detectable in commercial hen’s eggs. Horizontal spread of virus is naturally much rarer, requiring close contact, but virus can be readily spread from infected birds via contaminated needles during vaccination or through vaccines prepared from infected eggs or cell cultures. All isolates of ALV replicate efficiently in fibroblast cultures and in the bursa. Tropism for other tissues varies among isolates and is determined by both env and LTR sequences.

Clinical Features ALV’s of subgroup A and B cause B-cell lymphomas and other neoplasms. The B cell lymphomas originate in the bursa of Fabricius and metastasize to liver, spleen, and kidneys. They cause death in a few months. In contrast, ALV-J causes predominantly myeloid tumors and hemangiomas. Egg production was drastically reduced. Immunosuppression and weight loss also occurred. ALV’s with transduced oncogenes cause a variety of more rapidly occurring (acute) cancers, including sarcoma, myeloblastosis, and erythroblastosis by over-expression of mutated oncogenes from the viral LTRs.

Pathogenesis ALVs induce a wide spectrum of disease in naturally or experimentally infected animals. The prototypic disease induced by ALV is a B-cell lymphoma arising in the bursa of Fabricius starting a few months after infection and spreading to the liver and other organs during its course. Other malignancies, including myeloid tumors, erythroleukemia, sarcoma, and others, are not uncommon depending on the strain of virus and bird and the time and route of inoculation. The malignant diseases induced by viruses, which do not contain oncogenes, are the consequence of insertional activation of cellular proto-oncogenes (such as myc in the case of lymphoma, erb-B in erythroleukemia, etc.). This process is termed promoter insertion or insertional mutagenesis and was first observed by Bill Hayward with ALV integrations in myc. Additional integrations into the noncoding bic gene were observed in 1989. Bic was subsequently found to be the precursor of miR-155. When viruses infect 10–14-day embryos, more rapid lymphoma induction is observed with frequent integrations into myb and an antisense long noncoding RNA in the TERT promoter. In addition to malignancies, these viruses also induce hemangiomas, osteopetrosis, and wasting diseases. In some cases, an immune response against infected cells may be important; in others, cytopathic effects may play a significant role. A unique characteristic of ALV and a few other retroviruses is their ability to incorporate host sequences into their genome and to alter the function of proto-oncogenes to generate oncogenes. The presence of an oncogene renders the virus capable of inducing malignant transformation of cells in culture and one of a variety of malignant and rapidly fatal diseases in birds. At least 20 distinct cell sequences have been incorporated by ALV into a number of distinct isolates. RSV, which contains src, is the prototype oncogene-containing virus. Other notable oncogene-containing ALV variants include avian myeloblastosis virus (AMV; containing myb); avian myelocytomatosis virus-29 (MC-29; myc); avian erythroblastosis virus (AEV; erb-A and erb-B); Fujinami sarcoma virus (FSV; fps); and University of Rochester sarcoma virus-2 (RU-2; ros). Study of the genetic alterations that distinguish these oncogenes from proto-oncogenes, and the enzymatic and physiological function of the proteins they encode has been an important part of modern cancer research. Incorporation of oncogenes into the virus genome is usually at the expense of some viral genes and co-infection of a cell with a wild-type (helper) ALV is necessary to provide viral proteins for replication of oncogene-containing viruses. RSV, which exists as a replication-competent transforming virus, is the exception. It should be noted that the oncogenecontaining viruses are not efficiently transmitted from one animal to another due to their rapid pathogenicity. In most cases, they have probably arisen in the animal from which they were isolated and would have died out if not brought into the laboratory. Not all members of this group are highly pathogenic. RAV-0 (an endogenous virus) can infect susceptible chickens and induce viremia, but disease is rare and occurs only after a long latent period. The reduced virulence is probably an important feature of viruses inherited in the germline.

126

Avian Leukosis and Sarcoma Viruses (Retroviridae)

In infected birds, the only significant immune response is the appearance of type-specific neutralizing antibodies, which apparently recognize the regions of Env involved in receptor recognition. Group specific responses against Env or other proteins are not usually observed in infected chickens, although inoculation of virus into mammals induces antibodies capable of recognizing all virion proteins in the absence of subgroup-specific reactivity. The limited immune response observed in infected chickens has been attributed to the presence of endogenous proviruses whose expression (even at a low level) can induce tolerance to antigens in common with infecting virus. Indeed, it has been suggested that induction of tolerance might be a desirable feature for the animal, since it could prevent or limit immunopathological sequelae of infection. Postinfection immune response seems to be of little consequence in preventing subsequent malignant disease, since the cells, which will eventually form the tumor, are probably infected quite soon after infection, and the long latency reflects the necessity for subsequent rare events (such as mutations in other genes) rather than a continuing period of virus replication.

Diagnosis Avian retroviruses can be detected by ELISA assays or PCR assays.

Treatment Control of ALV infection is generally by detection and culling of infected individuals. No useful vaccination strategy has been developed. In principle, it should be possible to virtually eliminate the disease by breeding appropriate receptor alleles into commercial strains. Lymphoid leukosis caused by ALV (subgroups A and B) was eradicated in primary breeders in the US in the 1980s and 1990s, reducing the disease incidence in commercial layer hens. However, commercial broiler and layer chickens are still struggling with ALV-J and ALV-K infection in many countries, especially in Asia.

Endogenous Retroviruses ALV’s can become established in the germline and inherited stably as endogenous retroviruses (ERVs). Naturally occurring endogenous alpha proviruses form a distinct lineage of ALVs, showing a specific host range (subgroup E) for which many domestic chickens lack receptors (a phenomenon known as xenotropism), and a reduced replication capacity and pathogenicity relative to exogenous viruses. Endogenous viruses are usually expressed at a very low level and are usually defective in sequence. Analysis of the sequenced chicken genome revealed many endogenous retroviruses related to the gamma and beta retroviruses, with fewer alpha retroviruses. There were also proviral intermediates between alpha and beta genera. About 20% of these ERVs were transcribed and associated with ribosomes. In addition, there were thousands of solo LTRs.

Further Reading Bolisetty, M., Blomberg, J., Benachenhou, F., Sperber, G., Beemon, K., 2012. Unexpected diversity and expression of avian endogenous retroviruses. mBio 3 (5), e00344-12. Coffin, J.M., Hughes, S.H., Varmus, H.E., 1997. Retroviruses. New York: Cold Spring Harbor Laboratory Press. Hayward, W.S., Neel, B.G., Astrin, S.M., 1981. Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature 290, 475–480. Rous, P., 1911. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. Journal of Experimental Medicine 13, 397–411. Sefton, B.M., Hunter, T., Beemon, K., Eckhart, W., 1980. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell 20, 807–816. Stehelin, D., Varmus, H.E., Bishop, J.M., Vogt, P.K., 1976. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260, 170–173. Temin, H.M., Mizutani, S., 1970. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226, 1211–1213. Venugopal, K., 1999. Avian leukosis virus subgroup J: A rapidly evolving group of oncogenic retroviruses. Research in Veterinary Science 67, 113–119. Weiss, R.A., Vogt, P.K., 2011. 100 years of Rous sarcoma virus. Journal of Experimental Medicine 208, 2351–2355. Winans, S., Larue, R.C., Abraham, C.M., et al., 2017. The FACT complex promotes avian leukosis virus DNA integration. Journal of Virology 91, e00082-17.

Bluetongue Virus (Reoviridae) Raghavendran Kulasegaran-Shylini, Department of Pathogen Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom Polly Roy, Department of Pathogen Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom and University of Reading, Reading, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Introduction Bluetongue virus (BTV) infects a wide range of ruminants, including both domestic (sheep, cattle, goat, water buffaloes, camels etc.) and wild animals (white-tailed deer, elk, blesbuck etc.) and is common throughout the world, periodically causing serious outbreaks, particularly in sheep and cattle. BTV is transmitted by blood sucking biting midges (gnats) of the genus Culicoides. Bluetongue disease in sheep and cattle was first described in the late 18th century, but was recorded only in the late 19th century by Hutcheon, the Chief Veterinary officer of Cape Colony in South Africa. The clinical features were officially reported in 1902 as ‘malarial catarrhal fever’ of animals. However, since the diseased sheep generally exhibited distinctive lesions in their mouths and on the tongue, with the latter often turning blue, Spreull suggested the use of the common name ‘bluetongue’. In addition to this, Spreull also discovered that the causative agent was filterable and transmissible to cattle and goats causing subclinical infection. The disease was confined to Africa until 1943 when the first recognized outbreak outside of Africa occurred in Cyprus and soon after, in surrounding countries. Similar outbreaks were also observed in the USA around the same period and virus was isolated from one such outbreak in California in 1952. The disease then spread into southwest Europe and the Indian subcontinent causing severe outbreaks. Over the years, many serotypes of the virus have been isolated from all over the world including Australia, Central and South America and the Caribbean. In recent years, BTV has caused infections in most countries in central and southern Europe and has spread as far north as the United Kingdom and Denmark. Due to its serious economic importance, BTV has been studied extensively and substantial progress has been made in characterizing the virus at the atomic, molecular, and genetic levels. BTV classification, three-dimensional structure, genetics and the virus replication cycle are summarized here along with epidemiology, pathogenesis and diagnosis, as well as vaccination strategies.

Classification BTV is the prototype of the orbivirus genus, the largest of the genera within the Reoviridae family. Currently, 22 distinct virus species have been classified under the orbivirus genus. Orbiviruses are insect-borne viruses capable of infecting a wide range of hosts, including humans, animals, plants, and insects. Despite basic similarities, orbiviruses differ greatly in their structure, physicochemical properties, replication cycle, pathogenesis, and epidemiology. BTV includes 28 distinct serotypes identified worldwide, including novel isolates of atypical BTV, all of which can be genetically traced to a singular geographic origin.

Virion Structure and Genome BTV is the most extensively studied virus among the Orbiviruses. The particle has a diameter of 86 nm, and consists of two capsids of concentric protein layers, comprising seven structural proteins and a genome of 10 dsRNA segments. The outer capsid is made up of two proteins, VP2 and VP5, which surround the inner capsid, termed “core”. The core is composed of VP7 and VP3 in two concentric layers, encapsidating the dsRNA genome and the viral replicase complex of three minor proteins, VP1, VP4, and VP6. The complete genome of BTV-10 was the first sequenced in its entirety in 1989 and was the largest reported complete genome sequence of an RNA virus at the time. The total length of BTV-10 genome was determined to be 19,218 base pairs, with each segment varying in length, from 3954 bp to 822 bp. Based on the size differences, the RNA segments were numbered as S1-S10 and grouped into three size classes, large (S1-S3), medium (S4-S6) and small (S7-S10). All 10 RNA segments share unique 50 and 30 complementary ends and lack a 30 poly-A tail. Segments 1–8 each have only a single open reading frame, encoding six of the seven structural proteins (VP1-VP5 and VP7) as well as two non-structural proteins (NS1 and NS2). The two smallest segments, S9 and S10, have overlapping ORFs, S9 encodes the smallest BTV structural protein, VP6, and a small non-structural protein, NS4 encoded in the þ 1-reading frame, while S10, encodes the two isoforms of the non-structural protein NS3, namely NS3 and NS3A, with the latter lacking the N-terminal 13 amino acid residues (Table 1). A recent report suggests that S10 may also encode an additional NS protein, NS5, with unknown function. Mature BTV particles are relatively fragile, unlike for example, reovirus and rotavirus particles, and therefore pose a challenge to purify intact virion particles from infected cells. The initial transmission electron micrographs (TEM) of BTV suggested a doubleshelled arrangement of the virion particle. A low resolution (5 nm) cryo-electron microscopy (cryoEM) reconstruction of the virion particles and recombinant virus-like particles (VLPs) formed by the co-expression of four major capsid proteins (VP2, VP3, VP5 and VP7) revealed that the particles have distinct icosahedral morphology. The analysis also showed the specific arrangement of

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00106-5

127

128

Table 1

Bluetongue Virus (Reoviridae)

Proteins encoded by BTV segments and their functions

Genome No. of bpa Proteins No. of aa segment

Predicted size (Dalton)

Estimated no. of molecules

Location in virion

S1 S2

3954 2926

VP1 VP2

1302 956

149,588 111,112

12 180

Inner core Outer capsid

S3 S4

2772 2011

VP3 VP4

901 654

103,344 76,433

120 B24

S5 S6 S7

1639 1770 1156

VP5 NS1 VP7

526 552 349

59,163 64,445 38,548

360 NA 780

S8

1123

NS2

357

40,999

NA

S9

1046

VP6

328

35,750

60–72

S10

822

NS4 NS3

B79 229

NS3A

216

B 10,000 NA 25,572 NA/Low level 24,020

Function

RNA dependent RNA polymerase Receptor binding, virus entry, hemagglutinin, type specific neutralization Core Scaffold for VP7 trimers Inner core Capping enzyme with guanylyltransferase, methyltransferases 1 and 2, RNA 50 triphosphatase, inorganic pyrophosphatase NTPase activity Outer capsid Membrane penetration, virus entry Non Structural Upregulates viral protein translation, forms tubules Core surface Viral core protein, group-specific antigenic determinant layer Non Structural Phosphorylated, recruits viral ssRNA and core components, forms cytoplasmic VIBs, site of viral core assembly Inner core Involved in viral genomic RNA packaging, has hexameric configuration, helicase Non Structural Host-virus interaction, regulates interferon response Non Structural Glycoprotein, responsible for virus trafficking and egress

a

Based on BTV 10 genome sequence.

the two outer capsid proteins (VP2 and VP5) for the first time, one with 120 globular densities and the other with 60 spike-like triskelion structures. It was proposed that trimers of VP2 form the triskelion motifs while the globular configurations were the VP5 trimers. With further advancements in cryoEM data acquisition, density reconstitution, and image reconstruction, the structure of the virion particle was further refined to B24 Å and subsequently to 7 Å . These studies confirmed that the sail-like spikes of triskelion motifs are VP2 (111 kDa) trimers and the globular densities are VP5 (B59 kDa) trimers. The surface of the core forms a middle layer with 260 VP7 (B38 kDa) trimers and an inner layer formed by 60 VP3 (B103 kDa) dimers (detailed below). A recent 3.5 Å structure of BTV further revealed the secondary structural features of VP2 and VP5, and the specific sites of interaction between these two proteins and the scaffolding inner core, at atomic resolution (Fig. 1). Structurally each VP2 monomer (120 in total) is divided into four distinct domains, a hub, a hairpin, a pyramid-shaped body, and a highly flexible external tip. The exposed nature of this domain is consistent with its role as a key determinant of the host antibody response. The hub domain contains a lectin-like b-barrel flanked by helices that drive the trimerization of three monomers such that they sit above the VP7 layer of the core surface. A typical zinc-finger motif, CCCH, with tetrahedral coordination is located at the interface of the hub and the body domains. Each of the 120 VP5 trimers forms a compact globular structure. Each monomer (360 in total) forms a rectangular shaped structure with three distinct domains, an N-terminal flexible dagger sequestered in the canyons underlying the core surface, a helix-rich unfurling domain with a central coiled-coil motif that facilitates trimerization (analogous to a viral fusion protein), and an anchoring domain in the C-terminal region. This domain possesses four antiparallel b-strands with a potential membrane-binding motif. The VP5 trimers surround the VP2 trimers, underlying the gaps in the triskelion arms of VP2 such that the hairpin and hub domains of VP2 are in contact with the unfurling and membrane associating motifs of VP5. The core (B470 S), unlike the virion particle, is highly robust and was the first particle of BTV analyzed by cryoEM in 1992. The 75 nm structure revealed a T ¼ 13 icosahedral lattice and the presence of concentric layers of VP7, forming the core surface and VP3, forming the inner layer (subcore). The X-ray crystal structure of recombinant VP7 revealed an unusual organization of VP7 trimers, which provided a paradigm in the field and enabled the initial characterization of the assembly of the VP7 layer onto the VP3 layer at 23 Å resolution by cryoEM. These analyses further facilitated solving the subsequent X-ray crystal structure of the core at B3.5 Å resolution (Fig. 1). The surface of the core is made up of 780 copies of the VP7 monomer arranged as 260 trimers with a T ¼ 13 icosahedral lattice arranged around 132 channels as six-membered rings, with five-member rings at the vertices. In the T13 lattice, VP7 trimers occur as pairs of five quasi-equivalent trimers with each trimer containing two distinct domains, an upper b-rich domain, exposed in the core surface, making extensive contacts with the outer capsid and a helical lower domain that interacts with the VP3 layer below. The underlying subcore layer is composed of 60 VP3 dimers arranged as 12 decamers on a T ¼ 2 symmetry. The VP3 dimers are made up of two nearly identical isoforms (A & B) with a unique plate-like structure resembling a triangular wedge consisting of apical, carapace and dimerization domains. Each VP3 decamer consists of five ‘A’ molecules arranged around a five-fold apex with a central pore and five ‘B’ molecules arranged around ‘A’. In addition to the pores at the five-fold axes, there are pores at the three-fold axes that are blocked by VP7 trimers in the intact core. VP7 trimers in the core additionally make contact with the tip and hub domains of the VP2 spikes and extensively with all three VP5 domains with the VP5 domains bridging the channel formed by six VP7 trimers in the core. The VP3 layer has been suggested to be in contact with the enclosed viral dsRNA genome.

Bluetongue Virus (Reoviridae)

129

Fig. 1 Structure of BTV virion. (A) and (B) 3.5 Å cryo-EM density map of BTV virion, shown as radially colored surface representation (A) Outer layer showing VP7 triskelions (magenta), VP5 trimers (green) and VP7 trimers (yellow) (B) VP5 layer showing the two different VP5 trimer conformers (green and cyan) and VP7 trimers (yellow). (C) and (D) X-ray crystal structure of BTV core particles showing (C) 260 VP7 trimers arranged as five quasi-equivalent trimers in T ¼ 13 icosahedral symmetry (colored in red, orange, green, yellow and blue), (D) VP3 scaffold showing the unique A (green) and B (red) isoforms and highlighting VP3 decamer (dashed ellipse). Adapted from: (A) and (B) Zhang, X., Patel, A., Celma, C.C.P., et al., 2016. Atomic model of a nonenveloped virus reveals pH sensors for a coordinated process of cell entry. Nature Structure Molecular Biology 23, 74–80. (C) and (D) Grimes, J.M., Burroughs, J.N., Gouet, P., et al., 1998. The atomic structure of the bluetongue virus core. Nature 395 (6701), 470–478.

While these studies provided detailed structural descriptions of the VP7 and VP3 layers, the exact positioning of the three minor proteins and the dsRNA genome within the core remains unclear. However, X-ray diffraction patterns of the BTV cores suggest that most of the genomic dsRNA is packed as well-ordered layers surrounding putative transcription complexes at the apices of the core particle. Further, cryoEM reconstruction of recombinant core-like particles (CLPs) of VP3 and VP7 along with two minor proteins, VP1 (polymerase) and VP4 (capping), revealed a flower-shaped density at the 5-fold axes underneath the VP3 layer.

Life Cycle In both mammalian and insect cells, BTV follows the typical steps of virus replication, which begin with virus entry and release of intact viral cores, followed by synthesis of viral ssRNA, translation of the viral proteins, amplification of the viral genome, assembly of nascent virus particles and, finally, egress (Fig. 2).

Virus Attachment and Entry Into Cells In mammalian cells, BTV infection is initiated by attachment of the virus to the cell surface by the outer capsid protein, VP2, primarily via the tip domain that protrudes from the surface of the particle. While the identity of the receptor and the exact mechanism of cell entry have not been elucidated, it has been reported that sialoglycoproteins and sialic acids play roles in the virus entry process. BTV primarily uses the endocytic pathway and is transported to the cytoplasm via clathrin-coated vesicles. Once internalized, the clathrin coat is released and the endocytic vesicle fuses with an early endosome. The CCCH zinc-finger motif in VP2 acts as a pH sensor in the early endosome, triggering a conformational change, and the eventual disengagement of VP2 from the virus particle. The particle lacking VP2 is then transported to the late endosomal compartment, where a histidine cluster – located at the interface of the anchoring domain and the helices of the unfurling domain of VP5 – senses the late-endosomal pH. This triggers the release of

130

Bluetongue Virus (Reoviridae)

Fig. 2 Overview of BTV replication cycle. Virus entry occurs via sialic acid attachment by VP2 followed by clathrin-mediated endocytosis. VP2 senses the pH in the early endosome leading to its detachment. The core particle with exposed VP5 layer then moves to late endosome where VP5 senses the acidic pH leading to a conformational change and membrane permeabilization. Core is then released into the cytosol. Transcription and translation of viral proteins occurs leading to formation of tubules (NS1) and viral inclusion body (NS2) where components for core assembly are concentrated. Assembled core particles leave the VIB, leading to maturation by addition of the outer capsid proteins VP5 and VP2. The particles are trafficked using exocytic vesicles mediated by NS3 interaction with host factors leading to egress via budding or via host cell lysis.

the putative membrane-interaction element leading to the dramatic refolding of the dagger domain and the helical unfurling domain to form a new filamentous barb-like structure that protrudes out while the anchoring domain remains attached to the VP7 layer below. It is likely that the filamentous structures interact with the endosomal membrane and initiate permeabilization and destabilization of the endosomal vesicles, facilitating the release of the highly stable core, containing the dsRNA genome and the viral replication complex, into the host cytoplasm.

Transcription and Replication Once released into the host cytoplasm the transcriptionally active core remains intact and initiates replication of the viral genome. Single-stranded RNAs (ssRNAs) from the 10 genomic segments are repeatedly transcribed by the enzymes within the core and released into the host cell cytoplasm through the channels that connect the interior of the core with the exterior. The released transcripts (mRNAs) are capped at the 50 -end but lack the canonical 30 -poly-A, and can serve as templates for both translation and for negative-strand RNA synthesis to form new genomic dsRNA segments. The advent of a BTV reverse genetics (RG) system has enabled the dissection of genome replication within mammalian cells, which was found to occur in two stages. In the first, newly synthesized transcripts initiate the primary replication cycle by generating the primary replicase complex (PRC) consisting of the enzymatic minor proteins, VP1, VP4, and VP6, the inner core protein VP3, as well as two non-structural proteins, NS1 and NS2. NS1 is required for viral protein synthesis, whilst NS2 plays an essential role in selectively recruiting the four subcore proteins and the ssRNAs during particle formation (discussed below). In the second step, 10 ssRNAs segments are recruited to the PRC, forming the subcore. The viral replicase complex, composed of VP1, VP4, and VP6, then replicates the viral ssRNA to form dsRNA. VP1 (150 kDa) is the largest of the BTV proteins and has shown RNA dependent RNA polymerase activity in vitro. Recombinant VP1 is capable of de novo dsRNA synthesis when supplied with BTV or Rotavirus mRNAs. However, it cannot synthesize dsRNAs from non-viral RNAs unless they are fused with BTV specific 50 and 30 sequences, suggesting that the recognition of a specific sequence or secondary structure element is essential for the initiation of RNA replication. No high-resolution structures are available for VP1. However, molecular modeling of the sequence based on the known Reovirus l3 structure, suggests that VP1 has the typical RdRp organization with an N-terminal domain, a central polymerase domain, and a C-terminal domain. VP1 lacks the ability to cap the dsRNAs produced. This activity is instead performed by VP4. VP4 (76 kDa) functions as a single capping protein and contains all the necessary domains to catalyze the reactions required to generate type-1-like cap structures on ssRNAs in vitro, similar to those found on BTV mRNAs. VP4 was the first capping enzyme within the Reoviridae family to have its structure solved by X-ray crystallography. This revealed a unique multi-domain structure with an hourglass morphology with defined domains arranged in a linear layout (Fig. 3). While the atomic structure of VP4 did not account for its RNA-triphosphatase function, it provided the molecular basis for its guanylyltransferase and two methyltransferase activities (guanine-N7-methyltransferase, N7MTase and nucleoside-2-O-methyltransferase, 20 OMTase) activities characterized by in vitro experiments previously.

Bluetongue Virus (Reoviridae)

131

Fig. 3 Structure of BTV VP4 and NS1. (A) X-ray crystal structure of VP4 showing its domain organization. (B) Cryo-EM density reconstruction of NS1 tubule structure. (C) Atomic model of NS1 monomer showing the different domains.

VP6 (36 kDa) is the smallest of the proteins in the replication complex. In vitro analysis of recombinant VP6 suggests that it possesses helicase and nucleotide triphosphatase (NTase) activities and has high affinity for both ssRNAs and dsRNAs. Recent studies have shown that VP6 is essential for viral RNA replication and genome packaging. This attribute of VP6 has been utilized in the development of new replication defective vaccine strains, named ‘ECRA’ (Entry Competent Replication Abortive) vaccines (discussed below). Sedimentation analysis, size exclusion chromatography and EM analysis suggest that the VP6 forms hexamers with ring-like structures similar to those observed for other DNA and RNA helicases. While the VP6 structure is yet to be fully characterized, Nuclear Magnetic Resonance (NMR) analysis in 2014 suggested the presence of two large dynamic loops. In tissue culture, the first BTV-specific proteins are detectable 2 h post infection and the rate of protein synthesis increases rapidly until 12–13 h post infection, after which it slows but continues until cell death. The two NS proteins, NS1 (64 kDa) and NS2 (41 kDa), involved in primary replication cycle, are synthesized abundantly early in BTV infection forming two virus-specific intracellular structures, tubules, and viral inclusion bodies (VIBs), respectively. These two structures are the hallmarks of BTV infection. By contrast, synthesis of NS3 and NS3A (26 and 25 kDa) varies from barely detectable to highly expressed, depending on the host cells. NS1 has been suggested to positively influence the expression of BTV proteins and initial cryoEM analysis suggested that its tubules are made up of helically coiled NS1 dimers. Near-atomic resolution structure of two NS1 tubular forms has recently been obtained by cryoEM and the structure revealed that the same NS1 monomer gives rise to tubules with different helical assemblies and varying diameter (500 Å to 580 Å ). Each NS1 monomer structure consists of an N-terminal foot domain, a head domain, and a body domain connected to an extended C-terminal arm (Fig. 3). The C-terminal arm of an NS1 subunit wraps above the head domain of another NS1 subunit through hydrophobic interactions in the tubular form and deletion of this C-terminal arm perturbs tubule formation. It does not, however, inhibit virus recovery and NS1 is still functional, indicating that NS1 tubules are not necessary for mRNA translation. The structure also revealed the presence of two metal-bound zinc-finger motifs in the NS1 monomers. Biological analysis confirmed that while both are needed for tubule assembly, only one is involved in the regulatory function of NS1 during replication. NS2 is a phosphoprotein and is synthesized abundantly throughout infection. Initially distributed throughout the cytoplasm of infected cells, NS2 rapidly accumulates to form dense globular structures, known as virus assembly factories or virus inclusion bodies (VIBs). NS2 has a strong affinity for ssRNAs and viral ssRNAs accumulate within the VIBs soon after their formation. NS2 is phosphorylated at serine 249 and serine 259 by casein kinase II (CKII) and mutations of either one or both the serine residues influence VIB morphology. More recently, it has been shown that protein phosphatase 2A dephosphorylates NS2 and along with CKII modulates the phosphorylation status of NS2 in infected cells thereby regulating VIB morphology and virus replication. The N-terminal region of NS2 (160aa) was shown to have a b-sandwich motif in its monomeric form and forms homo-multimers composed of 10–11 monomers. The VIBs are the site of virus assembly and act as a scaffold for subcore formation. They contain viral RNAs in abundance along with the other viral proteins required for genome packaging, replication, and core assembly. Upon formation of the subcore in the VIBs, VP7 is added onto the VP3 layer to form the stable mature core particle. The viral core is then released into the cytoplasm, where the outer capsid proteins (VP2 and VP5) are layered on to the core surface. The mature progeny virions are then released from the host cell. NS3/NS3A, which is the only glycosylated transmembrane protein of BTV, traffics through the endoplasmic reticulum and Golgi apparatus, and is involved in virus budding at the plasma membrane (discussed below), while the smallest protein, NS4 (77–79 residues), acts as an interferon antagonist and appears to be a virulence factor in sheep.

Core Assembly NS2 plays a major role in recruiting each of the core components including the ssRNA transcript to facilitate the entire core assembly process within the VIBs. However, since core-like and subcore-like particles can be assembled in vitro by expressing the recombinant structural proteins, it has been possible to define the assembly pathway of VP3, from dimer to VP3 layer via the decamer intermediates and to show how 260 pre-formed VP7 trimers are layered onto the VP3 subcore. Recombinant core assembly data has further been supported by an in vitro cell-free assembly (CFA) system, which provided additional insights into

132

Bluetongue Virus (Reoviridae)

the entire core assembly process. In this assay, in vitro translated VP1, VP4, and VP6 were initially incubated with the ten ssRNA transcripts, followed by sequential addition of VP3 and VP7 to reconstitute a subcore and core structures with all ten ssRNA segments. The subcores were sensitive to RNase treatment prior to addition of VP7, suggesting that VP7 stabilizes the core. Furthermore, the assay also revealed an essential role for VP6 in genome packaging. In the presence of triphosphates the packaged ssRNA could serve as template for dsRNAs synthesis. Most importantly, in vitro reconstituted core particles were infectious in BTV permissive cells. Thus, NS2 was dispensable for core assembly in vitro suggesting its role in vivo is passive, acting as a condenser of the necessary components required for assembly.

Assembly of Genomic RNA and Packaging The mechanism by which BTV and related viruses with multipartite genomes package their genomes into capsids has proved elusive until very recently. The ten BTV RNA segments (3.95–0.8 kb) vary in sequence but share common complementary short 50 and 30 untranslated regions (UTRs) of variable length, which include highly conserved hexanucleotides at both ends. Recent evidence has shown that the sorting of BTV RNA segments starts by the recruitment of genomic ssRNAs via these UTRs. Further, intersegment interactions leading to complex RNA networks are the driving force for sorting and packaging of a correct set of genomic segments. There is a specific order of segment recruitment, the smallest segment, S10, triggers RNA-RNA interaction with other three small segments and forms the initial complex. The other six larger ssRNA segments are subsequently recruited in a size dependent manner to form the final complex. Highly specific segment assorted signals (SAS) dispersed across the ssRNA segments are responsible for driving the specific interactions between RNA segments and thereby promoting RNA complex formation. Perturbation of these SAS by mutagenesis or by the introduction of specific competitive oligoribonucleotides, disrupts the RNA assembly process and affects RNA packaging in vivo. These studies indicate how selective packaging is achieved for BTV and suggest that similar mechanisms may apply to other members of the Reoviridae family.

Virion Maturation and Egress Upon core assembly and dsRNA synthesis, nascent cores dissociate from the VIBs and the outer capsid proteins VP2 and VP5 are rapidly recruited to the core generating the mature virus particles. The precise mechanism behind the recruitment of VP2 and VP5 is not yet known, but it is likely that host factors are involved, VP5 with lipid raft and VP2 with vimentin and, in addition, both proteins have been found to concentrate on the surface of intracellular vesicles in preparation for their recruitment. The release of the mature virus particles can be either lytic or non-lytic, with the host cell influencing the mechanism of egress. Infection of mammalian cells results in extensive cytopathic effect, such that the egress in these cells is predominantly by cell lysis although non-lytic budding has been also documented. In contrast, infected insect cells show little or no cytopathic effect and infected cells are characterized by continual non-lytic budding of nascent virus. Non-lytic egress is primarily mediated by the viral glycoprotein NS3/3A, which is predominately found anchored to the plasma membranes and associated with the intracellular vesicles together with VP2 and VP5. It has been predicted that this protein forms homo-oligomers in infected cells and possesses a viroporin activity. The NS3/3A has two transmembrane domains with a short extracellular domain between them and the N and C termini project into the cytoplasm. The short C-terminal domain interacts with nascent viral particles via the outermost structural protein VP2, while the N-terminal domain of NS3/3A is capable of recruiting several host components of trafficking and exocytic pathways as well as the endosomal sorting complex required for transport (ESCRT) pathway. At the plasma membrane, NS3 directly binds to the membrane and facilitates the budding process. It has been shown that the NS3 protein is present in the envelope surrounding budding particles, and in cholesterol enriched membrane patches in close association with VP5 protein that also possessed an autonomous membrane-targeting signal. Finally, NS3 recruits TSG101, another ESCRT component, which eventually promotes scission of the budding viruses. Together these cellular factors are thought to traffic NS3/3A, along with the bound virus, to the plasma membrane. Thus, NS3/3A can be considered a bridge linking the virus particle to essential cellular trafficking machinery. The importance of NS3/3A to non-lytic egress was highlighted in studies using an NS3/3A defective virus. These viruses remained viable in mammalian cells, although with less efficiency, however, they were dramatically attenuated in insect cells. Altogether, these findings describe a striking role for the NS3 protein in mediating viral egress, using mechanisms similar to those described for enveloped viruses.

Epidemiology Bluetongue virus alternates its infectious cycle between the insect Culicoides vector and ruminant mammals. In many countries, the prevalence of BTV infection is high in ruminants although clinical disease is often not recorded. Due to the presence of Culicoides midges, the disease is prevalent throughout the tropics and subtropics, within separate ecosystems. The chance of infection depends in part on the genotype of the midge, the strain of virus, the level of viremia, and environmental factors. The epidemiology of bluetongue disease can be considered in the context of three major zones of infection: an endemic zone (generally subclinical infection), an epidemic zone (disease occurs at regular intervals), and an incursive zone (disease occurs usually only at extended intervals, but when it does occur, the epidemic is often extensive). Seasonal incursions of the virus into more temperate latitudes, sometimes accompanied by

Bluetongue Virus (Reoviridae)

133

disease, may occur under favorable climatic conditions at certain locations. Interestingly, cases of major epidemics in regions where disease occurs only sporadically, have often been traced to windborne transport of infected Culicoides from distant areas. Many BTV strains have been isolated from different sources since its first characterization in the early 1900s. Initial crossimmunity studies of multiple BTV strains and subsequent serum-virus neutralization tests confirmed the existence of multiple serotypes of BTV. The complete virion particle was purified in 1969, and subsequent gel electrophoresis analysis showed that the BTV genome contained 10 discrete dsRNA segments. The genetic diversity and variation in sequences of different genome segments within a single BTV serotype was identified initially by RNA oligonucleotide fingerprint analyses of BTV field samples, which demonstrated that both genetic drift and genetic shift contribute to BTV evolution. Nevertheless, some segments were clearly less variable within a single serotype, but more variable between types, than others (e.g., segments encoding outer capsid proteins). Further, field isolates from both animals and insect vectors consisted of mixtures of RNA segments from populations of circulating virus strains. High-frequency of genome segment reassortment was shown to occur readily between different BTV serotypes and within other orbivirus serogroups but not between viruses belonging to different serogroups. Early molecular hybridization studies demonstrated that genome segments 2 and 5 (encoding the two outer capsid proteins) exhibited little or no cross-hybridization between serotypes, whereas the other eight RNA segments gave consistently higher hybridization signals. Thus, genome segment reassortment may be an important factor in the generation of genetic diversity in orbivirus populations in nature. These results were initially confirmed by sequence comparisons of segments encoding VP5 and VP2 from six different serotypes in the 1980s. This was further affirmed by recent extensive phylogenetic analyses of more than 200 different isolates of different serotypes. The accumulating data suggested that while VP5 showed up to 70% sequence identity, VP2 sequences varied from 22% to 73% between serotypes. While BTV isolates can be separated into twenty eight distinct serotypes on the basis of genome segment 2, localized circulation of the virus in different regions throughout the world has led to evolution of distinct geographical variants broadly divided into Eastern (endemic in Asia, Indonesia and Australia) and Western lineages (endemic in Africa, the Caribbean and the Americas). Serotypes have additionally been classified into distinct nucleotypes based on phylogenetic analysis of segment-2 (VP2) and segment 5 (VP5). VP2 was classified into 12 nucleotypes: A-L that correlates with BTV serotypes while VP5 was classified into 10 nucleotypes: A-J and have partial correlation with BTV serotypes. The global distribution of different BTV serotypes has undergone drastic alteration over the recent years. Changes in climate and increased travel and trade have been implicated in the global distribution of BTV, and of Culicoides species susceptible to BTV infection. Molecular epidemiology studies indicate that individual strains can move large distances due to the windborne movements of infected insects and that a single bite from a fully infected adult Culicoides is sufficient to reliably transmit the virus to a susceptible mammalian host. Many serotypes of BTV previously not present, have now been detected in Europe, North America, Australia, Korea, India, the Middle East, and South America, with more than one BTV serotype identified in many regions. Particularly in Europe, the epidemiology of BTV has changed dramatically since 1998, when six different BTV serotypes emerged across southern European countries. Most of these serotypes have persisted and spread both westwards and northwards, providing evidence for an epidemiological ‘step-change.’. By 2002, different BTV serotypes were identified in Greece, Italy, Bulgaria, Kosovo, Serbia, Montenegro, Macedonia, Croatia, Albania, and Bosnia and Herzegovina. Several serotypes gradually spread north and in particular, a BTV-8 epidemic broke out in northern Europe in 2006 with the first case being reported in the Netherlands. Outbreaks of BTV-8 were then reported in Belgium, Germany, and Northern France although the exact mode and mechanism of these incursions remains unclear. BTV-8 subsequently spread across southern European countries and further north into Luxembourg, the Czech Republic, Switzerland, Denmark, and the UK. New outbreaks were also reported in Hungary, Austria, Sweden, and Norway. In 2008, BTV-6 was identified in Germany and the Netherlands and BTV-11 was identified in Belgium. The high number of cases reported for the BTV-8 outbreak in Belgium, Germany, Netherlands, France, and Luxembourg, led to the implementation of a vaccination strategy against the serotype across several countries in Europe, and the outbreak was finally controlled in 2009.

Pathogenesis and Clinical Features BTV is transmitted to vertebrate hosts through blood-feeding midges of the Culicoides genus. BTV infection has an incubation period of 4–12 days and produces a spectrum of conditions from sub-clinical infection to severe and fatal disease, depending on virus strain and host species. Clinical signs of infection are generally more severe in sheep, resulting in severe illness and high mortality (as high as 70%). The insect vectors carry the virus in their salivary glands and ducts and infect the host during a blood meal with virus inoculation at the site of biting. The regional lymphatic system underlying the infected skin laceration mediates the dissemination of the virus to the endothelial cells, and peripheral blood mononuclear phagocytes (PBMs). The virus proliferates in the PBMs and lymph nodes and is further disseminated via the blood stream to sites of secondary infection. In the final phase of infection, the virus begins circulating in the bloodstream. Viral RNA can persist for several months. BTV binds to glycophorins on the surface of bovine and ovine erythrocytes where it may remain in an infectious state as invaginations of the erythrocyte cell membrane. This minimizes contact with antibody and T cells and thus provides multiple opportunities for transmission by infection of blood-sucking midges. In sheep, viremia is usually detectable around 3–5 days post infection. During the initial stages the virus associates with both platelets and erythrocytes, however, in the late stages of infection, the virus is associated exclusively with erythrocytes, facilitating

134

Bluetongue Virus (Reoviridae)

the prolonged infection of ruminants and increasing transmission to the insect vectors that feed on them. BTV infection of mammalian cell lines induces cell death, either by triggering multiple apoptotic pathways or via necrosis. BTV replication in endothelial cells causes cell injury and infected ruminants develop lesions due to mononuclear cell infiltration. The accumulation of large acidophilic intra-cytoplasmic masses which, results in degeneration and necrosis. Infected ruminants suffer severe systemic damage, characterized by widespread edema and hemorrhage. In infected sheep with severe disease, swelling of the tongue and lack of oxygen make the tongue and mucous membranes appear blue (hence the name of the disease). Infection of pregnant cattle and sheep with BTV can result in maternal death, abortion, and fetal death, or congenital anomalies. BTV infection in most wild ruminant species is typically asymptomatic or subclinical, although in some cases severe disease in cattle and camelids has been described. Infected cattle and goats sporadically display clinical signs including nasal discharge, swelling, and, ulceration of the mouth.

Diagnosis Since bluetongue is a notifiable disease, animals diagnosed with clinical signs of bluetongue disease and pathology are required to be reported to local health officials. BTV infection is confirmed by virus isolation, detection, and characterization of the infecting serotype. Earlier methods relied primarily on serological tests to detect BTV antibodies in infected animals. Complement fixation, agar gel immunodiffusion (AGID) and haemagglutination-inhibition relied on virus/serum neutralization methods that enabled virus serotype identification based on the interaction of the outer capsid protein VP2 with serotype-specific antibodies. Among the various serogroup-specific assays for antibody detection, AGID and competitive enzyme-linked immunosorbent assay (cELISA) are the most widely used. However, although the AGID test is simple to perform and rapid, it is not highly sensitive or quantitative and has limitations in its specificity. In contrast, the cELISA, in which a group-specific monoclonal antibody based on BTV core protein VP7 is used, has proved to be the most sensitive and specific assay for detection of antibodies to most, if not all, serotypes and strains of BTV. Following extensive national and international validation, the cELISA became a universal test to certify ruminants for trade purposes and to diagnose BT infection in domestic and wild animals. The test is highly specific and sensitive detecting antibodies to most, if not all, serotypes and strains of BTV. For the detection of serotype-specific antibodies in animal sera, the cell culture-based microtiter serum neutralization (MTSN) is the most commonly used assay. MTSN is often used to monitor animal population for specific serotypes of BTV in epidemiological investigations. Recent advances in molecular and high-throughput sequencing technologies have enabled rapid identification of newly isolated strains by reverse transcription PCR (RT-PCR) assay, real-time RT-PCR, or sequencing specific segments that are highly variable across the different isolates. RT-PCR assays employing serotype-specific primers have provided the most rapid and specific information regarding isolate serotype. Selective sequencing of virus segments or whole genome sequencing provides serotype as well as other unique sequence information of isolates. These new approaches have enabled rapid and reliable confirmation of BTV serotype, and are potential methods that should be applied at the onset of an outbreak to allow for the early selection of vaccine.

Vaccination and Control Vaccination strategies in different countries have been developed according to their individual policies, the geographic distribution of the circulating BTV serotypes and the availability of appropriate vaccines. In South Africa, the country where bluetongue was first characterized, live attenuated virus vaccines have been used for over 50 years and are known to induce an effective and lasting immunity. Due to the high number of circulating serotypes however, live modified vaccines often have to be administered as a multivalent vaccine. Despite their success in endemic areas, these attenuated virus vaccines have exhibited some drawbacks, including abortion/embryonic death and teratogenesis in fetuses of pregnant females. In addition, the strength of the protective antibody response to attenuated vaccines correlates directly with their ability to replicate in vaccinated animals and when responses are sub-optimal viremia is observed that is sufficient for the vaccine strain to be transmitted to biting midges. Furthermore, there is a likelihood of reversion to virulence and/or reassortment of the vaccine strain with wild type viruses. In recent years, chemically inactivated BTV vaccines were used for mass vaccination of sheep and cattle in European countries during the BTV-8 and BTV-1 outbreaks. While these inactivated virus vaccines have been demonstrated to be safe and immunogenic in animals, they are only available for a limited number of serotypes. Additionally, in some cases adverse reactions have been associated with some of these inactivated virus preparations. Inactivated vaccines generally induce relatively transient immunity in comparison to live, attenuated vaccines and require booster immunizations. The high cost of vaccine production due to the need for large amounts of antigen required for each vaccination is another major drawback. The biggest limitation to the use of both the live attenuated and inactivated vaccines is the significant economic impact they have on agricultural trade. These vaccines do not allow the differentiation of naturally infected versus vaccinated animals (DIVA) as these vaccine strains are not free of viral protein components that interfere with serological tests. Thus, neither of these vaccines is DIVA compliant and restrictions are imposed on animal trade and movement if an animal is diagnosed positive, even if that positivity is the result of vaccination. Further, due to the presence of genomic RNA after vaccination and short-term BTV viremia in animals in the case of the live attenuated vaccine, a genetic DIVA is not always feasible. A number of recombinant protein technologies have been employed in order to provide DIVA compliant vaccine candidates and to reduce the potential adverse effects associated with inactivated and attenuated virus vaccines. Recombinant proteins have

Bluetongue Virus (Reoviridae)

135

predominantly been assessed in sheep in experimental vaccination challenge trials and shown to afford protection, but have yet to be commercialized. Recombinant outer capsid protein VP2, the serotype determining protein, could indeed elicit protective neutralizing antibody in vaccinated animals and later studies, which combined both outer capsid proteins (VP2 þ VP5) resulted in better protection than VP2 alone in vaccinated sheep. In addition, VP2-based vaccines have been developed using other live virus vector systems (i.e., poxviruses, herpesvirus, and adenovirus), which have been designed to express either a single or multiple proteins in animals following vaccination. Enhanced protective immunity afforded by recombinant VP2 þ VP5 led to the development of recombinant virus-like particles (VLPs) containing the VP2 þ VP5 and the two core proteins (VP3 and VP7), mimicking the virus particles without the viral genome or enzymatic proteins. The VLPs had the characteristic icosahedral structure with the four recombinant proteins in the same position and ratios as the native proteins. Multiple vaccination studies with homologous and heterologous VLPs (B10 mg) resulted in complete protection against virulent virus challenges in sheep. In recent years, extensive vaccination trials with VLPs of European serotypes had been undertaken in European breeds of sheep. These trials have also assessed VLPs delivered singly or in cocktail, as well as the evolutionary lineage of the virus strains, in order to assess their potential for commercialization. The influence of evolutionary divergence (Eastern and Western topotypes) on vaccine development has clearly highlighted that the development of serotype specific neutralizing antibodies was more critical than the topotype of the circulating strain. This suggested that isolate variation or evolutionary distance of the strain is not critical for BTV vaccines. Furthermore, the delivery of VLPs as a cocktail of different serotypes did not limit the ability of each serotype to raise a serotype specific neutralizing antibody response or to interfere with the protective efficacy of the vaccine when animals were challenged. These findings have indicated that multiple serotype vaccines could be developed using known serotypes rather than generating new vaccines for each BTV outbreak. Most importantly, it is possible to distinguish between VLP-vaccinated animals and those that are infected with the virus, thus addressing one of the major problems with current vaccines. Further, VLPs are entirely protein-based and there is no genetic component avoiding all issues of reversion or recombination. The use of the BTV reverse genetics system has further allowed direct introduction of mutations/deletions in the viral genome leading to the recovery of replication-defective virus strains using specific complementary cell lines. Entry Competent Replication Abortive (ECRA) vaccines, formerly known as Disabled Infectious Single-Cycle (DISC) vaccines were designed based on this principle. The ECRA vaccines are modified particles that contain only 9 of the 10 genome segments, they lack the essential catalytic gene VP6, and are hence replication incompetent. The defective virus can enter susceptible cells where they undergo a single cycle of replication but fail to make progeny virus due to the inability to package the dsRNA genome. Administration of ECRA vaccine results in expression of the viral proteins at the site of infection but does not cause infection in animals and hence combines the efficacy of live-attenuated vaccines with the safety of inactivated vaccines. Recent trials in cattle and sheep have shown promising results. A single dose of a DISC virus strain was sufficient to confer complete protection against a challenge in sheep 42 days after vaccination while a cocktail of six different vaccines protected animals as early as 21 days against any challenge with virulent strains. The animals were completely protected from challenge with individual virulent serotypes, even after 5 months of challenge without any clinical disease. ECRA vaccines are more cost-effective and since they are replication incompetent, the ECRA RNA segments are present in the bloodstream for a very short period thus minimizing the risk of transmission via insect vector, reassortment, or reversion to virulence. A second RG based candidate vaccine strain has been has been made against BTV known as Disabled Infectious Single-Animal (DISA), in which segment 10 is deleted, encoding NS3/NS3A. Unlike the ECRA strains, the DISA strain still replicates in normal cells and in sheep although it is significantly attenuated. The DISA vaccine strain is also DIVA compliant and is another promising candidate vaccine although rigorous animal studies are still lacking. While a completely new range of different vaccine candidates, showing promising levels of efficacy and safety, have been developed recently, none of them are near commercial production. The voluntary vaccination policy adopted in Europe and the acceptance of endemic disease in many parts of the world has resulted in a dearth of commercial organizations interested in producing these new vaccines. Given the likelihood of new BTV outbreaks across Europe, there is however, a need to develop suitable vaccines that can be produced efficiently as vaccination is the best way to control outbreaks.

Future Perspectives The advances in understanding BTV, at all levels, has led to it being one of the most well characterized RNA viruses, including the details related to its replication in both mammalian and insect hosts. Physically the resolution of the virion structure has increased substantially and the atomic detail of nearly all of the particle is now known. This level of understanding has provided insights on how the particle disrobes on entry and crosses the membrane to enter the cytoplasm. We also have a greater understanding of how transcription and replication proceed. Importantly, key insight has been obtained on how multiple segmented dsRNA genomes are incorporated into assembling virions, while cellular messenger RNAs are excluded. The role of cellular factors and virus encoded proteins in leading the virus out of the cell has never been better described. Yet despite this phenomenal progress, much remains to be learned. The structure of the particle, while instructive, does not represent the dynamic aspects of the capsid, that is less understood. The level of “breathing and the triggers” for channel opening to allow the initiation of transcription of the assembled particles are still unclear. How VP5 leads to membrane destablization is also not well understood. BTV is a large particle and it is difficult to envisage it crossing a membrane barrier with ease.

136

Bluetongue Virus (Reoviridae)

The dynamic nature of VIB is another area of uncertainty and while its description as a virus factory is true, it does not address how exactly all BTV components coalesce there or how, after assembly, the newly formed virions leave this amorphous mass in an orderly fashion. What are the triggers and timings of these processes and do they pertain to all cell types or only to some? Virions acquire their outer layer as it migrates the cell to the exterior. A role for an “enveloped” BTV has been proposed, but detail is sketchy. Not least, the astonishing difference in CPE observed in mammalian and insect cells is something that requires a much more work. Behind all experimental approaches, molecular, structural or immunological, is the desire to see new findings translated into improved options for disease control. Here, recent progress has been promising with some clever approaches to the generation of candidate vaccines that include DIVA compliance. Better yielding and genetically tighter variants will certainly be produced in the future opening the possibility that such intricately engineered vaccines may become the default for future outbreak control. Bluetongue is a disease with economic repercussions in both developed and developing countries. Hence, better control measures have the opportunity to improve also the quality of life for the livestock of subsistence farmers, which in turn can have knock-on benefits, to the communities that depend upon animal productivity.

Further Reading Belbis, G., Zientara, S., Bréard, E., et al., 2017. Bluetongue virus: From BTV-1 to BTV-27. In: Beer, M., Höper, D. (Eds.), Advances in Virus Research 99. Academic Press, pp. 161–197. Bhattacharya, B., Roy, P., 2010. Role of lipids on entry and exit of bluetongue virus, a complex non-enveloped virus. Viruses 2, 1218–1235. Boyce, M., Celma, C.C.P., Roy, P., 2008. Development of reverse genetics systems for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts. Journal of Virology 82, 8339–8348. Celma, C.C.P., Stewart, M., Wernike, K., et al., 2016. Replication-deficient particles: New insights into the next generation of bluetongue virus vaccines. Journal of Virology Dec 91 (1), e01892-16. Coetzee, P., Van Vuuren, M., Stokstad, M., Myrmel, M., Venter, E.H., 2012. Bluetongue virus genetic and phenotypic diversity: Towards identifying the molecular determinants that influence virulence and transmission potential. Veterinary Microbiology 161, 1–12. Huismans, H., 1979. Protein synthesis in bluetongue virus-infected cells. Virology 92, 385–396. Janowicz, A., Caporale, M., Shaw, A., et al., 2015. Multiple genome segments determine virulence of bluetongue virus serotype 8. Journal of Virology 89 (10), 5238–5249. Lourenco, S., Roy, P., 2011. In vitro reconstitution of bluetongue virus infectious cores. Proceedings of the National Academy of Sciences 108, 13746–13751. Maan, S., Maan, N.S., Samuel, A.R., et al., 2007. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes. Journal of General Virology 88, 621–630. MacLachlan, N.J., Drew, C.P., Darpel, K.E., Worwa, G., 2009. The pathology and pathogenesis of bluetongue. Journal of Comparative Pathology 141, 1–16. Matsuo, E., Roy, P., 2013. Minimum requirements for bluetongue virus primary replication in vivo. Journal of Virology 87, 882–889. Nomikou, K., Hughes, J., Wash, R., et al., 2015. Widespread reassortment shapes the evolution and epidemiology of bluetongue virus following European invasion. PLOS Pathogens 11 (8), e1005056. Roy, P., 2017. Bluetongue virus structure and assembly. Current Opinion in Virology 24, 115–123. Shaw, A.E., Hughes, J., Gu, Q., et al., 2017. Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses. PloS Biol 15, e2004086. Sung, P.Y., Roy, P., 2014. Sequential packaging of RNA genomic segments during the assembly of Bluetongue virus. Nucleic Acids Research 42, 13824–13838. Zhang, X., Patel, A., Celma, C.C.P., et al., 2016. Atomic model of a nonenveloped virus reveals pH sensors for a coordinated process of cell entry. Nature Structure Molecular Biology 23, 74–80.

Borna Disease Virus and Related Bornaviruses (Bornaviridae) Susan L Payne, Texas A&M University, College Station, TX, United States r 2021 Elsevier Ltd. All rights reserved.

Introduction The name bornavirus derives from Borna disease, a rare neurologic disease that mainly effects horses and sheep. The disease presents as an array of behavioral changes including ataxia, head tilt, muscle fasciculation, hind limb paresis, localized hypo- or hyperesthesia, disturbances in chewing and swallowing, and aggression. Borna disease is endemic in a fairly small geographic area encompassing central and southern Germany and neighboring countries. The disease name is linked to two major outbreaks that occurred from 1894 to 1896, among cavalry horses near the town of Borna, Germany. The infectious nature of Borna disease was not confirmed until the early decades of the 20th century and it would take approximately another 50 years before persistently infected cell cultures were established. However, even then the infectious agent remained uncharacterized because persistently infected cultures did not provide sufficient cell-free virus for convincing biochemical or structural studies. Finally, in the early 1990s molecular studies revealed the similarities of Borna disease virus (BoDV-1) to other unsegmented, negative strand RNA viruses.

Classification Until 2008 BoDV-1was the sole member of the family Bornaviridae. In 2008 novel bornaviruses were identified as the etiologic agents of proventricular dilatation disease (PDD) of birds. Identification of these avian bornaviruses was accomplished by using nucleic acid hybridization and unbiased high throughput sequencing techniques. Since 2008, polymerase chain reaction (PCR) and unbiased nucleic acid sequencing methods have been used to identify additional bornaviruses. Currently the International Committee on the Taxonomy of Viruses Master Species List contains 10 species of bornavirus. Two species have been isolated from mammals (Mammalian 1 orthobornavirus and Mammalian 2 orthobornavirus), 5 from birds (Passeriform 1 orthobornavirus, Passeriform 2 orthobornavirus, Psittaciform 1 orthobornavirus, Psittaciform 2 orthobornavirus and Waterbird 1 orthobornavirus) and 3 from snakes (Elapid 1 orthobornavirus, Queensland carbovirus, and Southwest carbovirus).

Virus Structure Bornavirus virions are enveloped and are assumed to have a helical nucleocapsid; their size is broadly estimated to be 80–130 nm in diameter. However, unlike many viruses, detailed ultrastructural analyzes of bornavirus virions have not been performed, due to the highly cell-associated nature of the virus. The illustration of a bornavirus particle shown in Fig. 1 is theoretical, based on relatively low resolution electron micrographs of virions and the known structures of other unsegmented negative strand RNA viruses.

Genome The genome of bornaviruses is a single negative stranded molecule of approximately 8.9 kb in length. The genome organization of mammalian and avian bornaviruses is highly conserved (Fig. 2) and similar to other members of the order Mononegavirales (Fig. 2). Bornaviruses contain 6 open reading frames and short non-coding regions are found at the 5′ and 3′ ends of the genome. Bornavirus genomes lack the discrete intergenic sequences characteristic of the paramyxoviruses and rhabdoviruses. Instead, transcription termination sequences for upstream transcripts are found just downstream of initiation sequences for the next transcript. A promoter near the 3’ end of the BoDV-1 genome initiates the N transcript, the most abundant viral transcript. The N transcript is used to produce two isoforms of N, p40 and p38 by use of alternative start codons. The X/P transcript produces two proteins, X and the phosphoprotein P. X and P are produced from overlapping reading frames. P is a 24 kDa phosphoprotein that serves as a co-factor for L, the catalytic subunit of the transcription/replication complex. The 10 kDa X protein interacts with P and has role in RNA replication. X also associates with mitochondria and prevents induction of apoptosis in infected cells. A third transcription start site is used to produce the M, G and L transcripts; both G and L are spliced. M (p16) is found in both the cytoplasm and nucleus of infected cells. M co-localizes with N, P, and X in punctate structures in the nucleus. As shown in Fig. 2, there are short overlaps of the M, G and L reading frames. The glycoprotein, G, is post-translationally modified by N-glycosylation to yield a 93-94-kDa primary product. G contains a proteolytic cleavage site and a portion of G is cleaved to produce amino terminal (G-N) and carboxyl terminal (G-C) products. Virus neutralization studies have demonstrated the importance of G in virus entry.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20976-X

137

138

Borna Disease Virus and Related Bornaviruses (Bornaviridae)

Fig. 1 Virion structure (left). Virus-like particle (83 nm in diameter) from the eye fluid of an Eclectus parrot with confirmed PDD and ABV0 infection (right). From: Payne, S., 2017. Chapter 22 – Family Bornaviridae. In Viruses (edited by S. Payne), Academic Press, pp. 191-196, https://doi.org/10.1016/B978-0-12-803109-4.00022-2.

Fig. 2 Bornavirus genome organization and transcription pattern. From: Payne, S., 2017. Chapter 22 – Family Bornaviridae. In Viruses (edited by S. Payne), Academic Press, pp. 191-196, https://doi.org/10.1016/B978-0-12-803109-4.00022-2.

Borna Disease Virus and Related Bornaviruses (Bornaviridae)

139

Life Cycle Most molecular studies of bornaviruses replication have been carried out using BoDV-1, but it is likely that mechanisms of transcription and genome replication elucidated for BoDV-1 will hold true for the avian bornaviruses as well. Bornaviruses are highly cell-associated, however some infectious particles are released from infected cells. In the case of cell-free virus, bornavirus G is the candidate for receptor attachment and subsequent fusion, but host receptors have not been determined. Although the molecular details are not yet known, it is clear that the nucleocapsids traffics to the nucleus where transcription initiates. L catalyzes RNA synthesis and likely serves to cap and polyadenylate viral mRNAs. Translation of N, X, P, M and L are cytosolic, while G is processed in the endoplasmic reticulum. All viral proteins with the exception of G are transported back into the nucleus where genome replication and assembly of ribonucleoprotein particles (RNP) occur. Immunostaining of persistently infected cells shows large punctate structures in the nucleus. These so-called vSPOTS are virus replication factories with a size of ~66 µm in diameter. vSPOTS are cage-like assemblies of the N protein with 59–180 nm pores. Viral genomes and the proteins X, P, M and L are also found in vSPOTS, therefore these structures contain all of the components necessary to be sites of transcription and/or genome replication. Filamentous viral RNPs, containing genomic RNA, N, P, X and L, associate with chromatin by using core histones as a docking platform. High mobility group box protein 1 (HMGB1), a chromatin- remodeling DNA architectural protein binds to viral P, stabilizes the RNP on chromosomes and appears to be required for efficient transcription. In mitotically active cells, the tight association of the RNP with chromatin facilitates transmission of RNPs to daughter cells. Within infected hosts, virus (in the form of RNPs) spreads long distances along neural networks to access the CNS. RNPs appear to move along axons in signaling endosomes. There is little to no free virus in the infected animal. In a cockatiel models of avian bornavirus infection, virus injected into pectoral muscle reached the spinal cord at about 20–25 days post-inoculation. From there virus spread quickly to the brain. From the CNS, virus spread to ganglia in the gastrointestinal tract, adrenal gland, heart, and kidneys. At late points of infection (80–100 days) viral antigen was also seen in smooth muscle and/or scattered epithelial cells of numerous tissues.

Epidemiology and Clinical Features Borna Disease The white-toothed shrew (Crocidura leucodon) is the animal reservoir for BoDV-1 in Borna disease endemic areas. The virus replicates in numerous tissues in the shrew and is excreted into the environment. In shrews the virus causes persistent infection without signs of disease. Borna disease, caused by infection with BoDV-1, is quite rare and predominantly affects horses and sheep in Central Germany. There is no evidence that the virus is naturally transmitted among infected livestock, therefore horses and sheep are ‘accidental’ and dead-end hosts. Disease is often fatal. Signs include ataxia, head tilt, muscle fasciculation, hind limb paresis, localized hypo- or hyperesthesia, disturbances in chewing and swallowing, and aggression. Grazing animals, in particular horses and sheep, likely inhale excreted virus and are infected by the olfactory route. The incubation period last from weeks to months as virus moves from the initial site of infection to the central nervous system.

Staggering Disease in Cats Beginning in the late-1970s, a neurologic disease characterized by stiffness, leading to paralysis and death was observed in cats in Central Europe and Scandinavia. Histopathology of affected cats revealed lymphohistiocytic inflammation of the CNS. In the 1990s BoDV-1 nucleic acids were isolated from some of cats with staggering disease, and challenge of pathogen-free cats with this virus induced neurologic disease. Staggering disease has been reported in cats world-wide and serologic surveys of healthy cats reveal that many are also positive for bornaviral antigens. Studies in Japan show approximately 25% of tested cats are positive for bornaviral antigens by Western blot assays. This is similar to findings in birds (see below) and underscores the fact that many BoDV-1 infections are asymptomatic. Epidemiology of bornavirus infection in cats in Japan points to vertical transmission, while reports from Europe suggest cats with outdoor access are at higher risk for developing staggering disease. For these cats, infection likely results from predation upon birds, rodents and/or other small mammals. In one European study, approximately 80% of cats with staggering disease tested positive for bornavirus-specific antibodies by immunofluorescence assays, compared to 16% of a healthy comparison group.

Bornavirus Associated Encephalitis in Humans Beginning in the 1980s there were efforts to link human bornavirus infection and psychiatric disorders. Many of the early studies claiming positive links relied on poorly described serologic techniques and many other studies failed to show links. However, while the relationship between bornavirus infection and human psychiatric disease remains unclear, three recent case reports provide striking evidence that mammalian bornaviruses can cause severe, fatal neurologic disease in humans. In all, 6 human deaths have been strongly associated with bornaviral infection. The first case report of fatal human bornavirus infection involved

140

Borna Disease Virus and Related Bornaviruses (Bornaviridae)

Fig. 3 Fluorescent antibody labeling of duck embryo fibroblasts infected with an avian bornavirus. A focus of infected cells (green) sits amidst uninfected cells. The uninfected cells are visualized by DAPI staining (blue) of their nuclei. From: Payne, S., 2017. Chapter 22 – Family Bornaviridae. In Viruses (edited by S. Payne), Academic Press, pp. 191-196, https://doi.org/10.1016/B978-0-12-803109-4.00022-2.

three breeders of squirrels in Germany that presented with encephalitis between 2011 and 2013. All three died within 2–4 months after onset of symptoms. After eliminating suspected causes of encephalitis, a genomic approach was used as a means of identifying novel pathogens. This approach revealed common bornaviral sequences in the brains of the three victims. Exchange of breeding squirrels linked the three human victims and an identical viral sequence was recovered from the brain of a variegated squirrel (Sciurus variegatoides). The squirrel bornavirus, (named variegated squirrel 1 bornavirus (VSBV-1) is phylogenetically distinct from BoDV-1 and is a member of the species Mammalian 2 orthobornavirus). Another case report from 2018 underscores the danger of handling squirrels. This report describes a fatal case of limbic encephalitis in a zoo caretaker in Germany. Molecular assays and immunohistochemistry revealed a limbic distribution of viral antigen in brain tissue. Sequencing and phylogenetic analysis of the brain-associated virus demonstrated a likely spillover infection from a Prevost’s squirrel (Callosciurus prevostii). Prompted by the human cases of VSBV-1, studies of squirrels have been carried out in order to assess the risk of infection. Small numbers of VSBV-1 infected squirrels have been detected among small breeders and zoos in Germany, the Netherlands and Croatia. The variegated squirrel is a native of southern Mexico and Central America while the Prevost’s squirrel is a native of Asia, thus the origins and natural reservoirs of VSBV-1 are unknown. Organ transplantation is another source of human bornavirus infection. A 2018 report presents strong evidence of BoDV-1 infection transmitted via solid organ transplant. As described in the case report, the organ donor was a 70 year old man from the Bavarian region of Germany who had no signs or symptoms of neurologic disease. Two recipients of kidneys developed neurologic disease that progressed to irreversible coma. A third patient, who received a liver graft, developed transient neurologic deficits but went into remission from the disease. A metagenomics analysis of brain tissues from the two deceased organ recipients was performed, revealing BoDV-1 sequences. Immunohistochemical analysis also revealed bornaviral antigens in brain tissues. Antibodies to BoDV-1 were detected in the third organ recipient. It is interesting to note that to date, these well-documented cases of bornavirus-associated encephalitis in humans have occurred in Central Europe, in Borna disease endemic areas. It remains to be seen if additional cases of fatal human encephalitis will be linked to bornavirus infection in the future.

Proventricular Dilatation Disease of Birds PDD is a fatal neurologic condition first recognized in the mid-1970s among captive parrots. A viral etiology was suspected early on, but the agent was not identified until 2008 when the genomes of novel bornaviruses were identified in the brains of affected birds. Infectious viruses were quickly recovered and used to produce PDD in experimental infections. While large parrots and macaws are the captive birds most likely to be diagnosed with PDD, the disease has also been described for other pet birds such as canary birds and finches. Clinical neurologic signs of PDD include weakness, ataxia, proprioceptive deficits, seizures, and blindness. Gastrointestinal malfunctions are also very common and result in severe weight loss, passage of undigested food, regurgitation and delayed crop emptying. Gastrointestinal malfunction results in the most common gross pathologic lesions associated with PDD – dilation and thinning of the walls of the proventriculus and ventriculus. Histopathologically, PDD is characterized by nonsuppurative inflammation in the central, peripheral, and autonomic nervous systems. ABV infects primarily the central and enteric nervous systems of birds. In experimental models, virus is inoculated into muscle tissue and onset of disease is highly variable, but is typically from several weeks to months. Although the virus is noncytopathic examination of brain tissue reveals selective loss of glial cells and neurons. Based on the known pathogenesis of Borna disease, it is believed that cell loss is due to T cell cytotoxicity rather than direct cytopathic effects of the virus. Treatment of affected birds with immunosuppressive agents can suppress or delay disease development in experimental models, supporting an immunopathologic basis for the disease.

Borna Disease Virus and Related Bornaviruses (Bornaviridae)

141

Fig. 4 Microscopic lesions from a birds with avian bornavirus infection and PDD showing lymphocytic infiltration (dark purple cells) (left) and presence of bornavirus antigen (discrete areas of dark brown staining) in neurons (right). From: Payne, S., 2017. Chapter 22 – Family Bornaviridae. In Viruses (edited by S. Payne), Academic Press, pp. 191-196, https://doi.org/10.1016/B978-0-12-803109-4.00022-2.

In birds, bornavirus replication is not restricted to neurons. Lymphocytic infiltrates and viral antigens and nucleic acids are abundant in the heart, kidneys, adrenals, gonads and skin (Fig. 4). Bird droppings contain viral nucleic acids and droppings have been used a source of infectious virus. Droppings are a likely vehicle for transmission of avian bornaviruses but it is interesting to note that attempts to experimentally infect birds via the oral or intranasal route have, to date, been unsuccessful. The first clues to bornavirus infection of waterfowl came from retrospective surveys of Canada geese and trumpeter swans suffering from neurologic disease of unknown origin but with neuropathology consistent with PDD. These studies were carried out at the Ontario Veterinary College, Guelph, Ontario, Canada. Bornaviral sequences and antigens were present in brain tissues of many affected birds. Subsequent surveys of healthy birds (hunter harvested, or culled as nuisance birds at airports and landfills) revealed that avian bornaviruses infection is widespread in North America and Europe. In North America, bornaviruses have been detected in the brains or urofeces of Canada geese (Branta Canadensis), snow geese (Chen caerulescens), Ross’s geese (Chen rossii), trumpeter swans (Cygnus buccinator), mute swans (Cygnus olor) and a variety of ducks and gulls. The majority of waterfowl infections are associated with the species Waterbird 1 orthbornavirus. The virus has also been detected in raptors suggesting that predation may be a source of infection. The overall impact of bornavirus infection of wild bird populations remains unknown.

Endogenous Bornavirus-Like Elements In recent years, whole genome sequencing has revealed insertions of many non-retroviral genome fragments into the genomes of diverse animal species. Among the viral genome fragments discovered in animal genomes are those of bornaviruses. Fragments of bornavirus genomes have been detected in fish, snakes, turtles, bats, rodents, squirrels, elephants, nonhuman-primates, and humans. Phylogenetic analyzes show that the establishment of endogenous Borna-like (EBL) elements took place took place as multiple independent events, as long as 65 million years ago. Bornavirus N, M, G and L genes are present in eukaryotic genomes. EBLs are derived from viral mRNAs and evidence supports a role for LINE-1 retrotransposition. Some EBLs comprise complete open reading frames and indeed, some are expressed. In humans there are seven EBL N elements (EBLNs) and all are expressed in at least one tissue. Robust protein expression of Homo sapiens EBLN-1 (hsEBLN-1) occurs in various cancer-derived cell lines and it functions in regulation of genome stability and cell cycle. Knockdown of hsEBLN-1 RNA results in abnormal cell growth, arrest of cell cycle at G2/M and increased apoptosis. In an experimental system, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) EBLN inhibits the replication of BoDV-1 in cell cultures by suppression of polymerase activity. Thus, some EBLs may play roles in intrinsic immunity to bornaviruses.

Pathogenesis Non-cytopathic infection and persistence are hallmarks of bornavirus infection, both in cultured cells and within the infected host. Relatively few infectious virions are produced as bornaviruses are highly cell-associated. Immunostaining of infected cell cultures reveals slow ‘spread’ of the virus as infected cells divide (Fig. 3). Infected cells undergoing mitosis reveal chromatin associated ‘particles’, suggestive of RNPs, being distributed to daughter cells. In the infected host, suppression of innate immune responses may favor establishment of infection and viral persistence. Bornaviruses have redundant mechanisms by which they interfere with innate responses. BDV N interferes with the IKK/NF-kB pathway by inhibiting the cleavage of NF-kB1 p105. N also interferes with IRF7 activation (prevents nuclear localization) and interferon (IFN) type 1 expression. P inhibits the activation of type I interferon (IFN) as well,

142

Borna Disease Virus and Related Bornaviruses (Bornaviridae)

through the TBK1-IRF3 pathway. BDV X interferes with IFN signaling and also localizes to mitochondria, blocking apoptosis mediated by the mitochondrial signaling protein MAVS. Other factors that favor persistence in the infected host include regulation of transcription and genome replication. An interesting and unique feature of bornavirus genome replication is truncation of genome strands. Many genomes cloned from persistently infected cells are truncated and lack sequences required for genome replication. It is speculated that this process leads to down-regulation of replication and or transcription. Genome truncation would also remove 5′ terminal triphosphates from RNA, leaving 5′ monophosphate ends, providing a mechanism for avoiding recognition by RIG-I. In horses and sheep, BoDV-1 is exclusively associated with neurons. Virus replication is non-cytopathic but in response to infection, large numbers of T-lymphocytes infiltrate the CNS and are found at sites of virus replication. Thus, there is an immune mediated component to BoDV-1 pathogenesis. Laboratory rodents are used as models for BoDV-1 infection (although rodents vary greatly in their susceptibility to BoDV-1 infection and different model systems employ specific rodent and virus strains). In the rodent model, virus replication is restricted to neurons and virus spreads through neural networks, similar to the case of rabies virus. BoDV-1 mainly targets hippocampal neurons, but also infects astroglial and oligodendroglial cells in the brain. The outcome of infection depends on the genetic background, age of infection and immune status of the animal. Infection of adult, immunocompetent rats results in an encephalitis similar to that seen in natural infections. It has been clearly demonstrated that encephalitis is immune-mediated, with cytotoxic T-cells being the major players. The pathology of the immunocompetent rat model is identical to the natural infection of horses and sheep. In contrast, experimental infection of neonatal rats (not yet immunocompetent) or immune-suppressed rats, leads to persistent infection, in the absence of acute disease. However, infection is not completely without consequence as persistently infected rats show behavioral, emotional and cognitive impairments. Neurological disease mediated by BoDV-1 comes in two forms: immune-mediated pathology and alterations in cell function as a direct result of viral proteins. Studies carried out in cultured cells and laboratory rodents reveal myriad effects of BoDV-1 infection at the cellular level. BoDV-1 proteins have various effects on cell signaling, apoptosis, and neurogenesis. Specific effects vary with virus isolate and cell types tested, however it is clear that BoDV-1 proteins cause significant changes in cell physiology and function. Some examples include: Up or down regulation of important signaling proteins such as PKC, Raf, MEK and ERK, changes in gene expression of (TGF)-b family members, and inhibition of histone acetylation. BoDV-1 infection of neuronal progenitor cells impairs neurogenesis and causes diminished neuritic outgrowth and synapse formation. Many of these cellular perturbations are linked to BoDV-1 P and it has been shown that expression of P in transgenic mice induces measurable behavioral abnormalities.

Diagnosis Clinical presentation alone is insufficient for a diagnosis of bornaviral disease in animals as differential diagnosis for an animal with encephalitis will include any number of viral agents (which will vary by species). Currently there is no single assay recommended for diagnosing bornavirus infection, however a number of serological assays for detection of antiviral antibodies have been described. These include immunofluorescence assays (targeting bornavirus-infected cells) and Western blot and ELISA (targeting recombinant N or P antigens). Results of serologic assays must be interpreted carefully as a negative antibody test does not rule out bornavirus infection, as some infected animals will be antibody negative. Conversely, antibody positive animals may be completely healthy and positive serology is not a predictor of disease among healthy animals. In birds with suspected PDD, serologic assays are highly problematic as antibody production in experimentally infected birds is highly variable. Detection of bornaviral RNA by reverse transcriptase (RT) PCR is a more reliable indicator of infection. Most PCR assays target the N or P genes. In cats, recommended samples include blood, serum, urine, conjunctival, oral, nasal and/or anal swabs. In birds, avian bornaviral RNAs can be detected in cloacal swabs and the urofeces but virus shedding is intermittent. While positive PCR results are indicative of bornavirus infection, negative results indicate only a current lack of viral shedding. Currently, PCR remains the best indicator test for avian bornavirus infection. Bornavirus induced encephalitis is usually fatal, thus many diagnoses are made post-mortem. Typical findings include nonsuppurative lymphocytic infiltrates in the brain or spinal cord. Perivascular cuffing is a common finding upon histological examination of brain tissues. Immunohistochemistry using N or P antisera can be used to stain bornavirus infected neurons to achieve a definitive diagnosis. RT-PCR of RNA extracted from brain tissue may also be used to confirm bornavirus infection.

Control and Prevention There are currently no bornaviral vaccines approved for veterinary use in either mammals or birds. Other than among pet birds, symptomatic bornavirus infections of animals are quite rare, even in confirmed endemic areas of Central Europe. Infections in this region are likely the result of contact with urine or feces of natural hosts, such as the white-toothed shrew. Currently the highest risk of human bornavirus infection appears to be contact with certain squirrels (S. variegatoides and C. prevostii). Four human deaths from Mammalian 2 orthobornaviruses have been reported in Germany, and all occurred among persons having contact with either Sciurus or Callosciurus squirrels which are exotic to Europe. The original source of the squirrel infections is unclear but

Borna Disease Virus and Related Bornaviruses (Bornaviridae)

143

keeping exotic squirrels as pets may be inadvisable. In 2018 two human deaths due to BoDV-1 infection were reported in Germany. These were associated with organ transplantation from a BoDV-1 infected donor. It is currently not clear if the risk of human bornavirus encephalitis is higher in Germany than in other locals, or if these cases were detected due to heightened awareness of the potential for bornavirus infection in this area. Unfortunately serologic assays will not detect 100% of bornavirus infections and even the most sensitive PCR assays will not detect virus if replication is restricted to the central nervous system. Controlling the spread of avian bornaviruses among pet birds might be achieved by testing birds using PCR assays of urofeces.

Future Prospects Since the advent of metagenomics analyzes and high-throughput sequencing, the list of known bornaviruses has grown from 1 to 5 recognized species, geographic range has expanded from an area of Central Germany to world-wide, and hosts have expanded to include birds and reptiles. Much of our understanding of bornavirus replication and pathogenesis comes from studies of BoDV-1 and it is likely that use of well-characterized laboratory rodent models will continue to add to our knowledge base. In recent years there has been debate over a role for bornavirus involvement in human psychiatric disease. The recognition of novel bornaviruses and the development of rigorous and validated methods for assessing the infection status of patients should allow clarification of this important issue. Indeed, recent reports of bornavirus-associated fatal encephalitis in humans clearly demonstrate the ability of humans serve as spillover hosts. We currently have only a rudimentary understanding of the natural reservoirs of bornaviruses and continued surveys are needed to gain a more complete understanding of the full host range and genetic diversity of these fascinating viruses. On a very different note, the study of EBLs should be pursued as a tractable model for understanding the ancient history of the association between eukaryotes and their viruses.

Further Reading Formisano, S., Hornig, M., Yaddanapudi, K., et al., 2017. Central nervous system infection with borna disease virus causes Kynurenine pathway dysregulation and neurotoxic quinolinic acid production. Journal of Virology 91, e00673. Hoffmann, B., Tappe, D., Höper, D., et al., 2015. A variegated squirrel bornavirus associated with fatal human encephalitis. The New England Journal of Medicine 373, 154. Horie, M., Kobayashi, Y., Suzuki, Y., Tomonaga, K., 2013. Comprehensive analysis of endogenous bornavirus-like elements in eukaryote genomes. Philosophical Transactions of the Royal Society London B Biological Sciences 368, 20120499. Hornig, M., Mervis, R., Hoffman, K., Lipkin, W.I., 2002. Infectious and immune factors in neurodevelopmental damage. Molecular Psychiatry 7, S34. Myers, K.N., Barone, G., Ganesh, A., et al., 2016. The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability. Scientific Reports 6, 35548. Tomonaga, K., Kobayashi, T., Ikuta, K., 2002. Molecular and cellular biology of borna disease virus infection. Microbes and Infection 4, 491.

Bovine Leukemia Virus (Retroviridae) Thomas Joris, Roghaiyeh Safari, Jean-Rock Jacques, and Luc Willems, Cellular and Molecular Epigenetics (GIGA), Liège, Belgium and Molecular Biology (TERRA), Gembloux, Belgium r 2021 Elsevier Ltd. All rights reserved.

Nomenclature AGID Agar Gel Immunodiffusion test BLV Bovine Leukemia Virus CAT1/SLC7A1 Cationic Amino acid Transporter 1/Solute Carrier family 7 member 1 EBL Enzootic Bovine Leukemia ELISA Enzyme-Linked Immunosorbent Assay FPPS Farnesyl Pyrophosphate Synthetase

Glossary Bidirectional transcription The long terminal repeats located at the 50 and 30 ends of the provirus contain promoter sequences that direct bi-directional transcription. Clonal expansion As a retrovirus, BLV replicates upon expression of an integrated provirus, budding of the viral particle and infection of a new cell (the infectious cycle). When integrated into the host chromosome, BLV replication

HTLV-1 Human T-cell Leukemia Virus type 1 ICTV International Committee on Taxonomy of Viruses LTR Long Terminal Repeat PBMCs Peripheral Blood Mononuclear Cells PCR Polymerase Chain Reaction PL Persistent Lymphocytosis PVL Proviral Load VPA Valproic Acid

also occurs by cell mitosis through a mechanism of clonal expansion. MicroRNA Besides genomic and subgenomic mRNAs, BLV also transcribes 10 viral microRNAs that are exported into the plasma of infected animals. Provirus BLV is a retrovirus whose RNA genome is reverse transcribed into DNA, the provirus, that integrates into the host chromosome.

Classification According to the International Committee on Taxonomy of Viruses (ICTV, 2019 release), BLV is classified in the Ortevirales order, Retroviridae family, Orthoretrovirinae subfamily and Deltaretrovirus genus.

Epidemiology and Clinical Features The bovine leukemia virus (BLV) is an exogenous, oncogenic retrovirus that naturally infects B-cells of cattle (Bos taurus), water buffalo (Bubalus bubalis), yak (Bos grunniens) and zebu (Bos taurus indicus) (Meas et al., 2000; Burny et al., 1988). BLV-associated disease was initially observed in 1871 in an enlarged spleen of a cow (Gillet et al., 2007). BLV is the etiological agent of enzootic bovine leukosis (EBL) characterized by lymphoma and lymphosarcoma (extranodular lymphoma in different organs such as eyes, heart, liver, lung, and skin) (Burny et al., 1985). While most are asymptomatic carriers, a minority of infected animals develop EBL several years after infection (Barez et al., 2015). The frequency of tumors and clinical latency depend on herd prevalence: a typical picture is 10% death after three years in a herd having 50% BLV prevalence. In approximately one third of BLV-infected cows, a benign condition called persistent lymphocytosis (PL) is characterized by an excessive accumulation of B-lymphocytes in the peripheral blood (Aida et al., 2013). In PL, the number of B cells can exceed that of T lymphocytes. Animals with PL have a higher risk of EBL although tumor development also occurs without persistent lymphocytosis. BLV infection is associated with immunosuppression that favors opportunistic infections (e.g., mastitis) (Suzuki et al., 2013).

Diagnosis BLV infection initiates a strong humoral and cytotoxic immune response that tightly controls viral replication (Meas et al., 2000; Kabeya et al., 1999; Pyeon et al., 1996). Since assays based on cytotoxic T-cell responses are not robust, diagnosis is primarily based on the presence of anti-BLV antibodies in the plasma or serum. The historical agar gel immunodiffusion test (AGID) is nowadays being replaced by the enzyme-linked immunosorbent assay (ELISA) (LaDronka et al., 2018; Gutiérrez et al., 2009; Portetelle et al., 1991). The viral antigens that elicit the best response belong to the capsid (p24) and envelope (gp51) proteins. Commercially available ELISAs have an excellent performance in terms of specificity and sensibility. The issue with these tests is their inability to discriminate between viral infection and maternally derived anti-BLV antibodies. Approximately 1–4 weeks after infection, the humoral immune response indeed produces anti-p24 and anti-gp51 antibodies that cannot be dissociated from passive immunity acquired through the colostrum

144

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00059-X

Bovine Leukemia Virus (Retroviridae)

145

(Ferrer and Piper, 1981; Kuczewski et al., 2019). This issue is solved by different PCR methods for detecting proviral sequences (e.g., end-point, real-time, nested, filter, CoCoMo and isothermal) (Jimba et al., 2010; Jaworski et al., 2018; Ruggiero et al., 2018).

Prevention and Treatment BLV is a major animal health problem worldwide causing important economic losses (Norby et al., 2016). A series of attempts were developed to reduce prevalence, chiefly by eradication of infected cattle or segregation of BLV-free animals (Rodríguez et al., 2011). The “test and eliminate” strategy requires identification of BLV-positive animals either by genomic or serological methods, removal of positive cases from the herd and slaughtering. Although having been instrumental in regions such as in Europe, this strategy was unsuccessful elsewhere mainly due to excessive costs (EFSA AHAW Panel, 2015). Since efficient transmission correlates with high proviral loads, an alternative is to identify and discard only the virus shedders. Although the impact on genetic and reproductive potential is reduced in the herd, BLV infection persists over longer periods (Ruggiero and Bartlett, 2019). The “test and segregate” approach detects and separates BLV-infected and healthy cattle in different areas. The need of separated operational structures duplicates housing facilities and equipment, thereby excessively increasing the costs. “Test and manage” BLV-infected and healthy animals in the same housing facilities requires permanent surveillance of the herd. This strategy is cost-effective, requires only minimal investment on facilities and does not need replacement of culled BLV-infected cattle (Bartlett et al., 2014). The “Test and manage” approach is nevertheless intensively laborious, requires strict adherence to the rigorous implemented measures and needs long-term commitment to the program. Management includes the single-use of disposable material (needles, syringes and obstetrical sleeves), the disinfection of reusable surgical instruments (dehorning, tattooing, castration or ear-tagging) and elimination of hematophagous insects (Rodríguez et al., 2011). The strategies of testing and slaughtering/segregating/managing could be implemented with a treatment that limits the proviral loads. For example, valproic acid (VPA), an HDAC inhibitor, has been successfully used to reduce the PVL in leukemic sheep (Achachi et al., 2005). Although VPA treatment does not clear infection, reduced PVL correlates with decreased transmission and pathogenesis. BLV transmission can also be prevented by feeding the calves with colostrum from BLV-infected cows (Ferrer and Piper, 1981; Kuczewski et al., 2019). Although anti-BLV antibodies from the colostrum efficiently prevent infection, there is a risk of transmission when plasma titers decrease. Vaccination against BLV has been quite difficult to achieve probably because of its ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is impossible once infection is established, the primary goal was to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. After a series of failures due to the fast decline of protective antibody titers and poor stimulation of cytotoxic response, an efficient and safe vaccine is now available (Gutierrez et al., 2014; Abdala et al., 2019). The concept of this vaccine is to establish a permanent infection with an attenuated strain that impedes wild-type challenge by activating the anti-viral immune response. Since the vaccine strain replicates at very low levels, is not pathogenic and does not transmit to uninfected sentinels or from cow-to-calf. Moreover, the low level of replication also prevents a significant impact on the host immunocompetence, thereby avoiding immunosuppression and opportunistic infections.

Life Cycle The infectious cycle of BLV replication initiates with the interaction of the viral particle with the cell membrane (Fig. 1). The BLV envelope glycoprotein gp51 binds to the cationic amino acid transporter 1 (CAT1)/solute carrier family 7 member 1 (SLC7A1) receptor (Bai et al., 2019). The transmembrane protein gp30 then promotes fusion between the lipid bilayers of the viral and cell membranes (Willems et al., 1995). Upon uncoating of the capsid, the BLV genomic RNA is reverse transcribed into a double stranded DNA provirus that is transported into the nucleus after circularization (Gillet et al., 2007). Integrase-mediated proviral insertion into the host cell genome is random albeit BLV proviral integration significantly favors transcribed regions of the genome (Gillet et al., 2013). Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. The integrated provirus can remain silent or be transcribed under the control of various stimuli (e.g., PKC, calmodulin) (Kerkhofs et al., 1996). Translation of viral proteins, encapsidation of genomic RNA and budding at the cell membrane release a new infectious virion. Besides this infectious cycle, BLV also replicates by mitosis of its host cell (i.e., clonal expansion) (Gillet et al., 2013). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. The mechanism that stimulate preferred mitosis of BLV-infected cells is still incompletely understood but involves oncogenic viral proteins such as Tax and G4 (Willems et al., 1990; Kerkhofs et al., 1998).

Virion Structure The BLV RNA genome is packaged in a capsid surrounded by an external envelope (Fig. 2). The p12 nucleocapsid (NC) protein is a proline-rich polypeptide that is tightly bound to the packaged genomic RNA. The p24 protein is the major constituent of the capsid

146

Bovine Leukemia Virus (Retroviridae)

A

B

Fig. 1 Schematic representation of the two modes of BLV replication. (A) In the infectious cycle, the virion enters the cell by envelope-mediated membrane fusion. After reverse transcription of the genomic RNA, the provirus integrates into the host chromosome. Upon transcription of the provirus and translation, viral proteins and genomic RNA form a virion that buds out of the cell (B) An infected cell harboring an integrated provirus undergoes mitosis. Further proliferation generates a population of cell clones characterized by a single proviral integration site.

Fig. 2 Structure of the BLV virion. Env, Gag and Pro-Pol proteins are indicated by orange, blue and green arrows. Gag ¼ group-specific antigen; Env ¼ envelope; MA ¼ matrix; NC ¼ nucleocapsid; CA ¼ capsid; PRO ¼ protease; RT ¼ reverse transcriptase; IN ¼ integrase; SU ¼ extracellular envelope protein; TM ¼ transmembrane protein.

Bovine Leukemia Virus (Retroviridae)

147

Fig. 3 Schematic representation of an integrated BLV provirus, viral RNAs and proteins. The long terminal repeats (LTR in green) flank the viral genes (in gray). The provirus is integrated into chromatin. Sense and antisense transcripts are colored in blue and purple, respectively. GAG ¼ group-specific antigen; ENV ¼ envelope; miR ¼ viral microRNAs; premiR ¼ precursor microRNA; U3 ¼ unique 30 end; R ¼ redundant region; U5 ¼ unique 50 end; PPT ¼ polypurine tract; PBS ¼ primer binding site; c ¼ packaging Psi sequence; AS1-S ¼ antisense 1 short; AS1-L ¼ antisense 1 long; AS2 ¼ antisense 2; pr ¼ precursor; p ¼ protein; gp ¼ glycoprotein; MA ¼ matrix; NC ¼ nucleocapsid; CA ¼ capsid; PRO ¼ protease; RT ¼ reverse transcriptase; IN ¼ integrase; SU ¼ extracellular envelope protein; TM ¼ transmembrane protein. Numbers are coordinates according to the GenBank AF033818.1 reference genome (1 is the transcription initiation site).

148

Bovine Leukemia Virus (Retroviridae)

(CA) of BLV virions and a major target for the host immune response. The matrix (MA) protein p15, which corresponds to the NH2terminal end of the gag precursor, is a myristylated and phosphorylated polypeptide. MA proteins bind the genomic viral RNA but also interact with the lipid bilayer of the viral membrane. The Gag proteins (MA, CA and NC) are derived from the proteolytic cleavage of the Pr44gag precursor carried out by the viral protease p14 (PRO). The BLV virion also contains two enzymes required to reverse transcribe the RNA genome into proviral cDNA (RT) and for its integration into the chromosome (IN). Finally, the capsid is surrounded by an envelope of cellular and viral origin. The extracellular gp51 (SU) and the transmembrane gp30 (TM) proteins are glycosylated polypeptides that associate through disulfide bonds. TM is embedded through a lipid bilayer acquired upon budding from the cell membrane. Insertion, in an oblique orientation, of the N-terminal fusion peptide of TM into the lipid bilayer of a target cell promotes cell fusion and viral entry. SU is involved in receptor binding and cell fusion. It should be emphasized that, although BLV virions are spontaneously expressed in ex vivo cultures from peripheral blood mononuclear cells (PBMCs), there is no viremia in infected animals, likely due to a very efficient immune response.

Genome Organization and Expression The BLV provirus integrated into chromatin is schematized in Fig. 3. The genome contains the classical gag-pro-pol-env structural and enzymatic genes required to produce the viral particle. As a delta-retrovirus, BLV also harbors 4 genes (Tax, Rex, R3 and G4) involved in different regulatory mechanisms (see the paragraph on pathogenesis). A series of non-coding RNAs are transcribed from a microRNA cluster (miRs) and in an antisense orientation (AS1-S, AS1-L, AS2). The provirus is flanked by two identical long terminal repeats (50 LTR and 30 LTR) that direct initiation and termination of transcription. The U3-R boundary of the 50 LTR identifies the transcription initiation site of the sense RNAs (Fig. 3). Transcription terminates by polyadenylation at the R-U5 junction of the 30 LTR. Sense transcription is initiated by a TATA box (GATAAT) and controlled by a series of enhancers. Among these, three 21 bp-long Tax-responsive elements (TxREs) contain imperfectly conserved cyclic-AMP responsive elements (AGACGTCA; TGACGGCA and TGACCTCA). These enhancers mediate activation of transcription by the viral Tax protein upon interaction with CREB/ATF (Calomme et al., 2004; Adam et al., 1994) (Fig. 4). Other regulators of sense transcription include binding sites for NF-κB, PU.1/Spi-B, glucocorticoid responsive element (GRE), E box and interferon regulatory factor (IRF) (see review (Gillet et al., 2007) for further details). The genomic RNA contains the Psi site (c) required for its packaging inside the virion and the tRNA primer-binding site (PBS) that is involved in initiation of reverse transcription. During reverse transcription, an RNA polypurine tract (PPT) resists digestion by RT and primes plus-strand DNA synthesis. The genomic RNA is capped with 7-methyl guanosine and is spliced into 4 transcripts (env, tax/rex, R3 and G4). The RT and IN proteins are translated from a 145-kDa polypeptide via a frameshift mechanism of the gag-protease precursor (pr66gagprt). Different Gag proteins (MA, CA and NC) are derived from the proteolytic cleavage of the Pr44Gag precursor. This post-translational maturation is carried out by the viral protease PRO (p14) that is encoded by a region located between the gag and the pol genes. p14 is synthesized from a Gag-protease precursor (pr66GagPrt) via a frameshift suppression of the gag termination codon by a lysine-specific tRNA. The sequences coding for the envelope partially overlap in a different frame the 30 end of the pol gene by 51 nucleotides (Fig. 3). A single-spliced RNA encodes the pr72Env precursor that is cleaved into gp51 (SU) and gp30 (TM) by the subtilisin/kesinlike convertase furin. The SU and TM glycosylated proteins associate through disulfide bonds and interact with the cell membrane.

Fig. 4 Regulatory elements of the LTR promoter. Transcription of the 50 -LTR is initiated by RNAPII at position þ 1. TxRE ¼ Tax responsive element; CRE ¼ cyclic-AMP response element; GRE ¼ glucocorticoid responsive element; E-box ¼ enhancer box; NF-κB ¼ nuclear factor κB; TATA ¼ TATA box; IRF ¼ interferon regulatory factor; U3 ¼ unique 30 end; R ¼ redundant region; U5 ¼ unique 50 end. All coordinates are indicated according to the GenBank: AF033818.1 reference genome.

Bovine Leukemia Virus (Retroviridae)

149

A

B

Fig. 5 Sequences of the BLV miRNAs. (A) Structural prediction of the transcribed BLV miRNA cluster, predicted from sequence 6163–6813 by the RNAfold WebServer of the Vienna RNA Websuite. Nucleotide 1 is at position 6163 of the GenBank AF033818.1 reference genome. 5p- and 3parms are highlighted in green and in red, respectively (B) Sequences, coordinates and references of the 10 BLV miRNAs.

150

Bovine Leukemia Virus (Retroviridae)

Among regulatory proteins, Tax is a phosphoprotein that activates directed transcription via CREB/ATF and TxRE elements located in U3 (Willems et al., 1987, 1998) (Fig. 4). Tax is oncogenic in immortalization assays in primary cells and in mouse models (Willems et al., 1990), a property that is shared with G4 (Kerkhofs et al., 1998). Localized in the mitochondria and the nucleus, the G4 protein interacts with farnesyl pyrophosphate synthetase (FPPS) involved in the mevalonate/squalene pathway and prenylation of Ras (Lefebvre et al., 2002). The role of nuclear R3 protein is still unclear but could pertain to regulation of Rex activity. The 18 kDa Rex nuclear phosphoprotein interacts with the RxRE responsive RNA element located between the AATAAA polyadenylation signal and the polyadenylation site (8156–8420) of viral transcripts (Derse, 1988). p18 mediates splicing and is required for the accumulation of viral genomic and env transcripts. Tax and Rex are essential genes while R3 and G4 are dispensable for infectivity (Willems et al., 1994; Florins et al., 2007b). BLV also transcribes two antisense RNAs (AS1 and AS2) initiating at two transcriptional start sites of an active RNAPII promoter located in the 30 LTR (Van Driessche et al., 2016; Durkin et al., 2016). The AS1 RNA is differentially polyadenylated, generating AS1-L (2200 bp) and, more abundantly, AS1-S (600 bp) (Fig. 3) The AS1-L isoform overlaps the microRNA cluster and is therefore cleaved in the cytoplasm by the RISC complex. The antisense transcripts are predominantly located in the nucleus and are predicted to be untranslated. Their possible role as long non-coding RNAs mediating mechanisms such as epigenetics remains to be determined. Finally, five precursor miRNAs are transcribed from a cluster located 30 of the env gene on the BLV genome (Rosewick et al., 2013; Kincaid et al., 2012) (Fig. 3). The pre-miRNAs are directly transcribed from RNA polymerase III (RNAPIII) promoters and are further processed in the cytoplasm into 10 mature miRNAs by Dicer (Fig. 5). The pre-miRNAs are therefore not processed by the Drosha enzyme, but still largely depend on the DUSP11 phosphatase and Dicer (Burke et al., 2016). In contrast to genomic and subgenomic RNAs, miRNAs are abundantly expressed in infected animals (i.e., up to 40% of all detected cellular miRNAs) (Rosewick et al., 2013; Gillet et al., 2016). Mechanistically, the BLV miRNAs target genes involved in proliferation, oncogenesis, cell signaling, apoptosis and immunity (e.g., GZMA, FOS, PPT1, ANXA1, MAP2K1 and PIK3CG) (Gillet et al., 2016). Deletion of the miRNA cluster reduces viral replication in the natural (bovine) and experimental (ovine) models. Ablation of miRNAs mainly affects proliferation and abolishes pathogenesis in BLV-infected sheep (Safari et al, 2020). It thus appears that BLV proviral transcription is characterized by a complex profile of miRNAs, sense and antisense RNAs driven by RNAPII and RNAPIII, respectively.

Pathogenesis Although the mechanisms associated with BLV pathogenesis still need to be further investigated, a series of experimental results generates a hypothetical but consistent model (Safari et al., 2017; Gazon et al., 2018; Florins et al., 2007a). The hypothesis is based on a tight equilibrium between a virus attempting to proliferate and an infected host that develops an efficient immunity to control viral replication. Evidence includes the following: (1) Despite a strong anti-viral immune response, BLV persists indefinitely throughout life; (2) BLV encodes oncogenes promoting cell proliferation and transformation (Kerkhofs et al., 1998; Twizere et al., 2003); (3) Expression of structural (gag, env), enzymatic (pol) and regulatory (tax, rex, R3 and G4) genes is barely detectable in vivo (Lagarias and Radke, 1989); (4) Viral miRNAs expressed abundantly in cells and in plasma are required to maintain high proviral loads (Rosewick et al., 2013; Gillet et al., 2016); (5) Non-coding antisense transcripts are silent to the immune response and may affect epigenetics (Durkin et al., 2016); (6) BLV integrates randomly but favors transcriptionally active sites nearby cancer drivers (Gillet et al., 2013; Rosewick et al., 2017); (7) Forced proliferation of infected cell clones generates replication-associated mutations that are predicted to drive oncogenicity (Rosewick et al., 2017); (8) Persistent infection induces inflammation and immunosuppression (Pyeon et al., 1996; Konnai et al., 2013) (9) The host's genetic background and viral determinants affect proviral loads (de Brogniez et al., 2015; Willems et al., 2000; Hayashi et al., 2017; Tajima and Aida, 2000; Takeshima et al., 2017) (10) Host immunity tightly controls viral replication (Florins et al., 2006; Florins et al., 2009; Florins et al., 2011; Frie et al., 2018) Regular switches between 50 and 30 transcription may allow transient Tax expression and fast silencing of viral expression. Associated with viral miRNAs, this mechanism would allow Tax-driven cell proliferation and synthesis of viral particles in presence of the host immunity. This model thus illustrates the dynamic equilibrium between a virus attempting to proliferate under a tight control exerted by the immune response.

Acknowledgments This work was supported by the “Fonds National de la Recherche Scientifique” (FNRS, grant PDR T.0261.20), the Télévie, “Crédits Sectoriels de Recherche en Sciences de la Santé” of the university of Liège and the Fondation Léon Fredericq (FLF). LW is research director of the FNRS.

Bovine Leukemia Virus (Retroviridae)

151

References Abdala, A., Alvarez, I., Brossel, H., et al., 2019. BLV: Lessons on vaccine development. Retrovirology 16, 1–6. Achachi, A., Florins, A., Gillet, N., et al., 2005. Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo. Proceedings of the National Academy of Sciences of the United States of America 102 (29), 10309–10314. Adam, E., Kerkhofs, P., Mammerickx, M., et al., 1994. Involvement of the cyclic AMP-responsive element binding protein in bovine leukemia virus expression in vivo. Journal of Virology 68 (9), 5845–5853. Aida, Y., Murakami, H., Takahashi, M., Takeshima, S.-N., 2013. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus. Frontiers in Microbiology 4, 328. Bai, L., Sato, H., Kubo, Y., Wada, S., Aida, Y., 2019. CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection. FASEB Journal 33, 14516–14527. Barez, P.Y., de Brogniez, A., Carpentier, A., et al., 2015. Recent advances in BLV research. Viruses 7, 6080–6088. Bartlett, P.C., Sordillo, L.M., Byrem, T.M., et al., 2014. Options for the control of bovine leukemia virus in dairy cattle. Journal of the American Veterinary Medical Association 244, 914–922. Burke, J.M., Kincaid, R.P., Nottingham, R.M., Lambowitz, A.M., Sullivan, C.S., 2016. DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs. Genes Development 30 (18), 2076–2092. Burny, A., Bruck, C., Cleuter, V., et al., 1985. Bovine Leukemia Virus, a versatile agent with various pathogenic effects in various animal species. Cancer Research 45 (suppl), 4578–4583. Burny, A., Cleuter, Y., Kettmann, R., et al., 1988. Bovine leukaemia: Facts and hypotheses derived from the study of an infectious cancer. Veterinary Microbiology 17 (3), 197–218. Calomme, C., Dekoninck, A., Nizet, S., et al., 2004. Overlapping CRE and E box motifs in the enhancer sequences of the bovine leukemia virus 50 long terminal repeat are critical for basal and acetylation-dependent transcriptional activity of the viral promoter: implications for viral latency. Journal of Virology 78 (24), 13848–13864. de Brogniez, A., Bouzar, A.B., Jacques, J.-R., et al., 2015. Mutation of a single envelope N-linked glycosylation site enhances the pathogenicity of bovine leukemia virus. Journal of Virology 89 (17), 8945–8956. Derse, D., 1988. Trans-acting regulation of bovine leukemia virus mRNA processing. Journal of Virology 62 (4), 1115–1119. Durkin, K., Rosewick, N., Artesi, M., et al., 2016. Identification and characterization of novel Bovine Leukemia Virus ( BLV ) antisense transcripts reveals their constitutive expression in leukemic and pre-leukemic clones. bioRxiv. 1–31. EFSA AHAW Panel, 2015. Scientific opinion on enzootic bovine leukosis. European Food Safety Authority. EFSA Journal 13, 4188. Ferrer, J.F., Piper, C.E., 1981. Role of colostrum and milk in the natural transmission of the bovine leukemia virus. Cancer Research 41 (12), 4406–4409. Florins, A., de Brogniez, A., Elemans, M., et al., 2011. Viral expression directs the fate of B cells in bovine leukemia virus-infected sheep. Journal of Virology 86 (1), 621–624. Florins, A., Gillet, N., Asquith, B., et al., 2007a. Cell dynamics and immune response to BLV infection: A unifying model. Frontiers in Bioscience 12, 1520–1531. Florins, A., Gillet, N., Boxus, M., et al., 2007b. Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep. J Virol 81 (18), 10195–10200. Florins, A., Gillet, N., Asquith, B., et al., 2006. Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in BLV-infected sheep. Journal of Virology 80 (24), 11998–12008. Florins, A., Reichert, M., Asquith, B., et al., 2009. Earlier onset of δ-retrovirus-induced leukemia after splenectomy. PLoS One 4 (9), 1–7. Frie, M.C., Droscha, C.J., Greenlick, A.E., Coussens, P.M., 2018. MicroRNAs encoded by bovine leukemia virus (BLV) are associated with reduced expression of B cell transcriptional regulators in dairy cattle naturally infected with BLV. Frontiers in Veterinary Science 4 (January), 1–16. Gazon, H., Chauhan, P., Hamaidia, M., et al., 2018. How does HTLV-1 undergo oncogene-dependent replication despite a strong immune response? Frontiers in Microbiology 8 (January), 1–6. Gillet, N., Florins, A., Boxus, M., et al., 2007. Mechanisms of leukemogenesis induced by bovine leukemia virus: Prospects for novel anti-retroviral therapies in human. Retrovirology 4, 18. Gillet, N.A., Gutierrez, G., Rodriguez, S.M., et al., 2013. Massive depletion of bovine leukemia virus proviral clones located in genomic transcriptionally active sites during primary infection. PLoS Pathogens 9 (10), e1003687. Gillet, N.A., Hamaidia, M., de Brogniez, A., et al., 2016. Bovine leukemia virus small noncoding RNAs are functional elements that regulate replication and contribute to oncogenesis in vivo. PLoS Pathogens 12 (4), e1005588. Gutiérrez, G., Alvarez, I., Fondevila, N., et al., 2009. Detection of bovine leukemia virus specific antibodies using recombinant p24-ELISA. Veterinary Microbiology 137 (3–4), 224–234. Gutierrez, G., Rodríguez, S.M., De Brogniez, A., et al., 2014. Vaccination against δ-retroviruses: The bovine leukemia virus paradigm. Viruses 6 (6), 2416–2427. Hayashi, T., Mekata, H., SekiguchiI, S., et al., 2017. Cattle with the BoLA class II DRB3*0902 allele have significantly lower bovine leukemia proviral loads. The Journal of Veterinary Medical Science 79 (9), 1552–1555. Jaworski, J.P., Pluta, A., Rola-Luszczak, M., et al., 2018. Interlaboratory comparison of six real-time PCR assays for detection of bovine leukemia virus proviral DNA. Journal of Clinical Microbiology 56, 1–11. Jimba, M., Takeshima, S.nosuke, Matoba, K., Endoh, D., Aida, Y., 2010. BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm. Retrovirology 7, 91. Kabeya, H., Ohashi, K., Sugimoto, C., Onuma, M., 1999. Characterization of immune responses caused by bovine leukemia virus envelope peptides in sheep. The Journal of Veterinary Medical Science 61 (5), 475–480. Kerkhofs, P., Adam, E., Droogmans, L., et al., 1996. Cellular pathways involved in the ex vivo expression of bovine leukemia virus. Journal of Virology 70 (4), 2170–2177. Kerkhofs, P., Heremans, H., Burny, A., Kettmann, R., Willems, L., 1998. In vitro and in vivo oncogenic potential of bovine leukemia virus G4 protein. Journal of Virology 72 (3), 2554–2559. Kincaid, R.P., Burke, J.M., Sullivan, C.S., 2012. RNA virus microRNA that mimics a B-cell oncomiR. Proceedings of the National Academy of Sciences of the United States of America 109 (8), 3077–3082. Konnai, S., Suzuki, S., Shirai, T., et al., 2013. Enhanced expression of LAG-3 on lymphocyte subpopulations from persistently lymphocytotic cattle infected with bovine leukemia virus. Comparative Immunology, Microbiology and Infectious Diseases 36 (1), 63–69. Kuczewski, A., Hogeveen, H., Orsel, K., et al., 2019. Economic evaluation of 4 bovine leukemia virus control strategies for Alberta dairy farms. Journal of Dairy Science 102 (3), 2578–2592. LaDronka, R.M., Ainsworth, S., Wilkins, M.J., et al., 2018. Prevalence of bovine leukemia virus antibodies in US dairy cattle. Veterinary Medicine International 2018, 5831278. Lagarias, D.M., Radke, K., 1989. Transcriptional activation of bovine leukemia virus in blood cells from experimentally infected, asymptomatic sheep with latent infections. Journal of Virology 63 (5), 2099–2107. Lefebvre, L., Vanderplasschen, A., Ciminale, V., et al., 2002. Oncoviral bovine leukemia virus G4 and human T-cell leukemia virus type 1 p13II accessory proteins interact with farnesyl pyrophosphate synthetase. Journal of Virology 76 (3), 1400–1414. Meas, S., Ohashi, K., Tum, S., et al., 2000. Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in draught animals in Cambodia. The Journal of Veterinary Medical Science 62 (7), 779–781.

152

Bovine Leukemia Virus (Retroviridae)

Norby, B., Bartlett, P.C., Byrem, T.M., Erskine, R.J., 2016. Effect of infection with bovine leukemia virus on milk production in Michigan dairy cows. Journal of Dairy Science 99, 2043–2052. Portetelle, D., Limbach, K., Burny, A., et al., 1991. Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection. Vaccine 9 (3), 194–200. Pyeon, D., O’Reilly, K.L., Splitter, G.A., 1996. Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus. Journal of Virology 70, 5706–5710. Rodríguez, S.M., Florins, A., Gillet, N., et al., 2011. Preventive and therapeutic strategies for bovine leukemia virus: lessons for HTLV. Viruses 3 (7), 1210–1248. Rosewick, N., Durkin, K., Artesi, M., et al., 2017. Cis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis. Nature Communications 8, 15264. Rosewick, N., Momont, M., Durkin, K., et al., 2013. Deep sequencing reveals abundant noncanonical retroviral microRNAs in B-cell leukemia/lymphoma. Proceedings of the National Academy of Sciences of the United States of America 110, 2306–2311. Ruggiero, V.J., Bartlett, P.C., 2019. Control of bovine leukemia virus in three US dairy herds by culling ELISA-positive cows. Veterinary Medicine International 2019. Ruggiero, V.J., Benitez, O.J., Tsai, Y.L., et al., 2018. On-site detection of bovine leukemia virus by a field-deployable automatic nucleic extraction plus insulated isothermal polymerase chain reaction system. Journal of Virological Methods 259, 116–121. Safari, R., Hamaidia, M., de Brogniez, A., Gillet, N., Willems, L., 2017. Cis-drivers and trans-drivers of bovine leukemia virus oncogenesis. Current Opinion in Virology 26, 15–19. Safari, R., Jacques, J., Brostaux, Y., Willems, L., 2020. Ablation of non-coding RNAs affects bovine leukemia virus B lymphocyte proliferation and abrogates oncogenesis, PLoS Pathogens 16 (5), e1008502. Suzuki, S., Konnai, S., Okagawa, T., et al., 2013. Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle. Microbiology and Immunology 57 (8), 600–604. Tajima, S., Aida, Y., 2000. The region between amino acids 245 and 265 of the bovine leukemia virus (BLV) tax protein restricts transactivation not only via the BLV enhancer but also via other retrovirus enhancers. Journal of Virology 74 (23), 10939–10949. Takeshima, S.N., Sasaki, S., Meripet, P., Sugimoto, Y., Aida, Y., 2017. Single nucleotide polymorphisms in the bovine MHC region of Japanese Black cattle are associated with bovine leukemia virus proviral load. Retrovirology 14, 1–7. Twizere, J.-C., Kruys, V., Lefèbvre, L., et al., 2003. Interaction of retroviral tax oncoproteins with tristetraprolin and regulation of tumor necrosis factor-a expression. Journal of the National Cancer Institute 95 (24), 1846–1859. Van Driessche, B., Rodari, A., Delacourt, N., et al., 2016. Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome. Scientific Reports 6, 31125. Willems, L., Burny, A., Collete, D., et al., 2000. Genetic determinants of bovine leukemia virus pathogenesis. AIDS Research and Human Retroviruses 16 (16), 1787–1795. Willems, L., Gatot, J.S., Mammerickx, M., et al., 1995. The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads. Journal of Virology 69 (7), 4137–4141. Willems, L., Gegonne, A., Chen, G., et al., 1987. The bovine leukemia virus p34 is a transactivator protein. EMBO Journal 6 (11), 3385–3389. Willems, L., Grimonpont, C., Kerkhofs, P., et al., 1998. Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation. Oncogene 16 (17), 2165–2176. Willems, L., Heremans, H., Chen, G., et al., 1990. Cooperation between bovine leukaemia virus transactivator protein and Ha-ras oncogene product in cellular transformation. The EMBO Journal 9 (5), 1577–1581. Willems, L., Kerkhofs, P., Dequiedt, F., et al., 1994. Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames. Proceedings of the National Academy of Sciences of the United States of America 91 (24), 11532–11536.

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae) Paul Becher and Volker Moennig, University of Veterinary Medicine, Hannover, Germany Norbert Tautz, University of Luebeck, Luebeck, Germany r 2021 Elsevier Ltd. All rights reserved.

Classification Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, Border disease virus (BDV) and Classical swine fever virus (previously called Hog cholera virus) belong to the genus Pestivirus within the family Flaviviridae. In addition, a growing number of additional pestiviruses has been identified from various hosts of the order Artiodactyla including pig, cattle, sheep, goat, wild boar, giraffe, Pronghorn antelope, and various other wild ruminant species. More recently, distinct pestivirus sequences have been detected in samples from several rodent and bat species. These viral sequences significantly differ from artiodactylous pestiviruses and form two separate clusters. The recently identified atypical porcine pestivirus (APPV) which causes congenital tremor in newborn piglets is most closely related to pestiviruses from bats. Taken into account the growing number and the expanded host range of genetically highly variable pestivirus species, the Flaviviridae study group of the International Committee on Taxonomy of Viruses (ICTV) has proposed a revision to the taxonomy of the genus Pestivirus. This included the modification of the criteria for pestivirus species demarcation, the creation of seven additional new pestivirus species and naming species in a host-independent manner using the format Pestivirus X. This revision is consistent with the recently revised format used for the genera Hepacivirus and Pegivirus of the Flaviviridae family. BVDV-1, BVDV-2, CSFV, and BDV are termed Pestivirus A, Pestivirus B, Pestivirus C, and Pestivirus D, respectively. The seven additional pestivirus species (with reference viruses or previously used designations provided in parentheses) are Pestivirus E (pronghorn pestivirus), Pestivirus F (Bungowannah virus), Pestivirus G (giraffe pestivirus), Pestivirus H (Hobi-like pestivirus), Pestivirus I (Aydin-like pestivirus), Pestivirus J (rat pestivirus) and Pestivirus K (atypical porcine pestivirus) (Fig. 1). The proposed changes only relate to the nomenclature of virus species names, while virus isolates and established pestiviruses are still be referred to their original names. Pestiviruses identified from pigs (Linda virus), sheep (MG770617, MK618726), sheep and goat (Tunisian sheep pestivirus, not included in Fig. 1) as well as from bats, rodents and whales (not included in Fig. 1) may represent additional species (Fig. 1). Demarcation of pestivirus species is based on sequence relationships and antigenic relationships, and is frequently associated with differences in host range and diseases caused by these viruses. For the bat and rodent pestiviruses, virus isolates are not available and therefore antigenic relationships have not been studied. Phylogenetic analyses of complete and partial genomic sequences revealed that CSFV, BVDV-1, BVDV-2, and BDV can be further segregated into defined genotypes and subgenotypes. The genetic variations identified in individual virus isolates may be implicated in disease control. It has been reported that vaccines and diagnostics that work well against homologous virus strains can be less efficacious for genetically different viruses. Moreover, genetic typing of CSFV and BVDV isolates can improve the understanding of epidemiological relations between outbreaks and may assist in tracing the source of disease outbreaks. CSFV can be divided into at least three major genotypes (1, 2 and 3), each comprising three to seven subgenotypes (1.1–1.7; 2.1–2.3; 3.1–3.4). Recent studies suggested that the genetic diversity of CSFV might be broader than previously reported and proposed up to seven subgenotypes within genotype 2. For assignment of newly identified CSFV isolates to a (sub)genotype, either full-length genomic sequences or partial sequences have to be determined and subsequently compared to the available sequences of other known CSFV isolates. In addition to full-length genomic sequences, complete E2 coding sequences provide a solid basis for phylogenetic analysis and assignment to a defined subgenotype. More than 500 complete E2 coding sequences are available for CSFV. Compared to BVDV-1 and BDV, the genetic variability of CSFV is lower. During long lasting epidemics, only a very limited number of nucleotide changes have been observed. BVDV-1 can be segregated into at least twenty-one subgenotypes (BVDV-1a-1u), while four subgenotypes have been described for BVDV-2. Epidemiological studies have demonstrated that various BVDV subgenotypes predominate in different countries and geographic regions. For BDV at least eight distinct and highly variable genotypes have been reported. Future investigations of BVDV-1, BVDV-2, BDV, and CSFV isolates may result in the identification of additional genotypes and subgenotypes.

Genome Structure and Polyprotein Processing The pestiviral genome is a single-stranded RNA with a length of about 12.3 kb but naturally occurring virus mutants with genomes up to 16.5 kb have been described. The single large open reading frame (ORF) is flanked on both ends by untranslated regions (UTRs). The 50 UTR folds into a complex structure which is critical for RNA replication and the formation of an internal ribosome entry site (IRES) mediating internal initiation of translation. In the 30 UTR, which also contains structural RNA elements of functional importance for viral RNA replication, highly conserved binding sites for the small cellular RNAs miR-17 and let-7 have been identified. Binding of these RNAs increases genome stability and stimulates translation. The polyprotein consists of approximately 3900 amino acids and is co- and posttranslationally processed by host- and virusencoded proteases into 12 mature proteins. However, also cleavage intermediates play a crucial role in the pestiviral life cycle. The organization of the polyprotein is NH2–Npro/C/Erns/E1/E2/p7/NS2/NS3/NS4A/NS4B/NS5A/NS5B–COOH (Fig. 2). The first protein encoded in the ORF is the N-terminal protease (Npro) which cleaves off itself from the remainder of the polyprotein

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21233-8

153

154

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

BVDV-1 KP313732 BVDV-1 AF526381 BVDV-1 U63479 Pestivirus BVDV-1 AB078950 84 BVDV-1 M31182 BVDV-1 M96751 84 BVDV-2 KJ000672 BVDV-2 AB567658 100 BVDV-2 GQ888686 Pestivirus BVDV-2 AF002227 82 BVDV-2 KT832818 74 BVDV-2 HQ258810 100 Giraffe AF144617 Pestivirus G PG-2 KJ660072 87 Hobi-like KC788748 Hobi-like AB871953 Pestivirus H 100 Hobi-like FJ040215 BDV U70263 BDV AF037405 BDV KF925348 Pestivirus D BDV KF918753 BDV GU270877 BDV AF144618 100

81

96 95

A

B

100

93

98

100

0.1

Ovine MG770617* Ovine MK618726* 99 CSFV AY646427 99 99 CSFV AF091661 100 CSFV X87939 Pestivirus C CSFV J04358 87 CSFV GQ923951 95 CSFV LC425854 Aydin JX428945 Pestivirus I 97 Burdur KM408491 Pestivirus E Pronghorn AY781152 Bungowannah EF100713 Pestivirus F Linda KY436034* Pestivirus J Rat KJ950914 100 Rodent KY370100* Bat MH282908* APPV KR011347 100 APPV KX950761 100 APPV MF167291 Pestivirus K APPV KU194229 72 APPV KU041639

Fig. 1 Phylogenetic relationship of pestiviruses. Phylogenetic trees were constructed using MEGA 6 and based upon distances between amino acid sequences for amino acid positions 3312–3899 (NS5B region) by maximum likelihood using a JTT þ G model as recommended by the Flaviviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV). Accession numbers of the individual sequences allocated to the eleven established pestivirus species (Pestivirus A-K) or representing tentative pestivirus species (Ovine MG770617 and Ovine MK618726, Linda KY436034, Bat MH282908 and Rodent KY370100; highlighted by astrisks) are indicated. Up to 6 sequences were used for each species. Numbers indicate branches supported by 470% of bootstrap replicates. The authors thank Gökce Nur Cagatay, University of Veterinary Medicine Hannover, Germany, for providing the phylogenetic tree.

thereby generating the N terminus of the core protein (C). The cleavages required for the release of the structural proteins C, Erns, E1 and E2 as well as p7 are mediated by the cellular signal peptidase residing in the ER. The C terminus of C is further processed by cellular signal peptide peptidase. NS2 acts as an autoprotease generating its own C terminus and thereby the N terminus of NS3. NS3 is multifunctional with proven protease, helicase and NTPase activities. NS3 is believed to generate autocatalytically its own C terminus. For full protease activity NS3 forms a complex with its cofactor NS4A which intercalates with one beta-strand into the protease domain of NS3. The NS3/4A complex catalyzes all cleavages further downstream in the polyprotein.

Virion Structure The virions of pestiviruses are enveloped and have a size of about 50 nm (Fig. 3). Early immuno-EM studies using monoclonal antibodies against Erns and E2 have shown for CSFV and BVDV the presence of both proteins at the surface of the virion. Already in

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

155

Fig. 2 Pestivirus genome organization. The genomic RNA contains one open reading frame flanked by 50 and 30 nontranslated regions (NTR). The proteinases involved in polyprotein processing are indicated. H, helicase; RdRp, RNA-dependent RNA polymerase. Modified from Becher, P., Thiel, H.J., 2011. Pestivirus. In: Tidona, C., Darai, G. (Eds.), The Springer Index of Viruses. Springer Science þ Business Media, LLC. doi:10.1007/978-0-387-95919-1.

Fig. 3 Extracellular virions on MDBK cells infected with giraffe pestivirus (Pestivirus G), 15 h p.i., bar: 100 nm. Reproduced with permission from Schmeiser, S., Mast, J., Thiel, H.J., König, M., 2014. Morphogenesis of pestiviruses: New insights from ultrastructural studies of strain Giraffe-1. Journal of Virology 88, 2717–2724.

those studies Erns was preferentially detected. A more recent, detailed structural and biochemical analysis of purified BVDV virions revealed a generally low envelope protein content with Erns being more abundant than the other envelope proteins. The averaged image of the virions did not show glycoprotein spikes even after acidification of the sample, indicating that these proteins may not form a regular structure at the virion. Alternatively, those structures are not visualized due to their low abundancy. The envelope proteins detected in the virions were covalent Erns homodimers as well as E1/E2 heterodimers but hardly any monomers or E2 dimers. Even though the virions bud from the endoplasmatic reticulum (ER), their lipid composition differed from the one of the ER membrane indicating lipid-sorting during virion morphogenesis. In contrast to hepatitis C virus (HCV), the virions of pestiviruses showed no apolipoprotein association. While E2 is the receptor binding protein and most neutralizing antibodies are directed against this component of the virion, a limited degree of neutralization was also observed for antibodies against Erns. Structural information for E2 of BVDV has been obtained in two studies. These structures revealed no similarity of E2 to the class II fusion proteins of the alphaviruses or members of the genus Flavivirus. Therefore, it remains to be finally determined whether E2 or E1, the least well investigated glycoprotein of pestiviruses, act as fusion protein in the process of cell entry. A duplication of the core protein as well as fusions of core with YFP (yellow fluorescent protein) did not lead to abrogation of virion morphogenesis. Surprisingly, even the deletion of C could be partially compensated by single mutations located in the C-terminal region of NS3. Taken together, the enormous flexibility of C with respect to formation of infectious virions may indicate that C does not form a rigid icosahedral structure in the virion but acts as an RNA binding protein with a potential RNA chaperone activity. This assumption is further corroborated by the fact that pestiviral genomes with massively increased genome size can still be efficiently packaged into infectious viral particles. For a final clarification of the architecture of the pestiviral capsids, a high resolution structure will be required. Erns is unique to pestiviruses and its most unusual feature is its RNase activity. Structural analysis further verified the postulated similarity to T2 RNases. The RNase activity is neither required for the infectivity of the virion nor for viral replication in cell culture. However, inactivation of the RNase led to an attenuation of BVDV in its natural host. Besides being part of the infectious virion, Erns is also present in significant amounts in the serum of infected animals. Detailed analyses have shown that an unusual in plane membrane anchor is critical for the shedding of Erns from infected cells. This alpha-helix also seems to play a distinct role in the formation of infectious viral particles. Furthermore, Erns was shown to act as an antagonist of the innate host response to infection. In cell culture experiments Erns was able to suppress the induction of interferon-b and interferon-induced protein expression after stimulation by extracellular dsRNA or ssRNA. The RNase activity of Erns is required for its immunosuppressive effect. Secreted Erns can bind to cells, be taken up by Clathrin-dependent endocytosis and interfere with IFN induction by extracellular dsRNA. Viral RNA shedded from pestivirus infected cells, most likely via exosomes, was shown to induce IFN secretion by plasmacytoid

156

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

dendritic cells (PDCs). This stimulation of PDCs could be antagonized by Erns via an RNase-dependent mechanism. For the in vivo function homodimerisation of Erns via a disulfide bridge seems to be of importance since a strong positive selection for the cysteine critical for dimerization has been observed. The cleavage of ss or dsRNA by Erns does, however, not depend on its dimerization.

Pestiviral Nonstructural Proteins and the Regulation of RNA Replication and Virion Morphogenesis Npro Npro is the only viral protein dispensable for RNA replication and virion morphogenesis. However, it is one of the major repressors of Interferon-1 (IFN-1) induction in pestivirus infected cells. Recently the crystal structures for Npro of a HoBi-like BVDV-3 strain and of CSFV strain Alfort 187 were reported. These studies did finally prove that the protease activity is based on a catalytic diad composed of His49 and Cys69. Moreover, the structural information depicted that the C terminus of Npro is locked into the catalytic site after autocleavage, thereby disabling any trans-cleavage activity of this enzyme. In line with these findings, the protease activity of Npro is not required for the suppression of IFN-1 response. Npro interacts directly with interferon regulatory factor 3 (IRF3), one of the major transcription factors responsible for the transcription of IFN-a/b mRNA. For this interaction a structurally important TRASH motif coordinating a Zn ion, is essential. Npro/IRF3 interactions finally lead to an ubiquitin-dependent proteasomal degradation of IRF3 which strongly interferes with the induction of an IFN-a/b response in pestivirus infected cells.

p7 The small hydrophobic protein p7 in part remains uncleaved at the C terminus of E2 while most p7 gets released by signal peptidase cleavage. p7 is so far not detected in virions and thus classified as nonstructural protein. The E2-p7 precursor is not essential for the viral life cycle, at least in cell culture. Due to its sequence characteristics a viroporin function was postulated for p7 and finally demonstrated. Together with results obtained by studying p7 of HCV the ion channel activity of p7 may be required to protect immature intracellular virions from inactivation by acidification. In addition to its viroporin function, p7 may be involved in critical protein/protein interactions during virion morphogenesis as observed in the HCV system.

NS2 NS2 has a length of about 450 amino acids and thus about twice the size of NS2 of HCV. NS2 is composed of a hydrophobic N-terminal half anchoring the protein to membranes and a C-terminal protease domain with a putative structural Zn-binding site residing in the cytoplasm. However, so far neither the membrane topology nor the structure of the protein has been clarified. The activation of the cysteine protease domain requires an interaction with the cellular chaperone DNAJC14 to become active thereby releasing itself from NS2-3. The finding that NS2 becomes only active for cleavage in trans after C-terminal truncation suggests that the C terminus of the protein remains after autocleavage in the active site rendering the cleavage product enzymatically inactive. Actually, the crystal structure of the HCV NS2 autoprotease revealed such an autoblockade, which is also described above for Npro. At the moment no function of the released NS2 has been identified. However, in the context of NS2-3 the NS2 part plays an essential role in virion morphogenesis (see below).

NS3 NS3 is an essential component of the viral replicase which can't be functionally complemented by uncleaved NS2-3. It is a multifunctional protein with an N-terminal serine protease domain and a helicase/NTPase domain connected by a flexible linker. The crystal structure of a single chain NS3-4A protease of CSFV verified that, analogous to findings from the HCV system, NS4A intercalates with its central peptide into the beta-barrel domain of the NS3 protease. Mutations based on the closed conformation structure of CSFV NS3/4A revealed a critical role for the interaction between helicase and protease domain in RNA replication. Only in complex with NS4A the NS3 protease gains its full protease activity and can process e.g., the NS5A/B site. Interestingly, for NS3/4A a surface interaction between NS3 and the part of NS4A located downstream of the central peptide could be visualized in the crystal structure. Mutagenesis studies demonstrated that inactivation of protease, helicase or NTPase function of NS3 interferes with viral RNA replication. For the most C-terminal part of NS3, namely helicase subdomain 3 a surprising compensatory role in CSFV variants with a core deletion was described. Single amino acid exchanges were able to partially rescue the core deletion variants with respect to virion morphogenesis. However, those viruses were attenuated in their natural host. These findings raise the question whether this domain may also be involved in virion morphogenesis in wild type pestiviruses, especially since in this virus system all nonstructural proteins, besides Npro, are required for this process. In this context it is remarkable that the members of the genus Pegivirus do not encode a core protein, but still produce infectious progeny. Moreover, also for other members of the Flaviviridae family mutations located in the helicase were able to rescue virion morphogenesis deficiencies.

NS2-3 Cleavage of NS2-3 is mediated by the autoprotease in NS2. Since NS3 but not NS2-3 is an essential component of the viral replicase the release of NS3 is of regulatory importance for viral RNA replication efficiency. The NS2 autoprotease depends in its activity on the stable interaction with DNAJC14. This chaperone is available only in low amounts in part due to the existence of an upstream ORF in the mRNA. Accordingly, NS2-3 produced at early times after infection will form a complex with DNAJC14 leading to activation of the NS2 protease and NS3 release. However, when translation of the viral polyproteins proceeds, NS2-3

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

157

translated at later time points will not be able to recruit DNAJC14 from the depleted intracellular pool and therefore will not undergo cleavage. Since NS3 is an essential component of the viral replicase this mechanism leads to a temporal downregulation of viral RNA replication which has been shown to be required for the noncp biotype of pestiviruses which is in turn a prerequisite for viral persistence in the animal. A shRNA-mediated knock down of the DNAJC14 mRNA in MDBK cells severely reduced the titers of BVDV-1. However, with this approach no conclusive data were obtained for other pestivirus species. By generating bovine and porcine DNAJC14 knock-out cells it was finally possible to prove that the replication of 6 distinct noncp pestivirus species, namely species A-D, F and G strictly depends on DNAJC14. Furthermore, this study allowed the conclusion that all cytopathogenic pestiviruses which are often products of RNA recombination replicate independent of DNAJC14. Those viruses are characterized by an overexpression of NS3 which is correlated with upregulation of viral RNA synthesis and cytopathogenicity. NS2-3 is a cleavage intermediate in polyprotein processing. A product-precursor relationship between NS3 and NS2-3 was observed for the noncp CSFV strain Alfort Tübingen. Incomplete cleavage results from DNAJC14 deprivation in the infected cells (see above). In all cells infected with noncp pestiviruses significant amounts of uncleaved NS2-3 are detected. This fact is in line with the observation that uncleaved NS2-3 is essential for the process of virion morphogenesis. This function of NS2-3 became apparent when ubiquitin was inserted between NS2 und NS3 in the context of an infectious cDNA clone of BVDV-1 strain NADL. In such a polyprotein cellular ubiquitin specific C-terminal hydrolases process the C terminus of ubiquitin resulting in complete cleavage of NS2-3 which correlated with a defect in virion morphogenesis. An analogous result was obtained for a virus mutant with an IRES insertion between the sequences coding for NS2 and NS3. These findings could be reproduced for CSFV. The latter study revealed that virion morphogenesis of these mutants could be rescued by authentic NS2-3/4A complexes supplied in trans, in which, however, none of the enzymatic activities of NS2 or NS3 was required. When a cell culture system for HCV became available similar studies led to the surprising result that virion morphogenesis of HCV does not depend on uncleaved NS2-3. This finding inspired research on pestiviral gain of function mutants capable of virion production in the absence of uncleaved NS2-3 which mimics the situation in the HCV system. These studies led to the identification of two gain of function mutations, one in NS2 and one in NS3 which were able to compensate the loss of virion morphogenesis due to an ubiquitin-insertion between NS2 and NS3 in BVDV. These mutants and the structural information obtained by the crystal structure of a NS3-4A single chain protease were used to address the question how NS3/4A complexes are functionalized for either RNA replication of virion morphogenesis. Reverse genetics and biochemical studies revealed that a surface interaction between NS3 and the C-terminal part of NS4A (kink region) is decisive whether a NS3/4A complex functions as a basic building block for viral replicase assembly or in the pathway of virion morphogenesis. Further studies are needed to understand those pathways in more detail.

NS4A NS4A is a small protein which acts in the NS3/4A serine protease complex as protease cofactor. The central domain as well as the kink domain are required for the stimulation of the NS3 protease activity. The N-terminal transmembrane domain of NS4A serves as membrane anchor for the NS3/4A complex. Trans-complementation studies demonstrated that NS4A is also involved in virion morphogenesis. Surface interactions between NS4A and NS3 further functionalize the protein (see above).

NS4B NS4B is a hydrophobic protein of about 35 kDa which is essential for RNA replication. However, structure and function of NS4B are largely unknown. Recently, a unique ER-like localization was described for NS4B of CSFV. In the N-terminal region of NS4B an amphipathic a-helix structure was determined which serves as membrane anchor. Mutations in this protein region could be correlated with RNA replication efficiency and virulence of CSFV. For mutations in a sequence motif indicative of an NTPase no conclusive in vivo data were obtained with respect to CSFV replication and virulence.

NS5A NS5A is a phosphoprotein with a size of about 58 kDa. It is anchored to membranes via an N-terminal amphipathic a-helix. A three domain structure with intervening low complexity sequences (LCS) was postulated for NS5A of BVDV. Domain I is essential for RNA replication and contains a structural Zn-binding motif. The downstream LCS I and domain II were more tolerant to deletions/insertions leading to the establishment of a reporter replicon with a mCherry-insertion in this part of the protein. Using those replicons BVDV NS5A was found to localize to lipid droplets, an analogy to the HCV system. NS5A is so far the only replicase component that can be complemented in trans. For NS5A a direct interaction with and a regulatory effect on NS5B has been described and relevant amino acids have been identified. Interestingly, those residues were found to be conserved between pestiviruses and HCV.

NS5B NS5B has RNA-dependent RNA polymerase (RdRp) activity. However, for replication of the viral RNA in vivo at least viral proteins NS3 to NS5B are required. The RdRp activity of NS5B can be stimulated by GTP and is capable of starting de novo RNA synthesis. Structural information has been obtained for NS5B of BVDV and more recently also for CSFV. The crystalized CSFV NS5B structure included a novel N-terminal domain. NS5B of BVDV has also been shown to be involved in virion morphogenesis.

158

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

Life Cycle Cell culture studies have shown that pestiviruses attach via Erns to heparan sulfate and use pH-triggered clathrin-mediated endocytosis for cell entry. Since pestiviruses are acid resistant, an additional activation step was postulated. CD46 was identified as receptor for BVDV-1 with E2 as viral interaction partner. However, expression of bovine CD46 did not confer susceptibility to non-susceptible cell lines indicating the existence of critical co-receptors. Remarkably, direct cell to cell transmission was described for BVDV even in the presence of neutralizing antibodies directed against E2. CD46 is dispensable for this process, which still employs clathrin-mediated endocytosis. Cell to cell spread may also be involved in the transmission of noncp pestiviruses over the placenta, which is required for establishing viral persistence in the fetus. For CSFV a Caveolar-dependent uptake into Alveolar macrophages has been reported involving Rab5, Rab7, and Rab11. Thus different entry pathways may be used depending on the cell type. Replication and virion formation are believed to take place at ER-derived membranes. Co-localization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections while NS5A was found by in vivo imaging at lipid droplets which also reside in close proximity to the ER. The efficiency of RNA replication is regulated via the extent of NS2-3 cleavage (see above) and correlates with the two biotypes observed in cell culture. While the noncp biotype is capable of persistence, the cp biotype produces higher amounts of intracellular RNA which correlates with viral cytopathogenicity, rendering this biotype incompetent for establishing persistent infections. Intracellular virions were found during exocytosis in transport vesicles and the Golgi apparatus. Significant titers of infectious particles are released at about 12 h post infection. The detection of core and double-stranded RNA in multi vesicular bodies may be an indication for an involvement of the endosomal sorting complexes required for transport (ESCRT) machinery in virion budding and secretion.

Epidemiology Classical Swine Fever The natural hosts of CSFV are domestic pigs and wild boar (Sus scrofa ferus). In endemic situations CSFV can be transmitted between domestic pigs and wild boar and vice versa. Other members of the Suidae family like common warthogs (Phacochoerus africanus) and bushpigs (Potamochoerus larvatus) are also susceptible to infection with CSFV. For a long time, it was commonly accepted that cattle can be infected with CSFV only under experimental conditions, while a recent report suggested the occurrence of natural CSFV infections of cattle in China. The significance of CSFV infection in cattle and, possibly, other ruminants remains to be elucidated. CSF is still endemic or sporadically occurs in large parts of the world. According to recent official reports to the OIE (since 2014), CSF is still present in China, Russia, South-East Asia, the Caribbean as well as several countries of South-America. While the African continent has no official OIE status for CSF, recent reports suggest the presence of the disease in at least some regions (e.g., Madagascar). A complete list of currently CSF-free countries is published by the OIE. Other regions of the world including Australia, New Zealand, United States of America, and Canada have been CSF-free for at least forty years. In the European Union (EU) there were only a few sporadic outbreaks of CSF in the Eastern parts of the EU during the past ten years. The last CSF outbreaks in the EU occurred in Lithuania (2009, 2011) and in Latvia (2012–2015). A detailed knowledge of the different routes of transmission is of particular importance to understand the epidemiology of CSF and to implement suitable and efficient control measures for eradication of the disease, surveillance and maintenance of a CSF-free status. CSFV can be transmitted by horizontal and vertical routes. Infected pigs shed high titers of infectious virus via saliva, lacrimal secretions, feces, urine, and semen. Meat, blood and other products of infected pigs can also contain high viral loads. After introduction into a pig farm CSFV spreads by direct contact. These horizontal infections mainly occur through the oronasal route. Spread of CSFV within a herd or within a wild boar population progresses rather slowly. Consequently, the disease may remain unrecognized for long time periods after introduction of CSFV into a holding. The occurrence of less virulent CSFV strains during the past decades is another important factor contributing to longer high risk periods. In addition to direct contact between infected and susceptible animals, CSFV can be efficiently transmitted via contaminated materials (e.g., cloth, shoes, vehicles, agricultural and veterinary equipment) and contaminated food and waste. While swill feeding is prohibited in most countries with industrial pig production, it still represents a common practice for feeding of backyard pigs in large parts of the world. The (illegal) import of virus-contaminated food represents a major threat for introduction of the disease in CSF-free countries. The recent re-occurrence of CSFV in Japan (2018) after 26 years of absence provides an illustrative example how easy CSFV can be introduced even in a country with high biosecurity measures and subsequently can spread among domestic pigs and wild boar populations. Vertical transmission of CSFV from pregnant sows to fetuses can occur throughout all stages of gestation and can result in the birth of persistently infected offspring. These persistently infected pigs shed the virus continuously or intermittently for the rest of their life and represent a permanent source for transmission of CSFV to susceptible animals of the same herd or to other pig holdings. The occurrence of persistently infected offspring is also known for wild boar and together with the decreased virulence of CSFV observed during the past decades may be implicated in the establishment of endemic situations in wild boar populations representing a constant risk of virus transmission to domestic pigs.

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

159

Bovine Viral Diarrhea and Border Disease The host range of BVDV-1, BVDV-2, and BDV comprises cattle, sheep, goats, domestic pigs, wild boar as well as many other animal species of the order Artiodactyla. BVDV-1 and BVDV-2 mainly infect cattle, while BDV has been detected most frequently in sheep. In some countries infections of sheep and goat with BVDV-1 or BVDV-2 are as frequent as infections with BDV. All these ruminant pestiviruses can cause Border Disease in sheep. Infections of cattle with BDV represent very rare events. Transmission of ruminant pestiviruses, in particular BDV, to pigs can interfere with CSF diagnosis, mainly due to the induction of cross-reacting antibodies. Bovine viral diarrhea is endemic and widely distributed in all parts of the world. In contrast to CSF, only a few countries in Europe have implemented effective BVD control and eradication programmes resulting in a BVD-free status (Sweden, Norway, Finland, Denmark) or in a significantly reduced prevalence of BVDV in cattle (Switzerland, Germany, parts of Austria and Northern Italy). As described above for CSFV, ruminant pestiviruses like BVDV-1, BVDV-2 and BDV can be transmitted horizontally and vertically. Horizontal infections can occur via direct and indirect contact. The viruses are excreted via nasal and oral fluids, lacrima secretions, urine, feces, and semen. The main route of infection is oronasally. Vertical transmission of BVDV-1, BVDV-2, BDV, and other ruminant pestiviruses from pregnant cattle (days 40–125 of gestation) and sheep (days 16–80 of gestation) to the fetuses can result in a specific acquired immunotolerance against the infecting virus leading to persistent infection of the offspring. These persistently infected (PI) animals are unable to mount an effective humoral and cellular immune response against the persisting virus. PI animals either develop severe disease (see below) or can survive without apparent clinical signs of disease for many years. They shed constantly large amounts of viruses until death and represent the main source of transmission of the virus to susceptible animals within a herd. Accordingly, these PI animals are significantly implicated in the spread of these viruses to other herds, other susceptible hosts, and to other countries.

Clinical Features and Pathogenesis Classical Swine Fever Infection with CSFV can result in acute, chronic, and late onset forms of CSF, which are characterized by a wide range of clinical symptoms. All forms of CSFV can be observed in both, domestic pigs and wild boar. CSFV infection usually results in fever, thrombocytopenia, leucopenia, and immunosuppression favoring secondary infections. The severity of disease mainly depends on age and immune status of the host animal and virulence of the virus. The disease is more severe and often fatal in young animals, while older breeding animals usually show milder symptoms. Highly, moderately and low virulent viruses can be distinguished. The incubation period of CSF in individual animals is three to ten days. After introduction of low and moderately virulent CSFV in large holdings the disease may remain unrecognized for several weeks. Slow transmission from animal to animal, absence of severe disease and lack of awareness represent significant factors implicated in such high risk periods and can contribute to unrecognized spread of CSF to other farms.

Acute CSF After infection with moderately and highly virulent CSFV strains piglets and young animals usually develop high fever (4401C) and various clinical signs. In the initial stage of the disease animals may show anorexia, lethargy, conjunctivitis, respiratory and gastrointestinal symptoms. In addition, incoordination of movement, weakness of hind legs, convulsions and other neurological symptoms can be frequently observed. In typical cases of CSF, hemorrhages of the skin complete the clinical picture of the disease. Hemorrhages can be observed during the second week and later stages after infection and most frequently occur on the ears, abdomen, tail, and distal parts of the limbs. In piglets, fatality is usually high and can be up to 100%. In fattening and breeding pigs clinical disease is less severe and less specific. Such older animals can mount an effective immune response resulting in overcoming the virus and recovery from the disease.

Chronic CSF A minority of infected animals is not able to develop an effective immune response and remain chronically infected. Such chronically infected animals constantly shed infectious virus. While virus-specific antibodies are produced by these animals, they fail to eliminate the virus. The animals show symptoms similar to those observed in acute CSF, but the disease in its initial stage is milder. Later, the disease is characterized by rather unspecific clinical signs including chronic enteritis, wasting, and intermitting fever. Secondary infections with other pathogens frequently occur and can contribute to the clinical signs of the disease. Chronic CSF is always fatal and the animals usually die after two to four months of infection.

Late Onset CSF As a common feature of pestiviruses, CSFV is able to cross the placenta and infect the fetus. The consequences of diaplacental infections mainly depend on the time of gestation and on viral virulence. Infections during early pregnancy result in abortion, stillbirth, malformations and mummification. Intrauterine infections between days 50–70 of pregnancy with low and moderately virulent CSFV strains which do not kill the pregnant sows can result in the birth of persistently infected offspring and late onset CSF. The persistently infected animals are characterized by an acquired immune tolerance against the infecting virus and shed high

160

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

amounts of infectious CSFV for the rest of their life. Animals can appear clinically healthy for longer time periods before they develop a chronic disease condition. Infected animals show poor growth, chronic wasting or occasionally congenital tremor and usually die within the first year of life. Late onset CSF is always fatal. Moreover, it has been reported that early post-natal infections can also result in CSFV persistence.

Bovine Viral Diarrhea and Mucosal Disease BVDV infection can result in a wide range of clinical consequences. Acute infections of immunocompetent non-pregnant animals usually lead to no or only mild enteric or respiratory disease and frequently remain unnoticed. However, a minority of highly virulent BVDV-1 and BVDV-2 strains can cause a hemorrhagic syndrome with high mortality. BVDV infections of pregnant animals result in transmission to the fetus and a number of severe consequences including embryonic death, abortion, stillbirth as well as malformations and persistent infection of the offspring (see below). Accordingly, the outcome of BVDV infections in cattle is mainly determined by the route of infection (horizontal versus vertical), the immunological status of the host animal, and the virulence of the virus.

Acute BVDV infection In immunocompetent cattle, acute BVDV infections result in a transient viremia accompanied by fever, transient leukopenia, lymphopenia and immunosuppression. Viremia starts at 3 days after infection and usually lasts up to two weeks until a specific immunity leads to elimination of the virus. In rare cases viremia can last up to three weeks. Clinical presentations of acute BVDV infections can include mild diarrhea or respiratory disease, but in many cases such infections remain unnoticed. BVDV induced immunosuppression decreases the capacity of the immune system to control other viral and bacterial pathogens. Consequently, infections with other infectious agents can result in more severe disease of the respiratory or gastrointestinal tract. In rare cases, highly virulent BVDV strains (mainly BVDV-2) can cause severe thrombocytopenia with hemorrhages and acute fatal disease. Field BVDV isolates can be categorized into noncp and cp biotypes according to their effects in cell culture. In contrast to cp BVDV, noncp BVDV isolates interfere with the host innate immune response by inhibition of interferon type 1 mediated antiviral defense. Furthermore, it has been reported that cp BVDV strains are faster and more efficiently eliminated by the host compared to noncp BVDV strains. Consequently, cytopathogenicity of BVDV is not associated with increased virulence in acutely infected immunocompetent animals.

Fetal infection with BVDV Horizontal transmission of BVDV from pregnant cattle to the fetus can result in different severe consequences depending on the age of the fetus at the time point of infection. During the first weeks of pregnancy, prior to the development of cotyledons, BVDV cannot infect the embryo as the virus is not able to penetrate the zona pellucida. After day 30 of gestation infection of the embryo can result in embryonic death which may represent an important cause for reduced conception rates of dams. Fetal death is most frequently observed after infection of the dam during early gestation, but can occur at all stages of gestation. Another important consequence after infection of the fetus with noncp BVDV between 40 and 125 days of gestation is the establishment of persistent infection of the fetus resulting in the birth of persistently infected (PI) calves. After infection of the dam between 80 and 180 days of gestation, the virus is transmitted to the fetus during the period of organogenesis and can cause cerebellar hypoplasia, ocular degeneration, brachygnathism, and skeletal malformations. After infection during the last trimester of gestation the fetus can mount an effective immune response including neutralizing antibodies capable of elimination of the virus. Such infections usually do not cause disease and the respective animals are apparently healthy at birth.

Persistent BVDV infection Infection of the fetus with noncp BVDV at a stage prior to immunocompetence of the fetus can result in a highly specific immunotolerance against the infecting virus and the birth of PI calves. As a consequence, persistently infected animals are not able to produce virus-specific antibodies and to eliminate the virus. The ability of noncp BVDV to inhibit the induction of type 1 interferon in the fetus is a crucial prerequisite for the establishment of lifelong persistent infection. PI animals are efficient virus producers, shed large amounts of viruses in all secretions and excretions and thus are significantly implicated in the spread of BVDV. PI animals can either appear clinically healthy or more frequently present as weak calves with various clinical signs due to increased susceptibility to secondary infections with other infectious agents. Another consequence is the onset of mucosal disease.

Mucosal disease Only persistently infected animals may develop mucosal disease. Affected animals show acute severe diarrhea, dehydration, as well as erosion and ulceration of the mucosa of the oral cavity, esophagus, and gastrointestinal tract. Mucosal disease frequently occurs within the first two years of life and is always fatal. Affected animals die within a few days or a few weeks after onset of disease. In mucosal disease affected animals, a cp BVDV variant is always present in addition to the persisting noncp BVDV. Accordingly, the emergence of cp BVDV in animals persistently infected with noncp BVDV is crucially implicated in the pathogenesis of mucosal disease. In most cases the cp viruses emerge in the PI animal by RNA recombination including deletions and duplications of viral sequences as well as insertions of cellular RNA sequences. In rare cases point mutations in the NS2 protein coding region can result

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

161

in the emergence of cp BVDV. Alternatively, superinfection with an antigenically highly similar cp BVDV strain can directly cause mucosal disease within a few weeks after superinfection. A so called late onset mucosal disease results from RNA recombination between a superinfecting antigenically less similar cp BVDV strain with the persisting noncp BVDV strain and usually occurs about 3–4 months after infection with a cp BVDV field strain or after vaccination with a live attenuated cp BVDV vaccine.

Border Disease Border disease (BD), also known as hairy shaker disease, was first described in the late 1950s. Again, intrauterine infections of pregnant sheep and goat are by far more important than acute infections of immunocompetent animals. Intrauterine infection during early pregnancy can cause fetal death, mummification, abortion, and stillbirth. Intrauterine infections during the 17th and 80th day of gestation can result in the birth of lambs with tremor, ataxia, brain malformations, hairy fleece, and poor growth. Alternatively, apparently healthy lambs can be born. Both clinically affected and apparently healthy lambs can be persistently infected. As described above for CSF and BVD, persistently infected animals constantly shed large amounts of infectious virus and therefore play an important role in virus transmission. In rare cases, PI lambs which recovered from disease can develop a syndrome similar to mucosal disease. Infections of immunocompetent sheep and goat mostly lead to either no or mild transient clinical signs.

Prevention and Control In order to prevent economic losses caused by pestiviral infections and to prevent spread of the disease in domestic or wild animals control measures are often applied. Vaccination, eradication or a combination of both are common methods. There are several ways to prevent pestiviral infections and different strategies have been developed. Reliable laboratory diagnostic tests and efficacious vaccines are prerequisites of efficient preventive control measures.

Diagnosis Acute infection with pestiviruses results in transient viremia. Since these viruses grow readily in cell cultures of their species of origin, classical diagnostic methods were based on the isolation of viruses from blood or organs of infected animals. Isolated cp BVDV can be detected using plaque assays, while noncp viruses can be visualized either by immunofluorescence or enzyme immuno techniques. Since tissue culture is time consuming and expensive, rapid screening tests have been developed. Immunohistochemistry using mono- or polyclonal antibodies can be applied in post-mortem samples of infected animals. Antigen-capture enzyme-linked immunosorbent assays (AgC-ELISA) are being used to diagnose cattle persistently-infected with BVDV. Since monoclonal antibodies are used in these test systems they are highly specific. Either blood or ear-notch samples are suitable for these tests. The most common method for the rapid diagnosis for ruminant and porcine pestiviruses is RT-PCR, which is applied using either individual or pooled samples of blood, milk, other body fluids, ear notches of cattle or organ suspensions. In order to achieve a more precise characterization, viruses are often sequenced. Either parts or the whole genome are used, respectively. By comparing the sequence data with published sequences in data bases the information can be used to obtain detailed taxonomic or epidemiological insights. As the result of acute infection or vaccination animals seroconvert. Classical methods for detecting virus-specific antibodies are virus neutralisation assays. These assays are highly specific and sensitive and they allow serological differentiation of viral strains. However, since they are based on the use of cell culture, they are expensive, time consuming and labor-intensive. Today these assays are still used for reference purposes. For the rapid and large scale screening of samples a number of alternative tests, in particular ELISAs for the detection of antibodies against structural and/or non-structural viral proteins are being used. Variants of these tests are direct, indirect or competition or double antigen ELISAs. Recombinant viral proteins and monoclonal antibodies have been used to improve sensitivity and specificity of these assays. All these tests have in common that they are relatively inexpensive and they are suitable for mass screening because they can be automated. Antibody detection can be performed in individual or pooled serum samples as well as in bulk milk samples of ruminants. Despite good characteristics of these ELISAs with respect to sensitivity and specificity, positive results are sometimes required to be confirmed by virus neutralization test.

Vaccines Vaccines have been used as one of the most important control tools against pestiviral infections. Today there are a number of vaccines available, in particular against BVD and CSF.

Vaccines against classical swine fever Currently modified live vaccines (MLV) are the vaccines of choice for the prevention of CSF. Conventional MLVs, e.g., the lapinised C-(China) strain of CSFV, are safe, highly efficacious and relatively inexpensive. However, when using these vaccines a serological distinction between vaccinated and infected animals is not possible. This might impede an organized effort for the control of the infection. Therefore, numerous efforts were made to develop DIVA (Differentiating Infected from Vaccinated Animals) vaccines, which would allow the distinction of naturally infected and vaccinated pigs. First DIVA vaccines were based on the immunogenic

162

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

E2 envelope protein of the virus, which had been expressed in baculoviruses. These vaccines were on the market for a few years, however, they have been abandoned because their efficacy was inferior to MLV. Nevertheless, at least one E2 subunit vaccine has recently been licensed in China. Moreover, considerable efforts have been undertaken to develop live DIVA vaccines. Current live DIVA vaccines have been developed using recombinant techniques, e.g., by constructing a chimeric virus using a BVDV backbone and the immunogenic E2 envelope protein of CSFV. Other vaccines are based on the use of adenovirus vectors. In parallel serological tests were developed to discriminate infected from vaccinated animals.

Vaccines against bovine viral diarrhea First MLVs against BVD were already developed in the 1960ties. They were highly efficacious, however, they could not be used in pregnant cattle, because transplacental transmission of vaccine virus often produced damage comparable to natural infection with BVDV. In response to these safety issues killed vaccines were developed, which in general had a very good safety record and animals of all ages could be vaccinated including pregnant animals. However, killed vaccines had to be administered repeatedly and the immune response was inferior to that of MLVs. In consequence fetal protection of pregnant animals was often insufficient. BVDV infection of vaccinated pregnant cattle often interfered with pregnancy and the whole spectrum of BVDV-related malformations was observed including the birth of PI calves. A rare phenomenon associated with one particular inactivated vaccine was observed in 2007 and subsequent years in Europe. A new disease “bovine neonatal pancytopenia (BNP)” leading to the death of newborn calves emerged. Epidemiological studies indicated a connection between this new syndrome and vaccination with an inactivated vaccine against BVD. In serum and colostrum of dams which had given birth to calves with BNP, alloantibodies reacting with blood leukocytes of calves were detected. Apparently impurities derived from bovine cells or fetal calf serum that was used for the production of the vaccine virus were presented by a powerful adjuvant in the vaccine thereby eliciting the production of alloantibodies. The vaccine was withdrawn from the European market. Today about 20 predominantly killed vaccines are licensed in the EU. All but one product are directed against BVDV-1 only. In North America close to 200 BVD vaccines, killed and live and sometimes in combination with other antigens, are on the market. Some of the vaccines are directed against BVDV-1 and BVDV-2. Since a few years, a novel MLV is on the market that is based on a combination of deletion mutants of noncp BVDV-1 and BVDV-2 both lacking Npro and the ribonuclease activity of Erns. This MLV is safe to administer to all age classes of cattle including pregnant animals and has been shown not to establish an infection in foetuses. The main purpose of vaccination against BVD is to achieve a strong fetal protection in order to prevent the production of new PI animals. Vaccines with this potency are well suited to also prevent all other consequences of acute BVDV infection, i.e. transient immunosuppression and subsequent clinical or subclincal symptoms.

Control of Classical Swine Fever in Domestic Pigs CSFV is arguably the most important pathogen of pigs. As mentioned above, domestic and feral pigs, as well as wild boar are equally susceptible. Suitable methods for the control of CSF in domestic pigs are stamping out of the infection, i.e., killing and destruction of infected and suspected contact animals in combination with movement restrictions and other strict biosecurity measures. A ban of swill feeding is a mandatory and very effective prophylactic control measure. Usually farmers are compensated for their losses. Stamping out is generally used in countries that are free from CSF and where vaccination is prohibited. Prophylactic vaccination can also be used as a control tool. It is applied, when new outbreaks can no longer be controlled by stamping out alone. In countries with endemic CSF vaccination is used in order to reduce economic losses. When used systematically, i.e., when the vast majority of a pig population is covered, the number of susceptible animals will decrease to an extent that the spread of the infection is no longer supported. The basic reproductive number of infection (R0) drops below 1. One infected animal infects less than one susceptible animal. As a result the infection fades out and CSF will eventually be eradicated. This strategy has been used successfully in domestic pigs in the 1970ties in some regions of The Netherlands and from 2007 to 2010 in the Romanian backyard pig population.

Control of Classical Swine Fever in Wild Boar CSFV can also infect wild boar. The epidemiology of the infection depends on a number of factors, in particular virulence of the virus and density of the animal population. From early experience it was thought that CSF outbreaks in wild boar are self-limiting. However, more recent outbreaks, e.g., in the 1990s in Germany became endemic. Possible reasons were high population densities and moderate virulence of the virus. CSF in wild boar is a severe threat to domestic pig holdings in the affected area. Therefore effective control measures are crucial. Key parameters for the successful control of CSF in wild boar are the number of susceptible animals, R0 (basic reproduction number of infection) and viral virulence. Fresh outbreaks result in a large number of deaths in younger age classes, while most older animals survive and will become seropositive. The latter are immune for the rest of their lives. This scenario may lead in low density populations to a drop of the basic reproduction number below the critical threshold (R0o1), and outbreaks are selflimiting. In contrast, when population density is high there will always be enough susceptible animals due to intense reproduction to maintain the infection (R041). CSF may become endemic for many years. Viral virulence influences these situations by the proportion of killed vs. surviving animals.

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

163

The prime purpose of any control measure is the reduction of susceptible animals to a threshold that R0 drops below one. In sparsely populated areas this might be the case after the initial phase of the outbreak when a part of the population has succumbed to the infection and the remaining animals are immune. In these situations hunting is often banned in order not to disturb the population and keep the infection in the affected area. Natural boundaries, e.g., major rivers or mountains are helpful in containing the infection. In dense populations the number of susceptible animals has to be reduced by external intervention:

• • •

Hunting activities should focus on the young age classes of wild boar, since older animals in an infected area are most likely to be immune. However, due to the high reproduction rate of wild boar experience has shown that hunting alone as a control tool is insufficient and does not result in eradication of the infection. Trapping might be considered a variant of hunting. Positive experiences using this control tool have been made, e.g., in Bulgaria. This strategy does not disturb the population and larger numbers of susceptible animals can be removed from the population. Oral immunization with baits containing MLV has been shown to be highly effective, and when applied over a period of a few years may significantly contribute to the extinction of the infection in endemic areas. A proven strategy is to vaccinate twice per year, i.e., in spring and autumn. Each vaccination is repeated once after four weeks. Baits are distributed in small restricted areas. Although very young age classes are not well reached by oral immunization, this strategy can reduce the number of susceptible animals sufficiently to reach R0o1. Sometimes a combination of the above strategies leads to success.

Control of Bovine Virus Diarrhea Systematic control of BVD including the removal of PI animals was long discussed controversially, because of the ubiquitous occurrence of the infection and the stealthy nature of the infection. In addition the economic impact of BVD was underestimated for a long time. Today the economic dimension of BVD is fully appreciated. Transient immunosuppression and fetal infections produce disease and economic losses can be dramatic. Meanwhile BVD is considered to be the most important viral disease of cattle. The infection is spread by direct contact of acutely infected animals and by fomites. However, the most important source of BVDV spread are persistently infected (PI) cattle. They are the virus reservoir in cattle populations and consequently they play a pivotal role in any control programme. The life span of PI animals ranges from a few hours after birth up to seven or eight years. During their lifetime they shed large amounts of virus at any time. This is unique and the number of new infections transmitted by PI cattle may be unlimited, depending on frequency of animal contacts. For more than fifty years vaccines have been used to prevent clinical BVD. Once the economic consequences of intrauterine infection were recognized, an additional requirement for licensing of vaccines was reliable fetal protection. However, despite widespread use of vaccination BVD prevalence did not change over the decades. The main reason for this failure was due to the remaining of PI animals as a powerful reservoir in cattle populations. Poor efficacy of some vaccines might have been an additional factor. In contrast to many other animal infections, e.g., CSF or rinderpest, BVD will never be eradicated using vaccination alone. With the advent of inexpensive laboratory screening methods, it was possible to identify and remove PI cattle. Scandinavian countries were the first in the early 1990s to implement systematic control programmes by searching with serological methods for herds with active BVD infections. PI animals were identified in seropositive herds and removed. After their removal herds were monitored in regular intervals and, when seronegative, they were declared BVD-free. Strict biosecurity measures and movement restrictions for nonnegative cattle were accompanying measures of these compulsory programmes. A few years of systematic control resulted in a BVD-free status of the Scandinavian countries. Vaccination was banned throughout the programme. Economic analyses showed that the cost benefit ratio of BVD control was highly positive. Austria followed a few years later with a similar control programme. In 2008 Switzerland chose a slightly different approach: The whole national cattle population was tested for PI animals. Thereafter all newborn calves were tested for BVD. The final phase of the programme is the constant serological surveillance of the cattle population. Within 3 years PI prevalence was reduced from 1.3% to 0.02%. Germany started a systematic effort to reduce PI prevalence in 2011 by testing newborn calves for BVDV. Vaccination as an additional control tool was allowed. PI prevalence was reduced from 0.48% (2011) to 0.01% (2017). Similar control programmes were initiated in Belgium, Scotland and Ireland. In countries or regions without a compulsory programme for systematic BVD control it is possible to prevent economic damage on a herd basis by removing PI animals, regular vaccination in order to prevent reinfection with BVDV and strict biosecurity.

Control of Border Disease of Sheep There are a few vaccines against BD available. Since the epidemiology of BD is very similar to BVD analogous strategies for its control can be used.

Further Reading Becher, P., Tautz, N., 2011. RNA recombination in pestiviruses: Cellular RNA sequences in viral genomes highlight the role of host factors for viral persistence and lethal disease. RNA Biology 8, 216–224. Blome, S., Moss, C., Reimann, I., Koenig, P., Beer, M., 2017. Classical swine fever vaccines-state-of-the-art. Veterinary Microbiology 206, 10–20.

164

Bovine Viral Diarrhea, Border Disease, and Classical Swine Fever Viruses (Flaviviridae)

Lindenbach, B.D., Thiel, H.J., Rice, C.M., 2013. Flaviviridae. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed., vol. 1. Philadelphia, PA: Lippincott Williams & Wilkins, pp. 712–746. Moennig, V., Becher, P., 2015. Pestivirus control programs: How far have we come and where are we going? Animal Health Research Reviews 16, 83–87. Moennig, V., Becher, P., 2018. Control of bovine viral diarrhea. Pathogens 7 (1). Nettleton, P.F., Gilray, J.A., Russo, P., Dlissi, E., 1998. Border disease of sheep and goats. Veterinary Research 29, 327–340. Postel, A., Austermann-Busch, S., Petrov, A., Moennig, V., Becher, P., 2018. Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges. Transboundary and Emerging Diseases 65 (Suppl 1), 248–261. Tautz, N., Tews, B.A., Meyers, G., 2015. The molecular biology of pestiviruses. Advances in Virus Research 93, 47–160. Yes¸ilbağ, K., Alpay, G., Becher, P., 2017. Variability and global distribution of subgenotypes of bovine viral diarrhea virus. Viruses 9 (6).

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae) Philippa M Beard, The Pirbright Institute, Pirbright, United Kingdom and The Roslin Institute, University of Edinburgh, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Introduction Poxviruses cause disease in farmed and free-living ruminants, ranging from mild, self-limiting lesions to systemic, fatal disease. The species of ruminant poxvirus covered in this article are listed in Table 1. The most common is Orf virus (ORFV) which is found worldwide and causes mild, transient, proliferative lesions on young sheep and goats. In contrast, the most severe poxvirus diseases of ruminants are caused by Sheeppox virus (SPPV), Goatpox virus (GTPV) and Lumpy skin disease virus (LSDV). These three viruses are recognised as causing high consequence transboundary diseases with a substantial morbidity and mortality burden.

Classification The family Poxviridae contains two subfamilies – Entomopoxvirinae and Chordopoxvirinae. Poxviruses that affect ruminants are found within the Chordopoxvirinae subfamily and include species within the genera Capripoxvirus, Orthopoxvirus and Parapoxvirus.

Virion Structure Poxviruses have a complex virion structure. The viral genome and transcription factors are believed to be contained in a folded tubular “nucleocapsid” structure. This is contained within the very recognisable biconcave core structure which has a lateral body in each concavity. The core structure is then enclosed within one to three lipid membranes. Orthopoxviruses produce four virion types that are distinguished by the number of membranes surrounding the core and their cellular location – the intracellular mature virion (IMV), the intracellular enveloped virion (IEV), the cell-associated enveloped virion (CEV) and extracellular enveloped virion (EEV). In some recent papers IMVs are referred to as mature virions, IEVs as wrapped virions, and EEVs as extracellular virions. Electron microscopy studies have shown parapoxviruses (PPV) produce IMVs, IEVs, CEVs and EEVs analogous to orthopoxviruses. These studies have also revealed a unique feature of the PPV virion – a raised surface tubule protein that is wrapped around the virion core to produce a “ball of wool” effect. This protein is encoded by the ORF104 gene; mutated viruses with this ORF disrupted produced morphologically aberrant particles and failed to create EEVs. Very little work has been reported on the virion structure of capripoxviruses (CPPV). Examination of LSDV, SPPV and GTPV purified virions using electron microscopy have identified CPPV virion size as length 293–299 nm and width 262–273 nm, consistent with other poxviruses, and the absence of any surface tubular proteins. Electron microscopy images from LSDV-infected tissue have revealed IMV and IEV-like structures (Fig. 1).

Genome The CPPV genome structure is similar to other poxviruses. It consists of a double stranded linear DNA genome that is 25% GC-rich, approximately 150 kbp in length, and encodes around 156 ORFs. Each end of the linear genome encodes for a Table 1

Poxviruses of ruminants

Genus

Species

Abbreviation

Capripoxvirus (CPPV)

Sheeppox virus Goatpox virus Lumpy skin disease virus Orf virus Bovine papular stomatitis virus Pseudocowpox virus Parapoxvirus of red deer in New Zealand Vaccinia virus Deerpox virus Mule deerpox virus

SPPV GTPV LSDV ORFV BPSV PCPV PVNZ VACV

Parapoxvirus (PPV)

Orthopoxvirus (OPXV) Unclassified

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20934-5

165

166

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae)

Fig. 1 Ultrastructural detail of LSDV morphogenesis. Tissue was collected from a cutaneous nodule on a calf experimentally inoculated with LSDV, then fixed and processed for electron microscopy using standard techniques. Acknowledgment: Beatriz Sanz-Bernardo, Philippa Hawes and Jennifer Simpson.

2200–2300 bp inverted terminal repeat sequence, and the linear ends of the genome are joined with a hairpin loop. The central region of the CPPV genome contains ORFs predicted to encode proteins required for virus replication and morphogenesis and exhibit a high degree of similarity with genomes of other mammalian poxviruses. In the outer regions of the CPPV genomes this similarity is lower. These ORFs contain genes encoding proteins likely to be involved in viral virulence and host range determinants. The CPPV isolates sequenced to date are genetically very similar, exhibiting 96% nucleotide identity. However phylogenetic analysis groups the isolates into the three species – SPPV, GTPV and LSDV. The genome of the PPVs is one of the smallest amongst the poxviruses at 134–139 kbp. It is also unusual in its high GC content, around 64%. The only other poxvirus with a similarly high GC content is the human pathogen molluscum contagiosum virus. The PPV genome encodes 130–132 ORFs and exhibits the standard poxvirus genome structure with inverted terminal repeats and hairpin loops. Genes encoding essential functions are found centrally and ORFs encoding proteins involved in virus pathogenesis towards the periphery. PPV species exhibit greater genetic variation than CPPV, for example the amino acid identity of PCPV compared to BPSV is only 72%.

Life Cycle It is widely presumed that the cellular life cycle of CPPV and PPV mirrors that of the well-studied orthopoxvirus genus. This is supported by microscopic and ultrastructural findings indicating cellular entry is followed by formation of cytoplasmic viral factories, and morphogenesis from immature virions to IMV, IEV, and CEV followed by virion release.

Sheeppox and Goatpox Virus Epidemiology Sheeppox and goatpox occur in Africa, the Middle East, and Asia including India and China. There are regular outbreaks on the periphery of these endemic zones, as for example Vietnam (2005 and 2008), Taiwan (2010), and Greece (2018). Participatory studies using farmer interviews have indicated the these diseases are widespread in low and middle income countries. For example, 80% of farmers in Sudan had experienced an outbreak of sheeppox at some time in the past, while 14% of sheep farmers in Algeria reported current cases of sheeppox in their flock. Serological studies to estimate the prevalence of SPPV and GTPV are few but support the theory that the diseases are widespread. 17% of sheep and 14% of goats tested in west Amhara region of Ethiopia were seropositive, while 17% of goats were seropositive in the Layyah Punjab region of Pakistan. Morbidity, mortality and case fatality rates (CFR) of sheeppox and goatpox vary between outbreaks. In an epidemic of sheeppox in Israel morbidity was 20.5% and CFR 19.3%. In an epidemic of sheeppox in Mongolia morbidity was 14.5% and CFR 6%. In imported, unvaccinated sheep in a feedlot

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae)

167

in Jordan an outbreak of sheeppox caused 21.5% morbidity and CFR 41%. In more endemic settings in Sudan morbidity due to sheeppox varied between flocks from 0.45%–15.9% and CFR 0%–33%. SPPV and GTPV are widely accepted to cause substantial economic hardship with particularly severe impacts on subsistence livestock owners in low and middle income countries. However the economic impact of SPPV and GTPV is poorly studied. A study from 2000 in India estimated it would take 6 years for a farm to recover from an outbreak of SPPV or GTPV, with average annual losses in income of 30%–43%, depending on flock type and the owner’s actions. SPPV and GTPV spread from animal to animal via direct contact or insect vectors such as biting flies (Stomoxys calcitrans). Aerosol infection of sheep has been achieved experimentally by spraying SPPV or GTPV into the close environment or directly into the face of sheep, although this method of transmission was found to be less effective than prolonged direct contact with a diseased animal. Under experimental conditions, 100% of animals inoculated intradermally with a virulent strain develop disease. The host tropism of SPPV and GTPV strains varies. Some SPPV and GTPV viruses are highly host specific and cause disease in the field only in sheep or goats respectively. Other strains cause disease in both sheep and goats. The host specificity of SPPV and GTPV has been tested experimentally. Two GTPV strains, one from Vietnam and one from Yemen, were inoculated into both goats and sheep. Both isolates caused more severe disease in goats, the Yemen isolate was also moderately pathogenic in sheep. However the Vietnam isolate caused only very mild disease in sheep. This supports evidence from the field that different SPPV and GTPV strains exhibit variable host preference. The genetic determinants of SPPV and GTPV strain virulence and host preference are unknown.

Clinical features and pathology Experimentally the incubation period of sheeppox and goatpox is 4–6 days following intradermal or subcutaneous inoculation. Both diseases are characterised by the appearance of round to oval, slightly raised, well demarcated lesions up to 2 cm in diameter on the skin. The appearance of skin lesions coincides with other clinical signs including severe pyrexia, purulent ocular and nasal discharge, anorexia, dyspnoea, lethargy, diarrhoea and superficial lymphadenopathy. The cutaneous lesions of sheeppox and goatpox are similar to the classic orthopoxvirus “pocks” and develop from macule to papule to pustule and then scab. Ulceration of the cutaneous lesions is a feature of some outbreaks. Lesions are often more numerous on sparsely haired areas of the body. Post mortem examination of cases of sheeppox and goatpox may reveal lesions in internal organs, described as firm red to white nodules in the lungs and intestines, liver and kidneys. Sheeppox and goatpox can occur in the field as a mixed infection concurrent with bluetongue virus, peste-des-petits-ruminants virus, orf virus or Mycoplasma capricolum subsp. capripneumoniae. These mixed infections often result in mortality over 50%.

Histopathology The dominant histopathological features seen in sheep and goats infected with SPPV and GTPV are keratinocyte hyperplasia and necrosis accompanied by vesicle formation in the epidermis, with large basophilic intracytoplasmic inclusion bodies often present in the degenerate and necrotic keratinocytes and in the epithelium of adnexal structures. Within the dermis oedema and haemorrhage is accompanied by infiltrating large histiocytic cells with a vacuolated nucleus and marginated chromatin. These cells are often found in perivascular areas, and can contain prominent large basophilic intracytoplasmic inclusion bodies. A necrotising vasculitis with histiocytic cells infiltrating the walls of blood vessels has been reported within the dermis and subcutis. In the lungs, a severe necrotising bronchointerstitial pneumonia is present, with large histiocytes and other mononuclear cells infiltrating the interstitial tissues, particularly in peribronchiolar and perivascular areas. Large histiocytic cells accompanied by necrosis and vasculitis have also been noted in the liver, kidney, lymph nodes and gastrointestinal tract. Immunolabeling of tissues from affected animals have identified CPPV antigen in histiocytic cells, keratinocytes, and bronchiolar epithelial cells.

Pathogenesis Most of the work on sheeppox and goatpox pathogenesis comes from time course studies following experimental infection. After intradermal or subcutaneous inoculation, virus disseminates from the inoculation site to distant cutaneous sites and other organs, possibly via a monocyte/macrophage associated viraemia. Virus is detected in the skin distant from the inoculation site 3-4 days later, and virus load increases up to day 7 or 8 and remains high until day 14 when it begins to gradually decrease. The skin and lung appear to be the major target organs for these viruses.

Diagnosis SPPV and GTPV are often strongly suspected based on the clinical signs displayed by the affected animals. Differential diagnoses include orf, dermatophilosis, dermatophytosis, bacterial pyoderma, and mange. Confirmation of the diagnosis of sheeppox or goatpox is usually made on the basis of detection of SPPV or GTPV DNA within lesions or blood. A PCR based on the CPPV-encoded G-protein-coupled chemokine receptor (GPCR) gene can used to differentiate between the three CPPV species. A CPPV neutralisation test can be used to detect neutralising antibodies in immune animals. Histopathology and electron microscopy are occasionally used for confirmation of the disease but are time consuming and expensive.

168

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae)

Treatment Due to their high consequence outbreaks of SP and GP are often treated by euthanasia of affected and in-contact animals (“stamping out”). In circumstances where treatment is appropriate and economically viable sheeppox and goatpox may be treated with supportive therapy and antibiotic coverage to reduce secondary bacterial infections.

Prevention Live attenuated strains of CPPV are widely used as vaccines to prevent sheeppox and goatpox. Similar to orthopoxviruses CPPVs are cross-protective within genus therefore one CPPV live attenuated vaccine can be used to protect against all three CPPV species.

Lumpy Skin Disease Virus Epidemiology Lumpy skin disease affects cattle and water buffalo and occurs in Africa, the Middle East, southeast Europe, the Caucasus, Russia and Kazakhstan. LSD was first described in southern Africa in the 1920s, from where it gradually spread northwards throughout Africa and entered the Middle East in the 1980s. Outbreaks occurred sporadically in this region until 2012 when the virus began to spread rapidly over long distances into new areas including Iran, Iraq, Jordan, Turkey, Russia and, in 2015, into mainland Europe (Greece). The disease has since been reported in many Balkan and Caucasian countries. The reasons behind the rapid and sudden spread of LSDV through the Middle East, Europe and western Asia since 2012 are unclear. Hypotheses include climatic conditions conducive to LSDV spread, or civil conflict hampering veterinary services and disrupting livestock movement patterns. LSD is highly host specific with extensive serological surveys of wildlife in southern Africa finding no evidence for LSDV infection of any species other than cattle and water buffalo. The mode of transmission of LSDV differs from SPPV and GTPV. Experimental studies found no evidence for direct transmission of the virus between animals, instead transmission is strongly believed to be via arthropod vectors. It is unclear if this vector borne transmission is biological or mechanical. LSD has been successfully transmitted experimentally via Aedes aegypti mosquitoes. LSDV was retained by the Ae. aegypti mosquitoes and transmitted to recipient cattle up to 6d after feeding on donor cattle. This work represents the only unequivocal demonstration of experimental transmission of LSD via vectors. In contrast two other species of mosquito (Culex quinquefasciatus and Anopheles stephensi) retained LSDV for only 24 h and did not transmit LSDV to recipients. Similarly, LSDV was detected in Stomoxys calcitrans (biting flies) and midges (Culicoides nubeculosus) after feeding the insects on a LSD-affected animal but transmission of the virus or disease to a naïve recipient animal was unsuccessful. Experimental transmission of LSDV has been demonstrated by transferring a Rhipicephalus appendiculatus ticks from donor to recipient animals mid-feed although the latter did not show clinical disease. LSDV has shown a propensity for the male reproductive tract, with virus isolated from necrotic lesions in the testes and epididymis, and from the semen of LSD-affected bulls. Clinical LSD accompanied by a severe necrohaemorrhagic vulvovaginitis and metritis was demonstrated in a group of heifers artificially inseminated with semen spiked with virulent LSDV, suggesting this could be a route of LSD transmission. A number of detailed epidemiological studies on LSD have been published. Communal grazing, proximity to water sources, temperature and rainfall have all been associated with outbreaks of LSD and are consistent with vector-borne transmission of virus. A well-documented LSD outbreak in a large dairy herd in Israel in 2006 was analysed and transmission by indirect or direct contact modelled. Indirect transmission was the only parameter that could explain the outbreak dynamics with an overall effect over 5 times larger than direct contact. Outbreaks in south-east Europe were strongly seasonal, occurring in spring and summer with very few, if any, outbreaks over winter. The median LSD spread rate in these outbreaks was calculated to be 7.3 km/week, consistent with vector-borne spread. Morbidity and mortality in LSD outbreaks are lower than sheeppox and goatpox. Recent reports from epidemic areas in the Middle East and Europe have indicated morbidity of 9%–26%, and mortality from 0.5% to 2%. In comparison, data from 1161 LSD outbreaks over 15 years in an endemic area in Uganda reported a lower disease impact with morbidity 4.77% and mortality 0.03%.

Clinical features LSD has an incubation period of approximately 7 days after experimental inoculation. The first sign of disease is pyrexia, followed by the appearance of distinctive and numerous raised cutaneous lesions from 0.5 to 5 cm in diameter which develop over 3–4 days from macule to papule to nodule. The cutaneous lesions are often accompanied by oral, nasal and ocular discharge, lethargy, anorexia, and in lactating animals a rapid drop in milk production. Brisket oedema and superficial lymphadenopathy are also reported. After 1–2 weeks the skin nodules become necrotic and the centre eventually sloughs, leaving a defect in the skin which can predispose the animal to secondary bacterial infection or myiasis.

Pathology Gross pathology of LSD is characterised by multifocal, acute to chronic nodular dermatitis, with necrosis and ulceration. Histologically there is mild acanthosis, ballooning degeneration of keratinocytes with intracytoplasmic intracellular inclusion bodies, histiocytic inflammation of the dermis and fibrinonecrotic vasculitis (Fig. 2).

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae)

169

Fig. 2 Microscopic image of the histopathological changes in the epidermis of a nodule from a calf experimentally inoculated with LSDV. The tissue was fixed in buffered formal saline, processed to a wax block, and stained with haematoxylin and eosin using routine methods. The arrows point to eosinophilic intracytoplasmic inclusion bodies present in the keratinocytes.

Pathogenesis Few details of the pathogenesis of LSD have been determined, and our understanding of how the virus causes disease is extrapolated from the more meticulously studied poxviruses such as the orthopoxviruses. It is believed that arthropod vectors inoculate LSDV into the skin of the animal, the virus then circulates in the blood stream via a low level viraemia and disseminates into distant cutaneous sites as well as the reproductive tract and other internal organs. Experimental models of LSD have consistently found that only around 50% of cattle inoculated with LSDV develop clinical disease; the host factors which control this variable susceptibility are unclear.

Diagnosis LSDV is often strongly suspected based on the characteristic clinical signs. PCR is used to detect viral DNA in fluids or tissues to confirm disease. Virus isolation and electron microscopy can be used for diagnosis but are relatively expensive and time-consuming techniques. A virus neutralisation test can be used to detect neutralising antibodies in immune cattle. ELISA-based serological tests to detect antibodies to LSDV have proved challenging to develop due to the complex and variable antibody response to poxvirus infection. Differential diagnoses for LSDV are bovine herpesvirus 2 infection (sometimes known as pseudolumpy skin disease), dermatophilosis, demodicosis, besnoitiosis, Hypoderma bovis infection, and cutaneous tuberculosis.

Treatment In epidemic situations treatment of LSD is rarely attempted, with stamping out of affected and in-contact cattle often mandated. In circumstances where treatment is appropriate and economically viable LSD may be treated with supportive therapy and antibiotic coverage to reduce secondary bacterial infections of the skin lesions.

Prevention LSD can be prevented by vaccination with a live-attenuated CPPV strain. The most commonly used vaccine for LSD is the “Neethling” attenuated LSDV strain. Vaccination can be accompanied by movement restrictions to prevent the spread of the disease. Detailed analyses of the recent outbreaks in Europe and neighbouring countries have found vaccination to be a highly effective method of disease prevention, while questioning the effectiveness of a comprehensive stamping out policy.

Orf Virus Epidemiology Orf, also known as scabby mouth or contagious ecthyma, is caused by infection with orf virus (ORFV) and found worldwide wherever small ruminants are reared. The disease can be readily recognised from its characteristic scabby lesions around the mouth and nares of lambs and kids. The morbidity can be high but mortality is rare, and the disease is usually self-limiting. ORFV is very robust and can survive months in dry conditions, likely promoting recurrence of the disease in the spring lambing season. Like many other PPVs, ORFV is zoonotic with regular reports of transmission of the virus to people where it causes a focal proliferative dermatitis. Cases in humans have been associated with high risk professions such as veterinarians and farm workers, and with cultural celebrations which involve animal slaughter. Molecular epidemiological studies of PPVs have been carried out, often targeting ORF011 (the B2L gene), which is the PPV orthologue of the vaccinia virus (VACV) Copenhagen strain gene F13L. A recent comprehensive study compared 23 isolates using ORF011 and ORF032 to enable categorisation of the PPV isolates to the correct genus.

170

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae)

Clinical features and gross pathology Orf is characterised by proliferative, focal to regionally extensive scabby lesions most commonly around the mouth of lambs and kids. The lesions develop from an initial erythema, to macules, papules, vesicles, pustules and proliferative scabs. The disease has no systemic phase and is usually self-limiting however it can cause reduced growth rates in affected animals, most likely by inhibiting feeding and/or suckling. Less commonly the infection can spread to the udder of the ewe or doe, causing a dermatitis of the teat. There are occasional reports of ORFV causing more severe outbreaks of disease. Reinfection can occur but the disease is milder and resolves more rapidly.

Histopathology The histopathology of orf is focal to regionally extensive moderate to marked proliferative dermatitis or cheilitis, with marked dermal vascular proliferation and dilation. Early lesions are characterised by severe acanthosis and ballooning degeneration of keratinocytes, with elongated rete pegs often a key feature. Dermal changes include a moderate inflammatory cell infiltrate (mainly lymphocytes and histiocytes) with oedema occasionally causing subepidermal vesicles.

Pathogenesis Orf virus (ORFV) infects broken or scarified skin and is therefore more often reported in animals grazing thistle, gorse or stubble. The virus initially replicates in regenerating keratinocytes immediately below the stratum corneum, before spreading laterally into adjacent epithelium. The inflammatory response consists of neutrophils, basophils and mast cells followed by abundant MHC class II+ histiocytic cells, T and B lymphocytes. A number of virulence factors encoded by ORFV have been studied and their mechanism of function elucidated. These include GIF and VEGF-E. GM-CSF and IL-2 inhibitory factor (GIF) is encoded by ORF117 of ORFV. This protein appears to be unique to PPVs with no orthologue found in any other Chordopoxvirus species. GIF is expressed as an immediate-late protein and binds sheep, but not human, GM-CSF and IL-2 and antagonises the bioactivity of both. More specifically GIF interferes with the myeloproliferative and chemoattractant properties of GM-CSF and the activation of lymphocytes and NK cells by IL-2. Recently, a structural biology approach was taken to identify how GIF targets these two different chemokines. This work revealed that GIF forms a dimer and binds two molecules of the target cytokine. GIF achieves this without sharing any structural similarity with either cognate cytokine receptor. GIF binds both GM-CSF and IL-2 with high affinity, but uses mutually exclusive interaction sites for each cytokine. GIF is the only such dual-purpose viral decoy receptor characterised to date. Orf virus encodes a fifth member of the vascular endothelial growth factor family, known as VEGF-E. This protein binds to VEGF-receptor 2 (VEGFR2) and neuropilin-1, but not VEGFR1 or VEGFR3. VEGF-E exhibits biological activity consistent with its receptor binding, causing increased mitogenic activity of endothelial cells and increase vascular permeability. An ORFV strain lacking VEGF-E has been constructed and characterised in vivo. This mutated strain exhibited reduced gross pathology with particularly striking loss of the characteristic erythema seen in early orf lesions. Histologically the strain lacking VEGF-E produced less inflammatory cell influx, reduced hyperplasia of the epithelium, smaller pustules which resolved more rapidly, and reduced neovascularisation in the dermis. VEGF-E is therefore a key factor driving the pathological changes associated with ORFV infection, with a strong angiogenic role contributing to the characteristic neovascularisation and capillary dilation. The biological activities of VEGF-E have been used in a mouse model to promote wound healing. Expression of recombinant VEGF-E in normal skin caused epidermal thickening and increased numbers of endothelial cells and blood vessels in the dermis. In wounded skin, VEGF-E enhanced wound re-epithelialisation and increased thickness of neovascularisation, suggesting a therapeutic application for VEGF-E in promotion of wound healing.

Diagnosis Clinical signs are often used to diagnose orf. Polymerase chain reaction (PCR) can be used for confirmation if needed. The main differential diagnoses for orf are sheeppox, goatpox, foot and mouth disease, bluetongue, peste-des-petits-ruminants, and mange.

Treatment Most cases of orf are self-limiting, however support therapy is indicated when the clinical signs are more severe.

Prevention Vaccination with a live attenuated strain of ORFV is the recommended method for reducing the disease. Vaccination is required on an annual basis.

Bovine Papular Stomatitis Virus Bovine papular stomatitis virus (BPSV) causes mild disease in calves, characterised by flat to slightly raised red to brown macules and papules, sometimes progressing to ulcers, in the mucus membranes of the oral cavity. Diagnosis, treatment and prevention measures are rarely required, as severe disease is very rare. In common with other PPVs, BPSV is zoonotic with transmission to animal handlers reported. Older publications suggest differentiation of BPSV and PCPV can be made based on lesion location,

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae)

171

however cases of BPSV affecting the teats of cattle have been reported. Hence, molecular techniques are required to correctly diagnose these diseases.

Pseudocowpoxvirus Pseudocowpoxvirus (PCPV) causes vesicular, pustular and scabby lesions most commonly on the teats and udders of milking cows. The clinical signs are often described as an incomplete ring of scabs in the shape of a horseshoe. The disease is mild and rarely of economic importance. PCPV should not be confused with the orthopoxvirus Cowpox virus which causes localised to systemic disease in cats, humans, rats, and other species.

Parapoxvirus of Red Deer in NZ Parapoxvirus of red deer in NZ (PVNZ) is recognised as a fourth species of PPV after being isolated from lesions on the skin of growing antlers (velvet) of farmed red deer in New Zealand. The disease was originally described on eight farms, and characterised by scabby lesions on the muzzle, lips, face, ears, neck and velvet of farmed red deer. Viruses with strong genetic similarities to PVNZ have been isolated from healthy deer in Bavaria and from proliferative lesions around the mouth of red deer in Italy.

Vaccinia Virus Vaccinia virus (VACV) is known to have a wide host range but rarely causes severe disease in an immunocompetent host. Sporadic cases of mild VACV infection of cattle (bovine vaccinia) have been reported in the literature for many years, however since the late 1990s bovine vaccinia in Brazil has increased to endemic levels in some regions. Bovine vaccinia presents as papules, vesicles and pustules, leading to ulcers and scabs, on the teat and udders of milking cows and oral cavity of suckling calves. There have been numerous reports of zoonotic transmission of the virus to animal handlers, mainly milking staff who develop vesicopustular and ulcerative lesions on their hands and arms, accompanied by fever, lymphadenopathy and lethargy. The disease can be severe enough to lead to hospitalisation. Human to human transmission of bovine vaccinia appears rare but has been documented. The pathogenesis of bovine vaccinia is unclear. Experimental intradermal inoculation of cattle with VACV in the skin of the teat showed evidence of local disease and systemic spread, with detection of virus antigen in gastrointestinal-associated lymphoid tissue, and excretion of virus in faeces. Molecular studies have shown at least two genetically distinct populations of VACV are responsible for the recent epidemic of bovine vaccinia in Brazil. The origins of the two VACV strains are unknown, hypotheses include a derivative of a VACV strain used during the smallpox eradication vaccination campaign in Brazil, or the emergence of a novel orthopoxvirus strain. Surveys of wild and domestic animals in Brazil have found serological and/or virological evidence of orthopoxvirus infection in a wide range of species including horses, rodents and marsupials, dogs, cats and wild-living primates. The role of any of these species in either the re-emergence or persistence of bovine vaccinia is unknown. A strain of VACV known as buffalopox virus causes sporadic outbreaks of disease in buffalo and, less commonly, in cows in India and other Asian countries. The disease is usually mild, causing typical pox lesions on the teats, udder, and extremities of the head of affected animals.

Cervidpoxvirus Cervidpoxvirus is a proposed poxvirus genera containing species provisionally named Deerpox virus or Mule deerpox virus. These viruses are associated with mucocutaneous ulcerative lesions in deer that are clinically dissimilar to proliferative PPV lesions. The full genome sequence of an isolate from skin lesions of a free-ranging mule deer has been reported and shown to be genetically distinct from other poxvirus genera. Experimental inoculation of black-tailed deer with an isolate of Deerpox virus resulted in mild clinical disease with gross and microscopic pathology consistent with poxvirus infection (focal to multifocal erythema, papules, pustules, ulcers and scabs), and confirmed by virus isolation. More extensive cutaneous and gastrointestinal lesions caused by Deerpox virus have been reported in captive and farmed white-tailed deer.

Further Reading Babiuk, S., Bowden, T.R., Parkyn, G., et al., 2009. Yemen and Vietnam capripoxviruses demonstrate a distinct host preference for goats compared with sheep. Journal of General Virology 90 (Pt 1), 105–114. Beard, P.M., 2016. Lumpy skin disease: A direct threat to Europe. Veterinary Record 178 (22), 557–558. doi:10.1136/vr.i2800. Carn, V.M., Kitching, R.P., 1995. The clinical response of cattle experimentally infected with lumpy skin disease (Neethling) virus. Archives of Virology 140 (3), 503–513. Deane, D., McInnes, C.J., Percival, A., et al., 2000. Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2. Journal Virology 74 (3), 1313–1320.

172

Capripoxviruses, Parapoxviruses, and Other Poxviruses of Ruminants (Poxviridae)

Felix, J., Kandiah, E., De Munck, S., et al., 2016. Structural basis of GM-CSF and IL-2 sequestration by the viral decoy receptor GIF. Nature Communications 7, 13228. Matos, A.C.D., Rehfeld, I.S., Guedes, M., Lobato, Z.I.P., 2018. Bovine vaccinia: Insights into the disease in cattle. Viruses 10 (3). Mercier, A., Arsevska, E., Bournez, L., et al., 2018. Spread rate of lumpy skin disease in the Balkans, 2015–2016. Transboundary and Emerging Diseases 65 (1), 240–243. Meyer, M., Clauss, M., Lepple-Wienhues, A., et al., 1999. A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases. EMBO Journal 18 (2), 363–374. Savory, L.J., Stacker, S.A., Fleming, S.B., Niven, B.E., Mercer, A.A., 2000. Viral vascular endothelial growth factor plays a critical role in orf virus infection. Journal of Virology 74 (22), 10699–10706. Wise, L.M., Veikkola, T., Mercer, A.A., et al., 1999. Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1. Proceedings of the National Academy of Sciences of the United States of America 96 (6), 3071–3076.

Chikungunya Virus (Togaviridae) Thomas E Morrison and Stephanie E Ander, University of Colorado School of Medicine, Aurora, CO, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Arbovirus Viruses that are maintained in nature in transmission cycles between susceptible vertebrate hosts and blood-feeding arthropod vectors. Arthralgia Pain in a joint. Autochthonous transmission Spread of a disease within a local population by local arthropod vectors. Conserved sequence elements (CSEs) Highly conserved genomic elements important for viral replication. Cytopathic vacuoles Collection of rearranged host membranes and spherules that are a site of replication for alphaviruses. Spherules Individual plasma membrane protrusions that serve as a site of alphaviral replication.

Sylvatic cycle of CHIKV transmission Transmission between nonhuman primates and Aedes species mosquitoes. Synovitis Inflammation of synovial membranes in the joints. Urban cycle of CHIKV transmission Transmission within a human population, where humans serve as the vertebrate reservoir and mosquitoes transmit the virus from human to human. Viremia High presence of virus in the bloodstream; required for the transmission of arboviruses from vertebrate to invertebrate hosts. Virus-like particles Non-infectious particles composed of viral structural proteins but containing no viral genome.

Classification Chikungunya virus (CHIKV) is an enveloped virus with a positive-sense, single-stranded RNA genome. CHIKV is classified within the Alphavirus genus of the family Togaviridae based on its antigenicity, genome sequence and organization, and replication strategy. The Alphavirus genus is divided into 7 antigenic complexes; determined by antibody cross-reactivity to the virion glycoproteins E1 and E2, epitopes of which share a high degree of conservation between genetically similar viruses. Along with Bebaru, Getah, Mayaro, o0 nyong ‘nyong, Ross River, Sagiyama, Semliki Forest (SFV), and Una viruses, CHIKV is a member of the Semliki Forest virus antigenic complex.

Virion Structure CHIKV particles are 60–70 nm in diameter and composed of an icosahedral nucleocapsid enclosed within a lipid envelope. The capsid is composed of 240 capsid monomers arranged in a T ¼ 4 symmetry, and it contains a single copy of the viral genome. The viral envelope is derived from the host cell plasma membrane. Embedded within the viral envelope are 80 trimeric spikes composed of viral glycoproteins E2 and E1. A single spike consists of a trimer of E2/E1 heterodimers, which facilitate viral attachment and entry. Also associated with the E2/E1 complex is E3. During glycoprotein trafficking through the ER-Golgi complex, E3 is covalently bound to the immature E2/E1 dimer, where it aids the folding and association of the dimer; after furin cleavage of E3 in the Golgi, E3 remains non-covalently associated to protect against inappropriate activation of the E1 fusion loop. In the related SFV, E3 dissociates from the E2/E1 complex at neutral pH, however, cryo-EM analysis of infectious CHIKV particles indicates that some E3 is retained in the virion. Also incorporated into CHIKV particles at substoichiometric levels is the structural protein TF, as determined by mass spectrometry of purified virus particles. As TF is produced by ribosome slippage during synthesis of the viral protein 6K and shares the same N-terminus, 6K may also be present at low levels in CHIKV particles.

Genome Organization and Features The CHIKV genome is a positive-sense, single-stranded RNA of approximately 12 kb in length. Like cellular mRNAs, the CHIKV genomic RNA (gRNA) and subgenomic RNA (sgRNA) possess both a 50 cap and 30 poly(A) tail. The capping and polyadenlyation of alphavirus RNAs are mediated by viral proteins and the cap type 0 is different from eukaryotic mRNAs. The first two nucleotides of the genome (AU) are highly conserved within the Alphavirus genus, and substitution of these nucleotides inhibits gRNA replication. The CHIKV genome is organized in a similar fashion to other alphaviruses, consisting of two sequential open reading frames (ORFs), encoding the nonstructural (ns) and structural proteins, flanked by untranslated regions (UTRs) and an internal noncoding region between the two ORFs (Fig. 1). The 50 ORF encodes the ns polyprotein, termed P1234, which is processed by the viral nsP2 protease into 4 mature ns proteins (nsP1 to nsP4). These ns proteins constitute the replicase that synthesizes new viral RNAs. The 30 ORF encodes the structural polyprotein composed of capsid, PE2 (E3 and E2), 6K/TF, and E1. As described in detail

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00019-9

173

174

Chikungunya Virus (Togaviridae)

nonstructural genes 5' UTR

nsp1

nsp2

nsp3

structural genes C E3

nsp4

E2

6K

E1

gRNA

3' UTR AAA

5' UTR

3' UTR C E3

E2

6K

E1

sgRNA

Type 0 5' Cap

Conserved Sequence Element (CSE)

Packaging Signal

AAA

Opal Stop Codon

Repeat Sequence Element (RSE)

Fig. 1 CHIKV gRNA and sgRNA organization. The CHIKV gRNA serves as a transcript that is translated into the nonstructural polyprotein and a template for negative-strand RNA synthesis. The sgRNA is transcribed from the negative-strand RNA and is translated into the viral structural polyprotein. In addition to the protein-coding regions (indicated in gray), the gRNA and sgRNA encode cis-acting elements important for the viral life cycle. To promote translation, both RNAs have a 50 cap and a poly(A) tail. Conserved sequence elements (CSEs) have roles in the evasion of host defense mechanisms, transcriptional enhancement, and transcription of viral RNAs. The packaging signal located within the nsP2 gene promotes encapsidation of gRNAs within the capsid of progeny virions. NsP4 is expressed by a readthrough mechanism of the opal stop codon. Repeat sequence elements (RSEs) at the 30 UTR vary across the CHIKV genotypes in number and length and are important for viral replication.

below, this structural polyprotein is processed by capsid autocleavage and cellular proteases into individual structural proteins that assemble into CHIKV virions.

Protein-Coding Regions The ns polyprotein is translated directly from the gRNA. A leaky opal stop codon near the 30 end of the nsP3 coding region results in the synthesis of two protein products, P123 and P1234. The structural polyprotein is translated from the sgRNA, which itself is transcribed from the subgenomic promoter active in the negative-strand viral RNA. As described in detail in the viral life cycle section, viral protein expression is temporally regulated by both the continual processing of the ns polyprotein and subsequent synthesis of viral RNA templates.

Non-Coding Elements In addition to the protein-coding functions of the genome, specific non-coding elements of the alphavirus RNA genome have important roles in genome stability, packaging, viral RNA replication, evasion of host defenses, and host range determination. These activities are mediated by cis-acting elements, such as conserved sequence elements (CSEs), packaging signals, and readthrough and frameshift elements. The function of these elements is dependent on the secondary RNA structure of the element, the nucleotide sequence, or a combination of both. These cis-acting elements are mostly localized to the 50 and 30 UTRs, but some also exist within the protein-coding regions. There are four CSEs in the CHIKV genome: the 50 CSE, the 51-nt CSE, the sgRNA promoter, and the 30 CSE. These CSEs are highly conserved amongst alphaviruses in both nucleotide sequence and genome location. (1) The 50 CSE of CHIKV and other alphaviruses consists of 60 nucleotides that fold into a stem-loop structure that effectively occludes binding by the mammalian anti-viral protein IFIT1, a key player in the type I interferon-induced antiviral state. Based on studies with Sindbis virus, this stemloop structure is also critical for RNA synthesis. (2) The 51-nt CSE in the nsP1 coding-region is another cis-acting element present in the 50 region of alphavirus genomes, and it forms two stem-loop structures. Both the structure and nucleotide sequence are important for this CSE to act as a transcriptional enhancer. (3) The promoter for the sgRNA is active in the negative-strand RNA, and it overlaps with the nsP4 coding sequence to encompass B19 nucleotides upstream and 2–5 nucleotides downstream of the sgRNA transcription start-site. Synonymous substitutions at this site inactivate the sgRNA promoter. (4) The 30 CSE consists of 19 nucleotides and is located just upstream of the polyA tail at the 30 end of the CHIKV genome. This element functions as the promoter for negative strand RNA synthesis. Specifically, the last 5 nucleotides of the 30 CSE are required for initiation of negative strand RNA synthesis, where the last nucleotide of the 30 CSE (a cytosine) is the site of transcription initiation. Furthermore, the function of the 30 CSE is complemented by the 50 UTR; as both are required for negative strand synthesis, it is hypothesized that viral RNA replication is assisted by genome circularization. In addition to the CSEs, the CHIKV genome also contains sequence regions important for genome packaging and viral mRNA translation. A packaging signal located within the CHIKV nsP2 coding region is required for genome encapsidation, and it permits the capsid protein to distinguish gRNA from sgRNA and cellular RNAs. Other functionally important regions of the CHIKV genome dictate nsP4 and TF gene expression. Translation of the gRNA predominately results in the synthesis of P123 instead of

Chikungunya Virus (Togaviridae)

175

P1234, however, low-frequency read-through of the opal stop codon near the junction of the nsP3-nsP4 coding regions results in the synthesis of P1234. A large stem-loop structure is predicted to be present immediately downstream of the opal stop codon, where it is positioned to promote readthrough events at the nsP3-nsP4 juncture. A mutation of the opal stop codon to form an arginine readthrough codon decreased CHIKV-induced disease in a mouse model. The  1 frameshift site (U UUU UUA) within the alphavirus 6K gene is responsible for the expression of the structural protein TF. Most alphavirus sequences downstream of this frameshift site form either an RNA pseudoknot or a hairpin that acts to further stimulate the  1 frameshift. However, the  1 frameshift in CHIKV and other members of the SFV complex appears to be independent of an RNA structural element. Instead, the frameshift is proposed to be stimulated by either the downstream RNA sequence or an unidentified trans-factor.

CHIKV 30 UTR The CHIKV 30 UTR is 498–723 nucleotides long, depending on the strain genotype (CHIKV genotypes are detailed in the Section “Epidemiology”), and is amongst the longest of the alphaviruses. Much of the length of the CHIKV 30 UTR is composed of repeat sequence elements (RSEs), and these vary in both number and length amongst the CHIKV genotypes. In mammalian cells, the third CHIKV RSE serves as a docking site for the cellular HuR protein which protects the viral RNA from degradation. However, most RSEs appear to be insignificant for viral replication in mammalian cells while they are required for optimal viral replication in mosquito cells. In the mosquito cell, the CHIKV 30 UTR RSEs are proposed to contain binding sites for host factors that regulate viral replication in a host-specific manner.

Life Cycle In general, alphaviruses have a similar life cycle, and much of what is understood of the CHIKV life cycle has been inferred from other alphaviruses. The alphaviral life cycle is generally composed of viral attachment and entry, ns protein expression, viral RNA replication, structural protein expression, and virion assembly (Fig. 2). Attachment & Entry CHIKV genome 5' UTR

3' UTR

nsP1

nsP2

nsP3 nsP4

C E3

nonstructural genes

Fusion & gRNA release

E2

6K

E1

virion assembly & budding

AAAA

structural genes

(+) gRNA (-) RNA

Translation of ns polyprotein

(+) gRNA

Processing in ER & Golgi

cis cleavage negative-strand replication complex (+) sgRNA

cis/trans cleavage positive-strand replication complex

Structural protein translation

trans cleavage

mature replication complex

Fig. 2 CHIKV life cycle. Upon attachment and endocytosis of CHIKV virions, the viral envelope fuses with the endosomal membrane and releases the gRNA into the host cell cytoplasm. The gRNA is translated to produce the nonstructural polyprotein P1234. Cis cleavage of the polyprotein by the nsP2 protease produces a negative-strand replication complex (P123 plus nsP4); leading to the synthesis of negative-strand RNA. The nsP2 protease then further processes P123 to yield a positive-strand replication complex (nsP1 plus P23 and nsP4), which transcribes the positivestrand gRNA and sgRNA. Complete processing of P23 produces the mature replication complex (nsP1, nsP2, nsP3, nsP4), capable of synthesizing positive-strand gRNA and sgRNA. The structural proteins are translated from the sgRNA as a polypeptide. However, capsid self-cleavage during translation yields the capsid and E3–E2–6K–E1 polypeptide. An N-terminal signal peptide in E3 directs the E3–E2–6K–E1 polypeptide into the ER. In the ER, E3–E2–6K–E1 is processed by host signal peptidases to yield PE2 (E3–E2), 6K, and E1. E1 and PE2 associate to form heterodimers. The cleavage of E3 occurs during trafficking in the Golgi complex, but E3 remains non-covalently associated with the E2/E1 heterodimer until their arrival at the plasma membrane. Meanwhile, nucleocapsid cores are formed in the cytoplasm and are transported to the plasma membrane. During budding, progeny virions acquire their E2/E1-studded lipid envelope as they egress from the host cell.

176

Chikungunya Virus (Togaviridae)

Attachment and Entry Initial CHIKV attachment to cells can be mediated by virion binding to cellular glycosaminoglycans (GAGs), a family of large, complex polysaccharides (e.g., heparan sulfate) that are present on the cell surface of most mammalian cells. Furthermore, the acquisition of enhanced GAG binding is a common cell culture adaptation of CHIKV and other alphaviruses. Virion-GAG interactions are mediated by residues in the domains A and B of the CHIKV E2 glycoprotein. For example, a mutation of CHIKV E2 domain A amino acid residues 79 and 82 to positively-charged residues enhances virion-GAG interactions, and results in an increased infectivity in cultured mammalian cells and decreased virulence in a mouse model of human disease. CHIKV attachment to cells also can be mediated by phosphatidylserine present in the viral envelope. Cellular phosphatidylserine receptors, such as T cell immunoglobulin and mucin domain-1 (TIM-1), can enhance the entry of CHIKV pseudovirions and virus-like particles in mammalian cells. Mxra8 was identified as a CHIKV entry receptor in a genome-wide CRISPR-Cas9 screen and the observation was confirmed in both cell culture and mouse models. Mxra8 is a cell adhesion molecule that also serves as a cell entry receptor for several other arthritogenic alphaviruses including Mayaro, Ross River, and o0 nyong nyong viruses. It possesses two immunoglobulin-like domains that form three contact points with E2/E1 heterodimers. Another putative mammalian cell entry factor for CHIKV is prohibitin, a multifunctional protein conserved in eukaryotes. Pretreatment of susceptible cells with anti-prohibitin-1 antibodies reduces CHIKV infection, and prohibitin-E2 interactions were shown with colocalization and immunoprecipitation studies. In mosquito cells, ATP synthase and heat shock protein 70 are the only putative CHIKV entry receptors identified to date. Following viral attachment and entry, cell-bound virus is internalized to release the gRNA into the cytoplasm. The particular details of CHIKV internalization appear to differ based upon cell type and the species from which the cell is derived. However, there are some common features regarding CHIKV internalization, including initial virion uptake by the endocytosis pathways of the host cell and the pH-dependent fusion of the viral envelope with the endosomal membrane. Regarding endocytosis of CHIKV particles, both clathrin-mediated endocytosis (CME) and clathrin-independent mechanisms may contribute. CME is a constitutively active cellular process that involves the formation of clathrin-coated pits at the cellular surface which pinch-off into the cell to form clathrincoated vesicles containing cargo such as transferrin and low-density lipoprotein. Microscopy studies examining CHIKV particle interactions identified viral interactions with both clathrin and clathrin-coated pits in human and mosquito cells, and selective targeting of endocytosis pathways can differentially affect CHIKV infection depending upon the cell type. Additional cellular factors including Eps15, dynamin, and FUZ can promote CHIKV internalization. Eps15 is required for the formation of clathrin-coated pits and dynamin mediates the pinching-off of endocytic vesicles from the plasma membrane. Each of these proteins can participate in both CME and clathrin-independent entry pathways. Finally, macropinocytosis also may mediate CHIKV internalization in some cells. Given that no study to date has been able to completely block CHIKV infection by targeting a single pathway, it may be that CHIKV particles exploit several cell-mediated internalization pathways to gain entry into host cells. Following internalization, the virion envelope fuses with the endosomal membrane to release the gRNA into the host cell. Upon endocytosis, cargo is typically delivered first to the early endosome then trafficked to late endosomes and lysosomes. In mammalian cells, CHIKV fusion occurs within Rab5 þ early endosomes. Viral fusion is a rapid process and can be completed within 40 s of virus particle delivery to the early endosome in mammalian cells. In mosquito cells, CHIKV particles associate with both Rab5 þ early endosomes and Rab7 þ late endosomes. Furthermore, knockdown of Rab7 reduces CHIKV infection of C6/36 Aedes (Ae.) albopictus cells, implying that viral fusion occurs in late endosomes in mosquito cells. Fusion is driven by the low pH of the endosome, and the differences between fusion in mammalian and mosquito cells may be due to species variation in endosomal pH. The mammalian early endosome has a pH of 6.8–5.5, whereas the pH threshold for CHIKV is pH 6.2–5.9 depending upon the specific virus strain. In the mammalian early endosome, low pH induces destabilization of the E2/E1 heterodimer and a conformational change in E2 that exposes the buried hydrophobic fusion loop of E1, which mediates membrane fusion. In brief, low pH-induced movement of the alphavirus E2 B domain uncovers the E1 fusion loop, which extends and inserts into the endosomal membrane. This allows E1 to trimerize via interactions between domains I and II of three E1 molecules in the virion spike. Afterwards, E1 domain III refolds, forming a hairpin trimer that forces the viral and host membranes together. A fusion pore forms and releases the nucleocapsid and its packaged genome into the cytoplasm where ns protein expression and genome replication can start. The endosomal membrane fusion is further promoted by the presence of cholesterol and sphingomyelin in the endosomal membrane. In addition, host factor TSPAN9 (tetraspanin 9) can indirectly promote CHIKV and SFV fusion in the early endosome, likely either by recruiting a proviral factor or sequestering an antiviral factor.

Non-Structural Protein Expression and Viral Replication Alphavirus RNA synthesis is associated with protrusions of the host cell plasma membrane. These rearranged membranes, called spherules, function as replication organelles and are the sites of RNA replication. Each spherule contains a single replication complex, and its formation is dependent on expression of viral nsPs and cleaved nsP4, in particular. NsP3 is hypothesized to contribute to spherule formation through association with host membrane curvature proteins. Later during the infection, individual spherules are endocytosed in bulk to form type-1 cytopathic vacuoles. In SFV, this assembly of cytopathic vacuoles is dependent on an endocytic process requiring PI3K and shuttling along actin and microtubule networks.

Chikungunya Virus (Togaviridae)

Table 1

General CHIKV viral protein functions

Viral protein

Enzymatic activity

Function in CHIKV replication

nsP1 nsP2

50 capping of gRNA and sgRNA; anchors replication complex to membranes Processing of ns polyprotein; RNA replication; 50 capping of gRNA and sgRNA

Capsid E3

Methyltransferase, guanylyltransferase Cysteine protease, helicase, NTPase/ RTPase ADP ribosylhydrolase RNA-dependent RNA polymerase (RdRp), adenylyltransferase Serine protease –

E2 6K/TF

– –

E1

–

nsP3 nsP4

177

Spherule formation; association with host factor FHL1 required for productive infection RNA replication; transcription of sgRNA Binds full-length gRNA; oligomerizes to form nucleocapsid Contains signal peptide for translocation of structural polypeptide into the ER; stabilizes E2-E1 heterodimers en route to surface expression Viral attachment protein; virion assembly Stability and infectivity of virions; putative role in mediating membrane permeability, viral budding, and ion channel formation Viral fusion protein

The first genes expressed during CHIKV infection are the ns polyproteins P123 and P1234. The latter is synthesized when the ribosome translating the gRNA reads through the opal codon near the nsP3-nsP4 junction (see above). Read-through to produce P1234 occurs at a low-frequency in alphaviruses, yielding lower quantities of P1234 than P123. During infection, the ns proteins are continuously processed by the nsP2 protease, and the timing and order of this processing regulates the progression of viral RNA synthesis. Upon translation, P1234 is cleaved in cis by nsP2 to form P123 and nsP4, which functions as a negative-strand replication complex. The newly synthesized negative-strand RNA is used as a template for both gRNA and sgRNA synthesis by the positive-strand replication complex. The positive-strand complex is produced when P123 is further processed by nsP2 in cis and trans to yield nsP1, P23, and nsP4. Subsequent cleavage of P23 in trans produces the mature replication complex. The mature replication complex is present within hours of infection and is markedly incapable of synthesizing negative-strand RNA. However, complete processing of the nonstructural polyprotein is not required for viral RNA replication. The individual ns proteins have distinct functions during viral replication (Table 1). The nsP1 protein encodes methyltransferase and guanylyl transferase activities that mediate 50 capping of the positive-sense gRNA and sgRNA. NsP1 also functions to associate the replication complex to host membranes and also may promote CHIKV particle release by downregulating the expression of tetherin, a host antiviral factor that traps virus particles at the cell surface during egress. As discussed above, nsP2 encodes a protease domain at the C-terminus that is required for the processing of the alphavirus nonstructural proteins. nsP2 also encodes an S-adenosyl-L-methionine (SAM) dependent RNA methyltransferase-like domain at the C-terminus and helicase and NTPase domains at the N-terminus. Both the helicase and NTPase domains play important functions in viral transcription and replication; the former recognizes CSEs important for gRNA and sgRNA synthesis, while the latter modifies the viral RNAs to have a 50 cap. Lastly, some CHIKV nsP2 protein also translocates to the host cell nucleus where it mediates transcriptional shutoff by promoting the polyubiquitination, and consequent degradation, of the RNA polymerase II catalytic subunit RPB1. This effect is mediated by a small, highly variable loop in the nsP2 SAM MTase-like domain. CHIKV nsP3 has three structural domains: a macrodomain, an alphavirus unique domain (AUD), and a hypervariable domain (HVD). The macrodomain in CHIKV has ADP-ribose hydrolase activity that promotes CHIKV replication and virulence. The AUD is capable of binding zinc, but possible other functions of this domain are presently not known. In contrast to the macrodomain and AUD, the sequence and length of the HVD, as the name implies, is highly variable among alphaviruses. This disordered region serves as a docking site for various host proteins in a virus-specific manner to promote viral replication complex assembly and function. Due to its disordered nature, this region permits large protein insertions, including fluorescent proteins and luciferase. During an infection, nsP3 associates with small, replication complexes at the plasma and endosomal membranes and large complexes associated with the cellular cytoskeleton. The HVD of CHIKV nsP3 also mediates binding with host factor FHL1; not only is FHL1 required for negative strand synthesis, but its expression determines cellular permissiveness for CHIKV. CHIKV nsP3 also sequesters cellular pools of stress granule proteins G3BP1 and G3BP2 and the mosquito homolog, Rasputin, which is important for viral replication complex formation. Other host factors known to interact with the CHIKV HVD of nsP3 include BIN1, CD2AP, MYBBP1A, NAP1L1, NAP1L4, and SH3KBP1. CHIKV nsP4 is expressed at much lower levels than the other ns proteins due to low frequency readthrough events (discussed above) and N-terminus rule degradation. Nsp4 functions as an RNA-dependent RNA polymerase (RdRp) required for the synthesis of viral RNAs. Poor in vitro expression of nsP4 has prevented structural studies, but sequence analysis predicts two functional domains. The predicted N-terminal domain is highly disordered and varies between alphaviruses. It is sensitive to point mutations and thought to mediate interactions with other viral and host proteins. The structure of the C-terminal domain is predicted to be similar to other RdRps, and this domain in SINV possesses an adenylyl-transferase activity, suggesting a mechanism for the presence of the poly(A) tail critical for viral RNA stability and function.

178

Chikungunya Virus (Togaviridae)

Structural Protein Expression and Virion Assembly The alphavirus structural proteins are expressed from the sgRNA and are initially translated as a polyprotein that is processed to produce five individual proteins (Table 1). The first cleavage of the polyprotein occurs at the capsid-E3 junction, mediated by the protease activity of the capsid protein, and it occurs before translation of the polyprotein is completed. Upon capsid self-cleavage and completion of translation, two structural polyproteins are produced due to a low frequency ribosome slippage causing  1 frameshift: E3–E2–6K–E1 and E3–E2–TF. E3 contains an N-terminal signal peptide that directs the polyprotein into the endoplasmic reticulum where complete processing will occur by host signal peptidase cleavage at both the N- and C-termini of 6K to yield PE2 (E3–E2), 6K/TF, and E1. E1 and PE2 form noncovalent heterodimers in the endoplasmic reticulum and are posttranslationally modified in the Golgi complex. Further processing of PE2 by furin in the Golgi complex produces mature E2 and E3. Following this cleavage, E3 remains noncovalently associated with E2 and prevents premature fusion of the E2/E1 dimers in the low pH of the secretory pathway. Upon arrival at the plasma membrane, E3 can dissociate from the E2/E1 spikes or remain non-covalently associated. Nucleocapsid cores, formed by the oligomerization of capsid protein around a single gRNA, are transported to the plasma membrane. Viral egress occurs via budding, whereby the nucleocapsid cores acquire a lipid envelope decorated with E2/E1 spikes.

Epidemiology CHIKV is an arbovirus that replicates in mosquitoes and mammals. The primary mosquito vectors that transmit CHIKV to humans are Ae. aegypti and Ae. albopictus. Adaptation of CHIKV to Ae. albopictus is a relatively recent event that arose due to the acquisition of an A226V point mutation in the E1 glycoprotein, and consequently, increases the risk of viral transmission in temperate climates. Transmission cycles of CHIKV can be sylvatic or urban. In the sylvatic cycle, the virus is primarily transmitted between nonhuman primates and arboreal Aedes species mosquitoes, and incidental spillovers can cause disease in humans. In the urban cycle, humans serve as the vertebrate reservoir, and mosquitoes transmit the virus from human to human. Historically, CHIKV was associated with localized outbreaks in Africa and Asia, but due to a dramatic re-emergence in 2004, CHIKV is now found in tropical and sub-tropical areas throughout the world. Phylogenetic analysis of extant strains suggest that CHIKV originated in Africa. The first confirmed epidemic of CHIKV infection in humans occurred in modern-day Tanzania in 1953, and the second recorded epidemic occurred in 1958 in Thailand. In 2004–2005, an outbreak in coastal Kenya spread to islands in the Indian Ocean region that had not previously been associated with CHIKV activity, resulting in hundreds of thousands of infections. In late 2005, CHIKV activity exploded in India, and viremic travelers continued to spread the virus to temperate regions, including Europe. As the virus strain implicated in this epidemic acquired mutations in the E1 and E2 glycoproteins capable of expanding the pool of mosquito vector species, autochthonous transmission was established in the temperate climates of Italy and France. In 2013, CHIKV arrived in the Caribbean region, marking the introduction of the virus to the western hemisphere. CHIKV spread rapidly across the Caribbean region and the Americas, resulting in millions of confirmed and suspected cases. In addition, locally transmitted cases occurred in Florida in 2014. Prior to 2004, three distinct genotypes of CHIKV had been described: Asian, East/Central/South African (ECSA), and West African lineages (Fig. 3). However, the 2005–2006 outbreak in the Indian Ocean was associated with a new CHIKV sublineage. This Indian Ocean Lineage (IOL) arose from the ECSA genotype, and it is notably characterized by the presence of the E1 A226V mutation in many isolates that increases CHIKV infectivity for Ae. albopictus mosquitoes. This point mutation occurred independently in different geographical regions and within different ECSA genetic backgrounds. During the 2005–2006 outbreak, the IOL genotype was globally disseminated by viremic travelers, including its introduction into Southeast Asia. However, the endemic Asian CHIKV strains were not displaced; instead these strains continued to be associated with CHIKV outbreaks in Asia and the Pacific region at the same time. Despite the explosive spread of the IOL/ECSA genotypes, the CHIKV strains that were first identified in the Americas were of the Asian genotype rather than of the IOL/ECSA lineage. Today, most of the CHIKV strains circulating in the Americas phylogenetically form a separate clade within the Asian genotype. However, in 2014 an ECSA CHIKV strain was introduced into Brazil. Given this genotype’s previously expanded vector range, it has elicited heightened concern over the risk of CHIKV spread to more temperate areas in the western hemisphere, including the United States.

Clinical Features CHIKV has an incubation period of 1–12 days, and in most cases, symptoms persist 1–2 weeks (Fig. 4). Unlike most other arbovirus infections, a high percentage (75%–95%) of CHIKV infections are symptomatic. For example, a case-control study during the 2009 epidemic in Thailand found 91% of laboratory-confirmed cases to be symptomatic. The acute disease presents with a sudden high fever, excruciating musculoskeletal pain (including polyarthralgia) in the periphery, headache, and rash; the afflicted joints are typically inflamed. Patients can have a very high viremia (up to 109 viral genome copies/mL) and typically remain viremic for about 1 week. In some cases, CHIKV disease is associated with atypical manifestations such as neurological and cardiovascular symptoms and even death in neonates, the elderly, and patients with pre-existing medical conditions.

Chikungunya Virus (Togaviridae)

179

Asian Lineage ECSA Lineage West African Lineage

Fig. 3 Geography of CHIKV genotypes. There are three genotypes of CHIKV strains: Asian, East/Central/South African (ECSA), and West African lineages. The geographic distribution of the ECSA and Asian genotypes has expanded dramatically since 2004, likely due to increased intercontinental travel, expansion of the CHIKV mosquito host range, and other factors.

Headache

Incubation Period

Acute Disease

Subacute Disease

Chronic Disease

High Fever

High Viremia

Rash Symptom Presentation

Viremia

1-12 d

Polyarthralgia

1-2 wks

3 wks - 3 mo

> 3 mo

Inoculation

Fig. 4 Clinical features of CHIKV infection. Unlike many other arbovirus infections, a high proportion of CHIKV infections are symptomatic. Many infected individuals develop a high titer viremia. The most common signs and symptoms include headache, fever, rash, and polyarthralgia. Symptoms typically appear 1–12 days after infection, and the acute disease can last for 1–2 weeks. Some patients can completely recover after the acute phase of the disease, however, many patients suffer from a subacute and chronic form of the disease. A subacute disease is characterized by the persistence of arthralgia for 3 weeks to 3 months after the initial infection. The chronic from of the disease can include a constant arthralgia over 3 months post-infection or a sudden relapse in clinical disease.

A hallmark of CHIKV infection is the relapse of symptoms or continued symptoms well after the acute infection period has passed. As many as half of all CHIKV patients will relapse or continue to experience arthralgia for weeks, months, or even years after the initial infection. This chronic form of CHIKV disease can be classified into two major types, a musculoskeletal disease and a chronic inflammatory rheumatic syndrome. During the relapse, pain is typically experienced in the same joints that were affected

180

Chikungunya Virus (Togaviridae)

during the acute phase of the infection, at an equal or a more intense level than previously experienced. The chronic disease form is associated with long-term sequelae, including synovitis and tenosynovitis in the wrists and ankles, joint effusion, and bone erosion/lesions. Analysis of human patients have identified some risk factors for the development of chronic CHIKV disease, which include increased age, severity and duration of initial joint pain, female sex, and pre-existing rheumatologic disease.

Pathogenesis In vitro, CHIKV is capable of productively infecting various mammalian cells, including those of human, primate, and rodent origin. A study examining CHIKV tropism for human cell types found productive infection of various adherent cells, including epithelial and endothelial cell lines, primary fibroblasts, and primary monocytes and macrophages; but no infection of T and B lymphocytes or monocyte-derived dendritic cells was seen. Productive infection is associated with a cytopathic effect and typically leads to apoptosis of virus-infected cells. Viral replication is completed within 8–16 h post-infection and results in titers of 105–108 PFU/mL depending on the cell type. In vivo, CHIKV replicates at the site of inoculation in the skin where it predominately targets fibroblasts, but it may also infect resident dendritic cells or macrophages. The virus then disseminates to lymph nodes, spleen, muscles, liver, and peripheral joints where it replicates in connective tissue fibroblasts, skeletal muscle cells, osteoblasts, and possibly in other cell types. During this acute phase of the disease, CHIKV potently induces type I interferon and other proinflammatory cytokines (including IL-1b, IL-6, IP-10, and RANTES), of which elevated levels have been correlated with increased disease severity in patients and experimentallyinfected animals. Virus replication results in musculoskeletal tissue damage and inflammation (including synovitis, tenosynovitis, and myositis) and immune cell infiltration leading to edema and degeneration of cellular and tissue architecture. Various leukocyte populations have been implicated as mediators of CHIKV-induced musculoskeletal tissue pathology in mouse models. Monocyte and neutrophil accumulation in musculoskeletal tissues is associated with CHIKV infection, and disrupting their recruitment to these sites can prevent more severe forms of pathology including the loss of bone. In addition, inoculation of CHIKV into the foot of mice causes a biphasic pattern of swelling with a small peak occurring 2–3 days after the infection and a larger peak occurring at days 6–8 postinfection. This second peak is associated with the influx of CD4 þ T cells into the foot tissue, and mice lacking CD4 þ T cells have reduced foot swelling. Thus, although CD4 þ T cells are required to develop an effective B cell response against CHIKV infection, these cells also exacerbate the joint disease. Post-acute and chronic CHIKV disease are typically defined as the persistence of disease signs and symptoms from three weeks to three months or for more than three months, respectively, after the acute disease onset. The underlying causes of chronic CHIKV disease are not well understood. The induction of autoimmunity and/or viral persistence in joint tissues have been hypothesized as contributing factors. However, chronic CHIKV disease also could be due to unresolved injury in joint tissue either caused or exacerbated by the acute infection. Animal models have provided evidence of viral persistence associated with chronic synovitis, perhaps as a result of prolonged immune activation. In mice, infectious virus can be recovered from aged mice at 60–90 days post infection, and CHIKV RNA was found in joint-associated tissues and feet up to 16 weeks after the initial infection. A recent mouse study demonstrated that muscle and dermal cells maintain CHIKV infection. In addition, CHIKV RNA can persist in these cells with no detectable surface expression of viral proteins. In nonhuman primates, infectious virus and viral RNA can be recovered weeks post inoculation from joints, muscles, lymphoid organs, and the liver. In aged macaques, RNA can be detected in the spleen as long as 5 weeks post infection. Moreover, CHIKV antigen has been detected in perivascular synovial macrophages and muscle satellite cells in tissue biopsies collected from patients diagnosed with chronic CHIKV disease.

Diagnosis, Treatment, and Prevention CHIKV infection can be misdiagnosed as dengue due to similarities in patient symptoms and geographic distribution of these two viruses, resulting in an under-diagnosis of CHIKV infection. Key clinical indicators for a CHIKV diagnosis are fever and arthralgia, and studies examining human cases have correlated these symptoms with a 74%–79% positive predictive value and with 73%–84% sensitivity. However, RT-PCR or CHIKV-specific serology is required to confirm a suspected CHIKV infection. Although studies are underway to identify CHIKV-specific antivirals, there is currently no specific treatment for CHIKV infection. Instead, patients receive general care in the form of anti-inflammatories and analgesics to reduce fever and to alleviate pain and other symptoms. Biological drugs have shown some promise; monoclonal antibodies against CHIKV have been shown to be protective against the disease in nonhuman primates and mice, even when administered at late stages of the infection. As with treatment, there are no specific prevention measures available against CHIKV. Mosquito control and methods to reduce risk of mosquito bites are the only preventative measures currently available. There is no approved vaccine available, but there are several in clinical trials. The first live, attenuated CHIKV vaccine strain (vaccine TSI-GSD-218; CHIKV strain 181/25) was developed by a serial passage of a clinical isolate in cell culture. The resulting 181/25 strain is immunogenic and protective against CHIKV challenge in animal models. While phase II clinical trials found it to be highly immunogenic, safety concerns regarding bulk production and reversion and instability of attenuation led to its discontinuation. Currently, there are two other CHIKV vaccines in phase II clinical trials: VRC-CHKVLP059-00-VP, a virus-like particle vaccine employing only the CHIKV structural proteins; and MV-CHIKV, another virus-like particle strategy using CHIKV pseudotyped, live-attenuated measles virus vaccine.

Chikungunya Virus (Togaviridae)

181

Further Reading Basore, K., Kim, A.S., Nelson, C.A., et al., 2019. Cryo-EM structure of Chikungunya virus in complex with the Mxra8 receptor. Cell 177, 1725–1737. Chen, R., Puri, V., Fedorova, N., et al., 2016. Comprehensive genome scale phylogenetic study provides new insights on the global expansion of Chikungunya virus. Journal of Virology 90, 10600–10611. Chen, R., Wang, E., Tsetsarkin, K.A., Weaver, S.C., 2013. Chikungunya virus 30 untranslated region: Adaptation to mosquitoes and a population bottleneck as major evolutionary forces. PLoS Pathogens 9, e1003591. Gardner, J., Anraku, I., Le, T.T., et al., 2010. Chikungunya virus arthritis in adult wild-type mice. Journal of Virology 84, 8021–8032. Hawman, D.W., Fox, J.M., Ashbrook, A.W., et al., 2016. Pathogenic Chikungunya virus evades B cell responses to establish persistence. Cell Reports 16, 1326–1338. Kendall, C., Khalid, H., Marietta, M., et al., 2019. Structural and phenotypic analysis of Chikungunya virus RNA replication elements. Nucleic Acids Research 47, 9296–9312. McCarthy, M.K., Davenport, B.J.J., Morrison, T.E., 2018. Chronic chikungunya virus disease. Current Topics in Microbiology and Immunology. Berlin, Heidelberg: Springer, pp. 1–26. Meertens, L., Hafirassou, M.L., Couderc, T., et al., 2019. FHL1 is a major host factor for Chikungunya virus infection. Nature 574, 259–263. Ooi, Y.S., Stiles, K.M., Liu, C.Y., Taylor, G.M., Kielian, M., 2013. Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry. PLoS Pathogens 9, e1003835. Teo, T.H., Lum, F.M., Claser, C., et al., 2013. A pathogenic role for CD4 þ T cells during Chikungunya virus infection in mice. Journal of Immunology 190, 259–269. Thiberville, S.D., Moyen, N., Dupuis-Maguiraga, L., et al., 2013. Chikungunya fever: Epidemiology, clinical syndrome, pathogenesis and therapy. Antiviral Research 99, 345–370. Tsetsarkin, K.A., Vanlandingham, D.L., McGee, C.E., Higgs, S., 2007. A single mutation in Chikungunya virus affects vector specificity and epidemic potential. PLoS Pathogens 3, e201. Voss, J.E., Vaney, M.C., Duquerroy, S., et al., 2010. Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature 468, 709–712. Young, A.R., Locke, M.C., Cook, L.E., et al., 2019. Dermal and muscle fibroblasts and skeletal myofibers survive Chikungunya virus infection and harbor persistent RNA. PLoS Pathogens 15, e1007993. Zhang, R., Kim, A.S., Fox, J.M., et al., 2018. Mxra8 is a receptor for multiple arthritogenic alphaviruses. Nature 557, 570–574.

Circoviruses (Circoviridae) Giovanni Franzo, Department of Animal Medicine, Production and Health (MAPS), Padua University, Padua, Italy Joaquim Segalés, Department of Animal Health and Anatomy, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain; Animal Health Research Center (CReSA) – Institute of Agrifood Research and Technology (IRTA), Campus UAB, Barcelona, Spain; and OIE Collaborating Center for the Research and Control of Emerging and Re-emerging Swine Diseases in Europe (IRTA-CReSA), Barcelona, Spain r 2021 Elsevier Ltd. All rights reserved. This is an update of A. Mankertz, Circoviruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00702-0.

Nomenclature

ISH In situ hybridization ORF Open Reading Frame PBMC Peripheral blood mononuclear cell PCR Polymerase Chain Reaction RCR Rolling circle replication RER Rough endoplasmic reticulum

CSB Chondroitin sulfate B cryo-EM cryo-electron microscopy ELISA Enzyme-linked immunosorbent assay HS Heparan sulfate IHC Immunohistochemistry

Glossary Apoptosis Programmed self-induced cell death. Botryoid Grape-like appearance. Bursa of Fabricius Specialized organ in birds, that is necessary for B-cell development. Cryogenic electron microscopy (cryo-EM) Cryogenic electron microscopy is an electron microscopy (EM) technique applied on samples cooled to cryogenic temperatures and embedded in an environment of vitreous water. It is an alternative to X-ray crystallography or NMR spectroscopy for

macromolecular structure determination without the need for crystallization. Open reading frame (ORF) Open reading frame is the part of a reading frame that has the ability to be translated. Phylogenetic tree Phylogenetic trees depict the evolutionary relationships among species that are believed to have a common ancestor. Rolling circle replication (RCR) Rolling circle replication is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA.

Classification The family Circoviridae was initially established in the mid-1990s and included animal viruses with circular, single-stranded DNA (ssDNA) genomes, distinct from other eukaryotic ssDNA viruses classified at the time. While originally only the genus Circovirus was defined, the variability in ssDNA genome sequences and structures prompted the creation of 2 genera (Virus Taxonomy: 2018b Release) within the Circoviridae family: Circovirus and Cyclovirus. Until 2010, only viral species responsible for relevant disease of avian species and swine were classified within the genus Circovirus, with the exception of a non-pathogenic porcine circovirus (PCV-1). However, metagenomic-based studies and degenerate PCR for circoviruses in unconventional hosts have since identified the presence of circovirus genomes in several other species including various other mammals and avian species, freshwater fishes and even ticks. However, the actual host has not been confirmed for some of these newly-detected circoviruses. A caution criterion should be followed, especially when viral sequences were obtained from samples associated to the digestive tract (e.g., feces or pharyngeal/rectal swabs) or from hematophagous parasites (i.e., tick-associated circoviruses) since the detection of viral DNA could be due to an infected meal. Members of Cyclovirus have been described since 2010 in both vertebrates and invertebrates. However, similarly to several circoviruses, a definitive host has been difficult to assign for most, if not all, cycloviruses. In fact, all of them have been identified through metagenomic analysis and degenerate PCR primers. Although they share several genomic features with circoviruses, the putative origin of replication (ori), which is marked by the nonanucleotide motif, is located on the Cap-encoding strand in cycloviruses while it is on the Rep-encoding strand in Circovirus genus members. Moreover, introns have been identified within Open Reading Frames (ORFs) of several cyclovirus genomes, but not in circoviruses. Based on genome-wide pairwise identities of Circoviruses and Cycloviruses, a species demarcation threshold of 80% genomic identity, well supported phylogenetically, has been proposed and accepted. Currently, 39 and 48 species have been classified within the Circovirus and Cyclovirus genera, respectively. (Fig. 1).

182

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00041-2

Circoviruses (Circoviridae)

183

Fig. 1 Maximum likelihood (ML) phylogenetic trees based on the complete genome of members of the viral species within the genus Circovirus (top) and Cyclovirus (bottom). The ML trees were inferred using Iq-Tree software with the GTR þ G þ I model of substitution after aligning complete genome sequences using the MAFFT algorithm. Branch support, estimated by 10,000 ultrafast bootstrap replicates, has been categorized and color-coded. The phylogenetic trees were midpoint-rooted.

184

Circoviruses (Circoviridae)

Fig. 2 Structure of the PCV-2 capsid estimated from cryo-EM of virus-like particles (PDB: 5ZBO). The surface of different capsomers has been colored with different nuances. In transparency, the ribbon structure has been depicted. The surfaces of the subunits constituting one of the pentameter clusters (center of the figure) are color-coded based on the corresponding amino-acid position within the protein. In the right insert, a single capsomer is displayed with the secondary structure highlighted in different colors (i.e., alpha helix and beta-sheets are displayed in red and purple, respectively).

Virion Structure The virion structure has been accurately characterized for some member of the genus Circovirus only, while no morphological data is available for the Cyclovirus genus members. Based on the available evidences, circoviruses are non-enveloped viruses with icosahedral T ¼ 1 symmetry. Virion sizes change depending on the viral species, although Porcine circovirus 1 and 2 (PCV-1 and PCV-2) and Beak and feather disease virus (BFDV) virions range from 15 to 25 nm in diameter. A similar viral structure can be expected based on the similarities in the capsid (Cap) protein among members of the same genus. In fact, the crystallographic three-dimensional reconstruction of PCV-2 and BFDV, despite their relative genetic distance (higher than approximately 55% and 65% at nucleotide and protein level, respectively), revealed a common organization, with 60 capsid protein subunits arranged in 12 pentameric clusters (Fig. 2). The PCV-2 capsid protein is responsible for viral attachment through binding to heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosamino-glycans on the cell surface. A recent structural study based on cryo-electron microscopy (cryo-EM) suggests that each subunit possesses up to five binding sites for heparin but only one can be occupied at a time. Therefore, each capsid might be composed of a unique combination of subunits ligated to heparin. Capsid protein also displays several basic amino acids in the N-terminal region, likely involved in DNA interaction and packaging.

Genome Recognized members of the family Circoviridae are featured by a monopartite ambisense single stranded circular DNA genome ranging from approximately 1.7 to 2.1 kb. Two main ORFs (ORF1 and ORF2), located on different strands in the double-stranded DNA (dsDNA) replicative form, are present in all circoviruses and encode the Rep proteins (encoded by ORF1), fundamental for genome duplication, and Cap protein (encoded by ORF2), the only constituent of the viral capsid. Although less characterized, other ORFs have been predicted in silico in several circoviruses. Unfortunately, experimental confirmation of their actual expression and function are often absent, with the most remarkable exception of BFDV and PCV-2. In the latter, up to 4 additional ORFs have been characterized and associated with several functions, including regulation of cell cycle, apoptosis and immune responses. Circoviridae genomes display a nonanucleotide motif marking the origin of replication (ori). The Rep proteins introduce a nick in the virion-sense strand between positions 7 and 8 of this motif, initiating circovirus genome replication through rolling circle replication (RCR). The two genera display some differences in the overall genome organization. Circovirus display two intergenic regions (IR) between ORF1 and ORF2. Also Cyclovirus genomes contain an IR between the 50 ends of Rep and Cap coding ORFs; however, the IR between the 30 ends of these ORFs is absent or significantly smaller than that observed in Circovirus. Additionally, as previously mentioned, the putative ori is located on the Rep-encoding strand of Circovirus, while in Cyclovirus is located on the opposite strand.

Circoviruses (Circoviridae)

185

Life Cycle Knowledge of the circovirus biological cycle and cell tropism is limited, and essentially obtained through studies on PCV-2. PCV-2 has been proven to display a wide cell tropism in vitro (i.e., PK-15, PEDSV.15, aortic endothelial cells, fetal cardiomyocytes, gut epithelial cells, fibrocytes, monocytes, and a few others) and it is detected in several cells and tissues in vivo. However, the capability of each cell to sustain a productive infection is limited and subject of debate. Likely, epithelial, lymphoblast and monocyte derived cells are the cell-types involved in the PCV-2 life cycle, although with different roles. In fact, while PCV-2 can be abundantly present in monocytes, macrophages and dendritic cells, its replication in these cells, if occurring, is limited. On the contrary, PCV-2 is able to actively replicate in certain subpopulations of lymphocytic cells, and mainly in epithelial cells both in vitro and in vivo. Viral binding is mediated by capsid interaction with sulfate-rich glycosaminoglycans like HS and CSB. The internalization process follows different paths depending on the cell line involved, at least in in vitro experiments. In the monocytic cell line 3D4/31, the process is clathrin mediated, while in epithelial cells the virus may follow two internalization pathways: a clathrin-mediated one and a dynamin-and cholesterol-independent, but actin- and small GTPase-dependent one. Remarkably, only the latter leads to a full infection. In monocytes, macrophages and monocyte-derived and bone marrow-derived dendritic cells, PCV-2 enters the cell through a mechanism that is not fully characterized. Similarly, virus disassembly and DNA release in the cytoplasm depend on the involved cell line. In the monocytic cell line 3D4/31, it is released at acid pH, while in epithelial cells the process seems to occur at neutral pH. In monocytes, macrophages and monocyte-derived and bone marrow-derived dendritic cells, disassembly appears to not occur. Therefore, the virus can be stored for a long time without replicating in those cells, which likely participate in spreading the virus through the body. After uncoating, viral DNA is transferred to the nucleus, where replication occurs. Since circoviruses do not possess a DNA polymerase, they depend completely on the cell machinery to replicate. PCV-2 DNA replication begins during the first S-phase and depends on both S and G2/M phases of the cell cycle. Viral RNA transcription is mediated by cellular enzymes after ssDNA is converted to dsDNA. The transcription strategy is complex and several RNAs have been detected, although the role of most is unknown. Two Rep proteins (Rep and Rep0 ) are translated from PCV-2 ORF1 RNA. While they share the same 50 end, they have different 30 (Rep0 is obtained through alternative splicing). While both proteins display domains involved in RCR and are fundamental for genome replication, some of their other functions are likely different. Additionally, the replicase proteins form homo- and hetero-dimers and the ratio of Rep versus Rep0 changes over the course of the infection, suggesting that different protein complexes could have a role in specific reactions. In contrast, only one Rep protein has been identified for other circoviruses. DNA encapsidation occurs in the nucleus and virions are released into the cytoplasm by budding. Viral factories observed in the cytoplasm are bounded by multiple membranes, suggesting that the rough endoplasmic reticulum (RER) or Golgi apparatus may play a role in virion assembly. Clusters of viral particles, potentially moving from organelle membrane to the cytoplasm in the final replication stages, are then released from the plasma membrane by exocytosis or by budding from the cell membrane. Finally, an increase in cell death during the last hours of infection coupled with the presence of large clusters of virions forming paracrystalline arrays suggests that cell lysis could be another path for virus release. Knowledge of other circovirus life cycles is essentially absent; however, the genome and protein resemblance, coupled with some similarity in the involved tissues and induced lesion (when present), could suggest a similar cycle. No consistent data are currently available on the biological cycle of cycloviruses. Since the putative cyclovirus ori and predicted Rep share conserved features with members of the genus Circovirus, cycloviruses are also thought to replicate through RCR.

Epidemiology The first Circovirus (later renamed PCV-1) was recognized in the 70s as a contaminant of porcine kidney cell lines. Thereafter, other circoviruses (i.e., BFDV, Pigeon circovirus (PiCV) and Goose circovirus (GoCV)) associated with clinical syndromes were reported in avian species in the 80s. However, despite overt clinical signs, the economic impact of these diseases remained limited. In the middle 90s, a dramatic change occurred with the emergence and discovery of PCV-2 as one of the most devastating viral diseases of the swine industry. This event drew attention towards this group of viruses. Since then, many Circovirus members have been reported in other avian and mammalian species (Fig. 1). Noteworthy, in most instances, no association with clinical signs could be demonstrated. Canine circovirus (CanineCV) and Porcine circovirus 3 (PCV-3) are two potential exceptions since some evidence of their potential role in overt clinical syndromes has been reported. Similar conclusions can be drawn for Cyclovirus members. Although some of these viruses were initially recognized in diseased subjects, including human beings, no causal association could be consistently established. Moreover, the actual host tropism is poorly known for most of them due to the lack of viral isolation and experimental models. In addition, several Cyclovirus DNAs have been detected and characterized from feces or digestive tract samples, which hinders the differentiation between contamination and actual infection. Paleovirology studies, investigating the presence of sequences related to circoviruses integrated in the genome of vertebrate species, suggest that the origin of this virus family may exceed 40–50 million years. Therefore, the very recent discovery of new species is likely attributable to the improved identification and diagnostic capabilities. However, at least in some instances, other factors likely played a role in the emergence and expansion of these viruses. PCV-2 is a particularly illustrative example.

186

Circoviruses (Circoviridae)

Retrospective studies demonstrated its presence in archived samples since 1962 and molecular clock-based studies dated it back at least at the beginning of the 20th century. Consequently, the switch from an apparently subclinical infection to a clinical one was likely due to the modern intensive farming conditions, which provided several cofactors (i.e., high animal density, stressing factors, management and husbandry related factors, co-infections, etc.) that enhanced viral replication and lead to signs and lesion appearance. A differential susceptibility of particular genetic lines of commercial pigs also seems to be involved. Finally, the evolution and adaptation of the virus is likely, as suggested by the emergence of new strains and genotypes over time, characterized by a partially different virulence and immunological features. The multifactorial nature of PCV-2 disease is supported by the frequent subclinical course of the infection and by the evidence that PCV-2 alone is typically not sufficient to cause disease under experimental conditions. A comparable scenario could be true for PCV-3, a virus that likely circulated in the swine population for decades and is now frequently detected in both symptomatic and asymptomatic animals. Even in non-intensively raised species like dogs and psittacine birds, CanineCV and BFDV infection can be detected both in severely ill and healthy subjects. The reasons behind this behavior are not fully understood. If a similar picture can be expected for other Circoviridae remains to be established. Unfortunately, most Circovirus and Cylovirus species have been detected only one or a few times. Therefore, data on their prevalence and distribution are largely lacking. The species for which adequate epidemiological and molecular epidemiology data are available, like BFDV, PCV-1, PCV-2, PCV-3, Duck circovirus (DuCV), GoCV and CanineCV, display a worldwide distribution with an absent-to-limited geographic clustering. However, these viruses affect domestic animals and their spreading patterns appear deeply connected to animal trade and movements. A different pattern could characterize circoviruses infecting wild species only. Significantly, some human cycloviruses initially considered to be limited to certain areas have demonstrated a wider geographic distribution than originally thought. Circoviruses were traditionally thought to have a narrow host range. Nevertheless, even if a “preferred” host can be identified, sporadic detection in other species is described with increasing frequency. PCV-2 has been detected in wild boars, feral pigs, peccaries, but also in mice (used as experimental models) and rats (especially captured on commercial swine farms), ruminants and in fur animals (minks, foxes and raccoon dogs). Similarly, PCV-3 was detected in cows, dogs and laboratory mice, as well as in wild animals including wild boar, chamois, roe deer and related ticks. The actual clinical impact and epidemiological role of those hosts remains unclear, with the only exception being wild boars whose high PCV-2 and PCV-3 infection frequency suggests they could be a PCV reservoir in nature. The close genetic similarity between strains detected in wild boar and domestic pigs testifies to a frequent strain exchange between these populations, although the directionality of the viral flux is still unknown. Detection of CanineCV in significant numbers of wild carnivores, including foxes, wolves and badgers, has been described, sometimes even with the presence of clinical signs. As a group, ssDNA viruses are considered to be rapidly evolving organisms, characterized by a substitution rate comparable to that of RNA viruses. Although few studies have been performed on circovirus evolution, they seem to make no exception. A certain heterogeneity in evolutionary rate characterize different species. For example, PCV-2 substitution rates approach 10–3 substitutions/site/year, while that of PCV-3 is about two orders of magnitude lower. A similar slower rate evolution was also estimated for PCV-1. Detailed studies explaining such differences are still lacking, but a combination of higher population size and stronger selective pressures imposed by the host could play a pivotal role. Within-host viral variability has been investigated for PCV-2 only, revealing the emergence of several subpopulations over the course of infection. Significantly, the higher within-host variability was observed in the capsid coding region, suggesting the immune response as one of the major drivers of viral evolution. A comparable tendency has been described at a population level, where the capsid coding gene is the most variable and the one under stronger selective pressure. Importantly, evidence of vaccine-driven immune-selection have been provided and the emergence of multiple genotypes (i.e., cluster of strains with an independent evolution) alternating over time is partially due to an escape from the herd immunity developed against previously circulating genotypes. In addition to mutation, frequent recombination has been reported within Circovirus member species, occurring especially in the intergenic regions. Epidemiological studies performed on PCV-2 have confirmed that recombination is a common phenomenon both within and between genotypes and demonstrated that recombinant viruses can have an evolutionary success comparable to parental ones. Viral persistence, shedding and infection routes have been investigated for a limited subset of circoviruses. Oronasal is the primary route of PCV-2 transmission, which was detected in most animal secretions and excretions, including saliva, nasal, tonsillar, bronchiolar and ocular secretions, milk, colostrum, semen, feces and urine. Vertical, transplacental infection can also occur. Even though experimental studies have not been performed for PCV-3, virus detection in the same excretions, secretions and in aborted fetuses suggests similar transmission modes. Orofecal transmission is likely for CanineCV. Horizontal transmission occurs for BFDV, PiCV, DuCV and GoCV, even though vertical transmission cannot be excluded; in fact, vertical transmission has been suggested for PiCV.

Clinical Features Circoviruses have been detected in the presence of several clinical syndromes in different species. However, a consistent association between infection and emergence of overt clinical signs is often lacking. Moreover, even when a causal nexus has been inferred, circovirus diseases are typically multi-factorial, infection being necessary but not sufficient to induce disease.

Circoviruses (Circoviridae)

187

PCV-2 is the most studied pathogen belonging to this family and represents an illustrative example. PCV-2 seroprevalence suggests that PCV2 infection is ubiquitous all over the world and most pigs come in contact with the virus during their life. Nevertheless, morbidity is much lower and variable among countries, farms and over time. Even if most of infections remain subclinical (PCV-2-SI), their relevance cannot be ignored since even asymptomatic cases have been associated to a decrease in productive performance. A number of clinical syndromes have been linked to PCV-2 infection, collectively named porcine circovirus diseases (PCVD). These can be broadly divided in systemic diseases and organ specific ones, although the differentiation can be only effectively made at a histological rather than clinical level. PCV-2 systemic disease (PCV-2-SD) is characterized by wasting or weight loss, pallor of the skin, respiratory distress, diarrhea, low grade fever and occasionally icterus. Enlarged subcutaneous lymph nodes are also a common finding. The onset of disease may be acute, leading to death within a few days in some pigs while others display a more chronic course and fail to gain weight or thrive. Morbidity in affected farms can reach 4%–30% (with a sporadic higher peak) and mortality ranges from 4% to 20%. PCV-2 lung disease (PCV-2-LD) and PCV-2 enteric disease (PCV-2-ED), postulated once as independent entities, are now believed to be part of PCV-2-SD. The frequent presence of co-infections and the role of PCV-2 in porcine respiratory disease complex (PRDC) further complicates the clinical scenario. PCV-2 is also involved in reproductive disease (PCV-2-RD), leading to mummifications, late term abortions and stillbirths. Return-to-estrus might also be linked to early embryo infection. However, the relevance of PCV-2-RD is apparently low under field conditions, likely because of the high seroprevalence and presumably immune coverage in adult sows. Actually, PCV-2-RD affected herds are typically start-up herds with high number of gilts or PCV-2 seronegative herds. Porcine dermatitis and nephropathy syndrome (PDNS) has also been linked to PCV-2 infection, although the role of PCV-2 in pathogenesis is debated. The most evident sign is the presence of irregular, red-to-purple macules and papules in the skin, primarily on the hind limbs and perineal area, but sometimes with a broader distribution, which over time evolve to dark crusts. The disease prevalence is low (less than 1%), but the lethality can reach 50%–100%. Pigs typically die within a few days after the onset of clinical signs due to acute renal failure while the survivors recover in 7–10 days. Similar clinical symptoms have been described in PCV-3 infected pigs, including PDNS, reproductive disorders, enteric and respiratory disease. Nevertheless, PCV-3 is often detected in healthy animals. To date, high amounts of PCV-3 have been found in few cases of reproductive disease and nursery pigs with periarteritis and myocarditis. CanineCV was initially identified in dogs with vasculitis and/or hemorrhagic gastroenteritis in the United States. Over time, it has been reported in several countries in presence of enteric signs, such as vomiting and diarrhea with hematochezia, and lymphadenitis. CanineCV has also been identified in histological lesions of the cerebrum from foxes with neurological disorders. Among avian species, psittacine BFDV is probably the most relevant. Like in PCVDs, mortality and clinical signs in birds infected with BFDV are variable and dependent on the species of bird, age at the time of infection, presence or absence of concurrent infections and possibly the viral strain involved. Typically, the disease is detected in young birds of less than 3 years and is clinically characterized by a chronic, progressive loss of feathers, and, in some species, deformities in beak and claw. The infection can last for years; however, more often the animals die after some months due to secondary infections, since the condition causes an immunosuppressive state. Circoviral infection in pigeons is associated with high morbidity and low mortality in most disease outbreaks. A broad range of signs and severity is observed in infected pigeons, including poor performance, diarrhea, and ill thrift. Circovirus particles were detected in a canary affected by a condition known as “black spot”, which is associated with abdominal enlargement, gall bladder congestion, failure to thrive, dullness, anorexia, lethargy, and feather disorder. Growth retardation, feathering disorders and increased losses have been reported in circovirus-infected geese. Clinical signs of circovirus infection in Mallard ducks include feather dystrophy along the dorsum (hemorrhagic shafts) and poor body condition with low weight gain. In one report, circovirus infected finches displayed nasal discharge, dyspnea, anorexia, and depression. A brief summary of most relevant clinical signs associated to circovirus infection are reported in Table 1. The clinical role of cycloviruses is essentially unknown. Of note, CyCVs have been detected in cases of respiratory, enteric and neurological infections in humans and the presence of the virus has been found in serum, stool or cerebrospinal fluid (CSF) of patients with acute flaccid paralysis, paraplegia or suspected central nervous system infections. However, a causal association has not been proven.

Pathogenesis Most circoviruses are considered able to cause disease in their corresponding hosts under certain conditions (not always well determined), although the majority of infections end up with subclinical scenarios. Circoviruses mainly replicate in epithelial and/ or endothelial cells and, to a limited extent, in immune cells such as macrophages and lymphocytes. These animals developing clinical disease tend to suffer from immunosuppression and damage in the lympho-reticular tissues. Among avian circoviruses, pathogenesis is mainly inferred from the knowledge generated on BFDV. This virus is considered to be epitheliotropic, targeting the basal epithelial layer of the feather and feather follicles. In fact, it is possible to detect circovirusspecific intranuclear inclusions in these cells, which are actively dividing. BFDV inclusions are also present in the cytoplasm of macrophages; although it has been suggested that most of the accumulated virions in the inclusions are due to the phagocytosing

188

Table 1

Circoviruses (Circoviridae)

Summary of the main criteria to diagnose the well-recognized circoviral diseases in different animal species

Disease

Diagnostic criteriaa Clinical signs and gross pathology Histopathology

Viral detection

PCV-2-systemic disease (PCV-2-SD)

Wasting Lymphadenopathy Interstitial pneumonia

Moderate to severe lymphocyte depletion with Moderate to high amount of PCV-2 in granulomatous inflammation of lymphoid tissues lymphoid tissues by IHC/ISHb

PCV-2-reproductive disease (PCV-2-RD)

Abortion Mummified fetuses Weak-born piglets Myocardial necrosis

Necrotizing and fibrosing myocarditis

Moderate to high amount of PCV-2 in myocardium by IHC/ISH

PCV-2-subclinical infection (PCV-2-SI)

No clinical signs Lesser average daily weight gain

None or mild lymphocyte depletion with or without mild granulomatous inflammation of lymphoid tissues

Low amount of PCV-2 in tissues by IHC/ISH (or lack of PCV-2 in tissues by IHC/ISH but positive PCR)

Fibrinous-necrotizing glomerulonephritis Systemic necrotizing vasculitis

PCV-2 definitive causality on PDNS has not been demonstrated, so, its detection is not yet part of the diagnostic criteria

Porcine dermatitis and Skin red papules and macules nephropathy Kidney generalized petechiae syndrome (PDNS) Nephromegaly PCV-3 associated diseasec

Abortion Mummified fetuses Weak-born piglets Congenital malformation

Non-suppurative systemic perivasculitis Non-suppurative myocarditis

Moderate to high amount of PCV-3 by ISH or low Ct values for qPCRd

Canine circovirus infection

Bloody diarrhea Vomiting Hemorrhagic enteritis

Systemic vasculitis Intestinal crypt necrosis Lymphocyte depletion of lymphoid tissues

Moderate to high amount of canine circovirus in tissues

Mink circovirus infection Beak and feather disease (BFD)

Diarrhea Loss of feathers Deformity of the beak Deformity of the claw

Gastro-enteritis Epidermal cell necrosis, epidermal hyperplasia and hyperkeratosis Macrophage intracytoplasmic inclusions in affected epidermis and feather pulp

Amount of virus not defined to establish a diagnostic criterion Moderate to high amount of BFDV by ISH or qPCR in tissues

Pigeon circovirus infection

Increased rearing losses Atrophy of thymus and bursa of Fabricius

Follicular hyperplasia and mild lymphocyte necrosis Moderate to high amount of PiCV by to lymphocyte depletion and histiocytosis ISH or qPCR in tissues Macrophage intracytoplasmic inclusion bodies

Duck circovirus infection

Feathering disorders Growth retardation and low body weight

Lymphocytic depletion, necrosis, and degeneration of lymphoid tissues

Moderate to high amount of DuCV in tissues by qPCR

Lymphocytic depletion and necrosis of the bursa Not well established criterion; Finch, goose, gull and Poorly described, but mainly canary circovirus growth retardation and low body of Fabricius presumed, moderate to high amount infections weight in all these species Macrophage intracytoplasmic inclusion bodies of these viruses clinical signs and gross lesions are indicative; the final diagnosis is established by means of histopathology and virus detection. immunohistochemistry/in situ hybridization. c although not yet clarified, PDNS has also been linked to PCV-3 infection. d quantitative PCR. a

b

activity of these cells rather than from endogenously replicated virus. The mechanism by which lymphoid organs such as the thymus and bursa of Fabricius become depleted in clinically affected birds is not entirely clear; it has been speculated that an indirect mechanism involving a cytokine-mediated reduction in lymphocyte differentiation might happen. Lymphocyte depletion is probably transient in subclinically infected animals, but persistent in diseased birds. Therefore, BFD-affected birds are susceptible to opportunistic secondary bacterial, chlamydial, and fungal infections. Pathogenesis of circovirus infection in mammals has been documented more thoroughly than that of avian viral species. Specifically, the vast majority of data derives from PCV-2, the most studied Circoviridae family member, which has been associated with a number of economically significant diseases (PCVD). The greatest amount of PCV-2 is found in the cytoplasm of monocyte/ macrophage lineage cells, both in PCV-2 diseased and subclinically infected pigs. In vitro studies have shown that PCV-2 is able to infect these cells, persisting for extended periods of time with apparently no or minimal active replication. Therefore, monocytic

Circoviruses (Circoviridae)

189

Fig. 3 Schematic representation of the pathogenesis of PCV-2 infection in post-natal swine. Adapted from: Porcine circovirus type 2: the virus, the disease and the vaccine, from Segalés, J. Grupo Asís Biomedia S.L., 2017.

cells seem not be the primary target for PCV2 replication and may represent a dissemination mechanism for PCV-2 throughout the host. Moreover, PCV-2 causes impairment of dendritic cell (DC) functionality in vitro, which may alter innate and virus-specific immune responses. However, in vivo information about these effects is minimal. Cell types that supports PCV-2 replication in vivo are not completely elucidated. Initial work suggested that macrophages and lymphocytes did not play a significant role in PCV-2 replication, but subsequent research indicated that those cells types (mainly macrophages) may support replication to a certain degree. Ultrastructural studies in cell culture and lymph nodes from PCV-2-SD affected pigs have suggested that mitochondria may play a role in PCV-2 replication. Further characterization of PCV2-infected leukocyte subpopulations from peripheral blood mononuclear cells (PBMCs) indicated that mainly circulating T (CD4 þ and CD8 þ ) and potentially B lymphocytes, support PCV-2 replication; PBMC-derived monocytes apparently do not. PCV-2 also replicates in fetal myocardiocytes in experimentally infected porcine fetuses and in zona pellucida-free morulae and blastocysts. Fig. 3 displays a proposed scheme for the post-natal pathogenesis of PCV-2 infections. PCV-2 viremia is first detectable around 7 days post-inoculation (PI), and viral load increases and reaches a peak between days 14 and 21 PI. Around 10–14 days PI, seroconversion against PCV2 is usually detected, especially if pigs remain subclinically infected. Under field conditions, the most likely age of infection is at the late nursery period and sero-conversion consequently occurs around 7–12 weeks of age; antibodies may last at least until 28 weeks of age. However, PCV-2 infection can occur at any age, and so evidence of infection and/or seroconversion must be monitored by means of viral detection (usually PCR or quantitative PCR) and/or serological (mainly ELISAs) tests, respectively. Besides its detection in serum samples, PCV-2 can be detected in several organs, with lymphoid tissues containing the highest viral loads. PCV-2 amounts measured by quantitative PCR methods are usually 2–4 logs higher in lymphoid tissues compared to serum. The virus can be detected in virtually all tissues of a viremic pig. Immunohistochemistry and in situ hybridization techniques have shown presence of PCV-2 in epithelial cells from the kidney and respiratory tract, endothelial cells, lymphocytes, enterocytes, hepatocytes, smooth muscle cells, and pancreatic acinar and ductular cells. The capacity of a pig to mount an adaptive immune response is key to prevent the progression of PCV-2 infection towards PCV-2-SD (Fig. 3). The onset of PCV-2 antibodies is followed by a decrease of viral loads and, eventually, resolution of viraemia. Long-lasting PCV2 viraemia and/or detection of PCV-2 in tissues, however, has been described in experimentally subclinically infected pigs despite the presence of high PCV-2 antibody titers. Inefficient humoral responses and, in particular, a poor PCV-2-neutralizing antibody response, are linked to increased viral replication, which results in severe lymphoid lesions and PCV-2-SD (Fig. 3). Under field conditions, from lactation to the growing-finishing period, the proportion of PCV-2 infected pigs and viral loads increase gradually, coincidentally with the waning of maternal-derived immunity. Porcine embryos are susceptible to PCV-2 infection and their susceptibility varies with developmental stage. Virological and clinical outcomes of direct intra-fetal inoculation of PCV-2 at 57, 75 and 92 days of gestation have been described. At 21 days postinfection it was found that PCV-2 replicated in all inoculated fetuses but virus replication was significantly higher in fetuses inoculated at 57 days of gestation. In such fetuses, the heart displayed myocarditis-like lesions and harbored the highest virus load and number of infected cells. Fetuses inoculated at 75 days of gestation were stillborn at birth, while fetuses inoculated at 92 days of gestation did not show any lesion at birth. Fetuses inoculated at 75 or 92 days of gestation, but not the fetuses inoculated at

190

Circoviruses (Circoviridae)

57 days, had antibodies against PCV-2 at delivery. PCV-2-infected cells identified in fetuses inoculated at different stages of gestation include cardiomyocytes, hepatocytes and cells of the monocyte/macrophage lineage. Intranasal inoculation of pregnant sows or artificial insemination with purposely contaminated semen have also caused PCV-2 infection of fetuses/newborn piglets and reproductive failure. In contrast, sows artificially inseminated with semen of boars experimentally infected with PCV2 did not cause reproductive problems or infection of dams. Therefore, it is believed that the amount of PCV-2 naturally shed in boar semen is not sufficient to infect sows or their fetuses. Little is known about the pathogenesis of PDNS, but it has been suggested that excessive PCV-2 serum antibody titers may be linked with the triggering of the condition. This hypothesis still awaits experimental confirmation: to date, studies on tissue sections from pigs with PDNS have failed to consistently demonstrate PCV-2 antigen or nucleic acid associated with PDNS lesions. Therefore, PCV-2 causality of PDNS is mainly based on circumstantial epidemiological and pathological evidences, but there is no experimental demonstration of such causation. No data is currently available regarding the pathogenesis of PCV-3 infection. The first lesion descriptions associated to PCV-3 resembled those traditionally described for PCV-2-SD and PDNS, including as well myocarditis and systemic inflammation. The latest reports have emphasized the presence of PCV-3 genome mainly in inflammatory infiltrates in animals displaying perivascular inflammation at a systemic level, affecting especially kidney, liver, lymph nodes, lung and brain. Moreover, viral nucleic acid has also been found in follicular areas of lymph nodes, but usually in a low amount, similar to the location and distribution observed in pigs subclinically infected by PCV-2. Detection of the virus in tissues of stillborns and pigs with congenital anomalies points to a potential role in reproductive disease and, therefore, vertical transmission as an important route of dissemination. Little is also known about the pathogenic role of CanineCV. On postmortem examination, some infected dogs showed histological lesions of necrotizing vasculitis and hemorrhage throughout the gastrointestinal tract, as well as granulomatous lymphadenitis of the lymph nodes. By in situ hybridization (ISH) analysis, abundant cytoplasmic viral nucleic acid has been detected in macrophages and monocytes in lymphoid tissues, including germinal centers and subcapsular and medullary sinuses of lymph nodes and ileal Peyer’s patches. However, CanineCV has been detected in feces of healthy dogs and dogs with hemorrhagic diarrhea with a similar frequency. Therefore, CanineCV does not seem to be involved as a primary agent in dogs, and other co-factors might be necessary to elicit disease, similarly to other circoviruses. The pathogenesis of cyclovirus infections is virtually unknown. All viruses of this genus have been detected in a number of samples by means of metagenomics and degenerate PCR methodologies, without an apparent association to a particular disease or condition. In fact, such detection has usually focused on the virome in the vertebrate and invertebrate species where they were detected. So far, the meaning of cyclovirus detection under disease scenarios if purely speculative. Importantly, techniques able to detect cycloviruses in situ in tissues or their putative humoral and cellular immune responses are still lacking.

Diagnosis Diagnosis is the investigation or analysis of the cause of a condition, situation, or problem. Regarding Circoviridae family members, the terminology diagnosis should be applied at two major levels: identification of a disease problem (causal association of the agent with the condition) and identification of an agent, its components or antibodies against it. Taking into account the ubiquitous distribution of circoviruses and cycloviruses, disease causality is difficult to be established since in most of the cases a multifactorial origin is involved. Moreover, for just a limited number of circoviruses (but not of cycloviruses), a relatively consistent association with disease has been demonstrated (Table 1). The consistency of the association between infection and clinical disorder has been established using three main criteria: presence of compatible clinical signs, characteristic gross and histopathologic lesions, and presence of the virus (ideally by immunohistochemistry and/or in situ hybridization methods) within the lesions. The most studied diseases have been by far PCVDs caused by PCV-2 and BFD caused by BFDV. In both cases, a significant clinical problem was first detected in swine and psitacine birds and the agent was subsequently discovered. Relevant gross and microscopic lesions were described in both diseases, being lymphocyte depletion and granulomatous inflammation of lymphoid tissues together with the presence of intracytoplasmic botryoid inclusion bodies common and very characteristic hallmarks. These histopathological lesions were subsequently confirmed in a number of circoviral infections in avian species (i.e., pigeon and geese). Importantly, avian circoviral diseases have been also associated to chronic, progressive loss of feathers, and, in some species, deformities of the beak and claws. The key diagnostic aspects of circoviral diseases are summarized in Table 1. Techniques able to detect the viral antigen or nucleic acid within tissues displaying histopathological lesions are key to establish a sound diagnosis of disease. Immunohistochemistry (IHC) and ISH methodologies have been successfully developed to detect a number of circoviruses. Most of the techniques have been used to investigate the viral tropism for pathogenesis research purposes. Nevertheless, a routine diagnostic activity using IHC or ISH is performed only on a limited number of circovirus infections of clinical relevance such as PCVDs and BFD. The study of Circoviridae family members has benefited enormously from the development of molecular techniques. As previously mentioned, Cycloviruses and most of Circovirus species have not been isolated yet. Therefore, the identification of their genome represents the only proof of their existence. In consequence, care must be taken in terms of interpretation of a given result.

Circoviruses (Circoviridae)

191

A positive result indicates the presence of nucleic acid of the virus in a sample but does not provide any clue about the effects of the agent on the vertebrate/invertebrate animal. Quantification of the genome implies a further step, since the amount of genome for a number of circoviruses has been correlated with disease occurrence. However, the number of genome equivalent copies is also difficult to interpret for these agents that have never been associated with a certain disease. Taking into account that their infection dynamics are unknown and a particular amount of nucleic acid for clinical infections versus being part of the normal virome has not been established, the significance of their presence is still to be established. A number of laboratory techniques have been developed to detect antibodies against circoviruses (not yet against cycloviruses). Some techniques such as the immunofluorescent antibody test and the immunoperoxidase monolayer assay have been used mainly in research, but implied the availability of a viral isolate, which limited their use almost exclusively for PCV-2. The development of ELISA tests using recombinant proteins widened the possibility of detecting antibodies against different circoviruses, such as PCV-2, BFDV, PiCV, GoCV, DuCV and mink circovirus (MiCV). However, only PCV-2 ELISA commercial kits are currently available for routine diagnosis. No serological tests have so far been developed for the detection of antibodies against cycloviruses.

Disease Control Control of circoviral diseases have been mainly focused on counteracting the specific clinical signs of pet animals by means of supportive treatments. Importantly, these signs can vary greatly among individuals due to the immunosuppressive nature of the disease in clinically affected animals. The initial control strategies for livestock species, mainly against PCV-2-SD, were developed to counteract the different infectious and non-infectious risks or triggering factors for the development of clinical disease. Noteworthy, most of the implemented measures were based on empirical results and not on contrasted and fully demonstrated efficacy. Overall, efforts were mainly directed to diminish/control the concomitant microbiological burden, decrease viral transmission and potentiate resistance against disease. Among these measures, the Madec’s 20-point plan (a list of general zootechnical actions to lower the impact of disease) was the most practiced one, since they significantly decreased the percentage of mortality in severely affected farms. These measures were designed to reduce the infection pressure of PCV-2 as well as other infections, improve hygiene and reduce stress at different production stages. The advent of commercial PCV-2 vaccines radically changed the clinical situation regarding PCV-2-SD and allowed the discovery of the so-called PCV-2-SI. The excellent efficacy of PCV-2 commercial vaccines surpassed the most optimistic expectations, to the point that nowadays it is the most used swine vaccine in the worldwide market. PCV-2 vaccines allowed a decrease in the impact of clinical signs and mortality, and more homogeneous animals in terms of body weight and improved average daily weight gain. Importantly, several commercial vaccines have appeared in the market and the different meta-analyzes published on the subject indicated no significant differences in their efficiency to control PCV-2-SD and a significant impact for all the products when compared to non-vaccinated animals. The most widely used vaccination scheme includes the use of the commercial products in piglets around weaning, using one (most often) or two doses. Also, several vaccines have been licensed to be applied in sows, which have been used to prevent PCV-2-SD in piglets (sows/gilts are vaccinated at the end of gestation) or to potentially prevent PCV-2-RD (sows/gilts are vaccinated pre-mating). So far, no commercial vaccines are available for other circoviral diseases. Although the lack of virus isolates may potentially jeopardize initial testing of potential vaccine prototypes, most of the experimental work is performed with recombinant proteins derived from the sequences of the Cap gene from circoviruses. Using this approach several vaccine prototypes have been developed against some avian circoviruses and tested under experimental conditions. Such developments include prototypes against BFDV, DuCV and PiCV. No vaccine products have been developed against the rest of circoviruses, mainly because they do not represent a significant threat to the corresponding infected animal populations. Also, it is not expected that vaccines will be developed against cycloviruses since their association with disease outcome is still to be demonstrated.

Conclusions The number of family Circoviridae members has been significantly expanded in the last 10 years, and the novel taxonomy implied the removal of the genus Gyrovirus and the addition of the genus Cyclovirus together with the genus Circovirus. A number of circovirus species are unequivocally linked to clinical disease in animals, mainly in pigs and birds, although the associated disorders are of multifactorial nature. A common denominator of these conditions is the affection of the immune system of the animals, leading to immunosuppression in severely affected ones. Curiously, a large number of circoviruses and all known cycloviruses to date have never been isolated, and the only evidence of their existence is genome detection. Contrasting with the simplicity of these viral genomes is the complex and still poorly understood pathogenesis. Progress in molecular biology techniques and phylogenetic studies will surely facilitate elucidating evolutionary relationships among classified as well as newly identified members of the family Circoviridae in the future.

192

Circoviruses (Circoviridae)

Further Reading Franzo, G., Cortey, M., Segalés, J., Hughes, J., Drigo, M., 2016. Phylodynamic analysis of porcine circovirus type 2 reveals global waves of emerging genotypes and the circulation of recombinant forms. Molecular Phylogenetics and Evolution 100, 269–280. Klaumann, F., Correa-Fiz, F., Franzo, G., et al., 2018. Current knowledge on Porcine circovirus 3 (PCV-3): A novel virus with a yet unknown impact on the swine industry. Frontiers in Veterinary Science 5, 1–13. Lv, Q., Guo, K., Zhang, Y., 2014. Current understanding of genomic DNA of porcine circovirus type 2. Virus Genes 49, 1–10. Meng, X.J., 2013. Porcine Circovirus Type 2 (PCV2): Pathogenesis and Interaction with the Immune System. Annual Review of Animal Biosciences 1, 43–64. Meng, X.J., 2012. Spread like a wildfire-The omnipresence of porcine circovirus type 2 (PCV2) and its ever-expanding association with diseases in pigs. Virus Research 164, 1–3. Nauwynck, H.J., Sanchez, R., Meerts, P., et al., 2012. Cell tropism and entry of porcine circovirus 2. Virus Research 164, 43–45. Rosario, K., Breitbart, M., Harrach, B., et al., 2017. Revisiting the taxonomy of the family Circoviridae: Establishment of the genus Cyclovirus and removal of the genus Gyrovirus. Archives of Virology 162, 1447–1463. Segalés, J., 2012. Porcine circovirus type 2 (PCV2) infections: Clinical signs, pathology and laboratory diagnosis. Virus Research 164, 10–19. Steiner, E., Balmelli, C., Herrmann, B., Summerfield, A., McCullough, K., 2008. Porcine circovirus type 2 displays pluripotency in cell targeting. Virology 378, 311–322. Todd, D., 2004. Avian circovirus diseases: Lessons for the study of PMWS. Veterinary Microbiology 98 (2), 169–174.

Relevant Website https://talk.ictvonline.org/ictv-reports/ictv_online_report/ssdna-viruses/w/circoviridae Circoviridae.

Coronaviruses: General Features (Coronaviridae) Paul Britton, The Pirbright Institute, Pirbright, United Kingdom © 2021 Published by Elsevier Ltd. This is an update of D. Cavanagh, P. Britton, Coronaviruses: General Features, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00370-8.

Glossary Infectious clone A full-length DNA copy of an RNA virus genome from which full-length viral RNA can be generated, leading to production of infectious virus. Nidovirales (nidoviruses) An order comprising positivesense RNA coronaviruses, toroviruses, arteriviruses, and roniviruses that have a common genome organization and expression, similar replication/transcription strategies, and

form a nested set of 30 co-terminal subgenomic mRNAs (nidus, Latin for nest). Ribosomal frameshifting Movement (shift) backward by one nucleotide of a ribosome that is on an RNA, caused by particular RNA structures and sequences. Subsequent continuation of the progress of the ribosome is in a different open reading frame.

Introduction Coronaviruses are known to cause disease in humans, other mammals, and birds. They cause major economic loss, sometimes associated with high mortality, in neonates of some domestic species (e.g., chickens, pigs). In humans, they are responsible for respiratory and enteric diseases. Coronaviruses do not necessarily observe species barriers, as illustrated most graphically by the recent spread of three zoonotic viruses in humans, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome (MERS) coronavirus and in 2019 SARS-CoV-2 responsible for the disease Covid-19 causing a global Pandemic infection. All of which appear to have originated from bats via an intermediate wild animal species before infecting humans, with lethal consequences. As a group, coronaviruses are not limited to particular organs; target tissues include the nervous system, immune system, kidney, and reproductive tract in addition to many parts of the respiratory and enteric systems. A great advance in recent years has been the development of systems (‘infectious clones’) for modifying the genomes of coronaviruses to study all aspects of coronavirus replication, and for the development of new vaccines.

Taxonomy and Classification Coronaviruses are part of the Order Nidovirales, which is divided into five Suborders; the Arnidovirineae, which contains the Family Arteriviridae; the Cornidovirineae, which contains the Family Coronaviridae; the Tornidovirineae, which contains the Family Tobaniviridae; the Mesonidovirineae, which contains the Family Mesonviridae; and the Ronidovirineae, which contains the Family Roniviridae. The viruses generically known as coronaviruses fall within the Coronaviridae Family which are divided into four genera, the Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Members of the Nidovirales Order have a similar genome organization and produce a nested set of subgenomic mRNAs and contain related specialized enzymes that are involved in replication of the RNA. Coronaviruses have been placed into different genera, initially on the basis of serological relationships which has subsequently been refined by gene sequencing.

Virion Properties Virions have a buoyant density of approximately 1.18 g ml1 in sucrose. Being enveloped viruses (Fig. 1(a)), they are destroyed by organic solvents such as ether and chloroform.

Virion Structure and Composition All coronaviruses have four structural proteins in common (Fig. 1(b)): a large surface glycoprotein (S; c. 1150–1450 amino acids); a small envelope protein (E; c. 100 amino acids, present in very small amounts in virions); integral membrane glycoprotein (M; c. 250 amino acids); and a phosphorylated nucleocapsid protein (N; c. 500 amino acids). Some Betacoronaviruses have an additional structural glycoprotein, the hemagglutinin-esterase protein (HE; c. 425 amino acids). This is not essential for replication in vitro and may affect tropism in vivo.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00139-9

193

194

Coronaviruses: General Features (Coronaviridae)

Fig. 1 (a) Electron micrograph of an IBV virion, showing the bulbous S protein. (b) Diagrammatic representation of the composition and structure of a coronavirus virion: S, spike glycoprotein; M, M0 , integral membrane glycoprotein; E, small envelope protein; N, nucleocapsid protein; NC, nucleocapsid (nucleoprotein) comprising the RNA genome and N protein. Cryoelectron microscopy of TGEV has indicated a core structure comprising the NC and the M protein. Two forms of M protein (M, M0 ) have been observed for TGEV (see main text). The coronavirus membrane proteins, S, E, M, and M0 , are inserted into a lipid bilayer (MEM) derived from internal cell membranes. (b) Reproduced from González, J.M., Gomez-Puertas, P., Cavanagh, D., Gorbalenya, A.E., Enjuanes, L., 2003. A comparative sequence analysis to revise the current taxonomy of the family. Coronaviridae. Archives of Virology 148, 2207–2235, with permission from Springer-Verlag.

Virions are c. 120 nm in diameter, although they can be up to twice that size, and the ring of S protein spikes is approximately 20 nm deep. When present, the HE protein forms a layer 5–10 nm deep. In some species, the S protein is cleaved into two subunits, the N-terminal S1 fragment being slightly smaller than the C-terminal S2 sequence. The S protein is anchored in the envelope by a transmembrane region near the C-terminus of S2. The functional S protein is highly glycosylated and exists as a trimer. The bulbous outer part of the mature S protein is formed largely by S1 while the stalk is formed largely by S2, having a coiled-coil structure. S1 is the most variable part of the S protein; some serotypes of the avian coronavirus, infectious bronchitis virus (IBV) differ from one another by 40% of S1 amino acids. S1 is the major inducer of protective immune responses. Variation in the S1 protein enables one strain of virus to avoid immunity induced by another strain of the same species. The M glycoprotein is the most abundant protein in virions. In most cases, only a small part (B20 amino acids) at the N-terminus protrudes at the surface of the virus. There are three membrane-spanning segments and the C-terminal half of the M protein is within the lumen of the virus. In transmissible gastroenteritis virus (TGEV), a proportion of M molecules have four membrane-spanning segments, resulting in the C-terminus also being exposed on the outer surface of the virus (M0 in Fig. 1(b)). The E protein is anchored in the membrane by a sequence near its N-terminus.

Coronaviruses: General Features (Coronaviridae)

195

Fig. 2 Schematic diagram representing the genomic expression of the avian coronavirus IBV. The upper part of the diagram shows the IBV genomic RNA, with the various genes highlighted as boxed regions. The black boxes represent the transcription regulatory sequences (TRSs) found upstream of each gene and direct the synthesis, via negative-sense counterparts, of the sg mRNAs (2–6 for IBV). The leader sequence, represented by a gray box, is at the 50 end of the genomic RNA and at the 50 ends of the sg mRNAs. The genomic RNA is translated to produce two polyproteins, pp1a and pp1ab, that are cleaved by virus-encoded proteases to produce the replicase proteins. The structural proteins, S, E, M, and E, and the accessory proteins, 3a, 3b, 5a, and 5b, produced from IBV genes 3 and 5, respectively, are translated from the sg mRNAs. The proteins produced by the sg mRNAs are represented by lines below the corresponding sg mRNA. All of the sg mRNAs, except the smallest species, are polycistronic but only produce a protein from the 50 -most gene. The ribosome frameshift (RFS) region, denoted as a black circle on the genomic RNA, directs the –1 frameshift event for the synthesis of pp1ab. Translation of the genomic RNA results in the production of pp1a. However, the translating ribosomes undergo the –1 frameshift about 30% of the time resulting in pp1ab. The 50 and 30 UTR sequences are represented as single lines downstream of the leader and N gene sequences, respectively.

Genome Organization and Expression Coronaviruses have the largest known RNA genomes, which comprise 28–32 kb of positive sense, single-stranded RNA. The overall genome organization being 50 UTR–polymerase gene–structural protein genes–30 UTR, where the UTRs are untranslated regions (Fig. 2). The first 60–90 nucleotides at the 50 end form a leader sequence. The structural protein genes are in the same order in all coronaviruses: (HE)–S–E–M–N. Interspersed among these genes are one or more other genes, often referred to as accessory genes as they have been demonstrated to be non-essential for replication in several coronaviruses using reverse genetic systems. The accessory genes encode small proteins of mainly unknown function. Some of these genes encode two or three proteins. In some cases (e.g., gene 3 of IBV and gene 5 of murine hepatitis virus (MHV)), translation of the third and second open reading frame (ORF), respectively, is affected by the preceding ORFs acting as internal ribosome entry sites. The proteins encoded by these small ORFs are mostly not required for replication in vitro; some of them might function as antagonists of innate immune responses. Following entry into a cell and the release of the virus ribonucleoprotein (genome surrounded by the N protein) into the cytoplasm, ribosomes translate gene 1, which is approximately 20 kb, into two polyproteins (pp1a and pp1ab). These are cleaved by gene 1-encoded proteases, to generate 15 or 16 proteins (Fig. 3). Translation of gene 1 results in two polyproteins, translation of the ORF 1a region results in pp1a and translation of the ORF 1b region results in pp1ab, the latter involves ribosomal frameshifting, which has two elements, a slippery site followed by an RNA pseudoknot. At the slippery site (UUUAAAC in IBV), the ribosome slips one nucleotide backward and then moves forward, this time in a –1 frame compared with translation ORF 1a, resulting in the synthesis polyprotein 1ab, in which the proteins encoded by ORF 1b are in effect fused with the proteins from ORF 1a. Proteins, including the RNA-dependent RNA polymerase (RdRp), from gene 1 associate to form the replicase complex, which is membrane associated. Coronavirus subgenomic mRNAs are generated by a discontinuous process. At the beginning of each gene is a common sequence (CUUAACAA in the case of IBV) called a transcription regulatory sequence (TRS). When the polymerase producing the nascent negative sense RNA, reaches a TRS, RNA synthesis is attenuated, followed by continuation at the 50 end of genomic RNA. This results in the addition of a negative copy of the leader sequence to the negative-sense RNA, resulting in a negative-sense copy of an sg mRNA. Of course, progress of the polymerase is not always halted at a TRS. Rather, it sometimes continues, producing a nested set of negative-sense sg mRNAs. These negative-sense copies of the sg mRNAs are the templates for the generation of the positive-sense sg mRNAs (Fig. 2). The amount of each sg mRNA does not necessarily decrease in a linear fashion; the efficiency of termination by a TRS is dependent on adjacent sequences, which are different for each gene. The leader sequence is found at the very 50 end of the genomic RNA and at the 50 ends of each sg mRNA.

196

Coronaviruses: General Features (Coronaviridae)

Fig. 3 Organization of the coronavirus replicase gene products. Translation of the coronavirus replicase ORF 1a and ORF 1b sequences results in pp1a and pp1ab; the latter is a C-terminal extension of pp1a, following a programmed –1 frameshift event (see legend to Fig. 2). The two polyproteins are proteolytically cleaved into 10 (pp1a; nsp1–11) and 16 (pp1ab; nsp1–16) products by the papain-like proteinases (PL1pro and PL2pro) and the 3C-like (3CLpro) proteinase. The PLpro proteinases cleave at the sites indicated with a black triangle and the 3CLpro proteinase cleaves at the sites indicated with a gray triangle. The nsp11 product of pp1a is produced as a result of the ribosomes terminating at the ORF 1a translational termination codon, a –1 frameshift results in the generation of nsp12, part of the pp1ab replicase gene product. Various domains have been identified within some of the replicase products: Ac is a conserved acidic domain; X ¼ ADP-ribose 10 -phosphatase (ADRP) domain; PL1 and PL2 the two papain-like proteinases; Y is a conserved domain; TM1, TM2, and TM3 are conserved putative transmembrane domains; 3CL ¼ 3CLpro domain; RdRp, RNA-dependent RNA polymerase domain; HEL, helicase domain; ExoN, exonuclease domain; NendoU, uridylate-specific endoribonuclease domain; MT, 20 -O-ribose methyltransferase domain. nsp’s 7–9 contain RNA-binding domains (RBDs).

Replication Cycle The N-terminal (S1) part of the S protein mediates attachment to cells. It is a determinant of host species specificity and, in some cases, pathogenicity, by determining susceptible cell range (tissue tropism) within a host. The C-terminal S2 part triggers fusion of the virus envelope with cell membranes (plasma membrane or endosomal membranes), which can occur at neutral or slightly acidic pH, depending on species or even strain. The virus glycoproteins (S, M, and HE, when present) are synthesized at the endoplasmic reticulum. Both subunits of the S protein are multiply glycosylated, while the M protein has one or two glycans close to its N-terminus. Interestingly, glycosylation of the M protein can be either N- or O-linked, depending on the type of coronavirus, although experiments using reverse genetics showed that conversion of an O-linked glycosylated M protein to an N-linked version had no effect on virus growth. Early and late in infection, formation of virus particles can occur in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) and endoplasmic reticulum, but most assembly occurs in the Golgi membranes. The M protein is not transported to the plasma membrane; its location at internal membranes determines the sites of virus particle formation. It interacts with the N protein (as part of the RNP) and C-terminal part of the S protein, retaining some, though not all, of the S protein at internal membranes. The E protein is essential for virus particle formation, though it is not known how it functions. It has a sequence that determines its accumulation at internal membranes, and its interaction with the M protein. The latter’s interaction with the N protein enables the formation of virus particles with spikes.

Genome Replication and Recombination Following infection of a susceptible cell, the coronavirus genomic RNA is released from the virion into the cytoplasm and immediately recognized as an mRNA for the translation of the replicase pp1a and pp1ab proteins. These proteins are cleaved by ORF1a-encoded proteases, after which they become part of replicase complexes for the synthesis of either complete negative-sense copies of the genomic RNA or negative-sense copies of the sg mRNAs. The negative-sense RNAs are used as templates for the synthesis of genomic RNA and sg mRNAs (Fig. 2). RNA synthesis appears to take place in replication complexes associated with rearranged host membranes generated by several pp1a derived proteins. Following synthesis of the sg mRNAs, the structural proteins are produced for the assembly and encapsidation of the de novo-synthesized genomic RNA, resulting in the release of new infectious coronavirus virions. The release of new virions starts 4–7 h after the initial infection. As indicated above, the synthesis of the sg mRNAs is the result of a discontinuous process in which the synthesis of a negative-sense copy of an sg mRNA is completed

Coronaviruses: General Features (Coronaviridae)

197

by the addition of the negative-sense leader sequence by a recombination mechanism. If a cell is infected with two related coronaviruses, the polymerase may swap between two RNA templates, in a similar way to addition of the leader sequence. This ‘copy-choice’ mechanism of genetic recombination results in a chimeric RNA. Such RNAs may give rise to new viruses with modified genomes with a capacity to infect a different cell and, in some cases, new host species.

Diseases and Host Range Probably all coronaviruses replicate in epithelial cells of the respiratory and/or enteric tracts, though not necessarily producing clinical damage at those sites. The avian coronavirus (a Gammacoronavirus), IBV, not only causes respiratory disease but can also damage gonads in both females and males, and causes serious kidney disease (dependent on the strain of virus, and to some extent on the breed of chicken). IBV is able to replicate at virtually every epithelial surface in the host. Some coronaviruses have their most profound effect in the alimentary tract (e.g., the porcine coronavirus transmissible gastroenteritis virus (TGEV) causes Z90% mortality in neonatal pigs). Human coronaviruses are known to be associated with enteric and respiratory diseases (e.g., diarrhea), in addition to respiratory disease. SARS-CoV was also associated with diarrhea in humans, in addition to serious lung disease. Other coronaviruses, for example, MHV and porcine HEV, spread to cells of the central nervous system, producing disease, for example, acute or chronic demyelination in the case of MHV. Coronavirus replication and disease are not necessarily restricted to a single host species. Canine enteric CoV and feline CoV can replicate and cause disease in pigs; these two viruses have proteins with very high amino acid identity to those of porcine TGEV. Canine respiratory CoV has proteins, including the S protein (which is the attachment protein and a determinant of host range), with very high amino acid identity (Z95%) to other group 2 viruses Hu CoV-OC43 and BCoV. This raises the possibility of co-infection in these hosts. There is evidence that pheasant CoV can infect chickens, and IBV infect teal (a duck), though without causing disease. The most dramatic demonstration that coronaviruses can have a wide host range was provided by SARS-CoV. This appears to have its origin in bats, was transferred to various other species (e.g., civet cat) that were captured for trade, and then caused lethal disease in humans. A similar zoonotic pathway has been hypothesized for the infection of humans by SARS-CoV-2, again with an origin in bats but via, as yet unknown, intermediator host or hosts, though generally accepted via a wild animal in a live animal market. Persistent infections in vivo are well known for MHV, and less well known for other coronaviruses (e.g., IBV). Following infection of very young chickens, IBV is re-excreted when hens start to lay eggs. The trigger for release is probably the stress of coming into lay. The S protein is a determinant of both tissue tropism within a host and host range. This has been elegantly demonstrated by genetic manipulation of the genome of MHV, which is unable to attach to feline cells. Replacement of the MHV S protein gene with that of CoV from feline coronavirus resulted in a recombinant virus that was able to attach, and subsequently replicate in, feline cells. However, other proteins can also affect pathogenicity. Research with genetically modified coronaviruses, using targeted recombination or ‘infectious clones’, has shown that modifications to proteins encoded in ORF1 and the small genes interspersed among the structural protein genes, result in attenuation of pathogenicity. Although the roles of these ‘accessory proteins’ are not known, this may offer a route to the development of a new generation of live vaccines. Currently, the most widely used prophylactics for control of IBV in chickens include killed vaccines and live vaccines attenuated by passage in embryonated eggs. However, disease control is complicated by extensive variation in the S1 protein which is the inducer of protective immunity.

See also: Coronaviruses: Molecular Biology (Coronaviridae). Enveloped, Positive-Strand RNA Viruses (Nidovirales). Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae). Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Coronaviridae)

Further Reading Britton, P., Cavanagh, D., 2007. Nidovirus genome organization and expression mechanisms. In: Perlman, S., Gallagher, T., Snijder, E.J. (Eds.), Nidoviruses. Washington, DC: ASM Press, pp. 29–46. Britton, P., Cavanagh, D., 2007. Avian coronavirus diseases and infectious bronchitis vaccine development. In: Thiel, V. (Ed.), Coronaviruses: Molecular and Cellular Biology. Norfolk, UK: Caister Academic Press, pp. 161–181. Cavanagh, D., 2003. SARS vaccine development: Experiences of vaccination against avian infectious bronchitis coronavirus. Avian Pathology 32, 567–582. Cavanagh, D., 2005. Coronaviridae: A review of coronaviruses and toroviruses. In: Schmidt, A., Wolff, M.H. (Eds.), Coronaviruses with Special Emphasis on First Insights Concerning SARS. Basel: Birkhäuser, pp. 1–54. Cavanagh, D., 2005. Coronaviruses in poultry and other birds. Avian Pathology 34, 439–448. Enjuanes, L., Almazán, F., Sola, I., Zuñiga, S., 2006. Biochemical aspects of coronavirus replication: A virus–host interaction. Annual Reviews in Microbiology 60, 211–230. González, J.M., Gomez-Puertas, P., Cavanagh, D., Gorbalenya, A.E., Enjuanes, L., 2003. A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae. Archives of Virology 148, 2207–2235. Masters, P.S., 2006. The molecular biology of coronaviruses. Advances in Virus Research 66, 193–292. Siddell, S., Ziebuhr, J., Snijder, E., 2005. Coronaviruses, toroviruses and arteriviruses. In: Mahy, B.W.J., ter Meulen, V. (Eds.), Topley and Wilson’s Microbiology and Microbial Infections, Virology. London: Hodder Arnold, pp. 823–856.

Coronaviruses: Molecular Biology (Coronaviridae) X Deng and SC Baker, Loyola University of Chicago, Maywood, IL, United States r 2021 Elsevier Ltd. All rights reserved. This is a reproduction of X. Deng, S.C. Baker, Coronaviruses: Molecular Biology, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B978-0-12-801238-3.02550-2.

Glossary Cell tropism Process that determines which cells can be infected by a virus. Factors such as receptor express can influence the cell type that can be infected. Discontinuous transcription Process by which the coronavirus leader sequence and body sequence are joined to generate subgenomic RNAs.

Double membrane vesicles (DMVs) Vesicles that are generated during coronavirus replication when viral replicase proteins sequester host cell membranes. These vesicles are the site of coronavirus RNA synthesis. Transcriptional regulatory sequences (TRSs) Sequences that are recognized by the coronavirus transcription complex to generate leader-containing subgenomic RNAs.

Introduction Coronaviruses (CoVs) were first identified during the 1960s by using electron microscopy to visualize the distinctive spike glycoprotein projections on the surface of enveloped virus particles. It was quickly recognized that CoV infections are quite common, and that they are responsible for seasonal or local epidemics of respiratory and gastrointestinal disease in a variety of animals. CoVs have been named according to the species from which they were isolated and the disease associated with the viral infection. Avian infectious bronchitis virus (IBV) infects chickens, causing respiratory infection, decreased egg production, and mortality in young birds. Bovine coronavirus (BCoV) causes respiratory and gastrointestinal disease in cattle. Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) cause gastroenteritis in pigs. These CoV infections can be fatal in young animals. Feline infectious peritonitis virus (FIPV) and canine coronavirus (CCoV) can cause severe disease in cats and dogs. Depending on the strain of the virus and the site of infection, the murine CoV mouse hepatitis virus (MHV) can cause hepatitis or a demyelinating disease similar to multiple sclerosis. CoVs also infect humans. Human coronaviruses (HCoVs) 229e and OC43 are detected worldwide and are estimated to be responsible for 5–30% of common colds and mild gastroenteritis. Interestingly, HCoV-OC43 and BCoV share considerable sequence similarity, indicating a likely transmission across species (either from cows to humans or vice versa) and then adaptation of the virus to its host. In contrast to the relatively mild infections caused by HCoV-229e and HCoV-OC43, the CoV responsible for severe acute respiratory syndrome (SARS-CoV) causes atypical pneumonia with a 10% mortality rate. Two additional HCoVs, HCoV-NL63 and HCoV-HKU1, were identified using molecular methods and are associated with upper and lower respiratory tract infections in children, and elderly and immunosuppressed patients. Most recently, a coronavirus likely endemic in the camel population has been associated with Middle East Respiratory Syndrome (MERS) in humans. MERS-CoV can cause a lethal respiratory disease and renal syndrome, and mortality is highest in older individuals with co-morbidities such as diabetes or immunosuppression. CoVs are grouped according to sequence similarity as alpha-, beta, gamma or deltacoronaviruses (see www.viprbrc.org for a complete list of reference genomes for coronaviruses). To date, the most infamous example of zoonotic transmission of a CoV is the outbreak of SARS in 2002–03. We now know that the outbreak started with cases of atypical pneumonia in the Guangdong Province in southern China in the fall of 2002. The infection was spread to tourists visiting Hong Kong in February, 2003, resulting in the dissemination of the outbreak to Hong Kong, Vietnam, Singapore, and Toronto, Canada. After attempting to treat cases of atypical pneumonia in Vietnam and acquiring the infection himself, Dr. Carlo Urbani alerted the World Health Organization (WHO) that this disease of unknown origin may be a threat to public health. The WHO rapidly organized an international effort to identify the cause of the outbreak, and within months a novel CoV was isolated from SARS patients and identified as the causative agent. Sequence analysis revealed that the virus was related to, but distinct from, all known CoVs. This led to an intensive search for an animal reservoir for this novel CoV. Initially, the masked palm civet and raccoon dog were implicated in the chain of transmission, since a SARS-CoV-like virus could be isolated from some animals found in wild animal markets in China. However, SARS-CoV-like viruses were never detected if these animals were captured from the wild, indicating that the civets may have only served as an intermediate host in the chain of transmission. Further investigation revealed that the likely reservoir for SARS-CoV is the Chinese horseshoe bat (Rhinolophus spp.), which is endemically infected with a virus, named bat-SARS-CoV, that is closely related to SARS-CoV (Fig. 1(a)). The bringing together of CoV-infected bats and susceptible civet cats and humans in live animal markets in China likely contributed to the emergence of SARS-CoV in 2002–2003. In 2012, a novel coronavirus eventually termed Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was isolated from a patient suffering from pneumonia in Saudi Arabia. MERS-CoV was then linked to multiple outbreaks of severe respiratory disease in the Middle East. Sequence analysis revealed that MERS-CoV was similar to but distinct from CoVs previously identified in bats in Europe and Asia. Further epidemiologic studies revealed that MERS-CoV could be isolated from the respiratory and

198

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.02550-9

Coronaviruses: Molecular Biology (Coronaviridae)

199

Fig. 1 Zoonotic transmission of emerging coronaviruses. (a) SARS-like coronaviruses are endemic in bats in Southern China. Virus transmission events may occur when animals are brought together in live animal markets. Replication of the virus in an intermediate host allows for the acquisition of adaptive mutations that allow for an expanded host range. SARS-CoV adapted to use the ACE-2 receptor in humans and can be spread by respiratory droplets. Infection with SARS-CoV generally results in symptomatic infection (red dots) characterized by fever and pneumonia. Super-spreader events (one person infecting many others) followed by global travel led to the pandemic of 2003. The quarantine of infected individuals and their contacts led to the cessation of the outbreak. (b) MERS-like viruses have been identified in bats and may be the progenitor of the virus now recognized to be endemic in camels in parts of the Middle East and Africa. MERS-CoV infection of camels seems to cause a respiratory disease with no significant mortality in young or adult camels. MERS-CoV infection in humans, proposed to be acquired from contact with camels or raw camel milk or meat, may cause pneumonia (red dot) or mild or asymptomatic illness (blue dot). Currently, there seems to be limited human-to-human transmission of MERS-CoV.

gastrointestinal tract of young camels and that the majority of adult camels tested had neutralizing antibodies to MERS-CoV, consistent with the idea that this virus was endemic in the camel population in the Middle East and in some regions of Africa. Currently, the majority of MERS-CoV infections are proposed to be acquired from either contact with camels or camel products such as raw milk or meat. There is some evidence supporting human to human transmission of MERS-CoV in hospital and home settings, however to date the virus has not evolved to transmitted easily from human to human (Fig. 1(b)). Estimates of mortality from MERS-CoV are in the range of 30–40%, so there is significant concern about the pandemic potential of this emerging coronavirus should it evolve to transmit efficiently between humans. The existence of animal reservoirs for SARS-CoV and MERSCoV presents the possibility for sporadic re-emergence of these significant human pathogens. By improving our understanding of the molecular aspects of CoV replication and pathogenesis, we may facilitate development of appropriate antiviral agents and vaccines to control and prevent diseases caused by known and potentially emerging CoVs.

Molecular Features of CoVs CoV virions (Fig. 2(a)) are composed of a large RNA genome, which combines with the viral nucleocapsid protein (N) to form a helical nucleocapsid, and a host cell-derived lipid envelope which is studded with virus-specific proteins including the membrane (M) glycoprotein, the envelope (E) protein, and the spike (S) glycoprotein. CoV particles vary somewhat in size, but average about 100 nm in diameter. The genomic RNA (gRNA) inside the virion, which ranges in size from 27 to 32 kb for different CoVs, is the largest viral RNA identified to date. CoV gRNAs have a broadly conserved structure which is illustrated by the SARS-CoV genome shown in Fig. 2(b). The gRNA is capped at the 50 end, with a short leader sequence followed by two long open reading frames (ORFs) encoding the replicase polyprotein. The remaining part of the genome encodes the viral structural and so-called accessory proteins. The structural protein genes are always found in the order S–E–M–N, but accessory protein genes may be interspersed at various sites between the structural genes. SARS-CoV has the most complex genome yet identified, with eight ORFs encoding accessory proteins. The expression of these ORFs is not required for viral replication, but they may play a role in the pathogenesis of SARS and MERS. In addition, the products of accessory genes may be incorporated into the virus particle, potentially altering the tropism or enhancing infectivity. For SARS-CoV, the proteins encoded in ORFs 3a, 6, 7a, and 7b have been shown to be incorporated in virus particles, but the exact role of these proteins in enhancing virulence is not yet clear. The features of CoV structural proteins are shown in Fig. 3. For each structural protein, a schematic diagram of the predicted structure of the protein is shown on the left and a linear display of the features is shown on the right. The CoV spike glycoprotein is essential for attachment of the virus to the host cell receptor and fusion of the virus envelope with the host cell membrane. CoV spike glycoproteins assemble as trimers with a short cytoplasmic tail and hydrophobic transmembrane domain anchoring the protein into the membrane. The spike glycoprotein is divided into the S1 and S2 regions, which are sometimes cleaved into separate proteins by cellular proteases during the maturation and assembly of virus particles. S1 contains the receptor-binding domain (RBD) and has been shown to provide the specificity of attachment for CoV particles. The cellular receptors and corresponding RBDs in S1 have been identified for several CoVs. MHV binds to murine carcinoembryonic antigen-related cell adhesion molecules (mCEACAM1 and mCEACAM2); TGEV, FIPV, and HCoV-229e bind to species-specific versions of

200

Coronaviruses: Molecular Biology (Coronaviridae)

Fig. 2 CoV virion and the genome of SARS-CoV. (a) Schematic diagram of a CoV virion with the minimal set of four structural proteins required for efficient assembly of the infectious virus particles: S, spike glycoprotein; M, membrane glycoprotein; E, envelope protein; and N, nucleocapsid phosphoprotein which encapsidates the positive-strand RNA genome. (b) Schematic diagram of the gRNA of SARS-CoV. Translation of the first two open reading frames (ORF1a and ORF1b) generates the replicase polyprotein. ORFs encoding viral structural and accessory (orange) ORFs are indicated at the 30 end of the genome. (a) Reprinted from Masters PS (2006) The molecular biology of coronaviruses. Advances in Virus Research 66: 193–292, with permission from Elsevier.

aminopeptidase N. Interestingly, both HCoV-NL63 and SARS-CoV have been shown to bind to human angiotensin-converting enzyme 2 (ACE2). MERS-CoV binds to dipeptidyl peptidase (DPP4 or CD26). ACE2 and DPP4 are expressed in both the respiratory and gastrointestinal tracts, consistent with virus replication at both of these sites. Once the S1 portion of the spike has engaged the host cell receptor, the protein undergoes a dramatic conformational change to promote fusion with the host cell membrane. Depending on the virus strain, this can occur at the plasma membrane on the surface of the cell, or in acidified endosomes after receptor-mediated endocytosis. The critical elements in the conformational change are the heptad repeats, HR1 and HR2, and the fusion peptide, F. After engaging the receptor, there is a dissociation of S1 which likely triggers the rearrangement of S2 so that HR1 and HR2 are brought together to form an antiparallel, six-helix bundle. This new conformation brings together the viral and host cell membranes and promotes the fusion of the lipid bilayers and introduction of the nucleocapsid into the cytoplasm. During infection, the spike glycoprotein is also present on the surface of the infected cell where it may (depending on the virus strain) promote fusion with neighboring cells and syncytia formation. The spike glycoprotein is also the major antigen to which neutralizing antibodies develop. The spike protein is a target for development of therapeutics for treatment of CoV infections. Monoclonal antibodies directed against the spike neutralize the virus by blocking binding to the receptor; synthetic peptides that block HR1-HR2 bundle formation have also been shown to block CoV infection. The membrane (M) and envelope (E) proteins are essential for the efficient assembly of CoV particles. M is a triple-membranespanning protein that is the most abundant viral structural protein in the CoV virion. The ectodomain of M is generally glycosylated, and is followed by three transmembrane domains and an endodomain which is important for interaction with the nucleocapsid protein and packaging of the viral genome. The E protein is present in low copy numbers in the virion, but is important for efficient assembly. In the absence of E protein, few or no infectious virus particles are produced. The exact role of the E protein in the assembly of virus particles is still unknown, but recent studies suggest that E may act as an ion channel. The nucleocapsid protein (N) is an RNA-binding protein and associates with the CoV gRNA to assemble ribonucleoprotein complexes. The N protein is phosphorylated, predominantly at serine residues, but the role of phosphorylation is currently unknown. The N protein has three conserved domains, each separated by highly variable spacer elements. Domains 1 and 2 are rich in arginine and lysine residues, which is typical of many RNA-binding proteins. Domain 3 is essential for interaction with the M protein and assembly of infectious virus particles. The N protein has been shown to be an important cofactor in CoV RNA synthesis and is proposed to act as an RNA chaperone to promote template switching, as described below.

Coronaviruses: Molecular Biology (Coronaviridae)

201

Fig. 3 Diagrammatic representation of the spike trimer assembled on membranes, with the S1 receptor binding domain (RBD) and S2 fusion domain indicated. The linear map of spike indicates the location of the RBDs for three CoVs, and the relative location of the heptad repeat domains 1 and 2 (HR1 and HR2) which mediate the conformational changes required to present the fusion peptide (F) to cellular membranes. The membrane (M), envelope (E), and nucleocapsid (N) proteins represented in association with membranes or viral RNA. The linear map of each protein highlights the transmembrane domains of M and E and the RNA-binding and M protein-binding domains of N. Domains 1 and 2 of N are rich in arginine and lysine (indicated by þ ). Reprinted from Masters PS (2006) The molecular biology of coronaviruses. Advances in Virus Research 66: 193–292, with permission from Elsevier.

Replication and Transcription of CoV RNA The replication and transcription of CoV RNA takes place in the cytoplasm of infected cells (Fig. 4). The CoV virion attaches to the host cell receptor via the spike glycoprotein and, depending on the virus strain, the spike mediates fusion directly with the plasma membrane or the virus undergoes receptor-mediated endocytosis and spike-mediated fusion with endosomal membranes to release the viral gRNA into the cytoplasm. Once the positive-strand RNA genome is released, it acts as a messenger RNA (mRNA) and the 50 end (ORF1a and ORF1b) is translated by ribosomes to generate the viral RNA-dependent RNA polymerase polyprotein, termed the viral replicase. Translation of ORF1b is dependent on ribosomal frameshifting, which is facilitated by a slippery sequence and RNA pseudoknot structure present in all CoV gRNAs. The replicase polyprotein is processed by replicase-encoded proteases (papain-like proteases and a poliovirus 3C-like protease) to generate 16 mature replicase products. These viral replicase proteins sequester host cell membranes to generate distinctive double-membrane vesicles (DMVs) that have been shown to be the site of CoV RNA synthesis. The replicase complex on the DMVs then mediates the replication of the positive-strand RNA genome to generate full-length and subgenomic negative-strand RNAs, and the subsequent production of positive-strand gRNAs and sgmRNAs. The sgmRNAs are translated to generate viral structural and accessory proteins, and virus particles assemble with positive-strand gRNA in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and bud into vesicles, with

202

Coronaviruses: Molecular Biology (Coronaviridae)

Fig. 4 Replication cycle of CoVs. The spike glycoprotein on the virus particle interacts with host cell receptors to mediate fusion of the virus and host cell membranes and release of the positive-strand RNA genome into the cytoplasm. The 50 -proximal open reading frames (ORF1a and ORF1b) are translated from the gRNA to generate the replicase polyprotein. The replicase polyprotein is processed by viral proteases into 16 nonstructural proteins which assemble with membranes to generate double-membrane vesicles (DMVs) where RNA synthesis takes place. A nested set of 30 co-terminal subgenomic (sg) RNAs is generated by a discontinuous transcription process. The sgRNAs are translated to generate the viral structural and accessory proteins. Viral gRNA is replicated and associates with nucleocapsid protein and viral structural proteins in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where virus particles bud into vesicles before transport and release from the cell. Reprinted from Masters PS (2006) The molecular biology of coronaviruses. Advances in Virus Research 66: 193–292, with permission from Elsevier.

subsequent release from the cell. Depending on the virus strain, this replication can be robust and result in destruction of the host cell or a low-level, persistent infection that can be maintained in cultured cells or infected animals. A hallmark of CoV transcription is the generation of a nested set of mRNAs, with each mRNA having the identical ‘leader’ sequence of approximately 65–90 nt at the 50 end (Fig. 5(a)). The leader sequence is encoded only once at the 50 end of the gRNA. Each subgenomic mRNA (sgmRNA) has the identical leader sequence fused to the 50 end of the body sequence. How are the leader-containing mRNAs generated during CoV transcription? Current evidence supports a model of discontinuous transcription, whereby the replicase complex switches templates during the synthesis of negative-strand RNA (Fig. 5(b)). The key sequence element in this process is the transcriptional regulatory sequence (TRS). The TRS is a sequence of approximately 6–9 nt (50 -ACGAAC-30 for SARS-CoV) which is found at the end of the leader sequence and at each intergenic region (the sites between the open reading frames encoding the viral structural and accessory proteins). Site-directed mutagenesis and deletion analysis has revealed the critical role of the TRS in mediating transcription of sgmRNAs. Deletion of any intergenic TRS results in loss of production of the corresponding sgmRNA. In addition, the CoV leader TRS and the intergenic TRS sequences must be identical for optimal production of the sgmRNAs. A three-step working model for template switching during negative-strand RNA synthesis has been proposed to describe the process for the generation of CoV leader-containing sgmRNAs (Fig. 5(b)). In this process, the 50 end and 30 end of the gRNA form a complex with host cell factors and the viral replication complex. The 30 end of the positive strand is used as the template for the initiation of transcription of negative-strand RNA. Negative-strand RNA synthesis continues up to the point of the TRS. At each TRS, the viral replicase may either read through the sequence to generate a longer template, or switch templates to copy the leader sequence. The template switch allows the generation of a leader-containing sgmRNA. In this model, alignment of the leader TRS, the newly synthesized negative-strand RNA, and the genomic TRS is critical for the template switching to occur. Disruption of the complex, or loss of base-pairing within the complex, will result in the loss of production of that

Coronaviruses: Molecular Biology (Coronaviridae)

203

Fig. 5 Model of SARS-CoV gRNA and sgRNAs, and a working model of discontinuous transcription. (a) Diagram of gRNA and the nested set of sgRNAs of SARS-CoV. The 50 leader sequence, the transcriptional regulatory sequences (TRSs), and the positive- and negative-sense sgRNAs are indicated. (b) A working model of CoV discontinuous transcription. I. 50 –30 complex formation. Binding of viral and cellular proteins to the 50 and 30 ends of the CoV gRNA is represented by ellipsoids. The leader sequence is indicated in red, the TRS sites are in orange. II. Base-pair scanning step. Minus-strand RNA (light blue) is synthesized from the positive-strand template by the viral transcription complex (hexagon). At the TRS site, base-pairing may occur between the template, the nascent negative-strand RNA, and the leader TRS sequence (dotted lines). III. The synthesis of negative-strand RNA can continue to make a longer sgRNA III, or a template switch can take place III0 to generate a leader-containing subgenomic negative-strand RNA, which could then serve as the template for leader-containing positive-strand sgRNAs. Modified from Enjuanes L, Almazán F, Sola I, and Zunia S (2006) Biochemical aspects of coronavirus replication: A virus–host interaction. Annual Reviews in Microbiology 60: 211–230.

sgmRNA. Further studies of the CoV replication complex may yield new insights into the role of the viral helicase and endoribonuclease in the generation of the leader-containing CoV RNAs. Another hallmark of CoV replication is high-frequency RNA recombination. RNA recombination occurs when a partially synthesized viral RNA dissociates from one template and hybridizes to similar sequences present in a second template. Viral RNA synthesis continues and generates a progeny virus with sequences from two different parental genomes. This RNA recombination event is termed copy-choice recombination. Copy-choice RNA recombination can be demonstrated experimentally when two closely related CoV strains (such as MHV-JHM and MHV-A59) are used to coinfect cells. Recombinant viruses with cross-over sites throughout the genome can be isolated, although sequences within the spike glycoprotein may be a ‘hot spot’ for recombination due to the presence of RNA secondary structures that may promote dissociation and reassociation of RNA. It has been proposed that copy-choice recombination is also the mechanism by which many CoVs have acquired accessory genes, and it has been exploited experimentally for the deletion or insertion of specific sequences in CoV genomes to assess their role in virus replication and pathogenesis.

CoV Accessory Proteins Sequence analysis of CoVs isolated from species ranging from birds to humans has revealed that all CoVs encode a core canonical set of genes, replicase (rep), spike (S), envelope (E), membrane (M), and nucleocapsid (N), and additional, so-called accessory genes (Table 1). The canonical genes are always found in the same order in the genome: rep-S-E-M-N. Reverse genetic studies (see below) have shown that this is the minimal set of genes required for efficient replication and assembly of infectious CoV particles. However, the genomes of all CoVs sequenced to date encode from one to eight additional ORFs, which code for accessory proteins. As the name implies, these accessory proteins are not required for CoV replication in tissue culture cell lines, but they may play important roles in tropism and pathogenesis in vivo. How were these additional genes acquired? Current evidence indicates that these additional sequences may have been acquired by RNA recombination events between co-infecting viruses. For example, the hemagglutinin-esterase (HE) glycoprotein present in four different CoVs (MHV, BCoV, HCoV-OC43, and HCoV-HKU-1) was

204

Coronaviruses: Molecular Biology (Coronaviridae)

Table 1

Coronavirus canonical and accessory proteins

Virus Alphacoronavirus TGEV FIPV HCoV-229E PEDV HCoV-NL63 BatCoV-HKU8 BatCoV-HKU2 Betacoronavirus MHV BCoV HCoV-OC43 HCoV-HKU1 SARS-CoV Bat-SARS-CoV MERS-CoV BatCoV-HKU5 BatCoV-HKU4 Gammacoronavirus Avian IBV Beluga whale CoV Deltacoronavirus Munia CoV Thrush CoV

Proteins: canonical (rep-S-E-M-N) and accessory rep-S-3a,3b-E-M-N-7 rep-S-3a,3b,3c-E-M-N-7a,7b rep-S-4a,4b-E-M-N rep-S-3-E-M-N rep-S-3-E-M-N rep-S-3-E-M-N-7 rep-S-3-E-M-N-7 rep-2a, HE-S-4–5a,E-M-N,7b rep-2a, HE-S-4a,4b-5,E-M-N,7b rep-2a, HE-S-5,E-M-N,7b rep-HE-S-4-E-M-N,7b rep-S-3a,3b-E-M-6–7a,7b-8a,8b-N,9b rep-S-3-E-M-6–7a,7b-8-N,9b rep-S-3–4a,4b-5-E-M-N-8b rep-S-3a,3b,3c,3d-E-M-N rep-S-3a,3b,3c,3d-E-M-N rep-S-3a,3b,3c-E-M-5a,5b-N rep-S-E-M-5a,5b,5c-6–7–8–9–10-N rep-S-E-M-5-N-7a,7b,7c rep-S-E-M-5-N-7a,7b,7c

likely acquired by recombination of an ancestral CoV with the HE glycoprotein gene of influenza C. Interestingly, the expression of the HE gene has no effect on replication of the virus in cultured cell lines, but has been shown to enhance virulence in infected animals. Other CoV accessory genes may have been acquired through recombination with host cell mRNA or other viral mRNAs. The specific role of the accessory proteins in CoV replication and pathogenesis is under investigation. For SARS-CoV, accessory protein 6 has been implicated as an important factor in viral pathogenesis. Researchers have shown that mice infected with murine CoV expressing SARS-CoV protein 6 rapidly succumb to the infection, indicating that the protein 6 enhances virulence. In addition, studies suggest that SARS-CoV accessory proteins may play a role in blocking host cell innate immune responses, which may enhance viral replication and virulence. Other accessory proteins, such as SARS-CoV 3a and 7a, have been shown to be packaged into virus particles, where they may enhance infectivity or alter cell tropism. Future studies will be aimed at elucidating how CoV accessory proteins modulate the innate immune response to coronavirus replication.

Manipulating CoV Genomes Using RNA Recombination and Reverse Genetics Genetic manipulation of CoV sequences is challenging because of the large size (27–32 kbp) of the RNA genomes. However, two approaches have been developed to allow researchers to introduce mutations, deletions, and reporter genes into CoV genomes. These approaches are (1) targeted RNA recombination and (2) reverse genetics using infectious cDNA constructs of CoV. The first approach exploits high-frequency copy-choice recombination to introduce mutations of interest into the 30 end of the CoV gRNA. In the first step of targeted RNA recombination, a cDNA clone encoding the region from the spike glycoprotein to the 30 end of the RNA is generated. These sequences can be easily manipulated in the laboratory to introduce mutations or deletions, or for the insertion of reporter or accessory genes, into the plasmid DNA. Next, RNA is transcribed from the plasmid DNA and the RNA is transfected into cells coinfected with the CoV of interest. RNA recombination occurs between the replicating CoV and the transfected substrate RNA, and viruses with the 30 end sequences derived from the transfected substrate RNA will be generated. The recombinant viruses are generated by high-frequency copy-choice recombination, but the challenge is to sort or select for the recombinant virus of interest from the background of wild-type virus. To facilitate selection of recombinant viruses, Masters and Rottier introduced the idea of host range-based selection. They devised a clever plan to use a mouse hepatitis virus (MHV) that encodes the spike glycoprotein from a feline CoV as the target for their recombination experiments. This feline-MHV, termed fMHV, will infect only feline cell lines. Substrate RNAs that encode the MHV spike and mutations of interest in the 30 region of the genome can be transfected into feline cells infected with fMHV, and progeny virus can be collected from the supernatant and subsequently selected for the ability to infect murine cell lines. Recombinant CoVs that have incorporated the MHV spike gene sequence (and the downstream substrate RNA with mutations of interest) can be selected for growth on murine cells, thus allowing for the rapid isolation of the recombinant virus of interest. This host range-based selection step is now widely used by virologists to generate recombinant viruses with specific alterations in the 30 end of the CoV genome.

Coronaviruses: Molecular Biology (Coronaviridae)

205

The second approach for manipulating CoV sequences, generating infectious cDNA constructs of CoV, has been developed in several laboratories. Full-length CoV sequences have been cloned and expressed using bacterial artificial chromosomes (BACs), vaccinia virus vectors, and from an assembled set of cDNA clones representing the entire CoV genome. The generation of a fulllength cDNA and subsequent generation of a full-length CoV gRNA allows for reverse genetic analysis of CoV sequences. Successful reverse genetics systems are now in place to study the replication and pathogenesis of SARS-CoV, MERS-CoV, MHV, HCoV-229e, and IBV. These reverse genetics systems have allowed researchers to introduce mutations into the replicase gene and identify sites that are critical for enzymatic activities of many replicase products such as the helicase, endoribonuclease, and the papain-like proteases. Reverse genetic approaches are also being used to investigate the role of the TRSs in controlling the synthesis of CoV mRNAs. Interestingly, the SARS-CoV genome can be ‘re-wired’ using a novel, nonanonical TRS sequence, which must be present at both the ends of the leader sequence and at each intergenic junction. This ‘re-wired’ SARS-CoV may be useful for generating a liveattenuated SARS-CoV vaccine. An important feature of this ‘re-wired’ virus is that it would be nonviable if it recombined with wildtype virus, since the leader TRS and downstream TRS would no longer match in a recombinant virus. The development of reverse genetics systems for CoVs has opened the door to investigate how replicase gene products function in the complex mechanism of CoV discontinuous transcription, and provides new opportunities to generate novel CoVs as potential live-attenuated or killed virus vaccines to reduce or prevent CoV infections in humans and animals.

Vaccines and Antiviral Drug Development Because of the economic importance of CoV infection to livestock and domestic animals, a variety of live-attenuated and killed CoV vaccines have been tested in animals. Vaccines have been developed against IBV, TGEV, CCoV, and FIPV. However, these vaccines do not seem to provide complete protection from wild-type virus infection. In some cases, the wild-type CoV rapidly evolves to escape neutralization by vaccine-induced antibodies. In studies of vaccinated chickens, a live-attenuated IBV vaccine has been shown to undergo RNA recombination with wild-type virus to generate vaccine escape mutants. Killed virus vaccines may also be problematic for some CoV infections. Vaccination of cats with a killed FIPV vaccine has been shown to exacerbate disease when cats are challenged with wild-type virus. Therefore, extensive studies will be required to carefully evaluate candidate vaccines for SARS-CoV or MERS-CoV. A variety of approaches are currently under investigation for developing CoV vaccines, including analysis of killed virus vaccines, live-attenuated virus vaccines, and viral vector vaccines (such as modified vaccine virus Ankara, canarypox, alphavirus, and adenovirus vectors). Studies have shown that removing the envelope (E) protein from either SARS-CoV or MERS-CoV is an effective approach for generating a live attenuated virus vaccine. In the absence of this structural protein, the virus replicates to lower titers in the lungs of mice, but still induces an immune response that is protective from challenge with wild type virus. The development of improved animal models for human coronaviruses will be essential for evaluating any candidate vaccines. Transgenic mice expressing human ACE-2 (for SARS-CoV) can be used as a model system for SARS, however the pathogenesis in ACE-2 expressing transgenic mice does not fully mimic the pathogenesis seen during SARS-CoV infection in humans. CoVs can be adapted for replication in small animal models. Passaging SARS-CoV in mice led to the generation of a mouse-adapted strain, SARS CoV-MA15, which induces a lethal respiratory tract infection of mice. CoV vaccine studies will benefit from an improved understanding of conserved viral epitopes that can be targeted for vaccine development. The use of neutralizing monoclonal antibodies directed against the SARS-CoV or MERS-CoV spike glycoprotein is another approach that may provide protection from severe disease. The success in the development and use of humanized monoclonal antibodies against respiratory syncytial virus (family Paramyxoviridae) to protect infants from severe disease indicates that this approach is certainly worth investigating. Studies have shown that patient convalescent serum and monoclonal antibodies directed against the SARS-CoV spike glycoprotein efficiently neutralize infectious virus. Further studies are essential to evaluate any concerns about potential antibody-mediated enhancement of disease and to determine if neutralization escape mutants arise rapidly after challenge with infectious virus. Studies evaluating monoclonal antibodies directed against a variety of structural proteins, and monoclonal antibodies directed against conserved sites in the spike glycoprotein will provide important information on the efficacy of passive immunity to protect against emerging coronaviruses. Currently, there are no antiviral drugs approved for use against any human CoV infection. With the potential for the emergence or re-emergence of pathogenic CoV from animal reservoirs, there is considerable interest in identifying potential therapeutic targets and developing antiviral drugs that will block viral replication and reduce the severity of CoV infections in humans. Two promising targets for antiviral drug development are the SARS-CoV protease domains, the papain-like protease (PLpro) and the 3C-like protease (3CLpro, also termed the main protease, Mpro) (Fig. 6). These two protease domains are encoded within the replicase polyprotein gene and protease activity is required to generate the 16 replicase nonstructural proteins (nsp1–nsp16) that assemble to generate the viral replication complex. The crystal structure of the 3CLpro was determined first from TGEV and then from SARS-CoV. Rational drug design, much of which was based on our knowledge of inhibitors directed against the rhinovirus 3C protease, has provided promising lead compounds for 3CLpro antiviral drug development. Interestingly, these candidate antivirals have been shown to inhibit the replication of SARS-CoV and other group 2 CoVs such as MHV, and the less related group 1 CoV, HCoV-229e. This indicates that the active site of 3CLpro is highly conserved among CoVs and that antiviral drugs developed against SARS-CoV 3CLpro may also be useful for inhibiting the replication of more common human CoVs such as HCoV-229e, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. Further studies are needed to determine if these inhibitors can be developed into clinically useful antiviral agents.

206

Coronaviruses: Molecular Biology (Coronaviridae)

Fig. 6 CoV proteases are targets for antiviral drug development; X-ray structures of the two SARS-CoV protease domains encoded in the replicase polyprotein. (a) The SARS-CoV papain-like protease (PLpro) with catalytic triad cysteine, histidine, and aspartic acid residues, and zinc-binding domain indicated. (b) The 3C-like protease (3CLpro, also termed main protease, Mpro) dimer with catalytic cysteine and histidine residues indicated.

Analysis of SARS-CoV papain-like protease led to the surprising discovery that this protease is also a viral deubiquitinating (DUB) enzyme. The SARS-CoV PLpro was shown to be required for processing the amino-terminal end of the replicase polyprotein and to recognize conserved cleavage site (-LXGG). The LXGG cleavage site is also the site recognized by cellular DUBs to remove poly-ubiquitin chains from proteins targeted for degradation by proteasomes. Analysis of the X-ray structure of the SARS-CoV PLpro has revealed that it has structural similarity to known cellular DUBs. These studies suggest that CoV papain-like proteases have evolved to have both proteolytic processing and DUB activity. The DUB activity may be important in preventing ubiquitinmediated degradation of viral proteins, or the DUB activity may be important in subverting host cell pathways to enhance viral replication. PLpro inhibitors are now being developed using structural information and by performing high-throughput screening of small molecule libraries to identify lead compounds. The recent development of a chimeric virus system which uses Sindbis virus to express the SARS-CoV or MERS-CoV papain-like protease, facilitates the study of protease activity and the efficacy of protease inhibitors. The chimeric virus system can be used in a biosafety level 2 environment, compared to the biosafety level 3 containment required for the study of SARS-CoV or MERS-CoV. Additional CoV replicase proteins, particularly the RNAdependent RNA polymerase, helicase, and endoribonuclease, are also being targeted for antiviral drug development.

Future Perspectives The development of targeted RNA recombination and reverse genetics systems for CoVs has provided new opportunities to address important questions concerning the mechanisms of CoV replication and virulence, and to design novel CoV vaccines. In the future, improved small animal models for testing vaccines and antivirals, and the availability of additional X-ray crystallographic structure information for rational drug design will be critical for further progress toward development of effective vaccines and antiviral drugs that can prevent or reduce diseases caused by CoVs.

See also: Coronaviruses: General Features (Coronaviridae). Enveloped, Positive-Strand RNA Viruses (Nidovirales). Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae). Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Coronaviridae)

Further Reading Almazán, F., DeDiego, M.L., Sola, I., et al., 2013. Engineering a replication-competent, propagation–defective Middle East respiratory syndrome coronavirus as a vaccine candidate. MBio 4, e00650. 13. Deng, X., Agnihorthram, S., Mielech, A.M., et al., 2014. A chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses. Journal of Virology 88 (20), 11825–11833. Enjuanes, L., Almazán, F., Sola, I., Zuñiga, S., 2006. Biochemical aspects of coronavirus replication: A virus–host interaction. Annual Reviews in Microbiology 60, 211–230. Ge, X.Y., Li, J.L., Yang, X.L., et al., 2013. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535–538. Graham, R.L., Donaldson, E.F., Baric, R.S., 2013. A decade after SARS: Strategies for controlling emerging coronaviruses. Nature Reviews. Microbiology 11, 836–848. Haagmans, B.L., Al Dhahiry, S.H.S., Reusken, C.B.E.M., et al., 2014. Middle east respiratory syndrome coronavirus in dromedary camels: An outbreak investigation. The Lancet Infectious Diseases 14, 140–145. Jiang, L., Wang, N., Zuo, T., 2014. Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein. Science Translational Medicine 6, 234ra59. Lau, Y.L., Peiris, J.S., 2005. Pathogenesis of severe acute respiratory syndrome. Current Opinion in Immunology 17, 404–410.

Coronaviruses: Molecular Biology (Coronaviridae)

207

Li, W., Wong, S.K., Li, F., et al., 2006. Animal origins of the severe acute respiratory syndrome coronavirus: Insights from ACE2-S-protein interactions. Journal of Virology 80, 4211–4219. Lu, G., Hu, Y., Qi, J., et al., 2013. Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature 500, 227–232. Masters, P.S., Rottier, P.J.M., 2005. Coronavirus reverse genetics by targeted RNA recombination. Current Topics in Microbiology and Immunology 287, 133–160. Perlman, S., Netland, J., 2009. Coronaviruses post-SARS: Update on replication and pathogenesis. Nature Reviews. Microbiology 7, 439–450. Raj, V.S., et al., 2013. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495, 251–254. Ratia, K., Kilianski, A., Baez-Santos, Y.M., et al., 2014. Structural basis for the ubiquitin-linkage specificity and deISGylating activity of SARS-CoV papain-like protease. PLoS Pathogens. http://dx.doi.org/10.1371/journal.ppat.1004113. Ratia, K., Pegan, S., Takayama, J., et al., 2008. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication. Proceedings of the National Academy of Sciences, USA 105, 16119–16124. Roberts, A., et al., 2007. A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice. PLoS Pathogens 3, e5. Scobey, T., et al., 2013. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proceedings of the National Academy of Sciences, USA 110, 16157–16162. Thiel, V., Siddell, S., 2005. Reverse genetics of coronaviruses using vaccinia virus vectors. Current Topics in Microbiology and Immunology 287, 199–228. Woo, P.C., et al., 2012. Discovery of seven novel mammalian and avian coronaviruses in the genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus. Journal of Virology 86, 3995–4008. Yang, H., Xie, W., Xue, X., et al., 2005. Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoS Biology 3, 1742–1751. Yount, B., Roberts, R.S., Lindesmith, L., Baric, R.S., 2006. Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome. Proceedings of the National Academy of Sciences, USA 103, 12546–12551. Zaki, A.M., van Boheemen, S., Bestebroer, T.M., et al., 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. New England Journal of Medicine 367, 1814–1820.

Relevant Websites http://www.cdc.gov/coronavirus Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) resource, Centers for Disease Control and Prevention, USA. http://www.viprbrc.org viral genome bioinformatics resource. http://www.who.int/csr/disease/coronavirus World Health Organization Global Alert and Response for emerging coronaviruses.

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae) Ali Mirazimi, National Veterinary Institute, Uppsala, Sweden and Karolinska Hospital University, Huddinge, Sweden Felicity Burt, University of the Free State, Bloemfontein, South Africa Anna Papa, Aristotle University of Thessaloniki, Thessaloniki, Greece r 2021 Elsevier Ltd. All rights reserved. This is an update of C.A. Whitehouse, Crimean–Congo Hemorrhagic Fever Virus and Other Nairoviruses, in Encyclopedia of Virology (Third Edition), Edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00620-8.

Introduction CCHFV is a tick-borne viral zoonosis found in Africa, Asia, eastern Europe and the Balkans. The geographic distribution of the virus correlates with that of ticks belonging to the genus Hyalomma, which are considered to be the principal vectors of the virus. CrimeanCongo hemorrhagic fever orthonairovirus (CCHFV) is a member of the Orthonairovirus genus of the family Nairoviridae within the Bunyavirales order of viruses. Species within the genus were grouped originally on the basis of antigenic relationships and morphological and genetic similarities. Recent taxonomic changes based on genomic characterizations, resulted in renaming of seven species within the genus and the addition of five new species to the genus. Taxonomic changes included revising the grouping of Hazara virus, historically considered to be the closest relative to CCHFV, and now placed in a separate species with Tofla virus. Neither of these viruses, Hazara and Tofla, are known to cause disease. Within the genus, CCHFV, the etiologic agent of mild to severe disease with fatalities, is considered to be the most significant public health concern. Nairobi sheep disease virus (NSDV), an important veterinary pathogen, causes severe disease in domestic sheep and goats in Africa and Ganjam virus, considered an isolate of NSDV, circulates in the Indian subcontinent. Lesser known human pathogenic orthonairoviruses include Dugbe virus and Kasokera virus, associated with febrile illness in Africa; Erve virus proposed to be the cause of thunderclap headache in Europe; and Issyk-Kul virus associated with febrile disease in central Asia. The remaining viruses within the Orthonairovirus genus have mostly been isolated from ticks, and the medical and/or veterinary significance of them is unknown. A hemorrhagic disease with symptoms suggestive of CCHF infection was described in eastern Europe and Asia as far back as the 12th century. The disease given the name Crimean hemorrhagic fever (CHF) was first reported in an outbreak involving 200 soldiers and peasants on the Crimean peninsula in 1944. The following year the disease was shown to be caused by a filterable agent, but was only isolated in mice in 1967. In 1956 a virus was isolated from blood samples collected from a febrile child in Stanleyville in the Belgian Congo (now referred to as Kisangani in the Democratic Republic of the Congo) and given the name Congo virus. The availability of a laboratory host made it possible to further charaterise the two viruses and in 1969 it was reported that the virus from Europe and Asian Republics of USSR and Bulgaria were antigenically identical to Congo virus from Africa. The names were combined and the virus is now referred to as Crimean-Congo hemorrhagic fever virus. Due to the high fatality rate, the propensity to cause nosocomial infections and a lack of specific anti-viral treatment or vaccine, the virus is classified among biosafety level 4 pathogens. This dictates that the virus can only be handled and cultured within the confines of a maximum containment facility. This requirement for a biosafety level 4 laboratory limits the number of facilities that culture the virus and has likely contributed to our incomplete understanding on the pathogenesis of disease and the correlates of protection. Development of vaccines was initially thwarted by the lack of suitable animal models however the identification of murine models, and more recently a non-human primate model, has contributed to recent development of candidate vaccines. The emergence and re-emergence of CCHFV in Balkan countries and south western regions of the Russian Federation, and more recently in southern regions of Europe, highlights the potential for this pathogen to spread to non-endemic regions where ticks of the Hyalomma genus are present.

Virion Structure, Genome and Life Cycle The CCHF virion is spherical and approximately 80–100 nm in diameter with a bilayered lipid envelope. The viral glycoproteins GN and GC protruding from the lipid envelop are likely associated with receptor recognition and are involved in the early steps of viral replication. The CCHFV contain the negative-sense small (S), medium (M) and large (L) genome segments, encapsidated by the nucleoprotein (N), and the RNA dependent RNA polymerase (RdRp), which are responsible to initiate transcription and genome replication in the host cell. The terminal complementary sequences 50 -UCUCAAAGA and 30 -AGAGUUUCU are conserved in all nairoviridaes members.

The S Segment The S segment encodes the N, which is the most abundant viral protein in the CCHFV particles, playing a key role in the encapsidation of viral genomic. The CCHFV N structure organize a large globular domain, to which N- and C-terminal portions of the polypeptide

208

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00036-9

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

209

a

b

Fig. 1 CCHF virus genome. CCHF virus consist of tri‐segmented negative sense RNA genome. The small (B1.6 kb), medium (B5.4 kb) and large (B12.1 kb) segments, code for the N, the GPC and the L protein, respectively. The small segment also codes for a non‐structural S protein (NSS) in opposite sense. The coding regions are flanked by non‐coding regions (NCRs).

contribute, plus a protruding ‘‘arm’’ containing a conserved caspase-3 cleavage site on the top of the arm. Most probably the globular region is responsible for RNA binding. The S segment can also codes for a non-structural protein in ambisense direction (Fig. 1).

The M Segment The M segment encodes a single polyprotein, the glycoprotein precursor (GPC) (Fig. 1). The maturation of CCHFV GPC is very complex and does not share much similarity with glycoprotein processing in other bunyaviruses. GPC maturation yields to two type I transmembrane glycoproteins, GN and GC which are spiked on the virus envelope. The GPC processing in the endoplasmic reticulum (ER) and Golgi results in two structural glycoproteins, GN and GC, and nonstructural proteins designated GP160, GP85, and GP38 [ and the non-structural M protein (NSM). The GPC is heavily glycosylated and subsequently cleaved by host proteases including the proprotein convertases.

The L Segment Nairoviridaes have an unusually large RNA dependent RNA polymerase protein (nearly twice the size) compared to those of other members (Fig. 1). This segment has a single open reading frame, and encodes an approximately 4000-amino acid polyprotein. The

210

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

amino acids 1–609 contain domains with the ovarian tumor (OTU) cysteine protease function. Sequences located between the OTU domain and the RdRp conserved motifs contain a potential leucine zipper and a C2H2 zinc finger motif.

Virus Life Cycle The CCHFV GN and/or GC are involved in early step of replication cycle of CCHFV. It has been suggested that GC is responsible for binding to the cellular receptors and mediates fusion in endosomes (Fig. 2). The processes of internalization, virion assembly

Fig. 2 CCHFV replication cycle. CCHFV binds to an unknown cellular receptor and penetrate into the cells in a clathrin-dependent manner. After fusion, the viral genome is released to the cytoplasma. The viral mRNAs are translated into the NP and L proteins at cytoplasma, and the glycoprotein precursor (GPC) translated into endoplasmic reticulum (ER). The NP and L protein helping to replicate the genomic RNA. The GPC is processed and maturate to Gn and Gc in the ER and the Golgi. The new CCHFV particles most probably assemble in the Golgi followed by virus release in Golgi-derived vesicles. The figure illustrated by Dr. J Vermeulen.

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

211

and egress are dependent on host-cell skeleton. The cellular receptors for CCHFV entry have not been identified. A functional interaction has been suggested between CCHFV GC and cell surface nucleolin. Following adsorption of CCHFV at plasma membrane, the CCHFV is endocytozed through a clathrin-mediated endocytosis mechanism. CCHFV entry has also been suggested to be dependent on cholesterol and a low pH. Following endocytosis, CCHFV particles are transported to early endosomes and subsequently to multivesicular bodies (MVB), where the CCHFV membrane is fused to the endosomes membrane. Following this process the genomic RNPs are released into the cytosol and transcription and replication process will be initiated. The replication of the RNPs minimally require the L protein and N. Since CCHFV is a negative strand RNA virus, genomic RNPs are used as template to synthesize capped mRNA. These process are followed by transcription and viral protein processing and subsequent maturation. The assembly and egress process for CCHFV is most probably like other bunyaviruses.

Epidemiology CCHF is the most geographically extensive viral hemorrhagic fever and the most widespread medically significant tick-borne viral disease. It is estimated that 3 billion people in Africa, Asia and Europe are at risk, and 10,000–15,000 infections and 500 fatalities occur annually. Ticks of the Hyalomma genus (mainly H. marginatum, H. rufipes, H. anatolicum, and H. asiaticum) are the principal competent vectors and reservoirs of CCHFV. The occurrence of the disease coincides with the geographic distribution of Hyalomma spp. ticks, with a 501 north latitude limit, which, however, is predicted to increase to northern areas due to warming climate. Although CCHFV has been detected or isolated from ticks in several other genera, their role in CCHF epidemiology remains unclear. CCHFV circulates unnoticed in nature between ixodid ticks and vertebrate hosts. The virus is maintained in ticks transstandially, transovarially, venereally and by co-feeding on infected or uninfected animals. Ticks remain persistently infected. The immature Hyalomma spp. ticks (larvae and nymphs) feed mainly on ground-feeding birds and small mammals and remain attached for up to 26 days. Adult ticks actively seek larger mammals, including hares, wild and domesticated ungulates, or humans. Overwintering infected ticks maintain the virus in nature. Transmission to humans occurs by bite or crushing of an infected tick or by direct contact (percutaneous and permucosal) with blood or tissues from viremic patients or livestock. Therefore, groups at higher risk are agricultural and abattoir workers, individuals involved in backyard slaughtering in endemic areas and health-care personnel. Nosocomial infections have been reported when exposure to the virus was substantial; the risk is higher when the patient presents hemorrhagic mainfestations. Isolation of the patient and barrier nursing should be applied. Epidemiological and behavioral factors that play a role in acquisition of the CCHFV infection differ among countries; e.g., while tick bite is the main mode of transmission in Turkey, the contact with the blood of infected livestock, seems to be the main route of CCHFV transmission to humans in Iran. Cases are seen from spring to autumn and they are usually sporadic. Non-nosocomial epidemics or outbreaks occur when environmental (e.g., mild winters, dry summers) and anthropogenic conditions (e.g., land fragmentation, socioeconomic changes) result in high tick populations leading to increased human exposure to ticks. Several biotic and abiotic drivers contribute to shape a favorable ecosystem. Given that CCHFV can be spread in long distances by migratory birds infested with infected ticks, or transported through international livestock trade, virus introduction in a suitable new habitat can lead to disease emergence and establishment. This was seen in Turkey, India and Spain where the disease emerged in 2002, 2011 and 2016, respectively. Based on environmental suitability, predictive risk maps have been constructed which can direct surveillance studies. The epidemiology of CCHF is changing, as the disease emerged in new places, while the incidence differs between years. Currently, the most CCHF-affected country is Turkey; since 2002, when CCHF emerged in the country, more than 10,000 cases have been registered, with the majority of them being observed in 2008 and 2009 (more than 1000 cases annually). The most endemic areas are in the Middle Anatolia-Black Sea region. Sporadic CCHF human cases and outbreaks have been reported in more than 30 countries in three continents. In Asia, CCHF has been reported from Afghanistan, China, India, Iran, Iraq, Kazakhstan, Kuwait, Oman, Pakistan, Saudi Arabia, Tajikistan, Turkey, Turkmenistan, United Arab Emirates, and Uzbekistan. In Europe, CCHF has been reported in Albania, Bulgaria, Georgia, Kosovo, Russia, Ukraine and Spain, while one single case has been reported in Greece. Among African countries, CCHF has been reported in Burkina Faso, Democratic Republic of Congo, Egypt, Kenya, Mauritania, Nigeria, Senegal, Somalia, Republic of South Africa, Namibia, Sudan, Tanzania and Uganda. Furthermore, detection of the virus in ticks or detection of viral antibodies in humans and/or livestock, in the absence of clinical case, has been reported in several countries. Wild and domestic animals infected with CCHFV develop only a transient viremia without presenting symptoms of the disease. Seroprevalence studies in humans, livestock and wild animals enable the identification of CCHFV endemic foci. CCHFV presents the most extensive genetic diversity among arboviruses. The highest level of diversity is seen in the M RNA segment, and especially in the mucin-like region. This phenomenon can be explained by the fact that the virus has to adapt to a variety of host (tick and vertebrate) cells, prompting for genetic changes and complex virus evolution. Based on S RNA segment sequences, seven genetic clades (three from Africa, two from Asia and two from Europe) can be identified which present a geographic correlation (Fig. 3). Phylogenetic studies show that the Greek strain AP92 (clade Europe 2) and West African viruses (clade Africa 1) are the oldest CCHFV lineages. The AP92 strain was initially isolated from Rhipicephalus bursa tick in 1975 in Greece, and only a limited number of human cases has been associated with this lineage. Several studies showed that this lineage is strongly associated with

212

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

Fig. 3 Neighbor-Joining phylogenetic tree based on whole open reading frame of CCHFV S RNA segment (1446 nucleotides) computed using the Kimura 2-parameter method. Evolutionary analysis was conducted in MEGA software version 7.0.

Rhipicephalus spp. ticks. Genetic recombination is rare, but reassortment events are common in CCHFV, especially in the M RNA segment which encodes the envelope glycoproteins, Gn and Gc, involved in the attachment of the virus to the host cells. Detection of genetic clades in unrelated geographic regions is common, and can be explained by the passive transportation of the virus to distant regions across continents through migratory birds and livestock trade. The increasing number of whole genome CCHFV sequences is expected to elucidate further the molecular epidemiology of CCHF and increase the knowledge on the evolutionary history of the virus.

Clinical Features Humans are the only vertebrates which present symptoms after CCHFV infection (Fig. 4), CCHFV-infected humans may present asymptomatic, mild, severe, or even fatal disease. It is estimated that more than 80% of CCHFV infections are asymptomatic or subclinical and remain unnoticed. The case fatality rate (CRF) in hospitalized patients is 10%–40%. The course and outcome of the disease depend on the inoculation viral dose, the virus strain and the immune status and response of the host. In severe and fatal cases the production of antibodies is usually low, delayed or absent. CCHF resembles Ebola virus disease in clinical symptoms, underlying mechanisms and cytokine production. Following initial virus amplification at the site of inoculation (dermal dendritic and Langerhans cells), the virus is spread through the lymphatic system to the target organs, mainly liver and spleen, and then is spread to additional organs. Endothelial damage leading to increased permeability and impaired immune response seem to play an important role in the clinical course and severity of the disease. Typically, the clinical course follows four distinct phases: incubation, pre-hemorrhagic, hemorrhagic and convalescence. The incubation period is 1–14 days and varies depending on the viral dose and the route of exposure (it is usually shorter after a bloodstream infection, e.g., tick bite or needle-stick injury). The pre-hemorrhagic phase (1–5 days) is characterized by abrupt onset of fever lasting for 4–5 days, chills, severe headache, photophobia, conjunctivitis, fatigue, dizziness, myalgia, flushed face and rash, as well as gastrointestinal symptoms, such as nausea, vomiting, abdominal pain, and diarrhea. Hepatomegaly, jaundice, splenomegaly, pleural effusions and cardiovascular symptoms, such as bradycardia or tachycardia and hypotension, can be also seen. Some patients present changes in mood, confusion and aggression, followed by depression and somnolence. Patients with severe disease enter the hemorrhagic phase (3–4 days) and develop hemorrhagic manifestations on the mucous membranes and skin ranging from petechiae, epistaxis, bleeding at venipuncture and injection sites, ecchymosis, hematomas, and gingival hemorrhage

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

213

Fig. 4 Large hematomas in a Crimean-Congo hemorrhagic fever patient (day 10 of illness), Greece, 5 June 2018.

to severe hemorrhages from various systems (melena, hemoptysis, hematemesis, hematuria, nose and vaginal bleeding, and cerebral hemorrhage). Hemophagocytosis is often detected in the bone marrow biopsy. Necropsy findings in a fatal case in Spain revealed massive hepatocyte necrosis, complete apoptosis of colonic enterocytes, hemorrhages in bone marrow without inflammatory infiltrates in any of the organs examined providing support for the view that unlike other viral hemorrhagic fevers, there is a primary cytopathic pathogenic effect in CCHF. Death occurs 5–14 days after symptom onset. The survivors show improvement signs on the 10th day onwards, but recovery may be delayed by one month or even longer. Some persisting symptoms include weakness, lethargy, dizziness, hearing loss, and amnesia. The hospitalization time is approximately 10 days. The clinical course of the disease is usually milder in children. The most frequently reported abnormal laboratory findings include thrombocytopenia, prolonged coagulation (prothrombin and activated partial thromboplastin) times, decreased fibrinogen, leucopenia, elevated levels of aminotransaminases, creatine phosphokinase, and lactate dehydrogenase. Collecting, handling and processing clinical samples require trained personnel and suitably equipped laboratories. Differential diagnosis of CCHF includes other viral hemorrhagic fevers, rickettsiosis and other tick-borne bacterial infections, leptospirosis, viral hepatitis, brucellosis and bacterial sepsis, while non-infectious diseases may also cause similar symptomatology. Additional diseases have to be included when symptoms from respiratory, gastrointestinal or central nervous system are present. The list of diseases depends highly on the geographic location of the case and the travel history of the patient. The differential diagnosis is difficult when the patient presents only a mild flu-like illness. Disease severity scoring systems have been developed. High viral load, severe thrombocytopenia, elevated liver enzymes and prolonged bleeding times are the most important predictors of severity and fatality in CCHF. Viral loads higher than 1  109 RNA copies/mL of plasma were strongly associated with fatality in CCHF patients. Specific HLA alleles and genetic polymorphisms in Tolllike receptors, as well as elevation of specific cytokines (e.g., IFN-inducible protein 10 and monocyte chemoattractant protein-1) and soluble adhesion molecules, have been also associated with severity and outcome of the disease.

Pathogenesis CCHFV infection in human can result in a severe hemorrhagic fever. Interestingly, serological evidence indicates that CCHFV can infect numerous vertebrate species. However, even within humans, CCHFV infection may result in a mild or sub-clinical infection.

214

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

The factors contributing to development of a severe disease are unknown. The incubation period ranges from a few days up to 1 week, the length most probably depending on the transmission route and the amount of inoculum. The severity of disease has been demonstrated to correlate to the amount of virus in the blood. Furthermore, it has also been shown that antibody responses have a highly significant inverse correlation with viral loads. The pathogenesis of CCHF is only poorly characterized due to several reasons such as (1) infections occur sporadically and in areas where facilities are limited for performing complete autopsies, (2) virus handling requires biosafety level 4 (BSL-4) containment laboratories, and (3) a lack of available animal models of disease. The limited knowledge about CCHF pathogenesis is mostly derived from blood analyzes and liver biopsies of patients, using materials e.g., from outbreaks in Turkey and South Africa. One of the most comprehensive study involving 50 CCHF patients from South Africa, which describe that cerebral hemorrhage, severe anemia, severe dehydration, and shock associated with prolonged diarrhea, lung edema, and pleural effusion are the factors causing fatal outcome. Almost all patients who died developed multiple organ failure. In the fatal cases, platelet counts can be extremely low from an early stage of illness. Increases in aspartate and alanine aminotransferase (AST and ALT) levels in the serum, prolongation of prothrombin and partial thromboplastin times have also been observed. A common suggested feature of agents causing hemorrhagic fever is the deregulation of host immune responses by combating and attacking cells involved in initiation of antiviral responses. The primary pathophysiological events appear to be leakage of erythrocytes and plasma through the vasculature into tissues. Endothelial damage can contribute to coagulopathy by deregulated stimulation of platelet aggregation, which in turn activates the intrinsic coagulation cascade, ultimately leading to clotting factor deficiency causing hemorrhages. For CCHFV, vascular leakage may be caused either by destruction of endothelial cells or by a disruption of the tight junctions which constitute the endothelial barrier between cells. Moreover, it is unclear whether these events are a direct consequence of infection or whether virus-induced host factors cause the endothelial dysfunction. However, in an epithelial cell line model CCHFV neither caused disruption of tight junctions nor necrosis or apoptosis of cells. This could suggest that the hemorrhages and coagulation disturbances may be caused indirectly, possibly by high levels of proinflammatory cytokines. Previous studies demonstrated higher level of IL-6 and TNF-a in CCHFV patients with fatal outcome compared to the nonfatal cases [38]. The ability of CCHFV to cause severe or lethal disease in mice deficient in the type I interferon system but not wild-type (WT) mice suggests that innate immune responses in vertebrate hosts play a substantial role in limiting CCHFV pathogenesis. Understanding the role of the adaptive immune response in the control of primary CCHFV infection has been limited by the lack of suitable animal models. However, a newly described humanized mouse model of CCHFV demonstrated that T cells were activated following CCHFV infection and may have a role in controlling the disease. In a recent study using mice treated with an interferon blockade antibody, demonstrating that adaptive immune responses can control CCHFV in mice. Recently a cynomolgus macaque model of CCHF for CCHFV has been described, in which cynomolgus macaques infected with a human clinical isolate of CCHFV, strain Hoti. These Infected macaques develop a spectrum of disease outcome similar to that of human CCHF cases from asymptomatic to severe or lethal infections. Histological analysis showed that CCHFV infection resulted mainly in pathological changes in the liver and spleen. In vitro studies have suggested retinoic acid-inducible gene I (RIG-I) as an innate immune sensor of CCHFV. However, it is most likely that more innate immune sensors contribute to sensing of CCHFV. The L segment of CCHFV encodes an ovarian tumor-like de-ubiquitinase (OTU) domain that recently has been shown to suppress innate immune responses. Several studies indicated that host apoptosis process may also be involved in the pathogenesis and controlling of CCHFV infection. There a few studies in humans have identified correlates between polymorphisms in Toll-like receptors (TLRs), nuclear factor-kappa B and severity of disease. CCHFV also antagonizes innate immune signaling.

Diagnosis Confirmation of infection early in the course of disease is important for isolation of patients to protect healthcare workers and for implementing appropriate supportive therapy. Although human cases can be identified using clinical criteria, laboratory confirmation is considered essential to distinguish the infection from other conditions that share similar manifestations. Laboratory confirmation during the acute phase is achieved by isolation of the virus, amplification of viral nucleic acid using reverse transcriptase polymerase chain reaction (RT-PCR) or detection of viral antigen. The virus is classified among biosafety level 4 pathogens and hence culturing the virus is restricted to high containment facilities of which there are limited numbers worldwide. In the absence of level four facilities, clinical samples can be inactivated prior to testing. Virus is most frequently isolated from clinical serum samples collected during the acute phase of illness from days 1–6 after onset of symptoms, although isolation has been reported from samples collected up to day 12. Although the virus replicates in a variety of mammalian primary and line-cell cultures, Vero cells are the most frequently used for routine isolation. The virus is poorly cytopathic and immunofluorescent assays are required to confirm infection of cells. Virus can also be isolated by intracerebral inoculation of 24 h old mice. Isolation of the virus in mice is usually more sensitive than cell culture but it requires 7–10 days for mice to succumb to infections whereas isolation in cell cultures can be confirmed within 3–6 days. Molecular techniques have distinct advantages over traditional virus isolation methods in that a result can be obtained far more rapidly and the assays can be performed using inactivated samples without the requirement for biosafety level four

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

215

facilities. The first diagnostic molecular assay was a conventional nested RT-PCR based on alignment of the NP gene of seven geographically distinct isolates of CCHFV and identification of consensus primer pairs. Since the development of the first in house RT-PCR, the availability of nucleotide sequence data for complete and partial genomes from various geographic regions and identification of genetic relationships, defining genotypes and their distribution, has contributed to further refinement of molecular assays. The availability of data allows design of molecular assays to accommodate nucleotide diversity and detect genetically different lineages. Real time RT-PCR platforms have been developed using simple intercalating dye and a melt curve analysis to detect amplicons; sequence specific probes with multiple primer pairs and probes to detect all lineages; low density macroarrays and padlock probes with colourimetric readout. Isothermal assays such as loop-mediated isothermal amplification (LAMP) and an isothermal recombinase polymerase assay have been described with potential for diagnostic application. Commercial molecular assays based on primer and probe technology are now available. Real time assays with probes allow quantification of viral load which has been used as a prognostic indicator and a high viral load can be considered to be a predictor of a fatal outcome. Detection of viral antigen using ELISA has been described and although it may have a role in low resource laboratories it lacks the sensitivity required for use as a diagnostic tool. With the availability of molecular assays, including isothermal RT-RPA that can be performed without the need for thermal cyclers, viral antigen ELISA are unlikely to be important in a diagnostic facility. However detection of viral antigen in paraffin embedded tissues using immunohistochemistry is a useful tool for confirming infection in fatal cases although molecular assays can also be used to detect viral nucleic RNA in post mortem samples. During the convalescent phase of illness, serological assays, such as in house or commercial ELISA and indirect immunofluorescent assays (IFA), are used for detection of IgG and IgM antibody. Human infections can confirmed by detection of a specific IgM response, or alternatively, demonstration of seroconversion or at least a four-fold increase in IgG antibody activity in paired serum samples. An antibody response is most commonly detectable from day 5 after onset of illness in non-fatal cases, whereas in fatal infections patients may not develop demonstrable antibody responses. Although assays such as targeted amplification and next generation sequencing based identification, Taqman cards and multiplexed Luminex based immunoassays have been investigated as diagnostic tools, the usefulness of these techniques will likely be limited by accessibility to suitable equipment and cost effectiveness in routine application. The need for rapid sensitive and specific point of care assays remains a requirement for increasing diagnostic capacity. In summary, diagnosis of human infections is usually achieved using a combination of assays including isolation of virus, detection of CCHF viral RNA and demonstration of antibody response. Interpretation of results for exclusion or confirmation of infection usually requires an accurate history of illness and consideration of kinetics of viremia and antibody responses.

Prevention The CCHFV constitutes a public health risk in endemic countries as well as neighboring areas. To date, WHO put CCHFV in their blue print and highlight the importance on developing vaccines and treatment for this virus. A vaccine against CCHFV will prevent infection in human populations at risk, and reduce the number of cases of CCHF. To date, there is a licensed vaccine against CCHFV available in Bulgaria. This vaccine has been developed in 1970 in Russia and is based on CCHFV cultivated in suckling mouse brain and subsequently inactivated by chloroform, heated at 581C, and absorbed on Al(OH)3. Data from the Bulgarian Ministry of Health suggest a fourfold reduction in reported CCHF cases since they used this vaccine in Bulgaria. However, this observation can be due a several factors which have decreased tick exposure in the region per se. The first detailed analysis of the cellular and humoral immune response in healthy individuals following vaccination with the Bulgarian vaccine demonstrated that vaccinated individuals developed anti-CCHFV immunity however, responses were low and required three booster vaccinations to improve immune responses. It should also be highlighted that the use of mouse brain derived vaccine unlikely gain widespread international regulatory approval due to several reseans such as possibility to autoimmune responses induced by myelin basic protein and etc. The most obvious problem for development of vaccines candidates against CCHFV, has been the lack of a good animal models. Whilst studies on the immunogenicity of vaccine candidates can increase our knowledge on immune response to these candidates, the outcome are difficult to interpret as no defined immune correlate of protection is available. Nevertheless, to date there are two mice model available which make it possible to assesse the protection studies. The data on vaccine candidates for CCHFV are presented in (Table 1), and the vaccine candidate which has demonstrated efficacy data are discussed in more detail below.

DNA Vaccines Recently, A DNA vaccine coding for CCHFV Gc, Gn and NP demonstrated induction of both humoral and cell-mediated immune responses which elicited complete protection from lethal disease in a mouse model. A DNA vaccine expressing only the M-segment glycoprotein precursor gene of CCHFV elicited strong antigen specific humoral immune responses with neutralizing titers after three vaccinations, demonstrated 75% protection and highlight that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV.

216

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

Table 1

Approaches for human vaccines against CCHFV

Vaccine candidate

Antigen

Immune response Antibody

Inactivated virus (mice brain) Whole Virus Positive Inactivated virus (cell culture) Whole Virus Positive DNA vaccine M-seg Positive M-seg Positive GC, GN and NPositive Transgenic Plant Protein

VLP Adenovirus

Protection

References

T cell

resultsPositive resultsNot tested resultsNot tested 80% resultsNot tested Not tested resultsPositive results75% resultsPositive results100%

Mousavi-Jazi, M., et al. Vaccine 2012; Canakoglu, N., et al. PLoS Negl Trop Dis 2015; Spik, K., et al. Vaccine 2006; Garrison, A.R., et al. PLoS Negl Trop Dis. 2017 Hinkula, J., et al. J Virol 2017.

GpC

Positive resultsNot tested

GN

Positive resultsNot tested

GC

Positive resultsNot tested

GC, GN M-seg N

Postive results Positive results40% Hinkula, J., et al. J Virol 2017. Postive results Postive results Negative results Sahib, M.M., et al. 2010 Postive results Not tested 75% Zivcec, M., et al. PLoS Negl Trop Dis. 2018

Modified Vaccinia Ankara (MVA)M-seg N

Not tested

Ghiasi, S.M., et al. Vaccine Immunol: CVI 2011;18: Negative results Kortekaas, J., et al. Vector Borne Zoonotic Dis 2015; Negative results Kortekaas, J., et al. Vector Borne, Zoonotic Dis 2015;

Postive results Postive results 100% Buttigieg, K.R., et al. PloS One 2014; Postive results Postive results Negative results Dowall, S.D., et al. Hum Vaccines Immunother 2016;

Inactivated Vaccines To date there is a report on a vaccine candidate based on a purified, formalin inactivated of CCHFV derived from cell culture which avoid the complications of a mouse brain derived vaccine (the Bulgarian licensed vaccine). Vaccination with this inactivated virus required the use of adjuvant. The efficacy in the IFNAR-/- mouse model of CCHF showed 80% protection using this vaccine candidate,. While inactivated virus vaccines have proven effective for protecting against many viral diseases, those based on highly pathogenic viruses such as CCHFV are difficult to manufacture in bulk within a high containment setting.

Modified Vaccinia Virus Ankara (MVA) There is a report describing a MVA poxvirus vector containing the glycoprotein encoding M segment ORF (MVA-GP). This vaccine candidate demonstrated 100% efficacy in animal studies. This MVA-GP vaccine induced antibody and cellular responses across a range of CCHFV epitopes. MVA platform has a proven safety record, having been used in the latter stages of the smallpox eradication campaign, and it is thermostable avoiding the requirement to maintain a cold chain and conferring an extra advantage for regions where such a vaccine might be used.

Adenovirus Vaccine A human serotype 5 adenovirus (AdHu5) vector carrying the M segment of CCHFV has been investigated as a vaccine candidate using IFNAR –/– mice. Despite the induction of both cellular and humoral responses, the vaccine failed to confer protection in a murine model. Recently, using a construct of human adenovirus type 5 expressing the CCHFV N protein, it was demonstrated that mice immunized with Ad-N developed an anti-N humoral immune response. It was shown that a prime-boost of this vaccine candidate regimen provided 78% protection.

Treatment The current medical management is largely supportive and on the treatment of symptoms. To date, there are no licensed specific antiviral compounds available for CCHF. Ribavirin, a broadspectrum antiviral drug, has been used as a common treatment approach. Several reports have suggested clinical benefits of using ribavirin, especially if administered early in infection. However, recently several studies have reported no effect on mortality rates. Recent studies have shown that T-705 favipiravir) has a higher efficacy than ribavirin in animal model. It should be also highlighted that a there are other reports demonstrating antiviral activity for several compounds against CCHFV, however, these studies are in vitro. The lack of efficient treatment and vaccines options, emphasizes the urgency for developing CCHF therapeutics.

Crimean-Congo Hemorrhagic Fever Virus and Nairoviruses of Medical Importance (Nairoviridae)

217

Further Reading Bente, D.A., Forrester, N.L., Watts, D.M., et al., 2013. Crimean-Congo hemorrhagic fever: History, epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antiviral Research 100 (1), 159–189. Hawman, D.W., Feldmann, H., 2018. Recent advances in understanding Crimean-Congo hemorrhagic fever virus. F1000 Research 7. Hoogstraal, H., 1979. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. Journal of Medical Entomology 15 (4), 307–417. World Health Organization. Crimean-Congo haemorrhagic fever (CCHF). Available from: https://www.who.int/emergencies/diseases/crimean-congo-haemorrhagic-fever/en/. Zivcec, M., Scholte, F.E.M., Spiropoulou, C.F., Spengler, J.R., Bergeron, É., 2016. Molecular Insights into Crimean-Congo Hemorrhagic. Viruses 8, 106. doi:10.3390/v8040106.

Dengue Viruses (Flaviviridae) Ashley L St. John and Duane J Gubler, Duke-NUS Medical School, Singapore, Singapore r 2021 Elsevier Ltd. All rights reserved.

Glossary Arbovirus A virus transmitted to vertebrates by hematophagous (blood-feeding) arthropods. Endemic A disease constantly present in a specific geographic region.

Extrinsic incubation time The time between infection and becoming infectious. Hyperendemicity The co-circulation of multiple dengue virus serotypes in the same population. Vertical transmission Transmission of a virus from a female arthropod to its progeny.

History Dengue fever is a very old disease; the earliest record of a dengue-like illness found to date is in a Chinese encyclopedia of disease symptoms and remedies, first published during the Chin Dynasty (AD 265–420) and formally edited in AD 610 (Tang Dynasty) and again in AD 992 during the Northern Sung Dynasty. There are reports of epidemics of dengue-like illnesses in the French West Indies in 1635 and in Panama in 1699. By the late 1700s, a disease clinically compatible with dengue had spread worldwide, with epidemics occurring in 1779 in Batavia (Jakarta), Indonesia and Cairo, Egypt, and in 1780 in Philadelphia, Pennsylvania, USA. From the late 1700s to World War II, repeated epidemics of dengue-like illness occurred in most tropical and subtropical regions of the world at 10- to 40-year intervals. There is no documentation, however, that dengue viruses were responsible for all of these epidemics because the diagnosis was based only on clinical reports. Clinical descriptions of some early epidemics were also compatible with chikungunya virus infection, which has a transmission cycle similar to that of the dengue viruses. It is likely that epidemic chikungunya did occur, but recent data show that the dengue viruses, not chikungunya virus, were responsible for the majority of epidemics in the past 60 years. The virus etiology of dengue fever was not documented until 1943–44, when Japanese and American scientists simultaneously isolated the viruses from soldiers in the Pacific and Asian regions during World War II. Albert Sabin isolated dengue viruses from soldiers who became ill in Calcutta (India), New Guinea, and Hawaii. The viruses from India, Hawaii, and one strain from New Guinea were antigenically similar, whereas three others from New Guinea were different. These viruses were called dengue 1 (DENV-1) and dengue 2 (DENV-2) and designated as prototype viruses (DENV-1, Hawaii, and DENV-2, New Guinea C). The Japanese virus, isolated by Susumu Hotta, was later shown to be DENV-1. Two more serotypes, called dengue 3 (DENV-3) and dengue 4 (DENV-4), were subsequently isolated by William McD. Hammon and his colleagues from children with hemorrhagic disease during an epidemic in Manila, the Philippines, in 1956. There have been viruses isolated that were suspected to represent new DENV serotypes, but characterization remains ongoing and identification of additional DENV serotypes has not been confirmed. Many early workers suspected that dengue viruses were transmitted by mosquitoes, but actual transmission was first documented by H. Graham in 1903. In 1906, T. L. Bancroft demonstrated transmission by Aedes aegypti, later known to be the principal urban mosquito vector of dengue viruses. Subsequent studies in the Philippines, Indonesia, Japan, and the Pacific showed that Aedes albopictus, Aedes polynesiensis and other members of the Aedes (Stegomyia) scutellaris complex were also efficient secondary vectors for dengue viruses. During and following World War II, A. aegypti greatly expanded its distribution in Asia, becoming the dominant day-biting mosquito in most Asian cities. Multiple dengue virus serotypes were also disseminated widely at that time. At the end of the war, both the vector (A. aegypti) and the viruses (all four serotypes) were widespread in Asia. A dramatic increase in human population growth and urbanization in the postwar years, especially in southeast Asia, created ideal conditions for increased transmission of urban mosquito-borne diseases. As a result, epidemics of dengue became widespread in the region. The first epidemics where dengue hemorrhagic fever (DHF) was reported occurred in the Philippines and Thailand in the mid 1950s. In the 1960s, but accelerating in the 1970s–80s, jet air travel increased dramatically. Population growth and urbanization combined with increased movement of people within and among countries of the region, resulted in increased spread of dengue viruses among population centers, increased frequency of epidemic activity, the development of hyperendemicity (co-circulation of multiple dengue serotypes), and the emergence of epidemic dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in the countries of southeast Asia and India during the 1960s. During the 1970s, epidemic DHF/DSS expanded geographically with an accelerated rate in Asia, to the Pacific Islands after an absence of 25 years, and to the Americas. A. aegypti had been eradicated from many countries in tropical America in the 1950s and 1960s as part of a program to control urban yellow fever. The program was terminated in the early 1970s and A. aegypti began reinfesting the countries where it had been eliminated. Increased epidemic dengue fever followed as the region was reinfested by A. aegypti and new viruses were introduced during the 1970s, 1980s, and 1990s. With the development of global hyperendemicity in the tropics, severe dengue has emerged as one of the world’s most important global public health problems in the 21st century. It is a leading cause of hospitalization and death among children in tropical developing countries and causes a high economic and social toll where the disease is endemic. Epidemics occur frequently.

218

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00015-1

Dengue Viruses (Flaviviridae)

219

Fig. 1 Global distribution of dengue fever and its principal epidemic mosquito vector, Aedes aegypti, 2019.

In 2020, dengue fever is the most important arboviral disease of humans, with more than half of the world’s population (an estimated 3.6 billion people) living in areas at risk for dengue in tropical and subtropical areas of the world (Fig. 1). An estimated 400 million dengue infections occur each year. The average case–fatality rate for patients with severe dengue is variable, depending on the country, but averages about 5%.

Taxonomy and Classification Dengue viruses belong to the family Flaviviridae, genus Flavivirus. There are four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. Dengue viruses belong to a larger, heterogeneous group of viruses called arboviruses. This is an ecological classification that requires transmission between vertebrate hosts, including humans, and hematophagous arthropod vectors. A discussion of the epidemiologic, evolutionary, biologic, and immunologic relationships of these viruses with each other and with other flaviviruses is beyond the scope of this article. As are other flaviviruses, dengue viruses are comprised of a single-stranded positive sense RNA genome surrounded by an icosahedral nucleocapsid. The latter is covered by a lipid envelope, which is derived from the host cell membrane from which the virus buds. The mature virion is approximately 50 nm in diameter (Fig. 2). The virus genome encodes three structural and seven nonstructural proteins in a single open reading frame that are expressed as one polyprotein. The open reading frame is flanked by two non-coding regions. The three structural proteins include the nucleocapsid core (C), a membrane associated protein (M), and an envelope (E) glycoprotein. The seven non-structural proteins include the NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Fig. 3). The functional domains responsible for virus neutralization, hemagglutination, fusion, and interaction with virus receptors are associated with the E protein. Epitope mapping has demonstrated three major domains on the E protein. Immunogenic epitopes have been mapped to all three domains, but domain II appears to be most important for dengue virus neutralization. There are 53 virus species in the genus Flavivirus recognized by the International Committee on Taxonomy of Viruses. The flaviviruses are divided into four subgroups based on the mode of transmission: (1) insect-specific viruses that have only been isolated from various mosquito species; (2) viruses that have no known arthropod vector, and which have been isolated only from rodents and bats; (3) mosquito-borne viruses; and (4) tick-borne viruses. The mosquito-borne and tick-borne groups are the largest, but many insect-specific viruses have been identified in recent years. The dengue viruses belong to the mosquito-borne subgroup, and are considered as one virus species with four serotypes instead of four distinct viruses according to the current classification. Other members of the mosquito-borne group that are of public health importance include the genus prototype yellow fever virus, and Japanese encephalitis, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Zika and others.

220

Dengue Viruses (Flaviviridae)

Fig. 2 Mature Dengue Virus (DENV) Structures. (A) A quarter of the central cross-section of a cryoEM map of DENV. The map is colored radially: red (1–130 Å), yellow (131–200 Å), green (201–234 Å), cyan (235–249 Å), and blue (4250 Å). The part of the DENV structure colored in red and yellow indicates the approximate radius that contains the RNA–capsid proteins complex. In green, the lipid bilayer membrane is shown with the E and M transmembrane regions; cyan indicates the E and M ectodomains, and blue indicates the glycosylation sites of E and M proteins. (B) Crystal structure of the prefusion dimeric E protein [Protein data bank (PDB) 1OAN]. DI, DII, and DIII are colored in red, yellow, and blue, respectively. The fusion loop on each E protein is circled in black. (C) Crystal structure of the postfusion trimeric E proteins (PDB 1OK8). One E protein is colored as indicated in (B), the other two are shown in gray. Fusion loops are assembled at one end (enclosed in the black box) and likely interact with the endosomal membrane. (D) Organization of E proteins on the compact mature DENV at 281C (PDB 3J27). Ribbon representation of an E protein raft is shown. One asymmetric unit contains three individual E proteins: mols A, B, and C. The neighboring symmetry-related E proteins within a raft are labeled as A0 , B0 , and C0 . All E proteins on the virus surface are also shown as gray surfaces. The 5- and 3-fold vertices are indicated by red pentagon and triangle shapes, respectively. (E) The cryoEM structure of the DENV2 class III particles at 371C (PDB 3ZKO). All of the E proteins have moved to higher radius. The E protein dimer near the 5-fold vertex (mols A–C0 ) has undergone rotation. The B and B0 dimer dissociate from each other. Source: Lok, S.-M., 2016. The interplay of dengue virus morphological diversity and human antibodies. Trends in Microbiology 24 (4), 284–293.

Dengue Viruses (Flaviviridae)

221

Fig. 3 Organization of the dengue virus genome.

Antigenically, the dengue viruses make up a unique complex within the mosquito-borne group of flaviviruses. Although the four dengue serotypes are antigenically distinct, there is evidence that serologic subcomplexes may exist within the group. For example, a close genetic relationship has been demonstrated between DENV-1 and DENV-3 and between DENV-2 and DENV-4. The sizes of the genomic open reading frames of DENV-1, DENV-2, DENV-3, and DENV-4 are 3392, 3391, 3390, and 3387 amino acids, respectively, the shortest among the mosquito-borne flaviviruses. An amino acid sequence positional homology of 63%–68% is observed among the DENV serotypes compared to 44%–51% between DENVs and other flaviviruses such as yellow fever and West Nile.

Geographic and Seasonal Distribution Dengue viruses have a worldwide distribution in tropical and subtropical areas (Fig. 1). The viruses are endemic in most urban centers of the tropics, with transmission occurring year-round, and epidemics occurring every 3–5 years. It is well documented, however, that dengue viruses are maintained for the most part by silent transmission during interepidemic periods in most tropical urban areas and, although the risk of infection is lower than during epidemic periods, there is still substantial risk for infection. Peak seasonal transmission of dengue viruses during both epidemic and interepidemic periods is usually associated with periods of high rainfall and moderated temperature in most endemic countries. Factors influencing seasonal transmission patterns of dengue viruses are not well understood, but obviously includes mosquito density, which may increase during the rainy season, especially in those areas where water level in larval habitats is dependent on rainfall. In areas where water in storage containers is not influenced by rainfall, however, other factors such as higher humidity and moderate ambient temperatures associated with the rainy season increases survival of infected mosquitoes, thus increasing the probability of secondary transmission. Virus strain, serotype, herd immunity, mosquito species, mosquito behavior and mosquito vector competence also influence the transmission dynamics.

Host Range and Virus Propagation There are only three known natural hosts for dengue viruses: Aedes mosquitoes, humans, and non-human primates. Viremia in humans may last 2–12 days (average, 4–5 days) with titers of infectious virus ranging from undetectable to more than 108 mosquito infectious doses 50 (MID50) per ml. Experimental evidence shows that several species of primates (chimpanzees, gibbons, and macaques) become infected and develop viremia titers high enough to infect mosquitoes, but do not develop illness. Viremia levels in lower primates are more transient, often lasting only 1–2 days if detectable, with titers seldom reaching 106 MID50 per ml. However, recent studies in Cambodia have suggested that humans with undetectable viremias by PCR were still capable of infecting mosquitoes. This has important implications for virus maintenance during interepidemic periods. Dengue viruses are known to cause clinical illness and disease only in humans and some non-human primates. Baby mice, which are used for the isolation and assay of many other arboviruses, may show no signs of illness after intracerebral inoculation with most unpassaged strains of dengue viruses. Experimentally, however, some strains can be adapted to produce illness and death in baby mice by serial passage. SCID, Rag2–18c1 and AG129 mice have been used for pathogenesis studies since these immunocompromised strains have defects either in innate or adaptive immune responses, allowing robust viral replication. Some strains of dengue, primarily low passage clinical isolates, are also capable of replicating transiently in immunocompetent mice.

222

Dengue Viruses (Flaviviridae)

Mosquito species of the genus Aedes appear to be the natural hosts for dengue viruses. Species of the subgenus Stegomyia are the most important vectors in terms of human transmission, and include A. (S.) aegypti, the principal urban vector worldwide, A. (S.) albopictus (Asia, the Pacific, Americas, Africa, and Europe), A. (S.) scutellaris, A. (S.) polynesiensis, A. (S.) hensilli, A. (S.) pseudoscutellaris (Pacific), A. (S.) africanus, A. (S.) taylori and A. (S.) luteocephalus (Africa). It is uncertain what role A. albopictus plays in transmission in areas where it has been recently introduced. Species of the subgenera Finlaya (Asia) and Diceromyia (Africa) appear to be important mosquito hosts involved in forest maintenance cycles of these viruses. Two other species, Ochlerotatus ( ¼Aedes) (Gymnometopa) mediovittatus (Caribbean) and Oc. ( ¼ Aedes) (Protomacleaya) triseriatus (North America), have been shown to be excellent experimental hosts of dengue viruses. Low passage or unpassaged dengue viruses can efficiently be propagated only in laboratory-reared mosquitoes and in mosquito cell lines. Mosquito species most commonly used for in vivo propagation include A. aegypti, A. albopictus, and Toxorhynchites spp., all of which can be reared with ease in the laboratory. Only three mosquito cell lines show high susceptibility to dengue viruses: C6/36 from A.albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from T. amboinensis. Dengue viruses can also be propagated in baby mice (see above) and in several vertebrate cell lines. These infection models have lower susceptibility to infection than the mosquito cells. However, dengue viruses must be adapted to each system by serial passage before consistent results can be obtained. Mammalian cell lines commonly used include LLC-MK2 and VERO (monkey kidney), BHK-21 (baby hamster kidney), FRhL (fetal rhesus lung), and PDK (primary dog kidney) cells. Propagation of dengue viruses in mosquito cell lines is associated with genetic stability due to slower accumulation of mutations than propagation in mammalian cell lines.

Genetics Laboratory and epidemiologic studies have demonstrated that genetic variants of dengue viruses occur in nature. This was first observed with DENV-3 isolated during the epidemics in Puerto Rico in 1963 and 1977, and in Tahiti in 1965 and 1969. The viruses in these epidemics were shown to be antigenically and biologically very similar to each other, but very different biologically from Asian strains of the same serotype. Antigenic differences were observed between Caribbean and Asian strains of DENV-4 as well. Oligonucleotide fingerprinting, restriction enzymes, primer extension, sequencing, and nucleotide sequence comparisons have been used to study the genetic variation among dengue viruses. The number of genetic subtypes identified in each serotype varies with the method used, but based on sequencing of the envelope protein, there are five distinct genotypes of DENV-1, five of DENV-2, four of DENV-3, and four of DENV-4. In general, viruses circulating in the same geographic region during the same time periods have shown genetic homogeneity, while differing from viruses of the same serotype from other regions. However, with increased transmission and spread of dengue viruses worldwide via airplane, they have increased in diversity, influencing both the virulence and epidemic potential. Since there is not an ideal animal model for dengue, it is not well understood how genetic changes or variation influences phenotypic expression of the viruses. That said, however, epidemiologic, clinical and virologic evidence is accumulating showing the importance of the virus strain in determining disease severity and epidemic potential. Genetic evidence of the diversity of dengue viruses influencing transmission dynamics comes from clinical and epidemiological studies in a series of epidemics in Asia, the Pacific and Caribbean Islands from the 1970s to the present time. In the earlier studies, viruses were isolated and stored unpassaged at  701C for years until technological advances allowed their study. Full-length genome sequencing was conducted on viruses isolated before, during and after these epidemics where DENV-2, -3 and -4 were predominant. Epidemiological and sequence data from three geographic regions (Asia, Pacific and Americas) and all three serotypes (DENV-2, -3 & -4) show limited genetic changes in the viruses (two-three amino acid changes mainly in the non-structural genes) correlated with phenotypic changes, including clinical severity, viremia levels in humans, virus replication in mosquitoes and cell culture, and epidemic potential. More recent studies have defined the molecular basis for phenotype changes of DENV-1 and -2. Collectively, the data suggest that dengue virus evolution via genetic drift and positive selection may result in the emergence of new virus clades or subtypes, often of the same genotype, that have greater epidemic potential, thus playing an important role in the epidemiology and pathogenesis of severe dengue. Epidemic potential, disease severity and pathogenesis of dengue are determined by a complex interaction of viral, host and environmental factors. Although there have been reports on intraserotypic recombination events among dengue viruses, the data suggest that this has not been an important factor in their evolution. However, with increased occurrence of the cocirculation of multiple serotypes in an area (hyperendemicity), there have been increased reports of concurrent infections with two serotypes increasing the probability that interserotypic recombination may occur.

Evolution The origin of dengue viruses is not known. It is not clear which of the four flavivirus groups (insect specific, vertebrate, mosquitoborne, tick-borne) is the oldest. Given the large number of insect-specific viruses identified in mosquitoes in recent years, which have tentatively been placed in the genus Flavivirus, however, it seems plausible that an ancestral flavivirus was a mosquito or a tick virus that diverged by adapting to a variety of vertebrate hosts, including rodents, birds, bats, and nonhuman primates. From these early introductions, the no-known vector, tick-borne encephalitis, and mosquito-borne (yellow fever, dengue, and Japanese encephalitis) subgroups arose. Speculation that tick-borne and mosquito-borne viruses had a common ancestor is supported by

Dengue Viruses (Flaviviridae)

223

the fact that several mosquito-borne flaviviruses (Koutango, Saboya, West Nile, St. Louis encephalitis and yellow fever) have all been isolated from ticks. Also, it has been reported that some tick-borne viruses replicate in mosquitoes or mosquito cell cultures. On the other hand, a comprehensive replication study of 66 flaviviruses in mosquito, tick, and vertebrate cell cultures showed conclusively that there is a strict host range specificity for viruses in the four subgroups, and that the conventional classification based on epidemiologic and phylogenetic relationships is correct. Regardless of the geographic and evolutionary origin, the data collectively suggests that the dengue viruses most likely evolved in mosquitoes before becoming adapted to lower primates and then to humans, an estimated 1500–2000 years ago. Biologically, dengue viruses are well adapted to their mosquito hosts, being maintained by vertical transmission (from female mosquito to its offspring) in mosquito species responsible for sylvatic cycles, with periodic amplification in lower primates. As noted above, forest cycles have been documented in South-East Asia and Africa, and possibly in Sri Lanka, India, Vietnam and China. These cycles involve several species of lower primates and three subgenera (Stegomyia, Finlaya, and Diceromyia) of canopy-dwelling mosquito species of the genus Aedes. At some point in the past, probably with the clearing of the forests and development of human settlements in Asia, these viruses moved out of the jungle and into a rural environment where they were, and still are, transmitted to humans by peridomestic mosquitoes such as A. albopictus. Ultimately, the migration of people moved the viruses into the cities of the tropics where they became “urbanized” and transmitted by the highly domesticated, urban A. aegypti mosquito, which had been spread around the world from Africa via sailing ships and increased commerce. Because of the rather slow rate of change (genetic drift) of the dengue virus genome, viruses isolated over long periods of time in the same geographic region still show striking homogenicity. The greatest genetic differences between dengue virus strains were observed between DENV-2 and DENV-3 isolated from forest mosquitoes in Africa and Asia, respectively, and viruses of the same serotype isolated from humans or mosquitoes in nearby urban areas. This suggests that there is limited gene flow between the forest and urban cycles.

Serologic Relationships and Variability Dengue viruses share a common morphology, genomic structure, and antigenic determinants with 52 other flaviviruses. Serologic tests most frequently used to determine antigenic relationships have included the hemagglutination-inhibition (HI), complement fixation (CF), and the plaque reduction neutralization (PRNT) tests. Because all flaviviruses share common antigenic determinants, identification of individual family members using these serologic tests is difficult. The dengue viruses make up one antigenic complex within the genus Flavivirus. They share complex-specific antigenic determinants on both structural and nonstructural proteins. Serotypes within the dengue virus complex are most accurately and easily identified with an indirect immunofluorescent antibody (IFA) assay using serotype-specific monoclonal antibodies which react with epitopes on the structural proteins, or by a polymerase chain reaction (PCR) using serotype specific primers. Both antigenic and biological variation among dengue viruses has been documented. As mentioned above, DENV-3 viruses isolated in the Caribbean and the South Pacific in the 1960s were found to be antigenically distinct from the prototype and Asian strains of DENV-3 using PRNT. They were also biologically unique in that they did not grow as well in baby mice and mosquitoes as did the Asian strains. DENV-4 viruses isolated in the Caribbean after the introduction of this serotype into that region in 1981 were antigenically distinct from DENV-4 viruses from Asia. Field and epidemiological evidence for natural strain variation among dengue viruses is more circumstantial. When DENV-2 was introduced into the South and Central Pacific islands in 1971 after being absent for more than 25 years, epidemics occurred on numerous islands. Marked variation was observed in disease severity, viremia levels, and epidemic potential in epidemics on different islands. This variation was observed with both DENV-1 and DENV-2 in the Pacific and with DENV-3 in Sri Lanka and Indonesia. Some DENV strains appeared naturally attenuated, causing mild illness with low viremia levels of short duration, whereas others caused rapidly spreading epidemics with severe hemorrhagic disease and high viremia levels. Factors that could influence epidemic transmission and disease severity, other than differences in the virus strain, were ruled out as a cause of this variation. Recent studies in Sri Lanka and Puerto Rico have shown that genetic mutations in strains of DENV-3, DENV-2 and DENV-4 have also influenced the epidemic transmission potential of the virus and the disease severity.

Epidemiology Dengue viruses occur in nature in three basic maintenance cycles. The primitive forest cycle involves canopy-dwelling mosquitoes and lower primates. A rural cycle, primarily in Asia and the Pacific, involves peridomestic mosquitoes (A. albopictus and A. scutellaris spp.) and humans. The urban cycle, which is the most important epidemiologically and in regard to public health and economic impact, involves the highly domesticated A. aegypti mosquito and humans. The viruses have fully adapted to humans and are maintained in most large urban centers of the tropics, with epidemics occurring at periodic intervals of 3–5 years. A combination of increased urbanization in the tropics, changing life styles, and the lack of effective mosquito control has made most tropical cities highly permissive for transmission of dengue viruses by A. aegypti. Increased air travel by humans provides an ideal mechanism to transport dengue viruses between population centers. As a result, in the past 50 years there has been a dramatic global increase in the geographic spread of dengue viruses within and among regions, resulting in increased

224

Dengue Viruses (Flaviviridae)

epidemic activity, development of hyperendemicity, and increased incidence of severe and fatal forms of the disease, especially DHF/DSS. After being observed only in Southeast Asia, epidemic DHF/DSS has spread to west Asia, the Peoples Republic of China, Taiwan, the Pacific islands, the Americas, Africa and the Middle East in the past 50 years. Factors responsible for the emergence and spread of the severe form of the disease, DHF/DSS, are not fully understood. The changing disease pattern described above provides support for the principal hypotheses regarding the pathogenesis of DHF/DSS, a secondary infection, and an increased virulence of the virus. Thus, increased transmission in urban areas and the development of hyperendemicity increases the probability of secondary infections and of genetic changes in the virus which may result in more severe disease due to antibody dependent enhancement (ADE) or to an epidemic virus strain with greater virulence. And increased spread of viruses between population centers via modern transportation increases the probability of introducing a virus strain with increased epidemic potential and virulence into new geographic areas. Increased transmission of multiple dengue serotypes thus increases the probability that severe disease will occur, regardless of whether the underlying cause is due to increased virulence, ADE, or, more likely, a combination of both. Dengue is primarily an urban disease. Most major epidemics of DHF/DSS occur in tropical urban cities where large and crowded human populations live in intimate contact with the principal mosquito vector, A. aegypti. This mosquito is a highly domesticated, day-biting species that lives and breeds in and around people’s homes. High mosquito densities often occur in tropical cities because of water-storage practices and the accumulation of domestic waste. Primary larval habitats for A. aegypti include a variety of domestic water-storage containers such as clay jars and pots, metal drums, cement cisterns, and many other artificial containers found in the domestic environment that collect and hold rain water. The latter include, but are not limited to flower vases and pots, used automobile tires, buckets, bottles, cans, old machinery, etc.

Transmission and Tissue Tropism Dengue virus transmission occurs mostly by bites of infective mosquito vectors. Any of the four serotypes may cause high levels of viremia in humans (Z108 MID50 per ml) that lasts on an average of 4–5 days (range, 2–12 days). If a competent mosquito vector takes a blood meal from a person during a viremic phase, virus is ingested with the blood meal and infects the cells of the mosquito mesenteron. After 8–12 days, depending on ambient temperature and the strain of mosquito, the virus will disseminate outside the midgut and infect other tissues, including the mosquito ovaries, nerve ganglia, brain and salivary glands. When the mosquito takes a subsequent blood meal, virus is injected into the person along with the salivary fluid. Dengue virus infection has no apparent effect on the mosquito, which is infected for life. A. aegypti is a highly competent epidemic vector of dengue viruses. It lives in close association with humans because of its preference to lay eggs in artificial water-holding containers in the domestic environment, and to rest inside houses and feed on humans rather than other vertebrates. It has a nearly undetectable bite and it is very restless in that the slightest movement will interrupt its feeding and make it fly away. It is not uncommon, therefore, for a single mosquito to bite several persons in the same room over a short period of time. If the mosquito is infective, every person bitten may become infected. In addition to transmitting the virus to humans or non-human primates, the female mosquito may also transmit the virus vertically through her eggs to her offspring. Although the implications of vertical transmission are not fully understood, it is thought to be an important mechanism in the natural maintenance cycles of dengue viruses, especially in rural and forest settings. The primary site of replication of dengue viruses after injection into humans by the feeding mosquito is not well understood. Infective mosquitoes will often probe the skin multiple times when looking for a blood vessel on which to engorge. Each time it probes, an infective mosquito injects salivary fluid infected with dengue viruses. Dengue viruses deposited in the epidermal and dermal layers likely infect a variety of cells, including dendritic cells, macrophages (Fig. 4). Additional responding cells such as mast cells, T cells, NK cells and others may be activated (Fig. 4). It is uncertain how systemic infection evolves, but it is likely that cells infected in the skin are recruited into the lymphatic system, allowing the infection to spread to the draining lymph nodes and beyond. Human tissues from which dengue viruses have been isolated or have had documented infection include liver, lungs, kidneys, lymph nodes, stomach, intestine, spleen and brain, but it is not known to what extent the virus replicates in these tissues. Infected cell types that have been identified include Langerhans cells, dermal dendritic cells, endothelial cells and macrophages and monocytes. Virus replication has also been detected in hepatocytes and Kupffer cells in the liver. Pathological changes similar to those observed in yellow fever, with focal central necrosis, have been observed in the liver of some patients who have died of dengue virus infection. There is some evidence that the viruses also replicate in endothelial cells and possibly in bone marrow cells. Encephalopathy has been documented in dengue infection but whether dengue viruses cross the blood–brain barrier and replicate in the central nervous system is still an open question. Dengue viruses have been transmitted by blood transfusion and organ transplantation.

Pathogenesis Dengue disease expression in humans is the result of the complex interaction between an individual with specific genetic and immunological characteristics and the strain of dengue virus with specific virulence and biological characteristics. The result may range from asymptomatic infection to a severe disease and death. Unfortunately, animal models do not fully replicate the symptoms and kinetics of human infections, which vary greatly. Most of what we know or think we know is based on in vitro

Dengue Viruses (Flaviviridae)

225

Fig. 4 Host responses to cutaneous dengue virus. Most infections by dengue virus (DENV) occur after subcutaneous injection of the virus into the skin. Released viral particles may infect nearby cells such as monocytes or dendritic cells (DCs) or activate resident immune cells like mast cells. A local inflammatory response to DENV in the skin induces the recruitment of leukocytes from the vasculature, including natural killer (NK) cells and T cells, which promote the killing of virus-infected cells at the site of injection. DENV is thought to then travel to draining lymph nodes via lymphatic vessels to establish systemic infection. These localized inflammatory responses occur days before there are any signs of severe infection. Source: St. John, A.L., Abraham, S.N., Gubler, D.J., 2013. Barriers to pre-clinical investigations of anti-dengue immunity and immunepathology. Nature Reviews Microbiology 11 (6), 420–426.

studies using highly passaged (adapted) viruses and artificial host systems, although low-passage clinical isolates are increasingly used for experimental studies. Therefore, there is still considerable controversy about the pathogenesis of severe dengue disease, including DHF/DSS. Disseminating virus is first detected in the draining and remote lymph nodes, leading to a systemic infection and viremia, which may be detectable 24–48 h prior to clinical symptoms. Leukopenia, neutropenia and thrombocytopenia are common, with altered bone marrow cellularity. Evidence suggests that at least two pathogenetic mechanisms are associated with severe dengue infection. Classical DHF/DSS is characterized by a vascular leak syndrome which, if not corrected, may rapidly lead to hypovolemia, shock, and death (Fig. 5). Multiple theories of dengue pathogenesis exist and these primarily focus on the role of immune-mediated pathology in the initiation of vascular leak syndrome. In secondary infections, the mechanisms implicating non-neutralizing antibodies, or weakly cross-reactive T cells in generating high viral titers have been proposed to contribute to severe disease. The underlying pathogenetic mechanism for this syndrome is thought to be an immune enhancement phenomenon in which the infecting virus complexes with non-neutralizing dengue antibodies, thus enhancing infection of mononuclear phagocytes. Phagocytes produce vasoactive mediators, which may result in vascular permeability and leakage. Serum anti-dengue virus antibody levels may also be a factor in antibody-mediated pathologies. Moderate concentrations of crossreactive antibodies are thought to promote enhanced disease severity, whereas low concentrations may be insufficient to induce the phenomenon and high concentrations could offer some cross-protection. Poorly neutralizing cross-reactive antibodies are also able to induce enhanced activation of mast cells, which release vasoactive mediators. Another hypothesis emphasizes the

226

Dengue Viruses (Flaviviridae)

Fig. 5 Clinical symptoms and pathogenesis of dengue in humans, across the spectrum of mild to severe disease. Source: St. John, A.L., Abraham, S.N., Gubler, D.J., 2013. Barriers to pre-clinical investigations of anti-dengue immunity and immune-pathology. Nature Reviews Microbiology 11 (6), 420–426.

importance of T cells in the clearance of dengue-infected cells. During secondary infection, memory recall responses by T cells that are only weakly cross-reactive for the secondary strain may exhaust immune resources and outcompete naïve T cells that would be more specific for the strain causing secondary infection. These activated T cells may also produce cytokines, in addition to infected cells, that could promote vascular leakage. Several vasoactive cytokines and host inflammatory factors have been identified in different human studies as being associated with severe dengue syndrome, including TNF, VEGF, and mast cell derived proteases. Loss of plasma from the vascular compartment may range from mild and transient to severe and prolonged, the latter often resulting in life threatening irreversible shock and death. Although there is a slightly higher risk (B10%) of developing classical DHF/DSS in persons experiencing secondary dengue infections, DHF/DSS may also occur in primary infections. This suggests that additional mechanisms of vascular pathogenesis exist that are not dependent on heterologous adaptive immune responses. Interestingly, DHF/DSS rarely or never occurs in the 3rd or 4th infection. In vitro studies have shown that not all dengue viruses can be enhanced and that not all antibodies can enhance dengue infection. That, in addition to epidemiologic and virological studies showing that dengue viruses in nature vary greatly in their epidemic potential, and virulence (see Section “Genetics” above), raises the question as to whether dengue virus strains vary in their ability to stimulate the production of enhancing antibodies, whether this is associated with virulence, and, if so, how this relates to the immune enhancement hypothesis. Unfortunately, animal models cannot explain the disease mechanisms of different dengue strains in humans. However, accumulating sequence and field data suggest the dengue viruses, like most other animal

Dengue Viruses (Flaviviridae)

227

viruses, vary in their virulence and in their epidemic potential. As already noted, both host immunity and virus virulence are likely playing a role in the pathogenesis of severe dengue disease. Patients infected with dengue viruses may also experience other forms of severe disease, including severe and uncontrolled bleeding, usually from the upper gastrointestinal (GI) tract. This severe hemorrhagic disease may be associated with multiple organ failure, and it is more difficult to manage than classical vascular leak syndrome. The underlying pathogenetic mechanism for this type of bleeding is not fully understood. A third type of severe and fatal dengue infection, which may or may not involve overt hemorrhagic disease, is neurological symptoms. Although many patients with this syndrome present clinically as viral encephalitis, conclusive evidence that dengue viruses commonly infect the central nervous system has not yet been obtained. Available data suggests that neurological symptoms may be secondary to cerebral hemorrhage, edema, or other indirect effects of dengue virus infection.

Clinical Features of Infection Dengue virus causes a spectrum of illness in humans ranging from subclinical to severe and fatal hemorrhagic disease with the latter representing only a small proportion of all infections. The incubation period may be as short as 3 days and as long as 14 days, but most often it is 4–7 days (Fig. 6). The majority of patients present with a mild, nonspecific febrile illness, or with classical dengue fever. The latter is generally observed in older children and adults, and is characterized by a sudden onset of fever, frontal headache, retro-ocular pain, and myalgias. Rash, joint pains, nausea and vomiting, lymphadenopathy and hemorrhagic manifestations such as nose and gum bleeding, purpura and petechiae are common (Fig. 5). The acute illness, which lasts for 3–7 days, is usually benign and self-limiting, but it can be very debilitating. Convalescence may be prolonged for several weeks. As noted, severe dengue may present in several forms. The most common form of severe dengue is a vascular leak syndrome, often observed in children under 15 years of age, although this syndrome may also occur in adults in areas of lower endemicity. Severe dengue is characterized by acute onset of fever and a variety of nonspecific signs and symptoms that may last 2–7 days (Fig. 6). During this stage of illness, dengue is difficult to distinguish from many other viral, bacterial, and protozoal infections. In children, upper respiratory tract symptoms caused by concurrent infection with other viruses or bacteria are not uncommon. The differential diagnosis should include other hemorrhagic fevers, hepatitis, leptospirosis, typhoid, malaria, measles, influenza and other diseases causing fever. The critical stage in severe dengue occurs when fever subsides to or below normal. This usually occurs 5–7 days after onset of fever (Fig. 6). Most patients recover without complications, but the patient’s condition may deteriorate rapidly with signs of circulatory failure, neurological manifestations, shock and death, if proper management is not available. The main pathophysiological features of severe dengue involve immune system activation, increased microvascular permeability and plasma leakage into the extravascular spaces. There may be altered hemostasis involving thrombocytopenia, impaired coagulation and vascular dysfunction. Skin hemorrhages such as petechiae, easy bruising, bleeding at the sites of venipuncture, and purpura/ecchymosis are the most common hemorrhagic manifestations; GI hemorrhage may occur, usually after, but in some cases before, the onset of shock. The World Health Organization (WHO) has recently redefined the definition of severe dengue. Previously limited to only DHF/DSS, other forms of severe disease not associated with vascular leakage are now included (Fig. 7). The strict criteria to define DHF remain unchanged with four major clinical manifestations: fever or recent history of fever, hemorrhagic manifestations, vascular leakage, and thrombocytopenia. Patients with DSS have DHF with objective signs of circulatory failure. Thrombocytopenia and hemoconcentration are constant features of DHF/DSS. As noted above, other severe forms of dengue include severe hemorrhage, myocardiopathy and neurological disease, which have been added to the new WHO guidelines. Patients may present with severe and uncontrollable upper GI bleeding with shock and death in the absence of hemoconcentration or other evidence of the vascular leak syndrome. Also, they may present with a variety of neurological and psychiatric disorders, including headache, dizziness, hysteria, and depression. In addition,

Fig. 6 A time course of dengue clinical symptoms during infection.

228

Dengue Viruses (Flaviviridae)

Fig. 7 Suggested dengue case classification and levels of severity; adapted from the 2009 WHO Dengue guidelines for diagnosis, treatment, prevention and control.

some patients present with clinical symptoms of viral encephalitis and Guillain–Barré syndrome, but as noted above, there is no conclusive evidence that CNS infection occurs. Treatment of dengue infection is symptomatic since there are no specific antiviral drugs. Management of vascular leakage syndrome, however, is very effective with a corrective fluid replacement therapy. The prognosis of patients with DHF/DSS depends on early recognition, monitoring, and management of vascular leakage. Good nursing care is critical. A definitive diagnosis of dengue virus infection can only be made in the laboratory by serologic and/or virologic methods.

Pathology and Histopathology The pathology of dengue virus infection is not well understood because systematic postmortem studies have not been done on patients representing all types and stages of severe dengue disease. The pathology may also be complicated by patient age, and co-morbidities. In general, the pathology of dengue is non-specific and difficult to distinguish from yellow fever, other viral hemorrhagic fevers, leptospirosis, and other diseases. Petechiae, serous effusion and organ hypoperfusion are common. Liver pathology is similar to that seen in yellow fever with centrilobular and mid-zonal necrosis, apoptosis and steatosis. There is often an increased proportion of immune cells seen in blood and in the spleen. Studies have not revealed destructive inflammatory vascular lesions, but some swelling and occasional necrosis has been observed in endothelial cells. Changes in the kidneys are suggestive of an immune complex type of glomerulonephritis. Complement deposition has also occasionally been identified in tissues. There is impaired bone marrow function, which improves when the patient becomes afebrile. Biopsy studies of the skin rash have demonstrated perivascular edema with infiltration of lymphocytes and monocytes.

Immune Response Persons infected with dengue viruses produce IgM and IgG antibodies, both of which appear 5–7 days after onset of illness in primary infections. The highest titers of IgM antibodies are detected in primary dengue infections, but an IgM response is also detected in secondary and tertiary infections. The IgM response is transient and generally disappears 30–90 days after onset of illness in primary infections and after shorter periods of time in secondary and tertiary infections. IgG antibodies, in contrast, may persist for at least 60 years and probably throughout the life span of the patient. In persons experiencing their first dengue or

Dengue Viruses (Flaviviridae)

229

flavivirus infection, peak IgG levels are detected 14–21 days after onset of illness and seldom exceed titer values of 640–1280 as measured by hemagglutination-inhibition or neutralization tests. There are exceptions, however, with some infections inducing antibody titers typical of secondary or tertiary infections during primary infection. In secondary infections, there is a rapid anamnestic IgG immune response to dengue complex-and/or other flavivirus-specific antigenic determinants. Both IgM and IgG antibodies neutralize dengue viruses, and infection provides life-long immunity to the infecting virus serotype. Both IgM and IgG antibodies to dengue viruses cross-react with other flavivirus antigens, including those of yellow fever, Japanese encephalitis, West Nile, Zika, and St. Louis encephalitis viruses. Those flavivirus cross-reactive antibodies are generally non-neutralizing. Cross-reactivity with viruses in the dengue complex makes the interpretation of serologic results difficult. In geographic areas where several flaviviruses are endemic, definitive laboratory diagnosis can only be made by virus isolation or nucleic acid detection, and in patients with a primary infection, by PRNT. Normally, a combination of laboratory (serologic and virologic), clinical, and epidemiologic data is used to confirm a diagnosis of dengue or other flavivirus infections. Because dengue virus-specific IgG antibodies persist for many years or decades, their presence in a single serum sample is not diagnostic of current or recent infection. Lower IgG titers simply indicate that the person has had a previous infection at some time in the past. Paired serum samples are required to confirm a current infection by demonstrating a fourfold or greater rise in IgG antibody levels. The presence of detectable IgM antibody in a single serum sample is considered to be diagnostic for a recent infection because this isotype does not persist for long periods of time. Increasingly, NS1 protein is used as a diagnostic marker of acute dengue infection since this viral protein is secreted from infected cells and can be detected in patient serum. NS1, like RNA detection by PCR, is a surrogate marker for the presence of infectious virus. Extensive work done in recent years has demonstrated that immunopathogenetic mechanisms play a major role in the pathophysiology of vascular leakage syndrome. Generally, CD4 þ and CD8 þ T-cell responses are directed against multiple viral proteins, including both structural and non-structural proteins. After primary infection, memory T cells proliferate in response to multiple dengue serotypes providing both specific and cross-reactive responses. After a secondary infection, T cell proliferation is of low-affinity and may be more reactive to the first infecting virus serotype than to the second virus strain, a phenomenon analogous to original antigenic sin seen in the antibody response. Whether T cell activation is involved in immune pathology is controversial and results from different human studies vary considerably. T cells have increasingly been suggested to contribute to infection control, both through the development of cytotoxic responses and through promoting high affinity antibody production. NK cells have also been identified to be activated during dengue infection; however, it is not yet clear whether they are primarily protective or potentially contribute to immune pathology. The vascular leakage syndrome is commonly associated with a cascade of immunopathologic events in which increased infection of dendritic cells, macrophages and other cell types lead to a cytokine storm that is thought to increase vascular permeability. The literature supporting this hypothesis is extensive, but highly variable with many studies lacking controls or performed in immune compromised animals, where there is a potential of enhancing the role of virus replication in immune pathology or promoting infection in cell types that are normally resistant to infection. There is strong evidence, however, that proand anti-inflammatory cytokines are elevated during the acute febrile phase of the disease. Aside from cytokines, additional vasoactive inflammatory mediators and inhibitors of coagulation are released during an acute infection, including products such as mast cell proteases, heparin, and matrix metalloproteinases. There is accumulating evidence that cell-mediated immunity also plays a role in terminating dengue infections. Dendritic cells (DCs) are likely the initial site of virus replication. Dengue virus infection stimulates DC maturation and activation, and the production of TNF-a, IFN-g and IFN-a/b that are known to inhibit virus infection. DCs migrate to T-cell-rich lymphoid organs where T cells are activated, stimulating memory responses and releasing cytokines and chemokines. Transcriptional activation of DCs has been associated with asymptomatic infections, supporting the concept that T cell responses can be protective. During secondary infections, T cells may be specific for a previous infecting serotype of dengue and cross-reactive for the secondary serotype. In that situation, T-cell responses are likely to be dominated by a subset of memory T cells, which produce IFN-g and CD40L, resulting in a better DC activation, T cell stimulatory capacity, IL-12 release, increased secretion of TNF-a and IFN-g, as well as potentially altered cytokine responses. When viremia is cleared, the cascade of events initiated by a Th1 cytokine response in the absence of effective viral control may contribute to the pathogenesis of DHF. Cross-reactive CD8 T cells that have low affinity to the second infecting virus serotype (see above) may be ineffective in clearing viremia, thus permitting a higher virus load and a more severe disease. However, cross-reactive CD4 T cells may contribute to crossprotection by promoting germinal center responses and enhancing the kinetics of antibodies that can neutralize the secondary serotype.

Prevention and Control of Dengue The options available for prevention and control of dengue have been limited, and dependent on controlling the mosquito vector, which has failed in most countries during the past 50 years. A highly effective vaccine against dengue is not available despite more than 70 years of research. However, recent progress in developing new mosquito control tools, vaccines and antiviral therapeutics has been promising, but it is unlikely that any single method or approach will be successful in reversing the trend of increased epidemic dengue. Most likely, a combination of vector control and vaccination will be needed to effectively control this disease.

230

Dengue Viruses (Flaviviridae)

Vaccines Effective vaccination to prevent dengue will likely require a live attenuated vaccine since this strategy ensures induction of both T and B cell memory responses. One vaccine produced by Sanofi Pasteur under the name Dengvaxia, has been licensed for use in people 9–45 years of age in some countries. In clinical efficacy trials Dengvaxia has been shown to be effective in preventing severe disease by 93% and decreasing hospitalization by 81%. The vaccine is most effective in individuals who have previously been exposed to the virus. However, 6 year follow up of vaccinees showed that there is a small increased risk of severe disease and hospitalization in vaccinees who were dengue-naïve at the time of vaccination, leading WHO to recommend that it not be used in seronegative individuals. The WHO recommended pre-vaccination screening for serological evidence of previous dengue infection, but also suggested that Dengvaxia could be used in highly endemic areas with a seroprevalence of 80% or higher. Currently, it is uncertain whether or how Dengvaxia will be used in dengue control programs. The dengue vaccine pipeline currently includes 2 candidate vaccines in phase 3 clinical trials, one in phase 2 and two in phase 1 (Table 1). In addition, there are several third generation vaccines under development using new molecular technology. The two most promising live attenuated candidate vaccines (CDC/Takeda and NIH/Merck) are expected to be licensed in the next 3–5 years. It is not known whether a balanced tetravalent protective response will be obtained. From a public health perspective, however, that may not be necessary. Current thinking is that a vaccine that protects against three or even two of the virus serotypes may be effective in protecting against a severe disease since 3rd and 4th infections are rarely if ever associated with a severe disease. A very promising issue has also been the rapid progress in developing antiviral drugs and therapeutic antibodies that can be used in the treatment of dengue infection, and perhaps, even in prevention and control programs. Research is currently focused on developing a safe and inexpensive drug that inhibits dengue virus replication that can be used in a manner similar to influenza antivirals. While most effort has been directed towards developing a small molecule drug, many studies are now screening drugs that were developed against other viruses and that have proven to be safe in humans. Monoclonal antibodies are also being developed for dengue therapeutics. Antibodies are able to limit virus infection through several known mechanisms including by aggregating virus particles, destabilizing the virus structure, preventing virus attachment to target cells, or by preventing fusion of the virus with the target cell membrane. Some monoclonal antibodies that are most effective in neutralizing DENV bind to the virus surface, locking the virus structure and preventing the dynamic movements required for virus attachment and fusion with the cell membrane. A DENV-1-specific therapeutic monoclonal antibody has shown promising results in early clinical trials and a DENV-2-specific antibody is being developed. These antibodies could be used for both treatment and for prophylaxis. Currently, the most widely used way to prevent dengue infection is to control the mosquito vector that transmits the virus. Unfortunately, with the exception of Singapore, programs to control A. aegypti mosquitoes have not been successful. For more than 50 years, the recommended method to control mosquitoes has been spraying ultralow volume (ULV) or thermal fog application of insecticides to kill adult mosquitoes. Field trials in Puerto Rico, Jamaica, and Venezuela, however, showed that this method was not effective in significantly reducing natural mosquito populations for any length of time. This supports epidemiologic observations that space spraying has little or no impact on epidemic transmission of dengue viruses. Unfortunately, however, the method is still the most widely recommended. Recent research has led to the development of a number of new and innovative chemical, genetic and biologic mosquito control tools. The insecticides include new classes of chemicals that will provide residual activity for 6 months to a year, a characteristic that is critical to control adult mosquitoes emerging from cryptic or hidden larval habitats. Other new insecticides can be formulated as spatial repellants to prevent mosquitoes from entering specific areas or to treat both natural and artificial oviposition sites. Both genetic and biological approaches to mosquito control are equally promising. The most promising genetic approach is the release of sterile male mosquitoes into a natural population to mate with wild female mosquitoes. Male sterilization can be achieved by genetic modification, irradiation or biologically by Wolbachia infection. Sterile males can be produced by the millions and released as eggs, pupae or adults into natural populations. If the sterile males are released in high enough numbers, egg hatch rate will decrease and the wild mosquito population will be suppressed. Field trials have been conducted in Malaysia, Cayman Islands, Panama, Brazil, Singapore, the United States and China, all with promising results.

Table 1

The dengue vaccine pipeline by stage of clinical development

Company

Vaccine strategy

Development phase

Sanofi Pasteur NIH/Merck CDC/Takeda WRAIR/GSK NMRC Merck/Hawaii Biotech

Chimeric, YF17-D backbone, DENV1–4 LAV, DENV-1,-3 & -4, D30/31 þ Chimeric, 2/4 LAV, DENV2 PDK53; þ Chimeric DENV1/2, 3/2 & 4/2 Inactivated, DENV1–4 DNA, DENV1–4 Subunit, DENV1–4

Licensed Phase 3 Phase 3 Phase 2 Phase 1 Phase 1 (on hold)

Source: YF17-D, Yellow fever vaccine strain 17-D; LAV, live attenuated virus; GSK, Glaxo-Smith Kline; NIH, National Institutes of Health; CDC, Centers for Disease Control and Prevention; NMRC, Navy Medical Research Center; PDK53, a live-attenuated DENV2 vaccine developed at Mahidol University, Thailand.

Dengue Viruses (Flaviviridae)

231

A promising biological control tool is the infection of A. aegypti mosquitoes with a common bacterium, Wolbachia, that naturally infects over 70% of all insects in nature, but for reasons that are unknown, not A. aegypti. Several strains of Wolbachia have been adapted to infect A. aegypti, causing their decreased survival and decreased susceptibility to dengue, yellow fever, Zika and chikungunya viruses. Field trials have been carried out in Australia showing effective spread of Wolbachia into the natural A. aegypti populations and effective control of dengue in one city. Other trials in Vietnam, Indonesia, Brazil and Colombia are in progress. An effective method of controlling A. aegypti is larval control, ie, to eliminate or control the larval habitats where the mosquitoes lay their eggs. Unfortunately, that is not possible in old cities that have expanded over the centuries and have many hidden or cryptic larval habitats. Fortunately, a number of these new mosquito control tools may be useful in reaching these sites, most of which are found in the domestic environment, where most transmission occurs. To have sustainability of prevention and control programs, some responsibility for mosquito control should be transferred from governments to citizens. For long-term sustainability, mosquito control programs must be regional, but operational control must be local, community-based and integrated. Persons living in A. aegypti infested communities have to be educated to take responsibility for their own health and help government agencies control vector mosquitoes, and thus prevent epidemic dengue. Effective sustainable prevention and control programs must involve a partnership between government agencies and the community. Countries with endemic dengue should develop active, laboratory-based surveillance systems that can provide some degree of early warning for epidemic transmission. Finally, prevention of excess mortality associated with DHF/DSS can be achieved by educating physicians in endemic areas on clinical diagnosis and management of severe dengue. As demonstrated in countries such as Thailand, early recognition and proper clinical management are the key factors reducing DHF/DSS case–fatality rates.

Future Continued population growth and urbanization of the tropical and subtropical areas, changing lifestyles, globalization and lack of effective mosquito control have been the most important factors responsible for the dramatically increased incidence and geographic expansion of dengue in the past 50 years. Epidemic dengue has become a global public health problem in the tropics and it is anticipated that this trend will continue unless more effective prevention and control strategies are developed and implemented worldwide. Fortunately, the dengue control pipeline includes a number of new and innovative tools, including vaccines, antiviral drugs, therapeutic antibodies, insecticides and other mosquito control tools, which if used in an integrated and synergistic way, could allow effective control of dengue in the near future.

Further Reading Bhatt, S., Gething, P.W., Brady, O.J., et al., 2013. The global distribution and burden of dengue. Nature 496, 504–507. doi:10.1038/nature12060. Gubler, D.J., Kuno, G., Markoff, L., 2007. Flaviviruses. In: Knipe, D., Howley, P. (Eds.), Fields Virology, fifth ed. Philadelphia: Lippincott Williams and Wilkins, pp. 1153–1252. Gubler, D.J., Ooi, E.E., Vasudevan, S., Farrar, J. (Eds.), 2014. Dengue and Dengue Hemorrhagic Fever, second ed. Wallingford: CAB International. Gubler, D.J., 1989. Aedes aegypti and Aedes aegypti-borne disease control in the 19900 s: Top down or bottom up. American Journal of Tropical Medicine and Hygiene 40, 571. Gubler, D.J., 2011. Dengue, urbanization and globalization: The unholy trinity of the 21(st) century. Tropical Medicine and Health 39, 3–11. Rico-Hesse, R., 2003. Microevolution and virulence of dengue viruses. Advances in Virus Research 59, 315–341. Simmons, C.P., Farrar, J.J., Nguyen, V.V., Wills, B., 2012. Dengue. New England Journal of Medicine 366, 1423–1432. St. John, A.L., Abraham, S.N., Gubler, D.J., 2013. Barriers to preclinical investigations of anti-dengue immunity and dengue pathogenesis. Nature Reviews Microbiology 11 (6), 420–426. St. John, A.L., Rathore, A.P.S., 2019. Adaptive immune responses to primary and secondary dengue virus infections. Nature Reviews Immunology. doi:10.1038/s41577-019–0123-x. World Health Organization, 1997. Dengue Haemorrhagic Fever: Diagnosis, Treatment and Control. Geneva: World Health Organization, p. 58. World Health Organization (WHO), 2009. World Health Organization (WHO) Dengue 2009: Guidelines for Diagnosis, Treatment, Prevention and Control. Geneva: World Health Organization.

Ebola Virus (Filoviridae) Andrea Marzi, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States Logan Banadyga, Public Health Agency of Canada, Winnipeg, MB, Canada r 2021 Elsevier Ltd. All rights reserved. This is an update of K.S. Brown, A. Silaghi, H. Feldmann, Ebolavirus, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00662-2.

Classification Ebola virus (EBOV) is the best known member of a relatively small but growing family of viruses known as Filoviridae, which itself belongs to the much larger order of negative-sense, single-stranded RNA viruses called Mononegavirales (Table 1). Within the family Filoviridae are five recognized genera—Ebolavirus, Marburgvirus, Cuevavirus, Striavirus, and Thamnovirus – each of which contains one or more species represented by specific type viruses. The genus Ebolavirus is composed of five species each with a single virus: Zaire ebolavirus (type virus: EBOV), Sudan ebolavirus (type virus: Sudan virus; SUDV), Bundibugyo ebolavirus (type virus: Bundibugyo virus; BDBV), Taï Forest ebolavirus (type virus: Taï Forest virus; TAFV), and Reston ebolavirus (type virus: Reston virus; RESTV). A sixth species of Ebolavirus, tentatively termed “Bombali ebolavirus” (type virus: Bombali virus; BOMV), and a sixth genus of Filoviridae, tentatively termed “Dianlovirus” (containing Měnglà virus; MLAV), have been proposed but have yet to be ratified by the International Committee on Taxonomy of Viruses. Informally, all viruses belonging to the Filoviridae family are referred to as filoviruses, while viruses belonging to the Ebolavirus genus are referred to as ebolaviruses. Only ebolaviruses and marburgviruses are known to cause human disease, which is generally referred to as EBOV disease (EVD) or Marburg virus disease (MVD), replacing the outdated terms Ebola and Marburg hemorrhagic fever (EHF and MHF). Considering the severity of the diseases they cause, and the threat they pose to global public health, filoviruses, including EBOV, are considered Category A Bioterrorism Agents and Tier 1 Select Agents by the US Centers for Disease Control and Prevention and the Department of Health and Human Services, respectively. All work with filoviruses is therefore restricted to maximum containment (biosafety level 4; BSL-4) laboratories.

Epidemiology Ebolaviruses were first identified in 1976 following two near-simultaneous outbreaks that occurred around Yambuku, Zaire (now the Democratic Republic of the Congo) and Nzara, Sudan (now South Sudan). Although these two outbreaks were originally attributed to a single virus—EBOV—years later it was determined that the etiological agent of the Sudanese outbreak was the highly related SUDV. Since then, four additional ebolaviruses have been discovered and dozens of outbreaks have occurred, mostly in Central Africa (Table 2). TAFV was discovered in 1994 following a single non-fatal infection in the Taï Forest reserve of Côte d0 Ivoire, and BDBV was discovered in 2007 following an outbreak in the Bundibugyo region of Uganda. RESTV was first identified in 1989 in a group of fatally infected nonhuman primates (NHPs) that had been imported from the Philippines to Reston, Pennsylvania, and it appears to be apathogenic in humans. Recently, full genome sequences for a putative sixth ebolavirus, BOMV, have been identified in free-tailed bats from Sierra Leone, Guinea and Kenya, although, to date, infectious virus has not been isolated and no known human cases have been reported. By far the largest and deadliest outbreak of any filovirus was that caused by EBOV in West Africa from 2013 to 2016. From a single index case in the village of Méliandou, Guinea, EBOV eventually spread throughout Western Africa to cause tens of thousands of cases. Guinea and the neighboring countries of Sierra Leone and Liberia bore the brunt of the epidemic, but several cases were exported to nearby African countries and numerous other countries around the world, including some European countries and the United States. Altogether the outbreak lasted nearly two and a half years and resulted in at least 28,616 cases and 11,310 deaths—although these numbers likely underestimate the true toll of the epidemic. In addition to the unprecedented number of human casualties, the West African EBOV epidemic also incited fear and anxiety around the world while at the same time devastating the public health infrastructure and economies of Guinea, Sierra Leone, and Liberia. Currently, the world’s second largest filovirus outbreak—also caused by EBOV—is occurring in the North Kivu and Ituri provinces of the Democratic Republic of the Congo (DRC). At the time of writing, over 2000 deaths and 3000 cases have been recorded. Although these are obviously not the only outbreaks that have been caused by ebolaviruses, they are the worst, and they serve as a reminder of the ongoing threat posed by these viruses to global public health and biosecurity. Ebolaviruses are considered to be zoonotic agents introduced into human populations either directly from the reservoir host or via intermediate hosts. The identity of this reservoir (or reservoirs) remains unknown; however, considerable circumstantial evidence suggests that this role may be fulfilled by bats. Antibodies specific to EBOV antigen, as well as EBOV-specific RNA sequences, have been identified in multiple species of bats (including Hypsignathus monstrosus, Epomops franqueti, and Myonycteris torquata), and epidemiological connections have been made between bats and index cases for certain outbreaks. Moreover, the complete genome of BOMV was isolated from bats (Chaerephon pumilus and Mops condylurus), and extensive lines of evidence

232

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00025-4

Ebola Virus (Filoviridae)

Table 1

233

Filoviridae family classification

Order

Family

Genus

Species

Viruses

Mononegavirales

Filoviridae

Marburgvirus

Marburg marburgvirus

Marburg Virus (MARV) Ravn Virus (RAVV)

Zaire ebolavirus Sudan ebolavirus Reston ebolavirus Taï Forest ebolavirus Bundibugyo ebolavirus “Bombali ebolavirus”

Ebola Virus (EBOV) Sudan Virus (SUDV) Reston Virus (RESTV) Taï Forest Virus (TAFV) Bundibugyo Virus (BDBV) Bombali Virus (BOMV)

Lloviu cuevavirus “Me ̌nglà dianlovirus” ̄ ̌ng striavirus XI la Huángjiāo thamnovirus

Lloviu Virus (LLOV) Me ̌nglà Virus (MLAV) ̄ ̌ng virus (XILV) XIla Huángjiāo virus (HUJV)

Ebolavirus

Cuevavirus “Dianlovirus” Striavirus Thamnovirus

Note: Quotation marks are used to indicate classifications that have not yet been ratified by the International Committee on Taxonomy of Viruses (ICTV).

Table 2

Ebolavirus outbreaks

Year

Location

Virus

Total Cases

CFR

1976 1976 1977 1979 1989 1989 1990 1992 1994 1994 1995 1996 1996–1997 1996 2000–2001 2001–2002 2002–2003 2003 2004 2005 2007 2007–2008 2008 2008–2009 2011 2012 2012–2013 2012 2013–2016 2014 2017 2018 2018-present

Yambuku, DRC Nzara, Maridi, Tembura, & Juba, South Sudan Tandala, DRC Nzara & Yambio, South Sudan Philippines Reston, Virginia ex Philippines Reston, Virginia ex Philippines Siena, Italy ex Philippines Ogooué-Invindo Province, Gabon Taï Forest, Ivory Coast Kikwit, DRC Mayibout, Gabon Booué, Gabon; Johannesburg, South Africa Alice, Texas ex Philippines Gulu district, Mbarrara, & Masindi, Uganda Ogooué-Invindo province, Gabon; Cuvette region, RC Ogooué-Invindo province, Gabon; Cuvette region, RC Mbomo & Mbandza, RC Yambio county, South Sudan Etoumbi & Mbomo, RC Kasai Occidental province, DRC Bundibugyo district, Uganda Philippines Kasai Occidental province, DRC Nakisimata, Uganda Kibaale, Uganda Luwero, Uganda Isiro, DRC Guinea, Liberia, & Sierra Leone Équateur province, DRC Bas Uélé province, DRC Équateur province, DRC North Kivu & Ituri provinces, DRC

EBOV SUDV EBOV SUDV RESTV RESTV RESTV RESTV EBOV TAFV EBOV EBOV EBOV RESTV SUDV EBOV EBOV EBOV SUDV EBOV EBOV BDBV RESTV EBOV SUDV SUDV SUDV BDBV EBOV EBOV EBOV EBOV EBOV

318 284 1 34 3 0 4 0 52 1 315 37 62 0 425 124 143 35 17 12 264 149 6 32 1 24 6a 36a 28,646b 69 8 54 3346c

88% 53% 100% 65% 0% 0% 0% 0% 60% 0% 81% 57% 74% 0% 53% 78% 90% 83% 41% 83% 71% 25% 0% 47% 100% 71% 50%a 36%a 40%b 71% 50% 61% 66%c

a

Laboratory-confirmed cases only. Includes cases exported to Italy (1), Mali (8), Nigeria (20), Senegal (1), Spain (1), the United Kingdom (1), and the United States (4). c Accurate as of 19 December 2019. Abbreviations: CFR, Case fatality ratio; DRC, Democratic Republic of the Congo; RC, Republic of the Congo. b

234

Ebola Virus (Filoviridae)

Fig. 1 Ebola Virus Genome Structure and Morphology. The Ebola virus (EBOV) genome is a single-stranded, negative sense RNA molecule, approximately 19 kb in length. The genome consists of seven genes, each indicated by a coloured box: nucleoprotein (NP), virion protein 35 (VP35), VP40, glycoprotein (GP), VP30, and the RNA-dependent RNA polymerase (L). Transcriptional editing of the GP gene at a conserved editing site (indicated by an arrow) produces two additional transcripts: soluble GP (sGP) and small soluble GP (ssGP). The grey regions of the diagram represent the 3’ and 5’ leader and trailer regions, respectively, as well as the untranslated and intergenic regions. Gene overlaps are indicated by arrows. A scanning electron microscope image depicts an EBOV virion, highlighted in yellow. A schematic depicts the structural proteins (NP, VP35, VP40, GP, VP30, VP24, and L), as well as the viral genome, that comprise the distinct, filamentous morphology of the virion.

suggest that the Egyptian rousette bat (Rousettus aegypticus) serves as a definitive reservoir for the related marburgviruses. Serological evidence of SUDV, TAFV, BDBV, and RESTV has also been reported in bats, although it is possible that cross-reactivity may complicate the specificity of these results. NHPs, such as gorillas and chimpanzees, along with other animals, such as duikers, are likely important dead end hosts. NHPs, in particular, are highly susceptible to ebolavirus infection and are common sources of bushmeat, the butchering and consumption of which has been linked to virus spillover into human populations. The majority of ebolavirus outbreaks appear to cluster in distinct environments depending on the viral species, suggesting that different ebolaviruses occupy distinct ecological niches, a hypothesis that is supported by mathematical modeling. Moreover, the relatively low genetic diversity among different ebolaviruses provides further evidence suggesting strong ecological adaptation. Interestingly, although all human-pathogenic ebolaviruses (EBOV, SUDV, TAFV, BDBV) seem to exist near equatorial Africa, RESTV is found in the Philippines, suggesting that ebolaviruses may be more widely distributed than is currently appreciated. Indeed, serological studies have found evidence of ebolaviruses in Bangladesh and China, Lloviu virus (the sole member of the Cuevavirus genus) was identified in bats from Spain, and Měnglà virus (genus “Dianlovirus”) was discovered in bats from Southern China. Ultimately, however, since the geographic distribution of ebolaviruses is directly related to the reservoir species, a complete understanding of ebolavirus epidemiology awaits the definitive identification of the reservoir host or hosts.

Genome The EBOV genome, like that of all ebolaviruses, comprises a single, linear strand of negative sense RNA approximately 19 kb in length (Fig. 1). The genome contains seven genes, each of which encodes one of seven structural proteins: the nucleoprotein (NP), the 35-kDa virion protein (VP35), VP40, the glycoprotein (GP), VP30, VP24, and the RNA-dependent RNA polymerase “Large” protein (L) (Table 3). As a result of transcriptional editing, the GP gene also encodes two non-structural proteins: soluble GP (sGP) and small soluble GP (ssGP). A small 40 amino acid peptide, known as D-peptide is also generated by a post-translational cleavage of sGP (Table 3). Each gene is flanked at its 30 and 50 end by highly conserved untranslated regions (UTRs), which contain transcription start (30 -CUnCnUnUAAUU-50 ) and stop (30 -UAAUUCUUUUU-50 ) signals, respectively, and incorporate a conserved pentamer (UAAUU). For all ebolaviruses except RESTV, three intergenic regions of variable length exist between NP/VP35, VP40/GP, and VP30/VP24, while three small gene overlaps (where the start site of one gene occurs prior to the stop site of the preceding gene) exist between VP35/VP40, GP/VP30, and VP24/L. Conversely, RESTV lacks the gene overlap between GP and VP30. The genome itself is flanked by a conserved 30 leader sequence, approximately 60 base pairs in length, and 50 trailer sequence, which varies in length depending on the virus, ranging from as little as 25 bp in RESTV to as long as 676 bp in EBOV. The leader and trailer sequences, which possess a high degree of complementarity and are thought to form secondary structures, contain

Ebola Virus (Filoviridae)

Table 3

235

Major defined roles of ebolavirus proteins Structural

Transcription/Replication

Immunomodulatory

NP

Major component of the nucleocapsid; binds and Critical component of the viral polymerase Not known protects viral genome RNA complex VP35 Critical component of the nucleocapsid Polymerase cofactor; critical component of the Binds and sequesters dsRNA; inhibits type viral polymerase complex I IFN gene expression VP40 Major matrix protein; critical for virion assembly Negative regulator of transcription/replication Not known and budding Critical component of virion; required for virus Inhibits tetherin activity GP1,2 attachment and fusion sGP Antibody decoy D-peptide Not known ssGP Not known VP30 Component of the nucleocapsid Critical transcription factor Not known VP24 Critical component of the nucleocapsid Negative regulator of transcription/replication Inhibits IFN gene expression and signaling L Component of the nucleocapsid RNA-dependent RNA polymerase Not known

conserved signals critical for genome replication and transcription. Importantly, because the genome of ebolaviruses—like all filoviruses, and, indeed, all mononegaviruses—is negative sense and therefore complementary to the mRNA sequences, it is not infectious. The viral polymerase complex is thus required to initiate replication and production of the (positive sense) antigenome prior to mRNA transcription. Overall, the genomes of viruses from different species within the Ebolavirus genus differ from each other, on the nucleotide level, by approximately 23%–36% while the genomes of ebolaviruses differ from those of other filoviruses by greater than 50%. For the most part, these differences are recapitulated at the level of the GP gene, and highlight the diversity within the Filoviridae family (Fig. 2). Intriguingly, however, within a given species of Ebolavirus, different variants are highly conserved, typically displaying less than 5% divergence.

Virion Structure Filoviruses derive their name from the Latin word filum, meaning “thread”, which accurately describes the overall morphology of the virion (Fig. 1). Ebolavirus virions, like those of all filoviruses, are long, filamentous particles that are often linear but can also bend into U-shaped, comma-shaped, or toroidal structures, indicating a high-degree of structural flexibility. Despite this flexibility, the EBOV virion maintains a uniform diameter of 96 nm, as measured from opposing outer edges of the viral envelope, and the length of a particle containing a single genome is approximately 980 nm long. However, approximately half of EBOV virions are polyploid, in which a single virion contains more than one copy of the viral genome, each arranged end-to-end continuously or separated from one another by a small gap. The result of this polyploidy is a population of exceptionally long virus particles of varying length, with some particles observed to contain as many as 22 genomes and measure over 21 mm in length. Empty virions containing no viral genome are also observed, possessing much smaller diameters (48–52 nm) and random lengths. Branched filaments are frequently observed in tissue culture supernatants, although the branches are typically empty. Virion morphology is ultimately determined by the seven ebolavirus structural proteins, most of which compose some part of the helical nucleocapsid (Fig. 1). Indeed, the EBOV nucleocapsid is among the most complex of all mononegaviruses and consists of the virus genome in complex with five viral proteins: NP, VP35, VP24, VP30, and L. NP binds directly to genomic RNA in a sequence-independent manner and oligomerizes to form a left-handed helical, loosely-coiled structure that is then more tightly condensed via interactions with VP24 and VP35, which are thought to form a heterodimeric “bridge” along the outer edge of the helix. Although NP appears to form vertical contacts along the inner edge of the helix with neighboring NP monomers on adjacent helical layers, no such connections are apparent along the outer edge of the helix, thus permitting the nucleocapsid’s characteristic flexibility. Overall, the nucleocapsid has an inner-edge diameter of B22 nm, an outer-edge diameter of B41 nm, and a pitch of B7 nm with 10.8 NP units per helical turn. Analysis of virion protein contents suggests that NP, VP35, VP24, and VP30 are all present at equimolar ratios, which is consistent with their involvement in forming repeating units of a highly ordered nucleocapsid. The role that VP30 plays in this structure remains unclear, however, since its presence or absence does not appear to affect nucleocapsid morphology. Likewise, although L is also considered to be a part of the nucleocapsid, it is present at such low copy numbers that it is unlikely to play a major structural role. VP40, the membrane-associated matrix protein, is not a part of the viral nucleocapsid, but instead serves to simultaneously drive the budding of nascent virions from the plasma membrane of the host cell and recruit nucleocapsids to the site of budding. In the virion, a layer of VP40 approximately 9 nm thick is thought to underlie the viral envelope, and VP40 extensions from this layer are thought to bridge the gap between the envelope and the nucleocapsid. In this way, VP40 keeps the nucleocapsid centered in the virion, with a spacing of 7–8 nm between the nucleocapsid and the

236

Ebola Virus (Filoviridae)

Fig. 2 Representative Filovirus Phylogeny. GP nucleotide sequences were aligned using MUSCLE, and a Neighbor-Joining phylogenetic tree was generated using Geneious Tree Builder. Percent consensus support is indicated at each branch, which was bootstrapped 1000 times. Genbank Accession Numbers: BDBV variant Butalya, KR063673.1; TAFV, KU182910.1; EBOV variant Mayinga, AF086833.2; BOMV, MF319185.1; SUDV variant Boniface, MH121162.1; RESTV variant Pennsylvania, AF522874.1; LLOV, NC_016144.1; MLAV, KX371887.2; MARV variant Angola, KU978782.1; RAVV, KU179482.1.

envelope. The outer surface of the viral envelope is studded with the heavily glycosylated viral glycoprotein (GP1,2), the virus’s only transmembrane surface protein and the sole structure responsible for receptor-mediated entry into host cells. GP1,2, a class I fusion protein B13 nm in length, is composed of a trimer of three identical disulfide-bound dimers consisting of proteins GP1 and GP2, which mediate receptor binding and membrane fusion, respectively.

Replication Cycle Virus Attachment and Fusion The virus replication cycle begins with virion attachment to a host cell (Fig. 3). Although ebolaviruses are thought to preferentially infect dendritic cells and macrophages early following infection, they have a broad cell and tissue tropism overall. Accordingly, with a notable exception of lymphocytes, ebolaviruses are capable of infecting many different cell types, including fibroblasts, epithelial and endothelial cells, and hepatocytes. Attachment to a host cell is mediated by at least two distinct mechanisms: (1) carbohydrate-binding receptors on the cell surface interacting with the heavily glycosylated GP1,2 and (2) phosphatidylserine (PS) receptors interacting with PS in the viral envelope. It is thought that the N- and O-linked glycans on GP1,2 interact promiscuously and non-specifically with a variety of carbohydrate-binding receptors, including C-type lectin receptors (such as DC-SIGN, L-SIGN, and LSECTin) and glycosaminoglycans, but that additional interactions between PS and PS receptors, such as those in the TIM (T-cell, immunoglobulin, mucin domain receptor) and TAM (Tyro3, Axl, Mer) families, are necessary for virus internalization. The broad distribution and redundancy of these attachment factors likely helps to explain the equally broad cell and tissue tropism

Ebola Virus (Filoviridae)

237

Fig. 3 The Ebola Virus Replication Cycle. Virion attachment to the host cell is mediated in part by non-specific interactions between EBOV GP and a number of different carbohydrate-binding factors, although interactions between phosphatidylserine in the virion envelope and phosphatidylserine receptors in the cell membrane also facilitate attachment. Following attachment, the virion is internalized by micropinocytosis, after which fusion between the viral and cellular envelopes is mediated by GP and Niemann-Pick C1 (NPC1), the cellular entry receptor for the virus. Primary transcription occurs following release of the NP-encapsidated viral genomic RNA into the cytoplasm and is mediated by the viral ribonucleoprotein complex (RNP) consisting of L, VP35, VP30. Host cell machinery translates the mRNA into protein. NP-encapsidated genome serves as a template for genome replication, carried out by L and VP35, via a positive-sense antigenomic intermediate. NP-encapsidated genomes are further encapsidated by VP35, VP24, VP30, and L, to generate highly ordered helical nucleocapsids that are trafficked to the cell membrane, where VP40 drives virus budding.

exhibited by ebolaviruses, and it also explains why a single surface-expressed cellular protein has yet to be identified as indispensable for virus entry. Following attachment, virions are thought to be internalized primarily via macropinocytosis, although other cellular uptake pathways may play a role, depending on cell type and the attachment factors engaged (Fig. 3). The internalized virion is then trafficked through the progressively-acidic endocytic pathway where cleavage by the cysteine proteases cathepsin L and B removes the GP1,2 mucin-like domain and glycan cap, exposes the receptor binding site, and prepares the molecule for fusion-dependent conformational changes. The cellular entry receptor for ebolaviruses was identified to be Niemann-Pick C1 (NPC1), a large, highly-conserved protein involved in cholesterol transport that is ubiquitously expressed and located on the interior membrane of late endosomes. NPC1 is critical for ebolavirus entry, and cells lacking this molecule are resistant to infection. Likewise, transgenic mice lacking the Npc1 gene are resistant to infection. Proteolytically cleaved GP1,2 binds to domain C of NPC1 via the exposed receptor binding site, and through a process that is still incompletely defined, a series of conformational changes are thought to occur in GP1,2 that release the internal fusion loop and lead to fusion between the virion and endosomal membranes. In addition to the requirement for NPC1, the acidic environment of the late endosome/early lysosome is also required for fusion, and additional but unidentified endosomal factors may also be required. Ultimately, fusion of the virion and endosomal membranes releases the viral nucleocapsid into the cytoplasm, from where primary transcription commences.

Primary Transcription & Translation Because the viral genome is in the negative sense and cannot by directly translated into protein, positive sense viral mRNAs must first be transcribed from the genome before the remainder of the replication cycle can take place (Fig. 3). The viral polymerase L binds to a conserved promoter in the 30 (Leader) end of the NP-encapsidated genome and moves successively towards the 50 end, initiating and terminating transcription at each gene’s transcriptional start and stop sites, respectively. Newly transcribed viral mRNAs are capped by the viral polymerase and polyadenylated by a polymerase stuttering mechanism at the transcription start site. mRNAs are monocistronic and, unlike the virus genome, are not encapsidated by NP. Because a certain proportion of the polymerase falls off the genome at each transcription stop site, the probability of the polymerase transcribing a given gene decreases with the gene’s distance from the 30 end of the genome. This results in a gradient of mRNA concentrations, with transcripts for NP being the most abundant and transcripts for L being the least abundant. Importantly, VP35 is an essential—but non-enzymatic—cofactor for the viral

238

Ebola Virus (Filoviridae)

polymerase that is thought to connect L to the NP-encapsidated genome by interacting with both proteins. VP30 is also required for transcription, particularly for activation of transcription from the leader sequence and re-initiation from each gene’s transcription start sites, and its function depends heavily on its phosphorylation state. Along with L, NP, and the virus genome, these components form the ribonucleoprotein complex (RNP), and they are necessary and sufficient for ebolavirus transcription. Subsequent translation of viral mRNAs is completed by host cellular machinery and is thought to occur at distinct sites within the host cell. One key feature of ebolavirus transcription that distinguishes it from that of the marburgviruses, is the use of co-transcriptional editing to produce multiple products from the GP gene. The primary product of the GP gene is the soluble glycoprotein (sGP), which is produced when the translational machinery encounters a stop codon roughly in the middle of the gene. Post-translational cleavage of sGP at its C-terminus produces the D-peptide, and both products are secreted from the infected cell. Thanks to a stretch of uridine residues before the stop codon, however, the viral polymerase occasionally stutters during transcription, resulting in the addition of a non-templated adenosine residue added to the mRNA. This additional residue results in a frameshift that allows translation to continue along the second half of the GP transcript, producing the pre-cursor protein to GP1,2. Post-translational cleavage of this protein by furin-like proteases produces the GP1 and GP2 subunits, which then dimerize and trimerize to form the functional GP1,2. Interestingly, the addition of two adenosine residues to the GP mRNA results in a different frameshift that creates a third stop codon ahead of the first two, thus producing the small soluble glycoprotein (ssGP). Like sGP and D-peptide, ssGP is also secreted from the cell. The function of these three products has yet to be clearly elucidated.

Replication As transcription and translation continue, increasing levels of viral protein—including NP and VP30—are thought to trigger the switch to replication, although the details remain unclear. While dephosphorylated VP30 is required for the transcriptional activity of the RNP complex, phosphorylation of VP30 causes its dissociation from the RNP complex and shifts RNA synthesis towards replication. NP-encapsidated genome also serves as a template for viral RNA replication, which requires L and VP35. The polymerase complex binds to a bipartite replication promoter in the leader sequence of the genome and synthesizes a complementary positivesense genome, referred to as the antigenome, which is immediately encapsidated by NP (Fig. 3). The antigenome then serves as a replication intermediate for the synthesis of additional copies of the viral genome. EBOV replication has been demonstrated to occur within peri-nuclear viral inclusion bodies, which appear to be dynamic and complex aggregates of viral proteins and RNA. Some evidence exists for the involvement of cellular proteins in replication/transcription, but further research is required.

Assembly & Budding Although the structure of the mature viral nucleocapsid has been well studied, the dynamics of assembly are incompletely understood. Following transcription and replication, the NP-encapsidated viral genomes are further encapsidated by VP35, VP24, VP30, and L, which together generate the highly ordered, helical nucleocapsid described above (Fig. 3). Based on the ability of VP24 to inhibit transcription and replication in in vitro experimental systems, it has been proposed that the addition of VP24 to the nascent nucleocapsid represents the final step in nucleocapsid assembly, serving to condense the structure and restrict access to the genome by the viral replication machinery. Mature nucleocapsids must then travel from the perinuclear region in which they are assembled to the plasma membrane, from where virus budding occurs. VP40, the matrix protein, is thought to be transported through the retrograde late endosomal pathway or in association with COPII vesicles to the underside of the plasma membrane, where it exists as a higher-order oligomer. Similarly, GP1,2 is transported to the plasma membrane via the ER-Golgi secretory pathway and collects in areas rich in VP40, likely due to interactions between the GP1,2 cytoplasmic domain and VP40. Actin-dependent transport mechanisms are thought to propel EBOV nucleocapsids along their long axis towards these sites on the plasma membrane, where interactions between NP and VP40 facilitate the envelopment of the nucleocapsid. Budding of the nascent virion from the cell is thought to require, at least in part, components of the endosomal sorting complex required for transport (ESCRT), which interact with two overlapping late domains present in VP40. Additional mechanisms may also be involved in virus budding, and the potential for direct cell-to-cell transmission remains a relatively under-investigated possibility.

Immune Evasion The successful completion of the virus replication cycle, and the subsequent dissemination of infection, relies on the ability of the virus to evade the host’s immune defences. Two of the most critical ebolavirus immunomodulatory proteins are VP35 and VP24. In addition to its roles in virion structure and transcription/replication, VP35 also functions to inhibit the induction of a type I interferon (IFN) response by binding to and sequestering double-stranded RNA (dsRNA) from intracellular nucleic acid receptors, such as RIG-I. Moreover, direct interactions between VP35 and components of the IFN-signaling cascade, including IKKe, TBK1 and IRF7, also serve to limit the induction of the IFN response. VP24, on the other hand, binds to members of the NPI-1 subfamily of karyopherin-a proteins and prevents their translocation of STAT1 into the nucleus. Since STAT1 nuclear translocation is a critical step in the induction of IFN-stimulated genes (ISGs), VP24 effectively blunts the antiviral response triggered by type I IFN signaling. VP24 has also been shown to inhibit RIG-I-dependent IFN gene expression. GP1,2 also plays a role at the plasma

Ebola Virus (Filoviridae)

239

membrane in preventing the activity of tetherin, a viral restriction factor that interferes with virus budding, and sGP, may serve as a decoy molecule for the antibody response. Given the multi-functional nature of most ebolavirus proteins, additional immunomodulatory activities of other proteins likely exist, although further investigation is required.

Clinical Features Prior to the West African epidemic, there was limited data available characterizing EVD, and, as a result, much of what we understood about this disease was obtained from animal models, usually in the context of EBOV infection. Different animal species, such as mice, guinea pigs, hamsters, ferrets, and NHPs have been used to model EVD and evaluate countermeasures—and each animal has its own set of advantages and disadvantages. Although inexpensive and widely accessible, immune-competent rodents are inherently resistant to EBOV infection, and virus must therefore be adapted through serial passaging in these animals to produce a lethal disease. Conversely, ferrets and NHPs are susceptible to wild type EBOV and exhibit disease remarkably similar to what is observed in humans. Rhesus and cynomolgus macaques, in particular, display a majority of the clinical signs of human disease, and they are considered the gold-standard model for research. Indeed, much of what is understood about EVD in humans was originally described in the NHP infection model. Our current understanding of human EVD is based predominantly on what we have learned from the West African EBOV epidemic, which infected more people than all previous EBOV outbreaks combined and produced a survivor cohort of thousands of people.

Early Disease Like many other infectious diseases, the early stages of EVD include an asymptomatic incubation period followed by the onset of nonspecific signs of disease. These symptoms include fatigue, malaise and myalgia often coinciding with the onset of fever. Because these symptoms are not specific for EVD, the disease can be mistaken for other, more prevalent diseases in Africa, such as malaria, yellow fever, and Dengue. Although diagnosis of EVD in this early stage may be possible, it remains realistic, since the early symptoms precede the detectable presence of virus and viral RNA in the blood.

Peak Disease Within the first week of illness, EVD patients become progressively weaker and develop nausea, vomiting, and diarrhea. Some cases also develop a maculo-papular rash and oozing from venipuncture sites or mucosal membranes. During this time, patients develop disseminated intravascular coagulation (DIC) along with thrombocytopenia. Patients are also extremely infectious, and the virus is present in all body fluids. If patients are not properly rehydrated at this point, loss of fluid from diarrhea and vascular leakage can result in severe dehydration and hypovolemic shock, ultimately leading to organ failure and death. Since EBOV replicates systemically during peak illness, almost all organs are affected. Liver damage is documented by an increase in liver enzyme levels, and an increase in creatinine levels is indicative of an acute kidney injury and subsequent renal failure. Viral antigens can be found systemically but is most abundant in the liver and spleen. In many EVD cases, central nervous system (CNS) dysfunction and life-threatening meningoencephalitis are observed.

Recovery The survival of EVD is associated with a decline in viremia and the development of a life-saving adaptive immune response towards the end of peak disease. However, EVD survivors can develop sequelae, as documented in limited reports from previous outbreaks. The EVD epidemic in West Africa resulted in the largest cohort of EVD survivors to date, and many have been followed to document late-onset sequelae. The most commonly reported sequela is uveitis, which occurs in B20% of EVD survivors and results in light sensitivity as well as blurred vision or temporary loss of vision. Other sequelae include hepatitis, myelitis, tinnitus, difficulty hearing and temporary hearing loss, cognitive difficulties, fatigue, muscle pain, and general weakness. These symptoms have been described to persist for one year or longer.

Virus Persistence Until the epidemic in West Africa, EBOV persistence had only been anecdotally described from the outbreak in Kikwit, DRC in 1995. Now, there is increasing evidence that EBOV persists in immune-protected sites and can cause late manifestations of disease months and years after initial infection. Indeed, infectious EBOV has been isolated from the eyes, CNS, and male reproductive organs (mainly the testes) from EVD survivors. During the epidemic, there were a couple examples of sexual transmission of EBOV originating from male EVD survivors. These events were discovered and documented based on EBOV sequence data. There is limited data available on what triggers the reactivation of EBOV from immune-protected sites and causes a disease; however, this topic is the focus of ongoing research, and several animal models are being used to model and analyze this phenomenon.

240

Ebola Virus (Filoviridae)

Fig. 4 Ebola Virus Pathogenesis. The primary target cells of Ebola virus infection are thought to be monocytes, macrophages, and dendritic cells. Infection of these cells not only permits virus dissemination, but also affects the immune response to infection, resulting in the production of inflammatory chemokines and cytokines as well as the impairment of maturation and lymphocyte co-stimulation. Immune dysfunction exacerbates virus dissemination and, along with rampant virus replication, contributes to organ damage. Damage to the adrenal cortex contributes to hypotension and hypovolemia, while damage to the liver contributes to coagulation dysfunction that, along with vascular dysfunction, can produce hemorrhage. Ultimately, death is precipitated by multi-organ failure and hypovolemic shock.

Pathogenesis Most of the information available regarding EBOV pathogenicity is derived from infections in animal models, as well as human cases from the West African epidemic. Fatal EVD cases appear to be the result of systemic shock due to vascular dysfunction and leakage, which is caused by a complex interaction of the immune system with vascular physiology. There are three major processes that are thought to lead to hypovolemic shock and death: (1) increased vascular permeability; (2) DIC; and (3) impaired protective immune responses. The first two processes result from a series of events starting with the infection of primary target cells, namely monocytes, macrophages and dendritic cells (Fig. 4). Infected macrophages are strongly activated—secreting pro-inflammatory molecules such as interleukin 6 (IL-6), tumor necrosis factor alpha (TNF)-a, IL-1b, nitric oxide (NO)—and serve to spread the infection systemically. In contrast, infection of dendritic cells results in their impaired activation, with no upregulation of co-stimulatory molecules, such as MHC class I and II molecules, CD80, CD86, and CD40. Activation of endothelial cells by inflammatory mediators such TNF-a and NO is believed to be the main cause for the decrease in their barrier function. TNF-a, NO, and pro-inflammatory cytokines increase vascular permeability and the expression of adhesion molecules on the surface of endothelial cells, which are necessary in extravasation of immune cells in inflamed tissue. Although these pro-inflammatory cytokines are an integral part of a normal, localized immune response by attracting and activating immune cells to the site of infection, in the context of a systemic and extensive response, they have a negative effect by inducing shock. Endothelial cells also serve as target cells for EBOV, and infection results in their activation—as indicated by the upregulation of adhesion molecule expression followed by cytolysis. However, the destruction of endothelial cells in vivo can only be observed at the end stage of the disease. Infection of macrophages also results in the expression of tissue factor (TF), which can impair the anticoagulant protein-C pathway by downmodulating thrombomodulin. TF is believed to be an important protein in the development of DIC, since

Ebola Virus (Filoviridae)

241

inhibition of TF delays death in NHPs and offers limited protection. TNF-a can also induce TF expression in endothelial cells, thus amplifying coagulopathy and DIC. Moreover, coagulopathy is known to enhance inflammation, leading to a vicious cycle where inflammation enhances coagulopathy, which in turn amplifies inflammation. Rapid systemic viral replication is also an integral part of EVD pathogenesis. EBOV infection in humans and NHPs results in rapid massive viremia and high titers in many organs, especially in the spleen and liver. This correlates with an impaired innate and adaptive immune response. Interestingly, high viremia and lethality are only seen in mice if a host-adapted virus or immune-deficient animals are used. These observations suggest that evasion of immune responses plays a pivotal role in pathogenesis. Indeed, mice lacking the interferon (IFN) a/b receptor or STAT1, the main signaling molecule in response to type I IFN, are extremely susceptible to EBOV infection and develop a lethal disease. Moreover, mouse-adapted (MA)-EBOV is less sensitive to IFN-a treatment in murine macrophages compared to wild type EBOV, and analysis of genomic mutations in MA-EBOV indicated that mutations in NP and VP24 are sufficient for a lethal phenotype. Although VP24 inhibits the interferon response in vitro, it remains unclear whether the mutations in MA-EBOV are required for type I IFN evasion or other functions. Nevertheless, while treatment of NHPs with IFN-a does not protect the animals, it does delay death, suggesting that the type I IFN response may be central to EBOV pathogenesis. EBOV is also able to inhibit IFN production through VP35, and deletion/mutation of the region in VP35 involved in its IFN antagonism results in a highly attenuated virus that does not cause disease in NHPs. Furthermore, comparative in vitro gene microarray analysis demonstrated a correlation between cytotoxicity and IFN antagonism, which was strongest with EBOV and weakest with RESTV. Adaptive immune responses are also impaired during infection. As mentioned above, upregulation of co-stimulatory molecules is inhibited in dendritic cells, which reduces activation of T cells. There is also a dramatic reduction in the number of T and natural killer cells, despite the fact that lymphocytes do not support EBOV replication. It is believed that lymphopenia is caused by “bystander” apoptosis, although the mechanism is not well understood.

Diagnosis Diagnosing EBOV infections in humans is difficult early on as the disease presents with symptoms common to many other infectious diseases. EBOV particles (viral antigens and viral RNA) and IgM antibodies specific to EBOV proteins are among the first parameters that can be detected in blood and serum samples generally at the same time as symptom onset. In the early outbreaks in Zaire (now DRC), Sudan and Gabon samples were shipped to Europe and the USA where antibodies were detected by ELISA and immunofluorescence; in addition, virus isolation was attempted. During the Kikwit, DRC outbreak in 1995, antigen detection was performed, and EBOV RNA was identified via conventional reverse transcription polymerase chain reaction (RT-PCR). The US CDC used both assays afterwards with good success in the field laboratories in rural areas. In the early 2000s, real-time RT-PCR assays using fluorescent probes were designed for the detection of EBOV from patient samples. Eventually, the real-time RT-PCR tests replaced all other tests since the application is manageable in the field, with a turn-around time of B2–3 h from the sample receipt to a test result. Several laboratory-developed and commercial tests are currently being used, but only limited data is available regarding their specificity and sensitivity. These tests have also been developed to work on automated diagnostic systems for high-throughput capacity. Other tests with a fast turn-around time include antibody-based lateral flow immunoassays designed for the detection of EBOV VP40 antigen or a combination of VP40, NP and GP antigens. Limited data is available regarding the sensitivity and specificity of these assays, and more testing is needed.

Treatment Monoclonal Antibody Therapy Shortly after the discovery of EBOV and SUDV, scientists started to develop potential treatment strategies. Convalescent serum therapy was among the first to be tested in animal models, but it demonstrated limited efficacy and was not developed further. However, during the EBOV outbreak in Kikwit, DRC in 1995 the treatment was used with a questionable success. Similarly, EBOVspecific equine IgG treatment was evaluated in NHPs with limited success. Despite these early setbacks, the scientific community continued to pursue antibody-based therapeutics. The human-derived monoclonal antibody KZ52 was tested in rodents with good efficacy but failed to protect NHPs from a lethal disease. In 2012 it was demonstrated that purified EBOV-specific IgG from NHPs could protect naïve NHPs from a lethal disease when multiple doses were administered even after infection. Based on this report significant efforts were made to develop poly- and monoclonal antibody therapies. Two antibody cocktails, MB-003 and ZMAb, each consisting of three monoclonal antibodies, were developed approximately the same time. MB-003 used plant-produced human-mouse chimeric antibodies, whereas ZMAb comprised a mixture of mouse monoclonal antibodies. Three doses of each cocktail protected NHPs from the lethal disease depending on antibody amount and time of administration. Notably, a cocktail composed of two ZMAb antibodies and one MB003 antibody resulted in what is now known as ZMapp and provided superior efficacy in NHPs. In 2014, during the West African epidemic, ZMapp was administered to two patients repatriated to the USA after they fell ill from EVD in Liberia, and it was

242

Ebola Virus (Filoviridae)

subsequently used on at least four additional patients evacuated from West Africa. ZMapp was also tested in phase 3 clinical trials towards the end of the West African outbreak, where it showed benefit compared to the standard care alone, although the results were not significant. More recently, a next-generation cocktail of three human antibodies, knowns as REGN-EB3, and a single human-derived monoclonal antibody, known as mAb114, have shown promising protective efficacy in NHPs. All three monoclonal antibody therapies are currently being used in clinical trials in the ongoing outbreak in the DRC, with an initial report showing superior efficacy and higher survival rates following a single intravenous dose of mAb114 or REGN-EB3 compared to three intravenous doses of ZMapp. Besides antibody therapeutics, other approaches have also been tried to counter EVD, including small interfering RNAs (siRNAs), phosphorodiamidate morpholino oligomers (PMOs), small-molecule compounds, immunomodulatory approaches, and post-exposure vaccination. The most successful ones are explained in detail below.

Small Interfering RNAs (siRNAs) The treatment potential of siRNAs against EBOV infection in NHPs was first described in 2010 when four intravenous doses of a cocktail of three siRNAs targeting VP24, VP35 and L packaged in a lipid nanoparticle protected animal from a lethal disease. The cocktail was further modified to contain siRNAs targeting L and VP35 and was termed TKM-Ebola. TKM-Ebola was administered to EVD patients evacuated from West Africa in 2014; however, because these patients received other care in addition to the siRNA, it was unclear whether the treatment was beneficial. The siRNA sequences were subsequently modified to match the outbreak strain (EBOV variant Makona), and this new product, now termed TKM-130803, was uniformly protective when seven doses were administered intravenously to EBOV-Makona-infected NHPs. In 2015, a small phase 2 clinical trial of TKM-130803 was conducted in Sierra Leone. Twelve patients received treatment, but only three individuals received the full seven doses and survived. Disease was too far advanced in the other nine patients, all of whom died before the remaining doses could be administered.

Small-Molecule Compounds Favipiravir, also known as T-705, has broad antiviral activity against a number of RNA viruses. It has shown partial protective efficacy in preclinical NHP studies against EBOV when administered intravenously twice daily for 14 days starting two days before a lethal EBOV infection. Treatment success was dependent on the dose, with the highest dose resulting in 60% survival. The drug inhibited virus replication with the virus mutating over time. Even though this data was published after the EBOV epidemic in West Africa, clinical trials of favipiravir were still conducted during the epidemic, since this drug was the only one available with a fairly well-known safety profile in humans, oral bioavailability, and demonstrated efficacy in EBOV-infected mice. The drug was also used in patients evacuated to Europe and the USA. Unfortunately, despite the clinical trial data and data obtained from repatriated cases, a beneficial effect of favipiravir in EVD patients could not be demonstrated. Remdesivir, also known as GS-5734, has been shown to prevent lethal EVD in NHPs when a relatively high intravenous dose of 10 mg/kg was initiated three days after infection and continued once daily for 12 days. The drug was used in a relapsed EBOV patient who had been evacuated to Europe and treated with other experimental drugs during the epidemic. Remdesivir is currently being used with good success in the ongoing outbreak in the DRC. A first report from the outbreak demonstrates that patients receiving an intravenous dose (200 mg) on day 1 followed by daily treatment with 100 mg until days 9–13, depending on virus load, had a B50% survival rate. Interestingly, B85% of people with a high viral load at the start of treatment survived, compared to 29% survival in patients with a low viral load. Although the efficacy of remdesivir is lower than those of mAb114 and REGNEB3, remdesivir has the potential for combination therapy. No preclinical data is yet available for combined approaches.

Prevention Treatment strategies are the best option during an outbreak; however, when an entire population of an endemic area needs to be protected from a disease, vaccination is the best preparedness strategy. For EBOV, vaccine development started shortly after the virus was discovered. Early vaccine approaches included inactivated virus and vaccinia virus-based vaccines. While these vaccines protected rodents from disease, NHPs were not protected. With additional funding becoming available in the early 2000s for countermeasure development against potential bioterrorism pathogens, efforts to develop an EBOV vaccine increased. Several vaccine platforms were developed most of which used EBOV GP1,2 as the antigen, including DNA-, protein-, and virus-like particle (VLP)-based vaccines, as well as virus-vectored vaccines based on alphavirus replicons, adenoviral vectors, modified vaccinia Ankara (MVA), human parainfluenza virus 3, rabies virus, and vesicular stomatitis virus (VSV). Although each of these vaccine approaches showed preclinical efficacy in NHP studies, there was limited interest from pharmaceutical companies to bring any of them to licensure. The situation changed in 2014, however, during the unprecedented West African epidemic, when most vaccination strategies with preclinical efficacy and an industrial partner for production were accelerated into human clinical trials worldwide. While these clinical trials involved several different vaccination approaches with demonstrated immunogenicity and safety in humans, this article will focus on the two most promising candidates that are currently being used in the ongoing

Ebola Virus (Filoviridae)

243

outbreak in the DRC. Notably, while China and Russia both licensed EBOV vaccines in 2018 (without human efficacy data), these vaccines are currently not being used in the DRC.

Adenovirus 26 (Ad26) & Modified Vaccinia Ankara (MVA) The deletion of genes essential for replication in the adenovirus genome produces a replication-deficient particle that can be used as a vector for an antigen of choice, such as EBOV GP. Indeed, human adenovirus 5 expressing EBOV GP1,2 was first used to generate an EBOV vaccine, but it was shown to have limited efficacy due to pre-existing adenovirus immunity in the human population. In order to circumvent this problem, several other adenoviruses, including human adenovirus 26 and 35, as well as chimpanzee adenovirus 3, were developed expressing EBOV GP1,2. Vaccination with an adenovirus 26-based vaccine (known as Ad26-EBOV) resulted in strong humoral immune responses and was able to protect NHPs from a lethal EBOV challenge. However, at least two doses were needed for protection and there were concerns with the longevity of the protective immune response. In order to address these concerns, a prime/boost strategy was implemented using Ad26-EBOV as a prime vaccination followed by a boost with the replication-incompetent MVA encoding GP1,2 (known as MVA-EBOV). This vaccination approach offered 100% protection in NHPs and has since been evaluated in several phase 1 human clinical trials, starting during the West African epidemic. The trials demonstrated a good safety profile and immune responses specific to EBOV GP. The Ad26-EBOV/MVA-EBOV vaccine approach is currently being used in the DRC in areas with no active EBOV cases to help increase the number of protected people with the hope to end the large ongoing outbreak in that country.

Vesicular Stomatitis Virus In contrast to Ad26 and MVA, the VSV-based EBOV vaccine is replication competent. The VSV glycoprotein was removed in this vector and replaced with EBOV GP1,2. Preclinical efficacy in NHPs was already demonstrated in 2005, yet no industry partner was found to produce human-grade vaccine until 2013. Further preclinical studies with NHPs demonstrated that a single dose of the VSV-EBOV vaccine is fast acting, protecting the animals within one week of vaccination; however, the longevity of the protective immune response remains unclear. During the West African epidemic, several phase 1–3 human clinical trials were conducted world wide, including a phase 3 trial in Guinea that demonstrated that a single dose of the vaccine was efficacious and prevented new infections. As this is a replication-competent vaccine, mild to moderate adverse effects after vaccination with a single dose of VSV-EBOV were reported. This vaccine has been used since the beginning of the ongoing outbreak in the DRC with remarkable success. Remarkably, the VSV-EBOV vaccine was licensed (under its commercial name ERVEBO) in November 2019 by the European Medicines Agency and a month later by the US Food and Drug Administration. With the long-awaited clinical licensure of VSV-EBOV, as well as the licensing of separate vaccines in Russia and China and the anticipated licensure of Ad26-EBOV/MVA-EBOV, there is increasing hope that future EBOV outbreaks will be rapidly contained and no longer pose a threat to global public health.

Acknowledgments We thank Ryan Kissinger (NIAID) for assistance with figure production. Filovirus research in the Immunobiology & Molecular Virology Unit, Laboratory of Virology is funded by the DIR, NIAID, NIH. This work was supported, in part, by the Public Health Agency of Canada.

Further Reading Agua-Agum, J., Allegranzi, B., Ariyarajah, A., et al., 2016. After Ebola in West Africa-unpredictable risks, preventable epidemics. The New England Journal of Medicine 375 (6), 587–596. doi:10.1056/NEJMsr1513109. Banadyga, L., Dolan, M.A., Ebihara, H., 2016. Rodent-adapted filoviruses and the molecular basis of pathogenesis. Journal of Molecular Biology 428 (17), 3449–3466. doi:10.1016/j.jmb.2016.05.008. Banadyga, L., Wong, G., Qiu, X., 2018. Small animal models for evaluating filovirus countermeasures. ACS Infectious Diseases 4 (5), 673–685. doi:10.1021/acsinfecdis.7b00266. Baseler, L., Chertow, D.S., Johnson, K.M., Feldmann, H., Morens, D.M., 2017. The pathogenesis of Ebola virus disease. Annual Review of Pathology: Mechanisms of Disease 12, 387–418. doi:10.1146/annurev-pathol-052016-100506. Broadhurst, M.J., Brooks, T.J.G., Pollock, N.R., 2016. Diagnosis of Ebola virus disease: Past, present and future. Clinical Microbiology Reviews. 773–793. doi:10.1128/ CMR.00003-16. Cross, R.W., Mire, C.E., Feldmann, H., Geisbert, T.W., 2018. Post-exposure treatments for Ebola and Marburg virus infections. Nature Reviews Drug Discovery 17, 413–434. doi:10.1038/nrd.2018.73. Feldmann, H., Feldmann, F., Marzi, A., 2018. Ebola: Lessons on vaccine development. Annual Review of Microbiology 72, 423–446. doi:10.1146/annurev-micro-090817062414.

244

Ebola Virus (Filoviridae)

Furuyama, W., Marzi, A., 2019. Ebola virus: Pathogenesis and countermeasure development. Annual Review of Virology 6 (1), 435–458. doi:10.1146/annurev-virology-092818015708. Hoenen, T., Brandt, J., Caì, Y., Kuhn, J.H., Finch, C., 2017. Reverse genetics of filoviruses. Current Topics in Microbiology and Immunology 411, 421–445. doi:10.1007/ 82_2017_55. Messaoudi, I., Amarasinghe, G.K., Basler, C.F., 2015. Filovirus pathogenesis and immune evasion: Insights from Ebola virus and Marburg virus. Nature Reviews Microbiology 13 (11), 663–676. doi:10.1038/nrmicro3524.

Relevant Website https://talk.ictvonline.org/ictv-reports/ictv_online_report/negative-sense-rna-viruses/mononegavirales/w/filoviridae Filoviridae.

Enteroviruses (Picornaviridae) Carita Savolainen-Kopra and Soile Blomqvist, National Institute for Health and Welfare, Helsinki, Finland Petri Susi, University of Turku, Turku, Finland r 2021 Elsevier Ltd. All rights reserved.

Nomenclature AFM Acute flaccid myelitis AFP Acute flaccid paralysis CNS Central nervous system CSF Cerebrospinal fluid CV Coxsackievirus E Echovirus EIA Enzyme immunoassay EV Enterovirus HFMD Hand-foot-and-mouth disease IgG Immunoglobulin G IgM Immunoglobulin M

Glossary Acute flaccid paralysis Any sudden weakness or loss of muscle tone. Onychomadesis Shedding of the nails.

IPV Inactivated polio vaccine IRES Internal ribosome entry site mRNA Messenger RNA NCR Non-coding region OPV Oral poliovirus vaccine ORF Open reading frame PCR Polymerase chain reaction PV Poliovirus RNA Ribonucleic acid VP Viral protein WHO World Health Organization

Recombination Process by which genetic material is broken and joined to other genetic material, genetic rearrangement.

Classification (Compact) Enteroviruses form one of the largest group of viruses, which infects both humans and other species. Currently, there are more than 150 enterovirus types in 15 species. Most types infect humans but enteroviruses have also been identified in many mammals including bovines, pigs, monkeys, dromedary camels and rodents. Within species different types share greater than 70% amino acid (aa) identity in the polyprotein, greater than 60% identity in the capsid protein coding region (P1), and greater than 70% aa identity in the non-structural proteins 2C þ 3CD. They also have a limited range of host cell receptors, a limited natural host range, a genome base composition within a 2.5% variation range as well as significant compatibility in proteolytic processing, replication, encapsidation and genetic recombination. Current EV typing system is based on genetic relationships of VP1 capsid protein. Human enteroviruses are currently classified based on their genome organization, sequence similarity and biological properties into four species: Enterovirus A (EV-A), EV-B, EV-C and EV-D (Fig. 1), and include representative members such as polioviruses, coxsackie A and B viruses and echoviruses. The most recently identified types have been named according to numbering and species convention, such as enterovirus B-100. Each human enterovirus species consists of types; 25 EV-A types, 63 EV-B types, 20 EV-C types including 3 poliovirus types and 5 EV-D types (Fig. 1). Types can be distinguished by nucleotide sequence identity in the VP1 protein coding region. Identity of at least 75% (85% aa identity) between a prototype strain and an isolated strain suggests serotypical identity given that the next highest identity with an other prototype strain is less than 70%. Recent evolutionary studies indicate, however, that for e.g., CVA13, CVA20, E1, E13 and E30 nucleotide sequence identity within type may even be 73%, and thus amino acid identity should also be used as the basis of type determination. Within types enterovirus can further be classified into subtypes or genotypes with demarcation cut-offs varying between 9%–20% in the VP1 protein coding region. Genomic regions outside the capsid do not correlate with serotype identity due to frequent recombination in the non-structural parts of the genome. The sequence relatedness in the P1 region coding for the capsid proteins correlates reliably with the traditional definition of a serotype. Antigenic groupings known as serotypes share similarities in immunologic response of the human host, protection from the disease, receptor usage and to some extent symptoms of clinical disease. Within a serotype isolates can be distinguished based on antigenicity measured with antisera raised in animals. Serotype still remains an important physical and immunologic property distinguishing different enteroviruses. Most of the prototype strains of enteroviruses were originally isolated in the 1940–1950s. Development of molecular methods has facilitated detection of new enterovirus types during the past twenty years. In addition, molecular epidemiological studies have indicated the need for re-classification of some types, e.g., human rhinovirus 87 as EV-D68 and closely related rhinoviruses as three different species under the genus Enterovirus.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21544-6

245

246

Enteroviruses (Picornaviridae)

Fig. 1 Phylogenetic tree depicting genetic relationships of enterovirus types in Enterovirus species A to D. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 116 nucleotide sequences coding for the complete VP1 capsid protein region. Evolutionary analyzes were conducted using MEGA vs. 10.0.5.

Enteroviruses evolve through mutations and recombination events. Substitution rates within types are in the range of 0.41  102 to 3.07  102 substitutions per site per year. According to recent evolutionary analyzes times of the most recent common ancestors of individual enterovirus types vary between 55 and 200 years ago. Both inter- and intratype recombination events are common. Recombination occurs more frequently in the non-structural protein coding region and is restricted to viruses belonging to the same species. Interspecies recombination has been detected in the 50 NCR and VP4 junction.

Virion Structure Enteroviruses were the first animal viruses for which structures with atomic detail were determined. Since 1985, when the X-ray structures of poliovirus and rhinovirus B-14 were determined, numerous high-resolution enterovirus particle structures with

Enteroviruses (Picornaviridae)

247

Fig. 2 Enterovirus structure. The capsid consists of 60 protomers, each consisting of four polypeptides that comprise the structural proteins VP1, VP2, VP3, and VP4 within capsid. VP4 faces the interior and is not visible on capsid surface. Cryo-EM model of coxsackievirus A7 (left). Reproduced from Seitsonen, J.J., Shakeel, S., Susi, P., et al., 2012. Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating. Journal of Virology 86, 7207–7215.

similar particle architecture have been resolved. Enterovirus particles are spherical with a diameter of about 28 nm and a density close to 1.34 g/ml, which is less than in most picornaviruses. Virus particles lack lipid membrane, which makes them insensitive to organic solvents and sturdy enough to ensure survival in harsh conditions in the gut where they replicate. Most enteroviruses are acid stable and retain infectivity at pH values lower than 3.0. However, virus particles are also flexible and able to undergo structural rearrangement during the entry and uncoating processes to release viral RNA. Core particle features include small and smooth icosahedral structure and modularity of viral subunits with conserved Swiss roll domains. Enterovirus particles are constructed of 60 repeated protomer units, which are composed of four structural proteins, VP1 to VP4, encoded by proteolytic processing of long viral polyprotein (Fig. 2). The protomers form an icosahedral shell with a pseudo-T ¼ 3 arrangement that encapsidates the viral RNA genome. External VP1 proteins are located around the five-fold axes, while VP2 and VP3 alternate around the 2- and 3-fold axes. These proteins have no sequence homology, but they share similar protein topology; each protein adopts eight-stranded, antiparallel b-barrel fold (Swiss roll) with C-termini located on the surface of the virion and N-termini facing the interior, which allows the formation of a dense and rigid protein shell. The main structural differences between VP1, VP2 and VP3 can be found in the loops that connect the b-strands and in the extended N- and C-termini. The amino acids within these domains define the morphology and antigenicity of the enterovirus types. VP4 is a short myristoylated capsid protein located in the interior of the virion. Viral RNA is tightly packaged within the capsid and in direct contact particularly with N-terminal amino acid residues of VP4. The N-termini of capsid proteins together with the myristate of VP4 have a major role in virus assembly and stability of the particle. Most enteroviruses have a deep surface depression – also known as “canyon” – encircling the five-fold axes of symmetry that often serves as the cellular receptor binding site. The surface area of receptor binding in the canyon has been proposed to allow neutralization by neutralizing antibodies, which block viral infectivity, but also immune evasion of enteroviruses via mutations. Since VP1 is located around the five-fold axes and accounts most of the accessible surface, it is the most important target for neutralizing antibodies. VP1 is also the most variable and immunogenic particle protein. However, not all enteroviral receptors bind to the canyon structure suggesting that other mechanisms of neutralization (and immune evasion) exist. Indeed, several mechanisms for antibody neutralization besides interference with receptor binding has been proposed; virus stability, uncoating or virus aggregation may be affected by neutralizing antibodies. The floor of the canyon within the VP1 b-barrel of all enteroviruses harbors a small, hydrophobic tunnel or pocket, which is in some enteroviral structures filled with a lipid moiety (for example, sphingosine, lauric acid or myristic acid) and likely to be involved in regulating particle stability. This pocket has been extensively studied for the development of novel antiviral substances. Several clinical trials have been performed with antiviral capsid binders, known as the WIN compounds (produced by Sterling-Winthrop and Janssen Pharmaceuticals). These molecules occupy the hydrophobic pocket and displace the pocket factor, thereby increasing particle stability and preventing receptor binding and/or genome release. However, antiviral effects per enterovirus type and clinical benefits have not been sufficient for clinical approval and use. As of January 2019, there are 147 structure depositions for enterovirus structures in the VIPERdb (see “Relevant Websites section”) including native structures and structures of mutant viruses, viruses complexed with receptors, antiviral compounds, peptides and antibody fragments determined by X-ray crystallography or cryo-electron microscopy. Progress in high-resolution cryo-electron microscopy techniques is likely to increase the number of high-resolution structures in the near future and to enhance our understanding of the structural diversity within the family Picornaviridae. Expedited structure determination is also likely to facilitate research on antiviral capsid binders.

248

Enteroviruses (Picornaviridae)

Fig. 3 Enterovirus genome organization and polyprotein cleavage patterns.

Genome Enterovirus genome is a single positive-stranded RNA molecule with a length of approximately 7500 nucleotides (Fig. 3). Enteroviral RNA can function as a messenger RNA (mRNA) and it is infectious. At the 50 end of the genome there is a covalently linked VPg protein (virion protein, genome linked). VPg is not required for infectivity, but it is suggested to act as a primer for RNA synthesis. The structure of the genome (Fig. 3) is common among many picornaviruses. The 50 non-coding region (NCR) with an approximate length of 700 nt controls genome replication and translation. It harbors the internal ribosome entry site (IRES) that directs translation of the mRNA by internal ribosome binding. In the 30 end of the genome there is a short, less than 100 nt, 30 NCR. It has also a secondary structure, a pseudoknot, that controls viral RNA synthesis. The genome ends with a poly-A tail in both virion and mRNA. The poly-A tail has an effect on infectivity; the genome is non-infectious if the poly-A tail is removed. Between the NCRs there is a single long open reading frame (ORF). The ORF is processed to form individual viral proteins. The gene expression is also similar in all picornaviruses including EVs. The polyprotein cleavage occurs during translation by virusencoded proteinases. The polyprotein is divided in three regions: P1-3 (Fig. 3). P1 encodes viral capsid proteins, P2 and P3 encode non-structural or functional proteins. 2Apro, 3Cpro, 3CDpro are involved in protein processing and 2B, 2C, 3AB, 3BVPg, 3CDpro, 3Dpol in genome replication.

Life Cycle To complete a successful infectious cycle, an enterovirus must proceed via several steps including attachment, internalization/ entry, endocytosis, replication, particle assembly and exit (Fig. 4). Recognition of cell surface receptor(s) by virus particle determines the host range and tropism of cell and tissue infection and is the first step in the virus replicative cycle. Currently, there are more than ten different receptors known to mediate enterovirus infection, which may explain the numerous diseases that are caused by enteroviruses. These include e.g., CAR (coxsackievirus-adenovirus receptor), CD54 (intercellular adhesion molecule 1; ICAM-1), CD55 (decay-accelerating factor, DAF), CD155 (poliovirus receptor, PVR), integrin (e.g., a2b1, aVb1, aVb3, aVb6), heparan sulfate and sialic acids. Cellular receptors are classified as true receptors (a cell surface protein or sugar moiety) that mediate internalization and/or genome release and co-receptor(s)/attachment factors (e.g., heparan sulfate) that act in the attachment of a virus to cell surface. For some enteroviruses, a single cellular receptor functions in both binding and entry (e.g., PVR) while other enteroviruses use several receptors in a coordinated fashion where binding to the “first” receptor leads to signaling cascade that allows the virus to bind to the true entry receptor (e.g., DAF-CAR). Virus binding to the cell surface receptor may initiate a cascade of events leading to extensive irreversible structural changes (externalization of the N-termini of VP1 and the release of VP4) or have no effect on virus structure. Irrespective of this, most enteroviruses are internalized via receptor-mediated endocytosis mechanism (receptor itself is not known to be endocytosed) and transported to the cytosolic replication site in endosomal vesicles for viral RNA release instead of direct release of viral RNA into cell cytosol upon contact with the receptor. Nevertheless, the receptor usage is highly dependent on cell type, virus strain or even virus passage number (due to adaptation), and therefore, the actual role of any of these molecules in clinical infection needs further studies. While the endocytic process has been analyzed in detail for only a few enteroviruses, it is evident that they can use several endocytic mechanisms. Previously, endocytosis was divided into clear-cut mechanisms, such as clathrin- or caveolin-mediated endocytosis, but more recently it has become evident that there are more exceptions than rules to enterovirus entry mechanisms. That is, the virus entry is defined by pathway-specific entry components. For example, poliovirus (PV) is internalized using actin

Enteroviruses (Picornaviridae)

249

Fig. 4 Enterovirus life cycle. Following the binding to cell surface receptor(s), virus is endocytosed into the cell cytoplasm, where its positivestrand ( þ ) RNA genome is released from viral capsid and used in translation and genome replication. Viral protein VPg (3B), which is used as primer in replication, is covalently linked to viral genome. Genome translation yields a single polyprotein that is proteolytically cleaved into replication proteins (2A–2C and 3A–3D) and capsid proteins (VP0, VP1 and VP3), which are used to make new viral capsids. Genome replication is mediated by viral RNA-dependent RNA polymerase (3D), which synthesizes negative-strand (  ) RNAs that serve as templates for synthesis of new ( þ ) RNA molecules. New ( þ ) RNA molecules can either enter a new round of replication or be packaged into progeny virions. Capsid proteins self-organize into protomers and pentamers and assemble into provirions that are converted into infectious, mature virions by genomeinduced cleavage of VP0 into VP4 and VP2. Mature virions exit the cell via non-lytic release of extracellular vesicles or via cell lysis.

and tyrosine kinases but not clathrin, caveolin or microtubules with rapid release of viral RNA in the periphery of HeLa cells. In contrast, in endothelial cells PV entry occurs slowly and is dependent on caveolin-1 and dynamin. There are also no rules for receptor-specificity and endocytosis. Echovirus 7 (E7) uses DAF as the primary receptor, but it is endocytosed via a clathrinmediated pathway, while coxsackievirus B3 (CVB3) entry via DAF is caveolin-1-dependent. In addition, the entry process of CVB3 requires occludin but not dynamin. Echovirus 1 (E1) uses specifically integrin a2b1 receptor in a process involving components of macropinocytosis, such as PKC, Pak1 and Rac1, and ESCRT proteins. Recent studies within Enterovirus B species have revealed remarkable similarities between virus types, which use a variety of cellular entry receptors, and it is apparent that many factors contributing to endocytosis are associated with macropinocytic uptake. These examples imply that enterovirus entry pathways are subject to virus type (and strain), cellular receptor and cell line used in the study. Viral RNA release or uncoating follow the endocytosis to the site of replication. This may result in conformational changes in the virus particles, which are detected by sedimentation of virus particles with different molecular weights (160S for native particles versus 135S) in a differential ultracentrifugation run. According to some studies, the resulting 135S particles have exposed N-terminus of VP1 and lack VP4 but contain viral RNA. It has been hypothesized that hydrophobicity of VP1 N-termini increase the affinity of 135S forms to vesicular membranes, and as a result a pore through which viral RNA is released to cytosol is formed. The directionality of enteroviral RNA release during uncoating is unknown but it has been shown for a related rhinovirus (RV-A2) that the 3' end of the genome exits the virion before the 5' end. The site of RNA release from the virions is under debate and subject of virus type; the release may occur both at the two-fold, near the quasi-three-fold and five-fold axes depending on the cues used to trigger the uncoating process (such as heat, acid or soluble receptor molecules). The mechanism of RNA replication is well conserved among all enteroviruses; it is mediated by an internal ribosome entry site (IRES), and requires several IRES trans-acting factors and a non-canonical replication machinery assembled on virus-induced ER-derived membrane structures enriched with phosphatidylinositol-4-phosphate and cholesterol lipids. These lipids regulate the activity of the replication machinery and its core enzyme, viral-encoded RNA-dependent RNA polymerase (3Dpol), and thus viral RNA synthesis. After delivery to the cytosol, viral RNA is directly translated by ribosomes into a single large polyprotein, which is proteolytically processed by viral proteinases, 2Apro, 3Cpro and 3CDpro, into functional proteins that mediate both genome replication and virus assembly. Genome replication by 3Dpol is initiated with the synthesis of negative-stranded RNAs of the viral genomic RNAs to generate double-stranded RNA

250

Table 1

Enteroviruses (Picornaviridae)

Clinical disease entities associated to enterovirus types

Disease

EV types most often detected

Comments

AFP/AFM

PV, EV-D68, EV-A71

PV cases almost ceased. Other types associated include CVA7, CVA9, CVB1-B6, echoviruses, EV-D70

AHC Encephalitis HFMD/herpangina Meningitis

EV-D70, CVA24 variant EV-A71 CVA6, CVA10, CVA16, EV-A71 Echoviruses, CVA and CVB viruses, EV-A71 CVB1–6 CVB2-B5, E11 CVB1–6 EV-D68, CVA21, CVA24

Myocarditis Neonatal sepsis Pleurodynia Respiratory disease

All EV types CVA1-A5, CVA8, CVA22 CVB types and echoviruses associated more rarely All EV types

E5, E6, E16, E19 have also been associated to severe sepsis CVA types and echoviruses rarely All EV types

replication intermediates. After strand separation, the negative strands serve as templates for the synthesis of new positive-stranded viral RNAs, which subsequently serve as templates for new rounds of replication or IRES-guided translation. Capsid proteins assemble into protomers and pentamers, which together with viral RNAs form mature virions. Thus, the particle assembly is directly coupled with the viral RNA replication. Besides cleaving the viral polyprotein, viral proteinases 2Apro and 3Cpro cleave several host proteins to optimize virus translation, replication and virus spread and to suppress antiviral cellular responses. This leads to morphological alterations and ultimately virus particle release by disruption of cellular membranes. However, enteroviruses can also egress non-lytically in phosphatidylserine enriched, non-canonical autophagosome-like vesicles. This exit strategy may provide a simultaneous (masked) route of transmission of multiple genomes to uninfected cells, which enables the virus to maintain the viral genetic integrity and evade the immune system.

Epidemiology Enterovirus infections are very common. Age, socioeconomic status and sex have an effect on the frequency and outcome of the infection. Enteroviruses are seen in people of all ages, but children are most prone due to lack of protecting antibodies from previous infections. Also the severity of infection, variety of clinical symptoms and prognosis varies between different age groups with children more likely to present with significant clinical symptoms. Encephalitis and aseptic meningitis are most frequently seen in children 5–14 years of age. Encountering the first enterovirus infection at an older age increases the severity of symptoms. Enterovirus infections are more prevalent among people with low socioeconomic status as well as in those living in urban areas. Males, in general, are more prone to enteroviral disease and severe infection than females. Enterovirus infections typically peak during summer and early autumn in temperate climates. In tropical environments enterovirus infections occur all year round or they are associated with rainy seasons. Enteroviruses are isolated from both lower and upper gastrointenstinal tract and are readily excreted to feces. They survive for extended periods, i.e., months, in the environment, e.g., sewage, in favorable conditions. Neutral pH, moisture, low temperature and presence of organic matter protect enteroviruses from inactivation. The main form of transmission of enteroviruses is via fecal-oral route; from respiratory/upper gastrointestinal tract to the gut and from there to the brain. Also, respiratory transmission occurs for certain types, e.g., EV-D68, and to a lesser extent, motherto-infant transmission. Hand contact of upper respiratory mucosa (mouth, nose, conjunctiva) may facilitate transmission. Transmission occurs readily within households, neighborhoods and communities. In addition, nosocomial transmission has also been reported, especially among newborns. Humans are thought to be the main reservoir of enteroviruses. Simian enteroviruses are genetically closely related and human enteroviruses have been detected in free-living non-human primates. In addition, swine vesicular disease virus is a porcine variant of CVB5. Based on seroprevalence studies neutralizing antibodies are readily found in different populations indicating high incidences of past infections. Some enterovirus types are considered to remain endemic in certain areas, e.g., EV-B type E11 in Europe, whereas other viruses cause outbreaks with 5–10 year intervals, e.g., E30. Nucleic acid sequencing and molecular epidemiology have been widely used as a tool for the detection of genetic relationships of enteroviruses and identification of epidemiological patterns. Molecular epidemiology has provided information on geographic and temporal origin of the strains to strengthen epidemiologic links, to observe similarities among isolates in an outbreak and differences among isolates in different geographic regions.

Clinical Features Most enterovirus infections are clinically unnoticeable. When symptoms occur, the prevailing clinical presentations are common cold, non-specific febrile illness and/or rash, which are indistinguishable from diseases caused by other viruses. However,

Enteroviruses (Picornaviridae)

251

enteroviruses can cause a variety of more specific clinical outcomes, e.g., hand-foot-and-mouth disease (HFMD), meningitis, encephalitis, acute flaccid paralysis/myelitis, conjunctivitis, sepsis-like disease in newborns and myocarditis (Table 1). Different enterovirus types can cause similar diseases during the same geographically restricted outbreak and, vice versa, one type can cause a variety of clinical symptoms. Hand-foot-and-mouth disease is the most significant form of clinical enterovirus infection globally. The WHO defines HFMD as a febrile illness with papulovesicular rash on palms and soles with or without vesicles/ulcers in the mouth. In herpangina, the patient has febrile illness with multiple oral ulcers on the posterior parts of the oral cavity. Causative agents are species A enteroviruses and usually multiple different types co-circulate in HFMD outbreaks. The incidence of CVA16 is high world-wide. CVA6 emerged in 2000s and alternates with CVA16 as the most common enterovirus in outbreaks. EV-A71 has caused extensive HFMD outbreaks especially in the Asia-Pacific region during last decades. In most cases, HFMD and herpangina are self-limiting diseases, recovery occurs within 1–2 weeks after onset, and skin blisters heal without scarring. Onychomadesis is a recently reported specific consequence of CVA6 infection. Severe HFMD complications are rare, but occasionally central nervous system (CNS) is involved, particularly in children under 5 years of age, and when the causative agent is EV-A71. During recent EV-A71 outbreaks in Asia, 10%–30% of hospitalized HFMD patients developed CNS complications such as meningitis, brainstem encephalitis, encephalomyelitis and/or acute flaccid paralysis. The most severe EV-A71 cases have acute refractory myocardial dysfunction and fulminant cardiorespiratory failure with relatively high mortality rate. The survivors are in high risk for long-term sequelae, which range from limb weakness to ventilator dependence and presence of multiple physical and psychobehavioral disabilities. The severity of the disease depends on multiple factors, including age of the patient, pre-existing immunity, co-infections and the type and (sub-) genotype of the outbreak virus strain. The susceptibility to a severe EV-A71 infection may be associated with certain HLA types of the host as well. Enteroviruses are the most important causes of meningitis, an inflammation of the tissue covering brain and spinal cord. Individuals of all ages can contract meningitis, but most prone are children younger than 5 years of age and people having impaired immune system. Typical symptoms are headache, fever, muscle and stomach aches, stiffness in the neck, photophobia and phonophobia. The prognosis is usually good and the infection is cleared in one to two weeks. The prominent causative enterovirus types are echoviruses and coxsackie B viruses. Large outbreaks may occur in the summer-fall season in temperate climates. Encephalitis is a more severe presentation of CNS infection. The direct inflammation of brain can be caused by many enterovirus types, but it is mostly associated with EV-A71. All enteroviruses can cause respiratory symptoms. The disease spectrum includes acute upper respiratory infection, pneumonia, bronchiolitis, wheezing, respiratory distress and asthma exacerbation. Enteroviruses have also been detected in the middle ear fluid in young children at the time of acute otitis media. EV-D68 has long been associated with respiratory diseases and, at least some of the strains, share biological properties, like acid-sensitivity, with human rhinoviruses. Clinical isolates of EV-D68 were rare up to 2005. Since then, EV-D68 has caused wide outbreaks with considerable proportion of severe respiratory infections. Especially young children may require hospitalization and intensive care due to bronchiolitis or pneumonia. In rare cases, and mostly in young children, EV-D68 infection has been lethal. Acute flaccid paralysis (AFP), is a case definition used in poliovirus surveillance, meaning any sudden weakness or loss of muscle tone. Acute flaccid myelitis (AFM), is a more specific definition for a paralytic case with lesions in the brainstem and spinal cord gray matter, which are detectable by magnetic resonance imaging. CSF findings in enterovirus AFM are non-specific, lymphocytic pleocytosis is mild to moderate, glucose is normal and protein levels are normal or elevated. Virus is not usually detected in CSF. In the past, the most important AFP/AFM causing enteroviruses were polioviruses. In the 2010s, when poliovirus eradication is almost complete, most of the AFP/AFM cases are caused by other, non-polio enteroviruses. During EV-D68 outbreak in the US and Canada in 2014, the incidence of AFM followed that of EV-D68 respiratory disease. EV-D68 was detected in CSF in only few cases, but more often in the respiratory specimens on the onset of AFM. The full recovery occurred in only a small percentage of the EV-D68 AFM patients, and the majority had residual weakness still one year after the onset. In EV-A71 HFMD cases, the onset of paralysis may occur in 2–6 days after the first symptoms. Newborns are susceptible to severe enterovirus diseases, although the majority of enterovirus infections are asymptomatic or mild in neonates as well. The most severe presentation of neonatal enterovirus disease is septic shock with multiorgan failure. The disease can include specific manifestations like pneumonia, hepatitis, pancreatitis, myocarditis, meningitis or encephalitis. Mortality is highest in neonates with both hepatitis and myocarditis. Neonates with symptomatic enterovirus infection usually have mother or other contact with recent symptomatic illness. Perinatal transmission is also possible. Severe disease has been associated especially with CVB2 – CVB5 and E11. Acute hemorrhagic conjunctivitis (AHC), first detected in 1969, is a highly contagious infection of eyes. EV-D70 and a variant of CVA24 are the leading causative agents of AHC and have caused pandemics as well as smaller outbreaks world-wide. In addition to general symptoms, like fever, sore throat and headache, the infection results in eye irritation, photophobia, discharge and subconjunctival hemorrhage, which ranges from discrete petechiae to large patches of hemorrhage covering the bulbar conjunctiva. Recovery is usually complete within less than 10 days without ocular sequelae. During EV-D70 pandemics, neurological symptoms were reported in about 1 out of 10 000 AHC patients. Viral myocarditis, pericarditis and myopericarditis are rare consequences of EV infections, usually caused by coxsackie B viruses. Bornholm disease or epidemic pleurodynia, also known as epidemic myalgia, is caused by coxsackie B viruses. The typical symptom of Bornholm disease is acute, severe pain in the chest, often only on one side. Other symptoms include headache, fever, sore throat and general malaise.

252

Enteroviruses (Picornaviridae)

There is also evidence that some enteroviruses may trigger onset of chronic diseases. Some coxsackie B viruses and echoviruses are associated to the onset of type 1 diabetes, but the exact mechanisms are not yet fully understood.

Pathogenesis Polioviruses were historically the model of enterovirus pathogenesis research. With the success of the on-going poliovirus eradication campaign (see “Relevant Websites Section”) pathogenesis research of other enteroviruses has gained increasing interest. The most current research on enterovirus pathogenesis is focused on coxsackie B viruses, particularly CVB3, and enterovirus A71 (EV-A71) in Asia. The prevalence, modes of transmission, genetic variability (quasispecies), spectrum of tropism, specific features in cellular life cycle and ability to cause both lytic and persistent infections in the human host make it challenging to not only recognize but also link prior enteroviral infections and subsequent chronic syndromes. Primary infection occurs via respiratory or gastrointestinal tract mucosa. Viremia infection occurs within a day after contact with the virus enabling virus dissemination to multicellular organs, and lasts for 7–14 days until it is cleared by virus-activated adaptive immune response. Enteroviruses also replicate in actively proliferating cells, which are evident during organ development in the young. In addition, infectivity in the young may be related to tropism, i.e., expression of virus-specific cellular receptor in target cells, and more specifically to age-related expression of virus receptors, such as CAR in case of coxsackie B viruses. Enteroviruses have been shown to infect muscle, heart, pancreas and CNS causing respective diseases (see Section Clinical Features), some of which can be fatal or result in long-lasting organ dysfunction. CVB3 has tropism to heart and pancreatic tissues and is thus causative agent of virus-induced myocarditis and pancreatitis while EV-A71 is considered to be a serious emerging CNS pathogen. Since EVs are transmitted via fecal–oral route of infection, a detailed understanding of how the virus travels from the gut to the brain is essential in order to understand the pathogenesis. It is possible that during viremia virus passively passes blood-brainbarrier (BBB) independent of receptor interactions. However, it is more likely that virus dissemination between organs is at least partially defined by tissue tropism. The current model favors the idea that virus infects CNS using “Trojan horse” tactic by infecting myeloid stem cells, which mask the virus from the immune system and enable long-distance dissemination from the gut to CNS. At cellular level the most prominent feature of EV infection is cytolysis, i.e., ability to cause direct cell lysis following virus replication. In cells, EVs shut-down host protein synthesis and inhibit host cell protein secretion by virus-specific 2A and 3C proteases and 2B and 3A proteins, respectively. In addition, enteroviruses benefit from a cellular degradation process known as autophagy, which is known to play an important role particularly in preventing cellular damage in neurons. EVs use autophagosomal membranes as scaffolds for viral replication, which eventually leads to cytopathic cell lysis. It has also been suggested that EV proteins possess pro-apoptotic properties promoting cell death. Enteroviruses are known to persist (i.e., to cause infection in which virus is not cleared but remains silent in specific cells) up to 90 days with occasional activation of virus with disease symptoms. The genetic mechanism by which enteroviruses persist is apparently related to the RNA genome; enteroviral genomes with the first 49 nucleotides deleted have been detected in samples from human heart and in murine model of chronic myocarditis. This results in virus variants that replicate slowly and express very low levels of genomic RNA. As a consequence, enteroviruses that do not induce cytolysis are generated in barely detectable amounts. During the persistent infection viral 2A protease is expressed. Viral 2A protein has been shown to cleave dystrophin, which is a vital heart protein that supports muscle fiber strength. Cleavage of dystrophin is thought to contribute to dilated cardiomyopathy. It is not known whether autophagy contributes to viral persistence. CVB3 infection in CNS tissue may be linked to its ability to establish persistent infection only in such target stem cells, which support sporadic reactivation whenever they become activated to generate downstream progenitor cells. Together with cellular effects, this data may explain why enterovirus is the most important causative agent of aseptic meningitis. Despite of vast number of cellular studies and epidemiological studies with phylogenetic analyzes, relatively little is known about the determinants of enteroviral virulence. In general, there are not many publications where a single amino acid mutation is linked to changes in disease symptoms. This is greatly related to quasispecies nature of enteroviruses, which makes it difficult to identify a uniform virus (population) with a disease-related point mutation. Nevertheless, differences may lie in either NCRs, capsid or functional regions. For example, CVB4-VP1 gene has been linked to CVB4-induced pancreatitis; VP1 mutation in a virulent strain increases both virus amounts and immunogenicity enhancing disease symptoms. Studies with chimeric viruses (generated by the exchange of fragments between the attenuated and the virulent strains) have suggested that the 5´-NCR plays a major role in poliomyelitis and myocarditis, latter of which is directly related to enterovirus persistence with 5´-terminal deletions. However, it is evident that many genomic regions contribute not only to symptom development in myocarditis but also to other disease phenotypes.

Diagnosis Enterovirus diagnosis is based on the detection of enterovirus or its genome from appropriate clinical specimens. Clinical diagnosis is rarely adequate enough because enterovirus symptoms are indistinguishable from other viral or bacterial diseases. Only in well-described community outbreaks, where circulation of a particular enterovirus type is confirmed, the diagnosis may rely solely on clinical and epidemiological findings.

Enteroviruses (Picornaviridae)

253

Stool specimens and rectal swabs are the most sensitive for enterovirus detection. Viruses are excreted in stools in large concentrations, and this may last few weeks or even months after the recovery. In AFP/AFM, two stool specimens should be collected and analyzed for poliovirus. Respiratory specimens are a good choice especially in the beginning of the disease and they are obligatory for detection of e.g., EV-D68. Challenging in the non-sterile respiratory and intestinal tract specimens is that the etiological connection to the disease cannot be assured, as a significant proportion of healthy individuals are positive for enteroviruses as well. Specimens collected according to the symptoms are recommended for confirmation of the diagnosis. CSF is analyzed when CNS is involved and, with the exception of EV-D68, EV-A71 and polioviruses, enteroviruses can be detected in most meningitis cases, but rarely in encephalitis. From newborns with a systemic sepsis-like disease, blood is used as a supplementary specimen for enterovirus detection. In AHC, enterovirus is most frequently detected in conjunctival and oropharyngeal specimens. Vesicle swabs contain a high viral load in cases of HFMD and herpangina. Biopsy samples, including nails in onychomadesis, can be analyzed as well. The specimens should be collected as soon as possible after the symptoms have appeared, but at least within four days of the disease onset is recommended. The virus can be detected e.g., in feces for weeks, but etiological connectivity to the disease decreases in time. The detection of enteroviruses is performed almost solely by molecular methods, and particularly by real-time PCRs. The PCR exceeds the traditional virus isolation both in speed and sensitivity. For enterovirus genome detection, the most sensitive assays rely on highly conserved sequence stretches in the 50 NCR of the genome. There are numerous published in-house protocols guiding the set-up of sensitive and specific assays. Commercial assays are available for either detection of enterovirus only or detection of enterovirus together with other major respiratory, neurotropic or gastrointestinal viruses by multiplexed PCR assays. The 50 NCR assays are highly sensitive, but allow the detection only in the pan-EV level, not in the type level. It is also noteworthy that some of the 50 NCR protocols do not separate enteroviruses from human rhinoviruses, which is a challenge when respiratory samples are analyzed. Enterovirus type-specific diagnosis is based on the sequences of the capsid coding parts of the genome. The VP1 coding region is the best one for EV typing as it has the most prominent antigenic sites, which are the basis for the classification of enteroviruses. Type-specific real-time PCRs have been designed for example HFMD causing strains CVA6, CVA10 and EV-A71 and these both confirm the presence of enterovirus and type the virus in one PCR reaction. Specific PCR assays are especially useful in large outbreaks, when rapid screening of large amounts of clinical specimens is needed. Sequencing of the VP1 coding region or its part is a common practice in many clinical and national laboratories. This can be done directly from the clinical specimens, but the assay is usually less sensitive than the PCRs targeting the 50 NCR and it is not recommended for the first choice in diagnostics. The appr. 350 nucleotide long sequence from the amino-terminal region of the VP1 gene is sufficient for reliable typing and molecular epidemiological studies. The complete VP1 sequence is needed for the classification of a new enterovirus type. In addition to VP1, occasionally coding regions for proteins VP4 and VP2 are used for typing. Interpretation of the results is, however, complicated due to possible recombination events between the VP4 and VP1 genomic parts. Next generation sequencing is used for sequencing the complete enterovirus genomes. In most cases, the complete genomic sequence is not relevant in diagnosing individual patients, but it gives important information e.g., on the emergence of new outbreak strains, recombinant types and evolutionary history. Although largely replaced by molecular assays in primary diagnostics, the golden standard in enterovirus diagnostics – isolation in cell culture – is used in national enterovirus/poliovirus laboratories globally. Most enteroviruses grow rapidly in continuous monkey and human cell lines. The typical cytopathic effect (CPE) appears in 2 to 3 days after inoculation, negative samples are followed for at least 10 days. The CPE is not specific for enteroviruses and the virus isolate should be confirmed as an enterovirus by a real-time PCR assay, sequencing or neutralization with type-specific antisera or pools made of them. Enterovirus-specific IgM and IgG antibodies are measured by commercial or in-house EIA assays. IgM antibodies are indicative of a recent symptomatic or asymptomatic infection. The IgG antibodies are determined from acute and convalescent sera, and a significant increase is an indication of an acute or very recent infection. The antigen in the IgM and IgG assays is mostly a mixture of VP1 antigens from multiple enterovirus types and gives group-specific, not type-specific results. In addition to IgM- and IgG -EIA, neutralizing antibodies can be measured against a specific virus type in a cell culture assay. The positivity is a reliable indication of a past infection – two serum specimens are needed for the detection of a rise or decline in titers to predict a recent infection. Although not practical in rapid diagnostics, both antibody assays are used widely in seroprevalence studies where large populations are screened for their infection history.

Treatment In general, enterovirus infections are cleared by host immune system in few days or at least in weeks. Anti-enteroviral drugs have not yet been approved for clinical use. The treatment of enterovirus disease is symptomatic and aims to prevent complications. Immune-modulating treatments, such as intravenous immunoglobulin (IVIG), have been used in severe neonatal cases, hypogammaglobulinemia patients and disease outbreaks, but the evidence of benefits is not conclusive. However, there is supporting evidence that early administered IVIG may reduce the risk of autonomic nervous system dysregulation and death in severe EV-A71 infections, and the treatment is recommended by the WHO. The other passive immunotherapy options are high-dose corticosteroids, plasmapheresis and convalescent serum.

254

Enteroviruses (Picornaviridae)

The need for broad-reactive, efficient and safe anti-enteroviral drugs is obvious and has led to an active development during the past decades. Some drugs have been tested in clinical trials but either the lack of efficiency or the emergence of side effects has prevented their licensing for clinical use. Any step of the enterovirus life cycle may be a target for antiviral therapy. The antivirals can affect the functions of either virus proteins or host factors essential to viral infection. The side effects are supposed to be less prominent if the goal is the virus itself, but targeting the virus may soon result in the emerge of drug-resistant virus strains. The drug candidates are screened from compound libraries and optimized chemically or synthesized de novo. As a new approach, drug repurposing is performed to screen for compounds that have already been proved to be safe and that are used in treatment of other diseases. So far the most promising antivirals have been capsid binders, which occupy the hydrophobic pocket in the capsid and interfere with receptor binding and uncoating by stabilizing the virus particle. The capsid binders can be either pan-enteroviral with broad specificity or targeted to a single type, e.g., EV-A71. In clinical trials, for example Pleconaril, Pirodavir, Vapendavir and Pocapavir have shown efficiency in inhibiting some enteroviruses, but not all types. The fast emergence of resistant viruses was also noticed. Promising targets have also been viral proteases 2A and 3C, which are involved in viral polyprotein processing. Rupintrivir targets the active site of 3C and inhibits rhinovirus and EV-A71 infection. Viral replication can be affected by inhibiting the 3D polymerase. This can be done either by using nucleoside analogs such as ribavirin and gemcitabine or non-nucleoside inhibitors like amiloride. 2C inhibitors have also been described, one of them being drug fluoxetine. Virus replication needs essential host factors, which may also be the targets for antivirals. For example, enviroxime, which is an inhibitor of phosphatidylinositol 4-kinase-b, was effective against all enterovirus species in clinical trials, but the drug caused adverse side effects.

Prevention The most effective prevention of enterovirus infection is achieved by good hygiene, which interrupts the transmission of the virus. Enteroviruses are spread in droplets by coughing and sneezing and they are excreted in feces. In addition, depending on the clinical picture, enteroviruses may be present in eye secretions and blisters. The guidelines emphasize the need for hand washing with soap and water as alcohol-based disinfectants do not have an effect on enteroviruses. Contaminated surfaces should be washed with chlorine or soap and water. Enterovirus diseases could be prevented efficiently with vaccines, as shown by almost completed eradication of polioviruses. Both inactivated poliovirus vaccine (IPV) and live, attenuated oral vaccine (OPV) have been safe and efficient in preventing poliomyelitis and diminishing virus circulation. The experience on the poliovirus vaccines is of great value when developing new vaccines against other enteroviruses. The major disadvantage of IPV is the weak prevention of transmission, which is better obtained by OPV, which induces better mucosal immunity. The disadvantage of OPV is the reversion of attenuated poliovirus strains to neurovirulent mutants, which may occasionally cause vaccine associated poliomyelitis or, in certain epidemiological situations, circulation of vaccine-derived polioviruses. Designing a broad-spectrum vaccine against many or all enterovirus types has been too challenging so far. However, attempts have been made to develop vaccines against prevalent enterovirus types such as EV-A71. The first formalin-inactivated whole-virus vaccine against EV-A71 was approved by the Food and Drug Administration of China in 2015. The second vaccine was licensed in 2016, and further products are in clinical trials at the moment. The EV-A71 strains used in the first two vaccines are from different origin, but they both belong to sub-genotype C4a, which has been the most important virus type in Chinese outbreaks. Phase III clinical trials have involved over 30,000 infants and showed that the vaccines are safe and can prevent over 90% of EV-A71associated HFMD or herpangina. Besides inactivated and live-virus-vaccines, attenuated whole-virus vaccines are under development against some other EV types as well. Other potential vaccine constructs include virus-like particles, recombinant proteins and DNA vaccines.

Further Reading Baggen, et al., 2018. The life cycle of non-polio enteroviruses and how to target it. Nature Reviews 16, 368–381. Bauer, et al., 2017. Direct acting antivirals and host-targeting strategies to combat enterovirus infections. Current Opinion in Virology 24, 1–8. Bitnun, A., Yeh, E.A., 2018. Acute flaccid paralysis and enteroviral infections. Current infectious disease reports 20, 34. CDC & WHO/EURO, 2015. Enterovirus Surveillance Guidelines. Guidelines for Enterovirus Surveillance in Support of the Polio Eradication Initiative (2015). WHO. (ISBN 9789289050814). Chen, et al., 2015. Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell 60, 619–630. doi:10.1016/j.cell.2015.01.032. Harik, et al., 2018. Neonatal nonpolio enterovirus and parechovirus infections. Seminars in Perinatology 42, 191–197. Harvala, et al., 2018. Recommendations for enterovirus diagnostics and characterization within and beyond Europe. Journal of Clinical Virology 101, 11–17. Jones, et al., 2018. Outcomes following severe hand foot and mouth disease: A systematic review and meta-analysis. European Journal of Paediatric Neurology 22, 763–773. Lukashev, et al., 2018. Molecular epidemiology and phylogenetics of human enteroviruses: Is there a forest behind the trees? Reviews in Medical Virology 28 (6), e2002. doi:10.1002/rmv.2002. Marjomäki, et al., 2015. Infectious entry pathway of Enterovirus B species. Viruses 7 (12), 6387–6399. doi:10.3390/v7122945. Rhoades, et al., 2011. Enterovirus infections of the central nervous system. Virology 411, 288–305. doi:10.1016/j.virol.2010.12.014.

Enteroviruses (Picornaviridae)

255

Seitsonen, J.J., Shakeel, S., Susi, P., et al., 2012. Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating. Journal of Virology 86, 7207–7215. World Health Organization. Regional Office for the Western Pacific, 2011. A Guide to Clinical Management and Public Health Response for Hand, Foot and Mouth Disease (HFMD). WHO. Yi, et al., 2017. Enterovirus 71 infection and vaccines. Clinical and Experimental Vaccine Research 6, 4–14.

Relevant Websites https://ecdc.europa.eu/en/enteroviruses European Centre for Disease Prevention and Control. http://polioeradication.org/ Global Polio Eradication Initiative: GPEI. https://www.cdc.gov/non-polio-enterovirus/index.html Non-Polio Enterovirus. http://www.picornaviridae.com/ Picornavirus Home. http://www.picornastudygroup.com/ Picornaviridae Study Group. http://viperdb.scripps.edu Welcome to VIPERdb. Scripps Research.

Enveloped, Positive-Strand RNA Viruses (Nidovirales) Luis Enjuanes, National Center for Biotechnology – Spanish National Research Council (CNB-CSIC), Madrid, Spain AE Gorbalenya, Leiden University Medical Center, Leiden, The Netherlands Raoul J de Groot, Utrecht University, Utrecht, The Netherlands Jeff A Cowley, CSIRO Livestock Industries, Brisbane, QLD, Australia John Ziebuhr, The Queen's University of Belfast, Belfast, United Kingdom Eric J Snijder, Leiden University Medical Center, Leiden, The Netherlands r 2008 Elsevier Ltd. All rights reserved. This is a reproduction of L. Enjuanes, A.E. Gorbalenya, R.J. de Groot, et al., Nidovirales, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00775-5.

Glossary 3CLpro or Mpro 3C-like proteinase, or main proteinase. ADRP ADP-ribose-100 -phosphatase. CS TRS Core sequence. ExoN 30 to 50 exoribonuclease.

NendoU Nidovirus endoribonuclease. O-MT Ribose-20 -O-methyltransferase. PLpro Papain-like cysteine proteinase. TRS Transcription-regulating sequence.

Taxonomy and Phylogeny The order Nidovirales includes the families Coronaviridae, Roniviridae, and Arteriviridae (Fig. 1). The Coronaviridae comprises two well-established genera, Coronavirus and Torovirus, and a tentative new genus, Bafinivirus. The Arteriviridae and Roniviridae include only one genus each, Arterivirus and Okavirus, respectively. All nidoviruses have single-stranded RNA genomes of positive polarity that, in the case of the Corona- and Roniviridae (26–32 kbp), are the largest presently known RNA virus genomes. In contrast, members of the Arteriviridae have a smaller genome ranging from about 13 to 16 kbp. The data available from phylogenetic analysis of the highly conserved RNA-dependent RNA polymerase (RdRp) domain of these viruses, and the collinearity of the array of functional domains in nidovirus replicase polyproteins, were the basis for clustering coronaviruses and toroviruses (Fig. 2). The more distantly related roniviruses also group with corona- and toroviruses, thus forming a kind of supercluster of nidoviruses with large genomes. By contrast, arteriviruses must have diverged earlier during nidovirus evolution. The current taxonomic position of coronaviruses and toroviruses as two genera of the family Coronaviridae is currently being revised by elevating these virus groups to the taxonomic rank of either subfamily or family. A comparative sequence analysis of coronaviruses reveals three phylogenetically compact clusters: groups 1, 2, and 3. Within group 1, two subsets can be distinguished: subgroup 1a that includes transmissible gastroenteritis virus (TGEV), canine coronavirus (CCoV), and feline coronavirus (FCoV), and subgroup 1b that includes the human coronaviruses (HCoV) 229E and NL63, porcine epidemic diarrhoea virus (PEDV), and bat coronavirus (BtCoV) 512 which was isolated in 2005. Within group 2 coronaviruses, two subsets have been recognized: subgroup 2a, including mouse hepatitis virus (MHV), bovine coronavirus (BCoV), HCoV-OC43, and HCoV-HKU1; and subgroup 2b, including severe acute respiratory syndrome coronavirus (SARS-CoV) and its closest circulating bat coronavirus relative, BtCoV-HKU3. A growing number of other bat viruses has been recently identified in groups 1 and 2. It is currently being debated whether some of these viruses (e.g., BtCoV-HKU5, BtCoV-133 (isolated in 2005), and BtCoV-HKU9) may in fact represent novel subgroups or groups. Avian infectious bronchitis virus (IBV) is the prototype of coronavirus group 3, which also includes several other bird coronaviruses. In arteriviruses, there are four comparably distant genetic clusters, the prototypes of which are equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) of mice, simian hemorrhagic fever virus (SHFV) infecting monkeys, and porcine reproductive and respiratory syndrome virus (PRRSV) which infects pigs and includes European and North American genotypes. Roniviruses are the only members of the order Nidovirales that are known to infect invertebrates. The family Roniviridae includes the penaeid shrimp virus, gill-associated virus (GAV), and the closely related yellow head virus (YHV). More than 100 full-length coronavirus genome sequences and around 30 arterivirus genome sequences have been documented so far, whereas only very few sequences have been reported for toroviruses, bafiniviruses, and roniviruses. Therefore, information on the genetic variability of these nidovirus taxa is limited.

Diseases Associated with Nidoviruses Coronavirus infections are mainly associated with respiratory, enteric, hepatic, and central nervous system diseases. In humans and fowl, coronaviruses primarily cause upper respiratory tract infections, while porcine and bovine coronaviruses establish enteric

256

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00775-X

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

257

Fig. 1 Nidovirus classification and prototype members. The order Nidovirales containing the families Coronaviridae (including the established genera Coronavirus and Torovirus, and a new tentative genus Bafinivirus), Arteriviridae, and Roniviridae. Phylogenetic analysis (see Fig. 2) has confirmed the division of coronaviruses into three groups. In arteriviruses, four comparably distant genetic clusters have been differentiated. To facilitate the taxonomy of the different virus isolates, the types Co, To, Ba, Ro, standing for coronavirus, torovirus, bafinivirus, or ronivirus, respectively, have been included. The following CoVs are shown: human coronaviruses (HCoV) 229E, HKU1, OC43 and NL63, transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV), porcine epidemic diarrhoea virus (PEDV), mouse hepatitis virus (MHV), bovine coronavirus (BCoV), bat coronaviruses (BtCoV) HKU3, HKU5, HKU9, 133 and 512 (the last two isolated in 2005), porcine hemagglutinating encephalomyelitis virus (PHEV), avian infectious bronchitis virus (IBV), and severe acute respiratory syndrome coronavirus (SARS-CoV); ToV: equine torovirus (EToV), bovine torovirus (BToV), human torovirus (HToV), and porcine torovirus (PToV); BaV: white bream virus (WBV); Arterivirus: equine arteritis virus (EAV), simian haemorrhagic fever virus (SHFV), lactate dehydrogenase-elevating virus (LDV), and three (Euro, HB1, and MLV) porcine reproductive and respiratory syndrome viruses (PRRSV); RoV: gill-associated virus (GAV) and yellow head virus (YHV). Human viruses are highlighted in red. Some nodes are formed by a pair of very closely related viruses (e.g., SARS-CoV and BtCoV-HKU3). Asterisk indicates tentative genus.

infections, often resulting in severe economic losses. In 2002, a previously unknown coronavirus that probably has its natural reservoir in bats crossed the species barrier and caused a major outbreak of SARS, which led to more than 800 deaths worldwide. Toroviruses cause gastroenteritis in mammals, including humans, and possibly also respiratory infections in older cattle. Bafiniviruses have been isolated from white bream fish but there is currently no information on the pathogenesis associated with this virus infection. Roniviruses usually exist as asymptomatic infections but can cause severe disease outbreaks in farmed black tiger shrimp (Penaeus monodon) and white pacific shrimp (Penaeus vannamei), which in the case of YHV can result in complete crop losses within a few days after the first signs of disease in a pond. Infections by arteriviruses can cause acute or persistent asymptomatic infections, or respiratory disease and abortion (EAV and PRRSV), fatal age-dependent poliomyelitis (LDV), or fatal hemorrhagic fever (SHFV). Arteriviruses, particularly PRRSV in swine populations, cause important economic losses.

Virus Structure In addition to the significant variations in genome size among the three nidovirus families mentioned above, there are also major differences in virion morphology (Fig. 3) and host range. Nidoviruses have a lipid envelope which protects the internal nucleocapsid structure and contains a number of viral surface proteins (Fig. 3). Whereas coronaviruses and the significantly smaller arteriviruses have spherical particle structures, elongated rod-shaped structures are observed in toro-, bafini-, and ronivirus-infected cells. The virus particles of the Corona- and Roniviridae family members carry large surface projections that protrude from the viral envelope (peplomers), whereas arterivirus particles possess only relatively small projections on their surface. Coronaviruses have an internal core shell that is formed by a nucleocapsid featuring a helical symmetry. The nucleocapsid (N) protein interacts with the carboxy-terminus of the envelope membrane (M) protein. The intracellular forms of torovirus, bafinivirus, and ronivirus

258

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

Fig. 2 Nidovirus phylogeny. Tree depicting the evolutionary relationships between the five major groups of nidoviruses as shown in Fig. 1 (Coronavirus, Torovirus, Bafinivirus, Ronivirus, and Arterivirus). This unrooted maximum parsimonious tree was inferred using multiple nucleotide alignments of the RdRp-HEL region of a representative set of nidoviruses with the help of the PAUP*v.4.0b10 software (AEG, unpublished). Support for all bifurcations from 100 bootstraps performed is indicated. The phylogenetic distances shown are approximate. For acronyms, see Fig. 1.

nucleocapsids have extended rod-shaped (helical) morphology. By contrast, mature (extracellular) toroviruses (but not bafini- and roniviruses) feature a remarkable structural flexibility, which allows them to adopt crescent- and toroid-shaped structures also. Unlike other nidoviruses, arteriviruses have an isometric core shell. In all nidoviruses, the nucleocapsid is formed by only a single N protein that interacts with the genomic RNA. Both the number and properties of structural proteins vary between viruses of the three families of the Nidovirales and may even vary among viruses of the same family. Nidoviruses usually encode at least three structural proteins: a spike (S) or major surface glycoprotein, a trans-membrane (M) or matrix protein, and the N protein (Fig. 3). Ronivirus particles are unique in that they possess two envelope glycoproteins, gp116 (S1) and gp64 (S2), but no M protein. Coronavirus and arterivirus particles possess another envelope protein called E that is not conserved in toroviruses, bafini-, and roniviruses. Toroviruses and subgroup 2a coronaviruses, such as MHV, have a hemagglutinin esterase (HE) as an additional structural protein, whereas the SARS-CoV has at least four additional proteins that are present in the viral envelope (encoded by ORFs 3a, 6, 7a, and 7b). The proteins may promote virus growth in cell culture or in vivo, but they are dispensable for virus replication. The major envelope proteins are the S and M proteins in coronaviruses and toroviruses, the GP5 and M proteins in arteriviruses, and the S1 and S2 proteins in roniviruses. Among these, only the corona-, toro-, and bafinivirus S and M proteins share limited sequence similarities, possibly indicating a common origin. Whereas S proteins can differ in size, they share an exposed globular head domain and, with the exception of roniviruses, a stem portion containing heptad repeats organized in a coiled-coil structure. The S proteins of corona- and toroviruses (and most likely those of bafiniviruses) form trimers that bind the cell surface receptor whereas receptor binding in roniviruses is probably mediated by gp116 (S1). The arterivirus envelope proteins form two higherorder complexes: one is a disulfide-linked heterodimer of GP5 and the M protein; and the other is a heterotrimer of the minor structural glycoproteins GP2, GP3, and GP4. Except for the E and M proteins, all arterivirus structural proteins are glycosylated. By contrast, the M proteins of corona- and toroviruses (and, most likely, bafiniviruses) are glycosylated, and they share a triplespanning membrane topology with the amino-terminus exposed on the outside of the virions and the carboxy-terminus facing the nucleocapsid. In TGEV, a proportion of the M proteins has a tetra-spanning membrane topology leading to the exposure of both termini on the virion surface. In the virion, the coronavirus E protein has a low copy number (around 20) and deletion of the E protein gene from the genome of the group 1 coronavirus TGEV blocks virus maturation, preventing virus release and spread. In the group 2 coronaviruses MHV and SARS-CoV, deletion of the E protein results in a dramatic reduction, of up to 100 000-fold, of virus infectivity. The coronavirus E and SARS-CoV 3a proteins are viroporins, that is, they belong to a group of proteins that modify membrane permeability by forming ion channels in the virion envelope.

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

259

Nidovirales GP2 GP3 GP4 E

Arterivirus

GP5 M N RNA Membrane

Coronavirus S M E N RNA Membrane

Torovirus S M HE N RNA Membrane

Torovirus rod shape

RNA S M

HE

N

Membrane

Bafinivirus

S M

N Membrane RNA

Ronivirus

(a)

(b)

gp116 gp64 Membrane

N RNA

Fig. 3 Nidovirus structure. Architecture of particles of members of the order Nidovirales: electron micrographs (a) and schematic representations (b) are shown. N, nucleocapsid protein; S, spike protein; M, membrane protein; E, envelope protein; HE, hemagglutinin-esterase. Coronavirus M protein interacts with the N protein. In arterivirus, GP5 and M are major envelope proteins, while GP2, GP3, GP4, and E are minor envelope proteins. Toro-, bafini-, and roniviruses lack the E protein present in corona- and arteriviruses. Proteins gp116 and gp64, ronivirus envelope proteins. Different images were reproduced with permission from different authors: arterivirus, E. Snijder (Leiden, The Netherlands); ronivirus, P. J. Walker (CSIRO, Australia); bafinivirus, J. Ziebuhr (Queen's University, Belfast) torovirus, D. Rodriguez (CNB, Spain); coronavirus, L. Enjuanes (CNB, Spain).

Genome Organization Nidovirus genomes contain variable numbers of genes, but in all cases the 50 terminal two-thirds to three-quarters of the genome is dedicated to encoding the key replicative proteins, whereas the 30 proximal genome regions generally encode the structural and, in some cases, accessory (group- and virus-specific) proteins (Fig. 4). Nidovirus genome expression is controlled at the translational and post-translational levels. Thus, for example, ribosomal frameshifting is required for the expression of ORF1b, and the two replicase polyproteins (pp1a and pp1ab) are proteolytically processed by viral proteases. The proteolytic processing occurs in a

260

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

Fig. 4 Nidovirus genome structure. Genome organization of selected nidoviruses. The genomic ORFs of viruses representing the major nidovirus lineages are indicated and the names of the replicase and main virion genes are given. References to the nomenclature of accessory genes can be found in the text. Genomes of large and small nidoviruses are drawn to different scales. Red box at the 50 end refers to the leader sequence. Partially overlapping ORFs have been drawn as united boxes. Spaces between boxes representing different ORFs do not mean noncoding sequences.

coordinated manner and gives rise to the functional subunits of the viral replication–transcription complex. By contrast, the expression of the structural and several accessory proteins is controlled at the level of transcription. It involves the synthesis of a nested set of 30 co-terminal sg mRNAs that are produced in nonequimolar amounts. As in cellular eukaryotic mRNAs, in general only the ORF positioned most closely to the 50 end of the sg mRNA is translated.

The Replicase The nidovirus replicase gene is comprised of two slightly overlapping ORFs, 1a and 1b. In corona-, toro-, bafini-, and roniviruses, ORF1a encodes a polyprotein (pp1a) of 450–520 kDa, whereas a polyprotein of 760–800 kDa (pp1ab) is synthesized from ORF1ab. Expression of the ORF1b-encoded part of pp1ab involves a ribosomal frameshift mechanism that, in a defined proportion of translation events, directs a controlled shift into the  1 reading frame just upstream of the ORF1a stop codon (Fig. 4). In arteriviruses, pp1a (190–260 kDa) and pp1ab (345–420 kDa) are considerably smaller in size. Proteolytic processing of coronavirus pp1a and pp1ab generates up to 16 nonstructural proteins (nsps 1–16), while processing of the arterivirus replicase polyproteins generates up to 14 nsps. It is generally accepted that most of the replicase nsps assemble into a large protein complex, called the replication–transcription complex. The complex is anchored to intracellular membranes and likely also includes a number of cellular proteins. Nidoviruses replicase genes include a conserved array of protease, RNA-dependent RNA polymerase (RdRp), helicase (HEL), and endoribonuclease (NendoU) activities. In contrast to other positive-strand RNA viruses, they employ an RdRp with a characteristic SDD rather than the usual GDD active site. The vast majority of proteolytic cleavages in pp1a/pp1ab are mediated by an ORF1a-encoded chymotrypsin-like protease that, because of its similarities to picornavirus 3C proteases, is called the 3C-like protease (3CLpro). Also the term ‘main protease’ (Mpro) is increasingly used for this enzyme, mainly to refer to its key role in nidovirus replicase polyprotein processing (Fig. 5). Nidovirus–Mproshare a three-domain structure. The two N-terminal domains adopt a two-b-barrel fold reminiscent of the structure of chymotrypsin. With respect to the principal catalytic residues, there are major differences between the main proteases from different nidovirus genera. The presence of a third, C-terminal domain is a conserved feature of nidovirus main proteases, even though these domains vary significantly in both size and structure. The C-terminal domain of the coronavirus Mpro is involved in protein dimerization that is required for proteolytic activity in trans. Over the past years, a large body of structural and functional information has been obtained for corona- and arterivirus main proteases which, in the case of coronaviruses, has also been used

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

Amino acids: 1 nsp:

1000 1

2

3

4

2000

3000

8 5 6 7α 7β 9 RFS

10

4000

5000

6000

261

7000

11 12

Arterivirus (EAV pp1ab, c. 345 kDa) PL1* PL2 PL1 TM1 nsp: 1 2

RdRp ZBD Hel NendoU

CL TM2 TM3 3

4

5

6

(11) 7 8 9 10 RFS

12

13

14

15

16

Coronavirus (MHV pp1ab, c. 800 kDa) Ac

PL1ADRP PL2

TM1

Y

TM2

CL TM3

RdRp ZBD

Rp RBPs

Hel ExoN

NendoU

MT

RFS

Torovirus (EToV pp1ab, c. 785 kDa) ADRP PL

TM1

TM2

CL TM3

CPD

RdRp

ZBD Hel

ExoN NendoU MT

RFS

Bafinivirus (WBV pp1ab, c. 770 kDa) ADRP

TM1

TM2

CL TM3

RdRp ZBD

Hel

ExoN NendoU MT

RFS

Okavirus (GAV pp1ab, c. 760 kDa) TM

CL TM

RdRp ZBD Hel ExoN NendoU MT

Fig. 5 Nidovirus replicase genes. Polyprotein (pp) 1ab domain organizations are shown for representative viruses from the five nidovirus genera. Acronyms as in Fig. 1. Arterivirus and coronavirus pp1ab processing pathways have been characterized in considerable detail and are illustrated here for EAV and MHV. N-proximal polyprotein regions are cleaved at two or three sites by viral papain-like proteases 1 (PL1) and 2 (PL2), whereas the central and C-terminal polyprotein regions are processed by the main protease, Mpro. PL1 domains are indicated by orange boxes and cognate cleavage sites are indicated by orange arrowheads. PL2 domains and PL2-mediated cleavages are shown in green and CL domains and CL-mediated cleavages are shown in red. Note that EAV encodes a second, but proteolytically inactive PL1 domain (PL1*; orange-striped box). For the genera Torovirus, Bafinivirus, and Okavirus, the available information on pp1ab proteolytic processing is limited and not shown here. Other predicted or proven enzymatic activities are shown in blue: ADRP, ADP-ribose 10 -phosphatase; Rp, noncanonical RNA polymerase (‘primase’) activity; RdRp, RNA-dependent RNA polymerase; HEL, NTPase/RNA helicase and RNA 50 -triphosphatase; ExoN, 30 -to-50 exoribonuclease; NendoU, nidoviral uridylate-specific endoribonuclease; MT, ribose-20 -O methyltransferase; CPD, cyclic nucleotide phosphodiesterase. Regions with predicted transmembrane (TM) domains are indicated by gray boxes. Other functional domains are shown as white boxes: Ac, acidic domain; Y, Y domain containing putative transmembrane and zinc-binding regions; ZBD, helicase-associated zinc-binding domain; RBPs, RNA-binding proteins. Expression of the C-terminal part of pp1ab requires a ribosomal frameshift, which occurs just upstream of the ORF1a translation stop codon. The ribosomal frameshift site (RFS) is indicated.

to develop selective protease inhibitors that block viral replication, suggesting that nidovirus main proteases may be attractive targets for antiviral drug design. In arteri-, corona-, and toroviruses, the Mpro is assisted by 1–4 papain-like (‘accessory’) proteases (PLpro) that process the less well-conserved N-proximal region of the replicase polyproteins (Fig. 5). Nidovirus PLpro domains may include zinc ribbon structures and some of them have deubiquitinating activities, suggesting that these proteases might also have functions other than polyprotein processing. Bafini- and roniviruses have not been studied in great detail and it is not yet clear if these viruses employ papain-like proteases to process their N-terminal pp1a/pp1ab regions (Fig. 5). The replicase polyproteins of ‘large’ nidoviruses with genome sizes of more than 26 kb (i.e., corona-, toro-, bafini-, and roniviruses) include 30 –50 exoribonuclease (ExoN) and ribose-20 -O-methyltransferase (MT) activities that are essential for coronavirus RNA synthesis but are not conserved in the much smaller arteriviruses (Fig. 5). The precise biological function of ExoN has not been established for any nidovirus but the relationship with cellular DEDD superfamily exonucleases and recently published data suggest that ExoN may have functions in the replication cycle of large nidoviruses that, like in the DEDD homologs, are related to proofreading, repair, and recombination mechanisms. NendoU is a nidovirus-wide conserved domain that has no counterparts in other RNA viruses. It is therefore considered a genetic marker of the Nidovirales. The endonuclease has uridylate specificity and forms hexameric structures with six independent catalytic sites. Cellular homologs of NendoU have been implicated in small nucleolar RNA processing whereas the role of NendoU in viral replication is less clear. Reverse genetics data indicate that NendoU has a critical role in the viral replication cycle. Two other RNA-processing domains, ADP-ribose-100 -phosphatase (ADRP) and nucleotide cyclic phosphodiesterase (CPD), are conserved in overlapping subsets of nidoviruses (Fig. 5). Except for arteri- and roniviruses, all nidoviruses encode an ADRP domain that is part of a large replicase subunit (nsp3 in the case of coronaviruses). The coronavirus ADRP homolog has been shown to have ADP-ribose-10 -phosphatase and poly(ADP-ribose)-binding activities. Although the highly specific phosphatase activity is not essential for viral replication in vitro, the strict conservation in all genera of the Coronaviridae suggests an important (though currently unclear) function of this protein in the viral replication cycle. This may be linked to host cell functions and,

262

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

L

REP 1a

S

REP 1b

3a E M 6 7a 8a

N AAA

+ 7b

3b + − + − + − + − + − + − + −

+ − + −

8b 9b AAA UUU

1

AAA UUU

2 S

AAA UUU

3 3a, 3b

AAA UUU

4 E

AAA UUU

5 M

AAA UUU

6

AAA UUU

7 7a, 7b

AAA UUU

8 8a, 8b

AAA UUU

9 N, 9b

Fig. 6 SARS-CoV genome organization and sg mRNA expression. A diagram of CoV structure using SARS-CoV as a prototype. Below the top bar a set of positive- and negative-sense mRNA species synthesized in infected cells is shown. Dark and light blue lines ( þ ), mRNA sequences translated and nontranslated into viral proteins, respectively. Light gray lines (  ), RNAs complementary to the different mRNAs. L, leader sequence. Poly(A) and Poly(U) tails are indicated by AAA or UUU, respectively. Rep 1a and Rep 1b, replicase genes. Other acronyms above and below the top bar indicate structural and nonstructural proteins. Numbers and letters to the right of the thin bars indicate the sg mRNAs.

particularly, to the activities of cellular homologs called ‘macro’ domains which are thought to be involved in the metabolism of ADP-ribose and its derivatives. The CPD domain is only encoded by toroviruses and group 2a coronaviruses. In toroviruses, the CPD domain is encoded by the 30 end of replicase ORF1a (Fig. 5), whereas in group 2a coronaviruses, the enzyme is expressed from a separate subgenomic RNA. The enzyme's biological function is not clear. Coronavirus CPD mutants are attenuated in the natural host whereas replication in cell culture is normal, suggesting some function in vivo. The available information suggests that nidovirus replicase polyproteins (particularly, those of large nidoviruses) have evolved to include a number of nonessential functions that may provide a selective advantage in the host. ORF1a of all nidoviruses encodes a number of (putative) transmembrane proteins, like the coronavirus nsps 3, 4, and 6 and the arterivirus nsps 2, 3, and 5. These have been shown or postulated to trigger the modification of cytoplasmic membranes, including the formation of unusual double-membrane vesicles (DMVs). Tethering of the replication–transcription complex to these virusinduced membrane structures might provide a scaffold or subcellular compartment for viral RNA synthesis, possibly allowing it to proceed under conditions that prevent or impair detection by cellular defense mechanisms, which are usually induced by the double-stranded RNA intermediates of viral replication. Finally, recent structural and biochemical studies have yielded novel insights into the function of a set of small nsps encoded in the 30 -terminal part of the coronavirus ORF1a. For example, nsp7 and nsp8 were shown to form a hexadecameric supercomplex that is capable of encircling dsRNA. The coronavirus nsp8 was also shown to have RNA polymerase (primase) activity that may produce the primers required by the primer-dependent RdRp residing in nsp12. For nsp9 and nsp10, RNA-binding activities have been demonstrated and crystal structures have been reported for both proteins. Nsp10 is a zinc-binding protein that contains two zinc-finger-binding domains and has been implicated in negative-strand RNA synthesis.

Structural and Accessory Protein Genes In contrast to the large genome of Coronaviridae, which can accommodate genes encoding accessory proteins (i.e., proteins called ‘nonessential’ for being dispensable for replication in cell culture (Fig. 6)), the smaller genomes of arteriviruses only encode essential proteins (Fig. 4). Coronaviruses encode a variable number of accessory proteins (2–8), while the torovirus genome contains a single accessory gene encoding a hemagglutinin-esterase (HE). Coronavirus accessory genes may occupy any intergenic position in the conserved array of the four genes encoding the major structural proteins (50 -S-E-M-N-30 ), or they may reside upstream or downstream of this gene array. Roniviruses are unique among the presently known nidoviruses in that the gene encoding the N protein is located upstream rather than downstream of the gene encoding the glycoproteins. Several members of the coronavirus group 1a are exceptional in that they contain genes downstream of the N protein gene, which has not been reported for other coronaviruses. The ronivirus glycoprotein gene is also unique in that it encodes a precursor polyprotein with two

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

263

internal signal peptidase cleavage sites used to generate the envelope glycoproteins S1 and S2 as well as an amino-terminal protein with an unknown function. The accessory genes are specific for either a single virus species or a few viruses that form a compact phylogenetic cluster. Many proteins encoded by accessory genes may function in infected cells or in vivo to counteract host defenses and, when removed, may lead to attenuated virus phenotypes. Group 1 coronaviruses may have 2–3 accessory genes located between the S and E genes and up to two other genes downstream of N gene. Viruses of group 2 form the most diverse coronavirus cluster, and they may have between three and eight accessory genes. In this cluster, MHV, HCoV-OC43, and BCoV form the phylogenetically compact subgroup, 2a, that is characterized by the presence of (1) two accessory genes located between ORF1b and the S gene encoding proteins with CPD and HE functions, (2) two accessory genes located between the S and E protein genes, and (3) an accessory gene, I, that is located within the N protein gene. Of this set of five accessory proteins, only three homologs are encoded by the recently identified HCoV-HKU1, which is the closest known relative of the cluster formed by MHV, HCoV-OC43, and BCoV. In contrast, the most distant group 2 member, SARS-CoV, has seven or eight unique accessory genes, two between the S and E protein genes, four to five between the M and N protein genes, and ORF9b which entirely overlaps with the N protein gene in an alternative reading frame. In group 3 avian coronaviruses, of which IBV is the prototype, several accessory genes, which are expressed from functionally tri- or bicistronic mRNAs, have been identified in the region between the S and E protein genes (gene 3) and between the M and N protein genes (gene 5). Some functionally dispensable ORF1a-encoded replicase domains may also be considered as accessory protein functions. For instance, MHV and SARS-CoV nsp2 turned out to be nonessential for replication in cell culture.

Replication Like in all other positive-stranded RNA viruses, nidovirus genome replication is mediated through the synthesis of a full-length, negative-strand RNA which, in turn, is the template for the synthesis of progeny virus genomes. This process is mediated by the viral replication complex that includes all or most of the 14–16 nsps derived from the proteolytic processing of the pp1a and pp1ab replicase polyproteins of arteriviruses and coronaviruses. The replication complex, which is likely to include also cellular proteins, is associated with modified intracellular membranes, which may be important to create a microenvironment suitable for viral RNA synthesis as well as for recruitment of host factors. Electron microscopy studies of cells infected with arteriviruses (EAV) and coronaviruses (MHV and SARS-CoV) have shown that RNA synthesis is associated with virus-induced, DMVs. The origin of DMVs is under debate and different intracellular compartments including the Golgi, late endosomal membranes, autophagosomes, and the endoplasmic reticulum have been implicated in their formation. Studies of cis-acting sequences required for nidovirus replication have mainly relied on coronavirus defective-interfering (DI) RNAs replicated by helper virus. Genome regions harboring minimal cis-acting sequences have been mapped to around 1 kb domains of the genomic 50 and 30 ends. Studies with MHV DI RNAs have indicated that both genome ends are necessary for positive-strand synthesis, whereas only the last 55 nt and the poly (A) tail at the genomic 30 end are required for negative-strand synthesis. It has been postulated that the 50 and 30 ends of the genome may interact directly during RNA replication, as predicted by computer-aided simulations of MHV and TGEV genomic RNA interactions in protein-free media. There is, however, some experimental evidence supporting proteinmediated cross-talk between both genome ends in the form of RNA–protein and protein–protein interactions. Several experimental approaches have implicated, in addition to the nsps encoded by the replicase gene, the N protein in coronavirus RNA synthesis. Early in infection, the coronavirus N protein colocalizes with the site of viral RNA synthesis. In addition, the N protein can enhance the rescue of various coronaviruses from synthetic full-length RNA, transcribed in vitro or from cDNA clones. In contrast, arterivirus RNA synthesis does not require the N protein. Host factors that may participate in nidovirus RNA synthesis have been identified mainly from studies of coronaviruses and arteriviruses. In coronaviruses (MHV and TGEV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been identified as a major protein binding to genomic RNA sequences complementary to those in the negative-strand RNA that bind another cellular protein, polypyrimidine tract-binding protein (PTB). hnRNP A1 and PTB bind to the complementary strands at the 50 end of coronavirus RNA and could mediate the formation of an RNP replication complex involving the 50 and 30 ends of coronavirus genomic RNA. The functional relevance of hnRNP A1 in coronavirus replication was supported by experiments showing that its overexpression promotes MHV replication, whereas replication was reduced in cells expressing a dominant-negative mutant of hnRNP A1. There is also experimental evidence to suggest that the poly(A)-binding protein (PABP) specifically interacts with the 30 poly(A) tail of coronavirus genomes, and that this interaction may affect their replication. Other cellular proteins found to bind to coronavirus genomic RNA, such as aconitase and the heat shock proteins HS40 and HS70, might be involved in modulating coronavirus replication. Similarly, interactions of cellular proteins such as transcription cofactor p100 with the EAV nsp1, or of PTB or fructose bisphosphate aldolase A with SHFV genomic RNA, suggest that, in arterivirus replication also, a number of cellular proteins may be involved.

Transcription RNA-dependent RNA transcription in some members of the Nidovirales (coronaviruses, bafiniviruses, and arteriviruses), but not in others (roniviruses), includes a discontinuous RNA-synthesis step. This process occurs during the production of subgenome-length

264

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

negative-strand RNAs that serve as templates for transcription and involves the fusion of a copy of the genomic 50 -terminal leader sequence to the 30 end of each of the nascent RNAs complementary to the coding (body) sequences (Fig. 6). The resulting chimeric sg RNAs of negative polarity are transcribed to yield sg mRNAs that share both 50 - and 30 - terminal sequences with the genome RNA. Genes expressed through sg mRNAs are preceded by conserved ‘transcription-regulatory sequences’ (TRSs) that presumably act as attenuation or termination signals during the production of the subgenome-length negative-strand RNAs. In arteriviruses and coronaviruses, the TRSs preceding each ORF are presumed to direct attenuation of negative-strand RNA synthesis, leading to the ‘jumping’ of the nascent negative-strand RNA to the leader TRS (TRS-L). This process is guided by a base-pairing interaction between complementary sequences (leader TRS and body TRS complement) and it has been proposed that template switching only occurs if the free energy (DG) for the formation of this duplex reaches a minimum threshold. This process is named ‘discontinuous extension of minus strands’ and can be considered a variant of similarity-assisted template-switching that operates during viral RNA recombination. The genome and sg mRNAs share a 50 -leader sequence of 55–92 nt in coronaviruses and 170–210 nt in arteriviruses. Toroviruses are remarkable in that they employ a mixed transcription strategy to produce their mRNAs. Of their four sg mRNA species, the smaller three (mRNAs 3 through 5) lack a 50 common leader and are produced via nondiscontinuous RNA synthesis. In contrast, sg mRNA2 has a leader sequence that matches the 50 -terminal 18 nt of the genomic RNA and its production requires a discontinuous RNA-synthesis step reminiscent of, but not identical, to that seen in arteri- and coronaviruses. Synthesis of torovirus mRNAs 3 through 5, and possibly of the two mRNAs in roniviruses, is thought to require the premature termination of negative-strand RNA synthesis at conserved, intergenic, TRS-like sequences to generate subgenome-length negative-strand RNAs that can be used directly as templates for sg mRNA synthesis. In the case of torovirus mRNA2, a TRS is lacking. Fusion of noncontiguous sequences seems to be controlled by a sequence element consisting of a hairpin structure and 30 flanking stretch of 23 residues with sequence identity to a region at the 50 end of the genome. It is thought that during negative-strand synthesis, the hairpin structure may cause the transcriptase complex to detach, prompting a template switch similar to that seen in arteri- and coronaviruses. In addition to regulatory RNA sequences, viral and host components involved in protein–RNA and protein–protein recognition are likely to be important in transcription. For example, the arterivirus nsp1 protein has been identified as a factor that is dispensable for genome replication but absolutely required for sg RNA synthesis. The identification of host factors participating in nidovirus transcription is a field under development and specific binding assays have recently identified a limited number of cellular proteins that associate with cis-acting RNA regulatory sequences. For example, differences in affinity of such factors for body TRSs might regulate transcription in nidoviruses by a mechanism similar to that of the DNA-dependent RNA-polymerase I termination system, in which specific proteins bind to termination sequences.

Origin of Nidoviruses The complex genetic plan and the replicase gene of nidoviruses must have evolved from simpler ones. Using this natural assumption, a speculative scenario of major events in nidovirus evolution has been proposed. It has been speculated that the most recent common ancestor of the Nidovirales had a genome size close to that of the current arteriviruses. This ancestor may have evolved from a smaller RNA virus by acquiring the two nidovirus genetic marker domains represented by the helicase-associated zinc-binding domain (ZBD) and the NendoU function. These two domains may have been used to improve the low fidelity of RdRp-mediated RNA replication, thus generating viruses capable of efficiently replicating genomes of about 14 kbp. The subsequent evolution of much larger nidovirus genomes may have been accompanied by the acquisition of the ExoN domain. This domain may have further improved the fidelity of RNA replication through its 30 –50 exonuclease activity, which might operate in proofreading mechanisms similar to those employed by DNA-based life forms. It has been suggested that the ORF1b-encoded HEL, ExoN, NendoU, and O-MT domains may provide RNA specificity, whereas the relatively abundantly expressed CPD and ADRP might control the pace of a common pathway that could be part of a hypothetical oligonucleotide-directed repair mechanism used in the present coronaviruses and roniviruses. The expansion of the replicase gene may have been associated with an increase in replicase fidelity, thus also supporting the further expansion of the 30 -proximal genome region to encode the structural proteins required to form complex enveloped virions.

Effect of Nidovirus Infection on the Host Cell Compared to other viruses, the interactions of nidoviruses with their hosts have not been studied in great detail. In many cases, information is based on relatively few studies performed on a limited number of viruses from the families Coronaviridae and Arteriviridae. Also, most studies have been performed with viruses that have been adapted to cell culture and therefore may have properties that differ from those of field strains. Coronaviruses and arteriviruses are clearly the best-studied members of the Nidovirales in terms of their interactions with the host. Coronavirus infection affects cellular gene expression at the level of both transcription and translation. Upon infection, host cell translation is significantly suppressed but not shut off, as is the case in several other positive-RNA viruses. The underlying mechanisms have not been characterized in detail, but data obtained for MHV and BCoV suggest that they may involve the 50 leader sequences present on coronavirus mRNAs. The viral N protein was reported to bind to the 50 -common leader sequence and

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

265

it has been speculated that this might promote translation initiation, leading to a preferential translation of viral mRNAs. Furthermore, host mRNAs were reported to be specifically degraded in MHV- and SARS-CoV-infected cells, further reducing the synthesis of cellular proteins. Another mechanism affecting host cell protein synthesis may be based on specific cleavage of the 28S rRNA subunit, which was observed in MHV-infected cells. Studies on cellular gene expression following nidovirus infections have mainly focused on the coronaviruses MHV and SARSCoV. For example, SARS-CoV infection was reported to disrupt cellular transcription to a larger extent than does HCoV-229E. Differences in cellular gene expression have been proposed to be linked to differences in the pathogenesis caused by these two human coronaviruses. Apart from the downregulation of genes involved in translation and cytoskeleton maintenance, genes involved in stress response, proapoptotic, proinflammatory, and procoagulating pathways were significantly upregulated. Both MHV and SARS-CoV induce mitogen-activated phosphate kinases (MAPKs), especially p38 MAPK. In addition, activation of AP-1, nuclear factor kappa B (NF-κB), and a weak induction of Akt signaling pathways occur after SARS-CoV infection and the N and nsp1 proteins were suggested to be directly involved in inducing these signaling pathways. Nidoviruses have also been reported to interfere with cell cycle control. Infection by the coronaviruses TGEV, MHV, SARS-CoV, and IBV was reported to cause a cell cycle arrest in the G0/G1 phase and a number of cellular proteins (e.g., cyclin D3 and hypophosphorylated restinoblastoma protein) and viral proteins (MHV nsp1, SARS-CoV 3b 7a, and N proteins) have been proposed to be involved in the cell cycle arrest in G0/G1. Many viruses encode proteins that modulate apoptosis and, more generally, cell death, which allows for highly efficient viral replication or the establishment of persistent infections. Infection by coronaviruses (e.g., TGEV, MHV, and SARS-CoV) and arteriviruses (e.g., PRRSV and EAV) have been reported to induce apoptosis in certain cell types. Apoptosis has also been reported in shrimp infected with the ronivirus YHV and is thought to be involved in pathogenesis. Both apoptotic and antiapoptotic molecules have been found to be upregulated, suggesting that a delicate counterbalance of pro- and antiapoptotic molecules is required to ensure cell survival during the early phase of infection, and rapid virus multiplication before cell lysis occurs. Coronavirus-induced apoptosis appears to occur in a tissue-specific manner, which obviously has important implications for viral pathogenesis. For instance, SARS-CoV was shown to infect epithelial cells of the intestinal tract and induce an antiapoptotic response that may counteract a rapid destruction of infected enterocytes. These findings are consistent with clinical observations of a relatively normal endoscopic and microscopic appearance of the intestine in SARS patients. Furthermore, SARS-CoV causes lymphopenia which involves the depletion of T cells, probably by apoptotic mechanisms that are triggered by direct interactions of the SARS-CoV E protein with the antiapoptotic factor Bcl-xL. Also the MHV E protein has been reported to induce apoptosis. The SARS-CoV 7a protein was found to induce apoptosis in cell lines derived from lung, kidney, and liver, by a caspase-dependent pathway. Apoptosis has also been associated with arterivirus infection but information on underlying mechanisms and functional implications is limited. Coronavirus and arterivirus infections trigger proinflammatory responses that often are associated with the clinical outcome of the infection. Thus, for example, there seems to be a direct link between the IL-8 plasma levels of SARS patients and disease severity, similar to what has been described for pulmonary infections caused by respiratory syncytial virus. In contrast, despite the upregulation of IL-8 in intestinal epithelial cells, biopsy specimens taken from the colon and terminal ileum of SARS patients failed to demonstrate any inflammatory infiltrates, which may be the consequence of a virus-induced suppression of specific cytokines and chemokines, including IL-18, in the intestinal environment. Innate immunity is essential to control vertebrate nidovirus infection in vivo. The induction of type I IFN (IFN-a/b) varies among different coronaviruses and arteriviruses. Whereas some coronaviruses such as TGEV are potent inducers of type I IFN, other coronaviruses (MHV and SARS-CoV) or arteriviruses (PRRSV) do not stimulate its production, thus facilitating virus escape from innate immune defenses. Type I interferon is a key player in innate immunity and in the activation of effective adaptive immune responses. Upon viral invasion, IFN-a/b is synthesized and secreted. IFN-a/b molecules signal through the type I interferon receptor (IFNR), inducing the transcription of several antiviral mediators, including IFN-g, PKR, and Mx. IFN-g is critical in resolving coronavirus (MHV and SARS-CoV), and also arterivirus (EAV, LDV, and PRRSV) infections. Like many other viruses, coronaviruses have developed strategies to escape IFN responses. For example, it has been shown that the SARS-CoV 3b, 6, and N proteins antagonize interferon by different mechanisms, even though all these proteins inhibit the expression of IFN by interfering with the function of IRF-3. In arteriviruses such as PRRSV, IFN-g is produced soon after infection to promote Th1 responses. However, PRRSV infections or vaccination with attenuated-live PRRSV vaccines cause only limited IL-1, TNF-a, and IFN-a/b responses. This then leads to IFN-g and Th1 levels that fail to elicit strong cellular immune responses.

See also: Coronaviruses: General Features (Coronaviridae). Coronaviruses: Molecular Biology (Coronaviridae). Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

Further Reading de Groot, R.J., 2007. Molecular biology and evolution of toroviruses. In: Snijder, E.J., Gallagher, T., Perlman, S. (Eds.), The Nidoviruses. Washington, D.C.: A.S.M. Press, pp. 133–146. Enjuanes, L. (Ed.), 2005. Current Topics in Microbiology and Immunology, vol. 287: Coronavirus Replication and Reverse Genetics. Berlin: Springer. Enjuanes, L., Almazan, F., Sola, I., Zuniga, S., 2006. Biochemical aspects of coronavirus replication and virus–host interaction. Annual Review of Microbiology 60, 211–230. Gorbalenya, A.E., Enjuanes, L., Ziebuhr, J., Snijder, E.J., 2006. Nidovirales: Evolving the largest RNA virus genome. Virus Research 117, 17–37.

266

Enveloped, Positive-Strand RNA Viruses (Nidovirales)

Masters, P.S., 2006. The molecular biology of coronaviruses. Advances in Virus Research 66, 193–292. Sawicki, S.G., Sawicki, D.L., Siddell, S.G., 2007. A contemporary view of coronavirus transcription. Journal of Virology 81, 20–29. Siddell, S.G., Ziebuhr, J., Snijder, E.J., 2005. Coronaviruses, toroviruses, and arteriviruses. In: Mahy, B.W.J., ter-Meulen, V. (Eds.), Virology, 10th edn., vol. 1. London: Hodder-Arnold, pp. 823–856. Snijder, E.J., Siddell, S.G., Gorbalenya, A.E., 2005. The order Nidovirales. In: Mahy, B.W.J., ter-Meulen, V. (Eds.), Virology, 10th edn., vol. 1. London: Hodder-Arnold, pp. 390–404. Snijder, E.J., Spaan, W.J.M., 2007. Arteriviruses. In: Knipe, D.M., Howley, P.M., Griffin, D.E., et al. (Eds.), Fields Virology, vol. 1. Philadelphia: Lippincott Williams and Wilkins, pp. 1205–1220. Spaan, W.J.M., Cavanagh, D., de Groot, R.J., et al., 2005. Nidovirales. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Elsevier Academic Press, pp. 937–945. van Vliet, A.L.W., Smits, S.L., Rottier, P.J.M., de Groot, R.J., 2002. Discontinuous and non-discontinuous subgenomic RNA transcription in a nidovirus. EMBO Journal 21 (23), 6571–6580. Walker, P.J., Bonami, J.R., Boonsaeng, V., et al., 2005. Roniviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Elsevier Academic Press, pp. 975–979. Ziebuhr, J., Snijder, E.J., 2007. The coronavirus replicase: Special enzymes for special viruses. In: Thiel, V. (Ed.), Molecular and Cellular Biology: Coronaviruses. Norfolk, UK: Caister Academic Press, pp. 31–61. Zuñiga, S., Sola, I., Alonso, S., Enjuanes, L., 2004. Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. Journal of Virology 78, 980–994.

Epstein–Barr Virus (Herpesviridae) Lawrence S Young, University of Warwick, Coventry, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Classification Epstein-Barr virus (EBV) is a member of the genus Lymphocryptovirus (LCV) which belongs to the lymphotropic Gammaherpesvirinae subfamily of the family Herpesviridae. EBV is closely related to the LCVs present in hominids (e.g., chimpanzees) and other Old World non-human primates (e.g., baboons and macaques). These viruses share homologous sequences and genetic organization, and infect the B lymphocytes of their host species resulting in the establishment of persistent infection in vivo, B cell lymphomagenesis and the ability to immortalize B cells from their own natural host in vitro. LCVs have been isolated from common marmosets which are New World monkeys but these viruses show considerable divergence from the genomes of EBV and Old World primate LCVs suggesting that these viruses represent a more primitive predecessor of the LCVs infecting higher-order primates. LCV-specific genes can be classified into three groups based on evolutionary analysis: (1) universal herpesvirus genes common to all herpesviruses including genes important for virus replication and structure, (2) ancestral genes specific to the LCV genus responsible for intrinsic properties of these viruses such as B cell tropism, B cell transformation and virus reactivation, and (3) acquired LCV-specific genes that evolved later in the LCV genus and therefore are present in EBV and Old World LCVs but not New World LCVs, these include genes associated with the evasion of host immune responses.

Virion and Genome Structure EBV is about 120–180 nm in diameter and has a similar structure to other herpesviruses with a core composed of linear dsDNA surrounded by a protein nucleocapsid comprising 162 capsomeres. There is a protein tegument which encompasses the nucleocapsid and is surrounded by a cell membrane-derived envelope containing both lipids and glycoprotein spikes which mediate virus infection (Fig. 1). The EBV genome is composed of linear double-stranded DNA, approximately 172 kilobase pairs (kb) in length. EBV has a series of 0.5 kb terminal direct repeats (TRs) and internal repeat sequences (IRs) that divide the genome into short and long, largely unique sequence domains (Fig. 2). EBV was the first herpesvirus to have its genome completely cloned and sequence. Since the EBV genome was sequenced from an EBV DNA BamHI fragment cloned library, open reading frames (ORFs), genes and sites for transcription or RNA processing are frequently referenced to specific BamHI fragments, from A to Z, in descending order of fragment size (Fig. 2). The virus has the coding potential for around 85 proteins, not all of which have been identified or characterized.

History In 1958 Dennis Burkitt described a lymphoma that represented the most common tumor affecting children in certain parts of East Africa. The geographical distribution of this malignancy suggested that the development of this tumor, named Burkitt lymphoma (BL), might be due to an infectious agent possibly related to malaria. In 1964 the successful establishment of cell lines from explants of BL enabled Tony Epstein and Yvonne Barr to identify herpesvirus-type particles by electron microscopy within a subpopulation of tumor cells in vitro (Fig. 1). Werner and Gertrude Henle subsequently demonstrated that BL-derived cell lines expressed antigens that were recognized not only by sera from patients with BL but also by sera from patients with infectious mononucleosis (IM) and, at lower levels, from normal healthy individuals. Similar seroepidemiological studies also suggested a link between infection with this virus (now called Epstein-Barr virus after its discoverers) and undifferentiated nasopharyngeal carcinoma (NPC) leading to the subsequent direct demonstration of EBV DNA in the tumor cells of NPC. The ability of EBV to efficiently immortalize B lymphocytes in vitro and to induce tumors in non-human primates established this virus as a putative oncogenic agent in humans. Over the last 50 years EBV has been implicated in a variety of other lymphoid and epithelial malignancies.

Epidemiology and Natural History of Infection EBV is the most common and persistent virus infection in humans with approximately 95% of the world’s population sustaining a life-long asymptomatic infection. In underdeveloped countries, primary infection with EBV usually occurs during the first several months to few years of life and is predominantly asymptomatic. However, in developed populations, primary infection is more frequently delayed until adolescence or adulthood, in many cases producing the characteristic clinical features of infectious mononucleosis (IM) otherwise known as glandular fever. EBV is orally transmitted and infectious virus can be detected in oropharyngeal secretions from IM patients, from immunosuppressed patients and at lower levels from healthy EBV seropositive individuals. The precise site of EBV replication in healthy individuals remains unknown but there is evidence that the virus replicates in differentiating epithelial cells in the oropharynx, in

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21278-8

267

268

Epstein–Barr Virus (Herpesviridae)

Fig. 1 Electron micrograph of the Epstein-Barr virus virion.

salivary glands and in gingival cells. The nasopharynx and oropharynx are associated with a ringed arrangement of lymphoid tissue (so-called Waldeyer’s ring) which may contribute to EBV shedding from virus-infected B cells being reactivated within the local mucosal environment and/or B cell-derived virus infecting and then replicating in differentiating epithelial cells. EBV can be completely eradicated by irradiation in bone marrow transplant recipients suggesting that B lymphocytes are the main site of EBV persistence. This is supported by the B lymphotropism of EBV which is mediated by the binding of the major viral envelope glycoprotein gp350 to the CR2 receptor on the surface of B cells. Virion penetration of the B cell membrane requires further interactions between the EBV glycoprotein gp42 (which forms a ternary complex with gH and gL viral glycoproteins) and HLA class II molecules. It appears that the presence or absence of HLA class II in virus producing cells influences the tropism of EBV for B cells or epithelial cells by affecting the availability of gp42. Other CR2-independent pathways are responsible for EBV infection of epithelial cells, including secretory component-mediated IgA transport, integrin interactions with polarized epithelium and direct cell-to-cell contact, but these are relatively inefficient and of unknown relevance to EBV infection in vivo. The B cell tropism of EBV and its ability to establish a latent infection in these cells both in vitro and in vivo further supports the contention that B lymphocytes are the reservoir of lifelong persistent infection. EBV can efficiently transform B cells in vitro converting approximately 3%–10% of all target B cells into permanently growing lymphoblastoid cell lines (LCLs) in which every cell carries multiple episomal copies of the EBV genome and expresses all latent genes (referred to as latency III or the “growth” program), including six Epstein–Barr nuclear antigens (EBNAs 1, 2, 3A, 3B and 3C) and EBNA leader protein (EBNA-LP), latent membrane proteins LMP1 and LMP2 (which encodes two isoforms, LMP2A and LMP2B) and the non-coding EBV-encoded RNAs (EBER1 and EBER2) and viral microRNAs (miRNA) (Fig. 2). When peripheral blood lymphocytes from healthy EBV seropositives are placed in culture, the few EBV-infected B lymphocytes that are present regularly give rise to spontaneous outgrowth of EBV-transformed LCLs provided that immune T lymphocytes are either removed or inhibited by the addition of cyclosporin A to the culture. LCLs can also be generated by direct infection of resting B lymphocytes with EBV derived from the throat washings of seropositive individuals or from producer B cell lines. LCLs have provided an invaluable, albeit incomplete, model of the lymphomagenic potential of EBV. Detailed examination of EBV infection in vivo has shown that the virus persists in the IgD-CD27 þ memory B cell subset and that these cells have down-regulated the expression of most, if not all, viral genes. The precise route of entry of EBV-infected B cells into the memory compartment remains a subject of much debate. This reservoir of infected cells is stably maintained thereafter, apparently subject to the same physiologic controls as the general mucosa-associated memory B cell pool. EBV persistence within this B cell population brings with it the possibility of fortuitous antigen-driven recruitment of infected cells into germinal centers, leading to progeny that either re-enter the circulating memory pool or differentiate to plasma cells that may migrate to mucosal sites (Fig. 3). The different forms of latency that are manifest in virus-associated malignancies (discussed below) may represent latency programs that have evolved to accommodate such changes in host cell physiology (Figs. 3 and 4). Thus germinal center transit appears to activate a latency program where only the genome maintenance protein EBNA1 is expressed (latency 0), while exit from germinal centers is possibly linked to the transient expression of the EBV-encoded latent membrane proteins, LMP1 and LMP2 (latency II). The ability of these proteins to mimic the key signals required for B cells to undergo a germinal center reaction, namely T cell help via the CD40 pathway (constitutively provided by EBV-encoded LMP1) and activation of the cognate B cell receptor (augmented by EBV-encoded LMP2A), supports this strategy whereby EBV exploits the physiological process of B cell

Epstein–Barr Virus (Herpesviridae)

269

Fig. 2 EBV and its latent genes. (a) Location and transcription of Epstein-Barr virus latent genes on the double-stranded viral DNA episome. The origin of plasmid replication (OriP) is shown in orange. The short green thick arrows represent exons encoding latent proteins: six nuclear antigens (EBNAs 1, 2, 3A, 3B and 3C, and EBNA-LP), latent membrane proteins (LMPs 1, 2A and 2B), BamHI fragment H rightward open reading frame (BHRF1) and BamHI-A fragment rightward reading frame (BARF1). The short blue arrows at the top represent the highly transcribed nonpolyadenylated RNAs, EBER1 and EBER2. The middle long green arrow represents EBV transcription during latency III, in which all the EBNAs are transcribed from either the Cp or Wp promoter; the different EBNAs are encoded by individual mRNAs that are generated by differential splicing of the same long primary transcript. The inner red arrow represents the EBNA1 transcript, which originates from the Qp promoter during latency I and latency II. Latency II is characterized by expression of EBNA1 together with the latent membrane proteins. Wp restricted latency is initiated from the Wp promoter and there is expression of all the EBNAs, except EBNA2, which is deleted in this form of latency (outer long blue arrow). (b) Location of open reading frames for EBV latent proteins on the BamHI restriction map of the prototype EBV B95–8 genome and including the viral microRNAs in sequence order. The BamHI fragments are named according to size, with A being the largest. Lowercase letters indicate the smallest fragments. TR refers to terminal repeats at each end of the genome. This region was often referred to as Nhet to indicate heterogeneity in this region according to the numbers of TRs within different virus isolates. BART, BamHI-A rightward transcript. Reproduced from Young, L.S., Yap, L.-F., Murray, P.G., 2016. Epstein-Barr virus: Over 50 years and still providing surprises. Nature Reviews Cancer 16, 789–802.

270

Epstein–Barr Virus (Herpesviridae)

Fig. 3 Virus persistence in the B cells of the human host and the origin of EBV-associated B cell lymphomas Upper panel: normal virus persistence. EBV resides in memory B cells of asymptomatic hosts. There are two models to explain how the virus enters memory B cells. In the germinal center model, EBV-infection of naïve B cells leads to a latency III ‘growth program’ in which the proliferation and expansion of the infected B cell pool is driven by expression of all EBV latent genes. Cells then enter the germinal center and express latency II (‘default program’), characterized by expression of EBNA1, LMP1 and LMP2. LMP1 provides a CD40-like signal and LMP2 a surrogate B cell receptor like signal; mimicking the same signals provided to the antigen-specific B cell in the normal germinal center. EBV-infected germinal center B cells leave the germinal center and enter the memory B cell pool. Here EBV protein expression is silenced (Latency 0); these cells are replenished by the same signals that normally induce the proliferation of memory B cells. Proliferating memory B cells require EBNA1 expression (Latency I) for viral episome segregation. Memory B cells can terminally differentiate into plasma cells (solid arrow) which triggers virus replication. EBV-infected germinal center B cells might also differentiate directly into plasma cells. (dashed arrow). In the ‘direct infection’ model (shown below), EBV accesses memory B cells following the direct infection of these cells which may involve a latency III intermediary. Lower panel: The origin of the EBV-associated B cell lymphomas. There is uncertainty about the exact stages of differentiation from which the EBV-positive B cell lymphomas arise (as indicated by the black dotted lines), as it cannot be assumed that the pattern of latency observed in the progenitor cell is recapitulated in the corresponding tumor. The figure illustrates the presumed (question marks indicate this uncertainty) cell of origin based on current evidence for post-transplant lymphoproliferative disease (PTLD), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma and Burkitt’s lymphoma. Reproduced from Young, L.S., Yap, L.-F., Murray, P.G., 2016. Epstein-Barr virus: Over 50 years and still providing surprises. Nature Reviews Cancer 16, 789–802.

differentiation to access and maintain persistent infection in the memory B cell pool (Fig. 4). The possible commitment of these cells to plasmacytoid differentiation may trigger entry into lytic virus replication thereby providing a source of low-level virus shedding into the oropharynx. There may also be circumstances in which infected cells in the reservoir can reactivate back to proliferative infections similar to those of an EBV-transformed LCL.

EBV Strain Variation The B cell-derived B95.8 strain of EBV was fully sequenced in 1984 and was, at that time, the largest DNA sequence (172 kb) ever determined. While variations in repeat regions of the EBV genome are observed amongst different EBV isolates, the genomes of viruses from different regions of the world or from patients with different virus-associated diseases are very similar. Strain variation over the EBNA2-encoding (BamHI WYH) region of the EBV genome permits all virus isolates to be classified as either “type 1” (EBV-1, B95.8-like) or “type 2” (EBV-2, Jijoye-like). This genomic variation results in the production of two antigenically distinct forms of the EBNA2 protein which share only 50% amino acid homology. Similar allelic polymorphisms (with 50%–80% sequence homology depending on the locus) related to the EBV type occur in a subset of latent genes, namely those encoding EBNA-LP, EBNA3A, EBNA3B and EBNA3C. These differences have functional consequences as EBV-2 isolates are less efficient in in vitro B lymphocyte transformation assays compared with EBV-1 isolates. A combination of virus isolation and sero-

Epstein–Barr Virus (Herpesviridae)

271

Fig. 4 EBV-associated lymphomas display different patterns of virus latent gene expression. While the post-transplant lymphomas associated with EBV infection expression a pattern of latent gene expression similar to that observed in virus-transformed B cells in vitro (latency III), Hodgkin’s lymphoma (latency II) and Burkitt’s lymphoma (latency I) express more restricted forms of EBV latency. Upper panels show in situ hybridization for the abundant non-polyadenylated EBER transcripts which are expressed in all forms of EBV latent infection. The middle panels depict immunohistochemical staining for the EBNA2 protein which is only expressed in post-transplant lymphoma and not either Hodgkin’s lymphoma or Burkitt’s lymphoma. The bottom panels show immunohistochemical staining for the LMP1 protein which is expressed in post-transplant lymphoma and HL but not in BL.

epidemiological studies suggest that type 1 virus isolates are predominant (but not exclusively so) in many Western countries, whereas both types are widespread in equatorial Africa, New Guinea and certain other regions. The evolutionary relationship between the type 1 and type 2 strains of EBV remain obscure. They may have evolved from a common progenitor virus or through recombination of the ancestral LCVs infecting Old World primates. The pronounced (but not exclusive) segregation of EBV-2 isolates within equatorial regions suggests that environmental factors (including immunological competence) may have influenced EBV evolution and may still be responsible for the effective competition between EBV-2 isolates and the ubiquitous EBV-1 family. In addition to this broad distinction between EBV types 1 and 2, there is also minor heterogeneity within each virus type. Individual strains have been identified on the basis of changes, compared with B95.8, ranging from single base mutations to extensive deletions. While infection with multiple strains of EBV was originally thought to be confined to immunologically compromised patients, more recent studies demonstrate that normal healthy seropositive individuals can be infected with multiple EBV isolates and that their relative abundance and presence may vary over time. The possible contribution of EBV strain variation to virus-associated tumors such as BL and NPC which is common in China and South-East Asia, remains unknown. A number of studies have failed to establish an epidemiological association between EBV strain variation and disease concluding that the specific EBV gene polymorphisms detected in virus-associated tumors occur with similar frequencies in EBV isolates from healthy virus carriers from the same geographic region. However, these studies focussed on specific regions of the EBV genome rather than comparing the entire viral DNA sequence. More recent work using hybrid capture technologies and next generation sequencing (NGS) have facilitated routine sequencing from primary material and have confirmed that, while there is a high level of overall similarity of tumor-derived EBV strains with the prototypical EBV genome, variation exists in viral genes that might result in functional differences that influence tumor development. A recent case-control study of EBV strain variation in NPC has found distinct polymorphisms in the virus genome associated with the tumor thereby defining a high risk EBV variant that could be used to screen at risk populations and develop more precise diagnostic and prognostic tests.

Immune Response EBV elicits both humoral and cell-mediated immune responses in infected hosts. Primary infection with EBV is associated with the rapid appearance of antibodies to replicative antigens such as VCA, EA and MA (gp350/220) with a later serological response to the EBNA proteins. In IM these responses are exaggerated and are accompanied by autoantibodies such as rheumatoid factor as well as a heterophile antibody response directed against antigens on the surface of sheep erythrocytes. These autoantibodies are the result of EBV-induced polyclonal B cell activation. In the chronic asymptomatic virus carrier, antibodies to VCA, MA and EBNAs are found, the titers of which remain remarkably stable over time. Of these antibodies those against MA are particularly important

272

Epstein–Barr Virus (Herpesviridae)

as they have virus neutralizing ability and can also mediate antibody-dependent cellular cytotoxicity. The levels of these EBVspecific antibodies are elevated in different EBV-associated diseases. As with other persistent viruses, cell-mediated immunity plays an important role in controlling EBV infection. Primary EBV infection elicits a robust cellular immune response and the lymphocytosis observed in IM is a consequence of the hyperexpansion of cytotoxic CD8 þ T cells with reactivities against both latent and lytic viral antigens. These reactivities are subsequently maintained at high level (up to 5% of the total circulating pool) in the CD8 þ memory T cell pool. An EBV-specific CD4 þ T cell response also contributes towards the control of EBV infection and, along with the CD8 þ response, appears to be important in preventing the uncontrolled proliferation of EBV-infected B cells. Thus, impairment of the T cell response, either by immunosuppressive drug therapy or HIV infection, is responsible for the development of polyclonal lymphoproliferations that can progress to frank monoclonal non-Hodgkin’s lymphomas (see below). These lesions can be controlled by adoptive therapy with EBV-specific T cells. The growth and survival of BL, NPC and Hodgkin’s lymphoma (HL) in immunocompetent individuals implies that the tumor cells can evade EBV-specific T cell surveillance. This may be achieved by restricting EBV latent gene expression to those viral proteins not efficiently recognized by the T cell responses (i.e., away from expression of the highly immunogenic EBNA3 family), and/or by downregulation of target cell molecules required for immune recognition such as MHC class I/II and the antigen processing machinery which is mediated by the production of immunosuppressive EBV-encoded proteins such as those expressed during the EBV lytic cycle that inhibit antigen processing (e.g., BNLF2a), suppress macrophage function (vIL-10) and disrupt MHC class II presentation (e.g., BGLF5).

EBV-Associated Diseases Infectious Mononucleosis Primary infection with EBV in childhood is usually asymptomatic but when delayed until adolescence or early adulthood can manifest clinically as IM, a self-limiting lymphoproliferative disease characterized by a quartet of symptoms – sore throat, cervical lymphadenopathy, fever and fatigue. IM is associated with the massive expansion of activated CD8 þ T cells that are reactive predominantly to EBV lytic cycle antigens with some limited responses against latent antigens. Immunodominant CD8 þ T cell responses in IM are focussed on immediate early proteins (e.g., BZLF1 and BRLF1) and early proteins (e.g., BALF2, BMRF1, BORF2) and these reactivities are subsequently maintained in the CD8 þ T cell memory pool at high levels. Activated CD4 þ T cell responses specific for EBV epitopes in EBV lytic and latent antigens are also elevated in IM patients but fall rapidly as the disease resolves. IM appears to result in the global down-regulation of the alpha chain of the IL-15 receptor on T cells and NK cells, an effect which lasts for years after acute infection and may influence other disease risks. The incidence of IM is low in developing countries where asymptomatic primary infection predominantly occurs in childhood. In certain poorly defined situations IM-like symptoms can persist resulting in chronic active EBV infection (CAEBV) associated with elevated antibody titers to virus lytic antigens but low titers to the EBV-encoded nuclear antigens (EBNAs) along with elevated EBV infection in circulating T cells and NK cells. CAEBV appears to originate from an EBV-infected lymphoid progenitor and is associated with intragenic deletions in the EBV genome.

Multiple Sclerosis Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), causing severe progressive disability. Genetic and environmental factors contribute to the development of MS and many studies implicate EBV including the universality of EBV infection in MS patients, elevated levels of antibodies against EBV in serum (particularly against the EBNA1 protein) during the course of MS and often years prior to the onset of the disease. Impairment of the EBV-specific CD8 þ T-cell response, increased shedding of EBV from saliva and accumulation of EBV-infected B cells and plasma cells in the brain are also found in MS patients. It has been hypothesized that a defect in the CD8 þ T cell control of EBV predisposes to MS by allowing EBV-infected autoreactive B cells and plasma cells to accumulate in the CNS. A recent phase I clinical trial using autologous EBV-specific T cell therapy demonstrated some clinical improvement in MS patients.

Lymphomas in Immunosuppressed Individuals Patients with primary immunodeficiency diseases such as X-linked lymphoproliferative syndrome (XLP) and Wiscott-Aldrich syndrome are at increased risk of developing EBV-associated lymphomas. Because these tumors are extremely rare little is known about the precise contribution of EBV and the associated pattern of viral gene expression in these lymphomas. Mortality from XLP is high with around 50% of patients developing fatal IM after primary infection with EBV and an additional 30% of patients developing malignant lymphomas. The defect responsible for XLP is mutation of an adaptor molecule, SAP, which mediates signaling in a wide range of immune cells and is involved, via its interaction with the SLAM family of receptors, in both innate and adaptive immune reactions. Allograft recipients receiving immunosuppressive therapy and patients with AIDS are also at increased risk for development of EBV-associated post-transplant lymphoproliferative disease (PTLD) and lymphomas. The incidence of non-Hodgkin’s lymphoma (NHL) in AIDS patients, prior to the advent of highly active antiretroviral therapy, was increased approximately 60-fold compared

Epstein–Barr Virus (Herpesviridae)

273

to the normal population. Around 60% of these tumors were large B cell lymphomas like those found in allograft recipients, 20% were primary brain lymphomas and 20% were of the Burkitt’s lymphoma (BL) type. EBV infection was present in approximately 50% of AIDS-related NHL, nearly all the primary brain and Hodgkin’s lymphomas, and in around 40% of the BL tumors. These NHLs range from polyclonal EBV-driven lymphoproliferations, much like that observed in vitro in virus-transformed LCLs (latency III, Figs. 2–4), to aggressive monoclonal non-Hodgkin’s B cell lymphomas (NHLs) in which additional cellular genetic changes (e.g., mutation of p53) are present. The incidence of both PTLD and NHL B cell lymphomas in allograft recipients varies with the type of organ transplanted and with the type of immunosuppressive regimen used. PTLD in solid organ recipients are derived predominantly from recipient B cells whereas PTLD following hematopoietic stem cell transplantation are mainly of donor origin. Allogeneic bone marrow or solid organ transplantation into EBV seronegative children is a particular risk factor for the development of these lesions. A proportion of these lesions in post-transplant patients resolve in response to a reduction in immunosuppression or to targeted therapies such as anti-CD20 monoclonal antibody (rituximab) or adoptive EBV-specific T cells.

Diffuse Large B-Cell Lymphomas EBV-positive diffuse large B-cell lymphomas (DLBCL) are monoclonal B-cell tumors that occur in patients over the age of 50 with no evidence of immunodeficiency or history of lymphoma. These lymphomas are thought to arise as a consequence of immunosenescence, present as two different morphologic variants (monomorphic and polymorphic) and are associated with extranodal involvement with the gastrointestinal tract, skin and bone marrow being the most commonly affected sites. EBV-positive DLBCL is more common in Asia than the West, representing around 8%–10% of DLBCL in Asian countries and 2%–5% of DLBCL in Western countries. The EBV-positive DLBCLs have a distinctive molecular profile with increased activity of certain cell signaling pathways (e.g., NF-kB, STAT3, MAP kinase), probably as a consequence of EBV latent gene expression which is either of the latency II (EBNA1, LMP1, LMP2A) or latency III type. Intragenic deletions of various regions of the EBV genome have been found in a proportion of DLBCL. EBV-positive DLBCL is an aggressive tumor with worse prognosis that the EBV-negative disease, particularly in Asian patients. EBV-positive DLBCL has also been found in younger patients but here the disease is very similar to EBV-negative DLBCL in terms of clinical outcome.

Burkitt’s Lymphoma The endemic form of BL which is found in areas of equatorial Africa and New Guinea represents the most common childhood cancer (peak age 7–9 years) in these regions with an incidence of up to 10 cases per 100,000 people per year. This high incidence of BL is associated with holoendemic malaria thus accounting for the climatic variation in tumor incidence first recognized by Dennis Burkitt. More than 95% of these endemic BL tumors (eBL) are EBV-positive compared with 20% of the low incidence, sporadic form of BL (sBL) which occurs worldwide. In areas of intermediate BL incidence, such as Algeria and Malaysia, the increased number of cases correlates with an increased proportion of EBV-positive tumors. Malaria is thought to contribute to endemic BL by inducing an intense polyclonal B cell activation, thereby increasing the target population for EBV infection, along with transient impairment of T cell immunity. Seroepidemiological studies have demonstrated elevated antibody titers to EBV capsid antigen (VCA) and early antigens (EA) in BL patients compared to children without the tumor. These elevated antibody titers have been found to precede the development of BL and can therefore be used to screen “at risk” individuals. While sBL in high-income countries responds well to intensive chemotherapy, such regimens are not feasible or safe in low-income countries where late presentation is common and severe treatment-associated toxicities are difficult to manage. Recently modified protocols using cyclophosphamide and doxorubicin in eBL have resulted in much improved disease-free survival. Nevertheless, treatment failure is common and often associated with incomplete treatment, treatment-related mortality, relapse or progression of disease. The pattern of EBV gene expression in BL is generally restricted to EBNA1 and the non-polyadenylated EBER transcripts (latency I) although broader virus gene expression involving the EBNA3 proteins and the viral Bcl-2 homolog BHRF1 has been observed in around 15% of tumors (Figs. 2–4). These so-called Wp-restricted BLs (named after the alternative virus Wp promoter that is used in these cells to drive EBV gene expression) carry an EBNA2-deleted EBV genome and, as a consequence of BHRF1 expression, are more resistant in vitro to apoptosis induced by cytotoxic drugs. A consistent feature of BL tumors, irrespective of geographical location or EBV status, is chromosome translocations involving the long arm of chromosome 8 (8q24) in the region of the c-myc proto-oncogene and either chromosome 14 in the region of the immunoglobulin (Ig) heavy-chain gene or, less frequently, chromosomes 2 or 22 in the region of the Ig light-chain genes. BL tumors display a phenotype reminiscent of germinal center centroblasts with lower mutational frequencies in cancer driver genes such as MYC, ID3, TCF3, CCND3 and TP53 as compared to sBL. In contrast, a greater overall mutational load including higher frequency of mutations in ARID1A and loss of the regulatory role of PTEN have been found in eBL versus sBL. Some of these effects can be attributed to the influence of EBV-encoded genes in driving proliferation and survival in eBL without the need for mutations in other genes such as TCF3 and ID3. Endemic BLs infected with EBV-2 appear to have a higher proportion of somatic mutations (similar to the levels observed in sBL) than those containing EBV-1. Thus, there are clear genetic and molecular distinctions between EBV-positive and EBV-negative BL that reinforce the crucial role of EBV in eBL pathogenesis.

274

Epstein–Barr Virus (Herpesviridae)

Hodgkin’s Lymphoma Epidemiological studies originally suggested a possible role for EBV in the etiology of Hodgkin’s lymphoma (HL). Thus, elevated antibody titers to EBV antigens have been detected in HL patients and these are present before the diagnosis of the disease. Furthermore, there is an increased risk of HL following IM. EBV has been demonstrated in around 40% of HL cases with both viral DNA, RNA and virus-encoded latent proteins (EBNA1, LMP1, LMP2 – latency II) localized to the malignant component of HL, the so-called Hodgkin’s and Reed-Sternberg (HRS) cells (Fig. 4). These malignant cells are of germinal center origin but lack a functional B cell receptor, implicating EBV infection in the rescue of these so-called crippled cells from their usual apoptotic fate. Genetic mutations in components of various cell signaling pathways (e.g., NF-κB, JAK/STAT, PI3K/Akt) are consistently found in EBV-negative HL as compared to the EBV-positive form of the disease. Thus, in EBV-positive HRS cells, the virus-encoded LMP1 oncoprotein is apparently sufficient to drive aberrant activation of these pathways in the absence of cellular mutations. Thus, EBV and cellular mutations probably provide mutually exclusive means to the same pathogenic end-point. The association of HL with EBV is age-related; pediatric and older adult cases are usually EBV-associated whereas HL in young adults is less frequently viruspositive. The proportion of EBV-positive HL in developing countries is high consistent with a greater incidence of HL in children and more frequent prevalence of the mixed cellularity histiotype; the histological subtype of HL in which EBV infection is most frequently detected. Although the incidence of HL is relatively low (1–3/100,000 per year), this tumor is not geographically restricted making its association with EBV significant in world health terms.

EBV-Associated T Cell and NK Cell Lymphomas EBV-positive monoclonal lymphomas of either CD4 þ or CD8 þ T cell origin are more frequently found in Southeast Asian populations arising as a consequence of either virus-associated haemophagocytic syndrome (VAHS) or in the setting of CAEBV. There is also a distinct spectrum of EBV-positive T/NK lymphoproliferative disorders ranging from cutaneous lymphoid proliferations to aggressive extranodal T/NK lymphomas such as lethal midline granuloma, an erosive lesion of the nasal cavity. These lesions often develop in individuals with CAEBV, exhibit a latency II pattern of EBV latency with some evidence of early virus gene expression and frequently carry EBV genomes with intragenic deletions particularly over the BART miRNA region of the virus genome.

Nasopharyngeal Carcinoma The association of EBV with undifferentiated NPC (WHO types II and III) was first suggested by serological evidence and then confirmed by the demonstration of EBV DNA in NPC biopsy material. NPC is particularly common in areas of China and SouthEast Asia, reaching a peak age-standardized incidence of around 25 cases per 100,000 per year. Incidence rates are particularly high in Cantonese males highlighting an important genetic predisposition as well as a role for environmental cofactors such as dietary components (e.g., salted fish). NPC tumors are characterized by a prominent stroma and the interaction between these stromal cells (activated lymphocytes, tumor-associated macrophages, cancer-associated fibroblasts etc.) and adjacent carcinoma cells appears to be crucial for the continued growth of the malignant component. EBV latent gene expression in NPC is restricted to EBNA1, the LMP2A/B proteins, the EBER, BARF1 and the BART miRNAs with a variable proportion of tumors also expressing the oncogenic LMP1 protein (latency II) (Fig. 2). While latent EBV infection appears to be a key driver in the development and progression of NPC, more detailed analysis of virus gene expression by RNA sequencing reveals a more complex pattern with expression of lytic genes associated with EBV replication. The precise levels and consistency of such EBV lytic gene expression in NPC remains unknown as does whether this has any impact on NPC pathogenesis. Extensive serological screening for EBV-specific antibody titers in high incidence areas, in particular IgA antibodies to VCA and EA, have proved useful in diagnosis and in monitoring the effectiveness of therapy. The quantitative analysis of cell-free tumorderived EBV DNA in the plasma of patients with NPC using real-time PCR (Q-PCR) is of both diagnostic and prognostic utility. A recent study has demonstrated that Q-PCR for plasma EBV DNA levels can also be used as a primary screening test, detecting early stage NPC which is more responsive to treatment. The precise contribution of EBV infection to the development of NPC remains unclear. The presence of monoclonal EBV episomes in NPC tumor cells indicates that virus infection precedes the clonal expansion of the malignant cell population. However, the lack of epithelial EBV infection in normal nasopharyngeal biopsies from individuals at high risk of developing NPC suggests that epithelial infection may not be the initiating event in virus-associated carcinogenesis. EBV infection as detected by in situ hybridization to the EBER RNAs is found in high grade (severe dysplastic and carcinoma in situ) preinvasive lesions in the nasopharynx but not in low grade disease or histologically normal nasopharyngeal epithelium. Both the high grade and carcinoma in situ lesions carry monoclonal EBV genomes. A scheme has been proposed whereby loss of heterozygosity occurs early in the pathogenesis of NPC possibly as a result of exposure to environmental co-factors such as dietary components (i.e., salted fish) creating low grade preinvasive lesions that after additional genetic and epigenetic events become susceptible to EBV infection (Fig. 5). Once infected, EBV latent genes provide growth and survival benefits resulting in the development of NPC. Additional genetic and epigenetic changes occur after EBV infection and contribute to metastatic disease. Analysis of the genomic landscape in NPC has confirmed a role for chromatin modification in the carcinogenic process and identified additional somatic events including mutations in the ERBB-PI3K signaling pathway. A crucial role for the NF-kB pathway in NPC pathogenesis has been recently identified and this appears to be either a consequence of LMP1 expression or of genomic aberrations in negative regulators of the NF-κB signaling.

Epstein–Barr Virus (Herpesviridae)

275

Fig. 5 Schematic representation of the pathogenesis of NPC. In nasopharyngeal carcinoma (NPC), the tumor cells carry monoclonal viral genomes indicating that EBV infection must have occurred prior to expansion of the malignant cell clone. EBV infection is detected in high-grade pre-invasive lesions in both NPC but not in low-grade disease. Multiple genetic changes have been found in NPC, with frequent chromosomal deletions and promoter hypermethylation of specific genes on chromosomes 3p, 9p and 11q. Deletions in both 3p and 9p have been identified in low-grade EBV-negative dysplastic lesions and in normal nasopharyngeal epithelium from individuals who are at high risk of developing NPC. In vitro studies demonstrate that CCND1 over-expression (a consequence of cyclin-dependent kinase inhibitor 2A [CDKN2A which encodes p16 INK4A ] deletion on chromosome 9p and amplification of the CCND1 locus on chromosome 11q) facilitates persistent EBV infection of immortalized nasopharyngeal epithelial cells. Increased expression of the anti-apoptotic protein BCL-2 and elevated telomerase activity in low grade dysplastic lesions may also promote the establishment of latent EBV infection. A scheme has been proposed whereby loss of heterozygosity (LOH) occurs early in the pathogenesis of NPC possibly as a result of exposure to environmental co-factors such as dietary components (i.e., salted fish) creating low grade preinvasive lesions that after additional genetic and epigenetic events become susceptible to stable EBV infection. Once infected, EBV latent genes provide growth and survival benefits resulting in the development of NPC. Additional genetic (e.g., LOH on chromosomes 11q, 13q, 14q and 16q) and epigenetic changes occur after EBV infection and contribute to metastatic disease. The precise timing of epigenetic events in the pathway of NPC development is unknown although some changes such as RASSF1A inactivation may occur early while others (e.g., inactivation of RARb2, p14ARF and DAPK) may be influenced by the ability of EBV to enhance the activity of the methylation machinery. Recent analysis of the genomic landscape in NPC has confirmed a role for chromatin modification in the carcinogenic process and identified additional somatic events including mutations in the ERBB-PI3K signaling pathway. The pathogenesis of EBV-associated GC is similar to NPC with pre-malignant lesions occurring before EBV infection and a distinctive hypermethylation phenotype. CIS, carcinoma in situ; EDNRB, endothelin receptor B. Reproduced from Young, L.S., Yap, L.-F., Murray, P.G., 2016. Epstein-Barr virus: Over 50 years and still providing surprises. Nature Reviews Cancer 16, 789–802.

EBV infection is also associated with the more differentiated keratinizing form of NPC (WHO type I) particularly in those geographical regions with a high incidence of the type III tumor Carcinomas with similar features to undifferentiated NPC have been described at other sites (e.g., tonsils, uterine cervix) but, other than stomach tumors, these so-called undifferentiated carcinomas of nasopharyngeal type (UCNT) are not consistently EBV positive. EBV has been demonstrated in thymic epithelial tumors from Chinese but not Western patients. Salivary gland UCNTs are EBV-associated in Greenland Eskimos and Chinese but not in Caucasian patients.

Gastric Carcinoma and Other Epithelial Tumors EBV infection is also present in around 10% of typical gastric adenocarcinomas, accounting for up to 90,000 cases per year worldwide. These EBV-GC tumors resemble NPC in carrying monoclonal EBV genomes, having a restricted pattern of EBV gene expression (EBERs, EBNA1, LMP2A, BARTs and BARF1) and in the appearance of virus infection as a relatively late event in the

276

Epstein–Barr Virus (Herpesviridae)

carcinogenic process. EBV-GCs have distinct phenotypic and clinical characteristics compared to EBV-negative GC tumors including loss of p16 expression, p73 promoter methylation, wild type p53, a different pattern of allelic loss and improved patient survival. Comprehensive genomic and molecular characterization of GC tumors has confirmed that EBV-GCs are a distinctive entity with a high CpG island methylator (CIMP) phenotype and apparent driver mutations in the PIK3CA and ARID1A genes.

Control and Prevention of EBV Infection While a number of nucleoside (e.g., acyclovir, ganciclovir, famciclovir) and nucleotide (e.g., cidofovir, foscavir) analogs are approved for the treatment of herpes simplex virus (HSV-1 and HSV-2), varicella-zoster virus and human cytomegalovirus, none are used routinely in the treatment of acute EBV infection. This may reflect difficulties in diagnosing IM, its long incubation period and, more significantly, the immunopathology of IM which results in symptoms that are related to the immune response to EBVinfected B lymphocytes rather than virus replication. Nevertheless, antivirals such as acyclovir and ganciclovir have been used in patients with CAEBV and in immunosuppressed patients where such prophylaxis reduces the incidence of PTLD. The conventional approaches of chemotherapy and radiotherapy are used to treat primary EBV-associated tumors. It is only in the context of relapsed, refractory or metastatic cancer that novel therapeutic strategies specifically targeting EBV proteins or exploiting the presence of the virus in malignant cells are being developed. Demethylating agents such as 50 -azacytidine or histone deacetylase (HDAC) inhibitors such as SAHA are able to induce the EBV lytic cycle including virus-encoded kinases (EBV thymidine kinase and BGLF4, a protein kinase) that can phosphorylate the nucleoside analog gancyclovir to produce its active cytotoxic form. These agents, which are continuing to be assessed in clinical trials, also induce expression of the more immunogenic EBV latent genes in NPC and HL as well as creating a tumor immune microenvironment that supports enhanced antitumor immune responses. Other experimental approaches are based on targeting individual EBV latent proteins either indirectly by inhibiting signaling pathways activated, for instance, by LMP1 (e.g., NF-κB, PI3-kinase) or more directly using specific inhibitors such as siRNA or dominant negative molecules. More recently specific inhibitors of the EBNA1 protein have been developed and these are showing therapeutic promise in pre-clinical models. The use of adoptive EBV-specific T cell therapy for the treatment of existing PTLD and in the prophylactic setting has been extremely successful. This approach is also showing signs of some clinical efficacy in NPC when T cells are enriched for reactivities to subdominant targets (e.g., LMP2A and EBNA1). More direct vaccine approaches to treat patients with EBV-associated tumors or to prevent disease development are being examined including: (1) whole EBV latent antigens delivered by a virus vector or in autologous dendritic cells; (2) peptide or polytope vaccination and (3) prophylactic vaccination against MA (gp350/220) on the virion surface or virus-like particles to generate a neutralizing antibody response.

Future Perspectives EBV was discovered over 50 years ago and its DNA was fully sequenced in 1984. It remains the most common persistent virus infection in humans – testimony to the intimate interaction between EBV and the immune host. This relationship relies on the ability of EBV to persist in the memory B cell pool of normal healthy individuals and corruption of this process leads to the development of virus-associated lymphomas. The role of EBV infection in NPC is less clear and may be a consequence of the aberrant establishment of virus latency in epithelial cells that have already undergone pre-malignant genetic changes. The development of more efficient in vitro systems for studying EBV infection and replication in different cell types cells along with the use of recombinant forms of EBV is shedding light on the complex interplay between the virus and host. Improvements in virus genome sequencing technologies as well as the ability to clone wild-type EBV strains for biological characterization will facilitate a better understanding of the natural history of EBV infection and how the virus contributes to the oncogenic process. These systems, along with detailed analysis of EBV-associated tumors, are already providing insights into the interaction of the virus with the tumor microenvironment and how this influences the local cytokine milieu to promote tumor growth and immune evasion. There are many interesting aspects of EBV biology that remain to be understood including the role of virus-encoded microRNAs and the possible contribution of lytic cycle antigens to virus persistence and oncogenesis. The challenge will be to exploit these new mechanistic insights both to gain a better understanding of EBV infection in vivo and to develop novel therapies for treating virus-associated disease.

Further Reading Dawson, C.W., Port, R.J., Young, L.S., 2012. The role of the EBV-encoded latent membrane proteins LMP1 and LMP2 in the pathogenesis of nasopharyngeal carcinoma (NPC). Seminars in Cancer Biology 22, 144–153. Farrell, P.J., 2015. Epstein-Barr virus strain variation. Current Topics in Microbiology and Immunology 390, 45–69. Feederle, R., Klinke, O., Kutikhin, A., et al., 2015. Epstein-Brr virus: From the detection of sequence polymorphisms to the recognition of viral types. Current Topics in Microbiology and Immunology 390, 119–148. Fitzsimmons, L., Kelly, G.M., 2017. EBV and apoptosis: The viral master regulator of cell fate? Viruses 9 (11), 339. Kimura, H., Cohen, J.I., 2017. Chronic active Epstein-Barr virus disease. Frontiers in Immunology 8, 1867. Lam, W.K.J., Chan, K.C.A., Lo, Y.M.D., 2019. Plasma Epstein-Barr virus DNA as an archetypal circulating tumour DNA marker. The Journal of Pathology 247, 641–649.

Epstein–Barr Virus (Herpesviridae)

277

Longnecker, R.M., Kieff, E., Cohen, J.I., 2013. Epstein-Barr virus. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology., sixth ed. Philadelphia: Lippincott, Williams & Wilkins, pp. 1898–1959. Shannon-Lowe, C., Rickinson, A.B., Bell, A.I., 2017. Epstein-Barr virus-associated lymphomas. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 372, 1732. Taylor, G.S., Long, H.M., Brooks, J.M., Rickinson, A.B., Hislop, A.D., 2015. The immunology of Epstein-Barr virus-induced disease. Annual Review of Immunology 33, 787–821. Thorley-Lawson, D.A., Allday, M.J., 2008. The curious case of the tumour virus: 50 years of Burkitt's lymphoma. Nature Reviews Microbiology 6, 913–924. Thorley-Lawson, D.A., Hawkins, J.B., Tracy, S.I., Shapiro, M., 2013. The pathogenesis of Epstein-Barr virus persistent infection. Current Opinion in Virology 3, 227–232. Tsao, S.W., Tsang, C.M., To, K.F., Lo, K.W., 2015. The role of Epstein-Barr virus in epithelial malignancies. The Journal of Pathology 235, 323–333. Vrzalikova, K., Sunmonu, T., Reynolds, G., Murray, P., 2018. Contribution of Epstein-Barr virus latent proteins to the pathogenesis of classical Hodgkin lymphoma. Pathogens 7 (3), 59.

Equine Herpesviruses (Herpesviridae) Gisela Soboll Hussey, Michigan State University, East Lansing, MI, United States Nikolaus Osterrieder and Walid Azab, Free University of Berlin, Berlin, Germany r 2021 Elsevier Ltd. All rights reserved.

Introduction The first disease attributed to a herpesvirus of horses, the agent that is now referred to as equid herpesvirus type 1 (EHV-1; equine abortion virus), was documented at the University of Kentucky Agricultural Experiment Station in Lexington, KY. EHV-1 was first shown to be associated with spontaneous abortions in pregnant mares in 1932. In 1941 and 1949, equine abortions and/or paresis were found to be associated with mild respiratory disease with clinical signs similar to those associated with equine influenza virus infections. EHV-1 was also shown to be the etiological agent of epizootic respiratory disease in young horses. Based on these investigations, the disease was termed viral rhinopneumonitis, and the agent was called equine rhinopneumonitis virus. In the 1980s, however, the two disease manifestations, viral abortion and viral rhinopneumonitis, were shown to be caused by two closely related but clearly distinct viruses that are now classified as EHV-1 (equine abortion virus) and EHV-4 (equine rhinopneumonitis virus). EHV-1 is the cause of respiratory disease in young animals, as well as equine abortions, highly fatal neurological disease, fulminating neonatal pneumonitis, ocular chorioretinal lesions and very rarely, an exanthematous condition involving the external genitalia of the mare. Equid herpesvirus type 2 (EHV-2) was first isolated from horses in 1963. The cytopathology caused by this virus closely resembled that of cytomegalovirus infections which were first described in 1921 in humans. EHV-2 is a ubiquitous slow-growing virus that infects horses at a very young age (1–2 years) and establishes a life-long chronic infection such that the virus is continually shed. To date, no major disease has been attributed to EHV-2. An association of EHV-2 with chronic throat infections (the lumpy bumpies) or recurrent eye disease (keratoconjunctivitis) has been established; however, causal relationships have not been unequivocally proven. In addition, EHV-2 may be a cofactor in EHV-1 and/or EHV-4 infections in that it may be able to modulate EHV-1 and EHV-4 replication by an immunosuppression causing general malaise or by modulation of EHV-1 gene expression through EHV-2-specific transcriptional transactivators. Equid herpesvirus type 3 (EHV-3; equine coital exanthema virus, ECE virus) was first isolated in 1968, concurrently in Canada, Australia and the US. EHV-3 is the etiological agent of equine coital exanthema, a generally mild genital infection of both mares and stallions that is transmitted venereally. Clinical signs include the formation of papules, vesicles, pustules, and ulcers on genital organs. Although mild, EHV-3 infection has negative impact on equine breeding and can cause temporary disruption of the mating season, artificial insemination, and embryo transfer. Sporadic cases of EHV-3 infection with periodic virus shedding and virus isolation are still reported. Equid herpesvirus type 4 (EHV-4; equine rhinopneumonitis virus), is associated mainly with respiratory disease, but has also been associated occasionally with equine abortions. EHV-4 is most commonly detected in weaned foals and yearlings. Adult horses are also susceptible to EHV-4 infection, however, they show milder clinical signs or infections remain subclinical. Equid herpesvirus type 5 (EHV-5) is closely related to EHV-2 and is frequently detected in (healthy) horse populations worldwide. In 1970, EHV-5 was first isolated from the nasal cavity of two horses in quarantine in Australia. It has been thought that the virus is harmless and cause no clinical disease but there is mounting evidence that EHV-5 is associated with equine multinodular pulmonary fibrosis (EMPF). More definite proof for direct causation of EHV-5 and EMPF and a clearer understanding of the pathogenesis is needed, however, before a solid conclusion can be reached. Equid herpesvirus type 9 (EHV-9), previously referred to as gazelle herpesvirus 1, was first isolated from a captive Thomson’s gazelle suffering neurological signs in 1993 in Japan. It has been shown to be closely related, but clearly distinct, from EHV-1. The natural host and the true host range of EHV-9 is still unknown but the virus has a relatively wide host range and infects equid and non-equid species in the wild and in captivity. EHV-9 was used for experimental infection of a number of animal species, including dogs, cats, mice, cattle, hamsters, goats, pigs, macaques and marmosets, in all of which it causes a highly lethal neurological disorders.

Classification EHV-1, EHV-2, EHV-3, EHV-4, EHV-5, and EHV-9 are all members of the Herpesviridae but belong to two different subfamilies (Alpha- and Gammaherpesvirinae) in this large virus family. EHV-6, 7 and 8 are also referred to as asinine herpesviruses 1, 2, and 3, respectively (AsHV-1 to AsHV-3), and will not be discussed in this article. The morphology of all six members is typical of the herpesviruses in that they are enveloped, contain an icosahedral capsid, and have a proteinaceous layer, the so-called tegument, which surrounds the nucleocapsid. Equine herpesviruses are composed of six distinct species: (1) EHV-1 is the major equine pathogen causing abortions, respiratory illness, neurological disease and ocular disease; (2) EHV-2 establishes asymptomatic longterm persistent infection and might be associated with keratoconjunctivitis; (3) EHV-3 is the causative agent of mild progenital

278

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20977-1

Equine Herpesviruses (Herpesviridae)

Table 1

279

The equine herpesviruses

Properties of the viral genome Member

Subfamily

Isomers

G þ C ratio (%)

Clinical manifestations

EHV-1 EHV-2 EHV-3 EHV-4 EHV-5 EHV-9

a-herpesvirinae g-herpesvirinae a-herpesvirinae a-herpesvirinae g-herpesvirinae a-herpesvirinae

2 1 2 2 1 2

57 58 68.1 50.5 55 56

abortion, respiratory infection, paralysis chronic throat infection; keratoconjunctivitis equine coital exanthema respiratory infections equine multinodular pulmonary fibrosis encephalitis in non-definitive hosts

exanthema; (4) EHV-4 is a major respiratory pathogen that differs significantly from EHV-1 at the DNA level, and is associated occasionally with equine abortions; (5) EHV-5 may be associated with equine multinodular pulmonary fibrosis; (6) EHV-9 can cause subclinical encephalitides in horses under experimental conditions. Experimental and accidental natural infections of nonequid species are mostly lethal due to severe neurological disorders. In this latter respect, EHV-9 possesses biological properties very akin to the alphaherpesvirus of pigs, pseudorabies virus. EHV-1, 3, 4, and 9 are Alphaherpesvirinae members and belong to the genus Varicellovirus. EHV-2 and EHV-5 are members of the Gammaherpesvirinae and are classified into the genus Percavirus.

Virion Structure Infectious EHV-1 and EHV-4 particles contain an envelope that is mainly composed of glycoproteins. To date, twelve glycoproteins have been identified and an association of gB, gC, gD, gG, gH, gL, gM, gp2, gI, gE, gK, ORF10 (UL49.5) with purified virions has been demonstrated. The glycoproteins were shown in other alphaherpesviruses to be involved in virus attachment and binding, penetration, egress and cell-to-cell spread of infection as well as immune evasion. Less is known for proteins that make up the third component of the mature virion, the tegument. In all cases, EHV-1 and EHV-4 encode homologs to most of the HSV-1 tegument proteins that have been surmised to exert the same function. Three different EHV-1 capsid species were identified and probably correspond to the capsid forms found in HSV-1, which are designated type A, B, and C capsids. The EHV-1 capsid species were designated: (1) L capsids, which appear to be empty capsids; (2) I capsids, which possess an electron lucent, immature core structure in the shape of a cross; and (3) H capsids, which contain an electron dense, mature core. All three capsids appear at approximately 6 h postinfection. I capsids are believed to be a major precursor in the formation of mature capsids. The major capsid protein has an apparent size of 148 kD, while other structural proteins have also been identified.

Genomes All six equine herpesviruses contain a linear, double-stranded DNA genome ranging between 140 and 184 kbp. The reported approximate molecular masses of the genomes are: EHV-1 (Ab4 strain), 150 kbp; EHV-2, 184 kbp; EHV-3, 152 kbp; EHV-4, 145 kbp; EHV-5, 182 kbp; and EHV-9, 148 kbp. The genomes of EHV-1, EHV-3, EHV-4, and EHV-9, exist in two isomeric forms, since the short region (S) can invert relative to the fixed orientation of the unique long region (UL). The S region is comprised of a central segment of unique sequences (US) bracketed by a pair of inverted sequences (IR). In contrast, the genomes of EHV-2 and EHV-5 exist as one isomer and are comprised of a large central segment of unique sequences that is bracketed by a pair of direct repeat sequences. The EHV-2 genome contains direct terminal repeats (TR) of 17,553 and 18,332 bp, respectively. While the TR of EHV-5 is much smaller (10 bp) in each terminal. Other characteristics of the genomes of EHVs are shown in Table 1 and Fig. 1. There are varying degrees of homology at the DNA level among the six equine herpesviruses. The sequences shared by the EHV1, EHV-3, EHV-4, and EHV-9 appear to be arranged colinearly. EHV-1 and EHV-9 exhibit the highest degree of nucleotide identity, ranging from 86% to 95%. EHV-1 and EHV-4 have 55%–84% sequence identity at the DNA level. EHV-1, EHV-4, and EHV-9 are antigenically very closely related and antibodies can cross-react. EHV-3 is the most divergent among the equid alphaherpesviruses and only shares an overall identity of approximately 60% with EHV-1, EHV-4 and EHV-9 (Fig. 2). EHV-2 and EHV-5 show approximately 62% identity on the DNA level. The EHV-1 and EHV-4 genomes have been cloned as bacterial artificial chromosomes (BAC). These infectious clones provide a basis for rapid and efficient mutagenesis in prokaryotic cells. A variety of recombinant viruses that lack non-essential genes or portions of an essential gene or with swapped genes have been generated and used in experiments to elucidate the functions of these genes in virus replication and/or pathogenesis. The complete EHV-1 (Ab4 strain) genome is 150,223 base pairs in size, while that of EHV-4 is 145,597. The EHV-1 genome has at least 78 unique corresponding genes [63 ORFs in the UL region, 6 ORFs in each inverted repeat (IR and TR), and 9 ORFs in the US segment]; however, the actual number of open reading frames (ORFs) in the genome is 84 due to duplication of the six genes

280

Equine Herpesviruses (Herpesviridae)

Fig. 1 Schematic presentation of the genomic organization of equine herpesvirus genomes.

Fig. 2 Phylogeny of equid herpesviruses. Genetic relationships of four equid Alphaherpesviruses and two equid Gammaherpesviruses are shown.

in the inverted repeat, and, based on recent discoveries in other herpesviruses, this number likely is a large underestimation. The EHV-4 genome harbors at least 76 unique genes and 79 ORFs due to duplication of three genes of the inverted repeats. The 63 open reading frames of the UL region are arranged colinearly between EHV-1 and EHV-4 and with those in the genomes of herpes simplex virus and varicella zoster virus. Several genes mapping within IR and US region differ in arrangement from those of other alphaherpesviruses. In addition, EHV-1 and EHV-4 contain a limited number of unique genes that are present neither in HSV-1 nor in VZV which might represent the viruses’ gene repertoire determining host specificity. A comparative genome sequencing study of three EHV-1 strains (Ab4: neuropathogenic strain; RacL11: abortogenic strain; KyA: non-pathogenic strain after serial passages in mouse fibroblast L-M cells) revealed that KyA genome is the shortest with 141,350 bp when compared to the RacL11 genome, 147,469 bp, and the Ab4 genome, 150,223 bp. Sequence analysis showed that ORF1 and ORF2 genes are deleted in both KyA and RacL11 strains, while other deletions of ORFs 17, 73, 74, and 75 were only detected in the KyA strain. Identical in-frame deletions in ORFs 14, 63, and 68 were observed in both RacL11 and KyA, while extra in-frame deletions in ORFs 61 and 71 were observed only in KyA strain. On the other hand, EHV-4 viruses appeared to be more conserved with minimal changes in their genomes, even when analyzing the genomes of EHV-4 that was suspected to be the cause of abortion in mares. Six genes and the origin (ORI) of replication have been mapped to the IR sequences of EHV-1, while the remainder of the genes locates in the unique regions of the genomes. Unique genes (ORFs 2, 3, 34, 58, 67, 75, and IR2 and IR3) of EHV-1 and some of EHV-4 with no homologs in HSV-1 have been identified and mapped to the UL, US, and repeat regions. In addition, another interesting gene (ORF71, gp2) was mapped in the US segment. This gene encodes a highly O-glycosylated protein referred to as gp2. Some size variations of gp2 were documented for EHV-1 strains KyA and wild-type strains Ab4 or RacL11. A gp2-null mutant was apathogenic in a murine model of EHV-1 infection. Replacement of the truncated gp2 gene of the apathogenic KyA virus with the gp2 gene of the pathogenic RacL11, which encodes for the full-size gp2 of 791 amino acids, resulted in a “transfer” of pathogenic properties, such that full-length gp2 is a major determinant of virulence. The EHV-9 genome is shorter, 148,371 bp, than the genome of EHV-1 (Ab4 neuropathogenic strain). The genome has in total 80 open reading frames that also present in EHV-1; however, no information about the presence of IR2 or IR3 is available. The

Equine Herpesviruses (Herpesviridae)

Table 2

281

Regulatory genes of EHV-1

Gene

Temporal class

Gene product

Function in replication

IE IR4 UL5 EICP0 IR2 IR3 ETIF

Immediate early Early Early Early Early Early Late

1,487aa 293aa 470aa

Trans-activates early genes and activates some late genes Enhances IE protein DNA binding and binds TBP Binds the IE protein, TFIIB, and TBP Promiscuous trans-activator, which Antagonizes IE protein function; binds IE protein, TFIIB, TBP Dominant negative regulator, which blocks IE protein binding to promoters Antisense to IE mRNA; precursor to microRNA? Trans-activates the IE promoter; essential for virus egress

1,165aa 0.9kb RNA 479aa

EHV-9 genome has a high degree of similarity with EHV-1, with ORFs 42, 52, and 53 showed the highest degrees of identity (95%) and ORF71 the lowest (86%). One frameshift in ORF68 was detected when compared to EHV-1. The genomic sequences of EHV-2, EHV-3, and EHV-5 have now been determined, but intensive research on gene functions has not yet been performed.

Life Cycle The initial attachment of EHV-1 and EHV-4 virions to cells occurs through the interaction of gC with cellular heparan sulfate, while gD is essential for virus entry after binding to specific cellular receptors. EHV-1 gD was shown to utilize unique and wide range of entry receptors that enables the virus to enter and infect cell types of different origins. Equine major histocompatibility (MHC) class I molecules were determined as the gD-binding receptors for both EHV-1 and EHV-4. Yet, these two viruses can still infect other non-equine cells independently of equine MHC-I. The interaction between EHV-1 gH and cellular a4b1-integrins have been shown to induce release of Ca2 þ from intracellular stores and facilitate fusion of the viral envelope with the plasma membrane. Transcription and translation of EHV-1 genes have been studied best for the KyA strain. The genes of the KyA virus are regulated at the transcriptional and translational levels in a temporal fashion, and three kinetic classes of genes designated immediate-early (IE), early (E) and late (L) have been described. The sole IE gene (IR1) maps in both IR segments and gives rise to a spliced 6.0 kbp mRNA. Multiple IE polypeptide species have been observed, and the major IE protein (IE1, 203 kD) is a nuclear-localized phosphoprotein that is capable of trans-activating other viral genes and auto-regulating its own transcription. Previous studies revealed that the IE protein harbors domains for binding general transcription factors, such as TFIIB and TATA-binding protein (TBP), and thus serves to promote the formation of pre-initiation complexes that mediate viral transcription. Following IE polypeptide synthesis, approximately 45 early transcripts can be detected. Four of these early proteins serve as regulatory proteins and are designated IR4 (EICP22), UL5 (EICP27), EICP0 (UL63) and IR2 (Table 2). The IR4 protein physically interacts with the IE protein and serves to enhance the DNA-binding of the IE protein to its target sequence (ATCGT) present within the promoters of EHV-1 genes characterized to date. IR4p also binds to TBP and is present at early viral promoters in association with the IE protein and TBP. These interactions explain the synergistic effect on the trans-activation of viral genes mediated by the IE and IR4 proteins. The EHV-1 homolog of ICP27 of HSV-1 is essential for virus replication in cell culture. It acts synergistically with either the IE protein or the EICP0 early regulatory protein to activate expression of both early and late viral gene expression. The third early regulatory protein is EICP0p, which is a powerful and promiscuous trans-activator that can independently activate expression of viral genes of all three temporal classes. Deletion of the EICP0 gene greatly impairs virus replication and severely retards late gene expression, suggesting that this regulatory protein is important in the switch from the early to late phase of viral gene programming. The fourth early regulatory protein is the IR2 protein that is a truncated form of the IE protein that lacks its essential trans-activation and serine-rich domains. The IR2 protein serves a negative regulatory role as it down-regulates viral gene expression by acting as a dominant negative protein that blocks IE protein binding to viral promoters and/or by squelching the limited supply of TFIIB and TBP. In addition to these four early auxiliary regulatory proteins, the EHV-1 unique IR3 gene contributes a regulatory role as it encodes a small transcript that is antisense to a portion of the IE transcript. In transient transfection assays, the IR3 transcript down-regulates IE gene expression and is only minimally expressed to a protein. Initial studies suggest that the IR3 transcript is processed to a microRNA, and thus the IR3 gene may use novel mechanisms to downregulate IE gene expression at late times of infection. Early gene expression is followed by viral DNA replication and the production of approximately 29 late transcripts. Although these 75 transcripts have been positioned on the viral genome, only a small number of protein products have been identified and characterized (see above). Viral DNA replication initiates at approximately 4 h postinfection and requires the virus-encoded DNA polymerase. DNA replication is thought to occur by the rolling circle mechanism whereby long concatemers of the viral genome are generated, cleaved and then packaged into the maturing virions. The UL15 homolog of HSV-1, one of the two spliced genes of EHV-1 known to date, appears to be essentially involved in the generation of unit-length genomes and their packaging into mature capsids. The start of viral DNA synthesis initiates the late phase of viral gene expression and the synthesis of the late regulatory protein ETIF, a counterpart to the alpha trans inducing factor of HSV-1. This 60 kDa protein is multifunctional and plays at least three roles in EHV-1 replication. ETIF in several molecular sizes is present in the tegument and thus contributes to the composition of this

282

Equine Herpesviruses (Herpesviridae)

virion component and overall virion structure. Secondly, after virus entry and uncoating, ETIF serves to trans-activate the IE promoter by binding to cellular factors that mediate its association with the TAATGARATT sequence at nt -630 to -620 in the IE promoter. This trans-activation function to initiate viral gene programming is important but is not essential as EHV-1 DNA is infectious and virus progeny are produced in cells transfected with plasmids carrying the EHV-1 genome. The activation of a viral promoter by ETIF is specific for the IE promoter as ETIF is not able to trans-activate any early or late promoter tested to date. ETIFdeleted virus (DETIF) and an ETIF complementing cell line revealed that ETIF plays a key role in secondary envelopment. As reported for other herpesviruses, EHV-1 maturation occurs by interaction of mature nucleocapsids with the inner portion of the nuclear membrane resulting in the formation of enveloped particles. Studies with DETIF and DUS3 EHV-1 support the model of the sequential envelopment/de-envelopment/re-envelopment for the egress of EHV-1. Nuclear egressed-nucleocapsids are transported to the TGN, where additional tegument proteins are recruited and the envelope with glycoproteins is acquired. The mature virions are released extracellularly through exocytosis.

Latency Like other members of the Alphaherpesvirinae, equid herpesviruses establish latency in different cell types in the horse after primary infection. Under stress or following immunosuppression, periodic reactivation from latency usually happens and leads to viral shedding and horizontal transmission. EHV-1 has been shown to establish latent state in leukocytes, lymphoid tissues, and neuronal cells of the trigeminal ganglion. EHV-4 can establish latency in trigeminal ganglia and lymphoid tissues. EHV-2 is thought to establish latency in B-lymphocytes, macrophages, lymphoid tissues, peripheral and central nervous systems, and possibly Langerhans cells. Potential sites of EHV-3 and EHV-5 latency are not yet identified; however, mononuclear cells are surmised to be the major site of EHV-5 latency. The location of EHV-3 latency is presumed to be the sciatic or sacral ganglia. Interestingly, latent state of EHV-1 and EHV-9 in the sensory ganglia of zebras has been identified.

Epidemiology In general, herpesviruses are species-specific and it has long been thought that these viruses have co-evolved with their hosts over millions of years; that is how equine herpesviruses acquired their nomenclature. Advances in sequencing technology and screening a wide range of species might now take the lead to change this general concept (i.e., herpesviruses are strictly species-specific) as there is clear evidence of frequent virus transfer beyond the definitive host. The horse is the natural host of EHV-1, -2, -3, -4, and -5, while the definitive host of EHV-9 is still unknown. Wild equids (zebras and onagers) and domestic donkeys can also serve as a host of EHV-1. Further, EHV-1 has a broad host range and the virus can infect several non-equid species including captive gazelles, antelopes, cattle, alpacas, llamas, black bears, polar bears, black and Indian rhinoceros, and Indian tapir. EHV-9 infections have been also documented in non-equid species, such as gazelles, giraffes and polar bears. A recombinant virus of EHV-1 and EHV-9 was detected in lung and brain tissues of an Indian rhinoceros and polar bears. Serological studies indicated the presence of EHV-1 antibodies in African white and black rhinoceroses, while EHV-9 antibodies were widespread high-titered in zebras and even more so in white and black rhinoceroses. It is generally thought, however, that both EHV1 and EHV-9 will not replicate to high levels in most non-definitive hosts and that no interindividual spread occurs. Epidemiological studies concerned with determining the host range of other equid herpesviruses are limited. The currently available reports show that the host range of EHV-2, EHV-3, EHV-4, and EHV-5 is more restricted than that of EHV-1. Domestic donkeys have been shown to be infected with EHV-2, EHV-4, and EHV-5. In addition to equine influenza virus, EHV-1 and 4 are considered to be the most relevant clinical equine viral respiratory pathogens. However, of the equine herpesviruses, subclinical detection of virus in respiratory secretion or blood samples is most common for EHV-5 and EHV-2 followed by EHV-4 and then EHV-1. For all of the equine herpesviruses the main epidemiologically relevant features include: (1) High incidence of respiratory infection early in life; (2) Establishment of latency in a high percentage of infected horses; (3) Frequent reactivation of latent virus with subsequent shedding, resulting in transmission to naïve hosts. Currently, it is estimated that as many as 80% of all horses are latently infected with EHV-1 and seroprevalence for EHV-4 is typically even higher. Prevalence may vary depending on geographic region, age and population. Furthermore, testing technology and tissues sampled for detection of latency affect sensitivity of testing and actual numbers of latently infected horses may be even higher. Rhinopneumonitis caused by EHV-1 and EHV-4 is spread by direct or indirect contact (ingestion and inhalation). The viruses are shed in nasal droplets for up to three weeks and are present in large amounts in aborted fetuses and the placenta. EHV-1 infection can also result in spontaneous abortions in pregnant mares between the eighth and eleventh months of pregnancy. The peak incidence is in the ninth and tenth months at which time approximately 70% of abortions occur. Presence of EHV-1 and histological lesions following infection with EHV-1 have also been reported in testis and semen of infected stallions but a venereal transmission has not been reported. Duration of shedding following recrudescence varies, but because prolonged shedding has been detected particularly in cases of equine herpesvirus myeloencephalopathy (EHM) outbreaks, current recommendations are to wait 28 days before lifting quarantine measures. EHV-2 and EHV-5 are endemic worldwide with high seroprevalence, and co-infections are common. The viruses have been isolated from the respiratory tract, conjunctiva, blood leukocytes, kidneys, spleen, testicles, genital tract and rectum, however

Equine Herpesviruses (Herpesviridae)

283

isolation is primarily reported from the respiratory tract, the conjunctiva and blood cells, which are presumed sites of primary infection and latency. Once infected, the horse is a life-long carrier and excreter of the virus. The exact mode of spread for EHV-2 and EHV-5 is unknown but horizontal transmission is the likely mode of transmission under field conditions. EHV-3 causes a mild coital exanthema that is spread by genital contact and – rarely – the respiratory route or via contact with contaminated objects. Information on seroprevalence is similarly limited and reported rates range from 6% to 48%. EHV-3 infection is usually cleared after 14 days and is not associated with equine abortions.

Pathogenesis and Immune Response Although EHV-1 and EHV-4 respiratory infections are clinically indistinguishable, their pathogenesis is quite different. Primary infection with EHV-1 and EHV-4 occurs via the respiratory tract by inhalation of infectious virus, nose-to-nose contact, or contact with contaminated tissues or fomites. Following infection and replication in the respiratory airway epithelium, EHV-1 can be detected by 24–48 h in the local lymph nodes of the respiratory tract and a cell-associated viremia is established between days 4 and 10 post infection. In contrast, a cell-associated viremia is not commonly observed with EHV-4, which stays restricted to the respiratory tract. For EHV-1, the cell-associated viremia transports the virus to sites of secondary infection where contact between infected leukocytes and the vascular endothelium leads to endothelial cell infection, inflammation, thrombosis and tissue necrosis and secondary disease manifestations directly following viremia. Secondary disease manifestations for EHV-1 include equine herpesvirus myeloencephathy (EHM), EHV-1 abortions, neonatal foal death and chorioretinopathies. Because a cell associated viremia is the pre-requisite for transport of the virus to sites of secondary infection, including EHM and late term abortions, EHV-4 is only occasionally associated with abortion and extremely rarely with neurological disorders. While a positive correlation between the duration and magnitude of viremia and incidence of EHM has been identified, and EHM is unlikely to occur in the absence of viremia, only a small percentage (B10%) of viremic horses subsequently develop EHM. A combination of host and viral factors have at this point be identified to be associated with incidence of EHM including age, breed, gender, season as well as a single nucleotide polymorphism in the viral polymerase gene that results in a coding change (D752 vs. N752) and is strongly associated with neuropathogenicity (D752) and high levels of viremia. However, strains containing each polymerase variant (D752 or N752) have the potential to cause EHM, and strict control measures should be taken regardless of the variant identified. Infection with EHV-3 occurs through direct skin-to-skin contact during coitus or via secretions containing live virus (i.e., contaminated materials during artificial insemination, hands or even lips and nose of horses nuzzling or sniffing each other). No disruption of the skin barrier is necessary to establish infection and viral replication is typically limited to the stratified epithelium, leading to a localized inflammatory response associated with the classical cutaneous lesions. Spread of the virus to the underlying tissues and dissemination via the blood stream does not occur preventing systemic dissemination. Primary infection with EHV-3 often leads to secondary infection with Streptococcus equi zooepidemicus affecting the severity and duration of clinical disease. Most horses clear the infection on their own within 2–3 weeks of infection but a high percentage of horses establish latency, and recrudescence of the virus and clinical signs can be observed in horses in consecutive breeding seasons. The exact pathogenesis of EHV-2 and EHV-5 is not fully understood. Infection with both viruses is likely acquired horizontally early in life usually by inhalation. The frequent isolation of EHV-2 from pharyngeal lymphoid tissue suggest that this might be a primary replication site from which the virus is then spread by peripheral blood leukocytes (PBL) to other organs. EHV-5 has recently been associated with EMPF. The pathogenesis is thought to mirror that of a murine model of lung fibrosis caused by MHV-68, marked by the progressive deposition of interstitial collagen, increased TGF-b and Th2 cytokine expression, and hyperplasia of type-2 pneumocytes. The six equine herpesviruses are antigenically distincts although they share certain antigens. EHV-1, EHV-4, and EHV-9 are closely related antigenically, such that cross-neutralizing antibodies are generated. Also, EHV-2 and EHV-5 are closely related. Almost no data are available on the relationship of EHV-3 to other members of the equine herpesviruses, however, all of the EHVs are believed to share complement-fixing antigens. Neutralizing antibodies are detected in the serum soon after an EHV-1 or EHV-4 infection. However, it has become clear that: (1) virus-neutralizing antibodies can reduce viral nasal shedding but do not correlate with protection from EHM or abortions, (2) protection from EHV-1 depends critically on induction of cytotoxic T-cell (CTL) responses and prevention of viremia, and (3) EHV-1 uses multiple mechanisms to evade the induction of protective immunity, providing a unique challenge for the development of preventatives. More recently there has been an increased interest in innate immunity to EHV-1 because early immunity is not only critically important for immediate protection but likely shapes subsequent adaptive immune responses. On the flip side, over the past years multiple immune evasion mechanisms employed by the virus have been identified. These include NK-cell lysis, alteration of cytokines regulating B- and T-cell responses, alteration of the chemokine network, interference with antigen presentation, inhibition of antibody dependent cytotoxicity, induction of T-regulatory cells and alteration of CTL responses. A number of EHV-1 candidate immunomodulatory genes have so far been identified. These include the UL49.5 and UL56/UL43 genes that interfere with MHC-I antigen presentation and induction of CTL responses, as well as glycoprotein G, which functions as a viral chemokine-binding protein that selectively inhibits IL-8 and CCL-3 mediated chemotaxis of phagocytes (neutrophils and macrophages). Other viral modulatory genes are likely present and may be identified in the future. Immunity to EHV-2, EHV-3, EHV-5, and EHV-9 is poorly understood; however, virtually all horses have antibodies to EHV-2 and EHV-5 confirming the general apathogenicity of these viruses.

284

Equine Herpesviruses (Herpesviridae)

Clinical Features and Pathology Clinical manifestations of EHV-1 and EHV-4 include primary respiratory disease, late term abortions, neonatal foal death, EHM and chorioretinopathy. EHV-4 infections are typically restricted to the respiratory tract but secondary disease has been reported occasionally. EHV-1 and EHV-4 respiratory disease can be mild or asymptomatic in older or previously exposed horses. In contrast, the respiratory disease observed in young immunologically naïve horses is often severe and lasts for 2–3 weeks. Infection is characterized by a short incubation time (o1 day), fever (39–411C), which can last between 2 and 5 days, sometimes with a second spike approximately one week after the primary pyrexia and coinciding with the onset of viremia. Animals also suffer from serous nasal discharge and congestion of the nasal mucosa and conjunctiva. Less frequently, one can detect a transitory period of anorexia, enlargement of the submandibular lymph nodes and edematous swelling of the lower parts of the body and the extremities. An initial leukopenia is followed by leukocytosis before the temperature falls. Coughing is reported in cases with severe respiratory disease and occasionally in milder cases depending on the environment. Lower respiratory disease associated with secondary bacterial infection, tachypnea, anorexia and depression can be observed in young foals. Clinically EHV-1 and EHV4 respiratory disease is difficult to distinguish, unless the virus is identified diagnostically. EHV-1 is also the cause of late-term abortions and premature delivery of foals that die soon after birth. Mares infected with EHV-1 commonly do not display respiratory disease and abort 2 weeks to several months after infection or re-activation of the virus. Abortion typically occurs in the last trimester of pregnancy but has been reported as early as the second trimester. Abortions happen without warning signs and the placenta is often found together with the fetus, which has died from asphyxia or dies shortly after birth. Sporadic abortions in individual mares are most common, but EHV-1 outbreaks with attack rates >50% “abortion storms” have been reported and depend on heard management, immune status and viral factors. Mares typically recover and deliver a healthy foal in the next breeding season. Occasionally apparently healthy foals are delivered that become ill within 2 days of delivery and show respiratory distress, fever, failure to nurse, weakness, diarrhea and leukopenia and do not respond well to treatment. Clinical signs of neurological disease caused by EHV-1 are highly variable and include head pressing, ataxia, and paralysis with complete recumbency. Both, EHV-1 and EHV-4 have been isolated from the central nervous system of infected horses, but neurological disease due to EHV-4 is extremely rare. EHV-1 does not invade the extravascular nervous tissue and is not neurotropic per se. Rather, the virus spreads from the respiratory tract to the CNS via infected leukocytes and infects endothelial cells of the blood vessels supplying the spinal cord. While the neurological form of the disease is usually lethal, some horses recover fully from neurological disease with no permanent neurological sequelae. EHV-1 infection can also lead to chorioretinopathy causing permanent “shotgun” lesions in a substantial proportion of infected horses. A recent study has shown that >50% of yearling horses exhibit classical shotgun lesions following experimental infection with EHV-1, but that lesions are not visible in vivo until 1–3 months after primary infection. Lesions can be focal, multifocal or in rare occasions diffuse affecting the whole eye. Clinically, only diffuse lesions have a significant impact and cause loss of vision. EHV-3 replication is restricted to the stratified epithelium of the skin or muco-cutaneous margins and lesions are restricted to the superficial skin of the external genitalia in both mares and stallions. An incubation period as long as 10 days can be observed following natural infection. The initial lesions are small (1–2 mm), raised, and reddened papules. The lesions then progress rapidly to the pustular form, and there is a general reddening of the vaginal mucosa in the mare. The number of lesions increases in the first few days, and by day 6, many of the lesions form ulcers up to 20 mm in diameter and 5 mm deep. Lesions can also be seen on the vulva, perineal skin, as well as the penis and prepuce of the stallion. The disease is usually mild such that the temperature, pulse, appetite and respiration remain close to normal. Occasionally, severely affected horses may be febrile and show depression and anorexia. Stallions may show a loss of libido and refusal to mount and mares may show frequent urination, arching of the back and vulvar discharge. The severity of the disease can be increased by secondary bacterial infections; however, uneventful cases are usually cleared within 2 weeks. EHV-3 is not abortigenic and does not lead to infertility. The role of EHV-2 and EHV-5 in clinical disease is less understood. EHV-5 has been implicated as the cause of EMPF in recent years. EMPF is typified by low-grade fever, weight loss, and progressive exercise intolerance, along with radiographic evidence of nodular pulmonary interstitial fibrosis and isolation of the virus from affected lungs. EHV-2 has been associated occasionally with chronic throat infections. However, pharyngitis as well as conjunctivitis, coughing and nasal discharge has so far only been experimentally induced in young immuno-suppressed animals. Infections with EHV-9 lead to encephalitis in horses under experimental conditions, but, more severely, in other species as well. Respiratory disease caused by EHV-1 and EHV-4 results in inflammation, congestion and sometimes necrosis of the upper respiratory tract. Extensive swelling of the nasal mucosa may occur, and in later stages, the lungs may become involved. One can find typical herpesvirus inclusion bodies in the nuclei of the respiratory epithelium. The respiratory infection can become more serious if followed by a secondary bacterial infection, which may lead to bacterial pneumonia. Fetuses that are aborted as a consequence of EHV-1 infection present with widespread hemorrhages and edema, as well as a yellowish discoloration of the fetal conjunctiva and splenomegaly – if virus is transmitted from mother to animal. Abortions without infection of the fetus also occur and the pathogenesis in the uterine vasculature largely resembles that of the vasculitis observed in the neurological form of the disease. The classic histopathology includes the presence of eosinophilic intranuclear inclusion bodies in various organs of the aborted fetuses. However, gross pathological and histopathological alterations are sometimes not as obvious and only sensitive methods (virus isolation, PCR, qPCR) are able to confirm the EHV-1 abortion. Classically, features associated with EHV-1 induced abortions differ in those fetuses aborted during the first 6 months of gestation

Equine Herpesviruses (Herpesviridae)

285

as compared with those aborted after 6 months. Those before 6 months present with widespread cell necrosis and inclusion bodies in the liver and lung. Those after 6 months exhibit jaundice, subcutaneous edema, excessive pleural fluid, pulmonary edema, splenomegaly and necrosis of the liver. Similar to histopathological findings in the CNS and the pregnant uterus, vasculitis is common in the choroid of the eye, as are mild or moderate perivascular and interstitial infiltrates of lymphocytes and histiocytes around the chorioretinal vasculature. Finally, vasculitis with perivascular to scattered interstitial lymphohistiocytic infiltrates can also be observed in the testis of intact males following experimental infection. In addition, occasionally, discrete lymphofollicluar structures within the interstitium of the testes, tubular degeneration and arteritis in the fascia surrounding the testes or fibrous interstitium maybe observed. Tissues affected following an infection with EHV-3 include the vaginal and vestibular mucosa, penis, prepuce and the skin of the perineal region. One of the characteristics of an infection with EHV-3 is the sloughing of the surface epithelial cells. On occasion, the skin of the lips and mucus membranes of the respiratory tract may become involved, but the exanthema is usually mild. EHV-2 and also EHV-5 infection becomes widespread throughout the body, and the viruses have been isolated from a variety of tissues. Horses affected by EMPF show coalescing or discrete nodules of fibrosis and an enlargement of bronchial lymph nodes. Histologically the lesions are centered on the alveolar parenchyma and are characterized by an expansion of collagen accompanied by infiltration of inflammatory cells into the interstitium.

Control Standard diagnostic methods for equid herpesviruses are well established, including virus isolation, serological assays, particularly serum neutralization tests (SNT) and type-specific ELISAs, as well as molecular assays that are now almost exclusively based on quantitative PCR protocols. A number of vaccines are available to combat EHV-1 infections, among them modified live vaccines (e.g., RhinomuneTM or PrevaccinolTM), inactivated vaccines (e.g., Pneumabort KTM, EquipTM EHV1,4, PrestigeTM, ProdigyTM), inactivated combination vaccines, which – among others – also contain EHV-4 (e.g., InnovatorTM, VeteraTM). Unfortunately, many EHV vaccines do not afford acceptable levels of protection, especially when inactivated combination vaccines are considered. All vaccines are given repeatedly to pregnant mares usually in the third, fifth, seventh and ninth months of pregnancy. To protect against viral rhinopneumonitis outbreaks, the vaccine is usually given to all horses every 3–6 months. There is considerable ongoing discussion as to proper vaccination against the neurological disease, which has become more prevalent during past years. Recent studies seem to suggest that modified live virus vaccines may be superior to especially inactivated combination vaccines with respect to duration of fever and virus excretion from the nasal mucosa; at this point there is however little evidence that vaccination can prevent EHM, and conflicting information is available on the use of vaccination for prevention of abortion. Clinical management often involves the use of antibiotics to prevent severe bacterial complications following the viral rhinopneumonitis. Because of the vaccine limitations, control of EHV-1 and EHV-4 infections involves isolation and quarantine of infected horses (up to 28 days) and sound hygiene for prevention of viral infection, since the viruses are highly contagious. Quarantine procedures are required with EHV-1 infections, since EHV-1 can lead to more serious disease of the CNS and the viruses can be spread easily in contaminated feed and water. In addition, minimizing stress and close contact of large groups of horses can prevent the spread of disease. It is important to recognize that EHV-1 infection is a reportable disease in some states of the U.S., and in these states, quarantine requirements will be determined by state officials. No vaccines are available for preventing EHV-3 infection at present and unless secondary bacterial infection develops, genital lesions caused by EHV-3 infection usually heal without therapeutic intervention.

Future Perspectives Considerable progress in unraveling the genetic makeup of EHV-1, EHV-2, EHV-3, EHV-4, EHV-5, and EHV-9 has been made during the last years, and the genomes have been sequenced in their entirety. With this information in hand, it will be possible to perform studies on gene expression and on proteins that are involved in virulence of each of the viruses. These studies will in turn open the possibility for a rational design of anti-EHV vaccines, especially against the most important pathogens, EHV-1 and EHV4. These goals may be achieved by the use of viral deletion mutants that carry targeted gene deletions, which is greatly facilitated by the advent of a number of infectious DNA clones (bacterial artificial chromosome – BAC – clones) during the past years. Using novel molecular approaches, a better understanding of the viruses’ biology will likely be possible in the near future and allow a finer definition of diseases caused by the different EHV.

Further Reading Abdelgawad, A., Damiani, A., Ho, S.Y., et al., 2016. Zebra alphaherpesviruses (EHV-1 and EHV-9): Genetic diversity, latency and co-infections. Viruses 8 (9), doi:10.3390/ v8090262. Azab, W., Kato, K., Arii, J., et al., 2009. Cloning of the genome of equine herpesvirus 4 strain TH20p as an infectious bacterial artificial chromosome. Archives of Virology 154 (5), 833–842.

286

Equine Herpesviruses (Herpesviridae)

Azab, W., Osterrieder, K., 2017. Initial contact: The first steps in herpesvirus entry. Advances in Anatomy, Embryology and Cell Biology 223, 1–27. doi:10.1007/978-3-31953168-7_1. Barrandeguy, M., Thiry, E., 2012. Equine coital exanthema and its potential economic implications for the equine industry. The Veterinary Journal 191 (1), 35–40. doi:10.1016/ j.tvjl.2011.01.016. Fortier, G., van Erck, E., Pronost, S., Lekeux, P., Thiry, E., 2010. Equine gammaherpesviruses Pathogenesis, epidemiology and diagnosis. The Veterinary Journal 186 (2), 148–156. doi:10.1016/j.tvjl.2009.08.017. Hussey, G.S., Goehring, L.S., Lunn, D.P., et al., 2013. Experimental infection with equine herpesvirus type 1 (EHV-1) induces chorioretinal lesions. Veterinary Research 44, 118doi:10.1186/1297-9716-44-118. Ma, G., Azab, W., Osterrieder, N., 2013. Equine herpesviruses type 1 (EHV-1) and 4 (EHV-4) – masters of co-evolution and a constant threat to equids and beyond. Veterinary Microbiology 167 (1–2), 123–134. doi:10.1016/j.vetmic.2013.06.018. Paillot, R., Ellis, S.A., Daly, J.M., et al., 2006. Characterisation of CTL and IFN-gamma synthesis in ponies following vaccination with a NYVAC-based construct coding for EHV-1 immediate early gene, followed by challenge infection. Vaccine 24 (10), 1490–1500. doi:10.1016/j.vaccine.2005.10.019. Rudolph, J., O’Callaghan, D.J., Osterrieder, N., 2002. Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC). Journal of Veterinary Medicine B, Infectious Diseases and Veterinary Public Health 49 (1), 31–36. Shakya, A.K., O’Callaghan, D.J., Kim, S.K., 2017. Comparative genomic sequencing and pathogenic properties of equine herpesvirus 1 KyA and RacL11. Frontiers in Veterinary Science 4, 211doi:10.3389/fvets.2017.00211. Soboll Hussey, G., Ashton, L.V., Quintana, A.M., et al., 2014. Equine herpesvirus type 1 pUL56 modulates innate responses of airway epithelial cells. Virology 464–465, 76–86. doi:10.1016/j.virol.2014.05.023. Soboll, G., Whalley, J.M., Koen, M.T., et al., 2003. Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes. Journal of General Virology 84, 2625–2634. doi:10.1099/vir.0.19268-0. Van de Walle, G.R., Sakamoto, K., Osterrieder, N., 2008. CCL3 and viral chemokine-binding protein gg modulate pulmonary inflammation and virus replication during equine herpesvirus 1 infection. Journal of Virology 82 (4), 1714–1722. Wilkie, G.S., Kerr, K., Stewart, J.P., Studdert, M.J., Davison, A.J., 2015. Genome sequences of equid herpesviruses 2 and 5. Genome Announcements 3 (2), doi:10.1128/ genomeA.00119-15. Williams, K.J., Maes, R., Del Piero, F., et al., 2007. Equine multinodular pulmonary fibrosis: A newly recognized herpesvirus-associated fibrotic lung disease. Veterinary Pathology 44 (6), 849–862. doi:10.1354/vp.44-6-849.

Equine, Canine, and Swine Influenza (Orthomyxoviridae) Janet M Daly, University of Nottingham, Sutton Bonington, United Kingdom Japhette E Kembou-Ringert, University of Tel Aviv, Tel Aviv, Israel r 2021 Elsevier Ltd. All rights reserved.

Nomenclature

M1/M2 Matrix protein 1/2 mRNA Messenger ribonucleic acid NP Nucleoprotein PA Polymerase acidic protein PB1 Polymerase basic protein 1 PB2 Polymerase basic protein 2 vRNA Viral ribonucleic acid

(H)/HA Hemagglutinin (subtype) (N)/NA Neuraminidase (subtype) HA0 Hemagglutinin polyprotein HA1, HA2 Hemagglutinin subunits HI Hemagglutination inhibition test IAV Influenza A virus

Glossary Antigenic drift The gradual accumulation of amino acid changes in the surface glycoproteins of influenza viruses. Antigenic shift The sudden appearance of a novel combination of surface glycoproteins in a host as a result of reassortment. Pandemic Outbreak of disease affecting a large region.

Reassortment The exchange of genome segments between distinct influenza A viruses (typically viruses of different subtypes). Subtype Classification of influenza A viruses based on the antigenicity of the hemagglutinin and neuraminidase proteins.

Classification (Compact) Most equine, canine and swine influenza viral infections are caused by influenza A viruses (IAV) in the Influenzavirus A genus of the family Orthomyxoviridae. Classification of viruses in this family is based on, but not limited to, the negative-sense and segmented nature of their RNA genome. Influenza A viruses (IAV) are further classified based on the antigenic reactivity of their surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Until recently, 16 HA (H1 to H16) and 9 NA (NA1 to NA9) subtypes of IAV viruses had been identified in different host species, with aquatic birds and primarily ducks being regarded as the reservoir hosts. Even though up to 144 possible subtype combinations of H1 to H16 and N1 to N9 are possible, only a few combinations have consistently been isolated from pigs, horses and dogs. Two additional subtypes of HA (H17 and H18) and NA (N10 and N11) have recently been identified in bats. Consistent isolation of H1N1, H1N3 and H3N2 IAV from pigs suggests that these subtypes are associated with endemic infections. In addition, H2, H4, H5, H7, and H9 subtypes have also been reported to cause sporadic infections. There are only two subtypes of IAV known to be endemic in horses, H3N8 and H7N7, although the latter is now believed to be extinct. An equineorigin H3N8 virus and avian-origin H3N2 virus are currently circulating in dogs. Although most cases of influenza virus infection reported in pigs, horses and dogs involve IAV, it appears that pigs at least can also be infected by other types of influenza viruses. Influenza C viruses (ICV), which are primarily human pathogens, have occasionally been isolated from pigs and the more recently discovered influenza D virus (IDV) was first isolated from pigs, although cattle are thought to be the major host. As a general rule, the nomenclature of influenza viruses includes: the antigenic type of the virus (A, B, C or D); the host of origin for non-human isolates (e.g., swine, equine, canine); the geographical location of origin of the isolate; the viral strain or isolate number and the year of isolation; and the HA and NA subtype in brackets. For example, an IAV isolate obtained from pigs in Hong Kong in 2010 with the laboratory reference number 35 will be named: A/swine/Hong Kong/35/10 (H5N1).

Virion Structure Mammalian IAV share the same morphological features. Virions are enveloped (with the viral envelope deriving from the host cell) and often pleomorphic. The diameter of spherical particles is around 80 and 120 nm, whereas filamentous forms of the virus often found in clinical isolates can reach up to 20 mm in length. The HA and the NA glycoproteins are inserted in the viral envelope in a ratio of approximately four to one (Fig. 1). The HA is a trimeric glycoprotein that binds to the cellular receptor during the entry stage of the virus. The NA protein, on the other hand, is a globular tetrameric glycoprotein. Additionally, another smaller integral matrix protein 2 (or M2 ion channel) is embedded in the envelope in a ratio of one M2 channel per 10–100 HA molecules. The lipid membrane is lined by another viral matrix protein (M1), which gives strength and rigidity to the viral envelope, which surrounds the viral genome.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20978-3

287

288

Equine, Canine, and Swine Influenza (Orthomyxoviridae)

Fig. 1 Influenza A virus structure of a virus particle and an RNA segment.

Genome The IAV genome consists of eight negative-sense, single-stranded viral RNA (vRNA) segments encoding for around 12–14 viral proteins depending on the viral strain. In addition to coding regions, each viral segment also contains non-coding regions of various length at both the 50 and 30 ends. The distal 10–12 nucleotides of these non-coding regions appear to be highly conserved among the genome segments. These conserved regions allow the formation of “panhandle” structures (helical hairpins) by partial inverted complementarity between the 50 and 30 ends; this unique feature serves as a promotor for viral mRNA transcription and plays a critical role during genome replication and packaging. Additionally, the 50 non-coding regions include the messenger RNA (mRNA) polyadenylation signal, a uridine-rich region that serves as a template on which the viral polymerase stutters to generate the poly-A tail at the 30 end of each viral mRNA. Viral ribonucleoproteins (vRNPs) consist of viral RNA segments covered with numerous copies of the nucleoprotein (NP) and a heterotrimeric RNA-dependent RNA polymerase complex composed of two “polymerase basic” (PB1 and PB2) proteins and one “polymerase acidic” (PA) protein. Within the virions, the vRNPs interact with each other and the M1 to form a “7 þ 1” configuration with seven segments of different lengths surrounding a central segment, which is thought to play an important mechanistic role during genome packaging and virus budding.

Life Cycle The virus life cycle can be broken down into six stages: (1) attachment and internalization; (2) fusion and uncoating; (3) mRNA synthesis and viral RNA replication; (4) translation; (5) assembly and packaging and (6) budding (Fig. 2). Binding of IAV to the target host cell mainly occurs via the HA protein, which recognizes and binds to sialic acid (N-acetylneuraminic acid, Neu5Ac) residues that are attached to an underlying galactose sugar chain by a2,3 or a-2,6 linkages on the surface of the host cell. After IAV particles are brought into the vicinity of the host cell surface, the HA and NA act co-operatively; HA binds cell surface sialic acid residues while NA locally degrades and removes unnecessary HA-sialic acid interactions. This creates iterative associations and dissociations of the HA with cell surface sialic acid residues, allowing the virus to ‘crawl’ across the cell surface. This probably ensures that the virus is directed to the right entry location on the cell surface, where HA-receptor mediated endocytosis will be triggered. Although it is still not clear whether binding of HA to sialic acid residues alone induces IAV internalization, induction of host cell signaling is required to direct IAV to its specific entry route and this certainly requires specific transmembrane receptors that mediate HA-receptor endocytosis. Additional host factors other than sialic acid residues have been identified as receptor determinants including C-type lectins and annexin V (A5). Virus particles are internalized via receptormediated endocytosis. Most virions are internalized via a clathrin-mediated endocytic process during which Epsin-1 is recruited to the binding sites of influenza viruses in synchrony with the assembly of clathrin-coated vesicles. Dynamin protein is then required for membrane fission, to pinch off the vesicles from the plasma membrane. Various studies have established that the virus can also infect cells via a clathrin-and caveolin-independent endocytic pathway showing all the characteristics of macropinocytosis. Recent

Equine, Canine, and Swine Influenza (Orthomyxoviridae)

289

Fig. 2 Influenza A virus life cycle. 1 ¼ Attachment and internalization; 2 ¼ fusion and uncoating; 3 ¼ mRNA synthesis and viral RNA replication; 4 ¼ translation; 5 ¼ assembly and packaging; and 6 ¼ budding. Adapted from Neumann, G., Noda, T., Kawaoka, Y., 2009. Nature 459, 931–939.

approaches based on single-virus tracking and imaging established that both clathrin-mediated and clathrin-independent pathways lead to efficient viral fusion. Following internalization of the virus, virions are initially transported from the cell periphery to early endosomes and then transferred to late endosomes where a late acidification step occurs. This low endosomal pH (E5) triggers a conformational change in the HA protein of the virus, which exposes the 23 residues near the N-terminus of HA2, also known as the fusion peptide. The HA is synthesized as a precursor protein (HA0) and must be cleaved into HA1 and HA2 by host proteases in order to be infectious. At neutral pH, the fusion peptide is hidden in a hydrophobic pocket in the HA2 trimeric interface. The exposed fusion peptide inserts into the endosomal membrane on the N-terminus of HA2 while its C-terminus remains anchored to the viral membrane. HA2 trimers then bend back on themselves in a hairpin-like conformation, bringing both membranes close to each other to merge the two outermost leaflets of both membranes (often referred to as hemifusion). Subsequently, the lipid stalk ruptures to form a fusion pore, which in turn activates the M2 ion channel. Hydrogen ions from the endosome are then pumped into the viral core via the M2 ion channel, creating an internal acidification of the virions, which disrupts protein–protein interactions that hold the viral core together and causes the release of the vRNPs from the M1 into the cytoplasm via the fusion pore. Replication of IAV takes place in the cell nucleus. After vRNPs are released into the cytoplasm, nuclear localization sequences (NLS) exposed on NP molecules recruit the cellular adapter protein importin-a. Importin-a then binds to the importin-b transport receptor and the entire complex directs the vRNPs to the nuclear pore complex, via which vRNPs are released into the nucleus. Replication and transcription are carried out by the viral RNA-dependent RNA polymerase complex made up of the PB1, PB2 and PA proteins. Because this complex is an integral part of each vRNP imported into the nucleus, each vRNP becomes an independent functional unit of replication and transcription. Given the negative sense nature of the genome, vRNA replication begins with an initial step of transcription, where vRNPs are transcribed into a complementary RNA (cRNA), which is then used as template for the synthesis of new vRNAs. As opposed to cRNA synthesis, influenza viral mRNA transcription is a unique primer-dependent transcription process during which the viral polymerase complex, recruited to the promoter-associated Pol II, binds the 50 cap of cellular mRNAs via its PB2 sub-unit. Subsequently, the PA sub-unit, containing the endonuclease activity, cleaves cellular mRNAs approximately 10 to 13 nucleotides from their 50 end. The PB2 cap-binding domain then rotates to position the stolen cellular capped primer sequences into the catalytic site of PB1, where it is then used as a primer for elongation of the mRNA, using vRNA as a template. Viral transcripts are then polyadenylated by a stuttering process of the polymerase on a short poly-U sequence located at the 50 end of the viral RNA. The coding capacity of IAV is enhanced by the presence of donor-acceptor splice sites. As a result, during mRNA transcription, these viral transcripts are capable of recruiting the cellular spliceosome machinery to produce additional spliced transcripts such as the M2 and the NEP. A third transcript of the NS gene segment (NS3) in which the donor/ acceptor splice site exhibits a novel GG/GUA motif was recently identified.

290

Equine, Canine, and Swine Influenza (Orthomyxoviridae)

The newly synthesized vRNPs are selectively exported out of the nucleus. It is thought that the viral nuclear export protein (NEP), via its leucine-rich nuclear export signals, plays a central role in targeting vRNP complexes to the chromosomal maintenance 1 (CRM1) nuclear export pathway, but nuclear export signals also exist in M1 protein and NP. Nuclear export of capped IAV mRNAs is believed to occur in an analogous manner to the nuclear export of cellular mRNAs. It is believed that following capping, the 50 caps of nascent viral transcripts bind to the cap-binding complex. The viral NS1 protein recruits proteins involved in nuclear export of cellular mRNAs but also forms inhibitory complexes with some essential export factors (such as Nxt1) to block the export of endogenous cellular mRNAs, as a way to ensure the preferential nuclear export of viral transcripts. Viral enveloped proteins (HA, NA and M2 for IAV) are synthesized on membrane-bound ribosomes into the endoplasmic reticulum where they are folded and trafficked to the Golgi apparatus for post-translational modifications, whereas PB1, PB2, PA, NP, NS1, NS2 and M1 synthesis is carried out by cytosolic ribosomes. NP, PB1, PB2 and PA proteins are synthesized during early infection. The NLS sequences on these proteins target them back into the nucleus by the recruitment of importin-a and -b to assist in viral mRNA transcription and viral replication. In addition to generating new transcripts by alternative splicing, IAV also generates so-called accessory proteins by either alternative translation initiation sites or ribosomal frameshift. Segment 2, for instance, additionally encodes for PB1-F2 and PB1-N40 (depending on the viral strain) by using alternative translation initiation sites. Likewise, segment 3 can also encode for PA-X (a fusion protein that results from a þ 1 ribosomal frameshift), while PA-N155 and PA-N182 are N-terminally truncated forms of PA. Viral RNPs are trafficked toward the apical plasma membrane in polarized epithelial cells. The M1 protein has also been identified as a key component in the assembly process of virions via protein-protein interactions. M1 is thought to interact with itself, the vRNPs, and the HA and NA surface glycoproteins to both facilitate the concentration of viral components at the budding site and allow the discrimination of viral proteins from host proteins. By interacting with the cytoplasmic tail and the transmembrane domain of envelope glycoproteins HA and NA, M1 functions as a bridge between the viral envelope and vRNPs. It is believed that budding of IAV occurs at distinct apical plasma membrane regions known as rafts. It has been suggested that IAV promotes budding by inducing membrane curvature. The expression and abundance of HA and NA on one side of the membrane are sufficient to induce budding, and the presence of M1 greatly increases budding efficiency. It is thought that M1 oligomerizes upon reaching the cellular membrane where it is modeled to form curved structures. This property could explain its role in the formation of either spherical or filamentous virion forms. To facilitate the release of virions from the plasma membrane, NA catalyzes the hydrolysis of glycosidic linkages that attach sialic acids to underlying sugar molecules. The sialidase catalytic site is located in a highly conserved deep pocket in the center of each monomer of the NA tetramer. The segmented nature of the genome means that IAV viruses can undergo reassortment, i.e., when more than one virus infects a single host cell a virus with a different combination of genome segments can arise. Antigenic shift occurs if the reassortant virus generated has a novel combination of HA and NA proteins. The lack of proofreading enzymes associated with RNA replication results in a high frequency of mutation. Together with high rates of replication, this enables IAV to evolve rapidly. The process of antigenic drift results from the accumulation of amino acid changes in the HA and NA proteins that enable the virus to escape neutralization by pre-existing antibody.

Epidemiology Three major subtypes of IAV circulate in the pig population worldwide. The H1N1, H3N2 and H1N2 subtypes are often associated with acute respiratory disease in pigs and, in some cases, with more chronic, multifactorial respiratory disease problems in combination with other viruses or bacteria. Although the same subtypes circulate in Europe and the USA, their genetic evolution is significantly different between continents. In Europe, H1N1 subtypes were introduced into the pig population from wild ducks. In contrast, two lineages of H1N1 circulate in the USA; the ‘classical’ H1N1 (cH1N1) lineage was first identified in 1930 is also found in Asia and the ‘reassortant’ H1N1 (rH1N1) viruses with HA and NA of cH1N1 and the internal proteins of a recently emerged H3N2 subtype virus. In European swine, circulating H3N2 viruses are reassortants of the classical H1N1 (avian-like) virus with the human H3N2 (avian-like HA and NA with internal genes from human H3N2 virus). In a separate reassortment event, the swine (avian-like) H1N1 viruses also gained the human N2 gene, generating a novel H1N2 reassortant, now endemic to Europe. Although different lineages of human H3N2 have also been detected in pig populations in Asia, these lineages seem to undergo less antigenic drift compared to their ancestral human lineages. In contrast to Europe, the H3N2 virus circulating in North American swine is a triple reassortant containing: HA, NA and PB1 of human origin; NP, M and NS genes originating from the classical swine H1N1; and PA and PB2 genes of north American avian virus origin. These triple reassortants are believed to have further acquired HA genes of diverse origins, leading to the appearance of the triple reassortant H1N2 virus (acquiring its HA from the classical swine H1N1). Other reassortant swine viruses have been reported in Europe (H1N7), Asia (H3N1) and the USA (H2N3) and some that only transiently affect pigs in other parts of the world (H9N2, H3N6 and H5N2). Novel reassortants of swine viruses with equine IAV have also been reported, highlighting the role swine play in the generation of new influenza viruses. The fact that most swine IAV are reassortants, usually of avian and human IAV, led to the hypothesis that swine serve as ‘mixing vessels’ for IAV through which novel viruses can be transmitted to man. The 2009 influenza pandemic caused by an H1N1 virus that originated in pigs and was a reassortant with genome segments from several viruses circulating in swine. Since the 2009 pandemic, further reassortant viruses of the 2009 H1N1 pandemic virus and other IAV strains circulating in pigs have been detected in many different countries.

Equine, Canine, and Swine Influenza (Orthomyxoviridae)

291

Two subtypes of IAV have circulated endemically in equids: H7N7 and H3N8. The prototype equine H7N7 virus was isolated from horses in Prague in 1956 when it was termed ‘equine subtype 1’. There is little evidence that H7N7 viruses are still circulating in the equine population. Virus of the H3N8 subtype was first isolated from horses in 1963. The virus is thought to have emerged in South America, spreading northwards and subsequently almost worldwide. The equine H3N8 viruses were initially a single lineage, which later evolved into two lineages (American and Eurasian). The Eurasian lineage, composed of viruses isolated from horses in Europe and Asia, continues to form a single lineage, whereas the American lineage has further diverged into three sublineages: ‘South American’, ‘Kentucky’, and ‘Florida’. Viruses in the Florida sublineage further evolved into two clades. Florida clade 1 (FC1) viruses predominate on the American continent but have also caused large outbreaks in Africa, Australia, Europe, Asia and South America. Florida clade 2 (FC2) viruses, on the other hand, predominate in Europe, but viruses from this clade have also been isolated in Asia and North America. Overall, viruses in the Florida sublineage have dominated since its emergence. Equine IAV of the H3N8 subtype is endemic in many American, European and Asian countries and occasionally causes epidemics. The most recent noteworthy epidemic of equine IAV was in Australia in 2007/2008. Australia had previously been free from equine IAV and succeeded, at great expense, in eradicating it within 6 months of its emergence. Sporadic infection of equids with other IAV subtypes occurs rarely. A highly pathogenic avian influenza of the H5N1 subtype was isolated from donkeys living in close contact with diseased birds. It was thought that transmission of IAV from horses to other species was unlikely until transmission of the equine H3N8 virus to dogs occurred. Here is some evidence that the equine H3N8 subtype may have occasionally caused human infection in people but experimental infection of human volunteers with equine IAV was largely unsuccessful. Historically, there were occasional reports of sporadic transmission and sub-clinical infection of dogs with IAV of human origin with no evidence of onward transmission. More recently, two subtypes of IAV (H3N8 and H3N2) have become established in dogs. The canine H3N8 virus is believed to have originated from horses. The virus was first isolated in 2004, but retrospective serological investigation suggested it had been circulating among greyhounds for some years prior to its identification. The canine H3N8 virus spread to other dog populations throughout the USA, mainly affecting dogs housed in large groups, e.g., dogs in animal shelters, boarding kennels and day-care centers. However, by 2018, the canine H3N8 virus seemed to have mostly died out, being limited to one large rescue center. A closely related virus was identified as the cause of an outbreak of respiratory disease among a group of foxhounds in the UK in 2002 and some dogs became infected during the large outbreak of equine H3N8 IAV in Australia in 2007/2008. However, the virus did not become established in dogs in Australia or the UK. The second canine IAV subtype originated from birds. The virus is reported to contain gene segments from several different avian subtypes. Infections with this subtype were first reported in South Korea, where dogs are farmed for meat, and spread to other Asian countries including China and Thailand. The virus was introduced into the USA in 2015, presumed to be due to importation of dogs rescued from the Far East. It was first identified in an animal shelter in Chicago and spread to other states within the USA far more rapidly than the H3N8 subtype had done. Dogs are sporadically infected with other types of IAV but these are not maintained in dog populations. An Asian lineage of a highly pathogenic avian influenza (HPAI) H5N1 virus was detected in a dog that had scavenged carcasses of chickens infected with the virus in Thailand. Antibodies were detected in other dogs, but no transmission of disease to in-contact dogs was observed. Other rare reports of avian subtypes of IAV affecting dogs include an HPAI H5N2 virus and H9N2 viruses. In a worrying development, an H3N1 virus was recently isolated from a dog with respiratory disease in Korea. This virus appeared to be a reassortant between an endemic canine H3N2 virus and the human ‘pandemic’ H1N1 subtype.

Pathogenesis Influenza A virus infection in pigs, horses and dogs is a disease of the upper respiratory tract. There is usually a short incubation period (2–3 days). The virus replicates in epithelial cells, leading to disruption of the mucociliary elevator, which is one of the factors that predisposes to secondary bacterial infection. Both host and virus factors (particularly the HA, polymerase proteins and NS1) contribute to IAV pathogenesis. As described above, proteolysis is required to cleave the inactive HA0 protein into the HA1 and HA2 subunits. In the endemic porcine, equine and canine viruses, the HA0 is cleaved by trypsin-like proteases found in the gut and respiratory tract that recognize a single arginine in the cleavage site (a monobasic cleavage site). The acquisition of a multibasic cleavage site in avian H5 and H7 subtype IAV during infection of chickens means that these viruses can be cleaved by ubiquitously expressed subtilisin-like proteases. This is one reason why HPAI viruses can cause severe and often fatal systemic infection in various species. Mutations in the polymerase complex proteins can contribute to IAV pathogenicity by enhancing transcription and replication of the viral genome. The NS1 protein is a highly multifunctional protein that is important for efficient virus replication and also modulates the host’s innate immune response. The type of sialic acid linkage in the host cell receptor of IAV has been shown to affect the HA receptor binding affinity in a host-specific manner leading to the suggestion that species-specific differences in the distribution of linkages on the respiratory epithelial cells influences the ability of IAV to transmit between species. The detection of both a2,3 and a-2,6 linkages in the pig respiratory tract was thought to be central to the role of the pig as a bridging host by enabling the co-infection of pigs with avian and human IAV leading to the generation of reassortant viruses. However, several studies have demonstrated that HA receptorbinding preference for a specific sialic acid linkage is not essential for infection but is more critical for virus transmission. Mutations in the HA can alter the receptor binding affinity and therefore adaptation of IAV to a new host. Indeed, a tryptophan (W) to leucine (L) mutation of residue 222 of the HA was associated with adaptation of both the equine H3N8 and avian H3N2

292

Equine, Canine, and Swine Influenza (Orthomyxoviridae)

viruses to dogs. However, interspecies transmission is not solely due to adaptive mutations in the HA and NA proteins. The virus must also overcome intrinsic and innate immune restrictions to replicate in a novel host. For example, interspecies transmission of avian IAV to mammalian hosts requires adaptation of the viral polymerase complex to a different importin-a isoform. Several recent studies have shown that differences in the pH stability of the HA protein reflect adaptation to different host species. A more pH-stable HA protein allows the virus to survive longer in mildly acidic environments and during infection, membrane fusion occurs in late rather than early endosomes. Avian IAV typically have a pH of activation of 45.5 whereas viruses that replicate in the upper respiratory tract of mammals generally have a pH of activation of r5.5. Swine IAV have a broad range of pH (5.3–6.0), suggesting that pH of activation of the HA is another factor contributing to the role of pigs as a bridging host for adaptation of avian viruses to human hosts. Furthermore, porcine respiratory epithelial cells have a lower interferon response to IAV infection than equivalent human cells, which may also contribute to the lower barrier to infection of pigs with IAV from other species.

Clinical Features The clinical signs of IAV infection are similar among pigs, dogs and horses. Typical signs include high fever, cough, nasal discharge, anorexia and apathy. Morbidity rates are usually high, but animals typically recover after 2–6 days. Although the mortality rate is typically low (less than 1%), the disease can be exacerbated by the development of secondary bacterial pneumonia.

Diagnosis Although a rapidly spreading febrile respiratory disease characterized by sudden onset of repeated coughing is very suggestive of IAV infection in pigs, horses and dogs, the clinical signs are not pathognomonic and are often suppressed in animals that become infected despite being vaccinated. Laboratory confirmation is therefore necessary. Detection of virus in clinical specimens (nasopharyngeal swab or nasal or tracheal wash samples) is usually achieved by RT-PCR, typically using primers for the more conserved matrix or nucleoprotein genes. This approach is more rapid and sensitive than virus isolation, but isolation of viruses remains important to fully characterize circulating viruses. Viral shedding is typically limited to day 2–4 post infection, declining rapidly during the clinical phase (Fig. 3). Therefore, to optimize the likelihood of obtaining a positive result, samples for virus detection or isolation should be obtained within 1–2 days of onset of clinical signs and/or from other animals in contact with the index case. Serological assays are important to confirm infection both as a diagnostic aid for ongoing cases and for surveillance purposes. The World Organization for Animal Health (OIE) recommends the hemagglutination inhibition (HI) test. The assay is based on binding of antibodies to a standardized dose of virus inhibiting its ability to agglutinate red blood cells. A variety of factors can affect the sensitivity and specificity of the test. For canine, equine and porcine influenza, receptor-destroying enzyme or periodate and heat treatment are used to inactivate non-specific inhibitors in serum and the test typically uses 0.5 or 1% chicken or turkey red blood cells. As there may be pre-existing antibodies (from previous infection or vaccination), paired serum samples should be taken, with a 4-fold increase of titer between an acute phase sample and a convalescent sample taken 10–21 days later usually taken to indicate recent infection (Fig. 3). Competitive ELISA for detection of antibodies to nucleoprotein are also increasingly used to diagnose influenza infection.

Treatment Although three classes of influenza antivirals (inhibitors of the M2 matrix protein, neuraminidase and PA polymerase) are licensed for human use, treatment for veterinary species is largely supportive. Treatments include fluids to help maintain hydration or

Fig. 3 Optimal sampling times for diagnosis of influenza A virus infection.

Equine, Canine, and Swine Influenza (Orthomyxoviridae)

293

correct dehydration, non-steroidal anti-inflammatory drugs to reduce fever and inflammation and antimicrobials for known or suspected secondary bacterial infections.

Prevention Inactivated virus vaccines are commercially available for use in pigs. In the US, it is recommended that autogenous vaccines are considered if the strains in commercially-available vaccines are not a close match for circulating strains, but autogenous vaccines are only permitted for use on the farms for which the vaccine was generated. Inactivated virus vaccines against equine IAV first became available in the late 1960s, shortly after the first human IAV vaccines were developed. Subsequently, subunit (‘split’) vaccines were introduced. Today, a range of different equine IAV vaccines is available; whole inactivated virus, subunit immune-stimulatory complex (ISCOM/ISCOMatrix), live attenuated and recombinant live vector. The live canarypox-vectored vaccine expresses only the HA protein of IAV HA enabling a differentiation of infected from vaccinated animals (DIVA) strategy to be adopted in combination with an ELISA to detect antibodies to NP, which is not present in the vaccine. Demonstration that vaccine effectiveness is compromised if vaccine strains are not updated periodically led to the formation of an OIE (World Organization for Animal Health) Expert Surveillance Panel on Equine Influenza Vaccine Composition, which meets annually. The first vaccine to protect dogs against the H3N8 IAV strain was launched in 2009 in the USA. Vaccines were developed against the H3N2 IAV soon after it emerged and there is currently a bivalent vaccine available containing both H3N8 and H3N2 strains in the USA.

Concluding Remarks Swine, equine and canine IAV have been referred to as ‘neglected influenza viruses’. It is unlikely that the characteristics of the next pandemic IAV, or where it may emerge from, can be accurately predicted therefore, surveillance of IAV strains circulating in these neglected species is of paramount importance.

Further Reading Barba, M., Daly, J.M., 2016. The influenza NS1 protein: What do we know in equine influenza virus pathogenesis? Pathogens 5, 57. Dou, D., Revol, R., Östbye, H., Daniels, R., 2018. Influenza A virus cell entry, replication, virion assembly and movement. Frontiers in Immunology 9, 1581. Mostafa, A., Abdelwhab, E.M., Mettenleiter, T.C., Pleschka, S., 2018. Zoonotic potential of influenza A viruses: A comprehensive overview. Viruses 10, 497. Smith, G.J., Vijaykrishna, D., Bahl, J., et al., 2009. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459, 1122.

Feline Calicivirus (Caliciviridae) Margaret J Hosie and Michaela J Conley, MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Classification Feline calicivirus (FCV) is a non-enveloped, single stranded, positive sense RNA virus classified within the Vesivirus genus of the Caliciviridae. Vesiviruses have been shown to infect and cause varying clinical signs among many animal species. FCV was originally isolated in 1957 in New Zealand from cells in culture and has been shown to cause respiratory illness among felids. FCV was originally classified as a picornavirus (known as feline picornavirus for some time) prior to amendment and inclusion within the vesivirus genus of the Caliciviridae family. The Caliciviridae family was first classified in the third report of the International Committee on Taxonomy of Viruses (ICTV) in 1979. Calicivirus is derived from the latin word for cup, calix. The classification was due to the use of a single capsid protein which assembled to form icosahedral particles of 30–40 nm in diameter with 32 cup-shaped depressions on their surface.

Virus Characteristics In the 1970s, small individual particles of 35 nm in diameter were observed in FCV infected cells by electron microscopy. FCV particles were also seen in association with smooth membranes within cells. The major capsid protein of FCV, VP1, assembles into capsomeres that form arch-like structures on the surface of the virus, giving rise to the characteristic cup-shaped depressions on the virus particle. The VP1 dimer conformations differ slightly, forming A, B and C isoforms. A/B (bent) and C/C (flat) capsomeres assemble at different positions in the viral particle which results in t¼3 icosahedral symmetry with 180 subunits (Fig. 1). A/B capsomeres are assembled around the five-fold symmetry axes while C/C capsomeres are located at two-fold symmetry axes (resulting in the alternation of A/B and C/C capsomeres around the three-fold symmetry axes). The major capsid protein VP1 comprises 3 domains: the shell (S) domain forms the floor of the capsid, P1 makes up part of the viral spikes and P2 (which is an insertion into P1) forms the outer surface of the capsid protrusions and has a role in binding the cellular receptor molecule as well as neutralising antibodies (Fig. 2). The S domain of VP1 contains an eight-stranded β-barrel structure, with strands designated B to I. These strands form two, four-stranded β-sheets (composed of strands BIDG and CHEF) as well as two alpha helices between strands C–D and E–F. The P domain is joined to the S domain via a flexible hinge region, allowing slightly different conformations to be adopted by the A, B and C VP1 isoforms. The P2 domain contains a six stranded β-barrel (A′ to F′) of varying lengths and positions, as well as a hyper-variable region that is responsible for the variation observed between strains. These hypervariable regions form neutralising epitopes which serve as targets for the host’s immune system. All members of the Caliciviridae family are morphologically similar to FCV, although the P2 domains of VP1 can differ, as shown in Fig. 3. For example, some capsomeres form rounded structures e.g., rabbit haemorrhagic disease virus and norovirus GII.10, while other caliciviruses, including FCV, form rhomboid structures.

Genome Organisation and Viral Proteins FCV encodes a 7.7 kb single stranded, positive sense RNA genome with a VPg protein covalently linked to the 5′ end and a 3′ polyA tail. An additional RNA species of 2.4 kb (known as subgenomic mRNA) is also produced during infection which

Fig. 1 Structure of FCV strain F9. Three-dimensional structure of FCV solved by cryo-electron microscopy and three-dimensional image reconstruction at 3.0 Å resolution. Domains of the major capsid protein VP1 are coloured: S domain (cyan), P1 domain (pink) and P2 domain (magenta). Image provided by Dr. Michaela J. Conley and Dr. David Bhella (MRC-University of Glasgow, Centre for Virus Research).

294

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20927-8

Feline Calicivirus (Caliciviridae)

295

Fig. 2 Structure of major capsid protein VP1. Ribbon diagram of FCV strain F9 major capsid protein VP1, comprising the N-terminal arm (NTA, yellow), the shell domain (S, orange), P1 domain (red) and P2 domain (purple). The hinge region between the S and P1 domains is also shown. Image provided by Dr. Michaela J. Conley and Dr. David Bhella (MRC-University of Glasgow, Centre for Virus Research).

Fig. 3 Comparison of three-dimensional structures of six caliciviruses. Vesivirus 2117 (A), a chimeric sapovirus (B), rabbit haemorrhagic disease virus (C), feline calicivirus F9 strain (D), norovirus genotype II.10 (E) and Norwalk virus (F). Reproduced with permission from Conley, M., Emmott, E., Orton, R., et al., 2017. Vesivirus 2117 capsids more closely resemble sapovirus and lagovirus particles than other known vesivirus structures. Journal of General Virology 98 (1), 68–76.

encompasses the coding region of the structural proteins of the virus. FCV encodes three open reading frames (ORFs), ORF1 encodes the non-structural proteins, ORF2 encodes the major capsid protein, VP1, while the minor capsid protein, VP2, is encoded on the third ORF (Fig. 4). The major capsid protein, VP1, forms the majority of the viral capsid (180 copies per particle) with the minor capsid protein, VP2, incorporated to a lesser extent. The subgenomic RNA forms the template for translation of VP1 which forms a 76 kDa precursor that is subsequently cleaved to yield the mature capsid protein of 62 kDa. This cleavage of VP1 is mediated by the viral protease and is essential for the life cycle of FCV. The leader of the capsid protein (LC) is cleaved post-translationally and has been shown to cause the cytopathic effect

296

Feline Calicivirus (Caliciviridae)

Fig. 4 Genome organisation of FCV. Diagrammatic representation of the open reading frames (ORF) and proteins encoded by the FCV genome and the two RNA species produced during infection.

observed in infected cells. Many viral products form protein-protein interactions. VP1 has been shown to interact with VPg, the protease-polymerase (NS6–7) as well as the minor capsid protein, VP2. The minor capsid protein, VP2, is crucial for viral replication and release of progeny viral particles. Each virion is predicted to incorporate one or two copies of VP2, which is involved in the self-assembly of the capsid.

Non-structural proteins Almost all of the non-structural proteins of FCV associate with membranous replication complexes in infected cells, where translation and viral genome replication are co-ordinated. These proteins include NS2, NS3 (helicase) and NS6–7 (protease-RNA dependent RNA polymerase). Virion protein g (VPg/NS5) is a small protein that is not required for FCV infection, although it is essential for translation of the viral RNA (unless replaced with a cap structure). Polypyrimidine tract binding protein (PTB) is redistributed by FCV to the cytoplasm (from the nucleus), where it interacts with both the genomic and subgenomic viral RNAs via their 5′ ends. PTB has been shown to partly co-localise with FCV replication complexes and provides a switch from translation to viral genome replication. VPg is also associated with NS4 in replication complexes which suggests a role in anchoring the small VPg protein near the complexes for close proximity during viral RNA replication. NS2 interacts with NS3, NS4 and NS6–7 in the membranous replication complexes. NS2, NS3 and NS4 have been proposed as possible transmembrane proteins which localise to the endoplasmic reticulum and may result in membranous rearrangements which are characteristic of FCV infection. NS3 has the ability to prevent the activation of IRF3 (interferon response factor-3) and therefore plays a role in the suppression the innate immune response during FCV infection. The FCV protease/polymerase (NS6–7) forms oligomers and interacts with VPg as well as ORF2 (encoding VP1). A weak interaction with VP2 has also been described. The viral polyprotein is cleavage in cis by the FCV protease to yield the individual non-structural proteins, however, the protease-polymerase (non-cleaved protein) is thought to be the active form of the RdRp. Nucleolin is essential for FCV replication and interacts with both viral RNA and the protease-polymerase. NS6, appears to reduce cellular translation through the cleavage of poly A binding protein (PABP) and has recently been shown to also impair the formation of stress granules within infected cells.

Life Cycle FCV infection is restricted to cells of feline origin although this can be overcome by transfection of viral RNA into cells, indicating that the restriction occurs at the stage of binding and entry rather than replication. Attachment of FCV to cells is most efficient at pH 6.5 and occurs as quickly as 15 min post cell exposure. The entry of FCV is pH dependent; the low pH generated by the

Feline Calicivirus (Caliciviridae)

297

Fig. 5 Three-dimensional reconstruction of FCV decorated with fJAM-A. Cryo-electron microscopy and three-dimensional reconstruction were used to solve the structure of FCV (F9 strain; cyan and pink) decorated with the soluble ectodomain of fJAM-A (magenta) at 3.5 Å resolution. Image provided by Dr. Michaela J. Conley and Dr. David Bhella (MRC-University of Glasgow, Centre for Virus Research).

acidification of endosomes is essential for the uncoating of FCV and the release of the viral RNA into the host cell before replication can ensue. The entry of FCV is dependent upon clathrin mediated endocytosis and the release of the viral genome is likely mediated by the permeabilisation of membranes during FCV entry. Many viruses utilise carbohydrate moieties (e.g., sialic acid) as attachment factors or as parts of their receptors. Neuraminidase treatment of feline cells (to cleave terminal sialic acid residues) reduced the binding of FCV to cells by approximately 23%, suggesting a potential role of sialic acid in FCV attachment and entry. FCV binds to α2,6-linked sialic acid (but not α2,3-linked), which is thought to be present on an N-linked glycoprotein on the surface of susceptible cells. This sialic acid linkage preference could contribute to tissue specificity and cell tropism. The functional receptor for FCV is feline junctional adhesion molecule A (fJAM-A), which is found at tight junctions between epithelial and endothelial cells. JAM-A can also be found on platelets and leucocytes, as described for both the murine and human JAM-A proteins. FCV infection results in the re-localisation of fJAM-A from tight junctions into the cytosol of the cell. Antibodies targeting JAM-A have been shown to reduce FCV binding to Crandell-Reese feline kidney cells, while the expression of fJAM-A in non-permissive cells rendered them susceptible to infection with FCV. JAM-A contains a short cytoplasmic tail, a transmembrane region and an ectodomain (composed of sub-domains D1 and D2). D1 of fJAM-A is sufficient for FCV attachment and feline JAMA shares over 75% amino acid identity with its human counterpart. Feline JAM-A contains a predicted N-glycosylation site at position 185, based on this sequence identity. All fJAM-A domains are necessary for infection with FCV although the PDZ-binding motif (within the cytoplasmic domain of fJAM-A) is not required for virus entry. FCV is able to interact with both monomeric and dimeric forms of fJAM-A with residues D42, K43 and S97 (in D1 of the fJAM-A ectodomain) being important for virus binding. FCV binds both monomeric and dimeric fJAM-A, since the virus binding site and fJAM-A dimerisation site are located at distinct regions/faces of the fJAM-A ectodomain. The structure of FCV bound to the fJAM-A ectodomain has been determined by cryo-electron microscopy and three-dimensional reconstruction (Fig. 5). Binding of fJAM-A to FCV appears to trigger a conformational change in the capsid resulting in the anti-clockwise rotation (15°) of the VP1 capsomeres. Two fJAM-A proteins bind to two VP1 proteins (a single VP1 capsomere) in a head-to-tail manner (i.e., the D1 domain of the first fJAM-A protein lies adjacent to D2 domain of the second fJAM-A protein). The fJAM-A proteins also appear to bind to FCV in their monomeric form. The conformational change observed upon fJAM-A binding likely acts as a priming step for virus entry into the host cell via clathrin-mediated endocytosis.

Epidemiology FCV is widespread in the domestic cat population, it has been isolated from up to 24% of healthy cats at cat shows and 10% of cats attending veterinary surgeries. The prevalence increases as the number of cats in a household increases, with the highest prevalence observed in large groups of cats housed together in multi-cat households. As many as 25%–40% of cats kept in colonies and shelters are infected, whereas the prevalence in small groups of cats is approximately 10%. The prevalence amongst colonies can range from 50% to 90%. FCV is host-specific; there are no known reservoirs or alternative hosts and the virus does not infect humans. FCV-like viruses have been isolated from dogs, although it is unlikely that such viruses play a role in the epidemiology of FCV infection. Following infection of cats, FCV is shed in the oral and nasal secretions. The majority of cats shed virus for more than 30 days, although shedding can last for weeks or even several years after the acute clinical signs have resolved.

Pathogenesis Cats become infected via the nasal, oral or conjunctival routes and the primary site of viral replication is the oropharynx. A transient viraemia occurs 3–4 days following infection and during this period the virus can be detected in many other tissues.

298

Feline Calicivirus (Caliciviridae)

Following acute infection, the virus induces necrosis of epithelial cells and vesicles are observed, typically on the margin of the tongue. The vesicles subsequently develop into ulcers and it has been demonstrated that the dermis becomes infiltrated with neutrophils in the affected regions before the mouth ulcers resolve 2–3 weeks later. Occasionally other tissues are infected, such as the lungs and joints. Focal alveolitis can occur, leading to acute exudative pneumonia and proliferative interstitial pneumonia and lameness has also be observed, with acute synovitis and thickening of the synovial membrane. The recent emergence of highly virulent systemic strains of FCV (VS-FCV) has been reported in the US, Europe and the UK. Unlike the majority of FCV infections, VS-FCV infected cats develop severe, diverse lesions, including cutaneous oedema and ulcerative lesions on the skin and paws, hepatic necrosis, pancreatitis, pulmonary oedema and disseminated intravascular coagulation. Outbreaks usually occur in multi-cat environments and have been characterised by rapid onset and high mortality. It has been reported that VS-FCV strains emerge independently from genetically distinct FCV strains but attempts to identify genetic patterns within the viral genome that define the highly virulent FCV biotype have been inconclusive so far. The pathogenesis of virulent systemic FCV infection might also include immune mediated components and/or environmental and management factors. Following recovery from acute disease, the majority of cats clear FCV infection within approximately 30 days, although some cats can shed virus for longer. In healthy carrier cats, FCV is localised within the tonsillar epithelium, but, since tonsillectomy does not terminate the carrier state, it is likely that the virus also persists in other sites. It has been postulated that amino acid mutations in the viral capsid protein might lead to the appearance of FCV variants that escape the host immune response and persist within infected cats.

Control and Prevention Disease Control FCV particles can survive for up to 2 weeks on solid surfaces and are stable between pH values ranging from 4 to 8.5 and the virus is resistant to many common disinfectants. Effective disinfectants include sodium hypochlorite (5% bleach diluted at 1:32), potassium peroxy-monosulfate, chlorine dioxide and commercial products that have been approved for their virucidal activity. FCV infections often arise in cat shelters, but virus transmission can be limited, or even prevented, by appropriate shelter design and by instigating management measures to avoiding cross infection of cats. For example, cats should be housed individually, only cats originating from the same household should be allowed to mix and flea control should be implemented to minimise the risk of transmission of FCV and other diseases. New cats arriving in shelters that are healthy should be vaccinated as soon as possible, preferably with a modified live virus vaccine to induce protection more rapidly. FCV can be a major problem in breeding catteries, characterised by upper respiratory disease in kittens as young as 4–8 weeks of age, coinciding with the waning of maternally-derived antibodies. FCV infection can be severe in young kittens, often affecting all kittens in affected litters and some kittens may develop pneumonia and die. Breeding queens should be vaccinated so that kittens acquire high levels of maternally-derived antibody via colostrum and milk, which will protect them from FCV infection for several weeks. It is recommended that breeding queens should be vaccinated prior to mating rather than during pregnancy. Modified live virus vaccines are not licensed for use in pregnant cats and only inactivated vaccines should be used. The earliest age for which FCV vaccines are licensed for kittens is six weeks, but vaccination could be considered in younger kittens if they are at risk of infection. Alternatively, early weaning from four weeks of age will protect kittens against infection from infected breeding queens.

Vaccination Both modified live and inactivated parenteral FCV vaccines are available. Modified live intranasal vaccines can still be used in US but are not available in Europe. It is thought that FCV vaccines provide protection mainly via the induction of virus neutralising antibodies, although cellular immunity has not been extensively examined following either FCV vaccination or infection. As FCV rapidly mutates and gives rise to viral variants, it has been suggested that field isolates could evolve and become resistant to vaccines based on strains that have been used for several decades; indeed, vaccine manufacturers are seeking to identify and introduce newer vaccine strains that might provide wider cross protection. At present, the most commonly used vaccine strains are F9 (the oldest strain that was isolated in the 1950s), FCV 255 and two more recent strains, G1 and 431. Several in vitro studies have provided evidence that older vaccine strains are less effective at cross-neutralising current circulating field strains isolated from sick cats, but these findings are controversial and further independent studies will be required to investigate vaccine efficacy against different FCV strains. Nevertheless, it is generally recommended that all healthy cats should be vaccinated against FCV, since vaccination provides good protection against acute oral and upper respiratory tract disease in the majority of cats. However, sterilising immunity is not induced, and so vaccinated cats can still become infected and subsequently shed FCV. It is important to consider that current vaccines are not able to protect cats equally well against all the currently circulating field strains of FCV.

Further Reading Conley, M.J., McElwee, M., Azmi, L., et al., 2019. Calicivirus VP2 forms a portal-like assembly following receptor engagement. Nature 565 (7739), 377–381. Coutts, A.J., Dawson, S., Willoughby, K., Gaskell, R.M., 1994. Isolation of feline respiratory viruses from clinically healthy cats at UK cat shows. Veterinary Record 135, 555–556.

Feline Calicivirus (Caliciviridae)

299

Hansman, S.G., Jiang, X.J., Green, K.Y. (Eds.), 2010. Feline Calicivirus by Christine Luttermann and Gregor Meyers in Caliciviruses: Molecular and Cellular Virology 2010. Caister Academic Press. Pedersen, N.C., Elliott, J.B., Glasgow, A., Poland, A., Keel, K., 2000. An isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus. Veterinary Microbiology 73, 281–300.

Relevant Websites http://www.abcdcatsvets.org/feline-calicivirus-infection-2012-edition/ Feline Calicivirus infection. https://icatcare.org/advice/cat-health/feline-calicivirusr-fcv-infection-0 Feline calicivirus (FCV) infection – International Cat Care.

Feline Leukemia and Sarcoma Viruses (Retroviridae) Brian J Willett and Margaret J Hosie, MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom r 2021 Elsevier Ltd. All rights reserved. This is an update of J.C. Neil, Feline Leukemia and Sarcoma Viruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00397-6.

History In the early 1960s, Harry Pfaff, a veterinary practitioner in the Glasgow area, observed a cluster of lymphoma cases in a local multicat household. Suspecting a possible infectious etiology, he contacted Professor William Jarrett, a veterinary pathologist at the University of Glasgow. Using electron microscopy, Professor Jarrett and his colleagues visualized virus-like particles in tumor material from the cats, and demonstrated that these particles bore a striking resemblance to the virus associated with murine leukemia, reporting their findings in Nature in 1964. Subsequent studies demonstrated that these feline leukemia viruses (FeLVs) could be transmitted to cats where they induced lymphosarcomas and a range of degenerative diseases, including anemias and thymic atrophy. Following these studies, Snyder and Theilen isolated a retrovirus from a feline fibrosarcoma that rapidly reproduced this tumor on inoculation into experimental cats. It is now recognized that feline sarcoma viruses (FeSVs) arise by recombination between FeLV and cellular proto-oncogenes and that, in contrast to FeLV, these viruses are not transmitted from cat to cat. FeLV remains an important virus clinically, affecting both domestic cats and wild felids. Several highly efficacious FeLV vaccines are available and are used widely. This, in conjunction with reliable testing and control programmes, has led to a significant decline in the prevalence of FeLV infection (o2%) across Europe and North America. Prevalence remains high in some areas, for example N.W. China (B11%) and Thailand (B25%). As a naturally occurring disease in an outbred host, FeLV has served as a paradigm for the natural history and molecular pathogenesis of the gammaretrovirus subfamily. It also played a foundational role in cancer genetics as a transducing agent which led to the discovery of novel cellular transforming genes.

Taxonomy and Classification FeLVs are RNA viruses and belong to the family Retroviridae. They are further classified in the genus Gammaretroviruses.

Virion and Genome Structure The virion particles are around 100 nm in diameter and consist of an outer membrane derived from the host cell surrounding a spherical core particle. The core encapsidates the viral genome which, as in other members of this viral family, is present as two linear single-stranded RNA molecules linked as a dimer. The virion RNA is positive-stranded and resembles cellular RNA having a 50 cap and a 30 poly(A) tract. As deduced from sequencing of proviral forms, the FeLV genome is around 8 kb long with a 67 base terminal redundancy and the gene order gag-pol-env. The particles have surface spikes composed of multimers of the two env-coded proteins, the gp70 surface glycoprotein (SU), and the p15E transmembrane anchor protein (TM). Inside the envelope are the structural gag gene products which form a spherical core particle composed of p27, the major capsid protein (CA), with an outer layer formed by the p15 matrix protein (MA). Another gag product, the p10 nucleocapsid (NC), is associated with the virion RNA. Other minor virion proteins encoded by the pol gene comprise the protease (PR), reverse transcriptase (RT), and integrase (IN) enzymes.

Replication Following attachment to specific host cell-surface receptors and fusion of the viral envelope with the cellular membrane, the viral core is internalized into the cytoplasm of the cell. Uncoating is followed by reverse transcription in which the virion RNA is converted into a double-stranded DNA form by the virion RT which uses a proline tRNA primer and carries a ribonuclease H function that degrades the virion RNA. After nuclear translocation, viral integration is catalyzed by the IN protein and involves the creation of a staggered cut in cellular DNA with a consequent 4 bp duplication of host DNA at the insertion site. The proviral form of FeLV is flanked by long terminal repeats (LTRs) of 480–560 bp. These are generated during reverse transcription by duplication of unique sequences at the 30 (U3) and 50 (U5) ends of the RNA genome. The LTRs contain promoters and enhancers that drive transcription of viral RNA and also processing signals for cleavage and polyadenylation of the RNA transcripts. The 50 LTR functions to initiate transcription while the 30 LTR acts primarily as an RNA processing signal.

300

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00043-6

Feline Leukemia and Sarcoma Viruses (Retroviridae)

301

The virion RNA can function as a messenger RNA for Gag and Pol products, while a spliced subgenomic mRNA of around 3 kb encodes the Env products. Most full-length RNA translation products terminate at the 30 end of gag to produce the Pr65gag precursor, while a small percentage read through into pol by misreading of a UAG termination codon, generating the Pr180Gag-pol precursor. During and after the budding process, the virion aspartyl PR catalyzes the cleavage of both precursor proteins to their mature forms. The envelope gene products are synthesized as a Pr80env precursor and processed by cellular PRs to the mature, disulfide-linked gp70 and p15E envelope proteins. The gag gene is also abundantly expressed in an alternative, glycosylated form via an upstream AUG codon. This product is expressed on the cell surface and shed after cleavage by cellular PRs. It is dispensable for in vitro virus replication but is highly conserved and may play a role in vivo, for example in counteracting intrinsic immune activity as has been described for the equivalent gene product in murine leukemia virus. Assembly of virus particles occurs at the cell surface by extrusion of cores which form at the budding site, concomitantly acquiring a host cell-derived outer membrane with virus-coded surface spikes. Virus replication and release is often noncytopathic.

Geographic Distribution FeLV occurs worldwide in domestic cat populations, although prevalence varies significantly and has declined in pet cats in areas where reliable testing, vaccination and control have been instigated. FeLV has also been isolated from several wild felids, including the European wild cat (Felis silvestris), Florida panther (Puma concolor coryi), Iberian lynx (Lynx pardinus) and Jaguar (Panthera onca). Endogenous retroviral sequences closely matched to FeLV are found in the sand cat (Felis margarita) and the jungle cat (Felis chaus). Although not a direct source of disease, these endogenous sequences may potentially participate in recombination with exogenous FeLV to generate variant viruses with altered host range.

Epidemiology The outcomes of FeLV infection fall into several categories, which vary in likelihood according to the age and immune status of the exposed host. The majority of cats undergo a transient infection lasting up to 3 months, during which they are viremic and shed virus. They then develop neutralizing antibody and concomitantly clear infectious virus and, a little later, virus antigen from the blood stream. Some cats appear to clear virus infection successfully, while others may harbor latent virus in the bone marrow for some years. A recent study based on analysis of virus loads by polymerase chain reaction (PCR) suggests that animals that control viremia may be further subdivided into either abortive, or regressive, sometimes known as latent, infection. Even where latent infection persists, reactivation is not a frequent event and the vast majority of these cats do not develop an FeLV-related disease. However, at this stage virus can be reactivated by immunosuppression. The final group (persistent or progressive infection) remains actively infected, shedding virus from epithelial surfaces and displaying high titer plasma viremia. Such cats may remain apparently healthy for 2–3 years before succumbing to an FeLV-related disease. The proportion of cases falling into these groups differs between multi-cat households and those households containing one or two free-ranging cats. In the former case, the introduction of an FeLV carrier results in repeated exposure of susceptible cats, often at a young age so that up to 30%–40% become persistently viremic and at risk of disease. Approximately 50% of free-ranging urban and suburban cats have serological evidence of exposure to FeLV but only 1%–5% of these cats are actively infected and the disease incidence is correspondingly lower. Dual infection with FeLV and feline immunodeficiency virus (FIV) occurs and is associated with rapid disease onset, particularly if cats with preexisting FeLV infection encounter FIV. Rapid death of dually infected animals may reduce the apparent overlap of these agents in the field, but the populations at risk of infection also differ. FIV infection rates increase directly with age while persistent FeLV infection has a peak incidence in young cats.

FeLV Subgroups and Host Range FeLV isolates were initially classified into three subgroups; A, B, C according to their viral interference properties in feline fibroblast cells in vitro. Viruses of a given subgroup prevent superinfection (interfere) with other viruses of the same subgroup (Table 1). This property is based on the use of three different host cell-surface receptors by FeLV-A, B, and C, and the blockade and/or downmodulation of these receptors in persistently infected cells by viral Env glycoproteins. Primary receptors for subgroups A, B, and C have been identified and shown to be transmembrane transporter molecules which the virus has subverted to gain entry to the host cell (Table 1). Subsequent studies have identified additional, less frequent viral variants with distinct interference properties and receptor usages, although the contributions of such viral variants to the pathogenesis of disease in the wider cat population remains unclear. The generation of novel variants occurs by either mutation or recombination. During the process of mutation, viruses may emerge with an intermediate phenotype, for example isolate FY981 can use either the FeLV-A or C receptors for infection. Notable variants include FeLV-T, a “lymphotropic” variant with complex entry requirements. FeLV-T does not replicate well in fibroblast cells and appears to use the FeLV-A receptor in conjunction with an auxiliary mechanism in which a truncated env gene product encoded by endogenous FeLV sequences (FeLIX) is used as a co-receptor through binding to the FeLV-B receptor.

302

Table 1

Feline Leukemia and Sarcoma Viruses (Retroviridae)

Properties of feline leukemia virus subgroups

Subgroup Origin

Receptor

Function

Pathogenesis

A

Exogenous

feTHTR1

Thiamine transport

B

Recombination FeLV-A  endogenous FeLV

Pit-1 (Pit-2)

Phosphate transport

C D

Mutation of FeLV-A (Env vrA) Recombination FeLV-A  endogenous retrovirus ERV-DC Mutation of FeLV-A (outside RBD) Mutation of FeLV-A (Env vrA)

FLVCR1 (FLVCR2) Unknown

Heme export Unknown

Minimally pathogenic to acute immunosuppression More common in leukemic cats Some isolate-specific diseases, e.g., FeLV-GM1 myeloid leukemia Erythroid hypoplasia Unknown

T TG35-2

FeLV-A receptor þ FeLIX, Pit-1 feRFC Reduced folate transport

Acute immunosuppression Unknown

FeLV-D is formed by recombination between exogenous FeLV and the endogenous cat retrovirus ERV-DC. FeLV-D appears to use a distinct receptor to the A, B and C subgroups. FeLV-TG35–2 is a virus isolated from a single cat in Japan and uses the feline reduced folate carrier as an entry receptor. Natural isolates contain either subgroup A alone, or mixtures of subgroups A þ B, A þ C, or A þ B þ C. FeLV isolates of subgroup A have a limited tropism in cell culture due to their use of the receptor THTR1 (e.g., FEA and HEK293 cells), whereas subgroup B and C isolates have a greatly expanded host range due to their use of Pit-1 and FLVCR1 respectively, infecting cell lines from diverse species, including cat, human, mink, and canine cells. FeLV infection is generally noncytopathic and persistent and the virus is commonly propagated in long-term cultures of embryo-derived fibroblasts. Some strains such as FeLV-T or FeLV-C are cytopathic or induce apoptosis in cells of lymphoid origin in vitro, reflecting their in vivo pathogenic properties.

Clinical Features and Pathology Of those cats that become persistently viremic following FeLV exposure, over 80% die within 3.5 years. Most young cats infected with FeLV die from degenerative diseases rather than from tumors. Profound immunosuppression associated with thymic atrophy is a common finding in kittens. Other diseases seen in FeLV-infected cats include enteritis, immune complex glomerulonephritis, pancytopenia, and hemolytic anemia. Erythroid hypoplasia, an acute disease involving failure of red cell development past the burst-forming unit (BFU) stage, is specifically associated with FeLV subgroup C. The most common neoplasm induced by FeLV is lymphosarcoma of T-cell origin, usually restricted to the thymus or sometimes occurring as a multicentric tumor in lymph nodes. The tumors often develop between 1 and 3 years after infection and the first signs may be chronic wasting and anemia. At presentation, the normal architecture of the lymphoid organ has usually been destroyed by a monomorphic infiltrate of lymphoblastic cells. The thymic tumors frequently display a rearrangement of the T-cell antigen receptor b-chain gene and may also express the co-receptor molecules CD4 and/or CD8. FeLV is also commonly associated with myeloid leukemias and a myelodysplastic syndrome-like disease, as well as with other forms of hematopoietic malignancy. Multicentric fibrosarcoma is a rare sequel to FeLV infection but these tumors are often associated with the de novo generation of a FeSV. FeLV is also found in association with 35% of alimentary tumors, primarily of B cell origin, but the virus is not always clonally integrated in these tumors and the role of the virus is, therefore, unclear. FIV can also increase the frequency of malignant diseases in cats, and some rare cases arise on a background of dual infection.

Envelope Gene Variation and Pathogenicity The common infectious form of FeLV is FeLV-A which is a remarkably highly conserved virus as shown by sequence analysis of several strains and serotypic analysis of a much larger number. FeLV variants frequently arise from FeLV-A by mutation and recombination, and such variants are often implicated in the acute diseases that develop in persistently infected cats. The variant viruses generated from FeLV-A are generally dependent on the continued presence of the prototype for their propagation in vivo. However, as the variants are transmitted less efficiently and are in some cases rapidly fatal, they tend to die out with the host while the prototypic FeLV-A continues to colonize new hosts (Fig. 1). The most commonly isolated FeLV recombinants are subgroup B viruses (Table 1). These are derived by recombination between FeLV-A and endogenous FeLV-related proviruses which are found in the genome of the domestic cat and related small feline species. Although the endogenous FeLV-related proviruses all appear to be replication defective, their envelope genes can be rescued by the recombination process leading to the generation of FeLV-B viruses. FeLV-B can infect cells refractory to, or already containing, FeLV-A by virtue of their distinctive receptor specificity.

Feline Leukemia and Sarcoma Viruses (Retroviridae)

303

Fig. 1 Life cycle of FeLV in its natural host, the domestic cat. FeLV-A is the most readily transmitted form and is found in 100% of isolates. In cats which become persistently viremic (progressive infection), the virus may evolve by recombination or point mutations to generate pathogenic variants that can lead to the rapid demise of the infected host animal. Multiple variants can arise in a single host. Few of these variants show any capacity for horizontal transmission to new hosts with the exception of FeLV-B. In this way, FeLV inflicts a substantial disease burden without significantly reducing host numbers.

Table 2a

FeLV gene transduction in neoplasia

Gene

Normal function of host gene product

Associated tumor

Examplesa

abl fes fgr fms kit myc Notch2 sis tcr

Plasmamembrane protein kinase Plasmamembrane protein kinase Plasmamembrane protein kinase Receptor protein kinase (CSF-1 receptor) Receptor protein kinase (SCF receptor) Transcription factor Transmembrane receptor Growth factor (B chain PDGF) T-cell antigen receptor (b-chain)

Fibrosarcoma Fibrosarcoma Fibrosarcoma Fibrosarcoma Fibrosarcoma T-cell lymphoma T-cell lymphoma Fibrosarcoma T-cell lymphoma

FeSV-HZ2 FeSV-GA,-HZ1,-ST FeLV-GR,-TP1 FeSV-SM,-HZ5 FeSV-HZ4 FeLV (T3, T17, FTT) (Inoculum FeLV-61E)a FeSV-PI FeLV-T17

a

Isolated from naturally occurring tumors apart from the indicated exception.

The anemia-inducing FeLV-C isolates are rarer and appear to be derived from FeLV-A by mutation of a single variable domain (VRA) of the env gene. The acute disease properties of these variants appear to be due to compromised viability of erythroid progenitor cells due to perturbation of the FeLV-C receptor, a vital heme exporter. Minor env mutations also appear to give rise to the acutely immunosuppressive FeLV-FAIDS variants. The prevalence of acutely immunosuppressive viruses in nature is unknown, but immunosuppressive disease is a common manifestation of FeLV infection. The relationship of subgroup variation to oncogenesis is complex. FeLV-B recombinants are more common in tumor-bearing than in healthy infected cats. This higher frequency might reflect merely longer-standing infection, but some FeLV-B-containing isolates have an altered spectrum of neoplastic disease. For example, FeLV-GM1, which contains a replication-defective FeLV-B component, induces mainly myeloid leukemia.

FeLV Oncogenesis: Virus Evolution and Mutagenesis of Cellular Oncogenes Two modes of virus-induced host gene mutation have been described in FeLV-associated cancers. The first is ‘transduction’, where recombination leads to the generation of an acutely oncogenic variant in which viral gene sequences are replaced by a host-derived insert. Such viruses are replication defective and are found in nature in association with a replication-competent FeLV helper virus. Multicentric fibrosarcomas of young cats are relatively rare, but are generally FeLV positive and frequently involve a novel sarcoma virus. Similarly, transduction of c-myc has been observed in up to 20% of naturally occurring thymic lymphosarcomas in FeLV positive cats. In all, nine different host cell genes have been shown to be transduced by FeLV (Table 2a). The transducing viruses induce tumors with short latency in cats and in the case of FeSVs may transform cells in tissue culture.

304

Feline Leukemia and Sarcoma Viruses (Retroviridae)

Table 2b

Insertional mutagenesis and FeLV oncogenesis

Common integration site

Gene function

Tumor

fit-1 flvi-1 flvi  2 (bmi-1) c-myc pim-1 flit-1

Transcription factor (c-myb) Unknown Transcription factor Transcription factor Protein kinase Growth factor receptor?

T-cell lymphoma non-T, non-B lymphoma T-cell lymphoma T-cell lymphoma T-cell lymphoma T cell lymphoma

Alternatively, host genes can be affected by proviral ‘insertional mutagenesis’ (cis-activation). Four known oncogenes and two uncharacterized novel integration loci have been identified as common tumor-specific insertion sites for FeLV in thymic lymphosarcomas (Table 2b). In this respect FeLV oncogenesis appears remarkably similar to that of the murine gammaretroviruses. Changes within the LTR are also a feature of tumor-associated FeLV. In thymic lymphosarcomas, sequence duplications of the core enhancer domain are found frequently and have been shown to arise de novo from infection with molecularly cloned FeLV isolates lacking such features. By analogy with the murine oncoretroviruses, the duplications are likely to increase the oncogenicity of the virus and reduce the latent period for tumor formation, possibly by increasing the potency of viral enhancer activity on nearby cellular promoters. There is evidence that these adaptive changes to the LTR operate tissue-specifically and proviruses carrying different duplications of LTR regions 50 and 30 to the core-enhancer region have been identified in myeloid leukemias and non-T, non-B splenic lymphomas, respectively. Chimeric murine retroviruses carrying the FeLV enhancer region have been generated, confirming that the tissue specificity is carried by this structure.

Immune Response Unlike infection with the lentiviruses HIV and FIV, FeLV infection of cats can lead to recovery. There is evidence that both neutralizing antibodies and virus-specific cytotoxic T cells (CTLs) play a role in resistance as either can be used to prevent or limit infection by passive transfer. In natural infection, CTLs to virus structural protein epitopes can be detected as early as 1 week after infection. In contrast, neutralizing antibodies may not be detected until approximately 6 weeks post infection. In the early literature on FeLV, a distinction was made between antiviral immunity and antitumor immunity. Cats with antitumor (FOCMA) antibody were thought to be protected from tumor development. This antibody response is now believed to be directed to endogenous FeLV env proteins and its role in modulating tumor development is uncertain.

Transmission Cats persistently infected with FeLV shed virus in their saliva, urine, and feces but, as the virus is fragile, close contact is required for transmission. The most frequent routes involve saliva and transplacental spread. Kittens infected in utero become persistently infected, but the consequences of infection in older cats depend on a number of factors. There is an age-related resistance to infection such that cats up to 12 weeks of age are highly susceptible, but above 16 weeks they are difficult to infect either naturally or experimentally. FeLV subgroup A is always found in field isolates while approximately half also contain FeLV-B, whereas FeLV-C is present in only 1%–2% of isolates. Although FeLV-B can arise de novo by recombination, it can also be transmitted between cats. This occurrence is dependent on pseudotype formation in which the genome of the B virus becomes enclosed in an envelope containing glycoproteins of the A subgroup.

Prevention and Control Successful control measures can be adopted in multi-cat households by removing or isolating persistently infected animals. Productively infected cats are identified routinely by the detection of soluble FeLV antigen (p27) by ELISA or immunomigration test, proviral DNA PCR on whole blood, RT-PCR for viral RNA in blood or saliva, or less frequently by direct virus isolation in culture. A few cats remain persistently antigenemic but nonviremic. These cats do not usually transmit the virus unless they are shedding virus in the milk or saliva. Numerous vaccine strategies have been shown to confer protection against FeLV under laboratory conditions and FeLV was the first retrovirus for which commercial vaccines were developed. Vaccines in current use include whole inactivated virus preparations, subunit vaccines comprising recombinant viral Env protein expressed in bacterial cells, and a canarypox recombinant virus

Feline Leukemia and Sarcoma Viruses (Retroviridae)

305

expressing Gag and Env. These vaccines offer significant protection against experimental challenge and are under evaluation for longer-term efficacy in the field. These vaccines do not generate sterilizing immunity but appear to prime the immune system to favor clearance of virus infection instead of persistent viremia.

Future Perspectives There is continuing interest in control of FeLV infection due to its importance in veterinary medicine. In the future we can look forward to improvements in vaccine efficacy and further dissection of the host responses that confer protection. While the focus of attention of cancer genetics has moved on to more easily manipulated models, FeLV remains as a useful touchstone for our understanding of retroviral pathogenesis in an outbred, naturally infected host.

Further Reading Anai, Y., Ochi, H., Watanabe, S., et al., 2012. Infectious endogenous retroviruses in cats and emergence of recombinant viruses. Journal of Virology 86, 8634–8644. Hanlon, L., Barr, N.I., Blyth, K., et al., 2003. Long-range effects of retroviral activation on c-myb over-expression may be obscured by silencing during tumor growth in vitro. Journal of Virology 77, 1059–1068. Lutz, H., Addie, D., Belák, S., et al., 2009. Feline leukaemia. ABCD guidelines on prevention and management. Journal of Feline Medicine and Surgery 11, 565–574. Mendoza, R., Anderson, M.M., Overbaugh, J., 2006. A putative thiamine transport protein is a receptor for feline leukemia virus subgroup A. Journal of Virology 80, 3378–3385. Miyazawa, T., 2002. Infections of feline leukemia virus and feline immunodeficiency virus. Frontiers in Bioscience 7, D504–D518. Roca, A.L., Nash, W.G., Menninger, J.C., Murphy, W.J., O’Brien, S.J., 2005. Insertional polymorphisms of endogenous feline leukemia viruses. Journal of Virology 79, 3979–3986. Studer, N., Lutz, H., Saegerman, C., et al., 2019. Pan-European study on the prevalence of the feline leukaemia virus infection – Reported by the European advisory board on cat diseases (ABCD Europe). Viruses 11, 993–1020. Torres, A.N., Mathiason, C.K., Hoover, E.A., 2005. Re-examination of feline leukemia virus: Host relationships using real-time PCR. Virology 332, 272–283. Tsatsanis, C., Fulton, R., Nishigaki, K., et al., 1994. Genetic determinants of feline leukemia virus-induced lymphoid tumors: patterns of proviral insertion and gene rearrangement. Journal of Virology 68, 8294–8303. Willett, B.J., Hosie, M.J., 2013. Feline leukaemia virus: Half a century since its discovery. The Veterinary Journal 195, 16–23.

Fish and Amphibian Alloherpesviruses (Herpesviridae) Maxime Boutier, Léa Morvan, and Natacha Delrez, University of Liège, Liège, Belgium Francesco Origgi, University of Bern, Bern, Switzerland Andor Doszpoly, Hungarian Academy of Sciences, Budapest, Hungary Alain Vanderplasschen, University of Liège, Liège, Belgium r 2021 Elsevier Ltd. All rights reserved.

Nomenclature

ICTV International Committee on Taxonomy of Viruses kbp kilobase pair ORF open reading frame PeHV Percid herpesvirus RaHV Ranid herpesvirus SalHV Salmonid herpesvirus SiHV Silurid herpesvirus TK thymidine kinase TR terminal repeat

AciHV Acipenserid herpesvirus AngHV Anguillid herpesvirus BAC bacterial artificial chromosome BfHV Bufonid herpesvirus CyHV Cyprinid herpesvirus dsDNA double-stranded DNA EsHV Esocid herpesvirus GaHV Gadid herpesvirus IcHV Ictalurid herpesvirus

Glossary Alloherpesvirus Herpesvirus classified in the family Alloherpesviridae, order Herpesvirales, grouping all herpesviruses infecting fish and amphibians described until now. Bacterial artificial chromosome Large DNA vector that can allow the stable maintenance and efficient mutagenesis of herpesvirus genome in Escherichia coli, followed by the reconstitution of progeny virions after its transfection into permissive eukaryotic cells. Behavioral fever Phenomenon by which ectotherms increase their body temperature by moving to warmer places in response to an infection.

Ectotherm Organism unable to internally self-regulate its body temperature, therefore relying on external sources of heat. Hyperplastic lesion Increase of the size of a tissue or organ as a result of cell proliferation. Latency Maintenance of viral genome as a non-integrated episome associated with minimal viral protein expression and absence of infectious particle production. Viral reactivation Phenomenon by which the state of viral infection switches back from latency to productive infection.

Introduction and Classification Herpesviruses form a diverse group of viruses initially described by a conserved virion structure containing a large, linear doublestranded DNA (dsDNA) genome. Indeed, the unifying property of herpesviruses is virion morphology rather than genetic content. Herpesviruses are now classified in the order Herpesvirales and share only one convincing conserved gene, i.e., the ATPase subunit of the terminase. Phylogenetic analysis of this gene has led to the current classification of herpesviruses by the International Committee on Taxonomy of Viruses (ICTV). The order Herpesvirales is now composed of three viral families, namely the families Herpesviridae, Alloherpesviridae and Malacoherpesviridae. Until now, herpesviruses of amniotes (reptiles, birds and mammals) have been grouped in the family Herpesviridae. Herpesviruses of molluscs are encompassed within the family Malacoherpesviridae. Finally, herpesviruses of fish and amphibians are classified in the family Alloherpesviridae. The family Alloherpesviridae contains four genera with 13 virus species currently accepted by the ICTV (Table 1). The alloherpesviruses of amphibians (Ranid herpesvirus 1; 2 and 3 [RaHV]) belong to the genus Batrachovirus, while fish alloherpesviruses have been clustered into three genera. The genus Cyprinivirus comprises the alloherpesviruses of cyprinids (Cyprinid herpesvirus 1; 2 and 3 [CyHV]) and that of eel (Anguillid herpesvirus 1 [AngHV]). The genus Ictalurivirus includes the alloherpesviruses isolated from catfish (Ictalurid herpesvirus 1 and 2 [IcHV]) and sturgeon species (Acipenserid herpesvirus 2 [AciHV]), while the genus Salmonivirus consists of herpesviruses of salmonids (Salmonid herpesvirus 1; 2 and 3 [SalHV]) (Fig. 1(A)). Interestingly, viruses of the genera Cyprinivirus and Ictalurivirus are able to infect fish from two different superorders (Elopomorpha, Ostariophysi) or subclasses (Chondrostei, Neopterygii), respectively. This observation supports a more recent evolution of the family Alloherpesviridae, associated with a lesser degree of coevolution of some alloherpesviruses with their hosts. In addition to the accepted viral species, more than 10 putative alloherpesviruses were detected by PCR from a wide range of fish species (Table 1). Unfortunately, only partial genome sequences are available. The official taxonomical classification of these viruses is still pending. However, according to phylogenetic analyses, these viruses undoubtedly belong to the family Alloherpesviridae. Some of them clearly cluster into already existing genera (Fig. 1(B)), e.g., SalHV-4 and SalHV-5 are likely going to expand the genus Salmonivirus. Other viruses (e.g., AciHV-1; EsHV-1) cannot be grouped with viruses of any existing genera (Fig. 1(B)). Further genomic data are needed from these virus species in order to establish their evolution and taxonomic classification. The

306

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20931-X

Fish and Amphibian Alloherpesviruses (Herpesviridae)

Table 1 Genus

Classification of fish and amphibian alloherpesviruses according to the ICTV (Order Herpesvirales, Family Alloherpesviridae) Viral name

Viral acronym Alternative viral name (s)

Susceptible host (s)

Ranid herpesvirus 1

RaHV-1

Lucké tumor herpesvirus

Ranid herpesvirus 2

Ranid herpesvirus 2

RaHV-2

Frog virus 4

Ranid herpesvirus 3

Ranid herpesvirus 3

RaHV-3

Northern leopard frog (Lithobates pipiens), green frog (L. clamitans), pickerel frog (L. palustris) Northern leopard frog (Lithobates pipiens) Common frog (Rana temporaria)

Anguillid herpesvirus 1

Anguillid herpesvirus 1

AngHV-1

European eel herpesvirus

Cyprinid herpesvirus 1 Cyprinid herpesvirus 2

Cyprinid herpesvirus 1 Cyprinid herpesvirus 2

CyHV-1 CyHV-2

Cyprinid herpesvirus 3a

Cyprinid herpesvirus 3

CyHV-3

Carp pox herpesvirus Goldfish haematopoietic necrosis virus Koi herpesvirus

Viral species (ICTV list)

Batrachovirus Ranid herpesvirus 1a

Cyprinivirus

Ictalurivirus

Acipenserid herpesvirus 2 Acipenserid herpesvirus 2 AciHV-2

White sturgeon herpesvirus 2 Channel catfish virus

Ictalurid herpesvirus 1a

Ictalurid herpesvirus 1

IcHV-1

Ictalurid herpesvirus 2

Ictalurid herpesvirus 2

IcHV-2

Ictalurus melas herpesvirus

Salmonivirus Salmonid herpesvirus 1a

Salmonid herpesvirus 1

SalHV-1

Herpesvirus salmonis

Salmonid herpesvirus 2

Salmonid herpesvirus 2

SalHV-2

Salmonid herpesvirus 3

Salmonid herpesvirus 3

SalHV-3

N/A

Acipenserid herpesvirus 1 AciHV-1

Oncorhynchus masou herpesvirus Epizootic epitheliotropic disease virus White sturgeon herpesvirus 1

N/A N/A N/A N/A N/A

Bufonid herpesvirus 1 Cyprinid herpesvirus 4 Esocid herpesvirus 1 Gadid herpesvirus 1 Percid herpesvirus 2

BfHV-1 CyHV-4 EsHV-1 GaHV-1 PeHV-2

N/A

Salmonid herpesvirus 4

SalHV-4

N/A N/A

Salmonid herpesvirus 5 Silurid herpesvirus 1

SalHV-5 SiHV-1

Unclassified

307

Sichel herpesvirus Blue spot disease virus Atlantic cod herpesvirus European perch herpesvirus Atlantic salmon papillomatosis virus Namaycush herpesvirus

Japanese and European eel (Anguilla japonica and A. anguilla) Common carp (Cyprinus carpio) Goldfish (Carassius auratus), gibel carp (Carassius gibelio) Common carp (Cyprinus carpio) Sturgeon (Acipenser spp.) Channel catfish (Ictalurus punctatus), striped catfish (Pangasius hypophtalmus) Black bullhead catfish (Ameiurus melas) Rainbow trout (Oncorhynchus mykiss) Salmon and trout (Oncorhynchus spp.) Lake trout (Salvelinus namaycush) White sturgeon (Acipenser transmontanus) Common toad (Bufo bufo) Sichel (Pelecus cultratus) Northern pike (Esox lucius) Atlantic cod (Gadus morhua) European perch (Perca fluviatilis) Atlantic salmon (Salmo salar) Lake trout (Salvelinus namaycush) Glass catfish (Kryptopterus bicirrhis)

a

Designates the type species of each genus (ICTV, 2018).

complexity and precision of the current classification are likely to rapidly evolve in the future based on the description of new alloherpesvirus members. Finally, many fish species have experienced integration of alloherpesvirus sequences fused with transposons into their genomes.

Virion Structure In terms of virion structure, alloherpesviruses are no exception to the remarkably conserved and morphologically distinct architecture found throughout the Herpesvirales order. It was demonstrated, for example, that the nucleocapsid of IcHV-1 was strikingly similar to that of human herpesvirus 1 (HHV-1 also known as herpesvirus simplex 1). The structure of herpesviruses is, from the center outwards, as follows: (i) a densely packed genome consisting of a single copy of linear dsDNA encapsulated in an icosahedral nucleocapsid (T ¼ 16) with a diameter of 100–130 nm; (ii) an amorphous proteinaceous tegument layer; and (iii) a lipid bilayer envelope acquired from the host and bearing various viral glycoproteins.

Genome Until now, 11 complete alloherpesvirus genomes have been sequenced and partial genome sequences are available from 11 more viruses. The full-length genomes of SiHV-1 (potential ictalurivirus) and BfHV-1 (potential batrachovirus) were released recently.

308

Fish and Amphibian Alloherpesviruses (Herpesviridae)

Fig. 1 Phylogenetic tree of the family Alloherpesviridae. The analyses were based on the Bayesian analysis (WAG amino acid model) of the deduced amino acid sequences of DNA polymerase genes (134 and 113 amino acid residues, respectively for panel A and B). High statistical values confirm the topology of the trees. The four main genera within the family Alloherpesviridae are designated by different colored lines on the trees. Panel A: phylogenetic tree of the classified viral species in the Alloherpesviridae family. Panel B: phylogenetic tree of the classified and unclassified potential members of the Alloherpesviridae family. AciHV: acipenserid herpesvirus; AngHV: anguillid herpesvirus; BfHV: bufonid herpesvirus; CyHV: cyprinid herpesvirus; EsHV: esocid herpesvirus; GaHV: gadid herpesvirus; IcHV: ictalurid herpesvirus; PeHV: percid herpesvirus; RaHV: ranid herpesvirus; SalHV: salmonid herpesvirus; SiHV: silurid herpesvirus. GenBank and RefSeq accession numbers: AciHV-1: EF685903; AciHV-2: FJ815289; AngHV-1: NC_013668; BfHV-1: MF143550; CyHV-1: NC_019491; CyHV-2: NC_019495; CyHV-3: NC_009127; CyHV-4: KM357278; EsHV-1: KX198667; GaHV-1: HQ857783; IcHV-1: NC_001493; IcHV-2: NC_036579; PeHV-2: MG570129; RaHV-1: NC_008211; RaHV-2: NC_008210; RaHV-3: NC_034618; SalHV-1: EU349273; SalHV-2: FJ641908; SalHV-3: EU349277; SalHV-4: JX886029; SalHV-5: KP686090; SiHV-1: MH048901.

Fish and Amphibian Alloherpesviruses (Herpesviridae)

Table 2

309

Data on complete genome sequences of fish and amphibian alloherpesviruses

Viral name

Genome size (kbp)

Genome structure

Structure sizes (kbp)

GC%

ORFs (No.)a

Genbank accession number

Anguillid herpesvirus 1 Cyprinid herpesvirus 1 Cyprinid herpesvirus 2 Cyprinid herpesvirus 3 Ictalurid herpesvirus 1 Ictalurid herpesvirus 2 Silurid herpesvirus 1 Ranid herpesvirus 1 Ranid herpesvirus 2 Ranid herpesvirus 3 Bufonid herpesvirus 1

248.53 291.14 290.3 295.15 134.23 142.92 149.34 220.86 231.8 207.91 158.25

TR-U-TR TR-U-TR TR-U-TR TR-U-TR TR-U-TR TR-U-TR TR-U-TR TR-U-TR TR-U-TR U U

U: U: U: U: U: U: U: U: U: U: U:

53.0 51.3 51.7 59.2 56.2 53.8 53,7 54.6 52.8 41.8 40.6

134 143 154 163 90 91 94 132 147 186 152

NC_013668.3b NC_019491.1b NC_019495.1b NC_009127.1b NC_001493.2b NC_036579.1b MH048901.1 NC_008211.1b NC_008210.1b NC_034618.1b MF143550.1

226 TR: 11 224 TR: 33 260 TR: 15 250 TR: 22 97 TR: 18 101 TR: 20 100 TR: 25 219 TR: 0.6 230 TR: 1 208 158

a

Predicted to encode functional proteins. Includes ORFs duplicated in TR. Designates Reference Sequences (RefSeq) in the GenBank. Note: U: Unique region; TR: Terminal repeat; kbp: kilobase pair.

b

The genome size of alloherpesviruses range between 134 and 295 kilobase pairs (kbp) containing 90–186 Open Reading Frames (ORFs). There are significant differences in the genome size of viruses belonging to different genera. The (potential) ictaluriviruses have the smallest genomes ranging from 134 to 149 kbp, while the estimated genome size of salmoniviruses is around 170 kbp. The genomes of the (potential) batrachoviruses are comprised within the range 158–232 kbp, and the largest genomes are presented by cypriniviruses ranging from 245 to 295 kbp (Table 2). The genome of CyHV-3 is the largest of all herpesviruses sequenced so far. The structure of alloherpesvirus genomes is simpler than that of most of the Herpesviridae. Most of their genomes contain only one unique region which is flanked by direct terminal repeats (TR). On the contrary, several of the members of the Herpesviridae family are more complex and composed of one long region associated with one or sometimes two short unique regions flanked by internal and terminal repeats. Fish alloherpesviruses have long TR, while frog herpesviruses have very short or even no detectable TR (Table 2). Alloherpesviruses differ from the members of the Herpesviridae family in the number of conserved genes. The latter have a subset of about 40 convincingly conserved genes in members of the family Herpesviridae. While alloherpesvirus genomes share 40–60 homologous genes within a genus, there are only 12 genes which have homologs in all completely sequenced alloherpesvirus genomes. These encode major structural proteins and enzymes involved in replication and packaging of viral DNA into virions.

Life Cycle Infection begins with viral entry, where surface glycoproteins attach to cellular receptors, leading to cell penetration. Two nonexclusive mechanisms of viral penetration have been described in the Herpesviridae family, i.e., direct fusion with the plasma membrane, or endocytosis followed by fusion of the viral envelope with the endosome membrane, both resulting in the release of the nucleocapsid, coated by tegument proteins, into the cytoplasm (Fig. 2). Nevertheless, the entry mechanisms of alloherpesviruses are still poorly known. Retrograde transport ensues along microtubules to the nuclear pores, where the viral linear genome is delivered into the nucleus. After circularization of the genome, cellular transcription machinery is used for expression of viral genes as a cascade, starting with immediate early genes, followed by early and late categories. As a result of early gene expression, replication of virus DNA is mediated by viral DNA polymerase, leading to the synthesis of branching concatemers. Finally, late gene expression results in the generation of structural proteins that assemble in the nucleus as capsids. Concatemers are then cleaved before the viral genomes are loaded into capsids. Nuclear egress begins with primary envelopment whereby budding at the inner nuclear membrane occurs, with release into the perinuclear space. From there, nucleocapsids exit the nucleus by fusion of the primary envelope with the outer nuclear membrane (de-envelopment). Naked capsids then get to sites of virion assembly and bud into vesicles belonging to the trans-Golgi network, where tegument addition and secondary envelopment with acquisition of the glycoprotein-studded envelope arise. Lastly, enveloped virions undergo egress via exocytosis. Increasing evidence points to alloherpesviruses displaying the capacity, as found in the Herpesviridae family, to establish latent infections. The balance between productive and latent infection is intricately linked to host biology, as latency may be interrupted by reactivation that reverts to a productive infection. In alloherpesviruses, water temperature could be a key factor in switching between productive and latent infections.

Epidemiology Fish Alloherpesviruses Alloherpesviruses share the general tendency of herpesviruses to possess a narrow host range, although limited infection can sometimes occur in non-natural host species. For example, CyHV-1 is usually restricted to common carp but was detected in

310

Fish and Amphibian Alloherpesviruses (Herpesviridae)

Fig. 2 Schematic representation of herpesvirus productive life cycle. Schematic representation and electron microscopy examination of CyHV-3 virion (upper left panel, bar represents 100 nm). Typical replication cycle of herpesviruses. Diagrammatic representation of the herpesvirus replication cycle, including virus entry and dissociation of the tegument, transport of incoming capsids to the nuclear pore, and release of viral DNA into the nucleus. Capsid assembly, DNA packaging, primary, secondary envelopment and egress are also illustrated. MTOC: Microtubule Organizing Center; ER: Endoplasmic Reticulum. Modified from Zeev-Ben-Mordehai, T., et al., 2014. Current Opinion in Virology 5 (100), 42–49. Electron microscopy picture adapted with permission from Mettenleiter, T.C., et al., 2009. Virus Research 143 (2), 222–234 Copyright Elesvier.

golden ide (Leuciscus idus) imported from Germany into North America. For CyHV-3, multiple vertebrate and invertebrate species have been identified as bearing viral DNA and being a potential epidemiological risk factor of viral spreading. The geographical distribution of alloherpesviruses is variable: for instance, whereas members of the Cyprinivirus genus are found worldwide, others have only been associated with restricted geographical areas and contexts. Such is the case for IcHV-2 that caused mass mortalities in black bullhead catfish farms in Italy in the late 1990s. In the case of CyHV-3, the first outbreaks in 1997 in Germany and its rapid worldwide distribution were attributed to international trade of common and koi carp and notably the methods used in koi carp competitions before awareness of this pathogen was commonplace. It can therefore be stated that the epidemiology of some alloherpesviruses is tightly interwoven with the precise use and trade of the domesticated host species. Deadly outbreaks of CyHV-3 have occurred in many natural habitats such as in some North American lakes, but have not caused any long-term decrease of common carp populations. Recently, the high virulence of CyHV-3 associated with its believed absence in Australia has led to the hypothesis of using this virus to control common carp populations which are considered as an invasive species in this country. However, the predicted safety and efficacy of this method have recently been under important scientific debate.

Amphibian Alloherpesviruses The real causes of alloherpesvirus emergence and spread in free-ranging amphibians are mostly unknown. However, the impact of human activities on their habitat and the climate may play a relevant role. Of the known batrachoviruses, RaHV-1 has been detected in free-ranging leopard frogs (Lithobates pipiens) in specific areas of the North American continent where, to the best of our knowledge, it has remained confined. The prevalence of RaHV-1 associated tumors ranged between 1% and 9% according to the collection site. A drastic reduction of the infected populations of leopard frogs was recorded in the 70's. Currently, no information is available concerning possible reemergence of the virus. Differently, RaHV-3 and BfHV-1 have been recorded in Europe only, in the common frog (Rana temporaria) and common toad (Bufo bufo), respectively. Their actual and current significance in the context of amphibian disease ecology is currently unknown.

Pathogenesis Fish Alloherpesviruses Fish alloherpesviruses are characterized by their temperature dependency, a consequence of the ectothermic nature of their hosts. The optimal temperature for viral replication and disease is variable among alloherpesviruses. Whereas AngHV-1 infection is

Fish and Amphibian Alloherpesviruses (Herpesviridae)

311

promoted by high temperatures (25°C), SalHV-3 outbreaks are reported when temperatures are low (8–10°C). In natural conditions, if the environmental water temperature is suboptimal for virus replication, the induced disease will be less severe or even asymptomatic, explaining the seasonal occurrence of alloherpesvirus diseases. Interestingly, expression of behavioral fever was described in common carp infected by CyHV-3. Studies of CyHV-3 pathogenesis mimicking natural conditions demonstrated that the skin is the major portal of entry of the virus after inoculation of carp by immersion, whereas the virus would penetrate through the pharyngeal periodontal mucosa following oral contamination. Viral spreading within the infected host is probably promoted by infected white blood cells allowing rapid spread in almost all tissues soon after inoculation. Horizontal transmission of fish alloherpesviruses can occur either directly between fish or indirectly, through several potential vectors (e.g., water, fish droppings). Salmoniviruses were detected in ovarian fluids, suggesting a possible vertical transmission. Vertical transmission was demonstrated for IcHV-1 after detecting the virus in offspring from IcHV-1 positive broodstocks. The hallmark of herpesviruses is their ability to establish latency in their host after initial productive infection. Some evidence of latency has been described for CyHV-1, CyHV-3, AngHV-1, SalHV-2 and IcHV-1 fish alloherpesviruses. The latency of alloherpesviruses in fish surviving the primary infection allows viral transmission to naïve fish upon disease reactivation. This aspect of the virus biology results in a certain spatial and temporal flexibility for viral maintenance in the host population.

Amphibian Alloherpesviruses Pathogenesis of the Batrachovirus associated disease is virtually unknown. RaHV-1 has been shown to be the etiologic agent of Lucké’s adenocarcinoma in leopard frogs. However, it is not known how the virus induces tumor development although environmental temperature may play a role. The disease could be reproduced by only infecting frogs during their larval stage with tumor extract. Viral replication appears to be dependent on low environmental temperatures, whereas tumor growth and metastasis would be promoted at higher temperatures. Regarding temperature and seasonality, the proliferative dermatitis associated with RaHV-3 and BfHV-1 are observed right after hibernation, during the mating season, between the end of winter and beginning of spring, when the environmental temperature is low. No virus-associated lesion has been reported during the later spring and summer. The molecular bases of the associated diseases are not known and Koch’s postulates have not been fulfilled for these two viruses.

Clinical Features Fish Alloherpesviruses Clinical aspects induced by fish alloherpesviruses can vary substantially among outbreaks or individuals. Indeed, variations are observed in the severity of clinical signs according to diverse factors including, but not restricted to, those related to the environment (temperature, density of the population) and host (age of the individuals, genetics, immune status). Primary infection involves productive viral replication that may result in viremia and acute disease associated with high morbidity and mortality. In general, fish exhibit non-specific behavioral changes including lethargy, hyperexcitability, erratic swimming or gasping at the water surface. Diseases associated with infection of naïve individuals by alloherpesviruses can include epithelial lesions, diffuse multisystemic hemorrhages, ascites, kidney and liver necrosis (Fig. 3(A), Table 3). Contrarily to their Herpesviridae family counterparts, alloherpesviruses usually induce severe diseases in their hosts. This may result from a milder degree of coevolution with their respective hosts. Alloherpesvirus infections might nevertheless be artificially enhanced by aquaculture methods. Among the most important fish alloherpesviruses, CyHV-2, CyHV-3 and IcHV-1 cause a systemic and lethal disease (Fig. 3(A); Table 3) characterized by mass outbreaks associated with high mortalities. During the summer, when temperature is optimal for the occurrence of IcHV-1 disease, 90% of fry and fingerlings may die in less than two weeks. During CyHV-3 infection, infected individuals may begin to die at 6–8 days post infection. Mortality rates are in general up to 70%–80% of infected individuals but they can reach 100% in some cases. Mass mortalities within 24 h were also recorded in CyHV-2 infected gibel carp. CyHV-1, IcHV-2, AciHV-2, SalHV-2 and SalHV-3 primary infections can also induce lethal diseases. Development of a benign epithelial hyperplasia, also called herpetic lesion or papilloma is another clinical pattern largely observed in alloherpesvirus infections. This clinical feature can be observed during primary productive infection with AciHV-2, SalHV-3 and SalHV-4 (Fig. 3(B)). In addition, this lesion can arise during recrudescence of a previous infection. Indeed, while CyHV-1 and SalHV-2 primary infection is typically characterized by an acute systemic disease, the recrudescence of the disease in survivors is associated with epithelial tumors. Papillomas associated with CyHV-1 infection appear 5–6 months after primary exposure in most survivors and are more commonly observed on the skin (body, fins, mouth) and at the site of intraperitoneal inoculation. Similarly, epitheliomas in SalHV-2 disease are observed around the mouth, at the caudal fin, operculum, and the body surface. These lesions may persist for one year. Experimental challenges show that tumor occurrence varies between species (12%, 40%–60%, and 100% in rainbow trout, chum salmon and masu salmon, respectively).

Amphibian Alloherpesviruses RaHV-1 causes adenocarcinomas in leopard frogs (Lucké’s adenocarcinoma). The tumor can grow extensively and metastasize, involving and invading other organs. Papillary projections are often observed in the neoplastic mass. Differently, the only reported

312

Fish and Amphibian Alloherpesviruses (Herpesviridae)

Fish and Amphibian Alloherpesviruses (Herpesviridae)

313

lesion associated with RaHV-2 is the occurrence of severe edema in frogs infected at larval stages. RaHV-3-associated disease is characterized by multifocal to coalescent gray skin patches scattered throughout the integument of affected frogs, which correspond to areas of prominent epidermal hyperplasia during microscopic examination (Fig. 3(B)). Large numbers of intranuclear eosinophilic inclusions are seen in the thickened epidermis. Toads infected with BfHV-1 show similar but distinct skin lesions. Relatively flat to slightly elevated, dark brown, cauliflower-like skin proliferations are observed grossly. Histologically, the lesions correspond to areas of epidermal hyperplasia, frequently associated with hyperkeratosis. Intranuclear inclusions are seen in infected toads, but less numerous and prominent than in frogs infected with RaHV-3. Interestingly, minimal to no associated inflammation is observed in both infections.

Diagnosis Diagnosis of alloherpesvirus infection can rely on various methods. First, diagnosis can be oriented by analysis of the clinical signs, anatomopathological and histopathological lesions. However, the latter are only indicative of the alloherpesvirus infection due to the absence of known pathognomonic signs and the high frequency of multipathogen co-infection. Still, hyperplastic epidermal lesions are typical and very indicative of some alloherpesvirus infections (SalHV-4, CyHV-1, RaHV-3, BfHV-1). Specific diagnosis involves the use of viral isolation using specific cell lines, viral detection using molecular methods or serological methods. Viral isolation of most alloherpesviruses has been achieved using cell lines. Nevertheless, viral isolation is still restricted by the absence of permissive cell lines: for example, among batrachoviruses, only RaHV-2 has been successfully isolated in cell culture. Specific molecular methods are now available for detection of most alloherpesviruses (PCR, qPCR, monoclonal and polyclonal antibodies). These methods can be tentatively used to diagnose productive, but also potentially latent infection (PCR, qPCR). Regarding the latter, the specific sites of alloherpesvirus latency are frequently unknown. Viral DNA can be detected in the brain and B lymphocytes of fish previously exposed to CyHV-3. Note that the sensitivity of the molecular tools developed could be decreased by the limited knowledge about genetic coalescence within each alloherpesvirus species. Detection of the antibody response against alloherpesviruses (ELISA, seroneutralization) can help to diagnose a previous (latent) infection (e.g., screening of broodstocks). Nevertheless, interpretation should be cautious, as specific antibody half-life is rather short in fish. Specific antibodies against CyHV-3 can barely be detected from sera of fish 280 days post infection. Finally, on-site diagnostic methods including lateral flow devices and loop-mediated isothermal amplification have been developed for some alloherpesviruses.

Management and Treatment Alloherpesviruses are not equally threatening the aquaculture sector. Some alloherpesvirus infections, like those induced by SalHV-1, were found to have limited impact on aquaculture. Other alloherpesviruses (e.g., IcHV-1), can be controlled by management procedures (screening of broodstocks, disinfection of facility and incoming water, microbiological independence of produced fish batches), improving rearing conditions (fish density, water quality, stress reduction) or by regulating the water temperature. As explained above, alloherpesvirus replication and disease expression correlates strongly with environmental temperature. This peculiarity has been exploited for the treatment of alloherpesvirus infection by changing environmental temperature to levels suboptimal or non-permissive for viral replication. For example, fish infected by CyHV-3 can be transferred to non-permissive Fig. 3 Illustration of typical clinical features observed during some alloherpesvirus infection. Panel A. Illustration of clinical features induced by fish alloherpesviruses. (a) channel catfish (Ictalurus punctatus) infected by IcHV-1, typical exophthalmia and distended abdomen caused by ascite. (b) Multiple plaques of epidermal proliferation (arrows) in a fingerling of Siberian sturgeon (Acipenser baeri) infected AciHV-2. (c) Severe papillomas caused by SalHV-4 in wild young Atlantic salmon (Salmo salar) captured at 10°C. (d) Proliferative cutaneous lesions in adult koi carp (Cyprinus carpio) infected by CyHV-1, multifocal to coalescing, slightly raised to nodular, white cutaneous proliferations along the dorsum, mouth, and pectoral fins. (e) Koi carp (Cyprinus carpio) infected by CyHV-3, extensive circular necrosis of the skin covering the body (black arrows), fin erosion (white arrowheads), and hemorrhages on the skin and fins (black arrowheads). (f) Diseased cultured European eel (Anguilla anguilla) with AngHV-1 infection. The eel shows a patchy pattern of hemorrhages in the skin, and severe hemorrhagic fins. Panel B. Illustration of clinical features induced by amphibian alloherpesviruses. (g) Multifocal to coalescent light tan to gray patches of skin are present, more frequently on the dorsum and along the flanks of a common frog (Rana temporaria) (black arrows). (h) Bufonid herpesvirus 1 infected common toad (Bufo bufo) associated gross skin lesions. Multifocal raised, dark brown, patchy areas of skin are scattered over the dorsum of the toad (black arrows). (i) Skin, Common frog (Rana temporaria) naturally infected with Ranid herpesvirus 3. The epidermis is focally, severely elevated (hyperplastic), forming a papillary projection (arrowhead) characterized by several clear spaces (epidermal vacuoles) partially filled with degenerating and necrotic keratinocytes. The underlying dermis shows loosely arranged collagen fibers (edema-two asterisks) and variably ectatic mucous glands (three asterisks). (j) Skin, Common toad (Bufo bufo) naturally infected with Bufonid herpesvirus 1. The epidermis is diffusely thickened (hyperplasia) up to 5 times as normal. Among the several nuclei are a number of intranuclear eosinophilic inclusions (inset, asterisks). The underlying dermis is characterized by loose collagen fibers (edema). (a) Courtesy of J. A. Plumb. (b) Adapted with permission from Shchelkunov, et al., 2009. Diseases of Aquatic Organisms 86 (3), 193–203. Copyright Inter-Research. (c) Adapted with permission from Doszpoly, et al., 2013. Diseases of Aquatic Organisms 107 (2), 121–127. Copyright Inter-Research. (d) Adapted with permission from Crossland, et al., 2018. Journal of Aquatic Animal Health 30 (3), 185–190. Copyright John Wiley and Sons. (e) Adapted from Michel et al., 2010. Emerging Infectious Diseases 16 (12), 1835–1843. (f) Adapted from Haenen, O.L.M., et al., 2002. Bulletin of the European Association of Fish Pathologists 22 (4), 247–257. (g, i, j) Courtesy of F.C. Origgi. (h) Adapted from Origgi, et al., 2018. Scientific Reports 8 (1), 14737.

314

Fish and Amphibian Alloherpesviruses (Herpesviridae)

Table 3

Clinical features caused by some alloherpesviruses

Viral name Batrachovirus Ranid herpesvirus 1 Ranid herpesvirus 2 Ranid herpesvirus 3

Susceptible host

Clinical signs (non-exhaustive)

Mortality

Northern leopard frog (Lithobates pipiens) Northern leopard frog (Lithobates pipiens) Common frog (Rana temporaria)

Renal adenocarcinoma

Unknown

Unclear, severe edema when infected at larval stage

Unknown

Proliferative dermatitis

Unknown

Cyprinivirus Cyprinid herpesvirus 1 Common carp (Cyprinus carpio)

Systemic disease in fry, hyperplastic cutaneous proliferation (papillomas, pox-like lesions) in surviving/ older fish Cyprinid herpesvirus 2 Goldfish (Carassius auratus), gibel Lethargy, anorexia, pale patches and hypersecretion of mucus in the gills, hemorrhages on the body and gills carp (Carassius gibelio) Cyprinid herpesvirus 3 Common carp (Cyprinus carpio) Lethargy, anorexia, hypersecretion of mucus on the skin, hyperemia of the fins, skin fin & gill necrosis, fin erosion, hemorrhages on the skin, neurological symptoms Anguillid herpesvirus 1 Japanese and European eel Decreased growth rates, hemorrhages and ulcerative (Anguilla japonica and A. anguilla) lesions on the skin (head, beak and caudal abdominal surface) fin and gills Ictalurivirus Ictalurid herpesvirus 1

Ictalurid herpesvirus 2 Acipenserid herpesvirus 2

Channel catfish (Ictalurus punctatus), striped catfish (Pangasius hypophtalmus) Black bullhead catfish (Ameiurus melas) Sturgeon species (Acipenser spp.)

Salmonivirus Salmonid herpesvirus 1 Rainbow trout (Oncorhynchus mykiss) Salmonid herpesvirus 2 Salmon and trout (Oncorhynchus spp.)

Salmonid herpesvirus 3 Lake trout (Salvelinus namaycush)

Unclassified Acipenserid herpesvirus 1 Bufonid herpesvirus 1 Esocid herpesvirus 1 Salmonid herpesvirus 4

White sturgeon (Acipenser transmontanus) Common toad (Bufo bufo) Northern pike (Esox lucius) Atlantic salmon (Salmo salar)

Salmonid herpesvirus 5 Lake trout (Salvelinus namaycush)

Reduced growth rates, erratic swimming, distended abdomen, hemorrhages, exophthalmia Erratic swimming, hemorrhages Erratic swimming, lethargy, hyperplastic skin lesions, hyperemia of the ventral scutes, mouth and anus

Up to 87.5% (fry)

Up to 100% (all ages) Up to 100% (all ages)

Up to 10%

490% (fry and fingerling channel catfish), 30%–40% (striped catfish) High mortality (fry and juveniles) Up to 80% (juvenile white sturgeon), up to 100% (juvenile Siberian sturgeon)

Darkened pigmentation, exophthalmia, abdominal Low distention, haemorrhages in the fins, pale gills Lethargy, anorexia, darkened appearance, skin ulcers, Up to 100%. High losses in focal epithelial proliferation on the mouth and body, juveniles exophthalmia Epithelial tumors (papilloma), preferably on the mouth, in surviving fish Lethargy, swim near the surface, behavioral Up to 100%. High losses in modification, mucoid patches on the skin, juveniles hemorrhages on eyes and jaw Few external clinical signs associated with a diffuse hyperplastic dermatitis Proliferative dermatitis Whitish circular plaques on the fins and dorsal parts White plaques of epithelial tumors (papilloma) on skin, or caudal peduncle Abnormal swimming, discolored musculature

Up to 35% (juvenile white sturgeons) Unknown NA 450% when secondary infections involved

water temperatures (430°C), allowing fish to clear viral infection and develop an adaptive immune response. This method was also used as a natural immunization method following iatrogenic CyHV-3 contamination. Unfortunately, fish infected with these methods may become latently infected by CyHV-3. Interestingly, fish infected by CyHV-3 have been shown to naturally seek for warmer environments (behavioral fever), thereby surviving viral infection; this feature could be exploited in aquaculture. The specific temperature range allowing alloherpesvirus replication and disease is variable among viral species. For instance, eel farms experiencing AngHV-1 infection usually decrease environmental temperature to decrease clinical signs and mortality. Antiviral treatments were tested against alloherpesviruses, but to the best of our knowledge, no commercial treatment is currently available. Extracts of the Clinacanthus nutans (Burm. f.) Lindau medicinal plant used as a prophylactic or therapeutic treatment against CyHV-3 infection showed positive effects on common carp survival. Nucleoside analogues such as acyclovir are able to limit IcHV-1, CyHV-3 or SalHV-2 replication in cell culture, and to be effective against SalHV-2 in vivo.

Fish and Amphibian Alloherpesviruses (Herpesviridae)

315

Prevention Facing the lack of efficient therapeutic methods, safe and efficacious vaccines represent the most promising control approach against alloherpesviruses. Vaccination of fish should involve delivery methods adapted to mass vaccination of the target species, minimizing the cost for vaccination and maximizing the duration of immunity with an early in life and limited number of vaccine administrations. Multiple vaccine candidates against alloherpesviruses have been studied but very few are commercially available nowadays. Inactivated vaccines consist of the entire killed viral particle, thus containing all structural viral antigens. Inactivated vaccine candidates have been described for SalHV-2, CyHV-2 and CyHV-3. Injection of inactivated CyHV-2 vaccines to gibel carp induced partial protection. A formalin-inactivated CyHV-3 vaccine, trapped within a liposomal compartment and used for oral administration, induced partial protection. The high amount of viral antigen required, the vaccine delivery method, and a generally low efficacy are the main concerns and restrictions for inactivated vaccine development. DNA vaccines consist of genetically engineered DNA plasmids encoding specific antigens from pathogens. DNA vaccines were tested against IcHV-1 and CyHV-3. Injectable DNA vaccines consisting of plasmids encoding CyHV-3 ORF25 envelope glycoprotein were shown to be partially efficacious against immersion challenge, but not against a cohabitation challenge. Attempts to deliver the DNA plasmids encoding ORF25 orally failed to induce an efficient protection. DNA vaccines against IcHV-1 showed equivocal results, even when cocktails of plasmid DNA encoding multiple ORFs were used. Studies on DNA vaccines against alloherpesviruses highlighted two main challenges for future research, namely selecting the best antigens for immunization and improving vaccine delivery methods. Attenuated vaccines consist of live viral strains with a virulence that has been reduced or suppressed by serial passages in cell culture (conventional attenuated vaccine) or by targeted viral genome editing (recombinant attenuated vaccine). Attenuated vaccines have the advantages of being compatible with mass vaccination and simulating a natural viral infection of the host. However, they raise safety concerns including residual virulence, reversion to virulence, and spread from vaccinated to naïve subjects. Conventional and recombinant attenuated vaccines were developed against IcHV-1 and CyHV-3. A conventional antiCyHV-3 attenuated vaccine has been developed by serial passages in cell culture and UV irradiation. This vaccine is commercialized in Israel for the vaccination of koi and common carp. The determinism of the attenuation is unknown, and consequently, reversions to a pathogenic phenotype cannot be ruled out. The attenuated strain has residual virulence for fish weighing less than 50 g. Due to scientific advances in molecular biology and molecular virology, the development of attenuated vaccines is evolving from empirical to rational design. A viral genome can be edited to delete genes encoding virulence factors in such a way that reversion to virulence cannot occur. This approach has been tested for CyHV-3 by targeting different genes such as the thymidine kinase (TK) and deoxyuridine triphosphatase. The previous cloning of IcHV-1 and CyHV-3 as infectious bacterial artificial chromosomes (BAC) allows viral mutagenesis. Recently, a vaccine candidate based on the double deletion of ORF56 and ORF57 was produced using BAC mutagenesis. This strain is safe for vaccinated fish, is limitedly transmitted to naive fish and induces full protection against a lethal cohabitation challenge. ORF57 was found to be the crucial virulence factor and is conserved in the genus Cyprinivirus. A recombinant attenuated vaccine candidate was also developed for IcHV-1 by deletion of the TK gene and was shown to be effective. Attenuated recombinant vaccines, associated with the tools provided by BAC mutagenesis, allow efficient development of vector vaccine strategies. Fish immunized with an IcHV-1 TK deleted recombinant attenuated strain expressing a reporter gene developed an antibody response against the transgene product.

Further Reading Adamek, M., Steinhagen, D., Irnazarow, I., et al., 2014. Biology and host response to Cyprinid herpesvirus 3 infection in common carp. Developmental and Comparative Immunology 43, 151–159. Boutier, M., Ronsmans, M., Rakus, K., et al., 2015. Cyprinid herpesvirus 3: An archetype of fish alloherpesviruses. In: Margaret Kielian, K.M., Thomas, C.M. (Eds.), Advances in Virus Research. Academic Press, pp. 161–256. Costes, B., Raj, V.S., Michel, B., et al., 2009. The major portal of entry of koi herpesvirus in Cyprinus carpio is the skin. Journal of Virology 83, 2819–2830. Hanson, L., Dishon, A., Kotler, M., 2011. Herpesviruses that infect fish. Viruses 3, 2160–2191. Hanson, L., Doszpoly, A., van Beurden, S.J., de Oliveira Viadanna, P.H., Waltzek, T., 2016. Chapter 9 – Alloherpesviruses of fish. In: Kibenge, F.S.B., Godoy, M.G. (Eds.), Aquaculture Virology. San Diego: Academic Press, pp. 153–172. Ilouze, M., Dishon, A., Kotler, M., 2006. Characterization of a novel virus causing a lethal disease in carp and koi. Microbiology and Molecular Biology Reviews 70, 147–156. Marshall, J., Davison, A.J., Kopf, R.K., et al., 2018. Biocontrol of invasive carp: Risks abound. Science 359, 877. McColl, K.A., Cooke, B.D., Sunarto, A., 2014. Viral biocontrol of invasive vertebrates: Lessons from the past applied to cyprinid herpesvirus-3 and carp (Cyprinus carpio) control in Australia. Biological Control 72, 109–117. Origgi, F.C., Schmidt, B.R., Lohmann, P., et al., 2018. Bufonid herpesvirus 1 (BfHV1) associated dermatitis and mortality in free ranging common toads (Bufo bufo) in Switzerland. Scientific Reports 8, 14737. Rakus, K., Ronsmans, M., Forlenza, M., et al., 2017. Conserved fever pathways across vertebrates: A herpesvirus expressed decoy TNF-α receptor delays behavioral fever in fish. Cell Host and Microbe 21, 244–253. Van Beurden, S., Engelsma, M., 2012. Herpesviruses of fish, amphibians and invertebrates. In: Magel, G.D., Tyring, S. (Eds.), Herpesviridae – A Look into this Unique Family of Viruses. Rijeka: InTech, pp. 217–242.

Fish Retroviruses (Retroviridae) TA Paul, RN Casey, PR Bowser, and JW Casey, Cornell University, Ithaca, NY, United States J Rovnak and SL Quackenbush, Colorado State University, Fort Collins, CO, United States r 2021 Elsevier Ltd. All rights reserved. This is a reproduction of T.A. Paul, R.N. Casey, P.R. Bowser, et al., Fish Retroviruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00670-1.

Glossary Cytopathic effect Morphologic changes in cells resulting from virus replication. Dysplasia An abnormality in the appearance of cells and indicative of a preneoplastic change. Hyperplasia An increase in the number of cells of an organ or tissue. Leiomyosarcoma Neoplasia of smooth muscle cells.

Malpighian cells Cells of the deepest layer of the epidermis. Neoplasia An abnormal growth of cells. Oncogene Cellular or viral gene whose products are capable of inducing a neoplastic phenotype. Proto-oncogene A normal cellular gene which when inappropriately expressed or mutated becomes an oncogene.

Introduction Retroviruses have been documented in a wide range of vertebrate species including lower vertebrates such as frogs, snakes, sharks, fish, birds, and turtles. To date, however, the majority of these reported sequences represent only partial fragments of the retrovirus. In recent years, the complete genomic sequence of exogenous fish retroviruses from Atlantic salmon (Salmo salar), walleye (Sander vitreus), and snakehead (Ophicephalus striatus), and an endogenous retrovirus of zebrafish (Danio rerio) have been determined. Analysis of these viral genomes indicates a high degree of diversity among the fish retroviruses as well as several unique features compared to mammalian and avian retroviruses. The etiological relationship between retroviruses and cancer is well established in mammalian and avian systems. Simple retroviruses can promote cell proliferation by inducing the ectopic expression of captured cellular oncogenes (viral transduction for acutely transforming viruses) in which an oncogene recombines into the viral genome, or by integration near or within the coding sequence of cellular proto-oncogenes (nonacute transforming viruses). Transcription factors encoded by some complex retroviruses, like human Tcell leukemia virus-1 (HTLV-1), also have nonacute oncogenic potential by deregulating cellular pathways controlling cell proliferation. Like their mammalian and avian counterparts, many fish neoplastic/proliferative diseases are suspected of having a retroviral etiology. Retroviruses are reported to be associated with 13 spontaneous proliferative diseases of fish based on the observation of retrovirus-like particles and, in some cases, reverse transcriptase activity in neoplastic lesions. Seven of these diseases with putative viral etiologies display a seasonal cycle, that is, they develop and regress annually. Retrovirus-associated tumors of the skin have received the most attention, since they are most visible and easily sampled. Skin tumors with suspected viral etiologies have been found in white sucker (Castostomus commersoni), walleye, yellow perch (Perca flavescens), and European smelt (Osmerus eperlanus). Retroviruses have also been linked to lymphoma in northern pike (Esox lucius), leukemia in chinook salmon (Oncorhynchus tshawytscha), and leiomyosarcoma of the swimbladder in Atlantic salmon. One noteworthy piscine retrovirus, the snakehead fish retrovirus (SnRV), has not been associated with tumor induction. In most of these systems, the etiologic relationship of retroviral infections and neoplasms rests on circumstantial evidence such as observations of viral particles by electron microscopy and the presence of reverse transcriptase activity. However, sequencing of fish retroviruses has facilitated use of molecular-based diagnostic reagents and, in some cases, shed light on potential modes of pathogenesis. In particular, the walleye retroviruses, walleye dermal sarcoma virus (WDSV) and walleye epidermal hyperplasia virus type 1 (WEHV-1) and type 2 (WEHV-2), express a subset of accessory genes exclusively during oncogenesis, specifically implicating the encoded proteins in the process of tumorigenesis.

Retroviruses and their Life Cycle Retrovirus particles are composed of viral structural and enzymatic proteins and two copies of the positive-sense single-stranded RNA genome. The proteins are surrounded by a lipid bilayer, acquired from the host cell, in which viral envelope glycoproteins are embedded. Retroviral infection is initiated when the surface glycoprotein binds to its cognate receptor on the surface of a susceptible cell. Upon fusion of the viral and cellular membranes, the viral core enters the cytoplasm where the virally encoded enzyme, reverse transcriptase, copies the RNA genome into DNA. The viral integrase protein binds reverse transcribed DNA and mediates its integration into the genomic DNA of the host. In rare cases, retroviral infection of host germ-line cells and subsequent integration into the chromosomes can establish retroviral sequences as heritable genetic elements, known as endogenous retroviruses.

316

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00670-6

Fish Retroviruses (Retroviridae)

317

Fig. 1 (a) Gross and histological images of walleye dermal sarcoma and (b) walleye epidermal hyperplasia.

Based on the complexity of the viral genome, retroviruses can be divided into two broad categories: simple and complex. Simple retroviruses, including members of the Gammaretroviruses and Alpharetroviruses, contain three genes: gag, pol, and env. Expression of a full-length unspliced messenger RNA (mRNA) and a spliced mRNA serve as the templates for translation of the retroviral structural and enzymatic proteins. These proteins are first translated as polyproteins from gag and pol, respectively, and are processed by a virally encoded protease. The viral envelope glycoprotein is encoded from a singly spliced viral transcript containing the env gene. Complex retroviruses are distinguished from simple retroviruses on the basis of their additional coding capacity and pattern of viral gene expression. This category of retroviruses includes Lentiviruses such as human immunodeficiency virus type 1 (HIV-1), Spumaviruses, and the Deltaretroviruses, human T-cell leukemia virus (HTLV-1), and bovine leukemia virus (BLV). Complex retroviruses produce multiply spliced transcripts that encode two classes of accessory/regulatory proteins. One class includes transcriptional regulatory proteins such as HTLV-1 Tax and HIV-1 Tat, which act in trans to directly regulate the activity of the viral promoter. A second class, including HTLV-1 Rex and HIV-1 Rev, act post transcriptionally, to facilitate transport of unspliced and singly spliced viral transcripts to the cytoplasm. The combined action of these proteins divides the replication cycle into two temporal phases: an early, regulatory phase, and a later, structural phase. During the regulatory phase, low levels of fully spliced transcripts encoding trans-activators increase levels of transcription leading to the accumulation of post-transcriptional regulatory proteins. These proteins allow production of unspliced and singly spliced mRNAs that encode the viral structural proteins. Both simple and complex retroviruses have been identified in fish species. Retroviruses isolated from walleye and snakehead fish resemble complex retroviruses based upon their genome structure and transcriptional profile, while simple retroviruses have been identified in Atlantic salmon and zebrafish.

Skin Tumors in Walleye and Their Associated Retroviruses Walleye dermal sarcoma (WDS) and walleye discrete epidermal hyperplasia (WEH) are neoplastic and hyperplastic skin lesions in walleye that have been etiologically associated with infection by three distinct retroviruses (Fig. 1). WDS and WEH were first reported in 1969 on fish from Oneida Lake in New York. Since then, WDS and WEH have been reported on walleyes throughout North America. These diseases have a seasonal cycle; tumor incidence is highest in the late fall and early spring months at frequencies of 27% and 5% for WDS and WEH, respectively. Lower tumor prevalence in summer months is associated with regression on individual adult fish. WDS appears as a cutaneous mesenchymal neoplasm arising multicentrically within the superficial dermis and overlaid with epidermis. Fall tumors appear highly vascularized due to a network of capillaries, while spring tumors are frequently white with ulcerated surfaces. Invasive tumors are rarely observed in feral fish. WEH lesions are benign mucoid-like plaques with distinct boundaries. They can range from 2 to 3 mm in diameter to large lesions with irregular borders that may be as large as 50 mm across. Histologically, this disease appears as an epidermal proliferation consisting primarily of Malpighian cells. Experimental transmission of WDS and WEH has been achieved with cell-free filtrates. Interestingly, only cell-free filtrates prepared from the spring, regressing WDS, not fall, developing WDS, are able to transmit disease. Infection of walleye fingerlings less than 9 weeks of age results in the frequent development of invasive tumors. Electron microscopic observation of retrovirus particles and cell-free transmission of both diseases suggest a retroviral etiology for WDS and WEH. WDSV was isolated from WDS tissue, and two independent retroviruses, WEHV1, WEHV2, were isolated from

318

Fish Retroviruses (Retroviridae)

12 kbp LTR

LTR

WDSV env

c gag

pro-pol

b a

env A-1 A-2 A-3 A-4 A-5 A-6 B WEHV1 env gag

a

pro-pol

b

c env A B WEHV2 c

env gag

pro-pol

a b

env A B-1 B-2 Fig. 2 Genomic and transcriptional maps of WDSV, WEHV1, and WEHV2. Genomic organization of viral DNA with retroviral genes and accessory genes orfa, orfb, and orfc is depicted under each virus heading. The envelope-spliced transcript (env) and additional spliced transcripts (A and B), which are capable of expressing accessory genes, are illustrated. The vertical dashed lines indicate the boundaries of the exons, introns, and open reading frames.

WEH lesions. Similar hyperplastic lesions on yellow perch in Oneida Lake are associated with two new retroviruses, perch epidermal hyperplasia virus types 1 and 2 (PEHV1, PEHV2).

Isolation and Sequencing of Retroviruses from WDS and WEH Lesions WDSV, WEHV1, and WEHV2 have been molecularly cloned and sequenced (Fig. 2). The genome structures of WDSV, WEHV1, and WEHV2 indicate large and complex viral genomes (12.7, 12.9, and 13.1 kbp, respectively). All three viruses have intact open reading frames (ORFs) capable of encoding the structural and enzymatic genes gag, pol, and env. gag (viral capsid) and pol (reverse transcriptase) are in the same reading frame and synthesized as a polyprotein through a termination suppression mechanism. pro (viral protease), responsible for cleaving the polyprotein, is located in the same reading frame as pol. A unique feature of all three viruses is the presence of additional orfs that have tentatively been called orfa, orfb, and orfc. The orfa and orfb genes are located between env and the 30 long terminal repeat (LTR), while orfc is located between the 50 LTR and the start of gag. Database searches with OrfA protein amino acid sequences suggest limited homology with D-type cellular cyclins within the cyclin-box motif, and are referred to as rv-cyclins. WEHV1 rv-cyclin has a 20% amino acid identity and 35% similarity with human cyclin D-3, whereas WEHV2 and WDSV rv-cyclin are most similar to human cyclin D-1 (22%/35% and 19%/29% amino acid identity/similarity). Similarities with known walleye and other piscine cyclins are not significantly greater. The WEHV cyclin homologs are 37% identical within the cyclin box motif and 21–28% identical with the WDSV cyclin. Sequence homologies between rv-cyclin and OrfB in the walleye retroviruses suggest they arose by a gene duplication event.

Fish Retroviruses (Retroviridae)

319

Roles of the WDSV Accessory Proteins in Pathogenesis Transcriptional mapping by RT-PCR and Northern blot analyses of developing and regressing WDS and WEH have demonstrated temporal gene expression profiles and complex splicing patterns analogous to those seen in the complex retroviruses of mammals. In developing WDS tumors, only low levels of multiply spliced subgenomic transcripts are detected. These transcripts predominantly contain the coding sequences for rv-cyclin and OrfB. Regressing spring tumors contain high levels of full-length and subgenomic transcripts as well as unintegrated viral DNA and transmissible virus. OrfC is likely encoded by the full-length transcript coincident with tumor regression. A detailed transcriptional analysis of WDSV showed an alternative splicing pattern of the orfa transcript (Fig. 2). In developing tumors, the orfa transcript contains the coding sequence for a full-length rv-cyclin protein that localizes to the nucleus of mammalian and walleye cells. Alternatively spliced forms of this transcript encode amino-terminal truncated forms of the rv-cyclin protein, which localize in the cytoplasm of cells. The different forms of rv-cyclin may play functionally different roles in developing and regressing tumors. A single, spliced transcript encodes the OrfB protein, which is predominantly localized in the cytoplasm in tumor explant cells, but is capable of shuttling into and out of the nucleus when expressed in piscine and mammalian cells. The OrfC protein from WDSV has been shown to localize to mitochondria and to disrupt mitochondrial function, which results in apoptosis. Only the full-length viral transcript found in regressing tumors is capable of encoding OrfC. Apoptotic cells are present in regressing, but not developing, WDS tumor sections. Therefore, the OrfC protein may be responsible for a direct viral mechanism of tumor regression. WDSV orfa and orfb are the only virus transcripts found in developing tumors, suggesting direct roles for rv-cyclin and OrfB in the process of tumorigenesis, and their oncogenic potential has been demonstrated by several experimental approaches. WDSV orfa supported growth of a yeast (Saccharomyces cerevisiae) strain conditionally deficient for the synthesis of the G1 to S cyclins that are necessary for cell cycle progression. WEHV orfa did not support yeast growth in this model. Transgenic expression of WDSV orfa in mice from a skin-specific promoter caused a moderate to severe squamous epithelial hyperplasia and dysplasia dependant on skin injury. This suggests that rv-cyclin is not oncogenic as a result of a ‘single-hit’ mechanism, but rather, that secondary genetic or epigenetic events (possibly wound repair) are necessary for tumor development and progression. A similar phenotype has been described in transgenic mice expressing v-jun oncogene from the H-2Kk major histocompatibility complex (MHC) class I antigen gene promoter. When expressed in mammalian or piscine cell culture, WDSV rv-cyclin localized in the nucleus and was found by co-immunoprecipitation to be associated with cyclin dependent kinase 8 and cyclin C, general transcription initiation factors, and RNA polymerase II transcription complexes. In piscine cells, rv-cyclin inhibited transcription from the WDSV promoter independent of cis-acting DNA sequences. However, rv-cyclin can activate other viral promoters in fish cells, and can activate the WDSV promoter in select mammalian cells; thus rv-cyclin activation and inhibition of transcription are dependent on both the promoter and the cell type. WDSV rv-cyclin contains a defined transcription activation domain in the carboxy end of the protein, and the isolated activation domain is capable of interacting with co-activators of transcription. WEHV rv-cyclins do not have a corresponding carboxy-region activation domain. WDSV rv-cyclin may exhibit oncogenic potential by differential regulation of host gene expression such as proto-oncogenes or tumor suppressors. Less is known about the capabilities of the OrfB protein, but initial studies indicate its direct association with the regulation of signal transduction pathways. It is concentrated, in tumor explant cells, at focal adhesions, and along actin stress fibers. Established OrfB-expressing lines are resistant to the chemical induction of apoptosis, suggesting a role in oncogenesis. The origins of the accessory genes of complex retroviruses are unclear. This includes the origins of the WDSV accessory genes encoding rv-cyclin, OrfB, and OrfC. In the case of rv-cyclin and OrfB, their extreme divergence from host cyclin sequences indicates that any transduction event was ancient and led, ultimately, to an exclusive, complex viral species. The OrfC protein, like the accessory proteins of other complex retroviruses, has no clear homology to host proteins.

Control of the Seasonal Cycle of Disease An interesting aspect of WDS is the control of the seasonal switch in viral gene expression and associated tumor regression. Potential modulators of viral gene expression include host immunity and endocrine activity (accompanying spawning) and environmental factors such as water temperature, physical trauma, and sunlight. A number of cis-acting elements important for transcription activation have been identified in the WDSV promoter. There is differential binding of proteins from developing and regressing tumor nuclear extracts to a 15 bp repeat region in the WDSV promoter. This element may be critical to the induction of high levels of virus expression. The WDSV rv-cyclin protein negatively regulates the WDSV promoter in tissue culture cells. Presumably, it is advantageous for the virus to have lower levels of gene expression during tumor development to avoid immune surveillance as well as the cytopathic effects associated with virus production. While rv-cyclin may function in the repression of virus expression during tumor growth, the host, environmental, or viral signals that switch on full virus expression are yet to be determined.

320

Fish Retroviruses (Retroviridae)

Fig. 3 Genomic and transcriptional map of SnRV. Predicted coding regions are shown as open or shaded boxes. The envelope-spliced transcript (env) and additional spliced transcripts (a, b, c), which are capable of expressing accessory genes, are illustrated. LP indicates the location of a predicted leader peptide. The vertical dashed lines indicate the boundaries of the exons, introns, and open reading frames.

Snakehead Retrovirus The SnRV was isolated from a productively infected cell line derived from a Southeast Asian striped snakehead fish. Cell culture supernatant from these infected cells demonstrated high levels of RT activity and the presence of type C-like retrovirus particles. Additionally, supernatant from the infected cell line induced cytopathic effects in cultures of a bluegill cell line (BF-2). Molecular approaches were utilized to identify and sequence the SnRV from infected cells. The large 11.2 kbp genome of SnRV contains intact coding regions for gag, pol, and env (Fig. 3). An arginine tRNA primer binding site used for reverse transcription initiation distinguishes SnRV from other retroviruses. The structure and transcriptional profile of SnRV suggests a complex expression pattern capable of encoding an ORF located between env and the 30 LTR and two very small ORFs termed ORF1 and ORF2. Within the leader sequence of SnRV resides a start codon located just upstream of the major splice donor site that could potentially encode a 14 amino acid peptide (LP). Expression of Env from a singly spliced transcript is predicted to utilize the LP initiation codon and fuses this 14 amino acid peptide in frame to downstream env sequences. A transcript with four exons and two initiator codons would encode ORF1 and ORF2 proteins. The 30 ORF would be expressed from a transcript containing three exons and would encode a protein of 24 kDa. Finally, the fourth spliced transcript may encode 30 ORF or possibly a LP-Env cytoplasmic domain (CD) fusion protein. The 30 ORF contains an N terminal acidic domain, cysteine residues, and a basic region, motifs commonly found in transcriptional activators. These small ORFs have no significant homology to any known proteins in the databases, and their role in the viral life cycle is unknown. No endogenous copies of SnRV have been identified in the snakehead genome and an uninfected cell line has been established. Although the presence of SnRV in wild populations has not been thoroughly examined, the virus has been independently isolated from two separate snakehead cell lines derived from whole fry tissue and from caudal peduncle tissue from juvenile fish. In all cases, fish from which these cell lines were derived appeared healthy.

Salmon Swimbladder Sarcoma-Associated Retrovirus An outbreak of neoplastic disease of the swimbladder of Atlantic salmon was first reported at a Scottish commercial marine fish farm in 1975. Affected salmon were sluggish and in poor condition. A viral etiology was suspected, and electron microscopic evaluation of tumors revealed the presence of budding viral particles with retroviral morphology. A second outbreak occurred between 1995 and 1997 in brood stock salmon held at the North Attleboro National Fish Hatchery in Massachusetts. The fish exhibited skin discoloration and hemorrhages on the fins and body and showed a general debilitation and lack of vigor. In May of 1997, significant mortality was noted at the facility such that 35% of the population was affected by late spring of 1998. All of the affected fish displayed swollen abdomens due to multinodular masses on internal and external surfaces of the swimbladder. In several cases, these multinodular masses occupied the entire swimbladder (Fig. 4). Histologic examination revealed that the tumors were composed of well-differentiated fibroblastic cells that were arranged in interlacing bundles, which were classified as leiomyosarcomas.

Isolation of a Retrovirus from Salmon Swimbladder Sarcoma An exogenous retrovirus, termed Atlantic salmon swimbladder sarcoma virus (SSSV), was initially identified in tumors by degenerate RT-PCR of tumor RNA, and the entire viral sequence completed by DNA sequencing of proviral DNA. In contrast to the complex walleye retroviruses, SSSV is a simple retrovirus. The viral genome contains ORFs capable of expressing gag, pol, and env (Fig. 5). Additionally, a short 25 amino acid leader peptide of unknown function is located upstream of the Gag-Pol polyprotein

Fish Retroviruses (Retroviridae)

321

Fig. 4 (a) Gross and (b) histological images of Atlantic salmon swimbladder sarcoma.

LTR

LTR11 kbp

SSSV LP

env gag

pro-pol env

Fig. 5 Genomic and transcriptional map of SSSV. Predicted coding regions are shown as open boxes. The envelope-spliced transcript (env) is illustrated. LP indicates the location of a predicted leader peptide. The vertical dashed lines indicate the boundaries of the exons, introns, and open reading frame.

ORF. SSSV differs from other simple retroviruses by not having related endogenous sequences in the host genome, and SSSV is the only retrovirus to use a methionine-tRNA as a plus-strand primer. Additionally, sequences in pol display homology to central polypurine tract regions identified in complex retroviruses. Central polypurine tracts facilitate the formation of a single-stranded DNA region called a central DNA flap as a result of reverse transcription priming from this internal site. In HIV-1, this feature plays a major role in complex retroviral replication and allows efficient infection of nondividing cells. It is intriguing that SSSV displays a high proviral copy number (greater than 30 copies per cell) with a polyclonal integration pattern in swimbladder tumors. SSSV must be capable of initiating multiple rounds of infection within the same cell. The specific mechanisms leading to the high copy number and its implications in the pathogenesis of disease are of significant interest for future research.

Sequence Comparisons of SSSV with Other Retroviruses A comparison of the sequence of SSSV viral proteins with other retroviruses indicates large regions of homology with mammalian type C and type D viruses in Pol and Env. Additionally, a 179 amino acid region in the C terminus of Gag and a 1064 amino acid region of Pro–Pol displays 23% and 33% identity to the related WDSV proteins, respectively. SSSV has striking homology to the sequence of the zebrafish endogenous retrovirus (ZFERV). BLAST analysis of the Gag, Pol, and Env ORFs of SSSV reveals a 25% identity with ZFERV over 533 amino acids within Gag, a 40% identity over 533 amino acids of Pol, and a 39% identity over 429 amino acids of Env.

Prevalence, Seasonality, and Transmission of SSSV A PCR diagnostic assay was developed using sequences within the pol gene of SSSV. Prevalence at the North Attleboro fish facility has been found to be 52% and 5% of a natural stock of Pleasant River salmon were found to harbor SSSV. Like other retroviral diseases of fish, observations suggest that salmon swimbladder sarcoma is seasonal. First, of the 34% fish mortality at the North Attleboro facility between 1997 and 1998, 57% occurred in June. Second, in the following year, the surviving salmon had an SSSV incidence (measured by PCR) that cycled with a peak in late summer to early winter and then diminished in late winter to early spring. The period of highest SSSV incidence correlated with salmon spawning runs in late fall.

322

Fish Retroviruses (Retroviridae)

SIV-agm HV-1

RSV

MMTV JSRV MPMV

HV-2

BLV

EIAV Visna

HTLV-1 FIV

HTLV-2

HFV

WEHV-2 WEHV-1

BFV

WDSV SnRV FELV GALV MLV

Xen-1 SSSV

0.2

ZFERV

Fig. 6 Unrooted phylogenetic tree of representative retroviruses based on an amino acid alignment of seven conserved domains in reverse transcriptase. Retroviruses are designated as follows: MPMV (Mason–Pfizer monkey virus), JSRV (Jaagsiekte sheep retrovirus), MMTV (mouse mammary tumor virus), RSV (Rous sarcoma virus), EIAV (equine infectious anemia virus), FIV (feline immunodeficiency virus), Visna (visna virus), HIV-2 (human immunodeficiency virus-2), HIV-1 (human immunodeficiency virus-1), SIV-agm (simian immunodeficiency virus-agm), HTLV-1 (human T-cell leukemia virus type 1), HTLV-2 (human T-cell leukemia virus 2), BLV (bovine leukemia virus), SSSV (salmon swimbladder sarcoma virus), SnRV (snakehead retrovirus), BFV (bovine foamy virus), HFV (human foamy virus), FeLV (feline leukemia virus), MLV (murine leukemia virus), GALV (gibbon ape leukemia virus), WDSV (walleye dermal sarcoma virus), WEHV-1 (walleye epidermal hyperplasia virus type 1), WEHV-2 (walleye epidermal hyperplasia virus type 2), ZFERV (zebrafish endogenous retrovirus), Xen-1 (Xenopus endogenous retrovirus-1).

Interestingly, WDSV prevalence, which peaks during late spring, also correlates with the time of spawning. This suggests that endocrine changes during spawning runs may be a critical factor in the observed seasonal variation in disease incidence.

Pathogenesis of SSSV The existence of SSSV sequences in association with an outbreak of the swimbladder sarcoma suggests a role for the retrovirus in pathogenesis of the disease. As a simple retrovirus with no transduced cellular oncogenes, one possible mechanism of tumorigenesis is the insertional activation of a cellular proto-oncogene. The high copy number of proviruses in tumors has made the analysis of insertional activation events difficult, but multiple insertions increase the likelihood of such a mechanism. It is also feasible that SSSV may express an oncogenic viral gene product. This mechanism of oncogenesis has been proposed for the Jaagsiekte sheep retrovirus (JSRV) in the induction of ovine pulmonary adenocarcinoma and in Friend spleen focus-forming virus (SFFV)-associated erythroid hyperplasia in mice. Additionally, the presence of common integration sites within JSRV-associated tumors suggests that insertional mutagenesis may act in concert with the envelope in tumor development. Thus, a multifactorial mechanism for the development of salmon swimbladder sarcoma must also be considered. In addition to sarcoma, a number of other distinct pathologies are associated with SSSV infection. These diseases are frequently debilitating and are, therefore, of significance to the aquaculture industry. Atlantic salmon at the North Attleboro hatchery, during the SSSV outbreak, presented with multifocal hemorrhages, sloughing of the epidermis, lethargy, wasting, and failure to mature sexually in addition to swimbladder tumors.

Zebrafish Endogenous Retrovirus Endogenous retroviruses have been identified in almost all vertebrate genomes, and most are defective due to mutations and deletions. An endogenous retrovirus, ZFERY with a genome of 11.2 kbp has been identified in the Tubingen stock of zebrafish, and contains intact coding regions for gag, pol, and env. The gag and pol genes are in the same reading frame. While the majority of endogenous retroviruses are transcriptionally silent because of mutation or methylation, ZFERV remains transcriptionally active. In addition to genomic transcripts that encode Gag and Pol proteins, an unusual multiply spliced env transcript is produced. Expression appears highest in the larval and adult zebrafish thymus, and no expression was detected in 2-day old embryos, suggesting that ZFERV expression may be tied to thymic development.

Fish Retroviruses (Retroviridae)

323

Phylogeny of Fish Retroviruses Retroviruses have been classified into seven genera based largely on highly conserved amino acid sequences in the retroviral reverse transcriptase gene. While the majority of the viral sequences employed in this classification represent mammalian and avian retroviruses, a new genus termed Epsilonretroviruses, representing the fish retroviruses WDSV, WEHV-1, WEHV-2, has been added to the most recent classification. As more retroviral sequences from lower vertebrates have been identified, it has become apparent that this classification scheme may be inadequate to represent the apparent diversity. Based on the unique characteristics of SnRV, including its genomic organization, tRNA primer, and complex transcriptional profile, the virus is yet to be definitively placed in the current classification. The large size of the genome, genetic organization, and presence of additional ORFs suggest that SnRV is closest to the spumaviruses and walleye retroviruses, but its limited sequence homology suggests SnRV is divergent from these groups. Phylogenetic analysis indicates that, while the walleye retroviruses cluster in a group representing the Epsilonretroviruses, SSSV and ZFERV appear to represent a new branch of piscine retroviruses between the walleye retroviruses and the Gammaretroviruses, a genera that includes the murine leukemia virus (MLV)-related retroviruses (Fig. 6). The SnRV appears quite divergent from the other fish retroviruses by its placement in a distinct branch near the Spumaviruses. This would suggest that SnRV is quite divergent from the genus Epsilonretrovirus and may represent yet another group of retroviruses. Interestingly, a more encompassing phylogenetic analysis using all known retroviral sequences from lower vertebrates, including partial endogenous retroviral pol fragments from Stickleback (Gasterosteus aculeatus), brook trout (Salvelinus fontinalis), Brown trout (Salmo trutta), freshwater whiting (Corogonus lavaretus), and puffer fish (Fugu rubripes), indicates that the majority of the fish viruses, excluding SnRV, cluster together with MLV-related viruses in a group separate from most non-MLV related mammalian retroviruses. This raises the possibility that some retroviral groups maybe restricted to particular vertebrate classes. However, it is evident from the diversity among the fish retroviruses, that there is a high degree of heterogeneity within this group.

Acknowledgment This research was supported in part by USDA grants 99–35204–7485 and 02–35204–12777 to J.W.C., National Oceanic and Atmospheric Administration award no. NA86RG0056 to the Research Foundation of State University of New York for New York Sea Grant to P.R.B., American Cancer Society grant RPG-00313–01-MBC to S.L.Q., and National Institutes of Health grant CA095056 to S.L.Q. T.A.P. was supported by National Institutes of Health training grant 5T32CA09682.

See also: Feline Leukemia and Sarcoma Viruses (Retroviridae). HIV Integrase Inhibitors and Entry Inhibitors. Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

Further Reading Bowser, P.R., Wolfe, M.J., Forney, J.L., Wooster, G.A., 1988. Seasonal prevalence of skin tumors from walleye (Stizostedion vitreum) from Oneida Lake, New York. Journal of Wildlife Diseases 24, 292–298. Coffin, J.M., Hughes, S.H., Varmus, H.E. (Eds.), 1997. Retroviruses. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Hart, D., Frerichs, G.N., Rambaut, A., Onions, D.E., 1996. Complete nucleotide sequence and transcriptional analysis of the snakehead fish retrovirus. Journal of Virology 70 (6), 3606–3616. Hernious, E., Martin, J., Miller, K., Cook, J., Wilkinson, M., Tristem, M., 1998. Retroviral diversity and distribution in vertebrates. Journal of Virology 72 (7), 5955–5966. Holzschu, D.L., Fodor, S.K., Quackenbush, S.L., et al., 1995. Nucleotide sequence and protein analysis of a complex piscine retrovirus, walleye dermal sarcoma virus. Journal of Virology 69 (9), 5320–5331. Lairmore, M.D., Stanley, J.R., Weber, S.A., Holzschu, D.L., 2000. Squamous epithelial proliferation induced by walleye dermal sarcoma retrovirus cyclin in transgenic mice. Proceedings of the National Academy of Sciences, USA 97 (11), 6114–6119. LaPierre, L.A., Casey, J.W., Holzschu, D.L., 1998. Walleye retroviruses associated with skin tumors and hyperplasias encode cyclin D homologs. Journal of Virology 72, 8765–8771. LaPierre, L.A., Holzschu, D.L., Bowser, P.R., Casey, J.W., 1999. Sequence and transcriptional analyses of the fish retroviruses walleye epidermal hyperplasia virus types 1 and 2: Evidence for a gene duplication. Journal of Virology 73 (11), 9393–9403. Paul, T.A., Quackenbush, S.L., Sutton, C., Casey, R.N., Bowser, P.R., Casey, J.W., 2006. Identification and characterization of an exogenous retrovirus from Atlantic salmon swimbladder sarcomas. Journal of Virology 80 (6), 2941–2948. Poulet, F.M., Bowser, P.R., Casey, J.W., 1994. Retroviruses of fish, reptiles, and molluscs. In: Levy, J.A. (Ed.), The Retroviridae, vol. 3. New York: Plenum, pp. 1–38. Quackenbush, S.L., Holzschu, D.L., Bowser, P.R., Casey, J.W., 1997. Transcriptional analysis of walleye dermal sarcoma virus (WDSV). Virology 237, 107–112. Rovnak, J., Hronek, B.W., Ryan, S.O., Cai, S., Quackenbush, S.L., 2005. An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter. Virology 342 (2), 240–251. Rovnak, J., Quackenbush, S.L., 2002. Walleye dermal sarcoma virus cyclin interacts with components of the mediator complex and the RNA polymerase II holoenzyme. Journal of Virology 76, 8031–8039. Shen, C.H., Steiner, L.A., 2004. Genome structure and thymic expression of an endogenous retrovirus in zebrafish. Journal of Virology 78 (2), 899–911.

Fish Rhabdoviruses (Rhabdoviridae) Gael Kurath, US Geological Survey, Western Fisheries Research Center, Seattle, WA, United States David Stone, Weymouth Laboratory, Centre for Environment, Fisheries and Aquaculture Science, Weymouth, United Kingdom r 2021 Elsevier Ltd. All rights reserved. This is an update of G. Kurath, J. Winton, Fish Rhabdoviruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00493-3.

Glossary Exophthalmia eyeball

Abnormal protrusion or bulging of the

Finfish Fish with fins, as opposed to shellfish Salmonid Fish in the family Salmonidae

Classification The family Rhabdoviridae currently has 18 genera accepted by the International Committee for Virus Taxonomy (ICTV), and three of those genera contain fish rhabdoviruses. In the genera Novirhabdovirus, Sprivivirus, and Perhabdovirus all viruses infect fish hosts, and there are no fish viruses in any of the other 15 rhabdovirus genera. In the overall phylogeny of the Rhabdoviridae the three fish virus genera are well separated from each other, and the novirhabdovirus genus occupies a position basal to all other genera (Fig. 1). The novirhabdovirus genus was established in 1998 and contains four viral species, as listed in Table 1. Two of these species contain infectious hematopoietic necrosis viruses (IHNV) and viral hemorrhagic septicemia viruses (VHSV), which are globally important fish pathogens that are well characterized in terms of molecular biology and genetic diversity, with many known genetic subgroups and strains within each species. Hirame rhabdoviruses (HIRRV) are also important pathogens but with a more restricted geographic and host range, and snakehead rhabdoviruses (SHRV) have not been extensively characterized. The sprivivirus and perhabdovirus genera were established more recently, in 2013 and 2012, respectively. There are two recognized species in the sprivivirus genus (Table 1). The species Carp sprivivirus contains a single virus, spring viraemia of carp virus (SVCV); another of the important fish pathogens that has multiple genetic subgroups distributed across the globe. The species Pike fry sprivivirus is comprised

Fig. 1 Phylogenetic analysis of 105 animal rhabdovirus sequences illustrating relationships among 14 established genera, two proposed genera, and several unassigned viruses. Viral species in the three fish rhabdovirus genera (see Table 1) are shown as individual taxa, while taxa within other genera are collapsed into triangles to conserve space. The three fish rhabdovirus genera are shown as colored branches, and an asterisk denotes the type species in each genus. Unlabeled branches and triangles represent individual viruses and proposed genera, respectively, that have not yet been accepted by the International Committee for Virus Taxonomy. The four genera of plant rhabdoviruses are not included here. The tree results from full length polymerase (L) amino acid sequences aligned and analyzed with gaps (2246 characters) under a Bayesian implementation of the Yule speciation model, using the LG substitution model and constant population size prior. Posterior probability (pp) values above 0.7 are shown for critical nodes, and taxa within each established genus clustered with pp of 1.0. Scale bar is substitutions per site. Tree provided by Dr. Rachel Breyta, University of Washington.

324

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20999-0

Fish Rhabdoviruses (Rhabdoviridae)

Table 1

325

Classification of fish rhabdoviruses into 3 genera and several unassigned virusesa

Genus Species Novirhabdovirus Salmonid novirhabdovirus Piscine novirhabdovirus Hirame novirhabdovirus Snakehead novirhabdovirus Sprivivirus Carp sprivivirus Pike fry sprivivirus

Perhabdovirus Perch perhabdovirus Sea trout perhabdovirus Anguillid perhabdovirus Unassigned

Virus(es)

General host rangeb

Infectious hematopoietic necrosis salmonid fish including rainbow trout virus (IHNV) Viral hemorrhagic septicemia virus diverse range of marine and freshwater species, rainbow trout (VHSV) olive flounder, ayu Hirame virus (HIRRV)

Geography

Genome

North America, Asia, GQ413939.1 Europe Europe, Asia, North KC778774.1 America Japan, Korea AF104985

Snakehead virus (SHRV)

snakehead

Southeast Asia

AF147498

Spring viremia of carp virus (SVCV) Pike fry rhabdovirus (PFR) Grass carp rhabdovirus (GrcRV) Tench rhabdovirus (TenRV)

wide range of cyprinid species

Europe, Asia, North America Europe

U18101

wide range of cyprinid species, pike and brown trout

JF872827 NC025376.1 KC113517.1

Perch rhabdovirus (PRV)

perch

Europe

JX679246

Lake trout rhabdovirus (LTRV)

brown trout varieties

Europe

AF434992

Eel virus European X (EVEX) Eel virus American (EVA)

eel

Europe, Asia

X827265

Starry flounder rhabdovirus

starry flounder

North American Pacific coast China China China

AY450644

Siniperca chuatsi rhabdovirus mandarin fish Chinese rice-field eel rhabdovirus Asian swamp eel Scophthalmus maximus turbot rhabdovirus

NC_008514.1 MH319839.1 HQ003891.1

a

virus listed under the 3 genera have been established as species by the ICTV (2018 taxonomy). Viruses listed as unassigned are examples of those that have been described in the literature but are not currently recognized by the ICTV. b formal classification for fish hosts: salmonid, Oncorhynchus and Salmo sp.; rainbow trout, Oncorhynchus mykiss; olive flounder, Paralichthys olivaceus; ayu, Plecoglossus altivelis; snakehead murrel, Channa striata; starry flounder, Platichthys stellatus; cyprinid, Cyprinidae sp.; pike, Esox lucius; brown trout, Salmo truttae; perch, Perca fluviatilis; eel, Anguilla sp.; mandarin fish, Siniperca chuatsi; Asian swamp eel, Monopterus albus; turbot, Scophthalmus maximus.

of three recognized viruses; pike fry rhabdovirus (PFRV), grass carp rhabdovirus (GrcRV) and tench rhabdovirus (TenRV). These viruses are confined in their geographical distribution and are generally considered to have less of a disease impact when compared to SVCV. The perhabdovirus genus has three virus species: Perch perhabdovirus, the type species which consists of a single virus member, perch rhabdovirus (PRV); Anguillid perhabdovirus which consists of two recognized viruses, eel virus European X (EVEX) and eel virus American (EVA); and Sea trout perhabdovirus which consists of two recognized viruses, the lake trout rhabdovirus (LTRV) and the Swedish sea trout virus (SSTV). These viruses are also confined in their geographical distribution, and the evidence that they have a significant disease impact is limited. Beyond the three established genera, several viruses were observed historically in a wide range of fish species and tentatively identified as rhabdoviruses based on the characteristic virion morphology. However, they remained unclassified due to the absence of genetic data, and most of the sample material in these cases has been lost so it is not possible to establish how they relate to the current taxonomy. Other more recently isolated fish rhabdoviruses are currently unassigned because they have not yet been sufficiently well characterized (Table 1).

Virus Structure Fish rhabdovirus virions have the bullet-shaped morphology that is characteristic of rhabdoviruses in general. Virus particles have a hostderived lipid envelope, and they are sensitive to inactivation by desiccation, as well as heat or UV exposure. Novirhabdovirus virions are approximately 150–190 nm in length and 65–75 nm in diameter (Fig. 2(A)). Sprivivirus virions are approximately 120–180 nm in length and 60–90 nm in diameter, and the perhabdovirus virions have a more compact morphology and measure approximately 115–130 nm in length and 85–95 nm in diameter. The unassigned starry flounder rhabdovirus is even more compact, with bullet-shaped virions measuring approximately 100
50 nm (Fig. 2(B)). Virions are comprised of five viral proteins. The single glycoprotein (G) on the enveloped surface of the virus particles has been shown to be the major antigenic protein and is by itself capable of eliciting a protective immune response. The matrix protein (M) is an inner component of the virions that binds both the cytoplasmic domain of the G protein

326

Fish Rhabdoviruses (Rhabdoviridae)

Fig. 2 Electron micrograph showing virions of fish rhabdoviruses. (a) Viral hemorrhagic septicemia virus (VHSV), in the genus Novirhabdovirus, (b) Starry flounder rhabdovirus (SFRV), an unassigned rhabdovirus of fish. Photos provided by J.R. Winton.

and the viral nucleocapsid, and it has been shown for IHNV to down-regulate host transcription. The major structural component of the nucleocapsid is the viral nucleocapsid (N) protein that associates with full length viral genomic RNA and facilitates its interaction with the viral polymerase. The large polymerase protein (L) is a complex protein that carries out the enzymatic functions of genome replication and transcription, and the phosphoprotein (P) is an essential co-factor that is required for polymerase function. These five virion proteins are found in all known rhabdoviruses and to date it appears that their roles are conserved across genera. In addition to the five familiar rhabdovirus proteins, the genus-specific hallmark of the novirhabdoviruses is the presence of a sixth protein, the nonvirion (NV) protein, which is not found in virus particles, but it is synthesized, in trace amounts, within infected cells. It has recently been demonstrated that the viral NV protein plays a role in abrogating host innate immune responses and suppressing apoptosis. Through the use of reverse genetics it has been shown that the NV gene and its encoded protein are not essential for replication in cultured cells or in fish, but in most cases it greatly enhances replication and pathogenicity. The absence of an NV gene in the genomes of both the spriviviruses and perhabdoviruses indicates that whatever the role of the NV protein is in novirhabdoviruses, it is not essential for rhabdovirus replication in fish.

Genome Fish rhabdovirus genomes are single stranded minus-sense RNA molecules of approximately 11,000–11,100 nt for novirhabdoviruses and spriviviruses, or 11,400–11,800 nt for perhabdoviruses. All contain the five canonical rhabdovirus genes in the order 30 N-P-M-G-L 50 , and novirhabdovirus genomes have the additional NV gene located between the G and L genes, as illustrated in Fig. 3. Genomic termini have leader and trailer regions ranging from 50 to 100 nt for 30 leaders, and 19–115 nt for 50 trailers, and these sequences have inverse complimentarity near the genome ends. Untranscribed intergenic regions are generally single nt for novirhabdoviruses, 2 nt for spriviviruses, and more variable for perhabdoviruses. Putative transcription initiation and termination/ polyadenylation signals (Fig. 3) are conserved across genes within each virus and among viruses within a genus. These signals are identical for spriviviruses and perhabdoviruses but they differ from those of novirhabdoviruses and other rhabdovirus genera. Within genes the untranslated regions upstream and downstream of the open reading frames are variable in length. For example, the putative transcript encoding the G gene of SVCV viruses differs by up to 85 nt due to variation in the 30 untranslated regions of the gene.

Genetic Diversity Molecular epidemiology and phylogenies have been described at local, regional, and global levels for all major fish rhabdoviruses. Hundreds or in some cases thousands of IHNV, VHSV, and SVCV field isolates have been characterized by genetic sequencing, providing a thorough understanding of the genetic diversity and evolutionary history of these viruses. Genetic typing of IHNV field isolates from North America, Europe, Russia and Asia has shown surprisingly low diversity, with no more than 9% nucleotide divergence globally in a variable region of the G gene. Despite this low diversity, phylogenetic analyzes clearly resolve IHNV into five major global genogroups that vary in geographic origin and host specificity. In North America the IHNV genogroups U, L, and M occur in partially sympatric ranges along the Pacific coast, and they show host-specific adaptation and virulence for sockeye salmon (Oncorhynchus nerka), Chinook salmon (O. tshawytscha), and rainbow trout (O. mykiss), respectively. The IHNV J and E genogroups arose in rainbow trout aquaculture in Asia and Europe, respectively, after intercontinental introduction from North America. Genetic typing of global collections of VHSV isolates has revealed greater diversity, with up to 18% nucleotide divergence in full length G gene sequences. Global VHSV phylogenies have 4 major phylogenetic groups designated genotypes I-IV. Genotypes I, II, and III occur in marine waters around Europe, mostly in wild fish, but within genotype I there are major subgroups that contain VHSV isolates from European rainbow trout farms, where they cause a severe disease burden. VHSV isolates in genotype IV are found in North America and Asia, where they cause disease outbreaks in several wild fish species and cultured flounder, but they are not virulent for rainbow trout.

Fish Rhabdoviruses (Rhabdoviridae)

327

Fig. 3 Genomes of representatives of the three genera of fish rhabdoviruses. All have minus-sense single-stranded RNA genomes of approximately 11,000–11,800 nucleotides (nt) containing nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and polymerase (L) genes as shown. Species in the genus Novirhabdovirus have an additional gene encoding a nonvirion (NV) protein at the G-L junction. Approximate sizes (in kDa) of encoded proteins are shown below each gene. Conserved gene junction sequences for each virus are shown in minus-sense (viral RNA) orientation below the genome with arrows indicating putative transcription stop (ending with seven U’s) and start signals.

Spriviviruses, particularly the SVCV isolates, are well characterized, and partial G gene sequences revealed up to 17.3% nucleotide divergence. Four SVCV genogroups (a-d) have been identified, and the clustering of the genogroups appears closely associated to geographical location, suggesting that the virus has evolved independently in different geographical regions. Genogroup a contains isolates from Asia, and more recently North America and the UK; genogroups b and c contain isolates from Eastern Europe; and genogroup d contains isolates from multiple European countries. To date, the pike fry spriviviruses have only been isolated in Europe, where they show relatively high diversity, with up to 28.5% divergence based on partial G gene sequences. The genetic diversity of Perhabdoviruses is less well defined, however, the limited studies have shown that nt diversity within species ranges from 7.9% for Sea trout perhabdovirus to 9.3% for Anguillid perhabdovirus and 14.2% for Perch perhabdovirus. Global phylogenies of fish rhabdoviruses show clear evidence of diversification into multiple lineages associated with geographic translocations, host jumps and adaptation to different fish hosts, and emergence events. Most recognized host jumps are into cultured rainbow trout, which has occurred multiple times in the histories of both IHNV and VHSV. Viral emergence events often occur after introduction of fish rhabdoviruses into a new geographic range. A classic example is the emergence of SVCV genogroup a in the USA, Canada and the United Kingdom following its potential introduction with infected carp from Asia in the early 1990s. Another example is introduction of VHSV into the Great Lakes region of North America in approximately 2003, resulting in emergence of a new VHSV genotype IVb, that caused large-scale die-offs in many wild fish species for several years. On a practical level molecular epidemiology of fish rhabdoviruses has been useful for fish health management decisions, tracking viral movements, defining viral transmission routes, and supporting risk assessment. Resulting viral phylogenies show clear evidence of anthropogenic influence on directions of virus evolution, with major impacts on fish health and aquaculture world-wide.

Life Cycle At the cellular level the replication cycle of fish rhabdoviruses is similar to that of rhabdoviruses in other vertebrate hosts. The viral glycoprotein (G) forms trimeric spikes on the virion surface that attach to receptors on fish host cells, facilitating entry by receptormediated endocytosis. Fibronectin has been identified as a primary cellular receptor for novirhabdoviruses but the receptor(s) used by most fish rhabdoviruses have not been identified. Once inside the cell the virus is uncoated, releasing the viral nucleocapsid, and the replication cycle occurs in the cytoplasm. Primary transcription of the negative-sense viral genome to produce mRNAs is

328

Fish Rhabdoviruses (Rhabdoviridae)

accomplished by viral polymerase complexes that contain the viral L, N, and P proteins and are carried in the virus particles. Viral mRNAs are produced in order from the N gene at the 30 end of the genome through the L gene at the 50 end, and translation by host cell machinery generates viral proteins. Based on knowledge from mammalian rhabdovirus systems it is assumed that viral proteins form more polymerase complexes and secondary mRNA transcription produces more viral proteins that accumulate and signal for the polymerase to switch from transcription to replication. Replication creates full-length anti-genomes that serve as templates for progeny RNA genomes. Viral proteins and genomes traffic to the cell surface and assemble, budding through the cell membrane to acquire the viral envelope as new virions are released.

Epidemiology IHNV, VHSV and SVCV are each currently distributed over several continents in the northern hemisphere, while many other fish rhabdoviruses are distributed more regionally, either in Europe or Asia (Table 1). IHNV was originally endemic to Western North America before spreading to Asia and Europe in the 1960s and 1980s, respectively. IHNV has a relatively narrow host range, naturally infecting various species of salmon and trout (Oncorhynchus sp., Salmo sp., Salvelinus sp.) and showing variation in host-specific virulence for individual salmonid species. HIRRV also appears to have a narrow host range, causing disease primarily in cultured and wild olive flounder (Paralichthys olivaceus) in several Asian countries, but there are rare reports of disease in other species and its host range has not been tested extensively. In contrast, VHSV has an extremely broad host range, naturally infecting over 80 species of fish from diverse taxonomic families. Oceanographic surveys have revealed that VHSV is broadly distributed in numerous species of wild marine fish in the Northern Atlantic and Northern Pacific Oceans, serving as a reservoir that occasionally spills over into cultured fish populations. For VHSV the concept of host specificity generally refers to whether virus strains are of low or high virulence for rainbow trout: VHSV strains from marine fish may cause disease in other host species but they have very low virulence in rainbow trout, while VHSV isolates from trout farms are highly virulent for rainbow trout. Although the host ranges for the spriviviruses and perhabdoviruses are not as great as for VHSV, they are found in a range of fish species including salmonids, cyprinids and percids. Naturally occurring SVCV infections have been recorded predominantly in cyprinid fish and non-cyprinid hosts include sheatfish (Silurus glanis), pike (Esox lucius), Siberian sturgeon (Acipenser baerii), largemouth bass (Micropterus salmoides) and bluegill sunfish (Lepomis macrochirus), and possibly rainbow trout (Oncorhynchus mykiss). The initial identifications were based on serological tests, but subsequent molecular characterization has shown that many of the original carp sprivivirus isolates may belong within the pike fry sprivivirus rather than carp sprivivirus. PFRV was initially reported in pike, sheatfish and grass carp (Ctenopharyngodon Idella), but given the recent findings, further genetic characterization is required to establish the full host range of the pike fry spriviviruses. PRV, the type species of the perhabdoviruses was first isolated from cultured perch (Perca fluviatilis) following mortality events in France and Norway, and antigenically similar viruses were then recovered from diseased pike perch (Stizostedion stizostedion), grayling (Thymallus thymallus) and largemouth bass (Micropterus salmoides) in France and pike in Denmark. SSTV was first discovered in brown trout (Salmo trutta m.lacustris) in Finland and sea trout (Salmo trutta trutta) from Sweden, but more recently, viruses assigned to the species Sea trout perhabdovirus have been isolated from perch, European eel (Anguilla anguilla) and brown trout (Salmo trutta) in the republic of Ireland. EVA was isolated from American elvers (Anguilla nostrata) and EVEX was isolated from European eels. Both field observations and controlled experimental studies clearly show that transmission of fish rhabdoviruses occurs horizontally by waterborne virus that is shed from infected fish and subsequently infects naive fish without the need for fish contact or vectors. Transmission can also occur vertically from parent to progeny fish by virus associated with the surface of fish eggs or milt. In aquaculture settings disinfection of fish eggs with commercial iodophores is generally effective in preventing vertical transmission. Common routes of virus introduction into fish culture facilities include import of infected fish or eggs, or virus in the water supply due to the presence of wild or feral infected fish in source waters. Within a fish farm or hatchery viruses spread with water flow, feed, or inadequate disinfection of equipment. On a regional scale virus can be spread due to general connectivity and flow of water in aquatic environments, interactions among fish culture facilities, or movement of wild fish populations. On a global scale the history of fish rhabdoviruses includes several examples of intercontinental spread of viruses such as IHNV and SVCV during the mid-1900s, due to movement of contaminated fish eggs or juvenile fish for aquaculture. Among fish that survive virus infection the existence of asymptomatic long-term carriers and reservoirs has been demonstrated in some cases and the biological role of viral persistence is an active area of research.

Clinical Features The majority of fish rhabdovirus disease outbreaks occur in cultured fish populations, including land-based fish farms, freshwater and marine netpen farms, and conservation hatcheries that rear fish for release into natural environments. In these settings fish infected with rhabdoviruses may exhibit petechial hemorrhages, accumulation of ascites, darkening of body color, or exophthalmia (Fig. 4), although many fish die without visible clinical signs. Mortality is frequently highest in younger fish and can be explosive in aquaculture settings, resulting in losses approaching 100% in some cases. The impacts of fish diseases include direct loss of fish due to mortality in disease outbreaks, culling of infected fish to avoid spread of disease, and costs of biosecurity

Fish Rhabdoviruses (Rhabdoviridae)

329

Fig. 4 Clinical signs of disease caused by fish rhabdoviruses. (a) skin hemorrhage in sockeye salmon (Oncorhynchus nerka) infected with IHNV (U genogroup strain), (b) exophthalmia (popeye) with eye hemorrhage in rainbow trout (O. mykiss) infected with IHNV (M genogroup strain). Photos by G Kurath.

and surveillance programs. Outbreaks of disease due to IHNV, VHSV, and SVCV have also been documented in wild and feral fish populations, with potential impacts on ecosystem health. The significance of fish rhabdovirus disease is evident in the OIE Aquatic Animal Health Code, which includes IHNV, SVCV, and VHSV as three of the 10 globally reportable pathogens of fish. Other fish rhabdoviruses, such as HIRRV, are regionally important pathogens. Due to the poikilothermic (cold-blooded) nature of their hosts, a unique aspect of fish rhabdoviruses is the strong influence of temperature on virus replication and severity of disease. Fish rhabdoviruses replicate with varying temperature optima typically between 10 and 251C, and they are inactivated at temperatures well below 371C. In SVCV infections, clinical disease is usually observed at water temperature between 51C and 181C, and is most severe at temperatures below 101C when it is believed the host's protective immune response is most likely suppressed or delayed.

Pathogenesis and Immune Response Fish rhabdoviruses gain entry into hosts through the gills and skin. A study using bioluminescence imaging of live fish infected with fluorescent-labeled IHNV indicated the skin at fin bases as a major portal of entry. Inside the host the viruses spread to multiple organs, with hematopoietic tissues of the kidney and spleen as common targets where virus accumulates to high levels. Virus is thought to shed from infected fish in urine, feces, and sloughing of mucus, but this process has not been characterized in detail. The diseases caused by fish rhabdoviruses are generally characterized as acute, hemorrhagic septicemias affecting multiple organs. Death is usually due to organ failure and subsequent loss of osmoregulation. Internally, necrosis of multiple organs is evident upon histological examination. The immune system of finfish has innate and adaptive immune responses typical of all vertebrates. The response to acute infection with fish rhabdoviruses is a strong interferon-based (IFN) innate response that is rapid and non-specific, resulting in transient cross-protection among different fish rhabdoviruses. It has been hypothesized that fish novirhabdovirus G proteins function as pathogen associated molecular patterns (PAMPs) to trigger signaling that activates the innate IFN response. In many cases this strong innate response is successful in controlling viral infection, leading to survival and clearance of virus. However, in other cases virulent strains of IHNV or VHSV are able to continue replicating despite the innate response, leading to disease and mortality, or viral persistence in some fish. Innate immune evasion mechanisms have been identified in novirhabdoviruses, including host cell shutoff by the viral M protein, and suppression of both the IFN-stimulated gene expression and apoptosis by the NV protein. Fish that survive rhabdovirus infection typically develop long-term protective immunity. Adaptive immunity in fish includes both humoral and cellular responses. It has been clearly shown that neutralizing antibodies produced in response to the rhabdoviral G protein are necessary and sufficient to provide long-term immunity against fish rhabdoviruses. These antibodies become detectable in fish sera several weeks after infection. Timing of the antibody response is dependent on environmental temperature, generally occurring more rapidly at higher temperatures. There is also evidence of a role for specific cellular immunity, such as cell mediated cytotoxicity, in response to fish rhabdoviruses. Recently new reagents have become available for distinguishing among T cell subsets, which will improve the ability to characterize these cellular immune responses.

Diagnosis Diagnostic methods for detection and identification of fish rhabdoviruses are well established. Definitive diagnosis of listed pathogens, VHSV, IHNV and SVCV is by isolation of the virus in cell culture followed with confirmation using serological and/or molecular methods. The World Organization for Animal Health (OIE), in its Manual of Diagnostic Tests for Aquatic Animals, includes

330

Fish Rhabdoviruses (Rhabdoviridae)

comprehensive methodology to diagnose and identify all three pathogens see “Relevant Websites section”. Many cell lines from finfish were developed beginning in the 1960s, and they support the replication of a broad range of rhabdoviruses from aquatic animals. Infection of these cell lines produces typical cytopathic effects (CPE) consisting of cell rounding and necrosis following the release of mature virions by budding. The rate of the appearance of CPE is related to the incubation temperature, which is typically set to mirror the temperature optimum of the fish host (e.g., 10–251C). Cell culture-based assays are also used routinely to detect the presence of infections or to quantify infectious virus. Serological methods using polyclonal or monoclonal antibody reagents have been commonly used for both diagnostics and research, but more recently molecular techniques such as real-time RT-qPCR are often used for surveillance and outbreak tracking, with conventional RT-PCR and sequencing for confirmatory diagnosis or identification of the infectious agent. In many regions of North America, Europe, and Asia cultured fish stocks are routinely surveyed for viral pathogens, including rhabdoviruses, by fish health professionals. For most fish rhabdoviruses surveillance and genotyping data are available as internet-accessible public sequence databases.

Prevention Control of fish rhabdovirus diseases in aquaculture is based primarily on avoiding infection through biosecurity programs designed to prevent introduction and spread of virus in fish culture facilities. Biosecurity includes strict hygiene practices, use of pathogen-free water sources (such as well water) for early life-stage rearing, application of water treatment technologies for some facilities using water from open sources, and frequent fish health inspections that make use of current technologies. In fish culture settings, the ability to break the cycle of vertical transmission between generations by the disinfection of eggs with an iodine solution has been important in controlling fish rhabdoviruses. Treatment options for fish showing signs of disease are limited to modifying husbandry conditions, typically by reducing fish density, or taking fish off feed to reduce stress. Due to the inadvertent intercontinental spread of several fish rhabdoviruses in the mid-1900s both national and international standards have been established that require fish health inspections for certain fish species that are involved in international or interstate transport. Several fish rhabdoviruses are on the list of pathogens for which inspections are indicated, and the global trade in aquaculture species is reasonably well regulated. However, the huge global trade in ornamental fish is essentially unregulated and is a documented source of virus movement. Research on vaccines and immunostimulants to protect fish from rhabdovirus infections has been conducted for more than 40 years. Early work focused on traditional vaccination strategies using inactivated viruses or live-modified vaccines, and immunostimulants such as interferon-inducer double-stranded RNA and bacterial lipopolysaccharide. Later, recombinant DNA technology produced experimental vaccines using bacterial or baculovirus expression systems as well as peptide immunogens. However, while some of these traditional and molecular vaccines were reported to be efficacious in laboratory and field trials, prohibitive costs, inconsistent efficacy, licensing barriers, safety concerns, and lack of mass delivery methods for juvenile fish prevented their use on a commercial scale. Since the 1990s, DNA vaccines expressing the viral G gene have been engineered for protecting fish against the novirhabdoviruses, IHNV, VHSV, and HIRRV. These vaccines have been studied extensively and show exceptionally high efficacy against severe viral challenges under a wide range of conditions. Although commercialization of these vaccines faces challenges similar to those noted above, a DNA vaccine against IHNV was licensed in Canada in 2005 for use in Atlantic salmon marine netpen aquaculture. This was one of the first DNA vaccines licensed for use in veterinary medicine and there is currently an industry requirement for 100% vaccination, so the IHNV DNA vaccine is used for millions of fish annually. Interestingly, DNA vaccines for SVCV also provide significant protection, but their efficacy is less than novirhabdovirus DNA vaccines for unknown reasons. In the absence of licensed vaccines for VHSV or SVCV, the national or international legislative controls and sound biosecurity strategies remain the most effective way of preventing the spread of these diseases. Also in the absence of commercially available vaccines some private aquaculture companies may use autogenous vaccines against fish rhabdoviruses, but the extent of this practice is not well documented.

Conclusions Due to their economic importance, the rhabdoviruses of finfish are among the best studied of all aquatic animal viruses, providing models of how aquatic animal pathogens function, spread, and persist in their environments. Extensive oceanographic surveys have revealed the role of wild fish as important reservoirs of virus for infection of fish in freshwater and marine aquaculture. Conversely, the potential for aquaculture fish to amplify pathogens that may impact wild fish is also a current concern. Field studies are providing insights into the ecology and evolution of fish viral disease, identifying anthropogenic impacts on fish health in altered environments, and supporting development of landscape transmission models for some fish rhabdoviruses. In laboratory studies fish rhabdoviruses also serve as useful components of model systems to study vertebrate virus disease, epidemiology, and immunology. The availability of a variety of established fish cell lines, the creation of new-generation reagents and tools, and the well-established laboratory challenge models that can use statistically robust numbers of relevant fish hosts make these model systems particularly attractive and powerful. New high throughput sequencing technologies have made the discovery, characterization and classification of viruses a simpler and more rapid process, and consequently the number of recognized rhabdovirus species is likely increase dramatically over the next decade. Experimental infections in vivo are increasingly

Fish Rhabdoviruses (Rhabdoviridae)

331

important for investigating viral pathogenesis, virulence, host-specificity of new emerging viral diseases and assessing the impact of environmental factors, or efficacy of control options such as vaccines. Reverse genetics systems for IHNV, VHSV and SHRV have been used to investigate the roles of individual viral proteins and the genetic determinants of virulence, as well as the use of recombinant viruses as gene vectors or attenuated vaccines. In the area of immunology, quantitative real-time PCR assays, microarray analyzes, and transcriptomics have been used to profile the fish immune gene response following either viral infection or vaccination. Collectively these studies provide essential information for managing fish rhabdoviruses in fish health and contribute to the basic science of virology.

Further Reading Ahne, W., Bjorklund, H.V., Essbauer, S., et al., 2002. Spring viremia of carp (SVC). Diseases of Aquatic Organisms 52, 261–272. Bootland, L.M., Leong, J.C., 2011. Infectious hematopoietic necrosis virus. In: Woo, P.T.K., Bruno, D.W. (Eds.), Fish Diseases and Disorders, vol. 3. Wallingford: CAB International, pp. 66–109. Einer-Jensen, K., Ahrens, P., Forsberg, R., Lorenzen, N., 2004. Evolution of the fish rhabdovirus viral haemorrhagic septicaemia virus. Journal of General Virology 85, 1167–1179. Harmache, A., LeBerre, M., Droineau, S., Giovannini, M., Bremont, M., 2006. Bioluminescence imaging of live infected salmonids reveals that the fin bases are the major portal of entry for Novirhabdovirus. Journal of Virology 80, 3655–3659. Kurath, G., 2008. Biotechnology and DNA vaccines for aquatic animals. Review Science Technology 27, 175–196. Kurath, G., Winton, J., 2011. Complex dynamics at the interface between wild and domestic viruses of finfish. Current Opinion in Virology 1, 73–80. Kurath, G., Garver, K.A., Troyer, R.M., et al., 2003. Phylogeography of infectious haematopoietic necrosis virus in North America. Journal of General Virology 84, 803–814. Lorenzen, N., LaPatra, S.E., 1999. Immunity to rhabdoviruses in rainbow trout: The antibody response. Fish and Shellfish Immunology 9, 345–360. Mork, C., Hershberger, P., Kocan, R., Batts, W., Winton, J., 2004. Isolation and characterization of a rhabdovirus from starry flounder (Platichthys stellatus) collected from the northern portion of Puget Sound, Washington, USA. Journal of General Virology 85, 495–505. Purcell, M.K., Laing, K.J., Winton, J.R., 2012. Immunity to fish rhabdoviruses. Viruses 4, 140–166. Salonius, K., Simard, N., Harland, R., Ulmer, J.B., 2007. The road to licensure of a DNA vaccine. Current Opinion in Investigational Drugs 8, 635–641. Smail, D.A., 1999. Viral haemorrhagic septicaemia. In: Woo, P.T.K., Bruno, D.W. (Eds.), Fish Diseases and Disorders, Volume 3: Viral, Bacterial and Fungal Infections. New York: CAB International, pp. 123–147. Stone, D.M., Ahne, W., Denham, K.L., et al., 2003. Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp viruses and pike fry rhabdovirus isolates reveals four distinct piscine vesiculovirus genogroups. Diseases of Aquatic Organisms 53, 203–210. Stone, D.M., Kerr, R.C., Hughes, M., Radford, A.D., Darby, A.C., 2013. Characterisation of the genomes of four putative vesiculoviruses: tench rhabdovirus, grass carp rhabdovirus, perch rhabdovirus, and eel rhabdovirus European X. Archives of Virology 158, 2371–2377. Vakharia, V.N., Li, J., McKenney, D.G., Kurath, G., 2019. The nucleoprotein and phosphoprotein are major determinants of the virulence of viral hemorrhagic septicemia virus in rainbow trout. Journal of Virology. in press. Walker, P.J., Blasdell, K.R., Calisher, C.H., et al., 2018. ICTV virus taxonomy profile: Rhabdoviridae. Journal of General Virology 99, 447–448. Wolf, K., 1988. Fish Viruses and Fish Viral Diseases. Ithaca: Cornell University Press.

Relevant Websites www.fishpathogens.eu Fish Pathogen Database. bioinfo/ihb.ac.cn Fish-Associated Virus Database (FVD). https://talk.ictvonline.org/taxonomy ICTV Virus Taxonomy 2018 Release. gis.nacse.org/ihnv Molecular Epidemiology of Aquatic Pathogens (MEAP)-IHNV. http://www.oie.int/en/standard-setting/aquatic-manual/access-online/ OIE Manual of Diagnostic Tests for Aquatic Animals.

Foot-and-Mouth Disease Viruses (Picornaviridae) David J Rowlands, University of Leeds, Leeds, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

History The earliest account that clearly describes foot and mouth disease was made by Fracastorius in 1546. Today, FMD is still one of the most important diseases of domestic livestock. It was the first animal disease shown to be caused by a filterable agent in 1897 by Loeffler and Frosch, who also demonstrated the presence of neutralizing antibody in serum. It is also the first virus for which serotype differences were recognized. Further milestones were the demonstration by Waldmann and Pope in 1921 that guinea-pigs could be infected by the virus and by Skinner in 1951 that it caused a lethal infection in suckling mice, thus providing small animal models for FMD research. Subsequently, cultivation of FMDV in tissue culture cells has enabled studies of viral structure and replication and also the large-scale production of vaccines. The combination of strict control measures and the use of vaccines has led to elimination of the disease from some parts of the world but many regions remain endemic for FMD.

Classification FMDVs comprise a species within the Aphthovirus genus of the Picornaviridae. The nature and organization of the genome, mode of replication and structure of the virion are, in general, similar to other viruses in the family. The original subdivision of the Picornaviridae into the four genera, Enteroviruses, Rhinoviruses, Cardioviruses and Aphthoviruses was based on physicochemical properties such as susceptibility to acid inactivation, buoyant density of CsCl solution and the nucleotide composition. Analysis of evolutionary relationships by nucleotide sequence comparisons have partly endorsed the original classifications; however, with further virus discovery the family has now expanded greatly and currently includes 110 species which are classified into 47 genera. The genus aphthovirus originally comprised the seven serotypes of FMDV (Fig. 1) With the discovery of more related viruses the genus was expanded to include 4 species; FMDV, bovine rhinoviruses A and B and equine rhinovirus A (Fig. 2). A combination of properties which distinguish the FMDVs include: (1) extreme sensitivity of the virion to acid inactivation (opH 6.8 in low ionic strength buffer); (2) high buoyant density in CsCl (1.43–1.50 g cm
3); (3) an exceptionally long multi-domain untranslated region (UTR) at the 50 end of the genomic RNA, including a large poly C tract (a property shared with some cardioviruses); (4) three separately encoded VPg proteins; (5) the use of two alternative in-frame protein translation initiation sites; (6) a leader protease protein located in the N-terminal region of the polyprotein; (7) interruption of translation (ribosome skipping) to facilitate polyprotein processing and; (8) very rapid replication.

Virion Structure As for other picornaviruses, FMDV particles are non-enveloped icosahedrons comprising 60 copies each of four structural proteins, VP1–4, encapsidating a single copy of the single-stranded positive-sense genomic RNA. Determination of the structure of the virus by X ray crystallography and cryo electron microscopy (Fig. 3) has shown that the three larger proteins (VP1–3) have the eightstranded antiparallel b-barrel folding motif seen in other picornaviruses and some plant viruses. VP4 is located on the inner surface of the particle. In common with other picornaviruses, VP1 molecules are disposed around the axis of five-fold symmetry whereas VP2 and VP3 alternate around the two-and three-fold symmetry axes. In contrast to many other picornaviruses, such as enteroviruses, in which the virion remains intact as an empty particle following thermally induced release of the RNA, heat or acid degradation of FMDV particles results in dissociation into pentameric subunits, consisting of five copies each of VP1–3, with release of the RNA and VP4. Several features of the structure are peculiar to FMDV. The protein shell is generally thinner and the external surface is smoother than in other picornaviruses. This is a result of the smaller sizes of VP1–3 (VP1 213, VP2 218, VP3 220 amino acids for virus O1) compared to other picornaviruses. The truncations are in the loop regions linking the core elements of the b barrels, which in other picornaviruses form prominent features at the outer surface. The deep grooves or pits encircling the five-fold axes of many picornaviruses are not present in FMDV and the position equivalent to these invaginations is occupied by the C-terminal portion of VP1. An important exception to the generally smooth contours of the surface of FMDV is provided by the G-H loop of VP1 (Fig. 3). In serotype O viruses this large loop extends from residue c.130 to 160 of which c. 135–158 are too disordered to be visible in electron density maps. The length of the VP1 G-H loop differs between different serotypes of the virus. In serotype 01 viruses the disorder of the VP1 G-H loop is induced by a disulfide bond between the cystine residues VP2 130 and VP1 134. Under reducing conditions this bond is broken and the G-H loop collapses onto the surface of the virus in an ordered configuration. In all serotypes the VP1 G-H loop includes an immunodominant antigenic site to which a high proportion of virus neutralizing antibodies are directed. Synthetic peptides representing sequences from this region are immunogenic and can induce

332

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00087-4

Foot-and-Mouth Disease Viruses (Picornaviridae)

Fig. 1 Genetic relatedness of FMDV strains and serotypes based on sequences within the VP1 gene (courtesy of N. Knowles).

Fig. 2 Genetic relationships within the genus Aphthovirus. (Courtesy of N. Knowles).

333

334

Foot-and-Mouth Disease Viruses (Picornaviridae)

Fig. 3 The FMDV O1M/avb6 receptor complex, both the O1M virus and integrin receptor are depicted using a surface representation. The capsid proteins are colored with VP1 in blue, VP2 green and VP3 salmon. The VP1 GH loop is drawn using semi-transparent magenta spheres in the “up” orientation to engage with receptor. For the integrin, the alpha subunit is green and the beta subunit red. (Courtesy of E. Fry & J. Ren).

Fig. 4 Genome map of FMDV. Boxed region is the polyprotein translation product with processing sites arrowed. (Courtesy of J. Ward).

protective immunity. The sequence of this region is variable both in composition and length between different viruses with the exception of a highly conserved receptor-binding motif which includes the triplet, Arg, Gly, Asp.

Genome The genome consists of a single molecule of single-stranded positive-sense RNA, which is infectious if transfected directly into cells in the absence of the protein components of the virion. The genome is longer than for most picornaviruses, comprising approximately 8500 nucleotides. The order of the gene products on the genome is basically similar to other picornaviruses but there are some unique/unusual features (Fig. 4). The genomic RNA terminates at the 50 untranslated end with a small protein, VPg

Foot-and-Mouth Disease Viruses (Picornaviridae)

335

Fig. 5 Secondary structure prediction for the 50 UTR of FMDV RNA with major functional motifs individually labeled.

(or 3B), linked by a phosphodiester bond through a Tyr at position 3 to the terminal uridine residue of the RNA genome. There is a variable length of poly(A) tract at the 30 end. Uniquely, the FMDV genome encodes three distinct VPgs, which are used with equal efficiency. This is a consistent feature of all FMDVs isolated to date (except for one which has only two copies of VPg) but the rationale for this gene triplication is unknown. In fact, virus derived from infectious cDNA clones from which one or two of the VPg copies are deleted is still infectious, although RNA synthesis is slower and viruses with reduced numbers of copies of VPg may have a competitive growth disadvantage. The 50 untranslated region (UTR) is exceptionally long (c. 1300 nucleotides) even by picornavirus standards and includes at least five distinct structural motifs (Fig. 5). The 50 end of the genome has a B400 nucleotide sequence, known as the S fragment, which is predicted to fold into an almost complete hairpin. Its function is unknown but by analogy to the highly structured 50 sequence of poliovirus RNA it is likely to be involved in the control of RNA replication. Deletion experiments have shown that viable virus can be recovered in cultured cells (BHK-21) from genomes in which a large proportion of the predicted hairpin structure has been removed. However, truncation of the S fragment alters the host susceptibility profile and there is evidence that it interacts with cellular proteins in a species-specific manner to down-regulate innate immune response mechanisms. The S fragment is followed by a poly(C) tract of 100–200 residues (exceptionally in excess of 400), depending on virus isolate. This tract is composed almost entirely of cytidine residues, occasionally interrupted with uridines. The function of the poly(C) tract is unknown and its presence in other aphthoviruses is equivocal. Its importance can be implied from its presence in all virus isolates and the observation that truncated poly(C) tracts increase in length following serial cell passage in cells. There is some evidence from FMDV and Mengovirus (a cardiovirus) that the length of poly(C) is related to pathogenicity. For example, deletion of the poly(C) tract from the highly virulent Mengovirus renders it completely non-pathogenic in mice. To the 30 side of the poly(C) tract there is a variable number of repeat domains, which are predicted to fold as pseudoknot structures. The number of pseudoknots varies between different virus isolates but none has been isolated with fewer than two copies. The number of nucleotides involved in these deletions/insertions corresponds closely to the number of nucleotides required to form the predicted pseudoknot structures, providing circumstantial evidence for their structural integrity. Although the role of the pseudoknots is unclear their evolutionary retention suggests that they provide the virus with a competitive advantage. To the 30 side of the pseudoknots there is a stem-loop structure known as the cre element (cis active replication element). This acts as a template for the uridylation of the VPg protein by 3Dpol, the viral RNA dependent RNA polymerase, in the replication complex. The uridylated VPg then functions as a primer for RNA replication. Although cre elements are found generally in picornaviruses, they are frequently present within the protein coding sequence and location within the 50 UTR is peculiar to FMDV. The 30 435 nucleotides of the 50 UTR fold into a series of stem loop structures similar to those present in the equivalent region of cardiovirus 50 UTRs and function as an internal ribosome entry site (IRES) to initiate protein synthesis. There is a short pyrimidine–rich tract between the IRES and the first of two in-frame alternative translation initiation codons. The 30 UTR is approximately 100 nucleotides in length and includes two stem-loop structures (SL1 and SL2) which make important interactions with features in the 50 UTR to facilitate translation and RNA replication. It terminates in a poly(A) tract of variable length. Bioinformatic analysis of FMDV genome sequences has revealed an underlying feature termed GORS (genome ordered RNA structure) not seen in other picornaviruses, such as enteroviruses. It is speculated that this inherent structural feature results in the production of short double stranded RNA products following genome degradation, which may be inhibitory to innate immune mechanisms. The high GORS score may contribute to the ability of FMDV to establish persistent infections and it is also a feature of hepatitis C virus which regularly induces chronic infections.

336

Foot-and-Mouth Disease Viruses (Picornaviridae)

Protein Products The protein-coding region is a continuous open reading frame of 6999 or 6915 nucleotides for FMDV serotype A10, depending on which of two functional in-frame initiation codons is used. The order of the gene products is shown in Fig. 4.

Leader protein The leader protein Lpro, which precedes the structural proteins, is a papain-like cysteine protease which cleaves the polyprotein at the L-P1 junction. It also cleaves a number of host proteins with profound consequences for protein translation (see “Section Translation”) and innate immune functions. Lpro invariably occurs as two forms, Lab and Lb, which are translated from different in-frame initiation sites separated by 84 nucleotides. Both forms of the protein are equally active proteolytically and the reasons for the conservation of two initiation sites and hence the expression of two forms of the protein are still unclear. The proportion of Lb produced exceeds that of Lab due to a less efficient Lab translation initiation site and the presence of low abundance codons in the Lab – Lb region. The Lpro proteins are not essential for virus viability per se since virus derived from an infectious clone with deleted Lpro domain can replicate. However, it has an attenuated phenotype and has been proposed as a candidate live vaccine.

P1 region The P1 region consists of the structural proteins. P1A, B, C and D are equivalent to VP4, 2, 3 and 1 respectively. The precursor protein P1 is cleaved by the viral protease 3Cpro into VP0 (VP4 þ VP2), VP3 and VP1 which together comprise the monomeric structural subunit. Five monomers assemble to form a pentameric subunit, which is the building block for icosahedral virion assembly. Cleavage of VP0 into VP4 þ VP2 is a final assembly maturation step and the mechanism is unclear.

P2 region The P2A protein is vestigial in size compared to other picornaviruses, being only 18 amino acids long, but it enables the nascent separation of the polyprotein at the P2A-2B junction. The amino acid sequence of 2A facilitates termination of translation of the polyprotein at this point without dissociation of the translation complex from the ribosome. Translation then continues but without the formation of a peptide bond between 2A and 2B. Cellular release factors determine the efficiency of this process i and it is proposed that changes in the levels of these during infection can alter the relative amounts of structural and non-structural proteins produced. There is some direct evidence for this in Theilers virus – a cardiovirus- which adopts a similar mechanism of interrupted translation. The rationale for such a mechanism is that it provides for the differential production of structural and nonstructural proteins as requirements change during the replication cycle. 2B multimerizes to form an ion channel (viroporin) and can alter the distribution of ions across cellular membranes. It antagonizes components of the cellular innate immune mechanisms. The precursor protein 2 BC influences membrane trafficking in infected cells, inhibiting transport from the ER to the Golgi complex. P2C has a nucleoside triphosphate binding motif and has ATPase activity. It appears to have a role in RNA replication since mutations in this protein can relieve the inhibition of RNA synthesis seen in the presence of guanidine hydrochloride. It forms hexameric complexes and is thought to function as an RNA helicase during viral replication.

P3 region The role of P3A in FMDV is unclear but it does not seem to alter cellular membrane architecture like 3A protein of poliovirus. The N terminal portion of the protein is conserved and includes both hydrophilic and hydrophobic domains through which it associates with membranes, where it functions as an important component of the RNA replication complex. The C terminal portion is variable between virus isolates and has an important influence on host specificity. Viruses with a significant deletion in 3A are highly pathogenic for pigs but are much attenuated in cattle. P3B or VPg occurs as three tandem copies of 23, 24 and 24 amino acids. Although differing in sequence each is rich in Pro, Arg and Lys residues and has a single Tyr at position three. Each of the individual VPg molecules is highly conserved between the A, O and C serotype viruses. The VPg molecules are post translationally modified to function as primers in RNA synthesis. All encapsidated RNA molecules terminate with a VPg molecule and each of the three forms is found in equal abundance in encapsidated viral RNA. Actively translating viral RNA lacks VPg, and cell extracts contain an enzyme that cleaves the phosphodiester bond to produce RNA terminating with a 50 -monophosphate. For poliovirus the cellular enzyme responsible for the removal of VPg has been identified as 50 tyrosyl-DNA phosphodiesterase 2 (TDP2) and this is likely to be also the case for FMDV. The observation that all virion associated RNA terminates in VPg suggests that it has a role in the selection of molecules for encapsidation. P3Cpro is the protease responsible for the majority of the processing cleavages. In common with other picornaviruses, sequence analysis suggests that the catalytic site of 3Cpro is related to trypsin, a serine protease, but with the replacement of the nucleophilic serine residue with cysteine. P3Dpol is the RNA-dependent RNA polymerase responsible for RNA replication and VPg uridylation. The crystal structure has been solved in the apo form and in complex with primer and template. As has been demonstrated for poliovirus, FMDV 3Dpol can form fibrillar structure in vitro but the significance of these in RNA replication is unknown. P3CD precursor has a distinct function as a catalyst for the P3Dpol mediated uridylation of VPg. A range of precursor proteins derived from the P3 region is present in infected cells resulting from the dynamics and ordering of the proteolytic processing cascade. It is likely that some of these intermediate products have distinct roles in the replication process. The molecular structures of virus particles, Lpro, 3Cpro and 3Dpol have all been determined by X ray crystallography.

Foot-and-Mouth Disease Viruses (Picornaviridae)

337

Life Cycle Cell Attachment and Entry The large mobile G-H loop of VP1 located at the surface of FMDV particles mediates their binding to susceptible cells. The sequence of the loop contains a highly conserved motif, Arg, Gly, Asp, which is a hallmark of ligands for a number of heterodimeric cell surface molecules called integrins. Synthetic peptides including this sequence can compete with virus for cell attachment and treatment of the virus with proteolytic enzymes such as trypsin, which cleave within the G-H loop, also prevents virus binding. Although the virus can bind to a number of integrins there is good evidence that avb6 is the preferred receptor in vivo. The structure of a complex of avb6 bound to FMDV has been determined by cryo electron microscopy (Fig. 3). Following attachment, the virus is internalized by endocytosis and release of the RNA is triggered by acidification in early endosomes. Reduction of the pH below c. 6.8 seems to be all that is required to initiate the infection process and the mechanism by which the genome is delivered to the cytoplasm is unknown. It is unlikely that cell entry requires complete dissociation of virions into pentameric subunits and genomic RNA triggered by acidification of the endosome since this would expose the RNA to the potentially hostile environment within the lumen. Transient empty particles are produced from aphthovirus particles under some conditions and these may represent intermediate structures that protect the genome during entry into the cytoplasm. Many tissue culture adapted strains of the virus have evolved in vitro to use heparin sulfate at cell surface as an alternative receptor.

RNA Replication RNA of infecting virus functions as a template for the synthesis of a negative-sense complementary strand, which serves as template for the synthesis of positive-sense strands, identical to the original infecting molecule. Positive-strands are synthesized in a complex structure [replicative intermediate (RI)] consisting of a single negative-strand template and several (c.6) nascent positivestrands. RNA synthesis is asymmetrical in favor of positive-strands. A proportion of the negative-strand templates occur as fulllength double-stranded hybrids [replicative form (RF)] and appear to take no further part in RNA synthesis. RF molecules accumulate in the cell during viral replication. Each RNA replication event is initiated by priming with an uridylated VPg molecule. A single molecule of viral RNA is sufficient to initiate infection, which implies that it can function sequentially as a template for translation, to produce the polymerase enzyme(s), and as a template for RNA replication. Single-stranded viral RNA is infectious in the presence of inhibitors of host cell DNA-dependent RNA polymerase. Double-stranded viral RNA is also infectious but not in the presence of inhibitors of the cellular polymerases. As for other positive sense RNA viruses, the RdRp of FMDV is error prone due to the absence of a proof-reading capability. Consequently, point mutations occur at a high rate and the viral population exists as a quasi-species – a swarm of variant molecules the sequence of which represents a consensus. In addition to a high replication error rate, recombination occurs readily. Both of these features contribute to the genetic flexibility of FMDV and enable it to respond rapidly to environmental selective pressures.

Translation FMDV RNA is translated efficiently in a cell-free system (e.g., rabbit reticulocyte lysate) to produce protein products similar to those found in infected cells. The 435 nucleotides upstream of the first AUG initiation codon is folded into a complex structure similar to that of the equivalent region of cardiovirus RNAs. This sequence acts as an IRES, allowing initiation of translation in the absence of the host cell CAP-binding protein, eIF4E. In addition to canonical translation initiation factors, IRESs recruit a number of cellular IRES trans-activating factors (ITAFs) which appear to act as chaperones to facilitate optimal conformational folding of the IRES sequence. The rate of total protein synthesis in virus-infected cells does not change until its decline towards the end of the growth cycle, when cytopathic effects (CPE) are apparent. There is, however, a marked change in the profile of proteins produced. When viral replication is maximal, virtually no host cell proteins are produced. This “swap over” of translation from host to viral products is similar to the situation in cardiovirus-infected cells and differs from the kinetics of translation following infection of cells with enteroviruses. In the latter, infection results in a rapid shutdown of host cell protein translation, which is followed later by a resumption of protein synthesis due to the increasing production of viral proteins. The shutdown induced by enteroviruses is largely, if not entirely, due to virus-induced cleavage of a host protein, p220 or eIF4G, an important component of the CAP-binding complex required for the initiation of translation of host mRNAs. In these viruses, eIF4G is cleaved by P2Apro. Cardioviruses do not induce eIF4G cleavage but downregulate host mRNA translation via P2A induced hypophosphorylation of initiation factor 4E-BP1 and so out-compete host mRNAs for utilization of the translation machinery. Although the kinetics of protein translation in FMDV-infected cells resemble those of cardiovirus-infected cells, eIF4G is cleaved. In contrast to enteroviruses, in FMDV eIF4G is cleaved by Lpro and not by P2Apro. 3Cpro can also cleave eIF4G but at a different site from Lpro and later in the infection cycle. Inhibition of host cell protein synthesis is likely to be of advantage to the virus both by removing competition for access to the translation machinery and also by inhibiting the expression of innate immunity response genes in infected cells. FMDV from which Lpro is deleted has a reduced ability to inhibit the interferon system.

338

Foot-and-Mouth Disease Viruses (Picornaviridae)

Post-Translation Processing The polyprotein translation product of FMDV RNA is proteolytically processed by three of four virus-encoded enzyme activities (Fig. 2). Three of the cleavages occur nascently on the growing polypeptide chain. The first separates Lpro from P1, the structural protein precursor, and is carried out by Lpro itself. The cleavage is within a Lys-Gly dipeptide and probably occurs normally in cis but can also occur in trans. L-P1 cleavage is the only processing step which is inhibited by the tripeptide D-Val-Phe-Lys-CH2Cl. Both Lb and Lab are proteolytically active. The second primary “cleavage” occurs at the junction of P2A and P2B and is facilitated by the 18-amino acid P2A sequence. There is good evidence that the separation of 2A from 2B and the remainder of the polyprotein does not occur by proteolytic hydrolysis of a peptide bond but rather involves a novel process of interrupted translation, referred to as ribosomal skipping. The third primary cleavage is between P2C and P3A and is catalyzed by 3Cpro. This protease is responsible for all other processing cleavages, apart from that which generates VP2 (P1B) and VP4 (P1A) from the precursor VP0 (P1AB). Many cleavages catalyzed by 3Cpro occur at Glu-Gly junctions but other dipeptides are recognized and 3Cpro of FMDV is the most promiscuous of the picornavirus proteases. The cleavage event to produce VP2 and 4 from the precursor, VP0, occurs in the final stages of virus maturation. The mechanism of this cleavage is not known. The production of viral proteins via cleavage from a polyprotein allows for alternative cleavage pathways and provides a range of intermediate precursor proteins. It is thought that some of the intermediate products have roles additional to those of the mature cleavage products, thereby expanding the functional capacity of a small viral genome.

Virus Assembly and Release A variety of assembly intermediates containing equimolar amounts of VP1, 2 and 0 are detected in infected cells. These correspond to monomer and pentamer subunits of the icosahedral capsid and 75S empty particles, which lack viral RNA but possess antigenic properties similar to mature viral particles. Following pulse-labeling experiments, empty particles can be “chased” into viral particles, but it has not been shown that they are on the direct morphogenetic pathway. All encapsidated RNA terminates with VPg, suggesting that this plays a role in selection for packaging and/or that only nascent RNA is encapsidated before VPg is cleaved off by TDP2. There is also evidence for the presence of multiple short RNA sequence motifs (packaging signals) distributed through the genome, which facilitate virion assembly. Paracrystalline arrays of virus are visible in infected cells and are released by lysis of the cell. In addition there is evidence that some viral particles are secreted prior to cell disruption or are released within exosomes.

Epidemiology FMD occurs widely and is endemic in many countries, especially in tropical regions. Many regions, including Europe, North America, Australia and New Zealand are free of the disease and maintain this status by rigorous application of import controls and quarantine. Mass vaccination campaigns have eliminated the virus from some areas, e.g., Europe, but have been less effective in others, due largely to logistical problems of vaccine distribution and the techniques of animal husbandry employed. The global distribution of the serotypes is shown in Figs. 6 and 7. There have been no reports of isolation of viruses belonging to serotype C since 2004 and these appear to now be extinct. In regions endemic for FMD, the virus is most likely maintained in persistently infected animals. It has been shown experimentally that infected bovines can secrete virus for long periods after the initial episode of disease. In some areas the wild animal population may act as a reservoir for infection (e.g., Cape buffalo in Africa). In non-endemic areas infection may be introduced from a variety of sources such as the importation of infected livestock, contaminated animal products such as carcasses containing bone (in contrast to meat, the post-mortem acidification of bone marrow and other non-muscular tissues is insufficient to inactivate the virus), or contaminated materials. More locally, transmission of infection is by direct transport of contaminated animals or materials or by wind-borne carriage of infectious aerosols. It is also suspected that the virus can be passively transmitted by migrating birds. High-throughput deep sequencing methodologies now allow virus transmission to be traced forensically at high resolution.

Host Range The virus typically infects cloven-hoofed species with domestic cattle being the most susceptible. Domestic pigs are also important hosts and are particularly effective in propagating the disease, since they secrete large quantities of virus in the form of aerosols. In sheep and goats, the clinical manifestations of infection are usually less severe than those seen in cattle and pigs making initial symptomatic diagnosis in these species more difficult. Natural infection of Indian elephants and of camels has been reported. Many wild species of deer and antelope are susceptible to infection and in African Cape buffalo infection is asymptomatic. Persistent infection with prolonged shedding of virus for months or years has been reported in wild and domestic bovine species. Although direct transmission of infection from carrier animals to naive animals has proven difficult under laboratory conditions

Foot-and-Mouth Disease Viruses (Picornaviridae)

339

Fig. 6 Global distribution of FMDV serotypes O. A, C and Asia 1 between 2009 and 2018. (the FAO World Reference Laboratory for FMD and the OIE/FAO Reference Laboratory Network).

Fig. 7 Distribution of FMDV serotypes SAT 1, SAT 2 and SAT 3 between 2009 and 2018. (the FAO World Reference Laboratory for FMD and the OIE/FAO Reference Laboratory Network).

there is epidemiological and some experimental data which supports the importance of persistent infection in maintenance and transmission of disease in the wild. A wide range of animals, including Australian marsupials and even birds, have been infected under laboratory conditions and, very rarely, infection of man has been demonstrated. The most important small animals for laboratory investigations are the guinea-pig and the suckling mouse. In the former, injection of virus intradermally into plantar pads results in the formation of vesicular lesions both at the site of injection and in the mouth and the remaining feet, and so resembles the lesion distribution in naturally infected susceptible species. Intraperitoneal infection of suckling mice results in rapid death. The viruses can be propagated in primary cells and cell lines of bovine or porcine origin. Cells derived from the BHK-21 line are most widely used for research or vaccine production purposes. The virus can be titrated by plaque assay in cultured cell monolayers or by cytopathic end-point dilution assay in microtiter plates. Primary bovine cells (e.g., bovine thyroid cells) are used for field virus isolation, as they are more susceptible to nonculture adapted virus strains.

Genetics and Evolution In common with other RNA viruses, the mutation rate of FMDV is extremely high and virus populations exist as quasispecies in which each individual genome is likely to differ from every other. Antigenic sites on the viral particle are tolerant of sequence variation, and antigenic diversity is a significant property of the virus. In addition to evolution by the accumulation of point mutation, genomic recombination occurs at a high rate in vitro and comparative sequencing of field isolates suggests that it is also a frequent event in vivo. The frequency and genomic location of recombinatorial events mirrors the genetic relatedness of the parental viruses.

340

Foot-and-Mouth Disease Viruses (Picornaviridae)

Serological Relationships and Variability Seven serotypes of FMDV are recognized, the definition of serotypes being that an animal convalescent from infection by virus of one serotype is fully susceptible to viruses of any of the remaining six. In addition to the major serotype differences, there is considerable antigenic variation between viruses within serotypes. The serological relationships between FMDV isolates are paralleled by genetic relationships as evidenced by RNA sequence analyzes (Fig. 1). Major advances in nucleotide sequencing technologies in recent years have resulted in these becoming the most accurate and rapid methods for evolutionary and epidemiological studies. For epidemiological studies and vaccine strain selection the serological relationships between field virus isolates or laboratory strains are expressed as r values, i.e., the ratio of the neutralizing titers of immune sera against heterologous and homologous viruses. The serological relationships between virus isolates are frequently nonreciprocal, showing that closely related viruses may induce broadly cross-reactive or narrowly specific immune responses.

Pathogenesis and Clinical Features The principal route of infection appears to be via aerosol impinging on the pharynx and respiratory tract. Aerosols may be produced locally during feeding on contaminated foodstuff or may be transmitted over considerable distances under appropriate meteorological conditions. Pigs secrete particularly high levels of virus-contaminated aerosols. In addition to mucosal secretion, high levels of virus are found in milk. Pasture may be contaminated with virus from urine and feces. Although the virus is labile when purified in laboratory conditions it can survive for long periods in certain environments (e.g., in slurry) at low temperatures. Vesicular lesions appear in the mouth on the tongue, gums and cheeks and later on interdigital mucosa and coronary bands of the feet. Virus can be isolated from many tissues in the body. The onset of clinical disease is usually very rapid and lesions can develop as early as 1–2 days after infection, depending on the virus strain and level of exposure. The pathogenicity of FMDV varies according to the virus strain, host species and age. The factors that govern the virulence of FMDVs are poorly understood and as with most viruses are probably multifactoral. Domestic cattle are the most susceptible species and morbidity is usually c. 100% in non-vaccinated animals. Wild bovines, such as African Cape buffalo, may produce no clinical manifestations. Although the disease is rarely fatal in adult domestic animals (o 5%), significant mortality may occur in young animals (c. 50%). Overt symptoms of disease are less obvious in pigs, although this species produces large volumes of infectious aerosol and are very important in disease transmission. Symptoms are much less severe in ovine species and can be unapparent, which can complicate surveillance for the presence of FMD. Infection of bovines typically produces a rapidly progressing febrile illness and the development of massive vesicular lesions in the mouth and on the feet. Infection primarily involves mucosal epithelia in the oropharynx, which correlates with expression of the principle receptor, integrin avb6. The lesions rupture with considerable loss of epithelial tissue (Fig. 8). The resulting discomfort discourages feeding until the lesions heal by the infiltration of fibrous tissue. The severity of the disease results in long-term loss of productivity in terms of meat and milk yield, and lameness may be a serious consequence for draft animals. Abortion and chronic subfertility are also common. Other organs infected include mammary glands, pancreas and heart.

Immune Response Infection elicits a vigorous humoral antibody response and, after recovery, immunity to re-infection with viruses of the same serotype is prolonged. The humoral antibody response is induced by both T-cell independent and T-cell dependent mechanisms. The role of cytotoxic T cells in recovery in unclear.

Fig. 8 Severe symptoms of FMD showing extensive sloughing of lingual epithelium.

Foot-and-Mouth Disease Viruses (Picornaviridae)

341

Four antigenic sites recognized by antibodies capable of neutralizing the virus have been described. One of these is an immunodominant linear sequence comprising the G-H loop of VP1, and synthetic peptides representing this tract are effective in inducing high levels of virus neutralizing antibody and can protect animals from infection. The remaining antigenic sites are conformational and one spans the interface between protomers in the assembled particles and so is lost on particle dis-assembly. Antibody responses to several of the non-structural proteins are induced during infection and these are being developed as markers to distinguish vaccinated from infected animals.

Diagnosis Initial diagnosis in the field is based on clinical observations, which are usually obvious for cattle but less so for other species, especially sheep. The presence of FMDV must then be confirmed using more specific virological and/or serological methods. Cultivation of virus from clinical samples is the gold standard for confirmation but this may be complicated by lack of containment facilities to perform the isolation, difficulties in growing field virus strains in cultures cells and poor quality of samples. It is also a lengthy process. Virus antigens can be detected using antibody based assays such as enzyme-linked immunosorbent assay (ELISA) using polyclonal sera or monoclonal antibodies. Genome sequence based methods relying on the reverse transcriptase – polymerase chain reaction (RT PCR) are used increasingly for diagnosis and for high accuracy epidemiological studies. Modern diagnostic techniques are becoming ever more sophisticated through the development of “pen side” rapid lateral flow devices, analogous to those used in pregnancy testing kits, and portable automated RT PCR apparatus.

Prevention and Control In disease free regions, natural protection afforded by geographical barriers is rigorously reinforced by strict controls on the importation of susceptible animals and potentially contaminated materials. Where outbreaks occur sporadically, due to occasional introduction from external sources, embargoes on animal movement and slaughter of infected herds have been successful in maintaining a disease-free national herd. In endemic areas, control is by mass vaccination. Ring or barrier vaccination is also used to limit the spread of infection during outbreaks. Dramatic demonstrations of the vulnerability of naive livestock to large-scale epidemics of FMD were provided by the outbreaks of serotype O virus in the UK and of serotype A virus in South America in 2001. The outbreak in the UK resulted in the slaughter of over 10 million animals (most of which were uninfected) to control spread of the disease and the cost to the country was many billions of pounds. In South America the outbreak was controlled by the resumption of blanket vaccination of all livestock. The first vaccines against FMD were produced by formalin inactivation of lymph drawn from lesions on the tongues of infected cattle. This source of immunizing virus was replaced by the Frenkel method of culture in fragments of epithelium stripped from the tongues of slaughtered cattle. Most vaccine in use today is produced by growing the virus in suspensions of BHK-21 cells in fermentation vessels of up to 10,000 liters capacity. Approximately 2  109 monovalent doses of vaccine are administered annually. Aziridines have largely replaced formalin as the inactivant, since the inactivation kinetics of the latter are non-linear and residual live virus in vaccines has occasionally been the source of outbreaks of disease. The serotype and strain composition of vaccines have to be tailored for local requirements. Inactivated virus is usually adjuvanted by adsorption onto aluminum hydroxide gel, and saponin may be added to enhance potency. In pigs, such vaccines elicit only immunoglobulin M responses and for this species vaccines are formulated with oil adjuvants. Oil adjuvanted vaccines are widely used for all species in S. America and China. Solid protection requires high levels of neutralizing antibodies and to achieve this with inactivated vaccines, immunization is repeated two to three times a year. The development of live attenuated vaccines has not progressed due mainly to the complexity of antigenic diversity and the fear of reversion to a virulent phenotype. However, greater understanding of the structure and function of the virus provides the basis for development of rationally designed live vaccines which may overcome the disadvantages inherent in earlier attempt to derive attenuated vaccine strains.

Future Perspectives There is great scope for developments in FMD vaccines to improve safety, stability, cross-protective efficacy and duration of immunity. Experimental peptide vaccines were demonstrated to elicit protective levels of neutralizing antibodies more than 30 years ago but poor responses in target species as compared to small animal models curtailed their development. However, research continues in this field. Production of virus-like particles (VLPs) via the co-expression of the structural precursor protein, P1, together with the protease, 3Cpro in recombinant expression systems, such as baculovirus transduced insect cells, is developing rapidly. The inherent instability of FMDV VLPs is being addressed by the identification and inclusion of stabilizing mutations. Adenovirus vectored P1/3Cpro constructs are being investigated as live recombinant delivery systems and naked DNA constructs capable of expressing the same proteins have also been shown to elicit anti FMDV responses. In addition, despite the failure

342

Foot-and-Mouth Disease Viruses (Picornaviridae)

through reversion to virulence of attenuated vaccines developed many decades ago, increased understanding of the structure and function of the viral genome at the molecular scale is leading to the development of rationally designed attenuated vaccine strains (virtually) incapable of reverting to a virulent phenotype. Massive outbreaks of FMD in the UK and in South America in 2001 stimulated interest in the development of drugs to halt the rapid spread of infection. Improved rapid diagnostic methods are being explored as is the ability to accurately distinguish infected from vaccinated animals. As to the molecular properties of the virus, both the determination of the near atomic structure of the particle and several of the non-structural proteins and the cloning and manipulation of full-length infectious cDNA molecules are important steps towards understanding the unique features of the virus.

Further Reading Bartling, S.J., Vreeswijk, J., 1991. Developments in foot and mouth disease vaccines. Vaccine 9, 75. Brooksby, J.B., 1982. Portraits of viruses: foot and mouth disease virus. Intervirology 18, 1–23. Diaz-San Segundo, F., Medina, G.N., Stenfeldt, C., Arzt, A., de los, Santos, T. Foot-and-mouth disease vaccines, 2017. Veterinary Microbiology 206, 102–112. Fry, E., Logan, D., Fox, G., et al., 1990. Architecture and topography of an aphthovirus. Semin. Virol. 1, 439–451. Foot-and-mouth disease virus. Mahy, B.J.W. (Ed.), Heidelberg: Springer. Foot-and-mouth disease virus. In: Rowlands, D.J. (Ed.), Virus Research 91. Elsevier. [Special Issue]. Foot-and-mouth disease – Current perspectives. Sobrino, F., Domingo, E. (Eds.), Great Britain: Horizon Bioscience. Foot-and-mouth Disease Virus – Current Research and Emerging Trends. Sobrino, F., Domingo, E. (Eds.), Norfolk, UK: Caister Academic Press. Yuan, Gao, Sun, Shi-Qi, Guo, Hui-Chen, 2016. Biological function of Foot-and-mouth disease virus non-structural proteins and non-coding elements. Virology Journal. [13/107].

Relevant Website http://www.picornaviridae.com/ The Pirbright Institute.

Fowlpox Virus and Other Avipoxviruses (Poxviridae) Efstathios S Giotis, Imperial College London, London, United Kingdom and University of Essex, Colchester, United Kingdom Michael A Skinner, Imperial College London, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Nomenclature APV Avipox viruses CNPV Canarypox virus CPE Cytopathogenic effect dsDNA Double-stranded DNA FWPV Fowlpox virus GC-content Guanosine cytosine content ICTV International Committee on Taxonomy of Viruses

Glossary Borell bodies Virus-containing granules that cluster to form “Bollinger bodies” and are found in cells infected with FWPV. Inclusion bodies (Or elementary bodies) are nuclear or cytoplasmic aggregates of stable substances, usually proteins.

PCR Polymerase chain reaction PEPV Penguinpox virus PGPV Pigeonpox virus PV Poxviruses REV Reticuloendotheliosis virus TKPV Turkeypox virus VACV Vaccinia virus

Passerine A perching bird of the order Passeriformes. Photolyase Enzymes that repair DNA damage caused by exposure to ultraviolet light. Psittacine A bird of the parrot family. Scab Skin lesion. Viroplasm (Or virus factories) cytoplasmic inclusion body where viral replication and assembly occurs.

Introduction Avipoxviruses (APV), of which fowlpox virus (FWPV) is the prototypic member, have received considerable attention due to their devastating effects to populations of domesticated, pet and wild birds, and due to their narrow host range, which allows them to be used as safe, non-replicating vectors for vaccination of birds and mammals. Despite their impact and increased frequency in wild birds, our understanding of the APV molecular and biological characteristics is restricted to FWPV and canarypox virus. Only few APV genomes are currently available and the viruses’ global distribution, phylogenetics and epidemiology are not as well understood as those of Orthopoxviruses.

Classification Avian pox viruses or avipox viruses (APV) form one genus of the Chordopoxvirinae subfamily. APV infect and cause diseases specifically in birds and have been isolated from more than 230 avian species worldwide. Traditionally, avipoxviruses have been assigned to species on the basis of original host, growth and morphological characteristics in the chorioallantoic membranes of embryonated eggs or cell cultures, and/or on the basis of clinical manifestations. Moreover, restriction enzyme and single-gene PCR (e.g., P4b gene) analyses have been used to detect and differentiate APV. Genome DNA sequencing has confirmed most provisional species demarcations, although some strains are now regarded as variants of other APV. According to the ICTV, ten APV species have so far been identified and classified. These include Fowlpox virus (FWPV), Canarypox virus (CNPV), Juncopox virus (JNPV), Mynahpox virus (MYPV), Pigeonpox virus (PGPV), Psittacinepox virus (PGPV), Quailpox virus (QUPV), Sparrowpox virus (SRPV), Starlingpox virus (SLPV), and Turkeypox virus (TKPV). Other viruses which may be members of the genus but have not yet been approved a separate species include Crowpox virus (CRPV), Peacockpox virus (PLPV) and Penguinpox virus (PEPV). APV fall into three major clades of phylogenetically related viruses. The first one (Clade A, 7 subclades) is related to FWPV which is the prototypic member of the genus, and the causal agent of a widespread, enzootic disease of domestic chickens and other gallinaceous birds. The second one (Clade B, 3 subclades) is related to CNPV which causes high mortality rates to songbirds such as canaries and magpies and the third clade (Clade C) comprises the psittacine PV.

Virion Structure Since the recognition of APV and orthopoxviruses as the causative agents of disease in the early decades of the nineteenth century, scientists have striven to elucidate their structure. Nevertheless, it has only recently become possible to acquire data on the precise location of poxvirus structural proteins at subviral resolution by using super-resolution microscopy and computational tools. Most

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00112-0

343

344

Fowlpox Virus and Other Avipoxviruses (Poxviridae)

Table 1

List of complete Avipoxviruses genomes

Strain

Organism

Host

Isolation source

GenBank Sequence Accession

Size (Kbp)

Open reading frames

ATCC VR 111 FGPV FGPVKD09 HP1 438 FP9

Canarypox virus Flamingopox virus Fowlpox virus

USA, 1948 South Africa, 2008 UK, 2003

NC_005309 NC_036582 AJ581527

359 293 266

328 256 241

FPVUS PEPVPSan92 PGPV FeP2 SWPV 1

Fowlpox virus Penguinpox virus Pigeonpox virus Shearwaterpox virus Shearwaterpox virus Turkeypox virus

Serinus canaria domestica (Canary) Phoenicoparrus minor (Lesser flamingo) isolate HP 438/Munich, passage 438, clone FP9 Iowa strain Spheniscus demersus (African penguin) Columba livia (Feral pigeon) Ardenna carneipes (Flesh-footed Shearwater) Ardenna pacificus (Wedge-tailed Shearwater) Meleagris gallopavo (Wild turkey)

USA, 2000 South Africa, 1992 South Africa, 2014 Australia, 2016

NC_002188 NC_024446 NC_024447 KX857216

288 306 282 326

261 242 224 310

Australia, 2015

KX857215

351

312

Hungary, 2011

NC_028238

188

171

SWPV

2

TKPV HU1124

structural studies in poxviruses (PV) have been focused on the archaetypal poxvirus vaccinia virus (VACV), which is the vaccine strain that eradicated smallpox (variola virus). In common with the other PV, APV are large viruses (about 330  280  200 nm) that can just be seen by light microscopy. Electron microscopy revealed that APV display the characteristic brick or ovoid morphology with a biconcave viral core. The viral core contains the linear dsDNA genome and enzymes for the virus uncoating and genome replication. It is enclosed by a lipoprotein envelope and flanked by two protein structures called the lateral bodies. All these structures are contained within an outer coat composed of randomly arranged surface tubules. The role of lateral bodies is not entirely clear and until recently it was proposed to be merely structural. Recent studies in VACV (and FWPV; unpublished) suggest that lateral bodies pack immune modulatory proteins, which can be delivered (upon virus uncoating) into the cytoplasms of target cells. They can therefore serve as facilitators of an early innate immune evasion strategy.

Genome APV have a single, unsegmented, linear molecule of double-stranded DNA (260–365 kbp) and encode on average more than 230 proteins. Their genome has a low GC-content (30%–40%) compared to 460% for parapoxviruses and molluscipoxviruses and 20% for entomopoxviruses. Similar to all PV, the central region of their genome is flanked by two identical inverted terminal repeats covalently linked by hairpin loops. The central region contains nearly 100 relatively conserved (among Chordopoxviruses) genes, which encode viral DNA replication, transcription and RNA modification, and virion formation. The terminally located genes are more prone to mutation and recombination, are thus more variable and encode proteins typically involved in host range restriction, immunomodulation and pathogenesis. Although APV have evolved to infect a wide range of avian species, to date only few complete APV genomes are available (Table 1); a pathogenic strain of Fowlpox virus (FPVUS), an attenuated strain of Fowlpox virus (FP9), a virulent Canarypox virus (VR-111), a pathogenic South African strain of Pigeonpox virus (FeP2), a Penguinpox virus (PSan92), a pathogenic strain of Turkeypox virus (HU1124), a Flamingopox virus (FGPVKD09) and two novel avipoxviruses from pacific shearwaters (Ardenna spp; SWPV-1 and SWPV-2). A comparison of the available APV genomes revealed overall synteny in their genome arrangement. There are however significant differences (i.e., gene duplications, translocations etc) both in the terminal and the internal regions in contrast to the high levels of conservation of central genomic regions in orthopoxviruses. Comparison of the plaque-purified tissue cultureadapted attenuated European strain FP9 strain with the US virulent FWPV strain revealed 118 differences, of which 71 genes were affected by deletion (26 of 1–9334 bp), insertion (15 of 1–108 bp), substitution, termination or frame-shifts. Only 110 (42%) of FWPV genes share significant similarity to those in other PV. The CNPV genome is about 80–100 Kbp larger than FWPV, with 39 genes lacking FWPV homologs and approximately 47% amino-acid divergence primarily in the terminal variable regions. The TKPV genome is considerably smaller than the other APV genomes. FeP2 and PEPV are more closely related to FWPV but the presence of whole or disrupted genes that are absent in FWPV but similar to CNPV sequences, suggests that they originate from a common ancestor of CNPV and FWPV. The DNA sequences of SWPV-1 and SWPV-2 are significantly different from each other but nevertheless have good similarity with CNPV (67% and 98% respectively). Genomic profiles of mynahpox, and quailpox viruses show marked differences from FWPV. Compared to mammalian PV, APV show considerable genome reorganization. The most closely related mammalian PV are the molluscipoxviruses and parapoxviruses with which APV share key aspects of gene organization as well as several aspects of immunomodulation and pathogenesis. Many field FWPV strains carry an active copy of the retrovirus reticuloendotheliosis virus (REV). REVs are closely related to mammalian retroviruses and have also been reported in the gallid herpesvirus 2 (GHV-2) that causes Marek’s disease in chickens

Fowlpox Virus and Other Avipoxviruses (Poxviridae)

345

Fig. 1 The replication cycle of APV. Certain elements of the schematic are from Motifolio.

and other galliform birds. The origin of REV insertion into the APV genome is a point of conjecture. Current theories propose that REV endogenization is either due to an ancestral insertion event following natural infection or that it has instead a iatrogenic origin. The REV insertion may account for the strains’ increased virulence and fitness. Certainly, REV has been considered one of the most important vaccine contaminants as it can interfere with vaccine-induced immunity. Several vaccine strains (i.e., FPV-S) have already been withdrawn from the market over fears that they are contaminated with REV. Nevertheless, most passaged laboratory and commercial vaccine strains appear to have lost most of the provirus, sometimes leaving only a single long terminal repeat sequence. In these cases the inserted REV sequences cannot give origin to a replication competent virus and therefore should not pose risks for the vaccinated animals.

Life Cycle The life cycle of PV, illustrated in Fig. 1, is a highly conserved (among Chordopoxviruses) and complex sequence of events that commences with the binding of PV onto the cell surface and ends with the release of virions from the cells. APV replicate exclusively in the host cell cytoplasm and have two distinct infectious virus particle types: the unenveloped mature virus (MV) or intracellular MV (IMV) and the extracellular enveloped virus (EEV). The MV and EEV virions differ in their surface glycoproteins and number of surrounding membranes and can both initiate infection. Viral entry involves either macropinocytosis followed by fusion of the viral envelope and the endocytic vesicle membrane or direct fusion of the virion with the plasma membrane. Upon fusion, the viral cores containing the viral DNA, viral transcription factors, and RNA polymerases pre-bound to early promoters, are liberated into the host cell cytoplasm and undergo a morphological modification termed activation. Core activation includes the switching of the virion shape from biconcave to oval, the uncoupling of the lateral bodies and the initiation of early viral gene expression under the control of viral early promoters. This step is followed by the rupture of the core structure, which releases the viral DNA into the cytoplasm. DNA replication is initiated immediately and is followed by two waves of transcription (intermediate and late), which are mediated by host-derived transcription factors. The newly synthesized viral DNA serves as a template for the subsequent cycles of genome replication and for the transcription of viral genes. Replication and virion assembly take place in cytoplasmic structures called virus factories or virosomes. APV generally undergo the various morphogenic stages similar to VACV but with some notable differences. Electron microscopy studies in FWPV showed that the first viral structures are crescent-shaped structures, formed in the virus factories, that begin to incorporate viral DNA. These structures develop into spherical immature virions (IV), which subsequently mature into the characteristic infectious brick- or ovoid-shaped IMVs. The two-lipid bilayer membranes of IMVs derive from the compartment between the endoplasmic reticulum and the cis-Golgi cisternae. In VACV, IMVs can either remain in the cells until cell lysis, bud through the plasma membrane or acquire an additional bilayer membrane by a process called “wrapping”. The VACV intracellular enveloped virions (IEV), formed after wrapping, migrate by actin tails to the cell surface where the external membrane of the virus

346

Fowlpox Virus and Other Avipoxviruses (Poxviridae)

fuses with the plasma membrane, releasing EEV. In contrast to VACV, electron microscopy and proteomic studies have demonstrated that FWPV, CNPV and PGPV lack some of the key genes involved in the wrapping step and the formation of actin tails. FWPV and CNPV appear to exit cells predominantly by budding of the IMV through the plasma membrane to form EEV.

Epidemiology The epidemiology of the APV is still unclear, due to a lack of studies, the scarcity of genomic data and consequent inability to accurately assign viral isolates into species. APV appear fairly host restricted, but the picture is complicated because many atypical species-species transmissions can occur e.g., prey to predator transmissions, close proximity of diverse animals in zoos and animal facilities etc. APV are considered a threat to both passerine and non-passerine birds including endangered species. Dramatic losses of passerine birds caused by CNPV-like viruses have been reported worldwide in wildlife ecosystems such as in Hawai, the Galapagos, Australia and New Zealand. Hosts included Andean condors, Australian magpie, Currawongs, Hawaiian crows and geese, palila, apapane and silvereyes. Since the 1950s, APV have been reported in a number of bird species including blackbirds, carrion crows, chaffinches, doves, dunnocks, goldfinches, greenfinches, starlings, jackdaws, pigeons, and sparrows. Since 2005, there has been a marked increase in reports of APV in great tits, with the disease confirmed in several central and northern European countries including the UK. Blue and coal tit populations have been less affected. APV outbreaks have been associated with wildlife translocations or human interventions. However, without long-term data on the (sero)prevalence of APV in wild birds, it is difficult to trace the infection sources and assess the associated risks for wild life. APV are transmitted via biting insects (e.g., mosquitos, mites, flies) and/or aerosols and invade birds through broken skin. The transmission potential of APV has been suggested to be correlated to the range of insect vector populations. In the face of climate change, seasonal and geographical shifts of insect populations may have broad repercussions in future outbreaks of APV. FWPV (and most likely all APV) is resistant and can survive in the environment for extended periods of time in desiccated scabs shed from infected hosts. Resistance is due to embedment of viruses within large cytoplasmic inclusions and production of photolyase which protect virions following cell lysis and during their environmental exposure.

Pathogenesis Studies in domestic poultry with FWPV and in canaries with CNPV have been responsible for most of the information currently available in APV pathogenesis. There is evidence that the effects of APV can vary in different avian species. The generalized pathogenicity model is that APVs replicate at the site of inoculation and cause dermal hyperplasia (pock lesions) and leukocyte infiltration. APV infection is typically found in one of two forms: the cutaneous and the diphtheritic form. Both forms can occur at the same time. A third rare systemic form (generalized) can also occur occasionally in canaries, causing primarily depression and anorexia. The cutaneous form of the disease (‘dry’ pox) is relatively mild and usually characterized by nodular, proliferative growths that are most commonly restricted to the eyes, beak or unfeathered skin of the body. Atypical forms of dry pox can be found in the feathered skin of the birds i.e., feather folliculitis. Infection is slow-developing and can become persistent and extensive although most often is self-limited. With few exceptions, dry pox only causes a drop in productive performance of birds. The nodular lesions are rarely fatal but may restrict the birds’ vision and ability to hunt/forage thus making them more susceptible to predation and secondary infections. In canaries the cutaneous lesions can be accompanied by often severe inflammation mainly in the periocular space that may be complicated by secondary infections. Inhalation or ingestion of aerosols from the dried scabs can lead to more severe infection the so-called “diphtheritic infections” (“wet pox”). This is characterized by fibronecrotic lesions on the mucous membranes of the respiratory and digestive tracts. These lesions resemble those of the avian herpesvirus infectious laryngotracheitis virus. Symptoms of wet pox also resemble those of other avian viruses that cause upper respiratory disease such as avian influenza, or Newcastle Disease virus, thus complicating initial diagnosis. Wet pox causes up to 15% mortality in chicken flocks by occlusion of the larynx and/or secondary bacterial/ fungal infections. Upon histological examination of FWPV-infected tissues, variably sized and often large, eosinophilic intracytoplasmic inclusions (Bollinger bodies) can be observed with elementary bodies called Borrel’s bodies. Other confirmatory techniques for laboratory diagnosis of FWPV include PCR, electron microscopy and viral culture. The mortality associated with FWPV disease in poultry is usually low, but in flocks under stress, mortality can reach up to 50%. CNPV is generally associated with higher mortality rates (approaching 100%) often without the characteristic skin lesions.

Host range FWPV affects mainly chickens and turkeys and has been reported to infect ducks, geese, pheasants, quail, canaries and hawks. Mammals are not susceptible to natural infection with FWPV (or any of the other APV). Nelson (1941) reported mild pathology in mice following intranasal inoculation with FWPV, with no virus replication. Atypical infections have also been reported (i.e., rhinoceros). More recent (albeit limited) studies have shown the presence of infectious viral particles and CPE of FWPV in mammalian cell cultures, such as embryonic bovine tracheal cells and baby hamster kidney cells. These studies challenge the general dogma that FWPV cannot undergo a full replication cycle in mammalian cells. Unlike the FWPV-like, the CNPV-like APV appear able to infect a wider range of avian species. CNPV causes mild infection to its most likely natural hosts, the native songbirds of temperate climes, and more severe infections in non-native canaries.

Fowlpox Virus and Other Avipoxviruses (Poxviridae)

347

The target cell determinants of PV binding are thought to be ubiquitously expressed glycosaminoglycans and no specific cell receptors are yet known to be required for virion fusion and cell entry. Host range restriction of PV is attributed to post-entry events associated with antiviral mechanisms such as the type I IFN response or T-cell responses.

Immunomodulation PV have developed strategies to prevent or evade early host innate responses induced as a consequence of the viruses’ binding/ fusing with the cell membrane and their progressive migration through diverse cell types and tissues. APV encode many gene homologs with possible host range functions such as chemokine-like molecules (e.g., fpv061, fpv121), putative chemokine receptors (e.g., fpv021, fpv206) as well as inhibitors and homologs of genes involved in apoptosis, autophagy, cell growth and various signaling cascades. Both FWPV and CNPV encode mimics of transforming growth factor (TGF)-b, and CNPV encodes an IL-10-like protein. These proteins are predicted to stimulate regulatory T-cells and suppress the host inflammatory responses. Viral mimicry of cytokines, chemokines and their receptors is well documented in orthopoxviruses. Whether APV use similar mechanisms to evade detection and destruction by the host immune system remains to be confirmed upon availability of chicken reagents. Our understanding of the strategies deployed by APV to disarm the interferon (IFN) response, the first line of host defense against invading viruses, remains rudimentary and is limited to few FWPV immunomodulatory proteins. FWPV and CNPV (unpublished), unlike other avian viruses (e.g., infectious bursal disease virus) do not induce type I IFN response in chicken embryo fibroblast culture. Moreover, both the wild-type pathogenic FWPV and the attenuated vaccine strain FP9 can block the induction of expression of the chicken interferon b (ChIFN2) promoter normally seen upon transfection with the dsRNA analog, polyI: C. A broad-scale loss-of-function genetic screen involving a library of 48 FWPV in vitro-generated mutants, each defective in a single, nonessential gene, identified fpv012 as a modulator of poly(I
C)-mediated ChIFN2 induction. fpv012 is a member of a family of genes that are highly expanded in the APV (31 in FWPV; 51 in CNPV), encoding proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs (ANK-PRANC proteins). Under ectopic expression, the first ANK of fpv012 is dispensable for inhibitory activity and the CNPV ortholog is also able to inhibit induction of ChIFN2. Conversely, a gain-offunction approach in which 4–8 kbp fragments of the FWPV FP9 strain were introduced into the vaccine strain modified vaccinia Ankara, identified that another ANK-PRANC protein (FPV014) contributes to increased resistance to exogenous recombinant chicken IFN-a.

Control & treatment FWPV infections in chickens and turkeys are generally well controlled with a standard vaccination program. The timing of vaccinations is dictated by the number of affected birds in a flock (when o20% of the birds have lesions), the onset of disease, the flock size and the type of commercial operation. Modified live FWPV, and PGPV vaccine strains of cell-culture origin are commercially available. The vaccines are usually applied by the wing-web stick method (or thigh-stick methods for turkeys), or injection between the age of 10–14 weeks of age. Birds can be vaccinated at any age if necessary. In some areas where mosquito populations are present throughout the year two vaccinations per year are used. For CNPV infections in pet canary birds and other passerine birds, a live attenuated CNPV vaccine is considered the best preventive measure. Vaccination could also play a role in the conservation management of other endangered avian species that are significantly affected by APV disease. Transmission prevention measures include an insect control program, the improvement of farm hygiene and animal husbandry practices (e.g., dust control, disinfection, ventilation, prevention of fighting of male birds in bird housing, quarantine procedures). Currently there is no treatment available and diseased birds are treated symptomatically i.e., by washing and sterilizing infected areas, removal of necrotic tissues from mouth and throat of infected birds etc. In the event of an outbreak, disinfecting water lines with a liquid iodine disinfectant, appears to reduce mortality. Broad-spectrum antibiotics and vitamin supplements are beneficial for the birds if there is evidence of secondary bacterial infection.

Use of APV as recombinant vaccine vectors There is no known zoonotic risk associated with APV. APV cause a productive infection in avian hosts and a non-productive or abortive infection in mammalian hosts. This feature allowed the development of host-restricted APV vectors as safe platforms for vaccines or gene delivery including CNPV (ALVAC) and FWPV (TROVAC). Other desirable features that make APV ideal vaccine vectors include their large genome size which supports a large coding capacity, their ability to readily enter most vertebrate cells to express antigens and their ability to modulate the host innate and adaptive immune responses. Numerous attenuated, live APVbased vaccine strains have been developed since the 1920s against diverse pathogens including avian influenza, Newcastle disease, rabies, cancer, some of which are currently tested or in use in both veterinary and human medicine. A landmark vaccination campaign using for the first time recombinant FWPV was employed successfully in Mexico in the mid-1990s to control high pathogenicity avian influenza H5N2 with almost a billion doses used. Since then, recombinant FWPV have been used in largescale vaccinations in China, Southeast Asia and elsewhere. The subject of APV vaccine-vector development is beyond the scope of this article and has been comprehensively reviewed by others. As more information is gathered on APV virulence, immune evasion and host range, it is likely that this knowledge will facilitate the engineering of safer and more efficient vectors for vaccines and gene therapy.

348

Fowlpox Virus and Other Avipoxviruses (Poxviridae)

Further Reading Bidgood, S.R., Mercer, J., 2015. Cloak and dagger: Alternative immune evasion and modulation strategies of poxviruses. Viruses 7, 4800–4825. Bolte, A.L., Meurer, J., Kaleta, E.F., 1999. Avian host spectrum of avipoxviruses. Avian Pathology 28, 415–432. Boulanger, D., Smith, T., Skinner, M.A., 2000. Morphogenesis and release of fowlpox virus. Journal of General Virology 81, 675–687. Boyle, D.B., 2007. Genus Avipoxvirus. Poxviruses. In: Mercer, A., Schmidt, A., Weber, O. (Eds.), Birkhäuser Advances in Infectious Diseases, first ed. Basel: Birkhäuser Verlag, pp. 217–251. [217-251]. Giotis, E.S., Skinner, M.A., 2019. Spotlight on avian pathology: Fowlpox virus. Avian Pathology 48, 87–90. Gubser, C., Hué, S., Kellam, P., Smith, G.L., 2004. Poxvirus genomes: A phylogenetic analysis. Journal of General Virology 85, 105–117. Gyuranecz, M., Foster, J.T., Dán, Á., et al., 2013. Worldwide phylogenetic relationship of avian poxviruses. Journal of Virology 87, 4938–4951. Herbert, M.H., Squire, C.J., Mercer, A.A., 2015. Poxviral ankyrin proteins. Viruses 7, 709–738. McFadden, G., 2005. Poxvirus tropism. Nature Reviews in Microbiology 3, 201–213. Nelson, J.B., 1941. The behavior of pox viruses in the respiratory tract: IV. The nasal instillation of fowl pox virus in chickens and in mice. Journal of Experimental Medicine 74, 203–212. Niewiadomska, A.M., Gifford, R.J., 2013. The extraordinary evolutionary history of the reticuloendotheliosis viruses. Plos Biology 11, e1001642. Pattison, M., McMullin, B., Alexander, D., 2008. Poultry Diseases, sixth ed. India: Elsevier, pp. 333–339. Schat, K.A., Skinner, M.A., 2014. Chapter 16: Avian immunosuppressive diseases and immunoevasion. In: Schat, Karel A., Kaspers, Bernd, Kaiser, Pete (Eds.), Avian Immunology, second ed. London: Academic Press, pp. 275–297. Trupkiewicz, J., Garner, M.M., Juan-Sallés, C., 2018. Chapter 33: Passeriformes, caprimulgiformes, coraciiformes, piciformes, bucerotiformes, and apodiformes. In: Terio, K.A., McAloose, D., St. Leger, J. (Eds.), Pathology of Wildlife and Zoo Animals, first ed. London: Academic Press, pp. 799–823. van Riper, C., Forrester, D.J., 2007. Avian Pox. In: Thomas, N.J., Hunter, D.B., Atkinson, C.T. (Eds.), Infectious Diseases of Wild Birds. Oxford: Wiley Blackwell Publishing. Weli, S.C., Tryland, M., 2011. Avipoxviruses: infection biology and their use as vaccine vectors. Virology Journal 8, 49.

Relevant Websites www.avianvirusresearch.org AvianVirusResearch. Bringing avian virologists together. http://www.ictvonline.org International Committee on Taxonomy of Viruses (ICTV). www.msdvetmanual.com Veterinary Manual. https://www.viprbrc.org/ Virus Pathogen Database and Analysis Resource. http://www.thepoultrysite.com The Poultry Site. Your poultry knowledge hub. http://www.violinet.org VIOLIN: Vaccine Investigation and Online Information Network.

Hantaviruses (Hantaviridae) Tarja Sironen and Antti Vaheri, University of Helsinki, Helsinki, Finland © 2021 Published by Elsevier Ltd. This is an update of A. Vaheri, Hantaviruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00411-8.

Glossary Coevolution Evolution of a virus together with its reservoir host.

Hantavirus A virus in the genus Orthohantavirus. Reassortant A virus having genome segments of two different viruses.

Historical Introduction Hantavirus infections are not new to humankind. The first description of a hemorrhagic fever with renal syndrome (HFRS)-like disease can be found in a Chinese medical account written in about AD 960 and the earliest definite description of HFRS comes from Far East Russian clinical records dating back to 1913. During World Wars I and II, HFRS became an important military problem; for example, ‘field nephritis’ in Flanders during World War I may well have been caused by a hantavirus. In Manchuria in the mid-1930s, 12,000 Japanese soldiers caught the disease and military researchers were investigating the cause of the disease, sometimes using prisoners of war in infection experiments. Finnish and German soldiers encountered an HFRS-like epidemic in Finnish Lapland in 1943–1944. During the Korean conflict in 1950–1953, the disease gained again much attention when about 3000 United Nations troops contracted it – since then known as Korean hemorrhagic fever – with a 5%–10% case–fatality rate. The milder form of HFRS, nephropathia epidemica (NE), common in Fennoscandia (Scandinavia and Finland), was first described by Swedish authors in 1934. However, the infecting agent of HFRS remained unknown until 1976, when the Korean researcher Ho Wang Lee discovered that cryostat-sectioned lungs of striped field mice (Apodemus agrarius), trapped near the Hantaan river, contained virus-specific antigen reactive with HFRSpatient sera. By a similar approach, the causative agent of NE was demonstrated in 1980 in bank voles, Myodes glareolus, trapped in Puumala, Eastern Finland. Another agent, which identified in urban rats in Seoul, Korea, was also found to cause HFRS. In the early 1990s, a further distinct European human-pathogenic hantavirus was isolated from the yellow-necked mouse, Apodemus flavicollis, near the village of Dobrava in Slovenia, and a few years later, a related, less pathogenic variant, Saaremaa virus from striped field mice on Saaremaa Island, Estonia. Thottapalayam virus from an insectivore (Suncus murinus, a shrew) in India, Prospect Hill virus from a meadow vole (Microtus pennsylvanicus) in USA, and Thailand virus from a bandicoot rat (Bandicota indica) had already been isolated in the early 1970s to the 1980s – apparently they all are nonpathogenic for humans. Human disease had not been known to be caused by a hantavirus in the Americas until a cluster of acute respiratory distress syndrome cases with a high (40%) case–fatality rate in the Four Corners area of Southwestern USA (where Arizona, Utah, Colorado, and New Mexico are contiguous) was recognized in May 1993. Subsequent studies led to the discovery of Sin Nombre virus and other hantaviruses that cause hantavirus cardiopulmonary syndrome (HCPS), all transmitted from sigmodontine rodents indigenous to the New World. An increasing number of pathogenic hantaviruses have been reported in South America, including Andes virus, which can be transmitted from person to person, and which possess high case–fatality rates. While only a few thousand HCPS cases have been reported so far, approximately 50,000 HFRS cases are estimated to occur worldwide annually. In the era of advanced sequencing methods, the rate of hantavirus discovery has accelerated. A great majority of the novel hantaviruses are carried by soricomorpha hosts, i.e., shrews and moles, and few of them by insectivorous bats. These variants appear to be non-pathogenic to humans. In 2016, the new order Bunyavirales was established, elevating the previous genus Hantavirus to the family Hantaviridae. Simultaneously, the species demarcation criteria were updated and the family was divided into 4 subfamilies, 7 genera, and 47 species. The hantaviruses causing human infections belong to the subfamily Mammantavirinae and genus Orthohantavirus.

Virology Hantaviruses are enveloped viruses with a tripartite negative-stranded RNA genome (Fig. 1). The 6.4 kb L (large) genome segment encodes a B250 kDa RNA polymerase, the B3.6 kb M (medium) segment, two glycoproteins 68–76 kDa Gn and 52–58 kDa Gc (formerly known as G1 and G2), and the B1.7 kb S (small) segment a 50–54 kDa nucleocapsid protein (N). In addition, the S segment of hantaviruses carried by cricetidae rodents has another overlapping ( þ 1) open reading frame (ORF) named NSs, which has been shown to inhibit the interferon response. Viral messenger RNAs of the members of the Bunyaviridae are not polyadenylated and are truncated relative to the genomic RNAs at the 30 termini. Messenger RNAs have 50 -methylated caps and 10–18 nontemplated nucleotides derived from host cell mRNAs. The termini of all three segments are highly conserved and complementary to each other, a feature that has assisted in cloning and discovery of new hantaviruses (Fig. 2).

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00137-5

349

350

Hantaviruses (Hantaviridae)

Fig. 1 Structure of a orthohantavirus. (a) The hantavirus particle contains the trisegmented viral RNA (vRNA) genome, comprising the small, medium and large ORFs. These are encapsidated by nucleocapsid (N) protein. The outer part of the virion consists of spikes comprising four units of each glycoprotein, Gn and Gc. The viral genome is replicated and transcribed by RNA-dependent RNA polymerase (RdRp). (b) | A hantavirus particle viewed by cryoelectron microscopy. The spike height is invariably 12 nm, and the median diameter of the virion is 135 nm. Part b image is courtesy of P. Laurinmäki and S. Butcher, University of Helsinki, Finland.

Unlike most other bunyaviruses, hantaviruses are not arthropod borne, their reservoirs are rodents, insectivores or bats. Each hantavirus is primarily carried by a distinct reservoir host species, although several host switches seem to have occurred during tens of millions of years of their co-evolution with their carrier hosts. We now know that the genetic diversity of hantaviruses is generated by (1) genetic drift (accumulation of point mutations and insertions/deletions) leading to mixtures of closely related genetic variants, quasispecies; (2) genetic shift (reassortment of genome fragments within a given virus genotype/species); and (3) to some extent, by homologous recombination.

Ecology and Epidemiology Hantavirus infections are prime examples of emerging and reemerging infections. Like most of these infections, hantaviral diseases are zoonoses. With the exception of the South American Andes virus, hantavirus infections are thought to be transmitted to humans primarily from aerosols of rodent excreta (feces, urine, saliva). Andes virus has also been shown to be transmitted human-to-human. Infections of hantaviruses in the rodents are asymptomatic and persistent, and have only a minor effect on the host. Some hantaviruses cause disease in humans. In Asia, Hantaan virus, carried by Apodemus spp. mice, cause severe HFRS, and Seoul virus, carried by rats, causes a milder disease. In Europe, there are two major hantaviral pathogens: Puumala virus carried by bank voles causes NE, while Dobrava virus (DOBV), carried by yellow-necked or striped-field mice, causes severe HFRS. Four different genotypes of DOBV circulate in Europe: the genotypes Dobrava and Sochi cause severe illness, while the genotypes Kurkino and Saaremaa a mild, NE-like disease. There also are reports that Seoul virus (SEOV), carried by rats (Rattus norvegicus and Rattus rattus), causes HFRS of moderate severity both in Europe and North America. Recent findings suggest that pet rats may also act as reservoirs of SEOV. In addition, European common voles (Microtus arvalis and Microtus rossiaemeridionalis) carry Tula hantavirus, which can asymptomatically infect humans on rare occasions. Hantavirus infections are quite common in Europe. Puumala virus occurs in Northern Europe, European Russia, and parts of Central and Western Europe. Dobrava genotype of DOBV is found mainly in the Balkans and the neighboring Central European areas. The genotypes Kurkino and Saaremaa have been detected in Eastern and Central Europe, while the genotype Sochi (carried by Apodemus ponticus) is circulating in the southern European Russia. Apart from infections of laboratory rats, Seoul virus has been detected in wild rats in France and in several city harbors. Seoul virus infections from pet rats have been increasing lately in Europe, but also in US. It is apparent that many parts of Europe, and most of Africa, remain completely or relatively unstudied with regard to hantaviruses. This suggests either that HFRS is rare or nonexistent in these regions or is not generally recognized and is not diagnosed by local biomedical communities.

Hantaviruses (Hantaviridae)

351

Fig. 2 Life cycle of a hantavirus in an infected cell. The hantavirus virion attaches to a receptor on the cell surface (step 1). This binding event induces endocytosis signaling (step 2), after which the virion enters the cell in clathrin-coated vesicles (step 3). Other entry pathways have also been observed for some hantaviruses. In the case of clathrin-mediated endocytosis, the clathrin coat of the vesicle is disassembled (step 4), and the virion-harboring vesicle enters the early endosome (step 5), which matures into a late endosome (step 6). Fusion between the viral and endosomal membranes is driven by acid-induced conformation changes in the viral fusion protein in the late endosome. This results in release of the viral ribonucleoproteins (RNPs) (step7). Initial transcription might take place at the site of release; alternatively, the RNPs might be transported to the ER–Golgi intermediate compartment (ERGIC) for transcription. It is also possible that the virus is directly transported to the Golgi complex from the late endosome, either before or after fusion. Viral replication is thought to occur in viral factories that might be located at the ERGIC or the cis-Golgi (step 8). The nascent viruses are thought to bud into the cis-Golgi (step 9), from where they are transported to the plasma membrane for release, presumably via recycling endosomes. The egress of progeny virions takes place at the plasma membrane (step 10).

In Northern Europe, HFRS as well as the carrier rodents exhibit peaks in 3–4 year cycles, while in Central Europe the HFRS incidence follows the fluctuations of “mast years”, that is, the abundance of beech and oak seeds for the hantavirus-carrying rodents. In Central Europe, HFRS peaks in the summer whereas in Northern Europe most cases occur in late autumn and early winter, from November to January. Risk factors for acquiring hantavirus infections and HFRS include professions such as forestry, farming, and military, or activities such as camping, and the use of summer cottages. Cigarette smokers and males are more likely to be infected than are females. In the Americas, the increased precipitation during El Niño/Southern Oscillation in South and North America has been suggested as the main reason for the peaks in rodent population densities and for the consequent increased number of HCPS cases. Sigmodontine-borne hantaviruses circulating in North America form three phylogenetically distinct groups: those associated with Peromyscus spp. and Reithrodontomys spp. rodents, and the third carried by Sigmodon spp. and Oryzomys spp. rats. The first group carries both human pathogens (Sin Nombre virus) and viruses not thus far associated with human disease (e.g., Blue River virus, Limestone Canyon virus). Reithrodontomys-borne viruses have not been shown to cause disease in humans. Hantaviruses discovered in South America are associated with several tribes of Sigmodontinae, mostly Oryzomyini-associated viruses, several of which are important human pathogens, including Andes virus. Results of phylogenetic analyzes of South American hantaviruses suggest that several host-switching events have occurred during coevolution with their rodent hosts. The phylogenetic split

352

Hantaviruses (Hantaviridae)

between murine and sigmodontine rodents presumably dates to a divergence that occurred between subfamilies 30 million years ago, when the precursors of the sigmodontine rodents crossed the Bering Strait into the Americas. Notably, sigmodontine rodents are found only in the Americas and thus it is unlikely that HCPS would occur in Eurasia, unless imported.

Clinical Picture and Pathogenesis Dobrava, Hantaan, Seoul and Puumala viruses all cause HFRS but the infections differ considerably in severity. All are characterized by acute onset, fever, headache, abdominal pains, backache, temporary renal insufficiency (first oliguria, proteinuria, and increase in serum creatinine, and then polyuria), and thrombocytopenia, but the extent of hemorrhages (hematuria, petechiae, internal hemorrhages), requirement for dialysis treatment, hypotension, and case–fatality rates are much higher in HFRS caused by Dobrava, or Hantaan viruses than in NE caused by Puumala or Saaremaa and Kurkino genotype viruses. About a third of NE patients experience temporary visual disturbances (myopia), which is a very characteristic if not pathognomonic sign of the Table 1

Orthohantavirus types

Virus

Host

Distribution

Disease

MURIDAE-BORNE Hantaan (HTNV)a Da Bie Shan Seoul (SEOV) Dobrava-Belgrade (DOBV) Sangassou (SANGV) Thailand (THAIV) Tigray (TIGV)

Mice and rats Striped field mouse (Apodemus agrarius) Chinese white-bellied rat (Niviventer confucianus) Rat (Rattus rattus, R. norvegicus) Yellow-necked mouse (A. flavicollis) African wood mouse (Hylomyscus simus) Bandicoot rat (Bandicota indica) Ethiopian white-footed rat (Stenocephalemys albipes)

Asia (Korea) Asia (China) Worldwide (Korea) Europe (Slovenia) Africa (Guinea) Asia (Thailand) Africa (Ethiopia)

HFRS NR HFRS HFRS NR NR NR

CRICETIDAE/ARVICOLINAE-BORNE Puumala (PUUV) Tula (TULV) Prospect Hill (PHV) Khabarovsk (KHAV) Fugong (FUGV) Fusong (FUSV) Luxi (LUXV)

Voles and lemmings Bank vole (Myodes glareolus) European common vole (Microtus arvalis) Meadow vole (M. pennsylvanicus) Reed vole (M. fortis) (Eothenomys eleusis) Reed vole (Microtus fortis) Yunnan red-backed vole (Eothenomys miletus)

Europe (Finland) Europe (Russia) North America (USA) Asia (Far East Russia) Asia (China) Asia (China) Asia (China)

HFRS NR NR NR NR NR NR

CRICETIDAE/SIGMODONTINAE BORNE Sin Nombre (SNV) Bayou (BAYV) Black Creek Canal (BCCV) Muleshoe (MULV) Andes (ANDV) Choclo (CHOV) Laguna Negra (LANV) Rio Mamore (RIOMV) Caño Delgadito (CADV) El Moro Canyon (ELMCV) Maporal (MAPV) Montano (MTNV) Necocli (NECV)

New World mice and rats Deer mouse (Peromyscus maniculatus) Rice rat (Oryzomys palustris) Hispid cotton rat (Sigmodon hispidus) Hispid cotton rat (S. hispidus) Long-tailed pygmy rice rat (Oligoryzomys longicaudatus) Pygmy rice rat (O. fulvescens) Vesper mouse (Calomys laucha) Small-eared pygmy rice rat (O. microtis) Cane mouse (S. alstoni) Western harvest mouse (Reithrodontomys megalotis) Delicate pygmy rice rat (Oligoryzomys delicatus) Orizaba deer mouse (Peromyscus beatae) Zygodontomys brevicauda

North America (USA) North America (USA) North America (USA) North America (USA) South America (Argentina) Central America (Panama) South America (Paraguay) South America (Bolivia) South America (Venezuela) North America (USA) South America (Venezuela) Central America (Mexico) South America (Colombia)

HCPS HCPS HCPS NR HCPS HCPS HCPS NR NR NR HCPS NR NR

SORICOMORPHA BORNE Asama (ASAV) Asikkala (ASIV) Bowe (BOWV) Bruges virus (BRGV) Cao bang (CBNV) Jeju (JJUV) Kenkeme (KKMV) Oxbow (OCBV) Seewis (SWSV) Rockport (RKPV) Yakeshi (YKSV)

Insectivores Japanese shrew mole (Urotrichus talpoides) Eurasian Pygmy Shrew (Sorex minutus) Doucet's musk shrew (Crocidura douceti) European mole (Talpa europaea) Chinese mole shrew (Anourosorex squamipes) Asian lesser white-toothed shrews (Crocidura shantungensis) Flat-skulled shrew (Sorex roboratus) American shrew mole (Neurotrichus gibbsii) Common shrew (Sorex araneus) Eastern mole (Scalopus aquatiqus) Sorex isodon

Asia (Japan) Europe Africa Europe Asia (Vietnam) Asia (Korea) Asia (Russia) North America Europe (Switzerland) North America (USA) Asia (China)

NR NR NR NR NR NR NR NR NR NR NR

a

Type species. Abbreviation: HFRS, hemorrhagic fever with renal syndrome; HCPS, hantavirus pulmonary syndrome; NR, disease not recorded.

Hantaviruses (Hantaviridae)

353

disease. Notably, the clinical consequences of all of the hantaviral pathogens in humans vary from none to fatal. Severe NE is associated with a certain haplotype, HLA-B8, DR3, DQ2 alleles, severe HCPS with HLA-B35, and mild NE with HLA-B27. Yet, although Puumala virus infection is generally associated with mild HFRS, NE may have significant long-term consequences. A 5-year follow-up study demonstrated that 20% of NE patients had a somewhat increased systolic blood pressure and proteinuria. This is important, since the infection is relatively common in many areas of Europe. In addition, in some patients, Puumala virus infection may infect the pituitary gland and lead to mortality or at least to hypophyseal insufficiency requiring hormonereplacement therapy. Hormonal deficiencies are common in acute NE and 5 years after the infection, 17% of the patients are diagnosed with a chronic, overt pituitary hormone deficiency. The pathogeneses of HFRS and HPS are not fully understood. However, it is known that b3 integrins can mediate the entry of pathogenic hantaviruses and that hantaviruses can regulate apoptosis. We and others have studied the pathophysiology of Puumala virus (PUUV) and other hantavirus infections and have come to the conclusion that vascular leakage induced by bradykinin (BK) is a key element in the pathophysiology. Orthohantaviruses predominantly infect microvascular endothelial cells of different organs in humans, and the vascular permeability can be a direct effect of viral infection in these cells or result of an imbalanced immune response. Other important elements of pathophysiology include thrombocytopenia, complement activation, circulating histones, cytotoxic and regulatory T cells (CTLs and T regs), certain cytokines (IL-6 and TNF-alpha in particular) and neutralizing antibodies. In the novel therapy we have successfully blocked vascular leakage in several very severely ill hantavirusinfected patients by the BK receptor antagonist icatibant. Recent developments of both primate and Syrian hamster models that mimic hantavirus diseases should assist in elucidating the mechanisms of pathogenesis. Hantavirus disease in the Americas is characterized by pulmonary edema but death often results from a cardiac failure; thus the term hantavirus cardiopulmonary syndrome (HCPS) has been proposed for the disease. HFRS and HCPS, although primarily targeted at kidneys and lungs, respectively, share a number of clinical features, such as capillary leakage, increased serum TNF-a levels, and thrombocytopenia; notably, hemorrhages and alterations in renal function occur also in HPS and pulmonary involvement is not rare in HFRS. Of the four structural proteins, both in humoral and cellular immunity, the nucleocapsid protein appears to be the principal immunogen. Cytotoxic T-lymphocyte responses are seen in both HFRS and HPS and may be important for both protective immunity and pathogenesis in hantavirus infections.

Diagnostics and Prevention The diagnosis of an acute hantavirus infection is primarily based on serology. Both immunofluorescence tests and enzyme immunoassays are widely used for the detection of specific IgM or low-avidity IgG antibodies, characteristic for an acute infection. In addition, immunochromatographic 5 min IgM-antibody tests have been developed. Hantaviruses show extensive serological cross-reactivity, especially within each of the three virus subgroups (murid, arvicolid, and sigmodontine borne; Table 1), but for accurate typing, neutralization tests are needed. For example, in Paraguay, a considerable seroprevalence of hantavirus antibodies, as high as 40%, is noted in people without a history of HCPS. Average seroprevalence in Finland and Sweden suggest that only 10%–25% of Puumala virus infections are diagnosed; thus, most infections are either subclinical or mild or atypical and remain undiagnosed. Viral RNA can usually be detected in the blood of HCPS and HFRS patients using polymerase chain reaction with samples collected during the first week of illness, which is useful because it also identifies the infecting virus genotype. Vaccines against hantaviral infections have been used for years in China and Korea, but not in Europe or the Americas. A candidate pan-hantavirus vaccine has been reported to be protective in animal models, but is not yet in clinical trials. No specific antiviral therapy is used in Europe, but both ribavirin and interferon-a have been administered in trials in China. A single dose of icatibant, a bradykinin receptor antagonist, has been successfully used to treat patients with severe HFRS caused by Puumala. A major problem is that at the time the patients are hospitalized, the rate of virus replication is already declining, thus the reduction of virus replication by antiviral drugs is no longer necessary for the patient. Thus, prevention of hantaviral infections continues to rely on reduced contact with excreta from infected rodents.

Further Reading Figueiredo, L.T., Souza, W.M., Ferres, M., Enria, D., 2014. Hantaviruses and cardiopulmonary syndrome in South America. Virus Research 187. doi:10.1016/j.virusres.2014.01.015. Forbes, K.M., Sironen, T., Plyusnin, A., 2018. Hantavirus maintenance and transmission in reservoir host populations. Current Opinion in Virology 28, 1–6. doi:10.1016/j. coviro.2017.09.003. Hepojoki, J., Strandin, T., Lankinen, H., Vaheri, A., 2012. Hantavirus structure – Molecular interactions behind the scene. Journal of General Virology 93, 1631–1644. Holmes, E.C., Zhang, Y.Z., 2015. The evolution and emergence of hantaviruses. Current Opinion in Virology 10, 27–33. doi:10.1016/j.coviro.2014.12.007. Jonsson, C.B., Figueiredo, L.T., Vapalahti, O., 2010. A global perspective on hantavirus ecology, epidemiology, and disease. Clinical Microbiological Reviews 23, 412–441. Kariwa, H., Yoshimatsu, K., Arikawa, J., 2007. Hantavirus infection in East Asia. Comparative Immunology, Microbiology and Infectious Diseases 30, 341–356. Laenen, L., Vergote, V., Calisher, C.H., et al., 2019. Hantaviridae: Current classification and future perspectives. Viruses 11 (9), 788. doi:10.3390/v11090788. Mills, J.N., Amman, B.R., Glass, G.E., 2010. Ecology of hantaviruses and their hosts in North America. Vector Borne and Zoonotic Diseases 10, 563–574. Noack, D., Goeijenbier, M., Reusken, C.B.E.M., Koopmans, M.P.G., Rockx, B.H.G., 2020. Orthohantavirus pathogenesis and cell tropism. Frontiers in Cellular and Infection Microbiology 10, 399. doi:10.3389/fcimb.2020.00399.

354

Hantaviruses (Hantaviridae)

Olsson, G.E., Leirs, H., Henttonen, H., 2010. Hantaviruses and their hosts in Europe: Reservoirs here and there, but not everywhere? Vector Borne and Zoonotic Infections 10, 549–561. Sironen, T., Plyusnin, A., 2011. Genetics and evolution of hantaviruses. In: Plyusnin, A., Elliott, R.M. (Eds.), Bunyaviridae, Molecular and Cellular Biology. Norfolk: Caister Academic Press, pp. 61–94. Vaheri, A., Vapalahti, O., Plyusnin, A., 2008. How to diagnose hantavirus infections and detect them in rodents and insectivores. Reviews in Medical Virology 18, 277–288. Vaheri, A., Henttonen, H., Voutilainen, L., et al., 2013a. Hantavirus infections in Europe and their impact on public health. Reviews in Medical Virology 23, 35–49. Vaheri, A., Strandin, T., Hepojoki, J., et al., 2013b. Uncovering the mysteries of hantavirus infections. Nature Reviews in Microbiology 11, 539–550. Yanagihara, R., Gu, S.H., Arai, S., Kang, H.J., Song, J.W., 2014. Hantaviruses: Rediscovery and new beginnings. Virus Research 187, 6–14. doi:10.1016/j.virusres.2013.12.038.

Henipaviruses (Paramyxoviridae) Lin-Fa Wang and Danielle E Anderson, Duke-NUS Medical School, Singapore, Singapore r 2021 Elsevier Ltd. All rights reserved. This is an update of B.T. Eaton, L.-F. Wang, Henipaviruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00653-1.

Glossary Biosafety level 4 A biosafety level is a set of biocontainment precautions required to isolate dangerous biological agents in an enclosed laboratory facility. The levels of containment range from the lowest biosafety level 1 (BSL-1) to the highest at level 4 (BSL-4). Pteropid bats These are among the largest bats in the world taxonomically placed within the genus Pteropus. They are commonly known as fruit bats or flying foxes and live in the tropics and subtropics of Asia (including the Indian

subcontinent), Australia, East Africa, and some oceanic islands in the Indian and Pacific Oceans. There are at least 60 extant species in the genus. Rule-of-six It describes a requirement for paramyxoviruses to have a genome length with a multiple of six nucleotides for efficient virus replication. Zoonosis A zoonosis is an infectious disease that is transmitted between species from animals other than humans to humans or from humans to other animals.

Introduction Hendra virus (HeV), the first henipavirus, was isolated in 1994 following an outbreak of acute respiratory disease in horses in a stable in Brisbane, Australia. In a period of less than three weeks, 21 of 30 horses became infected and 14 either died or were euthanized. Two people who worked at the stable also became infected and one died. Since the initial outbreak in 1994, there have been at least 60 recognized occurrences of HeV in Australia, with at least one occurrence per year since 2006. The second member of the genus, Nipah virus (NiV), emerged in 1998–99 in Perak State, Peninsular Malaysia as the cause of an outbreak of respiratory and encephalitic disease of low morbidity and mortality in pigs. The virus spread to humans causing febrile encephalitis among pig farmers and those who had a direct contact with pigs in Malaysia and Singapore, resulting in more than 100 human casualties. A highly related variant of NiV emerged in Bangladesh in 2001, and outbreaks of human NiV infections have been detected from that country almost every year since 2001. At least three separate NiV outbreaks have occurred in India, from east Bangladeshadjacent regions to the Kerala state on the west. In 2015, an outbreak of NiV or closely related virus occurred in the Philippines with lethal infections in both horses and humans. In addition to the six countries where henipavirus outbreaks have been detected in humans, molecular and serological evidence suggests that henipavirus or henipa-related viruses are present in bats from Australia and Indonesia in the east to Madagascar and west Africa. Three other henipaviruses have been discovered since 2009: Cedar virus was isolated from flying fox in Australia, the full-length genome sequence was determined for the Ghana virus from bats in Ghana and Mojiang virus from rats in China.

Classification When the full-length genome sequence of HeV was determined, it was clear that it represented a previously unknown member of the family Paramyxoviridae, subfamily Paramyxovirinae, but one with unique genetic features that precluded classification in the existing three genera Morbillivirus, Respirovirus or Rubulavirus in the subfamily at that time. Complete genome sequencing of NiV revealed a high degree of similarity to HeV and in 2002, the genus Henipavirus was created to accommodate these novel paramyxoviruses. In the latest report from the paramyxovirus study group released in 2019, the genus Henipavirus has been expanded to include three new species, Cedar henipavirus (Cedar virus, CedV), Ghanaian bat henipavirus (Ghana virus, GhV) and Mojiang henipavirus (Mòjiāng, MojV). Henipavirus is now classified as one of the seven ICTV-approved genera in the family Paramyxoviridae (Fig. 1).

Virion Structure Henipavirus particles are pleomorphic, varying from spherical to filamentous and ranging in size from 40 to 1900 nm. Nucleocapsids, composed of three major structural proteins (nucleocapsid protein, phosphoprotein and a large protein or RNA-dependent RNA polymerase), have a diameter of 18–19 nm with an average pitch of 5 nm. Examination by electron

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00100-4

355

356

Henipaviruses (Paramyxoviridae)

Fig. 1 Phylogenetic tree derived from the RdRP proteins of selected henipaviruses and prototype species of the other genera in the family Paramyxoviridae. The tree was constructed by using Neighbor-Joining approach with 1000 bootstrap replicates. Numbers at the nodes represent bootstrap values. Scale bar indicates amino acid substitutions per site. Genbank accession numbers are provided in brackets on the right.

microscopy revealed that HeV has a unique double-fringed appearance, caused by the presence of surface projections 15 7 1 nm and 8 7 1 nm in length. In contrast, NiV possesses a single layer of surface projections with an average length of 17 7 1 nm.

Genome Several molecular features distinguish henipaviruses from other paramyxoviruses. When the genome length of HeV was revealed at 18,234 nucleotides (nt), it was approximately 15% larger than all known paramyxoviruses at that time. The genomes of the Malaysian (MY, 18,246 nt) and Bangladesh (BD, 18,252 nt) strains of NiV differ by 6 nt because of a 6-nt insertion in the 30 untranslated region of the F gene in NiV-BD. The large genome size of 418 kb is a conserved feature of all henipaviruses know to date, including the more divergent CedV, GhV, and MojV with genome sizes varying between 18,162 nt (CedV) to B18,430 nt (GhV). It is not the additional length per se that is unique to henipaviruses. For example, we know now that the genomes of J virus and Beilong virus in the family Paramyxoviridae are over 19,000 nt in length. The novel henipavirus genomic feature is the length of the untranslated region at the 30 end of five of the six genes, which in most cases are from 3 to 13 times longer than their Paramyxoviridae counterparts. The function of these regions is unknown. Another unique feature of henipaviruses is the increased size of the P protein, at 707 and 709 aa for HeV and NiV, respectively. This is more than 100 aa larger than most of the known paramyxovirus P proteins. For all members of the family Paramyxoviridae, the genome length can be divided by six, due to the requirement of each N protein in the viral ribonucleoprotein to bind 6 nt residues. This “rule-of-six” is also true for henipaviruses despite their much larger genome sizes. The importance of this has been functionally demonstrated for henipaviruses using minigenome replicon assays. The genome organization of henipaviruses resembles that of the genera Respirovirus and Morbillivirus. As for other paramyxoviruses, the first 12 nt of the 30 and 50 genomic terminal sequences of henipaviruses are highly conserved. The first 3 nt of the genome termini are 50 -ACC-30 – a sequence that is fully conserved in the family Paramyxoviridae and different from that found in the family Pneumoviridae. There are six coding genes in the henipavirus genome coding for the following major proteins (from 30 to 50 order of the genome): nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), receptor binding protein (RBP, previously known as G) and RNA-dependent RNA polymerase (RdRp, also known as L). A comparison of henipavirus genome structure to other well known paramyxoviruses is provided in Fig. 2. Similar to other paramyxoviruses, the henipavirus P gene undergoes transcriptional editing to produce 50 -co-linear mRNAs which result in the production of V, W and C proteins in addition to the P protein. These additional accessory proteins play a role in antagonizing host innate immune responses. However, the CedV P gene lacks the coding capacity for these additional proteins.

Life Cycle Henipaviruses can replicate in a variety of mammalian cell lines. The rate of replication, the size of syncytia generated, and the location of nuclei in syncytia vary depending on the cell type and virus species. Infected Vero cell monolayers yield titers in excess

Henipaviruses (Paramyxoviridae)

357

Fig. 2 Genome size and organization of Hendra virus in comparison with the type species of the three other most characterized genera in the family Paramyxoviridae. Abbreviations: N ¼ nucleocapsid protein; P ¼ phosphoprotein; M ¼ matrix protein; F ¼ fusion protein; RBP ¼ receptor binding protein (formerly G protein); RdRp ¼ RNA-dependent RNA polymerase.

of 108 infectious virions per milliliter. The broad host range of henipaviruses has been demonstrated by the multiple natural susceptible hosts from bats, horses, pigs, humans to cats, dogs and rats. This is further corroborated by experiments with direct infection of cells of different species using live virus or pseudotyped viruses containing the F and G/RBP proteins of henipaviruses, and vaccinia virus–mediated cell surface expression of F and G/RBP glycoproteins. The wide host range of henipaviruses is considered uncommon for paramyxoviruses, which usually adopt a narrow host range. The ephrin B ligands, functioning in the cell-cell signaling with their cognate EphB receptors, have been identified as the entry receptor for henipaviruses with epherin-B2 being the main receptor and epherin-B3 and other homologs as alternative receptor(s). The ephrin proteins are highly conserved among vertebrates, which explains, at least in part, the unusually broad species tropism of henipaviruses. Ephrin-B2 is expressed in arteries, arterioles, and capillaries in multiple organs, including neurons, arterial smooth muscle and bronchiolar epithelial cells. In the brain, ephrin-B3 is expressed at a higher level than ephrin-B2. It should be noted that mice are less susceptible to henipaviruses although the level of ephrin B expression is similar, indicating that other factors, in addition to the receptor molecules, play a role in the overall susceptibility of a mammalian host to henipaviruses. For susceptible hosts, data obtained from field and laboratory studies indicate an oronasal or oropharyngeal route of entry. The initial site of replication is mostly localized within the respiratory system. Secondary infection can occur in most organs, but with key lesions associated with the respiratory and CNS system, most likely via hematogenous spread of the virus with replication occurring in vascular endothelium. Inflammation of blood vessels (vasculitis) occurs in most organs but is particularly prominent in the brain, lungs, heart, kidneys and spleen. It is important to note that while human lymphocytes (mainly T and B cells), and monocytes are not permissive for henipavirus infection, it has been shown that they can nevertheless bind and transfer NiV to permissive microvascular endothelial cells. This trans-infection phenotype may play an important role in the hematogenous spread of the virus in addition to the free form that makes up the plasma viremia seen in human patients in the acute phase of the lungs.

Epidemiology Human henipavirus infections in Australia and the Philippines and in Malaysia occurred as a result of transmission of the viruses from flying foxes via horses and pigs, respectively. In contrast, NiV may have been directly transmitted to humans in Bangladesh (and India, with less direct evidence) where it is believed that date palm juice contaminated with bat secretions constituted a potential transmission route. The mode of transmission from bats to spillover hosts is not known. It has been suggested that horses and pigs may have been infected by HeV and NiV through masticated fruit pulp discarded by flying foxes or by flying fox urine which contaminated pastures or pig sties. Alternatively, the source of virus may have been infected fetal tissues or fluids, a suggestion based on the fact that HeV outbreaks occurred during the birthing period of some species of flying fox, the isolation of HeV from a pregnant flying fox and its fetus, and the transplacental transmission of HeV in experimental infections. HeV has been transmitted from horses to humans in four occasions. The source of the virus may have been the saliva of infected horses, the nasal discharge commonly found at the terminal stages of the disease, or a wide range of infected tissues made

358

Henipaviruses (Paramyxoviridae)

Table 1

List of countries where infection of Nipah or Nipah-related virus in bats has been detected

Virus isolation

Molecular detection

Serological detection

Bangladesh Cambodia Malaysia

Bangladesh Ghana India Thailand

Annobón Bangladesh Bioko Cambodia Cameroon China Ghana India Madgascar Malaysia Malawi Príncipe Rio Muni São Tomé Tanzania Thailand Uganda Vietnam Zambia

accessible by necropsy. The paucity of virus in the bronchi or bronchioles of infected horses suggests transmission of HeV to either man or horse by aerosol is highly unlikely and experimental infection has confirmed the poor transmissibility of HeV in horses. Risk factors for human infection by NiV in Malaysia were contact with pigs or fresh pig products and because the virus was present in a wide range of organs, the greatest likelihood arose for those in direct contact with sick or dying pigs on farms during farrowing or slaughtering or in abattoirs. NiV is readily observed in the respiratory epithelium of naturally and experimentally infected pigs, a feature suggesting that the virus probably spread to humans and within the pig population by aerosol or by direct contact with oropharyngeal or nasal secretions. Although NiV was present in the urine and respiratory secretions of patients, human-to-human transmission in Malaysia was extremely rare. In contrast, in Bangladesh, epidemiologic evidence indicates the spread of the virus from one person to another. Fruit bats (flying foxes) in the genus Pteropus, family Pteropodidae, suborder Yinpterochiroptera (previously known as Megachiroptera) are the main reservoir hosts of HeV and NiV. In Australia, HeV has been shown to occur in all four flying fox species. In other parts of the world, serologic tests have shown that NiV or NiV-related virus is widely distributed in bats from Indonesia to the west border of Africa. In Australia, HeV has been isolated from at least bats of two different species, Pteropus alecto and Pteropus poliocephalus. NiV was first isolated from the urine of Island flying foxes and from the saliva on partially eaten fruit and has since been isolated from Lyle's flying foxes (Pteropus lylei) in Cambodia and Pteropus medius in Bangladesh. In addition to direct isolation, the presence of NiV in different bat populations has also been demonstrated by either molecular or serological detection in the following countries or regions: Annobón, Bangladesh, Bioko, Cambodia, Cameroon, China, Ghana, India, Madagascar, Malaysia, Malawi, Príncipe, Rio Muni, São Tomé, Tanzania, Thailand, Uganda, Vietnam, and Zambia (Table 1). Genome sequencing revealed that HeV strains isolated from equine and human sources during the outbreak in Brisbane are identical and differ only little from HeV strains isolated from flying foxes 2 years later. Subsequent sequencing of several additional horse and human HeV isolates confirmed the high genetic similarity. Similar observations were made in Malaysia between the human isolates from the outbreak and bat isolates several years later. In Bangladesh, four human isolates obtained in 2004 demonstrate significant genetic heterogeneity, suggesting multiple spillovers of NiV from flying foxes into the human population. The NiV sequences detected from human patients in India from 2001 to 2018 were more related to NiV strains from Bangladesh than the strains isolated in Malaysia.

Clinical Features Henipaviruses display either predominantly respiratory or neurological symptoms depending on the host. In natural infections of horses and young pigs with HeV and NiV, respectively, and in experimental infections of different animals with either virus, respiratory symptoms predominate. Neurological symptoms were also observed in a proportion of HeV-infected horses. For human infection with henipaviruses, the incubation period ranges from 2 to 45 days, but for 90% of patients, the incubation time is two weeks or shorter. Experimental infection of horses, ferrets and non-human primates is usually fatal, with death or euthanasia occurring 5–10 days post infection. However, observation following natural infection of horses during the initial outbreak indicated that some animals displayed respiratory symptoms but they survived.

Henipaviruses (Paramyxoviridae)

359

HeV-induced respiratory disease in horses may be accompanied by facial swelling and ataxia and in extremis, a copious frothy nasal discharge. The first two patients infected with HeV presented with fever, myalgia, headaches, lethargy, and vertigo. While one recovered, the other patient developed pneumonitis, respiratory failure, renal failure, and arterial thrombosis, and died of cardiac arrest. Findings at autopsy were consistent with a disseminated viral infection in various organs. Natural infection of pigs with NiV is usually asymptomatic, an outcome also observed after experimental administration of NiV by the ocular and oronasal route. When symptoms are present, they vary according to the age of the pig. Young animals present primarily with respiratory symptoms. Older animals display increased salivation and nasal discharge and occasionally develop neurological signs such as trembling, muscle spasms, and an uncoordinated gait. In Malaysia, pigs displaying symptoms of NiV infection, such as nasal discharge and rapid and labored respiration, had a harsh and nonproductive cough, which gave rise to the name ‘barking-pig-disease’. In contrast, NiV infection in humans is usually associated with a severe acute encephalitis and although a proportion of cases presented with respiratory symptoms, particularly in Bangladesh, the majority of the patients displayed fever, headache, drowsiness, dizziness, myalgia, vomiting, and a reduced level of consciousness. Clinical signs such as the absence of reflexes and the irregular twitching of muscles or parts of muscles and an abnormal doll’s eye reflex are indicative of brainstem and upper cervical spinal cord dysfunction. In the Malaysian outbreak, 105 of 256 patients died, a mortality rate of 41%. However, this figure is reduced to 30% when individuals who experienced either a mild or asymptomatic infection are considered. In Bangladesh, a combined case-fatality rate of multiple outbreaks is approximately 70%. For the most recent NiV outbreak in Kerala, India in 2018, the case-fatality rate was 94%. Both HeV and NiV can cause prolonged infection in humans before manifesting with severe neurological symptoms. In Malaysia, NiV persisted in a proportion of patients (c. 10%) who either recovered from encephalitis or who had experienced an asymptomatic infection. Such cases of relapsed and late-onset encephalitis, respectively, presented from months to years after the initial infection, with the longest reported case 11 years after an initial acute infection. In relapsed cases, although serum anti-NiV IgG antibody levels were elevated, no IgM response or vasculitis was observed, and no live virus was isolated from the throat and nasal secretions. Only four cases of human HeV infection have been recorded. Two displayed influenza-like symptoms and one died. A third, fatal case of encephalitis occurred over a year after a self-limiting episode of meningitis, attributed to HeV infection.

Pathogenesis As stated above, data from clinical and pathological studies of human cases as well as animal infection and cell tropism experiments all indicate that the initial site of replication is in the respiratory system. The respiratory disease caused by HeV in horses is characterized by pulmonary edema and congestion. Viral antigen is found in endothelial cells in a range of organs including the lungs, lymph nodes, kidneys, spleen, bladder, and meninges, and virus can be recovered from several internal organs, including the lungs, and from saliva and urine. Young pigs infected with NiV present primarily with respiratory symptoms characterized by tracheitis and bronchial and interstitial pneumonia. After experimental infection by the ocular and oronasal routes, virus replication occurs in the oropharynx and spreads to the respiratory tract and lymphoid tissues before appearing in the trigeminal ganglion and neural tissue. Viral antigen is found, particularly in clinically affected animals, in both lungs and meninges and virus is recovered from a range of tissues including tonsil, nasal, and throat swabs and lungs. The outcome of henipavirus infections can range from high mortality, as seen with HeV in horses and NiV in humans, to low mortality and morbidity, best exemplified by NiV infection in pigs, to asymptomatic infection in flying foxes. Experimental infections reveal that henipaviruses are infectious following either oronasal or parenteral administration. Figures for the minimum lethal dose of henipaviruses suggest that 200–300 plaque-forming units (pfu) is sufficient to produce a productive infection in some animal models. Transmission is mostly via the oronasal route. Infectious NiV was recovered from urine and the tracheal and nasopharyngeal secretions of infected patients in the early phases of their illness in the original Malaysian outbreak. However, human-to-human transmission in the Malaysian outbreak was extremely limited. In contrast, human-to-human transmission in Bangladesh has been well documented, probably due to more frequent and closer contact with familial caretakers and religious personnel. As stated above, henipaviruses are uncommon amongst the paramyxoviruses in respect to their broad host range. The ability to replicate, spread, and cause acutely lethal disease amongst multiple species suggest that these viruses, in addition of using a highly conserved molecules as entry receptor, have evolved effective means to antagonize or evade innate antiviral responses of evolutionally distant hosts from bats, horses, pigs to humans. As in other paramyxoviruses, the major anti-interferon (IFN) defences are encoded by the P gene of henipaviruses. It has been shown that the V, W and C proteins produced from the henipavirus P genes all play a role in modulating host immune system by directly interacting with multiple innate immune signaling molecules, including, MDA5, RIG-I, and TRIM25 which regulate IFN gene expression and STATs which are involved in IFN signaling. Interestingly, the NiV M protein has also been shown to inhibit type I IFN induction by indirectly antagonizing the activity of IKKe, one of the kinases responsible for IRF-3 activation. In this context, it is worth noting that CedV lacks the editing site in the P gene and the virus does not produce the V protein and fail to cause any overt disease in ferrets and guinea pigs, in which both HeV and NiV infection can lead to a severe disease. In susceptible non-pteropid species, henipaviruses elicit strong humoral immune responses. Antibodies to P, N, and M proteins are evident in Western blots and anti-F and anti-G/RBP antibodies are readily detected by various immunological methods. In

360

Henipaviruses (Paramyxoviridae)

contrast to a consistent antibody response in susceptible, nonvolant species, in flying foxes there appears to be less direct correlation between detectable virus replication and the appearance of antiviral antibodies. Antibody production is irregular and of uncertain longevity. Anti-NiV antibodies were present in most patients with clinical NiV encephalitis and IgM antibodies occurred more frequently than IgG antibodies in both serum and cerebrospinal fluid (CSF). The appearance of specific IgM antibodies in serum preceded their appearance in the CSF, a sequence consistent with virus replicating in primary infection site(s) prior to invasion into the central nervous system. The role played by cellular immunity in henipavirus infection is less known. In the most recent NiV outbreak in Kerala, careful analysis of humoral and cellular immune responses in the 2 survivors revealed that there was a dramatic increase in activated (Ki67 þ ) CD8 þ T cells with an acute effector phenotype (CD38 þ /HLA-DR þ /Granzyme B þ /PD1 þ ), suggesting that virus-specific naïve T cells were actively proliferating in response to the infection. It is interesting to note that clearance of viremia coincided with the peak of proliferating Ki67 þ CD8 þ T cells, which occurred before the peak IgG antibody levels.

Diagnosis Traditional diagnostic methods such as virus isolation, EM, immuno-EM, immunohistochemistry (IHC), serology, and polymerase chain reaction (PCR) played key roles in the initial discovery of HeV and NiV. As henipaviruses are BSL4 agents, it is important to note that while initial attempts to culture suspected henipaviruses may be conducted in a BSL3 laboratory, it is essential to continue further characterization inside a BSL4 facility once a cytopathic effect (CPE) is observed and the growth of henipavirus is confirmed by PCR or immune staining. All henipaviruses can replicate in different cell lines – a feature that contributed to the efficiency with which they were isolated during the initial disease outbreak investigations, but Vero cells remains the most commonly used cell line for initial virus isolation trials. More recently, different bat cell lines have been used with some success. For molecular diagnosis, PCR is still the method of choice. With the discovery of greater genetic diversity of henipaviruses in different parts of the world, and current PCR tests may not work for unknown viruses, next generation sequencing (NGS) is starting to be employed for outbreak investigation with suspected of henipaviruses or henipa-related viruses as well to any previously unknown pathogens. A probe-based NGS containing hybridization baits covering all known henipavirus genomes has recently been developed for more efficient NGS characterization, especially from clinical samples where host genetic materials are expected to give very background signal masking the low level of virus-specific NGS reads. For henipaviruses, serologic tests are important both during outbreak investigation and for disease surveillance. The virus neutralization test (VNT) is accepted as the standard reference method. Few laboratories, however, can conduct neutralization tests because of the requirement to handle live virus at BSL4. Two surrogate VNT test platforms which do not require BSL4 containment have been developed: (1) Luminex bead-based test: recombinant soluble G/RBP proteins of HeV or NiV are coated on Luminex beads and the binding of a soluble ephrin-B2 protein is used to mimic the virus-receptor interaction. When neutralizing antibodies are present, they will block this G-ephrin-B2 interaction, thus acting as a surrogate VNT; (2) Pseudotype virus: different pseudotype systems, based on Vesicular stomatitis or lentivirus, carrying the henipavirus F and G/RBP proteins have been developed as a surrogate VNT for detecting henipavirus-specific antibodies. Incorporation of reporter genes in these systems have resulted in a greater sensitivity and reproducibility.

Treatment At the present time, there is no licenced treatment available for henipavirus infections. Ribavirin, which inhibits the replication of HeV in vitro, was used during the NiV outbreak in Malaysia in 1998 and HeV outbreak in 2008 in Australia. While the trial in Malaysia demonstrated a 35% reduction (P ¼ 0.011) in mortality, it had no detectable therapeutic effect on the two HeV patients in Australia. In the absence of other therapies, ribavirin may be an option for the treatment of henipavirus infections with patient consent, but the efficacy of ribavirin as a therapy or prophylaxis in people remains uncertain at best. In different animal models, ribavirin either delayed but did not prevent the death or had no effect on infection caused by NiV or HeV. Chloroquine, an antimalarial drug, was shown to block the critical proteolytic processing needed for HeV F maturation and function. Although the drug inhibited NiV and HeV infection in vitro, it did not show any apparent clinical benefit in vivo. In contrast to existing small molecule antivirals, a henipavirus-specific therapeutic heptad peptide-based fusion inhibitor, analogous to the HIV-1-specific enfuvirtide (Fuzeon), has shown promising efficacy in animal studies. The a-helical heptad repeat (HR) domain of henipavirus F1 glycoprotein resembles other fusion glycoproteins which play a role the formation of a trimer-of-hairpins structure during the membrane fusion and virus infection process. In a hamster model of NiV infection, the administration of cholesterol tagged HR-derived peptides provided the first evidence of in vivo effectiveness of a fusion inhibiting peptide against NiV. Finally, passive immunotherapy based on recombinant human monoclonal antibodies to the henipavirus G/RBP protein also appear to have a promising therapeutic potential. In particular, the m102.4 mAb is shown to be effective in a non-human primate model against HeV or NiV infection. While human clinical application is yet to be proven, a phase I clinical trial has been completed, demonstrating the safety for human applications and an effective half-life in humans. The mAb has also been used in

Henipaviruses (Paramyxoviridae)

361

humans with high risk exposure to henipaviruses under emergency and compassionate grounds with no severe side effects observed.

Prevention A number of promising strategies to develop henipavirus vaccines are being explored. NiV F and G/RBP glycoproteins, either delivered via viral vectors or by administration as purified proteins with adjuvant, elicit neutralizing antibodies in many different animal models. The most studied and tested vaccine is based on the HeV-sG protein produced in CHO cells. The vaccine was licensed by Zoetis™ Inc., (formerly Pfizer Animal Health) and developed as an equine vaccine, under the trade name Equivac® HeV, for use in Australia. This remains to be the first and only licensed vaccine for any BSL4 agent. The vaccine is 100% effective in protecting horses under experimental challenge studies. Since the vaccine release in 2012, laboratory confirmed HeV infections in horses have only occurred in unvaccinated horses. NiV is one of the first three high priority targets set by the Coalition for Epidemic Preparedness Innovations (CEPI). Currently there are two CEPI-funded NiV vaccine projects ready for phase I clinical trial: the US-led HeV-sG vaccine platform (an extension of Equivac® HeV to NiV infection in humans) and the Japan-led measles viral delivery platform. Human phase I trials will be completed in the next few years and we are confident that a licenced human henipavirus vaccine will be available thereafter.

Further Reading Ang, B.S.P., Lim, T.C.C., Wang, L.-F., 2018. Nipah virus infection. Journal of Clinical Microbiology. 56. doi:10.1128/JCM.01875–17. Bean, A., Baker, M., Stewart, C.R., et al., 2013. Studying immunity to zoonotic diseases in the natural host – Keeping it real. Nature Reviews Immunology 13, 851–861. Broder, C.C., 2012. Henipavirus outbreaks to antivirals: The current status of potential therapeutics. Current Opinion in Virology 2, 176–187. Broder, C.C., Xu, K., Nikolov, D.B., et al., 2013. A treatment for and vaccine against the deadly Hendra and Nipah viruses. Antiviral Research 100, 8–13. Eaton, B.T., Broder, C.C., Middleton, D., Wang, L.-F., 2006. Hendra and Nipah viruses: Different and dangerous. Nature Reviews Microbiology 4, 23–35. Field, H.F., Mackenzie, J.S., Daszak, P., 2007. Henipaviruses: emerging paramyxoviruses associated with fruit bats. Current Topics in Microbiology and Immunology 315, 133–160. Luby, S.P., Gurley, E.S., 2012. Epidemiology of henipavirus disease in humans. Current Topics in Microbiology and Immunology 359, 25–40. Mahalingam, S., Herrero, L.J., Playford, G., et al., 2012. Hendra virus: An emerging paramyxovirus in Australia. Lancet Infectious Diseases 12, 799–807. Pernet, O., Wang, Y.E., Lee, B., 2012. Henipavirus receptor usage and tropism. Current Topics in Microbiology and Immunology 359, 59–78. Thibault, P.A., Watkinson, R.E., Moreira-Soto, A., Drexler, J.F., Lee, B., 2017. Zoonotic potential of emerging paramyxoviruses: Knowns and unknowns. Advances in Virus Research 98, 1–55. Wang, L.-F., Anderson, D.E., 2019. Viruses in bats and potential spillover to animals and humans. Current Opinion in Virology 34, 79–89. Wang, L.-F., Mackenzie, J.S., Broder, C.C., 2013. Henipaviruses. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed., vol. 2. Philadelphia: Lippincott Williams and Wilkins, pp. 286–313.

Relevant Websites https://cepi.net/ CEPI. New Vaccines For A Safer World. http://www.outbreak.gov.au/for-vets-and-scientists/hendra-virus Hendra virus. National pest & disease outbreaks. https://www.who.int/emergencies/diseases/hendra-virus/en/ Hendra virus infection. World Health Organiztion. https://gvn.org/ Home. GVN. https://www.who.int/csr/disease/nipah/en/ Nipah virus infection. World Health Organization. https://www.business.qld.gov.au/industries/service-industries-professionals/service-industries/veterinary-surgeons/guidelines-hendra/incident-summary Summary of Hendra virus incidents in horses. Business Queensland.

Hepatitis A Virus (Picornaviridae) Andreas Dotzauer, University of Bremen, Bremen, Germany r 2021 Elsevier Ltd. All rights reserved.

Glossary Antibody prevalence The percentage of a population with antibodies against a certain disease at a given time. Aplastic anemia Disease characterized by a decrease in blood cells resulting from underproduction due to bone marrow failure; called also hypoplastic anemia. COPII Coat protein complex II. cre Cis-acting replication element. ERGIG ER-Golgi intermediate compartment. Exosome Vesicle (50–120 nm) released from cells by fusion of late endosomal multivesicular bodies with the plasma membrane that contain various biologically active proteins and RNAs.

Incidence The rate of occurrence of new cases of a particular disease in a population in a certain period of time. Leukocytopenia Decrease in the number of white blood cells. Polyprotein processing Cascade of proteolytic cleavage events resulting in release of biologically active intermediate and finally mature proteins from the polyprotein. Ribosome stalling Transient translation stop. Serine-like protease Proteolytic enzyme characterized by a catalytic triad similar to that in serine-type proteases with Ser, His, Asp.

History Jaundice has been known as an epidemic disease for centuries, but the earliest outbreaks of what was almost certainly hepatitis A were documented by Rayrun "MRWmats_scpger, occurring in Preßburg, now Slovakia, in 1674 and 1697. Besides sporadic occurrence of the disease, characterized by a slow increase and slow decrease of the number of infected persons in the course of months (spread by person-to-person contact), with an overall small number of infections, larger vehement epidemics (spread by contaminated water and food) were reported in later times. The first pandemic HAV wave occurred in the 1860s and the first considerable record of the disease was registered during the American Civil War. In the second half of the nineteenth century, the disease became known as “icterus catarrhalis” (catarrhal jaundice; inflammation of the biliary tract was supposed) or “icterus epidemicus” (epidemic jaundice) as well as campaign jaundice, as epidemics are common in military medical history. At the turn of the nineteenth to the twentieth century, the disease was recognized as infectious and transmissible by person-toperson contact. The terms “hepatitis epidemica” (epidemic hepatitis) and “hepatitis infectiosa” (infectious hepatitis) were introduced as synonyms for catarrhal jaundice. Detailed epidemiologic recordings have been conducted since the beginning of the twentieth century. Two large pandemic waves were observed during the twentieth century, the first one originating during World War I and reaching its summit between 1918 and 1922, and the second one originating in the early 1930s reaching its widest distribution with the beginning of World War II. During World War II, hepatitis was demonstrated to be caused by at least two separate filterable agents, and the resulting diseases were called hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis), and the etiological agents hepatitis A virus (HAV) and hepatitis B virus (HBV), respectively. We now know three more viruses that have a specific tropism for the liver: HCV, HDV and HEV. These five viruses share no taxonomic and virological relationship. After World War II, epidemiologic studies in human volunteers showed that hepatitis A is spread by the fecal–oral route, and provided information on the duration of viremia and shedding of virus in feces. In the late 1960s and early 1970s, it was shown that chimpanzees and several small, non-human primates could serve as animal models for human hepatitis A. Meanwhile, mice genetically deficient for Ifnar1, a component of the type 1 interferon receptor, or for the mitochondrial antiviral signaling protein (Mavs) or the interferon regulatory factors (Irf3/Irf7), which are components of the induction pathway for interferon expression, as well as chimeric mice with human liver cells were found to be infectable by HAV and are also used as models for the disease. The replication of HAV in cell cultures was established between 1979 and 1981. The etiologic agent was identified through immune electron microscopy by Feinstone et al. in 1973. The molecular cloning of the HAV genome in the 1980s revealed that the genomic organization of HAV is similar to that of picornaviruses, and the first infectious cDNA clone of HAV was reported by Cohen et al. in 1987. The disease manifestations by hepatocellular destruction could be attributed to an immunopathogenic mechanism in the late 1980s by Vallbracht et al. An inactivated HAV vaccine has been available since 1992.

362

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21508-2

Hepatitis A Virus (Picornaviridae)

363

Taxonomy and Evolutionary Origin HAV is a member of the genus Hepatovirus within the family Picornaviridae. All human HAV strains known belong to only one serological group, but nucleotide sequence analysis of the VP1-2A junction region and the VP1 coding region, respectively, revealed that several distinct HAV genotypes (seven in the case of VP1-2A junction analysis, six in the case of VP1 region analysis; more than 15% nucleotide variation), which include several subgenotypes (nucleotide variation of more than 7.5%), can be distinguished. These genotypes correlate with the geographic origin of the virus isolates. Genotypes I and III are the most common types worldwide, particularly genotype IA. The cell culture-adapted viruses most commonly used are variants of the Australian strain HM175 (genotype IB) and the German strain GBM (genotype IA). Recently, multiple novel species, which have the same unique properties as human HAV, have been identified in bats and other small mammals, seals and even marsupials. Phylogenetic analyses indicate that these viruses have frequently jumped between the host species, and ancestral reconstructions suggest that human HAV descended from rodent viruses, which have originated in small insectivorous mammals. Based on the unique capsid structure of HAV, the HAV-like viruses of ancient insectivores might be direct descendants from a picornavirus-like insect virus.

Morphology and Physicochemical Properties The infectious spherical virion shed in feces is a nonenveloped particle with a diameter of approx. 27 nm. The pentakisdodecahedral capsid, which embodies the viral RNA genome, contains 60 copies of each of the 4 structural proteins, VP1 (also known as 1D), VP2 (1B), VP3 (1C) and VP4 (1A) (see Table 1). The mature capsid has a different structure than other mammalian picornaviruses, as the canyon surrounding the 5-fold symmetry axes, which is a prominent feature of picornaviruses and represents the attachment site to cellular receptors, is missing, and VP2 extends N-terminally across the 2-fold symmetry axis to the adjacent protomer, a feature seen in picornavirus-like dicistroviruses of insects. HAV forms a single serotype and the antigenic structure of the capsid is conserved among the human and the non-primate viruses. In blood as well as in supernatants of infected cell cultures HAV appears enveloped in exosomelike vesicles (eHAV). These vesicles may enclose up to four infectious virus particles, which still contain uncleaved VP1-2A. The HAV particle has a buoyant density of approx. 1.33 g cm
3 in CsCl (in iodixanol of approx. 1.22 g cm
3; eHAV particles band at approx. 1.08 g cm
3 in iodixanol gradients) and a sedimentation coefficient of approx. 160S. Empty capsids found in feces have a sedimentation constant of about 70S. HAV is extremely resistant to acid (pH 1.0 for 2 h at room temperature) and thermal inactivation (601C for 1 h). The isoelectric point of HAV is between values of 2.8 and 5.8.

Genome Organization and Expression, Replication, Morphogenesis The linear, single-stranded, positive-sense RNA genome of HAV is approx. 7500 nucleotides in length and is not capped but covalently linked by a tyrosine-O4-phospho-diester bond to the 2,5 kDa viral protein 3B (also known as VPg; see Table 1). It encompasses a structurally complex 50 nontranslated region (NTR) of approx. 740 bases, followed by a single open reading frame encoding a polyprotein (approx. 250 kDa; approx. 2230 amino acids) and a 30 NTR of approx. 60 bases which terminates with a poly(A) tail of about 60 nucleotides. In the polyprotein, the capsid proteins and those with functions during virion assembly represent the N-terminal third (VP4, VP2, VP3, VP1 (numbered according to molecular mass and also known as P1 region), and 2A) (see Table 1 and Fig. 1) with the remainder of the polyprotein comprising a series of proteins required for RNA replication (2B and 2C followed by 3A to 3D) (see Table 1 and Fig. 1). The viral polyprotein is translated directly from the messenger-sense genomic RNA, which is released into the cellular cytoplasm after uncoating of the virion. An internal ribosomal entry site (IRES) located within the 50 NTR (see Table 1) is involved in the capindependent initiation of protein synthesis. The IRES of HAV differs from that of other picornaviruses and forms its own group (type III picornavirus IRES). Translation from the HAV IRES requires all of the initiation proteins, including eIF4E and intact eIF4G, and infection with HAV does not result in cleavage of the translation initiation factor eIF4G to block the cap-dependent host protein synthesis, as featured by other picornaviruses. The HAV IRES-directed initiation of translation, which depends on the entire 50 NTR, is enhanced by sequences of the 50 -terminal coding region and by certain host cell factors, e.g., glyceraldehyd 3-phosphate dehydrogenase, poly(C) binding protein and polypyrimidine tract binding protein. The efficiency of translation is decreased by an extraordinarily high proportion of rare codons (particularly in the P1 coding region). But, as HAV does not induce shutdown of cellular protein synthesis, this not optimal codon usage might avoid strong competition for tRNAs and in addition may support proper protein folding by ribosome stalling. A further characteristic of the genome is a low G/C ratio and the avoidance of CpG dinucleotides. Proteolytic processing of the primary polyprotein (see Fig. 1) occurs simultaneously with translation and is largely carried out by the viral 3C protease, which is a serine-like protease in which cysteine replaces the nucleophilic serine in the catalytic triad of the active center. As cellular substrate, PABP (poly(A) binding protein) has been described for mature 3C, obviously resulting in late negative regulation of translation and contributing to the switch to replication. Furthermore, processing intermediates of 3C, in this case 3ABC and possibly 3CD, interfere with the induction of IFN-b transcription by 3C-mediated cleavage of MAVS (mitochondrial antiviral signaling protein), and of TRIF (TIR domain-containing adaptor inducing IFN-b), respectively (see Fig. 3).

7416–7478

735–803

804–1469 1470–2207 2208–3023/ 3026/3029

3024/3027/ 71/72/73 3030–3242 3243–3995 251

3996–5000

5001–5222

5223–5291

5292–5948

5949–7415

30 nontranslated region (30 NTR)

1A

1B 1C 1D

2A

2B

2C

3A

3B

3C

3D

Remarks

50 stem-loop region (nucleotides1–94): required for RNA Mechanisms regulating switch from translation to replication are not clear replication Polypyrimidine tract pY1(nucleotides 99–138): unknown function Internal ribsomal entry site (IRES) (nucleotides 152–734): directs Picornaviral Type III IRES cap-independent initiation of translation Involved in regulation of RNA replication

Proposed or known function

489

219

23

74

335

23

RNA-dependent RNA polymerase

VPg (virus protein genomic) Supposed protein primer for RNA replication Sole protease

As 3AB VPg precursor(pre-VPg)

Structural rearrangements of intracellular membranes for replication

Structural rearrangements of intracellular membranes for replication

Morphogenesis

Serine-like protease, with replacement of Ser by Cys in catalytic triad: Cys172, His44, Asp8 Involved in switching viral translation to replication by cleavage of PABP Intermediate 3ABC interferes with induction of beta interferon transcription by cleavage of MAVS Interferes with cellular COPII-mediated, classical secretory pathway cre-element consisting of 110 nucleotides with conserved AAACG motif at 5´ coding region required for replication: template for VPg (3B) uridylation

Mutations accompany adaptation to growth in cell culture Interferes with cellular macromolecular synthesis Interacts with membranes, may anchor pre-VPg to cellular membranes through a central region of 21 hydrophobic amino acids Induces structural alterations of intracellular membranes Tyr3 attached to 50 terminus of genomic RNA

Influence on membrane permeability Induces structural alterations of the ERGIG structure Mutations accompany adaptation to growth in cell culture Interferes with induction of beta interferon transcription by an unknown mechanism (affects MAVS and the IRF-3 kinases) Integral association with membranes

Peripheral association with membranes

During morphogenesis, precursor VP1–2A (also known as VP1pX), is likely cleaved by cellular protease So far not identified in infected cells

Extended by 40–80 adenylates Involvement in translation is not clear Capsid protein VP4 Not myristoylated Morphogenesis Pore-forming activity: may facilitate release of genome from the capsid 222 Capsid protein VP2 During morphogenesis, precursor VP4 - VP2 (VP0) is likely cleaved autocatalytically 246 Capsid protein VP3 272/273/274 Capsid protein VP1 Heterogeneous C terminus, depending on HAV strain and host cell

–

–

1–734

50 nontranslated region (50 NTR)

Length in amino acids

Nucleotide position

HAV regulatory genomic regions and HAV proteins; all numbering refers to HAV strain HM175 (accession no. NC_001489).

Regions/Proteins

Table 1

364 Hepatitis A Virus (Picornaviridae)

Hepatitis A Virus (Picornaviridae)

365

Fig. 1 HAV genomic RNA and HAV polyprotein processing including known or supposed functions of the viral proteins during morphogenesis and replication. The polyprotein that results from IRES-directed translation is cleaved by the viral protease 3C to release certain precursor proteins with biologically relevant functions as well as the mature nonstructural proteins. Primary cleavage results in the release of the structural precursor VP4-VP2-VP3-VP1-2A, which associate to assembly intermediates (pentamers), which are stabilized by 3C cleavage of the VP3 junctions (VP4-VP2 (VP0), VP3, VP1-2A)5. After assembly of the intermediate protein building blocks and the viral genome to provirions, maturation cleavages occur through an unknown proteolytic activity to release VP4 from VP2 and through a cellular, presumably a lysosomal protease to release 2A from VP1, resulting in the infective virion with 60 copies of each of the main structural proteins VP1 to VP4.

Synthesis of viral RNA by the viral 3D polymerase follows the accumulation of the nonstructural proteins spanning 2B to 3D, which induce the assembly of a macromolecular replication complex on membranes that are recruited from intracellular membranes. It is not clear which cellular membranes and which viral proteins are involved in this process. But it was demonstrated that the proteins 2B, 2C, 3A, 3B and their processing intermediates are able to interact with intracellular membranes and to induce structural membrane rearrangements. RNA transcription is protein-primed, with the uridylated VPg protein 3B (VPg-pUpU), which is produced in a reaction templated by a cis-acting replication element (cre) in the 50 -terminal coding region of 3D, representing the primer for the negative-sense RNA replication intermediate and the subsequent positive-sense genomic RNA synthesis. A participation of the 50 -terminal NTR structures in the switch from translation to replication on the same viral RNA is suggested. HAV morphogenesis is poorly understood. The primary polyprotein cleavage event occurs at the 2A/2B junction mediated by the 3C protease resulting in the structural precursor P1-2A. The steps resulting in particle formation are not entirely clear, but a model for this process has been suggested (see Fig. 1). The P1-2A structural precursors assemble to a pentameric structure and are further cleaved by the 3C protease to generate the precursors VP4-VP2 (also known as VP0) and VP1-2A (also known as VP1pX), as well as VP3 resulting in a structural building block (VP0,VP3,VP1-2A)5. The 2A C-terminal extension of VP1 is a critical structural intermediate in virion morphogenesis, probably clamping the pentamer at the fivefold symmetry axis. After assembly of 12 such pentamers with the genomic RNA to provirions (12  (VP0,VP3,VP1-2A)5-RNA), VP0 is cleaved into VP4 and VP2 by an unknown mechanism. These immature particles, still containing VP1-2A, are present in lysosome-like vesicles as well as in multivesicular

366

Hepatitis A Virus (Picornaviridae)

bodies (MVB). Entry into MVBs is supposed to be mediated by an interaction between VP2 and the cellular endosomal sorting complex required for transport (ESCRT) and from there still immature HAV is secreted non-lytically in exosome-like vesicles (eHAV) from the basolateral site of hepatocytes into blood. How immature particles get access to lysosome-like vesicles is unknown, but the maturation cleavage at the VP1-2A junction leading to the removal of 2A seems to result from the action of a lysosomal protease before mature HAV virions are apically released into bile non-lytically via a secretory lysosomal pathway.

Host Range, Transmission and Tissue Tropism Until recently, when several HAV species were discovered in diverse mammals, only humans and certain species of non-human primates were considered to be infectable by HAV under natural conditions. Non-human primates, which are also used as animal models, include chimpanzees, marmosets and owl monkeys. As mice with an impaired interferon system become permissive to human HAV infection, the ability of HAV to interfere with the species-specific interferon system (e.g., cleavage of MAVS) seems to contribute to HAVs host tropism. HAV is transmitted via the fecal-oral route. As the virus is excreted in feces, it is typically acquired by ingestion of fecescontaminated food or water. Direct person-to-person spread occurs under poor hygienic conditions. The site of HAV replication is the liver. The events that occur during the passage of HAV across the intestinal epithelium into blood, in which the virus reaches the liver via the portal circulation, are not clearly understood. Although HAV antigen could be detected in different organs, such as the kidneys, spleen and gastrointestinal tract, no extrahepatic sites of HAV replication have been clearly identified. The only evidence for replication within the gut comes from studies of orally inoculated owl monkeys, but it was demonstrated that replication of HAV in polarized intestinal cells does not result in basolateral release of the virus progeny and thereby not in penetration of the epithelium. A functional cellular receptor could not be identified so far. The ubiquitously expressed cell surface T cell immunoglobulin mucin 1 (TIM1) protein, which binds HAV and had been suspected to be a HAV receptor (also known as HAVcr1), is not essential for cellular entry. It can be supposed that the HAV receptor is expressed by different cell types, and conserved through evolution, and it is unlikely that the receptor is the main determinant of the strong hepatotropism. Some studies suggest that the hepatotropism of HAV is supported by fecal immunoglobulin A (IgA)-virus complexes (HAV/IgA), as HAV-specific IgA facilitates transcytosis of virus particles antivectorially through polarized epithelial cells via the polymeric immunoglobulin receptor (pIgR), and as subsequently HAV/IgA uptake via the hepatocellular asialoglycoprotein receptor (ASGPR) results in infection of hepatocytes (IgA-carrier hypothesis). As for eHAV, which is released into blood but not into the fecal transmission pathway, it is unknown how it enters hepatocytes and that selectively. The entry of eHAV might occur via an endocytotic pathway in which the virus is amenable to HAV-specific neutralizing antibodies, which develop in the course of the infection. After entry of HAV into susceptible cells, the structural protein VP4 might facilitate the release of the genomic RNA into the cytoplasm by its pore-forming ability (pores of 5–9 nm in diameter) in endosome-like membranes at acidic pH. The virus progeny produced in the liver is released back into the intestinal tract via bile for transmission.

Cell Culture and Growth Characteristics In vitro HAV can infect a variety of primate and nonprimate cell lines, including nonhepatic cells. The virus exhibits a protracted replication cycle and normally establishes a noncytolytic, persistent infection with low virus yields, and there is no evidence that HAV notably interferes with the macromolecular synthesis of the host cell. After infection of cultivated cells with the wild-type virus, a minimum of 8 weeks elapses before HAV can be isolated. Although a more rapid replication and higher virus titers are obtained after serial virus passages in cultivated cells resulting in cell culture-adapted viruses, even the replication of these virus variants is not detectable within the first days after infection. Adaptation of HAV to growth in cultivated cells seems to be achieved by varying sets of multiple interacting mutations, with adaptive mutations within the IRES enhancing viral translation in a cell-type-specific fashion, and mutations clustering in the 2B and 2C proteins (see Table 1) increasing replication regardless of the cell line used. The virus apparently downregulates its own replication and this may, for example by supporting the ability of HAV to inhibit innate cellular anti-viral defense mechanisms, be important for the establishment of persistent infections. A large proportion of the virus progeny remains cell associated, but extensive release of HAV and eHAV from the cells also occurs. Several cytopathogenic variants of HAV have been isolated which induce apoptosis resulting in cell death. These variants are highly cell culture adapted and characterized by a rapid replication phenotype and high virus yields. In these variants, both the downregulation of viral replication and the ability to inhibit the innate defense mechanisms are less effective. The molecular mechanisms resulting in apoptosis are not known.

Clinical Features and Pathology HAV infection may lead to a wide spectrum of manifestations ranging from a silent infection, over icteric courses to a fatal fulminant hepatitis. The acute icteric course of infection varies between common, over prolonged to relapsing hepatitis A. Persistent infections or chronic disease states have not been described.

Hepatitis A Virus (Picornaviridae)

367

The clinical presentation of the disease depends on the age of the infected individual. The likelihood of having symptoms and the severity of the disease increases with the age of the patient. Silent infections (asymptomatic or at least anicteric) are normally observed in very young children, under the age of 2. However, a clinically detectable disease can occur even in infancy (aged 2 weeks to 8 months) and it may be characterized by prolonged courses. In children under 5 years of age approximately 3%, in children 5–15 years old approximately 30% and in individuals over the age of 18 years as many as 70% may develop a clinically detectable disease. The heterogenous outcome of HAV infections seems to depend on individual physiological conditions and multiple independent factors, such as the amount of virus progeny, the strength of IgA and IgG responses or the activity of the cytotoxic T lymphocytes.

Common Course of Infection The incubation period ranges from 2 to 6 weeks with a mean duration of 4 weeks. The prodromal (preicteric) period of normally 4–6 days (which may vary from 1 day to more than 2 weeks) is characterized by nonspecific symptoms, like anorexia, nausea with vomiting, malaise, abdominal pain, loss of appetite, accelerated pulse, rash, headache and fever (38–391C) as well as by gastrointestinal symptoms, typically as obstipation, but diarrhea is also observed. The prodromal symptoms disappear with the onset of jaundice, which is seldom abrupt (in 15% of the cases no obvious prodromal symptoms are observed before the appearance of jaundice). The icteric phase, which ranges from 2 to more than 22 days (mean duration 3 weeks), is marked by jaundice, which may start with scleral icterus, dark-colored urine (conjugated bilirubinuria), clay-colored stools and clearly decelerated pulse. The reconvalescence period ranges from 3 to 6 weeks, but fatigue, dullness, right upper quadrant tenderness and fast exhaustion may remain for 2–4 months. In almost all cases the liver is enlarged. The clinical symptoms are accompanied by several abnormal biochemical parameters (see Fig. 2). The elevation of aminotransferase levels in serum (alanine aminotransferase (ALT) and to a lesser degree aspartate aminotransferase (AST)), which reflect hepatocellular damage with the release of liver enzymes into the circulation, roughly correlate with the severity of the disease. The elevation of serum alkaline phosphatase activity and serum bilirubin level relate to intrahepatic cholestasis. At the onset of symptoms, seroconversion to HAV occurs. Large amounts of HAV, which are produced in the liver and released into the gastrointestinal tract via bile, are secreted in the feces already during the late incubation period when no clinical symptoms are detectable and are shed for approximately 3 weeks until a few days after the onset of elevated levels of liver enzymes in the serum (see Fig. 2). Fecal shedding of HAV reaches its maximum just before the onset of hepatocellular injury and terminates around the time when the IgG antibody response is detectable. Viremia occurs a few days before and during the early acute stage of the clinical and biochemical hepatitis, in which it roughly parallels the shedding of virus in feces, but at a lower magnitude (see Fig. 2). More sensitive methods (especially nucleic acid amplification technologies) demonstrated that low levels of viral RNA may be present in feces and blood for many weeks.

Prolonged Hepatitis A In 8.5%–15% of the cases, jaundice lasts for up to 17 weeks. In general, the symptoms are initially stronger than during the common course of the infection and the antibody titers reached are higher and sustain for a longer period of time. Virus in feces and blood may be detectable for 2–3 months. The biochemical abnormalities last at least 5 months. Occasionally, prolonged courses are accompanied by exceptionally high serum aminotransferase levels (ALT up to 560 IU dl
1) and by high serum bilirubin levels (up to 7.4 mg dl
1) persisting for months (cholestatic hepatitis A). The cholestatic form of HAV infection is associated with extensive itching of the skin. The pathogenesis of prolonged hepatitis is not understood and different factors could contribute to the extended course: reinfections at different sites of the liver may occur by enterohepatic cycling of HAV, which may be supported by anti-HAV immunoglobulin A (IgA) (IgA-carrier hypothesis) and by serum eHAV (enveloped HAV), respectively. HAV/IgA is neutralized for infection of hepatocytes via the ASGPR only late in the course of infection by highly avid immunoglobulin G (IgG) and eHAV is resistant to neutralizing antibodies. Membrane enveloping of HAV might also facilitate viral spread in the liver supporting infection for a longer time. Also NF-κB, which is activated by HAV, may contribute to prolonged courses by attenuation of the effects of HAV-specific cytotoxic T cells. Furthermore, infection by different HAV genotypes resulting in an elongated course of the disease has been speculated.

Relapsing Hepatitis A After initial improvement in symptoms and liver test values (serum aminotransferase levels), one or more relapses of the disease (mostly biphasic) are described for up to 20% of the patients (the average age in adults is 30 years, in children 9 years). These relapses occur between 30 and 90 days after the primary episode, when high titers of neutralizing antibodies are already present. The severity of symptoms, the serum aminotransferase levels and the immunoglobulin M (IgM) response are similar or weaker than observed during the initial phase, with a higher tendency for cholestasis (30% of these patients). The relapses last longer

368

Hepatitis A Virus (Picornaviridae)

Fig. 2 Course of clinically relevant events in acute hepatitis A. This figure schematically shows the mean duration and intensity of certain parameters. The dotted lines indicate the duration and intensity of the events that may vary. NK T cells: Natural killer T cells.

(mean duration 6 weeks) than the initial phase, HAV is present in blood and the serum bilirubin level is higher. During the relapse the tendency for purpura and itching of the skin is increased. No specific risk factors could be identified and the pathogenesis of relapsing hepatitis is not understood. The relapses may represent a manifestation of endogenous reinfections by an enterohepatic cycling of HAV/IgA (IgA-carrier hypothesis) until the reinfection cascade is terminated by competitive replacement of the IgA in the HAV/IgA complexes by large amounts of the aviditymatured IgG response.

Fulminant Hepatitis A In rare cases, acute hepatitis A results in a fatal deterioration in liver function with massive destruction of liver cells. Surprisingly, no vigorous inflammatory response is observed. The fatality rate is below 1.5% of all hospitalized icteric HAV infection cases. This course of the disease is accompanied with fever over 401C. This outcome is more frequent in adults, especially in patients over 50 years of age, and the risk is increased in patients with underlying chronic liver disease.

Extrahepatic Manifestations Extrahepatic manifestations of the disease are rare and the etiology is unclear. Besides frequently observed transient suppression of hematopoiesis (a significant leukocytopenia occurs 1–7 days before the appearance of jaundice correlating with the viremic and the fever phases; see Fig. 2), rare cases of aplastic anemia (occurring 1.5–2 months after appearance of jaundice) with a lethality rate of over 90% are described, and it was demonstrated that HAV infects monocytes and inhibits their further differentiation. In some patients interstitial nephritis, pancreatitis, myocarditis or Guillain-Barré syndrom were observed. By statistical evaluation of medical records, it was suggested that HAV exposure may leave a protective effect on the development of asthma and allergic diseases. However, due to contradictory findings in different studies this is still a subject of debate.

Hepatitis A Virus (Picornaviridae)

369

Pathology The pathology of the liver in acute hepatitis A has been studied in humans and some animal models. The pathological lesions, which are caused by an immunopathogenic mechanism, are characterized by hepatocellular necrosis, which is most prominent in periportal regions, accompanied with large inflammatory infiltrates of mononuclear cells.

Innate and Adaptive Immune Response Innate Immune Response HAV, which does not interfere with the replication of other viruses, prevents the synthesis of beta interferon (IFN-b). However, the virus is sensitive to alpha and beta interferon (IFN-a/b) exogenously added to persistently infected cells. It can be demonstrated that HAV inhibits the transcription of the IFN-b gene by blocking effectively interferon regulatory factor 3 (IRF-3) activation due to a cooperative interaction of the HAV proteins 2B and 3ABC with the mitochondrial antiviral signaling protein MAVS, which is a component of the retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) signaling pathway (see Fig. 3). While MAVS is targeted for HAV protease 3C-mediated proteolysis by 3ABC, an intermediate of polyprotein

Fig. 3 Interference of HAV with the interferon system. This figure schematically shows the sites and effects of the interference of hepatitis A virus with IFN induction. The figure also displays that, besides inhibiting protein kinase R (PKR) activity, HAV does neither interfere with interferon signaling nor significantly with cellular macromolecular synthesis. dsRNA, Double-stranded RNA; IFN, Interferon; IRF, Interferon regulatory factor; ISGs, Interferon stimulated genes; ISRE, Interferon stimulated response element; MAVS, Mitochondrial antiviral signaling protein; MDA, Melanoma differentiation-associated gene; OAS, Oligoadenylate synthetase; RIG-I, Retinoic acid-inducible gene I; TLR, Toll-like receptor.

370

Hepatitis A Virus (Picornaviridae)

processing localized to mitochondria by 3A, 2B indirectly seems to interfere with MAVS as well as with the kinases responsible for IRF-3 phosphorylation (TBK1/IKKe) through interactions with cellular membrane structures. Signaling through the Toll-like receptor 3 (TLR-3) pathway is also impaired by an interaction with TRIF (TIR domain-containing adaptor inducing IFN-b) (see Fig. 3). Gamma interferon (IFN-g) produced by HAV-specific HLA-dependent cytotoxic T lymphocytes (CTL) may contribute to the elimination of the HAV infection by inducing an antiviral state in the later stages of the infection. The importance of NK cells for the elimination of HAV is controversially discussed. As CpG containing codons are avoided in the coding sequence, HAV evades recognition of unmethylated viral CpG by Toll-like receptor 9. Furthermore, it could be shown that HAV inhibits the activity of protein kinase R (PKR) by a so far unknown mechanism (see Fig. 3). Whether HAV induces or inhibits the activation of NF-κB is controversially discussed. HAV also has the ability to prevent apoptosis induced by accumulating dsRNA, but the underlying mechanism is not clear.

Adaptive Immune Response Anti-HAV IgM antibodies are present in almost all patients at the onset of the symptoms (see Fig. 2). These antibodies reach their maximum level 2 months post exposure, have only weak neutralizing activity and disappear in the course of 3 months, but in the case of prolonged infections IgM can be detected up to 1 year after onset of icterus. Anti-HAV IgA antibodies are also detectable at the onset of the symptoms (see Fig. 2). This response reaches its peak titer 50 days post infection and may last for more than 5 years. The majority of the IgA remains as serum IgA in circulation and is not secreted into the intestinal tract as secretory IgA by the polymeric immunoglobulin receptor (pIgR) pathway. But a significant fraction of this serum IgA is released into the gastrointestinal lumen via bile by liver functions under participation of the hepatocellular IgA-specific asialoglycoprotein receptor (ASGPR). Fecal samples contain IgA from 5 weeks till 3–6 months post infection. Salivary anti-HAV IgA is also detectable in HAV patients, its course parallels that of fecal IgA. The role of secretory IgA antibodies in the protection against HAV infection appears to be limited, as neutralizing activity is barely detectable. Studies suggest that HAV-specific IgA can serve as a carrier molecule for a liver-directed transport of the virus, supporting and enhancing the hepatotropic infection by uptake of HAV/IgA immunocomplexes via the ASGPR (IgA-carrier hypothesis). As IgA-coated HAV is translocalized antivectorially from the apical to the basolateral site of epithelial cells via the pIgR, it was assumed that fecal HAV/ IgA immunocomplexes, whose stability enables their fecal-oral transmission, are able to support the primary infection utilizing the IgA receptors. Furthermore, an enterohepatic cycling of HAV may be established during infection by HAV/IgA resulting in endogenous reinfections of the liver until large amounts of highly avid IgG replace IgA in HAV/IgA immunocomplexes. Depending on individual factors, this mechanism may play a role in the development of the different courses of hepatitis A. It is not clear by which processes and mechanisms the induction of the anti-HAV IgA response occurs. Neutralizing anti-HAV immunoglobulin G (IgG) antibodies are also detectable 3 weeks post infection for the first time, but this response develops slowly, reaching its peak titer approximately 4 months post infection (see Fig. 2), a time point late in the convalescence phase. Envelopment of HAV in blood (viremia) by cellular membranes (eHAV) unrecognizable for the immune system may contribute to the slow IgG antibody response, as well as the transient perturbation of hematopoiesis, which may result in an attenuation and retardation of the inflammatory response and of the induction of the adaptive IgG response. Anti-HAV IgG persists lifelong, although the titer may fall to undetectable levels after several decades. Neutralizing antibodies, which are effective in eliminating the virus from the blood, do recognize a conformational epitope clustered into a major, immunodominant antigenic site involving residues contributed by VP3 (Asp70, Gln74) and VP1 (Ser102, Val171, Ala176, Lys221). Neutralization of eHAV by antibodies is possible during an assumed endocytotic re-uptake from blood by hepatocytes in an endosomal compartment. HAV-specific, HLA-restricted cytotoxic CD8 þ T-lymphocytes (CTL) accumulate in the liver during the acute phase of the infection (after destruction of the infected hepatocytes, approximately 2–3 weeks after onset of the icterus, the CTLs leave the liver back into blood) and they play a prominent role both in eliminating the virus and in causing a liver injury (see Fig. 2). Gamma interferon (IFN-g), released by these CTLs, may stimulate HLA class I expression on hepatocytes and in the following promote upregulation of the normally low level display of antigen on liver cells. Multiple dominant T-cell epitopes could be identified in the proteins VP1, VP2, VP3, 2B, 2C and mainly 3D (amino acids 2025–2033). This multitude of T-cell epitopes combined with an inhibitory effect of HAV on CTL-suppressing regulatory T cells (by binding of HAV to HAVcr1 expressed on Tregs) during the acute phase of the disease seems to result in a strong activity of HAV-specific CTLs. Studies in experimentally infected chimpanzees suggest a more complex T cell response with the involvement of anti-viral cytokines producing CD4 þ helper T cells also. In addition, NK T cells and possibly bystander-activated CD8 þ T cells seem to be involved in the elimination of HAV and the destruction of hepatocytes. Therefore, multiple immune mechanisms seem to contribute to the acute liver injury in humans (immunopathogenesis), also leading to an efficient elimination of HAV, which might prevent persistence of the virus.

Diagnosis Since the clinical presentation of hepatitis A cannot be distinguished from hepatitis caused by the other hepatitis viruses, serologic tests or nucleic acid amplification techniques are necessary for a virus-specific diagnosis.

Hepatitis A Virus (Picornaviridae)

371

The routine diagnosis of acute hepatitis A is made by detection of anti-HAV IgM in the serum of patients (see Fig. 2). A further option is the detection of virus in the feces. In order to improve the safety of blood and blood products, blood screening with HAV-specific polymerase chain reaction (PCR) is performed, which reduces the window period of up to 3 weeks post infection during which HAV infection fails to be diagnosed by serologic assays.

Epidemiology HAV occurs worldwide and accounts for over 1.5 million clinical cases reported annually. The seroprevalence pattern ranges from high endemicity, such as in Africa, Southern Asia and Latin America, where infection normally occurs in childhood, over intermediate endemicity, such as in Eastern Europa and the northern parts of Asia, to low endemicity, such as in Western Europa and North America, where the majority of the population remains susceptible to HAV infection. However, the epidemiology pattern is complex and continuously changing, with considerable heterogeneity among different countries. In general, the anti-HAV antibody prevalence inversely correlates with the quality of the hygienic standards, and the incidence declines in many populations through improvements in public sanitation and living conditions. These improvements result in an increase of the pool of susceptible adults, with a shift in the age of infection to older age groups, in which a more severe disease form is observed, leading to an increased morbidity. HAV has been recognized as a main cause of fulminant hepatic failure in a growing number of countries. A minor seasonal variation of HAV infections is observed, with a peak occurring during the fall and winter months, mentioned in almost all earlier and contemporary reports, nowadays possibly as a result of exposure during summer vacations spent in endemic countries. At special risk for acquisition of hepatitis A are international travelers from areas of low endemicity to endemic areas, employees of child-care centers and sewage plants, gully workers, injecting drug users, homosexually active men and persons with an increased risk of developing a fulminant disease, such as persons with chronic hepatitis C virus (HCV) infections. The high physical stability of HAV provides a good opportunity for common-source transmission. Community-wide outbreaks are reported in association with infections of food handlers, and linked to contaminated food and drink, or uncooked clams from contaminated water. Hepatitis A is most commonly acquired by sharing the household with an infected person.

Prevention and Control There is no specific antiviral treatment for hepatitis A. As almost all HAV infections are spread by the fecal-oral route, good personal hygiene, high-quality standards for public water supplies, and proper disposal of sanitary waste are important measures to reduce virus transmission. Until the availability of an active prophylaxis, the disease could be prevented for up to 5 months with a certainty of 80%–90% by passive immunization with pooled IgG of at least 100 IU anti-HAV antibodies, a prophylaxis which is effective within hours. IgG is still used for postexposure prophylaxis. If administered within 2 weeks after exposure, either development of the disease is prevented or the severity of the disease as well as virus shedding is reduced. Since 1992, inactivated vaccines for active immunization have been available. These vaccines contain purified, attenuated, formalin-inactivated HAV produced in cell culture, which are absorbed to an aluminum hydroxide adjuvant. They are highly immunogenic and protect against both the infection and the disease caused by all strains of HAV with 100% efficacy for at least 10 years, which is consistent with the finding that all human HAV strains belong to one single serotype. HAV vaccines are also effective when given within 2 weeks after exposure. Live, attenuated HAV vaccines have been developed in China for a single-dose, subcutaneous injection using virus adapted to grow in cell culture, which protect with similar efficacy as the inactivated vaccines. No reversion of the attenuated virus to virulence has been reported so far. However, viral shedding is possible, which must be taken into account in vaccinations in populations with large numbers of susceptible adults (see Epidemiology section). Although the minimum level of neutralizing antibodies that protect against infection and disease is unknown, an estimate of a minimal protection level is approximately 20 mIU ml
1 blood.

Further Reading Bell, B.P., 2002. Global epidemiology of hepatitis A: Implications for controlstrategies. In: Proceedings of the 10th International Symposium on Viral Hepatitis and Liver Disease. Debing, Y., Neyts, J., Thibaut, H.J., 2014. Molecular biology and inhibitors of hepatitis A virus. Medicinal Research Reviews 34 (5), 895–917. Dotzauer, A., Kraemer, L., 2012. Innate and adaptive immune responses against picornaviruses and their counteractions: An overview. World Journal of Virology 1 (3), 91–107. Gerety, R.J. (Ed.), 1984. Hepatitis A. London: Academic Press. Gust, I.D., Feinstone, S.M., 1988. Hepatitis A. Boca Raton: CRS Press.

372

Hepatitis A Virus (Picornaviridae)

Jacobsen, K.H., 2010. The Global Prevalence of Hepatitis A Virus Infection and Susceptibility: A Systematic Review. Geneva: WHO Press. Lanford, R.E., Walker, C.M., Lemon, S.M., 2017. The chimpanzee model of viral hepatitis: Advances in understanding the immune response and treatment of viral hepatitis. ILAR Journal, 1–18. Lemon, S.M., Ott, J.J., Van Damme, P., Shouval, D., 2018. Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention. Journal of Hepatology 68, 167–184. Wasley, A., Fiore, A., Bell, B.P., 2006. Hepatitis A in the era of vaccination. Epidemiologic Reviews 28, 101–111.

Hepatitis B Virus (Hepadnaviridae) Peter Karayiannis, University of Nicosia, Nicosia, Cyprus r 2021 Elsevier Ltd. All rights reserved.

Nomenclature IU/ml

International units

Glossary Immunomodulation Changes in the body’s immune system, caused by agents that activate or suppress its function. Nucleos(t)ide analogue Antiviral medication that is incorporated into viral nucleic acid during synthesis causing chain termination.

Perinatal Of, relating to, or being the period around childbirth, especially the five months before and one month after birth. Percutaneous Through the skin following injury or puncture with a needle. Quasispecies A population of closely related variants of a virus isolated from an individual at a given time.

Introduction Since the discovery and recognition of HBV as a human hepatitis causing pathogen, great strides have been made in our understanding of its epidemiology, virology, life cycle, pathogenesis, treatment and vaccine protection. Discovered by Bloomberg and co-workers just over fifty years ago, it remains a major cause of morbidity and mortality in countries of the world where the virus is still endemic, in spite of effective regimens of antiviral drugs which suppress viral replication and vaccine campaigns to protect those at greater risk. Recent estimates suggest that 291 million people are chronically infected with HBV representing a global prevalence of 3.6%–3.9%. Accurate figures of chronic infection are incomplete from many parts of the world and even from Western countries entry data on death certificates for example may not be accurate. Thus there is a reliance on mathematical models that take into account various confounding factors. These suggest that as chronic infection over many years may lead to cirrhosis and hepatocellular carcinoma, the incidence of these two conditions for all viral hepatitis is 1.4 million annually, of which nearly 700,000 are HBV-related. This represents an increase of over 33% over the period 1990–2013, when viral hepatitis climbed from 10th to the 7th position of leading causes of mortality worldwide. Although, the prevalence of HBV infection is decreasing in several highly endemic countries due to actions which will be explained later, acute infections still occur primarily in high risk populations. What is more, recent population movements and migration from war-torn regions of the world to low endemicity countries may be changing the prevalence and incidence of HBV in these countries, particularly if such individuals evade surveillance. Vaccination programs and antiviral treatment remain the only means of reducing infections and halting progression of chronic liver disease to cirrhosis and HCC respectively. The study of the life cycle and molecular biology of HBV, in the absence of a reliable cell culture system for the propagation of the virus has been re-invigorated in recent years following the identification of its hepatocyte receptor. Nevertheless, studies in the early 1980s employing genetic engineering techniques led to the cloning of the viral genome and its sequencing. This allowed expression of its proteins, enabling the study of their function, and more importantly, unraveled the unique and fascinating mechanism of its replication strategy. In this respect, the contribution of animal models has been invaluable. These initially employed chimpanzees, and subsequently ducks, woodchucks, transgenic and more recently chimeric mice. This has allowed the study of many aspects of the life cycle, molecular biology and immuno-pathogenesis of the virus, and facilitated vaccine development and antiviral testing. What follows is what we know to-date about this very important pathogen.

Classification HBV is the prototype member of the hepadnaviridae which comprises a group of hepatotropic DNA viruses that have been steadily growing in numbers. These viruses are species specific and are broadly divided into two genera depending on their natural host. Thus, the mammalian Orthohepadnavirus genus includes members that infect rodents such as woodchucks (Marmota monax) and ground squirrels (Spermophilus beecheyi), bats (various species) and primates (humans, chimpanzees, gorillas, gibbons, baboons, orangutans, macaques and woolly monkeys). The Avihepadnavirus genus infects birds such as pekin ducks (Anas domesticus), herons, storks, geese and parrots with about 80% homology between them. The homology between the two genera is around 40% and the genetic relatedness of sequenced isolates from various species are evident in Fig. 1. Three branches are discernible within the orthohepadnaviruses, with human and primate isolates grouping together, and two other groups representing rodent and bat isolates respectively.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21228-4

373

374

Hepatitis B Virus (Hepadnaviridae)

Fig. 1 Phylogenetic tree based on complete genome sequences of mammalian, rodent and bat HBV isolates, showing three separate groupings. Human HBV genotypes A-J group together with primate isolates as shown. Similarly, rodent isolates from woodchuck (WHV), ground and arctic squirrel hepatitis virus (GSHV, ASHV) group together. Bat HBV isolates from different species group together on the third branch (e.g., LBHBV, long-fingered bat HBV). Reprinted by permission from Springer Nature, Rasche, A., Souza, B.F.C.D., Drexler, J.F., 2016. Bat hepadnaviruses and the origins of primate hepatitis B viruses. Current Opinion in Virology 16, 86–94.

Fig. 2 Structure of the hepatitis B virion. (a) Electron migrograph of HBV purified from plasma showing the infectious Dane particle and the spherical and filamentous subviral particles. (b) Cartoon of the virion and its components.

Virion Structure HBV has pleomorphic appearance under the electron microscope (Fig. 2) and this because other than the infectious virions, often referred to as Dane particles, there is an abundance of subviral particles which are known as filaments and spheres. The Dane particles were named after the scientist who led the team that discovered them and have a diameter of 42–45 nm. The virions have an outer glycolipid envelope made of hepatitis B surface antigen (HBsAg) in a lipid bilayer, which is thought to derive from the endoplasmic reticulum membrane. This in turn encloses the nucleocapsid or core of the virus consisting of hepatitis B core antigen (HBcAg) and measuring 30–32 nm in diameter. The cores contain the viral genome and a copy of its polymerase. Additionally the virus produces a soluble protein known as hepatitis B e antigen (HBeAg) which is not a structural protein, but rather constitutes a marker of active viral replication. The abundant sub-viral particles circulating in serum consist entirely of HBsAg and are devoid of cores and any viral nucleic acid. In this respect, these particles are not infectious. As mentioned above, these are the 25 nm spheres and the 22 nm diameter filaments, which outnumber infectious virions by 100–10,000-fold. Their production and secretion in such excess may serve to mop up anti-HBs antibodies or cause T cell anergy, both of which may contribute to immune tolerance and thus viral persistence. Similarly, HBeAg is thought to function as a tolerogen.

Hepatitis B Virus (Hepadnaviridae)

375

Fig. 3 Genome organization of the virus (a) and the transcripts encoding the various viral proteins (b), synthesized from cccDNA. All of them terminate at the common polyadenylation site. Reprinted (panel a) by permission from Wiley, Hunt, C.M., McGill, J.M., Allen, M.I., Condreay, L.D., 2000. Clinical relevance of hepatitis B viral mutations. Hepatology 31, 1037–1044.

Genome The partially double stranded DNA viral genome is a relaxed circular molecule (rcDNA) 3.2 kb in length, and is the smallest among DNA viruses. The strands overlap as shown in Fig. 3 with the minus (
)-strand being complete and the positive ( þ )-strand incomplete at its 30 end and of variable length (about 600 nucleotides missing). It is one of the most compact genomes as it contains 4 wholly or partially overlapping open reading frames (ORFs) (Fig. 3). What is more, every nucleotide of the genome lies within the coding region of one of the 4 ORFs. These ORFs also incorporate in them all of the regulatory elements of the virus, including two enhancers (Enh1, Enh2), the four promoters (core, S1, S2 and X), polyadenylation, encapsidation (e) and replication signals (DR1, DR2). The four ORFs are those of the Pre-S/S, Pre-core/core, X and polymerase and between them they encode for a total of 7 proteins which are translated from 6 co-terminal mRNAs. These are unspliced and capped, ending at a common polyadenylation signal embedded in the core ORF (Fig. 3). The synthesis of these mRNAs is regulated by the aforementioned enhancers and promoters which bear motifs which are recognized by transcription factors highly enriched in the nuclei of hepatocytes which are the target cells of the virus.

Transcription and Translation Surface proteins The Pre-S/S ORF encodes three proteins which are glycosylated and are part of the viral envelope. These are known as the large (L), middle (M) and small (S) HBsAgs, produced by differential initiation of translation at each of three in-frame initiation codons. Of these, the S-HBsAg constitutes the majority protein of the viral envelope. The proteins are translated from two mRNA transcripts of 2.4 kb and 2.1 kb in length, the synthesis of which is under the control of the S1 and S2 promoters respectively. The 2.4 kb transcript is utilized for the synthesis of L-HBsAg whilst the 2.1 kb transcript is responsible for the translation of M- and S-HBsAgs, the latter through leaky ribosome scanning. The proteins are co-terminal and the sequence of S-HBsAg consisting of 226 aminoacids is shared by the other two and constitutes their C-terminus. The M-HBsAg has an additional 55 amino-acids at its N-terminus

376

Hepatitis B Virus (Hepadnaviridae)

encoded by the Pre-S2 region, whilst the L protein has 107–118 (depending on genotype) further amino-acids encoded by the PreS1 region. A stretch of 75 amino-acids at the N-terminus of Pre-S1 appears to be responsible for the interaction of the virus with its hepatocyte receptor. As mentioned previously, the three proteins are glycosylated, although the L- and S-HBsAgs may be present in an unglycosylated form in particles also. In addition to glycosylation the L-HBsAg undergoes one other post-translational modification in the form of myristylation, a step that appears necessary for infectivity and envelopment of the core particles. These proteins are synthesized in close proximity to the ER membrane adopting a transmembrane configuration that facilitates budding of the virus into the lumen during virion maturation.

Core and polymerase The synthesis of two longer than genome length mRNAs (3.5 kb) which differ with respect to the start of their 50 end is under the control of the core promoter. The precore mRNA is longer by a small number of ribonucleotides amongst which the initiation codon for the synthesis of the precursor of the soluble HBeAg often referred to as the precore protein. This is synthesized when the first of two in frame initiation codons that the precore/core ORF contains is utilized. The N-terminus of this protein has a signal peptide that anchors the protein in the ER membrane. A signal peptidase in the lumen of the ER cleaves the first 19 amino-acids of the protein and what remains undergoes furin cleavage for the removal of an arginine rich domain at its C-terminus. The resulting protein constitutes the 15 kD HBeAg, which is secreted in serum. Initiation of translation at the second initiation codon results in the synthesis of the nucleocapsid or core protein (21 kD). The core has the capacity to dimerize and form nucleocapsids consisting of 240 copies (120 dimers) of the protein through self-assembly. The HBeAg and core proteins are translated from two separate transcripts known as the precore mRNA and the pregenomic RNA (pgRNA), respectively. The pgRNA is bicistronic in nature as it contains in addition the ORF encoding the polymerase (Fig. 3), a 90 kD protein that encompasses almost ¾ of the genome. The polymerase has multifunctional enzymic activities that include a reverse transcriptase (rt), DNA polymerase and RNase H activity. In addition, the pgRNA constitutes the template for synthesis of the (
)-DNA strand by reverse transcription during the replication of the viral genome, as explained later.

X protein Finally, the fourth ORF encodes for the 17 kD HBx protein that is translated from the shortest transcript of 0.7 kb in length. It consists of 154 amino-acids and has been implicated in a number of actions ranging from modulation of host-cell signal transduction pathways, gene transactivation under experimental conditions, activation of transcription factors and epigenetic control of the covalent closed circular DNA (cccDNA) minichromosome that is essential for transcriptional activation. Moreover, cccDNA can persist in hepatocytes indefinitely thus maintaining chronic lifelong infection until such time as the host cell is eliminated through immune lysis.

Life Cycle Attachment Other than the requirement of transcription factors enriched in hepatocytes, it is now evident that species specificity and hepatotropism are determined by the expression of the recently described sodium taurocholate co-transporting peptide (NTCP) on human hepatocytes, which is the HBV receptor (Fig. 4). NTCP is a bile acid transporter expressed at the basolateral membrane of hepatocytes. The receptor-virus interaction is effected by binding of the N-terminal end of L-HBsAg as described above. This may not be the only requirement as evidence suggests co-operative binding in the process of attachment and uptake involving in addition, heparan sulfate proteoglycans and glypican.

Penetration and Uncoating The virion is likely internalized through clathrin mediated endocytosis. The steps from uncoating to transfer of the nucleocapsid to the nuclear pore are not clear, but it seems that transport factors such as importin alpha and beta and component nucleoporin 153 assist in nucleocapsid delivery to the nuclear basket. The disassembly of the nucleocapsid is followed by the release of the rcDNA genome into the nucleoplasm with its covalently attached polymerase.

Transcription/Translation Within the nucleoplasm the rcDNA is converted into the cccDNA molecule involving at least three stages. The viral polymerase which is covalently attached to the 50 end of the (
)-DNA strand is removed, as is the short RNA oligomer from the 50 end of the ( þ )-DNA strand which is used to prime ( þ )-DNA strand synthesis. Next, the variable ( þ )-DNA strand is completed and the ends of the two now complete strands ligated to form a double stranded circular molecule, referred to as cccDNA. This is quite stable and functions as a minichromosome following association with a number of epigenetic factors that include histones H3 and H4, transcription factors that include CREB, ATF, STAT1 and STAT2, chromatin modifying enzymes, histone acetyltransferases and deacetylases, as well as the HBc and HBx proteins. Transcriptionally active cccDNA constitutes the template for viral transcript

Hepatitis B Virus (Hepadnaviridae)

377

Fig. 4 Diagrammatic representation of the stages of the life cycle of the virus as explained in the text: 1. Attachment; 2. Endocytosis; 3. Capsid release; 4. rcDNA entry into the nucleus; 5. cccDNA synthesis; 6. Transcription; 7. mRNA transfer to the cytoplasm; 8. Encapsidation; 9. (
)-DNA strand synthesis by reverse transcription; 10. ( þ )-DNA strand synthesis; 11. Budding of virions into the ER lumen; 12. Virus transfer by multivesicular bodies (MVB) to hepatocyte surface; 13. Release. Modified from Karayiannis, P., 2017. Hepatitis B virus: Virology, molecular biology, life cycle and intrahepatic spread. Hepatology International 11, 500–508, with permission from Elsevier.

synthesis by the host RNA polymerase II. Newly synthesized viral transcripts are exported to the cytoplasm for viral protein synthesis.

Encapsidation As mentioned above the bicistronic pgRNA encoding for the core and polymerase proteins, also serves as the template for DNA synthesis by reverse transcription. The pgRNA is longer than genome length by virtue of a terminal redundancy which duplicates the sequence at its 50 end by about 120 nt and terminates at the polyadenylation signal. The host RNA polymerase appears not to recognize this signal during the first pass but does so on the second encounter. Thus, the redundancy contains a second copy each of the direct repeat 1 (DR1) and the encapsidation signal e, preceding and incorporating the precore nucleotide sequence respectively. The encapsidation of the pgRNA into the nucleocapsid involves both viral and host factors. The polymerase has four domains which in sequence are the terminal protein involved in DNA priming, a spacer region of unknown function, the reverse transcriptase/polymerase (rt) and the RNAse H which are involved in DNA synthesis and degradation of the pgRNA respectively. The polymerase recognizes and binds to the secondary structure of e located at the 50 end of the pgRNA, an event that triggers encapsidation of the complex by the core protein (Fig. 5). This process is facilitated by the cap at the 50 end of the pgRNA, as well as eIF4E and heat shock proteins, which in addition support the stabilization and activation of the polymerase. The arginine rich C-terminus of the core protein is involved in pgRNA binding thus facilitating encapsidation also. The steps that follow are performed within the nucleocapsid. The secondary structure that e assumes consists of a lower stem, an upper stem, a side bulge and an apical loop, which are the result of base pairing of palindromic nucleotide sequences. Part of the sequence of the side bulge of e serves as a template for the synthesis of only a 4 nucleotide long DNA primer (Fig. 5(A)). A phosphodiester linkage between dTTP and the hydroxyl group of a tyrosine residue in the terminal protein (position 63), results in the covalent attachment of the primer to this domain of the polymerase. This is followed by a translocation event whereby the polymerase-primer complex localizes to the 30 of the pgRNA, where the primer hybridizes with a homologous region within DR1 (Fig. 5(B)). This is assisted by two other elements, f and o, which interact with e. Priming of the pgRNA is followed by the synthesis of the (
)-DNA strand initiated by reverse transcription as the complex proceeds towards the 50 end of the pgRNA, having a terminal redundancy of about 10 nucleotides (Fig. 5(C)). The RNAse H activity of the polymerase degrades in the process

378

Hepatitis B Virus (Hepadnaviridae)

Fig. 5 Replication strategy of the virus. (a) Primer synthesis; (b) translocation and binding to DR1; (c) synthesis of the (
)-DNA strand; (d) RNA primer fragment preserved from the degradation of the pgRNA; (e) ( þ )-DNA strand synthesis. Reproduced from Karayiannis, P., 2017. Hepatitis B virus: Virology, molecular biology, life cycle and intrahepatic spread. Hepatology International 11, 500–508, with permission from Elsevier.

the pgRNA except for the final 11–16 or so ribonucleotides which contain the DR1 region at the 50 of the pgRNA (Fig. 5(D)). ( þ )-DNA strand synthesis is primed by the ribonucleotide fragment which hybridizes with DR2 at the 50 end of the newly synthesized (
)-DNA strand with which it shares homology. This event necessitates a second translocation event which is followed by ( þ )-DNA strand synthesis that proceeds to the 50 end of the (
)-DNA strand. Circularization facilitated by the short terminal redundancy of the (
)-DNA strand allows template exchange and continuation of ( þ )-DNA strand synthesis along the 30 end of the (
)-DNA strand (Fig. 5(E)). The partially double stranded nature of the HBV genome is the result of the depletion of the nucleotide pool which is not replenished once the maturing nucleocapsids are enveloped by budding through the endoplasmic reticulum membrane. The passage of nucleotides is facilitated through pores within the nucleocapsid at the early stages of replication. As a result the ( þ )-DNA strand remains incomplete.

Maturation Early in infection and until sufficient amounts of HBsAg accumulate, nucleocapsids containing rcDNA are shuttled back to the nucleus in order to replenish the cccDNA pool. Hepatocyte nuclei may contain more than 50 copies of cccDNA through this amplification loop (Fig. 4). In the final stages, virions bud through the endoplasmic reticulum membrane where HBsAg proteins are already localized, into the lumen, acquiring in the process their outer envelope. This is assisted by the topology and conformational arrangement of L-HBsAg. Studies have shown that about half of the protein produced exposes its N-terminus on the cytosolic side of the ER membrane, facilitating binding of the nucleocapsid and initiation of budding. The rest of L-HBsAg has its N-terminus protruding in the lumen which enables exposure on the surface of the virion and thus accessibility for binding to the NTCP receptor.

Egress Virions that accumulate in the ER lumen rely on the multivesicular body (MVB) pathway for their export. This pathway depends on the endosomal sorting complex required for transport (ESCRT) system which uses ESCRT-0, -I, -II and -III complexes and associated proteins. SVPs on the other hand bud off independently and are processed via the ER-Golgi pathway.

Hepatitis B Virus (Hepadnaviridae)

379

Epidemiology In spite of the availability of an effective vaccine against HBV and concerted immunization campaigns in endemic areas, there are still about 291 million chronic carriers of HBV infection worldwide, representing 3.9% of the worlds population (estimates of 2016). The prevalence of HBV infection has been dropping in many endemic areas, but still this varies by geographical region. In North Western Europe, North America and Australia the prevalence is between 0.1% and 2%, in the Mediterranean region, Eastern Europe, Middle East, Indian subcontinent, Central and South America it ranges between 2% and 7%, whilst in Africa and the Far East it is between 2% and 14%. About two thirds of the worlds chronic carriers reside in Africa and the Far East. The geographical distribution of HBV infection is mirrored by the incidence of HCC in the same regions. In areas of high endemicity, the virus is transmitted perinatally from carrier mothers to their infants, or horizontally from infected siblings and other children in early childhood. In areas of intermediate endemicity apart from perinatal transmission, household and sexual contact, as well as percutaneous exposure, are likely routes of infection. Finally in Western countries transmission is nowadays through sexual contact or intravenous drug use. Following infection, the risk of becoming a chronic carrier is age dependent. Exposure to the virus perinatally and up to one year of age has the highest chronicity rate of 90%, an intermediate risk of 20%–50% for ages up to 5, and 5% for older children and adults.

Genotypes Whole HBV genome nucleotide sequencing of human isolates from various regions of the world, followed by phylogenetic analysis have identified 10 genotypes designated A-J based on sequence divergence of 48%. These have characteristic geographical distribution such that Genotypes A and D are frequently found in Africa, Europe, and India, genotypes B and C in Asia, genotype E confined to West Africa, and genotype F in Central and South America. Genotypes G and H have been isolated from Central America and Southern Europe but otherwise their distribution is less clear. Two new genotypes designated I and J have recently been described. Genotype I identified in Vietnam, Laos and Eastern India appears to be an intergenotypic recombinant between genotypes A, C, and G, whilst genotype J was isolated in Borneo, and is a recombinant between genotype C and gibbon HBV. Currently, 44 sub-genotypes also exist and are found in Genotypes A, B, C, D, F and I. These are based on nucleotide divergence of 4%; A1-6, B1-9, C1-16, D1-9, F1-4 and I1-2. Of the other recombinants, B/C and C/D types represent the majority, whilst other intergenotypic recombinants that occur less frequently involve most of the other genotypes. Likely differences between genotypes in relation to pathogenesis and response to antiviral treatment are beginning to emerge. Genotype C is more frequently associated than B with abnormal liver function tests, lower rates of seroconversion to anti-HBe, higher levels of serum HBV-DNA, cirrhosis and hepatocellular carcinoma. Moreover, there is a better sustained response to interferon treatment in patients with genotype B than those with C, and in patients with genotype A than those with D. Genotype A infection appears to be associated with biochemical remission and clearance of HBV DNA more frequently than genotype D, and has a higher rate of HBsAg clearance compared with genotype D. In contrast to the differences observed in response to interferon therapy, treatment with nucleos(t)ide analogs does not show differential responses between genotypes. Recently, quantitative measurements of HBsAg levels have been associated with potentially favorable responses to interferon treatment in both HBeAgpositive and -negative patients. For example, a reduction in HBsAg levels during treatment, particularly at 12 and 24 weeks, may be predictive of a favorable outcome in the case of Peg-IFN treatment in HBeAg positive patients.

Subtypes The nucleotide sequence encoding HBsAg is shared by all three envelope proteins. What is more, it contains the main virus neutralizing epitope which is referred to as the “a” determinant region. This epitope is hydrophilic in nature and thus displayed on the surface of the virion. It is conformational, assisted in this by a number of disulfide bridges, and thought to encompass aminoacids 110–160 of the protein. It is shared by all virus isolates. Subtypic specificities also exist and these can be defined either by specific antibodies or by the amino-acids present at positions 122 and 160 of the protein. Lysine (K) or arginine (R) at position 122 confers d or y specificity, respectively, whilst specificities w and r are conferred by the presence of K or R at position 160. Thus each subtype is given a 3 letter code e.g., adw, whereby the a is the group determinant, and d and w based on the amino-acids present at positions 122 and 160 respectively. The w sub-determinant is further divided into w1-w4 specificities. Nine subtypes of the virus are recognized in all (Table 1) depending on the presence of other subtypic determining amino-acids elsewhere in the “a” determinant region.

Variants Other than the above stable genotypes and subgenotypes, a number of genomic mutations have been described which arise during the natural history of the infection. As described above, the replication step in the HBV life cycle that entails the reverse transcription of the pgRNA replicative intermediate to synthesize the (
)-strand DNA of the virus is prone to errors by the viral

adw adw2/ayw1 adw1/ayw1 ayr/adrq þ /adrq-/adr

ayw2/ayw3/ayw4

ayw4 adw4q-/ adw2/ayw4 adw2 adw2 ayw3

A

D

E F G I J

K a a a – R R R a – – R

T – – – – – – – – – – –

C – – – – – – – – – – –

T – – – – – M – – – – –

T – – I I – – – – – – I

P – – – – – T L L – – T

A – – – – – – – – – – –

Q – – – – – – – – – – –

G – – – – – – – – – – –

N – T T T T T T T – – T

S – – – – – – – – – – –

M – – – – – – – – – – –

F – – – – – – – – Y Y –

P – – – – – – – – – – –

Amino acid sequence of ‘a’ determinant (positions 122–160) S – – – – – – – – – – –

C – – – – – – – – – – –

C – – – – – – – – – – –

C – – – – – – – – – – –

T – – – – – – S S – – –

K – – – – – – – – – – –

P – – – – – – – – – – –

T – – S S S S S S S S S

D – – – – – – – – – – –

G – – – – – – – – – – –

N – – – – – – – – – – –

C – – – – – – – – – – –

T – – – – – – – – – – –

C – – – – – – – – – – –

I – – – – – – – – – – –

P – – – – – – – – – – –

I – – – – – – – – – – –

P – – – – – – – – – – –

S – – – – – – – – – – –

S – – – – – – – – – – –

W – – – – – – – – – – –

A – – – – – – – – – – –

F – – – – – – – L – – –

A – – – V G G G G – – –

K – – R R – – – – – – –

Source: a ¼ R or K. Note: G and I share complete amino-acid homology over the “a” determinant. Distinction between the two is possible as R in genotype G is substituted by K at position 24 of S-HBsAg in genotype I. The latter is also distinguished by the presence of H, A, N, V and V at positions 56, 60, 87, 90 and 91 of L-HBsAg respectively and I at position 22 of M-HBsAg.

B C

Subtype/s

Genotype determining amino acid variation over the “a” determinant region of HBsAg, and relationship between genotypes and subtypes.

Genotype

Table 1

380 Hepatitis B Virus (Hepadnaviridae)

Hepatitis B Virus (Hepadnaviridae)

381

reverse transcriptase. It is estimated that the HBV genome evolves at a rate of 1.4–3.2  10–5 nucleotide substitutions/site/year. As a result, the virus circulates in the serum of infected patients as a population of very closely related genetic variants, referred to as quasispecies. The viability of these variants is dependent on the site of the nucleotide substitutions given the overlapping nature of the ORFs and the localization of the various regulatory elements. Therefore some of these mutations may be deleterious to the virus whilst others may be advantageous, offering either a replication advantage or facilitating immune escape. These are discussed later in the clinical settings in which they have been described.

Clinical Features HBV Infection may run an asymptomatic, acute icteric (jaundice) or in a minority of patients a fulminant hepatitis course (0.1%–0.5%). As already mentioned the risk of the infection progressing to chronicity is inversely related to age with approximately 5% of adults and 95% of young children becoming chronically infected. Other than age, outcome depends on genetic factors which likely influence the efficiency of the host immune response. These include polymorphisms of the MHC class II glycoproteins involved in viral peptide presentation during induction of the cellular immune response and mannin binding lectins which bind to mannose terminated carbohydrate residues, such as those present on the carboxy-terminus of the pre-S2 region of the middle envelope protein, facilitating phagocytosis.

Acute HBV Infection The incubation period following exposure is variable ranging from 3 to 6 months and may be dependent on the size of the incoculum and route of exposure. Before onset of symptoms some patients may experience a serum sickness-like syndrome including arthralgia, fever and urticaria. The clinical picture varies from asymptomatic anicteric infection to protracted icterus and, in some patients (o 1%), liver failure (fulminant hepatitis). The acute infection is self-limiting and most patients recover within 1–2 months after the onset of icterus.

Chronic HBV Infection This is defined as persistence of HBs antigenaemia of longer than 6 months duration and may or may not be accompanied by viraemia and hepatic inflammation. The latter is based on histological examination of liver biopsy material that is followed by assignment of a score for necro-inflammatory activity (out of 18) and stage of fibrosis (out of 6) which are used to assess disease progression and to determine whether a patient needs therapy.

Course of Chronic Infection Chronic HBV infection is quite variable and is typically characterized by four main phases. These have recently been renamed as the “HBeAg-positive chronic HBV infection” (previously immune tolerant), the “HBeAg-positive chronic hepatitis B” (immune clearance), the “HBeAg-negative chronic HBV infection” (non-replicative), and the “HBeAg-negative chronic hepatitis B” (reactivation phase) which may be seen in some patients, particularly in Southern Europe and the far East (Fig. 6). There may be a fifth phase in some patients, referred to as the “HBsAg-negative phase”, which is characterized by absence of serum HBsAg and positivity for anti-HBc with or without detectable anti-HBs. This phase is also known as “occult HBV infection”. During the HBeAg-positive chronic HBV infection phase the patient is HBeAg positive with high levels of HBV-DNA, but with near normal or minimally elevated alanine aminotransferase (ALT) levels and absent or minimal necroinflammation changes in the liver. This phase is more likely in children infected at birth or soon after, and may last 2–3 decades. Patients are highly infectious and HBeAg loss during this phase is very low indeed. Patients from this phase progress to the HBeAg-positive chronic hepatitis B phase which is more commonly seen in those infected in adult life. This phase is characterized by the presence of HBeAg and HBV-DNA the levels of which gradually decline, as well as increased ALT levels associated with necroinflammatory changes in the liver. Loss of HBeAg may be accompanied by an ALT flare, culminating in loss of HBeAg and seroconversion to anti-HBe in a large proportion of patients followed by entry to the HBeAg-negative chronic HBV infection. During this phase plasma HBV-DNA may remain undetectable, ALT levels return to normal whilst annual loss of HBsAg and/or seroconversion may occur spontaneously in 1%–3% of cases. A few patients from this phase and the rest from the previous may proceed to the HBeAg-negative chronic hepatitis B. These patients have raised ALT levels that may fluctuate from time to time, viraemia and continued necroinflammatory activity in the liver that may lead to fibrosis, and faster development of cirrhosis and HCC. Viral DNA integration into the host genome may take place early on in infection and HBsAg expressed from such sequences contributes to the total throughout chronic infection. Moreover, HBV DNA integration may contribute to the development of cancer in the long term. Finally, patients in the fifth HBsAg-negative phase have normal ALT values and usually, but not always, undetectable serum HBV DNA. cccDNA quiescent in the liver may cause viral reactivation following immunosuppression as may be the case for organ transplantation.

382

Hepatitis B Virus (Hepadnaviridae)

Fig. 6 Diagram of the natural history of HBV infection showing progression from acute to the newly renamed chronic phases of infection. Loss of HBsAg at any time during chronic infection as indicated, may lead to the establishment of occult B infection (OBI).

Mutant Viruses and Chronic Infection Patients in the HBeAg-negative chronic hepatitis B carry natural mutants of the virus which impact HBeAg production by either reducing levels (core promoter variants) or abrogating completely HBeAg (pre-core variants) production. These variants are selected at the time of, or soon after seroconversion, and become dominant during the reactivation phase. The most common precore mutation is the G1896A substitution, which creates a premature stop codon in the precursor protein from which HBeAg is elaborated. This mutation is located in the stem of the e encapsidation signal, which it does not destabilize but affords stronger base-pairing with the A1896 change in genotypes with a T at position 1858 of the precore region, such as B, C, D and E. A double mutation in the basal core promoter region (A1762T, G1764A) is suggested to result in decreased transcription of the precore mRNA, with a knock on effect on HBeAg production, whilst pgRNA synthesis remains unaffected. Additional mutations in this region may also contribute to this phenotype.

Pathogenesis The outcome of infection following exposure to HBV is variable with regards to probability of progression to chronic infection, the likelihood of a favorable outcome after antiviral treatment and the risk of progression to unwelcome chronic sequelae such as cirrhosis and HCC. All of these are dependent on viral, environmental, demographic and clinical factors, but also host genetics and other factors which have been identified such as defective viral mutants (pre-S/S deletion mutants), and more recently spliced mRNAs, miRNAs and exosomal vesicles. Acute self-limiting infection is characterized by necroinflammation in the liver, consistent with the rise in transaminase levels. It seems that the innate immune response in the initial stages of infection is not robust enough while viraemia decline prior to symptom appearance may indicate non-cytolytic suppression of virus replication, mediated perhaps by IFNg secreted by NK and NKT cells. Studies in animal models have indicated that an HBV-specific cytotoxic T cell response (CTL) is essential for clearance of infected hepatocytes. Such CTL responses are strong and polyclonal in nature, directed against viral proteins such as HBcAg, and are the prime cause of immunopathology in the liver as the virus is not directly cytopathic. Clearance of free virions and prevention of attachment are dependent on the development of neutralizing anti-HBs. In chronic HBV infection on the other hand, the virus and high levels of HBsAg or HBeAg may disrupt maturation of antigen presenting cells such dentritic cells (DCs), thus inducing tolerance. Studies have shown that NK cells and plasmacytoid DCs

Hepatitis B Virus (Hepadnaviridae)

383

(pDCs) are deficient or dysfunctional. Monocytes/macrophages, NK, NKT and T cells (including regulatory T cells) infiltrate the liver and cause chronic inflammation, as a result of induction of pro-inflammatory cytokines, resulting in immunopathology. CTL responses are narrow and monospecific and not sufficient to clear infected hepatocytes. Such cells are deficient in proliferation and production of cytokines such as IFNg. Intrahepatic CD8 þ T cells express programmed cell death protein 1 (PD-1) which suggests that they may become exhausted in the presence of cognate antigen expression. Long term infection with HBV associated with active viral replication can lead to cirrhosis and HCC. HBV is responsible for 30% of cases with cirrhosis and almost 50% of cases with HCC. The annual risk of HCC in patients with cirrhosis has been reported to be 2%–5%. Recurrent liver necro-inflammation due to the immunopathological events described above, accelerate hepatocyte turnover and deposition of collagen initially but gradually leading to fibrosis, cirrhosis and eventually HCC. This is a long term process, multifactorial in nature and implicating both host and viral factors.These include high HBV DNA levels (42000 IU/ml), high transaminase levels, prolonged HBeAg positivity, genotypes B, D and F, precore and basal core promoter variants, male sex and older age. Co-infections with HIV and hepatitis D virus, comorbid conditions such as diabetes mellitus and obesity, abuse of alcohol, carcinogen exposure such as smoking and aflatoxin B1, are also contributing factors. Integration of the viral genome into hepatocyte DNA can lead to chromosomal instability and expression of functional preS/S and X proteins which are truncated at their extreme carboxyl end. PreS/S proteins with deletions and mutations in HBx have been associated with increased risk of HCC. HBx is thought to promote viral replication and is a known transactivator in vitro, it inhibits TNF-a, Fas-induced apoptosis, whilst it promotes fibrogenesis and remodeling of extracellular matrix. In addition, spliced HBV mRNAs have been implicated in tumorigenesis as have microRNAs (miRNAs) which are involved in regulating gene expression, cell cycle progression, differentiation and apoptosis. Finally, exosomal vesicles are involved in virion export, cell-to-cell spread of the virus, miRNA export etc., which may be additional contributors to viral pathogenesis.

Diagnosis Acute or chronic infection with HBV is based on serological detection of HBsAg in serum. Its persistence longer than 6 months indicates progression to chronic infection, whilst appearance of anti-HBs (antibody to HBsAg) indicates recovery from infection, or acquired immunity after prophylactic vaccination. Detection of HBeAg indicates active viral replication, as does the detection of serum HBV-DNA by qualitative or quantitative polymerase chain reaction (PCR) tests. The latter is particularly useful in monitoring efficacy of antiviral treatment. Seroconversion to anti-HBe occurs after recovery from acute infection, and less often during the chronic phase, either spontaneously or after therapeutic intervention. In the latter case, this leads to a quiescent phase of disease that can be long-term, but does not necessarily mean that virus replication has stopped entirely (see below). Antibody to core antigen (anti-HBc) of IgM class at high level is a marker of acute infection, whereas total anti-HBc (primarily IgG) is detectable following acute and throughout chronic infection.

Treatment Antiviral treatment is the only means available to prevent progression of chronic infection to cirrhosis and eventually HCC. The primary aim is the long term suppression of viral replication to undetectable levels. Moreover, in HBeAg positive patients the loss of this marker and preferably the development of the respective antibody (anti-HBe) is a favorable outcome as it denotes immune control of the virus. These responses are accompanied by normalization of biochemical markers. A secondary outcome which is achieved less often is the loss of HBsAg with or without development of anti-HBs. Decision to treat is based on a viral load 42000 IU/ml, an ALT level above the ULN and the presence of moderate necroinflammation and fibrosis. Patients with HBV DNA levels 420000 and ALT levels more than 2xULN should be treated without histological assessment. Two approaches are currently available for antiviral treatment of chronic HBV Infection; nucleos(t)ide analogs (NAs) and immunomodulators, such as Pegylated Interferon a (PegIFNa). NAs are lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (TBV), tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). Of these ETV, TDF and TAF are the drugs of choice as they have a very high barrier to HBV resistance as opposed to the other three which have a low resistance barrier. NAs are taken orally and act as chain terminators at the stage of DNA synthesis. They are chemically modified so that their incorporation into nucleic acids prevents chain elongation and therefore cause premature termination of synthesis. These lend themselves for long-term treatment which effectively leads to suppression of HBV DNA to undetectable levels very quickly which is maintained whilst the patient remains on treatment. They are quite safe and can be used in any HBV infected patient whether HBeAg-positive or -negative, but also various subgroups including those with decompensated cirrhosis, liver transplant patients, acutely infected or those with severe flares during chronic HBV. NAs can also be used in patients with occult HBV to prevent reactivation if they are due to be immunosuppressed. 5 years of ETV or TDF treatment in HBeAg-positive patients results in nearly 99% and 97% cumulative virologic response and near 53% and 49% HBeAg loss respectively. HBsAg loss was achieved in 10% and HBsAg seroconversion in 8%. For HBeAgnegative patients, treatment with ETV and TDF over the same period achieved virological and biochemical responses of 98% and 95% and 99% and 88%, respectively. Very few such patients (1%) achieved HBsAg loss during treatment exceeding 5 years duration. Data on TAF treatment is available for the first two years since its licensing with comparable results to TDF. In HBeAg-

384

Hepatitis B Virus (Hepadnaviridae)

positive patients, the rates of virologic response at week 96 was 75%, whilst HBeAg loss and anti-HBe seroconversion were achieved in 22% and 18%, respectively. Only 1% of patients cleared HBsAg. In HBeAg-negative patients, TAF achieved a virologic response in 90% of them by week 96. PegIFNa acts by promoting cytotoxic T-cell activity for lysis of infected hepatocytes and by stimulating cytokine production for control of viral replication. Weekly intramuscular injections in HBeAg-positive patients for 1 year can lead to seroconversion to anti-HBe in 32% of patients. This is sustainable in over 95% of patients. In HBeAg-negative patients the intention is to suppress HBV- DNA levels followed by ALT normalization a goal which is achievable in about 44% of them. However, durability is only possible in around 20%. To manage such relapses following initial interferon treatment, nucleos(t)ide therapy should be started and continued long term in both HBeAg positive and negative patients not achieving a sustained response on PegIFN. Rates of HBsAg loss following 12 months of treatment are 3%–7%. Overall, among sustained responders whether HBeAg-positive or -negative 30% will clear HBsAg in the long-term.

Resistance Treatment with NAs of low genetic barrier to resistance over time can lead to increasing resistance levels. In the case of LAM for example, this is about 24% at year 1 rising to 470% by year 5. Adefovir resistance on the other hand is delayed being 0% at year 1, 3% at year 2 and rising to 29% by year 4. Entecavir resistance has only been seen so far in patients with lamivudine resistant strains. Resistance is associated with the acquisition of amino-acid substitutions in the rt domain of the HBV polymerase which has 6 spatially separated subdomains from A-F and which contribute to nucleic acid synthesis. Most importantly, amino-acid substitutions in subdomain C which contains the characteristic YMDD (tyrosine-methionineaspartate-aspartate) motif of the catalytic site of the enzyme can cause resistance. Resistance to LAM relates to substitution of the methionine (M) at position 204 to either valine (YVDD, rtM204V) or isoleucine (YIDD, rtM204I). The rtM204V substitution mutation is almost always associated with a second one involving a substitution of leucine with methionine at position 180 (rtL180M) in subdomain B. ADV resistance is conferred by mutations rtN236T in subdomain D and rtA181V in subdomain B, whilst the small number of entecavir resistant cases have in addition to the lamivudine resistant substitutions additional ones such as rtI169T, rtT184G, rtS202I and rtM250V.

Prevention Protection against HBV infection is afforded by prophylactic vaccination which was first introduced in the early 1980s. Current vaccines consist of recombinantly expressed HBsAg in yeast such as Saccharomyces cerevisiae. In adults, the vaccine is administered through intramuscular injection in the deltoid in a three dose schedule at 0, 1 and 6 months. In countries of high and medium sero-prevalence, universal vaccination programs have been instituted, and HBV vaccination is recommended for infants born to carrier mothers preferably within the first 12 h of life, given together with hepatitis B immune globulin (HBIg). Measuring anti-HBs levels 1–4 months after the last dose of the vaccine ensures development of protective immunity defined as a level 410 mIU/ml. Response rates to the vaccine in healthy individuals range from 90% to 95%, whilst in haemodialysed and hemophiliac patients these are lower (70%). The durability of the response to the vaccine with persistence of anti-HBs levels 410 mIU/ml has extended beyond 12 years in up to 80% of individuals immunized at a young age. Booster immunizations therefore may not be necessary for at least 10 years after vaccination. However, high risk professions such as healthcare personnel and individuals in high risk groups need to monitor antibody levels perhaps every 5 years. The impact of HBV vaccination on infection prevalence has become apparent from epidemiological studies around the world and in countries of medium to high endemicity. They have recorded a dramatic drop in HBV prevalence. For example, in Taiwan, 15 years after the start of the vaccination program, the prevalence of HBsAg in children under 15 years of age had decreased from 9.8% in 1984 to 0.9% in 1999 and by 2014 to 0.3%. Similarly, the incidence of HCC has been on the decline from 0.7 per 10,000 children between 1981–1986, to 0.57 and 0.36 between 1986–1990 and 1990–1994, respectively. In spite of vaccination, breakthrough infections in vaccinated subjects have been detected through monitoring in spite of the presence of a satisfactory antibody levels. Such infections may be due to the wild type virus or mutant viruses, the commonest of which involves a G145R substitution in the “a” determinant region of HBsAg. This and a number of other mutations in this region, which can occur in combination also, alter the antigenic structure of this epitope, causing failure in its recognition by neutralizing antibody. Mutant viruses have also been described in the setting of liver transplantation where use of HBIg or monoclonal anti-HBs is recommended in an attempt to prevent infection of the new liver graft.

Further Reading Cornberg, M., Wong, V.W., Locarnini, S., et al., 2017. The role of quantitative hepatitis B surface antigen revisited. Journal of Hepatology 66, 398–411. Lampertico, P., Agarwal, K., Berg, T., et al., 2017. EASL 2017 clinical practice guidelines on the management of hepatitis B virus infection. Journal of Hepatology 67, 370–398.

Hepatitis B Virus (Hepadnaviridae)

385

Ganem, D., Prince, A.M., 2004. Hepatitis B virus infection – Natural history and clinical consequences. New England Journal of Medicine 350, 1118–1129. Ginzberg, D., Wong, R.J., Gish, R., 2018. Global HBV burden: Guesstimates and facts. Hepatology International 12, 315–329. Hadziyannis, S.J., Papatheodoridis, G.V., 2006. Hepatitis B e antigen-negative chronic hepatitis B: Natural history and treatment. Seminars in Liver Disease 26, 130–141. Jazayeri, S.M., Alavian, S.M., Dindoost, P., Thomas, H.C., Karayiannis, P., 2014. Molecular variants of hepatitis B surface antigen (HBsAg). In: Thomas, H.C., Lok, A.S.F., Locarnini, S.A., Zuckerman, A.J. (Eds.), Viral Hepatitis, fourth ed. London: Wiley Blackwell, pp. 107–126. Karayiannis, P., Carman, W.F., Thomas, H.C., 2014. Molecular variants of the precore, core and core promoter regions of hepatitis B virus, and their clinical significance. In: Thomas, H.C., Lok, A.S.F., Locarnini, S.A., Zuckerman, A.J. (Eds.), Viral Hepatitis, fourth ed. London: Wiley Blackwell, pp. 127–142. Karayiannis, P., 2017. Hepatitis B virus: Virology, molecular biology, life cycle and intrahepatic spread. Hepatology International 11, 500–508. O’Brien, T.R., Yang, H.I., Groover, S., Jeng, W.J., 2019. Genetic factors that affect spontaneous clearance of Hepatitis C or B virus, response to treatment, and disease progression. Gastroenterology 156, 400–417. Rehermann, B., Thimme, R., 2019. Insights from antiviral therapy into immune responses to Hepatitis B and C virus infection. Gastroenterology 156, 369–383.

Relevant Websites https://www.cdc.gov/hepatitis/hbv/index.htm Hepatitis B Information. www.hepb.org Hepatitis B Foundation. https://www.who.int/news-room/fact-sheets/detail/hepatitis-b Hepatitis B World Health Organization.

Hepatitis C Virus (Flaviviridae) Ralf Bartenschlager and Keisuke Tabata, Heidelberg University, Heidelberg, Germany r 2021 Elsevier Ltd. All rights reserved. This is an update of R. Bartenschlager, S. Bühler, Hepatitis C Virus, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00416-7.

Glossary Chronic hepatitis C A liver disease with continued presence of HCV RNA in the blood for six months or more after primary infection. DAAs Direct acting antivirals, i.e., drugs targeting distinct viral proteins and inhibiting their function, thus disrupting virus replication. HCV viremia Presence of HCV RNA in the blood of an infected individual. Internal ribosome entry site (IRES) A folded RNA element that allows for cap-independent initiation of RNA translation. The specific type of IRES determines which subset of the canonical translation initiation factors is required for RNA translation. Polyprotein A precursor polypeptide from which distinct proteins are produced by proteolytic cleavage.

Positive-strand RNA virus A virus with a single-strand RNA genome that has the polarity of a cellular mRNA and therefore, can be used by host cell ribosomes for RNA translation to produce viral proteins. For that reason, the positive-strand RNA genome is infectious, i.e., it is sufficient to produce virus progeny in a permissive host cell. Replicon A DNA or RNA molecule capable of selfreplication in a cell or an adequate in vitro system (e.g., cell lysate). Sustained virological response (SVR) Continuous absence of virus from the serum, starting at a given time point after cessation of antiviral therapy. For instance, in HCV infection SVR6 means absence of HCV RNA 6 months after termination of therapy.

Classification HCV has been classified as a member of the genus Hepacivirus and is grouped together with the genera Pestivirus, Pegivirus, and Flavivirus in the familiy Flaviviridae. Based on genomic heterogeneity, 8 genotypes having more than 30% nucleotide sequence divergence and more than 80 subtypes differing from each other by 10%–30% at the nucleotide sequence level have been defined. Subtypes are designated by lower case letters following the number of the genotype (e.g., genotype 1 subtype b ¼ 1b). Genotype 1 is the most prevalent worldwide (B45% of all HCV cases), followed by genotype 3 (B30% of all HCV cases). Genotypes 2, 4, and 6 are responsible for B23% of all HCV infections, whereas infections with genotypes 5 constitute B1% of all HCV cases worldwide. Currently, there is no information available about the global prevalence of genotype 7. Infections with genotype 1–3 are found in almost all countries, whereas HCV genotypes 4–7 are to a large extent restricted to distinct geographical regions like Africa and Middle East (genotype 4), South Africa (genotype 5), and Southeast Asia (genotype 6). Genotype 7 has been isolated in Canada and Belgium from patients possibly infected in Central Africa. A novel genotype 8 was recently identified in 4 patients that migrated from Punjab State, India to Canada. Individual genotypes have not been ascribed to particular disease manifestations except for a higher prevalence of hepatosteatosis for patients infected with genotype 3 viruses. However, in the case of interferon (IFN)-based therapy, the infecting genotype is an important predictor of therapy outcome, although with the implementation of IFN-free antiviral drugs, the genotype has become much less relevant (see below).

Virion Structure and Properties HCV particles are enveloped and spherical with a very pleomorphic structure. They are most likely composed of a single copy of the viral RNA genome that associates with multiple copies of the core protein (Fig. 1). It is unclear whether a regular nucleocapsid is formed or a more unstructured ribonucleoprotein complex. Surrounding the capsid is a lipid envelope, probably derived from the endoplasmic reticulum (ER). Into this envelope, multiple copies of a heterodimeric complex of the two envelopeglyco proteins E1 and E2 are embedded. A hallmark of HCV particles is their association with various apolipoproteins of the host cell, most notably apolipoprotein E (ApoE). This association occurs within the cell, perhaps during assembly or virion egress, but also outside of the cell with released virus particles binding to apolipoprotein-containing very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL). This association is mediated most likely by the viral envelope glycoproteins binding to various apolipoproteins. Because of this tight association, infectious HCV particles have an irregular structure, a very low density and therefore, they have been designated as lipoviro-particles (Fig. 1). Moreover, HCV particles have a unique lipid composition containing an unusually high amount of cholesterol esters and a surprisingly low amount of phosphatidylethanolamine, thus resembling the composition of (V)LDL, but being very different from the lipid composition of e.g., HIV-1 particles. Because of the

386

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00032-1

Hepatitis C Virus (Flaviviridae)

387

envelope

A

core protein

RNA genome E1E2 envelope glycoproteins ApoE

B

ApoB

C

Fig. 1 Morphology and composition of HCV particles. (A) Schematic presentation of the HCV particle structure and composition. (B) Electron micrograph of negatively stained HCV particles. Virions were produced in cell culture and captured using electron microscopy grids coated via protein A with anti-HCV antibodies. Scale bar: 20 nm. (C) Cryo-electron microscopy image of an HCV particle at 78,000  magnification. Virions are heterogeneous in size ranging from 45 to 86 nm in diameter, with a mean diameter of 68 nm. The illustration is adapted from Bartenschlager, R., Penin, F., Lohmann, V., André, P., 2011. Assembly of infectious hepatitis C virus particles. Trends in Microbiology 19, 95–103. Copyright 2010, Elsevier Ltd. Images are reproduced from Catanese, M.T., Uryu, K., Kopp, M., et al., 2013. Ultrastructural analysis of hepatitis C virus particles. Proceedings of the National Academy of Sciences of the United States of America 110, 9505–9510. with permission of the authors.

association of HCV particles with apolipoproteins and (V)LDL and HDL components, size, density and composition of the virions differ, depending on the culture conditions. HCV produced in the human hepatoma cell line Huh7, which supports robust virus replication, have a density of 1.1 g/mL whereas virions produced in more authentic cell systems such as Huh7 cells cultured in human serum or primary human hepatocytes can have a much lower density (close to 1.0 g/mL). The association with lipoprotein components reduces sensitivity of the virus to neutralization by antibodies and increases virion infectivity.

Genome Organization and Viral Proteins The genome of HCV is a single-stranded RNA molecule of positive polarity and has a length of B9600 nucleotides (Fig. 2(A)). It is flanked by two highly structured non-translated regions (NTRs). The about 340 nucleotides long 50 NTR contains an internal ribosome entry site (IRES) (Fig. 2(B)), mediating cap-independent RNA translation giving rise to an around 3.000 amino acids long polyprotein. The 30 NTR is composed of an about 40 nucleotides long variable region, a polypyrimidine tract (heterogeneous in length) and a 98 nucleotides long highly conserved 30 terminal X-tail (Fig. 2(B)). Both the 50 NTR and the 30 NTR contain cis-acting RNA elements (CREs) that are required for viral replication. Within the 3´ terminal part of the NS5B gene 3 additional CREs are localized (5BSL3.1 to 5BSL3.3) with 5BSL3.2 being absolutely required for viral RNA replication by forming a long-distance RNA-RNA interaction with the middle stem-loop in the X-tail. Likewise, 4 CREs have been identified in the core protein coding region with two of them (SL47 and SL87) contributing to HCV RNA translation and robust replication in cell culture. The importance of the structural integrity of SL87 for robust HCV replication was confirmed in vivo using mice xenografted with primary human hepatocytes and in chimpanzees. Flanked by the two NTRs is the coding region of the HCV polyprotein that is cleaved proteolytically into 10 products (Fig. 2(A)). The structural proteins core, E1 and E2 are located within the N-terminal part of the polyprotein preceding the p7 protein, which appears to be an ion channel and therefore, was grouped into the viroporin protein family. The non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are encoded in the remaining part of the polyprotein. The NS2 protein, together with the N-terminal protease domain of NS3, is responsible for the autocatalytic cleavage of the NS2-NS3 junction (Fig. 2(A)). NS2 is a dimeric cysteine

388

Hepatitis C Virus (Flaviviridae)

Fig. 2 HCV genome organization, gene products and cis-acting RNA elements (CREs). (A) The HCV polyprotein coding region is flanked by two non-translated regions (50 and 30 NTR) represented in dark gray. The 50 NTR contains an internal ribosome entry site (IRES). Polyprotein cleavage by cellular signal peptidases is indicated by scissors, whereas cleavage by the viral NS3 serine protease is indicated by solid arrows. The cleavage at the NS2-NS3 junction by the NS2 cysteine protease is indicated by a dotted arrow. The star indicates the cleavage site for the cellular signal peptide peptidase removing the carboxy-terminal region of the core protein. Functions of the individual cleavage products are specified in the figure. (B) Schematic representation of CREs. The 50 NTR (left panel) contains the IRES as well as structures important for viral RNA replication. The middle left panel depicts the four CREs in the core coding region. Three CREs are located in the 30 -terminal part of the NS5B coding region (middle right panel). Stem-loop 5BSL3.2 forms a long-distance RNA-RNA interaction essential for RNA replication with the loop of the middle stem-loop in the X-tail. The right panel displays the organization of the 30 NTR with the variable region (v.r.), the poly U/UC-tract and the X-tail. Positions of start and stop codons are indicated with dots.

protease with a composite active site. The N-terminal NS3 domain carries a serine-type protease that after association with its cofactor NS4A, cleaves the four junctions between NS3 and NS5B. Two additional enzymatic activities (RNA helicase and nucleoside triphosphatase) reside in the carboxy-terminal two thirds of NS3. Structural integrity of intracellular membranes is altered by NS4B that has a complex and dynamic membrane topology. NS4B cooperates with NS5A, which is a zinc-binding phosphoprotein, composed of the following domains (from the N- to the C-terminus): an amphipathic a-helix serving as a peripheral membrane anchor, the structured domain I, low complexity sequence I, domain II, low complexity sequence II and domain III. Interestingly, domains II and III are intrinsically unfolded, but they also contain segments of transient secondary structure. The X-ray crystal structure of domain I was resolved and found to form various types of homodimers with one of them forming a basic RNA binding groove. NS5A is phosphorylated by several cellular kinases including the casein kinase I isoform alpha (CK1a), CKII, polo-like kinase 1 (PLK1) and calmodulin-dependent kinase II gamma (CaMKIIg) and CaMKIIδ. The phosphorylation state of NS5A affects its interaction with several host cell proteins such as VAP-A/B, arguing that distinct phosphorylation patterns confer different functions to NS5A, depending on an associating partner molecule. In this respect, NS5A exerts multiple functions; it is essential for viral RNA replication and virion assembly, but it is also involved in counteracting antiviral defenses such as the IFN response. The exact mechanism by which NS5A contributes to RNA replication has not been revealed, but it was found that the sole expression of NS5A can induce the formation of double membrane vesicles (DMVs), which are the presumed sites of HCV RNA replication (Fig. 3, inset). Moreover, drugs targeting NS5A block DMV formation, suggesting that the induction of the HCV replication organelle, designated “membranous web”, is one way by which NS5A contributes to HCV RNA replication. In addition, NS5A interacts with the core protein, for which phosphorylation of the carboxy-terminal region of NS5A is required. Thus, phosphorylation of NS5A at this site appears to trigger the assembly of infectious HCV particles, a process that may be supported by the interaction between RNA-loaded NS5A and the core protein. The 68-kDa protein NS5B is an RNA-dependent RNA polymerase (RdRp). It is a tail-anchored membrane protein with a structural organization similar to that of other polymerases, i.e., composed of palm, finger and thumb subdomains. However,

Hepatitis C Virus (Flaviviridae)

389

Fig. 3 HCV replication cycle. (Attachment) HCV binds to one or several receptors on the surface of the host cell. HSPG, SRB1, CD81 and presumably LDLR are required for or contribute to virus binding and entry. (Internalization) The virus particle enters the cell via clathrin-mediated endocytosis likely at tight junctions where Claudin-1 (CLDN1) and occludin (OCLN) reside. (Fusion) After a low-pH mediated fusion step from within an acidic endosome and uncoating, the HCV genome is released into the cytoplasm of the host cell. (RNA translation and replication) The viral RNA genome (red line) is translated at the rER and a membranous web, presumably originating from ER membranes in close proximity to lipid droplets (LD), is formed (DMV formation). DMV is the site of viral RNA amplification, which occurs via negative-strand RNA intermediates (blue line). Newly synthesized positive-strand RNA is used either for translation, or for replication, or the RNA is packaged into nascent capsids (Assembly). This most likely occurs at the ER where E proteins are retained with virus particles forming via budding into the ER lumen. (Maturation) During or after envelopment virus particles acquire ApoE, which is thought to be required for virion infectivity (indicated with the question mark). The association of HCV particles with ApoE and other apolipoproteins, occurs intra- and extracellularly. (Secretion) Virions are release after fusion of the transport vesicle with the plasma membrane. A three-dimensional reconstruction of the membranous web in HCVinfected cells is shown on the bottom left. The inner DMV membrane is indicated in light brown whereas ER is depicted in dark brown. Singlemembrane vesicles, cytoskeletal filaments and Golgi cisternae are shown in violet, blue and green, respectively. Scale bar: 100 nm. The illustration is adapted from Neufeldt, C.J., Cortese, M., Acosta, E.G., Bartenschlager, R., 2018. Rewiring cellular networks by members of the Flaviviridae family. Nature Reviews Microbiology 16, 125–142. The inset image is reproduced from Romero-Brey, I., Merz, A., Chiramel, A., et al., 2012. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLOS Pathogens 8, e1003056.

NS5B differs from most other RdRps by having a fully encircled active site, which is due to tight interactions between the finger and the thumb subdomain. Apart from the polyprotein a heterogeneous group of HCV proteins are expressed either by ribosomal frameshifting into the þ 1 ORF or by internal translation initiation. The resulting proteins, collectively designated core þ 1 (Fig. 2(A)), are not essential for replication and virus production in cell culture and they appear to be dispensable in vivo.

Life Cycle Hepatocytes are the primary target cells for HCV. However, viral RNA was also detected in PBMCs and bone marrow cells, but it is unclear whether a productive infection is possible in these cell types. In cell culture, HCV RNA replication was demonstrated in non-liver

390

Hepatitis C Virus (Flaviviridae)

cells like human T and B cell lines, human embryonic kidney cells (HEK293) and even neuroepithelioma-derived cell lines arguing that some of the extrahepatic manifestations observed with chronic hepatitis C patients, such as cryoglobulinemia or central nervous system abnormalities, might be caused by infection of these cell types. Furthermore, certain mouse cell lines can support the replication of engineered HCV replicons demonstrating that the viral replication machinery is also functional in a murine host cell environment. In hepatocytes, HCV infection starts with virion binding to host cell attachment factors such as heparan sulfate proteoglycans (HSPG) and low-density lipoprotein receptors (LDLR) located at the basolateral membrane (Fig. 3). Subsequently virus particles interact with the scavenger receptor class B member 1 (SRB1) and the tetraspanin CD81, which guide the lateral translocation of virion - receptor complexes towards tight junctions upon activation of the epidermal growth factor receptor (EGFR) and the EGFR/Shc/Grb2/HRas signaling pathway. Following these events, CD81 associates with claudin-1 (CLDN1) and occludin (OCLN) to trigger virus internalization through the clathrin-dependent pathway and a subsequent fusion step from within an acidic endosomal compartment (Fig. 3). Upon release of the RNA genome into the cytoplasm, the polyprotein is synthesized by IRES-dependent RNA translation occurring at the rough ER (rER) where host cell signal peptidases, signal peptide peptidases and viral proteases catalyze polyprotein cleavage. During or after polyprotein cleavage an ER-derived membranous web is formed where the positive-strand RNA genome is amplified via a negative-strand RNA intermediate (Fig. 3). Newly synthesized positive-strand RNAs are used for the synthesis of multiple polyprotein copies (probably 41000 protein copies per RNA molecule), or they serve as templates for further RNA synthesis, or they are incorporated into assembling virions. Viral envelope (E1-E2) glycoproteins are retained at rER membranes suggesting that viral envelopes are generated by budding into the lumen of this organelle. The envelopment of virus particles depends on components of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery. The ESCRT-0 complex component HRS (hepatocyte growth factor-regulated tyrosine kinase substrate) interacts with HCV NS5A and NS2 proteins, at least in case of the latter via recognition of polyubiquitinated residues. Other ESCRT proteins may interact with HRS to coordinate HCV assembly, as the depletion of the components of ESCRT-III or VPS4-VTA1 complexes causes assembly defects. Given the strong functional relationship between HCV and lipoproteins, it was speculated that HCV particles exit the cell via the canonical secretory pathway, comparable to VLDL. However, recent evidence suggests that HCV may also use an alternative secretion route bypassing the Golgi apparatus. In either case, virus particles are released after fusion of the transport vesicle with the plasma membrane as cell-free virions, or they are transmitted directly to neighboring cells via a cell-to-cell spread mechanism.

Formation of the HCV Replication Organelle The most abundant components of the membranous web are double membrane vesicles (DMVs) that have an average diameter of about 200 nm (Fig. 3, inset). Although the sole expression of NS5A suffices to induce DMVs, the efficiency of DMV formation is greatly increased upon the expression of an NS3–5B polyprotein. Morphologically, DMVs correspond to protrusions from the ER membrane with a fraction of DMVs being connected via the outer DMV membrane to the ER membrane. DMVs isolated from HCV-replicating cells contain active viral replicase arguing that DMVs are the site where HCV amplifies its genome. However, only a minority of DMVs has an opening connecting the DMV lumen with the cytoplasm, whereas the majority of DMVs appears to be completely “sealed”. Thus, viral RNA produced inside DMVs can either be released into the cytoplasm as long as the DMVs are not completely sealed or DMVs contain specific “pores” spanning both membranes and allowing RNA export out of the DMV interior. Interestingly, similar DMV-type viral replication organelles have been described for other positive-strand RNA viruses, including members of the distantly related Coronaviridae, Arteriviridae, and Picornaviridae families. At least in the case of HCV, the formation of DMVs may be linked to autophagy as deduced from the impaired RNA replication and reduced DMV number upon depletion of distinct factors of the autophagy machinery. Given the structural similarity of autophagosomes and DMVs, a link to autophagy appears plausible. However, in the case of poliovirus, it was shown that DMVs form via “secondary” invagination of single membrane vesicles, which appear to be the primary membrane structures induced by poliovirus early after infection. In contrast, single membrane vesicles have been observed only rarely in HCV-infected Huh7 cells, but they have been reported as sole structures in hepatocytes of HCV-infected individuals. Thus, it remains to be determined whether DMVs are bona fide HCV replication sites formed only in hepatoma cell lines or whether they are also found in vivo. Although HCV-induced DMVs are derived from the ER membrane, their lipid composition differs substantially from the originating membrane by having much higher levels of cholesterol and sphingolipids. This change in lipid composition is achieved by the NS5Amediated recruitment and activation of the lipid kinase phosphatidylinositol 4-kinase IIIa (PI4KA) that is enriched at (NS5A-containing) membranes to produce excessive amounts of PI4P. In addition, NS5A binds to and recruits, probably via VAP-A/B, lipid-transfer proteins such as oxysterol-binding protein that may deliver cholesterol into the DMV membrane in exchange for PI4P. Probably in a similar way, the glucosylceramide transfer protein FAPP2 may be recruited to DMVs to release lipids in exchange for PI4P into the DMV membrane. In this way cholesterol and sphingolipids would be enriched leading to the formation of lipid rafts likely required for HCV replicase activity and possibly the assembly of (lipidated) HCV particles.

Host Cell Factors Contributing to Various Steps of the HCV Replication Cycle During the last few years, numerous host cell factors affecting the HCV replication cycle have been identified, but only a few can be mentioned here. The first one is microRNA-122 (miR122). The best-known role of miRNAs, which are short single-strand, non-coding

Hepatitis C Virus (Flaviviridae)

391

RNA molecules (20–24 nucleotides long) is the downregulation of protein expression by translational repression of the target mRNA or promotion of its cleavage. The HCV genome contains several conserved binding sites for miR-122, the most abundant miRNA in the liver. However, in contrast to its canonical function in the case of HCV, binding of miR-122 to two sites in the 50 UTR increases viral RNA translation and replication primarily via two different mechanisms. First, miR-122 forms an extensive Argonaute-2containing oligomeric complex at the 50 end of the HCV genome and protects this RNA from nucleolytic degradation by the cellular 50 -to-30 exoribonuclease 1 (Xrn1) and Xrn2. Second, binding of miR-122 suppresses the formation of energetically favorable structures of the 50 UTR interfering with IRES activity, thus promoting RNA translation, which in turn interferes with viral RNA replication. Another host factor essential for HCV replication is the prolyl-peptidyl cis-trans isomerase cyclophilin A (CypA). Binding of CypA to the HCV NS5A protein appears to catalyze the isomerization of peptidyl-prolyl bonds in NS5A domain I and domain II, resulting in conformational changes that stimulate NS5A RNA binding and enhance viral replication. CypA activity also seems to be required for the formation of an HCV replication organelle, as deduced from the observation that CypA antagonists block de novo formation of the membranous web. A third important factor is SEC14L2, a cytosolic lipid-binding protein family member, which emerged as an important HCV host dependency factor. It was shown that ectopic expression of SEC14L2 enhances the replication of patient-derived HCV isolates in diverse human hepatoma cell lines that do not express this factor. This enhancement might be linked to the capacity of SEC14L2 to increase the accumulation of intracellular vitamin E protecting HCV from the detrimental effects of lipid peroxidation. As mentioned above, apolipoproteins, such as apolipoproteins A-I, B, C-I, and E are components of HCV virions. Of these, ApoE fulfills a dual function in the viral life cycle. First, ApoE interacts with HSPG, LDLR and SRB1 and facilitates virus entry; second, it interacts with NS5A and the envelope glycoproteins and contributes to infectious virus particle production. An association of ApoE with E1-E2 seems to be required for lipidation and maturation of HCV particles at a post-envelopment step (Fig. 3). However, this association can also happen extracellularly with secreted ApoE that is part of lipoprotein particles. Thus, it is possible that ApoE incorporation starts intracellularly during assembly and continues extracellularly after virus particle release.

Host Cell Lipids of Relevance to the HCV Replication Cycle To accommodate the net increase of intracellular membrane surface and the lipidation of HCV particles, a de novo synthesis of membrane lipids is induced in HCV-infected cells, along with an inhibition of lipid secretion. Indeed, lipidomic profiling of HCVinfected cells revealed distinct alterations of the lipid composition of host cells, including elevated levels of phosphatidylcholines and polyunsaturated fatty acids. At least two lipid biosynthesis pathways are activated by HCV: First, the transcriptional induction of lipid biosynthesis–related genes via the sterol regulatory element–binding protein (SREBP) pathway. In this case, HCV infection triggers trafficking of the ER-localized inactive SREBP precursor to the Golgi where it is proteolytically cleaved by site 1 protease (S1P) and S2P. The released N-terminal fragment is transported into the nucleus and it activates the transcription of lipogenic factors such as the fatty acid synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA), which is a rate-limiting enzyme of the cholesterol biosynthetic mevalonate pathway. Second, the 30 UTR of the HCV RNA genome binds to the cytosolic RNA helicase DEAD box polypeptide 3 X-linked (DDX3X), activating a signaling cascade that promotes SREBP-mediated transcription. In addition, HCV may mobilize fatty acids from lipid droplets. The strong dependency of HCV on lipids may explain the antiviral activity of inhibitors of distinct lipid biosynthesis pathways. For instance, treatment of cells containing replicating HCV RNA with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I induced the disintegration of the HCV replication organelle. Moreover, fatty acids can inhibit or stimulate HCV replication, depending on their degree of saturation. While saturated and monounsaturated fatty acids stimulate viral replication, polyunsaturated fatty acids impair replication but enhance the production of infectious virions.

Activation and Inhibition of Innate and Adaptive Immunity by HCV Both in cell culture, and in the majority of patients, treatment with IFN-a leads to a rapid and efficient block of HCV replication. This result is somewhat surprising given the high rate of persistence of HCV infections (50%–80%) and the finding that in the infected liver IFN-induced genes (ISGs) are activated. In several studies, it was concluded that HCV proteins such as the core or NS3 proteins interfere with various steps of IFN-a/b induced signaling and that some HCV proteins block individual IFN-a/b induced effectors. However, in primary human hepatocytes infected with HCV, a robust IFN response is induced and the virus is rapidly cleared. Thus, it is still uncertain whether HCV can block the IFN-induced antiviral defense. It also remains to be clarified by which mechanism IFNs block HCV replication. Much better established is the mechanism by which HCV aggravates the induction of innate antiviral defense. Several studies have shown that the NS3 protease proteolytically cleaves mitochondrial antiviral-signaling molecule (MAVS) relaying the activation of retinoic-acid-inducible gene 1 (RIG-I) to IRF-3 phosphorylation. As a result, IRF-3 dependent genes are not expressed and cells remain sensitive to virus infection. Indeed, it was shown that HCV controls ISG activation in infected cells, including the production of IFN-l, which is the predominant IFN type in hepatocytes. In contrast, in cells expressing a MAVS variant that cannot be cleaved by HCV ISG expression and IFN-l secretion are not affected, concomitant with reduced HCV replication. Although these

392

Hepatitis C Virus (Flaviviridae)

results clearly show the control of the MAVS signaling pathway by HCV, in infected cells the virus induces a robust IFN response. This may be due to activation of the Toll-like receptor 3 (TLR3) pathway that is less efficiently controlled by HCV. It was found that double-strand HCV RNA, a replication intermediate, is sensed by TLR3 in HCV-infected cells, but released via exosomes from infected cells. Inhibition of exosome release leads to intracellular accumulation of double-strand RNA, enhancing TLR3 activation and thus, induction of IFN gene expression. Apart from that HCV RNA-containing exosomes appear to be sensed by dendritic cells, contributing to the activation of IFN production. In addition to the innate cytokine response, a rigorous and multi-specific T cell response is critical for the control and elimination of HCV. Thus, a successful antiviral defense generally encompasses multiple MHC class-I and class-II restricted T cell epitopes and an efficient expansion of CD8 þ and CD4 þ T cells. In contrast, persistent infections are characterized by oligoclonal T cell responses, a low frequency of HCV-specific T cells and the induction of poorly neutralizing antibodies. The underlying reasons for the weak response in the majority of patients are not clear, but several possibilities are discussed. These include an impaired antigen presentation e.g., due to interference of HCV with dendritic cell function; a CD4 þ T cell failure due to deletion or anergy; a mutational escape in important T and B cell epitopes; a functional impairment of HCV-specific CD8 þ T cells. The mechanisms underlying T cell attenuation are unclear, but one attractive possibility is that the defect induced in innate immunity may result in a defect in CD4 þ T cell help. In fact, HCV-induced loss of T cell help appears to be the key event of immune evasion. In any case, CD8 T cells in chronic hepatitis C patients are exhausted, but this defect is partially restored in patients that have eliminated the virus in the course of antiviral therapy. This observation clearly shows that continuous HCV replication is required to suppress HCV-specific adaptive antiviral response, although an immunological “scar” remains upon virus elimination. The role of HCV-specific antibodies in controlling viral infection is much less clear. Antibodies are probably less important for viral clearance and in most cases they do not protect from reinfection, neither in experimentally infected chimpanzees nor in humans, even after multiple exposure to HCV. However, there is increasing evidence that the presence of HCV-specific antibodies at least partially attenuates infection. For instance, antibodies neutralizing HCV particles of different genotypes have been detected in sera of chronic hepatitis C patients but the frequency of these antibodies is low. Nevertheless, in-depth studies of selected individuals who spontaneously cleared HCV infection have led to the identification of broadly neutralizing antibodies (bNAbs). In combination with the recently solved crystal structure of the E2 ectodomain, important contact sites between bNAbs and regions in E2, which are of relevance for neutralization across genotypes and subtypes, can now be identified, which should pave the way for the development of a prophylactic HCV vaccine.

Epidemiology HCV is mainly transmitted by parenteral exposure to blood, blood products and objects contaminated with blood. The development of effective screening tests for blood and blood products and the implementation of viral disinfection procedures have almost excluded this route of transmission in countries where these measures are in place. Thus, the major remaining risk factor for acquiring HCV infection in developed countries is the use of contaminated needles in injection drug use and certain type of risk behavior. In some countries (e.g., Egypt) HCV infection has spread primarily by the use of inadequately sterilized medical instruments. In contrast to HBV infection, sexual transmission, and vertical transmission of HCV are much less frequent. Modeling of HCV prevalence indicates that 71 million people (62.5–79.4) in 2015 were living with chronic HCV infection, accounting for approximately 1% of the world population. Prevalence rates vary significantly between and within countries. China, Pakistan, India, Egypt, Russia, and the USA have the highest number of cases, corresponding to around 50% of global HCV disease burden. However, only 20% of HCV-infected individuals have been diagnosed. Although the total number of HCV infections is declining since 2007, there are regions in the world where the prevalence is increasing. The reasons for this increase include unsafe health-care-related injections (e.g., Azerbaijan, India, Iraq, Syria, Uzbekistan), i.v. drug use (e.g., Iran, Russia, Latvia, and the USA), or immigration from endemic countries (e.g., Qatar, United Arab Emirates). Of note, in 2015 the number of people dying from HCV-related liver cirrhosis and hepatocellular carcinoma (B400,000) and being cured (B850,000) was exceeded by the number of newly infected persons (B1.75 million). Therefore, the HCV pandemic is continuing unless efficient interventions are scaled-up and implemented globally.

Clinical Features Acute HCV infections are usually asymptomatic, or in about 30% of cases associated with non-specific symptoms such as abdominal pain, fatigue, weakness, poor appetite, nausea and icterus (Fig. 4). During an incubation period of 15–75 days, HCV RNA becomes detectable in serum by RT-PCR and virus titers usually peak to 105–107 genome equivalents/mL between week 6 and 10, irrespective of disease outcome (Fig. 4(A)). Two to four weeks after onset of viremia, serum ALT (alanine aminotransferase) levels begin to rise indicative of hepatocellular injury. Due to these uncharacteristic symptoms, acute HCV infection remains often unrecognized and therefore, undiagnosed. Only 20%–50% of infected individuals clear the infection, with those having a clinically overt acute infection having a higher chance to clear HCV, arguing that pathogenesis is driven mainly by the immune response.

Hepatitis C Virus (Flaviviridae)

393

Fig. 4 Course of acute and chronic HCV infection. (A) In acute HCV infection viral RNA is typically detectable between week 2 and 10 after virus exposure. ALT levels rise between week 4 and 10, peaking around week 6. By this time, the first symptoms manifest. HCV-specific antibodies arise late in infection. In self-limiting infection, HCV is cleared and anti-HCV antibodies may wane within years after resolution. (B) In chronic hepatitis C infection the early phase is similar to acute infection but later on, the virus persists. In the chronic phase, ALT levels fluctuate as does viremia. Antibodies against different HCV proteins are constitutively detectable.

While the duration of viremia in acute hepatitis C is unpredictable and can vary between 2 to 4–6 months, some patients even become HCV RNA negative during early convalescence, but later on viremia rebounds. Overall 50%–80% of HCV infections lead to a chronic carrier state (Fig. 4(B)). About 30% of these chronically infected persons progress to liver cirrhosis within 10–30 years after primary infection and hepatocellular carcinoma (HCC) occurs in up to 2.5% of these patients. In contrast to chronic hepatitis B, in persistent HCV infection HCC develops only to those patients, who have developed liver cirrhosis.

Pathogenesis HCV infection is most likely not cytolytic. Instead, hepatocyte destruction is mediated by the immune response, arguing that HCV, like HBV infection, causes an immune pathology. Since hepatocytes are the primary target of HCV infection, the histological alterations of chronic hepatitis C are hepatocellular injury, portal and parenchymal inflammation and necrosis. Liver damage is typically spotty and focal with accompanying chronic inflammatory cells, including macrophages, and the disease eventually develops to a variable degree of fibrosis. The progression rate to hepatic fibrosis is the major determinant for the outcome of chronic hepatitis C in terms of developing cirrhosis and HCC. Although the liver is the primary target, persistent HCV infection is often associated with extrahepatic symptoms like renal complications, lymphoma and diabetes. A high proportion of patients with chronic hepatitis C develop cryoglobulinemia, which may account for some of these extrahepatic manifestations. The mechanisms underlying HCV-associated tumorigenesis are poorly understood, but they are thought to arise from a combination of indirect and direct mechanisms. The most important indirect effect is a chronic inflammation, sustained at least in part by the antiviral immune response that attacks and destroys hepatocytes, yet this response is unable to eradicate the virus and eliminate the infection. Amongst the more direct effects are: (1) a possible reduction of the tumor suppressor miR-122 in infected cells due to binding to viral RNA and sequestration of miR-122 from normal cellular target RNAs; (2) a possible oncogenic effect of HCV core protein that has been observed in certain transgenic mouse models; (3) an elevated expression of EGFR, induced by HCV infection and playing an important role in HCC development; (4) the binding of NS5B to the tumor suppressor Rb, resulting in Rb degradation via the ubiquitin-proteasome pathway simultaneously with stimulation of hepatocellular proliferation. (5) persistant changes of the epigenome of infected cells.

Diagnosis Routine screening tests for detecting HCV infections are based on serological assays measuring HCV specific antibodies (most often by ELISA or similar immunoassay) and nucleic acid-based tests (NAT) such as RT-PCR to determine viral RNA. Current serological assays have a specificity of 499% and they are positive in 99% or more of immunocompetent patients where viral RNA is detectable. Once a patient is tested positive in a serological assay, the sample should be tested by RT-PCR. A seropositive person with a positive PCR test result may have an acute, or, much more likely, a chronic HCV infection. A seropositive person with a negative PCR test result might have had an early or eliminated infection, a false negative test result, or a false positive serologic test result, or a chronic HCV infection with a very low viremia at the time of blood sampling. Thus, repeated testing is required in those cases.

394

Hepatitis C Virus (Flaviviridae)

Given the higher sensitivity, the diagnostic window can be reduced by using NAT. Quantitative RNA detection assays have been implemented in many blood banks e.g., in the EU and the US. The risk to acquire a transfusion-associated hepatitis C in such countries has been reduced to less than 1 in a million blood donations. The determination of HCV genotypes, which is an important parameter for IFN-based antiviral therapy, is based on analyzing viral RNA either by hybridization or direct sequence analysis or by using genotype-specific primers for RT-PCR. In addition to NAT, other (direct) detection methods of HCV have been developed, including the quantitative detection of HCV core protein. However, these assays are not widely available.

Treatment For many years, antiviral therapy of chronic hepatitis C was based on the combination of a polyethylene glycol (PEG) conjugated form of IFN-a with ribavirin resulting in an overall sustained viral response (SVR) rate of B60% in eligible patients. However, the success rate depended very much on two important parameters. The first one was the genotype of the infecting virus. While up to 85% of genotype 2- and 3-infected patients developed SVR, only around 45% of patients infected with genotype 1 viruses did so. In addition, this therapy had numerous serious side effects, including neuropsychiatric symptoms and hemolytic anemia, and therefore, many patients were either not eligible for this treatment or had to discontinue the therapy. The second parameter determining the success of IFN therapy is 3 distinct SNPs near the IFN-l3 and -l4 genes. While the causality between HCV infection and these SNPs remains to be determined, they appear to be of predictive value for other diseases including nonalcoholic fatty liver disease or infections with cytomegalovirus or HIV. The limitations of IFN-based therapy of chronic hepatitis C have been overcome by the implementation of all-oral IFN-free direct acting antiviral drugs (DAAs) in 2014. These drugs allow virus elimination in 495% of individuals within a treatment period of 8–12 weeks; they are well tolerated and can be given to patients even with a decompensated liver cirrhosis. DAAs are targeting three viral proteins (Fig. 5): the NS3 serine protease, the NS5A replicase and assembly factor, and the NS5B RdRp. Numerous drugs against these three targets have been developed and although the drug names are complicated, the drug target is disclosed in the name. Protease inhibitors are ending with “-previr”, NS5A inhibitors with “-asvir”, and NS5B inhibitors with “-buvir”. While most of the first generation DAAs had a rather low resistance barrier, this limitation has been overcome with 3rd generation DAAs that are also rather pan-genotypic. Therefore, the first-line treatment of hepatitis C patients is based on a combination of at least two DAAs, e.g., a protease and a NS5A inhibitor or in addition a NS5B inhibitor (Fig. 5). Although IFN-free DAA treatment has been in use for just a few years, it is becoming clear that the incidence of end-stage liver disease caused by HCV infection is declining with patients that mount a SVR. However, it is also becoming clear that in patients with advanced cirrhosis, HCV elimination is of limited use to prevent HCC because these patients still have a high risk for liver tumor development. While this observation argues for a “point-of-no-return” beyond which the presence of HCV is no longer relevant for HCC development, at the same time it has become clear that antiviral therapy should be given as early as possible to HCV infected individuals and not be limited to people with advanced liver disease. Fortunately, the price of the DAA therapy is reducing enabling the treatment of a larger proportion of HCV infected individuals.

Prevention With the advent of highly efficacious DAAs global eradication of HCV has been considered as a reachable goal. While this is theoretically possible, major limitations need to be overcome. These include the limited availability of DAAs in many countries, the still high costs for these drugs, the insufficient health care systems in many high-endemicity countries and the fact that only a small proportion of HCV infections have been diagnosed. Moreover, reinfections with HCV are common, especially in high-risk populations such as intravenous drug users, and the number of new infections is rising, e.g., as a result of the opioid crisis in the USA (see above). For these reasons, global eradication strategies must not ignore the development of a prophylactic HCV vaccine. Although scientifically challenging, effective immunization against HCV appears to be feasible. Both in experimentally infected chimpanzees and in humans a rigorous and multi-specific T cell response can be induced and some individuals develop cross-neutralizing antibodies. What we need is a better understanding of the correlates of immune protection and how to induce a protective immune response with an ideal vaccine formulation. Thus far, only two vaccine candidates have been pursued in clinical trials. The first one is a recombinant envelope glycoprotein prophylactic vaccine that induced sterilizing immunity against experimental challenge of chimpanzees with a homologous HCV strain and reduced chronicity upon challenge with a heterologous strain of the same genotype. The second vaccine candidate is based on an adenovirus vector encoding an HCV NS3–5B polyprotein fragment used to prime the immune response, followed by a boost with a modified vaccinia Ankara (MVA) vector encoding the entire genome of the same HCV type. This combination, aiming to prime and boost CD4 and CD8 T cell responses, was found to be save and to induce polyfunctional, broad HCV-specific memory CD4 and CD8 T cells. However, in a recent phase 2 clinical trial this vaccine failed to reach clinical significance in reducing the incidence of chronic HCV infection compared to placebo among HCV-uninfected intravenous drug users. Therefore, further efforts are required to identify vaccine candidates inducing a protective immune response. Without such a vaccine, achieving global eradication of chronic HCV infection is questionable.

Hepatitis C Virus (Flaviviridae)

395

Fig. 5 Drug targets and direct acting antiviral drugs (DAAs) for treatment of HCV infection. Ribbon diagrams of the 3D structures of the NS3/4A protease complex, the homodimer of NS5A domain I and the NS5B RdRp domain are displayed on the top. Shown are the NS3 protease domain (light blue) complexed with the NS4A peptide (orange) and Boceprevir (red); the NS5A domain I dimer with monomers represented in red and green; the NS5B RdRp domain complexed with UTP (yellow). DAAs targeting each of these proteins and clinically used drug combination therapies are given below each viral protein. Protein structures are derived from Protein data bank (PDB) accession 2OC8 for NS3/4A, PDB accession 1ZH1 for NS5A and PDB accession 1GX6 for NS5B.

396

Hepatitis C Virus (Flaviviridae)

Acknowledgments The authors gratefully acknowledge Dr. Eliana G. Acosta (University of Heidelberg) for excellent assistance with the preparation of the manuscript and the figures.

Further Reading Bartenschlager, R., Lohmann, V., Penin, F., 2013. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nature Reviews Microbiology 11, 482–496. Bartenschlager, R., Penin, F., Lohmann, V., André, P., 2011. Assembly of infectious hepatitis C virus particles. Trends in Microbiology 19, 95–103. Catanese, M.T., Uryu, K., Kopp, M., et al., 2013. Ultrastructural analysis of hepatitis C virus particles. Proceedings of the National Academy of Sciences of the United States of America 110, 9505–9510. Choo, Q.L., Kuo, G., Weiner, A.J., et al., 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362. Lohmann, V., Körner, F., Koch, J., et al., 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatome cell line. Science 285, 110–113. Manns, M.P., Buti, M., Gane, E., et al., 2017. Hepatitis C virus infection. Nature Reviews Disease Primers 3, 17006. Neufeldt, C.J., Cortese, M., Acosta, E.G., Bartenschlager, R., 2018. Rewiring cellular networks by members of the Flaviviridae family. Nature Reviews Microbiology 16, 125–142. Ortega-Prieto, A.M., Dorner, M., 2017. Immune evasion strategies during chronic hepatitis B and C virus infection. Vaccines 5, E24.(Basel). Paul, D., Bartenschlager, R., 2015. Flaviviridae replication organelles: Oh, what a tangled web we weave. Annual Review of Virology 2, 289–310. Polaris Observatory HCV Collaborators, 2017. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: A modelling study. Lancet Gastroenterology & Hepatology 2, 161–176. Romero-Brey, I., Merz, A., Chiramel, A., et al., 2012. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLOS Pathogens 8, e1003056. Ross-Thriepland, D., Harris, M., 2015. Hepatitis C virus NS5A: Enigmatic but still promiscuous 10 years on!. Journal of General Virology 96, 727–738. Smith, D.B., Bukh, J., Kuiken, C., et al., 2014. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource. Hepatology 59, 318–327. Tu, T., Bühler, S., Bartenschlager, R., 2017. Chronic viral hepatitis and its association with liver cancer. Biological Chemistry 26, 817–837. Wrensch, F., Crouchet, E., Ligat, G., et al., 2018. Hepatitis C virus (HCV)-apolipoprotein interactions and immune evasion and their impact on HCV vaccine design. Frontiers in Immunology 21, 1436.

Relevant Websites https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi/56/hcv-classification HCV Classification. International Committee on Taxonomy of Viruses (ICTV). https://www.who.int/news-room/fact-sheets/detail/hepatitis-c Hepatitis C. World Health Organisation (WHO). https://www.cdc.gov/hepatitis/hcv/cfaq.htm Hepatitis C Questions and Answers for the Public. CDC. https://clarolineconnect.univ-lyon1.fr/uploads/webresource/189e3e07-df18-48b5-9164-8a6312cb8628.zip/hcv_life_cycle_site_web/index.html Hepatitis C Virus Life Cycle. https://www.hcvguidelines.org/ HCV Guidance: Recommendations for Testing, Managing, and Treating Hepatitis C. https://www.niaid.nih.gov/news-events/trial-evaluating-experimental-hepatitis-c-vaccine-concludes Trial Evaluating Experimental Hepatitis C Vaccine Concludes.

Hepeviruses (Hepeviridae) Xiang-Jin Meng, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Animal Reservoir A large animal population serving as a source of virus supply for the transmission of viruses to other animals including humans. Fulminant hepatitis A clinical condition characterized by massive necrosis of liver parenchyma and decrease in liver size that is usually caused by hepatitis virus infections or drug and toxin-induced liver injury. Quasi-enveloped virus particles Non-enveloped viruses but present as membrane-wrapped quasi-enveloped virions

in the blood of infected hosts. As a non-enveloped virus, hepatitis E virus is present in multivesicular body as membrane-associated quasi-enveloped particles with intraluminal vesicles resembling exosomes in infected cells and blood. Zoonotic infection Infectious diseases transmissible under natural conditions from vertebrate animals to humans.

Classification Hepatitis E virus (HEV) is classified in the family of Hepeviridae, which comprises two genera. The genus Orthohepevirus includes all mammalian and avian hepatitis E virus isolates, whereas the genus Piscihepevirus consists of the cutthroat trout virus. Within the genus Orthohepevirus, there exist four distinct species: Orthohepevirus A consisting of HEV isolates from human, wild and domestic pigs, deer, mongoose, rabbit, and camel, Orthohepevirus B comprising avian HEV isolates from birds, Orthohepevirus C containing HEV isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink, and Orthohepevirus D including HEV isolates from bat (Table 1). HEV isolates known to infect humans all belong to the species Orthohepevirus A, which consists of at least 8 distinct genotypes. Genotype 1 HEV (mostly Asian isolates) and genotype 2 HEV (a Mexican isolate and some African isolates) infect only humans, whereas genotypes 3 (human, pig, rabbit, deer, mongoose), and 4 (human and pig) HEVs infect humans and several other animal species. Genotypes 5 and 6 HEVs infect wild boars with unknown zoonotic potential. Genotype 7 HEV infects dromedary camels and reportedly a human, and the genotype 8 HEV infects Bactrian camel (Table 1).

Virion Structure HEV is an icosahedral, spherical virus particle of approximately 32–34 nm in diameter. HEV particles secreted in bile and stool are non-enveloped, however, in circulating blood and in supernatant of infected cell culture, HEV appears as membrane-associated quasi-enveloped particles with intraluminal vesicles resembling exosomes that are infectious but resistant to antibody neutralization. The capsid protein encoded by ORF2 is the only known structural protein on the virion, although the majority of ORF2 protein in serum and supernatant of HEV-infected cell culture exists as a secreted form (ORF2S) that is not associated with virions. The virion of HEV is a T ¼ 3 icosahedral capsid lattice consisting of 180 copies of capsid protein with three functional domains: S (shell), M (middle), and P (protruding). The S domain forms the icosahedral shell, while the P domain is the binding site for an unknown cellular receptor. The M domain interacts with S and P domains contributing to virion stability. The virion buoyant density is 1.35–1.40 g/cm3 in CsCl, and 1.29 g/cm3 in potassium tartrate and glycerol. The virion sedimentation coefficient is 183S. The HEV virion is more heat labile than is hepatitis A virus (HAV), another enterically transmitted hepatitis virus. Infectious HEV was still detectable after incubation at 371C for 21 days, at room temperature for 28 days, and at 41C for 56 days with a 2.7-log decrease of virus titer. Incubation of infectious virus at 601C for 1 min resulted in moderate decreases in infectivity, 2- to 3.5-log decreases in infectivity between 651C and 751C for 1 min, and complete inactivation at Z801C for 1 min. The fecal-oral route of transmission indicates that HEV is resistant to inactivation by acidic and mild alkaline conditions in the gastrointestinal tract.

Genome The genome of HEV, which possesses a 7-methylguanosine cap structure at its 50 end, is a single-stranded, positive-sense RNA molecule of approximately 7.2 kb in length. The genome consists of a short 50 noncoding region (NCR), three open reading frames (ORFs 1, 2, and 3), and a 30 NCR. ORF3 overlaps ORF2, but neither overlaps ORF1 (Fig. 1). The ORF1 encodes nonstructural proteins responsible for viral replication and protein processing. Functional domains within ORF1 have been identified, including methyltransferase (Met), papain-like cysteine protease (PCP), hypervariable region (HVR) or proline rich region (PRR), X (or

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00016-3

397

398

Table 1

Hepeviruses (Hepeviridae) Host range of hepatitis E virus and zoonotic infection in humansa

Natural animal host

Classification [genus/species, genotypes (gt)]

Experimental hosts for cross-species infection

Zoonotic infection in humans

Orthohepevirus A Human

gt 1, 2, 3, 4

Domestic swine

gt 3, 4

Wild boar Deer Rabbit Mongoose Dromedary camel Bactrian camel Moose

gt 3, 4, 5, 6 gt 3 gt 3 gt 3 gt 7 gt 8 unknown Orthohepevirus B

Chicken, wild birds

aHEV gt 1, 2, 3, 4 Orthohepevirus C

Rat Ferret Greater bandicoot Asian musk shrew Mink Common kestrel Red-footed falcon Bat Cutthroat trout

Orthohepevirus D Piscihepevirus

Non-human primates, pigs (gt3, gt4), rabbit (gt1, gt4), lamb (gt1), Wistar rats (gt1) Non-human primates, rabbit, Mongolian gerbil (gt4), Balb/C mice (gt3, gt4)

Pig Non-human primates Non-human primates

Turkey

Yes Yes (gt 3, 4), likely (gt 5, 6) Yes Yes Likely Yes Unknown Unknown No Yes Unknown Unknown Unknown Unknown Unknown Unknown No No

a

Modified from Meng, X.J., 2016. PLoS Pathogens 12(8), e1005695. doi:10.1371/journal.ppat.1005695, which was published under a Creative Commons License.

Fig. 1 A schematic diagram of comparative genomic organization of mammalian, avian, and fish HEVs. The three open reading frames (ORFs) are labeled and shown as boxes. ORF2 overlaps ORF3 but neither overlaps ORF1. ORF1 encodes nonstructural proteins with the putative functional domains indicated inside the box. ORF2 encodes the capsid protein, and ORF3 encodes a small multifunctional protein that is involved in virus replication. The HEV genome is capped (m7G Cap) at the 50 end, and contains a poly A tail at the 30 end. There are noncoding regions (NCR) at the 50 and 30 end of the viral genome. There is a junction region between ORF1 and ORF3 for mammalian and avian HEVs, which contains a stemloop structure and a cis-reactive element (CRE). The avian HEV genome is approximately 600 bp smaller than the mammalian and fish HEVs. Hel, helicase; HVR, hypervariable region; MT, methytransferase; NCR, noncoding region; P, a papain-like cysteine protease; RdRp, RNA-dependent RNA polymerase; X, macro domain. Reproduced from Meng, X.J., 2016. PLoS Pathogens 12(8), e1005695. doi:10.1371/journal.ppat.1005695, which was published under a Creative Commons License.

macro) domain, helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (Fig. 1). An RNA element located in the 50 end region of the ORF1 binds to capsid protein and is thought to be the RNA packaging signal. The viral genomic RNA contains two cis-reactive RNA elements (CREs) important for virus replication. CRE1 overlaps the 30 end of ORF2 and continues into the 30 NCR, and forms two stem-loop structures that interact with RdRp. CRE2 locates in the intergenic junction region between ORF1 and ORF3, and forms a stem-loop structure that is part of the promoter for synthesis of the subgenomic RNA. The intragenomic

Hepeviruses (Hepeviridae)

399

subgenomic promoter regulating subgenomic RNA synthesis is mapped to the junction region extending to the 30 end of ORF1. Both the sequence and the stem-loop structure in the junction region play important roles in HEV replication. ORF2 encodes capsid protein that contains a signal peptide sequence, and three glycosylation sites. The capsid protein contains an immunogenic epitope, induces neutralizing antibodies against HEV, and is the target for vaccine development. A secreted form of ORF2 protein (ORF2S) is translated from an AUG start codon that is 15 amino acids upstream to the N terminus of the capsid protein. The ORF2S does not block HEV cell entry but inhibits antibody-mediated neutralization. ORF3 encodes a small cytoskeleton-associated phosphoprotein. The N-terminus of ORF3 has a cysteine-rich region, binds to HEV RNA, and enters into a complex with the capsid protein. The C-terminus of ORF3 is a multifunctional domain involved in HEV virion morphogenesis and pathogenesis. The ORF3 is essential for virus infectivity in vivo, although it is not required for virion assembly or infection of hepatocellular carcinoma cells in vitro. Overlapping the macro and Hel domains in genotype 1 HEV, but not other HEV genotypes, is a small frameshifted ORF4 that is controlled by an internal ribosome entry site (IRES)-like RNA structure, which induces ORF4 protein translation under endoplasmic reticulum (ER) stress.

Life Cycle Attachment and entry: The attachment and entry of HEV into susceptible cells remains largely unknown, due in large part to the lack of an efficient cell culture system to propagate HEV. The capsid protein attaches to susceptible cells via heparin sulfate proteoglycans (HSPGs) to facilitate HEV entry, although a specific receptor for HEV has not yet been identified. The virions are internalized through a clathrin and dynamin-2 based pathway. The entry of quasi-enveloped HEV particles reportedly involves the small GTPases Rab5 and Rab7 and degradation of the quasi-envelope membrane within the lysosome. The uncoating process of the capsid is not understood, although the P domain of the capsid contains polysaccharide binding sites that may be involved in capsid disassembly to facilitate the uncoating process. Transcription and translation: Upon uncoating and delivering the viral genomic RNA to the cytosol, the ORF1 polyprotein is translated directly from the positive-sense viral genomic RNA by cap-dependent translation, and eukaryotic translation initiation factors 4A, 4G, and 4E are involved in HEV replication. Whether the ORF1 polyprotein requires further processing for functionality remains debatable, since contradictory results have been reported with some studies showing no processing at all while others reporting cleaved products corresponding to the sizes of Met, Hel, and RdRp. The ORF2 and ORF3 proteins are also translated in a cap-dependent fashion from a single bicistronic subgenomic mRNA by a process involving leaky ribosome scanning in which ribosomes bypass the ORF3 initiator AUG to initiate protein synthesis downstream at the ORF2 AUG initiator codon. Replication of viral genome: The replication of HEV genome involves the transcription of a negative-sense replicative RNA intermediate by viral RdRp. This negative-sense RNA subsequently serves as the template for the production of the positive-sense, progeny viral genomic RNA as well as the bicistronic subgenomic mRNA. In HEV-infected animal tissues, the negative-sense replicative RNA intermediate has been detected by negative strand-specific RT-PCR. A viral replication complex (VRC) forms on ER membranes or on vesicles consisting of ER-derived membranes, although the VRC composition is not well understood. For genotype 1 HEV, the VRC appears to contain the ORF4 protein, Hel, RdRp, and macro domains of ORF1, along with host eukaryotic elongation factor 1 isoform-1 (eEF1a1) and tubulin b. Assembly and release: Little is known about the assembly and release steps in the life cycle of HEV. The capsid protein packages the HEV genome through binding to a putative RNA packaging signal located in the 50 end region of the viral genome. Binding of viral RNA to the N-terminus of capsid leads to the formation of C-terminal/C-terminal dimers that are important for completing viral capsid assembly. It has been shown that ORF3 is involved in viral egress via interaction with host late domain proteins. Quasi-enveloped HEV has been detected within multivesicular bodies (MVBs) associated with trans-Golgi network protein 2 and CD63, suggesting that HEV capsids bud into intracellular vesicles requiring the MVB pathway to release virions. The ORF3 protein is a viroporin forming a functional ion channel at plasma membrane to facilitate the release of infectious virus particles.

Epidemiology Morbidity and mortality: In developing countries, a high seroprevalence of HEV antibodies was reported. For example, more than 70% of the general populations in Egypt are seropositive for HEV antibodies. Surprisingly, in some industrialized countries where clinical cases of hepatitis E are rare, the seroprevalence of HEV antibodies is much higher than expected, with up to 30% seropositivity in blood donors in the United States. The morbidity for hepatitis E is not very reliable due in large part to the lack of accurate etiology-specific data for acute viral hepatitis. Most sporadic cases of acute viral hepatitis are not routinely tested for HEV and thus are not reported. Historically, the incidence of hepatitis E in large outbreak settings was estimated at 1400–1650 per 100,000 population in India and Nepal. More recently, a large outbreak of 2621 cases of acute hepatitis E was reported in refugee camps in Darfur, Sudan with an attack rate of 3.3% and a case-fatality rate of 1.7%. The highest attack rate of clinical disease is generally in young adults of 20–29 years of age, although the seroprevalence of HEV antibodies is age-dependent and increases with age. HEV-associated mortality in the general population is o1% but it can reach up to 25% in infected pregnant women. According to the World Health Organization, annually there are an estimated 20 million HEV infections worldwide leading to approximately 3.3 million cases of hepatitis E with 444,000 HEV-related deaths.

400

Hepeviruses (Hepeviridae)

Animal reservoirs and cross-species infection: Since the discovery of the first animal strain of HEV, swine HEV, from pigs in the United States, genetically-diversified strains of HEV have now been identified from more than a dozen other animal species including chicken, deer, mongoose, rabbit, rat, ferret, moose, shrew, kestrel, egret, bat, goat, camel, and cutthroat trout. The ecology, natural history, zoonotic potential and pathogenicity for most of these animal hepeviruses are still largely unknown, although the genotype 3 HEV infecting pig, rabbit, deer and mongoose, and the genotype 4 HEV infecting pigs are known to be zoonotic and infect humans. Under natural conditions, zoonotic infections in humans by genotypes 3 and 4 HEVs from various animal species have been well documented. The sporadic and cluster cases of hepatitis E in humans from industrialized countries are caused primarily by the zoonotic genotypes 3 and 4 HEVs of animal origin. Cases of chronic hepatitis E in immunocompromised individuals are almost exclusively caused by the zoonotic genotype 3 HEV of animal origin. Pig caretakers such as farmers and swine veterinarians had a higher risk of zoonotic HEV infection. In addition to pigs, the zoonotic genotype 3 HEV has also been identified in rabbit, deer, and mongoose, suggesting that these animals are also reservoirs. Zoonotic transmissions of hepatitis E from deer to humans and from rabbit to humans have been reported. A genotype 7 camelid HEV also reportedly infected a human liver transplant recipient. More recently, human infections by rat HEV have also been reported. Under experimental conditions, genotypes 3 and 4 HEVs can infect across species barriers: genotypes 3 and 4 swine HEVs infect rhesus macaques and chimpanzee, and conversely, genotypes 3 and 4 human HEVs infect pigs. Genotype 3 rabbit HEV was also shown to infect pigs and cynomolgus macaques. Balb/c nude mice are reportedly susceptible to experimental infection with a genotype 4 swine HEV, although others failed to infect C57BL/6 mice with genotype 1, 3 and 4 HEVs. Rabbits have been reportedly infected with genotypes 1 and 4 HEV, although independent confirmation is still lacking. Avian HEV infects turkeys, but not rhesus macaques, suggesting that avian HEV is not zoonotic. Sources and risk factors of HEV infection: For epidemics, the main source of infection is HEV-contaminated drinking water in areas with poor sanitation such as villages in developing countries, and refugee camps. Hepatitis E epidemics are often associated with contamination of well water or leakage of untreated sewage into city water treatment plants, and poor sanitary conditions such as washing hands in a group basin. Person-to-person transmission of hepatitis E is not common during an epidemic. In contrast, the main sources for sporadic or cluster cases of hepatitis E appear to be contaminated animal meats, shellfish, and direct contact with infected animals. Hepatitis E epidemics are mainly in developing countries of Asia, Africa, and Latin America, particularly in regions with hot climates or poor sanitation conditions. However, sporadic or cluster cases of hepatitis E occur in both developing and industrialized countries. A high incidence of hepatitis E outbreaks is observed in seasons with monsoon rains and flooding. The use of river water for drinking and cooking, washing, and disposal of human excreta carries a higher risk of HEV infection. Pigs are reservoirs for HEV, and therefore pig farmers, swine veterinarians and other pig caretakers in both developing and industrialized countries are at increased risk of HEV infection. In the United States, swine veterinarians were 1.51 times more likely to be seropositive for HEV antibodies than normal blood donors. Individuals from traditionally major swine states are more likely to be seropositive for HEV antibodies than those from traditionally non-swine States. Individuals with occupational exposure to wild animals also have an increased risk of zoonotic HEV infection. Sewage workers in India had a significantly higher seroprevalence of HEV antibodies (56.5%) than controls (19%), although such an association was not found in Switzerland. In a large hepatitis E outbreak in 2004 in refugee camps in Darfur, Sudan, the risk factors included age of 15–45 years (OR ¼2.13) and drinking chlorinated surface water (OR ¼ 2.49). Transmission: HEV is primarily transmitted by fecal-oral route, although under experimental conditions it is rather difficult to infect non-human primates or pigs via oral route of inoculation. Waterborne transmission via sewage-contaminated drinking water and contaminated well or river water used for washing and drinking purpose is the main source of HEV infection in developing countries. Feces from infected animals contain large amounts of HEV leading to contamination of irrigation or coastal water with concomitant contamination of produce or shellfish. HEV strains of both human and swine origin have been detected in sewage water. Foodborne transmission is responsible for most sporadic and cluster cases of acute hepatitis E that were linked to the consumption of raw or undercooked animal meats. Sporadic cases of hepatitis E were associated to the consumption of raw or undercooked wild boar meat deer meat, and pork products. HEV RNA was detected in commercial pork products sold in grocery stores, and the contaminating virus in commercial pig livers from grocery stores remains infectious. Raw pig liver sausages (figatelli) were the source for some sporadic and cluster cases of hepatitis E in France. Consumption of raw deer meats was responsible for cluster cases of acute hepatitis E in Japan. Also, HEV infections were linked to the consumption of contaminated shellfish and strawberries. Zoonotic transmission through direct contact with infected animals occurs for genotypes 3, 4 and genotype 7 HEVs within the species Orthohepevirus A. Potential transmissions of hepatitis E from a pet cat and a pet pig to human owners were also reported. Vertical transmissions from mother-to-fetus associated with a high neonatal mortality has been reported. Among the 62 HEVinfected pregnant women, vertical transmission of HEV occurred in 33.3% of the cases. HEV RNA was detected in some cord blood and in colostrum of HEV-infected mothers, although breast-feeding appears to be safe for infants. Vertical transmission has been reported in HEV-infected human patients, but experimental evidence of vertical transmission of HEV in animal models is still lacking. Pregnant sows and pregnant rhesus monkeys experimentally-infected with HEV failed to transmit the virus to offspring. Egg whites from eggs of chickens experimentally infected with a chicken strain of HEV contain infectious virus, but evidence of complete vertical transmission is lacking. Blood-borne transmission through blood transfusions, although not common, has been reported in patients worldwide. HEV RNA has been detected in a small proportion of blood donors in Japan and some European countries, thus raising a potential

Hepeviruses (Hepeviridae)

401

concern of transfusion-transmitted hepatitis E. In Germany, HEV viremia was detected in approximately 0.12% of blood donors at a tertiary care center. In the United States, in one study HEV RNA was not detected in blood donors or recipients, while in a separate study three out of 128,020 blood donors from 27 states tested positive for genotype 3 HEV RNA. The incidence of viremia and viral loads in blood donors, and the total plasma volume the recipients receive are the determining factors for transfusionassociated HEV infection. It remains debatable as to whether donor blood should be screened for acute markers of HEV infection, although several European countries have already implemented HEV RNA screening of blood donors.

Clinical Features The majority of HEV infections are subclinical, and a proportion of the infected individuals do develop self-limiting acute viral hepatitis, but the acute disease usually does not go into chronicity. However, in immunocompromised individuals, HEV infections can progress into chronic hepatitis E, which has become an emerging and significant clinical problem. Numerous neurological diseases such as Guillain-Barrê syndrome and neuralgic amyotrophy have also been linked to HEV infection, although the underlying mechanism of neurological invasion by HEV remains unknown. Fulminant hepatitis E with a high mortality have been reported in HEV-infected pregnant women especially during the third trimester. Self-limiting acute hepatitis E: Only a proportion of HEV-infected individuals develop self-limiting acute viral hepatitis. The incubation period typically ranges from 2 weeks to up to 2 months. The clinical symptoms of acute hepatitis E, which include Jaundice, anorexia, abdominal pain, nausea, vomiting, and fever, are essentially indistinguishable from those of other types of acute viral hepatitis. Viremia and fecal virus shedding are detectable approximately 3 weeks post-infection and 1 week prior to the onset of clinical disease. A transient appearance of IgM HEV antibodies followed by long-lasting IgG antibodies appear late during the period of viremia and fecal virus shedding. The infected patients typically recover from acute hepatitis E and are protected against subsequent HEV infection. Chronic hepatitis E: Although HEV generally causes a self-limiting acute viral hepatitis in immunocompetent individuals, the majority of HEV infections in immunocompromised individuals such as solid organ transplant recipients and patients with HIV/ AIDS, lymphoma, or leukemia progress into chronic hepatitis E. Organ transplant recipients with chronic hepatitis E have persistently elevated aminotransferase levels, viremia, and hepatic lesions, and also shed HEV in feces for a prolonged period of time. Cases of chronic hepatitis E are almost exclusively caused by the zoonotic genotype 3 HEV, which infects pig, rabbit, deer and mongoose. Therefore, the source of chronic HEV infection is of zoonotic origin presumably through consumption of undercooked HEV-contaminated animal meats. Under experimental conditions, aimed at mimicking the conditions of immunocompromised organ transplant patients, pigs were treated with an immunosuppressive regimen and experimentally infected with genotype 3 HEV. The infected pigs developed chronic HEV infection with fecal virus shedding lasting for at least 22 weeks post-infection. Active suppression of cell-mediated immune responses under immunocompromised conditions facilitates the establishment of chronic HEV infection. Similarly, cynomolgus monkeys treated with tacrolimus, an immunosuppressive drug, and experimentally infected with a genotype 3 swine HEV also developed chronic HEV infection with pronounced hepatic lesions at 160 days after infection. Fulminant hepatitis with a high mortality during pregnancy: Accumulating evidence indicates that HEV infection during pregnancy causes fulminant hepatic failure with a high mortality. In India, one study showed that the mortality rate in HEV-infected pregnant women is 65.8% (25/38) due to fulminant hepatic failure. In another study, the mortality rate among the HEV-positive pregnant women was 27%, and approximately two-thirds of the infected pregnant women had preterm deliveries. However, in Egypt, a study of 2428 pregnant women did not link HEV exposure to a history of liver disease. Genotype 1 HEV is usually associated with fulminant hepatic failure in infected pregnant women, although zoonotic genotypes 3 and 4 HEVs are also associated with fulminant hepatic failure. Under experimental conditions, however, severe and fulminant hepatitis could not be reproduced in pregnant pigs or monkeys: pregnant sows experimentally infected with genotype 3 HEV had no clinical signs of hepatitis or elevation of liver enzymes. Similarly, pregnant rhesus macaques experimentally infected with genotype 1 HEV did not develop more severe hepatitis than non-pregnant monkeys. Recently, however, fulminant hepatitis with high mortality was successfully reproduced in genotype 3 rabbit HEV-infected pregnant rabbits. Fetal mortality rates ranging from 67% to 80% were observed. Miscarriages, high fetal and maternal mortality, and fulminant hepatitis were also reproduced in these genotype 3 rabbit HEV-infected pregnant rabbits. The underlying mechanism of fulminant hepatitis with high mortality during pregnancy is still unknown, although the social economic status of the patients, hormonal changes during pregnancy, immunological status of the patients, and the existence of other co-infecting agents in patients could all play a potential role. Neurological diseases: Neurological diseases such as Guillain-Barrê syndrome, neuralgic amyotrophy, encephalitis, and myelitis have been associated with zoonotic genotype 3 HEV infection and, to lesser extent, genotypes 1 and 4 HEV infections. GuillainBarrê syndrome and neuralgic amyotrophy are associated with HEV infection in approximately 5% and 10% of the cases, respectively. In some patients, HEV RNA was detected in cerebrospinal fluid, indicating an infection of central nerve system. The neurological disorders in humans are mostly associated with infection by genotype 3 HEV that infects pig, deer, rabbit and mongoose, and occur during both acute and chronic HEV infections. Under lab conditions, numerous human neuronal-derived cell lines have been shown to support HEV replication. In animals peripherally infected with HEV, viral RNA and antigen were detected in brain tissues. The available data thus far suggest that HEV infects cells in the central nerve system leading to manifestation of various neurological diseases, which has become an emerging and significant clinical problem.

402

Hepeviruses (Hepeviridae)

Pathogenesis The mechanism of HEV pathogenesis is not well understood. HEV enters the host through the fecal-oral route. It is thought that, after oral ingestion of HEV, the virus first replicates in the gastrointestinal tract before reaching its target organ, the liver, via viremia. In pigs experimentally infected with human and swine HEVs, virus replication has been identified in small intestines, colon, hepatic and mesenteric lymph nodes, although the clinical significance of these extrahepatic sites of virus replication remains unknown. HEV replication in the liver has been detected in non-human primates, pigs and chickens experimentally infected with HEV. It is believed that, after replication in liver, HEV is released to the gallbladder from hepatocytes and is excreted in feces. Fecal virus shedding and viremia proceeds the onset of clinical and biochemical hepatitis, and virus shedding ends when the level of serum liver enzyme returns to baseline. In pigs experimentally infected with a genotype 3 human HEV, mild-tomoderate multifocal lymphoplasmacytic hepatitis and focal hepatocellular necrosis were observed. In chickens experimentally infected with an avian strain of HEV, gross lesions including subcapsular hemorrhages and slightly enlarged right intermediate lobe of the livers, and histological lesions including foci of lymphocytic periphlebitis and phlebitis were also observed.

Diagnosis HEV serological assays are commercially available in some countries, although considerable variations in the specificity and sensitivity of these assays have been reported. These serological assays primarily use the recombinant ORF2 capsid protein or a mixture of both ORF2 and ORF3 proteins as the antigen. Some serological assays are approved for HEV diagnosis by regulatory agencies in Asia and Europe, however an FDA-approved diagnostic assay in the United States is still lacking. IgM anti-HEV is detectable in up to 90% of the acute HEV infections within 1–4 weeks after onset of the disease. IgG anti-HEV persists in infected individuals lasting from o1 year in some pediatric patients to 2–14 years in some adult patients. RT-PCR and quantitative RT-PCR assays have also been developed to detect and quantify HEV genomes. However, significant variations in the sensitivity and specificity of these PCR-based assays were reported, largely due to the extensive heterogeneity of HEV. At least five genotypes of HEV, genotypes 1–4 and 7, within the species Orthohepevirus A are known to infect humans, thus stressing the need for a standardized universal PCR-based assay for HEV detection.

Treatment Most HEV infections are self-limiting and acute in nature, and do not warrant antiviral treatment. However, a subpopulation of HEV-infected individuals, such as pregnant women with a high mortality and immunocompromised individuals with a high tendency of progressing into chronicity, do require antiviral treatment options. Currently there is no HEV-specific therapy, however broad-spectrum antivirals such as ribavirin and pegylated-interferon have been used with some success. Ribavirin monotherapy inhibits HEV replication and induces a sustained virus inhibition in patients with chronic HEV infections. Pegylated-interferon-a also induced a sustained antiviral response in HEV-infected patients. However, both ribavirin and interferon-a have severe side effects, and are contraindicated in pregnant women. Therefore, development of effective HEV-specific antivirals is warranted.

Prevention A commercial vaccine based on the recombinant ORF2 capsid protein of a genotype 1 HEV has been licensed for use only in China but not elsewhere. Other experimental HEV vaccines based on the capsid protein also appear to be very promising. However, significant challenges remain for a more cost-effective modified live-attenuated vaccine due to the lack of an efficient cell culture system to propagate HEV. The practice of good hygiene and avoiding drinking water of unknown purity or consuming raw or undercooked animal meats are important preventive measures.

Further Reading Cao, D., Cao, Q.M., Subramaniam, S., et al., 2017. Pig model mimicking chronic hepatitis E virus infection in immunocompromised patients to assess immune correlates during chronicity. Proceedings of the National Academy of Sciences of the United States of America 114 (27), 6914–6923. Dalton, H.R., Kamar, N., van Eijk, J.J., et al., 2016. Hepatitis E virus and neurological injury. Nature Reviews Neurology 12 (2), 77–85. Dalton, H.R., Kamar, N., 2016. Treatment of hepatitis E virus. Current Opinion in Infectious Diseases 29 (6), 639–644. Kamar, N., Selves, J., Mansuy, J.M., et al., 2008. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. The New England Journal of Medicine 358 (8), 811–817. Kenney, S.P., Meng, X.J., 2018. Hepatitis E virus: Animal models and zoonosis. Annual Review of Animal Biosciences 7, 427–448. doi:10.1146/annurev-animal020518–115117. Meng, X.J., Purcell, R.H., Halbur, P.G., et al., 1997. A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America 94, 9860–9865.

Hepeviruses (Hepeviridae)

403

Meng, X.J., 2014. Hepatitis E. In: Kaslow, R.A., Stanberry, L.A., LeDuc, J.W. (Eds.), Viral Infections of Humans: Epidemiology and Control, fifth ed. NY: Springer Publishing, pp. 439–454. Meng, X.J., 2016. Expanding host range and cross-species infection of hepatitis E virus. PLoS Pathogens 12 (8), e1005695. Meng, X.J., Shivaprasad, H.L., 2019. Avian hepatitis E virus infections. In: Swayne, D., Boulianne, M., McDougald, L., et al. (Eds.), Diseases of Poultry, fourteenth ed. Hoboken, NJ: John Wiley & Sons, Inc, pp. 528–534. Meng, X.J., Halbur, P.G., Opriessnig, T., 2019. Hepatitis E virus. In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., et al. (Eds.), Diseases of Swine, eleventh ed. John Wiley & Sons, Inc, pp. 544–549. Nimgaonkar, I., Ding, Q., Schwartz, R.E., Ploss, A., 2018. Hepatitis E virus: Advances and challenges. Nature Reviews Gastroenterology & Hepatology 15 (2), 96–110. Purcell, R.H., Emerson, S.U., 2013. Hepatitis E virus. In: Knipe, D.M., Howley, P.M., Cohen, J.I. (Eds.), Fields Virology, sixth ed. Lippincott: Williams and Wilkins, Philadelphia, PA, pp. 2242–2258. Purdy, M.A., Harrison, T.J., Jameel, S., et al., 2017. ICTV virus taxonomy profile: Hepeviridae. Journal of General Virology 98 (11), 2645–2646. Shukla, P., Nguyen, H.T., Torian, U., et al., 2011. Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus-host recombinant. Proceedings of the National Academy of Sciences of the United States of America 108 (6), 2438–2443. Yin, X., Ying, D., Lhomme, S., et al., 2018. Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection. Proceedings of the National Academy of Sciences of the United States of America 115 (18), 4773–4778.

Relevant Websites https://ecdc.europa.eu/en/hepatitis-e/facts Facts about hepatitis E European Center for Disease Control and Prevention. https://www.who.int/news-room/fact-sheets/detail/hepatitis-e Hepatitis E infection World Health Organization. https://www.cdc.gov/hepatitis/hev/index.htm Hepatitis E information Division of Viral Hepatitis CDC. https://discontools.eu/component/desease/desease.html?id=73 Hepatitis E virus Discontools. https://www.viprbrc.org/brc/home.spg?decorator=hepe Hepeviridae Virus Pathogen Database and Analysis Resources (ViPR).

Herpes Simplex Virus 1 and 2 (Herpesviridae) David M Knipe, Harvard Medical School, Boston, MA, United States Richard Whitley, University of Alabama at Birmingham, Birmingham, AL, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Encephalitis Infection of the central nervous system. Gingivostomatitis Infection of the oral cavity. Innate immunity Immune responses induced by recognition of pathogen-specific molecular patterns. Intrinsic resistance Host factors that are constitutively expressed and restrict infection. Keratitis Infection of the cornea.

Latent infection An infection in which the viral genome can be detected but no infectious virus can be detected, but in which infectious virus can be reactivated upon appropriate stimuli. Lytic infection An infection that produces progeny virus and leads to death and lysis of the infected cells. Pro-drug A compound that is metabolized in the body to an active drug molecule.

Classification Human herpes virus 1 (HHV-1), commonly known as herpes simplex virus 1 (HSV-1), and human herpes virus 2 (HHV-2), commonly known as herpes simplex virus 2 (HSV-2), are two species of viruses in the Simplexvirus genus in the Herpesviridae family of the Herpesvirales order. These two viruses are sometimes called type 1 and type 2, but they are formally species, so the word “type” is incorrect. Many of the genetic and phenotypic properties of HSV-1 and HSV-2 are similar, so we will describe them together as HSV where genes and mechanisms are shared by the two viruses.

Virion Structure The HSV virion (extracellular virus particle) consists of the core containing the double-stranded DNA genome surrounded by an icosahedral capsid, enclosed by a largely unstructured protein layer (tegument), surrounded by the outer lipid bilayer membrane (envelope) which contains embedded glycoprotein spikes (Fig. 1). The core consists of the DNA wrapped as a toroid in a liquid crystalline state. The DNA has no associated histones, and the phosphate charges on the DNA may be neutralized by polyamines. The capsid is a protein shell comprised of four viral proteins assembled with icosahedral symmetry based on 162 capsomeres or structural units defining the 16 triangular faces on the icosahedron. Twelve of the capsomeres form the vertices, of which 11 are pentons (five-fold symmetry) and one is the portal, through which virion genomic DNA is inserted, while 140 of the capsomeres are hexons (six-fold symmetry) that form the faces of the icosahedral structure. The tegument is comprised of at least 18 viral proteins and is largely unstructured, although there is some evidence for icosahedral organization imposed by interactions with the capsid and some granular and filamentous character. The envelope is a lipid bilayer membrane derived from host cell membranes that has as many as 13 viral proteins embedded in it.

Genome The HSV genome is a linear double-stranded DNA molecule of approximately 150,000 base pairs (bp) with a one bp 30 extension at each end. The virion DNA contains nicks and gaps and a G þ C content of 68% for HSV-1 and 69% for HSV-2 DNA. The HSV-1 and HSV-2 genomes are largely collinear with many of the lytic genes being highly homologous. HSV-1 X HSV-2 recombinants are readily isolated with cross-over events throughout the genomes, further demonstrating the similarity of the HSV-1 and HSV-2 genomes. The HSV genome can be considered to consist of two covalently linked components, the L (long) and S (short), each of which is bounded by inverted repeats. The termini of the molecules contain a short repeated sequence that is also present in inverted forms at the L-S junction. The inverted repeated sequences provide homology for recombination that results in inversion of the L and S components, relative to each other. As a result, virion and infected cell DNAs contain approximately equimolar populations of four isomers of HSV DNA that result from inversion of one component or the other or both. The reason for the inverted repeated sequences has not been defined, but they may play a role in recombination-mediated replication of the viral DNA genome.

Life Cycle HSV undergoes a lytic productive infection in epithelial cells and fibroblasts when it is inoculated onto the oral (usually HSV-1) or genital (usually HSV-2 but increasingly HSV-1) mucosa (Fig. 2). The virus spreads and causes the primary herpetic lesion. The virus

404

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21273-9

Herpes Simplex Virus 1 and 2 (Herpesviridae)

405

Fig. 1 Structure of the HSV virion. Electron micrograph of a negative-stained HSV-1 virion. The envelope, tegument, capsid, and core are indicated. Copyright, Travis Taylor and David Knipe.

Fig. 2 Stages of herpes simplex virus (HSV) infection of the host. A: HSV is introduced onto a mucosal surface or a break in the skin, and it replicates productively in epithelial cells at the site of inoculation and spreads through the tissue. Virus enters sensory neuron axons and is transported to the cell body in a ganglion. B: HSV establishes a latent infection in the neuronal cell nucleus. Viral DNA is circular and assembled in chromatin. C: Upon neuronal damage or activation, the virus reactivates and undergoes at least a limited productive cycle. Capsids are transported by anterograde transport to the axonal termini, and virions are released. Reactivated virus causes a recurrent infection of the mucosal tissue, causing the shedding of virus. Copyright, Patrick T. Waters and David Knipe.

406

Herpes Simplex Virus 1 and 2 (Herpesviridae)

enters the axons of sensory neurons and establishes a latent infection in the neuronal cell body. Upon stress or damage to the neuron, the virus reactivates and causes the recurrent infection.

Lytic Infection HSV lytic infection of epithelial cells or fibroblasts is initiated by binding of the virion to the cell surface and entry by fusion at the plasma membrane (Fig. 3, step 1a) or endocytosis followed by fusion in an endosome (Fig. 3, step 1b). The nucleocapsid and tegument are transported along microtubules (Fig. 3, step 2) to the nucleus where the capsid docks at the nuclear pore and releases the virion DNA into the nucleus. The input genome rapidly circularizes (Fig. 3, step 5) and is loaded with histones by host cell mechanisms. Virion protein 16 (VP16) localizes into the nucleus (Fig. 3, step 4) and assembles a complex of host proteins that promote transcription of the immediate-early (IE) mRNAs (Fig. 3, step 6). The IE mRNAs are transported to the cytoplasm where they are translated to yield the five IE proteins. Four of the five proteins localize to the infected cell nucleus where they promote early (E) and late (L) gene transcription. The E mRNAs are transported to the cytoplasm where they encode the E proteins, including seven viral proteins directly involved in viral DNA synthesis and other proteins that increase deoxynucleotide triphosphate levels for viral replication in resting cells. The E viral DNA polymerase and thymidine kinase (TK) are the targets of the nucleoside analogs used for antiviral therapy as described below. The seven essential viral proteins together with host proteins replicate the input viral genome to yield progeny viral genomes as concatemers (Fig. 3, step 8). Replication of the viral DNA is thought to occur in two stages: (1) Cairns circle replication of the input viral genome, and (2) rolling circle and/or recombination-based replication generating concatemeric progeny molecules of viral DNA. Following initiation of viral DNA synthesis, the replicative intermediates and progeny DNA molecules serve as substrates for

Fig. 3 Diagram of the replication cycle of herpes simplex virus. 1: The virus binds to the cell plasma membrane and the virion envelope fuses with the plasma membrane (1a) or the virus enters by endocytosis (1b), releasing the capsid and tegument. proteins into the cytoplasm. 2: The capsid is transported to the nuclear pore, where the viral DNA is released into the nucleus. 3: The vhs (virion host shutoff) protein causes degradation of host messenger RNAs (mRNAs). 4: VP16 localizes into the nucleus. 5: The viral DNA circularizes. 6: It is then transcribed by host RNA polymerase II to give first the immediate-early (IE) mRNAs. Gene transcription is stimulated by the VP16 tegument protein. Five of the six IE proteins act to regulate viral gene expression in the nucleus. 7: IE proteins transactivate early (E) gene transcription. 8: The E proteins are involved in replicating the viral DNA molecule. 9: Viral DNA synthesis stimulates late (L) gene expression. 10: The L proteins are involved in assembling the capsid in the nucleus and modifying the membranes for virion formation. 11: DNA is encapsidated in the capsid. 12: The filled capsid buds through the inner membrane to form an enveloped virion, and the virion exits from the cell by mechanisms described in the text. Copyright, Patrick T. Waters and David Knipe.

Herpes Simplex Virus 1 and 2 (Herpesviridae)

407

transcription of the L genes (Fig. 3, step 9). The L mRNAs are translated in the cytoplasm to yield structural proteins for assembly of progeny virions. The capsid proteins localize into the infected cell nucleus and assemble empty capsids (Fig. 3, step 10), and viral DNA is inserted into the capsid through the portal on the capsid (Fig. 3, step 11). Once the capsid is filled with approximately a unit-length of viral DNA, the concatemeric DNA is cleaved and the portal is plugged. Some tegument proteins are localized into the nucleus and inner nuclear membrane to promote budding of the nucleocapsid through the inner nuclear membrane. Egress of the virion from the host cell (Fig. 3, step 12) is thought to then involve a complicated pathway involving primary envelopment at the inner nuclear membrane, de-envelopment of the virus particle at the outer nuclear membrane, and then secondary envelopment at membranes of the Golgi apparatus. The enveloped virion then travels out of the cell by the secretory pathway. Viral DNA replication, late gene transcription, and viral DNA encapsidation take place within virus-specific nuclear structures called replication compartments (Fig. 3, steps 8–10). The concentration of viral host factors in these factories is thought to enhance the rate of the reactions, as well as possibly provide a platform for the viral processes. The accumulation of viral nucleic acid and capsids in the replication compartments gives rise to the nuclear inclusion bodies that show specific staining properties characteristic of herpesviral infection. The process of lytic infection involves the viral gene products inhibiting host cell metabolism, which eventually results in the death and lysis of the host cell. Shut off of the host cell protein synthesis involves the virion host shutoff (vhs) protein, a ribonuclease that targets to polyribosomes and degrades host cell mRNA (Fig. 3, step 3) and provides an opportunity for translation of host mRNAs. Shut off of host transcription and of host RNA splicing also reduces host gene expression.

Epigenetic Regulation HSV virion DNA is not associated with histones, but when the viral DNA enters the cell nucleus, host cell mechanisms rapidly load histones with heterochromatic post-translational modifications on the viral DNA so as to silence it. Virion protein 16 (VP16) from the tegument is released into the cytoplasm and localizes into the infected cell nucleus where it organizes a complex of host cell proteins that remove the heterochromatin marks and reduce the histone loading on the viral DNA, so that transcription factors and RNA polymerase II can have access to the viral DNA for its transcription. The IE ICP0 protein then acts to allow E and L gene expression. ICP0 is an E3 ubiquitin ligase that promotes the degradation of a number of host restriction factor proteins that increase heterochromatin on HSV DNA, including PML, IFI16, and ATRX. This leads to euchromatin across the HSV genome, allowing broad transcription of the genome.

Host Factors Like all viruses, HSV uses numerous host proteins and structures for its replication. Human cells have also evolved numerous mechanisms to restrict replication of HSV. First, the cells detect foreign viral macromolecules, and activate mechanisms by constitutively expressed host proteins that block the activity of the viral molecules, a process called intrinsic resistance. One of the first mechanisms of resistance is the rapid loading of heterochromatin on the incoming viral genome, as described above. Alternatively, recognition of HSV DNA, altered host RNAs, or viral proteins can induce innate responses which result in expression of host proteins that reduce viral replication in the infected cell and surrounding cells. These responses may involve induction of cytokines such as interferons and interleukins. Type 1 interferons such as interferon a and interferon b are produced in infected cells and bind to receptors on that cell and surrounding cells to induce the expression of multiple antiviral genes collectively called interferon-stimulated genes (ISGs). ISGs encode protein products that block or reduce viral gene expression or replication. Interleukins induced by viral infection may induce antiviral mechanisms or recruit immune cells to the sits of viral infection and that reduce viral replication. HSV has evolved a number of mechanisms to fight back against intrinsic resistance and innate immunity mechanisms. As described above, ICP0 promotes the degradation of host epigenetic restriction factors. ICP0 also blocks certain innate signaling pathways. In addition, the HSV-1 ICP34.5 protein blocks type 1 interferon signaling so the virus is less sensitive to type 1 interferons. The HSV-1 US3 protein kinase also blocks interferon signaling. Other examples can be found in the cited reviews.

Latent Infection HSV latent infection of sensory neurons involves the binding of the virion to the termini of the neuronal axons and entry, likely using the same entry molecules as for lytic infection described above, into the axon. The capsid moves along the axon by retrograde transport to the neuronal cell body, where the viral genome is released into the neuronal nucleus. The genome circularizes and is loaded slowly with histones due to the small pool of free histones in the resting neuron; nevertheless, lytic gene transcription is limited due to the lack of localization of VP16 to the neuronal nucleus and the cytoplasmic localization of at least one of the host proteins that forms a complex with VP16 to activate IE gene expression. The major viral gene that is transcribed is the gene encoding the latency-associated transcript (LAT), which is a long (greater than 10 kb) non-coding RNA primary transcript. LAT is processed by splicing to yield a stable 2 kb circular intron, and several microRNAs (miRNAs). LAT expression acts to promote the survival of the infected neuron by increasing heterochromatin on the viral genome and reducing viral lytic gene expression and inhibiting apoptosis. LAT increases the levels of the H3K9me3 (histone H3 lysine 3 residue trimethylated) facultative heterochromatin marker, possibly by

408

Herpes Simplex Virus 1 and 2 (Herpesviridae)

recruiting the Polycomb repressive complex 2 histone methyltransferase. Facultative heterochromatin seems ideal for latent HSV chromatin because it is silencing, but it would constitute a form of chromatin that is poised for reactivation. During maintenance of latent infection, there is minimal but detectable expression of HSV lytic genes. Because the neuron is a resting cell, the viral DNA genome can be maintained stably without replicating or being tethered to host chromosomes. Some have argued that CD8 þ T cells surrounding the latently infected neuron maintain latent infection, but an alternative explanation is that they are targeting reactivating neurons. In any event, neuronal damage, stress, and/or activation lead to kinase signaling pathways believed to phosphorylate histones in viral chromatin to allow generalized transcription of the latent genome to provide enough VP16 to trigger the lytic transcriptional cascade. This allows at least a limited amount of viral gene expression to form capsids, and capsids or virions move along the axon by anterograde transport to release reactivated at the axonal termini to cause a recurrent infection. It has been debated as to whether the reactivating neuron survives or not, but neuronal survival is likely inversely proportional to the level of reactivated gene expression and not an allor-none effect.

Epidemiology HSV infection is transmitted by direct contact with an infected person’s lesions, mucosal surfaces, or genital or oral secretions. The epidemiology of infection is best defined by the seroprevalence of HSV-1 and HSV-2. HSV infections remain common despite a recent steady decrease in prevalence (Fig. 4). The seroprevalence of HSV-1 and HSV-2 increases linearly with age and is higher in females. Primary HSV-1 infections usually occur in the young child and are most often asymptomatic. During the period of 1999–2002, HSV-1 seroprevalence in U.S.-born children ages 6–13 years was 31%, progressively increasing with age. In the most recent National Health and Nutrition Examination Survey (NHANES) of 2015–2016, the seroprevalence in people age 14–19 years was 27%, consistent with overall trends of decreasing prevalence of HSV-1 in early childhood over the last two decades. By adulthood, about half of the U.S. population (48%) has experienced HSV-1 infection. HSV-1 prevalence differs by race and ethnicity, with prevalence highest in the Mexican-American population and lowest in non-Hispanic whites, a finding that remains true despite a steady decline in overall prevalence in the U.S. The HSV-1 contribution to genital herpetic disease has increased over the past two decades. The percentage of people who randomly tested positive for HSV-1 only and had a diagnosis of genital herpes increased to 1.8% in NHANES survey 1999–2004 when compared to 0.4% in the NHANES survey 1988–1994. A retrospective evaluation of college students with newly diagnosed genital infection from 1993 to 2001 showed a striking increase of HSV-1 as a cause of symptomatic genital herpes from 30.9% in 1993 to 77.6% in 2001.

Fig. 4 Trends in age-adjusted prevalence of herpes simplex 1 among persons aged 14–49, for the total population and by race and Hispanic origin: United States, 1999–2000 through 2015–2016. 1Significant decreasing linear trend over time, p o 0.05. NOTES: Age adjusted by the direct method to the 2000 U.S. Census population, using age groups 14–19, 20–29, 30–39, and 40–49 years. Total population includes all race and Hispanic-origin groups including those not shown separately. Data for the Asian subpopulation are only available since 2011, so this subpopulation is not shown separately, but included in the total population. Access data table at https://www.cdc.gov/nchs/data/databriefs/db304_table.pdf#2. Source: NCHS, National Health and Nutrition Examination Survey, 1999–2016.

Herpes Simplex Virus 1 and 2 (Herpesviridae)

409

Geographic location, socioeconomic status, and age influence the occurrence of HSV infection, regardless of the mode of assessment. The global estimates of the incidence and prevalence of HSV-1 infections vary by region, being around 67%, and highest in African, Southeast Asian and Western Pacific countries. The prevalence of HSV-1 is higher and occurs earlier in childhood outside of Europe and the Americas, with estimates reaching 90% in Africa by 10 years of age. As noted, most of these infections are asymptomatic. The annual global incidence of HSV-1 infections is estimated to be 2% in 2012. The contribution of HSV-1 to the incidence of genital infections is less clearly defined in the developing world because prospective studies are lacking. Estimates of HSV-1 genital infections vary by region and are also impacted by both cultural sexual practices and the prevalence of childhood HSV-1 infections. In Africa, for example, the prevalence of genital HSV-1 is estimated to be 0%–0.1% (early childhood HSV-1 prevalence overall of 90%) compared to Eastern Mediterranean region with a 2%–3% prevalence of genital HSV-1 (early childhood HSV-1 prevalence close to 70%). Acquisition of HSV-2 usually occurs in association with onset of sexual activity and directly correlates with the number of lifetime sexual partners. Overall, seroprevalence of HSV-2 in the U.S. decreased in recent years after a significant increase in the mid-1990s. The most recent studies showed total prevalence of 12% during 2015–2016, which represents a decrease from 17% during 1999–2004. HSV-2 remains more common among women when compared to men and non-Hispanic blacks compared to other race and ethnic groups. HSV-2 prevalence is also affected by factors such as age (linear increase with age), poverty (higher in people below poverty line), social status (higher in those who are divorced, separated or widowed), and number of lifetime sexual partners (3.8% in people who reported one partner compared to 39.9% in people reporting Z 50 sexual partners lifetime). Previous studies had indicated that men who have sex with men (MSM) have an increased prevalence of HSV-2. More recently, the NHANES 2001–2006 study did not detect a difference in HSV-2 prevalence overall between homosexual, bisexual or heterosexual men; however, there was a slightly increased prevalence of HSV-2 in homosexual individuals with HIV infection compared to heterosexual men. Worldwide estimates of HSV-2 infection in 2012 indicated an overall prevalence of 11.3%. Estimates vary significantly by gender and geographic area. Prevalence in females is higher, compared to males (14.8% versus 8%) and was highest in Africa with a prevalence of 31.5%. The estimated incidence of HSV-2 infections also differs by gender and geographic areas, being typically higher in the younger age groups. A decline of incidence with age was observed in areas with higher prevalence, which could be attributed to age-related behavioral changes or decrease in susceptible people at older ages. In general, women acquire HSV-2 infection more frequently than do men, irrespective of the number of partners. For pregnant women, approximately 1% will excrete virus at the time of delivery. Nevertheless, the incidence of neonatal HSV infection is only approximately 1 in 1500 live born infants in the U.S. The incidence of neonatal HSV infection is affected by multiple factors, the most important of which is the maternal infection status. Pregnant women who contract genital HSV for the first time during the third trimester have the highest risk for transmitting infection to the fetus, as 57% of those neonates develop neonatal HSV infection. The rate of exposed neonates developing the disease decreases to 25% when pregnant women contract a first genital HSV infection but have a prior history of HSV infection of the other species. The risk drops significantly to 2% at most, for neonates born to women with established recurrent genital HSV of either species. The decrease in neonatal HSV infection with documented recurrent genital HSV infection is likely due to the protective transplacental antibodies. Cesarean delivery reduces the risk of neonatal HSV infection by avoiding exposure to viral shedding in maternal vaginal secretions. Other risk factors for neonatal HSV infection, particularly in women with established genital infection, include application of a fetal scalp electrode, virus species (higher risk with HSV-1 versus HSV-2), and duration of membrane rupture. Global estimates of annual neonatal HSV incidence is about 14,257 neonates from 2010 to 2015 with HSV-2 being responsible for about two thirds of the total number. The global rate of neonatal HSV is estimated to be around 10.3 per 100,000 live births. The estimated global annual incidence of neonatal HSV disease is significantly lower than that of the U.S. The reason behind this significant difference is not clear yet, and further studies are needed to identify potential explanations. Both HSV-1 and HSV-2 seem to contribute almost equally to neonatal HSV diseases in the U.S. This is not the case in other areas of the world where childhood HSV-2 prevalence is significantly higher, as in Africa where almost all neonatal HSV cases are thought to be due to HSV-2. Nosocomial HSV infection has been documented both in newborn nurseries as well as in intensive care units.

Clinical Features Mucocutaneous Infections Gingivostomatitis Mucocutaneous infections are the most common clinical manifestations of HSV-1 and HSV-2. Gingivostomatitis is usually caused by HSV-1 and occurs most frequently in children under five years of age. Clinical disease is characterized by fever, sore throat, pharyngeal edema, and erythema, followed by the development of vesicular or ulcerative lesions of the oral or pharyngeal mucosa. Skin lesions are usually grouped vesicles on an erythematous base. Mucous membrane vesicles usually rupture, resulting in a painful ulcerative lesion on an erythematous base, which is prone to bleeding. In children, clinical fussiness occurs and is associated with decreased oral intake that may lead to dehydration. Auto-inoculation of HSV from oral lesions to fingers results in herpetic whitlow, not uncommon in the pediatric population.

410

Herpes Simplex Virus 1 and 2 (Herpesviridae)

Another manifestation of mucocutaneous HSV is eczema herpeticum, which develops in patients with skin eczema and may not be limited to one area, but rather involves multiple patches of the skin affected by poorly controlled eczema. Mucocutaneous HSV lesions usually heal without leaving a scar at the site of infection. Recurrent HSV-1 infections of the oropharynx frequently manifest as herpes simplex labialis (cold sores) and appear on the vermilion border of the lip. Intraoral lesions, as a manifestation of recurrent disease, are uncommon in the normal host but do occur frequently in the immunocompromised host.

Genital herpes HSV-2 was the major cause of genital herpes in the past, but recent studies indicate that probably over half of new genital herpes cases are caused by HSV-1. Primary infection in women usually involves the vulva, vagina, and cervix. In men, initial infection is most often associated with lesions on the glans penis, prepuce, or penile shaft. In individuals of either sex, primary disease is associated with fever, malaise, anorexia, and bilateral inguinal adenopathy. Women frequently have dysuria and urinary retention due to urethral involvement. As many as 10% of individuals will develop an aseptic meningitis with primary infection. Aseptic meningitis, often presenting as recurrent disease known as Mollaret’s meningitis, usually presents with fever, photophobia, headache, and neck stiffness. It is more likely to happen in women with genital HSV-2 infections. Sacral radiculomyelitis may occur in both men and women, resulting in neuralgias, urinary retention, or obstipation. The complete healing of primary infection may take several weeks. The first episode of genital infection is less severe in individuals who have had previous HSV infections at other sites, such as herpes simplex labialis. Primary genital HSV is more likely to be asymptomatic regardless of viral species. Recurrent lesions with viral shedding could also be asymptomatic, explaining the high incidence of new infections. Recurrent genital infections in either men or women can be particularly distressing. The frequency of recurrence varies significantly from one individual to another. For those with HSV-2 infection, approximately one-third of individuals with genital herpes have virtually no recurrences, one-third have approximately three recurrences per year, and another third have more than three per year, while those individuals with HSV-1 infection are much less likely to suffer recurrences. By applying polymerase chain reaction assays to genital swabs from women with a history of recurrent genital herpes, viral DNA can be detected in the absence of culture proof of infection. Further, HSV DNA can be detected in over 20% of days in women. This finding suggests the chronicity of genital herpes in some women, as opposed to a recurrent infection. Epidemiological studies have also shown that HSV-2 infection increases the risk of human immunodeficiency virus (HIV) acquisition and transmission. It is postulated that inflammation in the genital tract caused by HSV-2 infection leads to increased activated CD4 þ T cells that are the host cells for HIV infection, and this would lead to increased acquisition and transmission of HIV. This is of importance because genital herpes is potentially a major risk factor for HIV infection.

Herpetic keratitis HSV keratitis is usually caused by HSV-1, either primary or recurrent and is accompanied by conjunctivitis in many cases. It is considered among the most common infectious causes of blindness in the U.S. The characteristic lesions of HSV keratoconjunctivitis are dendritic ulcers best detected by fluorescein staining. Deep stromal involvement has also been reported and may result in visual impairment. Acute necrotizing retinitis is a rare complication that leads to painless loss of vision. Chorioretinitis is another rare HSV ophthalmic complication that could develop in neonates or immunocompromised patients with HSV infections.

Other skin manifestations HSV infections can manifest at any skin site. Common among health-care workers are lesions on abraded skin of the fingers, known as herpetic whitlows. Similarly, because of physical contact, wrestlers may develop disseminated cutaneous lesions known as herpes gladiatorum. HSV infections have also been recognized as a trigger for erythema multiforme.

Neonatal Herpes Simplex Virus Infection The incidence of neonatal HSV is estimated to be 1 in 1500 live births in the U.S. Most (85%) affected neonates acquire HSV from exposure to infected secretions during birth (intrapartum acquisition), a small number (10%) acquire the virus postnatally, and rarely (B5%) the virus is acquired in utero, resulting in congenital HSV infection. Manifestations of neonatal HSV infection can be divided into three categories: (a) skin, eye, and mouth disease (SEM) (45% of all neonatal HSV cases); (b) CNS disease (30%); and (c) disseminated infection (25%). Most HSV infections during the neonatal period present with fever, lethargy, poor feeding, skin lesions, or seizures. In SEM, as the name implies, skin, eye, and mouth disease consists of cutaneous lesions (in 480%) and does not involve other organ systems. Involvement of the central nervous system may occur with encephalitis or disseminated infection and generally results in a diffuse encephalitis and results in seizures and lethargy. Cerebrospinal fluid analysis characteristically reveals an elevated protein and a mononuclear pleocytosis. Disseminated infection involves multiple organ systems and can produce disseminated intravascular coagulation, hemorrhagic pneumonitis, encephalitis, hepatitis, and cutaneous lesions. Diagnosis can be particularly difficult in the absence of skin lesions. The mortality rate for each disease classification varies from zero for skin, eye, and mouth disease to 15% for encephalitis and 60% or higher for neonates with disseminated infection in the absence of therapy. In addition to the high mortality associated with these infections, morbidity is significant in that children with encephalitis or disseminated disease develop normally in only approximately 40% of cases, even with the administration of appropriate antiviral therapy. Congenital HSV (acquired in utero, about 5% of neonatal HSV cases) manifests as a triad of skin

Herpes Simplex Virus 1 and 2 (Herpesviridae)

411

involvement (lesions, scarring and change of pigmentation), CNS involvement (calcifications and microcephaly), and ophthalmologic involvement (chorioretinitis and optic atrophy). Affected neonates are usually very ill.

Herpes Simplex Encephalitis HSV encephalitis is characterized by hemorrhagic necrosis of the inferomedial portion of the temporal lobe. Disease begins unilaterally, then spreads to the contralateral temporal lobe. It is the most common cause of focal, sporadic encephalitis in the U.S. today and occurs in approximately 1 in 250,000 to 500,000 individuals. Most cases are caused by HSV-1. The actual pathogenesis of herpes simplex encephalitis is unknown, although it has been speculated that primary or recurrent virus can reach the temporal lobe by ascending neural pathways, such as the trigeminal tracts or the olfactory nerves. Clinical manifestations of herpes simplex encephalitis include headache, fever, altered consciousness, and abnormalities of speech and behavior. Focal seizures may also occur. The cerebrospinal fluid formulae for these patients is variable, but usually consists of a pleocytosis of monocytes. The protein concentration is characteristically elevated, and glucose is usually normal. Red blood cells may or may not be present. Historically, a definitive diagnosis could be achieved only by brain biopsy, because other pathogens may produce a clinically similar illness. However, the application of polymerase chain reaction (PCR) for detection of virus DNA has replaced brain biopsy as the standard for diagnosis. The mortality and morbidity are high, even when appropriate antiviral therapy is administered. At present, the mortality rate is approximately 14%–19% even with appropriate intravenous (IV) acyclovir (mortality exceeds 70% without effective therapy). Approximately 50% of survivors will have significant neurologic sequelae.

Herpes Simplex Virus Infections in the Immunocompromised Host HSV infections in the immunocompromised host are clinically more severe, may be progressive, and require more time for healing. Manifestations of HSV infections in this patient population include pneumonitis, esophagitis, hepatitis, colitis, and disseminated cutaneous disease. Individuals suffering from human immunodeficiency virus infection may have extensive perineal or orofacial ulcerations. HSV infections are also noted to be of increased severity in individuals who are burned.

Pathogenesis Acquisition of HSV infections requires intimate contact between a person who is shedding virus and a susceptible host. After inoculation of HSV onto the skin or mucous membrane and an incubation period of 4–6 days, clinical disease may occur or, as is more common, the infection will be asymptomatic. HSV replicates in epithelial cells and fibroblasts. As replication continues, cell lysis and local inflammation ensue, resulting in characteristic vesicles on an erythematous base. Regional lymphatics and lymph nodes become involved; therefore, viremia and visceral dissemination may develop, depending upon the immunologic competence of the host. In all hosts, the virus generally ascends peripheral sensory nerves and reaches sensory ganglia. Replication of HSV within neural tissue is followed by anterograde axonal spread of the virus back to other mucosal and skin surfaces via the peripheral sensory nerves. Virus replicates further in the epithelial cells, reproducing the lesions of the initial infection, until infection is contained through both systemic and mucosal immune responses. The initial host response to HSV infection is derived mainly from the innate immune system including natural killer (NK) cells, neutrophils, B cells, and T cells, as well as production of cytokines and recruitment of adaptive and humoral immune response. HSV-specific antibodies and, subsequently, CD8 and CD4 lymphocytes develop with time. Prevention of latent infection is not possible; thus, latency is inevitable. The production of HSV-specific neutralizing antibodies contributes to the control of infection and is also used in laboratory testing to detect the viral infection. Latency is established when HSV reaches the dorsal root ganglia after retrograde transmission via sensory nerve pathways. The virus does not actively replicate during latency. Reactivation of latent infection results in one of two outcomes: asymptomatic viral shedding (more likely) or reactivation that results in disease with a clinical presentation similar to, but usually milder than, the original symptomatic infection. Reactivation of a latent infection may be symptomatic despite the asymptomatic nature of the original primary infection. Rarely, HSV can infect the central nervous system and cause encephalitis. The focality and temporal lobe affinity suggest direct extension of virus along neural tracts. Encephalitis caused by HSV is characterized by necrosis of the inferior medial portion of the temporal lobe, initially unilaterally and then contralaterally. This necrotic process accounts for the high morbidity and mortality of infection. Specific genetic mutations in the innate immune system make affected hosts susceptible to severe or recurrent disease. For example, toll-like receptor 3 (TLR-3) mutations have been reported to increase the risk for HSE in pediatric and young adults from the same family. Infection of the neonate is usually the consequence of direct contact with infected maternal genital secretions, accounting for approximately 85% of cases of neonatal herpes. The remaining 15% are caused by in utero infection (5%), secondary to viremia, or postnatal acquisition whereby the baby comes in contact with infectious virus in the environment.

Diagnosis The diagnosis of HSV infections is predicated on clinical evaluation of mucocutaneous manifestations. However, confirmation of the diagnosis requires isolation of HSV in appropriate cell culture systems or the detection of viral gene products or, alternatively,

412

Herpes Simplex Virus 1 and 2 (Herpesviridae)

the detection of viral DNA by polymerase chain reaction (PCR). HSV grows readily in tissue culture, producing cytopathic effects within a few days in a wide variety of mammalian cell lines. Routine testing to distinguish HSV-1 from HSV-2 in the isolate is important for genital isolates because HSV-1 is much less likely to recur. PCR is an important method for diagnosing HSV infections, particularly those involving the central nervous system. The detection of HSV DNA by PCR in the CSF has replaced brain biopsy as a method of diagnosis of central nervous system infections. Serologic assays that distinguish HSV-1-versus HSV-2-specific antibodies are commercially available. The utilization of immunoblot detection of specific glycoproteins that distinguish HSV-1 from HSV-2, namely, glycoprotein G-1 (gG-1) and gG-2, are available for determining prior exposure to HSV-1 and HSV-2 infections. Serologic testing is also used for population-based studies to identify the prevalence of the disease. Historically, Tzanck smears have been used to diagnose HSV infections. Tzanck smears are not sensitive enough for routine diagnostic purposes. However, immunofluorescent staining of cell trap preparations from lesions is both sensitive and specific for the diagnosis for HSV infections.

Treatment Infections due to HSV are amenable to treatment. Acyclovir and its pro-drug (valacyclovir) as well as famciclovir (pro-drug of penciclovir) are the mainstays of treatment. Acyclovir is mono-phosphorylated by the viral TK, and di- and tri-phosphorylates are added by cellular enzymes. Acyclovir tri-phosphate is recognized specifically by the viral DNA polymerase and incorporated into the growing viral DNA chain. Acyclovir causes chain termination and inhibits viral DNA synthesis. Valacyclovir is a pro-drug that metabolizes to acyclovir and results in good serum levels, similar to levels achieved with IV acyclovir. Famciclovir undergoes metabolic conversion to penciclovir which is active against HSV. Dose, route, and duration of therapy vary considerably depending on the site of infection and status of the patient. The use of high-dose acyclovir and addition of suppressive therapy has improved outcomes significantly in neonatal HSV infections. Primary mucocutaneous infections in the immunocompetent host are treated with oral acyclovir or valacyclovir for 7–10 days; famciclovir can also be used. Valacyclovir is FDA-approved in the pediatric population 12 years of age or older. Recurrent orolabial HSV infection can be treated with any of these three drugs. Suppressive therapy may be considered for patients with very frequent recurrent lesions. Patients with genital HSV infection can be treated with acyclovir, valacyclovir, or famciclovir. IV acyclovir should be considered in the initial phase of treating severe orolabial disease leading to decreased oral intake in a pediatric patient or in an adult with genital herpes associated with aseptic meningitis. Treatment of genital herpes in the HIV-infected patient may be followed by long term suppressive therapy with acyclovir, valacyclovir or famciclovir. Topical therapy with one of several antiviral ophthalmic preparations is appropriate for HSV keratoconjunctivitis. However, the treatment of choice is Viroptic or trifluorothymidine. Secondary choices include vidarabine ophthalmic or topical idoxuridine. Consultation with an ophthalmologist for confirmation of the diagnosis and management of HSV ocular disease is indicated. Neonatal HSV infection should always be treated with IV acyclovir. The duration of treatment varies: CNS and disseminated diseases are treated for 21 days and SEM only 14 days. All forms of neonatal HSV should be followed by a minimum of 6 months of suppressive therapy with oral acyclovir. Neonatal disease involving the eyes should also be treated with topical ophthalmic drugs (trifluridine or topical ganciclovir), and an ophthalmologist consulted. IV acyclovir is the mainstay of HSV encephalitis during adulthood. Encephalitis patients require longer treatment duration of 14–21 days as compared to 7–10 days for aseptic meningitis. Acyclovir resistance can develop. Resistance is usually related to viral TK mutations. The treatment of choice for infections caused by acyclovir-resistant strains is foscarnet. Consultation with specialists is recommended when resistance is known or suspected.

Prevention At the present, there is no licensed vaccine for the prevention for HSV infections. Vaccines in clinical testing include DNA vaccines, replication-defective viruses, and protein subunits with new adjuvants. Consequently, prevention of HSV infections resides in the most part on knowledge of the mechanisms of transmission, including person to person as well as in the hospital environment. Individuals with known recurrent HSV infections should be counseled on the possibility of transmission of infection while lesions are present. The use of condoms for individuals with recurrent genital herpes is encouraged in that detection of HSV DNA by PCR can occur even in the absence of lesions. Similarly, for individuals who have recurrent herpes labialis, kissing should be discouraged. There is a risk of nosocomial transmission of HSV within the hospital environment. Because many individuals shed HSV in the absence of clinical symptoms, it is impossible to exclude all workers from the hospital environment who could transmit infection. Thus, many authorities simply recommend strict hand washing and covering of lesions, should they exist. Finally, no data exist on the prevention of neonatal HSV infection. It has been theorized that anticipatory administration of acyclovir to babies delivered through an infected birth canal may prove of value, particularly for women who have first episode genital herpetic infection. However, no data exist to substantiate this hypothesis. Because over 1% of all women at delivery excrete HSV and the rate of neonatal HSV infection is only 1 in 1500 live born infants as noted earlier, the routine administration of acyclovir to all children born to HSV-positive women is not reasonable. Alternative approaches, namely administration of acyclovir to known HSV-2-infected women is gaining acceptance.

Herpes Simplex Virus 1 and 2 (Herpesviridae)

413

Further Reading Baines, J.D., 2011. Herpes simplex virus capsid assembly and DNA packaging: A present and future antiviral drug target. Trends Microbiology 19, 606–613. Dai, X., Zhou, Z.H., 2018. Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes. Science 360, eaao7298. Johnston, C., Gottlieb, S.L., Wald, A., 2016. Status of vaccine research and development of vaccines for herpes simplex virus. Vaccine 34, 2948–2952. Knipe, D.M., Lieberman, P.M., Jung, J.U., et al., 2013. Snapshots: Chromatin control of viral infection. Virology 435, 141–156. Kurt-Jones, E.A., Orzalli, M.H., Knipe, D.M., 2017. Innate immune mechanisms and herpes simplex virus infection and disease. In: Osterrieder, K. (Ed.), Cell Biology of Herpes Viruses. Advances in Anatomy, Embryology and Cell Biology 223. Springer, pp. 49–75. McQuillan, G., Kruszon-Moran, D., Flagg, E.W., Paulose-Ram, R., 2018. Prevalence of herpes simplex virus type 1 and type 2 in persons aged 14-49: United States, 2015–2016. NCHS Data Brief 304, 1–8. Orzalli, M.H., Knipe, D.M., 2014. Cellular sensing of viral DNA and viral evasion mechanisms. Annual Reviews of Microbiology 68, 477–492. Roizman, B., Knipe, D.M., Whitley, R.J., 2013. Herpes simplex virus. In: Knipe, D.M., Howley, P. (Eds.), Fields Virology, sixth ed. Philadelphia: Lippincott Williams & Wilkins, pp. 1823–1897. Whitley, R.J., Roizman, B., 2017. Herpes simplex viruses. In: Richman, D.D., Whitley, R.J., Hayden, F.G. (Eds.), Clinical Virology, fourth ed. Washington DC: ASM Press, pp. 415–445.

Relevant Websites https://www.cdc.gov/std/herpes/default.htm Genital Herpes. https://www.who.int/news-room/fact-sheets/detail/herpes-simplex-virus Herpes simplex virus World Health Organization.

History of Virology: Vertebrate Viruses F Fenner, Australian National University, Canberra, ACT, Australia r 2008 Elsevier Ltd. All rights reserved. This is a reproduction of F. Fenner, History of Virology: Vertebrate Viruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00424-6.

Introduction As in other branches of experimental science, the development of our knowledge of viruses has depended on the techniques available. Initially, viruses were studied by pathologists interested in the causes of the infectious diseases of man and his domesticated animals and plants and these concerns remain the main force advancing the subject. The idea that viruses might be used to probe fundamental problems of biology arose in the early 1940s, with the development of the knowledge of bacterial viruses. These studies helped establish the new field of molecular virology in the period 1950–70, and this has revolutionized the study of viruses and led to an explosion of knowledge about them.

The Word Virus Since antiquity the term virus had been synonymous with poison, but during the late nineteenth century it became a synonym for microbe (Pasteur's word for an infectious agent). It did not acquire its present connotation until the 1890s, after the bacterial or fungal causes of many infectious diseases had been discovered, using the agar plate, effective staining methods, and efficient microscopes. It then became apparent that there were a number of infectious diseases of animals and plants from which no bacterium or fungus could be isolated or visualized with the microscope. After the introduction in 1884 of the Chamberland filter, which held back bacteria, Loeffler and Frosch demonstrated that the cause of foot-and-mouth disease was a filterable (or ultramicroscopic) virus. The first compendium of all then known viruses was edited by T. M. Rivers of the Rockefeller Institute and published in 1928. Entitled Filterable Viruses, this emphasized that viruses required living cells for their multiplication. In the 1930s chemical studies of the particles of tobacco mosaic virus and of bacteriophages showed that they differed from all cells in that at their simplest they consisted of protein and nucleic acid, which was either DNA or RNA. Gradually, the adjectives filterable and ultramicroscopic were dropped and the word viruses developed its present connotation.

Early Investigations Foot-and-Mouth Disease Virus In 1898 F. J. Loeffler and P. Frosch described the filterability of an animal virus for the first time, noting that “the filtered material contained a dissolved poison of extraordinary power or that the as yet undiscovered agents of an infectious disease were so small that they were able to pass through the pores of a filter definitely capable of retaining the smallest known bacteria.” Although the causative agent of foot-and-mouth disease passed through a Chamberland-type filter, it did not go through a Kitasato filter which had a finer grain. This led to the conclusion that the causative virus, which was multiplying in the host, was a corpuscular particle. Loeffler and Frosch gave filtration a new emphasis by focussing attention on what passed through the filter rather than what was retained and established an experimental methodology which was widely adopted in the early twentieth century in research on viral diseases.

Yellow Fever Virus Following the acceptance of the notion of filterable infectious agents, pathologists investigated diseases from which no bacteria could be isolated and several were soon shown to be caused by viruses. One of the most fruitful investigations, in terms of new concepts, was the work of the United States Army Yellow Fever Commission headed by Walter Reed in 1900–01. Using human volunteers, they demonstrated that yellow fever was caused by a filterable virus which was transmitted by mosquitoes and that the principal vector was Aedes aegypti. They also showed that infected persons were infectious for mosquitoes only during the first few days of the disease and that mosquitoes were not infectious until 7–10 days after imbibing infectious blood, thus defining the extrinsic incubation period and establishing essentially all of the basic principles of the epidemiology of what came to be called arboviruses (arthropod-borne viruses).

414

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00424-0

History of Virology: Vertebrate Viruses

415

Physical Studies of Viruses Further advances in understanding the nature of viruses depended on physical and chemical studies. As early as 1907 H. Bechhold in Germany developed filters of graded permeabilities and a method of determining their pore size. Subsequently, W. J. Elford, in London, used such membranes for determining the size of animal virus particles with remarkable accuracy. In Germany, on the eve of World War II, H. Ruska and his colleagues had produced electron microscopic photographs of the particles of tobacco mosaic virus, bacteriophages, and poxviruses. Technical improvements in instrumentation after the war, and the introduction of negative staining for studying the structure of viruses by Cambridge scientists H. E. Huxley in 1957 and S. Brenner and R. W. Horne in 1959 resulted in photographs of the particles of virtually every known kind of virus. These demonstrated the variety of their size, shape, and structure, and the presence of common features such as the icosahedral symmetry of many viruses of animals. Following a perceptive paper on the structure of small virus particles by F. H. C. Crick and J. D. Watson in 1956, in 1962 D. L. D Caspar of Boston and A. Klug of Cambridge, England, produced a general theory of the structure of regular virus particles of helical and icosahedral symmetry. Structure of the virus particle became one of the three major criteria in the system of classification of viruses that was introduced in 1966.

The Chemical Composition of Viruses If the ultramicroscopic particles found in virus-infected hosts were the pathogens, what did these particles consist of? Following observations on plant and bacterial viruses in 1935, in 1940 C. L. Hoagland and his colleagues at the Rockefeller Institute found that vaccinia virus particles contained DNA but no RNA. Thus evidence was accumulating that viruses differed from bacteria not only in size and their inability to grow in lifeless media, but in that they contained only one kind of nucleic acid, which could be either DNA or RNA. The development of restriction-endonuclease digestion of DNA, based on studies of phage restriction by W. Arber (Nobel Prize, 1978) of Basel in the 1960s, and then elaborated by biochemist H. O. Smith of Baltimore in 1970, has simplified the mapping of the genomes of DNA viruses, a study initiated by D. Nathans (Nobel Prize, 1978), also of Baltimore, using Simian virus 40. The development of the polymerase chain reaction in 1985 by K. B. Mullis (Nobel Prize, 1993) revolutionized sequencing methods for both DNA and RNA, leading to the availability of the complete genomic sequences of most viruses and the ability to make diagnoses using minute amounts of material. Investigations of bacterial viruses were motivated by scientific curiosity, but animal virology was developed by pathologists studying the large number of diseases of humans and livestock caused by viruses, and it has retained this practical bias. Over the first two decades of the twentieth century, testing of filtrates of material from a number of infected humans and animals confirmed that they were caused by viruses. Among the most important was the demonstration in 1911 by P. Rous (Nobel Prize, 1966), at the Rockefeller Institute, that a sarcoma of fowls could be transmitted by a bacteria-free filtrate.

The Cultivation of Animal Viruses The first systematic use of small animals for virus research was Pasteur's use of rabbits, which he inoculated intracerebrally with rabies virus in 1881. It was not until 1930 that mice were used for virus research, with intracerebral inoculations of rabies and yellow fever viruses. By using graded dilutions and large numbers of mice, quantitative animal virology had begun. The next important step, initiated by E. W. Goodpasture in 1931–32, was the use of chick embryos for growing poxviruses. This was followed a few years later by the demonstration by F. M. Burnet that many viruses could be titrated by counting the pocks that they produced on the chorioallantoic membrane, whereas others grew well in the allantoic and/or amniotic cavities of developing chick embryos. Tissue cultures had first been used for cultivating vaccinia virus in 1928, but it was the discovery by J. F. Enders, T. H. Weller, and F. C. Robbins of Harvard University (Nobel Prize, 1954) in 1949 that poliovirus would grow in non-neural cells that gave a tremendous stimulus to the use of cultured cells in virology. Over the next few years their use led to the cultivation of medically important viruses such as those causing measles (by J. F. Enders and T. C. Peebles in 1954) and rubella (by T. H. Weller and F. C. Neva in 1962). Even more dramatic was the isolation of a wide variety of new viruses, belonging to many different families. Also the different cytopathic effects produced by different viruses in monolayer cell cultures were found to be diagnostic. The next great advance, which greatly increased the accuracy of quantitative animal virology, occurred in 1952, when the plaque assay method for counting phages was adapted to animal virology by R. Dulbecco (Nobel Prize, 1975), using a monolayer of chick embryo cells in a petridish. In 1958 H. Temin and H. Rubin applied Dulbecco's method to Rous sarcoma virus, initiating quantitative studies of tumor viruses. Biochemical studies of animal virus replication were simplified by using continuous cell lines and by growing the cells in suspension.

Biochemistry During the 1950s virus particles were thought to be ‘inert’ packages of nucleic acid and proteins, although in 1942 G. K. Hirst of the Rockefeller Institute had shown that particles of influenza virus contained an enzyme, later identified by A. Gottschalk of Melbourne as a neuraminidase.

416

History of Virology: Vertebrate Viruses

Further advances in viral biochemistry depended on methods of purification of virus particles, especially the technique of density gradient centrifugation. In 1967 J. Kates and B. R. McAuslan demonstrated the presence of a DNA-dependent RNA polymerase in purified vaccinia virus virions, a discovery that was followed the next year by the demonstration of a doublestranded (ds) RNA-dependent RNA polymerase in reovirus virions and then of single-stranded (ss) RNA-dependent RNA polymerases in virions of paramyxoviruses and rhabdoviruses. In 1970 came the revolutionary discovery by D. Baltimore of Boston (Nobel Prize, 1975) and H. Temin of Wisconsin (Nobel Prize, 1975), independently, of the RNA-dependent DNA polymerase, or reverse transcriptase, of Rous sarcoma virus. Many other kinds of enzymes were later identified in the larger viruses; for example, no less than 16 enzymes have now been identified in vaccinia virus virions. Just as investigations with bacterial viruses were of critical importance in the development of molecular biology, studies with animal viruses have led to the discovery of several processes that have proved to be important in the molecular biology of eukaryotic cells, although many of these discoveries are too recent for serious historical appraisal. Thus, in 1968 M. Jacobson and D. Baltimore of Boston showed that the genomic RNA of poliovirus was translated as a very large protein, which was then cleaved by a protease that was subsequently found in the viral replicase gene. RNA splicing was discovered in 1977, independently by P. A. Sharp (Nobel Prize, 1993) and L. T. Chow and their respective colleagues during studies of adenovirus replication. Work with the same virus by Rekosh and colleagues in London in 1977 resulted in the definition of viral and cell factors for initiation of new DNA strands using a novel protein priming mechanism. Capping of mRNA by m7G and its role in translation was discovered in 1974, during work with reoviruses by A. J. Shatkin. Other processes first observed with animal viruses and now known to be important in eukaryotic cells were the existence of 30 poly A tracts on mRNAs, the pathway for synthesis of cell surface proteins, and the role of enhancer elements in transcription. Analysis of viral nucleic acids progressed in parallel with that of the viral proteins. Animal viruses were found with genomes of ssRNA, either as a single molecule, two identical molecules (diploid retroviruses), or segmented; dsRNA; ssDNA, dsDNA, and partially dsDNA. The so-called unconventional viruses or prions of scrapie, kuru, and Creutzfeld–Jacob disease, which have been extensively investigated since 1957 by D. C. Gajdusek of the US National Institutes of Health (Nobel Prize, 1976) and S. B. Prusiner of the University of California at San Francisco (Nobel Prize, 1997), appear to be infectious proteins. Soon after the discovery that the isolated nucleic acid of tobacco mosaic virus was infectious (see below), it was shown that the genomic RNAs of viruses belonging to several families of animal viruses were infectious; namely those with single, positive-sense RNA molecules. Then in 1973 came the discovery of recombinant DNA by P. Berg of Stanford University and his colleagues, using the animal virus SV40 and bacteriophage l.

Structure of the Virion Using more sophisticated methods of electron microscopy, many animal virus virions have been shown to be isometric icosahedral structures, or roughly spherical protein complexes surrounded by a lipid-containing shell, the envelope, which contains a number of virus-coded glycoprotein spikes. X-ray crystallography of crystals of purified isometric viruses has revealed the molecular structure of their icosahedra; similar revealing detail has been obtained for the neuraminidase and hemagglutinin spikes of influenza virus.

Tumor Virology After the discovery of Rous sarcoma virus in 1911, there was a long interval before the second virus to cause tumors, rabbit papilloma virus, was discovered by R. E. Shope in 1933. However, after that viruses were found to cause various neoplasms in mice, and in 1962 J. J. Trentin of Yale University showed that a human adenovirus would produce malignant tumors in hamsters. Since then, direct proof has been obtained that several DNA viruses (but not adenoviruses) and certain retroviruses can cause tumors in humans. Molecular biological studies have shown that oncogenicity is largely caused by proteins they produce that are encoded by viral oncogenes, a concept introduced by R. J. Huebner and G. Todaro of the US National Institutes of Health in 1969 and corrected, refined, and greatly expanded since the mid-1970s by J. M. Bishop, H. Varmus (Nobel Prize, 1989), and R. A. Weinberg of Boston. Only one group of RNA viruses, the retroviruses, which replicate through an integrated DNA provirus, cause neoplasms, whereas viruses of five groups of DNA viruses are tumorigenic. Study of these oncogenic viruses has shed a great deal of light on the mechanisms of carcinogenesis. Oncogenic DNA viruses contain oncogenes as an essential part of their genome, which when integrated into the host cell DNA may promote cell transformation, whereas the oncogenes of retroviruses are derived from protooncogenes of the cell.

Impact on Immunology Immunology arose as a branch of microbiology and several discoveries with animal viruses were important in the development of important concepts in immunology. It was the discovery in the 1930s by E. Traub in Tübingen of persistent infection of mice with

History of Virology: Vertebrate Viruses

417

lymphocytic choriomeningitis virus that led to the development by F. M. Burnet (Nobel Prize, 1960) of the concept of immunological tolerance in 1949. It was work with the same virus that led to the discovery of MHC restriction by P. C. Doherty and R. Zinkernagel in Canberra (Nobel Prize, 1996) in 1974.

Vaccines and Disease Control From the time of Pasteur's use of rabies vaccine in 1885, a major concern of medical and veterinary virologists has been the development of vaccines. Highlights in this process have been the development of the 17D strain of yellow fever virus by M. Theiler (Nobel Prize, 1951) of the Rockefeller Foundation laboratories in 1937, the introduction of influenza vaccine in 1942, based on Burnet's 1941 discovery that the virus would grow in the allantois, the licensing in 1954 of inactivated poliovaccine developed by J. Salk and in 1961 of the live poliovaccine introduced by A. B. Sabin, both based on the cultivation of the virus in 1949 by Enders, and development of the first genetically engineered human vaccine with the licensing of yeast-grown hepatitis B vaccine in 1986. Finally, 1977 saw the last case of natural smallpox in the world and thus the first example of the global eradication of a major human infectious disease, the result of a 10-year campaign conducted by the World Health Organization with a vaccine directly derived from Jenner's vaccine, first used in 1796.

Recognition of HIV-AIDS In 1981 a new disease was described in the male homosexual populations of New York, San Francisco, and Los Angeles. It destroyed the immune system, and the disease was called the acquired immune deficiency syndrome (AIDS) and the causal virus, human immunodeficiency virus (HIV), a retrovirus, was first isolated by L. Montagnier, of the Pasteur Institute in Paris, in 1983. A sexually transmitted disease, it has now spread all over the world, with about 40 million cases worldwide, and without very expensive chemotherapy is almost always fatal. It is now clear that it arose from an inapparent but persistent infection of chimpanzees in Africa. In spite of enormous efforts over the past 20 years, it has so far been impossible to devise an effective vaccine.

Arthropod-Borne Viruses of Vertebrates Arthropods, mainly insects and ticks, were early shown to be important as vectors of virus diseases of vertebrates (the arboviruses) and of plants. Sometimes, as in myxomatosis, carriage was found to be mechanical, but in most cases the virus was found to multiply in the vector as well as the vertebrate concerned. The development of methods of growing insect cells in culture by T. D. C. Grace in Canberra in 1962 opened the way for the molecular biological investigation of the replication of insect viruses and arboviruses in invertebrate cells.

Taxonomy and Nomenclature After earlier tentative efforts with particular groups of viruses, viral nomenclature took off in 1948, with the production of a system of latinized nomenclature for all viruses by the plant virologist F. O. Holmes. This stimulated others, in particular C. H. Andrewes of London and A. Lwoff of Paris, to actions which resulted in the setting up of an International Committee on Nomenclature (later Taxonomy) of Viruses in 1966. Adopting the kind of viral nucleic acid, the strategy of replication, and the morphology of the virion as its primary criteria, this committee has now achieved acceptance of its decisions by the great majority of virologists. One interesting feature of its activities is that its rules avoid the controversies about priorities that plague taxonomists working with fungi, plants, and animals.

The Future The future will see an explosive expansion of understanding and knowedge of viruses of vertebrates, with the application of techniques of genetic engineering, nucleic acid sequencing, the polymerase chain reaction, and the use of monoclonal antibodies. All these discoveries are addressed in the appropriate sections of this encyclopedia. Molecular biology, which was initially conceived during studies of bacterial viruses, is now being used to study all the viruses of vertebrates and the pathogenesis and epidemiology of animal viral diseases. The expansion in knowledge of these subjects in the next decade can be expected to exceed that of the previous century.

See also: An Introduction to Plant Viruses. History of Virology: Bacteriophages

418

History of Virology: Vertebrate Viruses

Further Reading Bos, L., 1995. The embryonic beginning of virology: Unbiased thinking and dogmatic stagnation. Archives of Virology 140, 613. Cairns, J., Stent, G.S., Watson, J.D. (Eds.), 1962. Phage and the Origins of Molecular Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory of Quantitative Biology. Fenner, F., Gibbs, A.J. (Eds.), 1988. Portraits of Viruses. A History of Virology. Basel: Karger. Horzinek, M.C., 1995. The beginnings of animal virology in Germany. Archives of Virology 140, 1157. Hughes, S.S., 1977. The Virus. A. History of the Concept. London: Heinemann Educational Books. Joklik, W.K., 1996. Famous institutions in virology. The Department of Microbiology, Australian National University and the Laboratory of Cell Biology, National Institute of Allergy and Infectious Diseases. Archives of Virology 141, 969. Matthews, R.E.F. (Ed.), 1983. A Critical Appraisal of Viral Taxonomy. Boca Raton, FL: CRC Press. Porterfield, J.S., 1995. Famous institutions in virology. The National Institute of Medical Research, Mill Hill. Archives of Virology 141, 969. van Helvoort, T., 1994. History of virus research in the twentieth century: The problem of conceptual continuity. History of Science 32, 185. Waterson, A.P., Wilkinson, L., 1978. An Introduction to the History of Virology. Cambridge: Cambridge University Press.

Human Boca- and Protoparvoviruses (Parvoviridae) Maria Söderlund-Venermo, University of Helsinki, Helsinki, Finland Jianming Qiu, University of Kansas Medical Center, Kansas City, KS, United States r 2021 Elsevier Ltd. All rights reserved.

Nomenclature AAV adeno-associated virus, in genus Dependoparvovirus AGE acute gastroenteritis ARTI acute respiratory tract infection B19V human parvovirus B19, in Erythroparvovirus BocaSR bocavirus-transcribed small RNA BPV1 bovine parvovirus 1, in Bocaparvovirus BuV bufavirus, in Protoparvovirus CPV canine parvovirus, in Protoparvovirus CSF cerebrospinal fluid CuV cutavirus, in Protoparvovirus CTCL cutaneous T-cell lymphoma dsDNA double-stranded DNA EIA enzyme immunoassay HAE human airway epithelium HAE-ALI human airway epithelium cultured at an air–liquid interface HBoV human bocavirus, in Bocaparvovirus HEK293 human embryonic kidney 293 cells IgG immunoglobulin G IgM immunoglobulin M kDa kilo Dalton LEH left-end hairpin (of the genome) MVC minute virus of canines, in Bocaparvovirus

Glossary Cutaneous T-cell lymphoma (CTCL) A rare form of cancer affecting mature T cells in skin, forming skin lesions. CTCL is a class of non-Hodgkin lymphoma with unknown etiology, and is divided into several subtypes, including mycosis fungoides and Sézary syndrome. HAE-ALI Human airway epithelium cultured at an air-liquid interface: Human bronchial/tracheal epithelial cells are cultured on microporous membrane inserts in wells. After 3–4 weeks at an air–liquid interface, cells are polarized into apical side that is exposed to the air and basolateral side that is dipped into liquid media.

MVM minute virus of mice, in Protoparvovirus NCR noncoding region (of genome) NP1 nuclear phosphoprotein 1 NPS nasopharyngeal sample NS1–4 non-structural protein 1–4 nt nucleotide OAS original antigenic sin (an immunological phenomenon) ORF open reading frame (pA)d distal polyadenylation site (pA)p proximal polyadenylation site PCR polymerase chain reaction PLA2 phospholipase A2 Pre-mRNA precursor mRNA qPCR quantitative PCR REH right-end hairpin (of genome) RT-PCR reverse transcription PCR ssDNA single-stranded DNA TuV tusavirus, in Protoparvovirus VP1–3 viral capsid proteins 1–3, with long common C-termini VP1u a short unique N-terminus of VP1, which is not shared with VP2 or 3

IgG avidity The avidity, or binding force, of IgG to its antigen increases with the slowly maturing immune response after acute primary infection and can be measured by a protein-denaturing EIA disrupting the weak antigen-antibody bond in acute infection but not the stronger bond in past immunity. Original antigenic sin (OAS) An immunological phenomenon (long-known from influenza and dengue virus infections) observed when a prior virus infection of an individual fully or partly hinders the induction of antibodies to the currently infecting related virus, and instead the original virus-specific IgG response is enhanced.

Introduction Novel parvovirus sequences have been obtained after filtration, DNase treatment, and random PCR, followed by large-scale sequencing and bioinformatics. The first human bocavirus, HBoV1, was discovered in 2005 in airway samples of children with acute respiratory tract infection (ARTI). HBoV1 causes mild to life-threatening ARTI in young children worldwide. It is thus the second human-pathogenic parvovirus after parvovirus B19 (B19V). Three other closely related human bocaviruses, HBoV2, 3, and 4, have since been discovered in stool, but their association with acute gastroenteritis (AGE) is disputed. More recently, in 2012–2016, three novel human protoparvoviruses were discovered in stool samples of children with diarrhea and named bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV). BuV has been associated with gastroenteritis and CuV with cutaneous T-cell lymphoma (CTCL), while more studies are needed to reveal if TuV really is a genuine human virus.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21224-7

419

420

Human Boca- and Protoparvoviruses (Parvoviridae)

Classification Parvoviruses are small non-enveloped icosahedral (T ¼ 1) viruses with linear single-stranded (ss) DNA genomes forming double-stranded (ds) hairpin structures at both ends. The family Parvoviridae includes two subfamilies, Parvovirinae with 8 genera comprising viruses infecting vertebrates and Densovirinae with 5 genera comprising viruses infecting invertebrates, but with a third subfamily, Hamaparvovirinae, proposed with viruses of both vertebrates and invertebrates. Human parvoviruses are found in 5 different genera, Eryhtroparvovirus, Dependoparvovirus, Tetraparvovirus, Bocaparvovirus and Protoparvovirus, with human bocaviruses (HBoVs) and bufavirus (BuV), tusavirus (TuV), and cutavirus (CuV), belonging to the two latter genera, respectively. Virus species within the genera are monophyletic and their NS1 protein sequences (with some flexibility) are 430% identical to each other but o30% identical to those of other genera, which are general taxonomic criteria for parvoviruses. Characteristic for members of the Bocaparvovirus genus is a unique B25 kDa nuclear protein, NP1, encoded in the middle of the genome. For both boca- and protoparvoviruses, the encapsidated linear ssDNA genomes are heterotelomeric and Z90% of genomes are of negative sense. The founder viruses of Bocaparvovirus are bovine parvovirus 1 (BPV1) and minute virus of canines (MVC), from which the genus name was created. Protoparvovirus is an older, but recently renamed, well characterized genus, including one of the first known parvoviruses, Kilham rat virus, discovered already in 1959. New viral members are being discovered at a rapid pace, but at the time of writing this review, there are 21 species in Bocaparvovirus and 11 in Protoparvovirus, with viruses from two species each infecting humans. HBoV1, HBoV3, a gorilla and a chimpanzee bocavirus belong, according to the NS1 amino-acid sequence identities, to the species Primate bocaparvovirus 1, and HBoV2 and HBoV4 belong to the species Primate bocaparvovirus 2. Similarly, all three BuV genotypes belong to Primate protoparvovirus 1 and CuV to Primate protoparvovirus 3, while a rhesus monkey bufavirus founded the Primate protoparvovirus 2 species. TuV has been proposed to found the species Primate protoparvovirus 4. The current status of parvovirus taxonomy and phylogenetic trees, can be seen at the “Relevant Websites section”.

Virion Structure Parvoviruses are small (B25 nm) non-enveloped viruses with icosahedral T ¼ 1 capsid symmetry. The capsid comprises thus 60 copies of the common C-terminal part of the capsid proteins (VPs), the vast majority of which is provided by the shortest VP (VP3 for HBoVs and VP2 for protoparvoviruses), and the minority (5%–10%) by the longer VP1 (and VP2 for HBoV) proteins. Typical to most parvoviruses, the short unique N-terminus of VP1, VP1u, carries phospholipase (PLA2) activity, which is important in endosome escape. The capsid has multiple roles in parvovirus biology, including host cell recognition by binding to cellular receptors, intra-cellular trafficking by the activity of its PLA2 motif, nuclear entry by its signaling sequences, genome packaging through its 5-fold cylinder, and host immune recognition of its neutralizing epitopes. The HBoV major capsid protein is VP3 of B60 kDa, and all three VPs seem to be translated from the same transcript, in a ratio of 1:1:10. However, for use as antigens or for structural studies, virus-like capsids (VLP) can be assembled based on VP3 alone. The major capsid protein in BuV, TuV and CuV is VP2 of the same size. The structures of HBoV1, HBoV3, HBoV4 and BuV1–3 VLPs, obtained by the baculovirus expression system, have been determined to high (B3 Å ) resolution, by cryo-electron microscopy and 3D image reconstruction. The HBoV and BuV capsid cores are conserved and formed by an eight-stranded antiparallel b-barrel motif and an a-helix, similar to all other parvoviruses. The specific surface of the capsid is shaped by long amino acid loops between these secondary structures, containing ten variable regions (VR) of which VR-III and VR-IX have been identified as potential tropism determinants. Seemingly unique for the bocaparvoviruses, there is another a-helix within the surface loops. The HBoV and BuV capsids follow the typical parvoviral topology, including an open channel at the icosahedral 5-fold axes with a surrounding canyon, a depression at the 2-fold axes, and trimeric protrusions at the 3-fold axes. The HBoV 3-fold protrusions are less prominent than those of AAVs but more pronounced than those of parvovirus B19, and among the HBoVs, HBoV1 has the least prominent topology. Surprisingly, these trimeric 3-fold protrusions in BuV are separated, and therefore more similar to those of HBoV and B19V than those of animal protoparvoviruses, perhaps indicating a host-specific structural evolution. The conserved 5-fold channel is made by five b-strand ribbons forming a cylinder, through which DNA packaging is thought to occur. Unique for bocaparvoviruses is the presence of a density beneath the 5-fold axis that extends into the capsid interior. In both HBoV and BuV capsids, the location of the disordered VP1u and N-terminus of VP2 have not been structurally confirmed, but recent observations support the hypothesis that the N-termini would lie inside the capsid, from where a flexible VP1u could, when associated with a cell, be externalized through the 5-fold channel exposing its PLA2 moiety. Four HBoV-specific antigenic epitopes have been mapped in the capsid, three being specific for HBoV1 at the 3-fold protrusions and the wall between the 2- and 5-fold axes, and a fourth HBoV1–4 cross-reacting epitope at the 5-fold axis. Structural analyses of TuV and CuV are ongoing.

Genome All parvovirus virions have linear ssDNA genomes with dsDNA hairpin ends. Among viruses in the genus Bocaparvovirus, only the genomes of HBoV1, BPV1 and MVC have been sequenced in full length, including their terminal hairpins. The HBoV1 genome is 5543 nucleotides (nt) in length with nonidentical hairpin structures at the termini (Fig. 1(A)). In bocavirus virions, approximately over 90% of the viral genome are of negative sense. The HBoV1 left-end hairpin (LEH) is 140nt in length and is predicted to be ‘Y' shaped with short axial “rabbit ears” and mismatches that cause an unpaired “bubble”. The LEHs of both MVC and BPV1 have

Human Boca- and Protoparvoviruses (Parvoviridae)

421

Fig. 1 Gene expression profile of HBoV1. The ssDNA genome of HBoV1 is depicted to scale in negative sense with transcriptional and RNA processing units: the P5 promoter, 50 splice-donor sites (D1, D10 , D2, and D3) and 30 splice-acceptor sites (A1, A10 , A2, and A3), the proximal and distal polyadenylation sites ((pA)p1/2 and (pA)d1/REH, respectively), and the 30 noncoding region (NCR). The left-end hairpin (LEH) and right-end hairpin (REH) structures of the genome are shown. Seven groups of HBoV1 mRNA transcripts are detected during infection, with either long-form mRNA (RXL) that reads through the (pA)p site or short-form mRNA (RXS) that is polyadenylated at the (pA)p site. R1 mRNA has a minor species (R1m) that is unspliced at the central small intron (D3–A3). Major open reading frames (ORFs) are depicted in colored boxes with the nucleotide number (nt) and amino acid (aa) of the start and stop codons indicated. Proteins expressed from each mRNA are indicated beside their respective mRNAs with molecular weight in kDa. BocaSR is diagramed, and is transcribed from the 30 NCR of nt 5199–5388 that bears an intergenic RNA polymerase III promoter (Pol III).

been isolated in two configurations, called flip and flop, suggesting that also the HBoV1 LEH should exist in both configurations. The right-end hairpin (REH) of MVC and BPV1, but not HBoV1, harbors sequences that have the potential to be folded into a cruciform structure near the end tip, although it is thermodynamically less favorable for BPV1. Notably, HBoV1 REH shares an identical 21-nt sequence with the MVC REH at the end, and the HBoV1 LEH shares two identical sequences of 6nt and 8nt, respectively, with the BPV LEH. Both the HBoV1 and MVC REHs are perfect duplex (palindromic) structures without any nucleotide mismatches and, therefore, only one configuration is present. The intracellular duplex or replicative form of the HBoV1 genome is transcriptionally capable (Fig. 1(B)). The left half of the duplex genome encodes nonstructural, NS, proteins, and the right half encodes capsid, VP, proteins. The HBoV1 genome possesses two unique features among parvoviruses: (1) an additional NP1 protein is encoded from an open reading frame (ORF) lying in the middle of the genome, and (2) a viral noncoding RNA – bocavirus-transcribed small RNA (BocaSR), from the noncoding region (NCR) at the right-hand end of the duplex genome, after the VP ORF but before the REH. Parvoviruses utilize cellular DNA and RNA polymerases for replication and transcription, respectively. HBoV1 has only one (RNA polymerase II) promoter, P5, which transcribes a single precursor mRNA (pre-mRNA). The viral pre-mRNA is processed through alternative polyadenylation and

422

Human Boca- and Protoparvoviruses (Parvoviridae)

alternative splicing in the nucleus, which generates 7 groups of viral mRNA transcripts during infection (Fig. 1(B)). Notably, there is also an intergenic RNA polymerase III promoter at nt 5199–5338 that transcribes the 140-nt long BocaSR during infection. While NS1 is essential to viral DNA replication, both NP1 and BocaSR enhance viral DNA replication. In ex vivo human airway epithelium cultured at an air-liquid interface (HAE-ALI), also NS2 is essential to virus replication, whereas NS3 and NS4 are dispensable. NP1 also plays an important role in VP expression by overcoming a blockage of the internal polyadenylation in the middle of the genome, and is essential to the splicing of viral pre-mRNA at the A3 acceptor site that lies in front of the (pA)p sites (Fig. 1(B)). BocaSR not only plays a role in viral DNA replication but also in the expression of NS1, NS2, NS3, and NP1 (but not NS4). The VP proteins constitute the capsid and allow packaging of the viral genome. Notably, VP1–3 are likely translated from a single mRNA, R6 or R7, and VP2 utilizes a noncanonical translation initiation site, GUG, at nt 3422 (Fig. 1(B)). The human protoparvoviruses, BuV, TuV and CuV, have, similar to HBoVs, the same general genome organization as those of the other parvoviruses, with NS1 genes on the left half and VP genes on the right half of the negative-sense genome. In addition, BuV and CuV seem to have a small middle ORF, with unknown function. Contrary to HBoV and B19V, protoparvoviruses have two different promoters, one in the 30 end and the other in the middle of the genome, but only one termination site at the far-right end. The exact transcription maps for BuV, TuV and CuV have not been determined, however, they are most likely similar to those of the other better known animal protoparvoviruses.

Life Cycle HBoV1 is detected in nasopharyngeal samples (NPS) of patients with acute respiratory tract infections (ARTI), suggesting that HBoV1 naturally infects human airways. Productive HBoV1 infection has been observed only in HAE-ALI. Although HBoV1 does not productively infect monolayer-cultured (proliferating) human cells, including human embryonic kidney 293 (HEK293) cells, HEK293 cells fully support replication of the HBoV1 duplex genome – delivered by transfection of a full-length plasmid clone of HBoV1 (pIHBoV1) – and produces progeny virions that are fully infectious in HAE-ALI, but not in monolayer-cultured airway epithelial cell lines, such as A549 and BEAS-2B cells. HAE-ALI cultures generated from primary or reproliferating primary human bronchial/tracheal epithelial cells, as well as immortalized human airway epithelial cells (CuFi-7), support productive infection of HBoV1. Recently, it has been reported that Caco-2 cells, derived from human colon cancer, can be infected with HBoV1, but only after polarization/differentiation. HBoV1 can infect HAE-ALI cultures through both the apical and the basolateral sides, suggesting that the (unknown) virus receptor is expressed on both sides of the airway epithelia (Fig. 2, steps 1–2). As with other parvoviruses (e.g., B19V, CPV, AAV2, and MVM), HBoV1 likely enters the cells through receptor-mediated endocytosis, and intracellularly traffics from early endosome to late endosome (Fig. 2, steps 3). Virions may directly disrupt the nuclear envelope or be transported through the nuclear pore to translocate into the nucleus (Fig. 2, step 4). Within the nucleus, the viral genome is released and likely recognized by the cellular repair machinery (Fig. 2, step 6). Then, the ssDNA viral genome is converted into a duplex form (Fig. 2, steps 7a and 7b). In polarized HAE cells that are mitotically terminated (differentiated), cellular DNA repair DNA polymerases Z/κ are involved in HBoV1 DNA replication. This has not been shown for other parvoviruses. It is thus believed that the Y-family polymerases Z/κ synthesize the complementary strand, utilizing the 30 -OH hairpin end as primer. After expression from the duplex genome, NS and NP1 proteins are associated with the viral genome for viral DNA replication (Fig. 2, steps 12–16). NS or NP1 proteins are likely required for genome packaging as well (Fig. 2, steps 16–18); however, they are not packaged into the mature virions themselves. The viral DNA replication process involves complex hairpin structure transfers and protein-genome interactions, which produces viral DNA intermediates in various lengths and structures (Fig. 2, steps 12–16). Viral pre-mRNA is transcribed from the duplex form genome and further processed to generate NS-, NP-, and VP-coding mRNAs (Fig. 2, step 8). Capsid proteins VP1, VP2, and VP3, produced in the cytoplasm, are assembled into oligomers before translocating into the nucleus (Fig. 2, steps 10 and 11). Empty capsids are assembled in the nucleus after which viral ssDNA is encapsidated (Fig. 2, steps 17–18). Eventually, the mature virions are released from the nucleus into the cytoplasm and transported out of the infected cells (Fig. 2, steps 19–20), which undergo pyroptotic cell death. BuV, TuV and CuV have not yet been propagated in cell culture.

Epidemiology Human Bocaviruses HBoV1, being a respiratory virus, is most likely transmitted by the respiratory route, whereas HBoV2 and 3 are considered enteric and are most likely transmitted by the fecal-oral route. Findings supporting this are mainly that HBoV1 DNA is detected by quantitative (q)PCR in high amounts in airway samples during the acute phase of ARTI, whereas HBoV2–4 seldom are found in such samples but are detected in stool. HBoV1 is most common in children between 0.5 and 5 years of age, with a mean age of infection of 2 years. HBoV1 DNA is detected in 2%–20% of NPS of children with upper or lower ARTI, but due to its persistence in the airways for up to a year, it may be detected in lower DNA loads also in healthy children. The most common HBoV in stool samples is HBoV2 that has been found in 1%–20% of stools of both symptomatic and non-symptomatic subjects, followed by HBoV1, HBoV3 and HBoV4. None of these have, however, indisputably been shown to cause gastroenteritis. Further, HBoV4 is too

Human Boca- and Protoparvoviruses (Parvoviridae)

423

Fig. 2 The infection life cycle of HBoV1. HBoV1 infection of human airway epithelium cultured at an air–liquid interface (HAE-ALI) is illustrated with a polarized airway epithelial cell featured with the apical side towards the airway lumen, basolateral layers, columnar cilia, and adherent junction molecules. Each step of the virus life cycle is explained in the text. Ligase, an unknown cellular DNA ligase; -ssDNA, negative single-stranded DNA; pol Z/κ, DNA repair DNA polymerase Z and κ.

rare to state anything of its transmission, pathogenicity or tissue tropism. Contrary to B19V, none of the HBoVs seem to be transmitted by blood or blood products, even though a short viremic phase does occur during acute infection and HBoV1 DNA is infrequently detected in donated blood. Besides serum, respiratory and enteric samples, HBoV DNAs have been detected also in saliva, urine, cerebrospinal fluid (CSF), tonsils, and intestinal biopsies, as well as in environmental river water and in sewage. HBoV1 DNA is globally detected throughout the year with no obvious seasonality observed, however, the long persistence of HBoV1 DNA in the airways makes such studies problematic. HBoVs cause systemic infections, likely leading to immunity. Most HBoV1-specific antibody responses are strong and life-long, but B30% are weaker and some may be totally nonexistent. Further, the IgG responses against the enteric HBoVs are generally

424

Human Boca- and Protoparvoviruses (Parvoviridae)

weaker and even more prone to waning than those against HBoV1. This variation is due to both cross reactivity between the HBoVs and to an immunological phenomenon called “original antigenic sin” (OAS). If a person has been infected by HBoV2 earlier in life, his/her current HBoV1 infection might not induce HBoV1-specific IgG responses, leading to a false negative IgG result and a lower seroprevalence. Despite these challenges, the seroprevalence of HBoV1–4 in Finland already at the age of 6 have been determined to be 80%, 50%, 10% and B0%, and in China among adults to 67%, 50%, 40% and 1.4%, respectively, indicating that HBoV1 is the most common human bocavirus circulating worldwide. The median age of infection is 2 years for HBoV1 and slightly lower for the enteric HBoVs, while in adults and the elderly, HBoV infections are much less frequent.

Human Protoparvoviruses BuV, TuV, and CuV were all discovered by metagenomics in stools from children with diarrhea. The BuV DNA prevalence is between 0% and 4% in stools of diarrheic patients from Africa, Europe, Asia, and South America, and viral DNA can be found also in river water. In a study from Finland, also close to 1000 nasal swabs were studied for BuV DNA, only one being positive, indicating that BuV is not a respiratory virus. All BuV sequences typed to-date have been of genotype 1 or 3, while genotype 2 DNA has been identified only in the stool of a single child from Burkina Faso, described in the original discovery report. Likewise, TuV DNA has also been detected in a single pediatric stool sample, obtained from Tunisia, whereas the CuV genoprevalence was 1%–2% among stools from Brazil and Botswana. The three BuV genotypes, sharing only 65%–73% VP2 amino-acid identity, can be separately identified in EIAs and are thus also considered different serotypes. The global BuV seroprevalence pattern is strikingly different, varying among adults from 2% to 4% in Finland and the USA to 56%–85% in Kenya and the Middle East. The predominant genotype in Kenya was BuV-3 and that in the Middle East BuV-1, whereas BuV-2 was the second-most prevalent genotype on both continents. This finding contrasts the PCR findings, where BuV-2 DNA has been found in only one sample. TuV IgG was not found in the sera from any of the four continents, while CuV IgG was evenly distributed with a seroprevalence of 0%–6%. BuV-2 and CuV VP2s are more similar to each other than those of BuV-1 and -3, and they therefore need blocking by a competition EIA not to show considerable cross reactivity of IgG.

Clinical Features Human Bocaviruses HBoV1 is one of the most common respiratory viruses detected in NPS of children with ARTI. However, HBoV1 DNA is frequently (20%–80%) detected together with other respiratory pathogens and it is also detected in NPS of healthy children. In the early studies, researchers were therefore questioning the clinical significance of HBoV1 in ARTI and many considered it a bystander virus. Later, this high prevalence of HBoV1 DNA was shown to be due to virus persistence in the nasopharynx for several weeks to up to a year after acute infection, during which time the child may have several other respiratory tract infections. Consequently, the mere presence of this virus in the airways, determined by sensitive qualitative PCRs, cannot be a reliable marker for a HBoV1 etiology in the current ARTI. Comprehensive clinical studies, applying monodetection and/or more accurate diagnostic markers, including serology, qPCR, reverse transcription (RT)-PCR and antigen detection, have since provided convincing evidence of HBoV1 being a true respiratory pathogen in children. HBoV1 may cause both upper or lower ARTI, most often in children of 0.5–5 years of age, while smaller infants are mostly protected by passive maternal antibodies. The most frequent clinical features are those associated with the common cold, bronchiolitis, pneumonia, and exacerbations of asthma. The symptoms do not differ from those of other viral ARTIs; cough, fever, rhinitis, and wheezing. In addition, acute otitis media and diarrhea are often reported during HBoV1 infection and viral DNA has been detected in middle-ear fluids and stool. Typical signs of pneumonia, with patchy or interstitial infiltrates in chest radiographs, hyperinflation and atelectasis, as well as hypoxia and tachypnea are frequently seen in HBoV1-caused lower ARTI. HBoV1 has further been the sole finding also in several life-threatening infections with respiratory failure demanding intensive care with mechanical ventilation. In adults, HBoV1 DNA detections in airway samples are infrequent, possibly due to the high seroprevalence, but usually associated with respiratory symptoms. Summarizing 5 major studies in China with 412,000 adult ARTI patients, the prevalence of HBoV1 DNA in airway samples was 0.3%. In one study on Finnish elderly adults (465 years of age, with a mean age of 83 years), no acute HBoV1–4 infections were recorded among 438 episodes of ARTI, as diagnosed by serology and only 2 (0.5%) had HBoV1 DNA in their respiratory samples. Among these patients, 88% were HBoV1 IgG seropositive. A striking difference between these elderly adults and children was the low prevalence of HBoV1 (0%) and high prevalence of RSV (7%) genome detections in the samples of 283 asymptomatic elderly controls. The four HBoVs have also been found in up to 25% of diarrheic stool samples, but neither their DNA loads nor prevalence suggests a causal association with AGE. In most studies equally often HBoV DNA is detected in stools of symptomatic as of asymptomatic children. A prolonged excretion of HBoV1 and 2 in stool has furthermore been reported, making disease associations difficult. Nevertheless, one case is reported of a life-threatening hypovolemic shock due to diarrhea associated with a disseminated HBoV1 infection in an immunocompromised 9-year-old boy, with no other pathogens detected.

Human Boca- and Protoparvoviruses (Parvoviridae)

425

HBoV1, HBoV2, and HBoV3 have infrequently been detected, as sole microbes, in CSF of patients with severe encephalitis, indicating a possible role in infections of the central nervous system. In a HBoV2 DNA-positive CSF sample also typical parvoviral virions were detected by electron microscopy; the child was further seronegative but viremic. The severity of symptoms in the HBoV-positive patients did not differ from those of patients with other virus findings in CSF (dengue-, echo- or adenovirus). In addition, there are case reports of HBoV-associated myocarditis and hepatitis. A 13-month-old girl had a fatal subacute myocarditis with HBoV2 DNA detected in NPS, plasma, urine, ascites, mesenteric node, skeletal muscle, and lung, and she died of heart failure. A previously healthy 2.5-year-old girl had bronchiolitis and hepatitis with high-load HBoV1 viremia as sole finding, and another 7-month-old boy with an underlying T-cell deficiency, had hepatitis with HBoV1 DNA in consecutive NPA, feces, urine, and serum samples and with diagnostic increases in both HBoV1 IgM and IgG. These cases suggest that the full picture of the clinical impact of HBoVs is still to be revealed.

Human Protoparvoviruses Very little is known about the clinical pictures of BuV, TuV, and CuV infections. BuV has been shown to associate with diarrhea, since its DNA has been detected by PCR only in diarrheic stools, while non-diarrheic stools all have so far been PCR negative. However, the frequencies as well as viral loads have been low, suggesting a nonsignificant role of BuV in gastroenteritis. No further TuV and CuV PCR studies of stool samples have been reported, leaving their relationship with gastroenteritis open. CuV DNA has, however, in three separate studies been detected in skin lesions of cutaneous T-cell lymphoma patients, but not in skin biopsies of healthy subjects, reaching statistical significance. It cannot be proven at this time, whether or not this association is causal or consequential. Parvoviruses are usually not directly oncogenic, but they may cause chronic inflammation possibly leading to cell transformation. CuV DNA has infrequently been detected also in melanoma lesions and in both healthy and malignant skin biopsies of immunocomprimised transplant patients.

Pathogenesis As animal models of HBoV1 infection are currently not available, the respiratory pathogenesis of HBoV1 is not well understood and thus is limited to HBoV1 infection in the ex vivo HAE-ALI culture model. HBoV1 infection of polarized HAE-ALI cultures is persistent, and can last 45 days with release of progeny virions, or as long as the ALI cultures can be maintained. The infection induces progressive airway epithelial damage with disruption of the tight junction barrier, thinning of the epithelium, loss of cilia, and epithelial cell hypertrophy indicated by enlarged nuclei. HBoV1 infection induces pyroptotic cell death of airway epithelial cells, represented by activation of caspase 1 but not of caspase 3, as well as an increase in the expression of cytokines IL-1 and IL-18. The pyroptosis induced by HBoV1 infection is mediated by the NLRP3 (NLR family pyrin domain containing 3 protein)ASC (apoptosis-associated speck-like protein containing a CARD)-caspase-1 inflammasome-induced pathway. Mechanistically, HBoV1 infection upregulates the expression of the anti-apoptotic genes BIRC5 (Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5) and IFI6 (interferon alpha inducible protein 6), which inhibit HAE cell apoptosis, and therefore the infected cells die via pyroptosis. Notably, pyroptosis does not inhibit HBoV1 replication. HBoV1 infection-induced chronic inflammation may serve to promote its persistent infection. HBoV1-infected patients are mostly young children, still with maturing immune systems. If HBoV1-infected cells are not quickly eliminated by effective immune responses, the intensifying inflammation cycle could possibly even lead to lung tissue injury in HBoV1-infected young children. Pyroptosis is an inflammasome-mediated cell death with release of active IL-1, IL-18, and gasdermin D (GSDMD), which cause the cell to swell and eventually lyse. Therefore, HBoV1 infection-caused cell death is not limited to the infected cells but also spreads to the entire epithelia cultured on the insert, including uninfected cells of the inserts. It is currently unknown which viral components are sensed as pathogen-associated molecular patterns (PAMPs) by any pattern recognition receptors (PRRs) or which damage-associated molecular patterns (DAMPs) are generated into the cytosol during infection. Further studies are warranted to examine the nature of cell death in clinical specimens such as cells in airway washes from patients with monoinfection of HBoV1.

Diagnosis Diagnosis of HBoV1 ARTI is challenging because of viral persistence in the airways long after infection (Fig. 3). Qualitative PCR of NPS, the predominantly used diagnostics for ARTI, is therefore insufficient. For an accurate diagnosis of acute HBoV1 infection, a combination of many different approaches is needed. One such approach is quantitative PCR, which gives a better estimate of how recently the infection began; a high viral load (typically over 104 or 106 copies/ml) would therefore suggest an acute HBoV1 infection. However, as verified by serology, about a third of patients with low viral load and even a few symptomatic patients with HBoV1 PCR-negative NPS, still had an acute HBoV1 infection and conversely, some patients with high loads of HBoV1 DNA in NPA, yet do not have acute infection. Obviously, if no other pathogens are detected, it strengthens the probability of an HBoV1 etiology, but again, B60% of patients with other pathogens co-detected, experience also true acute HBoV1 infections. Another useful diagnostic method for respiratory samples is reverse transcription (RT)-PCR for detection of viral mRNA. Viral transcripts are

426

Human Boca- and Protoparvoviruses (Parvoviridae)

Fig. 3 Clinical course of HBoV1 infection. Appearance of respiratory symptoms, of HBoV1 DNA, mRNA and antigen in the nasopharynx, of HBoV1-specific DNA, IgM and IgG in blood, as well as increase of IgG avidity, after acute primary HBoV1 infection. The exact time of onset of symptoms related to time of infection (i.e., incubation time) is unknown, but has here been estimated based on the antibody kinetics and proportion of the other diagnostic markers in wheezing children with and without seroconversion. NPS, nasopharyngeal samples; IgM, immunoglobulin M; IgG, immunoglobulin G.

expressed mostly in the acute phase of HBoV1 infection and can thus be considered an indirect marker of virus replication and ongoing infection. By a novel commercial antigen assay, also viral capsid protein can be detected in NPS, which would indicate an acute phase of infection. The specificity is good but the sensitivity is lower than for the PCR-based assays. When an accurate diagnosis of HBoV1 respiratory tract infection is required, also serum (or plasma) is an important sample type. HBoV1 causes a systemic infection resulting in a short-phase viremia, and can therefore, during the very acute phase, be found by PCR in blood or serum. HBoV1 DNA has, however, been shown to persist longer in some individuals, slightly decreasing the clinical specificity. The most compelling evidence perhaps for an acute HBoV1 infection is, however, the detection of HBoV1specific IgM together with a seroconversion or a Z four-fold increase of IgG in paired serum samples, taken at the acute phase and at the convalescent phase two weeks later (Fig. 3). A low IgG avidity (even in a single IgG-positive sample) further verifies the acute HBoV1 infection, whereas a high IgG avidity indicates past immunity from a prior infection a few months to several years ago. The IgG-avidity EIA thus further increases the diagnostic value of serology. Nevertheless, two issues hamper the reliability of serology. First, the four HBoVs induce cross-reacting antibodies to common epitopes, showing reactivity in all four HBoV EIAs, despite only one of them is specific reactivity. Such cross-reactivity can, however, be avoided by blocking the HBoV1–4 common antibodies from binding by adding the heterotypic VLP antigens to the serum prior to the EIA, leaving only the antibodies to specific HBoV1 epitopes for measurement. Secondly, the four related HBoVs may occasionally interact immunologically resulting in OAS, a phenomenon described above (Epidemiology), perhaps leading to a slightly decreased negative predictive value. However, this OAS effect seems to occur more often in past immunity than in recent infection. Taken together, due to virus persistence in the airways, the detection of HBoV1 DNA by a qualitative PCR in NPS is insufficient. The most accurate diagnosis of acute HBoV1 respiratory tract infection is obtained by combining different diagnostic approaches; HBoV1-DNA monodetection, high viral-DNA load, and detection of viral mRNA or antigen in respiratory specimens, viral DNA detection in serum, and detection of HBoV1-specific IgM coupled with seroconversion or Z4-fold increase in paired sera of HBoV1-specific IgG antibodies with low avidity. For the human protoparvoviruses, only qPCR and IgG EIA methodology so far exist, but development of IgM EIAs is ongoing for future clinical studies.

Prevention Since HBoV1 is transmitted by close contact, droplets or aerosols, and HBoV2–4, BuV and CuV possibly by the fecal-oral route, standard hygienic practices like hand washing with soap may reduce spreading the virus. However, alcohol-derived gels are less effective against non-enveloped parvoviruses. Avoiding large crowds in closed areas during the cold seasons, may also diminish the risk of getting respiratory tract infections. Currently there are no vaccines or specific medication available for human parvovirus infections.

Further Reading Allander, T., Tammi, M.T., Eriksson, M., et al., 2005. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proceedings of the National Academy of Sciences of the United States of America 102, 12891–12896. Christensen, A., Kesti, O., Elenius, V., et al., 2019. Human bocaviruses and paediatric infections. Lancet Child and Adolescent Health 3, 418–426. doi:10.1016/S2352-4642(19)30057-4. Cotmore, S.F., Agbandje-McKenna, M., Canuti, M., et al., 2019. ICTV virus taxonomy profiles: Parvoviridae. Journal of General Virology 100, 367–368. doi:10.1099/jgv.0.001212.

Human Boca- and Protoparvoviruses (Parvoviridae)

427

Deng, X., Yan, Z., Cheng, F., Engelhardt, J.F., Qiu, J., 2016. Replication of an autonomous human parvovirus in non-dividing human airway epithelium is facilitated through the DNA damage and repair pathways. PLoS Pathogens 12, e1005399. Deng, X., Zou, W., Xiong, M., et al., 2017. Human parvovirus infection of human airway epithelia induces pyroptotic cell death by inhibiting apoptosis. Journal of Virology 91. pii: e01533-17. Huang, Q., Deng, X., Yan, Z., et al., 2012. Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia. PLoS Pathogens 8, e1002899. Jartti, T., Hedman, K., Jartti, L., Ruuskanen, O., Söderlund-Venermo, M., 2012. Human bocavirus – The first 5 years. Reviews of Medical Virology 22, 46–64. Mietzsch, M., Kailasan, S., Garrison, J., et al., 2017. Structural insights into human bocaparvoviruses. Journal of Virolology 12, 91. Ong, S.Y., Schuurman, R., Heikens, E., 2016. Human bocavirus in stool: A true pathogen or an innocent bystander? Journal of Clinical Virology 74, 45–49. Schildgen, O., Qiu, J., Söderlund-Venermo, M., 2012. Genomic analysis of the human bocaviruses. Future Virology 7, 31–39. Söderlund-Venermo, M., Brown, K., Erdman, D., 2017. Chapter 30. Human parvoviruses. In: Richman, D.D., Whitley, R.J., Hayden, F.G. (Eds.), Clinical Virology, fourth ed. ASM Press, pp. 679–699. Söderlund-Venermo, M., Lahtinen, A., Jartti, T., et al., 2009. Clinical assessment and improved diagnosis of bocavirus-induced wheezing in children, Finland. Emerging Infectious Diseases 15, 1423–1429. Qiu, J., Söderlund-Venermo, M., Young, N.S., 2017. Human parvoviruses. Clinical Microbiology Reviews 30, 43–113. Väisänen, E., Fu, Y., Hedman, K., Söderlund-Venermo, M., 2018. Human protoparvoviruses. Viruses 9, 354. Väisänen, E., Fu, Y., Koskenmies, S., et al., 2019. Cutavirus DNA in malignant and non-malignant skin of cutaneous T-cell lymphoma and organ transplant patients but not of healthy adults. Clinical Infectious Diseases 68, 1904–1910. Väisänen, E., Mohanraj, U., Kinnunen, P.M., et al., 2018. Global distribution of human protoparvoviruses. Emerging Infectious Diseases 24, 1292–1299. Xu, M., Arku, B., Jartti, T., et al., 2017. Comparative diagnosis of human bocavirus 1 respiratory infection with messenger RNA reverse-transcription polymerase chain reaction (PCR), DNA quantitative PCR, and serology. Journal of Infectious Diseases 215, 1551–1557.

Relevant Websites https://talk.ictvonline.org/taxonomy/ International Committee on Taxonomy of Viruses. www.ictv.global/report/parvoviridae Parvoviridae ssDNA Viruses International.

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae) Ding X Liu, Jia Q Liang, and To S Fung, South China Agricultural University, Guangzhou, China r 2021 Elsevier Ltd. All rights reserved.

Nomenclature ACE2 Angiotensin converting enzyme 2 APN Aminopeptidase N BAX Bcl-2-associated X protein BST2 Bone marrow stromal antigen 2 CNS Central nervous system CsA CyclosporinA CypA Cyclophilin A DMV Double-membrane vesicle DUB Deubiquitinating EIA Enzyme-linked immunoassays eIF2a Eukaryotic initiation factor 2 ER Endoplasmic reticulum ERGIC ER/Golgi intermediate compartment ERK Extracellular signal-regulated kinase ExoN Exoribonuclease GRP Glucose regulated protein HCoV Human coronavirus HR Heptad repeat IBV Avian infectious bronchitis coronavirus IC Ion channel ICTV International Committee on Taxonomy of Viruses IFITM Interferon inducible transmembrane protein IFN Interferon IRE1 Inositol requiring enzyme 1

Glossary Apoptosis A form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. Autophagy The natural, regulated mechanism of the cell that removes unnecessary or dysfunctional components. It allows the orderly degradation and recycling of cellular components. ER stress A cellular process that is triggered by a variety of conditions that disturb the folding of proteins in the endoplasmic reticulum. Innate immunity Refers to nonspecific defense mechanisms that come into play immediately or within hours upon pathogen recognition in the body. These mechanisms include physical barriers such as skin, chemicals in the blood, cytokine production and immune cells that attack foreign cells in the body.

JNK c-Jun N-terminal kinase MAP Mitogen-activated protein MDA5 Melanoma differentiation-associated 5 MERS-CoV Middle East respiratory syndrome coronavirus MHV Mouse hepatitis virus Mpro Main protease NendoU Endoribonuclease NF-κB Nuclear factor-κB nsp Non-structural protein ORF Open reading frames PERK PKR-like ER kinase PLpro Papain-like proteases RBD Receptor-binding domain RdRP RNA-dependent RNA polymerases RTC Replication-transcription complex RT-LAMP Reverse transcription loop-mediated isothermal amplification SARS-CoV Severe acute respiratory syndrome coronavirus sgRNA Subgenomic RNA TMPRSS2 Transmembrane serine protease 2 TRS Transcription-regulated sequences UPR Unfolded protein response UTR Untranslated region XBP1 X-box protein 1

Mitogen-activated protein kinase Serine-threonine protein kinases, important molecules in mediating the signal transduction from cell surface to nucleus, regulating cellular activities such as gene expression, mitosis, differentiation, and cell survival/apoptosis. Type I interferon A large subgroup of interferon proteins that help regulate the activity of the immune system, and a pleiotropic cytokine with antiviral, antitumor and immunoregulatory functions. Unfolded protein response A cellular stress response related to the endoplasmic reticulum stress. It has been found to be conserved between all mammalian species, as well as yeast and worm organisms. Viroporin A group of small hydrophobic viral proteins that tend to oligomerize to form hydrophilic pores or ion channels in cellular membrane.

Background The first human coronavirus (HCoV), strain B814, was isolated in 1965 from the nasal discharge of a patient with a common cold. Since then, more than 30 additional strains were identified. Among them, the prototypic stain HCoV-229E (named after a student specimen coded 229E) was isolated using standard tissue culture. HCoV-OC43 (Organ Culture 43) was later recovered using tracheal organ culture and found to be serologically distinct from HCoV-229E. These two viruses were the focus of HCoV research

428

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21501-X

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

429

Fig. 1 The updated classification scheme of HCoVs according to the ICTV.

in the following years, until the emergence of the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002–2003. In the post SARS era, two more HCoVs were identified. HCoV-NL63 (NetherLand 63) was isolated from the aspirate of a 7-month-old infant with bronchiolitis in 2004, whereas HCoV-HKU1 (Hong Kong University 1) was isolated from a Hong Kong patient with pneumonia in 2005. Since then, two more zoonotic HCoVs have emerged, namely as the Middle East respiratory syndrome coronavirus (MERS-CoV) and the 2019 novel coronavirus (2019-nCoV, a.k.a. SARS-CoV-2). Unlike SARS-CoV, MERS-CoV and SARS-CoV-2 that are associated with severe respiratory disease, the four common HCoVs (229E, OC43, NL63, and HKU1) generally cause mild to moderate upper-respiratory tract illness, presumably contributing to 15%–30% of cases of common colds in human.

Classification On the basis of the 10th International Committee on Taxonomy of Viruses (ICTV) report, coronaviruses are classified under the order Nidovirales, suborder Cornidovirineae, family Coronaviridae, subfamily Orthocoronavirinae (Fig. 1). According to serology studies and genomic analysis, Orthocoronavirinae is further divided into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Under the genus Alphacoronavirus, HCoV-229E and HCoV-NL63 belong to the subgenus Duvinacovirus and Setracovirus, respectively; under the genus Betacoronavirus, both HCoV-OC43 and HCoV-HKU1 belong to the subgenus Embecovirus.

Virion Structure Under the electron microscope, coronavirus virions are spherical or pleomorphic. Coronavirus particles are enveloped, about 80–120 nm in diameter, with club-like projections of the spike (S) protein decorating the surface. In some betacoronaviruses, including HCoV-OC43 and HCoV-HKU1, shorter projections of the hemagglutinin-esterase (HE) protein are also observed. The viral envelope is supported by the membrane (M) protein and contains a small amount of the envelope (E) protein. Inside the viral envelope, the genome is bound by the nucleocapsid (N) protein to form a helical symmetric nucleocapsid. The common structural and functional features of HCoV structural proteins are briefly summarized as follows. The S protein is a type I transmembrane protein, with a molecular weight of 128–160 kDa before glycosylation and 150–200 kDa after N-linked glycosylation. As a class I viral fusion protein, the S protein forms homotrimer and is cleaved by host proteases into a S1 subunit for receptor binding and a S2 subunit for membrane fusion. The ectodomain of the S protein is also modified by disulfide bonds, whereas the very short cytosolic tail is modified by palmitoylation. The S protein is the major determinant of host and tissue tropism, and may also contribute to viral pathogenesis by activating the endoplasmic reticulum (ER) stress response. The HE protein is also a type I transmembrane protein, about 48 kDa before glycosylation and 67 kDa after N-linked glycosylation. It forms homodimer via disulfide bonds. With its sialic acid-binding hemagglutinin activity, the HE protein may serve as a cofactor of S protein and facilitate virion attachment. Additionally, as it possesses esterase activity that removes acetyl groups from O-acetylated sialic acids, it has been postulated to have a role as a receptor-destroying enzyme that facilitates the

430

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

release of progeny virions from nonpermissive host cells, thereby enhancing virion spreading in the extracellular milieu. In fact, the HE protein of HCoV-HKU1 mediated receptor-destroying enzyme activity specific to the O-acetylated sialic acids recognized by its own S protein. The M protein (25–30 kDa) is the most abundant structural protein and possesses three transmembrane domains. The short N-terminal ectodomain of the M protein is modified by O-linked glycosylation in HCoV-OC43 and some animal coronaviruses including mouse hepatitis virus (MHV) and bovine coronavirus (BCoV). However, in HCoV-229E, HCoV-NL63, and most other coronaviruses, the ectodomain of M protein is modified by N-linked glycosylation. The M protein forms homodimer and interacts with other viral structural proteins to orchestrate the assembly of the coronavirus particle. This protein may also contribute to viral pathogenesis. For example, retinoic acid-inducible gene 1 (RIG-I)-dependent induction of type I interferon (IFN) is observed in cells overexpressing the M protein of SARS-CoV but not HCoV-HKU1. The E protein is a small (8–12 kDa) integral membrane protein found in low amounts in the virion. Current evidence strongly suggests that the E protein adopts an N-ecto/C-endo topology with one transmembrane domain. The SARS-CoV E protein is modified by N-linked glycosylation and three cysteine residues in its endodomain are modified by palmitoylation. Additionally, the E protein of SARS-CoV and avian infectious bronchitis coronavirus (IBV) has been shown to form homopentamers with ion channel (IC) activity. The IC activity may modulate the process of virion release and contribute to viral pathogenesis. Although the deletion of the E gene is not lethal for SARS-CoV, the mutant virus is severely defective in virion morphogenesis and attenuated in vivo compared with the wild type control. Underneath the viral envelope, the N protein (43–50 kDa) forms dimer and binds to the genomic RNA in a beads-on-a-string fashion, forming a helically symmetric nucleocapsid. In SARS-CoV and other coronaviruses, the N protein is phosphorylated by cellular kinases such as glycogen synthase kinase 3 (GSK3) and ataxia-telangiectasia mutated and Rad3-related. Other modifications such as SUMOylation, ADP-ribosylation, and proteolytic cleavage by caspases has also been demonstrated in the N protein of some coronaviruses. The N protein facilitates RNA packing and is involved in many other processes, including viral genome replication and evasion of the immune response.

Genome With a single-stranded, positive-sense RNA genome containing approximately 27 to 32 kilobases (kb), coronavirus has the largest viral RNA genome described so far. The genome sizes are approximately 27.5 kb for HCoV-229E and HCoV-NL63, and more than 30 kb for HCoV-OC43 and HCoV-HKU1 (Fig. 2). Because the genomic RNA harbors a 50 -cap structure and a 30 -polyadenylate tail, it can act directly as a messenger RNA (mRNA) encoding the viral replicase. Additionally, the genome also serves as a template for RNA replication and the genome is packed into progeny virions. There are two untranslated regions (UTRs) flanking the coding region. The 50 -UTR is 292, 210, 286, and 205 nucleotides long in HCoV-229E, -OC43, -NL63, and -HKU1, respectively, and contains a leader sequence (B70 nucleotides long) at its 50 -terminus. At the other end of the genome, the 30 -UTR is 462, 288, 287, and 281 nucleotides long in HCoV-229E, -OC43, -NL63, and -HKU1 respectively, and it contains a highly conserved octameric sequence B70 nucleotides upstream from the poly(A) tail. The HCoVs replicase gene comprises the 50 - two thirds of the genome and is made up of two overlapping open reading frames (ORFs)—ORF1a and ORF1b. ORF1a is directly translated from the RNA genome, producing the polyprotein pp1a; whereas the translation of ORF1b requires a programmed ribosomal frameshift near the 30 end of ORF1a, leading to the production of polyprotein pp1ab. The autoproteolytic cleavage of pp1a and pp1ab then gives rise to 16 non-structural proteins (nsp1–16).

Fig. 2 Genome structure of human coronaviruses (HCoVs) – HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1. The open reading frame 1a (ORF1a) and ORF1b are represented as shortened navy blue-boxes. The genes encoding structural proteins spike (S), envelope (E), membrane (M), nucleocapsid (N), and hemagglutinin-esterase (HE) are shown as orange boxes. The genes encoding accessory proteins are shown as dark gray boxes.

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

431

The remaining one third of the HCoV genome contains ORFs for viral structural proteins in the order of 50 -(HE)-S-E-M-N-30 , as well as several accessory proteins that are distinct among HCoVs of different species and genus. Specifically, HCoV-229E encodes two accessory proteins 4a and 4b; HCoV-OC43 encodes three accessory proteins ns2a, ns12.9 (a.k.a. ns5a), and protein I (a.k.a. N2 or N internal ORF protein); HCoV-NL63 encodes a single accessory protein 3; and HCoV-HKU1 encodes two accessory proteins: protein 4 and protein I. Although these accessory proteins are dispensable for viral replication in cell culture, they may be involved in viral pathogenesis and contribute to virulence in vivo. Unlike the viral replicase, structural proteins and accessory proteins are translated from a 30 -nested set of subgenomic RNA (sgRNA) species. The coding sequences of some accessory proteins overlap with those of the structural proteins, but are translated in distinct reading frames. In fact, the accessory gene encoding protein I of HCoV-OC43 and HCoV-HKU1 are encoded internally of the N genes.

Non-Structural Proteins The HCoV polyproteins pp1a and pp1ab are autocatalytically processed by viral proteases into 16 non-structural proteins. Nsp1, the most N-terminal cleavage product of the polyproteins, has been shown to suppress host protein synthesis and IFN response. Nsp3 encodes one or two papain-like proteases (PLpro). Nsp3 of the four common HCoVs contains two PLpro domains (PLP1 and PLP2), whereas the nsp3 of SARS-CoV and MERS-CoV contains only one PLpro domain. In general, PLP1 processes at cleavage site 1 to release nsp1, whereas PLP2 is responsible for the processing at both cleavage sites 2 and 3 to release nsp2 and nsp3. The deletion of PLP2 is lethal, whereas the proteolytic activity of PLP1 is dispensable for HCoV-229E replication. Notably, a recent study reported that ectopic expression of HCoV-NL63 PLP2 induces proteasomal degradation of p53, thereby inhibiting p53-dependent production of type I IFN and the innate immune response. Following the translation of ORF1a and ORF1ab, nsp5 is properly folded within the context of the replicase polyprotein and orchestrates its own autoproteolytic processing. Nsp5 cleaves pp1a/pp1ab at as many as 11 sites to produce a total of 13 mature proteins, and is therefore indispensable for virus replication. Nsp5 is also referred to as the coronavirus main protease (Mpro). Nsp6 of some coronaviruses, such as IBV, MHV, or SARS-CoV, activates the formation of autophagosomes from the ER via an omegasome intermediate. Nsp3, nsp4, and nsp6 are also responsible for remodeling cellular membranes to form doublemembrane vesicles (DMVs) or ER spherules, onto which the coronavirus replication-transcription complex (RTC) is assembled and anchored. The complex of HCoV-229E nsp7 and nsp8 is capable of synthesizing short RNA strands of B6 nucleotides. Coronavirus nsp8 protein also has template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases (RdRP). Current evidence suggests an essential cofactor function of nsp7 and nsp8 for the RNA-dependent RNA polymerase activity of nsp12. In a recent study, it is found that nsp8 of HCoV-229E has a metal ion-dependent RNA 30 -terminal adenylyl transferase (TATase) activity, when a partially double-stranded RNA with a short 50 oligo-U sequence is provided as the template strand. This supports the notion that nsp8 may catalyze the 30 -polyadenylation of HCoV genomes. Nsp9, a single-stranded DNA/RNA-binding protein which exists as a dimer in physiological situations, has been found to be indispensable for virus replication based on reverse-genetics experiments. Nsp10 is a double-stranded RNA-binding zinc-finger protein. Nsp7, nsp8, nsp9, and nsp10 are all closely associated with the replication complex built around the RNA-dependent RNA polymerase (nsp12). Nsp13 separates the double-stranded replicative intermediates to provide single-stranded templates for RNA synthesis. HCoV229E nsp13 contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. It exhibits a variety of enzymatic activities including NTPase, dNTPase, and RNA/DNA helicase activity. Using the NTPase active site, this protein also has an RNA 50 -triphosphatase activity, which may be involved in the capping of viral RNAs. Nsp14 demonstrates exoribonuclease (ExoN) activities. RNA viruses generally have high mutation rates that allow for rapid viral adaptation in response to selective pressure. Nsp14-ExoN is the first proofreading enzyme identified for an RNA virus, and it functions together with other CoV replicase proteins to perform the crucial role of maintaining CoV replication fidelity. Nsp15 is an endoribonuclease (NendoU) and a type I interferon antagonist. HCoV nsp15-NendoU can excise both single- and double-stranded RNA and specifically recognize uridylates to produce 2-30 -cyclic phosphodiester products, thereby preventing the activation of the host innate immune response. Nsp16 has a 20 -O-methyltransferase activity. The 20 -O-methylation capping protects viral RNA from recognition by melanoma differentiation-associated protein 5 (MDA5) and thus prevents MDA5-dependent production of type I interferon in virus-infected cells.

Accessory Proteins Although not essential for viral replication in cell culture, coronavirus accessory proteins may play in vivo functions that have not yet been fully elucidated. Most HCoV accessory proteins are genus-specific and show low homology to known proteins. But an accessory gene between the S and E gene is encoded by three HCoVs (3a of SARS-CoV, 4a of HCoV-229E, and ns12.9 of HCoV-OC43), suggesting a conserved role during HCoV infection. Indeed, all three proteins have been shown to serve as viroporins that regulate viral replication. Viroporins are oligomeric hydrophobic viral proteins that form and insert ion channels into the host cell membrane. The HCoV-OC43 ns12.9 protein is a recently identified viroporin that facilitates virion

432

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

Fig. 3 HCoV replication cycle. HCoV infection is initiated by binding of virions to cellular receptors, which drives the conformational change in the S2 subunit in S, promoting the fusion of the viral and cell plasma membrane. The gRNA serves as the template for translation of polyproteins pp1a and pp1ab, which are cleaved to form nsps. Nsps induce the rearrangement of cellular membrane to form DMVs, where the viral RTCs are anchored. Full-length gRNA is replicated via a negative-sense intermediate, and a nested set of sgRNA species are synthesized by discontinuous transcription. These sgRNAs encode viral structural and accessory proteins. The viral products produced will be assembled in the ERGIC, and bud out as a smooth-wall vesicle to the plasma membrane to egress via exocytosis. Host factors that promote infection and inhibit infection are highlighted in green and black, respectively.

morphogenesis and pathogenesis. The expression of important mediators of innate immune response was downregulated in cells expressing of HCoV-OC43 ns12.9, presumably due to inhibited promoter activity of ISRE, IFN-b, and NF-κB. Interestingly, because of a scenario in which HCoV-229E acquired an out-of-frame insertion or deletion, instead of an ORF4a and ORF4b, an intact ORF4 was present in some HCoV-229E clinical isolates. ORF4a of HCoV-229E was expressed in infected cells and localized at the ER/Golgi intermediate compartment (ERGIC). The ORF4a protein formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. The NS2 protein of HCoV-OC43 has cyclic phosphodiesterase activity, which may modulate cAMP-mediated signaling and important physiological processes such as lipid metabolism and apoptosis. Finally, although the accessory protein encoded by HCoV-NL63 ORF3 appears to be nonessential in cell culture, there were differences in RNA synthesis, protein expression, plaque morphology, and virus growth in cells infected with the ORF3-deleted mutant compared with the control.

Life Cycle The replication cycle of HCoV can be arbitrarily divided into five steps: attachment to host cells, viral entry and uncoating, expression of the viral replicase and formation of the replication-transcription complex, viral RNA synthesis, and virion assembly and release. Each step will be briefly introduced as follows (Fig. 3).

Attachment Coronavirus infection is initiated by binding of virions to cellular receptors. The S protein includes two functional domains: S1 (bulb) is the part binding to the receptor(s) and S2 (stalk) is responsible for fusion between virion and cell membranes. The receptor-binding domain (RBD) of S1 varies among different coronaviruses. RBDs of HCoV-229E, HCoV-NL63 and HCoV-HKU1 are located in the C-terminal region but not the N-terminal domains of the respective S1 subunits. Receptor binding is critical to initiate viral infection. HCoV has been shown to use either cellular proteins or carbohydrates displayed on the plasma membrane as receptors. Interestingly, all known protein receptors for HCoVs are cell surface peptidase, such as aminopeptidase N (APN) for HCoV-229E, dipeptidyl peptidase 4 (DPP4) for MERS-CoV, and angiotensin converting enzyme 2 (ACE2) for HCoV-NL63, SARS-CoV and SARS-CoV-2. On the other hand, HCoV-OC43 and HCoV-HKU1 employ glycan-based receptors carrying 9-O-acetylated sialic acid.

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

433

In addition to the receptor binding by the S protein, other HCoV structural proteins may also facilitate the early stage of attachment. For example, the M protein of HCoV-NL63 binds to target cells using heparan sulfate proteoglycans as the initial attachment factors. This is followed by the engagement of the S protein with the ACE2 receptor protein.

Viral Entry and Uncoating Specific binding between S1 and the cognate receptor leads to a conformational change in the S2 subunit and large-scale rearrangements of the S protein, resulting in the fusion between virus and cell membranes and the release of viral nucleocapsid into the cytoplasm. Many host factors are involved in the entry and uncoating of HCoVs. The cleavage of S protein into S1 and S2 subunits is mediated by one or more cellular proteases. HCoV-229E can enter host cells via two distinct pathways: one is mediated by surface proteases like type II transmembrane protease serine 2 (TMPRSS2), and another is mediated by endosomal cathepsin L. However, to avoid triggering an innate immune response, it is more likely that HCoV-229E enters cells via the TMPRSS2 pathway, as the endosome is a main site for recognition by toll-like receptors. Similarly, the entry of SARS-CoV depends on the endosomal cysteine protease cathepsin L and another trypsin-like serine protease to activate the S protein. Coated vesicles and the cytoskeleton are utilized by some HCoV for entry. For example, HCoV-OC43 employs caveolin-1-dependent pathway of endocytosis, and the scission of virus-containing vesicles from the cell surface is dynamin-dependent. Internalization of these vesicles also requires rearrangements of the actin cytoskeleton. The interaction between HCoV-NL63 S protein and the ACE2 receptor molecule triggers the recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. Additionally, virion release of HCoV-229E and IBV from early endosomes is shown to be dependent on the host factors valosin-containing protein (VCP), an AAA ATPase family protein that normally facilitates the export of misfolded proteins from the ER to the cytoplasm. On the other hand, some host factors could prevent the entry and uncoating of HCoVs. Interferon inducible transmembrane proteins (IFITMs) exhibit broad-spectrum antiviral functions against various RNA viruses. IFITMs restricted the entry of HCoV229E and HCoV-NL63, as well as SARS-CoV and MERS-CoV. Conversely, IFITM2 or IFITM3 serve as an entry factor to promote the infection of HCoV-OC43.

Formation of the Replication-Transcription Complex Among RNA viruses, the transcription of coronavirus RNA is unique. First, in order to maintain genetic stability, the large genome size requires unusual enzymatic activities, such as an exoribonuclease and an endoribonuclease activity. Also, the synthesis of a nested set of sgRNAs by discontinuous transcription demands a huge and complicated RTC. Following the release of viral nucleocapsid into the cytoplasm, the genomic RNA serves as a transcript encoding the viral replicase. The replicase gene includes two ORFs, ORF1a and ORF1b. ORF1a is translated into the polyprotein pp1a (440–500 kDa). Owing to a slippery sequence and an RNA pseudoknot near the end of ORF1a, a programmed  1 ribosomal frameshifting event can occur with a frequency of 25%–30%. This allows a continuous translation into ORF1b, producing a larger polyprotein pp1ab (740–810 kDa). Similar to likely all coronaviruses, the pp1a and pp1ab are autoproteolytically processed into 16 nsps, collectively forming the RTC for viral RNA synthesis. Notably, nsp3, 4, and 6 appear to be responsible for remodeling of cellular membranes to form DMVs or spherules, onto which the HCoV RTC is assembled and anchored. Host factors of the early secretory pathway, such as Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) and its effector ADP ribosylation factor 1, may also contribute to DMV formation and RTC assembly.

Viral RNA Synthesis The assembly of RTC sets a foundation for viral RNA synthesis. By using the genomic RNA as a template, new copies of fulllength viral genome are synthesized, using the full-length negative sense genomic RNA as the intermediate template. Meanwhile, the polymerase can switch templates at short motifs called transcription-regulated sequences (TRS) during negative sense RNA synthesis, producing a 50 -nested set of negative sense sgRNAs. These are in turn used as templates to synthesize a 30 -nested set of positive sense sgRNAs, which serve to encode the structural and accessory proteins. The core sequence of HCoV TRS is a conserved hexamer CUAAAC in HCoV-229E and HCoV-NL63, and a conserved heptamer UCUAAAC in HCoV-OC43 and HCoV-HKU1. Different numbers of sgRNAs are produced by the four common HCoVs. For instance, seven major viral RNA species are produced during HCoV-229E infection. The full-length genome (mRNA1) encodes the viral replicase, whereas mRNAs 2, 4, 5, 6, and 7 encode the S protein, accessory protein 4, E protein, M protein, and N protein, respectively. Notably, mRNA 3 is considered defective, because it contains a truncated version of the S gene that is not translated. Although the replication and transcription of viral genome is mainly carried out by the replicase, the involvement of other factors including viral structural protein and host proteins has been implicated. For instance, by serving as an RNA chaperone, the coronavirus N protein can facilitate template switching during sgRNA synthesis. Host proteins such as heterogeneous

434

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

nuclear ribonucleoprotein A1, polypyrimidine tract-binding protein, mitochondrial aconitase, and polyadenylate-binding protein have been suggested to participate in coronavirus RNA synthesis, presumably mediated by their RNA binding activity.

Assembly and Release of Virion Coronavirus structural and accessory proteins that are membrane-associated (such as S, HE, M, and E) are translated by ribosomes in the ER, whereas other viral proteins (such as N) are translated by free ribosomes. Most coronavirus structural proteins are also subjected to posttranslational modifications that modulate their functions. The building parts converge at the assembly site of ERGIC. Assembly is orchestrated by the M protein: homotypic interaction of M protein provides the scaffold for virion morphogenesis, whereas heterotypic interactions of M protein with other structural proteins, such as M-S and M-E, facilitate their recruitment and incorporation. Virion assembly is completed by the condensation of the nucleocapsid with the envelope components, a process mediated by M-N interactions. A small amount of E protein may provide the driving force for envelope morphogenesis by inducing membrane curvature. After assembly, progeny virions are transported in smooth-wall vesicles, trafficked to the plasma membrane via the secretory pathway, and released by exocytosis. The cytoskeletal system also participates in HCoV assembly and release. For instance, the interaction between tubulin and the cytosolic domain of S protein are required for the assembly and release of infectious virions during HCoV-229E and HCoV-NL63 infection. In addition, bone marrow stromal antigen 2 (BST2, a.k.a. tetherin), an interferoninducible antiviral protein, blocks the release of various envelope viruses by interfering with the budding step at the plasma membrane. Although budding of HCoV-229E occurs at the ERGIC, it is recently shown that BST2 can trapp HCoV-229E virions in the intracellular vesicles, thereby suppressing the release of progeny viruses. Interestingly, the S protein of SARS-CoV downregulates BST2 at the protein level by promoting its lysosomal degradation, thus antagonizing the BST2 tethering of SARS-CoV, HCoV-229E, and HIV-1 virus-like particles.

HCoV-Host Interactions As intracellular obligate parasites, HCoVs exploit the host cell machinery for their own replication and spread. Since virus–host interactions also form the basis of viral pathogenesis, knowledge about their interplay is of great research interest (Fig. 4).

Fig. 4 HCoV-host interaction. Schematic diagram showing the host signaling pathways activated during HCoV infection. Black pointed arrows indicate activation, and red blunt-ended lines indicate inhibition. Viral components modulating the pathway are in red, and host proteins are in blue. See text for detail.

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

435

Translational Control Viruses must utilize the host translation machinery to ensure efficient viral protein translation. In response to acute viral infection, host cell would shut down the protein translation system to cope with the infection stress, which is regarded as an integrated stress response. Integrated stress response is marked by the phosphorylation of the a-subunit of eukaryotic initiation factor 2 (eIF2a), downregulation of the general cap-dependent protein synthesis, and up-regulation of the expression of certain transcription factors, such as activating transcription factor 4 (ATF4). Due to the translation competition between cellular and viral mRNAs for limiting number of ribosomes and associated factors, coronavirus must hijack the host translational machinery to produce its own proteins. Some HCoVs, such as SARS-CoV and MERS-CoV, have been shown to induce host translation shutoff in susceptible cells. Specifically, SARS-CoV infection leads to sustained phosphorylation of eIF2a in 293T/ACE2 cells. Coronavirus nsp1 also suppresses host protein synthesis and IFN response. For example, SARS-CoV nsp1 suppresses the expression of host genes including type I IFN, thereby counteracts the host innate immune response and contributes to virulence. MERS-CoV nsp1 also negatively regulates host gene expression by inhibiting the translation and inducing the degradation of host mRNAs. Nsp1 of HCoV-229E and HCoV-NL63 inhibits the expression of reporter genes, probably by binding to ribosomal protein S6 and blocking the mRNA binding to the 40S ribosomal subunit.

ER Stress Response The ER is a cellular organelle important for protein synthesis, folding, and post-translational modifications. In normal circumstances, the ER can be loaded with a very high concentration of proteins without perturbing its unique luminal environment. However, when the protein load exceeds the ER folding and processing capacity, misfolded or unfolded proteins will accumulate within the ER, resulting in ER stress. ER stress activates the signaling pathways collectively known as unfolded protein response (UPR). These are initiated by three ER transmembrane sensors: protein kinase R (PKR)-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1) and activating transcriptional factor 6 (ATF6). The activation of UPR restores the ER homeostasis by enhancing protein folding, attenuating protein translation and ER-associated degradation (ERAD). HCoV infection triggers ER stress and activates UPR in virus-infected cells. Overexpression of the S protein of HCoV-HKU1 and SARS-CoV can activate PERK and the promoters of GRP78 and GRP94. Infection with HCoV-OC43 activates IRE1 and induces X-box protein 1 (XBP1) mRNA splicing, thereby upregulating downstream UPR effector genes. Introduction of two point mutations (H183R and Y241H) into the S protein of HCoV-OC43 induces a higher degree of XBP1 mRNA splicing and results in a more pronounced apoptotic cell death. It is also reported that when the E gene is deleted from SARS-CoV, the mutant virus also induces a higher level of XBP1 mRNA splicing and apoptosis, compared with the wild type control. This suggests that activation of the IRE1-XBP1 pathway may be generally pro-apoptotic during HCoV infection.

MAP Kinase Pathway The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular processes in response to extracellular stimuli. Four distinct subgroups within the MAP kinase family have been described: extracellular signal-regulated kinase 1/2 (ERK1/2), ERK5, c-Jun N-terminal kinase (JNK), and the p38 group of protein kinases. Activation of the ERK pathway has been observed in cells infected with a number of HCoVs, including SARS-CoV, MERS-CoV, and HCoV-229E. Phosphorylation of the 90-kDa ribosomal protein S6 kinase (p90RSK), a key substrate of ERK, was also observed in SARS-CoV-infected Vero E6 cells. Activation of p38 and its upstream kinases has been detected in cells infected with HCoV-229E, SARS-CoV, and MERS-CoV. In fact, the replication of HCoV-229E is suppressed by treatment with p38 inhibitor SB203580 or chloroquine, which attenuates p38 activation. As for JNK, phosphorylation of JNK and its upstream kinases was observed in cells infected with SARS-CoV or in cells overexpressing the SARS-CoV S protein. Notably, treatment with JNK inhibitor abolished persistent infection of SARS-CoV. Apart from their involvement in cell survival and apoptosis, MAP kinases also contribute significantly to the induction of proinflammatory cytokines during HCoV infection.

Autophagy Autophagy is a conserved cellular process involving self-eating. Specifically, cells under stress conditions, such as starvation, growth factor deprivation, or infection by pathogens, initiate autophagy in nucleation sites at the ER, where part of the cytoplasm and/or organelles are sequestered in DMVs (autophagosomes) and degraded is response to a fusion with lysosomes. Autophagy is regulated by highly conserved autophagy-related genes. Coronavirus infection activates the formation of autophagosomes, but inhibition of autophagy does not affect viral replication. The non-structural protein 6 (nsp6) is a transmembrane protein implicated in the formation of DMVs during SARS-CoV infection. Overexpression of nsp6 of IBV, MHV, or SARS-CoV induced the formation of autophagosomes from the ER via an omegasome intermediate. However, autophagosomes induced by IBV infection or overexpression of coronavirus nsp6 had smaller diameters compared with those induced by starvation, indicating that nsp6 may also restrict the expansion of autophagosomes. Notably, recent studies have shown that coronavirus employs the host machinery for COPII-independent vesicular ER export to derive cellular membranes for DMV formation. Although this process requires an autophagy-related gene called LC3, it is independent of host autophagy.

436

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

Apoptosis Apoptosis is a form of programmed cell death that is tightly regulated. When cells undergo apoptosis, they demonstrate specific hallmarks such as cell shrinkage, extensive plasma membrane blebbing, nuclear condensation, and DNA fragmentation. During viral infections, apoptosis is induced as one of the host antiviral responses to limit virus replication and production. Two main mechanisms of apoptosis have been established – the extrinsic and intrinsic pathways. The extrinsic pathway is initiated by the binding of extracellular death ligands to death receptors from the tumor necrosis factor super-family. The intrinsic pathway occurs internally in the cell and involves changes in the mitochondrial outer membrane permeability based on the ratio of pro-apoptotic and anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins. As HCoVs are known to infect tissue cultures, they have been associated with apoptosis induction in a wide spectrum of cell types during infection, including intestinal mucosal cells, kidney tubular cells and neuronal cells. Apoptosis in neuronal cells infected with HCoV-OC43 involved mitochondrial translocation of Bcl-2-associated X protein (BAX) but this phenomenon was independent of caspase activation. HCoV-229E infection resulted in massive cytopathic effect (CPE) and cell death in dendritic cells, albeit independent of apoptosis induction. Since dendritic cells are prevalent throughout the human body, it is possible that they are used as a vehicle to facilitate viral spread. Induction of apoptosis during HCoV infection is also regulated by cellular stress response pathways such as the UPR and MAP kinase pathways described above.

Pathogenesis HCoV-229E, -OC43, -NL63, and -HKU1 are considered as pathogens causing upper respiratory tract disease and responsible for up to 15%–30% of common colds in adults. Unlike SARS-CoV that spreads from the upper airway to cause a severe lower respiratory tract infection, HCoV-229E and HCoV-OC43 replicate principally in the upper respiratory tract epithelial cells, where they produce virus and cause local respiratory symptoms. There are striking differences in the extent of genetic variability when isolates of HCoV-OC43 and HCoV-229E are compared. HCoV-229E isolated at geographically distinct locations shows little genetic variability, whereas for HCoV-OC43 the opposite is true. The ability of HCoV-OC43 to tolerate mutations probably accounts for its ability to grow in mouse cells and to infect the mouse brain as well. As for HCoV-NL63, it shares homology with HCoV-229E and phylogenetic analyses suggest that HCoV-NL63 and HCoV-229E diverged approximately 1000 years ago. HCoVs attach to cellular receptors by the S protein on the surface of the virion. Internalization into host cells occurs by direct fusion with the plasma membrane or by endocytosis. Viral receptors, components that actively promote host cell entry, differ greatly from one virus to another, and each with its own distinct physiological functions. Both APN, the receptor for HCoV-229E, and ACE2, the receptor for SARS-CoV and HCoV-NL63, exist as prominent zinc-dependent peptidases on the plasma membrane. However, unlike SARS-CoV, HCoV-NL63 does not require cathepsin L or endosomal acidification to infect ACE2-expressing cells. In addition to the receptor ACE2, the entry of HCoV-NL63 also requires heparan sulfate proteoglycans on the cell surface, which act as attachment factors that increase the virus density and possibly facilitate receptor binding. Proteolytic cleavage of the HCoV S protein is also an important regulatory mechanism. In recent studies, the proteolytic activation of the HCoV-229E S protein is analyzed using trypsin-like serine proteases. It is found that fusion activation is not dependent on the cleavage of the S1/S2 site, but is highly dependent on the cleavage in the S20 region. This is very similar to the fusion activation of the IBV S protein, which requires furin-dependent cleavage at the S20 site. Compared with SARS-CoV and MERS-CoV, the ability of other HCoVs causing mild infections to suppress the host antiviral response is less well studied. HCoV-229E replication is shown to attenuate the inducible activity of the transcription factor nuclear factor kappa-B (NF-κB) and to restrict the nuclear accumulation of NF-κB subunits. Overexpression of structural or accessory proteins of HCoV-OC43 also leads to downregulation of over 30 genes related to innate immune response, including genes encoding MAP kinases, toll-like receptors, interferons, interleukins, and signal transduction proteins. Similar to the PLPro of SARS-CoV and MERS-CoV, the PLP2 of HCoV-NL63 possesses deubiquitinating (DUB) activity. This DUB activity may remove ubiquitins from critical intermediates in the innate immune signaling pathway, thereby suppressing host antiviral response. PLPro of SARS-CoV and MERS-CoV also recognizes and catalyzes the removal of another ubiquitin-like modifier called interferonstimulated gene 15 (ISG15). Such deISGylating activity has not been fully characterized for other HCoVs. HCoV infection is not always confined to the upper respiratory tract and can invade the central nervous system (CNS) under circumstances that are presently uncharacterized. Although evidence for a significant correlation between the presence of HCoV229E and HCoV-OC43 RNA and multiple sclerosis has not been demonstrated, accumulating evidence from cell culture and animal models highlights their neurotropic and neuroinvasive potential. HCoV-229E RNA is detected in about 44% (40 of 90) of human brains tested, with similar frequencies in brains from multiple sclerosis patients and patients who died from other neurologic diseases or normal control subjects. The detection of HCoV RNA in human brain samples clearly demonstrates that these respiratory pathogens are naturally neuroinvasive in humans and suggests that they may establish a persistent infection in the human CNS. Therefore, the close structural and biological relatedness of HCoV to the neurotropic animal coronaviruses has led to speculation of the possible involvement of HCoV in neurological diseases. Research proved that classical apoptosis associated with the BAX protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins that are usually associated with necroptosis, another form of regulated cell death when apoptosis is

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

437

not adequately induced. It is likely that in HCoV-OC43-infected neuronal cells, RIP1 and MLKL are activated to induce necroptotic cell death in an attempt to limit viral replication. However, this regulated cell death also leads to neuronal loss in the mouse CNS and accelerates the neuroinflammation process, reflecting the severity of neuropathogenesis.

Epidemiology and Clinical features HCoV-229E, -OC43, -NL63, and -HKU1 are distributed globally. By spreading via coughing and sneezing, they cause mild upper respiratory tract diseases in adults. However, in infants, young children, elderly, and immunocompromised individuals, they may sometimes cause life-threatening bronchiolitis and pneumonia. Apart from respiratory illnesses, they may also result in enteric and neurological diseases. Natural infection is probably acquired in a fashion similar to that of many other respiratory viruses, with primary infection of ciliated epithelial cells in the nasopharynx. Although HCoVs were found to cause epidemics every 2–3 years with a high probability of reinfections, there is a paucity of evidence on the epidemiology and clinical manifestations of these four HCoVs worldwide. Reinfection of HCoVs demonstrates that infection does not induce long-lasting protective immunity.

HCoV-229E First isolated in 1966, HCoV-229E is proposed to originate from African hipposiderid bats and adopt camelids as intermediate hosts. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic MERS-CoV. HCoV-229E infection is associated with common cold symptoms in healthy adults. But younger children and the elderly are vulnerable to lower respiratory tract infections. In particular, immunocompromised patients have been reported to suffer severe and life-threatening lower respiratory tract infections attributed to HCoV-229E. Moreover, the serological test suggested the possible involvement of HCoV-229E in the development of Kawasaki syndrome. The incubation period of HCoV-229E is approximately 2–5 days, followed by illness lasting 2–18 days. Symptoms of HCoV229E infection include headache, nasal discharge, sneezing, sore throat and general malaise. A few patients exhibit fever and cough. The clinical features can be distinguished from respiratory tract infections caused by other pathogens such as influenza A virus. HCoV-229E tends to be epidemic during winter in temperate-climate countries, and tests of a HCoV-229E laboratory strain suggested it is relatively stable in the environment.

HCoV-OC43 First isolated in 1967, HCoV-OC43 has no serological cross-reactivity with HCoV-229E. However, patients infected with HCoV-OC43 or HCoV-229E cannot be distinguished based on clinical symptoms alone; although coryza (inflammation of the mucous membrane of the nose) occurs more often during HCoV-229E infections, while sore throat manifestations occur more often during HCoV-OC43 infections. HCoV-OC43 is generally associated with mild upper respiratory tract infections, although it has also been shown to have neuroinvasive properties. In vivo studies in mice have shown that HCoV-OC43 can infect neurons and cause encephalitis. The virus has also been shown to cause persistent infections in human neural cell lines. Since the first isolation of HCoV-OC43 in the 1960s, seven genotypes (A–G) have been identified by phylogenetic analysis. HCoV-OC43 is also transmitted primarily during the winter in temperate climates.

HCoV-NL63 HCoV-NL63 was isolated from a 7-month-old girl with coryza, conjunctivitis, fever, and bronchiolitis in the Netherlands in 2004. HCoV-NL63 and HCoV-OC43 cause most of the respiratory infections leading to hospitalization. Although HCoV-NL63 infections often exists in mixed viral infections, the mixed infection does not usually increase the severity of the disease. In about 71% of the cases, patients are co-infected with other respiratory viruses, such as human rhinovirus, enterovirus and parainfluenza viruses. In most patients, HCoV-NL63 infection is associated with relatively mild symptoms like fever, cough, sore throat and rhinitis. Furthermore, HCoV-NL63 is one of the main causes of croup in children. HCoV-NL63 infects people in all ages, with the highest infection rate occurring before 5 years of age. It is estimated that 1%–10% of the population suffers annually from cold-like symptoms related to infection with HCoV-NL63. HCoV-NL63 epidemic shows a peak during the spring and summer in Hong Kong, indicating that the seasonality of HCoV-NL63 infection may not be restricted to the winter in tropical and subtropical regions. Surprisingly, studies show that HCoV-NL63 virions are exquisitely stable in liquid media and can be stored also without preservatives at ambient temperature for up to 14 days.

438

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

HCoV-HKU1 HCoV-HKU1 was isolated from an adult who had a chronic pulmonary disease in Hong Kong. The clinical symptoms of HCoVHKU1 infection included rhinorrhea, cough, nasal congestion, fever, sputum, sore throat, chills, postnasal discharge, and tonsillar hypertrophy. About 50% of patients infected with HCoV-HKU1 experience febrile seizures, but the symptoms of HCoV-HKU1 infections in the respiratory tract are not easily separable from those caused by other respiratory viruses. HCoV-HKU1 co-circulates with respiratory syncytial virus and an epidemic usually appears prior to influenza season. In addition, HCoVs have been associated with wheezing and exacerbations of asthma. Similar to other three mild disease causing HCoVs, HCoV-HKU1 is distributed worldwide. HCoV-HKU1 infection is relatively frequent in adults. One recent study has examined the circulation of HCoV in Israel during 2015–2016. In the winter influenza survey, 10.36% of patients were infected with the other three common HCoVs; among them, 43.43% were infected with HCoV-OC43, 44.95% with HCoV-NL63, and 11.62% with HCoV-229E. Although it is absent from the winter survey, 22.6% of the hospitalized patients were positive for HCoV-HKU1, mainly during the spring-summer period.

Diagnosis In most cases, HCoV infections, other than SARS-CoV or MERS-CoV, are not well identified by clinical diagnosis because they cause mild, upper respiratory tract disease, and no specific therapy is available. At the same time, HCoV detection is carried out in a limited number of virological laboratories, because HCoV infections cannot be easily distinguished clinically from other causes of upper respiratory tract infections. The presence of multiple genotypes and the co-infection with other respiratory viruses, such as human rhinoviruses, enteroviruses and parainfluenza viruses, render the detection of HCoV even more challanging. HCoVs are also sometimes detected in asymptomatic patients/individuals; so that the presence of these viruses may not be etiologically related to the illness. Electron microscopic examination of the clinical materials, although laborious, has contributed to the identification and characterization of many HCoVs. Electron microscopy has been extensively used in most of the initial studies, also because isolation of HCoVs from infected individuals and their propagation in tissue culture cells have been technically challanging. HCoV-229E has sometimes been isolated in human diploid cell lines. However, HCoV-OC43 initially required cell organ culture systems for isolation, although this virus can now be grown in tissue culture cells. HCoV-NL63 can infect monkey kidney LLC-MK2 cells or Vero cells, whereas HCoV-HKU1 has been grown only in primary human airway epithelial cells. Sometimes, these cell or organ culture techniques are labor intensive, time consuming, and relatively insensitive. HCoVs can be detected by RT-PCR with greater sensitivity than standard culture techniques. Nasopharyngeal aspirates are the most common respiratory samples for diagnosis. PCR primers can be designed to be broadly reactive or strain specific, based on primer binding sites (usually in the viral replicase and/or the N gene). A multiplex real-time RT-PCR assay capable of detecting all four common HCoVs (HCoV-229E, OC43, NL63 and HKU1) has become the diagnostic method of choice. A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) has been developed by colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method employs six specially designed primers that recognize eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 631C for 1 h. This RT-LAMP assay can achieve a high sensitivity of 1600 RNA copies per reaction with high specificity, and is suitable for clinical settings without conventional PCR equipment. Various serologic assays can also be used to detect HCoV infections, including complement fixation assays, neutralization assays, immunofluorescence assays (IFA), enzyme-linked immunoassays (EIA), and hemagglutination inhibition (HI) for viruses with an HE protein. Monoclonal antibodies have also been generated for direct detection of HCoV antigens in nasopharyngeal aspirates samples. Complement-fixing and enzyme-linked immunosorbent assays (ELISA) for HCoV-229E and HCoV-OC43 have been published, but are not yet available in clinical laboratories. Serologic tests for specific antibody responses against HCoVs are mainly reserved for epidemiologic studies. Initially, virus lysates or inactivated whole viruses were used as antigens for serologic assays. More recently, cloned expressed proteins, synthesized peptides, and pseudoviruses are also used. For example, recombinant N protein of HCoV-HKU1 expressed by E. coli and baculovirus has been used for IgG and IgM detection of sera from patients and normal individuals, using Western blot and EIA.

Treatment At present, there are no specific antiviral drugs available for treating HCoV infections, and therapy is therefore primarily supportive. Studies have shown that, if diagnosed early, HCoV-NL63 replication can be suppressed by treatment with PEG-IFN. The effect of various chemicals to inhibit HCoV replication has been studied in cell culture. Potential targets for anti-HCoV drugs include inhibiting virus entry, viral RdRP, and viral protease such as Mpro. Similar to SARS-CoV and MERS-CoV, peptide-based membrane fusion inhibitors targeting the heptad repeat 1 (HR1) and HR2 of HCoV-229E S protein have been developed. They effectively inhibit S-mediated membrane fusion and the replication of

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

439

HCoV-229E in cell culture. Moreover, when administered intranasally to a mouse model, these inhibitors could widely distribute in the upper and lower respiratory tracts and maintain fusion-inhibitory activity. Some polymers, such as a cationically modified chitosan known as N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride and its derivatives, have been shown to block HCoV-NL63 entry by inhibiting the receptor binding of S protein. Viral entry can also be inhibited using serine proteases (such as camostat) and cysteine protease inhibitor (such as K11777). These compounds inhibit the critical proteolytic activation of HCoV S protein, thereby blocking the entry step of HCoV infection. Chloroquine, a compound that inhibits acidification of endosomes, can also strongly inhibit the replication of HCoV-OC43 in vitro. The nucleoside analog ribavirin has been used to treat RSV infection, hepatitis C, and some viral hemorrhagic fevers. However, ribavirin only shows inhibition of HCoV-OC43 replication in cell culture at high concentrations (IC50 B10 mM), which may not be applicable to humans in clinical treatment. Remdesivir (GS-5734) is a novel adenosine analog prodrug. By targeting the viral RdRP, remdesivir exhibits potent antiviral activity against HCoV-OC43 and HCoV-229E with submicromolar IC50 values in cell culture. Targeting host factors required for replication may be another viable therapeutic approach against HCoV infection. A systemic investigation of HCoV-host interactome has identified cyclophilins as targets for pan-coronavirus inhibitors. In fact, knockdown of cyclophilin A (CypA) in Caco-2 cells inhibits the replication of HCoV-NL63, suggesting that CypA is required for virus replication. Notably, when cells are treated with cyclosporinA (CsA) or FK506, potent immunosuppressive drugs that bind to CypA or related immunophilins, the replication of HCoV-NL63, HCoV-229E, and SARS-CoV is inhibited. Importantly, recent studies also show that CsD Alisporivir, NIM811, and other novel non-immunosuppressive derivatives of CsA and FK506 can also strongly inhibit the growth of HCoV-NL63 in cell culture at low micromolar, non-cytotoxic concentrations. Therefore, nonimmunosuppressive CypA inhibitors may be promising candidates as antivirals against HCoV infection. Interestingly, the replication of HCoV-229E and MERS-CoV is also suppressed when exogenous arachidonic acid or linoleic acid is added to the culture medium, suggesting that the regulation of host lipid metabolism could also be a common and druggable target for coronavirus infections.

Prevention Due to the mild diseases associated with the infection by the four common HCoVs, no vaccines are currently available or being developed. Risk of infection can be reduced by good hand hygiene and coughing etiquette and avoiding close contact with infected individuals.

Acknowledgment This work was partially supported by National Natural Science Foundation of China grant 31972660 and grant 31900135, Natural Science Foundation of Guangdong Province grant 2018A030313472, and Guangdong Province Key Laboratory of Microbial Signals and Disease Control grant MSDC-2017-05 and MSDC-2017-06.

Conflict of Interest The authors declare no conflict of interest.

Further Reading Corman, V.M., Muth, D., Niemeyer, D., Drosten, C., 2018. Hosts and sources of endemic human coronaviruses. Advances in Virus Research. 163–188. doi:10.1016/bs. aivir.2018.01.001. Dubé, M., Le Coupanec, A., Wong, A.H.M., et al., 2018. Axonal transport enables neuron-to-neuron propagation of human coronavirus OC43. Journal of Virology 92. doi:10.1128/JVI.00404–18. Fung, T.S., Liu, D.X., 2019. Human coronavirus: Host-pathogen interaction. Annual Review of Microbiology 73. doi:10.1146/annurev-micro-020518–115759. Milewska, A., Nowak, P., Owczarek, K., et al., 2018. Entry of human coronavirus NL63 into the cell. Journal of Virology 92. doi:10.1128/JVI.01933–17. Ou, X., Guan, H., Qin, B., et al., 2017. Crystal structure of the receptor binding domain of the spike glycoprotein of human betacoronavirus HKU1. Nature Communications 8, 15216. doi:10.1038/ncomms15216. Su, S., Wong, G., Shi, W., et al., 2016. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends in Microbiology 24, 490–502. doi:10.1016/j. tim.2016.03.003. Tvarogová, J., Madhugiri, R., Bylapudi, G., et al., 2019. Identification and characterization of a human coronavirus 229E nonstructural protein 8-associated RNA 30 -terminal adenylyltransferase activity. Journal of Virology 93. doi:10.1128/JVI.00291–19. Woo, P.C.Y., Lau, S.K.P., Yip, C.C.Y., Huang, Y., Yuen, K.-Y., 2009. More and more coronaviruses: Human coronavirus HKU1. Viruses 1, 57–71. doi:10.3390/v1010057. Zeng, Z.-Q., Chen, D.-H., Tan, W.-P., et al., 2018. Epidemiology and clinical characteristics of human coronaviruses OC43, 229E, NL63, and HKU1: A study of hospitalized children with acute respiratory tract infection in Guangzhou, China. European Journal of Clinical Microbiology & Infectious Diseases 37, 363. doi:10.1007/s10096-017–3144-z. Zumla, A., Chan, J.F.W., Azhar, E.I., Hui, D.S.C., Yuen, K.-Y., 2016. Coronaviruses – Drug discovery and therapeutic options. Nature Reviews Drug Discovery 15, 327–347. doi:10.1038/nrd.2015.37.

440

Human Coronavirus-229E, -OC43, -NL63, and -HKU1 (Coronaviridae)

Relevant Websites https://www.cdc.gov/coronavirus/index.html Coronavirus Disease 2019. https://medlineplus.gov/coronavirusinfections.html Coronavirus Disease 2019 (COVID-19) MedlinePlus. https://www.medicalnewstoday.com/articles/256521.php Coronaviruses Medical News Today. https://www.who.int/csr/disease/coronavirus_infections/faq_dec12/en/ Frequently asked questions on novel coronavirus Update. https://www.sciencedirect.com/topics/neuroscience/human-coronavirus-229e Human Coronavirus 229E. https://en.wikipedia.org/wiki/Human_coronavirus_HKU1 Human Coronavirus HKU1. https://www.sciencedirect.com/topics/medicine-and-dentistry/human-coronavirus-nl63 Human Coronavirus NL63. https://www.sciencedirect.com/topics/veterinary-science-and-veterinary-medicine/human-coronavirus-oc43 Human Coronavirus OC43. https://talk.ictvonline.org/p/coronavirus-genomes ICTV.

Human Cytomegalovirus (Herpesviridae) Edward S Mocarski, Emory University School of Medicine, Atlanta, GA, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary allogeneic MHC-mismatched cell, tissue or organ. allograft A tissue or organ from someone other than an identical twin. alphaherpesvirus A subfamily of herpesviruses characterized by epithelial infection with latency in sensory neurons. antibody Secreted protein, called immunoglobulin, made by immune system plasma cells that protect by recognizing a specific antigen such as on the surface of viruses. antigen a specific pathogen component recognized by antibody, B cell receptor or T cell receptor. betaherpesvirus A subfamily of herpesviruses that includes the cytomegaloviruses and closely related salivary gland viruses. capsid The protein shell part of a virion into which viral DNA is packaged. capsomer A subunit of the capsid, also called a protomer. cleavage/packaging A process of inserting viral DNA into a capsid and cutting viral DNA during nucleocapsid maturation. concatemer A long repetitive head-to-tail structure, applied to replicating viral DNA prior to cleavage/packaging. congenital Evident at birth. core gene A subset of evolutionarily conserved common herpesvirus genes that carry out entry, replication, virion formation and maturation. cytopathology An alteration in appearance of cells, typically during infection. dense body A capsidless particle composed of tegument surrounded by an envelope. differentiation state-dependent A process that only occurs in cells at a particular point in their developmental or somatic differentiation. DNA A type of nucleic acid that encodes genes and forms the genome of some viruses and all organisms. early gene A gene expressed prior to the initiation of viral DNA replication. egress A process during viral maturation where the virion leaves the cell. encapsidation The process of packaging viral DNA into nucleocapsids. envelope A lipid bilayer membrane that surrounds the tegument of virions and dense bodies that contains viral glycoproteins responsible for binding to and entering into cells. envelopment The process of adding an envelope to nucleocapsid. epigenetic non-heritable. ERGIC Endoplasmic reticulum-Golgi intermediate compartment, the cellular site of envelopment.

Encyclopedia of Virology, 4th Edition, Volume 2

gammaherpesvirus A subfamily of herpesviruses characterized by oral transmission, oncogenicity and latency in lymphocytes. genome The nucleic acid molecule that encodes all of the genes of a virus or an organism. helicase-primase A replication enzyme that unwinds nucleic acid and synthesizes short primers that are extended by DNA polymerase. hematopoietic Blood-forming. herpesvirus A virus family characterized by a large DNA genome, similar-appearing enveloped virion particle and lifelong latency. hexon A type of capsomere/protomer forming most of the capsid structure. homologous gene products Proteins with evolutionary relatedness. immediate early gene The first set of genes expressed during infection. immunity Resistance to infection and disease through natural (innate) or acquired (adaptive) host defense mechanisms immunocompetent Having the capacity for immunity. immunocompromised Lacking the capacity for immunity. immunodeficiency An inborne or acquired lack of immunity. immunosuppression A reduction in immunity. inapparent Lacking in signs or symptoms. infectious Capable of person-to-person transmission (transmissible). late gene The last set of genes expressed during infection. latency Persistence in the absence of active replication with the potential to reactivate. leukodepletion Reducing the leukocytes. major histocompatibility complex (MHC) Tissue antigens that present antigen to T cells that vary in the population such that no two people are alike (except for identical twins). monoclonal antibody Antibody produced by a single B cell or plasma cell recognizing a specific antigen. mononucleosis The proliferation of mononuclear leukocytes (lymphocytes). neurodevelopmental Related to the development of the nervous system. nucleocapsid A DNA-containing capsid. opportunist A pathogen that infects and causes disease in immunocompromised hosts. opportunistic pathogen Causing disease the immunocompromised host. pathogen A disease-causing virus, microbe or multicellular organism.

doi:10.1016/B978-0-12-814515-9.00004-7

441

442

Human Cytomegalovirus (Herpesviridae)

pathogenesis The process of causing disease. penton A type of capsomere that composes capsid vertices. persistence The act of remaining. plaque forming unit A single infectious virus as determined by plaque assay. portal An entrance. promoter A segment of DNA that directs transcription of a gene. reactivation The process of transitioning from latency to active replication. reinfection The act of becoming infected with the same pathogen again. replication The process of making more viral DNA and virus. restriction factor A gene product that slows down or blocks a pathogen. roseolavirus A subset of betaherpesviruses that includes HHV-6A, HHV-6B and HHV-7. sequence identity Having similar nucleic acid (DNA or RNA) or amino acid (protein) order.

seronegative Lacking pathogen-specific antibody. seropositive Having pathogen-specific antibody. seroprevalence Proportion of seropositive individuals in a population. tegument The layer of protein between the capsid and envelope in a virion. transfusion The transfer of blood from one person to another. transplacental Crossing the maternal-fetal interface during pregnancy. transplant The transfer of a tissue or organ from one individual to another. tropism The likelihood of a particular tissue or cell to support virus replication. viral assembly compartment A cytoplasmic location where virus assembly and release takes place. virion A assembled, infectious virus particle, typically observed outside of a host cell.

Classification Cytomegaloviruses (CMVs) are defining members of the betaherpesviruses, one of the major subfamilies of herpesviruses infecting mammalian hosts. Herpesviruses are a biologically diverse, ancient group of viruses that are characterized by large DNA genomes (from just over 100,000 kilobase pairs to nearly 300,000 kilobase pairs), as well as a common virion structure, replication scheme and the biological property of lifelong latency. Betaherpesviruses generally, and CMVs in particular are more highly host restricted than other herpesviruses. CMVs were initially referred to as salivary gland viruses because they are sporadically shed into saliva which, along with urine and breast milk, represent primary sources for transmission. Although restricted to a single host species in nature, all CMVs share a common ancestor, biological features and characteristic cytopathology that includes a nuclear site of replication and a cytoplasmic assembly compartment where final envelopment and egress occur (Fig. 1). Despite species specificity, amino acid sequence similarity in proteins provides molecular evidence supporting the biological relatedness of betaherpesviruses that infect various animal species (Fig. 2). The cytomegalovirus (CMV) of humans (HCMV), officially classified Human herpesvirus (HHV)5 within the genus Cytomegalovirus, subfamily Betaherpesvirinae of the family Herpesviridae, has the largest and most complex genome of any human herpesvirus with a potential to encode hundreds of gene products. The other human betaherpesviruses, roseolaviruses classified as HHV6A, HHV-6B and HHV-7 have smaller genomes and retain homologs of approximately half of the canonical, annotated HCMV genes (shaded genes in Table 1). Betaherpesviruses of mice, rats, guinea pigs and monkeys are all species-restricted viruses that are more similar to HCMV than to roseolaviruses. Because they naturally infect laboratory animals, all have been used to model aspects of HCMV infection, immunity, latency and pathogenesis. Mammalian betaherpesviruses replicate slowly in cultured cells which must be from their natural host species, remain cell-associated within the host, and show genetic relatedness to each other more so than they do to either alphaherpesviruses or gammaherpesviruses.

Virion Structure Virions of HCMV appear similar to other herpesviruses (Fig. 1) despite a larger genome and a more complex coding capacity. The B230 nm enveloped virion includes a linear double-stranded DNA genome tightly packed within a 130 nm icosahedral nucleocapsid surrounded by a tegument layer with B30 virus-encoded phosphoproteins surrounded by a lipid bilayer envelope containing B22 viral membrane glycoproteins (Table 1). The most abundant tegument constituents are phosphoprotein (pp)65 (ppUL83) and pp150 (ppUL32). Although the most structurally complex of any characterized herpesvirus, the HCMV capsid is composed of herpesvirus-conserved major capsid protein (MCP), the primary constituent of capsomers (both hexons and pentons), as well as minor capsid proteins that form intercapsomeric triplexes, the smallest capsid protein (SCP) that decorates the hexon tips, and the portal protein (PORT) comprising one penton position responsible for conducting viral DNA encapsidation. In addition to a linear molecule of DNA, RNA associates with the origin of lytic DNA replication (oriLyt) in virions. The most biologically important HCMV envelope constituents are glycoprotein (g)B and complexes composed of gH:gL:gO (gH/gL trimer), gH:gL:pUL128:pUL130:pUL131A (gH/gL pentamer) and gM:gN which all play crucial roles in replication. Envelope gB and gH:gL complexes are very important targets of host humoral (antibody) immunity. Numerous additional envelope glycoproteins

Human Cytomegalovirus (Herpesviridae)

443

Fig. 1 Cryo and transmission electron micrograph (EM) of HCMV particles and HCMV-infected human fibroblast cells. A. Cryo-EM virion particle structure (strain AD169) showing the nucleocapsid, tegument and envelope with embedded gB (but without gH: gL pentamer) after sedimentation (note distortion of the envelope). B. Cryo-EM nucleocapsid particle from within a virion showing capsomeres and appearance of encapsidated viral DNA. C. Transmission EM of HCMV infection (multiplicity of infection ¼ 3) at day five postinfection showing a cell with nucleus and mature virions and dense bodies within the cytoplasmic assembly compartment where final envelopment precedes release by exocytosis through the plasma membrane to the extracellular space. Released mature extracellular virus particles accumulate between adjacent cells of the monolayer. Inset shows a close-up of mature virions (arrows) and dense bodies (arrow heads) within the assembly compartment. Intracellular virus particles are contained within vesicles. Panels A and B courtesy of Hong Zhou, UCLA.

(Table 1) are dispensable for replication in cultured cells although each likely contributes to virus biology in the host by influencing cell tropism, tempering host response to infection and dictating pathogenesis as well as persistence. Capsidless particles (called dense bodies), composed solely of tegument surrounded by an envelope, are produced in numbers equivalent to virions during infection (Fig. 1). Dense bodies as well as noninfectious capsid-containing particles predominate in all virus preparations, such that particle-to-plaque forming unit (PFU) ratios of HCMV stocks range from a low of 100:1 to over 1000:1.

Genome Structure The HCMV genome is a single B236,000 bp double-stranded DNA molecule composed of unique long (UL) and unique short (US) regions flanked by terminal and internal repeats in an arrangement depicted ab-UL-b0 a0 c0 -US-ca (alternative depiction,

444

Human Cytomegalovirus (Herpesviridae)

Human Cytomegalovirus (Herpesviridae)

445

TRL-UL-IRL-IRS-US-TRS). This structure is similar to human herpes simplex viruses but, with the exception of chimpanzee CMV, is not observed with nonhuman CMVs. Because recombination occurs between the inverted repeats of these herpesvirus genomes, virus particles carry one of four different sequence arrangements, or isomers, that are all infectious. The genome depicted in Fig. 2 includes conventional annotated and canonical protein-coding genes, four long noncoding (lnc) RNA genes and an origin of replicative DNA synthesis (oriLyt) located between UL57 and UL69, cleavage/packaging signals (pac1 and pac2 within terminal a sequences) and the major immediate early (IE) promoter (MIEP), a transcriptional enhancer that controls transcription of IE1 (p72) and IE2 (p86) genes (located between UL124 and UL128). The genome also encodes 23 micro (mi)RNA genes not depicted in Table 1 or Fig. 2. Importantly, HCMV is known to encode many more than the B200 annotated gene products shown in such conventional depictions. Hundreds of additional candidate viral proteins have been detected experimentally. The genome organization of human roseolaviruses (HHV6A, HHV6B and HHV7) as well as other nonhuman CMVs are co-linear with the HCMV genome, although their smaller genomes (145,000–162,000 base pairs) encode only B85 proteins where B70 genes are colinear with HCMV, from UL23 to UL124 (shaded rows in Table 1). Despite these evolutionarily conserved gene products, human roseoloviruses and HCMV do not exhibit significant DNA sequence identity. All characterized betaherpesvirus genomes also have a similarly positioned oriLyt, MIEP-enhancer and terminal direct repeats with conserved genome-terminal cleavage/packaging signals.

Evolution Based on the similarity to HCMV gene products and genome organization, CMVs have been estimated to have coevolved with common ancestors of mammals over the past 150 million years. The core genes that mediate the herpesvirus-conserved processes of viral DNA synthesis, capsid assembly and genome cleavage/packaging (nuclear events shown in Fig. 3), as well as a set of genes involved in transcription of true-late gene products, including a viral TATA binding protein (Table 1), likely have their evolutionary origin in a distant DNA bacteriophage ancestor. These and virion maturation events from nuclear egress through final envelopment and release require the function of herpesvirus core genes (Table 1 and Fig. 3). Because of their long evolution, betaherpesviruses exhibit a greater level of divergence than their respective host species. As such, non-human primate CMVs exhibit diversity in gene content and function evident even when comparing great ape and human CMVs. This extreme diversity in biologically related, but species-specific viruses is much greater than observed in alphaherpesviruses or gammaherpesviruses. The greatest conservation in genome organization is between viruses infecting closely related host species such as HCMV and chimpanzee CMV or murine CMV and rat CMV. There even appears to be a level of diversity generated during lifelong infection with HCMV, above and beyond the presence of multiple strains in a single host. Complete genome sequences have been determined for many circulating HCMV strains, including those that have been detected directly in secretions or infected tissues. An average of 495% DNA sequence identity is observed between HCMV strains. A similar range of viral strains appear to circulate worldwide. The presence of multiple strains and within-host variation as well as the detection of multiple distinct strains within a single individual are all features that may influence disease pathogenesis but most certainly document susceptibility to reinfection. Thus, in every animal species that has its own CMV, the virus enjoys both a widespread distribution and the ability to superinfect.

Life Cycle HCMV, and related mammalian viruses, cause systemic infection primarily targeting myelomonocytic, epithelial and endothelial cells. Like other herpesviruses, HCMV establishes chronic/persistent infection characterized by lifelong latency. Only a minor proportion of viral genes, mostly betaherpesvirus-common and herpesvirus core proteins, carry out DNA replication and cleavage/ packaging as well as virion assembly, maturation and release (Table 1). A majority of cytomegalovirus gene products modulate host defense mechanisms, acting within an infected cell (cell autonomous) as well as systemically to alter the course of innate and adaptive immunity, subverting some pathways and enhancing others (see Pathogenesis section). CMVs target conserved host defense pathways, evidence that these master manipulators co-evolved during the radiation of mammalian species as immune mechanisms were acquired.

Host Range HCMV only infects humans. There are no known susceptible nonhuman hosts, in nature or under experimental conditions. Furthermore, cultured human epithelial, endothelial, neuronal, myeloid and fibroblast cells are all susceptible. Chimpanzee Fig. 2 Genetic organization and annotated gene content of wild-type HCMV, based on low passage strain Merlin. The 236 kbp HCMV genome is depicted on eight lines with each gene arranged by orientation. Inverted repeats TRL/IRL (ab-b0 a0 ) and TRS/IRS (a0 c0 -ca) are shown in a thicker format than the UL and US components. Protein-coding regions (ORFs) are indicated by large colored arrows grouped according to the key shown at the bottom. 15 different gene families, core ORFs conserved across all mammalian and avian herpesviruses, betagamma ORFs conserved only in betaherpesviruses and gammaherpesviruses and other non-core ORFs are each depicted by a separate color. Noncoding RNAs are indicated by medium, white arrows (micro RNAs are not shown). Introns are shown as narrow white bars. Gene nomenclature is shown below the genome depiction. UL72 is both a core ORF and a member of the DURP gene family. Reproduced by permission of the Society for General Microbiology; modified by A. Davison.

446

Table 1

Human Cytomegalovirus (Herpesviridae)

Annotated cytomegalovirus gene products: Characteristics and functions

Human Cytomegalovirus (Herpesviridae)

Table 1

447

Continued

(Continued )

448

Table 1

Human Cytomegalovirus (Herpesviridae)

Continued

Human Cytomegalovirus (Herpesviridae)

Table 1

449

Continued

(Continued )

450

Table 1

Human Cytomegalovirus (Herpesviridae)

Continued

fibroblasts support HCMV replication, but cells from other species do not. Except for certain glioblastoma cell lines, undifferentiated or transformed cells are not susceptible to HCMV. Nonpermissive human cell lines as well as many nonhuman cells support viral entry but do not support replication owing to the presence of host restriction factors. Clinical isolates of HCMV show considerable sequence variability and sometimes represent mixtures of strains arising as a result of multiple infection or reinfection. Clinical strains undergo adaptation to cultured fibroblasts by losing expression of specific viral genes, mediated through point mutation, deletion and inversion, thereby confounding designation as well as experimental evaluation of wild type virus (Table 1). HCMV naturally initiates infection in mucosal epithelium, disseminates within myelomonocytic leukocytes that carry the virus in the bloodstream to other locations, such as endothelial and epithelial sites, in the host. In immunocompetent individuals, virus characteristically disseminates to and replicates in ductal epithelial cells of the salivary glands and kidney tubules, allowing shedding in saliva and urine that may continue for months to years, particularly in young children. Host innate and adaptive immune mechanisms dictate the level of systemic dissemination, with a much broader tissue distribution and greater levels in infected immunocompromised individuals or newborns compared to adults with fully mature immune systems. Levels of virus infection and extent of tissue involvement dictated by host immune status determine the risk for disease following infection. Tissue samples from immunocompromised individuals with acute HCMV disease affirm that HCMV replicates naturally in epithelial, fibroblast, endothelial, macrophage, neuronal and dendritic cells. Viral antigens are readily detected in infected tissues as well as in peripheral blood neutrophils of immunocompromised individuals with a risk of HCMV disease using monoclonal antibody reagents. One of the most important, yet poorly understood aspects of host cell range, is intrauterine infection and transplacental transmission where virus gains access to the developing fetus and results in congenital disease. HCMV infection during pregnancy starts with systemic dissemination to the maternal side of the placenta and continues with access to the maternal-fetal interface but little is known about the cells involved in this medically significant process. When transmission occurs during primary infection of a pregnant woman, there is a B40% chance that the fetus will become infected with about 10% of infected newborns showing signs of neurodevelopmental damage and disease at birth. In the case of transmission during recurrent infection, the chance of infecting the fetus has been estimated to be o1% with a similar proportion (10% of infected newborns) showing neurodevelopmental damage at birth. HCMV reactivates and is shed from ductal epithelium in mother’s milk, an important route of virus transmission in the human population worldwide. Entry into cells depends on specific envelope glycoproteins that recognize distinct cell surface receptors and lead to the fusion with plasma membrane and deposition of the HCMV nucleocapsid into the cytoplasm of the cell (Fig. 3). The cellular receptors mediating HCMV entry vary with cell type but include human platelet-derived growth factor receptor (PDGFR), epithelial growth factor receptor (EGFR), neuropilin-2, OR14l1 and various integrins. PDGFR binds to trimeric envelope (gH:gL:gO) complex, mediating efficient attachment to and entry into fibroblasts and some other cell types; whereas, neuropilin-2 and OR14l1 are cell surface receptors for a pentameric complex (gH:gL:pUL128:pUL130:pUL131A), that mediate attachment to and entry into

Human Cytomegalovirus (Herpesviridae)

gB (UL55) gH:gL (UL75:UL115) gH:gL:gO (UL75:UL115:UL74) gH:gL:pUL128:pUL130:pUL131

CYTOPLASM ppUL69 pp71 (UL82) pUL35 pUL26

Plasma Membrane

451

Golgi Body

Virion Vesicle Transport

ppUL48 pUL47 pUL48A

ER

Nuclear Pore

IE1p72 (UL123) IE2p86 (UL122) p84 & p43 (UL112-113) pUL21A ppUL29 Viral mRNA pUL34 pUL117 ppUL69

Viral Proteins and RNAs

Viral DNA

Viral Assembly MCP (UL86) Compartment Triplex (UL46:UL85) SCP (UL48A) AP (UL80.5) Pr-AP (UL80) Portal (UL104) TER (UL51:UL56:UL89)

ER-Golgi Intermediate Compartment

CVC (UL48:UL77:UL93) POL:PPS (UL54:UL44) pUL52; VPK (UL97) HP (UL70:UL102:UL105) SSB (UL57) pUL84 NUCLEUS NEC (UL50:UL53) (UL79:UL87:UL91:UL92:UL95) Viral mRNA

pUL88 pUL103

pp28 (UL99) pUL94 pUL71 gM:gN (UL100:UL73)

ppUL32 pUL47 ppUL48 pUL96 VPK (UL97)

Fig. 3 Summary of the HCMV replication pathway with focus on herpesvirus core (red) and betaherpesvirus-conserved (black) gene products playing known or predicted roles in the viral replication cycle from entry (upper left) to release of progeny virions and dense bodies (upper right). Major steps in the productive replication cycle are indicated in large red outlined font and major activities are indicated in large gray outlined font, with black arrows indicating the procession and individual functions identified by abbreviated names. The entry pathway shown employs direct fusion at the plasma membrane (attachment and penetration) although an endocytic pathway is important in some cell types. Entry requires gB, along with gH:gL:gO and/or gH:gL:pUL129:pUL130:pL131A, depending on the cell type. Virion tegument proteins (pUL69, pUL82, pUL35 and pUL26) are involved in regulation of the initial steps in replication. Nucleocapsid-associated pUL48, pUL47 and pUL48A are likely to control transport on microtubules, docking at nuclear pores and release of virion DNA from the capsid into the nucleus. Host cell RNA pol II is responsible for transcription of viral and host cell genes. Gene expression is regulated by products of IE genes (IE1-p72, IE2-p86) working together with E genes (UL34, UL112-UL113 UL69) and LL genes (UL21A, UL29, UL117), together with a late gene transcription complex that includes UL79, UL87, UL91, UL92 and UL95 gene products, all modifying the activity of RNA pol II. Viral DNA replication, which also takes place in the nucleus, depends on core proteins (POL: PPS, HP and SSB) as well as three betaherpesvirus-specific gene products (UL84, UL34 and UL112–113). Capsid assembly incorporates proteins (MCP, triplex and SCP) around an AP scaffold initiated by the portal protein, as occurs in other herpesviruses. Preformed capsids translocate to sites of DNA replication where maturational protease Pr-AP works in conjunction with a terminase complex (UL51, UL56 and UL89) and capsid vertex complex (UL48, UL77 and UL93), together with UL97 and UL52 gene products to cleave and package viral DNA. Nuclear egress and primary envelopment at the inner nuclear membrane is under the control of UL50 and UL53 gene products and capsid stability is provided by UL32, UL48, UL47 and UL96 gene products together with UL97 protein kinase. De-envelopment at the outer nuclear membrane, translocation on microtubules to the viral assembly compartment in the cytoplasm is followed by secondary (final) envelopment controlled UL99, UL94 and UL71 gene products with the UL97 protein kinase at specific cytoplasmic membranes. Virion envelope glycoproteins follow an independent vesicle transport pathway from the ER via the Golgi body to site of final envelopment (ER-Golgi intermediate compartment) in the cytoplasm (dashed gray arrows). HCMV modifies the cellular exocytic transport pathway from sites of secondary envelopment to release via the action of the UL88 and UL103 gene products.

epithelial, endothelial and certain myelomonocytic cell types. Envelope gB is responsible for fusing the viral envelope with cellular membranes and delivering the nucleocapsid to the cytoplasm during entry regardless of cell type (Fig. 3). Although crucial to cell tropism within the host, functions that control attachment and entry do not dictate the restricted host range characteristic of CMVs. For HCMV, this is primarily a consequence of a block to early post-entry step such that nonpermissive cells from distant host species (for example rodent) allow all steps through initial (IE) HCMV gene expression but do not support viral DNA synthesis or later phases necessary for production of progeny virus. Current evidence implicates early activation of cell death together with other host factors acting prior to viral DNA replication in restricting CMV replication within non-natural host species.

452

Human Cytomegalovirus (Herpesviridae)

Replication Steps The following categorical steps in replication are common across herpesviruses (depicted in Fig. 3): (1) virion binding to cell surface receptors through gH:gL complexes; (2) fusion mediated by virion envelope gB targeting the plasma membrane, either directly or after endocytosis into vesicles, and resulting in the release of nucleocapsids into the cytoplasm; (3) nucleocapsid association with microtubules with translocation to nuclear pores; (4) deposition of the viral genome out of the capsid into the nucleus; and, (5) coordinately regulated RNA pol II transcription that produces immediate early (IE), early (E) and late (L) gene products (Table 1). As with all herpesviruses, different temporal classes of genes are interspersed across the HCMV genome. For example, the 11 IE genes map to diverse locations in the UL, US and IRS/TRS regions of the viral genome (Table 1 and Fig. 2). Replication proceeds over several days before progeny virus are detected within cells or released extracellularly. Host RNA pol II transcription machinery becomes modified by virus-encoded proteins within the cell nucleus and synthesizes messenger (m)RNA as well as three types of noncoding RNAs, long noncoding RNAs, micro RNAs and oriLyt-associated vRNAs. Translation of virusencoded mRNAs into proteins is carried out by host cell polyribosomes in the cytoplasm where active translation of several hundred uncharacterized proteins that expand the coding potential of this virus well beyond annotated genes (Fig. 2). Initially, the HCMV MIEP-enhancer provides regulatory control over the decision whether to replicate, and epigenetic regulation dictates outcome in a cell type- and differentiation state-dependent manner that is influenced by viral gene products as well as the host cell immune environment. The MIEP generally confers very strong constitutive transcriptional activity, even in nonpermissive cells, enabling this transcriptional element to be widely adapted for experimental and commercial production of recombinant proteins. Epigenetic restriction of viral transcription is interrupted by several viral modulatory proteins, both from the input tegument as well as gene products produced during infection. These balance replication, persistence, latency and reactivation in a cell-typedependent fashion. Tegument proteins regulate viral gene expression and modulate the host response from earliest post-entry steps through late stages of replication. Input virion tegument proteins control the release of virion DNA into the nucleus, suppress intrinsic host defense mechanisms that would otherwise restrict viral replication and mediate activation of IE gene transcription. Later, tegument proteins modulate the cell-autonomous host response and support key steps in envelopment and egress from the nucleus and cytoplasm. During the initial steps of infection, the MIEP-enhancer is transactivated by virion tegument protein complex, enhancing RNA pol II transcription of IE genes. The IE gene products activate E and L gene transcription, including E and L products from the MIE regions itself, while suppressing the host cell response to infection, innate immune cell death and cytokinemediated immunity. Expression of B52 E genes requires functional IE1 and IE2 proteins. The replication cycle of HCMV is much slower than herpes simplex virus, requiring 24–36 hrs for the progression from E to L phase, 48–72 h until progeny start to accumulate, and maximum levels of virus release continuing from 96 hrs to a week post infection. Viral DNA synthesis initiates through events that depend upon a subset of regulatory IE, and E proteins (Table 1). Following initiation, the viral DNA polymerase complex assembles at replication forks together with single stranded DNA binding protein and helicase-primase complex that together produce long double-stranded DNA concatemers that are both a template for late transcription and a substrate for genome cleavage/packaging machinery (Fig. 3). All steps in replication depend on protein phosphorylation by a viral protein kinase together with host cell cycle kinases. At least two categories of L genes are encoded based on patterns of expression and dependence on viral DNA synthesis, known as leaky late (LL) and true late (TL). A late transcription complex that is also employed by gammaherpesviruses modifies the behavior of host RNA pol II to facilitate transcription of TL viral genes, likely in close collaboration with viral DNA replication machinery. HCMV capsid formation and maturation (capsid assembly, maturation and egress from the nucleus) are largely carried out by herpesvirus core genes (Table 1). Non-core gene products provide a means to evade and engage the host response to infection for the benefit of the virus. Viral DNA synthesis and capsid assembly proceed independently in the nucleus but converge at the point where concatemeric DNA is cleaved and packaged, or encapsidated, into preformed capsids (Fig. 3), filling each progeny capsid with a unit-length genome. Seven herpesvirus core encapsidation proteins process a concatemeric DNA template by recognizing sequence-specific cleavage/packaging (pac) sites located near genomic termini once a genome-length molecule has been inserted into a capsid. The resulting filled nucleocapsids become stabilized by the addition of capsid-proximal tegument proteins, and then proceed through the inner nuclear membrane under control of a nuclear egress complex. Transfer of the nucleocapsid into the cytoplasm most likely occurs by initial addition of a lipid bilayer envelope at the inner nuclear membrane and removal of this envelope through fusion with the outer nuclear membrane. The tegumented capsid gains a final (second) lipid bilayer envelope at ER-Golgi intermediate compartment (ERGIC) membranes decorated by virus-encoded glycoproteins within modified region of the cytoplasm called the viral assembly compartment (VAC), an area where viral egress also occurs (Fig. 1). Dense bodies form when tegument becomes enveloped in the cytoplasm and proceeds through cytoplasmic maturation steps along with virions. Thus, mature virions, noninfectious capsid-containing and capsidless dense body particles all accumulate at sites of final envelopment within the VAC prior to release from cells. Vesicle transport (exocytosis) machinery mediates the translocation and release of virus particles. Progeny virions are composed of B100 different virus-encoded proteins enclosing a single viral DNA molecule. The maturation process results in an excess of noninfectious and defective particles, such that only 0.1%–1% of progeny particles are able to initiate a new infection. Once maturation starts, infected fibroblasts remain viable and continue to release virus for days. A final burst of virus release accompanies the eventual death of infected cells after about five to seven days.

Human Cytomegalovirus (Herpesviridae)

453

Epidemiology Patterns of Infection HCMV is a ubiquitous virus with worldwide prevalence ranging from B60% to greater than 90%. Overall 95% of the world population becomes infected before old age. Contact with infected bodily secretions drives transmission without involvement of any intermediate host or any seasonal prevalence. Infection is typically subclinical in immunocompetent individuals. Transmission is more efficient in higher density living conditions, with larger family size and in settings of reduced hygiene. Transmission is reduced by simple measures such as avoiding salivary transfer hand washing. On a population level, HCMV is only rarely acquired congenitally (B1%). Breast milk and contact with other bodily secretions account for the majority of transmissions throughout life. Thus, infection posing a significant risk of disease when acquired in utero and is a certain risk when primary infection occurs during pregnancy. Developing areas (Eastern Europe, Africa, Asia and South America) exhibit higher rates of transmission at younger ages, than more developed areas (North America, Australia and Western Europe). Infants and young children infected with HCMV shed persistently in saliva and urine and represent important sources of new infections. In such settings, avoidance of direct contact with infected secretions and good hygiene practices reduces transmission from children to adults as well as between adults. During lifelong latency, virus may reactivate and be shed sporadically in saliva, urine and genital secretions. Blood and tissues from infected, HCMV seropositive individuals transmit virus to recipients of blood transfusions and transplants. Use of prescreened blood products from HCMV seronegative donors and leukodepletion of blood from seropositive donors has prevented transfusion-associated infections in many areas of the world. In US-specific surveys, overall HCMV seroprevalence has remained constant at B60% for decades, similar to other developed areas of the world. HCMV is acquired by 40%–50% of the US population by midlife, increasing to 90% by old age. Transmission patterns are influenced by host innate and adaptive immunity. Immunoprophylaxis and, particularly, vaccination would be expected to interrupt person-to-person transmission of this virus. Thus, transplacental transmission occurs much more frequently in pregnant women with primary infection than in HCMV-seropositive women with long-term infection and pre-existing immunity. The importance of adaptive cellular immunity against HCMV in preventing active infection is supported by the clinical experience in immunocompromised transplant recipients as well as AIDS. In solid organ transplantation, the HCMV seronegative recipient of an organ from a donor with long-term infection is most at risk of infection and disease. In this setting, the organ introduces the virus into a recipient. In allogeneic hematopoietic stem cell transplantation (HSCT), performed with either mobilized blood or bone marrow, the HCMV seropositive recipient of hematopoietic stem cells from a HCMV naïve host runs the greatest risk of HCMV disease. In this setting, the transplanted HCMV-naïve immune system cannot suppress virus. The nature of immune control in settings of congenital disease remains poorly understood. Whereas exogenously acquired primary infection is the sole source of virus in a HCMV-naïve woman, reactivation of latent virus, mixed infection with additional strains of virus have been observed in long-term seropositive women. All of these sources may contribute to intrauterine transplacental transmission, congenital infection and consequent disease in progeny of seropositive women. Serologic assays provide an accurate indication of lifelong infection with HCMV, but do not provide insights into viral shedding levels or susceptibility to reinfection. Levels of antiviral antibody and cellular immunity rarely correlate in seropositive individuals. Although strain differences are recognized using virus antigen-specific monoclonal antibodies that have facilitated characterization of mixed infections, distinct HCMV serotypes are not recognized. Together with various molecular genetic approaches that assess within host viral genome sequence variation, these approaches have provided evidence that different viral strains may predominate within a single individual at different times and that such mixed infections can arise from sequential reinfection.

Virus Propagation HCMV is most readily isolated from saliva or urine, sites of virus replication in ductal epithelial cells of salivary glands and kidney. Virus may be isolated on cultured susceptible cells from the original host species. Fibroblast cultures are most commonly used for virus isolation. The ability to replicate in epithelial and endothelial cells is lost when virus is propagated in fibroblasts because components of the gH/gL pentamer complex (UL128, UL130 and UL131) accumulate mutations. Primary or secondary cultures of epithelial, endothelial, macrophage and dendritic cells sustain gH/gL pentamer function. Additional mutations that disrupt genes that are either unnecessary or deleterious to virus replication occur. Fully wild type HCMV is very difficult to maintain in a culture. Several genes must be modified by conditional mutation. Variants of common laboratory strains, typically referred to by a single name (e.g., AD169 or Towne) have arisen as these strains have been serially propagated in different laboratories around the world. This makes comparisons between laboratories somewhat challenging even when using a strain that carries a common name. Clonal virus strains maintained as bacmids in bacteria currently predominate for experimental work; however, these strategies also generate unintended mutants and variants. Thus, clonal virus stocks should be prepared for which the full genome sequence has been determined. The difficulty of working with clinical isolates, the species specificity of replication and the rapid generation of variants when HCMV DNA is transfected into cells (or is propagated in cell culture) are well-recognized experimental confounders. HCMV is easiest to isolate from saliva or urine of infected individuals, virus may also be isolated from semen, cervical secretions, blood cells and breast milk, particularly during primary infection. Infectious virus or viral DNA is only rarely shed in HCMV seropositive adults with clinically silent long term latent/persistent infection. DNA PCR methods have been most successful for the detection of recurrent shedding, as a surrogate for virus in saliva, urine, milk, blood, tissues and other secretions. These

454

Human Cytomegalovirus (Herpesviridae)

methods have revealed that children with congenital or perinatal infection naturally shed virus for prolonged periods lasting up to years, that HCMV-seropositive adults shed less than 5% of the time and that shedding increases following pregnancy.

Clinical Features HCMV infection is generally inapparent. Infectious mononucleosis can be a rare consequence but is typically less severe than that caused by Epstein-Barr virus. Women of childbearing age often encounter HCMV primary infection without showing specific symptoms. Such asymptomatic infections are the major way this virus is transmitted to offspring during pregnancy. As a consequence of viral transmission during primary or recurrent infection, HCMV remains the single most important infectious cause of congenital disease in newborns, as significant as major genetic diseases such as Down syndrome and more frequent than other genetic congenital diseases. HCMV is a classic opportunist such that infection in immunocompromised hosts results in systemic HCMV disease affecting one or more organs, referred to as CMV syndrome. HCMV has been known to unveil immunodeficiency in individuals with a previously unrecognized combined or T cell-specific immune deficit, and as such is one of the most prominent AIDS-defining infections. As a ubiquitous herpesvirus that causes systemic infection, lifelong infection with HCMV correlates with chronic disease, immune defects and frailty, particularly in the elderly. There have been postulated contributions to atherosclerotic vascular disease, autoimmune vasculitis, systemic lupus erythematosus, Sjogren’s syndrome, periodontal disease, cancer, immunosenescence and risk of some specific diseases such as Crohn’s disease and glioblastoma multiforme. These associations between a common virus and chronic diseases require further scrutiny.

Congenital Disease Congenital disease represents the hallmark of HCMV pathogenesis. This disease follows transplacental transmission to the fetus during pregnancy. The highest incidence of mother-to-child transmission occurs from the first trimester through the gestational midpoint. Neurodevelopmental disease consequences such as hearing and eyesight compromise are most common. Broader neurological symptoms recognized at birth or during childhood are less common, but important. HCMV congenital disease remains a well-recognized, though medically underappreciated public health concern. HCMV is also a substantial cause of spontaneous abortion and still birth. The at-risk population is difficult to identify because infection in the pregnant woman is asymptomatic. HCMV congenital infection affects between 0.2% and 2% of pregnancies, where transmission remains negatively correlated with socioeconomic conditions. Nevertheless, congenital disease affects roughly 20% of HCMV-infected newborns regardless of socioeconomic status, with about half of babies with disease evident at birth and half developing in the newborn over the first years of life. Left untreated, disease generally worsens. Mild, but developmentally and medically significant congenital disease is often overlooked. Demographic characteristics contribute to transmission patterns, with younger (teenage) females more likely to transmit during pregnancy than women in their twenties or thirties. Severe congenital disease is relatively uncommon but involves extensive neurological symptoms such as retinitis, microcephaly, seizures and neurodevelopmental disabilities as well as damage to the reticuloendothelial system (petechial rash, hepatosplenomegaly, jaundice, extramedullary hematopoiesis) and low birth weight. This severe disease syndrome is known as CMV inclusion disease (CID). Antiviral drugs such as ganciclovir are used to treat CID. In addition, low birthweight, premature infants transfused with blood from an HCMV seropositive donor or breast fed by an actively shedding mother risk developing systemic disease (sepsis, hepatitis, pneumonitis) that is clinically distinct from CID. By the 1970s, the spectrum of congenital CMV disease was well-characterized, although medical awareness has not improved over the intervening decades primarily because of the belief that little could be done except to address the consequent disability. It is now clear that early intervention to improve sensorineural perception improves quality of life outcomes. Except for neurological disease and multiorgan CID, intervention with antiviral drugs, either in pregnancy or in newborns, has not yielded of significant benefit. Furthermore, the substantial contribution of recurrent maternal infection to transmission worldwide and the protean nature of neurodevelopmental damage has resulted in testing for HCMV infection at birth in some countries. Overall, the disease consequences of congenital CMV can be addressed through enhanced testing, increased clinical recognition and appropriate directed therapies. It is now very clear that, in addition to infection of HCMV-naïve women during pregnancy, long-term maternal infection also makes a substantial contribution to patterns of intrauterine infection, transplacental transmission and congenital disease. Transmission in women known to already be infected with HCMV is recognized to occur worldwide, predominating even in developed countries such as the US. Natural levels of acquired antiviral immunity provide a level of protection that apparently reduces, but that is insufficient to completely prevent transplacental transmission. The recognized benefit of natural maternal HCMV-specific immune memory has been difficult to quantify, although antiviral antibodies and T cell immunity likely impede reinfection, dissemination to the maternal-fetal interface and transplacental transmission to the fetus. Transplacental transmission occurs with a frequency less than 2% in women with long-term infection. In contrast, transmission occurs in upwards of B40% in women with primary infection during pregnancy. Thus, prior natural exposure to HCMV provides immunity that significantly reduces risk of congenital disease. While most newborns with congenital CMV infection survive, CID may be lethal and often results in permanent damage even when the infant is treated with antiviral therapy. Given estimated transmission frequencies of B0.2 to 2% of live births, HCMV causes congenital disease more frequently than any other pathogen. It is estimated that there are 41 million HCMV-infected infants born each year based on at total of 150 million births worldwide. HCMV-infected young children, regardless of whether

Human Cytomegalovirus (Herpesviridae)

455

infected in utero or postnatally, shed virus in saliva and urine for extended periods. Thus, children represent a silent, but major reservoir of virus transmitted to parents as well as other children. There is still a remarkable need to understand pathogenesis and immune control of congenital disease, including the processes of transplacental transmission and dissemination within the developing fetus. Premature infants who acquire HCMV infection during birth or post-partum (from breast milk, horizontal transmission or blood transfusion) are susceptible to systemic disease distinct from CID. Risk factors include very low birth weight, exposure to multiple units of blood and birth to a seronegative mother who has been recently infected but has not yet started producing antibodies. Transmission of HCMV from mother’s milk to newborn may be prevented by brief heat treatment (Holder Pasteurization) that inactivates infectivity but preserves nutritional benefits. Transfusion-associated HCMV disease in premature newborns is controlled by the use of blood products from seronegative (or leukocyte-depleted) donors.

Immunocompromised Host HCMV has become one of the most common and medically important opportunistic pathogens as allograft transplantation has become more common and as incidence of HIV infection has increased worldwide. HCMV is a highly significant pathogen in immunocompromised hosts receiving immunosuppressant therapy following solid organ transplantation, during the period of engraftment following allogeneic HSCT, in the course of cancer immunotherapy and anytime cellular immune function becomes compromised. HCMV is a very common complication during HIV/AIDS. Immunodeficient or immunocompromised individuals support higher levels of HCMV replication than immunocompetent hosts, predisposing to disease. Clinical manifestations of disease in solid organ transplant patients include systemic, febrile illness (CMV syndrome, with malaise, arthralgia and rash; neutropenia, thrombocytopenia and elevated liver enzymes), as well as a direct or indirect impact on specific organs. Disease risk has been reduced but not eliminated by preemptive therapy or prophylaxis with antiviral drugs. Clinical detection of viral antigens or nucleic acids without the need for virus culture increased the opportunity for preemptive intervention to prevent acute HCMV disease in the first few weeks following transplantation. Preemptive therapy has also benefitted allogeneic HSCT where late onset disease and indirect consequences of infection have subsequently emerged. Threshold detection nucleic acids (usually PCR detection of viral DNA in plasma or leukocytes) in blood is used to predict impending disease. Solid organ transplant recipients may develop HCMV pneumonitis, gastrointestinal lesions, hepatitis, retinitis, pancreatitis, myocarditis, and, in rare circumstances, encephalitis or peripheral neuropathy, whereas HSCT recipients typically develop HCMV pneumonitis and gastrointestinal disease. Establishing an etiologic role for HCMV requires detection of virus in affected tissue or bronchoalveolar lavage or quantification of HCMV in blood or plasma. The principle indirect effects of HCMV, particularly during solid organ transplantation, include allograft rejection, vascular disease complications and increased risk of opportunistic fungal and bacterial infections. Primary infection is most likely to lead to disease when a naïve recipient receives an organ from a HCMV seropositive donor, although the particular organ, level of major histocompatibility complex (MHC) match and immunosuppression regimen strongly influence outcome. HIV-associated destruction of CD4 T lymphocyte during AIDS removes critical antiviral immune control, resulting in retinal, gastrointestinal, pulmonary or multi-organ HCMV disease. Disease may follow primary infection, reactivation of latent virus, and reinfection with additional strains of virus. Disease pathogenesis is complicated because HCMV shedding and viremia are common in the face of impaired cellular immunity. Clinically inapparent HCMV infection may predominate even in the face of antiviral intervention and may only progress to disease as T cell-mediated immune surveillance becomes severely compromised, such as occurs in AIDS patients with declining CD4 T cell counts.

Pathogenesis HCMV infection and systemic dissemination precedes the development of disease; however, disease severity is not necessarily proportional to levels of virus, viral antigens or viral nucleic acid appearing in blood or tissues. Levels of virus shed in bodily secretions does not correlate with disease risk, whether considering transplacental transmission, congenital infection, transplantation or AIDS. With HCMV, direct viral damage to tissues may result from viral replication or from a combination of replication together with host inflammatory and immune response mechanisms. It is reasonable to expect that the pathogenic signature of HCMV in most settings is due to a combination of both. Direct tissue damage in severely immunocompromised patients is a hallmark of disease and develops as virus levels rise. The degree of immunosuppression matters such that HCMV disease risk increases with T cell depletion in HSCT but may be moderated by adoptive transfer of HCMV-specific T cells, by backing off therapeutic immunosuppressants in solid organ transplantation, or by the control of an immunosuppressive pathogen as seen with the rescue of HCMV immunity following highly active antiretroviral therapy (HAART) in AIDS. In such settings, the levels of HCMV DNA in the bloodstream provide an accurate indication of disease risk and is the basis of monitoring for pre-emptive therapy. Antiviral CD8 T cells are the primary immune mechanism for bringing HCMV under control in transplant settings. Shortcomings in this arm of immunity underlie late onset disease.

456

Human Cytomegalovirus (Herpesviridae)

In AIDS, antiviral CD4 T cells provide crucial support of overall immunity. HAART suppresses HIV replication in these T cells, reconstitutes or preserves cellular immune function, and thereby eliminates risk of HCMV retinitis, gastrointestinal disease and other problems associated with AIDS progression. This reconstitutes immunity and leads to a risk of uveitis where inflammatory disease is triggered by HCMV antigens in the affected tissues. It is settings of less dramatic immune compromise, including pregnancy, where transplacental transmission results in congenital disease in offspring, that the host response to infection is suspected of making contributions to pathogenesis. Pregnancy is a state of relative maternal immune compromise, resulting from maternal tolerance of fetal antigens in a complex and changing immune environment. Transmission and intrauterine replication may be enhanced by inflammatory damage. Fetal immune response patterns, though not fully developed, may contribute to virus control as well as risk of sensorineural damage in congenital disease.

Transmission HCMV transmission requires contact with virus-containing body fluids, as is the case with most human herpesviruses. HCMV is shed, often for prolonged periods of time, in urine, saliva, tears, semen and cervical secretions, as well as in breast milk. HCMV transmission rates are highest following direct contact with body fluids. Two distinct types of exposures are associated with horizontal transmission of HCMV: sexual transmission in adults and contact with urine or saliva from young children or saliva from adults. Infection is often transmitted among children and from young children to parents. One of the highest risk populations for acquisition of this virus is therefore an HCMV naïve pregnant woman who is a parent or caregiver of young children. This setting places the woman at risk of infection herself as well as of transmitting to the developing fetus. Adults may shed virus for weeks after primary infection as well as sporadically throughout life. General hygiene practices, such as regular hand washing, reduce transmission. Although HCMV shedding is common among hospitalized individuals, particularly immunocompromised patients, good hygiene practices by medical care professionals generally prevents HCMV transmission. Transmission via blood transfusion and HSCT has been reduced by donor screening. Transplantation of solid organs and tissues from seropositive donors to HCMV naïve recipients very efficiently transmits HCMV and remains clinically significant. HCMV is transmitted vertically during pregnancy, during birthing as well as via breast milk, a pattern that is distinct from roseolaviruses, alphaherpesviruses or gammaherpesviruses. In developed countries such as the US, primary infection occurs in a small proportion of pregnant women where there is a high risk for transplacental transmission. Approximately 40% of newborns delivered by women with primary infection show signs of HCMV infection, compared to only 0.2% to 2% of newborns delivered by long-term HCMV-seropositive women. Transmission at birth as well as through mother’s milk helps maintain HCMV infection in the population, with between 25% and 50% of infants nursed by seropositive mothers acquiring HCMV by one year of age. Importantly, disease sequelae are not generally associated with intrapartum or post-partum acquisition of HCMV in full-term newborns; however limited acute disease has been observed in rare cases.

Persistence and Latency HCMV infection is lifelong. HCMV seropositive individuals are at risk of disease due to virus reactivation should they become immunocompromised. Tissues and organs from these individuals can transmit infection to naïve transplant recipients, a clear indication that this virus persists in cell types with a wide distribution. Based on detection of viral DNA, natural latency occurs in CD34 þ stem cells and CD14 þ lineage-committed granulocyte-macrophage progenitors that give rise to macrophages and dendritic cells, but not in mature granulocytes or lymphocyte populations. In HCMV seropositive individuals, very few (0.004% to 0.01%) bone marrow-derived mononuclear cells carry a few genome copies of viral DNA. Somewhat surprisingly, HCMV gene expression during lifelong quiescent infection is characterized by the same subset of early and late genes that are expressed during acute infection, albeit at lower levels in the very small proportion of bone marrow-derived hematopoietic cells that carry viral DNA. On the one hand, this diversity of viral gene expression contrasts the restricted latent gene expression of other herpesviruses. On the other hand, the question of whether HCMV persists in a chronic active infection rather than as latent virus remains for experimental consideration. It may certainly be the case that the balance of persistence and latency is determined at the level of the individual infected cell. The presence of viral DNA and gene expression are strong indications that myelomonocytic lineage cells are the reservoir of lifelong HCMV infection. Sporadic shedding that is observed in immunocompetent individuals may be due to reseeding of ductal cells in the salivary gland by these myeloid cells; however, it remains possible that ongoing replication in ductal epithelium (or elsewhere) may seed the leukocytes and be responsible for the observed patterns of gene expression. Leaving aside the complexities, quiescent infection is observed naturally in progenitor (stem) cells from the bone marrow (myeloid lineage), characterized by an absence of IE gene expression with repressive chromatin structure metering epigenetic control over virus replication. Despite ambiguities, several viral protein and RNA gene products have been implicated in latency and differentiationdependent reactivation or in modulation of the host immune control over latency. HCMV infection of CD34 þ stem cells drives differentiation towards monocyte-like cells dependent on inflammatory nitric oxide and STAT3 transcription factor under the influence of the virus-encoded US28 chemokine receptor. Thus, even in the absence of immune control, immature myeloid cells carry viral DNA without supporting replication and retain the potential to reactivate upon differentiation. The generation of macrophages or dendritic cells from infected myeloid progenitors, typically achieved by stimulation with proinflammatory cytokines, results in viral IE gene expression and replication. Clinically relevant reactivation in allogeneic transplant settings appears to depend on the elaboration of

Human Cytomegalovirus (Herpesviridae)

457

proinflammatory cytokines that are themselves components of tissue rejection. Untreated, patients with compromised immunity to HCMV undergo a pattern of uncontrolled viral replication that results in disease. Clinical reactivation occurs in settings such as severe trauma or bacterial sepsis, where inflammation and the consequences of tissue damage suffice to induce replication possibly as a consequence of reperfusion injury. Severely immunocompromised patients, such as terminal AIDS patients, support high levels of HCMV replication with associated disease. Pregnancy in long-term seropositive women supports viral replication at levels that pose a small, but important risk of congenital infection in offspring.

Immunity HCMV is subject to innate immune control, including via cell death, cytokines and natural killer (NK) cell mechanisms. This virus is controlled by pathogen-specific adaptive immunity mediated by T cells and, to a lesser extent, antibodies. On one hand, virusspecific cellular immunity is crucial for lifelong control over HCMV. NK and T cells collaborate to control of acute infection. Memory T cell surveillance suppresses replication and sustains clinical latency. Immunocompromised hosts with defects in cellular immunity are particularly susceptible to HCMV, as illustrated by increased disease in long-term HCMV seropositive recipients of hematopoietic cell transplants from HCMV naïve donors as compared to allogeneic transplants from MHC-matched long-term HCMV immune donors. In such settings, despite sustained serum antibody levels, host control depends on donor memory T cells that may be enhanced by administration of MHC-matched CMV-specific CD8 T cell therapy. On the other hand, anti-HCMV antibody makes a contribution to host resistance, best illustrated by the long-standing observation that transferred maternal antibody prevents transfusion-acquired HCMV in low birthweight infants. In addition, an envelope gB subunit vaccine reduces rates of infection in HCMV naïve women that is likely due to an antibody-mediated mechanism. Finally, there is growing consensus that intravenous immune globulin with HCMV-specific antibody helps reduce intrauterine infection, transplacental transmission and congenital disease. HCMV emergence (whether amplification of persistent virus or reactivation from latency) is triggered by inflammatory signals and likely occurs sporadically throughout life even in fully immune individuals. This results in restimulation of HCMV-specific immunity such that memory T cells accumulate to exceptionally high levels with age. This cellular immunity affords protection because, once compromised, virus levels increase and disease consequences follow. The immunocompromised host provides the most convincing setting where HCMV control by T cell immune surveillance is crucial, supporting the widely held view that a cellular immune response, particularly by NK and memory T cells, maintain lifelong infection in immunocompetent individuals.

Modulation of the Host Response to Infection HCMV commits most of its genome coding capacity to altering the host response to infection within the infected host cell and organism (Table 1). All classes of viral gene products (IE, E and L, as well as those associated with latency) contribute to the modulation of the host response to infection. These viral functions (whether protein or RNA) are typically not required replication in cultured cells. There are three major categories of such functions: (1) those acting within the infected cell (cell autonomous), modulating gene regulation, pathogen sensing, cellular metabolism, cell cycle and cell death pathways; (2) those acting within the host organism at the innate immune level, modulating innate cytokine and cellular responses; and, (3) those acting within the host organism at the adaptive immune level, modulating T and B cell immune effector mechanisms and memory. The virus employs modulatory functions to either evade host clearance mechanisms or to exploit host response pathways. Either way, these viral functions serve to enhance viral infection, pathogenesis and persistence. As with other herpesviruses, most of these functions are committed to suppressing host immunity. However, HCMV encodes cytokines and chemokines as well as chemokine receptors that exploit cytokine-dependent signaling within infected cells to benefit systemic dissemination. One recent insight shows that viral chemokine receptor US28 signaling drives differentiation of hematopoietic stem cells to become a dedicated monocytic reservoir of infection. Such fundamental orchestration of cellular development serves to ensure lifelong persistence.

Modulation of cell autonomous response to infection HCMV gene products deflect intrinsic host cell antiviral responses mediated at the transcriptional level through activation of the cytokine pathways, cell stress, cell cycle, metabolism and cell death (Table 1). Protein-coding genes counteract host cell epigenetic downregulation of gene expression as well as interferon (IFN) activation. Similarly, epigenetic regulation involved in switching from latency to reactivation are influenced by viral regulatory proteins. Pathogen recognition receptor for DNA cGAS, NF-κB activation and IRF3 activation are all targeted by multiple viral proteins. Inflammatory transcriptional activation, IFN response, and IFN-dependent activation of protein kinase R and 20 ,50 oligoadenylate synthetase are all suppressed. During productive infection as well as latency and reactivation, HCMV enhances signal transduction through TNF receptors and encodes a chemokine receptor to interface with cellular chemokines and chemokine receptors and dramatically enhance cell activation, migration and differentiation, potentially to benefit replication as well as latency and reactivation. HCMV-encoded chemokine likely recruit leukocytes to benefit the virus and host FLT3R becomes activated and enhances viral persistence in stem cell models of latency. In a manner that has been compared frequently to tumor viruses, HCMV dramatically alters cellular metabolism and stress markers, downregulating ER and lysosomal stress, upregulating the unfolded protein/integrated stress response as well as tyrosine kinase signaling, activating certain cell cycle processes but imposing a striking block to cellular DNA synthesis. HCMV suppressed

458

Human Cytomegalovirus (Herpesviridae)

various cell death pathways via both protein and RNA gene products engaging mitochondrial targets such as BCL2 family members Bax and Bak as well as caspase-8 to prevent apoptosis and alternate death pathways driven by serine proteases and protein kinases. These functions appear to foster an environment in infected cells that sustains infection, whether the outcome is productive replication or persistence.

Modulation of the innate immune response to infection HCMV modulates cytokine signaling and encodes cytokine, chemokine, chemokine binding protein and chemokine receptor strategies to respond to host chemokines attract granulocytes and monocytes, apparently orchestrating the inflammatory response. This virus disarms the innate NK cell response through a large number of strategies sequestering ligands, impeding receptor engagement and mimicking MHC, all with the apparent goal of evading NK cell killing.

Modulation of adaptive immune response to infection Natural HCMV infection is highly immunogenic, resulting in broad humoral antibody as well as cellular T lymphocyte responses that persist for life and maintain control over the virus infection. Immune responses to certain viral antigens are slow to develop, likely due to virus modulation of adaptive antibody and T cell responses. The antibody response to HCMV is targeted by several virus-encoded IgG Fc receptors, some of which also enhance virus spread dependent on the presence of antiviral antibody. T cell immune surveillance provides crucial control preventing amplification of virus throughout life. Some of the first HCMV-encoded immunomodulatory functions discovered downmodulate the ability of T cells to recognize HCMV-infected cells. Thus, MHC class I antigen presentation is reduced, while cellular and virus-encoded cytokines act together to impede T cell activation and effector function to benefit the virus. An important immunomodulatory impact of HCMV infection is that the antiviral T cell response is sustained at very high levels and increases with age of the host, a phenomenon called “memory inflation”.

Diagnosis Diagnosis of HCMV progressively improved from the 1980s and 1990s as virus isolation methods gave way to rapid immunological and, particularly, nucleic acid detection that are more rapid, less costly, quantitative and potentially automatable. HCMV DNA is readily detected in the tissues, plasma, leukocytes and whole blood of individuals at risk of developing HCMV disease. Immunohistochemical detection of HCMV antigens in biopsy samples and blood can confirm infection. Commercial diagnostic tests that rely on quantification of viral DNA in whole blood or plasma are in common clinical use. DNA amplification (predominantly PCR) methods are used to monitor at-risk populations with the intention to preemptively treat when levels of viral DNA reach a threshold value predictive of impending disease. The levels of viral DNA in the bloodstream are predominantly cell-associated but detected proportionally in plasma as well, providing an accurate prediction of disease risk. Detection of HCMV DNA in saliva, urine or dried blood spots of newborns within the first three weeks of life is diagnostic of transplacental transmission during pregnancy; however, levels of shedding in this or any other clinical setting does not predict disease severity in the infected host. Widespread HCMV screening of newborn blood spots facilitates the recognition of newborns at-risk of HCMV congenital disease who should be monitored for progressive hearing loss during early childhood.

Prophylaxis and Treatment Antiviral prophylaxis has proven to be very effective in preventing HCMV disease in solid organ transplant recipients. However, late onset HCMV disease may follow prophylaxis in as many as 5% of patients. Prophylaxis or preemptive antiviral treatment in hematopoietic cell transplant recipients reduces the incidence of HCMV disease during the first 3–4 months from >80% to around 5%, significantly reducing mortality in adults and, particularly, in children. Late onset disease, usually more than 100 days after transplant, remains a clinical challenge. Long-term HIV antiviral therapy reduces incidence of HCMV disease in AIDS patients, although HCMV remains a significant risk in HIV-positive individuals when CD4 T cell counts drop to low levels, triggering HCMV-specific therapeutic intervention. The advent of antiviral drugs such as ganciclovir (and valganciclovir), cidofovir, foscarnet, and letermovir, has allowed better clinical management of HCMV disease. Despite therapeutic intervention, this virus remains medically significant. The incidence of HCMV pneumonia in allogeneic HSCT as well as the preponderance of HCMV retinitis AIDS contributed to the development of antiviral drugs ganciclovir, foscarnet and cidofovir as well as to the development of orally administered valganciclovir for therapy. Most recently, letermovir for has been approved for antiviral prophylaxis. Valganciclovir, ganciclovir, foscarnet and cidofovir are approved for treatment of HCMV disease in the USA. All of these target the viral DNA polymerase to block viral DNA replication, with ganciclovir, valganciclovir and cidofovir acting as nucleoside analogs, and foscarnet acting as a pyrophosphate analog. Intravenous ganciclovir and orally administered prodrug valganciclovir are first choices in most settings for prophylaxis, preemptive therapy or therapy of active disease. These antiviral drugs come with significant toxicities. The antiviral, letermovir targets viral genome packaging machinery and is orally administered like valganciclovir but lacks toxicity so enables earlier prophylaxis in allogeneic HSCT. Additional oral drugs such as maribavir, targeting the UL97 protein kinase, have been under development. Prolonged use of any investigational or licensed drug

Human Cytomegalovirus (Herpesviridae)

459

selects for resistant virus. Importantly, drug resistant mutants may complicate clinical management of the patient; however, fitness compromise of drug resistant mutants is such that they have not been found to circulate in the community.

Prevention The medical significance and impact of HCMV congenital disease is recognized worldwide, making the development of a vaccine a universal public health priority. Attempts to develop live, attenuated, killed and subunit vaccines over the past 50 years have not yet resulted in a licensed product. The Centers for Disease Control and Prevention currently recommends that parents and caregivers of young children be informed of how HCMV is transmitted and of hygienic measures that reduce transmission, particularly to women who care for young children and may become pregnant. The significantly lower rate of congenital disease following recurrent compared to primary maternal infection during pregnancy supports the potential benefits of universal vaccination. The limited success of a gB subunit vaccine strategy in reducing new infections by half in women of childbearing age lends a note of optimism that effective prevention may be possible. Additional subunit, mRNA and vectored vaccines incorporating gB along with the gH:gL pentamer plus antigens to stimulate T cell immunity are being evaluated as candidates for universal vaccination to prevent congenital infection or to protect stem cell and solid organ transplant recipients. Replication defective HCMV vaccines are also under clinical evaluation. Some vaccines are focused on recipients of organ and tissue transplants. Vaccines must either reduce transmission or suppress viral replication in tissues and organs depending on the clinical setting.

Future Perspectives Despite tremendous gains in knowledge of HCMV molecular biology and pathogenesis, this virus continues to be a medically challenging cause of disease especially for immunocompromised hosts and early in fetal development. To date, antiviral chemotherapy has been only partially successful in controlling HCMV infection in transplant and other immunocompromised patients. There remains a continued and pressing need for vaccination to reduce the incidence of congenital HCMV infection. An important goal for future research will be to translate the large and growing body of basic knowledge of HCMV biology into improved treatments and effective vaccines.

Further Reading Britt, W.J., 2019. Cytomegalovirus. In: Bennett, J.E., Dolin, R., Blaser, M.J. (Eds.), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, ninth ed. Oxford: Elsevier, pp. 1857–1872. Close, W.L., Anderson, A.N., Pellett, P.E., 2018. Betaherpesvirus virion assembly and egress. Advances in Experimental Medicine and Biology 1045, 167–207. Jackson, S.E., Sedikides, G.X., Okecha, G., et al., 2017. Latent cytomegalovirus (CMV) infection does not detrimentally alter T cell responses in the healthy old, but increased latent CMV carriage is related to expanded CMV-specific T cells. Frontiers in Immunology 8, 733. Jackson, S.E., Sedikides, G.X., Okecha, G., Wills, M.R., 2019. Generation, maintenance and tissue distribution of T cell responses to human cytomegalovirus in lytic and latent infection. Medical Microbiology and Immunology 208, 375–389. Mocarski Jr., E.S., Shenk, T., Griffiths, P., Pass, R.F., 2013. Cytomegalovirus. In: Knipe, D.M., Howley, P.W. (Eds.), Fields Virology, sixth ed. Philadelphia: Lippincott, Williams and Wilkins, pp. 1960–2014. Patel, M., Vlahava, V.M., Forbes, S.K., et al., 2018. HCMV-encoded NK modulators: Lessons from in vitro and in vivo genetic variation. Frontiers in Immunology 9, 2214. Patro, A.R.K., 2019. Subversion of immune response by human cytomegalovirus. Frontiers in Immunology 10, 1155. Schwartz, M., Stern-Ginossar, N., 2019. The transcriptome of latent human cytomegalovirus. Journal of Virology 93, e00047. Si, Z., Zhang, J., Shivakoti, S., et al., 2018. Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate. PLoS Pathogens 14, e1007452. Stern, L., Withers, B., Avdic, S., et al., 2019. Human cytomegalovirus latency and reactivation in allogeneic hematopoietic stem cell transplant recipients. Frontiers in Microbiology 10, 1186. Tandon, R., Mocarski, E.S., Conway, J.F., 2015. The A, B, Cs of herpesvirus capsids. Viruses 7, 899–914. Zhu, D., Pan, C., Sheng, J., et al., 2018. Human cytomegalovirus reprogrammes haematopoietic progenitor cells into immunosuppressive monocytes to achieve latency. Nature Microbiology 3, 503–513. Zuhair, M., Smit, G.S.A., Wallis, G., et al., 2019. Estimation of the worldwide seroprevalence of cytomegalovirus: A systematic review and meta-analysis. Reviews in Medical Virology 29, e2034.

Relevant Website https://www.cdc.gov/cmv/overview.html About Cytomegalovirus and Congenital CMV Infection. CDC.

Human Immunodeficiency Virus (Retroviridae) Blaide Woodburn, Ann Emery, and Ronald Swanstrom, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Combinatorial antiretroviral therapy (cART) Combination antiretroviral therapy is the current standard of care for those living with HIV-1. These regimens are often described as a “cocktail” of different antiretrovirals that keep the virus from replicating via varying modes of drug action. First line cART usually consists of two nucleoside reverse transcriptase inhibitors and an integrase inhibitor. Latent Reservoir Refers to the HIV-1 DNA that remains integrated in host cells following suppression of viral replication using cART. These viral genomes are generally not actively being expressed to produce infectious virus but do so stochastically and thus are responsible for viral rebound when antiretroviral therapy is stopped. Much of the latent reservoir is composed of defective viral genomes with intact genomes making up only a fraction of the viral sequences that persist as DNA in cells.

Reverse transcription The process of using single-stranded viral RNA as a template to produce full-length, doublestranded viral DNA. RNA splicing A form of RNA processing that allows the generation of multiple messenger RNAs from a single large precursor RNA leading to the translation of multiple different proteins. This process if often utilized by viruses that have smaller genomes to increase protein diversity. Virion A term used to describe a fully budded viral particle. 90–90–90 Initiative An initiative from the World Health Organization that aims to have 90% of all infected individuals aware of their infection status, 90% of those aware of their status in care and taking ART, and 90% of those taking ART to have the drugs fully suppressing viral replication.

Classification The human immunodeficiency virus type 1 (HIV-1) is an enveloped, plus-stranded RNA virus of the Retroviridae family, further classified into the orthoretrovirus subfamily. The name retrovirus implies a special feature of the viral life cycle - the reverse flow of genetic information. Retroviruses reverse the usual flow of genetic information where DNA is used as a template to synthesize RNA (transcription) to now include a step of reverse transcription where viral RNA is used as a template to synthesize viral DNA. Many of the drugs used to control an HIV-1 infection target the viral DNA polymerase (called Reverse Transcriptase or RT) to block the step of reverse transcription. There are a number of genera in the Retroviridae family, and HIV-1 is part of the Lentivirus genus. Lentiviruses have a worldwide distribution and have been found in cats, sheep, goats, horses, cows, and rabbits. Within the Lentivirus genus is also a lineage of primate lentiviruses, with HIV-1 being a member of this lineage.

The Origin of HIV-1 There is an important milestone that puts a time boundary as to when lentiviruses entered the primate lineage. Specifically, about one-half of the approximately 70 primate species in Africa are infected with a primate lentivirus (collectively called simian immunodeficiency viruses, or SIV) while none of the primates in the Americas are naturally infected. Thus we can infer that a lentivirus entered the primate population in Africa after the split of old world and new world primates and has spread through that population. In these naturally occurring infections the virus is rarely pathogenic, in contrast to the infection of humans with HIV-1. Sequence analysis of viral genomes has become a powerful tool for tracking the history of viruses, and this tool has been especially important for understanding the HIV-1 epidemic. The SIV most similar to HIV-1 is found in chimpanzees (Fig. 1). Using sequence analysis of the viral genome we can infer that two different SIV lineages (from two other primates: SIVrcm from red-capped mangabeys and SIVgsn from greater spot-nosed monkeys) infected a chimpanzee and recombined to create a virus that could grow and adapt in chimps (SIVcpz). Given the similarity between chimps and humans this meant that to a large extent this adaptation would also improve replication in humans. About 100 years ago a single transmission event between chimps and humans occurred that gave rise to what we know as the HIV-1 epidemic, a lineage of virus in humans known as the main or M group. Chimpanzees have been used as food sources for human populations, with hunting and dressing carcasses being an obvious path for the introduction of these viruses into humans. It is hypothesized that this is how the first transmission event of SIV from chimpanzees to humans might have occurred. In addition, several other closely related SIVcpz strains have been transmitted to humans (possibly with gorillas as an intermediate) but these have not reached epidemic proportions as the M group lineage has.

460

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00042-4

Human Immunodeficiency Virus (Retroviridae)

461

Fig. 1 Phylogenetic tree of the SIV and HIV lineages. Phylogenetic analysis of SIV and HIV sequences shows that each HIV-1 group (M, N, and O) originated from an independent transmission event of chimpanzee SIVcpz to humans followed by diversification into subtypes (e.g., Group M into Subtypes A, B, C, etc.). This analysis also demonstrate how HIV-2 has been transmitted multiple times to humans from its origin as SIVmm in sooty mangabeys. Figure adapted from Kuiken, C.L., Foley, B., Hahn, B.H., et al., 1999. Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Theoretical Biology and Biophysics Group. Los Alamos, NM: Los Alamos National Laboratory.

There is a second SIV that has infected humans, known as HIV-2. This virus is most similar to an SIV found in sooty mangabeys (SIVsm). The SIV of sooty mangabeys has been transmitted into humans a number of times giving rise to the smaller epidemic of HIV-2 in west central Africa with a virus that is significantly less pathogenic than HIV-1.

Organization of the Genome HIV-1 has a single-stranded RNA genome of about 9700 nucleotides that is packaged into the virion as two identical copies of RNA (Fig. 2(A and B)). The genomic RNA is plus stranded, meaning it is in the same polarity as its messenger RNAs (i.e., the strand that can be translated into protein). However, for retroviruses the genomic RNA is not translated when it enters the cell but rather copied into double-stranded DNA. Thus the genome exists in two states, genomic RNA that is packaged into the virion and a DNA copy of the genome that is integrated into the host cell genomic DNA. Once integrated into the host genome, transcription of viral DNA into RNA begins. Only one transcript type is made – a full length transcript equivalent to genomic RNA (Fig. 2(C)). The transcript is made by the cellular RNA Pol II and processed by cellular machinery, so it looks like a cellular mRNA with a 50 cap and a 30 poly A tail (Fig. 2(A)). The RNA and DNA forms of the genome are very similar, retaining the gene order. However, during DNA synthesis portions of the RNA sequence at the ends of the RNA are rearranged and duplicated at the ends of the DNA to give the DNA long terminal repeats (LTR; Fig. 2(C)). These duplications are mediated during DNA synthesis by a short direct repeat at the ends of genomic RNA called R. This allows the virus to place in its DNA form the transcription regulatory signals in U3 upstream of the transcription start site, signals that are duplicated near the 30 end of the RNA and thus not lost when they are excluded from the transcribed RNA at the 50 end. In the 50 untranslated region (UTR) the genome folds into extensive secondary structures with important regulatory functions. This region contains a packaging signal, c (Psi), that identifies the viral genome as the RNA that should be packaged into newly forming viral particles. Genomic RNA is packaged in dimers and a dimerization signal (DIS) initiates the dimeric folded structure. Downstream of the 50 UTR are the viral genes. HIV-1 has 10 genes (gag, pro, pol, vif, vpr, tat, rev, env, and nef). Like the 50 end of the genome, the 30 end of the genome also contains sequences that function in the RNA that are important for replication (such as the polyadenylation signal).

462

Human Immunodeficiency Virus (Retroviridae)

Fig. 2 HIV-1 virion structure and genome. (A) The HIV-1 RNA genome is approximately 9.7 kilobases and consists of 10 genes (shown with boxes) that code for 15 mature proteins. Each end of the RNA genome is comprised of a repeated sequence known as R with sequences inside of R labeled as U5 near the 50 end and U3 near the 30 end. The 50 and 30 untranslated regions (UTR) encode cis-acting replication signals as described in the text. Genomic RNA has the hallmarks of a eukaryotic RNA with a 50 cap and a 30 poly A tail. Also shown is the major splice donor site D1 and the other splice donors D2-D4, as well as the positions of the major splice acceptor sites A1-A5 and A7. (B) The HIV-1 particle is spherical, approximately 100 nm in diameter, and has a modest number of Env protein trimers (maroon) in its membrane envelope that facilitate entry into the host cell. The particle contains several structural proteins that dictate its geometry, including the matrix (MA; blue) and capsid (CA; green) proteins. Additionally, the particle is prepackaged with functional proteins reverse transcriptase (RT; orange) and integrase (IN; pink), and also includes the viral protease (PR; blue) which functioned during assembly/maturation. Two identical plus strands of viral RNA comprise the viral genome, which is contained in the capsid cone of the virion. (C) Through reverse transcription, HIV-1 RNA (red) is copied into doublestranded linear DNA (blue) that is then integrated into the host cell genome. DNA synthesis creates a direct repeat of sequences that in the RNA were discontinuous (U3 R U5, blue box), with this direct repeat called the long terminal repeat, or LTR. Following integration, the viral promoter within the LTR (U3) drives expression of the DNA form of the genome into full length RNA, creating new viral RNA to be packaged into assembling virions or used in the expression of viral proteins (with or without RNA splicing).

Expression of Viral Genes The full-length 9700 nucleotide-long transcript can be used either as genomic RNA and packaged into virions or as a messenger RNA. In eukaryotes only the 50 most reading frame can be translated on an mRNA. For HIV-1 this means that when the full-length transcript serves as an mRNA it is for the gag gene. About 95% of the time, a Gag protein is synthesized and translation stops at the end of the gag reading frame. However, embedded in the RNA near the end of the gag coding region is a stretch of uridines and a stem-loop structure that occasionally (5% of the time) causes the ribosome to slip back one nucleotide while decoding the RNA (  1 frameshift); this changes the reading frame the ribosome is in with translation now continuing into the pro and pol genes creating a Gag-Pro-Pol polyprotein precursor. This is one strategy the virus uses to get around the limitation of having translation limited only to the 50 most open reading frame of an RNA. The other strategy the virus uses to express its downstream genes is RNA splicing. Splicing is used in the expression of most cellular genes to excise introns from a long RNA precursor and piece together the exons that make up the final mRNA. This is what happens to the virus except here the internal regions of the genome serve as either exon or intron, depending on which gene is being expressed. The cellular splicing machinery generates over 40 spliced mRNAs from the full length genomic viral RNA, and a simplified scheme of these splicing events is shown in Fig. 2(A). If the full-length transcript is spliced, it always uses the main splice donor D1 (in the 50 UTR) and splices to one of the downstream acceptors, A1-A7. Splicing excises the section of the genome under the arc, so, for example, a splice to A1 removes the section between D1 and A1 (Fig. 2(A)). This joins the 50 UTR to the translation start site for the vif open reading frame (which is just downstream of the A1 splice site) to make a transcript that will be translated to generate Vif protein. Similarly, splicing to the other acceptors moves the translation start sites for each of the downstream genes next to the 50 UTR to allow it to be translated. There are still several twists. First, both the env and vpu open reading frames are

Human Immunodeficiency Virus (Retroviridae)

463

translated from the same mRNA (bending the rule of only the 50 most reading frame being translated). Second, RNAs that are spliced at D1 may or may not splice again from D4 to A7. The exons for Tat and Rev span this D4-A7 splice site, requiring the D4-A7 splice to form their complete open reading frames but at the expense of the intact env reading frame. The D4-A7 splicing event is regulated to allow the formation of mRNAs for Tat, Rev, and Nef when splicing occurs, and both the full length genomic RNA and mRNA for the Env protein when splicing is blocked. Cells typically require RNAs to be fully spliced in order to leave the nucleus, and the mRNAs for Tat, Rev, and Nef follow this rule (and the normal cellular mRNA export pathway). However, for the unspliced genomic RNA and partially spliced env mRNA (retaining the D4-A7 intron) the virus must trick the cell to export these RNAs. The viral Rev protein binds to a region of RNA secondary structure in the env intron called the Rev Response Element (RRE) and links the unspliced and partially spliced RNAs to an alternative RNA export pathway by binding to the host protein CRM1 to move these RNAs out of the nucleus and into the cytoplasm.

Virion Proteins Most of the viral proteins that are found in the virion are initially synthesized as larger polyprotein precursors that must be cleaved by a protease. Thus the gag gene encodes the Gag polyprotein precursor that is targeted to the cell plasma membrane where the virus buds out of the cell. During virion formation/maturation the viral protease (see just below) cleaves the Gag protein into its membrane-bound, N-terminal matrix (MA) protein, its capsid (CA) protein that will form the characteristic cone structure within the enveloped virus, its nucleocapsid (NC) protein which binds the viral RNA, and the C-terminal p6 protein which is involved in interacting with a host cell pathway to allow the membrane that surrounds the budding virus to pinch off and leave the cell. Approximately 5% of the time translation of Gag slips into the  1 reading frame to create the Gag-Pro-Pol polyprotein precursor. When the Pro domain dimerizes this creates the viral protease (PR), which is responsible for cleaving the Gag and GagPro-Pol polyprotein precursors. This happens during the budding/maturation process. The protease is responsible for cleaving itself out of the precursor, for cleaving Gag, and for releasing the viral DNA polymerase reverse transcriptase (RT). The protease cleavage event at the C terminus of RT releases the viral integrase protein (IN). PR and RT function as dimers, while IN functions as an even higher order oligomer. The p6 domain at the C terminus of Gag has a binding site for the viral Vpr protein. There are about 2000 copies of Gag in a virion, 100 copies of the Gag-Pro-Pol precursor, and 75 copies of Vpr. The other major viral protein is the envelope or Env protein on the surface of the virion. It takes a different path to the assembling virion. As a membrane protein, it is synthesized in the endoplasmic reticulum (ER) in the cell with the N terminus threaded into the lumen of the ER until the appearance of an encoded translocation stop signal about 200 amino acids from the C terminus of Env. The initial protein is called gp160 based on its apparent molecular weight and the fact that it is highly glycosylated. The protein trimerizes and migrates to the cell surface, always embedded in the membrane near its C terminus. In the Golgi, a cellular protease (furin) cleaves the protein into two subunits: an extracellular surface protein gp120 (SU) and the transmembrane protein gp41 (TM), with the gp120 and gp41 subunits maintaining noncovalent interactions. The final Env trimer is composed of three subunits of gp120 and three subunits of gp41. For HIV-1 there are only about 10–20 trimers on a virion, occupying only a small amount of the surface of the enveloped virus. gp120 interacts with the CD4 protein on the surface of helper T cells to determine which cell gets infected, i.e., CD4 is the primary receptor for the virus. After binding CD4, gp120 goes through a series of conformational changes to create a binding site for a coreceptor, usually a cellular surface protein called CCR5. After binding these two cell surface proteins, gp41 then mediates fusion between the viral and host cell membrane to allow entry of the capsid into the cytoplasm of the cell, where viral DNA synthesis occurs. Small amounts of other viral and cellular proteins have been reported to be associated with virions but these are largely of unknown significance.

The Other Viral Proteins Work Intracellularly All retroviruses encode the main replicative proteins Gag, Pro, Pol, and Env. For some retroviruses these are the only proteins encoded in the viral genome. Other retroviruses encode a variety of additional proteins, often called accessory proteins since they are in addition to the replicative proteins. However, adapter proteins might be a better name since most of them function to bring together proteins in the cell (think an adapter for an electrical plug) in ways that benefit the virus. HIV-1 encodes six such proteins: Tat, Rev, Nef, Vif, Vpr, and Vpu. Tat and Rev play critical roles in initiating the expression of the viral genome. The newly integrated viral DNA is transcribed by RNA Pol II from the host cell. However, most of the initial transcripts are short, with only a fraction being full length, with these few full length RNAs being spliced to form Tat and Rev mRNAs. The initial Tat protein that is synthesized binds to a feature of RNA secondary structure at the 50 end of the RNA, a structure called the TAR loop. In this way the Tat protein recruits the transcription and elongation factor P-TEFb. P-TEFb includes a protein kinase (CDK9) which phosphorylates the C terminus of RNA Pol II to increase its elongation efficiency allowing the synthesis of more full-length RNA. In parallel the early Rev protein binds to the RRE to allow partially spliced and unspliced viral RNAs to exit the nucleus thus allowing for all of the viral RNAs, including the unspliced genomic RNA, to exit the nucleus (as described above).

464

Human Immunodeficiency Virus (Retroviridae)

The viral Vif, Vpr, Vpu, and Nef proteins counteract cellular antiviral restriction factors and regulate cell processes to suit viral replication. These proteins have multiple targets and effects. However, a common theme is to bind a cellular protein that has a deleterious effect on viral replication and link it to the proteosome where it gets degraded. A clear example of how this group of viral proteins acts as an adapter is seen with Vif. Vif targets the cellular factor APOBEC3G for proteosomal degradation. In the absence of Vif, APOBEC3G is packaged into virions and causes cytosine deamination in the minus strand of viral DNA during reverse transcription in the next cell, which results in G to A mutations in the positive strand. The minus strand is initially present as single-stranded DNA and this is the target of APOBEC3G. Viruses lacking Vif are unable to grow in cells that produce APOBEC3G. Vif brings APOBEC3G to a cellular ubiquitin ligase where it is ubiquitinated, targeting it for degradation by the proteosome. Vpr also binds to a cellular ubiquitin ligase but its host cell target is still a focus of research. Vpr induces the infected cell to arrest in the G2 phase in the cell cycle, perhaps to induce DNA repair enzymes that ultimately assist in integration. However, such activity would have to be carried out by just the Vpr packaged in the virion since integration happens before viral gene expression. The extent to which the small amount of virion-associated Vpr is the effective form of the protein or if it has functions after expression of larger amounts of protein after integration is unknown. Vpu counteracts the cellular tetherin protein. Tetherin binds to both the cellular membrane and to the membrane of the newly budding virus, thus tethering the virus to the cell surface. Vpu reduces the amount of tetherin on the cell surface. Nef has multiple functions, such as down regulation of CD4 from the surface of the cell to reduce interaction with the newly synthesized Env trimers, down regulation MHC class I from the surface of the cell to reduce immune surveillance, reduction in the amount of SERINC5 which when incorporated into the virion inhibits subsequent fusion to the next cell, and interacting with a cellular kinase to affect cellular activation.

Life Cycle It is convenient to divide the viral life cycle into two phases: an early phase starting with binding of the virus particle to the target cell through the integration of viral DNA, and a late phase starting with transcription of the integrated DNA through virion budding and maturation.

Early Phase Attachment and Entry The first step of the virus life cycle is attachment to the host cell via the CD4 protein on the surface of helper T cells using the gp120/SU portion of the Env protein (Fig. 3). The normal form of HIV-1 requires a high density of CD4 on the surface of the cell, marking the CD4 þ T helper cell as its primary target cell. As noted above, fusion of the viral and host membranes requires the presence of the primary surface receptor, CD4, and one of two secondary surface receptors, usually CCR5 or sometimes CXCR4, two chemokine receptors that are found on a subset of cells. Engagement with the coreceptor exposes the hydrophobic gp41 fusion peptide, which then inserts into the host cell membrane. Insertion of each gp41 in the trimer brings the virus in even closer proximity to the host cell membrane; gp41 then forms a hairpin mediated by two helical domains in the protein, and since gp41 is in a trimer this results in a six helix bundle. This six helical bundle serves as the driving force behind membrane fusion by bringing the membranes close enough to form a fusion pore through which the virus can release its capsid into the cytosol of the host cell where viral DNA synthesis can begin. The need for a high density of CD4 for efficient entry and the use of CCR5 as the coreceptor make the predominant form (and the transmitted form) of the virus appropriately called HIV-1 R5 T cell-tropic.

Uncoating, Reverse Transcription, Nuclear Localization, and Integration Following release of the viral capsid into the host cell cytoplasm, the capsid core begins to uncoat, although the extent to which the p24 CA capsid protein is released is unclear. The partially uncoated core utilizes the host cytoskeletal network to traffic toward the host cell nucleus. Inside the viral capsid core, reverse transcriptase (RT) begins converting the single-stranded viral RNA into dsDNA, with DNA synthesis being primed initially using a host cell tRNA that was earlier packaged into the virion and placed near the 50 end of the RNA genome (Fig. 3). Linear, double-stranded DNA is fully synthesized in the cytoplasm. This replication complex contains integrase, and while in the cytoplasm the integrase protein removes 2 bases from each 30 end of viral DNA in preparation for integration in the nucleus, making the pre-integration complex (PIC). Once reaching the nucleus the PIC is transported across the nuclear membrane through the nuclear pore complex, in part mediated by interactions with residual CA protein. Inside the nucleus the PIC associates with host factors (such as LEDGF) that target the PIC to transcribed regions of the viral genome. These host factors help direct integration to regions of the host genome where the viral genome will be more likely to be transcribed. After interacting with the host chromatin, the integrase protein cleaves the host DNA and, in a strand transfer reaction, covalently links the viral DNA to the host DNA where it can be transcribed by the host transcription machinery (Fig. 3).

Human Immunodeficiency Virus (Retroviridae)

465

Fig. 3 Early phase of the HIV-1 life cycle. HIV-1 begins its life cycle by binding to its primary receptor CD4 and then its secondary receptor CCR5 (shown) or CXRC4 to facilitate membrane fusion. In the cytoplasm there is some level of “uncoating” to form the Reverse Transcription Complex (RTC) where reverse transcription of the viral RNA genome occurs while making its way to the nucleus using a vast array of microtubule networks. After completion of DNA synthesis the viral DNA is associated with integrase forming the Pre-Integration Complex (PIC), which moves through nuclear pore complex (NPC) to traverse the nuclear membrane. The PIC migrates to a host chromosome where integration occurs. Figure adapted from Campbell, E.M., Hope, T.J., 2015. HIV-1 capsid: the multifaceted key player in HIV-1 infection. Nature Reviews Microbiology 13, 471–483. with permission.

Late Phase Gene Expression and Assembly After integration, sequences in the LTR are recognized by host RNA polymerase II to synthesize viral RNA. However, initially the resulting transcripts are short and prematurely terminated, with only a small fraction being full length genomic RNA. The fraction that is full length is spliced by the host cell machinery initially to give mRNAs for Tat, Rev, and Nef. Tat recruits a host transcription factor complex P-TEFb with Tat binding to an RNA hairpin in the short transcript (TAR). Bringing P-TEFb close to the host RNA polymerase II leads to modification of the polymerase to allow more of the transcripts to be full length (i.e., making the polymerase more processive). Normally mRNAs have to be fully spliced to leave the nucleus. However, HIV-1 gene expression requires the use of partially spliced or even unspliced RNA (the form of genomic RNA). To accomplish this the viral Rev protein binds to another large RNA hairpin within the D4-A7 intron in the env gene. Rev links partially spliced and unspliced viral RNA to a different nuclear export pathway, mediated by the host protein Crm1, such that the RNA avoids the quality control the host applies to RNAs going through the mRNA nuclear export pathway. Over 40 RNA transcripts are generated from the full length viral genomic RNA transcript by alternate splicing patterns, which allow expression of all of the viral proteins and full length RNA for packaging into new virions. As noted above, some of the viral proteins function to manipulate the host cell (Tat, Rev, Nef, Vif, Vpr, Vpu) while others are used to make new virion particles. The Env protein migrates through the ER/Golgi apparatus of the cell to get to the cell surface membrane (Fig. 4). The Gag and Gag-Pro-Pol proteins are synthesized in the cytoplasm but are targeted to the inner face of the cell surface/plasma membrane. The Gag protein alone is sufficient to mediate budding of a particle from the cell membrane (with Gag-Pro-Pol being mixed in at a 1–20 ratio). The Env protein interacts with the N-terminal domain of Gag (the MA domain) to increase its incorporation into budding virion particles.

466

Human Immunodeficiency Virus (Retroviridae)

Fig. 4 Late Phase of the HIV-1 life cycle. Following integration, the HIV-1 genome is transcribed into full length RNA followed by complete splicing to produce Tat and Rev, which increase the level of expression and allow for unspliced and partially spliced RNAs needed for virion assembly and to function as mRNA for the structural proteins. HIV-1 Env is processed and trafficked to the host cell membrane via the secretory pathway (ER and Golgi) while Gag, with some bound to full length viral RNA, and Gag-Pro-Pol (here called GagPol) migrate through the cytoplasm toward the host cell membrane to form an immature, budding viral lattice. Membrane fission and release of the newly assembled virion from the host cell is mediated by ESCRT I, ESCRT-III, and ALIX proteins, and maturation of the virion is dependent on viral protease cleavage of Gag and Gag-Pro-Pol into their respective proteins. Figure adapted from Freed, E.O., 2015. HIV-1 assembly, release and maturation. Nature Reviews Microbiology 13, 484–496. with permission.

Budding and Maturation As a Gag lattice forms at the surface of the plasma membrane it creates a budding structure. However, the particle must undergo membrane fission to be released from the host cell in the form of an immature virion. Host proteins in the ESCRT pathway target the C-terminal domain of Gag (p6) to mediate this membrane fission allowing the enveloped virus to detach from the cell (Fig. 4). During this budding process, the viral protease PR dimerizes in the Gag-Pro-Pol precursor to become active, first cleaving itself out then cleaving the Gag and Gag-Pro-Pol precursor polyproteins into their respective proteins to initiate the maturation of the virion into its infectious form with its familiar cone-shaped capsid core.

Human Immunodeficiency Virus (Retroviridae)

467

Fig. 5 HIV-1 global subtype distribution and 2019 UNAIDS statistics. Countries forming a region are shaded in the same color on the world map. The surface area of each pi-chart corresponds to the total number of people living with HIV-1 in each region. CRF ¼ circulating recombinant form. URFs ¼ unique recombinant forms. Figure adapted from Hemelaar, J., Elangovan, R., Yun, J., et al., 2018. Global and regional molecular epidemiology of HIV-1, 1990–2015: A systematic review, global survey, and trend analysis. The Lancet Infectious Diseases 19, 143–155. with permission.

Epidemiology Currently we recognize distinct lineages of HIV-1 called subtypes or clades (designated subtypes A, B, C, etc. and also called subgroups; Fig. 5) which are all part of the M group. Virus isolates within a subtype are more similar at the sequence level than they are to virus isolates in different subtypes. We interpret the existence of these subtypes as the result of having a diverse (in sequence composition) population of viruses within the human population within western Africa around 60 years ago. The virus then spread in single transmission events to different areas of the world, creating a sequence or genetic bottleneck that defined the subsequent expansion of that virus in these different places. These are all strains of HIV-1 with either little or no biological differences but we can still see the evidence of the genetic bottleneck coming to North America for example, where we have viruses that are related to each other and called subtype B (or clade B), the same predominant viral lineage that is found in western Europe. We can see subtypes A and D in eastern Africa, and another jump occurred subsequently to create another A subtype cluster from the east African virus in eastern Europe. Subtype C virus entered southern Africa then spread through India and into China. These are broad stroke descriptions of the early spread of the virus into the larger human population. Where two subtypes are circulating in the same human population it is possible for a person to become infected with both subtypes where recombinant genomes between the two viruses can occur and get transmitted, making the inference of its history more difficult. In addition, as people have moved between different regions of the world over the years the different lineages of viruses have become more mixed.

Modes of Transmission and Risk Factors The modes of HIV-1 transmission are well-defined. HIV-1 can be transmitted through sexual activity, exposure to blood, and from mother to child during pregnancy, birth, and breast feeding. There are six bodily fluids by which HIV-1 transmission can occur, including blood, semen, pre-semen, vaginal secretions, anal secretions, and breast milk. Additionally, there are four common routes of transmission that include the vagina, the tip of the penis, anus, and open sores (especially in the genital tract). In general, exposure of mucosal surfaces to virus-containing fluids creates the risk of transmission. There is no epidemiological data to suggest that HIV-1 transmission can occur via exchange of saliva (kissing, sharing drinks and food) or mosquito bites, mostly because the virus in unable to reach transmissible titers in these fluids. Unprotected sexual contact is the primary mode of transmission worldwide. HIV-1 has a relatively low rate of transmission, with transmission rates at least 10–100 times less per sexual encounter than other sexually transmitted infections (STIs) such as gonorrhea, chlamydia, and syphilis. However, there are numerous factors that can enhance infectivity of the source partner such as a high viral load, other STIs (in either partner), and blood contact during sex. The probability of transmission increases with the likelihood of microtrauma during sexual contact and direct exposure of blood to infected fluids (Table 1). Outside of sexual contact, another common mode of transmission is through exposure of HIV-infected blood via injection drug use. Sharing of needles with HIV-contaminated blood has the highest rate of transmission of any exposure, aside from blood transfusion with HIV þ blood which has a transmission rate of essentially 100%. Mother-to-child transmission can take place

468

Human Immunodeficiency Virus (Retroviridae)

Table 1

Estimated per-act risk for acquisition of HIV-1, by exposure route

Exposure route

Risk per 100 exposures to an infected source

Blood transfusion Needle-sharing injection-drug use Percutaneous needle stick Receptive anal intercourse Receptive penile-vaginal intercourse Insertive anal intercourse Insertive penile-vaginal intercourse Receptive oral intercourse Insertive oral intercourse Mother-to-child transmission (without breast-feeding) Breast-feeding for 18 months

90 0.67 0.3 0.5 0.1 0.065 0.05 0.01 0.005 30 15

Source: Adapted from Smith, D.K., Grohskopf, L.A., Black, R.J., et al., 2005. Antiretroviral postexposure prophylaxis after sexual, injection-drug use, or other nonoccupational exposure to HIV in the United States: Recommendations from the US Department of Health and Human Services. MMWR Recommendations and Reports 54, 1–20. Kourtis, A.P., Lee, F.K., Abrams, E.J., Jamieson, D.J., Bulterys, M., 2007. Mother-to-child transmission of HIV-1: Timing and implications for prevention. The Lancet Infectious Diseases 6 (11), 726–732.

during pregnancy, during delivery, and during breastfeeding, although this transmission risk is essentially reduced to zero if the mother is on suppressive antiviral therapy.

Prevalence HIV-1 has been detected in every region of the world. Currently it is estimated that 37.9 million people are living with HIV. In 2019, 1.7 million people became newly infected and 770,000 died from AIDS-related illnesses (Fig. 5). Fortunately, 23.3 million individuals had access to antiretroviral therapy (ART), and this number will only continue to rise with global initiatives to increase sexual health education and access to treatment. The prevalence of HIV-1 varies globally, disproportionally affecting Sub-Saharan Africa. Eastern and Southern Africa contain B60% of the total global infections, with Asia and the Pacific (15%), West and Central Africa (13%), Latin America (2%), the Caribbean (0.9%), the Middle East and North Africa (0.6%), Eastern Europe and Central Asia (4%), and Western/Central Europe and North America (5%) comprising the other B40%. Prevalence is not uniform across the population in these regions. Both engaging in acts that can mediate transmission and the level of pre-existing infection within the subgroup where these acts are shared have led to significantly elevated levels of infection among certain subgroups of people, which has led to focused efforts in treatment and prevention within these subgroups.

Diagnosis A diagnosis of an HIV-1 infection is made based on one of three different measures. Most commonly the diagnosis is made based on the host antibody response to the virus. By examining a group of people with suspected time of exposure it is possible to determine that a person becomes antibody positive in an ELISA (enzyme-linked immunosorbant assay) test around 22 days after transmission. This test is usually confirmed with a Western Blot test where viral proteins transferred to a thin membrane are bound by host antibodies and the presence of the bound antibodies detected. While these tests are usually done using a sample of blood, antibodies are also shed into saliva which has allowed a “rapid test” to be developed based on a saliva sample to test for the presence of HIV-specific antibodies, although this test becomes positive later than the antibody blood test. The detection of antibodies is a measure of the host response to the replicating virus. The two other tests are designed to measure the presence of the virus itself. Virus is replicating in the body before the antibody response appears. Thus detection of the virus itself offers the possibility for detection of the infection even before the appearance of the host antibody response. The two tests that are used are a sensitive ELISA assay to detect the viral capsid protein (CA/p24), and a PCR-based assay to detect the presence of viral RNA. Most people who get infected are initially infected with a single or a few virus particles that then start replicating in the new host. Very early after infection there is not enough virus to measure with these methods so for about 7–10 days there is a period called the eclipse phase where the viral infection can't be seen by any of these tests. The detection of viral RNA in the blood is the most sensitive test and the first to be positive after the eclipse phase, followed within a week by the ELISA test for viral capsid protein, followed by one week for the detection of viral antibodies. In the initial absence of an immune response the virus multiplies rapidly, reaching a peak level of viremia in the blood at about 3 weeks. With the onset of the host immune response the virus is partially controlled and brought down to a “set point”, measured as copies of viral RNA per milliliter of blood (usually in the range of 10,000 to 100,000 copies of viral RNA per milliliter). This level of viral RNA is relatively constant or trends upward over time and represents a steady-state level of viral replication and the death of infected cells over time.

Human Immunodeficiency Virus (Retroviridae)

469

Table 2 AIDS Defining Illnesses. There are a set of illnesses often associated with HIV-1 infection and the onset of AIDS. These range from bacteria and other viral infections to virus-induced cancers. This is the list of AIDS defining illnesses as designated by the U.S. Center for Disease Control AIDS-defining illnesses Kaposi sarcoma Lymphoid interstitial pneumonia or pulmonary lymphoid hyperplasia complex Candidiasis of esophagus Lymphoma, Burkitt Cervical cancer Lymphoma, immuneblastic, primary brain Coccidioidomycosis, disseminated or extrapulmonary Mycobacterium avium complex or Mycobacterium kansasii, disseminated or extrapulmonary Cryptococcosis, extrapulmonary Mycobacterium tuberculosis of any site, pulmonary, disseminated, or extrapulmonary Cryptosporidiosis, chronic intestinal Mycobacterium, other species or unidentified species, disseminated† or extrapulmonary Cytomegalovirus disease (other than liver, spleen, or nodes), onset at age 41 month Pneumocystis jiroveci pneumonia Pneumonia, recurrent Cytomegalovirus retinitis (with loss of vision) Encephalopathy, HIV related Progressive multifocal leukoencephalopathy Herpes simplex: chronic ulcers (41 month’s duration) or bronchitis, pneumonitis, Salmonella septicemia, recurrent or esophagitis (onset at age 41 month) Histoplasmosis, disseminated or extrapulmonary Toxoplasmosis of brain, onset at age 41 month Wasting syndrome attributed to HIV Isosporiasis, chronic intestinal (41 month’s duration) Bacterial infections (recurrent) Candidiasis of bronchi, trachea, or lungs

Source: Schneider, E., Whitmore, S., Glynn, K.M., et al., 2008. Revised surveillance case definitions for HIV infection among adults, adolescents, and children aged o18 months and for HIV infection and AIDS among children aged 18 months to o13 years—United States, 2008. MMWR Recommendations and Reports 57 (RR-10), 1–12.

Once a person becomes positive for anti-HIV-1 antibodies they will remain antibody-positive for life. However, combination antiretroviral therapy (see below) can fully suppress viral replication such that the clinical tests for viral RNA and viral capsid protein become negative. For HIV-1, even though these two measures of virus turn negative the virus is still present in a latent state, and for most people the virus will "rebound" in the blood within 2–4 weeks if antiviral therapy is stopped. Thus people with HIV1 must initiate life-long therapy to stop viral replication (and disease progression) and keep the virus from reappearing in the blood.

Clinical Features There is an early feature of infection with HIV-1 that occurs shortly after transmission, although its frequency varies greatly among different cohorts. About 2–4 weeks after infection there can be transient flu-like symptoms associated with the initial host immune response. As the viral load is brought down to its set point these symptoms resolve. When symptoms appear at this stage they are referred to as acute infection syndrome. As these early symptoms resolve, a person transitions into a state of clinical latency where the virus is replicating and damaging the immune system but not yet to a point where symptoms occur. It is the cumulative damage to the immune system that leads to the defining feature of an HIV-1 infection, i.e., immunodeficiency. In the absence of antiviral treatment, there is an ongoing loss of CD4 þ T cells in the body, a cell type that is central to coordinating the host immune response. As the CD4 þ “helper” T cells are lost (we start with around 1000 such cells per microliter of blood) the host becomes increasingly susceptible to infections from other viral, bacterial, and fungal agents as well as virus-induced cancers (Table 2). Ultimately it is these other infectious agents or tumors that lead to death. The clinical state of immunodeficiency is defined as the appearance of one of these opportunistic infections or when the CD4 þ T helper cell count drops below 200 cells per microliter in the blood (which defines a state of enhanced risk of opportunistic infection). Two features of this process are highly variable. Some people progress rapidly to a state of immunodeficiency (1–2 years) while others progress slowly (15–20 years) or not at all, there being a small group of people (1%) who have a very low set point with no evidence of disease progression. Overall the average time from infection to the onset of immunodeficiency is in the range of 8–10 years. The other feature that is highly variable is what happens as a person becomes immunodeficient. Some of the infections are acquired from our environment and are agents that we are exposed to regularly but control in an immunocompetent state. Others are agents that we are already infected with and normally control in a latent state but then cannot. A person experiences only a subset of these infections but we have little understanding why the passage into an immunodeficient state can have such varied consequences. However, individually or together these infections present serious challenges for the body. Under certain circumstances HIV-1 itself may become the opportunistic infection. In untreated infection a condition named AIDS-associated dementia ensues that involves replication of the virus within the central nervous system and the adaptation of the virus to that environment.

470

Human Immunodeficiency Virus (Retroviridae)

In this case the virus expands from replication in blood and lymphoid cells into the CNS where it is associated with a distinct clinical outcome.

Pathogenesis There is one overarching principle in HIV-1 pathogenesis - the virus infects and kills CD4 þ T cells and these cells are lost over time until a state of immunodeficiency is reached. It is tempting to make viral infection and loss of these cells the sum total of viral pathogenesis. However, there are many complex changes in the body that occur over the course of an untreated infection and it has been difficult to determine whether these ancillary changes contribute to pathogenesis or are the result of the immunodeficient state induced by the cumulative loss of CD4 þ T cells. For example, it appears that too few CD4 þ T cells are infected and killed by the virus to account for the magnitude of loss of these cells over time. Thus there have been studies of “standby” effects where other mechanisms contribute indirectly to the loss of CD4 þ T cells and thus ultimately contribute to the immunodeficient state. Ideas that have been explored include a loss of the ability to produce new CD4 þ T cells after years of ongoing replacement, loss of normal architecture in lymphoid tissue where most T cells reside, and local effects where cells near an infected cell also die. Even the mechanism of how an infected cell dies is unclear. The viral protein Vpr induces cell cycle arrest which precludes the cell from dividing. The viral Env protein may continue to bind its CD4 protein receptor in the infected cells inducing aberrant signaling (several viral proteins down regulate CD4, perhaps for this reason). Budding of viral particles from the cell's plasma membrane could cause loss of membrane integrity. Virus replication may induce one of several “cell death” pathways within the cell. Viral evolution within an infected person is a key feature of viral pathogenesis. The virus replicates every one to two days within a cell and then must jump to a new target cell to continue replicating. At steady-state (i.e., with a constant set point) one new cell is infected for each cell that dies. If the immune system were fully competent we might expect it to win over the virus and clear the virus from the body, as happens with most RNA viruses (such as influenza virus). In contrast, HIV-1 sustains replication in the face of an active immune response. It is possible that the virus blunts the host immune response specifically as the virus replicates most efficiently in activated cells, such as those CD4 þ T cells responding to HIV-1 itself. Given the impaired host response, the virus is able to maintain a sufficiently large population size that, with its short replication time and relatively error prone replication apparatus, allows sequence diversity to accumulate in the viral population. Out of this diverse population variants that are able to escape the host's targeted immune response grow out, and this process is repeated over the course of the infection as the host response changes and the virus continues to evolve. Broadly, there are two types of adaptive (specific) immune responses the host mounts: CD8 þ T cells can target short sequences in viral proteins that are distributed among all of the viral proteins as they are “presented” on the surface of an infected cell (recognized as foreign resulting in the killing of that cell); alternatively antibodies target the surface proteins of the virus particle, for HIV-1 this is the Env protein. Since the Env protein is the target of both CD8 þ T cells and antibodies this explains why this protein is the most variable in the viral population, undergoing the most evolution to escape both arms of the host immune response. Viral evolution plays a role in several other features of replicating virus beyond immune escape: expansion of the host range of cell types that can be infected, and the evolution of drug resistance. In expanding its host range HIV-1 goes through distinct evolutionary pathways for its use of both its receptor CD4 and its chemokine coreceptor.

Coreceptor Switch The predominant form of HIV-1 uses the host chemokine receptor CCR5 as its coreceptor. Late in disease as the CD4 þ T cell population is lost the probability of encountering a preferred target cell decreases. Under these circumstances there is selective pressure to be able to replicate in a different cell type. There is a subpopulation of CD4 þ T cells that express CXCR4 but not CCR5. HIV-1 evolves to be able to use CXCR4 as its coreceptor thus expanding the number of cell types it can infect. Such viruses are typically seen late in untreated disease and correlate with more rapid disease progression, although it is not known if this is because of the expanded host range or as an indirect marker of the loss of CD4 þ /CCR5 þ T cells. The coreceptor switch involves evolution of sequences within the viral Env protein, which is responsible for interacting with the coreceptor during viral entry.

Macrophage-Tropic HIV-1 and Infection of the CNS One of the important AIDS-defining illnesses that became apparent shortly after the AIDS syndrome was identified was HIVassociated dementia (HAD), which appears in about 25% of people with untreated infections. The appearance of HAD focused attention on a potential role for HIV-1 in infection of the central nervous system (CNS). The brain/CNS is somewhat immune privileged and as a result there are not very many CD4 þ CCR5 þ T cells to support infection. However, there are two other cell types in the CNS that express CD4 and CCR5: the endogenous myeloid cell microglia and inflammatory macrophages that have trafficked across the blood-brain barrier into the CNS (perivascular macrophages that appear around blood vessels in the brain). However, there is one big and important difference with these cell types compared to CD4 þ T cells, the density of CD4 on

Human Immunodeficiency Virus (Retroviridae)

471

microglia and macrophages is about 25 times lower than it is on T cells. As a result, the normal form of HIV-1 (R5 T cell-tropic) is very inefficient at infecting these myeloid cells. This leads to the second evolutionary pathway to expand the host range of infected cells - evolution to efficiently use a low density of CD4 for entry, which we associate with macrophage tropism (R5 M-tropic). There are claims that HIV-1 can infect yet another cell type in the brain, astrocytes, although this is hard to reconcile with the fact that astrocytes do not express the viral receptor CD4. While CD4 þ T cells are not abundant in the CNS they are always trafficking from the blood at a low level and can be found in cerebral spinal fluid (CSF). Some of these cells are infected thus the brain is constantly being exposed to virus that is equivalent to the virus in the blood (R5 T cell-tropic). It has been hypothesized that monocytes can carry virus into the CNS, where they differentiate into tissue macrophages. However, the low density of CD4 on monocytes in the blood makes these cells poor targets for infection by the R5 T cell-tropic form of the virus found in blood. Thus trafficking CD4 þ T cells seem like the more likely culprit for introducing virus into the CNS. While the CNS gets exposed to virus in all people via infected T cell trafficking, it is only in a subset of people where there is detectable replication of HIV-1 in the CNS. There appears to be little movement of virus from the CNS back to the blood (at least that is detectable), thus when virus starts replicating in the CNS it appears as a new genetic lineage of the virus that is distinct from the virus in the blood. This is called a compartmentalized viral population. In the case of the CNS this is usually associated with the ability to use CD4 more efficiently for entry, i.e., the evolution of macrophage tropism. Between 10% and 20% of people have evidence of virus growing in the CNS within the first two years of untreated infection (seen as genetically compartmentalized virus in the CSF compared to the blood). This rises to 40% of people in late stage untreated infection, and includes most of the people who are diagnosed with HAD. An active area of research is determining the extent to which virus can persist in the CNS during suppressive antiretroviral therapy. HIV-associated dementia represents the end-stage of CNS pathogenesis, however it is now clear there is a broader spectrum of CNS-related disease states that can be measured and are collectively called HIV-associated neurocognitive defects (HAND). HAND is subclassified in the clinic based on symptomatic presentation and severity as either Asymptomatic Neurocognitive Impairment (ANI), Mild Neurocognitive Disorder (MND), or the most severe HIV-associated Dementia (HAD). These states are seen in people with untreated infections but there is concern that time spent untreated either leaves a person in one of these states even after therapy starts or makes it more likely they will progress even while on therapy. For people on antiretroviral therapy, the frequency of HAD has greatly decreased, occurring in only 2%–8% of treated people; however, ANI and MND have a prevalence of 30% and 20%–30%, respectively. As a general rule drugs are not efficient at crossing the blood-brain barrier leaving drug concentrations suboptimal in the CNS. With less potent drugs a phenomenon of CNS escape has been seen where virus is replicating within the CNS, sometimes with associated symptoms, while being suppressed in the blood. This is less of a problem with the recent more potent regimens.

Evolution of Drug Resistance With a large population size of virus within the host, a rapid rate of replication (300 times per year), and an error prone DNA polymerase (reverse transcriptase, about 1 nucleotide substitution in every three genomes synthesized), the virus generates a lot of sequence diversity. Most nucleotide substitutions (mutations) are deleterious and are lost from the population, but they are constantly reappearing with the error prone polymerase. However, as selective pressures change then minor variants in the population may suddenly become more fit and grow out. This is the case with drug resistance. If drug concentration or potency is too low and allows continued viral replication then resistant variants will be selected causing the drugs to lose potency. The success of highly potent drug regimens is that they quickly shrink the size of the pool of replicating virus which blocks the evolution of drug resistance to multiple drugs at one time. Ironically, the same three drugs that together block viral replication in a typical antiretroviral regimen, if given sequentially would simply select for resistance.

Latency Even when viral replication is completely blocked by suppressive therapy (see below), HIV-1 can persist in the body, referred to as a latent infection. In this state the virus exists as integrated DNA that is not transcribed. Expression of viral DNA into RNA and virus particle release happens stochastically. When suppressive antiviral therapy is present this transiently expressed virus cannot replicate and is lost. However, if therapy is discontinued this low level of stochastically released virus restarts the systemic infection. Latently infected cells appear to be a biological accident but with significant consequences. The virus has no "program" to become latent, its goal is to replicate as fast as possible. Thus when therapy almost clears all of the infected cells it is only a small number of long-lived cells that got infected but are not expressing the viral genome that accidently create this reservoir. However, this latent reservoir decays so slowly that it is present for the entire life of the infected person. There is great interest in the research community to find ways of removing this latent reservoir so that a person could stop taking antiretroviral therapy and not experience reappearance of the virus. This has been accomplished several times in the context of an allogeneic transplant of blood cells in the context of cancer therapy where the patient was given new blood cells from a donor who had both copies of their CCR5 gene mutated, making their new transplanted CD4 þ T cells resistant to infection. While this represents a "proof-of-concept" for an HIV-1 cure, this treatment is too extreme to use in the general population.

472

Human Immunodeficiency Virus (Retroviridae)

Table 3 FDA approved antiretrovirals for the treatment of HIV-1 infection. There are currently six classes of drugs approved for the treatment of HIV-1 infection, including Non-Nucleotide Reverse Transcriptase Inhibitors, Nucleotide Reverse Transcriptase Inhibitors, Protease Inhibitors, Fusion Inhibitors, Entry Inhibitors, and Integrase Inhibitors. Another drug class worthy of noting is Latency Reversing Agents, which aim to activate viral expression in latently infected cells to allow immune-mediated clearance, but these agents are still being investigated in the clinic and have yet to be approved by the FDA for the treatment of HIV-1 Nucleoside reverse transcripatase inhibitors

Protease inhibitors

Brand name

Generic name

Brand name

Generic name

Combivir Emtriva Epivir Epzicom Hivid Retrovir Trizivr Truvada

Agenerase Aptivus Crixivan Fortovase Invirase Kaletra Lexiva Norvir

amprenavir, APV (no longer marketed) tipranavir, TPV indinavir, IDV, saquinavir (no longer marketed) saquinavir mesylate, SQV lopinavir and ritonavir, LPV/RTV Fosamprenavir Calcium, FOS-APV ritonavir, RTV

Videx EC Videx Viread

lamivudine and zidovudine emtricitabine, FTC lamivudine, 3TC abacavir and lamivudine zalcitabine, dideoxycytidine, ddC zidovudine, azidothymidine, AZT, ZDV abacavir, zidovudine, and lamivudine tenofovir disoproxil fumarate and emtricitabine enteric coated didanosine, ddl EC didanosine, dideoxyinosine, ddl tenofovir disoproxil fumarate, TDF

Prezista Reyataz Viracept

darunavir atazanavir sulfate, ATV nelfinavir mesylate, NFV

Zerit Ziagen

Stavudine, d4T abacavir sulfate, ABC

Fusion inhibitors Fuzeon

enfuvirtide, T  20

Nonnucleoside revers transcirptase inhibiotrs Edurant

rilpivirine

Entry inhibitors selzentry

maraviroc

Intelence Rescriptor Sustive Viramune

etravirine delavirdine, DLV efavirenz, EFV nevirapine, NVP

Integrase inhibitors Isentress Tivikay Vitekta

raltegravir dolutegravir elvitegravir

Treatment The current standard of care for people living with HIV is combination antiretroviral therapy, or ART. Over a 15-year period we went from the identification of HIV-1 as the cause of AIDS (early 1980s) to the identification of the first active drug (AZT, late 1980s), to the discovery of potent three drug regimens capable of suppressing viral replication and stopping the disease course (late 1990s). Over the last 20 years drug potency has continued to increase and newer formulations allow drug delivery to become increasingly convenient, now one pill once a day for most people. Modern regimens allow people to live a near normal life with a full expected life span. However, HIV-1 is maintained as a latent infection even in the face of inhibitors that block all viral replication. This results in the need to take antiretroviral therapy throughout life since stopping typically results in the rebound/ reappearance of high levels of replicating virus in less than a month. There are four main classes of antiretrovirals used to treat HIV, each targeting a different stage of the virus life cycle. These include: Entry Inhibitors, Reverse Transcriptase Inhibitors, Protease Inhibitors, and Integrase Inhibitors. A full list of current FDA-approved drugs for the treatment of HIV-1 can be found in Table 3.

Entry Inhibitors Entry inhibitors block entry of the virus into the cell. One type of entry inhibitor binds to the host cell coreceptor CCR5 preventing its interaction with the viral Env protein (e.g., maraviroc) thus blocking the transition to the membrane fusion function of gp41. Another drug (enfuvirtide) is a peptide mimetic of a portion of the gp41 sequence that binds to block formation of the six helix bundle, thus preventing fusion. As a peptide this particular drug must be injected and thus is used only infrequently in people who have virus that is extensively drug resistant to other regimens.

Reverse Transcriptase Inhibitors Reverse Transcriptase Inhibitors (RTIs) target the viral DNA polymerase reverse transcriptase, which is packaged in the virus particle and is responsible for reverse transcribing the single-stranded viral RNA genome into double-stranded DNA in the newly infected cell. RTIs can be subdivided into two classes, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). NTRIs are nucleoside or nucleotide analogs that are missing the 30 -OH on the ribose. This OH- is used to extend the growing DNA chain as each nucleotide precursor is added. Thus when one of the NRTIs gets incorporated into the growing viral DNA chain it terminates synthesis since the growing point now cannot get extended with the

Human Immunodeficiency Virus (Retroviridae)

473

next nucleotide. NNRTIs are non-nucleoside analogs that bind in a pocket in RT to allosterically prevent enzymatic activity. NRTIs commonly comprise two-thirds of the three-drug cocktail prescribed as ART, with the third drug historically being a boosted protease inhibitor, an NNRTI, or more recently an integrase inhibitor.

Protease Inhibitors Protease Inhibitors (PIs) bind the active site of the viral protease, which is responsible for cleaving the HIV-1 polyproteins, namely the Gag and Gag-Pro-Pol precursors, into their mature and active protein domains. Protease inhibitors are peptide mimetics of the cleavage sites present in the precursor proteins. Inhibition of the viral protease prevents maturation of the viral proteins, leaving virions in either an immature state or a state of mixed processed and unprocessed proteins which results in a noninfectious virion. An early PI (ritonavir) had the fortuitous property of binding and inhibiting the enzyme responsible for its clearance, cytochrome P450–3A4, and in so doing greatly extended the half-life of the drug in the body. Ritonavir (or another drug with similar properties) is now often given in lower doses as a "booster" to increase the half-life of other drugs in the regimen that are metabolized by P450–3A4.

Integrase Inhibitors Integrase has a series of conserved acid amino acids that coordinate the binding of a Mg2 þ ion through an acidic and polar amino acid interface. This interface orients the Mg2 þ ion allowing it to be involved in a nucleophilic attack of a host DNA phosphodiester bond by the viral 30 hydroxyl, resulting in integration of viral DNA into the host DNA via a strand transfer reaction. Integrase strand transfer inhibitors, or ISTIs, bind to the same Mg2 þ to get oriented within the active site to inhibit the strand transfer reaction and, consequently, integration. ISTIs have recently emerged as the drug of choice over boosted protease inhibitors and NNRTIs in most ART regimens due to their increased potency, which results in less undesirable side effects and a lower probability that the virus will develop resistance mutations.

Prevention Of the 37.9 million people currently living with HIV-1, approximately 60% are currently receiving ART. Further, the percentage of people living with HIV-1 and on ART is expected to continually rise as governments and health organizations around the world increase efforts to achieve the ‘90–90–90 Goal’, an initiative from the World Health Organization that aims to have 90% of all infected individuals aware of their status, 90% of those aware of their status administered ART, and 90% of those administered ART to be virally suppressed. While diagnosis, treatment, and adherence are each crucial elements for controlling the HIV-1 epidemic, prevention efforts will also contribute significantly to making the 90–90–90 initiative successful. In this regard, Treatment as Prevention (TasP) has been one of the most effective prevention strategies in that people who are successfully suppressed on therapy have essentially a zero chance of transmitting the virus.

Biomedical Prevention Biomedical intervention is key to preventing the transmission of HIV-1 and further controlling the epidemic. There are numerous biomedical interventions that have been shown to decrease the rate of transmission. For example, circumcision has been shown to decrease a male’s risk of acquiring HIV-1 by 50–60%. HIV-1 prevalence tends to be higher in countries where male circumcision at birth is not standard medical practice. Thus, encouraging voluntary male circumcision could potentially prevent a significant number of new infections, if there was significant uptake. Additionally, female-oriented prevention strategies such as ARTcontaining vaginal gels as prophylaxis have been shown to decrease transmission rates up to 54% in clinical trials, although equivalent real-world efficacy of these gels has been harder to realize. For many viral diseases there is a vaccine that allows control of virus spread and is associated with a reduced disease course. The development of a vaccine for HIV-1 has proven to be especially challenging. One large trial showed about 30% efficacy in preventing infection, enough to inspire hope that there may be a path forward but too low to be useful in its current form. The search for an HIV-1 vaccine is currently driving an intense effort to understand how the body interacts with foreign antigens and to define how best to deliver these antigens to the immune system.

What is PrEP? The idea of Pre-Exposure Prophylaxis, or PrEP, as a prevention strategy grew from the observation that HIV-infected women on ART rarely transmit to their offspring during birth, as long as the women are virally suppressed. PrEP is treatment with antiretrovirals prior to exposure to decrease the risk of acquiring HIV-1 if exposed. During acute infection, HIV-1 first replicates in

474

Human Immunodeficiency Virus (Retroviridae)

mucosal tissue at the point of entry, such as the genital or rectal tract. The presence of antiretroviral drugs in these tissues prevents the initial replication of the virus leaving the person uninfected. PrEP typically includes one or two antiretroviral drugs rather than the three used for suppressive treatment of an infection. The ongoing development of long acting drug delivery systems (lasting weeks to months) is likely to have a significant impact on the uptake of PrEP in people at increased risk of exposure to HIV-1. Another new line of prevention research is to use broadly neutralizing monoclonal antibodies to neutralize incoming virus at exposure. If such an approach proves efficacious then genes for these antibodies could potentially be introduced using gene therapy vectors so that each person will be able to make these antibodies over an extended period of time.

Social and Educational Prevention While biomedical interventions are crucial tools for preventing HIV-1 transmission, there will continue to be an important role for social and educational prevention initiatives. It is clear that greater access to information regarding sexual health and safe sex practices, reducing risk associated with sharing injection equipment, and knowledge about basic HIV-1 transmission biology can significantly impact transmission rates. In countries such as Zimbabwe, Uganda, Zambia, Malawi, Kenya, and Thailand, initiating behavioral interventions has been successful in decreasing transmission rates. Particularly, improved sexual education courses, more thorough and informed HIV-1 testing and counseling, and well-organized, state-run prevention programs such as Thailand’s ‘100% Condom’ program, which promotes the use of condoms in sex work, have contributed to a decline in transmission. Unfortunately, many of these programs have yet to fully reach at-risk populations. Stronger political representation and funding opportunities will be crucial for expanding existing programs and creating new programs to aid these important populations. Ultimate success in fully controlling the HIV-1 epidemic will likely require some level of success in using all of these different approaches to prevention.

Further Reading Barré-Sinoussi, F., Chermann, J.C., Rey, F., et al., 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220, 868–871. Bushman, F., Nabel, G., Swanstrom, R., 2012. HIV: From Biology to Prevention and Treatment. Cold Spring Harbor Labroatory Press. Coffin, J., Hughes, S., Varmus, H., 1997. Retroviruses. Cold Spring Harbor Laboratory Press. Crawford, D., 2013. Virus Hunt: The Search for the Origin of HIV. Oxford University Press. Flint, J., Racaniello, V., Rall, G., Skalka, A., 2015. Principles of Virology: 4th Edition. ASM Books. Joseph, S., Swanstrom, R., 2018. The evolution of HIV-1 entry phenotypes as a guide to changing target cells. Journal of Leukocyte Biology 103, 421–431. Levy, J., 2007. HIV and the Pathogenesis of AIDS. ASM Press. Margolis, D., Garcia, V., Hazuda, D., Haynes, B., 2016. Latency reversal and viral clearance to cure HIV-1. Science 353, aaf6517.

Relevant Websites https://www.cdc.gov/ Center for Disease Control and Prevention. https://www.hiv.lanl.gov/content/index Los Alamos H.I.V. Database. https://www.unaids.org/en unaids. https://www.who.int/ World Health Organization.

Human Metapneumovirus (Pneumoviridae) Antonella Casola, Matteo P Garofalo, and Xiaoyong Bao, The University of Texas Medical Branch, Galveston, TX, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Capping Addition of a guanosine nucleotide to the 50 -end of mRNAs, which is then methylated by a methyl transferase. Glycosaminoglycan Long, unbranched, highly negativelycharged polysaccharides that consist of repeating disaccharide subunits that are located on the cell surface, or in the extracellular matrix. Lipid raft Membrane microdomains enriched in cholesterol and sphingolipids that modulate the membrane distribution of receptors and signaling molecules. PAMP Molecules (lipids, proteins, nucleic acids) derived from invading pathogens that trigger specific receptormediated host responses.

RDG Peptide motif responsible for protein interaction with adhesion proteins called integrins. Ribavirin A nucleoside analog (1-beta-D-ribofuranosyl1,2,4-triazole-3-carboxamide) that has antiviral activity against a number of RNA viruses, including hMPV. Syncytium A multinucleated cell, formed by fusion of the plasma membrane of an infected cell with those of its neighbors. Transcriptional elongation Process through which nucleotides are added to a growing RNA chain during gene transcription.

Classification Human metapneumovirus (hMPV) is a non-segmented, negative-sense (–), single-stranded RNA (ssRNA) virus, formerly a member of the Paramyxoviridae family. In 2016 it was reclassified into the family Pneumoviridae of the order Mononegavirales. This new family consists of two genera, Metapneumovirus and Orthopneumovirus, with the former including human and avian metapneumoviruses, and the latter human and bovine respiratory syncytial virus (RSV), as well as murine pneumonia virus (MPV). Based on genetic and phylogenetic analysis, hMPV is divided into two groups, A and B, with each group divided in two subgroups, i.e., A1 and A2; B1 and B2. Subgroup A2 is again subdivided into A2a and A2b. Despite differences in genetic sequences, there is significant antibody cross-reactivity and neutralization capacity and therefore protection against various genotypes. All hMPV strains share similar genome and virion structure and several different hMPV genotypes cocirculate each epidemic season, with the predominant genotypes varying from season to season.

Virion Structure and Virus Proteins In tissue culture, hMPV virions consist of enveloped pleomorphic, spherical or filamentous particles, ranging from 150 to 600 nm in size, with short protein spike projections of approximately 13–17 nM. HMPV encodes 9 proteins, classified as either integral membrane proteins, embedded in the virus envelope, or internal proteins, which associate with the virus nucleocapsid beneath the virus envelope, as illustrated in Fig. 1. The transmembrane proteins, which represent the virion spikes, are glycosylated and include the fusion protein F, glycoprotein G, and small hydrophobic protein SH. The F protein, which is well conserved among strains, mediates fusion of viral and cellular membranes during hMPV entry, it induces syncytium formation in infected cells, and determines the cellular host range. In addition, it is a major immunogenic protein. The functions of the G protein include cellular glycosaminoglycans binding (strain-dependent), and host immunity regulation. The SH protein, whose function is still unclear, also plays a role in modulating innate immune responses in airway epithelial cells (AECs) and dendritic cells (DCs). There are five internal proteins, four of which are part of the nucleocapsid and polymerase complex: the nucleoprotein N, the phosphoprotein P, the transcription regulatory proteins M2-1 and M2-2, and the RNA-dependent RNA polymerase L. The viral genome interacts with N, P, and L to form the virus ribonucleoparticle (RNP). The matrix protein M surrounds the RNP underneath the virus envelope, and, together with the F and G proteins, plays an important role in virus assembly and budding. A summary of hMPV proteins and their function is presented in Table 1. A further description of the various functions of hMPV proteins is presented in subsequent sections of this article.

Genome The hMPV genome consists of about 13,000 nucleotides, with a size ranging from 13,280 to 13,378 nucleotides depending on the genotype, and it contains 8 genes, which encode 9 proteins, with the order of viral protein expression as the following: 30 -N–P–M–F–M2–1–M2–2–SH–G–L-50 (Fig. 2). Among those, M2-1 and M2-2 are both encoded by hMPV M2 gene. The M2-1 open reading

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21507-0

475

476

Human Metapneumovirus (Pneumoviridae)

Fig. 1 HMPV virion structure. Schematic representation of name and location of hMPV proteins, either present in the envelope (F, G, SH) or underneath it (M), or associated with viral RNA (N, P, L, M2-1 and M2-2).

Table 1

hMPV proteins and function

Gene

Protein

Function

F G SH N P M2

Fusion protein Attachment glycoprotein Small hydrophobic protein Nucleoprotein Phosphoprotein M2-1 protein M2-2 protein

Cell binding, membrane fusion, virus assembly Binding to cellular glycosaminoglycans, virus assembly, innate immune response inhibition Innate immune response inhibition RNA genome encapsidation, virus intercellular spread Polymerase cofactor Essential for virus replication in vivo Viral RNA synthesis

L M

polymerase protein Matrix protein

RNA-dependent RNA polymerase responsible for mRNA generation and genome replication Viral assembly and budding

frame (ORF) is presumed to start with the first AUG codon of M2, while the M2-2 ORF possibly initiates with the second or third AUG (s) of M2, overlapping the M2-1 ORF by about 50 to 40 nucleotides. HMPV RNA synthesis comprises two independent events: viral replication and viral gene transcription, which are carried out by three essential proteins: the nucleoprotein N, phosphoprotein P, and large protein L. In addition to these three proteins, studies using recombinant hMPV lacking M2-2 demonstrated that the M2-2 protein is also important for viral RNA synthesis. Whether hMPV M2-2 is a key regulatory factor involved in the switch of the viral RNA polymerase from viral gene transcription to viral genome replication, as with the RSV M2-2 protein, is still unknown. Although RSV M21 functions as a transcriptional elongation and antiterminator factor, and is essential for viral replication, hMPV M2-1 protein was found to be dispensable for virus viability or replication in tissue culture, although it was very important for replication in vivo, as its deletion resulted in a highly attenuated virus.

Life Cycle The Entry of hMPV The major cell target of hMPV infection is the airway epithelial cell, although it can infect other cell types, like macrophages and DCs, leading to abortive replication in those cells. hMPV F is sufficient for mediating virus entry, as hMPV lacking G or SH is still infectious, although hMPV B strain also uses the G protein to bind to glycosaminoglycans to improve viral fitness. HMPV F protein shares conserved functional domains with other pneumovirus F proteins, and requires cleavage from a precursor protein F0 into F1 and F2 as a prerequisite for fusion activity. In cell culture, it also requires the addition of exogenous proteases, different from the orthopneumovirus RSV. HMPV F attaches to cellular receptors, including integrins, via interaction with its Arg-Gly-Asp (RDG) sequence, and catalyzes virus membrane fusion with cellular membranes during virus entry. In addition to plasma membranes, hMPV particles have been shown to be internalized by clathrin-mediated endocytosis and then fuse with endosomal membranes. Engagement of RGD-binding integrins has been shown to be required for endosomal trafficking and full virus membrane fusion with intracellular membranes. Although low pH exposure was initially thought to be necessary for hMPV fusion, inhibition of

Human Metapneumovirus (Pneumoviridae)

477

Fig. 2 HMPV genomic organization. Schematic representation of hMPV genome, illustrating gene order from the 30 to the 50 end and the M2-1 and M2-2 open reading frames both encoded by hMPV M2 gene.

endosomal acidification has only a modest effect on fusion for many hMPV viruses, and this event appears to be strain-specific. In addition to clathrin-mediated endocytosis, there is evidence that hMPV enters target cells via lipid rafts. When lipid raft inhibitors, such as cyclodextrin and nystatin were used, hMPV cell entry was inhibited. Virus internalization in immune cells, such as monocyte-derived DCs, seems to occur via macropinocytosis and also via clathrin-mediated endocytosis through the interaction with the C-type lectin receptor DC-SIGN.

Viral RNA Synthesis Upon entry, viral nucleocapsids are released into the cell cytoplasm, followed by synthesis of viral components. It is generally believed that the viral synthesis of hMPV is similar to the better known pneumovirus RSV. The viral RNA-dependent RNA polymerase (RdRp) complex, formed by the L and P proteins, is presumed to bind the N-encapsidated genome at the leader region, then sequentially transcribes each gene by recognizing the start and stop signals flanking the viral genes, leading to synthesis of mRNAs, which are capped and polyadenylated. Protein translation occurs in the endoplasmic reticulum, with transport of proteins through the Golgi apparatus, for post-translational modifications, mainly glycosylation, to sites of virus assembly. Genome replication presumably starts when enough nucleoprotein is present to encapsidate newly synthetized antigenome and genome copies. The negative-sense genome is replicated into a positive-sense antigenome, which serves as a template for synthesis of many copies of the viral genome. In addition to P and L, the M2-1 and M2-2 protein are also important for viral RNA synthesis, as mentioned above. Recent structural studies on P protein revealed it to be a multifunctional hub which interacts with numerous components of the viral RNA-synthesis machinery, including the RNA-free N, the M2-1, the L polymerase, and the nucleocapsid, demonstrating its importance in viral RNA synthesis. Experimentally, colocalization of the virus –RNA, þ RNA, mRNA, as well as, N and P proteins was found to occur in specific cytoplasmic structures called inclusion bodies (IBs), but not in others – such as stress granules, or processing bodies. This strongly suggests that IBs are the sites of active hMPV replication and gene transcription, similar to other negative sense RNA viruses, such as filoviruses and rhabdoviruses.

Viral Particle Assembly and Spread From IBs, newly synthesized nucleocapsids are transported to assembly sites, likely via actin cytoskeleton, to be incorporated into nascent virions. Similar to human RSV (hRSV) and paramyxoviruses, the M protein plays a fundamental role in hMPV morphogenesis, however, its presence is not sufficient for the formation of virus-like particles, which requires the presence of the F, G and possibly N proteins. The fully assembled virions are then released into the extracellular environment from the apical surface of polarized infected cells. The process of budding and the release of hMPV has not yet been elucidated. Many enveloped viruses, including several paramyxoviruses, use the endosomal sorting complex required for transport (ESCRT) machinery for budding. However, recent studies have shown that hRSV does not utilize this pathway, similar to influenza A virus and alphaviruses, but employes a Rab11-dependent mechanism, which is part of the apical recycling endosomal pathway. Similarly, the ESCRT is not required for budding of the avian metapneumovirus C, which shares about 80% homology with hMPV, suggesting that it is unlikely to be involved in hMPV budding, as well. Viral RNA, and other viral components, can also be transferred to other cells via cell-to-cell syncytia formation or via formation of intercellular extensions, which have been shown to be covered by branching filaments that contain viral proteins and RNA, likely corresponding to filamentous hMPV virions. A summary of the virus life cycle is presented in Fig. 3.

Epidemiology and Clinical Features hMPV was first discovered in the Netherlands in 2001. Through electron microscopy and real-time reverse transcriptasepolymerase chain reaction (RT-PCR), researchers identified this new viral agent in nasopharyngeal specimens collected from children with a respiratory illness. Since then, hMPV has been isolated in samples derived from a variety of age groups worldwide, with a higher prevalence in children younger than 5 years and adults older than 65 years of age. Although the disease burden for hMPV is lower compared to hRSV, the other important human virus member of the Pneumoviridae family, hMPV remains a cause of considerable morbidity and mortality, currently estimated at 33 million cases/year worldwide. The virus is believed to have circulated for a very long time, as hMPV-neutralizing antibodies have been detected in stored serum samples from the 1990s and as far back as the 1950s. From seroconversion studies, we know that in the first few months of

478

Human Metapneumovirus (Pneumoviridae)

Fig. 3 Schematic diagram of hMPV life cycle. Schematic representation of main steps of hMPV replicative cycle, from virus entry via endocytocis to RNA synthesis in specialized structures known as inclusion bodies.

life most children have maternally derived antibodies, and that primary infection occurs predominantly in the first two years of life, followed by common reinfections during childhood, with positive IgG and IgM detected during an acute respiratory illness. In a similar fashion to RSV, and most viruses associated with respiratory illnesses, hMPV infections show a seasonal cycle which spans between November and May in temperate climates, with the peak of disease mostly occurring during the months of March and April. In recent surveillance studies conducted in the US from 2008 to 2014, the data confirmed previous observations that hMPV season occrs later than that of RSV, with the RSV season occurring first, followed by influenza, and then hMPV. In addition, the data demonstrated a biannual pattern of early and late seasons, which did not occur for RSV. Different hMPV genotypes can circulate during the same epidemic year, and severe infection forms may be associated with different lineage viruses throughout the year. Some studies have suggested a correlation of specific genotypes with disease presentation, with hospitalization and diagnosis of pneumonia being more frequent in patients infected with genotype A, and wheezing with genotype B, however, there is no significant correlation with disease severity. The level of viral replication may also affect the clinical picture. Some studies have reported that, in the absence of a preexisting condition or immunosuppression, the disease severity and the duration of illness were associated with high hMPV viral load in the lower respiratory tract, although other studies showed conflicting results. The incidence of hMPV infection is higher early in life, in fact it is two times more common among children before 2 years of age, compared to those between 2 and 5 years of age. Hospitalization is also most common during the first year of life. Among infants and very young children, prematurity and pre-existing heart and lung conditions have been identified as predisposing factors for a severe disease. The estimated prevalence of hMPV infection in hospitalized children is between 5% and 7%, with some studies reporting up to 10% of all pediatric hospitalizations for respiratory illnesses, with a rate of 1.2 hospitalizations per 1000 children o5 years old per year. In the outpatient population, the impact of hMPV on acute respiratory illnesses in the pediatric population becomes as high as 15% during the peak season. HMPV infections in otherwise healthy young adults are usually mild and often go undetected, but it is known that reinfection occurs frequently during adult life, and the majority of adults have stable titers of neutralizing antibodies. Severity of disease increases again in the elderly, with higher morbidity and mortality rates, and hospitalization rates as high as 60%.

Human Metapneumovirus (Pneumoviridae)

479

In addition to children and the elderly, patients with preexisting conditions such as pulmonary neoplasia, lymphoma, chronic obstructive pulmonary diseases or asthma are also at greater risk of developing pneumonia and respiratory failure. The relationship of hMPV to asthma is still under investigation, but current evidence seems to support both higher prevalence of hMPV in patients hospitalized due to acute asthma exacerbations, and a higher risk of developing asthma later in life for those patients who experienced severe disease from hMPV infection during childhood. Coinfection with other respiratory viruses or bacteria may also occur. Dual infection with RSV has been more commonly reported, and there are conflicting results on whether virus co-infections lead to increased disease severity. Although less common than infection with influenza, superimposed bacterial pneumonia, whether a nosocomial infection or community-acquired, has been reported, which leads to increased mortality rate. Infection with hMPV is acquired through proximity or contact with infected subjects, mostly via respiratory secretions (droplets) or fomites. After an incubation period of 4–6 days, patients usually develop symptoms associated with upper respiratory tract infection (URTI), which may include fever, sore throat, cough, and development of acute otitis media. Patients who progress with the development of a lower respiratory tract infection (LRTI) show tachypnea, dyspnea, wheezing and hypoxia. High fever, found frequently among hospitalized children, is less frequent in adults, especially compared to infection with RSV. In rare cases, seizures and encephalitis have also been reported in association with hMPV. The virus can be isolated in secretions for up to 14 days after the onset of symptoms, making person-to-person spread within families or communities common. In the pediatric population, URTI symptoms last on average between 7 and 10 days, while LRTI requiring hospitalization have a longer duration. Bronchiolitis and interstitial pneumonia are usually confirmed by radiologic diagnosis. In systematic evaluations of the radiographic characteristics of acute respiratory illnesses associated with pediatric hMPV infection, peribronchial/perihilar opacities were the most commonly observed abnormality, occurring in 87% of children. Hyperinflation also occurred frequently (69%), with atelectasis (40%) and consolidation (18%) appearing less frequently. Subjects with different clinical diagnoses had different radiologic findings, e.g., children with pneumonia, were more likely to have consolidation and perihilar/peribronchial opacities, while children with asthma and bronchiolitis were more likely to have hyperinflation. When additional laboratory tests are performed, complete blood counts may show lymphopenia with or without neutrophilia, and abnormal chemistries, namely elevated transaminases on liver function tests. In immunocompromised patients, and the elderly, the presentation of hMPV LRTI is usually pneumonia, which may be severe. Because of increased transmission and severe disease, the mortality rate associated with hMPV infection is higher in elderly patients confined within long-term care institutions.

Pathogenesis Cellular Responses The airway epithelium represents the primary infection site of respiratory viruses, including hMPV. After infection, epithelial cells, together with lung resident cells – such as alveolar macrophages, secrete a variety of innate immune molecules, including antiviral substances and proinflammatory and immunoregulatory cytokines and chemokines, which initiates specific hMPV immune responses, both innate and adaptive, that are protective, but also in part, pathogenetic. For example, the depletion of alveolar macrophages have been shown to decrease hMPV replication, lung inflammation and disease severity in a mouse model of infection. Similarly, type I interferon (IFN) secretion, which plays a role in controlling hMPV replication, is also a determinant of a disease, as mice lacking type I IFN receptor have less lung inflammation and airway dysfunction. Increased levels of cytokines and chemokines have been shown to correlate with clinical disease in animal models of infection. For example, the thymic stromal lymphopoietin (TSLP) has been recently found to induce pulmonary inflammation and enhance hMPV replication in mice, and to correlate with wheezing and disease severity in young children with the infection. IL-17 has been shown to suppress regulatory T cell recruitment, increase Th1 and Th2 cells and neutrophil lung influx, and be involved in airway dysfunction in the course of hMPV infection, suggesting that IL-17 skews the immune response in the lung toward an inflammatory profile. Studies in mice have shown that neutrophils are the predominant cell type recruited to the airways early during hMPV infection. In this animal model, neutrophil depletion is associated with increased pulmonary inflammation and severe clinical disease, without changes in viral clearance, and with increased recruitment of lung gδ T cells, suggesting that the latter cells contribute to hMPV‐induced disease pathogenesis. T cell responses are required for virus clearance in hMPV-infected mice, together with neutralizing antibody production, however, they also contribute to pathology. Depletion of CD4 þ T cells reduces lung pathology and airway obstruction, without affecting viral replication, although it is associated with impaired generation of neutralizing antibodies. This impairment does not affect the protection against hMPV reinfection, suggesting that protection can be provided by an intact CD8 þ T cell compartment. Cytotoxic CD8 þ T lymphocytes have been shown to be capable of clearing hMPV in Rag-/- mice, underlying their importance in hMPV protection, however, they have also been shown to contribute to pathogenesis. Immunity to hMPV is incomplete, as reinfections can occur throughout the life. One mechanism utilized by hMPV to evade the adaptive immune response is the upregulation of programmed cell death-1 (PD-1) receptor on T cells and its ligand, PD-L1, in the lung, leading to functional impairment of hMPV-specific CD8 þ T cells. Inhibition of PD-1 signaling, or lack of the receptor in a

480

Human Metapneumovirus (Pneumoviridae)

Fig. 4 Schematic diagram of sites of antagonism of cellular signaling and IFN production/responses by hMPV proteins. Major cellular targets of hMPV proteins in the TLR4, RIG-I/MAVS and type I IFN signaling pathways. P indicates phosphorylation.

murine model of infection, result in a higher number of hMPV-specific CD8 þ T cells, both during primary and secondary infections, suggesting that this pathway could play an important role in the ability of hMPV to reinfect the host.

Pattern Recognition Receptors (PRRs) and Antagonism of Immune Responses Activation of the innate responses to infections is initiated by the recognition of specific pathogen-associated molecular patterns (PAMPs) derived from the invading pathogens, through a variety of host PRRs. Multiple PRR families, including RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), and NOD-like receptors (NLRs), and their corresponding signaling adapters, have been shown to detect hMPV and initiate cellular responses to infection. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are the two major RLRs activated by hMPV infection and they share a common adapter, the mitochondrial antiviral-signaling protein (MAVS), for signaling. The RIG-I/MAVS pathway has been shown to be important for the cytokine/chemokine/IFN induction by hMPV in AECs, while MDA5 is essential to induce IFN secretion in human and mouse DCs, as well as, in an experimental mouse model of infection, where it plays a protective role against a severe disease. The role of MAVS in hMPV-induced inflammatory and antiviral mediator secretion has also been investigated in mice, and it was found to be necessary for the generation of protective innate immune responses, through immune mediator production and recruitment and maturation of immune cells, which in turn, modulate activation of T cell responses. Several TLRs have also been shown to be important in hMPV-induced host responses. In human monocyte-derived DCs, TLR4 plays an important role in the induction of cytokines, chemokines and type I IFNs following hMPV infection. Its absence, in a mouse model of infection, is associated with significantly reduced lung inflammatory response, clinical disease severity, and airway dysfunction. TLR7 also plays a role in type I IFN production in response to hMPV infection, as TLR7-deficient plasmacytoid dendritic cells (pDCs) show reduced IFN-b secretion compared to wild type cells. Although the expression of TLR9 has been shown to be increased in a hMPV murine model of bacterial superinfection, its role in hMPV-induced host responses has not been revealed. MyD88 is a common TLR adapter, and its absence in DCs and in a mouse model of infection is associated with reduced proinflammatory mediators and type I IFN production, which results in amelioration of hMPV-induced pulmonary inflammation and clinical disease. The elucidation of the role of NLRs in hMPV infection has only recently been initiated, therefore, limited information is available. HMPV has been shown to activate NLRP3 inflammasome in an SH protein-dependent manner both in cells and mice, playing a detrimental role during infection, as inflammasome inhibition ameliorates clinical disease and lung inflammation without affecting viral replication. Furthermore, children experiencing a more severe disease form have significantly higher cellular and secreted levels of the inflammasome-regulated NLRP3 and IL-1b, respectively.

Human Metapneumovirus (Pneumoviridae)

481

Like many other viruses, hMPV has developed strategies to subvert cellular signaling and antiviral responses. The P protein of hMPV-B strains, but not the one from A strains, suppresses RIG-I-dependent type I IFN production. The hMPV G protein has also been shown to suppress RIG-I-mediated immune responses in AECs by disrupting the RIG-I-MAVS interaction and to inhibit TLR4-mediated innate antiviral signaling in DCs. The SH protein of hMPV downregulates type I IFN signaling pathway by reducing the expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1). SH also blocks type I IFN induction in pDCs by inhibiting the TLR7/MyD88/TRAF6 pathway, and suppresses the nuclear factor (NF)-ĸB transcriptional activity in AECs. The M2-2 protein is also an important immune regulatory protein by targeting the common adapter molecules MAVS and MyD88. A summary of the viral protein antagonism of cellular signaling pathways is presented in Fig. 4.

Diagnosis As hMPV infection shares clinical signs and symptoms with a variety of other respiratory viruses, it is not possible to make a diagnosis based solely on the patient’s clinical presentation, thus, laboratory testing is necessary to confirm the infection. HMPV can be detected by culture, nucleic acid amplification, antigen detection and serology, however, not all methods have clinical utility. Traditional virology methods, such as cell culture, are not very helpful with hMPV in clinical practice because isolation may take over 2 weeks, and the mild cytopathic effect caused by hMPV is not consistent among different genotypes. Furthermore, growing hMPV in cell culture requires exogenous trypsin and is only successful in a few specific cell lines. Real time PCR, targeting usually either the N or the F protein gene, is the method of choice in most medical facilities for hMPV diagnosis – especially with the current availability of multiplex assays, which allow simultaneous detection of several respiratory viruses, and therefore, establishes the presence of virus coinfections. Viral antigens can be detected in cells of respiratory secretions by immunofluorescence assay, but this method, despite being of value during seasonal epidemics, is less sensitive than RT-PCR. Enzyme-linked immunoassays (ELISA) can be used for serologic testing, however, as the majority of individuals test seropositive for hMPV after childhood, serology only yields useful information in cases of seroconversion, or when a four-fold increase in subsequent specimens is observed. Imaging studies, such as chest radiography, and computed tomography without contrast, are clinically useful for the overall evaluation of the patient and clinical decision-making process, but hMPV cannot be differentiated from other respiratory infections associated with bronchiolitis or pneumonia on the basis of imaging alone.

Treatment The treatment options available to those infected with hMPV are limited to supportive care with intravenous hydration and oxygen supplementation, if necessary. Similar to RSV infection, bronchodilators and corticosteroids have been used in patients with airway obstruction, with no clear benefits, therefore, they are generally not recommended for pediatric or adult patients. Ribavirin and nonspecific polyclonal intravenous immunoglobulins (IVIG) have been used with mixed results in some specific cases and case series. Though preclinical studies of ribavirin and IVIG for the treatment of hMPV did show promising results, only sporadic case reports have shown positive outcomes when they were used in adults and children mostly immunocompromised due to cancer or solid-organ transplant. Because of conflicting conclusions, and the lack of randomized controlled trials, there is no recommendation regarding the therapeutic use of ribavirin, nor IVIG. Several experimental products are under research and development for the treatment of hMPV, including specific monoclonal antibodies, fusion inhibitors, and small interfering RNAs. Among therapeutic immunoglobulins, monoclonal antibodies developed against hMPV are currently in the preclinical research pipeline, having shown efficacy in vitro and also in vivo, by reducing pulmonary viral load and severe disease in small animal models of infection. Additional therapeutic approaches under experimentation are fusion inhibitors and RNA interference, targeting primarily N and P proteins, which have been tested in vitro with a certain degree of effect.

Prevention Currently, the prevention of hMPV infection is mostly achieved through epidemiological countermeasures. Standard droplet isolation infection control practices are recommended in clinical settings where infected patients are being treated, as well as, in long-term care facilities when cases of hMPV infection are suspected. While a vaccine to prevent hMPV is not currently available, researchers are exploring different strategies to develop a safe and effective vaccine, including the generation of live-attenuated vaccines, vector and chimeric-based vaccines, as well as, subunit vaccines. Live-attenuated virus candidates in preclinical development include, cold-passaged temperature-sensitive strains and recombinant vaccine candidates generated by deletion (M2-2, G and/or SH proteins) or mutation (M2-1 or F proteins) of selected genes, which have shown good immunogenicity, and reduced viral replication in the lungs of various animal models of infection, with no enhanced disease – different from inactivated vaccines, both formalin and heat-inactivated, which increased disease pathogenicity. The recombinant virus lacking SH expression has been recently proved to be immunogenic in healthy adults, and it is now in the research pipeline as the parent virus for vaccine development.

482

Human Metapneumovirus (Pneumoviridae)

Chimeric, virus-like particle and subunit vaccines have also been developed and tested primarily in rodent models of infection. In general, those vaccines that including the F protein were shown to be immunogenic and protective, however, in the case of the F subunit vaccine, the antibody levels seemed to rapidly wane in nonhuman primates. Experimental monoclonal antibody preparations have also been studied for their prophylactic use to prevent severe disease. In rodent models, the use of these antibodies has shown promising results. In animals treated prior to hMPV challenge, viral load was low or undetectable in lung specimens and microscopic examination revealed reduced tissue damage compared to untreated or mock-treated animals.

Further Reading Biacchesi, S., Pham, Q.N., Skiadopoulos, M.H., et al., 2005. Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates. Journal of Virology 79, 12608–12613. Boivin, G., Abed, Y., Pelletier, G., et al., 2002. Virological features and clinical manifestations associated with human metapneumovirus: A new paramyxovirus responsible for acute respiratory-tract infections in all age groups. The Journal of Infectious Diseases 186 (9), 1330–1334. Chu, H.Y., Renaud, C., Ficken, E., et al., 2014. Respiratory tract infections due to human metapneumovirus in immunocompromised children. Journal of the Pediatric Infectious Diseases Society 3 (4), 286–293. Falsey, A.R., Erdman, D., Anderson, L.J., Walsh, E.E., 2003. Human metapneumovirus infections in young and elderly adults. The Journal of Infectious Diseases 187, 785–790. Jartti, T., van den Hoogen, B., Garofalo, R.P., et al., 2002. Metapneumovirus and acute wheezing in children. Lancet 360 (9343), 1393–1394. Kolli, D., Bao, X., Casola, A., 2012. Human metapneumovirus antagonisms of innate immune responses. Viruses 4, 3551–3571. van den Hoogen, B.G., de Jong, J.C., Groen, J., et al., 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nature Medicine 7, 719–724. Wen, S.C., Williams, J.V., 2015. New approaches for immunization and therapy against human metapneumovirus. Clinical and Vaccine Immunology 22 (8), 858–866. Williams, J.V., Wang, C.K., Yang, C.F., et al., 2006. The role of human metapneumovirus in upper respiratory tract infections in children: A 20-year experience. The Journal of Infectious Diseases 193, 387–395.

Human Norovirus and Sapovirus (Caliciviridae) Sumit Sharma and Marie Hagbom, Linköping University, Linköping, Sweden Lennart Svensson, Linköping University, Linköping, Sweden and Karolinska Institute, Stockholm, Sweden Johan Nordgren, Linköping University, Linköping, Sweden r 2021 Elsevier Ltd. All rights reserved. This is an update of K.Y. Green, Noroviruses and Sapoviruses. In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00555-0.

Introduction Acute gastroenteritis (AGE) remains a major cause of morbidity and mortality in people in all age groups. Enteric caliciviruses, including human noroviruses and sapoviruses, are today recognized as important etiologic agents of this illness. However, before 1970, it was assumed that bacteria or parasites caused almost all acute infectious diarrhea. It was not until the introduction of electron microscopy (EM) and negative staining in the early 1970s that several viruses were discovered and found associated with acute diarrhea. In 1972, virus particles measuring 27 nm in diameter, were detected by immuno-electron microscopy in the feces of volunteers who had received filtered fecal suspensions from an outbreak of winter vomiting disease that had originated from a primary school and spread to the local community of Norwalk in the US. The particle was therefore first named ‘Norwalk agent’; decades later, it was named norovirus and has a ‘star of David’ appearance under EM (Fig. 1). It has now been established as a causative agent for one-fifth of all acute gastroenteritis, with approximately 200,000 deaths annually worldwide. In 1976, Madeley and Cosgrove and Flewett and Davies, reported morphologically ‘typical human caliciviruses’ in the feces of children with acute gastroenteritis. In the years that followed, these viruses were detected by several investigators and were named ‘typical human caliciviruses’ or ‘Sapporo-like viruses’ after an outbreak in 1982 in Sapporo, Japan. Today, these viruses are known as sapovirus. Although human norovirus and sapovirus were first detected nearly five decades ago, several questions related to their pathogenesis and host–virus interactions still remain unanswered, partly due to the lack of a small-animal model and the inability to cultivate these viruses in vitro. However, most recently, human intestinal enteroids (HIEs) originating from stem cells in the small intestine have been established and are being used successfully to propagate human noroviruses. This recent major breakthrough has increased our knowledge of norovirus biology significantly. It is anticipated that the recent successful cultivation of sapovirus in HIEs will also lead to a similar advancement of understanding.

Norovirus Viral Classification Norovirus is a genus in the family Caliciviridae, which also includes the genera Bavovirus, Lagovirus, Minovirus, Nacovirus, Nebovirus, Recovirus, Salovirus, Sapovirus, Valovirus, and Vesivirus (Table 1). The norovirus genus contains a diverse group of viruses that infect several mammalian species including humans, dogs, cats, pigs, mice, and cattle. The classification within the norovirus genus has changed significantly through the years and was recently updated. Noroviruses are classified into 10 capsid genogroups (GI to GX) based on amino acid sequence diversity of the complete major capsid gene (VP1). Strains that infect humans belong to GI, GII, and GIV genogroups, with GI and GII causing the vast majority of human disease. Similarly, partial RNA dependent RNA polymerase (RdRp) nucleotide diversity is used to classify norovirus into eight P-groups (GI.P, GII.P, GIII.P, GIV.P, GV.P, GVI.P, GVII.P, and GX.P). These genogroups are further divided into capsid (VP1) or RdRp genotypes. This dual genotyping system can account for that recombination is a major factor in norovirus diversity, with the major recombination site present in the junction between the RdRp and VP1 genes (open reading frame [ORF]1–ORF2 junction). Today, VP1 genogroup GI has been divided into nine capsid genotypes (GI.1–GI.9) and GII into 27 capsid genotypes (GII.1–GII.27). The GI.P and GII.P RdRp genogroups are divided into 14 and 37 P-genotypes, respectively (GI.P1–GI.P14 and GII.P1–GII.P8, GII. P11–GII.P13, GII.P15–GII.P18, GII.P20–GII.P41). The division into VP1 capsid genotypes is based on the amino acid diversity of the complete VP1 gene, whereas the P-genotype is currently based on the nucleotide difference of 762 nucleotides at the 30 end of ORF1. The predominant human genotype GII.4 is further classified into variants based on phylogenetic clustering after becoming epidemic in at least two geographical locations.

Genome Human norovirus is a single-stranded positive-sense RNA virus [ssRNA( þ )]. The genome is B7.5 kb long and organized in three ORFs. The genome is covalently linked to a virus-encoded protein called VPg at the 50 end, and is polyadenylated at the 30 end

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21543-4

483

484

Human Norovirus and Sapovirus (Caliciviridae)

Fig. 1 Norovirus particles visualized by negative staining electron microscopy.

Table 1

Calicivirus classification detailing genus and type species

Genus

Type Species

Norovirus Sapovirus Lagovirus Vesivirus Nebovirus Recovirus Valovirus Bavovirus Nacovirus Salovirus Minovirus

Norwalk virus (NV) Sapporo virus (SV) Rabbit hemorrhagic disease virus (RHDV) European brown hare syndrome virus (EBHSV) Vesicular exanthema of swine virus (VESV) Feline calicivirus (FCV) Newbury  1 virus (NBV) Tulane virus St Valerian calicivirus Chicken calicivirus Turkey calicivirus Atlantic salmon calicivirus Fathead minnow calicivirus

Adapted from Desselberger, U., 2019. Caliciviridae other than noroviruses. Viruses 11, 286. Jan Vinjé, J., Estes, M.K., Esteves, P., et al., 2019. ICTV virus taxonomy profile: Caliciviridae. Journal of General Virology 100, 1469–1470.

(Fig. 2(A)). The untranslated regions (UTRs) at either end of norovirus contain conserved RNA secondary structures important for viral replication, translation and pathogenesis. ORF1 encodes a large polyprotein that is cleaved by the virally encoded cysteine proteinase into six mature non-structural proteins (Fig. 2(A)). ORF2 encodes the major structural protein, VP1, which in turn is organized into internal shell (S) domains and protruded domains (P). The S domain is the most conserved part of the VP1 gene. The P domain can be further divided into the P1 domain and the hypervariable P2 domain, which encodes epitopes that are targets for neutralizing antibodies as well as sites involved in binding to attachment factors/receptor. ORF3 encodes a minor structural protein called VP2, which has a putative role in assisting capsid assembly and genome encapsidation.

Virion Structure Early experiments to understand norovirus virion structure using electron cryo-microscopy and image processing techniques involving recombinant Norwalk-like virus (rNV) particles have shown that norovirus exhibits T ¼ 3 icosahedral symmetry,

Human Norovirus and Sapovirus (Caliciviridae)

485

Fig. 2 Norovirus and sapovirus genome organization. (A) The norovirus genome consists of three open readings frames. ORF1 (in grey) encodes the nonstructural proteins, with ORF2 (in green) and ORF3 (in blue) encoding the structural proteins VP1, the major capsid protein, and VP2, a minor structural protein, respectively. The non-structural polyprotein of ORF1 is processed by the cysteine protease (Pro), into six mature proteins: p48, an N-terminal protein proposed to be involved in replication complex formation; NTPase, a nucleoside triphosphatase; p22, a protein of unknown function but proposed to be involved in replication complex formation; VPg, covalently attached to the 50 end of the viral genome and involved in translation and replication; Protease, the cysteine proteinase, and RdRp, which is the viral RNA-dependent RNA polymerase. The major capsid protein is further divided into the shell and protruding domains (P), with the P domain subsequently divided into two different subdomains, P1 and P2. The approximate size of proteins expressed as number of amino acid (aa) are from the GII.4 MD-145 strain (AY032605). (B) Sapovirus genome consists of two open reading frames, ORF1 (non-structural proteins and VP1, in grey and green, respectively) and ORF2 (VP2, in blue). A subgenomic RNA is also produced during replication. A viral protein (VPg) is linked to the 50 end and a poly-A tail to the 30 end of both the genomic and subgenomic RNA. The approximate size of proteins expressed as number of amino acid (aa) are from the GI.1 Manchester strain (X86560).

wherein 180 capsid protein molecules form 90 dimers. X-ray crystallography studies have revealed that the capsid protein structure has S domain and a P domain, which are linked by a flexible hinge. The NH2 terminal part of the capsid protein (up to 225 aa) is involved in the formation of the icosahedral shell, while the P domain is formed by two subdomains, i.e., P1 (aa 226–278 and aa 406–520) and P2 (aa 279–405).

Life Cycle Until recently, there were no robust cell culture systems supporting human norovirus propagation, which has hampered the study and characterization of the viral life cycle. Hence, most information on the norovirus life cycle is obtained from surrogate virus systems such as murine norovirus and feline calicivirus, or the use of gnotobiotic pigs that can be infected with human norovirus. Different human norovirus replicon systems have also been used. These models have provided some insight into the life cycle of human noroviruses, but all with specific limitations. Murine norovirus, for example, is a common model for molecular studies due to the availability of cell culture and reverse genetics systems, but differs from human norovirus in terms of host interaction and pathogenesis. The recent development of human intestinal enteroids (HIEs) able to propagate human norovirus in vivo have already provided new insights into the human norovirus life cycle, and will be an essential tool for future studies. Enterocytes in the small intestine are likely the primary sites of norovirus infection. Biopsies from immunocompromised patients with chronic norovirus infections have shown the presence of viral antigens in small-intestine enterocytes. Small-intestine enterocytes were also norovirus antigen-positive in a gnotobiotic pig model of human norovirus infection, and human noroviruses can be propagated in enterocytes present in in vitro culture systems of HIEs. Moreover, B-cells can be infected with human norovirus in vitro, although evidence of B cell infection in vivo is lacking. Patients with severe combined immunodeficiency (SCID) who lack B cells can be infected with norovirus, albeit with a slightly lower viral load. Thus, B cells are not essential for infection and replication but may be important for norovirus pathogenesis, although further studies are needed to determine their exact role. Much is still unknown of the different steps of the live cycle of norovirus infecting a cell. However, an essential feature is that attachment factors for most human norovirus are cell-associated host glycans such as HBGAs (histo blood group antigens) present in epithelial surfaces of the small intestine, which can facilitate entry into the cell. For example, non-secretors, lacking the expression of a1,2-linked fucosylated HBGAs in the small intestine are not infected with the predominant GII.4 genotype. Furthermore, GII.4 strains of human norovirus only infect HIEs derived from secretors but not those from non-secretor individuals. The soluble form of HBGAs and bile salts also increase virus infectivity. The subsequent receptor engagement remains unknown for norovirus. Several models have been proposed, such as binding to glycosphingolipids on the cell surface, followed by the invagination and release of norovirus-containing vesicles by dynamin II-mediated scission of the invagination, and viral genome release into the cytosol. The VPg-linked RNA genome of norovirus will subsequently act as an mRNA, where the VPg

486

Human Norovirus and Sapovirus (Caliciviridae)

protein is important for mediating translation and replication. Translation of the ORF1 polyprotein is followed by co-translational and post-translational processing by a viral protease, resulting in replication complex formation. Translation of the viral proteins VP1 and VP2 occurs mainly from the subgenomic RNA (sgRNA), likely to produce higher levels of the major capsid protein for virus assembly, as the sgRNA is present at higher levels and each icosahedral capsid contains 180 copies of VP1. The processes behind norovirus assembly, encapsidation, and exit are largely unknown. The ability of VP1 to self-assemble into virus-like particles (VLPs) that are morphologically and antigenically highly similar to native virions suggests that capsid assembly during virus replication may occur without the involvement of cellular proteins. Non-enveloped viruses, including norovirus, were usually thought to exit infected cells through cellular lysis. Recent findings, however, have shown that cell destruction is not always necessary for norovirus exit. Stools from infected people show norovirus residing in vesicles of exosomal or plasma membrane origin, which indicates that at least some norovirus are shed non-lytically inside exosomes.

Epidemiology Norovirus causes sporadic and epidemic gastroenteritis in both children and adults and has been described from a variety of geographic regions and settings. The estimated global prevalence of norovirus is approximately 20% of all cases of AGE. Norovirus is also common in asymptomatic infections, with a globally estimated prevalence of 7%. All ages are affected, but the incidence is the highest in children aged o5 years. Norovirus is estimated to cause approximately 200,000 deaths annually worldwide, with 70,000 or more among children in developing countries. In children, norovirus is typically second only to rotavirus in causing acute gastroenteritis, and in several countries with national rotavirus immunization, it has become the most common etiology in children with AGE. Norovirus is mainly transmitted through the fecal-oral route. Besides sporadic cases, norovirus is also the most common agent in outbreaks of gastroenteritis due to its high stability in the environment and ease of spread through contaminated water and food (Table 2). Waterborne norovirus outbreaks have been linked to potable water, wells, recreational water use such as swimming, and fountains. Norovirus is further estimated to be responsible for almost 50% of foodborne outbreaks globally. Fruits, vegetables, berries, shellfish, cereals, spices, and oysters are common in foodborne norovirus outbreaks, as they are consumed raw and subject to contamination from water sources. Contamination of food can also occur during its preparation. Norovirus outbreaks occur frequently in settings with close human contact, such as schools, nursing homes, retirement communities, cruises, hotels, hospitals, or day care centers. The spread during an outbreak is predominantly via direct contact, exposure to aerosols or fomites. Many factors contribute to norovirus transmission and its ability to cause outbreaks. Norovirus is an extremely stable particle in the environment, and is highly resistant to disinfectants such as alcohols and to freezing and high temperatures. Furthermore, a low dose, i.e., 10–100 viral particles, is required for symptomatic infection, and norovirus can be shed in the stool at up to 1–1010 genome equivalents per g feces, and up to 106 genome equivalents per ml of vomit. Prolonged asymptomatic shedding further increase the risk of secondary spread (Table 2). Seasonality As the term ‘winter vomiting disease’ implies, norovirus infections exhibit a clear winter seasonality in many countries. This winter seasonality, however, is predominant in temperate regions, with peaks in December–February in the Northern Hemisphere or June–August in the Southern Hemisphere. In many tropical regions, such as Sub-Saharan Africa or South America, less data is available. Studies from several tropical countries have reported no or less clear patterns of seasonality. In some tropical countries Table 2

Characteristics of norovirus facilitating transmission in different outbreak settings and in person-to-person transmission

Characteristics

Observations

Stability

High survival in the environment. Resilient to freezing, heating and disinfectants such as alcohols

Consequence

Difficult to eliminate in water and surfaces, leading to infections from e.g., oysters, recreational water, food irrigated with sewage, and fomites. Increased risk of infections in closed settings such as hospitals Long-term/Asymptomatic Patients can shed norovirus up to three weeks after resolutionIncreased risk of secondary spread, can be particularly shedding of symptoms. immunocompromised patients shed the virus problematic concerning food handlers. even longer Viral diversity Multiple genetic and antigenic and strains exist, using Re-infections can occur more easily with different strains. different attachment factors in humans Diagnostic and detection methods may not be sensitive for all strains Low infectious dose Only B10–100 virus particles are needed for symptomatic Increases risk of person-to-person transmission, aerosols, High viral load in feces infection secondary spread, food, water and surface contamination and vomitus Short-term immunity Symptomatic re-infection with homotypic strains can occur Symptomatic re-infections occur. Adults are not protected although infected as children. May hinder development of effective vaccines with long-term protection Note: Modified from Nordgren, J., 2009. Norovirus epidemiology: Prevalence, transmission, and determinants of disease susceptibility. Linköping: Linköping University Electronic Press. Patel, M.M., Hall, A.J., Vinjé, J., Parashar, U.D., 2009. Noroviruses: A comprehensive review. Journal of Clinical Virology 44, 1–8.

Human Norovirus and Sapovirus (Caliciviridae)

487

such as Nicaragua, norovirus peaks during the rainy season, and its prevalence has been associated with average rainfall in other countries, whereas some studies have found an association with rainfall only for norovirus outbreaks, and not for sporadic cases. The impact of rainfall on norovirus infections is not understood, but may be linked with environmental spread of the virus. Molecular epidemiology Genotype GII.4 is the predominant genotype worldwide for both sporadic infections and outbreaks of norovirus. A global review of norovirus in children (aged o18 years) found GII.4 accounting for 70% of the capsid genotypes and 60% of the polymerase genotypes, followed by the capsid genotype GII.3 (16%). A further systematic review of norovirus in low-income countries found that GII.4 was the predominant genotype in all settings (47%), followed by GII.3 and GII.6. The most prevalent GI strain was GI.3. In some geographic settings, other capsid genotypes such as GII.17 and GII.2, can become temporarily predominant. Nevertheless, 450% of norovirus infections yearly are caused by norovirus GII.4, with pandemics occurring every B2–7 years. The first known human norovirus pandemic occurred in 1995–1997, with subsequent pandemics in 2002, 2004, 2006, 2009, and 2012. The reason GII.4 is the pandemic-causing genotype is not fully understood but could be related to higher biological fitness, seen as higher levels of viral shedding in infected patients, more diverse attachment receptor specificity and fast mutation frequency leading to antigenic drift and putative change of HBGA attachment factors, both leading to escape of previous herd immunity. The predominant genotype GII.4 has been linked to more severe symptoms and is less common in asymptomatic infections. Some studies have indicated that genotype GII.4 is relatively less common in food and waterborne outbreaks compared to other non-GII.4 or non-GI strains, which might be associated with higher resistance of some genotypes to environmental factors. Population genetics and norovirus epidemiology HBGAs, specifically the secretor, Lewis and ABO families, are strongly associated with susceptibility to norovirus in a genotypedependent manner. Secretor status, mediating the presence of a1,2 fucose-linked blood group antigens in epithelial surfaces of the small intestine, has been strongly linked to susceptibility to a wide array of norovirus genotypes. As expression of these HBGA glycans are linked to ethnicity and differ widely between populations, this likely has an effect on norovirus epidemiology. Many common norovirus genotypes such as the predominant GII.4, the recently emerging GII.17 and the common GII.6 genotype have all shown strong secretor specificity. Other genotypes, whose clinical importance in many cases is unknown, infect both secretors and non-secretors. As no genotype infects non-secretors exclusively, it is likely that populations with a high prevalence of secretors are more infected with GII.4 as well as other secretor specific or non-specific genotypes, which might lead to differences in disease burden, particularly as genotype GII.4 is associated with more severe symptoms.

Clinical Features Norovirus cause vomiting and diarrhea, both of which may happen together or independently. The incubation period is short, usually 1–2 days, with symptoms lasting 2–3 days. Besides vomiting and diarrhea, abdominal pain, fever, headache, chills, myalgia, and malaise can also occur. Other unusual symptoms include encephalopathy and convulsions. Convulsions among norovirus-infected children are reported more often than for rotavirus infections. Norovirus disease is more severe among the elderly and people with accompanying medical conditions. Besides old age, very young children are also vulnerable. A study from Japan reported that the duration of illness among children aged o2 years was twice that in children aged 2–4 years. Additionally, hematopoietic stem cell transplant (HSCT) recipients and other immunocompromised individuals can have persistent norovirus infection with a duration of months or even years.

Pathogenesis Until very recently, the lack of a cell culture system and an efficient animal model restricted our knowledge of norovirus pathogenesis. Our limited understanding has been obtained from histological or biochemical studies among human volunteers and data from porcine, bovine, and murine models. Histological examination of the small intestine of norovirus-infected human volunteers showed villi broadening and blunting, microvilli shortening, increased cytoplasmic vacuolization, and intercellular oedema. Like rotavirus infection, norovirus infection results in delayed gastric emptying. Besides epithelial cells, human norovirus capsid protein has also been detected in macrophages, T cells and dendritic cells (DCs) of norovirus-infected immunocompromised patients. Recently, in vitro studies have reported human norovirus propagation in differentiated human intestinal organoid/enteroid cultures, and in a few established human B cell lines. Mild inflammation of the lamina propria has also been reported in volunteer studies and animals. A large amount of understanding on norovirus pathogenesis comes from a murine infection model. Murine strains that cause both acute (MNV-1 and CW3) and persistent (CR6 and MNV-3) infection have been used to better understand norovirus pathogenesis. During the initial phase of MNV-1 infection in mice, the macrophages, DCs, B cells and T cells in the gut-associated lymphoid tissues become infected. It is suggested that murine noroviruses that cause acute infection involve M cells to overcome the epithelial barrier and cause initial infection. On the other hand, persistent murine norovirus strain CR6, which can be continuously shed in feces has been reported to infect tuft cells. Besides immune cells in the gut, MNV-1 infection also leads to extra-intestinal spread in immunocompetent mice with MNV-1 virus reportedly detected in spleen, liver, lungs, and lymph nodes. Furthermore, STAT1 (signal transducer and activator of transcription 1) not only limits MNV-1 virus replication in the intestines but also limits its spread to the peripheral tissues; however, MNV-1 infection in STAT1-deficient mice resulted in weight loss, gastric bloating, and

488

Human Norovirus and Sapovirus (Caliciviridae)

diarrhea. Differences in disease severity between MNV-1 and MNV-3 norovirus strains have been reported in wild-type mice, wherein MNV-1 results in modest to severe intestinal pathology, whereas MNV-3 infection is attenuated. Another interesting aspect was that no correlation between viral titres and disease severity was observed. Wild-type mice infected with MNV-1 showed intestinal pathology, while high viral load with no intestinal pathology was observed in MNV-3-infected wild-type mice. Although there are some similarities between human and murine noroviruses, such as involvement of carbohydrate attachment factors, and potential for continued viral shedding, there are certain differences as well, most notably is the inability of murine norovirus to produce severe diarrhea in mice. Recent studies have been addressing the role of the host microbiota in norovirus infections. It has been reported that human norovirus interacts with both commensal and pathogenic bacteria via norovirus capsid and HGBA-like carbohydrates expressed on bacterial surfaces. Antibiotic-administered mice prevented persistent murine norovirus infection. However, it should be noted that infection of HIEs with human norovirus clinical isolates does not require bacteria, warranting further research on the importance of commensal bacteria in human norovirus disease. It has also been suggested that norovirus might serve as a trigger for intestinal bowel syndrome or intestinal bowel disease. Norovirus infection increases intraepithelial cytotoxic T cells in the duodenum for up to 6 days after the onset of symptoms. Additionally, apoptosis of norovirus-infected epithelial cells has also been suggested.

Immunity Protection from norovirus can be divided into genetic and immunological components. Briefly, the genetic factors for norovirus susceptibility are associated with expression of HBGAs in the small intestine. Secretor status is defined by the presence of a functional fucosyltransferase 2 (FUT2) enzyme which determines the presence of a1,2 fucose linked HBGAs (A, B, H-type 1, and Lewis b). Lack of a functional FUT2 enzyme (yielding the so-called secretor-negative phenotype) is a strong restriction factor for several norovirus genotypes, including the predominant genotype GII.4. The overall prevalence of the non-secretor phenotype is approximately 20%, but large differences exist between populations. Immunologically, humoral responses have an important role in the protection against norovirus infection. Higher serum levels of antibodies blocking norovirus VLP binding to HBGAs correlate with a lower risk of developing illness to some norovirus genotypes. The potential mechanism of action is blocking of the HBGA binding site of the virus capsid hindering the attachment to HBGAs on the cell surfaces of the small intestine. Higher levels of norovirus-specific memory B cells also correlate with lower risks of developing illness. This in combination with that immunodeficient persons lacking B-cells can develop chronic norovirus infections, suggest B cells to be important for preventing norovirus infection. Cross-reactive antibodies can be developed after infection, and immune response following infection of one genotype can provide protection against other genotypes. However, norovirus infections do not elicit long-lasting immunity, and a person can develop symptoms upon reinfection even with the same norovirus strain. The estimates of the degree and duration of homotypic and/or heterotypic protection after infection varies widely.

Diagnosis Since the initial detection of norovirus using electron microscopy in the early 1970s, various diagnostic approaches have been used to detect noroviruses in clinical and environmental samples. Currently, both antigen and molecular (for detecting viral genome) tests are widely used. (1) Antigen tests Enzyme immunoassays Enzyme immunoassays have been developed for norovirus GI and GII antigens in feces, but these assays have varying sensitivity (32%–92%) and specificity (65%–100%). Although most kits include cross-reactive monoclonal and polyclonal antibodies, variability in norovirus load in clinical specimens, the circulation of antigenically distinct genotypes and variability within the genotype due to antigenic drift affect their performance. However, their biggest advantage is the high throughput and feasibility for use in laboratories with minimum infrastructure. Besides conventional enzyme immunoassays, Immunochromatographic (ICG) lateral flow assays can be used for rapid detection as they do not require specialized laboratory equipment or highly trained personnel. However, their low sensitivities, warrant further screening of the negative samples. (2) Molecular tests Reverse transcription-PCR (RT-PCR)-based assays are today considered the gold standard for detecting norovirus and have been continuously updated over time to ensure high specificity and sensitivity. Conventional RT-PCR One of the first RT-PCR assays used primers to the first detected norovirus strain, the Norwalk virus. Subsequent generations of RT-PCR assays used primers from a more conserved region (the viral polymerase gene). Although having higher performance than that of enzyme immunoassays; hybridization or sequencing was required to ensure high specificity and sensitivity. Another

Human Norovirus and Sapovirus (Caliciviridae)

489

shortcoming of the conventional RT-PCR over real-time RT-PCR is the processing required for visualizing the amplicon after genome amplification. Real-time RT-PCR and multiplex molecular assays Real-time RT-PCR assay today is the most common method for norovirus detection. It is usually applied in a multiplex fashion to distinguish GI and GII genogroup noroviruses. Most real-time PCR assays use primers and probes targeting the most conserved part of the norovirus genome, the ORF1-ORF2 junction. Recently, the use of large multiplex molecular assays (Biofire FilmArray Gastrointestinal Panel, Luminex xTag Gastrointestinal Pathogen Panel, and Taqman Array Card) for simultaneous detection of known gastrointestinal pathogens, including viruses, has increased. This reduces the turnaround time for accurate results and aids in identifying infections and/or co-infections that remained undiagnosed by routine test methods for single pathogens. Single-unit tests Besides multiplex assays, there also exist single-unit, easy-to-use norovirus automated molecular detection tests that require very little time, and staff training, and one such assay is the Xpert Norovirus assay (Cepheid, Sunnyvale, CA, USA).

Treatment In immunocompetent individuals, norovirus gastroenteritis is self-limiting, and the treatment of norovirus gastroenteritis is generally supportive. Administering oral rehydration solutions (ORS), or intravenous rehydration, is the primary treatment strategy for correcting the altered electrolyte and fluid imbalance. Antiemetics, such as ondansetron can also be given to reduce vomiting and facilitate oral rehydration therapies. Loperamide, an anti-motility drug, is often used by travellers; however, its use in children is not recommended in all countries. Therapeutics for persistent norovirus infection is of clinical importance and studies involving antiviral therapy to norovirus are limited. Nitazoxanide, a broad-spectra antiparasitic and antiviral agent, shortened the duration of norovirus illness, and resolved diarrhea in a norovirus-infected patient with refractory acute myelogenous leukemia and HSCT. Similarly, enteric administration of human immunoglobulin resolved chronic diarrhea in a transplant patient. Besides oral immunoglobulins and nitazoxanide, the anti-viral drug ribavirin has been linked to viral clearance in common variable immunodeficiency (CVID) patients.

Prevention Norovirus spreads form person-to-person, contaminated food, water and other environmental contaminants. Thus, epidemic outbreaks as well as food and waterborne outbreaks are common, with norovirus being responsible for B50% of foodborne AGE outbreaks. Various fruits and ready-to-eat vegetables that come into contact with contaminated water are often the source of norovirus-related food borne outbreaks. To date, there is no consensus regarding what viral load should be considered as a cut-off for contamination. However, a recent study using HIE model reported that a ct (threshold cycle) cutoff of 30 in a fecal sample could be considered as infectious. One of the ways of preventing norovirus infection is to ensure that water used for irrigation, washing vegetables or culturing shell fish is not contaminated with norovirus. Another major issue to be considered is that the water used for irrgation be pre-treated and not contaminated with fecal wastewater. Besides food itself, the norovirus can also be transmitted via restaurant personnel infected with norovirus, who are often the index source of foodborne norovirus outbreaks in restaurants or cruises. Hence, appropriate disinfection measures are needed to prevent this contagious disease. Good hand hygiene remains an important measure for controlling norovirus spread. Cleaning with soap and running water is better for removing norovirus than alcohol-based hand disinfection. Until very recently, the lack of a cell culture system for propagating human norovirus hindered assessment of norovirus inactivation systems. A recent study using HIEs as a model to examine the effect of chlorine and alcohols (ethanol and isopropanol) on inactivating human norovirus infectivity. The authors reported that, irrespective of alcohol treatment duration/concentration, complete norovirus inactivation could not be achieved. On the other hand, chlorine treatment resulted in a complete norovirus inactivation. In norovirus outbreaks in hotels, restaurants or cruise ships, besides isolating infected individuals and maintaining personal hygiene, the disinfection of contaminated surfaces is equally important. These include tables, chairs, commodes, doorknobs, bathroom, floors, and carpets. Hypochlorite at a minimum concentration of 1000 ppm and quaternary ammonium compounds are preferred for decontaminating surfaces and objects although the latter are less effective. In health care settings, limiting norovirus spread at the earliest instance is of utmost importance. Besides following the above routine disinfection procedures, additional measures such as discarding all unused disposable items present in the patient’s room should be considered. Vaccination There is no licensed norovirus vaccine available till date; however, there are vaccine candidates at various stages of clinical development. Most of these are VLP-based vaccines administered through an oral, intranasal, or intramuscular route. Recently, a safety and immunogenicity study of a bivalent (GI.1/GII.4) VLP-based vaccine (administered intramuscularly) showed that it was well tolerated and elicited pan-immunoglobulin and HBGA-blocking antibodies. Furthermore, a single-site, randomized, doubleblind, placebo-controlled clinical trial of an oral non-replicating adenovirus-based vaccine expressing VP1 gene of the GI.1

490

Human Norovirus and Sapovirus (Caliciviridae)

norovirus strain with a double-stranded RNA (dsRNA) adjuvant showed that it was well tolerated and generated both systemic and mucosal immune responses.

Sapovirus Classification The family Caliciviridae consists of 11 genera including sapovirus (Table 1). Human sapoviruses are highly heterogeneous, and genetic classification is usually based on phylogenetic analyses of the sequence of the major capsid gene VP1, the most diverse region of the genome. Today, sapoviruses are divided into 15 genogroups, of which viruses within genogroups GI, GII, GIV and GV infect humans. Viruses in the other genogroups have been detected in swine (GIII and GV–GXI), sea lions (GV), mink (GXII), dogs (GXIII), bats (GXIV, GXVI–GXIX), and rats (GXV). These genogroups are further divided into genotypes, but a standardized genotyping method is still lacking, although this would be important in order to facilitate worldwide comparison of the data. The International Calicivirus Scientific Committee in 2010 proposed that new sapovirus genotypes should only be assigned based on the complete capsid sequence.

Virion Structure Human sapoviruses are about 30–38 nm in diameter and have an icosahedral structure, with characteristic cup-shaped depressions on the surface, and are morphologically distinguishable from other gastroenteritis viruses (e.g., rotavirus, astrovirus or adenovirus) by the ‘star of David’ appearance. The surface structure is formed from 180 copies of the VP1, arranged in a T ¼ 3 icosahedral symmetry. Each VP1 subunits contains two principal domains: the S and P domains, which are connected via a flexible hinge region. The major component of the complete virus, VP1, is a B60-kDa protein, while the VP2 protein has not been identified in the virion but is predicted to be a strong basic protein in the interior of norovirus particles. VP1 expression results in the spontaneous assembly of VLPs.

Genome The human sapovirus genome is a 7.4–7.5 kb ssRNA( þ ). The genome contains two ORFs (Fig. 2(B)). A sgRNA consisting of VP1 and VP2 genes is also produced during replication. The viral protein VPg is covalently attached to the 50 end of genomic RNA and sgRNA, and the 30 end is polyadenylated. ORF1 encodes a polyprotein that is cleaved post-translationally into non-structural proteins (NS1, NS2, NS3, NS4, NS5, and NS6–NS7) and a major capsid protein VP1, whereas ORF2 encodes the minor capsid protein VP2. A third ORF (ORF3) has been predicted in several human and bat sapovirus strains; however, its function is unknown.

Life Cycle There is yet no cell culture model for the human sapovirus, which has limited our knowledge of its replication cycle. Most of replication cycle studies of the Caliciviridae family arise from feline and murine norovirus studies and from porcine sapovirus in porcine cell lines. It is thought that sapovirus has a similar replication cycle to norovirus, with receptor binding, endocytosis, uncoating and release of genomic RNA into the cytoplasm. The endocytosis of porcine sapovirus has been shown to be clathrin- and cholesterol-mediated. Bile acids likely support porcine sapovirus replication via escape from the late endosome into the cytoplasm to initiate virus replication, as in the absence of bile acid, the virus remains in the late endosomes/lysosomes and is destined to be degraded. Recently, it was shown that porcine sapovirus-infection induced an early activation of the PI3K-AKT (phosphatidylinositol4,5-biphosphonate 3-kinase catalytic subunit alpha-AKT) and MEK-ERK (extracellular signal-regulated kinase) pathways that facilitated early to late endosome trafficking as well as the acidification and release of virus from the late endosome. The dsRNA genome is synthesized from the genomic ssRNA( þ ), and transcribed/replicated, thereby providing viral mRNAs/new ssRNA( þ ) genomes. Calicivirus genomes do not have a 50 cap structure; instead, a small virus-encoded protein (VPg) is covalently linked to the 50 end of the genome. Similar, the porcine sapovirus genome is covalently linked to VPg, and this linkage is required for the translation and infectivity of viral RNA. The VPg protein is bound directly to the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells, which was required for initiating viral translation and infectivity. Lytic or non-lytic mechanisms encapsidate the genomic RNA during virus assembly and release. Moreover, the cyclooxygenase/prostaglandin E2 (COX-PGE2) pathway is induced during porcine sapovirus replication, a mechanism proposed to be used by the virus for efficient viral growth and which could provide a potential future target for the control of sapovirus replication.

Epidemiology Sapovirus has been detected all over the world, both in outbreaks and sporadic cases and is a public health concern. The virus infects and causes disease in people of all ages but is most common in young children and the elderly. Similar to norovirus, sapovirus shows winter predominance in many countries. A recent 1-year study of out-hospitalized patients in Spain found that

Human Norovirus and Sapovirus (Caliciviridae)

491

sapovirus was the second major gastroenteritis pathogen, with a prevalence of 15%, and was most prevalent in infants, young children and the elderly. Similarly, in Nicaragua, having introduced rotavirus vaccination, sapovirus was the second most commonly detected enteropathogen among children aged o5 years and was observed in 15% of hospitalizations due to gastroenteritis.

Clinical Features Sapovirus infection manifests with typical viral gastroenteritis symptoms, characterized by vomiting, watery diarrhea and abdominal pain. Other symptoms, including fatigue, nausea, headache, myalgia and chills can also be present. The clinical picture is very similar to norovirus, but the symptoms tend to be milder than those of norovirus and rotavirus infections. Laboratory diagnosis is essential for virus detection. Similar to norovirus infection, there is usually no or low fever. The incubation period is 1–4 days, but asymptomatic cases have been reported. Although the person does not show symptoms, they are still shedding high amounts of infectious virus and are capable of spreading the virus through the fecal-oral route. In immunocompetent individuals, diarrhea is usually resolved within one week.

Pathogenesis Knowledge of the pathogenesis and cell tropism of human sapovirus is limited due to a lack of a small-animal model. Pathological lesions have only been studied in pigs infected with porcine sapovirus, and visualization by histopathological staining showed that the virus infects cells of the small intestine. Tissue sections show histological changes with villus atrophy in the duodenum and/or jejunum of inoculated pigs at day 3–7 post infection. These results were confirmed by scanning EM which also revealed villi shortening, blunting, fusion, or absence of villi.

Cell Culture None of the human sapoviruses had previously been propagated in cell culture. However, porcine enteric calicivirus (PEC) grows in primary porcine kidney cells and in a porcine kidney cell line (LLC-PK), in the presence of porcine intestinal contents or bile acids to permit virus replication. It was thought that the bile acid or intestinal contents support growth in vitro and in vivo by downregulating innate immunity, through cyclic AMP/PKA signaling, which downregulates STAT1 and antiviral interferon (IFN) signaling. However, sapovirus growth in cell lines deficient in their ability to induce or respond to IFNs showed a 100–150-fold increase in infectious virus production, indicating that the primary role of bile acids is not inactivation of the innate immune response. Although the addition of bile acids is an essential co-factor for porcine sapovirus replication in cell culture, the mechanism has yet to be fully elucidated. Recently, the stem-cell derived HIE culture system was used for successful replication of human norovirus. After this historical success, the non-transformed cell culture system has been used for other human enteric viruses such as enterovirus, rotavirus, adenovirus type 41 and astrovirus. This human intestinal non-transformed cell culture method is being currently explored for cultivation of human sapovirus.

Immunity Seroprevalence studies of human sapoviruses have demonstrated gradually increasing seroprevalence during the early years, reaching high levels in school-aged children that remain high in adults. Reinfection with the same genogroup is common, but is rare with the same genotype, suggesting that infection generates a genotype-specific immunity from each infection. The fact that sapovirus gastroenteritis occurs more frequently in young children than in older children and adults suggests that natural infection likely provides long term protection against sapovirus and provides hope for success of a possible future sapovirus vaccine. In contrast to norovirus infection, sapovirus infection has not been associated with host HBGA phenotypes. Neither the fucosyltransferase 2 (Fut2)/ non-secretor mutation (428A) nor any of the ABO blood groups or Lewis phenotypes protected children against sapovirus-infection.

Diagnosis Visually, sapoviruses are distinguishable from other gastroenteric viruses, except norovirus, by their ‘Star of David’ morphology in EM, but EM has low sensitivity. Enzyme-linked immunosorbent assay (ELISA) can be used for antigen detection of human sapovirus, but the method is less sensitive than the current commonly and routinely used RT-PCR methods. Due to the high genetic diversity of sapoviruses, primers targeting well-conserved regions, may also amplify other gastroenteritis viruses. Multiple primers are often used for detecting sapoviruses of different genogroups. Targeting the RdRp-VP1 junction region, the overlapping part of the RdRp and VP1 genes, has the highest detection rate and is often the first choice for screening of clinical samples. For genotyping, VP1-targeting primers are preferred, as they allow reliable genotyping of the sequenced PCR products, since the RdRp-VP1 junction region is too short and conserved for sequence analysis.

492

Human Norovirus and Sapovirus (Caliciviridae)

Prevention Sapoviruses can be transmitted from person to person via contacts with sapovirus-positive feces, vomitus, or sapoviruscontaminated materials/surfaces, or via contaminated food and drinking water. Sapovirus, like norovirus, is probably transmitted efficiently by aerosol from vomiting. To date, no vaccines or antiviral drugs are available for preventing or treating human sapovirus infections. Infectivity and stability testing of the porcine sapovirus has shown that the virus is stable at pH 3.0–8.0 at room temperature (RT) for 1 h, sensitive to ethanol treatment (60% and 70%) at RT for 30 s, inactivated by 200 mg/L (or ppm) sodium hypochlorite at RT for 30 min and inactivated by heating at 561C for 2 h. The stability of human sapovirus needs to be further investigated.

Further Reading Ahmed, S.M., Lopman, B.A., Levy, K., 2013. A systematic review and meta-analysis of the global seasonality of norovirus. PLoS One 8, e75922. Atmar, R.L., Ramani, S., Estes, M.K., 2018. Human noroviruses: Recent advances in a 50-year history. Current Opinion in Infectious Diseases 31, 422–432. Bartnicki, E., Cunha, J.B., Kolawole, A.O., et al., 2017. Recent advances in understanding noroviruses. F1000Research 6, 79. Chhabra, P., de Graaf, M., Parra, G.I., et al., 2019. Updated classification of norovirus genogroups and genotypes. Journal of General Virology 100, 1393–1406. Costantini, V., Morantz, E.K., Browne, H., et al., 2018. Human norovirus replication in human intestinal enteroids as model to evaluate virus inactivation. Emerging Infectious Diseases 24, 1453–1464. Desselberger, U., 2019. Caliciviridae other than noroviruses. Viruses 11. Estes, M.K., Ettayebi, K., Tenge, V.R., et al., 2019. Human norovirus cultivation in nontransformed stem cell-derived human intestinal enteroid cultures: Success and challenges. Viruses 11. Ettayebi, K., Crawford, S.E., Murakami, K., et al., 2016. Replication of human noroviruses in stem cell-derived human enteroids. Science 353, 1387–1393. Nordgren, J., Svensson, L., 2019. Genetic susceptibility to human norovirus infection: An update. Viruses 11. Oka, T., Wang, Q., Katayama, K., et al., 2015. Comprehensive review of human sapoviruses. Clinical Microbiology Reviews 28, 32–53. Robilotti, E., Deresinski, S., Pinsky, B.A., 2015. Norovirus. Clinical Microbiology Reviews 28, 134–164.

Relevant Website https://www.cdc.gov/norovirus/index.html Norovirus. Home. CDC.

Human Papillomaviruses (Papillomaviridae) Alison A McBride, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Samuel S Porter, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States and University of Maryland, College Park, MD, United States Published by Elsevier Ltd. This is an update of P.F. Lambert, A. Collins, Papillomaviruses: Molecular Biology of Human Viruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00458-1. and G. Orth, Papillomaviruses: General Features of Human Viruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00457-X.

Taxonomy, Classification, and Evolutionary Relationships Human papillomaviruses (HPVs) belong to the family Papillomaviridae that was established by the International Committee on the Taxonomy of Viruses in 2005 (previously, they had been classified with Polyomaviridae under the now defunct Papovaviridae). Papillomaviridae has two sub-families, the Firstpapillomavirinae and Secondpapillomavirinae (the latter contains only one genus). Firstpapillomavirinae are further classified into genera, based on nucleotide sequence homology of the L1 gene. Viruses with 61% or greater L1 nucleotide identity are considered to be within the same genus. Firstpapillomavirinae has 52 genera that are named by a Greek letter prefix. Five genera from the Firstpapillomavirinae contain the human papillomaviruses: Alpha-, Beta-, Gamma-, Mu-, and Nupapillomavirus. Within each genus, viral genomes are further separated into species that share between 71% and 89% nucleotide identity within the L1 gene. For example, within the Alphapapillomavirus genus, the species are named a-1, a-2, a-3, etc. At the next level, viral genomes are divided into types and the L1 sequence of each type must differ 410% from any other type. Within each type, there are usually distinct variant lineages. For example, the oncogenic virus HPV16 can be further separated into genetic lineages A1–4, B1–4, C1–4, and D1–4. In the HPV community, a virus receives an official designation as a reference genome only if the complete viral genome has been sequenced and a viral genomic DNA clone has been sent to the HPV Reference Center in Sweden for validation. To date, reference genomes have been officially designated up to HPV226. However, an even greater number of additional novel HPV types have been identified and sequenced but have not been submitted to the HPV Reference Center. The sequences of these Nonreference genomes can be found in the Papillomavirus Episteme database (see “Relevant Websites” section). Fig. 1 shows a phylogenetic tree of the named reference HPV types found in the Alpha-, Beta-, Gamma-, Mu-, and Nu- genera. Papillomaviruses are an ancient group of viruses that have co-evolved with their hosts for millions of years. When Homo sapiens emerged they were likely already infected with multiple ancestral HPV types related to those in the genera we recognize today. The HPVs have continued to slowly evolve, adapting to different anatomical and biological niches. Current thinking is that niche adaptation to susceptible host cells inadvertently led to the emergence of HPV-associated oncogenesis.

Virion Structure HPVs have non-enveloped icosahedral capsids with T ¼ 7d symmetry and are approximately 55 nm in diameter. The capsid is composed of two proteins, L1, the major structural protein, and L2, the minor protein. Each capsid consists of 360 L1 proteins arranged into 72 pentamers (or capsomeres) and a variable number of L2 monomers (observed to range from 12 to 72 molecules). HPV particles are very stable, and are resistant to degradation by various chemical and enzymatic processes. The L1 proteins can self-assemble into Virus-Like Particles (VLPs), and these VLPs are the main components of the current HPV prophylactic vaccines (See Fig. 2). The HPV capsid encloses a circular, double-stranded DNA genome of approximately 7–8 kbp in length. A relatively unusual feature of papillomaviruses is that the capsids package the viral DNA in the form of chromatin; host core histones organize the genome into a chromatinized minichromosome.

Genome The circular dsDNA viral genome is organized into the following three parts: the upstream regulatory region (URR) contains elements that regulate transcription and replication; the early region contains genes expressed in the early and maintenance phases of infection; and the late region encodes the proteins expressed at late stages of the viral life cycle. Fig. 3 shows the genomic arrangement of an oncogenic Alpha-HPV genome. All viral mRNA is expressed from one strand of the viral genome. In the oncogenic Alpha-HPVs, the early viral genes are transcribed from promoters located in the URR (just upstream from the early region) and terminate at the early polyadenylation site. An intermediate class of transcripts (that encode E1, E2, and E1̂ E4) use the late promoter (located in the E7 open reading frame (ORF) in the oncogenic Alpha-HPVs) and terminate at the same early polyadenylation site. Finally, late transcripts initiate from the late promoter and terminate at the late polyadenylation site at the end of the coding region. Other HPVs have additional

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00018-7

493

HPV72 HPV61 HPV102 HPV83 HPV89 HPV87 6 HPV8 14 HPV1 84 HPV 8 6 HPV 9 3 HPV 70 HPV V85 HP 9 7 V HP 18 V HP V 4 5 HP V59 HP V82 HP V51 HP V69 6 HP V2 4 HP V5 8 HP V5 3 H P P V 3 67 H PV H

HPV62 HPV81 HPV71 HPV90 HPV106 HPV57 HPV2 HPV2 7 HP V 2 HPV 9 77 HPV HPV 160 HP 78 V HP 28 HP V3 V HP 125 HP V10 HP V94 HP V11 HP V11 7 HP V1 8 H V1 2 4 H PV 52 HP PV2 1 9 H V 5 PV 20 14

Human Papillomaviruses (Papillomaviridae)

H P H V1 HP PV 75 H P V1 60 H P V 1 80 HP V 1 2 1 4 HP V13 2 HP V13 3 HP V16 0 7 HP V197 V HP 178 V2 02 HP V HP 140 V HPV 154 HPV 141 1 HPV 36 1 HP V 71 169 HPV1 26 HPV2 16 HPV12 9 HPV215 HPV116 HPV137 HPV213 HPV153

494

3 12 5 PV 22 H PV 109 H V 38 HP PV1 39 H PV1 55 H V1 9 HP V14 8 HP V21 1 HP V20 HP V144 HP 212 V HP 163 V HP 1 7 3 V H P 205 HPV 158 HPV 5 9 HPV 4 HPV 5 HPV6 46 HPV1 5 HPV13 HPV179 HPV161 HPV162 HPV166 HPV128

H HP PV5 HP V3 2 V HP 1 5 HP V3 6 HP V73 1 HP V34 HP V91 HP V43 V HP 40 HP V7 V HP 74 V HP 44 V HPV 13 11 HPV HPV 6 42 HPV 32 HPV6 6 HPV56 HPV53 HPV30 HPV63 HPV1 HPV204 HPV41 HPV157 HPV148 HPV199 HPV127 0 HPV21 2 HPV13 65 1 V P H 200 HPV 8 4 HPV 1 13 HPV 50 HPV 64 V1 HP 112 V HP 119 V H P 168 V HP V147 HP V211 7 HP V18 4 HP V22 8 8 HP PV 23 H 2 2 V 7 HP V1 56 HP PV1 84 H PV1 H

1 V2 93 HP PV 24 H PV 8 H PV9 H V5 7 HP V4 HP V36 3 HP V14 HP V105 HP V8 HP V99 HP 12 V HP 206 V HP 92 HPV 96 HPV 50 1 HPV 5 11 HPV 9 V HP 4 HPV75 76 HPV HPV104 HPV107 HPV209 HPV38 HPV23 HPV120 HPV100 HPV22 HPV151 HPV14 5 HPV17 4 HPV9 HPV 1 H P V 59 1 HPV 22 1 1 HPV 1 HP 113 V HP 17 V HP 37 HP V110 HP V217 HP V15 H P V80 HP V10 HP V2 1 HP V1 14 H V1 03 H PV 08 HP PV1 203 V1 34 70

beta

nu mu

ha alp

ga m ma

Fig. 1 Phylogenetic tree of human papillomaviruses. Phylogenetic tree of reference HPVs based on the nucleotide sequence of the L1 gene. The five genera of human viruses are shown and the names of oncogenic Alpha-HPVs are shown in red.

promoters in the early coding region of the viral genome. The use of different viral promoters and polyadenylation signals results in a diverse assortment of polycistronic transcripts, many of which are further alternatively spliced, thus allowing efficient use of the limited coding capacity of the small genome. All papillomaviruses contain four conserved open reading frames that encode two replication proteins, E1 and E2, and two capsid proteins, L1 and L2. All HPVs also express two additional proteins encoded from spliced transcripts: E8̂ E2 is a truncated E2 protein that antagonizes the function of full-length E2 and E1̂ E4 is an intermediate protein encoded primarily from an alternative open reading frame in the E2 gene. Almost all HPVs express E6 and E7 proteins. In addition, the oncogenic AlphaHPVs (and those that are evolutionarily closely related) express a spliced E6 transcript, E6*. In the Alpha-HPVs, short, hydrophobic, transmembrane proteins (called E5) are encoded at the end of the early region. Sometimes more than one evolutionarily distinct E5-like protein is encoded in this region and they are named E5a, E5b, E5g, or E5δ.

Host Range and Tissue Tropism Papillomaviruses are highly species specific and tissue tropic. HPVs will only infect and replicate in the cutaneous and mucosal epithelial surfaces of Homo sapiens. HPVs enter and establish a persistent infection of keratinocytes and rely on the differentiation

Human Papillomaviruses (Papillomaviridae)

495

Fig. 2 Structure of an HPV L1 capsid. Electron cryo-microscopy of HPV16 L1 capsid, created from the PDB structure 3J6R using the NGL viewer. The icosahedral capsid is composed of 360 L1 monomers arranged into 72 star-shaped pentamers.

Fig. 3 Viral genome. A genome map of the oncogenic Alpha-papillomavirus HPV18. The core early genes shown in green, and those encoding the late, capsid proteins in blue. The E4, E5, E6, and E7 genes are shown in purple. The URR (upstream regulatory region) contain transcriptional regulatory elements and the origin of replication (ori). The early and late promoters (PE and PL) and polyadenylation sites (pAE and PAL) are indicated.

496

Human Papillomaviruses (Papillomaviridae)

virion release virion assembly

keratinocyte differentiation

L1 L2

E1 E2 E4

genome amplification E6 E7

genome maintenance

Fig. 4 Differentiation-dependent Life Cycle of Papillomaviruses. The stages of the HPV lifecycle in a stratified epithelium. The virus enters the basal cells through a fissure in the epithelium and the genome replicates here at low levels. As the infected cells move up to the surface of the epithelium during the differentiation process, high levels of viral gene expression and genome amplification are induced. Viral particles assemble in the upper layers and are shed from the surface in squames.

program of these cells to support viral production. In addition, each HPV replicates in specific types or anatomical regions of the host epithelium. This tropism is not well understood and does not appear to be due to entry receptors; rather it is thought that each virus relies on specific cellular processes (e.g., transcription, tissue renewal, differentiation) to support the viral life cycle. Likely, the functions of the E5, E6, and E7 proteins of each HPV evolved to take advantage of these niches. Cancers of the cervix often arise at the transformation zone, which is the junction between squamous and columnar epithelial cells. Cells that reside here are especially susceptible to HPV-associated carcinogenesis.

Life Cycle The HPV life cycle is intricately and inexorably tied to the differentiation program of the host keratinocyte (Fig. 4). In a stratified epithelium, only the basal cells divide. Keratinocyte division can occur symmetrically to produce two basal cells, or asymmetrically to produce one basal cell and a second daughter cell that differentiates and moves slowly towards the surface of the epithelium as part of the terminal differentiation process. Infection of the basal cells ensures that the viral genome is maintained for long periods of time and at the same time will continually yield cells that eventually produce progeny viral particles. The different stages of viral replication and gene expression are temporarily and spatially regulated at the different stages of keratinocyte differentiation. Viral gene expression and DNA replication are very low in infected basal cells but are activated in suprabasal cells to amplify the viral genome to high levels. In even more differentiated cells, late viral gene expression produces the L1 and L2 proteins to package the viral genome and form infectious progeny virions. Compared to many viruses, the HPV infectious cycle is relatively long, with at least three weeks from initial infection to the first production of progeny virions.

Entry The HPV life cycle begins when an infectious virion accesses the basal keratinocytes exposed by microabrasions in the host epithelium. The major L1 capsid protein initially interacts with heparin sulfate proteoglycans on the exposed basement membrane. Here, L1 is cleaved by Kallikrein-8 (KLK8), causing a conformational change in the capsid. Subsequent interaction with cyclophilin

Human Papillomaviruses (Papillomaviridae)

Table 1

497

Functions of the HPV Proteins

Protein

Function

E1 E2 E8̂E2 E1̂E4 E5 E6 E7 L1 L2

Origin binding replicative helicase DNA binding protein; E1 helicase loader, transcriptional regulator, tethering and partitioning of viral genome DNA binding protein; repressor of viral transcription and replication Late protein; disrupts keratin filaments; aids virion release Regulates cell cycle, contributes to viral egress Immune evasion, manipulates cell pathways to produce a cellular environment conducive to persistent viral replication Immune evasion, manipulates cell pathways to produce a cellular environment conducive to persistent viral replication Major structural protein Minor structural protein, trafficking of viral DNA during entry, packaging viral DNA

B triggers a second conformational change in the virion, exposing a furin cleavage site in the minor capsid protein L2. Furin cleavage of L2 exposes the RIG-1 epitope and facilitates the transfer of the virion to an unknown secondary receptor on the basal keratinocyte. The viral particle is internalized by endocytosis and the low pH in the endosome, and the activity of cyclophilin B, causes L1 to disassemble from the particle, leaving a complex of the L2 protein and viral chromatin. L2 is inserted into the endosomal membrane resulting in virus containing vesicles that move through the trans golgi network. These vesicles enter the nucleus at the onset of mitosis when the nuclear membrane breaks down. They bind to condensed mitotic host chromosomes until cell division is complete.

Early Stages of Infection After cell division is complete, the L2-viral minichromosome complex is released from the membrane vesicles and localizes to ND10 bodies to begin the early part of the life cycle. Many viruses associate with ND10 bodies early in infection, even though these bodies are thought to be involved in anti-viral defense. However, viruses often disrupt and reorganize ND10 bodies by displacing repressive components and taking advantage of positive factors. Similarly, the HPV L2 protein displaces the host Sp100 protein from ND10 bodies to promote a local nuclear environment conducive to early viral replication and transcription. The viral early promoter drives transcription of early genes, including those of the replication proteins E1 and E2. E1 and E2 bind the replication origin, unwind the viral DNA, and work in concert with the cellular DNA replication machinery to amplify the viral genome to a low copy number (just a few copies per cell). The new viral genomes establish a persistent infection by localizing to beneficial regions of the host nucleus that will support low level gene expression, and evade host defenses.

Maintenance Phase of Infection During this phase of the life cycle, the late structural genes are not transcribed and no infectious particles are formed, thus it is dubbed the “non-productive” phase. To maintain a long term non-productive infection, the virus must keep a low immunogenic profile and faithfully pass the genome onto successive daughter cells as the basal keratinocytes proliferate. The E2 protein binds to sites in the viral genome and tethers the extrachromosomal viral genome to the host chromosomes. This ensures that the viral genome is retained in the nucleus and distributed to daughter cells in mitosis. In this way, the viral genome persists as a low copy extrachromosomal circular DNA.

Late Stages of Infection When cells maintaining HPV genomes detach from the basement membrane and begin their journey upward through the epithelium, the virus begins the late (or “productive”) phase of its life cycle. In these terminally differentiating cells, expression of the viral E5, E6, and E7 proteins delay the normal keratinocyte differentiation program and disrupt cell cycle regulation. High levels of the E1 and E2 proteins induce massive amplification of the viral genome in differentiated cells that are in a G2-like phase. HPVs rely on the cellular DNA replication machinery to replicate viral DNA but this is not present in G2 phase or differentiated cells. Therefore, HPVs induce a DNA damage response and repair factors traffic to the viral genome to replicate viral DNA in nuclear foci using what is presumed to be recombination-directed replication mechanisms. Following this, transcription and translation of the late structural proteins L1 and L2 occur; these proteins assemble around chromatinized progeny genomes to generate infectious virions. Further maturation of the virions occurs in the oxidizing environment of the uppermost, dying keratinocytes. These cells, containing arrays of mature virions, are sloughed off the surface of the epithelium by desquamation. Prior activities of the E4 and E5 proteins increase the fragility of these squames, so that they will more easily release viral particles.

498

Human Papillomaviruses (Papillomaviridae)

Functions of Individual Viral Genes (Table 1) The E1 Protein The E1 gene product is a sequence-specific origin binding replicative helicase that is essential for viral DNA replication. Homodimers of the E1 and E2 proteins initially bind cooperatively to their respective binding sites in the viral origin of replication. A conformational shift in the E1 protein displaces E2 and recruits additional E1 proteins to form a double hexamer, which is the functional replicative helicase that unwinds the DNA bidirectionally and allows cellular replication machinery to bind and process the template.

The E2 Protein The viral E2 protein is multifunctional; it is involved in replication, transcription, and maintenance of the viral genome. E2 consists of an N-terminal transactivation domain linked by a flexible “hinge” sequence to the C-terminal DNA binding/dimerization domain. E20 s main role in initiation of replication is to assist in loading the E1 helicase onto the origin of replication, but it further contributes by displacing nucleosomes and recruiting cellular replication proteins. E2 additionally functions in maintenance replication by partitioning the viral DNA to daughter cells by tethering the genome to host chromatin. E2 is also the primary regulator of HPV transcription, functioning predominantly by recruiting cellular activating or repressing factors to promoters and enhancers in the viral genome through its E2 binding sites (E2BSs).

The E8̂E2 Protein The E8̂ E2 protein arises from a spliced transcript that encodes a short peptide from the E8 ORF fused to the C terminal DNA binding and dimerization domain of E2. E8̂ E2 can competitively bind to E2 binding sites and recruits repressive cellular NCoR/ SMRT complexes which restricts HPV transcription and replication.

The E1̂E4 Protein The E4 protein is expressed as an E1̂ E4 fusion protein from a spliced transcript encoding the first few codons of E1 joined to the E4 ORF. The resulting protein is a small, mostly cytoplasmic protein that interacts with a variety of cellular partners. Despite its location in the early region of the viral genome, high levels of E1̂ E4 expression correlate with the start of productive viral DNA synthesis in the later stages of the life cycle, where it is among the most abundant protein of the infected cell. The exact role of E1̂ E4 in viral genome replication is not fully understood. E1̂ E4 induces G2/M cell cycle arrest by sequestering cyclins and cyclin dependent kinases, and interacts with many other cellular proteins. The major function of E1̂ E4 may be its contribution to the release of progeny HPV virions by associating with and destabilizing cornified cell envelopes and keratin filaments, thus aiding virion egress from infected, desquamated cells.

The E5 Protein The E5 proteins are small hydrophobic membrane proteins that are encoded only by the human Alpha-papillomaviruses (as well as many animal papillomaviruses). At the sequence level, there are several different types of E5 protein (named alpha, beta, gamma and delta) that are each evolutionarily distinct. For the most part, the oncogenic Alpha-HPVs encode alpha-E5 proteins and these are the best studied. E5, together with E6 and E7, promotes proliferation of infected cells. Alpha-E5 proteins increase cell growth by enhancing the signaling capacity of the epidermal growth factor receptor (EGFR). Additionally, E5 forms a complex with the 16K subunit of the V-ATPase and this may impair keratinocyte cell-cell communication. E5 also hinders cellular antigen presentation by blocking HLA-I from trafficking to the cell surface and by inhibiting the maturation of MHCII. The alpha-E5 proteins encoded by oncogenic high-risk HPVs inhibit apoptosis and maintain the proliferative capacity of differentiating keratinocytes. Moreover, HPV E5 (along with E6) has been proposed to contribute to viral egress by inducing koiliocytic vacuoles in the nucleus of infected cells, thus increasing cell fragility and aiding virion release from desquamated cells.

The E6 Protein The E6 proteins contain two zinc finger domains linked together by an alpha helix. Together, these domains give E6 proteins the ability to interact with different cellular proteins that contain acidic, helical LxxLL motifs. The Alpha-HPV E6 proteins bind to the cellular E3 ubiquitin ligase, E6 Associated Protein (E6AP). In the oncogenic Alpha-HPVs this complex ubiquitinates p53, leading to p53 degradation by the proteasome and allowing the cell cycle to proceed with G1/S progression. This action by E6 to degrade p53 is required for the long-term maintenance of the viral genome. On the other hand, the Beta- and Mu-HPV E6 proteins bind LxxLL motifs on mastermind-like (MAML) proteins. MAML proteins are transcriptional coactivators for Notch signaling and inhibit the ability of Notch to promote squamous epithelial differentiation. The oncogenic Alpha-HPV E6 proteins contain a

Human Papillomaviruses (Papillomaviridae)

499

C-terminal region that allows them to bind and degrade PDZ domain proteins involved in cell polarity. These E6 proteins also induce telomerase expression, allowing proliferating cells to escape senescence due to eroding telomeres. In contrast, many Beta-HPV E6 proteins increase tolerance to UV-mediated DNA damage and in this way could act as a cofactor in the development of squamous cell skin cancer. The multifunctional HPV E6 proteins also inhibit innate immune response pathways.

The E7 Protein Almost all HPVs encode an E7 protein, a small early protein that binds multiple cellular targets. The main functions of E7 are to ensure that the cellular environment is favorable for HPV replication. In the high-risk Alpha-HPVs, the E6 and E7 proteins manipulate the cell in such a way as to promote genetic instability and oncogenic progression of the infected cell are thus considered to be oncogenes. For example, high-risk E7 promotes cell cycle progression by binding and degrading the tumor suppressor retinoblastoma protein (pRb), thus overriding its ability to inhibit the progression of the cell cycle from G1 into S phase and promoting cellular proliferation. This stabilizes p53, which in turn is degraded by high-risk E6. E7 also causes global, epigenetic reprogramming by upregulating Lysine Demethylase 6 (KDM6) A and B. E7 also induces cellular DNA damage signaling to promote viral DNA amplification in differentiated cells. Similar to the E6 proteins, E7 proteins inhibit the host innate immune responses.

The L1 Protein L1 is the major structural protein of the HPV capsid. The L1 protein forms pentamers called capsomeres, and 72 of these selfassemble into an icosahedral particle with T ¼ 7 symmetry. The L1 protein is sufficient to form virus-like particles (VLPs). L1 initiates the infection process by binding to the extracellular matrix (ECM) at the base of a stratified epithelium, and then facilitates entry of the virion into the cell by interaction with a secondary receptor on the basal keratinocyte. While most of the L1 dissociates from the viral genome throughout the trafficking to the nucleus, some L1 remains associated with HPV DNA. In the late stage of the viral life cycle, L1 is expressed in the uppermost layer of the epithelium where, together with L2, it encapsidates viral DNA to produce progeny virions.

The L2 Protein L2 is the minor capsid protein and it has several important functions during viral entry and trafficking of the viral genome to the nucleus. After endocytosis, L2 becomes transmembranous and redirects the viral genome complex to the nucleus in a membrane vesicle via the retromer pathway. When the infected cell undergoes mitosis, the L2-genome containing membrane vesicle associates with host mitotic chromosomes. After the nuclear envelope reforms, the viral complex localizes to the ND10 nuclear bodies. Here, L2 displaces Sp100, a restriction factor of HPV, to facilitate an environment conducive to viral transcription. At late stages of infection L2 is expressed in the upper layers of the epithelium where it assists in packaging of the viral genome.

Natural History, Transmission and Epidemiology HPVs are ubiquitous and, in most cases, cause asymptomatic or transient, self-limiting infections. The viral particles are very stable and are released from the desquamating surface of infected epithelia. Transmission of cutaneous types are by direct host to host contact, or through contact with a contaminated surface. Anogenital and oral HPV types are usually transmitted by sexual contact. In both cases, the virus enters the epithelium and accesses the basal epithelial cells through small scratches or microabrasions. HPV infection is extremely common. Children and adolescents have the highest prevalence of warts of the hands and feet, and a high proportion of individuals become infected with anogenital HPVs when they become sexually active. In most cases, the infections are self-limiting and regress without treatment. However, individuals persistently infected with oncogenic HPV types are at risk of progression to cancer. Widespread vaccination with the current HPV vaccine can prevent infection with the most common oncogenic types.

Clinical features, Pathogenesis and Histopathology of HPV Infection There are hundreds of different HPV types and they each infect different regions of the cutaneous epithelium (skin), the anogenital mucosal epithelium, and the epithelium of the oral cavity. These infections give rise to common, plantar, flat plane or anogenital warts (papillomas). Lesions and pre-cancers are also found in the oral cavity and anogenital regions. Many HPV types (in particular those from the Beta and Gamma genera) persist asymptomatically in the cutaneous epithelium, resulting in lesions only in individuals with immunodeficiencies. The cell layers of the epithelium are greatly expanded in many common warts resulting in exophytic lesions with rough, cauliflower-like appearances. Plantar warts (verruca) are endophytic and infected cells grow deep into the sole of the foot. Plane warts present as flat papules on cutaneous skin surfaces. These warts often show characteristic histological features of acanthosis

500

Human Papillomaviruses (Papillomaviridae)

(thickening of the epithelium), parakeratosis (nuclei retained into the cornified layer), and hyperkeratosis (a thickened cornified layer). Lesions often contain koiliocytes, which are cells with shrunken hyperchromatic nuclei that are displaced by a large perinuclear vacuole. The presence of koiliocytes is usually indicative of HPV infection. Anogenital warts (condyloma acuminata) present as discrete, small papules on the genital regions. Laryngeal papillomatosis (recurrent respiratory papillomatosis) is a rare condition associated with low-risk HPV infection (possibly due to an unidentified immunodeficiency). Here, benign papillomas form in the respiratory tract and tend to recur despite multiple surgeries.

Oncogenic HPVs A subset of Alpha-papillomaviruses are associated with cancer. The E6 and E7 proteins (oncogenes) of these viruses promote unscheduled cellular proliferation and inactivate cell cycle checkpoints. This leads to genetic instability as infected cells accumulate mutations that promote carcinogenesis. Infection with oncogenic HPVs is extremely common and most infections resolve without treatment. Only long term, persistent infection with oncogenic HPVs is associated with oncogenic progression. The International Agency for Research on Cancer (IARC) has reported that 13 Alpha-HPVs are oncogenic and that close, evolutionarily related types are “probably” oncogenic. Fig. 1 shows a phylogenetic tree of human papillomaviruses with the oncogenic Alpha-HPVs indicated in red. In many cancers associated with HPV infection, the viral genome is integrated into the host chromosomes. Integration is not part of the normal viral life cycle and is a dead end for virion production. The only viral proteins consistently expressed in cancers are the E6 and E7 oncogenes. It is thought that the viral genome accidently integrates into regions of the host genome that promote dysregulated expression of the E6 and E7 oncoproteins. The resulting increase in cellular proliferation, absence of cell cycle checkpoints, and promotion of genomic instability drives infected cells toward oncogenesis.

HPV-associated Cancers About 5% of human cancers can be attributed to oncogenic Alpha-HPV infection. Persistent HPV infection is the causative agent of almost all cancers of the uterine cervix and the majority of vaginal, vulvar, penile, anal and rectal cancers. A large proportion of oropharyngeal cancers are also due to HPV infection. Beta-HPVs cause asymptomatic infection of the skin and are often found in squamous cell carcinomas. However, they are not essential for the maintenance of the tumors and may play a role in the development of cancer by impairing the repair of UV damage. Conversely, these infections may also enhance CD8 þ T cells antitumor activity and suppress cancer.

Immune Response HPVs must avoid host immune defenses and the virus replication cycle itself is a strategy of immune evasion. There is no viremia or cell lysis associated with HPV infection and high levels of viral genome replication, transcription and protein synthesis are confined to terminally differentiated cells. Viral particles are shed from the surface of the epithelium and so do not induce an immune response. The virus also suppresses interferon and other cytokine responses and downregulates innate immune sensors. Most infections are self-limiting and regression is due to a cell-mediated immune response. Infection does not always lead to seroconversion and levels of natural antibodies to HPV antigens are low. This is in contrast to the immune response to HPV vaccines, which induce extremely high levels of anti-L1 neutralizing antibodies. The levels of these antibodies contained in serum exuded at epithelial microabrasions are sufficient to prevent infection. However, HPV infection is not completely invisible to host immune defenses as evidenced by the great increase in pathogenic HPV lesions and cancers in cases of immunosuppression and immunodeficiency. Individuals with uncontrolled HIV infection, or immunosuppressed organ transplant recipients, often have prolific HPV disease. Some genetic primary immunodeficiencies also give rise to uncontrolled HPV infections. For example, individuals with WHIM syndrome (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) are very susceptible to HPV warts, but treatment of the underlying immune defect results in resolution of HPV lesions. In the rare genetic disease Epidermodysplasia verruciformis, Beta-HPVs that usually cause asymptomatic infections in healthy individuals give rise to plaques of HPV infection on the cutaneous epithelium with a high propensity to develop into squamous cell carcinomas on UV-exposed skin.

Diagnosis Dermatologists and gynecologists diagnose common, plantar, plane and genital warts from clinical and histological appearance; evaluation of HPV type is usually unnecessary. However, because cervical HPV infections are common (and can lead to precancerous and cancerous lesions), a routine gynecological exam incorporates a Pap smear (invented by George Papanicolaou). In this test, cells are brushed from the surface of the cervix and analyzed by cytopathologists for signs of HPV infection or other abnormalities. For follow up, cervical (or anal) pre-cancerous lesions are treated in situ with acetic acid so that they can be visualized by colposcopy or anoscopy. Small biopsies of these lesions are further analyzed histologically to determine the stage of cervical or anal squamous intraepithelial lesions (ASIL or CSIL). HPV types that infect the anogenital region can be classified as

Human Papillomaviruses (Papillomaviridae)

501

“low oncogenic risk” or “high oncogenic risk” and tests are available to determine the HPV DNA type and thus the relative risk of disease progression.

Treatment HPV infections are very common, and in most healthy individuals are self-limiting and require no treatment. If bothersome, common warts of the hands and feet can be treated with salicylic acid (available as over-the-counter treatments). Topical application of creams containing imidazoquinoline-based IRMs (Immune Response Modifiers) can enhance the host immune response and induce regression of various types of warts. Persistent pre-cancerous lesions of the cervix are removed by ablation techniques such as laser vaporization, cryotherapy or LEEP (loop electrosurgical excisional procedure), while HPV associated cancers are treated by surgery, chemotherapy, and radiotherapy.

Prevention Vaccination with prophylactic HPV vaccines is the most effective means of prevention. To date, three highly effective vaccines have been developed against the most common HPV types that cause anogenital infection and associated cancers. Each of these vaccines contains the HPV L1 protein, self-assembled into VLPs (Virus-Like Particles) and an adjuvant. Cervarix contains HPV16 and HPV18 VLPs, Gardasil-4 contains HPV6, 11, 16, and 18 VLPs, and Gardasil-9 contains HPV6, 11, 16, 18, 31, 33, 45, 52, and 58. The VLPs are extremely immunogenic and when delivered intramuscularly induce high titers of neutralizing antibodies. The efficacy of these vaccines in preventing warts and cancer precursor lesions in the anogenital region is excellent and there is hope that in countries with high rates of vaccination uptake, HPV-associated cancers could eventually be eliminated.

Acknowledgments The research of the authors is supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Further Reading Cubie, H.A., 2013. Diseases associated with human papillomavirus infection. Virology 445, 21–34. de Oliveira, C.M., Fregnani, J., Villa, L.L., 2019. HPV vaccine: Updates and highlights. Acta Cytologica 63, 159–168. Lambert, P.F., Mcbride, A., Bernard, H.U., 2013. Special Issue: The Papillomavirus Episteme. Academic Press. Mcbride, A.A., Munger, K., 2018. Expert views on HPV infection. Viruses 10. Schiffman, M., Doorbar, J., Wentzensen, N., et al., 2016. Carcinogenic human papillomavirus infection. Nature Reviews Disease Primers 2, 16086. Strickley, J.D., Messerschmidt, J.L., Awad, M.E., et al., 2019. Immunity to commensal papillomaviruses protects against skin cancer. Nature 575, 519–522. van Doorslaer, K., Chen, Z., Bernard, H.U., et al., 2018. ICTV virus taxonomy profile: Papillomaviridae. Journal of General Virology 99, 989–990. Vande Pol, S., 2015. Papillomavirus E6 oncoproteins take common structural approaches to solve different biological problems. PLoS Pathogens 11, e1005138.

Relevant Websites https://hpvcentre.net/ HPV INFORMATION CENTER. https://www.cdc.gov/hpv/ HPV, the Vaccine for HPV, and Cancer Caused by HPV. CDC. https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-agents/hpv-and-cancer Human Reference clones. hpvcenter. https://www.hpvcenter.se/human_reference_clones/ Human Reference clones. hpvcenter. https://pave.niaid.nih.gov/ PaVE: Papilloma virus genome databse. NIH.

Human Parainfluenza Viruses (Paramyxoviridae) Elisabeth Adderson, St. Jude Children’s Research Hospital, Memphis, TN, United States and University of Tennessee Health Sciences Center, Memphis, TN, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of E. Adderson, A. Portner, Parainfluenza Viruses of Humans, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00459-3.

Classification The family Paramyxoviridae, in the order Mononegavirales, consists of large negative-sense, single-stranded, enveloped RNA viruses belonging to 4 subfamilies and 14 genera. The 4 human parainfluenza viruses (HPIVs) belong to 2 separate genera within this family. HPIV-1 and HPIV-3, as well as bovine, murine, and porcine parainfluenza viruses, are members of the Respirovirus genus. HPIV-2 and HPIV-4 are members of the Orthorubulavirus genus, which also includes mumps virus and several pathogens of non-human mammalian species. HPIV-4 is further divided into two subtypes, HPIV-4a and HPIV-4b, on the basis of serologic reactivity.

Virion Structure HPIVs are roughly spherical viral particles 150–250 nm in diameter consisting of a host-cell plasma membrane–derived lipid bilayer surrounding an 18-nm diameter internal helical nucleocapsid containing the negative-sense, non-segmented, single-stranded RNA genome. Projecting from the surface of the virion are 2 transmembrane glycoproteins, hemagglutinin–neuraminidase (HN) and fusion (F) protein. These glycoproteins are anchored to the plasma membrane of the infected cell or the virion envelope by a hydrophobic transmembrane region. A schematic representation and an electron micrograph of a naturally occurring parainfluenza virus (PIV) are shown in Fig. 1.

Genome The genomes of HPIVs range from 15,400–17,000 nucleotides and encode 6 common structural proteins, linked in tandem in the order 30 -nucleocapsid protein (NP)–phosphoprotein (P)–matrix protein (M)– F–HN–large protein (L)  50 . In general, the genome organization of HPIV is remarkably similar, but differences exist in gene sequences, intergenic regions, and nonstructural proteins expressed from the P gene. Sequence and immunological analyzes suggest that human HPIV-1 and murine Sendai virus are closely related, as are human- and bovine-origin PIV-3 Fig. 2. The RNA nucleocapsid, a complex consisting of approximately 2000 molecules of NP bound to the RNA genome, rather than the RNA genome itself, serves as a template for RNA synthesis. The viral RNA-dependent RNA polymerase complex consists of a heterocomplex of about 200 P and 20 L molecules. The P gene is unique in its capacity to express not only P protein but also alternative proteins. This varying expression is determined by either use of internal initiation codons in the same or different reading frames or by insertion of non-templated G residues during the editing of the P gene specific mRNA. These alternative proteins have been designated V, C, and D, depending on the PIV. The approximate molecular weights in Daltons (Da) of the HPIV proteins, as exemplified by HPIV-3, are as follows: NP (58,000 Da); P (68,000 Da); M (40,000 Da); F (63,000 Da); HN (72,000 Da); and L (256,000 Da) Table 1.

Life Cycle HPIV replication starts in ciliated respiratory epithelial cells, initially in the nose and throat, and may subsequently progress to affect ciliated and alveolar cells of the lower respiratory tract. Infection is initiated by the attachment of viral particles to the host cell, the energy-dependent fusion of the viral envelope and target cell membrane, and the formation of pores through which the viral genome is introduced into the cell. The HN and F glycoproteins cooperatively mediate the direct binding of the virion to sialic acid–containing glycoprotein or glycolipid receptors on upper and lower respiratory tract epithelial cells. The same process is responsible for the hemagglutination of avian and mammalian erythrocytes. HN also causes the enzymatic (neuraminidase) cleavage of sialic acid residues from the carbohydrate moiety of glycoproteins and glycolipids, which functionally serves to prevent the self-aggregation of virus during release and likely aids in the spread of virus from infected cells. HN possesses an N-terminal stalk region of approximately 130 amino acids anchoring a large glycosylated hydrophilic globular head region to the viral envelope. A small uncharged hydrophobic peptide near the N-terminus spans the viral envelope, and a small hydrophilic domain is internal to the membrane. The globular head contains the active site for virus attachment, neuraminidase activity, and antigenic determinants that induce neutralizing antibodies for HPIVs. HN exits on the surface of the virion as disulfide-linked dimers of homodimers Fig. 3.

502

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21547-1

Human Parainfluenza Viruses (Paramyxoviridae)

503

Fig. 1 HPIV genome. The genome consists of 6 common structural proteins, linked in tandem in the order 30 -nucleocapsid protein (NP) – phosphoprotein (P) – matrix protein (M) – fusion protein (F) – hemagglutinin-neuraminidase (HN) – and large protein (L)  50 .

Fig. 2 Parainfluenza virus structure.

Table 1

HPIV-3 proteins

Name

Function

Size (Da)

Nucleocapsid protein (NP) Phosphoprotein (P) Matrix protein (M) Fusion protein (F) Hemagglutinin–neuraminidase (HN) Large protein (L)

Structure, RNA binding RNA-directed RNA polymerase Structural, virion assembly Fusion of virus membrane with host cell membrane Viral attachment and entry into host cell, sialidase RNA-directed RNA polymerase

58,000 68,000 40,000 63,000 72,000 256,000

Besides being needed for attachment, HN is essential to or enhances the fusion activity of the F protein. F proteins are class I fusogenic viral proteins that are synthesized as inactive precursors, then post-translationally cleaved by a host cell trypsin-like protease to form prefusion homotrimers. Following receptor binding, a conformational change in HN activates the F protein, triggering the insertion of hydrophobic membrane attack domains into the target cell membrane. The cell and viral membranes are then approximated by refolding of F into a hairpin structure. Membrane fusion precedes the formation of a pore through which the viral nucleocapsid is deposited into the host cell cytoplasm. After the infecting nucleocapsid is introduced into the host cell, the polymerase complex transcribes viral mRNAs and initiates genome replication by generating an intermediary positive-sense RNA antigenome template. HN and F proteins are synthesized, post-translationally modified, and transported to the host cell membrane, which becomes the envelope for progeny virions. Once protein synthesis is underway, N protein encapsulates newly synthesized genomes that, in association with P, form new nucleocapsids that are also transported to the cell plasma membrane. The M protein lines the inner surface of the viral envelope. During infection, M associates with the inner leaflet of the plasma membrane, where it orchestrates the release (budding) of progeny virus into the respiratory tract lumen by interacting with specific sites on the cytoplasmic tail of the viral glycoproteins and the nucleocapsid and by transporting viral components to the budding site. HN facilitates the release of new virus particles by cleaving sialic acid residues on the host cell membrane. Embryonated hen’s eggs can support the growth of some strains of HPIV-1, HPIV-2, and HPIV-3. All 4 HPIV types grow well in primary monkey or human kidney cells, which are also used to isolate virus from clinical samples. LLC-MK2, a rhesus monkey kidney cell line, offers an efficient system for isolating PIVs and an experimental tissue culture system. HPIV-2 and HPIV-1 require trypsin in the medium to cleave the F glycoprotein for cell growth. Viral infection of tissue culture can be detected by hemadsorption with chicken and guinea pig erythrocytes and by the cytopathic effects produced by the viruses.

Epidemiology The HPIVs are important causes of upper and lower respiratory tract infections worldwide (Table). Between 25%–40% of HPIV detections are in children aged 2 years or younger; and half are in children aged 7 years or younger. By 5 years of age, almost all persons have serological evidence of HPIV-3 infection, and 75% have been exposed to HPIV-1. HPIV-2 infection, but not infection

504

Human Parainfluenza Viruses (Paramyxoviridae)

Fig. 3 Structure of an HPIV-3 hemagglutinin–neuraminidase dimer shown in colored ribbon representation, complexed with b-Sialic acid in red and green ball-and-stick representation. Reproduced from Lawrence, M.C., Borg, N.A., Streltsov, V.A., et al., 2004 Structure of the hemagglutininneuraminidase from human parainfluenza virus type III. Journal of Molecular Biology 335, 1343–1357, used with permission.

Table 2

Epidemiological and Clinical Features of HPIV Infections

Seasonality

HPIV-1

Croup Other URTIa Pneumonia in young infants, elderly adults

Autumn, biannual in odd-numbered years

HPIV-2

Croup Other URTIa Pneumonia in young infants, elderly adults

Autumn, biannual in even-numbered years

HPIV-3

Bronchiolitis, pneumonia in young infants, elderly adults Severe respiratory infection in immunocompromised patients Croup Other URTIa

Spring-summer, annual

HPIV-4

Other URTIa Pneumonia in young infants

Autumn, annual

a

Other URTI includes rhinitis, pharyngitis, sinusitis, otitis media.

due to other HPIVs, is more common in males. In the United States, HPIV-1 and HPIV2 infections generally peak in the autumn of alternate years (odd- and even-numbered, respectively), whereas HPIV-3 infections are more common in the spring and summer. The circulation of HPIV-4 infections has been less well studied, but detections appear to peak annually in the winter time. Overall, HPIV-3 is the most commonly detected HPIV, followed by HPIV-1, HPIV-2 and, lastly, HPIV-4. The simultaneous detection of more than one HPIV in an individual is unusual, but about one-third of HPIV are co-detected with other respiratory viruses, most commonly with rhino- and enteroviruses and influenza. Reinfections are possible, but generally cause milder disease than primary infection does. Outbreaks in healthcare and long-term care settings have been reported Table 2.

Clinical Features HPIVs are highly communicable and are transmitted person-to-person by respiratory droplets and by direct contact with e.g., hands and surfaces contaminated by respiratory secretions. The incubation period is 2–6 days; persons are most contagious in the early stages of infection. Virus is typically shed for 4–21 days, longer in children than in adults. Infections can cause rhinitis, pharyngitis, otitis media, sinusitis, infectious laryngotracheobronchitis (croup), bronchitis, bronchiolitis, and pneumonia. Less common manifestations include apnea, febrile seizures, aseptic meningitis, encephalitis, parotitis, pericarditis, myocarditis, disseminated infection, and Guillain-Barré syndrome. Although most infections are mild and self-limited, severe disease may occur in young children, the elderly, and immunocompromised persons. HPIVs are responsible for about 7%–17% of pediatric hospitalizations for febrile or acute respiratory infections. Certain serotypes are associated with specific clinical syndromes: notably

Human Parainfluenza Viruses (Paramyxoviridae)

505

HPIV-1 and HPIV-2, with infectious laryngotracheobronchitis (croup), and HPIV-3, with bronchiolitis and pneumonia in infants. HPIV-4 causes milder disease than other HPIV types do. HPIV, particularly HPIV-3, may cause severe and protracted infections among persons with cellular immunodeficiencies. Rates of progression of upper respiratory tract infection (URTI) to lower respiratory tract infection (LRTI) may be up to 40% in patients with combined immunodeficiency and recipients of hematopoietic stem cell transplants and reported mortality rates range from 10% to 40%. HPIV LRTIs are associated with allograft dysfunction and rejection among lung transplant recipients and cryptogenic organizing pneumonia and persistent airflow obstruction in hematopoietic stem cell recipients, particularly those with graft-versus-host disease. Secondary bacterial and fungal infections may complicate HPIV, and these co-infections are associated with increased morbidity and mortality. HPIV infections may also trigger exacerbations of reactive airways (asthma), chronic obstructive pulmonary disease, and post-infectious olfactory dysfunction. HPIV-1 and HPIV-2 cause 60%–75% of croup in pediatric outpatients in the United States, resulting in up to 65,000 hospitalizations annually. The illness, which is most common in children aged 6 months to 2 years, is characterized by inflammation of the larynx and trachea, subglottic edema, and airway obstruction. Children typically present with inspiratory stridor and barking cough, generally preceded by a brief history of coryza or other upper respiratory symptoms. Respiratory failure develops in up to 3% of affected children, but mortality is rare.

Pathogenesis HPIVs bind to specific sialic acid-containing receptors on upper respiratory tract epithelial cells. The virus and host factors contributing to disease pathogenesis are not completely understood. Both viral (F and HN structure) and host features (genetic susceptibility, host cell proteases, immunocompetence, previous infection) are likely to influence the severity of infection. Viral shedding is prolonged, and disease is more severe in persons with T-cell immunodeficiencies, implying that cellular immunity is important in the control of an acute infection. Severe infection occurs following the spread of the infection to the lower respiratory tract. HPIV has direct cytopathic effects, causing ciliary damage, epithelial necrosis, and recruitment of a mononuclear inflammatory response. Recent studies suggest that host inflammatory responses, rather than the direct effects of viral replication, are important determinants of the severity of signs and symptoms of HPIV infection. Alterations in HN receptor binding or activity may influence host inflammatory responses independent of viral replication or the ability to infect epithelial cells. Infection may also trigger long-term bronchial hyperreactivity in genetically susceptible persons. HPIV infection triggers cellular and innate responses that reduce viral replication and the persistence of virus in the respiratory tract. Protection from reinfection is associated with the development of serum and secretory antibodies against F and HN. Local IgA responses appear to be particularly important for sustained immunity. Various HPIVs can interfere with host immune responses by inhibiting interferon responses, for example by the accessory C protein. HPIVs share certain antigenic determinants, but structural and antigenic variation occurs between serotypes and, to a lesser degree, within strains belonging to the same type. Antigenic diversity is not generally progressive but may contribute to the propensity of HPIV to cause recurrent infections. The duration of protection after acute illness is relatively short, especially in infants, and protection from infection by heterotypic stains is incomplete.

Diagnosis The gold standard for diagnosis of HPIV infections is culture on primary monkey kidney cells, human embryonic kidney cells, or human lung carcinoma cells, but this process is labor-intensive and slow, requiring incubation for 4–7 days. Nucleic acid amplification tests (NAATs), performed as single assays or as components of multiplexed respiratory panels, are several-fold more sensitive than is culture and are the diagnostic testing modality of choice where available. The sensitivity and specificity of these assays is 495%. Recent study results suggest a potential role for quantitative viral load testing to identify patients at high risk of disease progression or poor outcomes. Antigen testing and enzyme immunoassays have also been developed for HPIV diagnostics; these methods are rapid but less sensitive and specific than NAATs are. Appropriate samples for diagnostic testing include nasal or nasopharyngeal washes or swabs and bronchoalveolar wash fluid. Detecting specific IgM and IgG in paired acute and convalescent sera has limited utility in diagnosing acute infections, but these approaches are useful in research and epidemiological studies.

Treatment and Prevention Most HPIV infections are mild and self-limited. Treatment is mainly supportive, concentrating on the provision of adequate hydration and supplemental ventilatory support, if needed. Oral or intramuscular corticosteroids reduce airway edema and reduce the duration and severity of symptoms and the risk of respiratory failure in children with croup. Nebulized epinephrine may have similar effects of short duration. Bronchodilators may be useful in patients with wheezing. In immunocompromised patients with severe infections, reductions in immunosuppressive therapy should be considered if feasible. Some experts also recommend using intravenous gammaglobulin in this population, especially those with hypogammaglobulinemia, although its efficacy in this setting is unproven. No licensed antivirals active against HPIV are currently available. Although ribavirin, a synthetic

506

Human Parainfluenza Viruses (Paramyxoviridae)

guanosine analog, has in vitro activity against HPIV, it has not been effective in reducing the progression of URTI to LRTI or in reducing mortality in immunocompromised patients. DAS181 is a recombinant sialidase fusion protein that inhibits HPIV replication by removing cell-surface sialic acids. In early-phase clinical trials of inhaled DAS181 in immunocompromised patients with severe HPIV pneumonia, treatment was well-tolerated and appeared to reduce morbidity; a phase III trial is ongoing. Other antiviral therapies in development include selective inhibitors of HN, fusion, and IMP dehydrogenase and small-molecule inhibitors of RNA polymerase. Adoptive T-cell transfer using donor lymphocytes specific for HPIV is another strategy being investigated for use in severely immunocompromised patients. Paramyxoviruses are labile and readily inactivated by heat, organic solvents, detergents, light, and low pH. Droplet and contact infection prevention precautions are appropriate in the health care setting. Despite considerable effort, no licensed vaccines to prevent HPIV exist. Early formalin-inactivated vaccines were poorly immunogenic and were scrutinized because of the potential of similarly designed vaccines for respiratory syncytial virus to enhance pulmonary inflammatory responses to infection with wildtype virus. Vaccines currently in early clinical trials include live attenuated HPIV-3 and HPIV-2, bovine PIV-3, human-bovine chimeras, bovine PIV or Sendai virus expressing heterologous HPIV F and HN proteins, and oligomannose-coated liposomal HN. These vaccines have, in general, been safe and immunogenic, although multiple doses are likely to be required, and immunity may be incomplete or of short duration.

Further Reading Batista, M.V., El Haddad, L., Chemaly, R.F., 2018. Paramyxovirus infections in hematopoietic stem cell transplant recipients. Current Opinion in Infectious Diseases 31, 542–552. Branche, A.R., Falsey, A.R., 2016. Parainfluenza virus infection. Seminars in Respiratory and Critical Care Medicine 37, 538–554. DeGroote, N.P., Haynes, A.K., Taylor, C., et al., 2011–2019. Human parainfluenza virus circulation, United States. Journal of Clinical Virology 124, 104261. doi:10.1016/j. jcv.2020.104261. Hijano, D.R., Maron, G., Hayden, R.T., 2018. Respiratory viral infections in patients with cancer or undergoing hematopoietic cell transplant. Frontiers in Microbiology 9, 3097. doi:10.3389/fmicb.2018.03097. Pawelczak, M., Kowalski, M.L., 2017. The role of human parainfluenza virus infections in the immunopathology of the respiratory tract. Current Allergy and Asthma Reports 3, 16. doi:10.1007/s11882-017-0685-2. Tang, J.W., Lam, T.T., Zaraket, H., et al., 2017. Global epidemiology of non-influenza RNA resiratory viruses: Data gaps and a growing need for surveillance. Lancet Infectious Disease 17, e320–e326.

Human Pathogenic Arenaviruses (Arenaviridae) Sheli R Radoshitzky, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States Juan C de la Torre, The Scripps Research Institute, La Jolla, CA, United States © 2021 Published by Elsevier Ltd.

History and Classification The first arenavirus, lymphocytic choriomeningitis virus (LCMV), was isolated in 1933 from a suspected case of St. Louis encephalitis. Shortly after LCMV was found to cause aseptic meningitis in humans, and the house mouse was identified as its natural reservoir. By the late 1960s the Arenaviridae family was recognized to include a group of viruses that shared with LCMV common morphology, serology, biochemical features, and a natural history of establishing long-term chronic infections in their natural rodent hosts. The pathogenic potential of arenavirus infections in humans was underscored with the discovery of Junín virus (JUNV) in the late 1950s as the causative agent of Argentinian hemorrhagic fever (AHF), and the subsequent identification of Machupo virus (MACV) and Lassa virus (LASV) in 1962 and 1969 as the etiologic agents of Bolivian hemorrhagic fever (BHF) and Lassa fever (LF), respectively. In the following years, additional pathogenic arenaviruses were discovered, including Guanarito (GTOV), Sabiá (SBAV), and Chapare (CHAPV) viruses in South America, and Lujo virus (LUJV) in Africa. During the last decade the use of the new technologies of deep sequencing and genome discovery has resulted in the discovery of many new arenaviruses, but their contribution to human disease remains to be determined. Notably, arenaviruses have been also discovered in reptiles (snakes) and fish, representing a significant expansion of the host range of arenaviruses. Current arenavirus classification is based on pairwise sequence comparisons (PASC) of coding-complete genomes. Based on the most current sequence dataset, S segment and L segment nucleotide sequence identities for viruses within the same genus need to be higher than 40% and 35%, respectively, This analysis and additional characteristics, such as genome architecture, host range, modes of transmission and/or sites of replication in the cell, led to the establishment of four genera within the family Arenaviridae: Antennavirus, Hartmanivirus, Mammarenavirus, and Reptarenavirus. The hosts of mammarenaviruses are rodents, with the exception of Tacaribe virus (TCRV) that has been found in phyllostomid bats and ixodid lone star ticks. Reptarenaviruses and hartmanivirus infect reptilian hosts, and these infections can cause boid inclusion body disease (BIBD). Antennaviruses have a piscine host. Based on antigenic properties, mammarenaviruses have been divided traditionally into two distinct groups. Old World (OW) mammarenaviruses (“Lassa–LCMV serocomplex”) include viruses indigenous to Africa and the worldwide distributed LCMV, whereas New World (NW) mammarenaviruses (“Tacaribe serocomplex”) include viruses indigenous to the Americas. This classification is largely consistent with phylogenetic data and muroid rodent host phylogeny, with OW mammarenaviruses infecting murid rodents primarily in Africa and NW mammarenaviruses infecting cricetid rodents primarily in the Americas. This article focuses on arenaviruses known to cause disease in humans, all of them within the Mammarenavirus genus.

Mammarenavirus Virion Structure Mammarenavirions are pleomorphic, ranging in size from 40 to more than 200 nm in diameter, with dense lipid envelopes. The virion’s surface is decorated with evenly spaced spike projections composed of heterotrimer complexes of the viral glycoproteins GP1 and GP2 and the stable signal peptide (SSP) (Fig. 1(A)). Cryo-EM studies have shown that surface GP complexes are aligned with subjacent Z and ribonucleoprotein (RNP) densities, which are packed into a two-dimensional lattice at the inner surface of the viral membrane. Virions contain the L and S RNP complexes organized in circular configurations. The L and S genomic RNAs are not present in equimolar amounts within virions (L:S ratios B 1:2), and low numbers of both L and S antigenomic RNAs are also present within virions. In addition, host ribosomes are documented to be incorporated into virions, but the biological significance of this incorporation remains uncertain.

Mammarenavirus Genome Organization and Proteins Mammarenaviruses have a bi-segmented negative-stranded RNA genome (Fig. 1(B)). Each genome segment, L (large, 7.3 kb) and S (small, 3.5 kb) uses an ambisense coding strategy to direct the synthesis of two proteins from two non-overlapping open reading frames (ORF) of opposite polarities that are separated by non-coding intergenic regions (IGRs). The S RNA encodes the viral NP and the glycoprotein precursor (GPC), whereas the L RNA encodes for the viral RNA dependent RNA polymerase (RdRp) (L protein) and the matrix Z protein. Mammarenavirus genomes exhibit high degree of sequence conservation at their 30 -termini and similarly to other viruses with segmented negative strand (sNS) RNA genomes, they exhibit 50 - and 30 -inverted complementary sequences at their L and S genome segments that are predicted to form panhandle structures. This prediction is supported by EM data showing the existence of circular RNP complexes within arenavirions. For several arenaviruses, a non-templated G residue has been detected at the 50 end of progeny genomic RNAs during replication. The IGRs are predicted to fold into stable hairpin structures. Transcription of the

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00014-X

507

508

Human Pathogenic Arenaviruses (Arenaviridae)

Fig. 1 A. Schematic illustration of an arenavirus particle. Shown is the pleomorphic shape of the enveloped virion particle decorated by spikes composed of heterotrimers of GP1 and GP2 associated with the SSP (no shown in the figure). The surface glycoproteins are aligned with subjacent matrix Z protein that serves also as a bridge between the surface glycoproteins and the S and L vRNP complexes inside the particle. Each vRNP consists of the NP (red) and L polymerase (blue) together with the S or L genome RNA species. Mammarenavirus virions are thought to incorporate ribosome particles. B. Mammarenavirus genome organization. Each genome segment uses an ambisense coding strategy to direct the synthesis of two different proteins in opposite direction and separated by a non-coding intergenic region (IGR) that serves as a bona fide transcription termination signal. The S segment encodes for the virus NP and GPC precursor, whereas the L segment encodes for the L polymerase and the matrix Z protein.

S–derived NP and GP mRNAs was shown to terminate at multiple sites within the predicted distal stem of the IGR, supporting the view that a structural motif rather than a sequence-specific signal promotes the release of the arenavirus polymerase from the template RNA. There are significant differences in sequence and predicted folded structure between the S and L IGRs, but among isolates and strains of the same arenavirus, the S and L IGR sequences are highly conserved. In addition to their role in control of transcription termination, IGRs have been shown to play a critical role in production of infectious particles. GPC is co-translationally cleaved by the signal peptidase to generate a 58-amino acid stable signal peptide (SSP), and posttranslationally processed by the cellular protease subtilisin kexin isozyme-1 (SKI-1)/ site 1 protease (S1P) to generate the mature virion surface glycoproteins GP1 and GP2. GP1, GP2 and SSP form the GPC complexes that decorate the surface of virions and mediate virus receptor recognition and cell entry. GP1 mediates virion interaction with host cell-surface receptors, whereas GP2 directs fusion of virus and host cell membranes. The fusion process depends on a low pH-driven conformational change of GP2 from a metastable prefusion structure to a more stable postfusion six-helix bundle. Notably, the SSP contributes to trafficking and processing of GPC as well as to the GP2-mediated pH-dependent fusion process. NP is the most abundant viral polypeptide both in infected cells and virions. It is the main structural component of the viral RNP responsible for directing RNA genome replication and gene transcription. The C-terminus of NP exhibits a type I interferon (IFN-I) counteracting activity. Crystallographic studies identified distinct N- and C-terminal domains within NP of LASV, LCMV and JUNV, features that are expected to be present in other mammarenavirus NPs. The N-terminal domain of NP contains an RNAbinding site and plausible cap-binding activity. The C-terminal part of NP contains a functional DEDDH 30 ‒50 exoribonuclease folding domain similar to the one described for the non-structural 14 protein of severe acute respiratory syndrome (SARS) coronavirus. This 30  500 exoribonuclease activity of NP plays critical roles in NP’s anti-IFN activity and in other additional steps of mammarenavirus multiplication yet to be elucidated. Mammarenavirus L proteins have a central region that includes conserved motifs characteristically found in the RdRp domains of negative-sense RNA viruses. Residues critical for LASV L function have been shown to be located both within and outside the predicted RdRp domain. Bioinformatic analysis and structural studies revealed that the N-terminus of LASV L has an endonuclease domain of similar structure to the cap-snatching endonuclease domains of influenza A virus polymerase acidic (PA) and La Crosse virus L proteins. In addition, EM characterization of a functional MACV L has revealed a core ring-domain decorated by appendages, which likely reflects a modular organization of the arenavirus polymerase. Mammarenavirus Z proteins exhibit a modest degree of overall conservation. However, three regions within Z have a higher degree of conservation: (1) an N-terminal myristoylation site marked by a highly conserved G motif that is critical for membrane anchoring and interaction with other viral proteins; (2) a RING domain that binds two zinc ions through three conserved motifs; and (3) a C-terminal region containing proline-rich motifs that serve as functional late budding motifs. Structural studies revealed that the N- and the C-terminal arms flanking the RING domain are disordered, which may enable Z to recruit a variety of cellular partners to modulate virus multiplication. In addition, Z assembles in a dodecameric manner through a head-to-tail dimerization of the RING domain. These findings are consistent with Z playing a key role in virion assembly, which is further supported by EM

Human Pathogenic Arenaviruses (Arenaviridae)

509

Fig. 2 Mammarenavirus life cycle. Mammarenavirus enter cells via receptor-mediated endocytosis (RME). There are some differences on the specific steps of the pathway used by different mammarenaviruses to reach the late endosome where the acidic environment triggers a pH-dependent fusion event between viral and cell membranes that releases the vRNP into the cytoplasm of the infected cells where viral RNA replication and gene transcription take place. Correct processing of GPC by the cellular signal peptidase and S1P protease is required for the formation of infectious progeny. The matrix Z protein plays critical roles mediating interactions between the vRNP and surface GP complex required for the formation of infectious progeny. Z is also the main driving force of mammarenavirus budding.

studies indicating that Z bridges the viral RNP to the viral envelope proteins. Z is also involved in suppression of the host cell type I IFN response via its interaction with RIG-I-like receptors.

Mammarenavirus Life Cycle Cell Attachment and Entry Receptor-mediated endocytosis is the main cell entry pathway used by mammarenaviruses. The acidic environment of the late endosome facilitates a pH-dependent conformational change in the GP complex and subsequent GP2-mediated fusion step between viral and cell membranes. Following fusion, the viral RNP is released into the cytoplasm where it directs both replication and transcription of the viral genome (Fig. 2). The conserved and widely expressed cell-surface receptor for extracellular matrix proteins a-dystroglycan (aDG) is a main receptor for the OW mammarenaviruses LCMV and LASV. Posttranslational glycosylation modification of aDG by the like-acetylglucosaminyltransferase (LARGE) is critical for aDG’s function as a mammarenavirus receptor, and specific LARGE alleles have been shown to be positively selected among the Yoruba of Western Africa where LASV is endemic. However, non-pathogenic mammarenaviruses also use aDG-mediated cell entry, indicating that aDG does not play a unique direct role in mammarenavirus pathogenesis. Secondary alternative receptors, including members of the Tyro3/Axl/Mer and T-cell immunoglobulin (TIM) and mucin receptor families may account for LASV and LCMV infection of cells lacking fully glycosylated aDG. Cell entry of the OW hemorrhagic fever (HF) mammarenavirus LUJV is mediated by neuropilin (NRP)-2, a cell-surface receptor for semaphorins. NRP-2 is highly expressed in microvascular endothelial cells, which may contribute to LUJV-induced coagulopathy. Human transferrin receptor 1 (TfR1) is the main cellular receptor used for cell entry of pathogenic NW mammarenaviruses, including JUNV and MACV. Consistent with the use of TfR1 as a primary receptor, JUNV enters cells through clathrin-mediated endocytosis.

510

Human Pathogenic Arenaviruses (Arenaviridae)

Fig. 3 Mammarenavirus RNA replication and gene transcription. The basic steps of mammarenavirus RNA replication and gene transcription are illustrated for the S segment. Following the pH-dependent fusion event between the viral and cell membranes in the late endosome, the vRNPs are delivered in to the cytoplasm where they initiate transcription from the genome promoter located at the genome 30 -end. Primary transcription results in synthesis of NP and L mRNA from the S and L segments, respectively. Subsequently, the virus polymerase adopts a replicase mode and moves across of the IGR to generate copies of the full length antigenome RNA (agRNA) species that serve as template for the synthesis of the GPC (agS) and Z (agL) mRNAs. The agRNA species serve also as templates for the amplification of the corresponding genome RNA species.

Notably, completion of the cell entry process for LASV and LUJV involves a late endosomal receptor switch mechanism. LASV uses the late endosomal resident proteins LAMP1 and LUJV uses CD36 in these final entry stages.

Expression and Replication of the Viral Genome NP and L coding regions are transcribed into a genomic complementary mRNA. However, the GPC and Z coding regions are not translated directly from genomic RNA, but rather from genomic sense mRNAs (Fig. 3). These genomic sense mRNAs are transcribed from templates of the corresponding antigenome RNA, which also function as replicative intermediates. Transcription initiation is carried out through a mechanism shared by all negative sense viruses called cap-snatching, in which short fragments of 50 -capped host cell mRNAs are used by virus polymerase complex to prime the synthesis of viral mRNAs. Transcription termination occurs within the distal side of the IGR and generates non-polyadenylated viral mRNAs. Virus replication proceeds in two steps: first, the de novo (primer-independent) synthesis of an intermediate RNA with positive polarity (cRNA) and second, using this cRNA as template, the synthesis of progeny genomic vRNA with negative polarity. The 50 -end of arenavirus genome and antigenome RNAs each contain a non-templated G residue that has been proposed to reflect a prime-and-realign mechanism for RNA replication mediated by L. Using cell-based minigenome (MG) assays, NP and L have been identified as the minimal viral trans-acting factors required for efficient RNA synthesis mediated by the virus polymerase. For LCMV, both genetic and biochemical evidence indicate that oligomerization of L is required for polymerase activity. Z is not required for RNA replication and gene transcription mediated by the virus L polymerase, but rather exhibits a dose-dependent inhibitory effect on both RNA biosynthetic processes. Consistent with these findings, studies using in vitro reconstitution of RNA synthesis directed by MACV polymerase have provided evidence that Z, via direct interaction with the polymerase, is able to lock the polymerase in a promoter-bound, catalytically inactive state.

Human Pathogenic Arenaviruses (Arenaviridae)

511

Mutation-function analysis of the genome 50 -and 30 -termini using cell-based MG assays for LCMV, LASV, and MACV indicate that the activity of the arenavirus genomic promoter is dependent on both sequence specificity within the highly conserved 30 -terminal 19 nt of arenavirus genomes and on the integrity of the predicted panhandle structure. This panhandle is formed via sequence complementarity between the 50 - and 30 -termini of viral genome RNAs. Mammarenavirus RNA replication and gene transcription are regulated in a coordinated manner but intracellular levels of NP do not determine the balance between virus RNA replication and transcription. MG-based assays confirmed the IGR role as a bona fide transcription termination signal, but synthesis of translation-competent viral mRNAs does not strictly require the presence of the IGR.

Assembly and Budding Production of infectious mammarenavirus progeny requires both Z and GPC and the correct processing of GPC into GP1 and GP2. Z is a structural component of the virion and cryo-electron microscopy structural studies reveal a location of LMCV Z within virions consistent with its role as a matrix protein. Consistent with its matrix protein function, Z is the main driving force of mammarenavirus budding, a process mediated by the interaction of Z’s late domain budding motifs, PTAP or PPPY, or both, with components of the endosomal sorting complexes required for transport (ESCRT) within the vacuolar protein sorting (Vps) pathway. Myristoylation of Z is strictly required for its targeting to the plasma membrane, the location of arenavirus budding (Fig. 2).

Epidemiology of Human Pathogenic Mammarenaviruses Typically, mammarenaviruses cause persistent, frequently asymptomatic infections in their reservoir hosts, which are characterized by virus multiplication in many different tissues and chronic viremia and viruria. Both, vertical transmission (exposure to infectious virus early in ontogeny) and horizontal transmission (exposure to virus later in life through aggressive or venereal behavior) may contribute to the mammarenavirus chronic carrier state in rodents. Human infections occur via exposure to rodent fomites, ingestion of contaminated food, exposure to broken skin or mucous membranes, or by inhalation of aerosolized virions from contaminated material.

Lassa Fever (LF) LF is caused by LASV, which is endemic in vast areas of western sub-Saharan Africa. Imported cases of LF have been reported in the United States, Canada, the United Kingdom, Japan, Germany, Netherlands, and Israel. The case-fatality rate (CFR) of LF is about 1%–2% in the endemic areas, with estimated 300,000 infections annually. Most LASV infections in Africa are asymptomatic, mild or subclinical, but the CFR in hospitalized confirmed cases of LF can be as high as 69%. The disease is especially severe late in pregnancy with fetal death, miscarriage, or spontaneous abortion occurring in nearly all cases. The main reservoir host of LASV is the Natal multimammate mouse (Mastomys natalensis). Maintenance of LASV in Natal mastomys is thought to occur via vertical transmission but recent studies suggest the existence of an additional horizontal transmission mechanism. Other rodents might also serve as LASV hosts or have roles in LASV transmission. Humans become infected with LASV through direct contact with infected rodent tissues, excreta, or blood or via inhalation of aerosolized virus. Infected meat could contribute to virus transmission in populations that include peridomestic rodents as part of their diet. Personto-person transmission of LASV can occur in nosocomial settings via direct contact with body fluids from symptomatically infected individuals or corpses. Human infections tend to be more common in the dry season, which may reflect a higher prevalence of Natal mastomys within human dwellings. LASV prevalence is focal and varies greatly between geographical regions. For example, in Sierra Leone, seroprevalence ranges from 8% to 52% and in Nigeria from 13% to 37%. LASV exhibits a high degree of genetic diversity; nucleotide differences can reach up to 32% for the L segment and 25% for the S segments. The genetic diversity among LASV strains correlates with geographic distribution rather than time. This high genetic diversity of LASV might explain the observed variability of LF’s clinical presentation and possible regional differences associated with CFRs and disease symptoms.

Lujo Hemorrhagic Fever (LHF) LHF, caused by LUJV, was identified in 2008 in South Africa. The only known nosocomial outbreak involved five cases of viral HF disease with clinical symptoms remarkably similar to LF, despite LASV and LUJV being distantly related. LHF disease has an abrupt onset and exhibits disseminated intravascular coagulation (DIC), features that distinguish LHF from LF. The overall lack of epidemiological data on the prevalence of LUJV in South Africa and the limited number of verified cases prevents any conclusion on whether the high (80%) CFR associated with the LHF outbreak reflects a more severe disease compared to LF. The natural reservoir of LUJV remains unknown.

Lymphocytic Choriomeningitis (LCM) The primary host of LCMV is the house mouse. Other rodents, including pet hamsters and domesticated guinea pigs can become infected and transmit the virus to humans. LCMV is transferred vertically from one generation to the next and chronically infected

512

Human Pathogenic Arenaviruses (Arenaviridae)

rodents shed the virus in their urine, saliva, nasal secretions, and droppings throughout life. Mounting evidence indicates that LCMV is a neglected human pathogen of clinical relevance as an under-recognized cause of neurologic disease in the fetus, and adult, and important threat to immune compromised individuals. Humans become infected with LCMV through close contact with infected rodents, solid organ transplantation, or by vertical transmission associated with congenital infections. The seroprevalence of LCMV within house mouse populations and in humans is highly variable, even within the same geographic region, resulting in the focal and uneven spatial distribution of the virus. As with LASV, LCMV exhibits high genetic diversity. Up to 18% and 25% nucleotide divergence was observed within the S and L segments, respectively, of LCMV lineages. The high genetic diversity of LCMV and the lack of clear correlation of virus genetic lineages to particular geographic locations likely reflect the long and complex phylogeographic history of the common house mouse host.

Argentinian Hemorrhagic Fever (AHF) Junín virus (JUNV), the etiologic agent of AHF, is endemic to the Pampas in Argentina. In the absence of treatment, the CFR rate can reach 20%–30%. Pregnant women have higher CFR and many miscarry, especially if infection occurs during the third trimester. AHF is typically a seasonal disease, with a peak of frequency occurring during the corn-harvesting season, and infected cases are primarily rural male agricultural workers. The drylands laucha (Calomys musculinus) is the main reservoir of JUNV but other animals can also become infected. The patchy spatial distribution of the drylands laucha has been suggested to account for the focal distribution of AHF. Annual increase in the number of these rodents coincides with the corn harvesting season providing opportunities for rodent-to-man transmission. Human transmission is thought to occur predominantly by inhaling aerosolized viral particles from contaminated soil and plant litter, which are disturbed during the mechanized harvesting process, or by exposure to primary aerosols of rodent urine or contact with contaminated nesting materials in border habitats. Horizontal transmission via aggressive encounters among adult, male rodents is the primary mode of viral persistence in nature, which is facilitated by the high viral load in saliva of drylands lauchas. Vertical transmission might contribute to the maintenance of JUNV via intra-generational infection by horizontal transmission when population numbers are reduced. Seroprevalence in humans range from 4.7% to 12.3% in AHF-endemic areas, and significantly lower (0.44%) in non-endemic areas. In rodents, prevalence of infection can be as high as 10.9% with highest prevalence observed in current epidemic areas and lowest (0.2%) in non-endemic areas. In contrast to LASV and LCMV, Nucleotide similarity between JUNV strains is high, reaching up to 94.5% and 95.4% for GP and NP, respectively.

Bolivian Hemorrhagic Fever (BHF) The CFR of BHF, caused by Machupo virus (MACV), is approximately 5%–30% with the highest rates occurring among those under 5 and over 55 years of age. BHF cases are more common at the peak of agricultural activity, during the dry season, with males over 15 years of age more frequently affected. The big laucha (Calomys callosus) is the reservoir host of MACV. Both horizontal and vertical transmission have been shown as possible maintenance mechanism of MACV within its reservoir rodent. Person-to-person transmission of MACV is possible but rather uncommon, likely reflecting the infrequent and low virus detection in blood or from throat and oral swabs of infected patients.

Venezuelan Hemorrhagic Fever (VeHF) Guanarito virus or GTOV emerged in 1989 as the cause of VeHF, a severe hemorrhagic illness with CFR of E30%. The disease has focal distribution in the southern and southwestern portions of Portuguesa state and in adjacent areas of Barinas state. VeHF cases peak during the period of agricultural activity in these regions and involve mainly male agricultural workers. The short-tailed zygodont (Zygodontomys brevicauda) is the main reservoir host of GTOV. The virus is also commonly found in Alston’s cotton rats (Sigmodon alstoni). These hosts have not been reported within houses or farm building. Therefore, infections are assumed to occur outdoors, in rural areas, and persons that have frequent contact with rodent-infested grassland habitats are at higher risk.

Pathogenesis and Pathology The mechanisms underlying pathogenesis of mammarenaviral disease in humans are not well understood. Pathological findings in patients’ autopsies do not account for the severity of symptoms seen in many cases of human mammarenaviral disease. Mammarenaviruses typically enter humans in an aerosolized form and are deposited in the lung, where initial viral replication occurs. Antigen-presenting cells (APCs), DCs and macrophages, are prominent targets in the initial stages of infection, and facilitate virus access to the lymphoid system and subsequent systematic spread to other organs and tissues including liver, kidneys, lungs, adrenal glands, and heart. LASV has also been recovered from placenta, mammary glands, and aborted fetal tissues, and in cases of AHF, virion-like particles were detected in the central nervous system (CNS), ovaries, and testes. LCMV load can reach high levels in meninges, choroid plexus, and ventricular ependymal linings, where the inflammatory response produces the characteristic LCM pathology. LCMV exhibits a strong tropism for the fetal brain, where LCMV congenital infection produces its

Human Pathogenic Arenaviruses (Arenaviridae)

513

most common and severe pathologic effects, including microencephaly, periventricular calcifications, hydrocephalus, cerebellar hypoplasia, focal cerebral destruction, and gyral dysplasia. The extent of hemorrhagic and coagulopathy manifestations differs between human pathogenic mammarenaviruses. Hemorrhages are common in AHF cases, but are rare and mainly limited to mucosal surfaces in LF cases. Bleeding is usually associated with thrombocytopenia and platelet dysfunction. Fatal cases of LF are associated with lower levels of platelet activating factor (PAF) and PAF-like molecules, as well as hemoglobin breakdown products D-urobilinogen and I-urobilin. Disseminated intravascular coagulation (DIC) or complement activation do not appear to play a role in mammarenavirus pathogenesis. Impaired vascular endothelium function, including increased permeability, likely plays a central role in LF and AHF pathogenesis. However, only minimal vascular histological lesions are detected in fatal human LF and AHF cases and infected nonhuman primates (NHPs), and the mechanisms responsible for virus induced increase in vascular permeability remain to be elucidated. In contrast to other HF-causing viruses, LASV and JUNV do not trigger a “cytokine storm” that could interfere with the integrity of the vascular endothelium, but infection of the endothelium might cause changes in endothelial cellular function leading to increased fluid flow and subsequent edema. Pathological findings in LF patients include both macroscopic and microscopic abnormalities. Gastric mucosal, renal, and subconjunctival hemorrhages, petechiae, and increased vascular permeability are common macroscopic observations, whereas microscopic observations include multifocal hepatocellular necrosis with modest inflammatory cell involvement, splenic necrosis, necrosis of renal tubular cells, adrenocortical cell necrosis, focal renal interstitial lymphocytic infiltrates, mild mononuclear interstitial myocarditis, alveolar edema with capillary congestion, interstitial pneumonitis, and rhabdomyositis. The most consistent pathological hallmark of LF in humans is multifocal hepatocellular necrosis. High virus titers in liver tissue correlate with severe hepatitis. However, the degree of hepatic tissue damage is insufficient to cause hepatic failure, and there is no correlation between the degree of hepatic necrosis and chemical indicators of liver damage, indicating that LASV-induced hepatitis is unlikely to be the primary cause of death in fatal cases of LF. The most common macroscopic abnormality observed in severe cases of NW mammarenaviral disease is widespread hemorrhage affecting the skin and mucous membranes, Virchow-Robin space, kidneys, pericardium, spleen, adrenal glands, and lungs. Microscopic lesions include acidophilic bodies, focal liver necrosis, acute tubular and papillary necrosis in the kidneys, and reticular hyperplasia of the spleen and lymph nodes. Pathognomonic symptoms of AHF illness can vary depending on the specific JUNV strain and can be “hemorrhagic”, “neurologic”, “mixed”, and “common”. Infection with “hemorrhagic” strains results in a pronounced bleeding tendency with disseminated cutaneous and mucous membrane hemorrhage. In contrast, infection with “neurologic” strains show little or no hemorrhagic manifestations but result in overt and progressive signs of neurologic dysfunction. LCM disease correlates with detection of viral antigen in meninges and cortical neurons, as well as mononuclear cell infiltrates in the meninges and around vessels and glial nodules in the deeper structures, suggesting an immune-mediated pathology associated with LCMV infection. An animal model of congenital LCMV infection revealed a very strong tropism of LCMV for neuroblasts and altered neuron migration, findings that may explain the location of periventricular calcifications and the gyral malformations in children with congenital LCMV.

Clinical Features LF and LHF LF is mild or asymptomatic in about 80% of infected individuals, but 20% develop acute LF. The incubation period can range from 5 to 21 days but is typically 10–14 days. Within 2–4 days of infection, many patients experience an array of symptoms including headache, myalgia, arthralgia, lower back, abdominal and retrosternal chest pain, dizziness, nausea, tinnitus and sore throat. Cough, vomiting, diarrhea and constipation are also common, whereas lymphadenopathy, oliguria, tachycardia, vertigo, splenomegaly, hepatomegaly, and jaundice have been reported occasionally. Disease progression is often associated with pharyngitis, conjunctivitis, respiratory distress, pleural and pericardial effusions, and facial and neck edema. In most cases of LF recovery begins 8–10 days after disease onset, and 4–11 days later viremia is undetectable. In contrast, in severe cases of LF, worsening of symptoms and hemorrhagic and neurologic manifestations are more common. Hemorrhagic manifestations can affect skin and mucosal surfaces. Neuroglial findings including diffuse encephalopathy, confusion, tremors, coma, and convulsions are common prior to shock and death. Widespread edema can be observed in children with LF, which usually has a fatal outcome. Common clinical findings in LF cases include proteinuria, albuminuria, and elevated AST levels, whereas moderate leukopenia has been reported only in some patients. Survivors of LF often recover without sequalae. However, long-term unilateral or bilateral sensorineural deafness can affect a significant (E13%–30%) number of survivors. Known cases of LHF disease presented with similar clinical symptoms to those documented for LF cases. Initial symptoms of nonspecific febrile illness increase in severity over 7 days with the development of diarrhea, pharyngitis, and facial and neck edema. Terminal features are shock and multi-organ system failure often with evidence of DIC that resulted in death of four of the five LHF cases.

LCM In most healthy individuals, LCMV infection course is asymptomatic, or manifested as a mild febrile illness. Following 1–2 weeks of incubation, some infected individuals may develop a flu-like illness that could include anorexia and gastrointestinal

514

Human Pathogenic Arenaviruses (Arenaviridae)

symptoms. Sore throat, cough, pharyngitis, and other symptoms of respiratory tract involvement are less common. Leukopenia, moderate thrombocytopenia, mild elevations of AST, and infiltrates on chest radiographs can be seen in some patients. Additional, but rather infrequent, reported clinical findings are lymphadenopathy, dysesthesia, conjunctivitis, arthralgia, arthritis, rash, testicular or parotid pain, as well as abdominal, back, and chest pain. LCMV invasion of the CNS can occur in some infected individuals, usually resulting in symptoms of classic aseptic meningitis. In some cases, encephalitis, encephalomyelitis, meningoencephalitis, acute hydrocephalus, ascending or bulbar paralysis, or transverse myelitis may develop. CSF pleocytosis is characteristically observed during this CNS phase of illness and LCMV can be isolated from the spinal fluid. Infections are rarely fatal, and most patients recover without sequelae, but some might experience asthenia, headaches, confusion and difficulty in concentration. LCMV Infection during pregnancy, especially during the first trimester, increases the risk for miscarriage, in utero fetal death, fetopathy, and severe neurologic sequelae. Congenital LCMV can result in severe CNS or ocular malformations and can mimic the signs/symptoms of classic TORCH syndrome. In immunosuppressed organ recipients, LCMV infection can be fatal due to multi-organ system failure with LCMV-associated hepatitis as a prominent feature. In most immunosuppressed patients CNS manifestations are also observed. Clinical findings in these patients include elevated transaminases levels, leukopenia, and thrombocytopenia, as well as increased protein levels and leukocyte numbers in CSF.

NW Mammarenaviral HFs AHF has an incubation period of 1–2 weeks, followed by a prodromal phase characterized by fever and malaise, headache, myalgia, epigastric pain, and anorexia. After 2–4 days, signs become increasingly severe including prostration and GI disturbances. In some cases, dizziness, photophobia, retro-orbital pain, or disorientation may also occur. Initial signs of vascular damage may appear at this early phase of AHF. During the second week of illness, about 20%–30% of patients develop severe hemorrhagic and neurologic manifestations, or secondary bacterial infections. Hemorrhagic manifestations include bleeding from mucous membranes and ecchymosis at needle puncture sites, but only very modest blood loss is observed. Neurologic manifestations include seizures, convulsions, tremor of the hands and tongue, and less frequently, delirium, coma, encephalitis and meningoencephalitis. Following shock, death usually occurs 7–12 days after disease onset. Clinical laboratory findings include leukopenia and thrombocytopenia and non-specific electrocardiogram abnormalities, whereas chest radiography is usually normal in the absence of secondary infections. Patients’ clinical improvement during the second week of AHF correlates with the appearance of neutralizing antibodies. Convalescence often lasts several weeks with polyuria, fatigue, alopecia, and dizziness. BHF and VeHF caused by MACV and GTOV, respectively, as well as NW mammarenaviral HF caused by CHAPV and SBAV, display clinical symptoms similar to those described for AHF. Proteinuria and elevated hematocrit during the peak of hemorrhagic manifestations are characteristic of severe cases of BHF. Early symptoms of VeHF disease are indistinguishable from dengue fever, also common in Venezuela. CNS manifestations of VeHF including encephalitis are associated with poor prognosis. Hearing loss has been reported in convalescence cases of VeHF. The only reported case of a naturally acquired infection with SBAV caused disease with symptoms similar to those of other NW mammarenaviral HF, but also included extensive liver necrosis.

Diagnosis LF LASV can be isolated from blood during the febrile phase of LF disease, as well as from autopsy tissue samples. Serum detection of LASV antigen by ELISA is robust, reliable, and can be completed in a short time. LASV-specific antibodies can be detected by immunofluorescence (IF) and ELISA. ELISA IgM titers appear earlier and persist longer than IF IgM titers. Virusspecific IgG ELISA antibody detection persists for long periods, whereas IF antibody appears to wane over time below detectable limits. Reverse transcriptase-PCR (RT-PCR) can detect virus RNA in blood with high sensitivity. Nanopore sequencing using the MiniON device was successfully used to genetically characterize LASV isolates during the 2018 upsurge of LF cases in Nigeria. The ability of this portable sequencing technology to genetically characterize in situ RNA viral samples in real-time cases of LASV infection associated with disease symptoms will represent a major breakthrough in the study of LF epidemiology. LF presents with symptoms indistinguishable from those of other febrile illnesses including malaria, and therefore it is difficult to diagnose LF clinically, but it should be suspected in patients with fever (Z381C) not responding to antimalarial and antibiotic drugs. Useful clinical findings for diagnosis of LF are fever, pharyngitis, retrosternal pain, and proteinuria. Viremia levels, fever, sore throat, vomiting, edema, and bleeding are the predictors of poor prognosis. CNS signs, face and neck edema, jaundice, bleeding, hematuria, and proteinuria were shown to be associated with a fatal outcome. Several biomarkers including increased levels of blood urea nitrogen and creatinine (biomarkers for kidney function), serum AST (liver malfunction), and serum electrolyte potassium, have been shown to have a strong correlation with CFR, highlighting the role of liver and renal dysfunction and electrolyte disturbance in the severity of LF.

Human Pathogenic Arenaviruses (Arenaviridae)

515

LCM LCMV can be isolated from blood during the febrile phase of LCM and during meningitis symptoms, but CSF contains higher viral load, and PCR-based tests using CSF samples have been successfully used. LCMV-specific IgM antibodies can be detected by ELISA and IF in serum and CSF in acute cases of LCM. Neutralizing antibodies appear late after onset of disease and their diagnostic value is limited.

NW Mammarenaviral HFs During the acute febrile phase of disease, virus can be isolated from blood samples by inoculation of newborn hamsters or mice, but cocultivation of patient’s peripheral blood mononuclear cells with Vero cells may offer higher level of sensitivity. Virus can also be isolated from autopsy tissues, with the exception of brain. Viral antigen in blood and tissues from patients with JUNV, MACV, SBAV, or GTOV infection can be usually detected by antigen-capture ELISA. Serologic diagnosis of AHF and BHF is usually made by IF and CF, but the limited specificity and sensitivity of these tests pose problems. The ELISA test is the most useful and practical for rapid detection of IgM and IgG antibodies in a clinical setting and sero-epidemiologic surveys. The plaque neutralization test can be valuable for evaluation of convalescent plasma units intended for therapeutic use in AHF.

Prevention and Control Medical Management Supportive therapy including hydration and pain relief management represent standard of care in suspected cases of mammarenaviral HFs. Platelet transfusions and factor replacement could help to control bleeding. Infusion of crystalloids to counteract the commonly observed moderate degree of vascular permeability should be carefully considered due to a high risk of pulmonary edema. The low cardiac output observed in human cases of AHF support the use of Swan-Ganz catheterization. LCMV ependymal infection and inflammation may cause acute hydrocephalus and a need for surgical shunting. Management of patients that undergo shock is difficult. Patients with mammarenaviral HF pose in general a low risk of contagion. However, nosocomial outbreaks and infection of multiple contacts have occurred when the index case was severely ill with high viremia and viral load in tissues. Patients may excrete virus in urine or semen for weeks after recovery from disease. Therefore, body fluids should be monitored for infectivity before the patient is released, and counseling should be provided emphasizing protection of sexual partners and the use of disinfectant prior to use of toilets. The highest exposure risk is parenteral and can be minimized through staff training. Respiratory protection against small-particle aerosols should be implemented with caregivers and people in close proximity to patients. Special precautions are indicated when blood and other body fluids are handled in the clinical laboratory.

Antiviral Drugs The prophylactic and therapeutic value of the nucleoside analog ribavirin (Rib) against human pathogenic mammarenaviruses is well supported by results from both cell culture and animal models of infection. Importantly, Rib reduced both morbidity and mortality in human cases of LF, and evidence suggests that Rib treatment can have also clinical benefits in cases of JUNV, MACV and SABV infections. However, the need of intravenous administration for optimal efficacy, poor penetration into the CSF, and side effects, including anemia and congenital disorders, pose some limitations to the use of Rib. In addition to Rib, several compounds have been reported to have anti-mammarenaviral activity in cultured cells, but their safety and efficacy in vivo remain to be determined. The broad-spectrum antiviral favipiravir and the mammarenaviral GP-mediated fusion inhibitor ST-193 have yielded very promising results in different animal models of mammarenaviral disease. Progress in mammarenavirus molecular genetics has facilitated the development of screens to identify drugs targeting specific steps of the arenavirus lifecycle. These drugs include direct-acting antivirals (DAAs) that target specific viral gene products and functions, and drugs that target host-cell factors (HTAs) required for the completion of virus replication cycle. Combination therapy of DAAs and HTAs should counteract the emergence of drug resistant variants often observed with monotherapy strategies. Moreover, related viruses are likely to rely on the same host machinery, thus providing an opportunity for the development of broad-spectrum antiviral therapeutics. Limited market opportunities are an obstacle for the development and licensing of new antiviral drugs against human pathogenic mammarenaviruses. It would be therefore beneficial to use the newly developed screening platforms to implement drug repurposing strategies that can significantly reduce the time and resources required to advance a candidate antiviral drug into the clinic. Moreover, drug repurposing can generate new knowledge on virus biology by uncovering previously unexplored pathways and specific host cell factors contributing to virus multiplication, knowledge that can be harnessed to identify new targets and therapeutics.

Antibody Therapy Treatment of AHF cases with convalescent plasma from survivors of AHF has been shown to be quite effective in reducing AHF-associated mortality. About 10% of patients treated with convalescent plasma may exhibit usually self-limited neurologic signs including fever,

516

Human Pathogenic Arenaviruses (Arenaviridae)

headache, cerebellar tremor, and cranial nerve palsies, which may reflect the prior invasion of the CNS by the virus. Studies in animal models of infection suggest that passive antibody therapy could be also helpful to treat BHF, VeHF and LF. Experimental studies of passive protection by monoclonal antibodies have been successful in LCMV. Notably, LASV GPC-specific recombinant human monoclonal antibodies derived from survivors of LF with strong neutralization profiles in cell-based assays have shown robust therapeutic efficacy in guinea pig and non-human primate (NHP) models of LF, supporting the feasibility of antibody-based therapy against LF.

Vaccines The safety, immunogenicity, and protective efficacy of the JUNV live-attenuated Candid #1 strain was demonstrated in preclinical studies in both guinea pig and NHP models of AHF. Vaccination campaigns focused on agricultural workers in the JUNV-endemic area showed Candid #1 to be an effective and safe vaccine in humans, and it was licensed in 2006 for use exclusively in Argentina. However, comparison of Candid#1 clonal isolates from blood of vaccinated NHPs revealed a 1000-fold range of virulence among them, raising some concerns about the stability of the Candid #1 vaccine. LASV is the mammarenavirus that poses the highest concern to human health due to morbidity and lethality associated with LF together with the vast LASV-endemic regions and size of the population at risk in Western Africa. Accordingly, LF has been included on the revised list of priority diseases of the WHO R&D Blueprint for which the development of a vaccine is encouraged. Studies involving LF survivors and animal models of LASV infection indicate that early and robust virus-specific CD4 þ and CD8 þ T cell responses, rather than the presence of neutralizing antibodies, which appear late after infection and at low titers, are the best correlates of recovery and protection. Although antibodies do not appear to contribute to viral control and recovery during acute LASV infection, genetically engineered neutralizing monoclonal antibodies can be successfully used for immunotherapy. These data suggest that an ideal LASV vaccine should trigger both long-term robust cell-mediated immunity (CMI) and humoral responses following a single immunization, features frequently displayed by live-attenuated vaccines (LAV). A number of LASV vaccine platforms have shown promising result in animal models of LASV, including LAV candidates based on vaccinia virus, vesicular stomatitis Indiana virus (VSIV), Mopeia virus (MOPV) an OW arenavirus closely related to LASV, yellow fever virus 17D vaccine strain, measles virus (MV)-based vector, alphavirus replicons and the ML29 reassortant carrying the L segment of the non-pathogenic MOPV and the S segment of LASV. The Coalition for Epidemic Preparedness Innovations (CEPI) was created as non-profit organization to accelerate vaccine development against emerging epidemic infections for which the commercial market is insufficient to justify private investment. LASV vaccine candidates supported by CEPI include recombinant VSV and MV expressing LASV GPC and DNA-based vaccines. A single injection of rVSV/LASV-GPC experimental vaccine, in which VSIV G was replaced with LASV GPC, fully protected guinea pigs and NHPs against LASV strains from the same clade. Despite these promising results, there are still some safety concerns because of reported side effects in individuals vaccinated with an Ebola virus vaccine based on the same platform (rVSVDG/EBOVGP), which included post-vaccination arthritis, vector RNAemia, and detection of infectious vaccine virus in the skin of vaccinated individuals. The MV-based vector vaccine platform has shown promising results in human clinical trials providing antibody-based protection. Whether this platform would be effective for development of a vaccine against LASV with predominant T cell-mediated mechanism of protection remains to be determined. Currently there is no human licensed preventive DNA vaccine for any infectious disease, which poses great technical obstacles for developing an DNA-based vaccine against LASV. The LASV candidate vaccine reassortant ML29 induced a robust cross-reacting and protective, sterilizing cell-mediated immune response against strains from distantly-related LASV lineages in a guinea pig model of LF. In addition, ML29 was shown to be stable and safe in animal models of LF, including immunocompromised NHPs. This finding is relevant because the LASV vaccine target population will likely include individuals with some degree of undiagnosed immune suppression due to the high prevalence of malaria and HIV-1 infections in Western Africa. Progress in arenavirus molecular genetics can also facilitate the implementation of novel strategies for the development of safe and effective LF vaccines based on the use of codon deoptimization and reorganization of the coding and non-coding intergenic regions.

Perspective Since the discovery of LCMV more than 70 years ago, mammarenaviruses have served as important model systems for the study of host–virus interactions. Specifically, studies with LCMV in mice, the virus’s natural reservoir, have uncovered a wide range of principles in the fields of virology and immunology that apply universally to other viral infections, including virus-induced immunopathology, MHC restriction, the contribution of negative immune regulators to viral persistence, and the concept of T cell exhaustion and demonstration that PD-1/PD-L1 blockade can rescue T cell function. In addition to their impact on basic science, mammarenaviruses include several human pathogens that remain a significant public health risk in much of the world. Control of human pathogenic mammarenaviruses faces significant obstacles including current limited diagnostic protocols and sparse resource allocation for vaccine development and other medical countermeasures. However, progress in molecular genetics has facilitated novel approaches for the investigation of mammarenavirus molecular and cell biology, whereas the application of next generation sequencing technologies has brought a major breakthrough to the investigation of mammarenavirus epidemiology including their prevalence and distribution, as well as genetic diversity. This new knowledge has had also great implications for the development of new approaches to antivirals and vaccines to combat human pathogenic mammarenaviruses.

Human Pathogenic Arenaviruses (Arenaviridae)

517

Disclaimer The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the US Department of the Army, the US Department of Defense, the US Department of Health and Human Services, or of the institutions and companies affiliated with the authors.

Further Reading Andersen, K.G., Shapiro, B.J., Matranga, C.B., et al., 2015. Clinical sequencing uncovers origins and evolution of Lassa virus. Cell 162 (4), 738–750. Basler, C.F., 2017. Molecular pathogenesis of viral hemorrhagic fever. Seminars in Immunopathology 39 (5), 551–561. Bederka, L.H., Bonhomme, C.J., Ling, E.L., Buchmeier, M.J., 2014. Arenavirus stable signal peptide is the keystone subunit for glycoprotein complex organization. mBio 5 (6), e02063. Boisen, M.L., Hartnett, J.N., Shaffer, J.G., et al., 2018. Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever. Scientific Reports 8 (1), 5939. Buchmeier, M.J., Peters, C.J., de la Torre, J.C., 2007. Arenaviridae: The virus and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, fifth ed., vol. 2. Philadelphia, USA: Wolters Kluwer Health/Lippincott Williams & Wilkins, pp. 1792–1827. Golden, J.W., Hammerbeck, C.D., Mucker, E.M., Brocato, R.L., 2015. Animal models for the study of rodent-borne hemorrhagic fever viruses: Arenaviruses and hantaviruses. BioMed Research International 2015, 793257. Hastie, K.M., Kimberlin, C.R., Zandonatti, M.A., MacRae, I.J., Saphire, E.O., 2011a. Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 30 to 50 exonuclease activity essential for immune suppression. Proceedings of the National Academy of Sciences of the United States of America 108, 2396–2401. Hastie, K.M., Zandonatti, M.A., Kleinfelter, L.M., et al., 2017. Structural basis for antibody-mediated neutralization of Lassa virus. Science 356, 923–928. Jae, L.T., Raaben, M., Herbert, A.S., et al., 2014. Virus entry. Lassa virus entry requires a trigger-induced receptor switch. Science 344, 1506–1510. Kranzusch, P.J., Schenk, A.D., Rahmeh, A.A., et al., 2010. Assembly of a functional Machupo virus polymerase complex. Proceedings of the National Academy of Sciences of the United States of America 107, 20069–20074. Lukashevich, I.S., 2013. The search for animal models for Lassa fever vaccine development. Expert Review of Vaccines 12 (1), 71–86. Raaben, M., Jae, L.T., Herbert, A.S., et al., 2017. NRP2 and CD63 are host factors for Lujo virus cell entry. Cell Host Microbe 22, 688–696. Radoshitzky, S.R., Buchmeier, M.J., Charrel, R.N., et al., 2019. ICTV virus taxonomy profile: Arenaviridae. Journal of General Virology 100 (8), 1200–1201. doi:10.1099/jgv.0.001280. Sarute, N., Ross, S.R., 2017. New world arenavirus biology. Annual Review of Virology 4 (1), 141–158. Vela, E., 2012. Animal models, prophylaxis, and therapeutics for arenavirus infections. Viruses 4, 1802–1829. Watanabe, Y., Raghwani, J., Allen, J.D., et al., 2018. Structure of the Lassa virus glycan shield provides a model for immunological resistance. Proceedings of the National Academy of Sciences of the United States of America 115, 7320–7325.

Human Polyomaviruses (Papillomaviridae) Melissa S Maginnis, The University of Maine, Orono, ME, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of M. Safak, Polyomaviruses of Humans, In Reference Module in Biomedical Sciences, Elsevier Inc., 2018, doi:10.1016/B978-0-12801238-3.02646-5.

Nomenclature

NCCR Non-coding control region NJPyV New Jersey polyomavirus PML Progressive multifocal leukoencephalopathy PyV Polyomavirus PyVAN Polyomavirus-associated nephropathy STAg Small T (tumor) antigen STLPyV St. Louis polyomavirus TSPyV Trichodysplasia-spinulosa polyomavirus VP Viral protein WHO World Health Organization WUPyV Washington University polyomavirus

BKPyV BK polyomavirus HIV Human immunodeficiency virus HPyV Human polyomavirus JCPyV JC polyomavirus KIPyV KI polyomavirus LIPyV Lyon IARC polyomavirus LTAg Large T (tumor) antigen MCC Merkel cell carcinoma MCPyV Merkel cell polyomavirus MWPyV Malawi polyomavirus

Glossary IHC Immunohistochemistry, an experimental method used in diagnosis to test for the presence of antigens, such as viral antigens, in a tissue section through the use of antigenreactive antibodies. Receptor A molecule that is expressed in the cell membrane and serves as an attachment site for a ligand such as a virus.

Seroprevalence The frequency of positive occurrence of a particular disease or condition (viral infection) within a population as measured by serological (blood) tests. Tissue tropism Specific cells and tissues that are able to support the replication of a particular virus.

Introduction Human polyomaviruses (HPyVs) commonly infect the majority of the population, and serological studies suggest that transmission occurs during childhood but seroprevalence continues to increase into adulthood. Transmission of HPyVs is thought to occur via direct person-to-person contact, contact with fomites, or through the ingestion of contaminated food or water. HPyVs demonstrate a high degree of species-specificity, and thus tractable animal models for studies of HPyVs are lacking, and the majority of the clinical data is from observations of human subjects. In addition, this group of viruses exhibits a narrow tissue tropism, generally targeting specific tissues within the host. HPyVs establish life-long persistent infections in target host cells resulting in asymptomatic infections. However, under conditions of immunosuppression such as HIV infection or use of immunosuppressive agents for immune-mediated diseases, HPyVs can become reactivated and cause a range of serious clinical diseases. BK and JC polyomaviruses were first identified in the 1970s after isolation from patients with nephropathy and progressive multifocal leukoencephalopathy (PML), respectively, and are named after the patients. Another human polyomavirus was not identified until the 2000s, and since that time, there have been 11 newly-identified human polyomaviruses isolated from various tissues and fluids including respiratory samples, urine, stool, blood, skin, and liver (Table 1). The increase in HPyV isolation and identification was ignited by improved technologies such as rolling circle amplification (RCA) and high-throughput viral sequencing. Karolinska Institute polyomavirus (KIPyV) and Washington University polyomavirus (WUPyV) were both isolated from nasopharyngeal tissue and named for the research institute credited with the identification. Merkel cell polyomavirus (MCPyV) was isolated from individuals with Merkel cell carcinoma (MCC) and was identified as a causative agent of MCC. Shortly thereafter, HPyV6 and HPyV7 were identified from normal skin tissues, although they are now linked to pathologies of the skin including dermatoses. The eighth polyomavirus identified was Trichodysplasia Spinulosa-associated polyomavirus (TSPyV) named for the TS skin lesions from which it was isolated. HPyV9 was isolated from blood, skin, and urine of a kidney transplant recipient, and HPyV10 or Malawi (MWPyV) was isolated from stool samples and from condylomas from a patient with warts, hypogammaglobulinaemia, infections, and myelokathexis (WHIM) syndrome. St. Louis polyomavirus (STLPyV)/(HPyV11) was identified in stool samples from children in Malawi, The gambia and St. Louis. HPyV12 was isolated from human liver tissue and stool, and New Jersey polyomavirus (NJPyV)/(HPyV13) was isolated from a muscle biopsy and skin lesions in a pancreas transplant recipient with vasculitis and necrotic plaques. The most recently identified polyomavirus, Lyon IARC polyomavirus

518

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21316-2

Human Polyomaviruses (Papillomaviridae)

Table 1

519

Human polyomaviruses and associated diseases

Number Designation

Polyomavirus

Isolation source

Year of Disease association ID

HPyV1

BK polyomavirus (BKPyV)

Urine

1971

HPyV2 HPyV3 HPyV4 HPyV5 HPyV6 HPyV7 HPyV8

JC polyomavirus (JCPyV) Karolinska Institute polyomavirus (KIPyV) Washington University polyomavirus (WUPyV) Merkel cell polyomavirus (MCPyV) Human polyomavirus 6 Human polyomavirus 7 Trichodysplasia spinulosa-associated polyomavirus (TSPyV) Human polyomavirus 9 Malawi polyomavirus (MWPyV) St. Louis polyomavirus (STLPyV) Human polyomavirus 12 New Jersey polyomavirus (NJPyV)

Brain Nasopharyngeal tissue Nasopharyngeal tissue Tumors, skin Skin Skin Skin lesions

1971 2007 2007 2008 2010 2010 2010

Polyomavirus-associated nephropathy, hemorrhagic cystitis Progressive multifocal leukoencephalopathy Potential mild respiratory illness Potential mild respiratory illness Merkel cell carcinoma Pruritic and dyskeratotic dermatitis Pruritic and dyskeratotic dermatitis Trichodysplasia spinulosa

Blood, skin, urine Stool, condylomas Stool Liver, rectum, stool Muscle, skin

2011 2012 2012 2013 2014

? ? ? ? ?

HPyV9 HPyV10 HPyV11 HPyV12 HPyV13

(LIPyV), discovered in 2017, was isolated from skin swabs and oral fluids. LIPyV has not yet been formally assigned as a human polyomavirus species, yet it could be the fourteenth HPyV. HPyVs within the family of Polyomaviridae share remarkable similarities in structure and genome composition and organization. In addition to their overall similarities in virion structure and genome organization, there are many parallels in the general schema of the infectious viral cycle including viral attachment, entry, trafficking, and the temporally regulated gene expression. However, the specific cellular factors required for a productive infectious cycle for each virus are disparate. Furthermore, polyomaviruses demonstrate a broad tissue tropism in the human host infecting skin, kidney, and brain cells, yet pathogenic viral disease is generally limited to those with an underlying immunosuppression. Polyomavirus disease ranges from nephritis to a fatal infection of the brain to skin cancer. The commonalities across structure, genome, and life cycles of HPyVs is described herein as well as detailed sections on specific differences in viral life cycles and clinical diseases of select HPyVs, including BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), Merkel cell polyomavirus (MCPyV), and other recently-discovered HyPVs associated with significant human disease manifestations.

Virion Structure Human polyomaviruses are classified by a nonenveloped protein capsid that is B40–45 nm in diameter (Fig. 1). The virion capsid is comprised of two to three structural proteins, viral protein 1 (VP1), VP2, and VP3. VP1 is a pentameric protein that associates with either a VP2 or a VP3 in the interior of the virion capsid (Fig. 1). VP1 is the only capsid protein expressed on the exterior of the surface and serves as the viral attachment protein. Polyomavirus capsids are made up 360 molecules of VP1 arranged into 72 pentamers. The pentameric VP1 proteins associate with neighboring VP1 pentamers via C-terminal extensions in which the 72 pentamers are organized as 60 hexamers and 12 pentamers allowing for the formation of a spherical, capsid with a T ¼7d skewed icosahedral structure. VP1 pentamers from all human polyomaviruses exhibit a b barrel jelly roll structure of VP1 monomers, formed by anti-parallel b strands, and the connecting loops of which serve as the binding site for many HPyV sialic-acid containing receptors. The virion capsid is stabilized by intra- and inter-pentameric disulfide bonds and calcium ions, and assembly occurs entirely in the nucleus where the concentration of calcium ions is highly regulated. The N-terminal region the VP1 pentamers faces the interior of the virion and binds the minor capsid proteins, VP2 or VP3, which can interact with genomic DNA (Fig. 1).

Genome Encased within the virion capsid is the circular double-stranded (ds) DNA genome that is approximately 5000 bp in size and packaged with cellular histones. The simple genome is comprised of 5–6 protein-coding genes resulting in a relatively limited protein repertoire, yet the polyomaviruses genome structure and organization are fairly conserved among HPyVs (Fig. 2). Polyomavirus genomes are divided into three regions organized in a bidirectional manner into early and late regions separated by a non-coding control region (NCCR) or regulatory region (RR). The early region of the genome encodes for the early viral proteins, the nonstructural proteins large T antigen (LTAg), small t antigen (STAg), and in some cases middle t antigen (MT), alternative T (ALT), and alternatively spliced T0 proteins are encoded from an alternative splicing of a single mRNA transcript. The late viral

520

Human Polyomaviruses (Papillomaviridae)

Fig. 1 Icosahedral structure of nonenveloped human polyomavirus virion. (A) High resolution structure of BKPyV resolved by cryo-electron microscopy (EM) at 3.8 Å demonstrating. (B) A view of the BKPyV virion interior with VP1 pentons (gray), VP2 (blue), and VP3 (green) and packaged dsDNA (yellow, pink). (C) Enlargement from structure (in (B)) demonstrates VP2/VP3 interactions with dsDNA, which is wrapped in cellular histones. Modified and reproduced from Hurdiss et al., 2018. Structure. 26 (5), 839–847. and Hurdiss et al., 2016. Structure. 24 (4), 528–536. under Creative Commons licenses.

Fig. 2 General schematic of human polyomavirus genome. The dsDNA genome size is B5000 bp and is organized into early and late regions separated by the non-coding control region (NCCR). The NCCR contains the origin of replication (ORI), and replication is driven through several functions of TAg, including helicase activity to unwind the viral dsDNA genome. The early region encodes for T antigen proteins including large T antigen (LTAg), small T antigen (STAg), and other splice variants of T antigen (indicated by dashed line, varies by HPyV). Not pictured are middle T Ag and truncated T Ag, present in some HPyVs. The late region encodes for structural proteins viral protein 1 (VP1), VP2, and VP3 (lacking in MCPyV). Some HPyVs, including JCPyV and BKPyV also encode for the nonstructural protein agnoprotein. Figure artwork generated by Michael Wilczek.

genes include the viral structural proteins VP1, VP2, and VP3 (except for MCPyV). JCPyV and BKPyV encode for an additional protein, agnoprotein, which is expressed predominantly in the cytoplasm of infected cells and is required for release of infectious progeny into the supernatant. JCPyV Agno protein has been demonstrated to play a role in egress through the nuclear membrane. The NCCR demonstrates genomic diversity among polyomaviruses and contains promoters for early and late genes, the origin of replication (ORI), a binding domain for LTAg, and promoters and enhancers. HPyV NCCRs undergo rearrangements within the host during active viral replication, yielding insertions, deletions, and duplications. The rearranged NCCR of polyomaviruses are generally associated with enhanced viral replication, expansion of tissue tropism, and thus viral pathogenesis. Viral strains with rearranged NCCRs are generally only found during viremia or pathogenic states. A number of HPyVs, including BKPyV, JCPyV, and MCPyV have been demonstrated to also encode for microRNAs (miRNAs) complementary to LTAg. These miRNAs can function by binding to LTAg mRNA and regulating expression of early genes and contribute to viral immune evasion.

Human Polyomaviruses (Papillomaviridae)

521

Fig. 3 General schematic of human polyomavirus life cycle. (1) Viral attachment is mediated by VP1 binding to sialic-acid containing receptors or alternate receptors such as glycosaminoglycans or serotonin receptor. (2) Viral entry is mediated via endocytosis (mechanism varies by HPyV). (3) HPyVs traffic through the endocytic compartment to the endoplasmic reticulum (ER), and (4) the virions are partially disassembled in the ER. (5) Transcription begins with viral early genes followed by replication of the dsDNA genome then transcription of viral late genes. (6) Virions are assembled in the nucleus before (7). release, possibly through cell lysis. Figure generated by Jeanne DuShane and created in BioRender.com.

Infectious Cycle Despite differences in tissue tropism and broad effects on host cells, the current information for human polyomaviruses suggests they follow the same general schema for infection of host cells (Fig. 3). HPyVs bind to cell surface receptors on host cells, and for most HPyVs this has been demonstrated to be a carbohydrate sialic acid receptor. The nature of the carbohydrate receptor differs among polyomaviruses but can include a2,3-, a2,6-, or a2,8-linked sialic acids or alternate receptor such as glycosaminoglycans. In fact, a number of HPyVs attach to a sialic acid receptor expressed on a ganglioside, a glycosphingolipid comprised of a ceramide in a lipid-rich region of the membrane with an extracellular carbohydrate moiety with sialic acid residues. The utilization of gangliosides results in a lipid-mediated caveola- or lipid raft-mediated internalization process for some HPyVs. However, JCPyV requires a unique sialic acid receptor a2,6-linked lactoseries tetrasaccharide c (LSTc) and also requires a secondary receptor, the 5-hydroxytryptamine family of receptors (5-HT) in the 2 subfamily and clathrin-mediated endocytosis viral internalization. BKPyV has also been demonstrated to enter specific cell types via a caveolae- and clathrin-independent mechanism. Regardless of the internalization strategy, polyomaviruses are internalized into the endocytic compartment and traffic to the endoplasmic reticulum (ER). Within the ER, resident chaperones, disulfide isomerases and reductases, and components of the ER-associated protein degradation (ERAD) pathway facilitate partial uncoating of the viral capsid. The partially disassembled virion is then deposited into the cytoplasm and retrotranslocates to the nucleus where transcription and replication ensues in a temporally-regulated process. Transcription of viral early genes is initiated through host cell RNA polymerase II and transcription factors binding to the promoter region of early genes. A precursor mRNA is generated, which is then alternatively spliced to produce large tumor (T) antigen (LTAg), small T antigen (STAg) and TAg splice variants. LTAg is considered a master regulator of polyomavirus infection, exerting multiple functions necessary for the infectious process. The expression of LTAg and STAg drives cell cycle progression from G0 or G1 into S phase by binding to cell cycle regulators pRb, p53, and PP2A. LTAg binds pRb family members through the conserved LXCXE motif. Some HPyVs have additional open reading frames (ORFs) that encode for proteins although their functions are not well described. TSPyV and MCPyV encode for additional T antigens, middle T antigen (MTAg) and alternative tumor antigen (ALT), respectively. While the function of MTAg and ALT are not yet well characterized, they are expected to play roles in cellular proliferation and transformation due the presence of Rb binding motifs. Viral replication is carried out through reliance on the cellular replication machinery and proceeds in a bidirectional manner. LTAg binds to the ori in the NCCR, unwinds the dsDNA genome through helicase activity, and drives viral replication. Finally, late viral gene transcription ensues on the complementary strand in the opposing direction from the early genes to produce the late viral genes VP1, VP2, and, in most cases, VP3, which then encapsidate the virion in the nucleus. MCPyV lacks a VP3, which in other HPyVs is translated from an internal start site within the VP2 transcript. JCPyV and BKPyV also produce the late viral gene product agnoprotein, which has a number of reported properties including roles in transcription and replication of the viral genome and viral release through viroporin activity. Polyomaviruses are thought to be released from infected cells upon viral lysis of the host cells, such as through the viroporin activity of JCPyV agnoprotein. However, nonlytic release for polyomaviruses, such as BKPyV, has also been reported and thus the process is not well understood.

522

Human Polyomaviruses (Papillomaviridae)

BKPyV BKPyV Clinical Features BKPyV, also classified as HPyV1, was first identified in 1971 when it was isolated from the urine of a renal transplant patient with ureteric stenosis. BKPyV is shed in urine and is usually acquired in childhood, and 50%–90% of individuals are seropositive by adulthood. Transmission is thought to occur via a peroral or respiratory route, as BKPyV can be detected in the respiratory tract and tonsils, but could also occur through semen, transfusion, or organ transplantation (renal allografts). Initial infection results in either an asymptomatic infection or a mild respiratory illness before dissemination to the kidney and urogenital tract, where the virus establishes a persistent infection. BKPyV is shed in the urine of both healthy and immunosuppressed individuals, yet those with immune suppression have higher viral loads, which could be due to viral reactivation. Thus, BKPyV can be diagnosed by qPCR of urine or plasma samples. It is also detected through urine cytology as non-cell associated particles, and virions in infected “decoy cells” via electron microscopy. In immunosuppressed individuals, BKPyV can become reactivated resulting in clinical disease, most commonly polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystisis (PyVHC). PyVAN results from BKPyV infection in renal tubular epithelial cells in kidney transplants and causes renal dysfunction. BKPyV reactivation due to immunosuppression is most common in kidney transplant (KT) recipients, occurring in up to 10% of KT recipients, which can result in 50%–70% allograft loss. Due to a high incidence of allograft rejection due to PyVAN, kidney transplant patients are regularly screened for BKPyV by viral load in the urine or plasma, and PyVAN is diagnosed by tissue biopsy. However, other BKPyV-associated clinical diseases including ureteric stenosis, encephalitis, meningoencephalitis, pneumonia, and vaculopathy have been reported in immunocompromised patients. In individuals with PyVAN, BKPyV can also be detected in the urothelial cells of the bladder. On the other hand, PyVHC can occur in up to 15% of individuals who receive an allogenic hematopoietic stem cell transplantation or those otherwise immunosuppressed due to damage to the bladder. Additionally, the WHO International Agency for Research on Cancer (IARC) Working Group has classified BKPyV as Group 2B for “possibly carcinogenic to humans,” as increasing evidence suggests a role for BKPyV in the development of bladder cancer and other cancers of the urinary epithelia in organ transplant recipients, yet causation has not been established. Ureteric stenosis has been observed in transplant patients, characterized by damage to the ureteric epithelium due to inflammation. Finally, some cases of BKPyVinduced CNS disease have been observed including meningoencephalitis in an individual with AIDS and viral encephalitis in a pediatric heart transplant recipient. A case of BKPyV-induced progressive multifocal leukoencephalopathy (PML) has also been reported, in which samples were positive for BKPyV and not JCPyV, which is the etiological agent for PML. Treatments for BKPyVinduced disease varies depending upon the disease pathogenesis and stage of disease. Treatment for PyVHC is supportive and involves palliative care and analgesia. The first line in PyVAN treatment involves altering the dosage or type of immunosuppressive drugs administered to transplant patients. Antiviral drugs are often used in addition to immunosuppressive agents to treat PVAN including nucleoside analog cidofovir or the lipid conjugate brincidofovir.

BKPyV Infectious Cycle BKPyV attaches to ganglioside receptors containing a2,3- or a2,8-linked sialic acid of the b-series gangliosides GD2, GD3, GD1b, and GT1b. Following attachment to sialic acid receptors, BKPyV is internalized into cells through either a caveolae- or lipid-mediated pathway or through a clathrin- and caveolae-independent pathway, suggesting cell-type dependent differences in viral entry mechanisms. BKPyV then traffics through the endocytic compartment along microtubules to eventually arrive in the ER where the capsid is partially disassembled. In addition, autophagic and Rab-18 þ vesicles are reported to play a role in trafficking of BKPyV. Similar to other polyomaviruses, BKPyV utilizes ER reductases, disulfide isomerases, and components of the ERAD pathway, including Derlin and heat shock proteins (Hsp), for particle disassembly. Derlin-1, Hsp105, and small glutamine rich tetratricopeptide repeat containing alpha (SGTA) mediate the movement of BKPyV from the ER to the cytosol. BKPyV is capable of transport into the nucleus through the nuclear pore complex via the importin a/b pathway through the nuclear localization signals (NLS) in VP2 and VP3. Like most DNA viruses, once in the nucleus, BKPyV hijacks the cellular transcription and replication machinery. In order to reprogram the cell into a viral factory, BKPyV activates the cellular signaling pathways mitogen activated protein kinase (MAPK) and Akt/ mTOR (mammalian target of rapamycin), which leads to increased cellular proliferation, and thus aids in the production of virus. Viral transcription begins with production of the early genes, yielding the production of T antigens including LTAg, STAg, and truncated TAg (truncTAg). LTAg binds to pRb and p53, pushing the cell into S phase, further inducing cell division and preventing apoptosis. In addition, BKPyV activates the DNA damage response signaling pathway by activating the ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-Related) kinases, which protect the host cells during viral replication. This counteraction of the DNA damage induced by BKPyV infection and activation of ATR is critical for production of viral progeny. Production of TAg and regulation of the cell cycle drive viral replication. The BKPyV archetype strain is isolated from urine samples and is the transmissible form of the virus. The archetype strain has five sequences blocks (O, P, Q, R, and S) with the ORI, TATA boxes, and binding sites for cellular transcription factors necessary for infection including Ap-1, Ets-1, NF1, NFAT, nuclear factor kappa B (NF-κB), and Sp1. The NCCR of viral isolates from kidney and other sites during disease states exhibit variation in the NCCR with rearrangements of the sequence blocks. The rearranged strains usually contain deletions of P, Q, R, or S blocks and multiplication of the P block. The Gardner strain is the prototype of a rearranged BKPyV strain, and the Dunlop strain represents another rearranged strain commonly used in

Human Polyomaviruses (Papillomaviridae)

523

BKPyV studies. BKPyV rearranged strains have been isolated from the kidneys of patients with BKPyVAN and from the blood, and those strains are associated with increased viral replication and higher viral loads. The rearrangements of the NCCR likely confers enhanced cellular tropism, allowing for increased replication in additional tissue types within the host and in vitro, as archetype strains replicate poorly in most cell culture models. BKPyV produces the late genes VP1, VP2, VP3, and agnoprotein. BKPyV release from cells following virion assembly has been shown to occur via lytic and non-lytic egress, and again may reflect cell-type dependent differences. Agnoprotein may play a role in virion release from cells, as proposed for JCPyV.

JCPyV JCPyV Clinical Features JC polyomavirus (JCPyV), or HPyV 2, was also identified in 1971 when it was isolated from the brain of a patient with PML. JCPyV infects the majority of the population with seroprevalence ranging from 30% to 80%, depending upon geographical location and sampling. JCPyV is shed in the urine and can be found in untreated wastewater, and thus is predicted to spread perorally. Initial infection is thought to occur in the tonsillar tissue but virus has not been detected in the saliva. JCPyV then spreads to secondary sites of replication including the kidneys and B cells of the bone marrow, where it establishes a lifelong persistent infection. While infection of the kidney is asymptomatic, periodic viral shedding occurs as virus can be detected in the urine of healthy individuals. JCPyV remains in the kidney in healthy individuals with full immune function, yet in individuals with severe immunosuppression, JCPyV can become reactivated and spread to the central nervous system (CNS) possibly crossing the blood-brain barrier through B lymphocytes or another mechanism. Another model proposes that JCPyV may persist in the brain and then become reactivated locally in the CNS. Within the brain, JCPyV infects glial cells, astrocytes and oligodendrocytes, which are responsible for production and maintenance of myelin in the brain. JCPyV infection of these cells results in cytolytic destruction resulting in demyelination and the causation of the fatal, neurodegenerative disease PML. Symptoms of PML include impaired cognitive function, motor dysfunction such as paralysis, hemiplegia, and speech and vision disturbances. In addition, JCPyV replication in neurons of the cerebellum has been associated with development of granule cell neuronopathy (GCN), which results in clinical symptoms including ataxia and progressive cerebellar atrophy. GCN can occur in conjunction with PML or independently, and has been associated with mutations in the C-terminus of VP1. PML is diagnosed by magnetic resonance imaging (MRI) and the presence of JCPyV DNA in the cerebrospinal fluid (CSF). The development of PML is rapid and progressive, leading to demyelination within the white matter of the brain resulting in multifocal lesions. Individuals at risk for development of PML include those with HIV or those receiving immunomodulatory therapies. In the HIV-infected population, PML develops in B5% of individuals. While the use of highly active antiretroviral therapy (HAART) has lessened the burden on HIV-infected individuals, PML is still considered an AIDS-defining illness with a 50% mortality rate in this population. The second most at-risk population are those receiving the immunomodulatory therapy natalizumab for immunemediated diseases such as relapsing remitting multiple sclerosis (MS). Natalizumab is a monoclonal antibody specific for very late antigen-4 (VLA-4), a4b1 integrin, which blocks T cell extravasation to the brain, where autoreactive T cells can attack myelin, the hallmark of MS pathogenesis. In natalizumab treatment of a JCPyV-seronegative patient, the incidence of PML development is 1:1000, yet the incidence can increase to as great as 1:100 dependent upon JCPyV serostatus, duration of natalizumab treatment, and prior immunosuppressive use. Further, there have been cases of PML in individuals receiving humanized monoclonal antibody treatments (rituximab, efalizumab, and infliximab) or dimethyl fumarates and other fumaric acid ester-containing drugs for immunomodulatory therapies such as rheumatoid arthritis (RA), Crohn’s disease, and psoriasis. However, a particular predilection for PML in MS patients is illuminated by the nearly 800 cases of natalizumab-induced PML in this patient subset since 2006. Thus, natalizumab remains on the market but contains a black box warning of the potentially fatal outcomes due to PML. PML is fatal within 1–2 years of symptom onset, and can be fatal within 3 months of symptom onset if left completely untreated. Currently the treatment options for JCPyV are very limited. In most cases, PML treatment begins with addressing the underlying immunosuppression. Treatment of the immunosuppression for HIV-infected individuals includes initiation or modifications to HAART regimens, while those on immunosuppressive therapies will undergo cessation of therapy. While these treatments can help to slow PML progression, they do not halt PML development. Further, there is currently no way to reverse demyelination, and thus these individuals often suffer severe debilitation. Moreover, removal of the immunosuppressive therapy can result in immune reconstitution inflammatory syndrome (IRIS), which can cause neurological worsening and result in fatality. Novel treatments for JCPyV are currently under development and have yielded some promising results under “compassionate use” trials. JCPyV VP1 vaccines in combination with immune boosting adjuvants cytokine interleukin 7 and toll-like receptor (TLR) 7/9 agonist led to clinical improvement and slowed PML progression. In addition, monoclonal antibodies capable of neutralizing PML variants with VP1 mutations also demonstrated promising results in the clinic. Use of selective serotonin reuptake inhibitors (SSRIs), such as mirtazapine, in an off-label use, has resulted in mixed clinical results, suggesting that this avenue of treatment requires further investigation. Additionally, JCPyV infection has been detected in the brain in other non-PML disease states. For instance, JCPyV-associated encephalopathy has been reported, in which cortical pyramidal neurons of the gray matter were infected. Viral meningitis with the presence of JCPyV and absence of other viral infections have also been reported. JCPyV infection in the kidney is generally not associated with clinical disease, yet JCPyV has been reported to cause PyVAN in rare cases. Further, JCPyV has also been designated as Group 2B for “possibly carcinogenic to humans”, due to potential associations with colon cancer, yet definitive causation has not been established.

524

Human Polyomaviruses (Papillomaviridae)

JCPyV Infectious Cycle JCPyV infection is mediated by VP1 binding to cellular sialic acid receptor, a2,6 LSTc. The X-ray crystal structure of VP1 in complex with LSTc revealed specific VP1 residues (L54, S266, S268) that make key contacts with LSTc. Mutation of these residues in vitro results in reduced infectivity and specifically reduced viral attachment. Following viral attachment to sialic acid receptors, JCPyV enters cells through clathrin-mediated endocytosis (CME) through the 5-hydroxytryptamine (5-HT) 2 subfamily of receptors. The internalization of JCPyV via CME is dependent upon the activation of clathrin scaffolding proteins adapter protein 2 (AP2), b-arrestin, and the GTPase dynamin. Following internalization, JCPyV traffics into the endocytic compartment through the Rab 5-positive endosomes and caveolin 1-positive vesicles and traffics along microtubules and microfilaments. The virus eventually arrives at the ER before retrotranslocation to the nucleus for transcription and replication of the viral genome. JCPyV infection also induces the activation of the cellular signaling pathway, MAPK and activation of the terminal kinase extracellular signal-regulated kinase (ERK), which can reprogram cells to induce proliferation and growth. The activation of the MAPK pathway is necessary for viral transcription and infection in culture. JCPyV relies on the expression and activation of specific cellular transcription factors in order to complete the infectious process. Transcription and production of the JCPyV early gene product, LTAg regulates the JCPyV infectious cycle. LTAg binds to Rb and p53 to drive the cell into S phase inducing cell cycle progression and inhibiting apoptotic responses, respectively. Initial infection of the kidney and subsequent transmission of JCPyV in the urine of infected individuals occurs with the archetype (CY) strain. However, within the infected individual, the virus undergoes significant mutations in the NCCR, during high levels of replication, yielding rearranged NCCRs. Isolates from the brain, denoted “PML-types” contain rearranged NCCRs in which the six sequence blocks (a, b, c, d, e, and f) have undergone deletions of repressor elements and duplication of enhancer elements. The Mad1 prototype strain is a PML-type strain that was isolated from the brain of a PML patient in Madison, WI. Mad1 contains deletions of b and d and duplication of an enhancer element (a, c, e, a, c, e, f). These rearrangements result in duplication of the TATA box and transcription factor binding sites for transcription factors that have been reported to play a critical role in JCPyV infection including DDX-1, Sp1, Spi-B, NFAT4, NF-1X, NF-κB, Oct-6, and YB-1/Pura. While the NCCR rearrangements facilitate additional transcription factor binding sites, these alterations are also necessary for neurotropism. Interestingly, mutations in the sialic-acid binding sites in VP1 are also associated with neurotropism in the host, as 90% of viral isolates from individuals with PML contain at least one mutation in one of the sialic acid-binding sites: L54, S266, and/or S268. The mutation of VP1 and NCCR rearrangements suggest that mutations may occurs during times of enhanced replication, ultimately resulting in increased neurovirulence.

MCPyV MCPyV Clinical Features MCPyV is a common polyomavirus of the skin with B60%–80% seroprevalence in the population, and the majority of individuals exposed during childhood. While MCPyV persists in the skin of healthy individuals, it also has been identified as a causative agent of Merkel cell carcinoma (MCC). To date, MCPyV is the only polyomavirus with a clear association with human cancer. MCC is a rare, yet aggressive form of skin cancer that occurs in sun-exposed regions of the body including the head, neck, arms, and legs. Elevated risk for MCC is associated with advanced age, exposure to sunlight and UV radiation, and immunosuppression. The link to immunosuppression guided the search for an infectious cause of MCC, especially given that risk of MCC development is 10-fold higher in individuals with HIV/AIDS. Immunosuppression due to autoimmune conditions such as rheumatoid arthritis (RA), solid organ transplants, and other co-morbidities such as chronic lymphocytic leukemia and non-Hodgkin lymphoma also increase the risk of MCC development. MCC is most common in those with advanced age, as the median age of diagnosis is 76 years old (National Cancer Data Base). However, HIV-positive individuals can experience an earlier age of onset and development of MC tumors in areas not exposed to the sun such as the oral or anogenital mucosa. MCC has a 46% rate of disease-associated mortality, and prognosis is especially poor in AIDS patients that can have advanced tumor stage and reduced survival. Diagnosis of MCC is usually carried out by skin biopsy and pathological examination. Biopsy samples are stained with antibodies such as CM2B4, which detects MCPyV T antigen, and the cytokeratin 20 antibody that is a marker of Merkel cells. In addition, individuals with MCC may undergo further testing such as sentinel lymph node biopsy and/or a CT scan to determine if the cancer has metastasized. Further, serology testing for MCPyV oncoprotein antibody titer has been recommended for determining patient prognosis, as MCPyV-positive tumors usually have a better prognosis in comparison to MCPyV-negative tumors. Thus, tracking antibody titers can help in MCC disease management and risk stratification for treatments. It is recommended that PCR testing for the presence of MCPyV should be used in conjunction with other methods, as PCR results can include false positives due to contamination as well as false negatives due to strain-specific differences. The treatment options for MCC vary greatly depending upon the stage of cancer, MPCyV status, and underlying immunosuppression. In some cases of early state MCC, surgical excision or Mohs micrographic surgery may be the only course of treatment necessary. Yet, in other cases, additional radiation therapy is warranted. In metastasized MCC, chemotherapy may be the treatment course, and while responsive in the short term, there is usually developed resistance and also immunosuppression, yielding reduced immune reaction to the tumors. Another treatment option for MCC, which has yielded successful results, include immune checkpoint inhibitors (ICIs), programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) antibodies. PD-L1 is upregulated in MCPyV-positive MCC tumors, and when PD-L1 is recognized by PD-1 receptors on T lymphocytes, the immune

Human Polyomaviruses (Papillomaviridae)

525

response to the tumor is down-regulated, resulting in tumor immune evasion. Thus, ICIs targeting either PD-1 or PD-L1 stimulate the anti-tumor immune response so immune cells can recognize and destroy tumor cells. This type of treatment is only possible in individuals who have a stable, robust immune system, and thus is not effective in those who are immunocompromised due to HIV or immunosuppressive therapy for underlying immune-mediated disease. In fact, patients with higher levels of CD8 þ and CD3 þ T lymphocytes have a better prognosis, reduced metastases, and improved survival rates. MCPyV was first detected in 2008 when it was isolated from tumors of individuals with Merkel cell carcinoma. MCPyV was identified using digital transcriptome subtraction. The presence of MCPyV in tumors was confirmed by sequencing and Southern blot analysis. Of the 10 tumors tested, 8 were positive for MCPyV, confirming a strong association (80% MCPyV positive), which led to the WHO IARC classification as a Group 2A, “carcinogenic to humans.” The genome of MCPyV was found to be clonally integrated into MCC tumors, making MCPyV the only human polyomavirus that is known to integrate into the host genome. The integration occurs before clonal expansion of the tumor and is hallmarked by expression of small T antigen (ST) and a mutated form of large T antigen (LT), which inhibits viral replication. However, the viral structural proteins VP1 and VP2 are not expressed in tumors suggesting that active viral replication is not occurring in these tissues. The mutations that arise in LT include a truncation, deletion of the DNA binding region (ORI), and deletion of the helicase domains and growth suppressing domains. The LXCXE motif that binds to the tumor suppressor pRb remains intact, and thus when LT binds to Rb the tumor suppressor functions are inactivated contributing to tumor development. Despite the role of LT in tumorigenesis, MCPyV ST is the major oncogenic protein leading to tumor progression in MCC. In fact, expression of ST can lead to cellular transformation in vitro and in vivo independent of LT expression. ST can increase levels of LT through the stabilizing domains, and ST can also alter the alter the function of FBXXW7 to inhibit Ub ligase activity leading to accumulation of LT and transcription factors that regulate cell cycle progression including c-Myc and cyclin E. The ST protein also induces hyperphosphorylation of 4E-binding protein (4E-BP1) to promote protein translation and increase cellular proliferation and transformation.

MCPyV Infectious Cycle The mechanism of MCPyV pathogenesis and resultant MCC development is not fully understood, and the cell types infected in the host are not clear. MCC was originally described in 1972 as a trabecular carcinoma of the skin. The name was later changed to MCC named for the Merkel cells, mechanoreceptor cells that aid in perception of touch, within the tumor tissue. However, Merkel cells do not support MCPyV replication. While this might indicate that a lack of replication could facilitate viral integration and the development of MCC tumors, the cells of origin of MCC tumors remains unclear. Epithelial cells and fibroblasts, including human dermal fibroblasts, are infected by MCPyV. Thus, it has been proposed that tumors may be of dermal fibroblast origin or perhaps Merkel cells are infected through a bystander mechanism. Further, expression of particular cell markers of pro/pre-B cells suggest that MCC tumors may originate from B cells. Importantly, tissue tropism of viruses in the host is dictated by the expression of specific host-cell factors necessary for infection and the virus-host cell interactions that ensue. However, a lack of a tractable cell culture model for MCPyV replication has limited the understanding of the MCPyV infectious cycle. Infection is initiated by attachment to cellsurface glycosaminoglycans (GAGs) such as heparan sulfate (HS) proteoglycans. The viral capsid protein VP1 also binds specifically to sialic-acid containing ganglioside receptors with the Neu5Ac motif, such as GT1b, presumably to facilitate a post-attachment cell entry step. The subsequent steps in the infectious cycle remain to be elucidated and may differ greatly in cell types that support active replication in comparison to the integration in pre-tumor cells.

Other HPyVs Since the beginning of the 2000s, there have been 11 (possibly 12), human polyomaviruses identified. The HPyVs represent broad sites of isolation, tissue tropism, and disease outcomes. Due to the relative novelty of these viruses, researchers are still working to understand more about the infectious cycle and viral pathogenesis associated with infections. In some cases, a tissue culture model or an infectious experimental form of the virus have not been established, and thus alternatives are utilized such as molecular, biochemical, or in silico analyses along with utilization of virus like particles (VLPs) or pseudoviruses (PsV). VLPs are generated by the expression of viral capsid proteins, while psuedoviruses are comprised of the viral capsid proteins along with a reporter. Each of these particle types have demonstrated an overall similar size and structure to native polyomavirions and have be useful in experimental studies for the early steps in the infectious cycle. While many HPyV experimental models are still under development, the clinical understanding and seroprevalence of HPyVs has been a focus of study to understand the medical importance of HPyV infections.

Polyomaviruses of the Skin Of the thirteen HPyV species, seven of the HPyVs have been isolated from skin including MCPyV, TSPyV, HPyV6, HPyV7, HPyV9, MWPyV (HPyV10), and HPyV13, suggesting common circulation of these skin HPyVs in the population. As described above, MCPyV is a causal agent in the development of MCC. The other HPyVs of the skin have been associated with other asymptomatic or non-malignant diseases.

526

Human Polyomaviruses (Papillomaviridae)

TSPyV has been isolated from individuals with TS, which is characterized by spiny follicular papules and hyperkeratotic spicules usually localized to the face in immunocompromised individuals. The progression of TS can result in thickening of the skin, disfigurement, and facial spines. The underlying immunosuppression in TS patients has generally been the result of immunosuppressive therapies, organ transplants, or leukemia. Approximately 70% of the population is seropositive for TSPyV, and there is a higher seroprevalence in solid organ transplant patients, including renal transplant recipients. Interestingly, samples obtained from skin show low levels of seroprevalence in the general population. TS is diagnosed through physical examination in combination with biopsy and histopathological analysis. Samples can be analyzed by immunohistochemistry (IHC) with VP1-specific antibodies, electron microscopy for viral particles, or through PCR analysis of purified viral DNA. Transmission of TSPyV is thought to occur via peroral or respiratory route, and viral persistence within tonsillar tissue has been suggested. While TSPyV has been mostly linked to the skin pathology of TS, additional studies have demonstrated that TSPyV can be isolated from blood, feces, urinary tract, and respiratory samples. A case of a respiratory sample from an immunocompromised child with respiratory illness without facial papules spines has been described, suggesting a potential for respiratory illness. TS has been treated with a topical cidofovir cream leading to partial resolution of symptomatic disease. Studies using a TSPyV PsV demonstrated that TSPyV binds to sialylated glycans with an a2,3- and a2,6-linked sialic acid. While the replication cycle for TSPyV has not been characterized, the viral genome encodes for five ORFs: STAg, LTAg, VP1, VP2, and VP3. STAg contains the conserved binding motif for PP2A while LTAg has binding sites for pRb and p53 but a strong interaction of LTAg with p53 has not been confirmed. HPyV6 and HPyV7 were identified from skin swabs from healthy individuals, and the seroprevalence of HPyV6 is reported to be B80% and HPyV7 is B66%. Given the high rate of seroprevalence, HPyV6 and 7 infections are likely asymptomatic in healthy individuals, yet clinical disease has been noted in individuals with immunosuppression due to immunosuppressive therapy, solid organ transplantation, HIV infection, or uncharacterized immunosuppression. Additionally, HPyV6 has also been isolated from tonsils, CSF from a PML patient, feces, and the respiratory tract. HPyV7 has been identified in tonsils, urine, feces, and the respiratory tract. HPyV6 and 7 are can infect keratinocytes in the epidermis of the skin, and both been associated with pruritic and dyskeratotic dermatitis. Additionally, HPyV6 has been associated with epithelial neoplasm, angiolymphoid hyperplasia, and keratoacanthomas. HPyV7 has been associated with thymic epithelial tumors. Both viruses are diagnosed by PCR and IHC, as TAg and VP1 can be detected in samples with specific antibodies. HPyV6 and 7 express LTAg, STAg, and VP1, 2, and 3. The receptors for HPyV 6 and 7 have not been identified but structural studies demonstrate that the sialic acid-binding site present in the VP1 attachment protein of many HPyVs is occluded in these viruses, and thus a receptor remains to be identified. LTAgs of HVPyV 6 and 7 have been shown to bind specifically to p53. HPyV9 was isolated from a kidney transplant patient but has also been found from skin swabs, with a low seroprevalence of B20%–50%. HPyV10, MWPyV, and Mexico polyomavirus (MXPyV) represent the different isolates of the same virus, demonstrating a 95%–99% sequence identity. HPyV10 was identified from condyloma samples from a patient with WHIM syndrome, which is characterized by uncontrolled human papillomavirus (HPV) infections. Additionally, HPyV10, MWPyV, and MXPyV have been isolated from stool samples, and seroprevalence is B75% of the population based on a VP1 ELISA. The MWPyV LTAg can bind to pRb and p53, and STAg can bind to PP2A. Interestingly, the recently identified LIPyV, which was isolated from skin swabs and oral fluids has a seroprevalence of B5%, similar to that of NJPyV. LIPyV has been detected in the stool of diarrheatic cats, suggesting a possible feline origin and a host jump mode of transmission.

Polyomaviruses of the Other Tissues KIPyV and WUPyV were originally isolated from the respiratory samples from children with acute respiratory illness. Seroepidemiology studies suggest that transmission occurs during childhood and either causes a mild respiratory illness or is asymptomatic and remains as a persistent infection, possibly in the tonsils. Seroepidemiological studies suggest that 70% of the population are seropositive for KIPyV and 80% are seropositive for WUPyV, as detected by VP1 antibodies by ELISA. The prevalence of both KI and WU in respiratory secretions is higher in immunosuppressed individuals, such as those with HIV. Additionally, KIPyV has been isolated from respiratory and stool samples of a patient with hematopoietic stem cell transplantation. STLPyV was isolated from stool samples, and can be detected in B70% of the population. TAg of STLPyV has a unique splice variant, referred to as 229T. Additionally, the STLPyV represents genetic mosaicism with the closely related MWPyV. HPyV12 was isolated from resected liver tissue but is also found in rectum tissues and fecal samples, with a seroprevalence of B20%. NJPyV was isolated from a pancreatic transplant recipient with retinal blindness and vasculitis myopathy, with a seroprevalence of B5%. A high degree of sequence identity to NJPyV, suggests a possible transmission of this virus from shrews to humans, yet this remains to be confirmed.

Perspectives Human polyomaviruses represent a diverse group of human pathogens that generally cause asymptomatic infection in healthy individuals and persist in an asymptomatic state at varying frequencies in the population. However, HPyVs can cause serious disease in immunocompromised individuals ranging from skin cancer to allograft rejection to a fatal neurodegenerative disease.

Human Polyomaviruses (Papillomaviridae)

527

HPyVs share many commonalities including virion structure, organization of the genome and protein-coding regions, binding sites for particular host-cell transcription factors, conserved mechanisms of viral replication and virion assembly. Less is known about the recently identified polyomaviruses, which also have the propensity to cause serious disease and could represent viral emergence into the population from other species. Thus, additional research on polyomaviruses will continue to provide insights into these disease mechanisms. Lastly, given the species-specificity of polyomaviruses, animal models are lacking. Development of animal models would revolutionize the polyomavirus field and provide tremendous opportunities to explore viral infection and pathogenesis in vivo.

Further Reading Ambalathingal, G.R., Francis, R.S., Smyth, M.J., et al., 2017. Clinical Microbiology Reviews 30 (20), 503–528. Assetta, B., Atwood, W.J., 2017. The biology of JC polyomavirus. Journal of Biological Chemistry 398 (8), 839–855. DeCaprio, J.A., 2017. Merkel cell polyomavirus and Merkel cell carcinoma. Philosophical Transactions of the Royal Society B 372, 20160276. DeCaprio, J.A., Garcea, R.L., 2013. A cornucopia of human polyomaviruses. Nature Reviews Microbiology 11 (4), 264–276. Feltkcamp, M.C.W., Kazen, S., van der Meijden, E., et al., 2013. From Stockholm to Malawi: Recent developments in studying human polyomaviruses. Journal of General Virology 94, 482–496. Ferenczy, M.W., Marshall, L.J., Nelson, C.D., et al., 2012. Molecular biology, epidemiology, and pathogenesis of progressive multifocal leukoencephalopathy, the JC virusinducing demyelinating disease of the human brain. Clinical Microbiology Reviews 25 (3), 471. Haley, S.A., Atwood, W.J., 2017. Progressive multifocal leukoencephalopathy: Endemic viruses and lethal brain disease. Annual Review of Virology 4, 439–467. Harms, P.W., Harms, K., Moore, P.S., et al., 2018. The biology and treatment of Merkel cell carcinoma: Current understanding and research priorities. Nature Reviews Clinical Oncology 15, 763–776. Helle, F., Brochot, E., Handala, L., et al., 2017. Biology of the BKPyV: An update. Viruses 9, 327. Kamminga, S., van der Meijden, E., Feltkamp, M.C.W., et al., 2018. Seroprevalence of fourteen human polyomaviruses determined in blood donors. PLoS One 13. e0206273. Liu, W., MacDonald, M., You, J., 2016. Merkel cell polyomavirus infection and Merkel cell carcinoma. Current Opinion in Virology 20, 20–27. Mayberry, C.L., Nelson, C.D.S., Maginnis, M.S., 2017. JC polyomavirus attachment and entry: Potential sites for PML therapeutics. Current Clinical Microbiology Reports 4 (3), 132–141. Moens, U., Krumbholz, A., Ehlers, B., et al., 2017. Biology, evolution, and medical importance of polyomaviruses: An update. Infection, Genetics and Evolution 54, 18–38. Rinaldo, C.H., Tylden, G.D., Sharma, B.N., 2013. The human polyomavirus BK (BKPyV): Virological background and clinical implications. APMIS 121, 728–745. Sheu, J.C., Tran, J., Rady, P.L., et al., 2019. Polyomaviruses of the skin: Integrating molecular and clinical advances in the emerging class of viruses. British Journal of Dermatology 180, 1302–1311.

Relevant Websites https://merkelcell.org/about-mcc/. About Merkel Cell Carcincoma. Merkel Cell Carcinoma. https://talk.ictvonline.org/ictv-reports/ictv_online_report/dsdna-viruses/w/polyomaviridae. Polyomaviridae. Polyomaviridae. dsDNA Viruses. ICTV. https://www.ninds.nih.gov/Disorders/All-Disorders/Progressive-Multifocal-Leukoencephalopathy-Information-Page. Progressive Multifocal Leukoencephalopathy. NINDS. NIH.

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae) Amanda R Panfil and Patrick L Green, The Ohio State University, Columbus, OH, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Clonality The number or abundance of HTLV-infected T-cell clones in an infected individual. Immortalization Process by which cells evade normal cellular senescence and begin to proliferate or grow indefinitely. An early step in oncogenesis. Latency Period of restricted or altered viral gene expression. Leukemia Cancer of the body’s blood forming tissues; cancer of the blood or bone marrow. Lymphoma Cancer of cells in the lymph nodes. Oncogenesis Development or induction of cancer.

Proliferation An increase in the number of cells caused by a disturbance in the balance between cell division and cell death. Proviral load Viral DNA burden in an HTLV-infected individual. The number of proviral copies per set number of peripheral blood mononuclear cells. Provirus Viral genome that is integrated into the host cell DNA or chromosome. Transformation Change in the growth or reproduction of cells. Tumorigenesis The formation of tumors or cancer.

Classification Human T-cell leukemia viruses (HTLVs) are classified within the retroviridae family. To date, four distinct types of HTLVs have been identified: HTLV-1, HTLV-2, HTLV-3, and HTLV-4. HTLV-1 was first detected and isolated in the laboratory of Dr. Robert Gallo. The virus was discovered in a T-cell line established from a cutaneous T-cell lymphoma patient. Around the same time, researchers in Kiyoshi Takatsuki’s laboratory in Japan defined a new, distinct form of leukemia with unusual clinical features and cellular morphology. This new leukemia was called adult T-cell leukemia (ATL) and subsequent studies would identify and isolate HTLV-1 from this tumor. Over the past several decades, several epidemiological and molecular studies have shown that HTLV-1 has the ability to disrupt growth regulation and immortalize human T-cells, linking this virus to several types of human disease (to be discussed). HTLV-2 is genetically and immunologically related to HTLV-1, with around 70% nucleotide identity and several overlapping gene functions. HTLV-2 was originally isolated from a patient with a variant form of hairy T-cell leukemia. Like HTLV-1, HTLV-2 can also infect and immortalize human T-cells in vitro. However, there is no strong disease association related to this virus. Indeed, a vast majority of HTLV research has been focused on HTLV-1 due to its significant disease connection. Nevertheless, comparative studies between the related HTLV-1 and HTLV-2 have been, and continue to be, extremely informative for identifying specific virus-host interactions associated with the pathogenic process (Table 1). More recently, HTLV-3 and HTLV-4 have been identified in bush meat hunters in Cameroon. While HTLV-3 and HTLV-4 have similar genomic organization to HTLV-1 and HTLV-2, their biology and disease association remain less clear. Soon after the discovery of HTLV-1, highly related novel viral sequences were found in T-cells of several non-human primates and termed STLVs (simian T-cell leukemia viruses). Like HTLV-1, STLV-1 can cause T-cell leukemia/lymphoma in non-human primates and these tumors display many of the same hallmarks found in ATL patients. According to several different phylogenetic algorithms, it is suspected that many HTLV-1 subtypes arose from zoonotic interspecies transmission between monkeys and humans. Thus, STLV

Table 1

Summary and comparison of various aspects of HTLV-1 and HTLV-2 viruses HTLV-1

HTLV-2

Genome Infection Viral entry Transcription Transmission

Complex deltaretrovirus Cell-to-cell HSPG and NRP1 for binding, GLUT1 for entry Tax-1-mediated LTR activation; antagonized by HBZ Breastfeeding, contaminated blood products, sexual transmission

Immortalization Transforming proteins Disease

CD4 þ T-cells Tax-1, HBZ Yes; ATL, HAM/TSP, HTLV-1-associated uveitis, dermatological conditions 5–10 million people worldwide; areas of endemic infection CTL against viral gene products Chemotherapy, antiviral therapy, targeted therapies, alloHSCT

70% nucleotide similarity to HTLV-1 Cell-to-cell GLUT1 and NRP1 for both binding and entry Tax-2-mediated LTR activation; antagonized by APH-2 Mainly contaminated blood products, some mother-to-child and sexual transmission CD8 þ T-cells Tax-2 No; some neurological abnormality

Prevalence Immune response Treatment

528

800,000 people worldwide CTL against viral gene products None reported

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21225-9

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

529

is a useful animal model to study infection, seroconversion, and disease pathogenesis. STLV-2 and STLV-3 share close sequence similarity with HTLV-2 and HTLV-3, respectively. However, there is no known simian counterpart to HTLV-4 identified to date.

Virion Structure HTLVs, like all retroviruses, contain two single-stranded positive-sense copies of RNA as their genetic material. The RNAs are packaged with various viral enzymes (reverse transcriptase (RT), integrase, and protease) and surrounded by capsid proteins forming a polymorphic, roughly spherical viral nucleocapsid. Also contained within the nucleocapsid is a cellular factor called tRNApro that serves as the primer for viral RT initiation. A lipid bilayer envelope of host-cell origin containing viral envelope proteins surrounds the capsid. HTLV-1 virions measure approximately 105–115 nm in diameter based on electron tomography of naturally infected T-cells and cell lines.

Viral Entry Like most retroviral envelope proteins (Env), HTLV Env is a type-I transmembrane glycoprotein synthesized as a precursor protein in the endoplasmic reticulum, delivered through the Golgi to the plasma membrane, and subsequently acquired on the surface of virions during budding from infected cells. The Env glycoprotein precursor is cleaved by a cellular protease to produce surface (SU) and transmembrane (TM) subunit proteins. Specific interactions between Env and particular receptors on the surface of target cells are the initial and essential step in HTLV infection. Of note, infection can also occur following internalization via an endocytic pathway. In both instances, entry is initiated by SU binding to a cellular receptor, followed by fusion of viral and cellular membranes directed by the TM subunit. Prior to binding of SU to a receptor, the TM subunit is maintained in close association with the SU subunit in a fusogenic-inactive metastable conformation. This conformation of SU/TM keeps a fusion peptide found on the TM domain buried, thus preventing premature membrane fusion, Env inactivation, and cell toxicity. TM activation occurs when SU binds to a cellular receptor, triggering alterations of the SU/TM interaction and enabling TM to attain its fusogenic state. The receptors and co-receptors for HTLV virion binding and entry are expressed on a broad range of cell types. Indeed, both HTLV-1 and HTLV-2 have been detected in a variety of hematopoietic cells from virus-infected individuals. The primary HTLV receptor is glucose transporter type 1 (GLUT1). Loss of GLUT1 expression inhibits virion binding and infection in cell culture, while ectopic expression of GLUT1 restores HTLV infectivity. GLUT1 binds SU and promotes Env-mediated virion entry via SU residues D106 and Y114. Two additional factors, neuropilin-1 (NRP1) and heparan sulfate proteoglycans (HSPG), function as co-receptors for HTLV. NRP1 is overexpressed in transformed cells and upregulated in activated T-cells, the preferential cell target of HTLV infection. NRP1 binds SU, and together with GLUT1, form a stable tripartite complex when co-expressed in cells. Interestingly, HTLV-2 SU binding to target cells does not depend on HSPGs. Based on this collective body of work, a potential model for HTLV entry posits interaction of SU with heparan sulfate chains, presumably on cell surface HSPG, initiates virus attachment (at least for HTLV-1). The SU is then able to bind NRP1, which commonly associates with heparan sulfate chains, to allow virus binding. The binding of SU to HSPG/NRP1 allows a conformation change in SU that exposes the GLUT1-binding domain, thus triggering virus/cell fusion and release of the HTLV nucleocapsid into the cytoplasm. HTLV-1 and HTLV-2 SU proteins share 65% identity at the amino acid level and share many of the same cell receptors (GLUT1, NRP1). However, the in vivo tropism of HTLV-1 and HTLV-2 are distinct with HTLV-1 primarily detected in CD4 þ T-cells and HTLV-2 found primarily in CD8 þ T-cells. This discrepancy in T-cell tropism has been investigated using HTLV-1/HTLV-2 recombinant viruses and mapped to the Env region in the viral genome. An obvious explanation for this could be differences in the receptors the viruses use to enter T-cells. However, as previously discussed, HTLV-1 and HTLV-2 share many of the same receptors/co-receptors. An alternative explanation could therefore be the expression level of these receptors on the surface of T-cells. Indeed, HSPGs are only essential for HTLV-1 SU binding, not HTLV-2 SU. Interestingly, activated CD4 þ T-cells express high levels of HSPG and low levels of GLUT1, while activated CD8 þ T-cells express low levels of HSPG and high levels of GLUT1. Overexpression of HSPG in CD8 þ T-cells increases HTLV-1 internalization, while overexpression of GLUT1 in CD4 þ T-cells enhances HTLV-2 internalization. This suggests HTLV-1 is more dependent on HSPG, while HTLV-2 relies heavily on GLUT1 levels. In summary, HTLV-1 Env requires HSPG and NRP1 for binding and GLUT1 for entry, while HTLV-2 Env requires GLUT1 and NRP1 for both binding and entry. Intriguingly, recent studies found both HTLV-1 and HTLV-2 proviruses were detected as early as 1 week post-infection in both CD4 þ and CD8 þ T-cells using an in vivo rabbit model of infection. This would argue that the tropism and transformation by HTLV-1: CD4 þ T-cells and HTLV-2: CD8 þ T-cells is not solely conferred by the viral envelope at the level of entry. Indeed, this same study showed early proliferation of both CD4 þ and CD8 þ T-cells irrespective of HTLV-1 or HTLV-2 virus type using in vitro cell culture immortalization assays. A late selection and outgrowth of the preferred T-cell type was observed several weeks after initial viral infection, indicating the predominance of T-cell type occurs during the clonal expansion process. This also highlights the potential role of HTLV Env not only in viral entry, but also during transformation and clonal proliferation.

Genome HTLV-1 and HTLV-2 are complex deltaretroviruses that contain the standard retroviral structural and enzymatic genes; gag, pol, and env (Fig. 1). In addition, there is a unique region in the 30 end of the genome that encodes several regulatory and accessory genes

530

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

Fig. 1 HTLV genomes. Schematics of (A) HTLV-1 and (B) HTLV-2 proviral genomes. The viral gag, pro, pol, and env structural/enzymatic genes are flanked by 50 and 30 LTRs. The pX region at the 30 end of the viral genome contains several regulatory and accessory genes. Drawing is intended to be illustrative and not to exact scale.

on both the sense and antisense genomic strands. Originally this region was termed ‘pX’. The HTLV proviral genome is roughly 9 kb in length and is flanked by 50 and 30 long terminal repeats (LTRs). The LTRs are exact duplicates comprised of a U3, R, and U5 region. These regions contain promoter elements, polyadenylation signal sequences, and other regulatory sequences necessary for proper viral transcription. HTLV-1 expresses 8 major mRNA species, while HTLV-2 expresses 7 major mRNA species from the sense strand. Both viruses express 1 major mRNA species from the antisense strand. Like other retroviruses, the unspliced mRNA serves as both genomic RNA and mRNA that encodes Gag (MA-CA-NC) and Gag-Pol (MA-CA-NC-RT-IN) polyproteins. The HTLV protease (Pro) is generated as part of the fusion protein Gag-Pro or Gag-Pro-Pol, which are dependent on ribosomal frameshifts for their generation. The Env glycoprotein precursor is encoded by a major singly spliced mRNA. Tax and Rex regulatory proteins are encoded by a doubly spliced, or completely spliced, mRNA. They share partial overlapping reading frames and their start codons lie in the 2nd exon. Tax translation is preferentially favored over Rex due to a strong Kozak consensus sequence. Tax is a potent activator of HTLV mRNA transcription through activation of the viral promoter. Rex regulates the export of unspliced and singly spliced or incompletely spliced viral mRNA to the cytoplasm. The major role of HTLV-1 p30 is to sequester Tax/Rex mRNA in the nucleus and thus downregulate their expression. p30 has also been shown to affect the cell cycle and interfere with Tax transcriptional activation, thus favoring a latent cell environment. The HTLV-2 genomic and functional equivalent to p30 is p28. The viral proteins p12, p13, p10, and p11 are not required for viral replication in vitro, but are suspected to play a role in viral persistence in vivo. For a detailed description and summary of these accessory genes, please see a review by Edwards, et al. The antisense encoded HBZ protein (HTLV-1 bZIP transcription factor) functions to

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

531

antagonize Tax transcriptional activation through sequestration of cellular transcription factors, thus regulating viral transcription/ replication and facilitating the establishment of latency. The HTLV-2 equivalent to HBZ is called APH-2 (anti-sense protein of HTLV-2) and it shares significant amino acid homology and functional similarities (to be discussed).

Life Cycle The HTLV capsid core contains two copies of viral genomic RNA along with reverse transcriptase, integrase and viral protease. Similar to HIV-1, HTLV is believed to undergo complete reverse transcription of its viral genome after intracellular uncoating of the capsid core. The partially uncoated viral core, which contains the preintegration complex, is then translocated to the nucleus where it can integrate into the host cell chromosome to form the provirus. As with other retroviruses, integration is catalyzed by integrase and facilitated by virus-specific host factors. Recently, an integration host factor for HTLV-1 and HTLV-2 was identified as cellular serine/threonine protein phosphatase 2A (PP2A). HTLV-1 does not appear to integrate into preferred sites of the cellular genome in HTLV-1-infected asymptomatic individuals. However, in patients with HTLV-1-associated diseases such as HAM/TSP, clinical disease diagnosis has been correlated with proviral integration into transcriptionally active regions of the cellular genome. Integration site selectivity studies with HTLV-2 have not been performed to date.

Viral Gene Transcription From the integrated provirus, viral mRNAs are produced to generate an array of HTLV proteins. Transcription is driven from the 50 LTR which contains the promoter/enhancer, while the polyadenylation signal is found in the 30 LTR. The first mRNA produced after integration is the doubly-spliced mRNA encoding Tax and Rex. The HTLV-encoded transactivating transcriptional regulatory protein, Tax, functions to enhance viral transcription. Thus, generating a positive transcriptional feedback loop. Briefly, Tax recruits CREB to a set of conserved 21-base pair repeats in the U3 region of the LTR known as Tax-responsive elements (TREs). The TREs consist of 3 domains; A, B, and C. The A and C domains are C/G rich, while the B domain is similar to the cAMP response element (CRE). Tax does not directly bind DNA, but instead facilitates the binding of CREB to the TREs. CREB then recruits cofactors p300 and CBP to the promoter. Together, this protein complex promotes transcription of viral gene products. The anti-sense derived protein HBZ inhibits Tax-1-mediated viral transcription. HBZ interacts with CBP and p300 and competes these essential cofactors away from the Tax-1 complex, thereby decreasing Tax-driven viral transcription. The HTLV-2 equivalent to HBZ is termed APH-2. APH-2, unlike HBZ, lacks a bZIP (basic leucine zipper) domain but still interacts with CREB to repress Tax-2-mediated viral transcription. The other gene product encoded from the doubly-spliced mRNA encoding Tax, is Rex. Rex is involved in post-transcriptional gene regulation. During normal cellular gene transcription, intron-containing mRNAs are retained in the nucleus until they are fully spliced or degraded. HTLV expresses several gene products from unspliced mRNA (genomic and gag/pol) and singly spliced mRNA (env) containing introns. Therefore, the virus must overcome the default cellular splicing pathway to efficiently replicate. Rex exports unspliced or incompletely spliced viral mRNA from the nucleus to the cytoplasm by binding target viral mRNA within the Rex response element (RxRE). Previous studies revealed Rex-1, Rex-2 and their RxREs are structurally similar and functionally interchangeable. The HTLV-1 RxRE is a 205 nt sequence located in the U3 and R regions of the 30 LTR, while the HTLV-2 RxRE is 226 nt in length and is located in the R/U5 region of the 30 LTR. The secondary structure of RxRE contains four stem loops, which are critical for Rex-dependent mRNA export. Also contained within the RxRE is a cis-acting repressive sequence (CRS). Experimental data suggests the CRS retains the unspliced mRNA in the nucleus to presumably ensure sufficient amounts of Tax and accumulation of Rex substrate. Once Rex binds to the RxRE, this overcomes the CRS effects and Rex can then export the mRNA to the cytoplasm. Rex also interacts with the nuclear export factor CRM1. The Rex/CRM1/RxRE containing mRNA can then be shuttled out of the nucleus through the nuclear pore. Once released from the viral mRNAs, Rex binds importin b and is transported back to the nucleus. Several studies have identified phosphorylation sites at serine and threonine residues within Rex. Many of these modified sites are necessary to regulate Rex function. The viral accessory proteins p30 (HTLV-1) and p28 (HTLV-2) also play a role in post-transcriptional regulation by inhibiting the export of doubly spliced tax/rex mRNAs. HTLV-2 p28 is functionally homologous to HTLV-1 p30. p30/p28 bind to and retain tax/rex mRNA in the nucleus, thereby decreasing the export and expression of completely spliced tax/rex mRNA. Consequently, this RNA redistribution promotes the expression of other viral gene products.

Viral Translation Translation of accessory and regulatory HTLV gene products proceed in a similar manner to cellular translation. However, the structural and enzymatic gene products follow an alternative translation pathway. For every ten Gag proteins that are translated, a Gag-Pro-Pol fusion protein is produced. This fusion protein is generated via two successive ribosomal frameshifts. A  1 frameshift occurs near the 30 end of Gag open reading frame (ORF) that allows the ribosome to continue into Pro. A second  1 frameshift occurs near the end of Pro ORF that allows the ribosome to continue reading through Pol. This alternative translation helps ensure the correct ratios of gene products are generated.

532

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

Assembly, Budding, and Maturation Like other retroviruses, the unspliced mRNA serves as both genomic RNA and mRNA that encodes Gag (MA-CA-NC) and Gag-Pol (MA-CA-NC-RT-IN) polyproteins. Gag (p55) precursor protein is myristoylated and targeted to the cellular membrane. Gag is cleaved by the viral protease into matrix (MA, p19), capsid (CA, p24), and nucleocapsid (NC, p15) proteins. This cleavage of Gag is essential for the assembly of infectious virions. The MA protein contains amino acid residues necessary for Gag homodimerization, the amino terminus of the CA protein is responsible for particle formation, and the NC protein coats the viral gRNA. After assembly, the virus particles bud from the surface of the host cell and mature into infectious virions via proteolytic cleavage.

Epidemiology HTLV-1 is estimated to infect 5–10 million people worldwide with areas of endemic infection. This data is based strictly on reliable epidemiologic data of people in HTLV-1 endemic areas. Epidemiologic researchers do not yet have reliable data from some of the more densely populated areas of the world; therefore, this infection rate may be much higher. Regions with prevalent infection include the Southwestern region of the Japanese archipelago, parts of the Caribbean, foci in South America (Columbia, French Guyana, Brazil), areas of intertropical Africa, the middle East (Mashhad region in Iran), clusters in Australo-Melanesia, and Romania. The uneven worldwide distribution of HTLV-1 ranges from infection rates of 1%–2% in high endemic regions, although these rates can reach 20%–40% in people older than 50 in some particular areas. Recently, a hospital and community-based cohort study in Central Australia reported HTLV-1 infection rates of 33.6% in certain communities. The infection rates increased with age, reaching over 48% in men older than 50. This uneven geographical and ethnic divide is not well understood. It is hypothesized to be due to a founder effect in some cases, followed by the persistence of a high viral transmission rate due to various local environmental and cultural customs/traditions. The prevalence of HTLV-1 infection in an endemic area has been linked to age (especially in women), sex, and economic level. HTLV-1 is genetically stable, which is highly unusual for a retrovirus given the nature of reverse transcriptase. The genetic stability is mostly due to viral amplification that occurs via clonal expansion of infected cells vs. viral replication and subsequent new infections (which is the case for HIV-1). As a result, HTLV-1 has low sequence variation that can be utilized to track the migrations of infected populations worldwide. There are 4 major geographic subtypes, or genotypes, of HTLV-1: Cosmopolitan a-subtype, the Central African b-subtype, the new Central African d-subtype, and the Australo-Melanesian c-subtype. A small number of rare subtypes are also found in Central Africa (e, f, and g). The most widespread subtype is the Cosmopolitan a-subtype and it has very little sequence variation in the envelope gene and the LTR region. There is no strong evidence to suggest specific mutations or genotypes are associated with the development of HTLV-1-associated diseases. HTLV-2 is estimated to infect 800,000 individuals worldwide based on healthy blood donor rates of infection. The largest region of infection is the United States at 400,000–500,000 infected people, followed by Brazil at 200,000–250,000 infections, and finally 20,000–40,000 people in Europe. High rates of HTLV-2 infection have been reported in Amerindian populations and are widespread in injection drug users (IDU). Overall, the global burden of HTLV-2 infection is 6–12 fold lower than HTLV-1.

Transmission HTLV infection of new cells is heavily dependent upon cell-to-cell transmission (as opposed to cell-free transmission). Therefore, new infections require the transfer of virus-infected cells and transmission primarily occurs by 3 routes: mother-to-child, sexual intercourse, and contaminated blood products. Mother-to-child transmission via breastfeeding occurs in 10%–25% of breast fed children born to HTLV-1 infected mothers. A high proviral load in the mother’s milk or blood cells, high antibody titers in the mother’s serum, and duration of breastfeeding (46 months) are all increased risk factors for mother-to-child transmission. Vertical mother-to-child transmission due to transplacental transmission or during delivery is more rare (o3% risk). Sexual transmission of HTLV is usually more prevalent in male-to-female transmission than female-to-male, but this is not exclusive. On-going sexual transmission of HTLV-1 is prevalent in central Australia, Japan, and South America. The exposure to HTLV-infected blood products by blood transfusion, organ donation, and sharing of blood-contaminated needles can lead to viral infection with variable success (12%–44%) in blood recipients. Although HTLV is endemic in many regions around the world, many countries still do not screen their blood supply or organ transplants for HTLV. HTLV-2 is mainly transmitted via contaminated blood products by IDU, and to a lower degree sexual and mother-to-child transmission.

Clinical Features HTLV-1 is the causative infectious agent of several human diseases and conditions, including ATL, HAM/TSP, HTLV-1-associated uveitis, and various dermatological conditions. Hereafter, HTLV-1-associated disease will be focused on ATL and HAM/TSP. These diseases develop in roughly 5%–10% of infected individuals and generally only after a lengthy clinical latency period of several decades. Although very similar to HTLV-1 in nucleotide sequence and coding similarities, the clinical data associating HTLV-2 with malignancy have not been conclusive to date.

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

533

ATL HTLV-1 was first identified as the cause of an aggressive non-Hodgkin’s peripheral T-cell malignancy called adult T-cell leukemia/ lymphoma (ATL). ATL is most commonly a CD4 þ T-cell disease; however there have been rare examples of CD8 þ disease origin. The clinical features that classify ATL include lymphadenopathy, skin lesions, increased abnormal lymphocytes, frequent blood and bone marrow involvement, hypercalcemia, and lytic bone lesions. The abnormal lymphocytes detected in ATL patients are called ‘flower’ cells. These cells have multilobular nuclei with homogeneous and condensed chromatin, small or absent nucleoli, and an agranular and basophilic cytoplasm. Prospective studies have indicated HTLV-1 infected patients with a higher proviral load (44 copies/100 peripheral blood mononuclear cells) are at a higher risk for developing ATL. ATL is a heterogeneous disease and is thus divided into four clinical subtypes based on the 1980s Shimoyama classification: acute, lymphoma, smoldering, and chronic. Patients who develop smoldering or chronic ATL present with a skin rash and minimal blood involvement. These two subtypes are generally less aggressive and are collectively known as indolent ATL. The ‘aggressive’ ATL subtypes, acute and lymphoma are characterized by a large tumor burden, lymph node and blood involvement, and hypercalcemia. Patients who develop aggressive ATL typically display 45% of their total T-cells as flower cells. These subtypes are also accompanied by multiorgan failure and frequent opportunistic infections due to intrinsic T-cell immunodeficiency. Aggressive ATL subtypes also frequently display multidrug and chemotherapy resistance, lending to their poor prognosis. In a retrospective analysis of 1594 patients diagnosed and treated for ATL in Japan between 2000 and 2009, the median survival rate and four year survival rate (OS4) were as follows: acute ATL, 8 month median survival, 11% OS4; lymphoma ATL, 11 month median survival; 16% OS4; chronic ATL, 32 month median survival; 36% OS4; and smoldering ATL, 55 month median survival; 52% OS4.

HAM/TSP HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a slow progressing, chronic neurodegenerative disease caused by HTLV-1. HAM/TSP is characterized by painful stiffness and weakness of the legs (lower limb spasticity), bowel and bladder disturbances, and slow but steady progression over several years. These symptoms develop due to inflammation or swelling that occurs in the spinal cord. HTLV-1-infected cells infiltrate the central nervous system and cause persistent immune activation against proliferating HTLV-1-infected T-cells. HAM/TSP patients frequently display high proviral loads and high levels of HTLV-1-specific antibodies in the cerebrospinal fluid (CSF). Many symptoms of HAM/TSP are also found in patients with MS. In fact, the clinical course of HAM/TSP and MS are similar, with steady worsening of neurological function without any distinct relapse or period of remission.

HTLV-2-Associated Symptoms HTLV-2 has the ability to transform T-cells in culture, but clinical data demonstrating HTLV-2 association with malignancy have not been conclusive. There are however several reports throughout the 1990s correlating HTLV-2 infection with a few cases of neurological disease. HTLV-2-associated neurological symptoms include leg weakness, reflex abnormalities, sensory abnormalities, urinary tract abnormalities, sphincter disturbance, spastic paraparesis, and ataxia spastic. Interestingly, these symptoms appear after several decades of infection and closely mimic the neurological symptoms of HTLV-1-associated HAM/TSP. Ultimately, a clear link between HTLV-2 and neurological disease has been difficult to determine, mainly due to lower worldwide prevalence and concomitant infections with HIV-1, hepatitis B and C, and human herpes virus.

Pathogenesis HTLV-1 and HTLV-2 are transmitted efficiently in a cell-to-cell mediated fashion. Both viruses immortalize T-cells in vitro and in vivo and induce T-cell proliferation. However, these two viruses have distinct oncogenic properties. A large body of work was initially focused on the Tax proteins and the role they play in triggering transforming activity. Loss of Tax by mutation or deletion prevents HTLV-mediated cellular transformation during in vitro cell culture assays. Reciprocally, Tax over-expression, in the absence of other viral factors, can induce cellular transformation in cell culture models. The ability of Tax to contribute to cellular transformation is dependent on the interaction of Tax with several important signaling pathways involved in cell survival, proliferation, and genomic stability. Recently, the anti-sense derived HBZ and APH-2 proteins have garnered more attention. A brief description and summary of the Tax and anti-sense derived proteins for both HTLV-1 and HTLV-2 follows.

Tax Tax-1 and Tax-2 share approximately 85% amino acid similarity. Tax-1 is 353 amino acids long, while Tax-2 is 356 amino acids in length. Tax-1 contains a CREB-binding domain and nuclear localization signal at its N-terminus. The central portion of Tax-1 contains two leucine zipper-like motifs (LZ) at amino acids 116–145 and 225–232. These motifs are required for DNA interactions and for protein dimerization. The amino terminal LZ is important for Tax activation of the classical NF-κB pathway, while the carboxyl terminal LZ is important for alternative NF-κB pathway activation. The C-terminus of Tax-1 contains an ATF/CREB

534

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

Fig. 2 Tax proteins activate the NF-κB pathways. (A) Brief schematic outlining the classical (left) and alternative (right) NF-κB pathways. (B) Tax-1 (left panel) activates both the classical and alternative NF-κB pathways at various steps within the pathway (shown in yellow). Tax-2 (right panel) only activates the classical NF-κB pathway (shown in green).

activation domain, p300/CBP binding domain, and PDZ binding domain. Tax-2 is less well characterized compared to Tax-1. Like Tax-1, Tax-2 also contains a CREB-binding domain at its N-terminus. However, instead of a NLS, Tax-2 contains a nuclear localization determinant (NLD) within the N-terminus. Also similar to Tax-1, Tax-2 contains an ATF/CREB activating domain at its C-terminus. Unlike Tax-1, Tax-2 lacks a PDZ domain. While both Tax-1 and Tax-2 possess transcriptional transactivating activity and transforming capacity, Tax-2 functions less efficiently than Tax-1. One of the major cellular pathways affected by Tax is the NF-κB pathway, a pathway of transcription factors involved in the regulation of cell survival and proliferation. Briefly, the NF-κB family of transcription factors are controlled by inhibitory IκB proteins which sequester NF-κB/Rel in the cytoplasm as multiprotein complexes (Fig. 2(A)). In the classical NF-κB pathway, upon activation by extracellular stimuli such as TNF-a and IL-2, the IκB proteins become serine phosphorylated by the IKK complex composed of NEMO, IKKa, and IKKb. This targets the IκB proteins for ubiquitination and degradation by the proteasome, freeing the p50/RelA heterodimer to translocate to the nucleus where it can activate transcription of NF-κB responsive genes. The classical pathway is quickly activated in response to stimuli, with IκB degradation occurring within minutes of stimulus recognition. However, this pathway is also short lived and is quickly repressed when stimulus is withdrawn or lost. Tax activates the classical NF-κB pathway via its interaction with, and activation of, the IKK complex. Tax expression leads to constitutive activation of the IKK complex and thus continuous degradation of IκB protein. Tax maintains this activation via interaction with NEMO. Activation of the IKK complex is increased by ubiquitination of Tax, which promotes the translocation of Tax to the cytoplasm. Both Tax-1 and Tax-2 have been extensively shown to activate the classical NF-κB signaling pathways through various protein–protein interactions at various stages of the signaling pathway (Fig. 2(B)). Tax-1 can also activate the alternative NF-κB pathway through its ability to induce the processing of p100 to p52. Briefly, the alternative NF-κB pathway is activated by stimulus, such as RANKL signaling (Fig. 2(A)). Upon stimulation of the cell, an IKKa homodimer is phosphorylated and activated by the NF-κB inducing kinase (NIK). Activation of the IKKa homodimer leads to the phosphorylation of p100. p100 is usually dimerized with RelB. Upon phosphorylation and subsequent ubiquitination, p100 is cleaved into the transcriptionally active p52 protein. This cleavage removes an inhibitory ankryin repeat and allows the p52/RelB dimer to translocate into the nucleus and drive expression of target genes. The alternative pathway is slowly activated due to the kinetics involved in processing p100 to p52. Complete activation can take up to several hours. However, the alternative pathway is

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

535

also long-lived with cellular transcription remaining active after stimulus is lost. Interestingly, only Tax-1, but not Tax-2, is able to efficiently activate the alternative pathway (Fig. 2(B)). Although NF-κB activation should lead to cell proliferation, HTLV-infected cells cease to proliferate one cell division cycle after infection. Tax activation of NF-κB signaling increases the cyclin-dependent kinase inhibitors, p21cip1/waf1 and p27kip1 and thus become arrested in senescence. Therefore, in order for Tax to promote oncogenesis of the infected cell, the cellular senescence response would have to be circumvented. In addition to being a potent activator of NF-κB signaling, Tax-1 has also been shown to inactivate cell cycle checkpoints, alter DNA repair pathways, induce micronuclei formation, and disrupt spindle assembly and cytokinesis. This helps establish an environment of genetic instability that contributes to oncogenesis. Tax-1 also uses multiple mechanisms to promote cellular proliferation, including activation of the Akt pathway. In contrast, Tax-2 lacks micronuclei inductive ability. By comparison, Tax-1 has a more robust ability to modulate cell cycle progression and apoptosis compared to Tax-2. The Tax-1 protein is essential for cellular transformation and contributes to genomic instability within the infected cell. HTLV-1-associated diseases progress very slowly and are characterized by a long clinical latency period. Thus, with each round of DNA replication, Tax-1 further increases genomic DSBs (double-strand breaks) and accumulation of mutations. Tax-1 is crucial for its ability to drive viral replication and immortalize T-cells. However, Tax-1 is frequently lost from ATL tumor cells through epigenetic silencing, 50 LTR deletion, or abortive protein mutations within the Tax-1 gene. This suggests other HTLV-1 proteins contribute to the transformation process.

Hbz The other HTLV-1 product that is involved in oncogenic transformation and the pathogenic process is the anti-sense derived Hbz gene. Unlike Tax, the Hbz transcript is detected in all ATL cells. Hbz transcription initiates in the mostly epigenetically unmodified 30 LTR and is regulated by viral CREs and several SP1 binding sites. There are both spliced and unspliced Hbz transcript variants and the proteins encoded by these transcripts have nearly identical amino acid sequence and function. However, spliced Hbz is more abundant in infected cells and therefore has been well-studied. Subsequent discussion of Hbz refers to the major spliced variant. Hbz mRNA possesses proliferative potential in addition to serving as a protein-coding template. Hbz mRNA alone is able to support proliferation of an IL-2-dependent T-cell line when cultured at suboptimal concentrations of IL-2. However, the precise mechanism is unclear. Recently, Hbz mRNA was shown to inhibit apoptosis in mouse CD4 þ T-cells and increase the transcription of the anti-apoptotic gene survivin. Thus, Hbz mRNA serves both as a proliferative and anti-apoptotic factor. HBZ is a 206 amino acid nuclear protein comprised of three functional domains: an N-terminal activation domain, a central basic region, and a C-terminal bZIP domain (Fig. 3(A)). The activation domain contains two well-studied LXXLL-like motifs which enable HBZ to bind the KIX domain of CBP/p300. The LXXLL motifs are also required for HBZ to activate TGF-b signaling. Through interactions with CBP/p300, HBZ is able to sequester these factors away from Tax-1 and repress Tax-1-mediated LTR-activation. The bZIP domain allows HBZ to heterodimerize with cellular bZIP proteins of the AP1 superfamily (CREB2, c-Jun, JunB, JunD, CREB, MafB, and ATF3) and affects their binding to DNA recognition sites. HBZ mutant proviral clones are able to immortalize T-cells in vitro, but a rabbit model of infection revealed that HBZ is required for efficient HTLV-1 infection and persistence in vivo. Viral persistence and immune evasion are key determinants for a successful HTLV lifecycle. To ensure survival, the virus employs a period of latency, or restricted viral gene expression. HBZ helps to maintain a persistent latent infection in cells through several mechanisms (Fig. 3(C)). In contrast to Tax-1, HBZ does not affect the alternative NF-κB pathway and actually inhibits the classical NF-κB pathway. HBZ interacts with p65 (RelA) and induces its proteasomal degradation. Therefore, HBZ prevents NF-κB hyperactivation (induced by Tax) and therefore prevents cellular senescence. Like Tax-1, HBZ also affects genomic integrity through the induction of DSBs. These HBZ-induced DSBs are dependent on the miR17 and miR21 HBZ-inducible miRNAs. miR17 and miR21 target and suppress expression of the gene that encodes hSSB2, a single-stranded DNA-binding protein that prevents genomic instability. HBZ also enhances hTERT expression through its JunD interaction. hTERT, the enzyme responsible for maintenance of telomere length, is often upregulated in HTLV-1-infected cells. Upregulated hTERT translates to increased cellular proliferation and further accumulation of deleterious mutations. HBZ is able to usurp cellular apoptosis through various pathways. HBZ suppresses expression of Bim, a pro-apoptotic gene by repressing its transactivation. HBZ also activates the mTOR pathway via interactions with GADD34. This pathway regulates cellular metabolism and promotes cellular proliferation in response to environmental stimuli. HBZ promotes proliferation of infected T-cells in a number of ways. HBZ interacts with ATF3, thus preventing ATF3 activation of p53 tumor-suppressor signaling. HBZ also interacts with JunD and enhances its transcriptional activation. Loss of JunD inhibits HBZ-induced cellular proliferation, highlighting the significant role JunD plays in HTLV-1 biology. HBZ upregulates transcription of the noncanonical Wnt ligand Wnt5a while suppressing the canonical Wnt pathway. Wnt5a enhances proliferation and migration of ATL cells. Finally, IRF-1, a known tumor suppressor, is negatively regulated by HBZ. HTLV-2 also encodes an anti-sense transcript named APH-2 (anti-sense protein of HTLV-2) (Fig. 3(B)). Like HBZ, the APH-2 mRNA is transcribed from the 30 LTR, spliced, and polyadenylated. APH-2 mRNA is expressed in a majority of HTLV-2-infected patient PBMCs (peripheral blood mononuclear cells). However, loss of APH-2 has no effect on in vitro cell immortalization and contrary to HBZ, its loss enhances in vivo viral replication in rabbits.

536

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

Fig. 3 The anti-sense derived proteins of HTLV. (A) HBZ is encoded from the antisense strand of the HTLV-1 proviral genome. Domain structure of HBZ is depicted. (B) APH-2 is encoded from the antisense strand of the HTLV-2 proviral genome. Domain structure of APH-2 is depicted. Brief summary of the various cellular pathways and processes affected by (C) HBZ and (D) APH-2 proteins.

APH-2 protein shares less than 30% homology to HBZ. However, the two proteins share several functional similarities. Although it lacks a classical bZIP domain, APH-2 still interacts with CREB (through an LXXLL motif and LXXLL-like motif) and thus inhibits Tax-2-mediated viral transcription. APH-2 inhibits p65 (RelA), like HTLV-1 HBZ, but not as strongly. Conversely, unlike HBZ, APH-2 enhances transcriptional activity of IRF-1 – suggesting HTLV-2 cells are more prone to IRF-1-mediated apoptosis. This finding is supported by data that shows APH-2 deficient proviral clones replicate more efficiently compared to WT in a rabbit model of in vivo infection (Fig. 3(D)). Taken together, this data suggests APH-2 does not affect proliferation or infectivity to the extent of its counterpart HBZ.

Viral Clonality DNA analyses of asymptomatic HTLV-1-infected individuals show little variation in HTLV-1 proviral DNA sequence – unlike HIV1. This suggests HTLV replication occurs not by error-prone RT, but through mitotic cell division. Therefore, HTLV exists predominantly in a latent form where the genome is maintained by mitotic host cell division, followed by occasional re-activation and de novo infection. Clonality is the number and abundance of HTLV-1-infected T-cell clones in an individual. Initial research estimated that in asymptomatic HTLV-1-infected individuals, there were as many as 100 clones in circulation. However, more recent high throughput techniques have estimated the number of clones in circulation in a single host is 420,000. Interestingly, 91% of ATL cases have a predominant, malignant T-cell clone with a single proviral genome. Clonality studies of non-malignant HTLV-1-carriers who developed ATL suggest a malignant clone does not necessarily develop from the largest pre-existing infected T-cell clone, but often from a clone of previously low abundance. These recent advances in clonality studies have also shown there are several less abundant clones which underlie the largest malignant clone. This data reaffirms the risk of malignant transformation depends on the longevity of the cell and subsequent number of cell divisions that enable accumulation of DNA damage (Fig. 4). Unlike other retroviruses,

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

537

Fig. 4 HTLV-1 immortalization and transformation of infected cells. HTLV-1 infection (Left panel) occurs primarily in a cell-to-cell mediated fashion. The HTLV-1 viral genome is integrated into the host chromosome in newly infected CD4 þ T-cells (Center panel). Expression of the viral genes Tax-1 and HBZ from the proviral genome affects a variety of important processes within the cell. These cellular changes lead to eventual immortalization and transformation. The development of disease (ATL) occurs after a prolonged clinical latency period of several decades (right panel). ATL cells consistently express HBZ and have variable intermittent expression of Tax-1.

HTLV-1 is a trans-activating retrovirus. The virus carries no dominant viral oncogene and does not cause cis-activation of a cellular oncogene. Instead, viral proteins (Tax-1, HBZ) activate transformation with very slow tumor formation kinetics.

In Vivo Animal Models Several different animal models have been established and utilized since the discovery of HTLV almost 40 years ago. New Zealand white rabbits have been used to study both early immune response and viral persistence. Inoculation of rabbits with lethally irradiated HTLV producer cells results in seroconversion and T-cell infection, but the absence of disease. Mutational analysis of various viral genes and elements using proviral clones can be utilized with this system. Infection of rabbits with cloned mutant viruses allows investigators to address how specific alterations affect viral replication, proviral load, and alter persistence in the presence of a functional immune system. In addition to rabbits, rats and non-human primates have proved useful for studying HTLV-1 viral transmission, viral infection, neurologic HTLV-1 disease, and vaccine efficacy studies. The mouse model is the most popular animal model used in HTLV-1 studies. Transgenic mice expressing Tax-1 or HBZ have been used to study the role of these gene products during disease development. NSG mice (NOD.Cg-PrkdcSCIDIL2rgtm1Wjl/SzJ) can be used in a tumor transplant model for HTLV-1. Following transplantation of HTLV-1-transformed cell lines, the mice will develop tumors. Interestingly, only certain HTLV-1 infected cell lines are tumorigenic in NSG mice. Immortalized cell lines generated in culture from a proviral clone are non-tumorigenic compared to cell lines generated in culture from co-cultured ATL patient leukemic cells. This confirms that although HTLV-1 can immortalize T-cells in vitro, there are additional molecular events that occur, contributing to the development of tumorigenesis. The NSG tumor transplant model is useful for testing therapeutic reagents and investigating the importance of individual viral genes in the context of the intact viral genome. The recent advancement of a ‘humanized’ mouse offers a model system for ATL development in a human microenvironment. However, while the reconstituted immune cells are phenotypically similar to human, they lack full functionality.

Immune Response The host immune response – both antibody and cell-mediated – against HTLV occurs within a few weeks of exposure to the virus. The first two months after infection, Gag antibodies predominate the body. Shortly after this, antibodies to Env appear. In approximately 50% of infected individuals there is a detectable Tax antibody response. The infected host also develops a strong cytotoxic T-cell response. The majority of cytotoxic T lymphocytes (CTLs) are against Tax, with a smaller percentage directed against Gag, Env, and other nonstructural gene products. The immune response helps control, but does not eliminate the virus. Little to no cell-free HTLV virions are found in the plasma. Therefore, proviral load is used to quantify viral infection or viral burden. Proviral load is the number of proviral copies per set number of PBMCs. A high proviral load is associated with both inflammatory and malignant disease caused by HTLV-1. Although antibody titer can vary between infected patients, it directly correlates with the proviral load. The immune response plays an important role in reducing proviral load and thereby protecting the infected person against inflammatory disease. Most of what is currently known concerning antibody responses against HTLVs

538

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

have been carried out with HTLV-1. The immune response to HLTV-2 is less well understood. What is known is that high frequencies of CTLs specific for Tax-2 are often found in patients. Several studies have suggested the immune response can determine the course of HTLV-1-associated disease. The development of ATL is observed more frequently in patients who acquired HTLV-1 early in life (e.g., via breastfeeding). ATL is characterized by a late onset and it is believed the initial reduced anti-viral immune response favors viral persistence. HAM/TSP patients typically acquire their HTLV-1 infection via infected blood products. This elicits a vigorous immune response and these patients have 10–100 fold higher proviral load than asymptomatic patients. The humoral and cell-mediated CD4 þ and CD8 þ T-cell responses are highest in HAM/TSP compared to ATL and asymptomatic carriers. Indeed, a vigorous expansion of CD8 þ T-cells, the presence of Tax specific CTLs in the cerebral spinal fluid (CSF), and high levels of anti-HTLV-1 antibodies in the serum and CSF characterize HAM/TSP. There has been a suggested role of Tax-specific CTLs in the cellular destruction and inflammation in the central nervous system and spinal cord of HAM/TSP patients.

Diagnosis and Prevention The diagnosis of HTLV infection is based on the detection of antibodies by ELISA, or enzyme-linked immunosorbent assay. However, this type of assay is unable to distinguish HTLV-1 from HTLV-2. A follow-up confirmation test by immunoblot and/or PCR is required. The PCR analysis will also provide the patient’s proviral load. This type of test is not convenient for routine screening and makes it difficult to make a more accurate approximation of HTLV prevalence worldwide. Many HTLV-1 infected individuals are asymptomatic for many years and have the capacity to transmit the virus. There is currently no cure, preventative vaccine, or prophylactic vaccine for HTLVs; therefore infected individuals remain capable of passing infectious virus for their lifetime. However, since HTLVs are blood borne pathogens, prevention strategies that are effective for HIV and Hepatitis B & C also reduce the risk of HTLV transmission. Blood and organ donor testing have been shown to decrease HTLV-1 prevalence and HTLV-1 diseases in endemic regions. In Japan, HTLV-1 prevalence rates decreased from 20% in 1987 to 2.5% in 2017 after the implementation of universal antenatal care and avoidance of breastfeeding in HTLV-1 carriers.

Treatment HTLV-1 and HTLV-2 are lifelong infections and there is currently no cure or recommended treatment strategy for asymptomatic HTLV-infected patients. The possibility of a vaccine against HTLV-1 exists because the virus displays low antigenic variability and natural immunity occurs in humans. Experimental vaccination with attenuated vaccinia virus-derived candidates encoding Env provided partial protection from HTLV-1 challenge in squirrel monkeys, offering promising potential for vaccine possibilities. Overall, a variety of vaccine approaches have been attempted for the prevention of HTLV-1 infection, including DNA, live vectors, synthetic peptides, or combinations of these approaches. Unfortunately, despite progress in HTLV research, there is currently no licensed vaccine against HTLV-1. Therefore, HTLV-infected individuals should avoid blood and organ donations, practice safe sex, discourage needle sharing, and avoid breastfeeding in efforts to prevent the spread of the virus. Treatment strategies for HTLV-1-associated diseases vary depending on the severity of symptoms and diagnostic criteria presented. There is no uniform standard of care for ATL. Several treatment options include ‘watchful waiting’, chemotherapy, antiviral therapy, allogeneic hematopoietic stem cell transplantation, and molecular targeted therapies. Patients diagnosed with the less severe, indolent forms of ATL (chronic and smoldering subtypes) generally display few symptoms. These patients are often advised to utilize ‘watchful waiting’ until disease progression to a more aggressive phenotype. When following this standard of care, patients with chronic or smoldering types of ATL have a median survival of only four years. When treated with an antiviral therapy regimen of zidovudine (AZT) and interferon-alpha (IFNa), patients with indolent forms of ATL have a better prognosis. AZT/IFNa therapy of more aggressive ATL subtypes has also been shown to improve patient survival. Patients with acute ATL, treated with first-line AZT/ IFNa therapy, had an 82% 5-year survival rate. Cell culture studies suggest AZT/IFNa treatment causes HTLV-1-infected cells to undergo apoptosis. Conventional chemotherapy has been used for the treatment of aggressive ATL subtypes with varying degrees of success. Although chemotherapy regimens continue to be prescribed as a treatment for ATL, overall patient survival has been minimally improved. ATL tumors are frequently chemotherapy resistant and contribute to patient relapse after therapy. Chemotherapy resistance arises from p53 mutations – a common feature of ATL cells – and immune deficiency due to early stages of disease. Several alternative therapies against ATL have been reported, including allogeneic stem cell transplantation, proteasome inhibitors, HDAC inhibitors, JAK 1/2 inhibitors, arsenic trioxide, monoclonal antibodies directed against ATL/T-cell surface molecules, anti-folates, nucleotide phosphorylase inhibitors, antiangiogenic therapy, and immune-modulatory drugs. Several of these therapies are part of ongoing or proposed clinical trials and will not be described in more detail due to space constraints. Many of the treatment options available for HAM/TSP patients are directed at the treatment of symptoms associated with the disease. Baclofen or tizanidine is used to treat muscle stiffness, urinary symptoms are treated with medications which reduce the activity of the bladder muscle, constipation is treated with increased fiber and laxatives, non-steroidal anti-inflammatory medication is used for nerve pain and inflammation of the spinal cord can be controlled by corticosteroids. Unfortunately, no therapy has been shown to alter the long-term disability associated with HAM/TSP.

Human T-Cell Leukemia Virus-1 and -2 (Retroviridae)

539

Further Reading Ciminale, V., Rende, F., Bertazzoni, U., Romanelli, M.G., 2014. HTLV-1 and HTLV-2: highly similar viruses with distinct oncogenic properties. Frontiers in Microbiology 5, 398. Doueiri, R., Green, P.L., 2012. Human T-cell leukemia virus type 2 (HTLV-2) biology and pathogenesis. In: Robertson, E.S. (Ed.), Cancer Associated Viruses. Springer. Enose-Akahata, Y., Vellucci, A., Jacobson, S., 2017. Role of HTLV-1 Tax and HBZ in the pathogenesis of HAM/TSP. Frontiers in Microbiology 8, 2563. Fochi, S., Mutascio, S., Bertazzoni, U., Zipeto, D., Romanelli, M.G., 2018. HTLV deregulation of the NF-kappaB pathway: An update on tax and antisense proteins role. Frontiers in Microbiology 9, 285. Fujii, M., Matsuoka, M., 2013. Human T-cell leukemia virus types 1 and 2. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology. sixth Ed., Lippincott Williams & Wilkins. Giam, C.-Z., 2012. HTLV-1 and oncogenesis. In: Robertson, E.S. (Ed.), Cancer Associated Viruses. Springer. Giam, C.Z., Semmes, O.J., 2016. HTLV-1 infection and adult T-cell leukemia/lymphoma – A tale of two proteins: Tax and HBZ. Viruses 8. Higuchi, M., Fujii, M., 2009. Distinct functions of HTLV-1 Tax1 from HTLV-2 Tax2 contribute key roles to viral pathogenesis. Retrovirology 6, 117. Ilinskaya, A., Heidecker, G., Jones, K., 2010. Interaction between the HTLV-1 envelope and cellular proteins: Impact on virus infection and restriction. Future Medicinal Chemistry 2, 1651–1668. Mahieux, R., Gessain, A., 2011. HTLV-3/STLV-3 and HTLV-4 viruses: Discovery, epidemiology, serology and molecular aspects. Viruses 3, 1074–1090. Matsuoka, M., Green, P.L., 2009. The HBZ gene, a key player in HTLV-1 pathogenesis. Retrovirology 6, 71. Nasr, R., Marcais, A., Hermine, O., Bazarbachi, A., 2017. Overview of targeted therapies for adult T-cell leukemia/lymphoma. Methods in Molecular Biology 1582, 197–216. Panfil, A.R., Al-Saleem, J.J., Green, P.L., 2013. Animal models utilized in HTLV-1 research. Virology: Research and Treatment 4, 49–59. Yamagishi, M., Fujikawa, D., Watanabe, T., Uchimaru, K., 2018. HTLV-1-mediated epigenetic pathway to adult T-cell leukemia-lymphoma. Frontiers in Microbiology 9, 1686.

Relevant Websites http://hamtsp-net.com/english/about/index.html HAM-net HTLV-1 Assochiated Myelopathy (HAM) registration. https://www.htlvaware.com HTLV AWARE HTLV Home. https://www.nationalmssociety.org/What-is-MS/Related-Conditions/HTLV-I-Associated-Myelopathy-(HAM) HTLV-I Associated Myelopathy (HAM) National MS Society. https://rarediseases.org/rare-diseases/htlv-type-i-and-type-ii/ HTLV Type I and Type II National Organization for Rare. https://htlv.net International Retrovirology Association HTLV. https://www.cdc.gov/mmwr/preview/mmwrhtml/00021234.htm Recommendations for Counseling Persons Infected.

Infectious Bursal Disease Virus (Birnaviridae) Daral J Jackwood, The Ohio State University/OARDC, Wooster, OH, United States r 2021 Elsevier Ltd. All rights reserved.

Nomenclature cvIBDV Clinical virulent pathotype of IBDV hvVP2 Hypervariable region of the protein VP2

scIBDV Subclinical pathotype of IBDV VP Viral protein vvIBDV Very virulent pathotype of IBDV

Glossary

Icosahedron

A polyhedron shape with 20 faces.

Bursa of Fabricius The organ in a chicken responsible for the maturation of B-lymphocytes.

Introduction and Classification Infectious bursal disease virus (IBDV), a member of Birnaviridae, genus Avibirnavirus, causes infectious bursal disease (IBD) also known as Gumboro disease, as it was first described in Gumboro, Delaware, USA. IBDV causes an immunosuppressive disease, through infection of immature B-lymphocytes in the bursa of Fabricius of chickens. The immune suppression caused by IBDV can lead to secondary infections from opportunistic pathogens that exacerbate the disease. IBD is often not recognized in chickens until these secondary infections cause an immune suppression related disease such as gangrenous dermatitis or respiratory disease. In addition, the immune suppression can cause clinical reactions from live-attenuated vaccines, and reduced efficacy of vaccination programs for other diseases. Economic losses result from morbidity, mortality, poor feed efficiency, slower growth, uneven bird weights within a flock, longer times to market and increased condemnations at the processing plant. Isolates of IBDV have been given names based on a variety of criteria including clinical observations, the person that first described a given isolate, antigenic differences, alpha-numeric designations and geographic locations. Establishing a nomenclature system based on the antigenic and pathogenic types of IBDV is difficult because standard assays for determining these characteristics are not possible on a worldwide basis. As a result, nucleotide sequence comparisons and phylogenetic analysis of the VP2 gene have been used to categorize the viruses into genogroups. These procedures are universally available and have identified at least seven genogroups to date (Fig. 1). In addition, a new system for naming historic and future IBDV isolates by their genogroup classification has been also proposed. There are two major serotypes of IBDV. Serotype 1 viruses were the first strains isolated from chickens. Serotype 2 viruses were originally isolated from turkeys but have also been found in other avian species including penguins. No disease has been attributed to the serotype 2 viruses in any avian species. Nucleotide sequencing of the VP2 gene and virus-neutralization assays can be used to distinguished serotype 2 from serotype 1 viruses. Antigenic drift and genetic recombination have contributed to the development of several antigenic subtypes of serotype 1 viruses. The first IBDV strains identified in Gumboro Delaware are known as classic viruses. The term variant was used to describe those viruses that were able to replicate in the presence of antibodies to the classic strains. Although classic and variant are still used to describe the antigenic diversity among serotype 1 IBDV, there are numerous antigenic variations of these viruses. In some cases, partial cross-neutralization and protection is observed between antigenic strains of IBDV. However, there are other strains that have substantial antigenic drift resulting in little or no cross-neutralization or protection between the strains. The antigenicity of serotype 1 IBDV is dictated by the amino acids in the hypervariable region of VP2 and the VP2 epitopes are dependent on the proper folding of this protein. Neutralizing monoclonal antibodies bind to the P domain amino acids in VP2 and amino acid substitution mutations in the PBC and PDE domains were found to contribute to the antigenic drift of IBDV strains.

Virion Structure and Genome Infectious bursal disease virus is a non-enveloped icosahedron (Fig. 2). The genome of IBDV consists of two segments of doublestranded RNA. The RNA-dependent RNA polymerase, VP1 is encoded by the smaller genome segment B. The larger segment A encodes a polyprotein that is self-cleaved by the viral encoded protease VP4. Cleavage of this polyprotein yields two capsid proteins, pVP2 and VP3, and the VP4 protease (Fig. 3). The pVP2 protein is further processed at the COOH terminus to yield the mature capsid protein VP2. VP2 is comprised of base (B), shell (S) and projection (P) domains. The P domain contains four loop structures designated PBC, PDE, PFG, and PHI that make up the surface of the virion. Trimers of VP2 comprise the structural units of

540

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20975-8

Infectious Bursal Disease Virus (Birnaviridae)

541

Fig. 1 IBDV genogroups. Phylogenetic analysis of 105 IBDV nucleotide sequences of the hypervariable region of VP2 identifies 7 genogroups. The analysis was conducted using neighbor-joining method with 1000 bootstrap replicates. The evolutionary distances were computed using the maximum composite likelihood method. Reference strains are identified by GenBank accession numbers. From: Michel, L.O., Jackwood, D.J., 2017. Classification of infectious bursal disease virus into genogroups. Archives of Virology. doi:10.1007/s00705-017-3500-4.

100 nm

Fig. 2 Electron micrograph of IBDV.

542

Infectious Bursal Disease Virus (Birnaviridae)

Fig. 3 Schematic presentation of genome segments A and B of IBDV and the proteins they encode.

the T3 viral capsid and the P domain of VP2 is responsible for inducing neutralizing antibodies and a protective immune response in chickens. The larger genome segment also has an overlapping reading frame with the polyprotein that encodes a non-structural protein VP5 which is involved in apoptosis.

Pathogenesis and Clinical Features Infectious bursal disease is highly contagious. Pathogenicity ranges from attenuated to highly virulent and strains are typically placed in one of three pathogenic groups; subclinical (sc)IBDV, clinical virulent (cv)IBDV and very virulent (vv)IBDV. Immune suppression can be caused by viruses in all three of the pathogenic groups. Infections with the cvIBDV may be subclinical in chicks before 3 weeks of age due to only partial protection by maternal immunity to the infecting IBDV strain. Chickens are most susceptible to clinical disease at 3–6 weeks of age when maternal antibodies have waned. The vvIBDV usually cause clinical disease in young chickens and have caused severe clinical infections in Leghorn chickens up to 18 weeks of age. Early IBDV infections, even though they may be subclinical, can cause severe, long-lasting immunosuppression. Following an early IBDV infection, immature B-lymphocytes in the bursa of Fabricius are destroyed before they can populate secondary lymphoid organs such as the spleen and Harderian gland. Consequently, chickens immunosuppressed by early IBDV infections do not respond well to vaccination and are predisposed to infections by opportunistic viruses and bacteria. The IBDV induced immune suppression can exacerbate the severity of disease caused by other poultry pathogens such as Newcastle Disease virus, infectious bronchitis virus and avian influenza. In clinical infections, the onset of clinical signs occurs after an incubation of 3–4 days. Chickens can exhibit severe prostration, incoordination, watery diarrhea, soiled vent feathers, vent picking, and inflammation of the cloaca. The bursa of Fabricius will become edematous, yellowish, and occasionally hemorrhagic. Congestion and hemorrhage of the bursa and skeletal muscles is sometimes seen in vvIBDV infected birds. Chickens that have recovered from IBDV infections have small bursas. Microscopic lesions in the bursa include inflammation, severe lymphocyte necrosis, atrophy of the follicles and follicular depletion of lymphocytes.

Diagnosis and Control IBDV can cause morbidity, mortality and gross lesions in the bursa are usually indicative of an infection. However, it can also be difficult to diagnose IBD because some strains cause a sub-clinical disease. In these cases, the recognition that IBD has infected a flock often occurs when opportunistic microorganisms cause secondary infections resulting from the IBDV induced immune suppression. Gross lesions in the bursa can signal an IBD outbreak but some strains cause atrophy of the bursa without gross lesions. The diagnosis of IBDV using traditional virus isolation methods can yield variable results. Many of the pathogenic strains do not replicate in cell culture unless they are passed multiple times which can lead to genetic and phenotypic changes. Passage in 9 day old chicken embryos has been used successfully to isolate IBDV but there is the risk that an IBDV vaccine or subpopulation of the field virus will be selected using this method. Most diagnostic laboratories use reverse transcriptase-polymerase chain reaction (RT-PCR) followed by nucleotide sequencing of the VP2 gene to detect and identify IBDV strains. As described above, genogroups of the virus have been defined and are based on the sequence of the hypervariable VP2 sequence region (Fig. 1).

Infectious Bursal Disease Virus (Birnaviridae)

543

There is no treatment for IBD. The virus is fastidious and thus disinfection of contaminated farms after depopulation has had limited success. The first line of defense is achieving high levels of maternal antibody to IBDV in young chicks. High titers of anti-IBDV antibodies in breeder hens are transferred through the egg yolk to the young chicks. It is important to use IBDV vaccines that are very similar or an antigenic match to the pathogenic IBDV endemic to the region where the chicks will be housed. Vaccinating broiler chicks or pullets with live-attenuated vaccines is practiced to protect them after maternal immunity wanes. It is important to administer these vaccines at the proper time so residual maternal antibodies to IBDV do not interfere with their replication. To help solve this problem, recombinant herpesvirus of turkeys (HVT) vectors containing the VP2 gene of IBDV (HVT-IBD vaccines) have been produced and are used to vaccinate broilers and pullets in ovo or at one-day of age. These live HVT-IBD vector vaccines are not neutralized by maternal antibodies to IBDV and thus can be administered when maternal immunity is high. The immunity induced by HVT-IBD vector vaccines can be slow to develop in young chicks. Therefore, it is important to make sure the maternal immunity adequately protects the birds until active immunity to the IBDV strain is achieved by these vaccines.

Further Reading Becht, H., 1980. Infectious bursal disease virus. Current Topics in Microbiology and Immunology 90, 107–121. Cosgrove, A.S., 1962. An apparently new disease of chickens-avian nephrosis. Avian Diseases 6, 385–389. Coulibaly, F., Chevalier, C., Delmas, B., Rey, F.A., 2010. Crystal structure of an Aquabirnavirus particle: Insights into antigenic diversity and virulence determinism. Journal of Virology 84, 1792–1799. Jackwood, D.J., 2017. Recent advances in vaccine research against economically important viral diseases of food animals: Infectious bursal disease virus. Vaccines Veterinary Microbiology 206, 121–125. (special issue). Jackwood, D.J., 2019. Overview of infectious bursal disease in poultry. In: Line, S., Moses, M.A. (Eds.), The Merck Veterinary Manual. Whitehouse Station, NJ: Merck & Co., Inc. Michel, L.O., Jackwood, D.J., 2017. Classification of infectious bursal disease virus into genogroups. Archives of Virology. doi:10.1007/s00705-017-3500-4. van den Berg, T.P., 2000. Acute infectious bursal disease in poultry: A review. Avian Pathology: Journal of the W.V.P.A 29, 175–194. van den Berg, T.P., Morales, D., Eterradossi, N., et al., 2004. Assessment of genetic, antigenic and pathotypic criteria for the characterization of IBDV strains. Avian Pathology: Journal of the W.V.P.A 33, 470–476.

Relevant Websites https://www.oie.int/doc/ged/d9315.pdf Infectious Bursal Disease (Gumboro disease) – OIE. https://www.merckvetmanual.com/poultry/infectious-bursal-disease/overview-of-infectious-bursal-disease-in-poultry Overview of Infectious Bursal Disease in Poultry. https://www.youtube.com/watch?v=-Op4z86V3l8, https://www.youtube.com/watch?v=jOiyVM15Z-Q Poultry Health Today.

Infectious Pancreatic Necrosis Virus (Birnaviridae) Øystein Evensen, Norwegian University of Life Sciences, Oslo, Norway r 2021 Elsevier Ltd. All rights reserved. This is an update of Ø. Evensen, N. Santi, Infectious Pancreatic Necrosis Virus, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00772-X.

Glossary Fingerlings A life-cycle stage when young salmonids are one finger in length. Fry Newly-spawned fish that has fully absorbed their yolk sack. Milt The sperm from male fish. Parr A young salmonid with parr-marks (dark blotches) on the sides, before migration to the sea.

QTL Quantitative trait loci. Smolts Salmonid fish going through smoltification; physiological changes that will allow fish to change from life in freshwater to life in seawater. The smolt state follows the parr state. Start-feeding The time when the fry take up feed for the first time.

Introduction Infectious pancreatic necrosis (IPN) is an acute and contagious disease of juvenile and post smolt salmonids which include the genus Salmo, Oncorhynchus, and Salvelinus, typically occurring at time of start-feeding of fry and after sea transfer in post-smolt Atlantic salmon. Virus characteristics such as variations between virus serotypes and strains, and viral infection loads can also play an important role in determining mortality rates. The clinical manifestation of the disease has changed over the last two decades. While IPN outbreaks were frequently seen in Atlantic salmon farming at start-feeding and after sea transfer, both manifestations of disease have declined with the introduction of IPN-QTL fry in major salmon-producing countries, Norway, UK, and recently also in Chile. When occurring, outbreaks are seen during the first few weeks after start-feeding of fry. Disease outbreaks can also be seen in 10–20 g salmon in freshwater but are less frequent. In the mid-1980s the number of outbreaks in post-smolt Atlantic salmon after seawater transfer emerged as a significant disease problem. Mortality rates in freshwater in genetically susceptible fish vary considerably from negligible to almost 100%. Disease outbreaks in seawater typically result in 5%–10% cumulative mortality but can reach 70% at cage level in genetically susceptible fish. This variation in mortality has been ascribed to factors related to the host species (e.g., the age and/or genetic resistance of fish) and environmental stressors. IPN-QTL (homozygous) fish are in principle resistant to IPN.

Classification Infectious pancreatic necrosis virus is the type species of the genus Aquabirnavirus within the family Birnaviridae. IPN in salmonid fish is characterized by necrosis of the exocrine pancreas, giving the name to the disease, and the name of the type species. IPN in Atlantic salmon also causes necrosis of the liver parenchyma. Aquabirnaviruses have been isolated from a number of freshwater and marine fish species as well as from marine invertebrates worldwide and are associated with a wide range of host pathologies. Aquatic birnaviruses are an antigenically diverse group of virus. Serological classification based on neutralizing antibodies separates IPNV isolates into serogroups A and B. Serogroup A contains nine serotypes (A1-A9) comprising most of the isolates, whereas serogroup B so far consists of only one serotype. Serological classification has not been standardized and serotyping is today replaced by genetic classification. This includes phylogenetic analysis based on sequencing the VP2-coding region of segment A clusters IPNV isolates into six genogroups. Genogrouping based on 310 basepairs (bp) at the VP2/NS junction region yields seven genogroups with an additional genogroup that includes a Japanese aquabirnavirus isolates. (Fig. 1). There are no agreed species demarcation criteria for the genus Aquabirnavirus, and this has led to difficulty in nomenclature. Distantly related strains may cause similar pathologies, whereas closely related strains may range from avirulent to very virulent. Closely related strains are found in both farmed fish and marine species in the same geographical region. Therefore, the species concept does not apply very well to members of the genus aquabirnaviruses. Another aspect contributing to this is the intrinsic variability of RNA viruses like the aquabirnaviruses, derived from the error-prone process of replication. For future characterization of aquabirnavirus isolates, it should be sufficient to classify them according to genus and genogroup.

Virion Structure and Genomic Organization The IPNV virion is 60 nm in diameter and a single-shelled icosahedral structure with no envelope (Fig. 2). The virus genome consists of two segments of double-stranded RNA. For serotype Jasper, segment A comprises 3097 base pairs (bp) and the smaller segment B comprises 2784 bp.

544

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00076-X

Infectious Pancreatic Necrosis Virus (Birnaviridae)

545

Fig. 1 Molecular phylogenetic tree based on nucleotide sequences of the VP2/NS junction among 93 worldwide isolates of IPNV and other aquabirnaviruses (corresponding GenBank/DDBJ accession numbers are shown). Bootstrap values form 100 replicates are shown at major nodes. Scale: 0.02 replacement nucleotides per site. Reproduced from Nishizawa, T., Kinoshita, S., Yoshimizu, M., 2005. An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region. Journal of General Virology 86, 1973–1978, with permission from Society for General Microbiology.

Genome segment A contains a large open reading frame (ORF) encoding a 106 kDa polyprotein (NH2-VP2-protease (VP4)-VP3-COOH), which is co-translationally cleaved to generate pVP2 (the precursor of VP2), VP4 and VP3. The processing of the large polyprotein is controlled by VP4, which is a viral protease with a serine/lysine catalytic dyad. The cleavage sites have been identified at the pVP2-VP4 and VP4-VP3 junctions, and the structure of VP4 has been elucidated by X-ray crystallography. The

546

Infectious Pancreatic Necrosis Virus (Birnaviridae)

Fig. 2 Negative staining of IPNV serotype Sp grown on Chinook salmon embryonic (CHSE-214) cells and purified by density gradient centrifugation.

Table 1 Point mutations in VP2 result in a marked reduction to almost complete loss of virulence whereby 217 and 221 are key determinants. Substitution of threonine for proline at residue 217 (T217P) reduces the virulence, producing a mortality rate approx. 50% lower than the highvirulent variant (TAT). Similarly, single amino acid substitutions of alanine for threonine at residue 221 render highly virulent strains (TAT) almost avirulent. Passage in culture of high virulent strains is associated with motif changes in position 221 (A to T) for TAT ant PAA variants, each of which results in avirulence Amino acid residues of the VP2 protein 217 T T P P

221 A T A T

In vivo virulence characteristics 247 T T A A

Highly virulent Avirulent Moderately virulent Avirulent

pVP2 precursor is processed further into the mature VP2, which is the major capsid protein. In addition, three small peptides are generated, that can be detected in viral particles. VP2 spontaneously self-assembles into particles with a diameter of about 25 nm, which suggests that late maturation avoids premature assembly during particle morphogenesis. Some studies have indicated that elements of VP3 may be exposed on the surface of the virion, serotype specific and neutralizing antibodies are almost exclusively directed against continuous and discontinuous epitopes of VP2. Indeed, VP2 is the type-specific antigen and the neutralizing sites reside within the hypervariable region of VP2 (Table 1). The region covering amino acid residues 183–335 has been found by several authors to contain the neutralizing epitopes, although it has so far not been possible to identify their exact localization or sequence. This part of the VP2 protein carries several loops that stick out from the surface over this stretch (Fig. 3). VP3 is an internal capsid protein associated with the viral genome. Studies using yeast two-hybrid systems in combination with co-immunoprecipitation, showed that VP3 binds to VP1. In addition, VP3 was shown to specifically bind to dsRNA in a sequence independent manner. The binding between VP3 and VP1 is not dependent on the presence of dsRNA. Genome segment A also contains a small ORF that proceeds and partly overlaps the large ORF encoding the 106 kDa polyprotein. The smaller ORF encodes a highly basic, arginine-rich polypeptide of varying size. The protein has been detected in infected cells but has not, to date, been conclusive demonstrated to be present in purified viral particles. This apparently non-structural protein, designated VP5, is encoded by most but not all IPNV strains examined. In an Asian IPNV strain, an anti-apoptotic effect has been demonstrated for VP5 and this may aid viral replication in the initial phase of the infection. However, the amino acid sequence of VP5 is not well conserved and the anti-apoptotic effect does not occur in all IPNV strains encoding the polypeptide.

Infectious Pancreatic Necrosis Virus (Birnaviridae)

547

Fig. 3 Structural layout of the VP2 protein showing the PBC, PDE, PFG, and the PHI loops which represents amino acid residues, 210–247, 254–262, 282–288, and 314–334, respectively.

Fig. 4 Schematic presentation of the IPN virus particle. VP2 is located on the surface, VP3 bound to viral RNA and VP1 bound to the dsRNA or free inside the virus particle.

Segment B encodes a 90 kDa protein (VP1) which functions as an RNA-dependent RNA polymerase (RdRp). This enzyme directs the transcription of non-polyadenylated mRNA from genome segments A and B. Within the viral particle, VP1 occurs as a free polypeptide and covalently linked to the 50 terminus of each genome segment (Fig. 4).

Life Cycle IPNV replicates in the cytoplasm and a single cycle of replication takes 16–20 h. IPNV infects a wide range of cultured fish cells, including CHSE-214, RTG-2 and BF-2 cells. These cell lines are routinely used for laboratory propagation the virus. IPNV replicates well at temperatures ranging from 15–221C, resulting in a characteristic cytopathic effect. Available information on attachment, penetration and uncoating is limited. Purified virus attaches to CHSE-214 cells by specific and non-specific binding to cell membrane components. After binding, IPNV can be observed in endosomes after 20–30 min, suggesting that the entry of IPNV is attained by endocytosis. The entry does not seem to be dependent on the acid pH of the endosomes, and the virion RdRp VP1 is active without proteolytic treatment of the virus, indicating that uncoating may not be a precondition for virus replication. VP1 can be guanylylated in vitro whereupon it becomes a primer for in vitro RNA synthesis. Unlike other viral VPg polypeptides, VP1 can guanylylate itself in vitro in a template-independent manner, and the guanylylation site has been mapped to serine 163. Segment A

548

Infectious Pancreatic Necrosis Virus (Birnaviridae)

specific mRNA is synthesized in larger amounts (2–3 times) than segment B, reflecting the relative abundance of the viral proteins. The assembly of virions takes place in the cytoplasm of infected cells, and virus release occurs via cell destruction. Apoptosis is induced in CHSE-214 cells following IPNV infection and the so-called “McKnight” cells associated with sloughing of mucosal cells in the intestines of juvenile salmonids suffering from IPN have morphological resemblance to apoptotic cells. Similar apoptotic bodies are also found in the liver of Atlantic salmon post-smolts suffering from IPN.

Epidemiology It has long been known that survivors of an IPN outbreak become carriers and surviving fish continue to shed virus without any apparent clinical symptoms. In addition, sub-clinical infection is important for horizontal transmission of the virus. The efficiency by which the virus is spread in a population of susceptible individuals varies between strains and there are indications this might correlate with virulence characteristics whereby highly virulent variants are more efficient at spreading. Transmission of the virus to progeny has been shown to occur vertically via eggs in brook trout and rainbow trout. The virus is possibly present inside the eggs since there are indications that surface disinfection of the eggs is not fully effective in preventing the introduction of IPNV to hatcheries. One possible route of intra-ovum introduction is via the milt, by adsorption of virus to the sperm head. Surveys for the prevalence of IPNV in susceptible strains of salmon, have concluded that most North Sea salmon farms harbor IPNV carrier fish, making selection for IPNV-free brood fish difficult. Once introduced to a hatchery, the virus is difficult to eliminate, as it is stable in water and relatively resistant to many disinfectants, and this has coined the term house-strains to these hatchery specific IPNV strains. Sea water outbreaks of IPN are the result of persistent infection obtained during the freshwater period.

Pathogenesis and Clinical Features IPNV causes acute disease of juvenile salmonids and is the major pathogen of concern in brook trout (Salvelinus fontinalis), rainbow trout (Oncorhyncus mykiss), and Atlantic salmon (Salmo salar L.) hatcheries. Today, outbreaks also occur in fingerlings and parr later in the freshwater phase for Atlantic salmon, as well as in post-smolts after transfer to seawater. Salmonid fish with acute IPNV infection show darkened skin, abnormal swimming behavior (whirling or swimming on the side), and a distended abdomen. Whitish threads of debris are often seen hanging from the anal orifice. At necropsy, common findings are ascites and hemorrhages in perivisceral adipose tissue. The liver is often pale macroscopically. Histopathologically, IPN is characterized by acute multifocal necrosis of the exocrine pancreas and liver necroses. In cases of more protracted disease, evidence of a cellular response can be observed. Multifocal hepatic necroses are typical and acute catarrhal enteritis with necrosis and sloughing of the intestinal epithelium have been reported. Viral antigen can be detected within lesions by immunohistochemistry. A high proportion of fish surviving an IPN outbreak become persistently infected without apparent signs of disease. These subclinically infected individuals are of major importance as sources of horizontal transmission of the virus. The carrier state has no direct negative impact on infected individuals, although there are some indications of suppressed immune response. Recent studies have shown that there are differences in the ability of different strains of IPNV Sp serotype to establish persistent infections in Atlantic salmon fry. Carrier fish shed virus in their feces but titers fluctuate over time and increase during periods of stress. In addition, persistently infected brood fish can transfer IPNV to their progeny. Recently it has been documented that persistently infected fish carry IPNV from the hatchery to the grow-out farm. This was shown by sequence analysis of strains from different stages of the production. Virus can be isolated from peripheral leukocytes a short time after experimental infection but not during chronic stages. The virus is located in head kidney leukocytes in fish that recover from an acute infection and most likely in macrophages or macrophage-like cells of the head kidney in persistently infected fish. It has also been shown that IPNV multiplies in adherent leukocytes isolated from carriers, although a lytic infection is not seen and, concordantly, virus is not found in the supernatant medium from cultures of isolated head kidney macrophages. Similarly, IPNV is also able to induce persistent, non-lytic infections in cell cultures in which a high proportion of the cells are infected but produce relatively low quantities of virus. Different strains of IPNV appear to vary in their ability to induce persistence in cell culture. Infected cells are resistant to superinfection by IPNV, both homologous or heterologous strains or serotypes, and cultures can be cleared of infection by several passages in the presence of virus neutralizing antiserum. The underlying mechanisms of the establishment and maintenance of IPNV persistence in vivo and in vitro are largely unknown.

Molecular Determinants of Virulence The mortality rate during an IPN outbreak can vary, ascribed to host factors and traits related to the virus. Viral factors influencing mortality rates are dose of infection as well as virulence variations between serotype and strains. IPNV stains of different serotypes are highly heterogenic and the Sp serotype is considered to be more virulent than the Ab serotype. Within the Sp serotype different genotypes exhibit different virulence characteristics studied by the use of reverse genetics. In vitro transcribed positive-sense cRNAs of IPNV are infectious when transfected into permissive cell cultures, allowing the recovery of genetically modified isolates. The reverse genetics technique is a valuable tool for studying the IPNV life cycle and the role

Infectious Pancreatic Necrosis Virus (Birnaviridae)

549

of individual viral proteins in the pathogenesis of infection. Experimental studies using such recombinant IPNV strains have demonstrated that VP5 expression is not required for efficient replication in vitro or in vivo, or for virulence or persistence of the virus. Reassortant viruses containing segment A from a high-virulence strain and segment B from a low-virulence strain, prepared by either reverse genetics or co-infection techniques, have also been used to demonstrate that segment B had no direct influence on IPNV virulence. For IPNV serotype Sp, it has been shown that certain amino acid sequence motifs in VP2 strongly correlate with virulence. Residues 217 and 221 appear to be critical determinants of virulence, and a genetic signatures involving these amino acids are strongly associated with avirulent, moderately and highly virulent strains, as shown in Table 1. Furthermore, serial passage of the Sp serotype in cell culture leads to the attenuation of virulence as a result of an A- T mutation at amino acid residue 221. In summary, the neutralizing domain, the virulence signatures and the cell culture adaptation with subsequent loss of in vivo virulence, seem all to be located in the VP2 hypervariable region for IPNV. A limited number of serotypes and strains of IPNV have been characterized for their virulence signatures, and the same applies to identifying the immunogenic domain but indications are that the stretch between amino acids 183–337 of VP2 are important.

Control Good management includes the environment of fish to minimize stress and to reduce the likelihood of transmission of the pathogen, particularly important at the time of first feeding of fry, when smolt are transferred to sea and during the first few months after sea water transfer. Transfers of fish between farms are considered as one of the highest risks in the transmission of IPNV. Another factor is the movement of eyed eggs and fry between hatcheries, which is common practice in Norway and other salmon producing countries. Vertical transmission has not been conclusively demonstrated in Atlantic salmon but there is evidence from other salmonid species to indicate that vertical transmission is a strategy utilized by IPNV. Diagnosis of IPN is made by combination of clinical signs, histopathological examination combined with immunohistochemistry for in situ detection of virus antigen in exocrine pancreas or liver. Combinations of clinical signs and real-time PCR methods are also frequently used to document IPN virus infection. A number of studies have addressed the efficacy of IPN vaccines, most of these based on inactivated vaccine preparations delivered by injection (intraperitoneally). In addition, plasmid-based vaccines have also been tested under experimental conditions although with limited efficacy. Currently, the majority of vaccines used are inactivated whole virus vaccines (IWV) while vaccination with an E. coli-expressed VP2 recombinant protein is used less frequently. Reliable challenge models for IPN in (genetically) susceptible Atlantic salmon post-smolts have been established and high level of protection can be obtained with high antigen dose experimental vaccines (Fig. 5). Plasmid vaccines expressing the structural proteins of IPNV and delivered by the oral route to rainbow trout, confer high level of protection against lethal challenge (480% relative percent survival). This is in contrast to the level of protection obtained in Atlantic salmon vaccinated with plasmid vaccines. Mode of delivery might play a role but has not been defined.

Fig. 5 Susceptible Atlantic salmon parr were vaccinated with two doses of inactivated whole virus vaccines (oil-adjuvanted) and challenged at 450 degree days. Protection against mortality coincided with antigen dose in the vaccine with close to 90% relative protection in the high dose group while less than 50% in the low-antigen dose group. Control mortality at 87%.

550

Infectious Pancreatic Necrosis Virus (Birnaviridae)

Fig. 6 Number of reported cases of IPN in Atlantic salmon and rainbow trout farming in Norway between 2000 and 2018. IPN-QTL fish were introduced in 2009–2010 and there has been a steady decline in number of reported outbreaks of IPN since.

In addition, a number of studies have been published over the last years related to genetic resistance to IPN virus infection in Atlantic salmon. Currently, only IPN-QTL eggs are available from breeding companies. Recent studies have shown that epithelial cadherin (cdh1) determines IPN virus infection in Atlantic salmon, and putatively a single nucleotide polymorphism within the full-length cdh1 gene was identified as a causal factor but not conclusively determined. Infected salmon carrying the QQQTL genotype appear with very low levels of virus in target organs, like liver. There are indications that the cdh1–1 gene product serves a function as receptor or co-receptor for IPNV, based partly on a co-localization of IPNV and cdh1–1 proteins in infected liver tissue. Since IPN-QTL eggs were introduced, the number of reported cases of IPN over the last 6–7 years in Norway have declined markedly (Fig. 6). In Norway, the number of reported cases peaked in 2009 (223 reported cases) and in 2017, 23 cases were reported. In addition to introduction of IPN-QTL fish, eradication programs in individual hatcheries have also contributed to the reduced number of reported cases.

Further Reading Ahmadivand, S., Soltani, M., Behdani, M., et al., 2017. Oral DNA vaccines based on CS-TPP nanoparticles and alginate microparticles confer high protection against infectious pancreatic necrosis virus (IPNV) infection in trout. Developmental and Comparative Immunology 74, 178–189. Coulibaly, F., Chevalier, C., Gutsche, I., et al., 2005. The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120, 761–772. Dobos, P., 1995. The molecular biology of infectious pancreatic necrosis virus (IPNV). Annual Review of Fish Diseases 5, 25–54. Duncan, R., Nagy, E., Krell, P.J., Dobos, P., 1987. Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a virus-encoded protease, and fine structure mapping of genome segment A coding regions. Journal of Virology 61, 3655–3664. Hill, B.J., Way, K., 1995. Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Annual Review of Fish Diseases 5, 55–77. Hong, J.R., Wu, J.L., 2002. Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection. Cell Death and Differentiation 9, 113–124. Houston, R.D., Haley, C.S., Hamilton, A., et al., 2010. The susceptibility of Atlantic salmon fry to freshwater infectious pancreatic necrosis is largely explained by a major QTL. Heredity 105, 318–327. Kristoffersen, A.B., Devold, M., Aspehaug, V., et al., 2018. Molecular tracing confirms that infection with infectious pancreatic necrosis virus follows the smolt from hatchery to grow-out farm. Journal of Fish Diseases 41, 1601–1607. Liao, L., Dobos, P., 1995. Mapping of a serotype specific epitope of the major capsid protein VP2 of infectious pancreatic necrosis virus. Virology 209, 684–687. Moen, T., Torgersen, J., Santi, N., et al., 2015. Epithelial cadherin determines resistance to infectious pancreatic necrosis virus in Atlantic salmon. Genetics 200, 1313–1326. Mutoloki, S., Jøssund, T.B., Ritchie, G., Mungang’andu, H.M., Evensen, O., 2016. Infectious pancreatic necrosis virus causing and subclinical infections in Atlantic salmon have different genetic fingerprints. Frontier Microbiol 7, 1393. Nishizawa, T., Kinoshita, S., Yoshimizu, M., 2005. An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region. Journal of General Virology 86, 1973–1978. Santi, N., Sandtrø, A., Sindre, H., Song, H., et al., 2005. Infectious pancreatic necrosis virus induces apoptosis in vitro and in vivo independent of VP5. Virology 342, 13–25. Santi, N., Song, H., Vakharia, V.N., Evensen, Ø., 2005. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. Journal of Virology 79, 9206–9216. Song, H., Santi, N., Evensen, Ø., Vakharia, V.N., 2005. Molecular determinants of infectious pancreatic necrosis virus virulence and cell culture adaptation. Journal of Virology 79, 10289–10299.

Influenza A Viruses (Orthomyxoviridae) Laura Kakkola, University of Turku, Turku, Finland Niina Ikonen, Finnish Institute for Health and Welfare, Helsinki, Finland Ilkka Julkunen, Institute of Biomedicine, University of Turku, Turku, Finland r 2021 Elsevier Ltd. All rights reserved.

Nomenclature

NLRP

ARDS Acute respiratory distress syndrome bp Base pair cRNA Complementary ribonucleic acid cRNP Complementary ribonucleoprotein GISR Global Influenza Surveillance and Response System HA Hemagglutinin IAV Influenza A virus IFITM Interferon-induced transmembrane protein IFN Interferon Ig Immunoglobulin IL Interleukin IRF Interferon regulatory factor ISG Interferon stimulated gene M1 Matrix protein 1 M2 Matrix protein 2 MDA5 Melanoma differentiation-associated protein 5 mRNA Messenger ribonucleic acid NA Neuraminidase NEP Nuclear export protein Nf-κB Nuclear factor kappa-light-chain-enhancer of activated B cells

Glossary Antigenic drift Mutations occurring to influenza genes during virus replication triggered by immunological pressure or RNA polymerase errors. Antigenic shift Reassortment of viral segments when a cell or a host is simultaneously infected with two or more different IAV strains or subtypes.

Nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing NOD Nucleotide-binding oligomerization domain NP Nucleoprotein NPA Nasopharyngeal aspirate NS1 Non-structural protein 1 PA Polymerase acidic protein PB1 Polymerase basic protein 1 PB2 Polymerase basic protein 2 PKR Protein kinase R RdRp RNA-dependent RNA polymerase RIG-I Retinoic acid-inducible gene I RNA Ribonucleic acid RT-PCR Reverse transcription polymerase chain reaction STAT Signal transducer and activator of transcription TLR Toll-like receptor TNFa Tumor necrosis factor alpha vRNA Viral ribonucleic acid vRNP Viral ribonucleoprotein WHO World Health Organization

ARDS Acute respiratory distress syndrome is a lifethreathening condition caused by a breakage in endothelialepithelial barrier due to IAV infection killing the epithelial cells, resulting in the production of inflammatory mediators leading to accumulation of leukocytes and fluid in the lungs leading to an acute respiratory failure.

Classification of Influenza A Viruses Influenza A viruses (IAV) belong to the family Orthomyxoviridae, together with two other human-infecting influenza viruses, influenza B and C viruses. In addition to humans, influenza A viruses infect various animals, including ducks, chickens, pigs, whales, horses, seals, cats, and bats. Influenza A viruses are subtyped based on the hemagglutin (HA) and neuraminidase (NA) proteins. To date, eighteen different HA proteins (H1-H18) and eleven NA proteins (N1-N11) have been identified. IAV strains are named according to HA and NA subtype, the location or country of isolation and the year of isolation. For example A/Finland/9/2019 (H1N1) describes IAV strain isolated in 2019 in Finland with a strain number of 9, and the isolated virus is a subtype H1N1.

Virion Structure Influenza virions are 100–140 nanometers in diameter. Virions are pleomorphic and mainly elliptical in shape. The enveloped IAV virion consists of cell-derived lipid bilayer, with NA and HA proteins sticking out from the membrane and forming trimers (HA) and tetramers (NA). HA is cleaved into two subunits, HA1 and HA2, that are attached by disulfide bridges. Matrix protein 2 (M2) forms ion channels through the lipid bilayer, and matrix protein 1 (M1) forms a lining underneath the lipid bilayer (Fig. 1).

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00046-1

551

552

Influenza A Viruses (Orthomyxoviridae)

Fig. 1 Structure of influenza A virus. Influenza A virus particles are composed of a viral envelope with trimeric HA and tetrameric NA envelope glycoproteins, envelope-associated matrix protein and eight separate viral ribonucleotide protein complexes (vRNP) that include separate vRNA segments covered by nucleoprotein (NP) and polymerase proteins attached to viral 30 and 50 ends. M2 ion channel protein is embedded into the viral membrane and some nuclear export protein, NEP is also found in virus particles.

Nucleoproteins (NP) wrap eight genomic RNA segments, and the polymerase subunits PA, PB1 and PB2 that form the RNA-dependent RNA polymerase complex (RdRp) are attached to each segment. NP prevents vRNA segments from forming secondary structures and assists in virus replication. Nuclear export protein (NEP) is packaged within the virion.

Genome Genomic Organization Influenza genome is approximately 13,500 bp long. IAV genome consists of eight segments of negative sense single-stranded RNA. Segments are numbered from 1 to 8 based on the molecular weight. Each genomic segment encodes proteins (Fig. 2 and Table 1): segment 1 encodes RNA polymerase subunit PB2, segment 2 encodes via different reading frames RNA polymerase subunit PB1 and PB1-F2 protein, segment 3 encodes RNA polymerase subunit PA and by a ribosomal frameshift PA-X protein, segment 4 encodes HA, segment 5 encodes NP, segment 6 encodes NA, segment 7 encodes by different reading frames and splicing matrix proteins M1 and M2, and segment 8 encodes by different reading frames and splicing non-structural proteins NS1 and NEP. Each RNA segment has non-coding regions at both ends that vary in length, containing poly-adenylation and packaging signals. The ends of the segments are highly conserved and partially complementary. This allows the segments to form helical hairpin structures which serve as promoter areas for RNA replication and transcription, and onto which viral polymerase subunits are bound. Polymerase subunits PB1, PB2 and PA are bound to the ends as a single heterotrimeric complex. The entire viral genomic RNA segments are covered by NP and vRNAs with the associated proteins form viral ribonucleoprotein complexes (vRNPs). NP wrapped around the vRNA segments prevents RNA in forming secondary structures and opens the vRNA for viral polymerasedirected RNA synthesis.

Reassortment and Mutagenesis Due to a segmented genome, influenza virions are capable of exchanging segments between virus strains. This genomic shuffling occurs when two or more different strains or subtypes infect the same cells in a host. This phenomenon is called antigenic shift. Due to error prone viral RNA-dependent RNA polymerase (RdRp), IAVs mutate rather fast. The HA and NA, in particular, are under constant immunological pressure and mutations in HA or NA genes can create new escape mutants. The mutation rate has been estimated to be ca. 0.8%–1.0% per year at the amino acid level of the HA molecule. The phenomenon when mutations in virus genes occur during virus replication is called antigenic drift. Both antigenic drift and antigenic shift can result in new IAV subtypes or strains creating new pandemic or seasonal epidemiccausing viruses.

Influenza A Viruses (Orthomyxoviridae)

553

Fig. 2 Proteins encoded by the eight IAV genomic RNA segments.

Table 1

IAV genome segments, their sizes and functions of the IAV encoded proteins in the virus life cycle and some cell regulatory functions

Genomic segment

Protein

Nucleotides, amino acids

Function

1 (2341 nt)

PB2

2280 nt, 759 aa

2 (2341 nt)

PB1 PB1-F2 PA HA NP NA

2274 nt, 757 aa 264 nt, 87 aa 2151 nt, 716 aa 1698 nt, 550 aa 1497 nt, 498 aa 1365 nt, 454 aa

M1 M2 NS1 NEP

759 294 693 366

Polymerase subunit, 50 -cap snatching, transcription and replication initiation, type I IFN induction inhibition Polymerase subunit, RNA-dependent RNA polymerase Apoptosis inducer, type I IFN induction inhibition Polymerase subunit, RNA synthesis and replication, endonuclease activity Surface glycoprotein, binding to receptor, fusion with endosome membrane vRNA encapsidation, nuclear import of vRNPs Surface glycoprotein, releases HA from sialic acids, cleaves sialic acids on cell surfaces and in mucus, assists in budding Matrix protein, vRNP interaction, RNA nuclear export regulation, viral budding Ion channel, virus uncoating and assembly Interferon antagonist, splicing regulator Nuclear export of vRNPs

3 4 5 6

(2233 (1778 (1565 (1413

nt) nt) nt) nt)

7 (1027 nt) 8 (890 nt)

nt, nt, nt, nt,

252 aa 97 aa 230 aa 121 aa

Life Cycle Influenza A viruses infect lung cells and immune cells residing in the lungs. New virions are produced locally in the tissues, and coughing or sneezing spreads various size aerosol particles which transmit the viruses to new recipients. Influenza virus is mainly spread via larger aerosol particles within 1.5–2 m from an index cases as well as with direct contact with upper respiratory tract secretions.

Attachment and Entry A schematic representation of the life cycle of influenza A virus is shown in Fig. 3. IAV attaches to the sialic acid residues of the cell surface glycoproteins and glycolipids. Attachment to these receptors on the target cell surface occurs by the viral HA protein. Seasonal (human) IAVs specifically bind to sialic acids with a2,6-linkage to galactose in the cell surface glycoproteins that function as viral receptors. Virus is internalized by a receptor-mediated endocytic pathway. At low pH of the endosomes, the M2 ion

554

Influenza A Viruses (Orthomyxoviridae)

Fig. 3 Life cycle of IAV in infected cell. IAV attaches to terminal sialic acid residues on the host surface via its HA molecule (phase 1). The virus enters the cell via clathrin-mediated endocytosis (2). In endosomes, due to a conformational change in the trimeric HA molecule triggered by low pH, the membranes of the virus and endosomes are fused and vRNPs are released into the cell cytoplasm (3). vRNPs are transported into the nucleus where the primary transcription, replication and secondary transcription are taking place (4). Viral mRNAs are transported into the cytoplasm and viral proteins are synthesized. Newly synthesized Ps, NP, M1, NS1 and NEP proteins are transported into the nucleus where they regulate vRNA replication and packaging of newly synthesized vRNPs that are exported from the nucleus by the aid of M1 and NEP (4a, 4b). vRNPs and viral envelope glycoproteins HA and NA are transported onto the plasma membrane where the assembly (5) and budding of new viral particles takes place (6).

channel opens and proton influx through the M2 ion channel acidifies the interior of the viral capsid. The subsequent acidification of the endosome causes conformational change in the HA protein, resulting in the exposure of a fusion peptide in the N-terminal end of the HA2 subunit. The insertion of the HA2 fusion peptide into the endosomal membrane causes the viral and endosomal membranes to fuse. Low pH inside the virus particle causes M1 proteins to degrade and dissociate. This membrane fusion and virus capsid acidification results in the release of vRNP complexes into cytoplasm. In the cytoplasm, the released vRNPs are transported into the nucleus via cellular importin transport machinery. This transport is facilitated by the nuclear localization signal on NP onto which cellular importin a subtypes bind and with the aid of importin b molecule vRNPs are translocated into the nucleus through nuclear pore complexes. All eight vRNP complexes are transported into the nucleus within an hour after virus attachment. In the nucleus, three forms of viral RNAs are produced: complementary viral RNA (cRNA) that is used as a template for the new viral genomic RNA (vRNA) and for the viral mRNA.

Transcription and Translation Viral RNA transcription and RNA processing take place in the nucleus. Transcription of viral mRNAs is initiated by the PB2 subunit of the polymerase complex that binds to the host pre-mRNAs. The PA subunit of the polymerase complex is an endonuclease that cleaves 50 -cap structures from cellular pre-mRNAs in a process called cap snatching. The cap-structure is needed later to promote viral mRNA translation by host cell ribosomes. The PB1 subunit ligates the 50 -cap structure to be used as a primer for the synthesis of viral mRNAs. PB1 is also an RNA-dependent RNA polymerase that incorporates nucleotides to the newly synthetized viral mRNAs. Polyadenylation of viral mRNAs takes place at the 50 -end of the vRNA segment by reiterative stuttering of the polymerase complex. All eight viral RNA segments are transcribed to mRNAs. Transcripts for M and NS contain splicing sites, and cellular spliceosome is used to produce mRNAs encoding M2 and nuclear export protein NEP. Processed viral mRNAs are transported to the cytoplasm for translation. Translation of viral proteins takes place on cytosolic ribosomes (for PB1, PB2, PA, NP, NS1, NS2, and M1) and on endoplastic reticulum associated ribosomes (for HA, NA and M2). Cellular ribosomes recognize the 50 -cap structures and translate the viral mRNAs into proteins. Of the newly synthetized viral proteins, the polymerase subunits PB1, PB2 and PA, and NP, NEP, M1, NS2 and NS1 are transported into the nucleus where the proteins regulate viral RNA replication, mRNA processing and downregulate innate immunity (NS1). HA, NA and M2 are incorporated into the membranes of endoplasmic reticulum (ER) during translation. HA and NA form trimers (HA) and tetramers (NA) on ER membranes and these molecular complexes are glycosylated at the Golgi

Influenza A Viruses (Orthomyxoviridae)

555

complex. HA is cleaved into HA1 and HA2 by cellular proteases before entering the plasma membrane. This cleavage is a prerequisite for the progeny virions to be infectious and have a functional HA2-associated fusion peptide to initiate the next round of infection.

Genome Replication In addition to mRNA transcription, the new viral genomic RNA segments are replicated in the nucleus. First, viral cRNA is synthesized by an unprimed process by viral RNA-dependent RNA polymerase using the incoming vRNAs as templates. These newly-synthesized cRNAs are wrapped by newly-produced viral NPs and a polymerase (PB1, PB2 and PA) complex to form a cRNP structure. This cRNP is subsequently used as a template to produce more vRNA by RNA-dependent RNA polymerase. Newly synthesized vRNA segments are wrapped by NPs, newly-produced polymerase subunits are added, and these vRNPs are ready to be exported from the nucleus.

Viral Assembly Viral ribonucleoprotein complexes (vRNA, NPs and polymerase subunits) are assembled in the nucleus. NEP exports the vRNPs and M1 complexes out from the nucleus. vRNP-M1 complexes created by a nuclear egress process are transported to the plasma membrane by an unknown mechanism that involves cellular protein Rab11. Viral M1 directs the formation of the progeny virions on the plasma membrane and viral surface glycoproteins HA and NA associate with vRNP-M1 complexes.

Budding of New Virions Progeny virus assembly and virus budding occur on distinct areas on the plasma membrane. HA and NA, along with M2, localize on the plasma membrane to raft regions that are rich in cholesterol and sphingolipids. Viral proteins HA, NA and M1 from the incoming vRNP-M1 complexes induce plasma membrane curvature and new progeny viruses are formed by wrapping of the plasma membrane around the 8 segments of vRNPs. M2 on the plasma membrane is localizing to the budding site and assists in membrane bending and scission. These events result in the budding of the new virions from the plasma membrane. Viral NA protein cleaves sialic acid from the cell surface glycoproteins and glycolipids facilitating progeny virions to be released from the host cell. This sialidase activity of NA also facilitates the virus movement and entry into new host cells by cleaving the sialic acids in the mucus of respiratory track.

Immunological Responses and Immune Evasion Non-specific structural protective barriers, such as mucosa, ciliated epithelial cells, and protease inhibitors at the airways, restrict influenza viruses to gain access to epithelial cell surfaces. If IAV bypasses these barriers and is able to enter the target cell, various types of immunological responses are initiated in the host.

Innate Immune Responses The entry of IAV initiates innate immune responses in the target cell leading to the production of interferons and cytokines. The RNA structures of the incoming and replicating virus are recognized by cellular pattern recognition receptors. In influenza infection, the viral structures are recognized by cytoplasmic cellular dsRNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA-5), and by cellular membrane-associated Toll-like receptors (TLR3 and TLR7). The recognition of viral RNAs initiates a signaling cascade in the cell, leading to the activation of transcription factors such as interferon regulatory factor 3 (IRF3), IRF7 and nuclear-factor-kappa-B (NF-κB). These transcription factors translocate into the nucleus and activate the transcription of type I and type III interferons (IFN-a/b and IFN-l, respectively) and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a), interleukin 6 (IL-6) and IL-1b. Another innate immune response mechanism, inflammasome, is activated through NOD-like receptors, such as NOD-like receptor family pyrin domain containing 3 (NLRP3), leading to transcription of pro-IL-1b, pro-IL-18, and pro-caspase-1, which are subsequently cleaved into active forms of the proteins. Interferons are secreted from the cell, and they act both as para- and autocrine signaling molecules to activate antiviral and inflammatory responses in infected cells and in neighboring cells. Secreted type I or III IFNs bind to their specific receptors IFNAR or IFNLR, respectively, on the cell surface and a signaling cascade is initiated. Through activation of cellular Janus kinase (JAK) pathways transcripton factors called signal transducer and activator of transcription (STAT) are activated. STAT1 and STAT2 form heterodimers, translocate into nucleus where they associate with interferon regulatory factor 9 (IRF9). In the nucleus these activators initiate the transcription of antiviral and proinflammatory genes, that are called interferon-stimulated genes (ISGs). During virus infection, several hundred of ISGs can be induced. The main antiviral proteins produced in IAV infection include

556

Influenza A Viruses (Orthomyxoviridae)

protein kinase R (PKR), Mx-proteins and interferon-induced transmembrane protein family proteins (IFITMs). These antiviral proteins inhibit the early steps of IAV infection. Interferons and interleukins, i.e., cytokines, regulate immune responses and inflammatory reactions, and act as mediators in initiating the activation of adaptive immune responses.

Adaptive Immune Responses Adaptive immune responses are specifically targeted to various antigenic structures of the invading pathogen. Adaptive immune responses include antibody-mediated response (B cells) and cell-mediated response (T cells). IAV infection leads to both B cell and T cell-mediated immune responses. On the site of infection, dendritic cells take up IAV particles, maturate and migrate to local lymph nodes to present virus-derived antigens to T cells. Activated virus-specific lymphocytes migrate from the lymph nodes to the site of infection where locally secreted cytokines further enhance the development of adaptive immune response. Cell-mediated immunity is important in the recovery from influenza infection. Cytotoxic T cells (CD8 þ ) kill virus-infected cells and secrete cytokines to restrict virus replication. T cells also develop to memory T cells that are specific for conserved IAV epitopes found e.g., in NP, M1, and PA proteins. However, the cytotoxicity of the memory T cells fades in time. Helper T cells are important in promoting B cell responses against IAV infection. Viral antigens in lymph nodes are taken up by B cells and with additional B cell stimulatory signals IAV-specific antibody response is activated. B cells produce IgA and IgG antibodies that act on mucosal membranes and in tissues, respectively. The majority of antibodies are targeted against HA, NA and NP proteins. Anti-HA and antiNA antibodies are protective and neutralizing towards the reinfection with IAV. However, due to relatively fast mutation rate of IAVs, this protection is not always effective against infections with new IAV strains. In addition, the capability of B cells to produce antibodies vanes with age.

Immune Evasion Strategies of IAV IAVs, as with other pathogenic viruses, have several strategies to evade or delay host cell immune responses. The main player in this is the viral NS1 protein. In infected cells, NS1 is rapidly expressed to high levels and the protein is found mainly in the nucleus, but some protein is also found in the cell cytoplasm. The RNA-binding domain of NS1 interacts with viral double-stranded RNA to protect it and to interfere with the interaction of viral RNA with cellular RNA recognition receptors RIG-I-like receptors and TLRs. In addition, the effector domain of NS1 interacts with host cell mRNA processing. This leads to shut-down of host protein synthesis facilitating virus replication and viral protein translation. NS1 interferes also with immune response pathways with several mechanisms: it interferes with cell antiviral gene transcription in the nucleus, it inhibits RIG-I pathway and interferoninduced pathway, it inhibits the function of antiviral proteins, it affects the signaling pathways, and it inhibits adaptive immune response by affecting the maturation of dendritic cells. Another immune response antagonist, PB1-F2, activates apoptosis and cell death by targeting mitochondrial membranes. It also interferes with the RIG-I pathway by interacting with mitochondrial antiviral signaling (MAVS) protein on the mitochondrial membrane.

Evolution and Epidemiology Evolution of IAV Surface glycoproteins, HA and NA, are under constant immunological pressure. These proteins mutate rather fast due to antigenic drift when the RNA-dependent RNA polymerase makes mistakes during RNA replication (Fig. 4). IAV attaches to the cell surface receptor with HA protein and the antibodies targeting especially receptor-binding part of HA restrict the binding IAV on the cell surface receptors and reduce the likelihood of infection. However, amino acid changes in the major antigenic sites and changes in glycosylation pattern reduce the ability of anti-HA and anti-NA antibodies to bind to the virus, and return the ability of virions to infect the cells. IAV NA protein acts as a sialidase and cleaves sialic acids enabling the virions to be released from the cell surface after budding from the plasma membrane. The antibodies targeting the catalytic sites of NA prevent the release of progeny virions. However, mutations that retain catalytic activity but hinder antibody binding again assist the infectious cycle of virions. This constant mutation of HA and NA proteins is referred as antigen evolution. Due to this antigen evolution, pre-existing immunity to other IAV strains does not fully protect from new epidemic strains. Hence new influenza epidemics occur yearly, and the influenza strain composition of vaccines has to be constantly evaluated.

IAV Epidemics and Pandemics Influenza A viruses have caused severe pandemics and yearly epidemics. Of the potential HA/NA combinations, three influenza A subtype viruses (H1N1, H2N2, and H3N2) have caused pandemics or epidemics in humans. In addition, avian influenza A virus subtypes H5N1, H7N7, H9N2, and H7N9 have been able to infect humans. The most variable part of the HA protein has 25%–80% homology between the IAV subtypes (Fig. 5). Currently two main subtypes, H1N1 (pdm09) and H3N2, are circulating

Influenza A Viruses (Orthomyxoviridae)

557

Fig. 4 Major antigenic sites of H1 virus (Ca, Cb, Sa and Sb) shown on a trimeric (on left) and on a monomeric (on right) HA molecule. Different antigenic sites are shown in different colors and the receptor binding pocket is shown in purple.

Fig. 5 Phylogenetic trees based on HA sequences of various IAV subtypes. A. The tree of five different subtypes was constructed by the Neighbor-Joining method with Mega software version 7. The length of branches are propotional to the evolutionary distances. B. Phyologenetic tree based on HA sequences of H1N1 subtypes. The tree was constructed by the Neighbor-Joining method. The horizontal lines are propotional to the number of nucleotide changes.

in the human population world wide. Influenza epidemics occur in the Northern hemisphere usually in November-April, and in the Southern hemisphere in June-October. In countries near the equator this seasonality is not that distinct, however, the peak activity of IAV infections is mainly during the rainy season.

558

Influenza A Viruses (Orthomyxoviridae)

Fig. 6 Timeline of IAV pandemics and global epidemics in the 20th and 21st centuries.

WHO has estimated that seasonal influenza virus infections (both IAV and IBV) cause 3–5 million cases of severe repiratory illness annually and 290,000–650,000 deaths. This accounts for roughly 2% of all annual respiratory illness-related deaths. The intensity of the epidemic varies between countries due to the heterogenicity of the epidemic virus strains and differences in the immune status of a given population. H3N2 subtypes of IAV have caused seasonal epidemics since 1968 when the H3N2 subtype caused a pandemic. H3N2 subtypes readily infect all age groups, and they are the cause for the most of the IAV related hospitalizations and deaths. H1N1 subtypes caused seasonal epidemics less frequently until 2008. H1N1 epidemics usually affect younger age groups than the elderly ones. In 2009 a new pandemic H1N1 subtype (“swine flu”) caused a world wide pandemic and since then this subtype has been circulating and causing annual epidemics. IAVs have caused several pandemics (Fig. 6). New pandemics arise when the virus has a new set of reassortant segments (antigenic shift). The first identified pandemic was caused by an H1N1 subtype (Spanish flu) in 1918. It has been estimated that the 1918 virus infected one third of the world population causing at least 50 million deaths. The high mortality among healthy adults was exceptional for this virus. However, the 1st World War, low hygienic conditions and lack of antibiotics and intensive care may have contributed to the poor prognosis of the patients which was often due to secondary bacterial infections. The next two pandemics, in 1957 (Asian flu) and 1968 (Hong Kong flu), were caused by H2N2 and H3N2 subtypes, respectively. The Asian flu strain was a reassortant between human H1N1 and avian H2N2, and this subtype caused annual epidemics for the next ten years, being then replaced by the H3N2 Hong Kong flu subtype. Both pandemics resulted in an estimated 1 million deaths each. In 1977 the reappearance of a previous 1950s-like H1N1 subtype (Russian flu) caused a wide global epidemic, affecting the younger population since older individuals had protective immunity from infections caused by the descendants of the 1918 H1N1 virus. The H1N1 subtype causing the pandemic in 2009 and resulting in roughly 400,000–800,000 deaths, was exceptional in that sense that it originated from North America. The virus had accumulated genomic segments from IAVs of birds, swine and humans. The 2009 pandemic, like the one in 1918, affected the younger generations since the elderly individuals (born before 1950s) had pre-existing immunity due to infections caused by the 1918 descendant H1N1 viruses. The 2009 subtype was named as the H1N1pdm09 virus. Avian influenza strains have caused limited epidemic clusters or spradic cases of infection in humans. Avian IAV subtype H5N1 has caused almost 900 human cases with 50% mortality in ca. 30 countries. Subtype H7N7 caused an outbreak in the Netherlands with mild symptoms in humans (though one died), as also H9N2 in Hong Kong and China. Subtype H7N9 has caused nearly 1600 cases with 35% mortality in China and this subtype is causing new human cases annually. These avian influenza strains are transmitted to humans mainly directly from birds, and human-to-human transmission is either absent or extremely rare. As for future, the avian IAVs are posing a threat of starting a new deadly pandemic. To prevent this from happening, global measures have been put in place (instructions on farming the birds and culling of poultry if infection is detected) and the appearance of IAV strains with amino acid changes enhancing the likelihood of human-to-human transmission is intensively monitored.

Clinical Features Seasonal IAV infections can cause asymptomatic or mild disease, or a more pronounced disease which may even lead to death. Incubation time from the time of exposure until the onset of the disease is usually 1–4 days, but can be up to 7 days. Virus is shed in the respiratory secretions a day before symptoms, and shedding usually continues for up to five days. Symptomatic disease often begins with a sudden high fever that lasts for several days. Other common symptoms are headache, chills, muscle pain, dry cough, sore throat, fatigue and loss of appetite. Nasal congestion and runny nose follow later. Cough and fatigue can last for a few weeks. In young children another common symptom is diarrhea. Common complications to follow IAV infection in adults are bronchitis and sinusitis, and in children otitis media. Most infected people recover from the infection but some develop more severe, sometimes even life-threatening conditions. Usually people belonging to high risk groups develop these complications: 465 years of age, pregnant women, children o2 years of age, and people with certain medical chronic conditions such as disease of the lungs, diabetes, or cardiovascular diseases. The most severe complication is pneumonia, caused either by the virus or by concurrent bacterial infection. Other severe complications may include myocarditis, encephalitis, myositis, rhabdomyolysis, ARDS or multi-organ failure of, for example, respiratory or kidney organs. Influenza infection can also worsen the underlying disease.

Influenza A Viruses (Orthomyxoviridae)

559

Pathogenesis Virus initially replicates in the epithelial cells of the trachea and bronchia, and can disseminate into other parts of the respiratory system. Replication of the virus and host inflammatory responses cause cellular damage to the epithelium. The recovery process of the epithelium begins in few days, but can last up to a month. Viral pneumonia occurs in some patients when the virus replicates in alveolar epithelial cells. This causes the walls of the alveoli and bronchioles to rupture, leading to pneumonia. Often pneumonia is a combined viral-bacterial pneumonia. The patient appears to recover from IAV infection, but then a few days later symptoms of pneumonia develop. This is caused by a secondary infection with bacteria such as Streptococcus pneumoniae, Staphylococcus aureus, or Hemophilus influenzae. This condition can be treated with antibiotics, but can be fatal. In some patients IAV infection can lead to an acute respiratory distress syndrome (ARDS). This life-threathening condition is caused by a breakage in the endothelial-epithelial barrier due to IAV infection killing the epithelial cells. This results in the production of cytokines that attract leukocytes and activate endothelial cells, leading to infiltration of leukocytes into the lungs. The responses triggered by immune cells then further break the barrier, resulting in the accumulation of fluids into the lungs and a respiratory failure. Another severe pathological outcome in IAV infection is a cytokine storm. In addition to lung epithelial cells, IAV infects also endothelial cells and lung-resident macrophages. All these infected cell types secrete cytokines leading to the expression of ISGs. This host response is tightly autoregulated, however, in some patients the regulation fails. As a result, there is an excessive inflammatory response in the lungs, leading to permeabilization of lung endothelium and leakage of fluids into the lungs.

Diagnosis IAV infections are diagnosed based on the presence of virus or viral RNAs or proteins in the sample (acute infection) or by detection of an increase in influenza antibodies in paired serum specimens. IAV can be cultured from nasal swabs or nasopharyngeal aspirates (NPAs). For treatment decisions, fast diagnosis is required. Thus virus cultivation is not an optimal method of choice, although it is important in epidemiological surveillance of circulating strains and in selecting vaccine strains. IAV particles in the samples can be detected by antigen detection methods, such as rapid point-of-care tests that are fast but robust, and give accurate results only in the early phases of the infection when the viral load is the highest. Presently, the most commonly used method is the detection of IAV RNA in a sample by RT-PCR. Different PCR methods are fast, some can quantitate the amount of viral RNA in the sample and can subtype the IAV strain causing the infection.

Treatment and Prevention Antivirals More detailed description of antivirals and vaccines is available elsewhere in the Encyclopedia. Briefly, commercially available and widely used drugs against influenza viruses are of three categories. Neuraminidase inhibitors, such as zanamivir and oseltamivir, target viral surface NA proteins. Adamantane compounds, amantadine and rimantadine, block the M2 ion channel protein. Amantadine compounds are effective only for IAV and not for IBV. Baloxavir marboxil, a new antiviral against influenza, is a cap-endonuclease inhibitor. Antivirals are effective when started within 48 h after the onset of infection, making the treatment at the population level challenging. In addition, wide use of antivirals creates viral escape mutants that are resistant to drugs, and in fact, all currently circulating IAV strains are resistant to amantadines. Resistance to NA inhibitors is presently not very common. Treatment with antivirals given in due time or prophylactically can reduce the severity of symptoms and occurrence of secondary infections, reduce the duration of the disease, and prevent the IAV infection.

Vaccines The most effective countermeasure for IAV infections is vaccination. Yearly updating of influenza vaccines (against influenza A and B) is needed due to constant immunological pressure on influenza viruses that gives rise to new progeny virions with altered protein sequences with ability to escape immune responses elicited by vaccines. Thereby, WHO estimates twice a year the composition of the annual vaccines based on viral strains obtained and characterized by WHO Global Influenza Surveillance and Response System (GISRS). To manufacture influenza vaccines, the whole viruses are inactivated (split vaccines), viruses are attenuated or vaccines include only the HA and NA proteins of influenza virus (subunit vaccines). Presently, influenza vaccines include four influenza strains: two influenza A (H1N1 and H3N2) and two influenza B (Victoria and Yamagata) strains. In addition to influenza virus components, different types of adjuvants may be included in the vaccine to strengthen the immune response. There is two ways to administer influenza vaccines, by injection (inactivated) or inhalation (live attenuated). Influenza vaccination is recommended for risk groups, i.e. mainly infants, elderly people and those with predisposing underlying conditions, such as lung and cardiovascular

560

Influenza A Viruses (Orthomyxoviridae)

diseases, diabetes, impaired immunity or cancer. The vaccine efficacy varies every year, it depends on the timing of the vaccination campaign, the age and immune status of the vaccinees, the match of vaccine strains to circulating virus strains, and the testing procedures of the vaccine efficacy. Usually the efficacy is 40%–70%. In addition to prevention of infection, vaccination has been shown to reduce the severity of the influenza disease and to prevent secondary bacterial infections.

See also: Avian Influenza Viruses (Orthomyxoviridae)

Further Reading Chen, X., Liu, S., Goraya, M.U., et al., 2018. Host immune response to influenza A virus infection. Frontiers in Immunology 9, 320. Dou, D., Revol, R., Östbye, H., Wang, H., Daniels, R., 2018. Influenza A virus cell entry, replication, virion assembly and movement. Frontiers in Immunology 9, 1581. Ferhadian, D., Contrant, M., Printz-Schweigert, A., et al., 2018. Structural and functional motifs in influenza virus RNAs. Frontiers in Microbiology 9, 559. Gounder, A.P., Boon, A.C.M., 2019. Influenza pathogenesis: The effect of host factors on severity of disease. Journal of Immunology 202 (2), 341–350. Hsu, A.C., 2018. Influenza virus: A master tactician in innate immune evasion and novel therapeutic interventions. Fronties in Immunology 9, 743. Kash, J.C., Taubenberger, J.K., 2015. The role of viral, host, and secondary bacterial factors in influenza pathogenesis. American Journal of Pathology 185 (6), 1528–1536. Klemm, C., Boergeling, Y., Ludwig, S., Ehrhardt, C., 2018. Immunomodulatory nonstructural proteins of influenza A viruses. Trends in Microbiology 26 (7), 624–636. Krammer, F., 2019. The human antibody response to influenza A virus infection and vaccination. Nature Reviews Immunology 19 (6), 383–397. Krug, R.M., 2015. Functions of the influenza A virus NS1 protein in antiviral defense. Current Opinion in Virology 12, 1–6. Kuiken, T., Taubeberger, J.K., 2008. Pathology of human influenza revisited. Vaccine. 26 (Suppl 4), D59. [66]. Lowen, A.C., 2017. Constraints, drivers, and implications of influenza A virus reassortment. Annual Review of Virology 4 (1), 105–121. Meineke, R., Rimmelzwaan, G.F., Elbahesh, H., 2019. Influenza virus infections and cellular kinases. Viruses. 11 (2). Padilla-Quirarte, H.O., Lopez-Guerrero, D.V., Gutierrez-Xicotencatl, L., Esquivel-Guadarrama, F., 2019. Protective antibodies against influenza proteins. Frontiers in Immunology 10, 1677. Taubenberger, J.K., Morens, D.M., 2008. The pathology of influenza virus infections. Annual Review of Pathology 3, 499–522.

Relevant Websites https://www.gisaid.org/ GISAID. Initiative. https://www.cdc.gov/flu Influenza (Flu). CDC. https://www.ecdc.europa.eu/en/seasonal-influenza Seasonal influenza. ECDC. Europa EU. https://www.who.int/influenza/en/ WHO. Influenza. World Health Organization.

Influenza B, C and D Viruses (Orthomyxoviridae) Thorsten Wolff, Robert Koch Institute, Berlin, Germany Michael Veit, Free University of Berlin, Berlin, Germany r 2021 Elsevier Ltd. All rights reserved.

Glossary EC50 Half maximal effective concentration. EC50 describes the concentration of a substance that gives half maximal response of a biological pathway. GISRS Global Influenza Surveillance and Response Systems. GISRS is the joint collaboration of WHO with 6 WHO Collaborating Centers, four Essential Regulatory Laboratories and National Influenza Centers in more than 140 countries dedicated to protect people from the threat of influenza. It was established in 1952. HA Hemagglutinin. HEF Hemagglutinin-esterase-fusion protein. HEF mediates host cell attachment and fusion of the incoming virions as well as progeny virion release after completion of the replication cycle. IBV Influenza B virus. IC50 Inhibitory concentration 50. IC50 describes a concentration of a compound sufficient to limit virus titers by 50% when added to a host or a host cell.

ICV Influenza C virus. IDV Influenza D virus. NA Neuraminidase. NEP Nuclear export protein. NLS Nuclear localization signal. NLS describes a topogenic amino acid sequence that directs the import of a protein into the nucleus of a eukaryotic cell. NS1 Non-structural protein. PKR DsRNA-activated protein kinase. RIG-I Retinoic acid induced gene I. SC35 Serine/arginine-rich splicing factor 35. vRNP Viral ribonucleoprotein. The genomic RNAs of influenza viruses are complexed with the viral nucleoprotein and polymerase into vRNPs that represent the smallest unit for expression of the encoded protein(s). WHO World Health Organization.

Classification (Compact) Influenza B virus (IBV) was identified for the first time during an outbreak of an acute respiratory disease in 1940 in Northern America, in which it was found to lack antigenic cross-reactivity to influenza A virus (IAV). IBV is classified as the only member of the genus influenza virus B within the order of the Orthomyxoviridae. IBV was estimated to have diverged from IAV around 4000 years ago based upon the calculated rate of amino acid substitutions within HA proteins, but the mechanisms of replication and transcription, as well as functionality of most viral proteins appear to be largely conserved, with some unique differences. IBV has been detected in rare occasions in seals and pigs, but humans are the major host species of IBV, in which it causes typically the same spectrum of symptoms of influenza-like illness as IAV although pathologies outside the respiratory tract have been determined in a minority of IBV infections. A large recent metagenomic study detected an IBV-like virus in a fish species suggesting that the genus may have additional vertebrate host reservoirs outside mammalian species. Sentinel surveillance detects IBV in some seasons as the dominating circulating influenza virus type highlighting its significant socio-economic impact. There are currently two lineages of IBV (B/Yamagata-like and B/Victoria-like viruses) with limited antigenic cross-reactivity. Viruses of both lineages circulate to varying extents and ratios in all parts of the world. The lack of a large animal host reservoir able to foster the appearance of pandemic variants, a lower evolutionary rate and generally considered milder courses of disease in comparison to IAV infection are typical features of IBV. Influenza C virus (ICV) was first isolated in 1947 from a human patient having mild respiratory symptoms. Since the virus showed no cross-reactivity with antisera against influenza A or B viruses, it was classified as a new genus of the Orthomyxoviridae (influenza C virus). The virus has a worldwide distribution and the majority of humans develop antibodies against ICV early in life. Humans are the main reservoir of ICV, but occasionally the virus may also infect dogs and pigs. In 2011 a new Influenza C-like virus was isolated from clinically ill pigs. Since this isolate is unable to reassort with ICV it is now officially named as influenza D virus (IDV). Subsequent studies showed that IDV can also infect small ruminants and cattle, the latter of which is apparently its main reservoir. The first part of this article reviews current knowledge on type B influenza virus, whereas the second part addresses important aspects of type C and D viruses.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21505-7

561

562

Influenza B, C and D Viruses (Orthomyxoviridae)

Influenza B virus Virion Structure IBV virions show pleomorphic structures that can have spherical, irregular or filamentous appearances and are surrounded by a membrane, in which the viral membrane proteins HA, NA, NB and BM2 are integrated. Type A and B influenza viruses cannot be distinguished based upon their morphology or size as determined by electron microscopy (EM) (Fig. 1). The average size of spherical IBV virions of the prototypic B/Lee/40 strain grown in embryonated chicken eggs was determined to be 137 7 27 nm. Apparently, different organizational patterns have been reported for the eight viral negative-strand RNA gene segments, which together with the viral nucleoprotein and polymerase exist as viral RNPs inside IBV virions. Analysis of ultrathin sections by transmission electron microscopy suggested that virions of laboratory and clinical IBV strains package mostly eight RNP structures that are organized in a 7 þ 1 configuration with a central long RNP encircled by 7 others. In contrast, 3D reconstructions by cryoEM tomography were rather indicative for a disordered organization of IBV vRNPs. Studies of purified virions by electron microscopy revealed the presence of 400–500 spike-like projections per spherical virion corresponding to the viral glycoproteins. Quantitative analysis of purified virions by mass spectrometry showed that M1 is the most abundant IBV virion component followed by NP, HA and NA, whereas the level of NB and P proteins were the lowest.

Genome The genome of IBV is organized into eight single-stranded RNA molecules that are of negative polarity and vary in length from 1.1 to 2.4 kb (Table 1). Each of the segments carries characteristic highly conserved sequences of 10 (AGUAG (A/U) AACA) and 9 nucleotides (UCGUCUUCG) at their 50 and 30 -termini, respectively, which themselves flank short segment-specific non-coding regions (NCR) that encompass the coding sequences and short U-rich elements directing polyadenylation. Each IBV gene segment

Fig. 1 EM pictures of influenza B and C viruses. Left: Influenza B virus. Negative staining EM. (B/Harbin/7/94, Source: RKI, ZBS4). Right: Influenza C virus. Cryo EM of C/Johannesburg/1/66. Source: Kai Ludwig (Core Facility BioSupraMol of the Free University Berlin). Scale bar ¼ 100 nm.

Table 1 Identities and lengths of genome segments and encoded gene products from prototypic influenza B virus, influenza C virus and influenza D virus strains Segment

B/Lee/40a vRNA (nts)

B/Lee/40a Protein(s) (aa)

B/Colorado/06/2017 (Vic)a Protein(s) (aa)

B/Phuket/3073/2013 (Yam)a Protein(s) (aa)

C/Jhb/1/1966a Protein(s) (aa)

D/swine/Ok/1334/2011b Protein(s) aa

1 2 3 4 5 6

2396 2368 2304 1882 1841 1557

7

1191

PB2 (770) PB1 (752) PA (726) HA (584) NP (560) NA (466) NB (100) M1 (248) BM2 (109) NS1 (281) NEP (122)

PB2 (772) PB1 (753) P3 (710) HEF (664) NP (552) M1 (246) CM2 (?) NS1 (243) NS2 (184)

1096

PB2 (770) PB1 (752) PA (726) HA (583) NP (560) NA (466) NB (100) M1 (248) BM2 (109) NS1 (282) NEP (123)

PB2 (774) PB1 (754) P3 (709) HEF (655) NP (565) M1 (242) CM2 (115) NS1 (246) NS2 (182)

8

PB2 (769) PB1 (752) PA (726) HA (584) NP (560) NA (466) NB (100) M1 (248) BM2 (109) NS1 (281) NEP (122)

a

source: Influenza Virus Database, NCBI (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database). source: Hause, B.M., Collin, E.A., Liu, R., et al., 2014. Characterization of a novel influenza virus in cattle and Swine: Proposal for a new genus in the Orthomyxoviridae family. Mbio 5, e00031-14. Abbreviations: nts, nucleotides; aa, aminoacids.

b

Influenza B, C and D Viruses (Orthomyxoviridae)

563

Fig. 2 Scheme of an IBV particle and its genome segments. Adapted from viralzone https://viralzone.expasy.org/80?outline=all_by_species with permission.

encodes one or two of the eleven known viral gene products (PB1, PB2, PA, HA, NP, NA, NB, M1, BM2, NS1, NEP) utilizing distinct strategies to expand viral coding capacity Fig. 2. The nomenclature of the viral gene segments resembles those of IAV, with the largest three encoding the polymerase subunits PB2 (segment 1), PB1 (segment 2) and PA (segment 3), followed by the monocistronic HA (segment 4) and NP genes (segment 5). The residual three segments each encode for two viral proteins, which is achieved by different strategies: Segment 6 differs from its IAV counterpart by encoding not only the viral neuraminidase (NA), but also the fourth viral transmembrane glycoprotein NB in an overlapping reading frame. Its translation is initiated at the first AUG start codon, just four nucleotides upstream of the NA start codon. The NA and NB open reading frames overlap for 292 nucleotides. Translational initiation at the NA start codon is efficient despite being located downstream of the NB start. Also the expression strategy of segment 7 encoding the viral M1 matrix and the BM2 ion channel protein differs from the one used by IAV, in which the primary mRNA transcript is processed by cellular splicing machinery proteins. In contrast, the IBV M1 and BM2 proteins are both translated from a collinear transcript in which the M1 stop codon and the BM2 start codon are assembled into a UAAUG pentanucleotide, which facilitates ribosomal re-initiation of protein synthesis following completion of M1 translation. Translation of BM2 coding sequence depends on the presence of a conserved cis element of 45 nucleotides preceding the stop-start signal. This element contains a nucleotide stretch complementary to helix 26 of 18S rRNA, which has been suggested to facilitate the recruitment of the 40S ribosomal subunit to the mRNA upon termination at the M1 stop codon. The NS segment (segment 8) of IBV encodes NS1 and NEP proteins, which are expressed either from a collinear mRNA (NS1) or from a spliced transcript, from which an intronic sequence is removed (NEP). NEP exon 1 mRNAs utilizes the AUG start and ten codons of the NS1 reading frame, which is directly spliced to exon 2 that overlaps with the NS1 coding region for 155 nucleotides

564

Influenza B, C and D Viruses (Orthomyxoviridae)

in a þ 1 reading frame. No homologs of accessory protein genes identified in IAV such as PB1-F2 or PA-X have been reported for IBV. Reassortment, i.e., the exchange of gene segments between two viruses infecting one cell, is a general evolutionary mechanism of segmented RNA viruses, which is also a major driving force for the appearance of novel epidemic IBV strains both from within and in between the Victoria- and Yamagata lineages (see below). Classical coinfection experiments indicated that there can be phenotypic mixing of type A and B viruses generating particles that contain antigenic determinants derived from both parental types. However, IBV has never been observed to produce stable reassortant viruses harboring a gene segment(s) of IAV and vice versa. At least some part of this restriction appears to operate on the level of genome incorporation. Recombinant viral gene segments carrying the IBV HA or NA reading frames could be incorporated together with six other IAV segments into infectious chimeric influenza A/B viruses, when the coding regions were flanked with the packaging signals of the type HA and NA segments. The unique packaging signals of each of the viral gene segments have been mapped for IAV to contain the non-coding regions (NCRs) as well as terminal parts of the coding sequences, but have been less well studied for IBV. The appearance of defective interfering (DI) RNAs derived from viral gene segments containing the conserved terminal promoter sites with large internal deletions of the coding regions, especially in cells infected at high multiplicity, has also been described for IBV. However, the precise mechanism(s) by which the viral polymerase generates viral DI RNA remain to be elucidated.

Life Cycle and Functions of Viral Proteins Virus Entry and Genome Expression and Replication by Polymerase Proteins The main targets of IBV are the epithelial cells lining the upper respiratory tract. The available evidence indicates that the replication cycles of IBV and IAV follow very similar or even identical principles, which are, however, executed by divergent and a few type-specific proteins, respectively, resulting in some virus type-specific mechanisms that are described in detail below. Hence, IBV and IAV were shown to enter a permissive host cell by receptor-mediated endocytosis, which is initiated by binding of the viral hemagglutinin (HA) to sialic acid residues that are present either as glycoprotein or glycolipids on the host cell surface. Intracellular release of the viral genome requires both acidification of the interior of the virion through the viral BM2 ion channel, which is followed by HA–mediated fusion of the viral and endosomal membranes in the acidic environment of the late endosome. The pH optimum for fusion activity of the IBV HA has been determined at pH of 5.4–5.6, which is slightly above the range known for HA of human IAV (pH ¼ 5.0–5.4). The viral RNPs are transported from the cytosol into the nucleus, where the viral RNA polymerase, a trimeric enzyme of ca. 270 kDa initiates transcription and replication of the viral gene segments. These events have been less well studied in cells infected with IBV compared to type A viruses. However, the high conservation of structural elements within the three polymerase subunits PB1 (enzymatic core), PB2 (cap binding domain, aa 318–480) and PA (endonuclease site, aa 1–195) as determined by X ray crystallography, as well as functional studies of recombinant polymerases strongly argue for a conserved mode of action. Within the nucleus each vRNP acts as an independent unit that can either be transcribed into polyadenylated positive-sense viral mRNA using a cap-snatching mechanism, or serves as the template for synthesis of a positive-sense complementary RNA (cRNA) which in turn directs the replication of another virion RNA (vRNA) molecule. The IBV PB2 subunit binds not only to methylated G-capped RNAs like its IAV counterpart, but also to unmethylated GpppG caps indicating a larger flexibility of the IBV PB2 cap binding domain. The viral RNA polymerase complex binds the vRNA or the cRNA at the promoter sites located on the highly conserved stretches of nearly complementary nucleotides at the 30 - and 50 -termini. Successful expression and purification of soluble recombinant IBV polymerase has recently facilitated detailed in vitro studies of binding parameters to viral vRNA and cRNA mimics as well as characterizations of the enzymatic activities of IBV P proteins. It is generally believed that influenza viruses express an error-prone RNA polymerase which contributes to high viral evolutionary rates, but this has not been determined experimentally for the IBV polymerase. The major component of the vRNPs is the viral nucleoprotein (NP) that is a basic (pI 4 9.0) 62 kDa protein and wraps around the viral RNA. It is an essential cofactor for viral transcription and replication and it consists of a head domain, a body domain and a tail loop like its IAV counterpart, to which it has sequence identity below 40%. The tail loop of one NP monomer can insert into a neighboring monomer, which drives the formation of homo-oligomers. A distinct feature of the IBV NP protein is an extended N-terminal region of 70 amino acids that appeared intrinsically disordered in structural analyzes. In a study by NMR and smallangle X ray scattering the N-terminal NP region was bound to nuclear import factor importin-a7, which together with biochemical and mutational analyzes suggested that the N-terminal region of NP that is enriched in basic amino acids between positions 30 and 71 directs its nuclear targeting. Intracellular expression of viral mRNA directs the synthesis of eleven known IBV proteins that facilitate amplification and nucleo-cytoplasmic export of the viral genome, the production of progeny virions and/or manipulate innate antiviral defenses. The newly synthesized vRNPs are exported to the cytosol with the support of the viral NEP and, possibly, the M1 protein, which both contain nuclear export signals. The nuclear export of vRNPs is sensitive to inhibition by leptomycin B indicating a dependency on the exportin 1-dependent pathway. IBV encoded NS1 protein suppresses the stimulation of cytosolic pattern recognition receptors such as RIG-I and PKR through newly replicated vRNPs. IBV replication triggers an apoptotic response in infected cells and the activation of both extrinsic and intrinsic induction pathways has been described. The assembly and budding of IBV progeny

Influenza B, C and D Viruses (Orthomyxoviridae)

565

virions occurs at the plasma membrane, which is followed by virion release that depends on NA activity. In the following we will discuss the remaining structural and regulatory IBV gene products with a focus on their virus type-characteristics.

Hemagglutinin (HA) The HA of IBV is synthesized in the ER as a HA0 precursor of approx. 63 kDa with a predicted leader peptide of 15 amino acids that needs to be proteolytically cleaved by the cellular signal peptidase. HA0 precursor is cleaved by trypsin-like proteases into HA1 (approx. 346 amino acids) and HA2 subunits (approx. 223 amino acids) to become biologically active. HA has an essential role in the viral life cycle as it mediates binding of the virion to host cell surface receptors as well as promoting the release of the viral genome from the late endosome. The HA of IBV is heavily glycosylated containing 10–12 N-linked glycosylation sites and it is assembled into homotrimers. The high number of glycan attachment sites may reflect the prolonged circulation of IBV in humans as glycosylation is believed to be a strategy, by which influenza viruses mask antigenic sites. Cleavage activation of the HA occurs within a motif that is highly conserved among viruses of the Yamagata and Victoria lineages (PAKLLKER↓GFFGAIAGFLE), which liberates the hydrophobic fusion peptide at the N-terminal end of HA2. Trypsin and the serine proteases TMPRSS2 and TMPRSS11d were shown to execute HA cleavage activation in vitro. TMPRSS2 knock out mice were shown susceptible to IBV infection suggesting the existence of additional proteases that can activate the HA. Several X ray structures have been reported for the neutral pH form of HA expressed by the divergent IBV lineages (Fig. 3). Despite a low degree of sequence identity of approx. 25% to IAV HA these studies revealed an overall conserved fold of the trimeric HA ectodomain composed of a membrane-distal globular head domain (mainly located on HA1) and an elongated stem (mainly located on HA2) formed by each monomer. Each head domain carries a receptor binding pocket that is built by the 190 helix (aa 193–202) on the top, the 240 loop (aa 237–242) as the left boundary and the 140 loop (aa 136–143) as the right edge with additional contributions by the conserved Phe 95, Trp 158 and Tyr 202 residues. The principal receptor determinants of IBV HA are sialic acid (SA) conjugated to cellular surfaces. An interesting variation among clinical IBV isolates has been noted in terms of their receptor specificity by glycan array analysis. 19 of 21 Yamagata-like viruses isolated between 2001 and 2006 in Taiwan preferentially bound to a-2,6 linked sialic acid that is also the

Fig. 3 Crystal structure of an HA (left) and HEF (right) trimers. Polypeptide chains are displayed as green (HA1, HEF1) and magenta cartoons (HA2, HEF2). HA (left): The amino acids contributing to receptor-binding (Phe-95, Ser-140, Trp-158, His-191, Tyr-202, Pro-238 and Ser-240) are shown as red stick in one HA1 subunit. The fusion peptide at the N-terminus of the HA2 subunit is displayed as blue sticks. HEF (right): The esterase domains shown in blue within one HEF1 subunit. The catalytic triad of the esterase domain (serine 57, aspartate 352, histidine 355) is shown as red sticks. The amino acids contributing to receptor-binding (Tyr 127, Thr 170, Gly 172, Leu 184, Thr 186, Phe 225, Tyr 227, Arg 236, Phe 293) are shown as red stick in one HEF1 subunit. The fusion peptide at the N-terminus of the HEF2 subunit is displayed as a blue line. Figures were created with Pymol2.2 (https://pymol.org/2/) from pdb file 4M40 showing HA from influenza virus B/Yamanashi/166/1998 and from pdb file 1FLC containing C/Johannesburg/1/66.

566

Influenza B, C and D Viruses (Orthomyxoviridae)

major receptor determinant for IAV in the human upper respiratory tract. In contrast, 12 of 23 Victoria-like viruses additionally bound a-2,3 linked sialic acid, which is the receptor for avian IAV and can be found in humans mainly in the lower respiratory tract (LRT). 6 of 23 Victoria-like IBV recognized specifically sulfated glycans. Receptor specificity is considered an important determinant of influenza virus pathogenesis, but it remains to be established whether IBV strains with dual receptor specificity are more prone to infect the LRT which is usually associated with more severe symptoms. The HA is a main target for antibodies elicited after infection or immunization. Phylogenetic and structural analyzes revealed four major canonical antigenic sites reflecting the strong immune pressures on the IBV HA. Antigenic sites have been mapped to the 120 loop (116  137), the 150 loop (141  150), the 160 loop (162  167), and the 190 helix (194  202) which contain sequence-variable immunodominant epitopes. The IBV HA protein contains a highly conserved cytosolic tail domain of 10 amino acids (RDNVSCSICL) with important regulatory roles in virion assembly and budding. The two cysteine residues are modified by palmitoylation thereby increasing hydrophobicity of the tail. Removal or shifting of palmitoylation sites affected membrane fusion activity of the HA. Studies by reverse genetic analyzes suggested that the cytosolic tail is important for the incorporation of IBV HA into virions and localization of viruses in lipid raft microdomains during entry. A deletion in the HA tail did not affect the fusion activity of HA, but yet it strongly reduced viral propagation and incorporation of HAs into virions. Interestingly, the phenotype of the HA truncation mutant was partially rescued by a compensatory E136K mutation in M1 suggesting that the assembly of HA into budding virions depends on an interaction with M1.

Neuraminidase (NA) The neuraminidase, the receptor destroying enzyme of influenza A and B viruses, is a type II transmembrane glycoprotein found on the surface of virus-infected cells and in the membrane of the virions. Its main function is thought to be the removal of inhibitory receptor determinants from mucus in the respiratory tract to facilitate virion docking to the host cell, as well as to mediate the release of progeny virions from the host cell, which is the last step of the virus replication cycle. Hence, inhibition of NA by small molecules or antibodies diminishes virus propagation. There are many structural similarities between the NA molecules of type A and B viruses. In both viruses NA matures into a tetrameric structure despite the low degree of sequence identity of 30%. The monomers form a head domain composed of six-bladed b-propellers that are linked to membrane-proximal elongated stalks, which together build a box-like tetramer. The enzymatic and antigenic sites are located in the head domain. The eight NA amino acids that directly bind to sialic acid and are important for the catalytic activity of NA (R118, D151, R152, R224, E276, R292, R371, and Y406; N2 numbering) are highly conserved among type A and B viruses. NA is the main target for current antiviral therapy of human infections by circulating influenza A and B viruses (see below).

NB NB is an enigmatic second transmembrane glycoprotein encoded by segment 6 with an unknown function. It consists of an N-terminal ectodomain of 18 amino acid, a predicted 22 amino acid transmembrane domain and a cytosolic part of 60 amino acids. It has two glycosylation sites in the ectodomain. Mature NB forms a tetrameric structure and is incorporated into virions, but only in low amounts as determined by quantitative mass spectrometry. NB transport to the cell surface was suggested to depend on palmitoylation of cysteine 49. Most interestingly, a recombinant influenza B/Florida/04/2006 virus engineered to lack NB expression by introduction of an early stop codon not affecting NA expression replicated well in stable cell lines or primary tissue cultures. The NB-deficient mutant virus had no specific phenotype in mice and propagated in a ferret model in virtually identical manner to the wildtype virus with a retained (low) capacity for respiratory droplet transmission. These findings partially contrast with a weakly delayed viral growth for a B/Lee/40 mutant virus in which NB palmitoylation at cysteine 49 was abrogated, which may have affected trafficking of NB to the cell surface. Yet, the likely essential function of the NB protein in IBV replication, pathogenesis or transmission, which is expected by the strong conservation of the NB reading frame, remains to be discovered.

Matrix protein (M1) The M1 proteins are the most abundant morphogenic virion proteins of IBV and IAV. The IBV M1 protein has a conserved length of 248 amino acid and it shares about 30% sequence identity with its type A virus counterpart. The physicochemical properties, domain structure and regulatory roles of the IAV M1 protein in facilitating nucleocytoplasmic export of vRNPs late in infection have been well studied, but considerably less is known about the characteristics of the IBV M1. However, IBV M1 has been suggested to engage into a binding interaction with the viral BM2 protein and that this interaction is important for incorporation of M1 and vRNPs into virions. Mutational analysis identified within the IBV M1 protein a bipartite nuclear localization signal (amino acids 74–94) and two leucine-rich exportin 1-dependent nuclear export signals at positions 3–14 and 124–133. However, the role of these topogenic sequences in the viral replication cycle remain to be determined. In case of IAV the M1 protein is thought to bridge an interaction between vRNP and the viral nuclear export protein (NEP), which facilitates nuclear export of the viral genome via the exportin 1-pathway. However, the IBV NEP has a reported ability to bind directly to vRNPs suggesting that M1-mediated complex formation would be dispensable or redundant for IBV.

BM2 BM2 is a type III transmembrane protein that provides a proton channel function essential for the intracellular release of vRNPs from late endosomes. BM2 consists of a short 7 amino acid ectodomain, a 19 amino acid transmembrane domain and a cytosolic domain of 83 amino acids, and it exists in the form of a homotetramer. BM2 is a functional homolog with the IAV M2 protein to

Influenza B, C and D Viruses (Orthomyxoviridae)

567

which it has little sequence identity apart from a conserved 19-HxxxW-23 proton channel motif. BM2 was recently shown to efficiently complement the multi-cyclic growth of a recombinant M2-deficient influenza A virus, which was otherwise confined to a single cycle infection. BM2 differs from the M2 protein in being insensitive to channel blockers such as amantadine resulting in natural resistance of type B influenza viruses to this first generation class of anti-influenza compounds. The resistance to amantadine was explained by differences in the interior part of the proton channel, which is covered by polar amino acids and not hydrophobic residues as in the case of the IAV M2 protein, as well as by a reduced size of the channel pore. Apart from serving as a proton channel BM2 protein has also important morphogenic activity since recombinant viruses with C-terminal BM2 deletions of as little as six amino acids displayed irregularly formed virions and the virus was strongly attenuated in its replication ability. The large cytosolic BM2 domain adopts a coiled-coil tetramer. Biochemical analyzes showed that the C-terminus of BM2 is important for efficient membrane association of the M1 protein in flotation centrifugation, as well as incorporation of M1 and vRNPs into virion particles. Within the cytosolic BM2 domain, there is an electrostatic interaction with a positive charged N-terminal part (residues 44–71) and a negative C-terminus (72  103), the latter of which has been suggested to bind to the IBV M1 protein. From these analyzes, a model emerges in which the BM2 protein is a central factor that recruits viral RNPs and the M1 protein to virion assembly sites as a prerequisite for efficient budding. Whether BM2 also contributes to scission of virus particles from the plasma membrane as described for M2 for IAV remains to be investigated.

Non-structural protein 1 (NS1) The NS1 protein is abundantly expressed in virus-infected cells and the main antagonistic protein enabling IBV to interfere with the innate type I interferon (IFN) defense and to propagate efficiently in mammalian hosts. Hence, there are strong parallels to the functions of the IAV NS1 protein although they share a sequence identity of less than 25%, and there are also type-specific activities of the two NS1 proteins. The IBV NS1 protein has a conserved length of 281 amino acids with a predicted molecular mass of 32 kDa. It is described as a non-structural protein although low levels of NS1 were detected in purified virions by mass spectrometric analysis. Early biochemical analyzes identified in the N-terminal NS1 domain an RNA binding activity (amino acids 1–93) and the ability to bind to the IFN-induced protein ISG15 (1  103), a ubiquitin-like modifier of nascent proteins. Later on, it was found that the C-terminal part of NS1 (94  281) can also bind RNA. Reverse genetic analysis confirmed that in epithelial cells the NS1 protein inhibits the accumulation of IFN-b transcripts and activation of IRF3, a central transcriptional regulator of IFN-b expression. For this activity the C-terminal NS1 domain was essential. However, IBV is a stronger activator of type I and III IFN expression early in infection compared to IAV. This depends on the cellular pattern recognition receptor RIG-I and it is possibly caused by a more rapid release of vRNPs from endosomes. Interestingly, the IBV NS1 protein does not bind RIG-I, but rather targets the ubiquitin E3 ligase TRIM25 that activates RIG-I by ubiquitination, whereas the type A NS1 protein binds RIG-I and TRIM25 directly. Phenotypic analyzes of recombinant mutant IBV demonstrated that NS1 facilitates efficient viral propagation in vitro and in vivo by blocking the activation of the central immune kinase PKR that can shut-off protein synthesis in infected cells. Silencing of PKR strongly depended on a binding interaction of the N-terminal NS1 domain with the kinase domain of PKR. Structural information from X-ray crystallography has been reported for the N-terminal domain (15  93) of NS1 that adopts a six helical dimeric fold and a large C-terminal fragment (141  281). It is uncertain whether the IBV NS1 protein forms higher order structures as was recently shown for the type A NS1. ISG15 has been shown to regulate influenza A and B virus infections. It was therefore not surprising to find out that the IBV NS1 protein not only binds ISG15, but it can also prevent its conjugation to target proteins. Interestingly, the IBV NS1 protein binds strongly to the human ISG15 and only poorly to ISG15 homologs in other mammalian species, which provides an important example of a species-specific viral antagonism of the type I IFN response. The intracellular localization of the IBV NS1 protein is highly dynamic throughout the infection. At early times of infection, NS1 accumulates in nuclear SC35-positive speckle domains enriched in RNA processing factors and leads to a rounding of their usually irregular appearance. This activity was localized within the N-terminal 90 amino acids which also displays a monopartite NLS at position 46–57 inside the N-terminal RNA binding domain. However, speckle association of NS1 is resolved in late phases of infection when vRNPs are exported to the cytosol, and is accompanied by a cytosolic relocalization to downregulate cellular immune receptors such as PKR and RIG-I. It has been speculated that the transient interaction of the IBV NS1 protein with speckles reflects a role in promoting export of viral mRNAs. The IBV NS1 protein lacks two important properties, which are found in IAV NS1 proteins: First, the absence of interference with the cellular polyadenylation machinery, which is mediated by binding to a 30 kDa subunit of cleavage and polyadenylation factor. Second, IBV NS1 has the inability to activate phosphatidylinositol-3-kinase (PI3K) signaling by binding to its p85beta subunit, which has been suggested to prevent premature apoptosis in infected cells.

NEP The IBV NEP protein has a conserved length of 122–123 amino acids. In spite of its similar size the sequence identity of IBV NEP with IAV NEP is below 25%. However, both NEPs have conserved functions as vRNP export factors and regulators of the viral RNA polymerase. The IBV NEP has been shown to interact with human nucleoporins and exportin 1, and this interaction has been mapped in the hydrophobic N-terminal region at positions 10–19 of NES. In contrast to IAV, the IBV NEP has been reported to bind directly to vRNPs, and may, hence, be able to facilitate nucleocytoplasmic export of the viral genome independently of the M1 protein. An additional activity of NEP in regulating polymerase protein functions was observed in minigenome reporter assays, in which increasing amounts of NEP reduced the accumulation of viral mRNA and enhanced cRNA levels without affecting vRNA levels. Neither the precise target of IBV NEP within the viral RNPs nor the mechanism underlying this regulation have been established.

568

Influenza B, C and D Viruses (Orthomyxoviridae)

Epidemiology IBV is considered to cause seasonal influenza epidemics every 2–4 years, but less comprehensive systematic analyzes on its epidemiology are available compared to IAV. Two IBV lineages derived from the prototypic B/Victoria/2/87- and B/Yamagata/16/ 88-strains co-circulate with the IAV H1N1 and H3N2 subtypes to cause seasonal influenza. However, the extent of human infections for each of the four viruses is highly variable and can differ from one geographic region to the other even in the same epidemic season. Sentinel surveillance data in Europe since 2001 showed that the detection of IBV among circulating influenza viruses ranged from 1% to 70% through all age groups and that IBV was in five seasons the most prevalent virus type. A long-term global comparison suggested that IBV is the most prevalent influenza virus type affecting older children (5–17 years) and is second behind A(H3N2) in the elderly (4 65 years). Moreover, in Germany the rates for hospitalization attributable to IBV infection were estimated to be 81 per 100.000, which was in between the rates for A(H3N2) and A(H1N1) influenza (99 and 56 per 100.000). Significantly, IBV infections were the main cause of mortality attributable to influenza in the US in four epidemic seasons between 1997 and 2009. IBV was detected in 1222 (73%) of the 1674 fatal influenza cases reported through the German Public Health notification system for infectious diseases in the severe 2017/2018 season. Taken together, studies show that IBV imposes a substantial burden of disease. The IBV HA genes evolve at a rate of 2.0  103 substitutions/site/year, which is somewhat slower compared to the seasonal IAV HA subtypes H3N2 (5.5  103 substitutions/site/year) and H1N1 (4.0  103 substitutions/site/year). This finding correlates with a slower antigenic drift of IBV and may indicate higher structural and/or functional constraints on its HA. The B-Yamagata and B-Victoria lineages split in the 1970s and the lineages were distinguished based upon genetic and antigenic differences of their HA and NA genes (Fig. 4). During their co-circulation multiple reassortment events occurred either within or in between the lineages. Recent molecular genetic analyzes indicated that only the HA, PB1 and PB2 genes continue to evolve in a lineage-specific manner. Current viruses with the Yamagata-like HA have diverged into clades 2 and 3, whereas most strains with a

Fig. 4 Phylogenetic tree of IBV HA genes with representatives of B-Yam and B-Vic lineage using the Neighbor-joining method: Vaccine viruses (red), reference viruses (blue) and circulating isolates (black) are shown. The continued evolution of IBV HA genes is presented on https://nextstrain.org.

Influenza B, C and D Viruses (Orthomyxoviridae)

569

Victoria-like HA belong to clade 1A. There is low antigenic cross-reactivity between the two IBV lineages and often circulating strains and the IBV vaccine component in trivalent vaccines have not matched well.

Clinical Features Infections by type B or A viruses cause febrile influenza-like illness characterized by a sudden onset of fever accompanied by cough, nasal congestion, headache, myalgia and other types of malaise that appear after a typical incubation period of 1–3 days. There is a large spectrum in the severity of symptoms ranging from an inapparent infection with no virus shedding to a fulminant disease, which may depend on the immune status, underlying risk factors, and age of the patient. Some studies on pediatric populations reported slightly increased frequencies of myalgia, sore throat and hoarseness in IBV infection compared to IAV, but other studies have not confirmed this observation. From outpatient-attended influenza infections or experimental analyzes there are no data suggesting that the clinical features of IBV infection are different from the ones caused by IAV. Complications of IBV infection affect the lungs (e.g., bronchiolitis or pneumonia), but have also been observed in other organ systems leading to neurological (encephalitis), muscular (myositis) or cardiologic presentations (myocarditis).

Pathogenesis Clinical symptoms of seasonal influenza are in general similar for IBV and IAV and no unique pathogenetic mechanism has been ascribed for IBV, when it infects and spreads through the human respiratory tract. One autopsy study of 45 fatal influenza B cases showed that both lineage viruses caused inflammation of the trachea and bronchi with necrotic cells, pulmonary edema and hemorrhages as common findings. Viral antigen was mainly found in the columnar epithelium of the trachea and bronchi as well as in submucosal glands even in the absence of bacterial coinfection. These findings were similar to the ones seen in fatal influenza A virus infection. Yet, it is unclear how representative these findings from post mortem tissue are to the pathogenesis in uncomplicated influenza for which there is only little data available. The pathogenesis of IBV infection in experimental animal models depend on virus adaptation (e.g., mice), the use of specific virus strains (ferrets, guinea pigs) or in some models there is no pathological changes (e.g., Syrian golden hamsters).

Diagnosis It is not possible to diagnose an influenza B virus infection based on clinical presentation alone as there is a large range in symptom severity and a number of other respiratory pathogens that cause similar or identical illness. Hence, laboratory testing of respiratory patient samples such as nasopharyngeal swabs or broncho-alveolar lavage fluid is required to confirm a suspected IBV infection by the presence of viral RNA or antigen. In principle, reverse transcription-dependent real-time PCR assays, viral culture or rapid point-of-care tests detecting viral antigen or the viral genome are used, which differ in sensitivity and testing time. RT-PCR assays have a high sensitivity close to 100% and can be completed within a few hours, but usually RT-PCR analyzes require the samples to be transported to a diagnostic laboratory operating the required equipment (e.g., thermocyclers). PCR primers and probes often target the M gene to detect IBV and utilize the HA sequence for discrimination between the B-Yamagata and Victoria lineages. Results from rapid antigen detection or isothermal nucleic acid amplification may be available within 30 min, but these assays have on an average slightly lower sensitivity and specificity.

Treatment There are two classes of compounds licensed for the treatment of acute influenza A and B virus infections, which target either the viral NA or the PA subunit of the viral RNA polymerase. The neuraminidase inhibitors (NAI) include the oral oseltamivir (given as the prodrug oseltamivir phosphate), the inhaled zanamivir and the intravenous peramivir, which all target the substrate binding pocket of NA and reduce enzymatic activity in the nanomolar range to stall the spread of newly synthesized virions to other host cells. It is a consistent finding that IC50 values for IBV NA are up to tenfold higher compared to concentrations needed to inhibit IAV NA. For oseltamivir this observation has been explained by a lower flexibility of the active site residue E276 that impedes high affinity binding to the hydrophobic pocket. The development of resistance towards NAI in IBV has been observed in experimental studies as well as in patients undergoing antiviral treatment. However, the genetic barrier to acquire resistance mutations appears to be high, possibly due to a loss of viral fitness. Global surveillance by the GISRS detected NAI resistance in circulating IBV in five consecutive seasons between 2012–13 and 2016–17 at frequencies between 0.2% and 2.0% using phenotypic assays suggesting that these compounds remain suitable for clinical use. Single amino acid mutations conferring NAI-resistance in clinical isolates have been identified either directly at the catalytic site (R152K), at the NA framework (D198N) or near the sialidase center (G109E, G402S).

570

Influenza B, C and D Viruses (Orthomyxoviridae)

Baloxavir marboxil, the prodrug of an oral anti-influenza compound, was licensed for the treatment of seasonal influenza in Japan and the US in 2018. It targets the cap-dependent endonuclease activity located on the PA polymerase subunits of IAV and IBV, and the drug can be given as a single dose. The EC50 of Baloxavir acid for the propagation of IBV in MDCK cells was reported in the range of 5.6–8.5 n mol. Analyzes of A(H1N1) and A(H3N2) viruses isolated from patients in the treatment studies showed that single mutations of the conserved residue I38 to T, F or M reduced the sensitivity to Baloxavir by more than 10-fold. No resistant IBV were reported in the clinical studies, but reverse genetics showed that PA I38 mutations increase the EC50 of IBV by 2- to 7-fold. Structural analyzes showed that resistance mutations reduced the affinity of the inhibitor to PA. Strong conservation of the PA I38 position in current human influenza B viruses suggests only a low background level of resistance to Baloxavir. Importantly, no cross-resistance has been found for Baloxavir and NAI.

Prevention Vaccination is the most effective measure to protect against seasonal influenza A and B, for which either inactivated or liveattenuated vaccines are available. Since IAV and IBV strains circulate in unpredictable patterns, killed vaccine formulations contain a mixture of inactivated whole viruses or purified HA and NA proteins from one A(H1N1), one A(H3N2) and one (trivalent influenza vaccine, TIV) or two IBV strains (quadrivalent influenza vaccine, QIV) designed to elicit a protective humoral immune response. The specific strains to be included into the vaccine of the forthcoming season are recommended by WHO twice a year based upon extensive genetic analysis and antigenic profiling of circulating virus strains. Live-attenuated vaccines contain a mixture of three or four attenuated virus strains with the same antigenic profiles. High vaccine effectiveness depends on a good antigenic match between the vaccine strains and the circulating epidemic viruses. The elderly may also have weaker immune responses against the vaccine. Since there is only limited antigenic cross-reactivity between the two IBV lineages that co-circulate since 2001 with unpredictable frequencies, it has turned out a formidable challenge to predict the specific IBV lineage component for TIV. In fact, TIV was in five of ten seasons from 2001 to 2010 reported to confer little or no protection against the circulating IBV strains in the US. Still, recent meta-regression analysis quantified that there was reduced but substantial IBV vaccine effectiveness against laboratory confirmed infection even in seasons with a type B lineage mismatch (67% for matched seasons, 35% for mismatched seasons). The suboptimal performance of TIV led to a first recommendation in 2012/2013 for the inclusion of both IBV lineages into a seasonal QIV, which have been increasingly used by different countries. Modeling studies suggested a good potential that the simultaneous coverage of both IBV lineages by quadrivalent formulations can substantially reduce the burden of influenza B infection if vaccine uptake is sufficiently high.

Influenza C and D viruses Virion Structure and Genome Influenza C (ICV) and D virus (IDV) particles exhibit various morphologies: elliptical, spherical with a diameter of 80–120 nm or filamentous with a similar diameter but with lengths in the mm range (Fig. 1). A peculiar feature of ICV and IDV particles not observed for IAV virions is the arrangement of its unique spike, the hemagglutinin-esterase-fusion glycoprotein (HEF) in a reticular structure that consist mainly of hexagons. The regular polymeric structure can also be observed in isolated HEF molecules indicating that the hexagonal arrangement is an intrinsic feature of the protein likely involving lateral interaction between its ectodomains. The interior of the virus particle contains the viral ribonucleoprotein (RNP) complexes, which consists of negative-sense, singlestranded RNA bound to the nucleoprotein NP and to three polymerase proteins (Fig. 5). In contrast to IAV and IBV, the genomes of ICV and IDV consist of only seven (not eight) different segments (Table 1), but most virus particles nevertheless package eight RNPs arranged in the typical 1 plus 7 pattern in which a central RNP is surrounded by seven circularly arranged RNPs. It is still not known how specific packaging of genome segments is achieved, but the segmented genome provides an evolutionary advantage of allowing the exchange of individual genome segments with those of other strains. All RNA segments are flanked by non-coding sequences, which are more variable in length than those of influenza A and B viruses. The first twelve nucleotides at each 30 end as well as the first eleven nucleotides at each 50 end are conserved between genome segments and are partially complementary to each other, which enables the single stranded RNAs to form “panhandle” structures. This unique structure serves as the promotor for transcription of mRNAs and plays a pivotal role in genome replication and packaging. A uridine-rich region at the 50 end of each segment serves as the poly A tail template for mRNA transcription. The longest three gene segments encode the proteins PB2, PB1 and PA/P3 that form the trimeric polymerase complex, which is bound to one end of each RNP. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain cap-binding and endonuclease activities, respectively, required for transcription initiation by cap-snatching, a process during which a short nucleotide sequence is cleaved from the 5´ end of cellular pre-mRNAs. The 3D-structure of polymerase proteins and the active site of the polymerase and endonuclease as determined by X-ray crystallography are conserved between influenza viruses suggesting that the mechanism of transcription and replication is very similar in all influenza virus types. The fourth segment of ICV encodes the glycoprotein HEF, the only spike of the viral membrane, which is discussed in more detail below. The fifth segment encodes

Influenza B, C and D Viruses (Orthomyxoviridae)

571

Fig. 5 Scheme of an ICV particle and its genome segments. Adapted from viralzone https://viralzone.expasy.org/81?outline=all_by_species with permission.

the nucleoprotein NP that associates with the viral genome segments in a double-helical conformation in which two NP strands of opposite polarity are associated with each other. The sixth segment encodes two proteins, the matrix protein M1, a peripheral membrane protein that covers the viral envelope on its inside, and CM2, a short transmembrane protein with proton-channel activity required for virus entry. M1 contributes to the morphology of virus particles and also to another unique feature of ICV, the formation of cord-like structures, very long (up to 500 mm) tubules containing HEF and M1, but no RNPs emanating from the surface of virus-infected cells. In contrast to IAV, the larger M1 protein of ICV and IDV is translated from a spliced RNA, but the mechanisms of splicing differ between ICV and IDV. In ICV the removal of an intron generates a stop codon whereas in IDV splicing generates a second exon encoding a 4-residue peptide that is added to the primary M1 transcript. The unspliced mRNA encoding CM2 translates into a long precursor protein called M10 or p42. P42 contains an internal signal peptide, which targets the protein into the ER. Residues Cterminal to the signal peptide are translocated into the lumen of the ER until translocation is stopped by a second hydrophobic region, the transmembrane region of CM2. The signal peptide is then cleaved by signal peptidase yielding the mature CM2 and the p31 protein, which is rapidly degraded. The seventh vRNA encodes the two non-structural proteins NS1 (246 amino acids) and NS2 (182 amino acids) that are generated via mRNA splicing: the unspliced mRNA is translated into NS1 and the spliced mRNA translates the shorter NS2 protein. The N-terminal residues of NS1 and NS2 are identical in sequence, splicing then generates a shift in the ORF in such a way that the remaining residues are translated from a different reading frame. Similar to IAV and IBV, it is believed that NS1 counteracts the cellular interferon response and NS2, which is also designated nuclear export protein (NEP), mediates the nuclear export of RNPs. Small amounts of NEP may be incorporated into virus particles.

Functions of the HEF Glycoprotein During the Life Cycle Research on ICV and IDV has concentrated mainly on its only glycoprotein HEF that combines the function of HA and NA of IAV and IBV, i.e., it possesses receptor-binding, receptor-destroying and membrane fusion activities. HEF (like HA) is a typical type 1

572

Influenza B, C and D Viruses (Orthomyxoviridae)

transmembrane protein with a short N-terminal, cleavable signal peptide, a long ectodomain, a transmembrane region and a very short, cytoplasmic tail which contains (in contrast to HA of Flu B) the longer acyl chain stearate. HEF is synthesized as a precursor HEF0, which is cleaved into the N-terminal HEF1 subunit and the membrane-anchored HEF2 subunit. Although there is only 12% amino acid identity between HA and HEF, crystallography of their ectodomains revealed that the overall structures as well as the folding of individual domains are quite similar. Both glycoproteins form a mushroom-shaped trimer consisting of a membranenear stalk (containing the regions involved in membrane fusion) and a globular head domain carrying the receptor-binding site. HEF contains an additional bulge located at the lower part of the globular domain that carries the receptor-destroying region that is not present in HA (Fig. 3). Influenza C virus does not use sialic acid as its receptor on the target cell, but an acetylated derivative, namely 9-O-acetyl-Nacetylneuraminic acid. 9-O-Ac-Neu5Ac as the terminal sugar on both glycoproteins and glycolipids can be used as a receptor, regardless of whether it is attached to galactose via an a-2,3 or a-2,6 linkage. There is some evidence that the abundance of 9-O-AcNeu5Ac influences the tropism, since MDCK II cells (a standard cell line used for growth IAV and IBV) is resistant to ICV infection due to insufficient number of receptors. Instead, ICV is usually grown and plaque-titrated in another subline of Madin-Darby canine kidney cell, namely MDCK I cells. The receptor binding site is located near the top of the globular head in a shallow groove surrounded by residues from four secondary structure elements: the 170-loop, 190-loop, 230-helix and 270-loop and thus exhibits a similar structure as the binding site for Neu5Ac in HA (Fig. 3). Interestingly, the crystal structure of HEF of IDV, which is otherwise almost identical to the structure of HEF of ICV, revealed a more open receptor-binding cavity, which may be the basis for the broader cell tropism of IDV. Some coronaviruses, such as the prototype member mouse hepatitis virus and human and bovine coronaviruses, contain a hemagglutinin esterase (HE) protein that also binds 9-O-Ac-Neu5Ac, but the ligand is bound in an opposite orientation. Intrinsic temperature sensitivity is another peculiar feature of HEF of ICV that distinguishes it from HA. Folding of the protein in the endoplasmic reticulum (and hence cell surface expression) is less efficient at 371C than at 331C. Probably as a consequence, ICV grows to higher titers in cells cultured at 331C compared with 371C. This temperature sensitivity may be one of the factors that prevents the replication of ICV in the lungs that has a higher temperature compared with the upper respiratory tract. On the contrary, HEF of IDV exhibits an exceptional temperature and acid-stability and hence IDV is the most stable of the four types of influenza viruses, a feature that likely affects the tissue tropism and cross-species transmission of the virus. Influenza C virus is proposed to enter cells via the endocytic pathway and to subsequently fuse with the membrane of the endosome. This process is also mediated by HEF and requires its proteolytic cleavage into HEF 1 and HEF 2 subunits and subsequent exposure to mildly acidic pH. Although the fusion mechanism of HEF has not been investigated, it is believed to be similar to HA where well-studied conformational changes catalyze membrane fusion. This assumption is based on the very similar structures of HA and HEF at neutral pH and especially the presence of a long central a-helix in HEF2 and a smaller helix on the outside packed in an antiparallel fashion, which undergo large acid-induced conformational changes that are the main drivers for membrane fusion (Fig. 3). The long a-helix contains a predicted heptad-repeat, a characteristic element of most viral fusion proteins that interact in the low pH structure to form a stable 6-helix coiled-coil domain. A notable difference between HA and HEF is the fusion peptide, a hydrophobic region at the N-terminus of the small HEF2 subunit which contains hydrophobic, aromatic, but also a few negatively charged residues. It is fully conserved between ICV and IDV HEF (IFGIDDLI), but very different from the respective, strictly conserved amino acids of HA2 (GLFGAIAGFIE). In addition, while the fusion peptide of HA is completely buried in the interior of the molecule, the fusion peptide of HEF has a loop-like structure, which is partly exposed at the surface of the virus. HEF may therefore be regarded as having an internal fusion peptide, similar to virus fusion proteins that do not require cleavage activation. HEF from both ICV and IDV possess a monobasic cleavage site (one arginine at the C-terminus of HEF1) and it is in this respect similar to the HAs from mammalian and low pathogenic avian IAVs. The replication of these viruses is restricted to the site of virus infection, usually the respiratory tract. Interestingly, the amino acids surrounding the monobasic cleavage site of HEF differ between ICV and IDV. HEF from ICV contains three closely spaced basic residues at the C-terminus of HEF1 (TVTKPKSR), whereas HEF1 from IDV contains only two, widely spaced basic residues (RTLTPATR). The molecular features of the cleavage site in viral glycoproteins determine which of the many trypsin-like serine proteases recognize the precursor protein and the expression of this protease determines whether a certain cell type produces infectious virus particles. Thus, differential cleavage activation of HEF may contribute to the differences in host range of ICV and IDV, i.e., human versus cattle. The last step in the viral replication cycle is the release of virus particles from infected cells, which requires the removal of all putative receptors. In influenza A and B the receptor-destroying activity is carried out by the second viral glycoprotein, the neuraminidase NA, whereas HEF performs this activity for ICV. In accordance with its receptor binding specificity, HEF is not a neuraminidase, but an esterase that cleaves acetyl from the C9 position of terminal 9-O-Ac-Neu5Ac. The esterase activity, which belongs to the class of serine hydrolase, is included in an additional domain of HEF1. The esterase domain is structurally the most conserved part of HEF; the position of the catalytic triad characteristic for serine hydrolases (serine 57, aspartic acid 356 and histidine 359) is identical in ICV and IDV HEFs, which exhibit 55% amino acid identity and an almost identical 3D-structure. The structure of the esterase site is also very similar between HEF and coronavirus HE and it has been proposed that the HE arose from Influenza C-like HEF by a lateral gene transfer of the middle part of the HEF gene encoding mainly the receptor-binding and esterase domains.

Influenza B, C and D Viruses (Orthomyxoviridae)

573

Epidemiology Although influenza C virus infections occur primarily in a pattern of sporadic cases or in limited outbreaks, serological studies indicated that this virus is widely distributed around the world and that the majority of humans develop antibodies against the virus early in life, which prevent subsequent infections with ICV or mitigate the symptoms. According to a phylogenetic analysis of their HEF genes the existing strains are divided into six genetic and antigenic lineages. The average rate of nucleotide substitution in HEF has been calculated to be 4.9  10–4 substitutions/site/year, an order of magnitude slower compared to the corresponding rate for the HA gene of IAV. Only 40 residues (B5%) are not completely conserved through all lineages of HEF, the variable amino acids are mainly located in the globular head domain of HEF1. Reassortment between viruses of different lineages occurred frequently, and newly emerged reassortants replaced previously circulating viruses and became epidemic strains. The primary host and reservoir of influenza C virus are humans, but antibodies against influenza C virus are present in dogs and especially in pigs. A number of influenza C virus strains were isolated from pigs in China and these strains could be experimentally transmitted from pig to pig. Infection of humans by IDV has not been reported, but based on serological studies it is assumed that the virus circulates widely in cattle, especially in calves and the virus may spread to pigs and small ruminants (sheep and goat). Based on the comparison of the nucleotide sequence of the HEF gene, two distinct co-circulating lineages are currently described, which frequently reassort with each other, but not with ICV. HEF of IDV is also stable in evolution, but the substitution rate for HEF from IDV is 1.54  10–3 per site and year, and thus faster than in the HEF of ICV. The reason for the slow evolutionary rate of HEF from ICV is unknown. It may be either a low error rate of the polymerases and/or that HEF does not tolerate many amino acid changes without compromising its functionality.

Clinical Features and Pathogenesis Influenza C virus usually causes inflammation of the upper respiratory tract, especially in children from one to six years of age. Clinical symptoms, such as cough, fever and malaise are typically mild. Occasionally the virus can spread to the lower respiratory tract, causing symptoms such as bronchitis and pneumonia, particularly in children younger than two years. Experimental infection of pigs and cattle with IDV causes only mild symptoms (rhinitis and tracheitis) and virus replication is usually restricted to the upper respiratory tract. This is somewhat in conflict with the observation that IDV is associated with a more pronounced disease in the field and it was thus proposed that the manifestation of clinical symptoms requires coinfection with other respiratory viruses or bacteria that colonize the respiratory tract.

Diagnosis Due to the usually mild clinical symptoms no rapid diagnostic tests are in place for ICV. These infections are usually identified in respiratory patient samples in retrospective studies using RT-PCR targeting the viral M or NP genes and/or virus isolation in tissue culture. Also, infection by IDV is mainly detected by RT-PCR analyzes of respiratory samples such as nasal and pharyngeal swabs or lung tissue.

Treatment and Prevention Drugs developed against influenza A virus are not effective against ICV or IDV, since their target molecule is not present (neuraminidase inhibitors) or there are different amino acids at the binding site (M2 inhibitors). Since ICV is not thought to be a significant concern for human health, no vaccines have been developed. Prototype vaccines against IVD are currently under development.

Further Reading Burnham, A.J., Baranovich, T., Govorkova, E.A., 2013. Neuraminidase inhibitors for influenza B virus infection: Efficacy and resistance. Antiviral Research 100, 520–534. Carrat, F., Vergu, E., Ferguson, N.M., et al., 2008. Time lines of infection and disease in human influenza: A review of volunteer challenge studies. American Journal of Epidemiology 167, 775–785. Hause, B.M., Ducatez, M., Collin, E.A., et al., 2013. Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to human influenza C viruses. PLoS Pathogens 9, e1003176. Herrler, G., Klenk, H.D., 1991. Structure and function of the HEF glycoprotein of influenza C virus. Advances in Virus Research 40, 213–234. Herrler, G., Rott, R., Klenk, H.D., et al., 1985. The receptor-destroying enzyme of influenza C virus is neuraminate-O-acetylesterase. The EMBO Journal 4, 1503–1506. Langat, P., Raghwani, J., Dudas, G., et al., 2017. Genome-wide evolutionary dynamics of influenza B viruses on a global scale. PLoS Pathogens 13, e1006749. Muraki, Y., Hongo, S., 2010. The molecular virology and reverse genetics of influenza C virus. Japanese Journal of Infectious Diseases 63, 157–165. Nakatsu, S., Murakami, S., Shindo, K., et al., 2018. Influenza C and D viruses package eight organized ribonucleoprotein complexes. Journal of Virology. 92.

574

Influenza B, C and D Viruses (Orthomyxoviridae)

Paul Glezen, W., Schmier, J.K., Kuehn, C.M., Ryan, K.J., Oxford, J., 2013. The burden of influenza B: A structured literature review. American Journal of Public Health 103, e43–e51. Pflug, A., Lukarska, M., Resa-Infante, P., Reich, S., Cusack, S., 2017. Structural insights into RNA synthesis by the influenza virus transcription-replication machine. Virus Research 234, 103–117. Reich, S., Guilligay, D., Pflug, A., et al., 2014. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516, 361–366. Rosenthal, P.B., Zhang, X., Formanowski, F., et al., 1998. Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus. Nature 396, 92–96. Su, S., Fu, X., Li, G., Kerlin, F., Veit, M., 2017. Novel influenza D virus: Epidemiology, pathology, evolution and biological characteristics. Virulence 8, 1580–1591. Wang, J., Pielak, R.M., Mcclintock, M.A., Chou, J.J., 2009. Solution structure and functional analysis of the influenza B proton channel. Nature Structural & Molecular Biology 16, 1267–1271. Wang, Q., Tian, X., Chen, X., Ma, J., 2007. Structural basis for receptor specificity of influenza B virus hemagglutinin. Proceedings of the National Academy of Sciences of the United States of America 104, 16874–16879.

Relevant Websites https://www.gisaid.org/ GISAID. Global Initiative on Sharing All Influenza Data. https://www.fludb.org/brc/home.spg?decorator=influenza Influenza Research Database. https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database Influenza Virus Database. https://nextstrain.org Nextstrain.org. https://reactome.org/PathwayBrowser/#/R-HSA-168254 Reactome Pathway Database. https://viralzone.expasy.org/ ViralZone root. ExPASy. https://www.who.int/influenza/en/ WHO. Influenza. World Health Organization.

Jaagsiekte Sheep Retrovirus (Retroviridae) James M Sharp, University of Zaragoza, Zaragoza, Spain and Edinburgh, United Kingdom Marcelo De las Heras, University of Zaragoza, Zaragoza, Spain Massimo Palmarini, MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom Thomas E Spencer, University of Missouri, Columbia, MO, United States r 2021 Elsevier Ltd. All rights reserved.

History Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a naturally occurring lung adenocarcinoma of sheep known as ovine pulmonary adenocarcinoma (OPA, also known as jaagsiekte or sheep pulmonary adenomatosis). OPA was recognized for the first time in South Africa in the nineteenth century as a cause of dyspnea in herded sheep, hence the origin of the Afrikaans name ‘jaagsiekte’, meaning driving ( ¼ jaagt) sickness ( ¼ziekte). OPA is one of the original ‘slow diseases’ of sheep (along with scrapie, maedi-visna and paratuberculosis) originally described in the 1930s by the Icelandic physician Björn Sigurdsson. The slow diseases of sheep were of great biological importance as they allowed, for the first time, the recognition that an infectious agent could cause clinical disease many months or years after the initial infection of the host. Studies on JSRV were hampered for years by the lack of a suitable tissue culture system for the cultivation of JSRV. The isolation of full-length JSRV molecular clones (JSRV21 and JS7) allowed the in vitro generation of infectious viral particles by a transient transfection system, which has sparked a variety of studies that have elucidated many aspects of the molecular biology of JSRV. JSRV is a remarkable virus in many respects. It is the only known virus that induces a naturally occurring lung adenocarcinoma. In addition, the JSRV envelope glycoprotein is an oncoprotein. This is a unique example among retroviruses and oncogenic viruses in general in which a structural protein functions also as a dominant oncoprotein.

Classification JSRV belongs to the genus Betaretrovirus within the family of the Retroviridae. Retroviruses are divided on the basis of their modality of transmission as “exogenous” and “endogenous” viruses. Exogenous retroviruses are horizontally transmitted between infected and uninfected hosts. Endogenous retroviruses are stably integrated in the genome of the host species from which they derive, are usually defective, and are transmitted vertically like any other Mendelian gene. JSRV is an exogenous retrovirus and it is highly related to enzootic nasal tumor virus (ENTV) of sheep (ENTV-1) and goats (ENTV-2). JSRV and ENTV have common pathogenic characteristics, and both cause low-grade adenocarcinomas of secretory cells in different portions of the respiratory tract of small ruminants. Interestingly, sheep, goats, and other members of the Caprinae have several copies of nonpathogenic JSRV-related “endogenous” retroviruses (commonly referred as enJSRVs) stably integrated in their genome. The phylogenetic relationship between JSRV, ENTV, and enJSRVs is indicated in Fig. 1.

Genetic Organization and Virion Proteins The JSRV virions are enveloped particles of approximately 100 nm in diameter and a density by isopycnic centrifugation in sucrose gradients of 1.15 g ml1 for particles obtained from cell cultures. Virions purified from lung secretions of OPA-affected sheep have a slightly higher density (1.16–1.18 g ml1). JSRV has the typical genomic organization of a simple retrovirus; the genomic RNA of 7455 nt (in the JSRV21 infectious molecular clone) contains the canonical retroviral genes gag, pro, pol, and env (Fig. 2). Apart from Env, few studies have been undertaken to assign functions to the JSRV proteins and these are assumed to be the same as other retroviruses. The gag gene encodes the structural proteins of the viral core. Gag is expressed as an immature polyprotein that is cleaved upon exit by the viral protease. The JSRV Gag is cleaved into at least five proteins: MA(p23), p15, CA (p26), NC, and p4. MA is myristoylated and presumably interacts with the cell membrane during viral egress. CA is the major capsid protein, while NC interacts tightly with the genomic RNA although no specific studies have been conducted on the JSRV NC. The pro gene overlaps gag and encodes most probably a dUTPase (DU, deoxyuridine tryphosphatase) and the viral protease (PR). The main function of DU is to avoid misincorporation of uracil into DNA during reverse transcription (see below). As mentioned above, PR cleaves the Gag polyprotein upon exit and it is absolutely required in order to obtain mature infectious viral particles. The pol gene overlaps pro and is predicted to encode the viral reverse transcriptase (RT) and integrase (IN). Both RT and IN are virion-associated enzymes; RT copies the viral single-stranded RNA genome into double-stranded DNA, while IN serves to join the proviral DNA into the host genome. RNaseH is a subdomain of RT and serves to degrade the viral genomic RNA once has been copied into DNA. An additional open reading frame (orf-x) overlaps pol and has some unusual features, including a codon usage different from other genes within JSRV, and a very hydrophobic predicted amino-acid sequence that shows no strong similarities to any known

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00079-5

575

576

Jaagsiekte Sheep Retrovirus (Retroviridae)

JSRVJS7 JSRV809T

UK North America

JSRV21 JSRV84r528 JSRVSA

Africa

JSRV92K3 enJS5F16 enJSRV-14

enJSRVs

enJS56A1 enJSRV-20 ENTV-1

Fig. 1 Phylogenetic analysis of sheep betaretroviruses. A phylogenetic tree of representative small ruminant betaretroviruses was derived by the neighbor-joining method using JSRV, ENTV-1, and enJSRVs env aligned using Clustal W. JSRV, enJSRVs, and ENTV-1 cluster in three distinct phylogenetic groups. Note that JSRV isolates from Africa cluster in separate branches from European and North American isolates.

protein and only weak homology to a member of the G protein coupled receptor family. The role of this open reading frame is unknown. Orf-x is conserved among all of the exogenous JSRVs examined to date, but it does not seem to be required for JSRV replication in vitro nor for cell transformation either in vitro or in vivo. The env gene encodes the glycoproteins of the viral envelope (Env). The JSRV Env is formed by two subunits, the surface domain (SU) which interacts with the cellular receptor and mediates viral entry, and the transmembrane domain (TM), which fixes SU to the lipid bilayer. A unique feature of the JSRV env is that it functions essentially as a dominant oncogene, and its sole expression is sufficient to induce cell transformation in vitro and in vivo (see below). According to some studies, an additional protein, named Rej or JSRV Env signal peptide, is also encoded with in the env gene. It moderately enhances nuclear export of unspliced viral RNA and considerably increases viral particle release by favoring a posttranscriptional step of the replication cycle. Noncoding regions are present at the 50 and 30 end of the genome. The R region is repeated at the 50 and 30 end of the genome. U5 is present uniquely at the 50 end, and the U3 is present at the 30 end of the genome. The viral promoter and enhancer regions are present in the U3 (see below). Genomic sequence variability among JSRV strains is very low. For instance, the infectious molecular clones JSRV21 and JSRVJS7 are 99.3% identical along the entire genomic sequence, and they were derived from naturally occurring OPA cases from Scotland collected many years apart. JSRV21 and JSRVJS7 Env proteins are 100% identical. JSRV isolates from Africa can be distinguished phylogenetically from the UK and North American isolates, although there is still a high degree of homology (B93% along the entire nucleotide genomic sequence) among the two groups.

Replication Cycle In general, the replication cycle of JSRV is not thought to be markedly different from other betaretroviruses. The lack of a suitable tissue culture system for the propagation of JSRV has not allowed detailed studies on the replication of this virus. JSRV interacts with a specific cellular receptor to enter the cell. The use of retroviral pseudotypes identified hyaluronidase 2 (HYAL2) as the cellular receptor for JSRV entry. HYAL2 is a glycosylphosphatidylinositol (GPI) linked membrane protein with a low hyaluronidase activity and is widely expressed on many different cell types. Besides ovine HYAL2, the human ortholog also allows JSRV entry, while mouse and rat Hyal2 do not. Detailed steps of JSRV entry and post-entry would need more clarification. However, using pseudovirus of Moloney leukemia murine virus (MoMlv) it was shown that JSRV-mediated entry is a relatively slow and non-classical pH-dependent process that requires dynamin-associated endocytosis. Fusion activity of the JSRV Env protein is dependent on low pH and it is modulated by the cytoplasmic tail. Despite this particular aspects of JSRV other steps are likely to be similar to those of other retroviruses including uncoating, reverse transcription of the viral genome into double-stranded DNA, entry into the nucleus of the pre-integration complex, and stable integration of the proviral DNA into the host DNA. During the process of reverse transcription, the noncoding regions at the 50 and 30 ends of the genome (R, U5, and U3) are duplicated and give origin to the viral long terminal repeats (LTRs). The retroviral LTRs are major determinants of retrovirus tropism, as the 50 LTR of the provirus initiates transcription and the U3 region contains the majority of cis-acting sequences interacting with the cellular RNA polymerase II and with cellular transcription factors. The exogenous JSRV LTRs are particularly active in reporter assays using type II pneumocytes/Clara cell lines and interact with lung-specific transcription factors such as forkhead box A2 (FOXA2; alias HNF-3b). These features may explain the preferential expression of exogenous JSRV in the transformed cells of the lungs, which have phenotypic characteristics of type II pneumocytes and Clara cells.

Jaagsiekte Sheep Retrovirus (Retroviridae)

577

ORFs pol pro

orf-x

env

gag U3

R U5

U3

Proviral DNA

R U5

LTR

LTR (a) R U5

U3

Full-length mRNA

CAP

R AAA

Gag

M MA p15

CA

NC p4

Gag-Pro

M MA p15

CA

NC DU PR p4

Gag-Pro-Pol

M MA p15

CA

NC DU PR p4

R U5

RT

IN

U3

Spliced (env) mRNA

R

CAP

AAA Env LP R U5

SU

TM U3

Spliced (orf-x) mRNA

CAP

R AAA

Orf-X

Orf-x ? Internal promoter (?) orf-x AAA

(b)

Orf-X

Orf-x ?

Fig. 2 Genomic organization, mRNAs and viral proteins of JSRV. (a) Schematic organization of the JSRV open reading frames (ORFs) and their relative position in the JSRV provirus. The JSRV LTR, repeated at both ends of the provirus genome, is divided into U3, R and U5. (b) Major JSRV RNAs and proteins. The JSRV provirus transcribes various mRNAs. A full-length mRNA encodes for the Gag, Pro and Pol proteins and serves as genome for the newly synthesized viral particles. A spliced mRNA encodes the viral Env. The Env glycoprotein is glycosylated, and putative glycosylated sites are indicated by full circles. Two additional mRNAs have been detected and encompass the orf-x reading frame, but their biological significance is unknown at present. MA, matrix; CA, capsid; NC, nucleocapsid; DU, dUTPase; PR, protease; RT, reverse transcriptase; IN, integrase; LP, leader peptide; SU, surface; TM, transmembrane.

The expression of JSRV, as in all retroviruses, is believed to follow the basic transcriptional events of cellular mRNAs including capping of the 50 end and polyadenylation at the 30 end. JSRV encodes a full-length mRNA that serves as genome of the viral progeny and is also translated into the Gag polyprotein and the proteins encoded by pro and pol. The latter are expressed as fusion proteins with Gag. As in all betaretroviruses, pro and pol are in different open reading frames to gag and are expressed by ribosomal frameshifting. The viral Env is produced from a mRNA which is spliced using the splice donor in the untranslated gag region and a splice acceptor immediately before env. Another spliced mRNA has been found to use the same splice donor of the env mRNA and a splice acceptor before the orf-x reading frame. This mRNA presumably expresses a protein encoded by orf-x, but there are no published data supporting this assumption. A possible mRNA expressing orf-x has been found to lack the R-U5 regions (typical of all other mRNAs starting from the U3) and is thought to derive from the expression of an internal promoter that has not been characterized. Other mRNAs deriving from the use of secondary splice acceptors and non-conventional polyadenylated sites have been detected but their biological significance, if any, is unknown.

578

Jaagsiekte Sheep Retrovirus (Retroviridae)

Fig. 3 Electron and confocal microscopy of cells expressing JSRV. Electron microscopy in 293T cells transiently transfected with the JSRV21 infectious molecular clone showing intracytoplasmic particles (a; scale ¼ 200 mm). In panel b, intracellular particles in the vicinity of the cell membrane (white arrow) and extracellular particles complete with viral envelope (black arrow) are also visible (scale ¼ 100 mm). (c–e) Confocal microscopy in HeLa cells transiently transfected with JSRV and probed with an antiserum towards the JSRV matrix (c) and g-tubulin (d). The JSRV Gag concentrates in the pericentrosomal area (e).

JSRV assembles in the cytoplasm (Fig. 3(a) and (b)) like all betaretroviruses, most likely in the pericentriolar region (Fig. 3(c)–(e)). Other retroviruses such as lentiviruses and gammaretroviruses assemble instead mostly at the cell membrane. The JSRV intracellular particles interact with Env at the cell membrane or in a cellular compartment not yet fully identified and egress from the cell. Upon exit, the Gag polyprotein is cleaved by the viral protease into the mature proteins described above. A more detailed description of the replication cycle of retroviruses is discussed elsewhere in this encyclopedia.

enJSRVs The sheep genome contains at least 27 copies of endogenous retroviruses highly related to JSRV (hence the name enJSRVs). Endogenous retroviruses are believed to derive from integration events of ancestral exogenous retroviruses into the germline of the host. enJSRVs have a high degree of homology with JSRV. For example, JSRV21 and enJS56A1 are 92% identical at the nucleotide level along the entire genome. Major differences are located in the U3 region, in two regions in Gag (termed variable regions 1 and 2), and in the cytoplasmic tail of the transmembrane domain of the Env (termed variable region 3). At least some of these highly divergent regions are the basis of important biological differences between JSRV and enJSRVs. enJSRV transcripts have been detected in most tissues by sensitive reverse transcriptase polymerase chain reaction (RT-PCR) assays. However, high levels of enJSRVs (both mRNA and proteins) are found specifically in the epithelia of the genital tract of the ewe and, particularly, in the epithelia of the uterus (Fig. 4). In the placenta, enJSRVs are expressed in the mononuclear trophectoderm cells of the conceptus (embryo/fetus and associated extra-embryonic membranes), but are most abundant in the trophoblast giant binucleate cells (BNCs) and multinucleated syncytial plaques of the placentomes. The temporal expression of enJSRVs envelope (env) gene in the trophectoderm is coincident with key events in the development of the sheep conceptus. Indeed, using a morpholino antisense oligonucleotide to induce loss-of-function in utero, enJSRVs Env knockdown caused a reduction in trophoblast outgrowth and inhibited/prevented trophoblast giant binucleate cell differentiation during blastocyst elongation and formation of the conceptus. Thus, enJSRVs have been proposed to be essential in sheep for peri-implantion growth and differentiation. enJSRVs expression appears to be regulated by progesterone and expression of the progesterone receptor. However, the specific enJSRV loci that are transcriptionally active are not known at present. enJSRVs are also expressed in the sheep fetus, supporting the idea that sheep are tolerized toward JSRV. Indeed, JSRV-infected naïve sheep (with or without lung adenocarcinoma) have no

579

Uterus

Vagina

Cervix

Oviduct

Jaagsiekte Sheep Retrovirus (Retroviridae)

Fig. 4 enJSRVs are highly expressed in epithelia of the genital tract of the ewe. In situ hybridization analysis of enJSRVs env mRNA in the oviduct, uterus, cervix, and vagina. Note the specific expression of enJSRVs env mRNA in the epithelia lining the oviduct, cervix, and vagina of the cycling ewe. During the estrous cycle and pregnancy (P), enJSRVs mRNA is particularly abundant in the luminal and glandular epithelia of the uterus. LE, luminal epithelium; GE, glandular epithelium; M, myometrium; S, stroma. Modified from Palmarini, M., Gray, C.A., Carpenter, K., et al., 2001. Expression of endogenous betaretroviruses in the ovine uterus: Effects of neonatal age, estrous cycle, pregnancy, and progesterone. Journal of Virology 75 (23), 11319–11327.

detectable specific humoral or cellular immune responses, although recombinant JSRV CA, in the presence of adjuvants, can induce antibody production and specific T-cell responses in vaccinated sheep. Three full-length enJSRV loci have been cloned and characterized (enJS56A1, enJS5F16, and enJS59A1). All three loci have deletions or stop codons that make them replication incompetent. However, enJS56A1 and enJS5F16 maintain intact ORFs for gag and env. By using retroviral vectors pseudotyped by the enJSRVs Env, it was found that they too use HYAL2 as a cellular receptor and interfere by receptor competition with JSRV entry. One of the enJSRVs loci, enJS56A1, is defective for viral exit when overexpressed in transfected cells, although abundant intracytoplasmic Gag is detected and intracytoplasmic viral particles are visible by electron microscopy. The replication defect of enJS56A1 is determined by its Gag protein, which is trans-dominant over the exogenous JSRV Gag if co-expressed in the same cell. A tryptophan residue in position 21 of the enJS56A1 Gag (replacing an arginine in JSRV) is the main determinant for the block induced by enJS56A1. Thus, enJS56A1 exerts a unique mechanism of retroviral interference, which occurs at a late step of the replication cycle. The mechanism and timing of the block induced by enJS56A1 are not yet understood. However, the observation of viral particles by electron microscopy in cells expressing enJS56A1 (or co-expressing enJS56A1 and JSRV) suggests that enJS56A1-induced interference depends on a defect in Gag trafficking. Recent studies suggest that enJS56A1 appears to block JSRV, most likely in trans, by hampering the ability of the latter to reach the centrosome, the proposed site of assembly for betaretroviruses. enJSRVs are not expressed in the differentiated epithelial cells of the lungs but are expressed in the epithelium of the genital tract. As mentioned above, enhancer regions are located in the U3 and indeed enJSRVs transcription is regulated by progesterone via the progesterone receptor. Interestingly, the enJSRVs LTRs do not respond to lung transcription factors (such as FOXA2) unlike the exogenous JSRV LTRs. Thus, the LTR appears to be a major contributor to the different tropisms (genital tract vs. respiratory tract) shown by enJSRVs and JSRV. The enJSRVs Env (or at least the Env of those enJSRVs loci cloned so far) is not able to transform cells in vitro, unlike the highly related JSRV Env. Indeed, the cytoplasmic tail of the JSRV Env is where major determinants of transformation are located. In particular, an SH2 binding domain is present in the exogenous JSRV Env, but is absent in the enJSRVs Env sequenced to date. It is hypothesized that enJSRVs have been selected in the sheep for their ability to confer resistance to infection of the host by the related exogenous betaretroviruses. This innate resistance could have provided a selection pressure for betaretroviruses with tropism towards the respiratory tract (i.e., the current JSRV) rather than the genital tract.

Ovine Pulmonary Adenocarcinoma OPA is a naturally occurring lung cancer of sheep that has been reported in most sheep-rearing countries. It is absent from Australia and New Zealand and has been eradicated from Iceland. The disease is characterized by a progressive respiratory condition caused by the growth of the lung adenocarcinoma.

580

Jaagsiekte Sheep Retrovirus (Retroviridae)

Fig. 5 JSRV-induced tumors in vivo. Histology from a lung tumor section of naturally occurring cases of OPA (a) and experimentally reproduced tumor in young lambs (b). Sections were stained with routine hematoxylin and eosin. Note the presence of papillary to acinar neoplastic lesions that replace the normal alveolar and bronchiolar structures of the lungs. Immunohistochemistry (IHC) in lung tumor sections from natural OPA affected sheep showing the expression to the viral Env (characterized by membrane and intracytoplasmic brown color) (c). Double IHC in the lung sections from the same tumor combining surfactant protein C marker (black dots), a marker for alveolar type II cells, and Env JSRV protein marker (light brown) (d). Carazzi´s hematoxylin was used as a counterstain.

In vivo JSRV infects differenciated and undifferenciated epithelial cells of the terminal bronchioli and alveoli. Natural tumors are heterogenous variably containing cells expressing markers of Club and ATII together with stem/progenitor cells of the lung airway epithelia (Fig. 5). In addition, mesenchymal growths with intense expression of vimentin and desmin are also found. OPA is invariably fatal once the disease is diagnosed, and affected sheep die as a result of compromised respiratory function caused by tumor enlargement or from secondary bacterial infections. The incubation period of the naturally occurring disease can be very long lasting several months to years. Susceptibility/resistance to JSRV of some breeds has been suggested. A single genetic association study associated CCR5 and MX1 genes with resistance and susceptibility, respectively, in Spanish Latxa diary sheep. OPA can be transmitted experimentally only with material that contains JSRV, such as lung secretions collected from affected animals or virions obtained by transiently transfecting cells with JSRV infectious molecular clones. The experimental OPA model is highly reproducible. JSRV infection can be induced in most, if not all, inoculated lambs aged 1–6 months at the time of inoculation and a high proportion of them develop clinical signs and OPA lesions. Neonatal lambs are most susceptible and, in contrast to naturally occurring OPA, the incubation period for experimentally induced OPA can be as short as a few weeks. Although most experimentally inoculated lambs develop clinical signs and OPA lesions, under natural conditions the majority of JSRV-infected sheep do not develop OPA during their commercial lifespan. Aerogenous transmission is well recognized but, recently, the importance of milk and colostral transmission has been highlighted. JSRV in these secretions can reach the intestinal lymphoid tissues of lambs nursed by infected mothers. Interestingly, most infected animals harbor the virus as a disseminated infection of their lymphoid tissues and do not show detectable pulmonary lesions. Although lymphoreticular cells appear to serve as the principal reservoir of virus infection, viral antigens are detected only rarely in this compartment where sensitive PCR assays are necessary to detect viral RNA or proviral DNA. The short incubation period in young lambs experimentally infected with JSRV may be explained by the combination of high virus infectious doses present in the inoculum and the higher abundance of the permissive target cells (type 2 pneumocytes and Clara cells) in lambs compared to adult animals. In natural infections, the mechanisms involved in converting the stable persistent lymphoid infection in peripheral tissues to a progressive pulmonary epithelial tumor are not clear. In addition, it is not known whether and how efficiently sheep infected by JSRV, but with no neoplastic lesions, are able to transmit the virus to uninfected sheep. In an affected flock, control of OPA is very difficult as no vaccines or effective diagnostic tests are available.

Jaagsiekte Sheep Retrovirus (Retroviridae)

581

Fig. 6 Expression of the JSRV Env transforms cells in vitro. Transformation of the rat fibroblast cell line 208F by the JSRV envelope glycoprotein. 208F cells were mock-transfected (a) or transfected with an expression plasmid for the JSRV Env (c). Mock-transfected 208F cells are morphologically flat, possess a strong contact-inhibition and do not grow in soft agar (b). On the other hand, 208F cells transfected with an expression plasmid for the JSRV Env show the onset of foci of transformed cells (c), which are able to form colonies in soft agar (d).

Mechanisms of Virus-Induced Cell Transformation The mechanisms used by JSRV to induce cell transformation are different from those followed by the majority of oncogenic retroviruses. OPA tumors are multiclonal, and common proviral integration sites have been rarely observed. However, no cellular oncogenes have been found to be transduced by JSRV. The oncogenic properties of JSRV are due directly to one of its structural proteins. Transfection of a variety of fibroblast and epithelial cell lines with expression plasmids for the JSRV Env leads to efficient cell transformation (Fig. 6). Moreover, experimental infection of mice and lambs, with replication incompetent vectors expressing JSRV Env, induced tumors in the inoculated animals. Thus, the JSRV Env is a dominant oncoprotein both in vitro and in vivo and viral spread is not necessary for tumorigenesis. The mechanisms involved in JSRV Env-induced transformation have not been fully elucidated; however, signal transduction involving the PI3K-Akt and H/N Ras-MEK-MAPK pathways are important. Additional pathways including mTOR/p70S6K, EGFR and Wnt signaling and AGR-2-YAPI-AREG axis are active in OPA and may contribute to oncogenesis in this disease. Conflicting reports are available on the involvement of other cellular oncogenes such as the Stk/Ron tyrosine kinase in the onset of JSRV Env-induced cell transformation. One of the major determinants of JSRV Env transformation is the transmembrane domain (TM), although other regions may be important. In particular, a putative docking site (Y-X-X-M) for phosphatidylinositol 3-kinase (PI-3K) is critical for JSRV-Env induced cell transformation. Within this motif, Y590 is crucial for JSRV Env-induced cell transformation, although Y590 mutants maintain a reduced ability to induce cell transformation in some cell lines. In summary, the JSRV Env acts as a dominant oncogene in vitro and in vivo and its expression is sufficient to induce lung adenocarcinoma in the target species.

Enzootic Nasal Tumor Virus ENTVs of sheep and goats are distinct betaretroviruses highly related to each other and JSRV. ENTV causes a contagious tumor of the mucosal nasal glands, as well as respiratory and olfactory mucosa, in their respective target species known as enzootic nasal adenocarcinoma (ENA). Clinically, ENA is characterized by respiratory distress caused by the enlargement of the tumor, nasal discharge, and skull deformation. Like OPA, ENA has a long incubation period.

582

Jaagsiekte Sheep Retrovirus (Retroviridae)

The molecular biology of ENTVs (ENTV-1, ENTV2) has not been studied in great detail, but the virus displays many features in common with JSRV. The cellular receptor for ENTVs is also HYAL2 and the ENTVs Env, like its JSRV counterpart, is a dominant oncogene that appears to follow the same mechanisms of cell transformation used by JSRV. The main differences between the ENTVs and JSRV appear to be in LTR, small regions of Gag, Orf-x and the transmembrane region (TM) of Env (concentrated in the U3 region of the viral LTRs). The ENTV-1 LTR, unlike the JSRV LTR, does not bind lung-specific transcription factors such as FOXA2, which is likely the basis of tropism differences between the two viruses.

Future Perspectives Small ruminant betaretroviruses are a fascinating group of viruses with unique characteristics that are of broad interest through their veterinary, comparative medical and biological importance. The veterinary importance arises from the economic impact in many sheep rearing countries of the diseases induced by JSRV and ENTVs combined with the absence of any effective control tools or mechanisms. Their comparative medical interest stems from the striking similarity of some forms of human lung adenocarcinoma to OPA. Investigations looking for the JSRV in human lung cancer have not found evidences for its presence in human pulmonary adenocarcinoma but data do not rule out a role for a unknown retrovirus. In addition, the comparison of the transcriptomes of OPA and human lung adenocarcinomas indicate close similarity, but highlights that there remain many differences between the sheep and human diseases. OPA is considered an excellent outbred large animal model for these tumors with opportunities to investigate issues that are not available from other systems. Lung cancer is the main cause of death among cancer patients and effective therapeutic strategies are greatly needed to improve patient survival and well-being. OPA is a large animal model that can identify and test the efficacy of new therapeutic interventions in a highly reproducible system. enJSRVs are an especially active group of endogenous retroviruses and offer insights into several areas of general biological interest, such as viral replication, interference, and reproductive biology. Understanding the mechanisms of the enJS56A1-induced block could inspire the design of novel anti-retroviral strategies and shed light on early events in retroviral assembly and/or trafficking. In particular, this unique viral block provides additional clues on the variety of mechanisms shaping co-evolution of endogenous/exogenous retroviruses and their hosts. enJSRVs are highly expressed in the genital tract of the ewe and are intimately involved in early placental development in this animal species. The sheep/enJSRVs model can be useful to experimentally address the hypothesis that endogenous retroviruses have shaped and are essential for mammalian biology.

Further Reading Armezzai, A., Varela, M., Spencer, T.E., Palmarini, M., Arnaud, F., 2014. Menage a Trois: The evolutionary interplay between JSRV, enJSRVs and domesic sheep. Viruses 6, 4926–4945. Borobia, M., De las Heras, M., Ramos, J.J., et al., 2016. Jaagsiekte retrovirus can reach Peyer´s patches and mesenteric lymph nodes of lambs nursed by infected mothers. Veterinary Pathology 53, 1172–1179. Fan, H. (Ed.), 2003. Jaagsiekte Sheep Retrovirus and Lung Cancer. Berlin: Springer. Karagianni, A.E., Vasoya, D., Finlayson, J., et al., 2019. Transcriptional response of ovine lung to infection with Jaagsiekte sheep retrovirus. Jouranl of Virology 93 (21). Maeda, N., Palmarini, M., Murgia, C., Fan, H., 2001. Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA. Proceedings of the National Academy of Sciences of the United States of America 98, 4449–4454. Martineau, H.M., Cousens, C., Ilmach, S., Dagleish, M., Griffiths, D., 2011. Jaagsiekte sheep retrovirus infects multiple cell types in the ovine lung. Journbal of Virology 85, 3341–3355. Palmarini, M., Gray, C.A., Carpenter, K., et al., 2001. Expression of endogenous betaretroviruses in the ovine uterus: effects of neonatal age, estrous cycle, pregnancy, and progesterone. Journal of Virology 75 (23), 11319–11327. Palmarini, M., Sharp, J.M., De las Heras, M., Fan, H., 1999. Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep. Journal of Virology 73, 6964–6972. Wootton, S.K., Halbert, C.L., Miller, A.D., 2005. Sheep retrovirus structural protein induces lung tumours. Nature 434, 904–907.

Japanese Encephalitis Virus (Flaviviridae) Sang-Im Yun and Young-Min Lee, Utah State University, Logan, UT, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of A.D.T. Barrett, Japanese Encephalitis Virus, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00434-9.

Classification Japanese encephalitis virus (JEV) is a species belonging to the largest genus Flavivirus (53 species) of the family Flaviviridae, which includes three other genera, Pestivirus (11 species), Hepacivirus (14 species), and Pegivirus (11 species), according to a report published by the International Committee on Taxonomy of Viruses in 2018. Like many other species within the Flavivirus genus, JEV can infect both arthropods (mosquitoes), which serve as vectors, and vertebrates (including humans), which act as animal hosts. JEV is a clinically important mosquito-borne zoonotic flavivirus representing the JEV antigenic complex, a group of virus species that show a high degree of interspecies cross-neutralization and phylogenetic relatedness. This group also includes West Nile (WNV), Murray Valley encephalitis, Saint Louis encephalitis, Usutu, Koutango, Yaounde, and Cacipacore viruses. Moreover, JEV is genetically related to other medically significant but antigenically distinct mosquito-borne flaviviruses, such as dengue (DENV), yellow fever (YFV), and Zika (ZIKV) viruses, as well as tick-borne flaviviruses, including tick-borne encephalitis (TBEV), louping ill, Langat, Powassan, Omsk hemorrhagic fever, and Kyasanur forest disease viruses.

Virion Structure JEV is an enveloped virus, 50–60 nm in diameter. It comprises three virus-encoded structural proteins: capsid (C), membrane (M) or its precursor prM, and envelope (E). Conventional thin-section electron microscopy (EM) has revealed that the intracellular virions in JEV-infected cells have an inner electron-dense core, B30 nm in diameter, surrounded by an electron-lucent narrow interspace and an outer electron-dense thin layer (Fig. 1(A)). The inner core of the virus is the nucleocapsid, a poorly-defined structure consisting of the viral genome and multiple C proteins. This nucleocapsid is surrounded by a host cell-derived lipid bilayer membrane that is coated with an outer protein shell containing two transmembrane proteins, prM/M and E. Based on the protein composition and architecture of this shell, two different forms of the virion are recognized: (a) extracellular mature virions (fully infectious), with a smooth protein shell containing M and E proteins; and (b) intracellular immature virions (noninfectious), with a spiky protein shell containing prM and E proteins. Occasionally, partially mature virions are released from JEV-infected cells; these forms are still infectious but presumably less so than completely mature virions; their protein shell appears partly smooth and partly spiky, depending on the local protein composition of either M þ E or prM þ E. Also, subviral particles are secreted from JEV-infected cells; these are rather heterogenous in size but generally smaller than the viral particles and contain only the prM/M and E proteins on the viral membrane, without the inner nucleocapsid.

The Extracellular Mature Virion The structure of the B50 nm-sized mature JEV has recently been solved at 4.3 Å resolution by cryo-EM and three-dimensional image reconstruction (PDB ID 5WSN), showing a stoichiometry and arrangement of the M and E proteins on the viral membrane that agrees well with all the previously reconstructed cryo-EM structures of mature DENV (PDB IDs 1K4R and 3J27), WNV (PDB ID 3J0B), ZIKV (PDB IDs 5IRE, 6CO8, and 5IZ7), and TBEV (PDB ID 5O6A). The outer protein shell of the mature JEV is made up of 60 icosahedral asymmetric units (Fig. 1(B)), each containing three antiparallel M:E heterodimers, arranged into 30 diamond-shaped “tiles” by positioning two asymmetric units in opposite orientations. Each tile therefore contains three E:M:M:E heterotetramers, with the two adjacent M proteins being hidden under the antiparallel E:E homodimer. Each E monomer consists of an N-terminal banana-shaped ectodomain, a membrane-proximal three helix-containing stem, and a C-terminal double-spanning membrane anchor. The E ectodomain is divided into three b-barrel domains (DI-DIII), with a hydrophobic fusion loop located at the distal end of DII and fitted into a pocket at the DI-DIII interface of the neighboring E ectodomain. The M protein consists of an N-terminal flexible loop, an amphipathic a-helix, and a C-terminal double-spanning membrane anchor. Overall, the outer protein shell of the mature virion is stabilized by multiple molecular interactions: (a) electrostatic and hydrophobic interactions between the M-loop and E-DI and DII; (b) hydrogen-bond interactions between the M-helix, E-DII, and the loop of a neighboring M; and (c) charge interactions between E-DI, DIII, and the stem region.

The Intracellular Immature Virion The structure of the B60 nm-sized immature JEV has not yet been determined at the atomic level, but those of immature DENV (PDB IDs 3C6D and 4B03), WNV (PDB ID 2OF6), YFV (PDB ID 1NA4), and ZIKV (PDB ID 5U4W) have already been reported at

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21503-3

583

584

Japanese Encephalitis Virus (Flaviviridae)

Fig. 1 Virion structure of JEV. (A) Thin-section EM images of BHK-21 cells uninfected or infected with JEV strain CNU/LP2 for 18 h. Insets #1 and #2 are the enlarged views of virus-induced vesicles containing the intracellular immature JEV particles. (B) Three-dimensional cryo-EM structure of the extracellular mature JEV particles (strain P3). The inner nucleocapsid core, a complex of the viral genomic RNA and multiple copies of the C protein, is enveloped by a lipid bilayer that is coated with an outer protein shell composed of 180 copies each of two membraneanchored proteins, M and E. On the viral membrane, the 180 M:E heterodimers are organized into 60 icosahedral asymmetric units (open black triangle), each containing three antiparallel M:E heterodimers (medium purple, sky blue, and dark khaki), which then form 30 diamond-shaped tiles (open gray diamond) as two asymmetric units oriented in opposite directions. The 2-, 3-, and 5-fold axes of symmetry are marked with the corresponding numbers (2, 3, and 5). The E monomer comprises a three-domain-containing ectodomain (E-DI, red; E-DII, yellow; and E-DIII, blue), followed by a three-helix-containing stem (E-H1, E-H2, and E-H3, deep sky blue) and a two-transmembrane helix-containing anchor (E-TM1 and E-TM2, cornflower blue). The fusion loop (green) is located at the tip of E-DII. The M monomer comprises a flexible loop (M-Loop, pink), followed by an a-helix (M-H, hot pink) and a two-transmembrane helix-containing anchor (M-TM1 and M-TM2, orange). The pdb data file for the virion structure was retrieved from the Protein Data Bank (PDB ID 5WSN), and its virion structure was visualized using Chimera software.

high resolution. Given their close relatedness, immature JEV is likely to have an overall virus structure similar to that of these other immature flaviviruses. In all of the currently available cryo-EM structures of immature flaviviruses, the inner nucleocapsid is poorly defined, as is true of their respective mature virions; however, the outer protein shell is well defined, composed of 60 trimers of prM:E heterodimers, with each trimer appearing as a protruding spike on the surface of the viral membrane. Each spike has a tripod-like structure holding the three E ectodomains together, in each of which E-DII forms a tripod leg, and E-DI and DIII shape a tripod foot. In this structure, three hydrophobic fusion loops come together to form the tripod head, with each loop exposed at the apical tip of E-DII but capped by the N-terminal pr portion of prM, thereby preventing the virions from prematurely fusing with intracellular membranes during viral morphogenesis. The C-terminus of the pr fragment is connected to the N-terminal loop segment of M that extends downward toward the viral membrane along the lateral surface of E-DII.

Japanese Encephalitis Virus (Flaviviridae)

585

Fig. 2 Genome structure and gene expression of JEV. (A) Schematic representation of the single-stranded positive-sense RNA genome of JEV strain CNU/LP2. The genomic RNA has a cap structure at the 50 end, followed by a 50 NCR, an ORF, and a 30 NCR with no poly(A) tail at the 30 end. (B) Schematic representation of the two polyprotein precursors (ppC-NS5 and ppC-NS10 ) translated from the JEV single ORF. While the ppC-NS5 precursor is the full-length polyprotein, the ppC-NS10 precursor is a C-terminally truncated polyprotein produced by a  1 ribosomal frameshift (FS) occurring between codons 8 and 9 of NS2A. The two polyproteins are processed at the specific cleavage sites by cellular and viral proteases (yellow, cyan, and gray arrowheads) into three structural proteins (red) and at least seven nonstructural proteins (blue). Of these 10 mature proteins, two possess a bipartite structure: NS3, which has an N-terminal protease (PRO) domain and a C-terminal helicase (HEL) domain; and NS5, which has an N-terminal methyltransferase (MET) domain and a C-terminal RNA-dependent RNA polymerase (POL) domain. Green asterisks indicate four N-linked glycosylation sites located in the pr portion of prM (Asn15), E (Asn154), and NS1/10 (Asn130 and Asn207). (C) Predicted membrane-spanning regions and their orientation in all 10 major JEV proteins associated with the ER membrane. (D) Detection of viral proteins in BHK-21 cells infected with JEV strain CNU/LP2. At 18 h after infection, total lysates of uninfected (U) and JEV-infected (I) cells were examined by immunoblotting with each of 14 JEV region-specific rabbit polyclonal antibodies, as indicated. In each blot, purple and yellow dots indicate the major viral proteins reacting with the given antibody. Panels A–C are modified from a review article (Yun, S.I., Lee, Y.M., 2018. Early events in Japanese encephalitis virus infection: Viral entry. Pathogens 7, 68), and panel D is reproduced from a research article (Kim, J.K., Kim, J.M., Song, B.H., et al., 2015. Profiling of viral proteins expressed from the genomic RNA of Japanese encephalitis virus using a panel of 15 region-specific polyclonal rabbit antisera: Implications for viral gene expression. PLoS One 10, e0124318), with permission.

Genome Genome Structure JEV contains a linear single-stranded positive-sense RNA genome, B11,000 nucleotides in length (Fig. 2(A)). The 50 end of the genomic RNA has a type 1 cap and begins with the conserved dinucleotide AG (abbreviated m7GpppAmG, where the “m” indicates a methyl group added on the terminal nucleobase guanine at the N-7 position and the first nucleotide adenosine at the ribose 20 -O position). In contrast, the 30 end of the genomic RNA lacks a poly(A) tail but terminates instead with the conserved dinucleotide CU. In flaviviruses, a methylated 50 -cap on the genomic RNA plays multiple roles in preventing RNA degradation, promoting translation initiation through a canonical cap-dependent mechanism, and protecting viral RNAs from recognition by a cellular factor(s) that senses the presence of non-self RNAs; however, these viruses can also utilize a non-canonical translation initiation mechanism that depends minimally on the presence of a methylated 50 -cap. The viral genome consists of an B95-nucleotide 50 noncoding region (NCR), an B10,299-nucleotide open reading frame (ORF), and an B574-nucleotide 30 NCR.

586

Japanese Encephalitis Virus (Flaviviridae)

The 50 NCR and the following B100-nucleotide 50 -terminal region of the ORF contain a cluster of cis-acting primary sequences and secondary/tertiary structures that are involved in regulating viral translation and RNA replication through a long-range intramolecular RNA–RNA interaction with the 30 NCR, which also comprises an array of cis-acting linear sequences and higher-order structures.

Gene Expression The genomic RNA of JEV is infectious on its own, since it can be directly translated by the protein synthesis machinery of its host cells. Upon JEV infection, the single long ORF encoded in the viral genomic RNA is translated into a full-length polyprotein precursor (designated ppC-NS5) that is then cleaved co- and post-translationally to generate at least 10 functional proteins: C-“anchor”-prM-E-NS1-NS2A-NS2B-NS3-NS4A-“2K”-NS4B-NS5, where NS denotes “nonstructural” (Fig. 2(B) and (C)). Mainly based on previous research with YFV, DENV, and WNV, the flavivirus full-length ppC-NS5 is shown to be processed in a spatially and temporally controlled manner by four different proteases: (a) the host signal peptidase, which mediates cleavage at the junctions of anchor-prM, prM-E, E-NS1, and 2K-NS4B on the lumen side of the endoplasmic reticulum (ER) membrane; (b) the viral two-component serine protease NS2B þ NS3, which recognizes the cleavage site at the junctions of C-anchor, NS2A-NS2B, NS2B-NS3, NS3-NS4A, NS4A-2K, and NS4B-NS5 on the cytoplasmic side of the ER membrane; (c) the host furin, which cleaves prM into the pr fragment and M protein in the trans-Golgi network (TGN); and (d) an as-yet unidentified host protease, which mediates the cleavage at the NS1-NS2A junction. In the case of JEV, WNV, and presumably other members of the JEV antigenic complex, the viral single ORF is translated into not only the full-length ppC-NS5 but also a C-terminally truncated polyprotein precursor (designated ppC-NS10 ) by a programmed  1 ribosomal frameshift event occuring at codons 8–9 of NS2A (Fig. 2(B) and (C)). As with the full-length ppC-NS5, the truncated ppC-NS10 is also processed to yield C, prM/M, E, and a unique NS10 , an extended form of NS1 that has 52 additional amino acids added to the C-terminus of the protein. This frameshifting takes place at a slippery heptanucleotide sequence from 3554 Y_CCU_UUU (0 frame) to YCC_UUU_U (  1 frame), which is stimulated by a pseudoknot structure located six nucleotides downstream of the slippery sequence. The pseudoknot consists of two immediately adjacent, possibly coaxially stacked stem loops (SL1 and SL2), with a five-nucleotide loop region (3585YUGGC) of SL1 base-paired with its complementary sequence (3621GCCAG) six nucleotides downstream of SL2. Using a collection of 14 JEV region-specific rabbit polyclonal antibodies, we conducted a comprehensive immunoblot analysis that presented a genome-wide expression profile of the viral gene products accumulated in JEV-infected cells, enabling us to label all the mature viral proteins except NS2A, together with a significant number of related species that are presumably produced as a result of further viral protease-mediated processing, post-translational modification, and/or intracellular protein degradation (Fig. 2(D)). Described below are the 12 biologically active JEV proteins, with a short account of their structure and function in the viral life cycle.

Structural proteins The structural proteins required for infectious virus production are C, prM/M, and E: (1) C, a highly basic B12-kDa cytosolic protein, forms a compact homodimer, with each monomer folded into four a-helices (Fig. 3(A)). It binds to the viral genomic RNA for nucleocapsid formation through charge interactions, probably with both the unstructured N-terminal region (residues 1–25) and the last C-terminal a-helix that are rich in basic residues. For viral assembly, C associates with the cytoplasmic face of the ER membrane through hydrophobic interactions, possibly with the first and second a-helices. Interestingly, C is also found in the nucleolus of JEV-infected cells, and Gly42 and Pro43 at the beginning of its second a-helix are critical for the nuclear and nucleolar localization that plays a role in promoting viral replication in a cell type-dependent manner (i.e., Vero vs. C6/36). Also, full-length C is further cleaved internally between Lys18 and Arg19 in the unstructured N-terminal region by the host cysteine protease cathepsin L, which is important for cell type-specific viral replication (e.g., N18 vs. Vero). Although the nature of their cell type dependency is poorly understood, both the nuclear/nucleolar localization and internal cleavage of the C protein affects JEV’s virulence in mice. (2) prM, a glycosylated B24-kDa membrane protein, is the precursor molecule of M; it has an N-glycan covalently linked to Asn15 in its pr portion, which has a b-barrel fold, as inferred from the crystal structure of the DENV pr fragment in complex with its E ectodomain (PDB ID 3C6E). In the intracellular compartments along the secretory pathway, prM acts as a chaperone to assist in the proper folding and oligomerization of the main surface glycoprotein E and the efficient secretion of viral particles. In the immature virion, the pr portion of prM shields the hydrophobic fusion loop of E to prevent premature fusion during transit through the secretory pathway as part of viral morphogenesis. (3) M, a nonglycosylated B8-kDa membrane protein, is the proteolytic product of prM; it consists of an N-terminal 19-aminoacid flexible loop, a perimembrane a-helix, and a C-terminal double-spanning membrane anchor (Fig. 3(B)). In the mature virion, M is the minor component of a viral envelope complex E:M:M:E heterotetramer, in which the two adjacent M proteins are buried underneath the antiparallel E:E homodimer. A recent study has shown that a single Ile36-Phe mutation introduced at the end of the M-helix has little effect on viral entry but fails to produce infectious particles in mammalian cells, although not in mosquito cells, and it results in full attenuation in mice. Despite this knowledge of its structural details, the functional role of M in viral replication, particularly at the early and late stages of JEV infection, remains unclear.

Japanese Encephalitis Virus (Flaviviridae)

587

Fig. 3 Ribbon representation of JEV C, M, E, NS1, NS3, and NS5 proteins. Displayed are the atomic structures of a JEV C:C homodimer (A), a JEV E:M:M:E heterotetramer (B), a C-terminal fragment of JEV NS1 (C), a WNV NS1:NS1 homodimer as a reference for the as-yet experimentally unsolved JEV NS1:NS1 homodimer (D), the PRO and HEL domains of JEV NS3 (E), and a JEV NS5 monomer (F). The pdb data files for all the protein structures were retrieved from the Protein Data Bank (PDB) database, as indicated by their four-character identifiers, and their protein structures were visualized using Chimera software. If a dimer or tetramer is shown, only one monomer is used to color-code the structurally or functionally important domains/regions/motifs/residues. Black italic numbers indicate the amino acid positions of the N- and C-termini of each protein. A detailed description of the protein structures is provided in the main text.

(4) E, a glycosylated B53-kDa membrane protein, forms an antiparallel E:E homodimer lying horizontally on the surface of the mature virion, with each monomer composed of an N-terminal ectodomain, a membrane-proximal stem, and a C-terminal membrane anchor (Fig. 3(B)). The E ectodomain folds into three b-barrel domains (PDB IDs 3P54, 5MV1, and 5MV2): (a) DI, a centrally located globular domain, with an N-glycan covalently attached to Asn154 in the E0-F0 or glycan loop and a stretch of closely distributed basic residues (Lys279–Lys297) on the I0 strand and its flanking region; (b) DII, an elongated dimerization domain, with a glycine-rich hydrophobic peptide (Gly100‒Gly109) in the c-d or fusion loop and several functionally important loops, such as h-i, i-j, and k-l loops; and (c) DIII, an immunoglobulin-like domain, with an RGD motif (Arg387-Gly388-Asp389) in the F-G loop. DIII is then connected to the stem region, which includes three a-helices lying nearly flat on the viral membrane, followed by the membrane anchor region, which includes two antiparallel a-helices traversing the viral membrane. E is the major component of the viral envelope complex E:M:M:E heterotetramer, which directs both receptor-mediated endocytosis and pH-dependent membrane fusion during virus entry into host cells. Therefore, E is the primary target for neutralizing antibodies during infection.

Nonstructural proteins The nonstructral proteins involved in viral polyprotein processing, RNA replication, and assembly/release, as well as evasion of the host’s innate immunity, are NS1/10 , NS2A, NS2B, NS3, NS4A, NS4B, and NS5: (1) NS1, a glycosylated B45-kDa multi-domain protein with two N-glycans each conjugated at Asn130 and Asn207, exists not only as a membrane-associated cruciform homodimer in the secretory compartments and on the surface of JEV-infected cells but also as a secreted barrel-shaped homohexamer composed of three dimers with lipid molecules loaded inside. The

588

(2)

(3)

(4)

(5)

(6)

Japanese Encephalitis Virus (Flaviviridae) crystallized C-terminal half of JEV NS1 forms a central “b-ladder” domain, with an N-glycan at Asn207 and a long “spaghetti loop” on one side (Fig. 3(C)), which is preceded by its as-yet uncrystallized N-terminal half that is likely folded into a small “b-roll” dimerization domain on the other side of the central b-ladder and a bulging “wing” domain with an N-glycan at Asn130 projecting from it, as inferred from the reported crystal structures of full-length NS1 of WNV (Fig. 3(D)), DENV (PDB ID 4O6B), and ZIKV (PDB IDs 5K6K and 5GS6). In these flaviviruses, the b-roll domain, together with a “connector” subdomain of the wing domain, creates a hydrophobic surface on one side of the dimer for membrane association and faces towards the interior of the hexamer for lipid association. Although the molecular mechanisms are not well understood, the cell-associated dimeric form of NS1 is known to be involved in viral RNA replication and particle production, whereas the secreted hexamer is associated with multiple components of the innate and adaptive immune systems, thereby modulating host immune responses. The secreted hexamer was originally described as a soluble complement-fixing agent present in the blood and tissues of infected hosts. Thus, NS1 is a major immunogen, capable of inducing protective immunity during flavivirus infection. NS10 , a glycosylated B58-kDa multi-domain protein, is the frameshift product of NS1 that acquires a 52-amino-acid C-terminal extension corresponding to the N-terminal nine amino acids of NS2A before the frameshift site and the unique 43 amino acids in the shifted  1 reading frame afterwards. Although the structure of its C-terminal extension is undefined, NS10 is dispensible for viral replication in vitro but plays a role in enhancing viral virulence in vivo. Further investigation is required to elucidate the differential roles of NS10 (apart from the multiple known functions of NS1) in JEV biology, particularly related to viral pathogenesis and host antiviral responses. NS2A, a hydrophobic B25-kDa transmembrane protein, is predicted to have eight a-helices; five span the ER membrane, and the other three are exposed to the ER lumen. NS2A is a component of the viral replication complex that can inhibit the host’s interferon (IFN)-mediated antiviral response in cells infected with JEV, WNV, or DENV. In the case of DENV, NS2A inhibits the RIG-I‒MAVS signaling pathway by preventing TBK1/IRF3 phosphorylation. In JEV, NS2A is degraded by TRIM52, a tripartite motif-containing cellular protein with antiviral activity. In YFV, full-length NS2A (224 amino acids) is cleaved internally by the viral two-component serine protease NS2B þ NS3 to generate its C-terminally truncated variant (190 amino acids). A subsequent mutagenetic study has shown that loss of this internal cleavage site in YFV blocks the production of infectious particles without altering the level of viral RNA replication; also, this defect can be restored by introducing a suppressor mutation in NS3, suggesting a role for NS2A and NS3 in viral morphogenesis. Similarly, mutations introduced into WNV NS2A block viral particle production. Further studies are warranted to elucidate the precise membrane topology and function of NS2A. NS2B, a hydrophobic B14-kDa transmembrane protein, has three potential membrane-spanning a-helices, with a central B45-amino-acid hydrophilic loop (residues 51–95) located on the cytoplasmic side of the ER membrane that forms a stable complex with NS3. This hydrophilic loop serves to anchor the NS2B þ NS3 complex to intracellular membranes and acts as a cofactor for the protease activity of NS3. Also, recent genetic and biochemical studies have shown that the transmembrane domains of JEV NS2B play a role in both viral RNA replication and particle formation, likely through interactions with the viral protein NS2A and the host factor SPCS1, a component of the signal peptidase complex. Moreover, it is intriguing to note that the B14-kDa NS2B protein is barely detectable in JEV-infected cells; instead, its smaller variant, B12-kDa NS2B’, is predominantly found. The nature and functional significance of NS2B’ in JEV biology are unknown. NS3, a cytoplasmic B69-kDa bi-domain protein, contains an N-terminal serine protease (PRO) domain, a flexible interdomain linker, and a C-terminal RNA helicase (HEL) domain (Fig. 3(E)). The PRO domain has a catalytic triad of His51Asp75-Ser135 positioned along a groove on its surface and requires a hydrophilic cofactor region of NS2B for its protease activity, with a specific cleavage recognition sequence of two basic residues at positions P2 and P1 and a small unbranched amino acid at position P10 . As a member of the DEAH/D-box helicase family within superfamily 2, the HEL domain folds into three subdomains (S1‒S3), with the S1-S2 interface containing seven sequence motifs: I (also known as Walker A; 197 G‒201T), Ia (222L‒232M), II (also known as Walker B; 285D‒288H), III (316T‒321G), IV (362F‒367K), V (409T‒417N), and VI (457Q‒464R). These motifs are associated with three catalytically related activities: (a) RNA helicase, which unwinds RNA duplexes and remodels RNA-protein complexes in an ATP-dependent manner; (b) nucleoside triphosphatase (NTPase), which provides energy from NTP hydrolysis; and (c) RNA 50 -triphosphatase (RTPase), which removes the g-phosphate of a nascent 50 -triphosphorylated RNA to generate its 50 -diphosphorylated form as an initial step for RNA capping. Interestingly, the full-length NS3 of JEV is further cleaved into smaller products, including an B34-kDa NS3N-term and an B35-kDa NS3Cterm , but the functional significance of these cleavage products is unknown. In addition to its roles in viral RNA synthesis, the HEL domain is also involved in viral assembly/packaging of YFV and WNV. In JEV, NS3 is associated with cellular microtubules and the TSG101 protein, which are potentially involved in viral assembly. Moreover, NS3 can induce cell death via caspase-dependent and/or -independent pathways in the case of JEV, WNV, DENV, and Langat virus. In DENV, the NS2B þ NS3 protease inhibits the activation of type I IFN signaling by cleaving the human adapter protein MPYS (also known as STING and MITA), but not its murine version. NS4A, a hydrophobic B14-kDa transmembrane protein, consists of three potential a-helices, with the first and third spanning the ER membrane and the second facing the ER lumen. NS4A, together with NS4B, is believed to serve as a major scaffold capable of inducing membrane rearrangements during the formation of viral replication complexes. Recent studies have shown that a single Lys79-Arg substitution in NS4A causes a defect in JEV RNA replication that can be rescued by introducing a compensatory Tyr3-Asn mutation in NS4B, suggesting that a genetic interaction between NS4A and NS4B is

Japanese Encephalitis Virus (Flaviviridae)

589

crucial for JEV RNA replication. In YFV, NS4A has been found to interact with NS1, which is essential for viral RNA replication. JEV NS4A is also reported to interact with cyclophilin B during RNA replication, which can therefore be suppressed in vitro by cyclosporin A treatment. JEV NS4A is further thought to participate in the inhibition of IFN signaling, as seen in WNV and DENV, and its interaction with the host DEAD-box RNA helicase DDX42 has been implicated in viral pathogenesis. (7) NS4B, a hydrophobic B27/25-kDa transmembrane protein, is predicted to have five a-helices; the first two are located in the ER lumen, and the other three traverse the ER membrane. NS4B appears as a doublet on gel electrophoresis under denaturing and reducing conditions, indicating that a post-translational modification occurs in the protein, although its nature and significance for JEV biology are unknown. In DENV, NS4B co-localizes with NS3 and double-stranded RNA in the perinuclear region, where viral RNA replication presumably takes place, and it specifically binds to NS3, promoting NS3 helicase activity. In other mosquito-borne flaviviruses (WNV, DENV, and YFV), NS4B is known to play an important role in innate immune evasion, inhibiting the JAK‒STAT signaling pathway by reducing STAT1 phosphorylation and/or inhibiting the RIG-I‒MAVS signaling pathway by blocking TBK1/IRF3 phosphorylation, thereby suppressing IFN gene expression and the activation of IFNstimulated response element promoters. Further research is needed to clarify the structure and function of NS4B in JEV biology. (8) NS5, a cytoplasmic B103-kDa bi-domain protein, contains an N-terminal S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MET) domain, a flexible interdomain linker, and a C-terminal RNA-dependent RNA polymerase (POL) domain (Fig. 3(F)). The MET domain, crystallized with S-adenosyl-L-homocysteine (SAH, the demethylated form of SAM), has a catalytic tetrad of Lys61-Asp146-Lys182-Glu218, with their side chains pointing toward the center of the domain. This domain is associated with two enzymatic activities: (a) guanylyltransferase (GTase), which transfers a guanosine monophosphate (GMP) moiety from GTP in a backward orientation to the 50 end of the 50 -diphosphorylated RNA generated by the NS3 RTPase, thereby creating a unique 50  50 triphosphaste linkage (GpppA-RNA); and (b) methyltransferase (MTase), which transfers a methyl group from SAM sequentially to the terminal nucleobase guanine at the N-7 position and to the first nucleotide adenosine at the ribose 20 -O position, thereby creating a type 1 cap (m7GpppAmRNA). Mutagenetic studies have shown that N-7 methylation is essential for viral translation and RNA replication, whereas 20 -O methylation is involved in evasion of the host’s innate immunity. The POL domain adopts a classical “right-hand” fold comprising fingers, palm, and thumb subdomains with seven sequence motifs, i.e., A (533Y‒545T), B (601Q‒615F), C (662R‒673K), D (690S‒694K), E (712P‒717H), F (453I‒479M), and G (404K‒413A), as well as the “priming loop” (790V‒812E) that initiates viral RNA synthesis de novo with two catalytic Asp residues (Asp536 in motif A and Asp668 in motif C) located on the palm subdomain. As noted in JEV and other flaviviruses, NS5 can shuttle between the nucleus and the cytoplasm, suggesting that it has additional functions other than viral RNA synthesis. In DENV, this nucleocytoplasmic shuttling is mediated by the importin and exportin transport system through a nuclear localization signal (residues 322–370) and a nuclear export signal (residues 329–345), respectively; however, these two signals overlap each other at the interface between the MET and POL domains, where a potential binding site for NS3 is also located. Thus, the role of the nuclear localization of NS5 remains unclear. NS5 is also a potent antagonist of IFN-stimulated JAK‒STAT signaling, capable of (a) suppressing STAT1/TYK2 phosphorylation (in JEV, WNV, and TBEV), (b) binding to STAT2 and promoting its degradation (in DENV, YFV, and ZIKV), or (c) interacting with type I and II IFN receptor complexes and blocking JAK1/TYK2 phosphorylation (in Langat virus). JEV NS5 can also suppress the induction of type I IFN by inhibiting the nuclear translocation of IRF3 and NF-κB by interrupting their interaction with two nuclear transport proteins, importins a3 and a4.

Life Cycle The life cycle of JEV and other flaviviruses can be divided into three major phases (Fig. 4): entry (pre-RNA replication), translation and RNA replication, and morphogenesis (post-RNA replication).

Viral Entry JEV infects a wide range of cell types in mosquitoes, humans, and various animal species, demonstrating its broad host cell tropism. The first step in the infection process is the attachment of a virion to the surface of a susceptible cell (Fig. 4(A)). This step involves multiple molecular interactions, each characterized by the binding of a part of the viral surface glycoprotein E to a certain host factor. Thus far, three such interactions have been documented: (a) A string of closely spaced positively charged residues in E-DI binds to one of the negatively charged glycosaminoglycans, such as a heparan sulfate proteoglycan. (b) An N-linked glycan in E-DI binds to one of the membrane-bound mammalian C-type lectins (e.g., DC-SIGN/L-SIGN, MR, CLEC5A, or LSECtin) or one of the secreted mosquito C-type lectins (e.g., mosGCTL-7) that is subsequently captured by its cell-surface receptor. (c) A tripeptide RGD motif in E-DIII binds to one of the RGD-binding integrins, such as avb3. Since these interactions are rather nonspecific, they are likely to play a role in recruiting the virions on the cell surface to promote a specific interaction of the viral E glycoprotein with its asyet unidentified bona fide receptor(s) that directs the virions to a receptor-mediated clathrin-dependent/independent endocytic pathway for internalization. The cellular proteins implicated in JEV entry to date include HSP70, HSP90, the 37/67-kDa laminin receptor, CD4, CD14, vimentin, and the low-density lipoprotein receptor; however, their precise roles remain to be determined.

590

Japanese Encephalitis Virus (Flaviviridae)

Fig. 4 Life cycle of JEV. Shown at the bottom are confocal microscopic images of BHK-21 cells uninfected or infected with JEV strain CNU/LP2 for 20 h, then immunostained with a primary rabbit anti-JEV NS4B antibody and a secondary Cy5-conjugated goat anti-rabbit IgG. Nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). Illustrated above the immunofluorescent images are three phases of the JEV life cycle: (A) Entry phase, which includes the steps of viral attachment, endocytosis, membrane fusion, and uncoating at the early stage of viral replication. A rectangle-shaped inset depicts the low pH-induced structural reorganization of the viral envelope complex E:M:M:E heterotetramers occurring in an endosomal compartment. (B) Translation and RNA replication phase, which includes viral polyprotein synthesis and processing, as well as RNA synthesis and capping occurring in virus-induced specialized membranous compartments around the nucleus. An oval-shaped inset outlines the replication of the viral genomic RNA occurring in a semiconservative and asymmetric manner. (C) Morphogenesis phase, which includes the steps of viral assembly, maturation, and release at the late stage of viral replication. A rectangle-shaped inset depicts the low pH-triggered structural rearrangement of the icosahedrally arranged spikes (3  prM:E) on the surface of immature virions occurring during transit through the secretory pathway from the ER to the plasma membrane. The schematic diagram is modified from a review article (Yun, S.I., Lee, Y.M., 2018. Early events in Japanese encephalitis virus infection: Viral entry. Pathogens 7, 68).

Extensive structural and biochemical studies of other flaviviruses (DENV, WNV, and TBEV) have demonstrated that once the internalized virions reach an endosomal compartment, an acidic pH induces irreversible structural reorganization of the viral envelope complex E:M:M:E heterotetramers (Fig. 4(A), rectangle-shaped inset). This reorganization initially involves dissociating the metastable antiparallel E:E homodimers (the effect on the two adjacent M proteins is unknown), exposing their fusion loops to insert them into the inner leaflet of the endosomal membrane, and then reassociating the E monomers into stable parallel E:E:E homotrimers. This E trimerization requires the “folding back” of DIII against DII and the “zipping up” of the stem extended from DIII along the side of DII and toward the fusion loop embedded halfway into the endosomal membrane, thus driving the membrane fusion. This fusion process precedes a poorly understood uncoating step that may be mediated by one or more cytoskeletal structures (e.g., microtubules), resulting in the release of the viral nucleocapsid/genome into the cytoplasm.

Viral Translation and RNA Replication After being released into the cytoplasm, the viral genomic RNA encoding a single long ORF is translated on ER-bound ribosomes into two polyprotein precursors, ppC-NS5 and ppC-NS10 (Fig. 4(B)). These precursors correspond to the full-length product and its C-terminally truncated form resulting from a  1 frameshift event occuring during the translational elongation step at codons 8

Japanese Encephalitis Virus (Flaviviridae)

591

and 9 of NS2A, respectively (Fig. 2(B)). During and after translation, the two precursors are proteolytically processed by a combination of the viral serine protease (NS2B þ NS3) and three host cell proteases (signal peptidase, furin, and an unknown protease) to produce three structural (C, prM/M, and E) and at least seven nonstructural (NS1/10 , NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. Based on recent electron tomography studies of several flaviviruses (DENV, WNV, ZIKV, and TBEV), a subset of the viral nonstructural proteins, particularly NS4A and NS4B, have been shown to be able to induce the proliferation and invagination of the ER to create clusters of B90-nm-wide pouch-like vesicles (known as “vesicle packets”). Each vesicle often appears to have a pore connecting its interior with the cytosol (Fig. 4(B)). These vesicles can be seen in close proximity to highly folded membranes known as “convoluted membranes” or “paracrystalline arrays.” Inside these vesicles, most, if not all, of the viral nonstructural proteins, together with as-yet poorly defined host factors, are assembled on the membrane into a replication complex. Within the complex, viral genomic RNA is replicated in a semiconservative and asymmetric manner by dynamic coordination of the two most conserved multienzymatic nonstructural proteins, NS3 and NS5 (Fig. 4(B), oval-shaped inset). Initially, the linear positive-sense genomic RNA is converted to a circular form through an intramolecular long-distance RNA–RNA interaction between the 50 - and 30 -terminal regions. The cyclized RNA serves as a template for the synthesis of its complementary negativesense RNA, with the NS5 polymerase recognizing its promoter element (known as the stem-loop A) at the 50 end, and in turn, initiating negative-sense RNA synthesis at the 30 end. The nascent negative-sense RNA remains base-paired with the positive-sense RNA template, forming a double-stranded RNA molecule (called a “replicative form”). Subsequently, the negative-sense RNA serves as a template for the synthesis of multiple copies of new positive-sense RNAs (B10-fold more than negative-sense RNAs), each continuously displacing a preceding positive-sense RNA and thereby forming a partially single-stranded and partially doublestranded RNA molecule (called a “replicative intermediate”). The synthesis of both negative- and positive-sense RNAs is mediated by NS5 polymerase activity, coupled with the NS3 helicase and NTPase activities that are apparently required during the synthesis of positive-sense RNA. During or shortly thereafter, the nascent positive-sense RNA undergoes RNA capping, which involves removal of the g-phosphate from the 50 end to generate its 50 -diphosphorylated form through NS3 RTPase activity and subsequent transfer of a GMP group from GTP to the 50 end of the 50 -diphosphorylated RNA in a 50  50 triphosphate linkage via NS5 GTase activity. The 50 capped RNA then goes through cap methylation, involving the initial transfer of a methyl group from SAM to the terminal nucleobase guanine at the N-7 position and then to the first nucleotide adenosine at the ribose 20 -O position by the NS5 MTase, thereby creating a type 1 cap at the 50 -end of the viral genomic RNA. In addition to the three major RNA species (the single-stranded positive-sense genome, the double-stranded replicative form, and the partially single-stranded and partially double-stranded replicative intermediate), a set of subgenomic flavivirus RNAs (sfRNAs) of B0.2–0.5 kb is produced in cells infected with JEV or another flavivirus. These sfRNAs correspond to the 30 -terminal region of the viral genomic RNA, produced by the cellular 50 -30 exoribonuclease XRN1, which stops at a higher-order RNA structure within the 30 NCR. Primarily on the basis of previous work with other flaviviruses (e.g., WNV), it has been suggested that the accumulation of sfRNAs in infected cells alters the homeostasis of cellular mRNA concentrations to promote viral replication and pathogenesis.

Viral Morphogenesis Following viral translation and RNA replication, the assembly process is believed to begin in the vicinity of vesicle packets through the binding of viral C proteins to the newly made genomic RNA. This binding is followed by budding of the C protein-genomic RNA complex into the lumen of the ER, where the viral surface glycoproteins prM and E are incorporated (Fig. 4(C)). The budding process appears to be driven by oligomerization of the prM and E glycoproteins, which can lead to budding of subviral particles in the absence of the C protein-genomic RNA complex (nucleocapsid). Each of the nascent immature virions accumulated at the neutral pH of the ER lumen has an outer protein shell containing 60 icosahedrally arranged spikes; each spike comprises three prM:E heterodimers, with the three E proteins protruding from the viral membrane (Fig. 4(C), rectangle-shaped inset). As a notable feature of immature particles, the fusion loop at the apical tip of E-DII is protected from prematurely fusing with the ER membrane because it is covered with the globular pr portion of prM, rendering the particle noninfectious. These immature virions then undergo the maturation process during their transit through the secretory pathway to the plasma membrane, from which the mature virions are released. Comprehensive structural and biochemical studies of other flaviviruses (DENV, WNV, and TBEV) have established that in the TGN, an acidic pH triggers an extensive structural rearrangement in the outer protein shell (Fig. 4(C), rectangle-shaped inset), converting the 60 protruding spikes into 60 asymmetric units that are organized into 30 diamond-shaped tiles each composed of three E:prM:prM:E heterotetramers (forming a total of 90 heterotetramers per virion). This structural change exposes the hidden cleavage site on prM for furin, a TGN-resident protease, and the furin-mediated cleavage of prM yields a soluble pr fragment and a membrane-anchored M protein. The resulting two pr fragments remain associated with the E:M:M:E heterotetramer at the acidic pH of the TGN, but they become dissociated from the mature virions when released into the extracellular space, which has a neutral pH. Both the fully and partially mature virions, together with the subviral particles and the NS1 hexamers, are exocytosed from virus-infected cells. A significant number of host factors are likely to be involved in viral morphogenesis, but at present they remain largely elusive.

592

Japanese Encephalitis Virus (Flaviviridae)

Epidemiology Geographic Distribution and Epidemiologic Patterns JEV is the causative agent of Japanese encephalitis (JE), an acute encephalitis syndrome that is prevalent in the Asia-Pacific region, including South, East, and Southeast Asia, as well as the northern part of Oceania, affecting more than half of the world’s population (Fig. 5). Suspected JE outbreaks were recognized in Japan as far back as the early 1870s. In 1924, the disease was initially named Japanese B encephalitis to distinguish it from encephalitis lethargica, known as Japanese A encephalitis. In 1934, a filterable agent from the brain of a deceased patient was used to demonstrate experimentally that it causes the encephalitis in nonhuman primates, and the virus (strain Nakayama) was then isolated in 1935. For over half a century, JEV was mainly confined within originally defined boundaries in Asia. In the early 1990s, however, its geographic range expanded southward into the Torres Strait islands and the Cape York Peninsula in the northern Australia, westward into Pakistan, and eastward into Saipan in the western Pacific Ocean. Surprisingly, JEV RNA/proteins were detected in Italy from tissue samples of both healthy and dead birds in 1997–2000, as well as from a pool of Culex pipiens mosquitoes in 2010. More startlingly, a resident in Angola was found to be locally coinfected with YFV and JEV in 2016. These pieces of recent evidence therefore indicate that JEV is circulating not only widely in the Asia–Pacific region but also possibly locally in Europe and Africa, with the potential for further spread. JEV shows two epidemiological patterns, largely depending on regional climate (Fig. 5): (a) an endemic pattern, observed in the tropics, where viral transmission takes place throughout the year but increases during the rainy season; and (b) an epidemic pattern in the subtropical and temperate zones, in which viral transmission tends to occur seasonally during the summer and early fall. Although only 0.1%–4.0% of people infected with JEV are symptomatic, 50,000–175,000 clinical JE cases are estimated to

Fig. 5 Geographic distribution and epidemiologic patterns of JEV. Marked on the map are the countries or areas (mosquito) where Cx. tritaeniorhynchus has been reported, the countries or areas (orange) where JE has been reported in humans, and the areas (khaki) where JEV has been detected in birds and mosquitoes with no clinical JE case reported. Also indicated across the map are two major epidemiological patterns of JE: endemic (light red belt) and epidemic (light blue belt). The map is created based on the data from: (1) Jeffries, C.L., Walker, T., 2015. The potential use of Wolbachia-based mosquito biocontrol strategies for Japanese encephalitis. PLoS Neglected Tropical Diseases 9, e0003576. (2) Platonov, A., Rossi, G., Karan, L., et al., 2012. Does the Japanese encephalitis virus (JEV) represent a threat for human health in Europe? Detection of JEV RNA sequences in birds collected in Italy. Eurosurveillance 17, 20241. (3) Preziuso, S., Mari, S., Mariotti, F., Rossi, G., 2018. Detection of Japanese encephalitis virus in bone marrow of healthy young wild birds collected in 1997–2000 in Central Italy. Zoonoses and Public Health 65, 798–804. (4) Ravanini, P., Huhtamo, E., Ilaria, V., et al., 2012. Japanese encephalitis virus RNA detected in Culex pipiens mosquitoes in Italy. Eurosurveillance 17, 20221. (5) Simon-Loriere, E., Faye, O., Prot, M., et al., 2017. Autochthonous Japanese encephalitis with yellow fever coinfection in Africa. New England Journal of Medicine 376, 1483–1485. (6) World Health Organization, Japanese encephalitis. Available from: https://www.who.int/news-room/fact-sheets/detail/japanese-encephalitis.

Japanese Encephalitis Virus (Flaviviridae)

593

occur annually worldwide. The fatality rate can reach up to B30% of JE cases, with a wide range of long-term neuropsychiatric sequelae developing in up to B50% of JE survivors.

Genetic Diversity All JEV isolates can be classified into one of the five genotypes (GI‒GV), based on the complete nucleotide sequence of the viral genome; the sequence divergence is estimated to be as high as B20% at the nucleotide level and B10% at the amino acid level when their ORFs are compared. Of the five genotypes, GIII had been the major genotype detected from its first isolation in 1935 until the 1990s. In 2003, the first comprehensive phylogenetic study reported that the prevalence of the five genotypes varied by geographic locations in the Asia–Pacific area: (a) all five genotypes were found in the Indonesia–Malaysia region, (b) GI, GII, and GIII in the Thailand–Cambodia–Vietnam region, (c) GI and GIII in the Japan–Korea–China region, (d) GII and GIII in the Taiwan–Philippines region, (e) GIII in the India–Sri Lanka–Nepal region, and (f) GI and GII in the Australia–New Guinea region. It should be noted that since the early 2000s (or presumably even earlier), GIII has been gradually replaced by GI, which is now the dominant genotype, occasionally co-circulating with GIII in many Asian countries, including Thailand, Cambodia, Vietnam, Japan, Korea, China, and India. In the late 2000s and early 2010s, GV also emerged for the first time outside of the Indonesia–Malaysia region, in Korea and China. With respect to the origin of JEV, it has been proposed that the virus initially evolved from its ancestral virus into the five genotypes in the Indonesia-Malaysia region, where they either circulate in that region or spread across the Asia–Pacific area. A closer look at the successful emergence and establishment of the currently dominant GI has suggested that it originated from the Indonesia–Malaysia region, diverged in the Thailand–Cambodia–Vietnam region, and then dispersed northward to southwestern China, from which it subsequently spread to other JE-affected countries (e.g., Japan, Korea, and Taiwan). Although JEV presents only a single serotype, a considerable degree of antigenic variation has been noted not only between two different genotypes but also among strains of the same genotype. In line with this situation, relatively low levels of cross-neutralizing antibody response against heterologous genotypes (e.g., GI and GV) have been observed among people who previously received a GIII-based killed or live JE vaccine. Also, GI strains have been isolated from JE patients who had already been immunized with a GIII-based live JE vaccine. Thus, the genetic and antigenic variation of JEV may have a negative impact on the development of broadly effective JE vaccines.

Transmission Vector-Borne Transmission JEV is an arthropod-borne virus (arbovirus) transmitted by a hematophagous mosquito vector between animal hosts (Fig. 6). The most competent mosquito vectors are Culex species, of which Cx. tritaeniorhynchus is primarily responsible for the recurrence and expansion of JEV in Asia, and Cx. annulirostris mainly accounts for the introduction and spread of JEV in northern Australia. Notably, the primary vector Cx. tritaeniorhynchus is widely distributed not only in Asia, where JEV has been circulating regularly, but also in Europe and Africa, where JEV may have been introduced recently (Fig. 5). Other Culex species implicated in local JEV transmission include Cx. annulus, Cx. bitaeniorhynchus, Cx. fuscocephala, Cx. gelidus, Cx. orientalis, Cx. pipiens, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. sitiens, Cx. tarsalis, and Cx. vishnui, from each of which the virus has been detected. JEV infection has also been documented in a small number of field-caught or laboratory-inoculated non-Culex species, such as four Aedes species (Ae. albopictus, Ae. detritus, Ae. japonicus, and Ae. vexans), three Anopheles species (An. minimus, An. sinensis, and An. tessellatus), one Armigeres species (Ar. subalbatus), and one Mansonia species (Ma. uniformis). However, these JEV-susceptible Culex and non-Culex mosquitoes are not all equally competent for JEV transmission, and thus their competency needs to be examined. JEV is maintained in an enzootic cycle involving several vertebrate hosts (Fig. 6), of which suids (e.g., domestic pigs) and ardeid birds (e.g., herons and egrets) are known as the main virus-amplifying hosts, since they generally develop asymptomatic infection but produce a relatively prolonged high-titer viremia sufficient to infect engorging mosquitoes. In particular, domestic pigs play a major role in transmitting the virus to humans in many Asian countries, where pig farms are usually located near human residences; thus they can serve as a sentinel animal. In the mosquito-vertebrate-mosquito cycle of JEV, not only migratory birds but also hibernating/migrating bats are considered as reservoir hosts that can contribute to the overwintering and long-range dispersal of the virus. Another possible overwintering mechanism for JEV is transovarial transmission, passing the virus directly from a female mosquito to its eggs. On the other hand, humans and equids (e.g., horses and donkeys) are recognized as incidental deadend hosts, since they typically produce a level of viremia insufficient for further transmission but develop clinical illness. Limited data from serosurveys, along with experimental infection studies in some cases, have suggested that JEV can cause subclinical infection in many other vertebrates (e.g., cows, goats, sheep, chickens, ducks, dogs, cats, rabbits, buffaloes, raccoons, and raccoon dogs), as well as in some reptiles and amphibians; however, their roles in JEV transmission remain undefined.

Non-Vector-Borne Transmission Although JEV is a mosquito-borne pathogen, it can be transmitted in the absence of a mosquito vector via several non-vector-borne routes: (a) direct contact transmission, which has been demonstrated experimentally in pigs and mice; (b) aerosol transmission,

594

Japanese Encephalitis Virus (Flaviviridae)

Fig. 6 Vector-borne transmission cycle of JEV. In nature, the virus is horizontally maintained primarily by culicine mosquito vectors between vertebrate animal hosts, with domestic pigs and ardeid birds serving as the most important virus-amplifying hosts/reservoirs. Typically, symptomatic infections are observed in humans, horses, and pregnant sows (fetuses). Also, an infected female mosquito is able to vertically transmit the virus to her eggs. The schematic drawing is modified from a review article (Yun, S.I., Lee, Y.M., 2014. Japanese encephalitis: The virus and vaccines. Human Vaccines & Immunotherapeutics 10, 263–279).

which has been shown in several laboratory animals, including rodents and squirrel monkeys; (c) transplacental transmission, which is known to occur commonly in pregnant sows but rarely in pregnant women and is reproduced experimentally in pregnant mice; (d) blood-borne transmission, which has been reported to occur in humans during blood transfusion and organ transplantation; and (e) artificial insemination, which is thought to be a potential mode of transmission from infected boars to breeding sows. Further studies are needed to address the impact of these instances of vector-free transmission on JEV ecology and epidemiology.

Clinical Features JEV is a zoonotic pathogen that can cause a spectrum of neurological diseases in both people and livestock, such as horses, and reproductive problems in pigs (Fig. 6). In humans, the clinical signs after JEV infection typically appear within a week, but the incubation period may be as long as 2 weeks. While most infections are asymptomatic, some present a range of clinical manifestations, from mild febrile illnesses that may remain undiagnosed to severe neuroinvasive diseases that are often fatal, depending on the composite interaction between viral virulence factors and host risk factors (e.g., age and genetic makeup). In particular, the potentially life-threatening neurological disease caused by JEV infection is JE, acute encephalitis that is characterized by extensive inflammation in the central nervous system (CNS). The encephalitis begins with prodromal flu-like symptoms, including fever, anorexia, nausea, vomiting, malaise, myalgia, headache, and back pain. During this period, liver dysfunction, gastric hemorrhage, and thrombocytopenia have also been observed in some patients. The prodromal phase may progress to an acute encephalitic phase, marked by decreased alertness and various neurological signs such as a mask-like expression, muscle rigidity, tremulous eye movements, coarse extremity tremors, abnormal involuntary movements, and pathological reflexes. Also, a polio-like acute flaccid paralysis has been described in JE patients. Patients with more severe and profound neurological signs during the encephalitic phase are more likely to experience long-term sequelae, such as persistent seizures, motor neuron weakness, cerebellar signs, extrapyramidal disorders, arm flexion deformities, leg hyperextensions, cognitive deficits, language impairment, learning disabilities, and behavioral problems. Many animals are believed to be susceptible to JEV infection, but horses and pigs are known to show overt clinical signs of JEV infection. Like humans, horses may develop a variety of clinical conditions ranging from self-limiting illnesses with nonspecific signs (e.g., fever, lethargy, anorexia, and congested mucous membranes) to mild or severe forms of encephalitis with neurological signs (e.g., dysphagia, ataxia, wandering, neck stiffness, radial paralysis, vision impairment, aggressive behavior, and tremors). Occasionally, neurological deficits can persist for a long period of time after recovery. Although uncommon, encephalitis is also reported to occur in cows and can eventually lead to death. In pigs, JEV infection during pregnancy commonly leads to reproductive losses, such as abortion, stillbirth, and mummification. Even though infected piglets are born alive, they often display neurological signs (e.g., tremor and convulsion), die soon after birth, and present with neuropathological lesions (e.g., hydrocephalus, cerebellar hypoplasia, and spinal hypomyelinogenesis). However, nonpregnant pigs generally show no symptoms or only a low-grade fever but occasionally develop neurological conditions, especially at a young age. In boars, JEV infection can disturb spermatogenesis, leading to a decrease in sperm count and motility.

Japanese Encephalitis Virus (Flaviviridae)

595

Pathogenesis JEV is a neurotropic virus that affects the CNS, highlighted by neuronal cell death. For all neurotropic viruses, virulence is determined by a set of viral factors that interact with their host targets during infection and define two pathogenic properties: (a) neuroinvasiveness, the ability of the virus to penetrate the CNS from peripheral sites where the virus is inoculated by a mosquito bite; and (b) neurovirulence, the ability of the virus to establish a lethal infection within the CNS. Over the years, significant progress has been achieved in determining the structure and function of the viral proteins that are critical for the various stages of JEV replication, but limited progress has been made in identifying the viral factors required for JEV virulence and the sequence of events involved in JEV pathogenesis. Based on our current understanding of viral pathogenesis, JEV may replicate initially in leukocytes (e.g., lymphocytes, monocytes, and/ or macrophages/dendritic cells) in peripheral lymphoid organs (e.g., lymph nodes and spleens) prior to penetration of the CNS. JEV can invade the CNS by crossing the blood-brain barrier (BBB) as cell-free or cell-associated virions or by directly infecting vascular endothelial cells. The replication of JEV in endothelial cells can compromise the integrity of the BBB. Within the CNS, JEV infects not only neurons but also non-neuronal cells, such as microglia and astrocytes. The viral replication in neurons directly causes cell death, which then activates the adjacent non-neuronal cells to produce a variety of inflammatory mediators (e.g., IL-1, IL-6, IL-8, TNF-a, MCP-1, IP-10, and RANTES) capable of promoting leukocyte migration and infiltration into the brain. A massive inflammatory response causes an overproduction of reactive oxygen/nitrogen species and cytokines (e.g., TNF-a), which then triggers bystander cell death in uninfected neurons. Thus far, a multitude of viral genetic factors, including proteins and RNA elements, are shown to contribute to JEV virulence, but the viral surface glycoprotein E is believed to be the key factor that determines viral virulence, including both neuroinvasiveness and neurovirulence. Further research is required to define the viral factors required for JEV virulence, their functions, and the necessary virus-host interactions.

Diagnosis Clinical Diagnosis JE patients may have some abnormalities in (a) complete blood count, showing an elevated leukocyte count (10  35  109 cells L1) with a high percentage of neutrophils; (b) cerebrospinal fluid (CSF) analysis, revealing an elevated opening pressure (4250 mm), moderate pleocytosis (10–100 cells mm3), and slightly increased protein level (50–200 mg dL1); and (c) electroencephalogram, displaying diffuse theta and delta waves, a burst suppression pattern, epileptiform activity, and alpha coma. The early clinical signs of CNS involvement include abnormal oculocephalic reflex, acute onset hemiparesis with hypertonia, and decorticate and decerebrate posturing. Molecular imaging techniques such as X-ray computed tomography (CT), magnetic resonance imaging (MRI), and singlephoton emission computed tomography (SPECT) are applicable to the diagnosis of JE. In JE patients, CT and MRI scans show brain lesions most often in the thalami, and less often in the basal ganglia, hippocampus, substantia nigra, cerebral cortex, cerebellum, brain stem, and white matter. MRI is generally more sensitive than CT at detecting brain lesions, particularly in the early phase of JE. SPECT scans can detect a significant level of hyperperfusion in the thalamus and putamen in the acute encephalitic stage, which can be extended to the frontal lobes at an advanced stage. Overall, these clinical diagnostics and neuroimagings are informative but nonspecific, so the etiological agent needs to be identified by laboratory diagnosis.

Laboratory Diagnosis JEV infection is diagnosed by the detection of infectious virions, viral antigens/RNAs, and viral antibodies: (a) Infectious JEV can be isolated from clinical specimens (e.g., blood, CSF, and tissues) by inoculating them in a culture of susceptible primary cells or continuous cell lines, or more efficiently by injecting them into the brains of suckling mice. However, JEV isolation from clinical samples is generally challenging because of the low level of circulating virions, the rapid development of neutralizing antibodies, and the rapid clearance of transient viremia. (b) JEV-specific antigens can be identified using JEV-specific antibodies and a variety of immunological methods based on hemagglutination, coagglutination, immunofluorescence, or antigen-capture immunoassay. JEV RNAs can be amplified using JEV-specific primers and a range of molecular techniques, including conventional reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and reverse transcription loop-mediated isothermal amplification (RT-LAMP). (c) JEV-specific IgMs can be detected in the blood and CSF of JE patients by an IgM antibodycapture enzyme-linked immunosorbent assay (MAC-ELISA) or its derivatives, including a biotin-labeled antigen sandwich ELISA, a nitrocellulose membrane-based IgM-capture dot enzyme immunoassay, and an antibody-capture solid-phase radioimmunoassay. Other traditional serological tests such as hemagglutination inhibition, complement fixation, and neutralization can also be applied to the detection of JEV-specific IgM. It should be noted, however, that all the serology-based data for the detection of both JEV antigens and antibodies need to be interpreted with caution because there is some cross-reactivity with other flaviviruses.

Treatment There is no clinically approved drug or therapy available for the treatment of JEV infection. Supportive care is the only current option to relieve the symptoms caused by JEV infection.

596

Japanese Encephalitis Virus (Flaviviridae)

Prevention JEV can be prevented by reducing the population of mosquito vectors (e.g., using insecticides), protecting people from mosquito bites (e.g., applying mosquito nets and repellents), and immunizing susceptible individuals with a vaccine. Of these measures, vaccination is the only one available for sustainable long-term protection against JEV. Thus far, four types of JE vaccines have been commercialized in various countries for humans: (a) The mouse brain-derived killedinactivated vaccine JE-VAX was developed using the Japanese Nakayama strain, isolated from the brain of a patient in 1935. First licensed in Japan in 1954, JE-VAX was widely used internationally until the late 2000s, when the new cell culturederived vaccines became available (see below). In Japan, a similar mouse brain-derived killed vaccine was also produced using the Chinese Beijing-1 (P1) strain, recovered from a patient’s brain tissue in 1949 and distributed domestically from 1989 to the mid-2000s. (b) The cell culture-derived live-attenuated vaccine SA14-14-2 was produced in primary hamster kidney (PHK) cells using the SA 14-14-2 strain, an attenuated derivative of the virulent parental SA14 strain isolated from a collection of Cx. pipiens larvae in China in 1954. Since its initial licensure in China in 1988, SA14-14-2 has become the most widely used JE vaccine in many Asian countries, including Cambodia, India, Laos, Myanmar, Nepal, South Korea, Sri Lanka, and Thailand. (c) The first cell culture-derived killed-inactivated vaccine was generated in PHK cells using the Chinese Beijing-3 (P3) strain, isolated during a JE outbreak in 1949. In China, this vaccine was licensed in 1968; it was then adapted by production in African green monkey kidney (Vero) cells, a method that was licensed in 1998 but has more recently been replaced by the PHK cell-produced live SA14-14-2 vaccine. Also, two other Vero cell-produced killed vaccines were commercialized: one produced with the Beijing-1 strain and marketed in Japan as JEBIK V or ENCEVAC in 2009–2011; and the other produced with the SA 14-14-2 strain and marketed since 2009 in many countries, including Australia, Canada, Hong Kong, India, Switzerland, the US, and Europe, under one of four trade names (IC51, IXIARO, JESPECT, and JEEV). (d) The cell culture-derived live chimeric vaccine ChimeriVax-JE (also known as IMOJEV, JE-CV, and THAIJEV) was created by genetically engineering the live-attenuated YFV 17D vaccine as a vector, replacing its prM-E coding region with the corresponding region of JEV SA14-14-2. The Vero cell-produced ChimeriVax-JE vaccine is currently available in Australia and Thailand. In addition to humans, pigs and horses are also vaccinated in some countries to prevent JEV-related disease. Given the fact that all of the discontinued and currently available JE vaccines are based on only one of four GIII strains (Nakayama, Beijing-1, Beijing-3, and SA14-14-2), the genetic and antigenic variations among all five genotypes should be taken into consideration when developing next-generation JE vaccines, to provide broad protection against all the JEV strains belonging to different genotypes.

Further Reading Brinton, M.A., Basu, M., 2015. Functions of the 30 and 50 genome RNA regions of members of the genus Flavivirus. Virus Research 206, 108–119. Chao, L.H., Klein, D.E., Schmidt, A.G., Pena, J.M., Harrison, S.C., 2014. Sequential conformational rearrangements in flavivirus membrane fusion. eLife 3, e04389. Chen, S., Wu, Z., Wang, M., Cheng, A., 2017. Innate immune evasion mediated by Flaviviridae non-structural proteins. Viruses 9, e291. Dong, H., Fink, K., Zust, R., et al., 2014. Flavivirus RNA methylation. Journal of General Virology 95, 763–778. Hasan, S.S., Sevvana, M., Kuhn, R.J., Rossmann, M.G., 2018. Structural biology of Zika virus and other flaviviruses. Nature Structural & Molecular Biology 25, 13–20. Hegde, N.R., Gore, M.M., 2017. Japanese encephalitis vaccines: Immunogenicity, protective efficacy, effectiveness, and impact on the burden of disease. Human Vaccines & Immunotherapeutics 13, 1–18. Klema, V.J., Padmanabhan, R., Choi, K.H., 2015. Flaviviral replication complex: Coordination between RNA synthesis and 50 -RNA capping. Viruses 7, 4640–4656. Lindenbach, B.D., Murray, C.L., Thiel, H.-J., Rice, C.M., 2013. Flaviviridae. In: Knipe, D.M., Howley, P.M., Cohen, J.I., et al. (Eds.), Fields Virology. Philadelphia, PA: Wolters Kluwer Health, pp. 712–746. Luo, D., Vasudevan, S.G., Lescar, J., 2015. The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development. Antiviral Research 118, 148–158. Mackenzie, J.S., Barrett, A.D., Deubel, V., 2002. The Japanese encephalitis serological group of flaviviruses: A brief introduction to the group. Current Topics in Microbiology and Immunology 267, 1–10. Neufeldt, C.J., Cortese, M., Acosta, E.G., Bartenschlager, R., 2018. Rewiring cellular networks by members of the Flaviviridae family. Nature Reviews Microbiology 16, 125–142. Pijlman, G.P., Funk, A., Kondratieva, N., et al., 2008. A highly structured, nuclease-resistant, noncoding RNA produced by flaviviruses is required for pathogenicity. Cell Host & Microbe 4, 579–591. Turtle, L., Solomon, T., 2018. Japanese encephalitis – The prospects for new treatments. Nature Reviews Neurology 14, 298–313. Villordo, S.M., Carballeda, J.M., Filomatori, C.V., Gamarnik, A.V., 2016. RNA structure duplications and flavivirus host adaptation. Trends in Microbiology 24, 270–283. Wang, X., Li, S.H., Zhu, L., et al., 2017. Near-atomic structure of Japanese encephalitis virus reveals critical determinants of virulence and stability. Nature Communications 8, 14.

Relevant Websites https://www.cdc.gov/japaneseencephalitis/ Centers for Disease Control and Prevention. https://talk.ictvonline.org/ictv-reports/ictv_9th_report/positive-sense-rna-viruses-2011/w/posrna_viruses/257/flaviviridae International Committee on Taxonomy of Viruses.

Japanese Encephalitis Virus (Flaviviridae)

https://www.aphis.usda.gov/animal_health/emergency_management/downloads/disease_strategy_jev.pdf United States Department of Agriculture. https://www.who.int/news-room/fact-sheets/detail/japanese-encephalitis World Health Organization. http://www.oie.int/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/Disease_cards/JAPANESE_ENCEPHALITIS.pdf World Organization for Animal Health.

597

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae) Anne K Cordes, Hannover Medical School, Institute of Virology, Hannover, Germany Thomas F Schulz, Hannover Medical School, Institute of Virology, Hannover, Germany and German Center for Infection Research, Hannover-Braunschweig Site, Braunschweig, Germany r 2021 Elsevier Ltd. All rights reserved. This is an update of T. Toptan, Y. Chang, P.S. Moore, Kaposi’s Sarcoma-Associated Herpesvirus: General Features, Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B978-0-12-801238-3.02612-X and E. Gellermann, T.F. Schulz, Kaposi’s Sarcoma-Associated Herpesvirus: Molecular Biology, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00686-5.

Nomenclature ALR AIM2-like receptor ATP Adenosine triphosphate AZT Azidothymidine Bub1 Budding uninhibited by benzimidazoles 1 CDK-6 Cyclin-dependent kinase 6 CENP-F Centromeric protein F cGAS Cyclic GMP-AMP Synthase CIB1 Calcium and integrin-binding protein-1 cKS Classical Kaposi’s Sarcoma DBD DNA-binding domain DC-SIGN Dendritic cell-specific ICAM-grabbing nonintegrin DNA deoxyribonucleic acid ELISA Enzyme linked immunosorbent assay EphA2R Ephrin receptor A2R ESCRT Endosomal sorting complexes required for transport FAK Focal adhesion kinase HAART Highly-active antiretroviral therapy HHV-8 Human herpesvirus-8 HIV Human immunodeficiency virus IFIT Interferon induced proteins with tetratricopeptide repeats IFN Interferon IKK IκB kinase IL Interleukin IRF3 Interferon regulatory factor 3 K-bZIP Basic domain-leucine zipper KCP Complement control protein kDa Kilodalton KICS KSHV inflammatory cytokine syndrome KS Kaposi’s sarcoma KSHV Kaposi’s sarcoma herpesvirus LANA Latency-associated nuclear antigen LBS LANA binding site LUR Long unique coding region MCD Multicentric Castleman’s Disease MCM Minichromosome maintenance MeCP2 Methyl CpG binding protein 2 MHC Major histocompatibility complex MIR Modulator of immune recognition miRNA Micro RNA mRNA Messenger RNA

598

MSM Men who have sex with men NEMO NF-κB essential modifier NF-κB Nuclear factor kappa-light-chain-enhancer of activated B-cells NLR Nucleotide-binding oligomerization domain-like receptors NLS Nuclear localization signal NPM Nucleophosmin NuMA Nuclear mitotic apparatus protein Ori Origin of replication PAMP Pathogen-associated molecular patterns PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction PEL Primary effusion lymphoma PI3-K Phosphoinositide 3-kinase PML Promyelocytic leukemia POD Promyelocytic leukemia (PML) oncogenic domains PPR Pathogen recognition receptors pRb Retinoblastoma protein Pre-RC Pre-replication complex RFC Replication factor C Rho Ras homolog RLR retinoic acid-inducible gene-I-like receptors RNA Ribonucleic acid RTA Replication and transcription activator RV Rhadinovirus SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis STING Stimulator of interferon genes SUMO2 Small ubiquitin-related modifier 2 TBK1 TANK-binding kinase 1 TLR Toll-like-receptor TR Terminal repeat vbcl2 viral B cell lymphoma homolog vCyc Viral cyclin VEGFA Vascular endothelial growth factor A vFLIP Viral Fas-associated protein with death domain-like interleukin-1b-converting enzyme-inhibitory protein vGCR Viral G protein-coupled receptor vIAP Viral inhibitor of apoptosis protein vIRF Viral interferon regulatory factor vORF Viral open reading frame VR Variable region

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00017-5

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

Glossary Concatamer A long DNA molecule consisting of multiple identical subunits arranged in a head-to-tail linear conformation. Episome A circular viral DNA. Kaposi’s Sarcoma An endothelial cell-derived tumor caused by KSHV. Kaposi’s Sarcoma-associated herpesvirus (KSHV/HHV-8) An oncogenic herpesvirus and the causative agent of Kaposi’s Sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. Latency The phase of the herpesvirus life cycle in which the virus does not replicate in a productive manner but persists in the host cell.

599

Multicentric Castleman’s Disease (MCD) A group of rare lymphoproliferative disorders (Non-Hodgkin’s lymphoma). The plasma cell variant of MCD is associated with KSHV. Primary effusion lymphoma A B cell lymphoma (NonHodgkin’s lymphoma), caused by KSHV that is characterized by a plasma cell differentiation profile and immunoglobulin mutations, indicating its origin in a postgerminal center B cell. Tegument The space between virus capsid and envelope containing viral proteins.

Classification Kaposi’s sarcoma herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) is a Rhadinovirus and the only member of this genus that can infect humans. Other members of the two rhadinovirus lineages (RV1 and 2) infect New and Old World primates. Rhadinoviruses (g2-herpesviruses) belong to the subfamily Gammaherpesviridae in the family Herpesviridae. KSHV DNA was first discovered in Kaposi’s sarcoma (KS) biopsies from AIDS patients suffering from KS in 1994. It is an oncogenic virus and Kaposi’s sarcoma (KS) is one of the three known malignant diseases that can be caused by this virus. In addition to KS, these include two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman’s Disease (MCD). In 2016, KSHV infection was also linked to the clinical syndrome with sepsis-like features and involving the overexpression of cytokines, which was named KSHV inflammatory cytokine syndrome (KICS). Based on the sequence of the hypervariable regions (VR1 and VR2) of the K1 gene, seven different KSHV subtypes (A – F and Z) are known today. In addition, three variants (P, M, N) of the K15 gene at the other end of the viral genome occur in several KSHV subtypes and variants M and N are thought to be the result of recombination events with related rhadinoviruses. Some KSHV subtypes are typically found in certain geographic regions (e.g., subtype B in Africa, subtype D in Asia, subtype E in indigenous American populations).

Virion Structure KSHV has an enveloped icosahedral capsid with 162 capsomers and a total diameter of about 150–200 nm. The capsid is surrounded by the tegument and the virus envelope which is studded with multiple glycoproteins (gB, gH, gL, gM, gN, ORF4 and gpK8.1A) that play a crucial role in virus binding and entry into the host cell.

Genome The KSHV genome is made up of a double stranded DNA of 165–170 kb that consists of a long unique coding region (LUR) of 140 kb, which is flanked by multiple GC-rich terminal repeat subunits of 801 bp, resulting in a total length of the terminal repeat region of approx. 20–35 kb (Fig. 1). The viral LUR contains at least 86 viral open reading frames (vORFs), of which more than 20 are genes that were unique for KSHV when it was first sequenced and are therefore termed K genes (K1 – K15); since then similar genes have been found in other New World primate rhadinoviruses that were discovered subsequently. Many KSHV genes are conserved among some or all members of the herpesvirus family (ORF4 –ORF75). Furthermore, the virus genome encodes for 12 microRNAs (miRNAs) as well as for multiple non-coding RNAs and antisense RNAs (Fig. 1). Most of the viral genes are expressed during the lytic replication phase. Four KSHV genes and the miRNAs form the so called KSHV latency locus (ORF71, 72, 73, K12 and viral miRNAs) (Fig. 2), as they are expressed in latently infected cells. There are also viral genes that are expressed only in latently infected B cells (ORF K10.5), or are frequently expressed in tumor biopsies as part of a “relaxed latency” gene expression program (e.g., vIL6, K15). In the virion, the KSHV genome is found in a linear form. After infection of the host cell and once the viral DNA has reached the nucleus, the viral DNA is circularized (episome) by host enzymes and associates with host cell histones. These chromatinized episomes are attached to the host chromosomes by the latency associated protein (LANA) and can thereby be transferred to latently infected dividing daughter cells.

600

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

Fig. 1 Schematic map of the KSHV genome. Protein-coding ORFs are denoted ORF4–ORF75 and K1–K15, and the names of encoded proteins are given for some (see RefSeq NC_003409 and NC_009333). ORFs typical of Old World or New World Primate rhadinoviruses are shaded gray and other marked features are described in the text. The scale is in kbp. Reproduced from Gellermann, E., Schulz., T.F., 2008. Kaposi’s sarcomaassociated herpesvirus: Molecular biology. In: Mahy, B.W.J., Van Regenmortel, M.H.V. (Eds.), Encyclopedia of Virology, third ed. Elsevier. pp. 195–201.

Life Cycle KSHV shows a broad host cell tropism. In vivo, the virus has been found in B cells, endothelial and epithelial cells, monocytes and also in keratinocytes. Two of the cell lineages play a major role in the pathogenesis of the three KSHV-associated tumors: infected endothelial cells are the origin of Kaposi’s sarcoma, while infected B cells can progress to primary effusion lymphoma and multicentric Castleman’s disease.

Binding and Entry of the Virus to the Host Cell The first interaction of the virus with its potential host takes place between glycoproteins that are embedded in the KSHV envelope and heparan sulfate proteoglycan on the cell surface. In addition, specific host cell glycoproteins on the host cell membrane serve as ligands for several viral glycoproteins, such as gH/gL, gpK8.1A, gM and gB. Host cell entry receptors include integrins a3b1, aVb3 and aVb5, ephrin receptor A2R (EphA2R), DC-SIGN and xCT/CD98. In addition to attaching the virus to the cell surface, engagement of these cell surface receptors can induce an intracellular signal cascade that is important to facilitate virus entry and that may vary between different cell types. In HFF cells, the interaction of integrins with EphA2R induces the phosphorylation and activation of FAK, Src and PI3-K signal molecules. pPI3-K induces the activation of c-Cbl which in turn leads to polyubiquitination of EphA2R and the recruitment of Eps15 and AP2 resulting in the formation of clathrin-coated pits and uptake of the virus. Also in BJAB and HEK293 cells the uptake of the virus follows clathrin-mediated endocytosis. In HMVEC-d and HUVEC cells, interaction between integrins and xCT leads to activation of FAK, Src, PI3-K signal molecules, RhoA-GTPase and ROS signal molecules and to the recruitment of c-Cbl, CIB1, Crk and p130C as proteins. C-Cbl in turn induces monoubiquitination of integrins a3b1 and aVb3 and leads to the translocation of these receptors and

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

601

Fig. 2 The latency gene cluster in the KSHV genome. The KSHV latency locus encodes four proteins indicated by black arrows (K12/kaposin, ORF71/ vFLIP, ORF72/vcyc, and ORF73/LANA-1), as well as 12 miRNAs (white boxes), and is flanked by lytic genes (striped arrows). Lytic promoters are indicated by white arrows and latent promoters by black arrows. Readthrough of a viral polyadenylation signal located at nucleotide 122,070 leads to miRNA expression. P1 (127880/86) gives rise to a precursor RNA that is spliced to produce 5.7 and 5.4 kb tricistronic (ORF71, ORF72, and ORF73) and 1.7 kb bicistronic (ORF71 and ORF72) mRNAs. Additionally, P1 drives two transcripts that could function as precursors for the miRNAs. As soon as RTA is expressed, P2 (127610) is induced and gives rise to a 5.5 kb mRNA encompassing all three ORFs. Lytic transcription from P4 (118758) leads to a 1.3 kb transcript coding for K12/kaposin. P3 (123751/60) controls a 1.7 kb transcript encoding vFLIP and vcyc and a 1.5 kb transcript that has the potential to encode the miRNAs. Sequence coordinates are derived from GenBank NC_003409. Reproduced from Gellermann, E., Schulz., T.F., 2008. Kaposi’s sarcoma-associated herpesvirus: Molecular biology. In: Mahy, B.W.J., Van Regenmortel, M.H.V. (Eds.), Encyclopedia of Virology, third ed. Elsevier. pp. 195–201.

xCT together with KSHV into the lipid raft region where the endosomal sorting complexes required for transport (ESCRT) is recruited; this in turn induces a signal cascade resulting in macropinocytosis of the virus. In THP-1 cells (monocytes), endocytosis was found to be mediated by clathrin as well as by caveolin.

Transport of the Virus in the Host Cytoplasm and Entry of Viral DNA Into the Nucleus Following endocytosis/pinocytosis the virus lipid envelope fuses with the endosome membrane which releases the viral capsid into the cytoplasm. The viral capsid then takes advantage of the cellular microtubule system and host cell dynein motor proteins in an ATP dependent retrograde manner. Rho-GTPases that were activated by FAK and integrins acetylate host microtubules which in turn enable the transport of the viral capsid towards the nucleus. Once the virus has reached the nucleus, the double stranded linear viral DNA enters into the nucleus through a nuclear pore. After entry of the viral DNA into the nucleus, there is a short phase in which a small number of lytic genes are expressed, among them ORF50 that encodes the Replication and Transcription Activator (RTA), the key regulator of the productive (lytic) replication cycle. The ensuing short burst of lytic replication is quickly switched off and it is thought that epigenetic mechanisms, in particular the gradually increasing deposition of histones with repressive chromatin marks are responsible for this transition to a latent stage of infection.

Latency During latency most viral genes are silenced. Only a small number of genes are expressed from the latency locus which consists of ORFs 71, 72, 73, K12 and viral miRNAs (Fig. 2). These latent genes are under the control of a latent promoter located upstream of ORF73 as well as promoters located downstream of ORF73; several splicing events help to generate mRNAs for ORF73, ORF72/71 and K12, as well as for the precursor RNA that gives rise to the viral miRNAs (Fig. 2). An internal ribosomal entry site between ORF72 and ORF71 allows translation of ORF71 from the bi-cistronic ORF71/ORF72 mRNA (Fig. 2). In addition, a lytic promoter located within an intron immediately upstream of ORF73 is active and can direct LANA expression during the lytic replication cycle. In latently infected B cells ORF K10.5 is also expressed. The proteins encoded by these viral genes are crucial for the

602

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

maintenance of latency, since their silencing by siRNA inhibits growth of KSHV-infected primary effusion cell lines. These gene products and their specific functions will be discussed in the following. ORF71 (K13) encodes for the protein vFLIP (viral Fas-associated protein with death domain-like interleukin-1b-converting enzyme-inhibitory protein). Similar to cellular FLIPs, vFLIP inhibits death receptor-mediated apoptosis by inhibiting caspase 8 and 10. However, it is also a strong activator of the NF-κB pathway as a result of its ability to bind to IKKg/NEMO, an important NF-κB pathway regulator. Activation of the NF-κB pathway by vFLIP is important for cell proliferation and cell survival. vCyclin is the protein encoded by ORF72. The protein is known to associate with the host cyclin-dependent kinase 6 (CDK-6). Together, these proteins phosphorylate a variety of substrates (pRb protein, histone H1, CDK inhibitor, nucleophosmin (NPM), p21 and p27 (Kip1)) resulting in the regulation of cell cycle and cell proliferation. Expressed on its own, vcyclin causes DNA damage. It is thought that the antiapoptotic property of vFLIP, which is translated simultaneously with vcyclin from a bi-cistronic mRNA (see above), may mitigate these properties of vcyclin. The latency associated nuclear protein (LANA) is encoded by ORF73 and is a constitutively expressed protein that plays a major role in maintaining and replicating the latent viral episome in latently infected cells. Although ORF73 is part of the major latency locus that during latency is under the control of a latent promotor, in the lytic phase the transcription of LANA is inducible by RTA via a second, bidirectional promotor that lies downstream of the constitutively active promotor. This bidirectional promotor regulates the transcription of LANA and a bi-cistronic mRNA for ORFK14 and the viral chemokine receptor homolog, vGCR, which is translated in the opposite direction from LANA. The LANA protein contains an acidic, highly repetitive internal repeat domain that is flanked by an amino terminal domain (amino acid 1–312) and a basic carboxy terminal domain (amino acid 931–1162 in the prototypic KSHV strain) and has a deduced molecular mass of about 130 kDa, but an apparent molecular weight on SDS PAGE gels of approximately 220–250 kDa. Due to the varying length of the repetitive internal repeat domain in different KSHV strains, the length of the protein differs in different KSHV isolates. During latency, LANA is mainly found in the nucleus, where the full-length protein is found in characteristic nuclear microdomains referred to as “LANA nuclear speckles”. The nuclear localization signal (NLS) is located in the N-terminal domain of the protein (amino acid 24–30). LANA is known to form dimers via its C-terminal DNA-binding domain (DBD). These LANA dimers attach the viral episome to host cell chromosomes, with the N-terminal domain binding to histones H2A and H2B on host chromosomes and the C-terminal domain binding to the LANA binding site within the terminal repeat sequence of the KSHV genome. This LANA-mediated linkage of the episome to the host chromosome is vital for segregation and the persistence of the virus in the host cell during latency. LANA is also required for the replication of the latent episome during the S-phase of the cell cycle by recruiting cellular factors such as minichromosome maintenance (MCM) proteins. In addition to these basic roles as origin-binding protein, replication of the latent viral episome and its partitioning to daughter cells during mitosis, LANA has also been shown to act as a transcriptional regulator and is involved in other cellular nuclear processes, such as the modulation of DNA repair mechanisms and of innate immune responses. The ORF K12 encodes three different proteins: Kaposins A, B and C. Kaposin A and B have been shown to effect endothelial cells. Kaposin A induces cell proliferation and Kaposin B has been shown to induce RhoA dependent angiogenesis. Moreover, Kaposin B stabilizes cytokine mRNAs resulting in an increase in cytokine expression. The role of Kaposin C is yet unknown. The major latency locus further comprises viral microRNAs (miRNAs). These miRNAs are short single stranded RNA fragments of 19–23 nucleotides in length. The miRNA cluster of the major latency locus comprises 12 pre-miRNAs that are processed into 25 mature miRNAs that are expressed during latency. Even though they are non-coding, these viral miRNAs are important for establishing latency in several aspects. Some interfere with cellular signal cascades such as IFN signaling (miR-K12-11) or the NFκB pathway (miR-K1 targets IκBa, an NFκB regulator) and this may result in an inhibition of the lytic phase. Other miRNAs (miR-K2 and miR-K5) have been shown to be involved in KSHV induced angiogenesis through an induction of the expression of VEGFA and may thus contribute to KS oncogenesis. Furthermore, miR-K9-5p, miR-K12-7 and miR-K12-9 have been found to directly inhibit the expression of ORF 50 (RTA) and thereby prevent the switch from latency into the lytic phase. ORF K10.5 encodes vIRF3, one of four viral interferon regulatory factor homologs, and is expressed constitutively in latently infected B cells. Also termed LANA-2, this protein has been shown to inhibit the NFκB pathway by interacting with the b subunit of the IKK complex and was found to disrupt the promyelocytic leukemia (PML) oncogenic domains (PODs) by increasing the level of SUMO2-ubiquitin modification of the PML subunit of PODs. Furthermore, vIRF3 is known to inhibit p53 induced apoptosis and to interfere with the cellular interferon type 1 response.

Replication of the Latent KSHV Episome In PEL cell lines, 30–80 copies of viral DNA persist in each latently infected cell; however, the number of latent viral genomes in an infected primary endothelial or B cell in vivo is not known. To avoid a loss of viral genomes during cell division each viral genome is replicated once per cell cycle and the viral genomes are then distributed equally onto the two daughter cells. This replication and distribution requires an accurate multi-step process. DNA replication is initiated in the G1 phase of the cell cycle by the binding of LANA to the origin of replication (ori-P) via its LANA binding sites (LBS1–3) which in turn triggers the formation of a pre-replication complex (pre-RC) that associates at ori-P. The interaction of LANA with multiple other proteins is required to promote the viral DNA replication at ori-P and the

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

603

maintenance of the viral genome. The interaction of LANA with replication factor C (RFC) ATPase enables the loading of the sliding clamp protein PCNA onto the DNA which in turn promotes the DNA polymerase in its functions. Furthermore, LANA and PCNA form a complex together with the cellular mitotic kinase Bub1 that enhances the monoubiquitination of PCNA that also results in an increase in episome replication and maintenance. Moreover, the interaction of LANA with cellular Topoisomerase-2 b was described. The recruitment of these proteins by LANA to the TR region was reported to be crucial for viral DNA replication. The replication of the viral DNA not only starts at ori-P, but several other origins of replication are expected to exist. At least one additional ori was identified and is thought to be LANA independent.

Genome Segregation During mitosis, the latent virus genomes are tethered to host chromosomes to ensure a controlled segregation. LANA is a protein that plays a central role in this process. LANA provides the direct link between the histones (H2A and H2B) on mitotic chromosomes and the viral TR region. In addition, its interaction with other host chromatin proteins such as MeCP2 (Methyl CpG binding protein 2) has been reported to contribute to this tethering of viral episomes to mitotic chromosomes. Furthermore, LANA has been shown to recruit several other cellular proteins that are known to be involved in the control of mitosis: these include nuclear mitotic apparatus protein (NuMA), centromeric protein F (CENP-F), kinetochore protein, Bub1, and these may also play a role in promoting the segregation of viral episomal DNA.

Lytic Activation The persistence of latent episomes in dividing cells with the help of the mechanisms described in the preceding paragraph is essential for the persistence of the virus in its host. However, the distribution of latent episomes dividing daughter cells is not 100% effective. It is therefore thought that reactivation of the virus from latency and the occasional production of viral progeny contributes to the long-term persistence of KSHV in infected individuals by periodically reinfecting new cells. In addition, infectious viral progeny is needed for the virus to spread to new hosts. Many more viral genes are expressed during the lytic than during the latent phase. The lytic gene expression pattern follows the classical pattern of immediate-early, early and late viral genes. The transcription of late genes occurs after the amplification the viral genome by the herpesviral DNA polymerase and is therefore susceptible to inhibitors of this viral enzyme, such as foscarnet. In contrast, early gene expression occurs in cells treated with DNA polymerase inhibitors. The early proteins are synthesized between 10 to 24 h, and the late lytic genes transcribed about 48 h, after the induction of the lytic phase. Several stimuli (e.g., chemical compounds like histone deacetylase inhibitors, hypoxia, oxidative stress) can induce this switch from latency to the lytic phase. Crucial for the lytic phase is the immediate-early lytic protein RTA (regulator of transcriptional activation), a nuclear protein encoded by ORF 50. Expression of RTA is sufficient to induce the lytic cycle and also required in many stimulation conditions. RTA-independent modes of reactivating KSHV have however been described. In addition, a limited lytic gene expression program involving e.g., the vIL6 protein does not depend on RTA. RTA can activate both its own promotor and can also stimulate the transcription of multiple other lytic proteins that are necessary for lytic DNA replication and the assembly of new virions. The amplification of the viral DNA starts at two origins of lytic replication ori-Lyt-L (located between K4.2 and K5) and ori-Lyt-R (located between K12 and ORF71) and is initiated by the formation of the pre-replication complex that is induced by binding of RTA and K8 (K-bZIP) that again recruit further proteins. Binding of this pre-replication complex to ori-Lyt leads to a change in conformation of the viral DNA and to the formation of the replication initiation complex (complex of further viral and cellular proteins). The DNA amplification itself takes place via a rolling circle mechanism that leads to the synthesis of KSHV genome concatemers. Cleavage at the TR region leads to linear DNA genomes of defined length which are then packaged into new KSHV capsids that are assembled in the nucleus. The capsids most likely leave the nucleus via an envelopment (gaining an envelope by passing through the inner nuclear membrane)-de-envelopment (losing the envelope while passing through the outer nuclear membrane)-mechanism. Re-envelopement of the mature virus particle is poorly understood but is thought to take place after assembly of the virus particles in the Golgi apparatus by budding.

Interaction of KSHV With the Host Immune System Infection of a host cell with KSHV leads to activation of the innate immune response via an interaction between pathogenassociated molecular patterns (PAMPs) and pathogen recognition receptors (PRRs) that results in an increased expression of interferons and pro-inflammatory cytokines. Several PPRs (e.g., TLRs and RLRs that activate RNA and/or DNA sensor proteins and NLRs and ALRs that can form an inflammasome complex) are known and PRRs can vary between different cell types. Together the combination of innate responses results in the expression of a range of different effector molecules. During latency, KSHV evades the host immune system by expressing only a limited number of virus proteins. Some of these are able to modulate the immune system: thus LANA has been shown to interfere with the processing and MHC-mediated presentation of peptides to T cells. To evade the host immune system during viral lytic replication, KSHV possesses multiple mechanisms. Two viral proteins, modulator of immune recognition 1 and 2 (MIR1; MIR2), which are expressed in the early phase of lytic replication, interfere with MHC molecules and thereby abrogate the presentation of viral antigens on the cell surface.

604

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

Furthermore, several KSHV proteins, including LANA and ORF52, antagonize cGAS and thereby abrogate the activation of STING, TBK1 and IRF3 that results in a reduced expression of interferons. In addition, vIRF1, vIRF3, RTA and ORF45 also inhibit the interferon- and/or NFκB-induced cytokine expression. Moreover, ORF63 inhibits NLRP1 and NLRP3 which results in a reduced production two pro-inflammatory cytokines IL-1b and IL-18. KSHV ORF4 encodes a complement regulatory protein (KCP) that antagonizes the deposition of activated complement factors C3b and C4b on the surface of the infected cell. There are also examples of KSHV taking advantage of the innate immune response to promote latent persistence. Thus vIRF2 suppresses lytic KSHV replication by activating certain cellular interferon-induced genes such as IFIT 1–3, which in turn restrict lytic replication.

Epidemiology The prevalence of KSHV varies in different geographic regions of the world. Serological studies employing either immunofluorescence assays on latently infected or lytically induced PEL cell lines or ELISAs based on recombinant proteins or synthetic peptides have shown that the seroprevalence of KSHV in HIV-negative blood-donors in Western and Northern Europe as well as North America is generally less than 3%, although different results have been obtained with different serological assays. However, in Sub-Saharan Africa the sero-prevalence in HIV-negative adults is much higher and in the range of 35%–50%. Seroprevalence rates may be even higher in some East African countries, where even young children (0–4 years) have been reported to show seroprevalence rates of up to 25%. In countries bordering on the Mediterranean Sea, such as parts of Italy, Turkey and Israel seroprevalence rates in the range of 10%–20% have been reported, with considerable regional variation. Sexual orientation and lifestyle can affect KSHV seroprevalence, which is higher among men who have sex with men (MSM) and individuals with a high number of sexual partners. Other infections may increase the risk of infections, in particular HIV, but also malaria and helminths, which are considered risk factors for KSHV infection in East Africa and may explain the higher seroprevalence rates in rural populations in this geographic region. These epidemiological findings suggest that sexual contact provides an important route of infection among adults. KSHV can be detected in saliva by PCR and transmission by saliva is considered an important route of transmission, including during sexual contact. Prior to puberty, having a KSHV-seropositive sibling is a very strong risk factor for a child to be infected with KSHV. Transmission via saliva represents a likely route of the high prevalence in children living in KSHV-endemic areas. In addition, transmission via breast milk and under birth have been postulated. Studies in populations in endemic countries also suggest that there may be a genetic predisposition to become infected with KSHV before puberty. KSHV is thought to also be transmittable via blood transfusions and there is some evidence that intravenous drug use may represent a risk factor.

Clinical Features Kaposi’s Sarcoma Kaposi’s sarcoma (KS) lesions often appear as dark red spots or nodules on the skin, but also the mucosa in the oral cavity as well as internal organs may be affected. In the early stages, the skin alterations are referred to as patch lesions. These progress to raised plaques and then firm nodules, which can grow, disturb the lymph drainage and result in edema as well as ulcerate. Kaposi’s sarcoma metastasizes rarely, and individual distinct nodules are thought to have resulted from the independent infection of multiple precursor cells. KS lesions can infiltrate and destroy the local tissue. Epidemiologically, Kaposi’s sarcoma can be subdivided into four forms that carry a different clinical prognosis. Classical Kaposi’s sarcoma mainly occurs in elderly men. It is often found on the lower limbs and shows only a slow progression. Due to the patients’ higher ages and slow tumor progression, classic KS is not normally the cause of death for these patients. Post-transplantation or iatrogenic Kaposi’s sarcoma usually occurs more than a year after an organ transplant and is associated with a suppressed immune system. The African endemic form of KS occurs in East and Central Africa, is not associated with HIV, and can run a clinically more aggressive course than classic KS, with the involvement of internal organs such as the lung and intestine. The HIV-associated form, defined as KS in an HIV-positive patient, is today the most common form. Its severity can vary, but rapid progression with the extensive involvement of multiple visceral organs is often seen and can rapidly lead to the death of affected patients. Thanks to effective antiretroviral combination therapy, HIV-associated KS can be controlled in most patients; however, KS may emerge or reemerge and progress dramatically in rare patients whose HIV viral load is well controlled.

Primary Effusion Lymphoma The KSHV associated non-Hodgkin’s Lymphoma PEL occurs mainly in HIV infected men. Clinically, it manifests itself as a pleural or abdominal effusion containing high numbers of malignant cells, often in the absence of an obvious solid tumor mass, which may later be found to line the pleural of abdominal cavity. Progression is often rapid and the disease is often fatal.

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

605

Multicentric Castleman’s Disease MCD also belongs to the group of non-Hodgkin’s Lymphomas. KSHV is mainly associated with the plasma cell variant of MCD. As in the case for PEL, the plasma cell variant of MCD is a rare disease that often occurs in combination with HIV infection. It is associated with splenomegaly, lymphadenopathy and polyclonal hyperimmunoglobulinemia.

Pathogenesis KSHV is an oncogenic virus that causes Kaposi’s sarcoma and the two HIV associated non-Hodgkin’s Lymphomas PEL and MCD. Several viral proteins, as well as the KSHV miRNAs, are thought to be involved in its oncogenic properties, but their precise contribution to the development of the three KSHV-associated tumors is still under investigation and likely varies in different tumor entities. Based on siRNA silencing experiments and experiments in transgenic mice it is thought that the three latent viral proteins LANA, vcyc and vFLIP play an essential role in the development of KSHV-associated lymphoid neoplasia. Some of their properties discussed above are likely to play a role in this context, in particular the ability of LANA to antagonize cellular DNA repair pathways including p53-dependent responses, the vcyc-mediated dysregulation of the cell cycle and DNA damage and the anti-apoptotic properties of vFLIP. In addition, viral proteins that are expressed during the early stages of the lytic replication cycle or as part of a “relaxed latency” transcriptional program are thought to be important for the development of these three tumors. Transcriptome analyzes of KS tumors have shown that, in addition to the viral genes located in the latency locus (LANA, vcyc, vFLIP, K12, viral miRNAs; see above), others, in particular the viral protein encoded by ORF K15, vIL6, vGCR are frequently expressed in KS tumors. Experimental evidence obtained for these three proteins in tissue culture experiments or transgenic mice suggests that they may contribute to the atypical angiogenesis seen in KSHV-infected lesions, B-cell proliferation and/or the growth of vascular tumors. In addition, several other viral proteins, including the K1 protein, several of the viral interferon regulatory factor homologs (vIRFs) and the anti-apoptotic viral proteins vbcl2 and vIAP have cell biological properties that are compatible with a role in oncogenesis.

Kaposi’s Sarcoma Kaposi’s sarcoma is the most common tumor caused by KSHV. It has its origin in endothelial cells and, in advanced tumors, is characterized by an abundance of KSHV-infected atypical endothelial spindle cells. Infiltration of inflammatory cells is another typical histological feature. Transcriptome and immunohistochemistry studies using antibodies to viral proteins indicate that viral gene and protein expression in Kaposi’s sarcoma lesions can vary. While viral genes encoded in the latency locus (see above) are most frequently expressed, other commonly expressed genes include for example the viral K15 gene and the number of endothelial spindle cells staining for the K15 protein varies in different tumors. Expression of other viral genes or proteins linked to oncogenesis (see above), including for example K1, vGCR and vIL6 has been documented in fewer cells in some tumors.

Primary Effusion Lymphoma This rare lymphoproliferative disorder is characterized by immunoglobulin mutations in B cells, indicative of a post-germinal center differentiation stage. These cells accumulate mainly in the fluid of the pleural, pericardial or abdominal cavity. In some cases, a solid tumor mass can be found lining the pleural or peritoneal membranes. The expression of CD138/syndecan-1 is typical for PEL cells and indicates their plasma cell – like differentiation. LANA, vcyc, vFLIP, vIRF3 and viral miRNAs were found to play a major role in the proliferation of PEL cells and by implication in PEL pathogenesis. PEL cells carry multiple episomal (and in some cases integrated) KSHV genomes (about 50–100 KSHV genome copies per cell in established PEL cell lines). The majority of PEL cells are latently infected. The proportion of cells that have spontaneously switched on the lytic replication cycle varies in different PEL tumors and cell lines and ranges from less than 1% to 10%–15% of these KSHV infected cells.

Multicentric Castleman’s Disease The overproduction of cytokines, especially interleukin-6 and -10, as well as the increase in vascular endothelial growth factor seems to be responsible for the progression of the disease. MCD is often accompanied by productive viral replication with high viral loads detectable in the peripheral blood of affected patients. Lytic KSHV replication is therefore thought to play an important role in the pathogenesis of this disease. Attempts to treat MCD with a combination of high-dose ganciclovir and AZT (azidothymidine) have been successful in some cases, underlining the importance of viral lytic replication at least in some cases (see below).

KSHV Inflammatory Cytokine Syndrome The KSHV Inflammatory syndrome (KICS) was first described in 2016 by Polizzotto et al. as a KSHV related clinical appearance that seems to be associated to Kaposi’s Sarcoma and HIV co-infection and that goes along with a systemic inflammation and a

606

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

wide variety of symptoms (e.g., gastrointestinal disturbance, edema, respiratory symptoms, anemia and others) and that leads to an increase in mortality. KSHV lytic replication is thought to play an important role in the pathogenesis of this disease.

Diagnosis Kaposi’s Sarcoma The diagnosis is currently based on the clinical appearance of KS lesions on the skin, the standard histopathology of KS biopsies, combined with the immuno-histochemical detection of KSHV LANA in histological sections of KS tumors.

Primary Effusion Lymphoma The diagnosis is mainly based on cytological or histopathological examinations, immunohistochemical staining for LANA and the detection of KSHV DNA in the tumor cells.

Multicentric Castleman’s Disease The diagnosis is mainly based on histological examinations of lymph node biopsies, including the immunohistochemical staining for KSHV LANA.

Treatment In tissue culture, KSHV replication can be inhibited by several competitive inhibitors of the herpesviral DNA polymerase, such as foscarnet, ganciclovir, brivudine and cidofovir. However, in infected patients, these drugs are not effective against established KSHV-associated tumors, supporting the notion that the latent viral gene expression program and latent viral replication, which does not rely on the viral DNA polymerase, plays an essential role in the pathogenesis of KS, PEL and MCD. Interestingly, preemptive treatment of HIV-infected MSM with ganciclovir to prevent disease caused by human cytomegalovirus reduces the number of KS in these patients, indicating that productive replication of KSHV contributes to the development of this tumor, possibly by increasing the number of KSHV-infected cells. However, in the case of MCD, which is frequently associated with KSHV lytic replication and an increased KSHV load in peripheral blood (see above), a combination treatment of high dose ganciclovir and AZT has been shown to be effective in some patients. The rationale behind this regimen is that ganciclovir and AZT, both prodrugs that need to be activated by phosphorylation, are phosphorylated, respectively, by the viral ORF36 kinase and the viral thymidine kinase to generate cytotoxic drugs that are thus preferentially active in KSHV-infected cells.

Kaposi’s Sarcoma Classical Kaposi’s sarcoma The classical Kaposi’s sarcoma (cKS) is responsive to radiation and local or systemic chemotherapies. No antiviral therapies with activity against cKS are in use.

Post-transplantation or iatrogenic Kaposi’s sarcoma The tumor can completely regress if the immunosuppressive medication is stopped. If it is not possible to discontinue the patients’ immunosuppressive therapy, the tumors can be treated with radiation therapy.

HIV associated Kaposi’s sarcoma When treating the HIV associated form, it is crucial to reduce the HIV load and to increase the CD4 þ -cell count of the patients by HAART (highly-active antiretroviral therapy). This therapy can lead to a regression of the tumor. If the HAART therapy is not sufficient though, an additional chemotherapy based on liposomal daunorubicin is the appropriate choice. A few studies have reported that the tyrosine kinase inhibitor imatinib (Gleevec) may be partially effective and can cause disease regression in a few cases.

Primary Effusion Lymphoma The therapy includes a combined chemotherapy along with the treatment of the HIV infection. However, the prognosis of patients suffering from PEL is poor, with an average survival time of about six months after diagnosis.

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae)

607

Multicentric Castleman’s Disease MCD can be treated with chemotherapy. Experimental approaches to therapy include a combination therapy with high dose ganciclovir and AZT (see above) as well as with antibodies directed against IL-6 and its receptor.

Prevention No Vaccination Against KSHV is Available Today Since the development of KSHV-associated tumors is often associated with uncontrolled HIV infection, an effective HAART therapy should be pursued. However, HIV-associated KS, MCD and PEL can occur in patients with a well-controlled HIV viral load.

Further Reading Aneja, K.K., Yuan, Y., 2017. Reactivation and lytic replication of kaposi's sarcoma-associated herpesvirus: An update. Frontiers in Microbiology 8, 613. Dittmer, D.P., Damania, B., 2016. Kaposi sarcoma-associated herpesvirus: immunobiology, oncogenesis, and therapy. Journal of Clinical Investigation 126 (9), 3165–3175. Kumar, B., Chandran, B., 2016. KSHV entry and trafficking in target cells-hijacking of cell signal pathways, actin and membrane dynamics. Viruses 8 (11). Kumar, B., Roy, A., Veettil, M.V., Chandran, B., 2018. Insight into the roles of E3 ubiquitin c-Cbl ligase, ESCRT machinery, and host cell signaling in Kaposi's sarcomaassociated herpesvirus entry and trafficking. Journal of Virology 92 (4). Mariggiò, G., Koch, S., Schulz, T.F., 2017. Kaposi sarcoma herpesvirus pathogenesis. Philosophical Transactions of the Royal Society London B: Biological Sciences 372 (1732). Purushothaman, P., Dabral, P., Gupta, N., Sarkar, R., Verma, S.C., 2016. KSHV genome replication and maintenance. Frontiers in Microbiology 7, 54. Schulz, T.F., Cesarman, E., 2015. Kaposi sarcoma-associated herpesvirus: Mechanisms of oncogenesis. Current Opinion in Virology 14, 116–128. Uppal, T., Jha, H.C., Verma, S.C., Robertson, E.S., 2015. Chromatinization of the KSHV genome during the KSHV life cycle. Cancers 7 (1), 112–142. Veettil, M.V., Bandyopadhyay, C., Dutta, D., Chandran, B., 2014. Interaction of KSHV with host cell surface receptors and cell entry. Viruses 6 (10), 4024–4046. Yan, L., Majerciak, V., Zheng, Z.M., Lan, K., 2019. Towards better understanding of KSHV life cycle: From transcription and posttranscriptional regulations to pathogenesis. Virologica Sinica 34 (2), 135–161.

Marburg and Ravn Viruses (Filoviridae) Courtney Woolsey, Thomas W Geisbert, and Robert W Cross, The University of Texas Medical Branch, Galveston, TX, United States r 2021 Elsevier Ltd. All rights reserved.

Epidemiology Marburg virus (MARV) was first identified in 1967 from a contemporaneous cluster of cases of severe hemorrhagic fever in Germany and the former Yugoslavia (now Serbia). Laboratory workers became infected after processing African Green Monkey (Chlorocebus aethiops) tissues for the production of poliovirus vaccines. The monkeys were freshly imported from Uganda, suggesting the virus was of African origin. Detailed virological analysis including electron microscopy demonstrated a unique filamentous morphology and a lack of serological cross-reactivity to known viruses at the time, indicating this was a member of a new family of virus, a filovirus. In subsequent years, a relatively small number of cases and sporadic outbreaks of Marburg virus disease (MVD) surfaced primarily in the vicinity of the greater Lake Victoria area in Central Africa, including the eastern Democratic Republic of Congo, Uganda, and Kenya. Case fatality rates (CFR) ranged from 27% to 100% for these episodes (Fig. 1 and Table 1). In 2004–2005, the largest and most lethal outbreak of MVD occurred outside of this region in northwestern Angola, wherein over 250 cases were confirmed with a B90% case fatality rate (CFR). The high mortality rate suggested something unique in this outbreak or enhanced transmissibility or virulence of the virus variant responsible. In 2008, exported cases in the Netherlands and United States were reported from travelers that visited a bat cave in the Lake Victoria region of Africa and later returned to their home countries prior to experiencing symptoms of MVD (Table 1). Subsequent cases in 2012, 2014, and 2017 have been limited to Uganda.

Fig. 1 Distribution of Rousettus aegyptiacus bats in Africa. Red areas depict estimated range of Rousettus aegyptiacus bats. Yellow stars indicate confirmed MARV outbreaks. Green stars indicate sites where MARV has been detected in wild Rousettus aegyptiacus populations. Inset: Rousettus aegyptiacus in flight. Photo courtesy of Jonathan Towner (CDC).

608

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00077-1

Marburg and Ravn Viruses (Filoviridae)

Table 1

Outbreaks of Marburg virus disease

Year

Location

1967

Germany (Marburg, Frankfurt), Yugoslavia (now Serbia-Belgrade) ex Uganda (importation of African green monkeys) 1975 South Africa (Johannesburg) ex Zimbabwe (visited Chinhoyi caves) 1980 Kenya (visited Kitum Cave in Mt. Elgon National Park/Nzoia; nosocomial transmission) 1987 Kenya (visited Kitum Cave in Mt. Elgon National Park/Mombassa) 1988 U.S.S.R. (Koltsovo) 1990 U.S.S.R. (Koltsovo) 1998–2000 Democratic Republic of Congo (a continuum of overlapping outbreaks in workers at a gold mine and their families in the Durba-Watsa region) 2004–2005 Angola (Uige province) 2007 Uganda (Kakasi Forest Reserve, Kitaka gold mine in Kamwenge District) 2008 Netherlands, United States ex Uganda (visited Python Cave in Queen Elizabeth National Park) 2012 Uganda (Kabale) 2014 Uganda (Kampala) 2017 Uganda (Kween District)

609

Cases/Fatalities (CFR)

MARV/ RAVV

31/7 (23%)

MARV

3/1 (33.3%) 2/1(50%) 1/1 (100%) 1/1(100%) 1/0(0%) 154/128 (83.1%)

MARV MARV RAVV MARV MARV MARV

252/227 (90.1%) 4/1 (25%) 2/1 (50%) 15/4 (27%) 1/1 (100%) 3/3 (100%)

MARV MARV MARV MARV MARV MARV

Most outbreaks of filovirus disease are associated with at least some type of exposure to infected wildlife, through the capture, preparation, and/or consumption of bushmeat, or visiting or working in areas inhabited by bats. The strongest evidence for the role of bats in the maintenance of filoviruses in nature is serological and molecular evidence of MARV in sub-Saharan African bats indicating previous or active infection. In 2009, MARV was first isolated from a colony of Egyptian rousette bats (ERBs), Rousettus aegyptiacus, cave-dwelling fruitbats ubiquitous to sub-Saharan Africa (Fig. 1). Genetic sequence comparisons of viruses isolated from ERBs in the Kitaka Cave of Uganda closely resembled the viruses responsible for an outbreak among mine workers that had been working in the same cave. Later reports indicating active infection of ERBs in Sierra Leone and high seropositivity among ERBs in South Africa should serve as a warning that MARV is not solely endemic to the Lake Victoria region of Africa. Studies addressing the mechanisms of maintenance of the virus in colonies of R. aegyptiacus support a seasonal cycle of enzootic pulses that align with breeding and birthing cycles that create periods of increased risk of human spillover. Human-to-human transmission has been documented in nearly every outbreak of MARV and is a result, in part, of a failure to abide by universal barrier precautions. The virus is detectable in all bodily fluids and excretions. Virus titers tend to increase as the disease progresses. These features put health care workers at an elevated risk for nosocomial transmission. Animal studies have suggested that as little as 1 virion of MARV is sufficient to start the infection and induce a lethal disease. Further, the virus can persist in immune-privileged sites including the testes, a troubling issue when one considers persons who have survived the infection but continue to shed the virus. Indeed, sexual transmission has been documented in outbreaks of MVD.

Classification MARV is taxonomically classified within the order Mononegavirales, a large group of enveloped, nonsegmented, negative-sense, single-stranded RNA viruses known to cause significant disease in humans. Within this order, MARV belongs to the family of Filoviridae, which comprises five genera according to the latest International Committee for Taxonomy of Viruses (ICTV) report (See Relevant Websites Section): Ebolavirus, Cuevavirus, Striavirus, Thamnovirus, and Marburgvirus. While there is a large degree of speciation for Ebolavirus, only a single species of Marburgvirus exists: Marburg marburgvirus. Bayesian phylogenetic analyses of full-length genome sequences indicate two distinct evolutionary lineages within this sole species corresponding to MARV and Ravn virus (RAVV). These viruses vary by B22% at the nucleotide level. MARV is further subdivided into two major clades. One clade consists of Ugandan isolates, including the Poppinga isolate from the 1967 Germany outbreak and bat isolates from Python Cave in Queen Elizabeth National Park (the location in which the Dutch and American tourists likely acquired MVD). Other isolates within this clade include a single specimen from Durba, DRC (1999), the Kenyan Musoke isolate (1980), and all of the available specimens from the 2004–2005 Angola epidemic. The other clade consists of a single 1975 isolate from Zimbabwe (Ozolin isolate), most of the isolates acquired during the 1998–2000 DRC outbreak, and several 2007–2009 Ugandan specimens (a Kitaka miner and Kitaka mine and Python cave bats). Recent isolates from ERBs in South Africa and Zambia, as well as the 2014 and 2017 Ugandan human cases, are related to the same clade. Bayesian coalescent phylogenetic estimates suggest the most recent common ancestor of MARV originated ca. 240 years ago, whereas RAVV emerged 58 years ago. The molecular evolutionary rate for MARV is estimated to be 5.67
104 nucleotide substitutions/site/year, which is slower than in Ebola and Reston viruses, but more rapid than in the Sudan virus.

610

Marburg and Ravn Viruses (Filoviridae)

Fig. 2 Negatively contrasted MARV virions recovered from the fluid of infected MA-104 rhesus macaque cells (scale bar ¼ 360 nm). Courtesy of Thomas W. Geisbert.

Virion Structure The nomenclature for filoviruses was inspired by their distinctive thread-like appearance (latin filum, thread). In tissue culture, MARV is highly pleomorphic with filamentous, shepherd’s crook, U-shaped, 6-shaped, circular (tori), or branched forms (Fig. 2). Each virion is composed of a tubular host-derived envelope with B10 nm peplomer protuberances studding its surface and an interior ribonucleoprotein complex, or nucleocapsid (NC), surrounded by a matrix layer. For filamentous and six-shaped particles, a striated density exists between the electron-dense envelope and the NC central core, which is partially visible with other particle forms. The NC has an outer diameter of 45–50 nm that runs the length of the particle and a central axis of 19–25 nm. CryoEM studies demonstrate MARV filamentous particles have a mean diameter of 91 7 6 nm and a mean length of 892 7 63 nm, which is shorter than that of Ebola virus (EBOV) particles. These dimensions are slightly larger than prior estimates presumably due to shrinkage from conventional EM thin-section processing, i.e., dehydration and plastic embedding, as well as truncation during sectioning. Particles as long as 14 mm have been observed; however, the majority of virions measure B1 mm and this size corresponds with optimal infectivity in human and monkey tissues. Length discrepancy is attributed to the unique ability of filoviruses to express multiple copies of their genome in a single particle. Empty particles devoid of NC are frequently evident, and are smaller in diameter with varying lengths. The buoyant density of MARV in a potassium tartrate gradient is B1.14 g/ml. Morphological heterogeneity has previously hampered efforts to resolve the ultrastructure of MARV, particularly the ribonucleoprotein complex. Cryo-electron tomography combined with sub-tomogram averaging has since provided a well-defined structural arrangement of the NC. The NC is composed of five proteins that collectively package the viral RNA genome: the nucleoprotein (NP), viral protein 35 (VP35), viral protein 30 (VP30), viral protein 24 (VP24), and the polymerase catalytic domain (L). MARV possesses a highly flexible, left-handed, helical NC with a pitch of 7.52 7 0.19 nm and boomerang-shaped protrusions of variable symmetry with 13.8–15.8 (average 14.96) protrusions per turn. Two lobes of density exist for each NC: an innermost layer corresponding to NP subunits that enwrap the viral RNA and an outer layer composed of a ring of VP35-VP24 heterodimer bridges that independently interact with the C-terminus of NP monomers to form the protrusions. Each turn contains 29.92 copies of NP and each NP monomer packages 6.0 7 0.2 RNA bases. The NC interacts weakly with the matrix/envelope layers in a pliable, “Velcro-like” fashion. The NC has a pointed and barbed end. Virions bud from filopodia-like cell extensions with the pointed end facing outward.

Genome Filoviruses have the largest genomes in the Mononegavirales order. MARV has a marginally larger genome (B19.1 kb) than EBOV (B18.9 kb). The negative-sense, single-stranded RNA genome consists of seven monocistronic genes that encode seven respective proteins: NP-VP35-VP40-GP-VP30-VP24-L (Fig. 2). Each gene contains a single open reading frame (ORF), 30 and 50 untranslated regions (UTRs) flanking the ORFs, and a highly conserved UAAUU transcription start/stop signal. In contrast to EBOV, MARV expresses a single mRNA molecule per gene. Cotranscriptional editing of the EBOV GP gene results in the synthesis of several GP mRNA species. On the contrary, deep sequencing of in vitro and in vivo virus populations suggests an increased coding capacity for the Angola variant of MARV. Quasispecies analysis, or the examination of mutant population dynamics that are generated upon replication of RNA viruses, identified hot spots in NP and L mRNAs, as well as U-to-C and A-to-G substitutions in the 30 -untranslated region (UTR) of the NP mRNA. Clustered U-to-C substitutions indicate potential cellular adenosine deaminase (ADAR) activity within the viral genomic RNA. The functions of these editing sites have yet to be determined (Fig. 3). To enable transcription and replication, the viral RNA-dependent RNA polymerase (L and VP35 proteins form the RdRP) recognizes cis-acting regulatory elements located at the 30 leader and 50 trailer ends of the genome. The RdRP uses a start-stop mechanism to transcribe mRNAs sequentially; consequently, it produces more positive-sense mRNA transcripts at the 30 end of the

Marburg and Ravn Viruses (Filoviridae)

611

Fig. 3 MARV genome and structural organization. Each gene color corresponds to the encoded viral protein. The gene order is arranged 30 to 50 . Intergenic regions are denoted by a straight line. Note the VP30-VP24 gene junction contains an overlapping segment.

Table 2

Summary of physical and functional attributes of MARV proteins Molecular weight (kDa)

Localization

Function

695

96

Inner nucleocapsid

1557

329

32

Outer nucleocapsid

Viral protein 40 (VP40)

1405

303

38

Envelope matrix

Glycoprotein (GP) Viral protein 30 (VP30) Viral protein 24 (VP24)

2846 1249 1287

681 281 253

170–200 28 24

Envelope spike Nucleocapsid Outer nucleocapsid

Polymerase (L)

7745

2331

B200

Nucleocapsid

Core component of the nucleocapsid, RNA encapsidation, budding, vital for transcription and replication Polymerase cofactor, outer layer nucleocapsid component, antagonism of innate immunity Matrix protein, assembly, budding, host adaptation, IFN antagonism Entry, fusion, immune evasion, cytotoxicity Nucleocapsid formation Outer layer nucleocapsid component, budding, cytoprotective responses, host adaptation, modulation of transcription and replication Catalytic component of the RNA-dependent RNA polymerase

Protein

Gene (nt)

Nucleoprotein (NP)

2796

Viral protein 35 (VP35)

Protein (aa)

Abbreviations: nt, nucleotides); aa, amino acids; kDa, kilodaltons; IFN, interferon. Gene and ORF lengths were derived from the GenBank Accesssion Number NC_001608 MARVMusoke reference sequence.

genome than at the 50 end. Short intergenic regions segregate genes, except for an overlapping region at the VP30-VP24 gene junction. The structure of the overlapping segments are exclusive to filoviruses as they do not resemble other members of the Mononegavirales order. The filovirus replication promoter has a bipartite structure similar to paramyxoviruses. While MARV and EBOV do not have genome lengths that are a multiple of six, each NP protomer is in contact with six nucleotides in the NP–RNA complex implying they abide by the “rule of six” similar to paramyxoviruses. The first element of the MARV replication promoter is at the 30 end of the genome and is predicted to adopt a secondary stem-loop structure. The second promoter element consists of a (UN5) x 3 hexamer motif located within the 30 UTR of the NP gene. The 50 non-coding region of the genome is thought to contain the antigenomic replication promoter. MARV mRNA transcripts are translated into individual proteins that contribute to the structural integrity of the virion or serve a role in transcription, replication, or immune evasion (Table 2).

NP The primary role of NP is to encapsidate the viral genomes and antigenomes. This function protects the viral RNA from degradation and detection by host pattern recognition receptors (PRRs). Purified NP self-assembles into a loosely coiled helix that resembles the NC structure, confirming its importance in NC formation. A coiled-coil motif in a central region of NP is thought to facilitate this helical arrangement. The abundance of NP determines the RdRP switch from transcription to replication and is governed by phosphorylation levels. NP also participates in budding by recruiting endosomal sorting complexes required for

612

Marburg and Ravn Viruses (Filoviridae)

transport (ESCRT) protein, Tsg101, via a C-terminal late PSAP domain motif. Recruitment of Tsg101 is required for actin-driven transportation of NC to viral inclusion sites in filopodia.

VP35 VP35 along with the large protein (L) catalytic domain forms the RdRP complex. Oligomerization of VP35 is required for the polymerase cofactor activity. VP35 assists in transcription and replication by inhibiting NP oligomerization and RNA binding, allowing the polymerase to access the RNA template. VP35 participates in immune evasion by blocking IFN gene expression and dendritic cell maturation. MARV VP35 is a comparatively less-efficient antagonist of IFN signaling than EBOV VP35. However, RAVV VP35 IFN-suppressing activity is as potent as that of EBOV VP35.

VP40 The matrix protein, VP40, is the most abundant protein in the virion and coats the inner viral membrane. VP40 is responsible for the filamentous morphology of MARV. Transfection of VP40 into cells results in the release of virus-like particles (VLPs) that bud from the cell membrane, emphasizing its vital role in assembly and budding. VP40 recruits the glycoprotein (GP) to budding sites at the plasma membrane and forms a flexible, weak interaction with the nucleocapsid. A PPPY late domain motif in the VP40 N-terminus interacts with proteins of the host ESCRT machinery, namely tumor susceptibility gene 101 (Tsg101) and neural precursor cell expressed developmentally down-regulated 4 E3 ubiquitin ligase (NEDD4), to facilitate particle release. VP40 can also hijack the cellular coat protein complex II (COPII) vesicular transport system to enhance cell egress. BCL2-associated athanogene 3 (BAG3), a host chaperone, was found to inhibit EBOV and MARV VP40-associated budding. VP40 also regulates MARV replication and transcription. VP40 is a species-specific virulence factor that plays an integral part in pathogenesis. Serial adaptation of MARV and RAVV is required to produce lethal disease in immunocompetent rodents. However, STAT2 knockout Syrian hamsters are susceptible to infection with various wild-type variants, and humanized or IFN-alpha/beta receptor knockout mice are susceptible to disease with non-adapted filovirus. Mutations in VP40 are required for adaptation to mice and guinea pigs. Many of these amino acid changes correspond with the ability of the virus in these hosts to antagonize the type I IFN response. MARV VP40 interferes with the IFN response by blocking the phosphorylation of Janus kinases to inhibit IFN alpha- or beta-induced tyrosine phosphorylation of STAT1 and STAT2.

GP MARV GP is the sole surface protein and is expressed on the virion envelope as homotrimeric spikes. GP performs the following functions: attachment, entry, fusion, and immune evasion. These vital roles make GP an attractive target for vaccines and therapeutics. The precursor GP undergoes various posttranslational modifications in the endoplasmic reticulum (ER) including glycosylation, phosphorylation, and acylation. N-linked and mucin-type O-linked glycans that form the glycan cap and mucin domain can contribute up to B50% of the apparent molecular mass. Following modification, GP is cleaved by furin or furin-like proteases in the trans-Golgi network into two disulfide-linked subunits, GP1 (160 kDa) and GP2 (38 kDa). The GP1 ectodomain facilitates receptor binding, whereas the transmembrane subunit, GP2, mediates fusion. The GP of MARV can subvert the immune system via a myriad of mechanisms. GP can counteract its sensitivity to antiviral tetherin, which normally restricts the release of virions. In addition, putative immunosuppressive motifs in GP were shown to induce lymphocyte death and cytokine suppression. Finally, MARV GP can sterically shield cell surface proteins, e.g., major histocompatibility complex class I, Fas, and integrin b1, to enhance viral replication and spread.

VP30 VP30 associates with the nucleocapsid via NP binding following phosphorylation and is essential for replication. EBOV and MARV VP30 share high structural similarity. While EBOV VP30 is important for viral transcription and replication, the role of MARV VP30 is not fully understood as it is not required for these functions in a minigenome system. Interestingly, VP30 is required for successful recovery of infectious MARV from a full-length cDNA clone. The mechanism is possibly attributed to nucleocapsid maturation, yet VP30 is not essential for NC-like particle formation. In vitro RNA interference (RNAi) silencing of VP30 reduces synthesis of viral proteins and particle release.

VP24 VP24 helps form the outer NC layer and is involved in the maturation of nucleocapsids. This protein functions as an interface between nucleocapsids and budding sites. VP24 also regulates transcription and replication. Unlike EBOV VP24, MARV VP24 is

Marburg and Ravn Viruses (Filoviridae)

613

unable to block type I IFN signaling. However, an extended b-sheet of MARV VP24 recruits Kelch-like ECH-associated protein 1 (Keap1), a suppressor of the antioxidant response and regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. Presumably, this leads to transcription of cytoprotective genes in cells to prolong infection.

L The major and catalytic component of the MARV RdRP, L protein, is essential for both transcription and replication. MARV L is hypothesized to carry out RNA synthesis, capping, and polyadenylation of viral mRNAs.

Life Cycle The MARV life cycle begins when GP binds to lectins (e.g., DC-SIGN, L-SIGN, hMGL ASGP-R, LSECtin) and gylcosaminoglycans on the surface of host cells. Other attachment factors include the receptor tyrosine kinase Axl, and T cell Ig mucin (TIM) proteins TIM-1 and TIM-3. The latter group of proteins is thought to interact with phosphatidylserine molecules on the viral envelope. Next, MARV virions enter cells by a macropinocytosis-like mechanism and are compartmentalized in an endosome. The GP1 subunit is cleaved by an endosomal cysteine protease to remove heavily-glycosylated domains, allowing GP1 to bind the entry receptor, Niemann-Pick C1 (NPC1). Other surface proteins may also serve as receptors to enhance the infection. Endosomal acidification causes the MARV GP2 subunit to undergo a pH-dependent conformational change, releasing the fusogenic loop and permitting fusion of the viral and late endosomal membranes. Following nucleocapsid release into the cytosol, the genome is uncoated and mRNAs are sequentially transcribed, co-transcriptionally capped, and polyadenylated by the viral RdRP. Host cell machinery then translates these positive-sense mRNAs into viral proteins. The genome serves as a template to generate positive-sense antigenomes. Antigenomes then serve as templates to produce progeny genomes that are packaged into virions or transcribed to produce more virus-specific mRNAs. The abundance of nucleoprotein determines when the polymerase switches from gene transcription to genome replication. For assembly, nucleocapsids are transported along actin filaments from viral inclusion bodies to budding sites with the aid of viral and cellular proteins, such as VP40 and ESCRT machinery. GP is recruited via a tubulin-dependent process. The lipid envelope is host-derived and acquired from the virion budding off the host cell membrane.

Clinical Features MVD symptoms in humans are primarily based upon clinical data acquired during outbreaks in Germany, Yugoslavia, DRC, and Angola. There are three stages of the disease: a generalization phase, an early organ phase, and late organ phase or convalescent period. The incubation period ranges from 3 to 21 days with an average of 5–10 days, depending on the route of transmission and infectious dose. The generalization phase (day(s) 1–4) is characterized by fever, severe headache, malaise, myalgia, pharyngitis, and gastrointestinal symptoms (nausea, abdominal pain, diarrhea, anorexia). These symptoms may persist for the entire course of the infection. By day 4–5, patients develop a maculopapular rash, the earliest distinguishing feature indicating infection by a filovirus. Other common symptoms include thrombocytopenia, leukopenia, and lymphadenopathy. At days 5–13 (early organ phase), multiple organs are affected including the liver, kidneys, and the pancreas. Most patients show hemorrhagic manifestations at this point, such as petechiae, ecchymoses, bloody diarrhea, and mucosal bleeding. Infected individuals may appear irritable, aggressive, and confused. Patients may also experience dyspnea, increased vascular permeability, edema, or conjunctival infection. From day 13 on, patients enter the late organ phase characterized by preagonal symptoms such as severe disseminated intravascular coagulation (DIC), multiorgan failure, coma, shock, and eventually death. Alternatively, survivors enter an extensive convalescent period characterized by partial amnesia, sweating, myalgia, exhaustion, sweating, and peeling of skin at rash sites.

Pathogenesis Animal models have advanced the understanding of virus infection and dissemination. Filovirus pathogenesis studies have largely been conducted in rodents and NHPs. The latter animal model most accurately recapitulates human infection, as rodents do not typically exhibit the immunological or hemorrhagic manifestations of MVD. MARV particles enter the body through compromised skin or mucosal membranes and infect monocytes, macrophages and dendritic cells (DCs). These early target cells then migrate to regional lymph nodes and spread through the lymphatic system to major organs. The liver and spleen are preferred sites of replication and contain high numbers of monocytes and macrophages. In the late stages of the disease, nearly every organ is affected. Monocytes and macrophages appear highly activated and secrete reactive oxygen species and proinflammatory cytokines/ chemokines, such as IL-1-beta, IL-6, IL-8, TNF-alpha, and MCP-1. These soluble factors recruit other inflammatory cells and increase vascular permeability. The secretion of tissue factor by macrophages contributes to coagulopathy and disseminated

614

Marburg and Ravn Viruses (Filoviridae)

Fig. 4 Virulence factors for MARV. Binding of cytokines to the cell surface receptor activates the JAK/STAT pathway. This interaction increases the kinase activity of JAKs, promoting tyrosine phosphorylation of the receptor complex and mobilization of STAT proteins and association with IRF9. STAT proteins then form hetero- or homodimers and translocate into the nucleus to induce transcription of antiviral genes. MARV VP40 blocks STAT signaling by preventing the phosphorylation activity of JAKs. In the cytoplasm, RIG-I and MDA-5 recognize short and long viral dsRNA, respectively. A complex of proteins then signals recruitment of interferon regulatory factor (IRF) proteins that migrate to the nucleus to stimulate the production of ISGs and IFN-beta. MARV VP35 prevents immune signaling by interfering with these pathways. VP24 binds cellular Keap1, leading to Nrf2 accumulation and persistent upregulation of cytoprotective responses. GP sterically shields epitopes and host surface proteins of cells, e.g., MHC-I and integrins, to induce immunopathology. MHC-I is essential for presenting epitopes to killer T cells and inducing cellular immunity. GP can also antagonize the IFN-inducible protein tetherin. Tetherin traps virus particles at the cell surface to prevent them from being released and infecting other cells. Abbreviations: JAK, Janus kinase; STAT, signal transducer and activator of transcription; RIG-I, retinoic acid-inducible gene I protein; MDA-5, melanoma differentiation-associated protein 5; IFNs, interferons; ISGs, interferon-stimulated genes.

intravascular coagulation (DIC). Consequences of DIC include widespread deposition of fibrin resulting in ischemia, hemolytic anemia, as well as hemorrhagic diathesis due to consumption of platelets and clotting factors. In culture, infected human primary macrophages weakly upregulate T cell costimulatory molecules CD40 and CD80, fail to upregulate CD83, and only express low levels of CD86 and HLA class I and II molecules. In contrast to monocytes and macrophages, MARV-infected dendritic cells (DCs) upregulate co-inhibitory molecules and fail to undergo maturation or elicit cytokine production. Costimulatory molecules of DCs are downregulated, and infected cells fail to induce proliferation of allogenic T cells. The lack of support from DCs is thought to contribute to lymphocyte apoptosis and an impaired adaptive response. Direct interaction with viral proteins or induction of Fas death receptor pathways may additionally advance filovirus-induced lymphocyte apoptosis. Due to extensive necrosis and apoptosis of lymphocytes in secondary lymphatic tissues, an appropriate adaptive immune response is delayed or not induced. Other cell types permissive to MARV infection include endothelial cells, fibroblasts, hepatocytes, and adrenal cortical and medullary cells. In contrast, neutrophils and lymphocytes are spared. Infected endothelial cells are observed in low numbers in the NHP model suggesting that vascular changes are caused by paracrine signaling. Coagulopathies are likely exacerbated by an impaired synthesis of clotting factors in the liver, given the prominent pathology seen in this organ. Additionally, MVD causes necrosis of the adrenal glands leading to decreased steroid synthesis. As steroids help to control blood pressure, MARV infection may eventually lead to hypovolemia. The systemic virus spread and replication, dysregulation of the immune response, coagulopathies, and hypotension ultimately result in shock and multiorgan failure. MARV proteins broadly dyregulate host antiviral responses to cause pathogenesis (Fig. 4). An IFN-induced cellular protein, tetherin, blocks the release of several viruses including filoviruses from the cell surface at lipid-raft sites. To counter this response, MARV GP antagonizes tetherin restriction to allow egress of virus particles. Furthermore, GP sterically shields epitopes and host surface proteins of cells, e.g., MHC-I and integrins, which contributes to the lack of an adaptive response. Steric shielding of host proteins is more pronounced for MARV Angola GP than for MARV Musoke GP, indicating its potential role in variant-specific pathogenicity. MARV VP24 has an unusual role in that it interacts with host Keap1 to modulate cytoprotective antioxidant pathways and cell survival pathways, presumably to prolong infection. Two viral proteins are considered as the main players in

Marburg and Ravn Viruses (Filoviridae)

615

MARV IFN antagonism: VP40 and VP35. MARV VP40 blocks tyrosine phosphorylation of JAK proteins to disrupt JAK/STAT signaling, whereas MARV VP35 prevents the activation of RIG-I and MDA-5 signaling. In turn, MARV viral proteins downregulate several ISGs. MARV VP40 strongly suppresses ISG54, ISRE, IRF1, and IFN-beta induction in cells stimulated with type I IFNs or by a potent and broad spectrum ISG-inducer, Sendai virus. MARV VP35 also attenuates the expression of these genes, but to a much lesser extent compared to EBOV VP35. This difference is likely attributed to a differential dsRNA binding ability of each virus protein. MARV VP35 coats the backbone of dsRNA to inhibit RLR activation, whereas EBOV VP35 can also bind the blunt ends of dsRNAs (end capping). At a higher multiplicity of infection (MOI) of Sendai virus, MARV VP35 and VP40 were less effective IFN antagonists. As mentioned, most studies show that MARV infection suppresses innate immunity. MARV-infected immortalized human liver cells (Huh7) secrete low levels of IFN, and treatment of infected cells with polyinosinic-polycytidylic acid (poly I:C) or exogenous IFN does not lead to an expected transcription of classical ISGs. If type I or II IFN is added to cultured cells prior to filovirus infection, viral replication is substantially reduced. Accordingly, supplemental IFN-alpha or beta ameliorates disease in mice and prolongs survival, but does not prevent death in NHPs. These results suggest that at least some ISGs restrict filovirus replication and that MARV has a decreased ability to suppress antiviral gene expression once a host initiates an immune response. Transcriptomic analyses demonstrate that virus infection leads to substantial immune activation including ISGs, upregulation of complement system genes, and genes involved in neutrophil and monocyte recruitment. Thus, activation of innate immunity is dysregulated or only temporarily delayed. Alternatively, only uninfected bystander cells may initiate an antiviral response. Temporal characterization of gene expression in MARV-Angola-infected cynomolgus macaques further supports this concept. Upregulation of common innate response and pro-inflammatory genes in peripheral blood are observed early after infection (days 1–3) and sustained throughout the disease course. Many of these genes function in RLR and IFN signaling, for instance, MX1, RIG-I (DDX58), PARP14, STAT1, IRF3, IRF7, IRF9, IFITs, RNA helicases, and ISG15. However, increases in IFN-alpha and IFN-beta gene expression are not detected, nor are genes involved in other innate defense pathways, such as complement activation or antigen presentation. ISG upregulation does not appear to reduce viral load, or postpone death or clinical signs of the disease in these animals, proving MARV-Angola tolerates a high level of IFN-mediated antiviral activity. It is possible that MARV may weaken or overcome the effects of ISG activation by interfering with alternative signaling pathways or modulating key proteins in these antiviral pathways. For example, it has also been shown that infection of macaques leads to early signaling of T helper 2 (Th2; humoral response)-associated genes, IL-4 and IL-5, and serum levels of immunosuppressive IL-10. No appreciable changes in T helper 1 (Th1; cell-mediated response) cytokines, such as IFN-gamma and TNF-alpha, are noted until late in the disease. Th1 cytokines may contribute to a favorable prognosis since the survivors of a 2012 MARV outbreak in Uganda exhibited a Th1 response. IL-4 and IL-10 suppress Th1 type cytokine, IFN-gamma and IL-2 production, so it is possible that these cytokines exacerbate pathogenesis by skewing immunity towards a detrimental Th2 response. Thus, MARV-mediated immunopathology is multifaceted.

Diagnosis Early in the course of the disease, MVD cases present as a non-specific, febrile illness which can easily be confused with the symptoms of other infections endemic to central Africa including influenza, malaria, and Dengue fever. This is a major confounder especially in the early phases of an outbreak as there would otherwise be limited suspicion for MARV infection unless certain risk factors (e.g., exposure to bats) were addressed upon presentation to the health center. If MARV infection is suspected, isolation of the patient is paramount to allow for expedient containment and safe testing under proper containment. Antigen-capture enzymelinked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and IgM-capture ELISA testing have been routinely utilized to confirm MVD. Virus isolation is often considered as the “gold standard” of any diagnosis; however, the biosafety level 4 (BSL4) facilities needed to safely cultivate filoviruses are rarely available. In cases of an ongoing outbreak investigation, the use of IgG-capture ELISA can be useful for testing recovered individuals as well as their contacts for estimation of asymptomatic cases within an outbreak. The rapid progression of disease in MVD coupled to the resource-limited environment of most filovirus outbreaks often complicates and delays the diagnosis. Unfortunately, some patients may succumb prior to seeking health care or before receiving a confirmed diagnosis. In the case of deceased patients, immunohistochemistry on autopsy tissues, virus isolation, or PCR from cadaver samples may be performed.

Treatment Currently there are no regulatory-agency approved therapeutics or treatments for MVD. Palliative support should be offered under strict barrier conditions to reduce the risk of nosocomial infections. Based on the resources available and abilities of the treatment facility, patients should be provided with fluids and electrolyte replacement to prevent dehydration and maintain proper oxygenation and blood pressure. The risk for secondary infections is high due to an impaired immune system; attempts to treat any complicating infections should be made. Several experimental treatments have been in development over the last few decades and have shown promise in animal models. Commercial interest has impeded further development, although nucleoside analog

616

Marburg and Ravn Viruses (Filoviridae)

phosphorodiamidate morpholino oligomers (PMOs) have entered phase I human clinical trials for safety testing (see Relevant Websites section). Although rodent studies may yield proof-of-concept efficacy and inform dosing regimens, protective immunity in these models may not translate to the more robust NHP models. As the latter models most reliably recapitulate the pathophysiology observed in humans, only NHP studies are discussed here. Some of the first interventions against MVD involved immunomodulatory treatment with IFN or an anticoagulant (recombinant nematode anticoagulant protein c2 (rNAPc2). These approaches proved unsuccessful in the NHP model. Subsequent medical countermeasure evaluations, such as twice-daily delivery of a nucleoside analog (BCX4430), or daily administration of phosphorodiamidate morpholino oligomers (PMOs), protected 100% of NHPs up to 2 or 4 days, respectively. However, the majority of animals exhibited clinical signs and had detectable viremia with these antivirals. There is also evidence for the usefulness of antibody therapy against MVD. Three doses of convalescent purified IgG from the serum of immunized NHP survivors fully protected rhesus macaques from a lethal MARV-Ci67 challenge and prevented 67% from disease when given up to 2 days post-infection. Presently, the most encouraging therapeutic candidates against the most pathogenic Angola variant of MARV (associated with 90% case fatality rate) are stable nucleic acid-lipid particles (SNALPs) and MR191-N monoclonal antibodies. These treatments can rescue NHPs from MVD and lethality after viremia and clinical signs have already developed. SNALPs employ short-interfering RNA (siRNA) technology, which relies on host RNA interference (RNAi) machinery to target and degrade viral transcripts. Multiple doses of SNALPs directed against MARV NP RNA protected rhesus monkeys from a highly lethal 1000 PFU Angola challenge whenever treatment was delayed up to 5 days. Remarkably, treatment with MR191-N antibodies has a similar therapeutic window, but requires only two doses. Intravenous (i.v.) administration of MR191-N protects 100% of NHPs from advanced stages of disease when treated on days 4 and 7, and 80% of macaques when treated on days 5 and 8. MR191-N was isolated from a human survivor and is a neutralizing antibody that blocks the receptor binding site. The use of live attenuated vaccines as post-exposure prophylaxis has also shown limited therapeutic potential in NHP models, although the treatment window is shorter. This possibility might be of interest as some MVD vaccine candidates utilize a similar vaccine platform as the recombinant Vesicular stomatitis virus (rVSV)-based Ebola vaccine, which was deployed and highly effective at preventing disease during the 2013–2016 West African and 2018–2020 EBOV outbreaks. An rVSV construct expressing a Musoke variant GP was 100% effective when administered at 20–30 min after a 1000 PFU challenge of MARV-Musoke, B83% effective at 24 h, and 33% effective at 48 h. For high and low-dose challenges with the most pathogenic Angola variant, a total of 25% and 80–89% of treated macaques survived, respectively (Table 3).

Table 3

Summary of post-exposure treatment studies for Marburg virus disease in the non-human primate model

Treatment

Time post-exposure

Treatment doses

MARV challenge

Survival

Illness

Viremia

IFN-beta rNAPc2 BCX4430 PMOplus (pool) PMOplus (NP) Convalescent IgG (purified)

1h 10 min 1–48 h 30–60 min 1 h4 d 15–30 min 48 h

15 15 26–30 14 3

B1000 PFU Musoke i.m. B1000 PFU Angola i.m. 1275 PFU Musoke s.c. B1000 PFU Musoke s.c. i.m. B1000 PFU Ci67 i.m.

33% 17% 83%–100% 100% 83%–100% 100%

100% 100% 0%–33%? 100% 100% 0%–33%

100% 100% 100% 100% 83%–100% 0%–33%

siRNA SNALPs

30–45 min 24 h 48 h 3d 4d 5d 3d 6d

7

B1000 PFU Angola i.m.

7

B1000 PFU RAVV i.m.

100% 100% 100% 100% 100% 50% 100% 100%

50% 25% 50% 50% 100% 100% 25% 100%

33% 0% 0% 17% 75% 100% 25% 100%

MR191-N mAbs

4–5 d 5d

2 2

B1000 PFU Angola i.m. B1000 PFU RAVV i.m.

80%–100% 100%

80%–100% 60%

100% 100%

rVSV

20–30 min 24 h 48 h 20–30 min 20–30 min

1

B1000 PFU Musoke i.m.

1 1

B1000 PFU Angola i.m. B50 PFU Angola i.m.

100% 80% 33% 25% 80%–89%

60% 17% 67% 100% 22%–40%

0% 0% 80% 100% 33%–60%

Abbreviations: IFN, interferon; rNAPc2, recombinant nematode anticoagulant protein c2; PMO, phosphorodiamidate morpholino oligomers; NP, Marburg virus nucleoprotein; IgG, immunoglobulin G; siRNA, short interfering RNAs; SNALPs, stable nucleic acid lipid particles; mAbs, monoclonal antibodies; rVSV, recombinant Vesicular stomatitis virus; RM, rhesus macaques; CM, cynomolgus macaques; PFU, plaque-forming units; MARV, Marburg virus; RAVV, Ravn virus; i.m., intramuscular; s.c., subcutaneous. Illness is defined as an animal having fever and/or showing significant clinical signs of a disease.

Marburg and Ravn Viruses (Filoviridae)

617

Prevention Similar to other viral hemorrhagic fever infections, secondary, or human-to-human, transmission is a common feature of MVD in humans. Therefore, suspected and confirmed patients should only be addressed under strict barrier techniques utilizing, at minimum, a universal precautions approach. Furthermore, efforts to ensure targeted public health outreach and education of affected and surrounding communities should be undertaken to ensure proper awareness of immediate risks associated with MVD. Cases of laboratory-acquired infections were noted in the former Soviet Union (Table 1) underscoring the need for stringent high containment practices to be in place when manipulating the virus in the laboratory setting. Accordingly, only basic diagnostic efforts limited to molecular or antigen based detection of inactivated samples should be attempted at containment levels below BSL-4. While several promising vaccine candidates are currently in development, there are no vaccines or prophylactic measures approved by regulatory agencies. An overview of prospective vaccines for MVD is shown in Table 4. Originally, scientists explored formalin-inactivated whole MARV particles as a vaccine. This approach failed to induce a protective immune response in rhesus macaques. Thereafter, Venezuelan equine encephalitis virus replicons expressing the Musoke variant GP, NP, or a combination of both antigens, were evaluated. Replicon-based vaccines protected 67–100% of cynomolgus monkeys against a high dose (8000 PFU) subcutaneous (s.c.) Musoke challenge, but did not protect NHPs against RAVV. Another vaccine expressing MARV VLPs fully protected cynomolgus macaques and was cross-protective against Musoke and Ci67 variants, as well as against RAVV. It would be of interest to see whether VLPs are also effective against the most pathogenic variant, the Angola strain, as this was not reported. The safety profile of DNA subunit vaccines makes them an attractive option. However, DNA vaccines afforded only partial protection of cynomolgus macaques against MVD using MARV GP as a primary immunogen. Specifically, three doses of vaccine resulted in 67% and 100% survival of monkeys against a 1000 PFU i.m. Musoke and Angola challenge, respectively. All immunized animals became sick when challenged with the Musoke variant and approximately half of them when challenged with the Angola variant, indicating lack of sterile immunity for this vaccine platform. The most promising vaccines against MARV-Angola use a live attenuated vaccine platform. These include recombinant Adenovirus 5 (rAd5) and VSV (rVSV) vectors, which both employ MARV GP as an immunogen. Both vaccines are safe, immunogenic, and require a single injection to elicit complete protection. Moreover, these vectors provide cross-protection against multiple MARV variants. Added benefits of the rVSV-based vaccine are its proven defense against aerosol infection and post-exposure treatment potential. Vaccination with rAd5 or rVSV results in robust antibody production and cellular responses that are thought to contribute to protection. Safety is a significant issue for any replication-competent vaccine, particularly in the immunocompromised, a major concern in an HIV-endemic region. Nevertheless, immunization with a rVSV expressing EBOV GP resulted in a merely transient viremia in simian/human immunodeficiency virus-infected macaques, and NHPs intrathalamically inoculated with MARV GP- or EBOV GP-expressing rVSVs lacked neurovirulence. Another concern for live attenuated vaccines was that pre-existing immunity against the vector would influence protective efficacy. However, this concern is not

Table 4

Summary of vaccine studies for Marburg virus disease in the non-human primate model

Vaccine

Immunogen

Vaccine doses MARV challenge

Survival Illness

Inactivated MARV VEEV replicon

Irradiated whole virus GP (Musoke) NP (Musoke) GP þ NP (Musoke) GP þ NP (Musoke)

1? 3 3 3 3

200 LD50 Popp parenteral 8000 PFU Musoke/RAVV s.c. 8000 PFU Musoke s.c. 8000 PFU Musoke s.c. RAVV

50% 100% 67% 100% 0%

50% 0%? 100% 0% ?

VLPs DNA plasmid

GP þ NP þ VP40 (Musoke) QS-21 Adjuvant 3 3 GP (Musoke) 4 GP (Angola)

1000 PFU Musoke/Ci67/RAVV s.c. 1000 PFU Musoke s.c. 1000 PFU Angola i.m.

100% 67% 100%

0–33% 100% 50%

100% 100% 100%

25% 0% 0%

DNA plasmid prime þ rAd5 boost GP (Angola) rAd5 GP (Angola) Blend GP (Z þ S þ Ci67 þ RAVV) þ Z NP

3þ1 1 2

1000 PFU Angola i.m. 1000 PFU Angola i.m. 1000 PFU Musoke/Angola s.c.

rVSV

1 1

1000 PFU Musoke/RAVV/Angola i.m. 100% 1000 PFU Musoke aerosol 100%

GP (Musoke) GP (Musoke)

0% 0%

Abbreviations: MARV, Marburg virus; VEEV, Venezuelan equine encephalitis virus; VLPs, virus-like particles; rAd5, recombinant Adenovirus 5; rVSV, recombinant Vesicular stomatitis virus; RM, rhesus macaques; CM, cynomolgus macaques; GP, glycoprotein; NP, nucleoprotein; VP40, viral protein 40; Z, Zaire ebolavirus; S, Sudan ebolavirus; RAVV, Ravn virus; PFU, particle-forming units; s.c., subcutaneous; i.m., intramuscular. Illness is defined as an animal having fever, viremia, and/or exhibiting significant clinical signs of disease. A question mark (?) indicates the data was unclear or not provided in the literature.

618

Marburg and Ravn Viruses (Filoviridae)

relevant since previous vaccination with a Lassa virus GP-expressing vaccine did not abrogate immunity when NHPs were sequentially immunized with an EBOV GP-expressing rVSV and subsequently challenged with EBOV. The rVSV vaccine is also durable, with protection lasting as long as 14 months after immunization. While the scarcity of MVD outbreaks and lack of commercial interest have hindered further vaccine development, vaccines in phase I human clinical trials include a DNA plasmid vaccine (VRC-MARDNA025–00-VP) and the multivalent Modified Vaccinia Ankara - Bavarian Nordic MVA-BN(R)-Filo vaccine (see Relevant Websites section). Although MARV outbreaks are generally rare, those that do surface are generally associated with high morbidity and mortality rates (Table 1). Preventative measures to limit exposure should be taken with extreme stringency. Given the clear association of MARV with fruit bats, avoidance of known bat roosts or wildlife that commonly comes in contact with these species or their excreta (i.e., non-human primates) is advised unless proper personal protective procedures are carried out.

Further Reading Albariño, C.G., Wiggleton Guerrero, L., Spengler, J.R., et al., 2015. Recombinant Marburg viruses containing mutations in the IID region of VP35 prevent inhibition of Host immune responses. Virology 476, 85–91. Bharat, T.A., Riches, J.D., Kolesnikova, L., et al., 2011. Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells. PLoS Biology 9, e1001196. Cross, R.W., Mire, C.E., Feldmann, H., Geisbert, T.W., 2018. Post-exposure treatments for Ebola and Marburg virus infections. Nature Reviews Drug Discovery 17, 413–434. Feldmann, H., Sanchez, A., Geisbert, T.W., 2013. Filoviridae: Marburg and Ebola Viruses. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed. Philadelphia, PA, USA: Lippincott Williams & Wilkins. Kortopeter, M.G., Dierberg, K., Shenoy, E.S., Cieslak, T.J., Medical Countermeasures Working Group of the National Ebola Training and Education Center's (NETEC) Special Pathogens Research Network (SPRN), 2020. Marburg virus disease: A summary for clinicians. International Journal of Infectious Diseases 99, 233–242. Mehedi, M., Groseth, A., Feldmann, H., Ebihara, H., 2011. Clinical aspects of Marburg hemorrhagic fever. Future Virology 6, 1091–1106. Messaoudi, I., Amarasinghe, G.K., Basler, C.F., 2015. Filovirus pathogenesis and immune evasion: Insights from Ebola virus and Marburg virus. Nature Reviews Microbiology 13, 663–676. Olejnik, J., Mühlberger, E., Hume, A.J., 2019. Recent advances in marburgvirus research. F1000Research 8. Pattnaik, A.K., Whitt, M.A., 2015. Biology and pathogenesis of rhabdo- and filoviruses. New Jersey: World Scientific. Towner, J.S., Khristova, M.L., Sealy, T.K., et al., 2006b. Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola. Journal of Virology 80, 6497–6516.

Relevant Websites https://clinicaltrials.gov/ct2/show/NCT02891980?cond=Marburg þ Virus þ Disease&draw=2&rank=6 A Safety Trial to Test MVA-BN(R)-Filo and Ad26.ZEBOV Vaccines in Healthy Volunteers. www.ictv.global/report/filoviridae Filoviridae. International Committee on Taxonomy of Viruses (ICTV). https://vhfimmunotherapy.org/ Viral Hemorrhagic Fever Immunotherapeutic Consortium.

Measles Virus (Paramyxoviridae) Roberto Cattaneo, Mayo Clinic, Rochester, MN, United States Michael McChesney, University of California, Davis, CA, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of R. Cattaneo, M. McChesney, Measles Virus, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B9780-12-801238-3.02619-2.

Glossary Oncolytic virotherapy The treatment of cancer patients based on the administration of replication-competent viruses that selectively destroy tumors but leave healthy tissue unaffected.

Subacute sclerosing panencephalitis (SSPE) A rare but always lethal brain disease that can occur several years after acute measles. Syncytia Fused cells with multiple nuclei characteristics of measles virus infection.

Classification Measles virus (MeV) is an enveloped nonsegmented negative-strand RNA virus of the order Mononegavirales, family Paramyxoviridae, genus Morbillivirus. Measles is the only medically relevant Morbillivirus; Paramyxoviridae of other genera include prevalent mumps and parainfluenza viruses, and emerging bio-threats to humans and economically important livestock like Nipah and Hendra viruses. Among negative-strand RNA viruses, the Paramyxoviridae are defined by a membrane fusion apparatus that can trigger fusion of viral and cell membranes at neutral pH. The genus Morbillivirus consists of closely related, but host-specific viruses that cause age-old diseases of humans and animals like measles, rinderpest, and canine distemper. In addition, newly recognized morbilliviruses of aquatic mammals cause emerging diseases. The defining characteristics of the Morbilliviruses is that the attachment protein hemagglutinin (H) does not have neuraminidase activity, but interacts with two different receptors: first, the primary receptor signaling lymphocyte activation molecule (SLAM, CD150), and then the adherens junction protein, nectin-4. Morbillivirus infection of SLAM-expressing immune cells explains immunosuppression, and infection of upper airways epithelial cells expressing nectin-4 accounts for extremely efficient transmission of the virus.

Virion Structure MeV particles are enveloped by a lipid bilayer derived from the plasma membrane of the cell in which the virus was grown. Fig. 1 illustrates a MeV particle (top), which components are color-coded as and mapped on the viral genome (bottom). The particles of MeV and the other members of the Paramyxoviridae have been visualized as pleomorphic or spherical, depending on the methods used for their purification. Their diameter ranges from 120 to 300–1000 nm, implying that their cargo volume may differ by a factor 30. Indeed, large particles can contain multiple encapsidated genomes, as deduced initially from sedimentation and ultraviolet inactivation studies. Three RNA genomes are drawn schematically in the MeV particle of Fig. 1. In cultivated cells, MeV particles accumulate below the plasma membrane, and the ratio of intracellular to secreted infectious virus is approximately 10:1. Therefore ultrastructural studies have been performed mainly in virus-infected cells. Fig. 2 visualizes by cryo-electron tomography a MeV particle being assembled in infected HeLa cells. Glycoprotein spikes (H and F proteins) that extend approximately 8–12 nm from the surface of the membrane are inserted into the viral envelope. The two viral glycoproteins form the membrane fusion apparatus: the H protein contacts the cellular receptors, whereas F fuses the viral and cellular membranes. The F protein spike is trimeric, whereas the H protein forms covalently linked dimers that form noncovalently linked tetramers. In the assembly process, the viral glycoproteins are preferentially incorporated into nascent viral particles, whereas the majority of host proteins are excluded. The matrix (M) protein is observed as an organized two-dimensional para-crystalline array associated with the inner leaflet of the plasma membrane. It is the assembly organizer, and bridges the envelope with the ribonucleocapsids (RNP). The helical RNP is the replicative complex: the RNA genome is tightly encapsidated by a helically arranged nucleocapsid (N) protein. Two other proteins, a polymerase (L for large) and a polymerase cofactor (P for phosphoprotein), are associated with the RNP. In monolayers of most immortalized cell lines, MeV infections spread mainly through fusion of an infected cell with recipient cells expressing an appropriate receptor, rather than through particle budding. Receptors on recipient cells trigger the viral membrane fusion apparatus expressed on the surface of infected cells to form fusion pores; pore expansion then results in coalescence of plasma membranes, and formation of large multinucleated syncytia. This mechanism promotes the spread of progressively larger genome populations, without selection for individual genomes. From this vantage point, the MeV infectious “unit” appears to be highly complex and diverse. Indeed, genetic analyses of MeV infections have provided independent evidence of multi-genome MeV transmission.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21506-9

619

620

Measles Virus (Paramyxoviridae)

Fig. 1 Diagram of a MeV particle, and of MeV transcription and replication. (A) The particle is drawn with its six main components: the nucleocapsid (N) that covers the genomic RNA and interacts with the phosphoprotein (P), and polymerase (large, L), forming the ribonucleocapsid complex; the fusion (F) and hemagglutinin (H) proteins forming the membrane fusion apparatus; and the matrix (M) protein controlling particle assembly as well as transcription and membrane fusion. Particles can contain multiple encapsidated genomes, of which three are drawn schematically in the particle. (B) The encapsidated negative strand RNA genome, which serves as template for transcription. The coding regions of the proteins are color-coded, non-coding regions are black. Transcription starts with a short, uncapped leader RNA (not shown) from the 30 end of the genomic RNA; this is followed by the transcription of 50 capped and polyadenylated mRNAs (wavy lines), which encode the viral proteins. The polymerase complex stops at the end of each transcript, polyadenylates (A), ignores the 3-nucleotide intergenic region, and restarts transcription. However, re-initiation of transcription is not always successful, resulting a gradient in the direction of 30 to 50 . In the middle of the P mRNA, the polymerase complex adds a single G residue (G) to about half of the transcripts, allowing access to the V reading frame. The C reading frame is accessed by an alternative ribosome initiation codon. (C) During replication, the polymerase complex ignores the transcription stop/start signals, rendering a full-length antigenomic RNA, which is also encapsidated. The synthesized antigenome serves then as template for the synthesis of additional copies of genomic RNA. Reproduced from Cattaneo, R., Donohue, R.C., Generous, A.R., Navaratnarajah, C.K., Pfaller, C.K., 2019. Stronger together: Multi-genome transmission of measles virus. Virus Research 265, 74–79.

Genome The 15,894 nucleotides (nt) MeV negative-strand RNA genome begins with a 56 nt 30 region, called leader, and ends with a 40 nt region, called trailer. These control regions are essential for transcription and replication and flank the six genes. The term gene refers here to contiguous transcription units separated by three untranscribed nucleotides. There are six genes coding for eight

Measles Virus (Paramyxoviridae)

621

Fig. 2 Cryo-electron tomography of a MeV assembly site in HeLa cells. (a) Tomographic slice (1.18 nm thick) of the assembly site. (b) Three-dimensional reconstruction of the data. Color coding: glycoproteins (purple), viral membrane (red), M (green), RNP (gold), and actin filaments (magenta). Black arrows indicate areas where M is absent on the viral membrane. Scale bars are 100 nm. Reproduced from Ke, Z., Strauss, J.D., Hampton, C.M., et al., 2018. Promotion of virus assembly and organization by the measles virus matrix protein. Nature Communications 9, 1736.

proteins, in the order (positive strand): 50 -N-P/V/C-M-F-H-L-30 (Fig. 1, bottom). The P gene uses overlapping reading frames to code for three proteins, P, V, and C. Complete sequences of several MeV wild type and vaccine strains have been published in GenBank.

Replicative Complex The first gene codes for the N protein. Each N protein interacts with and covers 6 nt, and about 13 N proteins constitute a turn in the RNP helix (Fig. 3). RNPs are formed when N is expressed in the absence of other viral components, suggesting that N–N interactions drive RNP assembly. Two domains have been identified in the N protein: a conserved amino-terminal NCORE (about 400 amino acids) and a variable carboxyl-terminal NTAIL (about 100 amino acids). N core is essential for self-assembly, RNA binding, and replication activity, whereas N tail interacts with a carboxyl-terminal domain of the P protein (Fig. 3, upper right). N protein exists in at least two forms in infected cells: one associated with RNA in an RNP structure, and a second unassembled soluble form called N0 that may encapsidate the nascent RNA strand during genome and antigenome replication. The second gene codes for three proteins implicated in transcription or innate immunity control: P, V, and C proteins. Two of these proteins have a modular structure: the 231 amino-terminal residues of V are identical with those of P, but its 68 carboxylterminal residues are translated from a reading frame accessed by insertion of a pseudo-templated G residue through polymerase stuttering. This V domain is highly conserved in Paramyxoviridae, with cysteine and histidine residues binding two zinc atoms per protein. The main function of the V protein is to counteract the innate immune response: V interacts with both STAT2 and MDA5, thereby antagonizing both type I interferon signaling and interferon production. The main function of the P protein (Fig. 3, red) is to support viral replication and transcription; it is an essential component of the polymerase, and of the protein complex mediating RNA encapsidation by N0 protein. The P protein amino-terminal segment, identical with the V protein amino-terminus, is phosphorylated on serine and threonine residues and it contains regions of high intrinsic disorder, possibly facilitating interactions with multiple proteins. P self-assembles as a tetramer through a central region in its unique domain, and interacts with NTAIL through its carboxyl-terminus. The third protein expressed from the P gene is called C. Its reading frame is accessed by ribosomes initiating translation 22 bases downstream of the P AUG initiation codon. The MeV C protein not only inhibits the interferon response but also enhances infectivity. The third, fourth, and fifth genes code for envelope associated proteins that are discussed below in the context of receptor recognition and membrane fusion. The sixth and last gene codes for the RNA-dependent RNA polymerase (large protein, L), that possesses all enzymatic activities necessary to synthesize mRNA: nucleotide polymerization, capping and methylation, and polyadenylation. L adds poly-A tails to nascent viral mRNAs co-transcriptionally, by stuttering on a stretch of U residues occurring at the end of each viral gene. Sequence comparison identified six highly conserved domains in the L protein that were tentatively

622

Measles Virus (Paramyxoviridae)

Fig. 3 Proteins involved in MeV replication, and possible positioning of the N, P and L proteins. The solvent-accessible 30 end of the RNA genome (orange surface) is positioned near the amino-terminus of the first N protomer (adjacent N protomers are shown in different shades of blue). Only NTAIL of the terminal N protomer is shown for simplicity. The protomer can bind P via two possible mechanisms, via an interaction between the NTAIL molecular recognition element and the XD domain of P, and via the N-terminal peptide of P (PNTD). Reproduced with permission from Desfosses, A., Milles, S., Jensen, M.R., et al., 2019. Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication. Proceedings of the National Academy of Sciences of the United States of America 116, 4256–4264.

assigned different catalytic functions. Fig. 3 uses the polymerase of vesicular stomatitis virus (LVSV, yellow) to represent the MeV L protein, for which no atomic resolution structure is available.

Transcription and Replication After cell entry, the polymerase transcribes the viral genome with a sequential “stop–start” mechanism. The polymerase accesses the genome through an entry site located near its 30 end, transcribes the first gene (N) with high processivity, polyadenylates the N mRNA, and reinitiates P mRNA synthesis. The frequency of re-initiation is less than 100%, resulting in a gradient of transcript levels; N is transcribed at the highest levels, the most promoter-distal L gene at the lowest. The gene order and transcription strategy are fundamental characteristics that MeV shares with all other Paramyxoviridae. The negative-strand genome is then used to synthesize positive-strand “antigenomes” that produce more genomes, completing one amplification cycle. Amplification produces the genomic templates for secondary transcription.

Envelope Complex The third gene codes for the M protein, a moderately hydrophobic protein that can associate with membranes. M also binds the RNP, and interacts with the F and H protein cytoplasmic tails, modulating cell fusion. M is the assembly factor and may also drive viral particle release.

Measles Virus (Paramyxoviridae)

623

The fourth gene codes for the F protein. An interesting peculiarity of the F mRNA is its long (almost 500 nt) 50 untranslated region (Fig. 1). The function of this region has not been characterized, but it may regulate protein expression. The F protein is synthetized as a precursor F0, which folds into a trimer that is cleaved into two subunits, F1 and F2 by the ubiquitous intracellular protease, furin. Only activated trimers can unfold and cause membrane fusion, a process which occurs after the attachment protein H contacts a receptor and transmits the fusion triggering signal to the F trimer. The fifth gene codes for the attachment protein. Since this protein hemagglutinates certain nonhuman primate red blood cells, but lacks neuraminidase activity, it is called H instead of HN. Other Paramyxoviridae use sialic acid as their receptors, and they need neuraminidase to destroy receptor activity while budding from a host cell. Morbilliviruses do not use sialic acid as a receptor and do not need a neuraminidase. The nature and cell-specific expression of the two cellular MeV receptors, SLAM and nectin-4, explains the tissue specific tropism on MeV infections.

Life Cycle MeV begins its circuit through selected organs of the human body within alveolar macrophages and dendritic cells (DC), which express the primary receptor SLAM. These cells first transfer the infection through the epithelial barrier, and then spread it into lymphoid tissues (Fig. 4, left panel). SLAM was originally identified on activated B and T lymphocytes, but it is also expressed constitutively on immature thymocytes, memory T cells, and certain B cells. Subsets of other cell types, including monocytes and DC, also express SLAM. This cellular distribution overlaps with the susceptibility of different cell types to wild-type MeV infection. Another strong argument for the central role of SLAM in MeV tropism is the fact that three morbilliviruses (MeV, canine distemper virus, and rinderpest virus) enter cells via SLAM (human, canine, or bovine, respectively). Experiments in macaques revealed that the earliest target cells after intratracheal MeV inoculation are DCs and alveolar macrophages. More recently nectin-4, also called poliovirus receptor-like-4 (PVRL4), was shown to serve as the receptor for MeV spread in the upper airway epithelium (Fig. 4, right panel). This adherens junction protein interacts with H with five times higher affinity than SLAM. Nectin-4 sustains basolateral entry of MeV, and of all animal morbilliviruses examined, into upper airway epithelial cells, including those of the trachea. Contrary to the infections with other respiratory viruses, MeV enters the airway epithelium “en masse”: several days after hostto-host transmission, highly infected immune cells synchronously deliver large amounts of virus to the upper airways, for secondary amplification. Since the upper airways are the anatomical location most useful to support particle aerosolization, this two-phase mechanism of host invasion may account for the extremely contagious nature of MeV infection. The live attenuated MeV vaccine strain, Edmonston, can also use the regulator of complement activation membrane cofactor protein (MCP; CD46) as a receptor. The primary function of CD46 is to bind and promote inactivation of the C3b and C4b complement products, a process protecting human cells from lysis by autologous complement, a function that requires ubiquitous expression. Only tissue-culture adapted MeV interact with CD46, and indiscriminate cell entry through this protein correlates with MeV attenuation. A chimeric MeV expressing a vaccine strain H protein, which binds to CD46, is attenuated in the nonhuman primate, and productive infection is still confined to SLAM-expressing cells in vivo.

Epidemiology Humans in all age groups are susceptible to measles, but because the immunity of natural infection is protective life-long, measles occurs only once in a lifetime, as documented by Panum in the 19th century. This clearly differs from infections with other respiratory tract viruses (e.g., respiratory syncytial virus or influenza). Morbidity from measles complications is highest in infants and immunocompromised hosts (non-immune, pregnant women or patients with cancer or AIDS). Measles remains one of the most contagious of infectious diseases, with attack rates approaching 99% in susceptible populations. Using several epidemiological modeling studies in the recent past, a consensus on the level of herd immunity required to prevent a measles epidemic is up to 94%. One explanation for the high level of contagion is that, contrary to the infections with other respiratory viruses, MeV enters selectively the upper airway epithelium after strong amplification of the infection in immune cells. MeV is then transmitted by respiratory aerosol from infected hosts by coughing and sneezing in the prodromal phase through several days after onset of the measles rash. In the past, measles epidemics targeted young children. Since the widespread vaccination campaigns began in 1963, measles outbreaks have occurred in older children and young adults. The shifting pattern of measles outbreaks may be due to failure to vaccinate, and to variable rates of primary or secondary vaccine failures.

Clinical Features Measles, one of the most contagious infectious diseases of man, was recognized clinically by the rash and other signs from early historical times. It was differentiated from smallpox by Rhazes in the tenth century, who noted that it could be more fatal than smallpox. Transmitted by aerosol droplets, the infection has an incubation period of 7–10 days, with onset of fever, cough, and

624

Measles Virus (Paramyxoviridae)

Fig. 4 How MeV spreads in its host. Measles and the other morbilliviruses have developed an immunosuppressive host invasion strategy: the airways epithelium is initially bypassed within carrier immune cells (left panel and inset). After replication in cells of lymphoid organs, epithelial invasion occurs from the basolateral side (right panel and inset). Left half: bypass of the airway epithelium. MeV (red dots), once inhaled, infects alveolar macrophages or dendritic cells that express SLAM (blue protein on the surface of an immune cell, top left). Virus-carrying immune cells traverse the airways epithelium (inset, top to bottom) and deliver the infection to the local lymph nodes (red to indicate infection). The infection spreads rapidly to the primary lymphoid organs, thymus and spleen, where the virus efficiently replicates. Right half: basolateral entry and spread through the airway epithelium. Inset, right side: after viral amplification in lymphoid organs, circulating immune cell delivers the infection to epithelial cells. The plasma membrane of the infected cells is drawn as thick red line to indicate the presence of viral glycoproteins. The membranes of the immune and epithelial cell fuse when the viral attachment protein binds to nectin-4 in the adherens junction (AJ, green, shown only in the lower epithelial cells). Then the infection spreads laterally in the epithelium via adherens junction (black arrows). Infected epithelial cells (top two cells with red plasma membranes) release progeny viral particles in the airway lumen. Preferential nectin-4 expression in the tracheal epithelium (shown in red) facilitates host exit at a strategic location for efficient aerosol particle formation, and explains extremely efficient transmission of MeV. Apical secretion of MeV particles is inefficient, but infectious centers detaching from the epithelium may also be expulsed by coughing and sneezing. TJ: tight junction. Reproduced with permission from Mateo, M., Generous, A., Sinn, P.L., Cattaneo, R., 2015. Connections matter  How viruses use cell–cell adhesion components. Journal of Cell Science 128, 431–439.

coryza, followed in about 4 days by the skin rash which begins on the face and spreads to the whole body. The skin rash fades in about 5 days and clinical resolution is usually uneventful. Measles is typically an infection of childhood and protective immunity is lifelong, such that a second case of measles in a child or adult is highly unusual. Prior to widespread vaccination against measles in the 1960s, the infection had a case–fatality rate of less than 5% in children, higher in infants and in children in low income countries, where fatality rates of up to 20% can still occur.

Complications Complications include pneumonia, encephalitis, otitis media, blindness, and secondary infections by common bacteria and viruses. In low income countries, common complications are diarrhea and wasting. “Atypical measles”, a severe MeV infection that occurred following the use of whole, inactivated virus vaccine from 1963 to 1967, was characterized by high and persistent fever, a

Measles Virus (Paramyxoviridae)

625

different body rash that resembles Rocky Mountain spotted fever, and lobar pneumonia with effusions. This unusual clinical manifestation is discussed in the section on Pathogenesis. Compared to infections by other respiratory viruses, measles has more serious clinical potential, possibly because the other infections are usually confined to the respiratory tract and not systemic; but measles in man causes less disease and death than infections due to the most closely related morbilliviruses, rinderpest in cattle, or canine distemper in dogs.

Immunosuppression Latent tuberculosis may be reactivated following MeV infection. This predictable complication of MeV-induced immunosuppression was initially studied by von Pirquet, who observed that the tuberculin skin test response is suppressed during MeV infection. MeV-induced immunosuppression has an onset near to the peak of viremia, such that the primary immune responses to MeV are not impaired, but the immunosuppression is severe and lasts for several months to years. The severe and sustained nature of immunosuppression may be mainly the result of strong replication of the virus in SLAM-expressing cells, which include T and B memory cells. Infections by other morbilliviruses are also associated with immunosuppression and this complication was a hallmark of epidemic disease in harbor seals that resulted in the discovery of a new morbillivirus, the phocine distemper virus. In the extreme case, immunosuppression with massive lymphocyte depletion is fatal in canine distemper virus-infected ferrets and rinderpest-infected cattle.

Immune Responses The primary immune responses to MeV, initially IgM antibody response, type 1 CD4 and CD8 þ T-cell responses, followed by neutralizing IgG antibodies, are completely effective in controlling viral replication and resolution of the infectious process. As originally observed by Macfarlane Burnet, both primary and secondary immunodeficiencies that impair T cell responses, for example, the DiGeorge syndrome or advanced HIV infection, are a significant risk for failure of the host to control MeV infection, resulting in persistent infection and death or serious disease in the lower respiratory tract and the central nervous system. In contrast, deficiency of the antibody response does not impair the immune control of MeV replication. These fundamental observations in antiviral immunity were confirmed by experimental infections of rhesus monkeys with MeV. CD8 þ T cell depletion of monkeys at the time of viral inoculation resulted in prolonged viremia until the T cells repopulated, but depletion of B cells had no significant effect on MeV infection. A persistent and/or a fatal MeV infection has occurred in HIV-infected children, with CD4 þ T cell counts being below 200/ml, who contracted measles. This happened to a young, HIV-infected man who was vaccinated with live, attenuated MeV vaccine as required for college admission. However, the risk of a serious disease form of measles in immunocompromised hosts (50% or greater case fatality) outweighs the risk of vaccination, and measles vaccination is recommended for HIV-infected infants and children unless they have severe immunodeficiency with low, age-adjusted CD4 þ T cell counts in blood.

Persistent Infections Persistent MeV infection is rare, but it can result in giant cell pneumonia or two different neurological diseases: measles inclusion body encephalitis and subacute sclerosing panencephalitis (SSPE). In SSPE, MeV infection spreads slowly by cell contact within the central nervous system; however, the virus is typically defective in its expression of M and/or F proteins and the infection does not lead to the formation of progeny virus particles. Another rare neurologic complication, acute demyelinating encephalomyelitis, not due to continuing viral replication in the brain, is associated with autoimmunity to myelin. Several other diseases, including multiple sclerosis, inflammatory bowel disease and certain behavioral disturbances, have been linked anecdotally to MeV infection, but causal connections have not been established.

Pathogenesis The histopathological hallmark of MeV infection is the formation of syncytia, or multinucleated giant cells. Syncytial cells are not unique to MeV infection but they are characteristic. MeV infects cells of ectodermal, endodermal, and mesenchymal origin and syncytial cells have been observed in all of these cell types. Multinucleated giant cells were observed in lymphoid organs and in mononuclear cell aggregates in many tissues, including the inflamed lung, where they are referred to as Warthin–Finkeldey giant cells. These syncytia are of lymphocyte, macrophage, dendritic, or reticular cell origin. Syncytial cells are also readily observed in epithelia, including the columnar epithelium of the trachea and bronchi, the stratified squamous epithelium of the skin and buccal mucosa, and the transitional epithelium of the urinary bladder and urethra. Endothelial syncytial cells were observed in small pulmonary arteries of monkeys infected with MeV. Eosinophilic cytoplasmic and nuclear inclusion bodies can be seen in measles giant cells. The syncytial cells of measles are not long lived and they disappear with resolution of the infection.

626

Measles Virus (Paramyxoviridae)

The major pathologic changes in measles infection are due to inflammation and necrosis followed by tissue repair without fibrosis. Secondary infections by bacterial or other viral pathogens are common and they alter the pathologic process accordingly, especially in the respiratory and gastrointestinal tracts. Pathology in the lower respiratory tract is mainly peribronchiolar inflammation and necrosis with a mild exudate, but interstitial pneumonitis with mononuclear cell infiltrates may occur. In the brain and central nervous system, perivascular mononuclear infiltrates can occur with a few necrotic endothelial cells, microglia, and neurons. In lymph nodes, spleen, and thymus, the major changes are mild to moderate lymphocyte depletion and multinucleated giant cells. Lymph node and splenic follicular hyperplasia are not seen in primary measles infection but are present if the host was previously infected or vaccinated against measles. The unexpected occurrence of “atypical measles” following vaccination with whole-inactivated virus in the early 1960s and subsequent infection with wild-type virus is thought to be due to an aberrant, anamnestic host immune response resulting in an Arthus reaction or delayed hypersensitivity. Similar immunopathology was seen in children exposed to respiratory syncytial virus (RSV) following vaccination with whole-inactivated RSV. Atypical measles was experimentally induced in monkeys vaccinated with a whole-inactivated MeV vaccine followed by challenge with a wild type virus. Compared to monkeys vaccinated with a live, attenuated MeV vaccine, the aberrant immune responses associated with atypical measles resulted in immune complex deposition and eosinophilia. A marked skewing of T cell cytokine responses toward type 2 response, with abnormally high interleukin 4 production was also observed.

Diagnosis As measles became a rare illness in regions with high vaccine coverage where the virus has been eliminated or controlled, the diagnosis of MeV infection became a challenge. The typical punctate, maculopapular skin rash, and the prodromal signs and symptoms are not specific for this viral infection and measles diagnosis requires differentiation from other pathogens causing exanthems in children, including Epstein–Barr Virus and cytomegalovirus infections, rubella, dengue, Zika virus infection, scarlet fever, typhus, toxoplasmosis, meningococcus, and staphylococcus. An enanthem with small white lesions on the buccal, labial, or gingival mucosal (Koplik’s spots) is considered specific for MeV infection and it typically occurs a few days before the skin rash. A buccal swab smeared on a glass slide, fixed and stained (e.g., Wright–Giemsa), may show multinucleated syncytia of epithelial cells. Secondary infections due to MeV-induced immunosuppression are caused by the common pathogens in a geographic region. Serological diagnosis of MeV infection is based on early IgM response in blood by antibody-capture ELISA or by other methods detecting increase in serum IgG antibodies between paired serum specimens. MeV can be cultured from peripheral blood mononuclear cells and from oral or nasal aspirates. Rescue of wild-type MeV is readily done using B95a cells, or Vero cells expressing human SLAM. The virus can be detected by reverse transcription polymerase chain reaction (RT-PCR) from these samples and from urine. Genetic sequencing of MeV isolates worldwide has yielded clustering of viral genomes into ca. 24 distinct genotypes, based on a variable carboxyl-terminal region of the N protein, which have been used to track imported cases of MeV infection and to distinguish wild type virus from vaccine strains. As of 2018, only 4 MeV genotypes have been circulating globally (Fig. 5). The World Health Organization (WHO) hosts a laboratory manual and website with descriptions of measles diagnostic samples and assays.

Fig. 5 Global distribution of MeV genotypes, 2016–2018. The size of the circles reflects the numbers of identifications reported for each genotype. Source: World Health Organization. Reproduced from Brown, K.E., Rota, P.A., Goodson, J.L., et al., 2019. Genetic characterization of measles and Rebella viruses detected through global measles and Rubella elimination surveillance, 2016–2018. Morbidity and Mortality Weekly Report 68, 587–591.

Measles Virus (Paramyxoviridae)

627

Treatment No specific antiviral treatment is recommended for the typical case of measles in a child or adult. In addition to supportive care, vitamin A can prevent disease complications and WHO recommends its preventive use. In a measles outbreak, passive antibody, in the form of human immune globulin given by the intramuscular or intravenous route, can protect non-immune, immunocompromised hosts from serious complications of measles. The antiviral, ribavirin, may attenuate severe measles pneumonia or disease complications in an immunocompromised patient. Novel viral polymerase inhibitors may become available to treat severe measles in the near future.

Prevention A live attenuated MeV strain, called Edmonston, developed by John Enders and further attenuated by Maurice Hilleman in the 1950s, was licensed in the US in 1963. In 1971, the US government licensed Merck to produce MMR (for Measles, Mumps and Rubella) vaccine by combining MeV with mumps and rubella live, attenuated vaccine viruses. MMR remains one of the safest and most effective human vaccines. Some other attenuated measles vaccine strains are used in other countries. It is remarkable that for this RNA virus, vaccine failure due antibody escape has never occurred. Measles remains one of the leading causes of vaccine-preventable diseases and death in the world. The WHO and other international bodies have proposed the global eradication of MeV in the twenty-first century, following the current campaign to eradicate poliovirus. This is an achievable goal as humans are the only known host species for this virus, although nonhuman primates are susceptible hosts. Regional elimination of MeV has been achieved: MeV circulation has been interrupted for decades in the Americas, and in many European countries, by the vaccination of children with the live, attenuated vaccines in current usage. However, decline of vaccination coverage has led to recent measles re-emergence in Europe and North America. Many countries are planning to increase vaccination coverage as they progress toward targeted reduction of measles mortality, or elimination of transmission. Recent experience in high and low income countries has shown that the maintenance of effective herd immunity against MV requires a two-dose vaccination strategy. Because of the highly contagious nature of this infection, about 95% of children require vaccination to prevent the circulation of MeV in a population of more than several hundred thousand. Greater than 90% measles vaccine coverage has been achieved in many countries but more than 25% of countries have not reached this level of vaccination yet. The HIV pandemic presents a potential obstacle to MeV eradication globally, especially in Africa and Asia, but HIV-infected children that are not immunocompromised can be safely vaccinated with live, attenuated MeV (see above). The importation of MeV into countries where elimination has been achieved is a very real challenge for diagnosis and control which has been successfully met by molecular genetic methods of rapid viral genomic sequencing and taxonomy.

MeV-Based Vaccines and Cancer Therapeutics The live, attenuated measles vaccine, which has an outstanding efficacy and safety record, is being developed as a pediatric vaccine eliciting immunity against additional microbial infections. Vectored MeV-expressing the hepatitis B surface antigen (HBsAg) has been generated by reverse genetics. One of these vectored MeV vaccines induced protective levels of HBsAg antibodies while protecting rhesus macaques against measles challenge. Another vectored MeV expressing the capsid and envelope protein from chikungunya virus is currently in a phase III clinical trial. Another advantage of immunization with a di- or multivalent MV-based vaccine is the delivery of an additional immunization safely without additional cost. The knowledge gained from basic research has also been applied to the development of vectors for targeting and eliminating cancer cells. Oncolytic virotherapy is the experimental treatment of cancer patients based on the administration of replicationcompetent viruses that selectively destroy tumor cells but leave healthy tissue unaffected. MeV is one of several human vaccine strains, or apathogenic animal viruses, currently being genetically modified to improve oncolytic specificity and efficacy. These modifications include targeting cell entry through designated receptors expressed on cancer cells. Moreover, cancer cell specificity can be enhanced by silencing the expression of proteins that counteract the interferon system, which is often non-functional in cancer cells. Clinical trials of MV-based oncolysis for ovarian cancer, myeloma, and glioma are ongoing.

Further Reading Bankamp, B., Hickman, C., Icenogle, J.P., et al., 2019. Successes and challenges for preventing measles, mumps and rubella by vaccination. Current Opinion in Virology 34, 110–116. Griffin, D.E., 2013. Measles virus. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Lippincott Williams and Wilkins, pp. 1042–1069. Katz, S.L., 2004. Measles (rubeola). In: Gershon, A.A., Hotez, P.J., Katz, S.L. (Eds.), Krugman’s Infectious Diseases of Children, eleventh ed. Philadelphia, PA: Mosby, pp. 353–372. Lamb, R.A., Parks, G.D., 2013. Paramyxoviridae. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Lippincott Williams and Wilkins, pp. 957–995. McChesney, M.B., Miller, C.J., Rota, P.A., et al., 1997. Experimental measles. I. Pathogenesis in the normal and the immunized host. Virology 233, 74–84.

628

Measles Virus (Paramyxoviridae)

Miest, T.S., Cattaneo, R., 2014. New viruses for cancer therapy: Meeting clinical needs. Nature Reviews Microbiology 12, 23–34. Mühlebach, M., Mateo, M., Sinn, P.L., et al., 2011. Adherens junction protein nectin-4 (PVRL4) is the epithelial receptor for measles virus. Nature 480, 530–533. Navaratnarajah, C.K., Generous, A.R., Yousaf, I., Cattaneo, R., 2020. Receptor-mediated cell entry of paramyxoviruses: Mechanisms, and consequences for tropism and pathogenesis. Journal of Biological Chemistry 295, 2771–2786. Panum, P., 1939. Observations made during the epidemic of measles on the Faroe Islands in the year 1846. Medical Classics 3, 829–886. Pfaller, C.K., Bloyet, L.-M., Donohue, R.C., et al., 2020. The C protein is recruited to the measles virus ribonucleocapsid by the phosphoprotein. Journal of Virology 94, e01733. Rota, P.A., Moss, W.J., Takeda, M., et al., 2016. Measles. Nature Reviews Disease Primers 2, 16049. von Pirquet, C., 1908. Das Verhalten der kutanen Tuberkulinreaktion wahrend der Masern. Deutsche Medizinische Wochenschrift 34, 1297–1300.

Relevant Websites https://www.who.int/immunization/monitoring_surveillance/burden/laboratory/manual/en/ Immunization, Vaccines and Biologicals. https://www.mayoclinic.org/diseases-conditions/measles/symptoms-causes/syc-20374857 Measles. https://www.cdc.gov/measles/index.html Measles (Rubeola).

Molluscum Contagiosum Virus (Poxviridae) Joachim J Bugert and Rosina Ehmann, Bundeswehr Institute of Microbiology, Munich, Germany r 2021 Elsevier Ltd. All rights reserved. This is an update of J.J. Bugert, Molluscum Contagiosum Virus, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B9780-12-801238-3.02621-0.

Nomenclature H&E stain

Histological staining method using the two colorants hematoxylin and eosin

Glossary Acanthoma Benign tumor of the stratum spinosum of the dermal epithelium. Curettage Scoop-like surgical instrument for the removal of tissue by scraping or scooping. Detritus Accumulation of dead biological material, e.g., dead cells from tissue.

Florid Disease manifestation in its fully developed form. Histology The study of microscopic structures of tissues often with the help of fixed and stained sections. Lobule Anatomical term for a clear division or extension of an organ. Stratum spinosum Prickle cell layer in the dermal epithelium.

Virion Structure MCV particles have a brick-shaped ovoid morphology similar to other vertebrate poxviruses with an average size of 360  210 nm and the tubular core protein structures typical for MCV (Fig. 1).

History “Molluscum contagiosum” was first described as a separate disease entity and transmissible infection of the human skin by T. Bateman (1778–1821) in 1814. W. Henderson subsequently observed viral inclusion bodies in microscopical sections of MCV infected skin, which he named “molluscum bodies”, in 1841, also known as “Henderson–Paterson bodies”. Molluscum bodies were later compared to “Borrel bodies”, inclusion bodies found in avipoxvirus associated infections, and a viral etiology was therefore proposed by Goodpasture, Woodruff and King around 1930. MCV was first transmitted between human volunteers using sterile filtered (cell free) extracts of human MCV lesion material in the laboratories of Juliusberg, Wile and Kingery in the early 20th century.

Fig. 1 MCV electron microscopy. Ammonium molybdate negative stain of an MCV particle demonstrating the typical core protein pattern and envelope structure (scale bar 200 nm). Electron microscopy: Bugert and Hobot, Cardiff University School of Medicine, 2005.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21229-6

629

630

Molluscum Contagiosum Virus (Poxviridae)

Genome The genomes of sequenced MCV isolates vary slightly in size around 190,000 bases and show the classical structure of poxviruses with a “conserved core” harboring the genes that code for the proteins of the replication and transcription machinery. The conserved core region is flanked by genes that target interactions with the host immune system. The left and right genome termini contain “inverted terminal repeats”, important structures needed for the replication process of poxviruses. To date there are 15 full length genomes available for MCV subtypes 1 and 2. Two other subtypes named 3 and 4 are rarely described and so far have not been characterized on the genome level. MCV subtypes 1 and 2 are genetically very similar and all subtypes 1–4 of MCV cause the same clinical picture. MCV is predicted to encode a total of 170–181 proteins with many of them not yet characterized.

Life Cycle MCV is an extremely specialized virus that targets only human keratinocytes. After attachment of MCV particles to target cells and subsequent entry, genomic DNA is released into the cytosol by a process called uncoating. mRNA synthesis of viral genes is regulated into three temporal phases: early, intermediate and late. This allows the virus to first produce viral proteins that interact with the host immune system to create a favorable environment for virus replication and transcription in the cell while proteins needed for the assembly of complete virus particles may be produced at a later time point of infection. After formation of progeny virus the particles have to undergo complex maturation steps until they are released from the cell ready to initiate the next replication cycle (Fig. 2). Interestingly, MCV does not replicate in standard cell culture systems. It causes cytopathogenic effects in human fibroblasts and expresses early genes in a large range of human and animal cells. However, the infection does not yield virus in vitro, and seems to be abortive due to what appears to be a second stage uncoating defect (Fig. 2).

Epidemiology MC is a frequent disease with worldwide distribution. Reported incidence rates vary between 0.14%–7% and seem to be highest in children aged 2–5 years. However, infection can occur at any age and all ethnicities are affected. Hot and humid climates might favor infection with MCV. Serological studies in several countries suggest that MCV has a surprisingly high prevalence in many populations. Non-neutralizing antibodies are found in 5%–30% of populations studied so far. The discrepancy to the considerably lower incidence rates could be explained by a high number of subclinical or very mild infections. Risk groups for MC

Fig. 2 MCV life cycle in standard single cell culture (green) on the background of the full poxviral lifecycle.

Molluscum Contagiosum Virus (Poxviridae)

631

include groups of people living under crowded and poor hygienic conditions or closed communities like nurseries or military barracks. as well as immunocompromised individuals. The disease is transmitted via smear infection with the virus-containing chalky substance released from MC lesions either directly between humans (self-inoculation of different skin areas of the same patient is also commonly seen) or indirectly via fomites. MC is also a sexually transmissible disease. Productive MCV infection is presumably specific for humans. Attempts to transmit MCV to laboratory animals were unsuccessful, unless MCV infected human skin was transplanted into SCID mice. MC-like infections have been reported in various animal species, however, characterization on the molecular level was so far achieved only for a molluscum contagiosum-like virus which causes lesions in horses, donkeys and other equids. Though clearly related to MCV, this virus was found to form a separate species.

Clinical Features After a variable incubation time of 2 weeks to 6 months MCV causes benign tumors of the human skin. Because of their limitation to the epidermal layers these tumors are classified as acanthomas. The typical epidermal tumors are described as pearly, pinkish protrusions with an average diameter of 1–5 mm (Fig. 3). The lesions have a solid consistency and a central indentation or umbilication. The indentation is formed by regions of cellular decay releasing a white chalky substance composed of large amounts of MC virions and cellular debris. MC lesions are mainly located on the face, neck, arms and in the genital areas. Lips, tongue, and mucous membranes are rarely affected. MC is not known to infect the skin of the palms or the soles of feet. If MC affects the eyelid it often leads to subsequent conjunctivitis and keratitis by mechanical irritation. Patients can have several hundred lesions which develop by self-inoculation over time. These lesions persist for several weeks to months and can last up to years in some cases. MC lesions are usually painless if not manipulated by scratching. Signs of inflammation often occur only shortly before regression of the skin proliferations. If fever occurs it is usually linked to bacterial superinfection of the lesions. Immunosuppressed patients such as HIV-infected individuals or patients after splenectomy are frequently reported to develop higher numbers of MC lesions and often with a larger size (10 mm and more in diameter, also called molluscum giganteum). In such patients, MC lesions can be florid and often superinfected. This is known as “eczema molluscatum” in analogy to similar conditions observed in herpes simplex virus and vaccinia virus infections. While self-limiting in otherwise healthy individuals, MC may be persistent and hard to manage in the immunocompromised.

Pathogenesis After transmission by smear infection, MCV enters basal keratinocytes and induces a marked hyperproliferation of the stratum spinosum. Infected cells form large eosinophilic inclusion bodies containing large numbers of virions that compress the nuclei of

Fig. 3 MCV clinical picture. Forearm of a patient with approximately 50 MC lesions, some of which are bacterially superinfected. The lesions are in different stages of development suggesting autoinoculation and local dissemination.

632

Molluscum Contagiosum Virus (Poxviridae)

the keratinocytes, often dislocating them to the cellular periphery. Due to the hyperproliferation the epithelium starts to wrinkle and form lobules. After initial proliferation more and more cells in the upper layers of the epithelium die and form the chalky mass that contains large numbers of virions and cellular detritus. This zone of cellular decay leads to the central indentation from which the chalky mass can be readily expressed in mature lesions. Healing of these lesions is often associated with signs of inflammation that include swelling and reddening. A unique trait of MCV is its outstanding ability to evade and subvert the host immune system enabling the virus to persist for many weeks to months in the infected tissues. Several viral proteins play a major role in the molecular pathogenesis of MC. These include: (1) MC005L – interferes with the modulatory protein NEMO and its interaction with ubiquitin thereby inhibiting NF-κB activation. (2) MC007L – targets retinoblastoma protein (pRb). Dysregulation of this protein is associated with tumor pathogenesis and enables the virus to perturb the cell cycle. (3) MC54L – exhibits sequence similarity to human interleukin 18 binding protein (IL-18BP) and acts as a decoy receptor for IL-18. This effectively decreases the IL-18 mediated synthesis of interferon gamma, activation of NK cells and Th1-mediated immune response. (4) MC66L – encodes a homolog of selenocysteine-containing glutathionine peroxidase. This protects infected cells from peroxide radicals and ultraviolet-mediated apoptosis. It may also explain how MCV replicates exclusively in the epidermis. MC66L mediated anti-apoptotic activity may also explain the increased survival time of MCV-infected cells in the UV exposed epidermis, allowing MCV a longer productive cycle. (5) MC80R – this protein shows moderate homology to human major histocompatibility complex (MHC) class I and mediates ER-retention of host MHC I by a so far unique mechanism. ER-retention of MHC I leads to a reduced cell surface presentation of viral antigens to cells of the immune system. (6) MC132 – also inhibits NF-κB activation, however, the target protein for interaction is the cellular transcription factor p65. (7) MC148R – encodes a secreted protein which has a homology with CC family chemokines. A candidate gene for a MC148R human homolog was discovered mapping to chromosome 9p13. Subsequent studies showed that MC148R protein is a highly selective antagonist to CCR8 in human (but not mouse) monocytes and lymphoid tissues, and can conceivably interfere with monocyte invasion and dendritic cell function. However, the observation that MC148R can inhibit allograft rejection in transgenic mice supported the theory that it may possess anti-inflammatory properties. MC148 is expressed early in the MCV lifecycle, and may provide an anti-inflammatory activity gradient in the transition zone between epidermal and dermal tissue as a form of stand-off defensive device for the virus before other mechanisms engage. (8) MC159L and MC160L – known as vFLIPs (virus Fas-linked ICE-like protease), are inhibitors of death receptor-induced apoptosis and programmed necrosis. They share homology with caspase-8 and caspase-10 in the tandem death effector domains (DEDs) at the amino termini. MC159L blocks apoptosis induced by FAS and TNF. A recent study on vaccinia virus infected MC159 transgenic mice revealed enhanced innate inflammatory reaction characterized by increased expression of the chemokine monocyte chemotactic protein-1 (MCP-1) and infiltration of gδT cells into peripheral tissues. Thus, MC159L is considered a dual-function immune modulator. MC160 protein inhibits NF-κB activation either by its DED-containing N-terminus binding to procapase-8 or its C-terminal region interacting with Hsp90, leading to a degradation of IKK (IκB kinase)1 and inhibition of phosphorylation of the p65 subunit. (9) MC163L – was found to be a regulator of apoptosis. Expression of this protein dampened cellular apoptotic responses by interaction with several pathways.

Diagnosis The pathognomonic appearance of the umbilicated lesions with a central discharge of the chalky cellular detritus is often sufficient for clinical diagnosis. In doubt, histological sections stained with H&E can assist diagnosis. Histologic presentation of MC is characterized by hyperproliferation of the stratum spinosum with a typical formation of lobules. Large numbers of eosinophilic inclusion bodies can be found in the hyperproliferating keratinocytes and the cellular debris which is discharged from the central indentation (Fig. 4). Other diagnostic methods include electron microscopy to demonstrate MCV particles, PCR and real-time PCR for MCV nucleic acid detection and ELISA for detection of antibodies against MCV. Differential diagnoses include infectious (cutaneous cryptococcosis, furuncles, herpesvirus infections, histoplasmosis and papillomavirus infections) and non-infectious diseases (adnexal tumors, basal cell carcinoma, fibrous papules, naevi).

Treatment In immunocompetent patients – especially in children – it is advisable to wait for spontaneous disease regression. Although MC lesions may persist for several months the disease is usually self-limiting and heals without scar formation. If treatment is needed because of cosmetic reasons or complications like bacterial superinfection, MC lesions in mechanically stressed locations (eyelid, inguinal region) or persistent infection in immunocompromised, there is a broad choice of surgical and medical treatment options available. Surgical intervention includes curettage, cryotherapy, laser treatment, electrosurgery or occlusive duct tape. Medical options can be divided into topical and systemic approaches. Topically applied drugs include acantholytic and keratolytic substances like

Molluscum Contagiosum Virus (Poxviridae)

633

Fig. 4 Histologic presentation of a typical MC lesion. Histological section (HE stain) of an MC lesion demonstrates the typical lobular organization and hyperproliferative character of the skin affections in MC. Virus particles and lipid debris are discharged from the central indentation.

cantharidin, potassium hydroxide (KOH) and salicylic acid. Other topical solutions reported to alleviate MC lesions include drugs and plant extracts like podophyllotoxin, Australian lemon myrtle (Backhousia citriodora), diphencyprone, 5-aminolaevulinic acid and nitric oxide (NO). Systemic treatments include antiviral agents like Cidofovir, treatment of underlying HIV infection with antiretroviral drugs, as well as interferon–alpha. The immunomodulatory drug imiquimod received a lot of attention during the last few years, however, the results of clinical studies led to controversial conclusions and there is so far no reliable evidence for its effectiveness. In general, it has to be stated that although MC is a relatively frequent disease, high-quality clinical studies to compare different therapeutic options for MC are scarce leaving no consensus on best practice. The 2017 Cochrane review on intervention for cutaneous molluscum contagiosum comes to the conclusion that there is no reliable evidence-based recommendation for the treatment of MC so far and that awaiting spontaneous resolution is favored if possible.

Prevention There is no vaccine available. The best prevention is avoidance of smear infection by following good hygiene practices: covering of MC lesions with clothing or bandages, avoidance of scratching and manipulation of the papules.

Further Reading Bugert, J., Alikhan, A., Shwayder, T., 2019. Molluscum contagiosum. In: Hoeger, P., Kinsler, V., Yan., A. (Eds.), Harper’s Textbook of Pediatric Dermatology, fourth ed. © 2019 John Wiley & Sons Ltd. Chen, X., Anstey, A.V., Bugert, J.J., 2013. Molluscum contagiosum virus infection. The Lancet Infectious Diseases 13, 877–888. doi:10.1016/S1473-3099(13)70109-9. Forbat, E., Al-Niaimi, F., Ali, F.R., 2017. Molluscum contagiosum: Review and update on management. Pediatric Dermatology 34, 504–515. doi:10.1111/pde.13228. Harvey, I.B., Wang, X., Fremont, D.H., 2019. Molluscum contagiosum virus MC80 sabotages MHC-I antigen presentation by targeting tapasin for ER-associated degradation. PLoS Pathogens 15 (4), e1007711. doi:10.1371/journal.ppat.1007711. Katz, K.A., Swetman, G.L., 2013. Imiquimod, molluscum, and the need for a better “best pharmaceuticals for children” act. Pediatrics 132, 1–3. doi:10.1542/peds.2013-0116. Katz, K.A., 2014. Imiquimod is not an effective drug for molluscum contagiosum. The Lancet Infectious Diseases 14, 372–373. doi:10.1016/S1473-3099(14)70728-5. Nichols, D.B., De Martini, W., Cottrell, J., 2017. Poxviruses utilize multiple strategies to inhibit apoptosis. Viruses 9, 215–250. doi:10.3390/v9080215. Senkevich, T.G., Koonin, E.V., Bugert, J.J., Darai, G., Moss, B., 1997. The genome of molluscum contagiosum virus: Analysis and comparison with other poxviruses. Virology 233, 19–42. doi:10.1006/viro.1997.8607. Sherwani, S., Farleigh, L., Agarwal, N., et al., 2014. Seroprevalence of molluscum contagiosum virus in German and UK populations. PLoS One 9, e88734. doi:10.1371/journal. pone.0088734. Shisler, J.L., 2015. Immune evasion strategies of molluscum contagiosum virus. Advances in Virus Research 92, 201–252. doi:10.1016/bs.aivir.2014.11.004. Trcˇko, K., Hošnjak, L., Kušar, B., et al., 2018. Clinical, histopathological, and virological evaluation of 203 patients with a clinical diagnosis of molluscum contagiosum. Open Forum Infectious Diseases 5. doi:10.1093/ofid/ofy298. van der Wouden, J.C., van der Sande, R., Kruithof, E.J., et al., 2017. Interventions for cutaneous molluscum contagiosum. Cochrane Database of Systematic Reviews 5, CD004767. doi:10.1002/14651858.CD004767.pub4. Zorec, T.M., Kutnjak, D., Hošnjak, L., et al., 2018. New insights into the evolutionary and genomic landscape of molluscum contagiosum virus (MCV) based on nine MCV1 and six MCV2 complete genome sequences. Viruses 10. doi:10.3390/v10110586.

Relevant Websites https://www.cdc.gov/poxvirus/molluscum-contagiosum/index.html Molluscum Contagiosum. https://4virology.net Viral Bioinformatics Research Centre.

Mumps Virus (Paramyxoviridae) Stephen A Winchester and Kevin E Brown, Frimley Park Hospital, Frimley, United Kingdom and Immunisation and Countermeasures Division, Public Health England, London, United Kingdom Crown Copyright r 2021 Published by Elsevier Ltd. All rights reserved This is an update of B.K. Rima, W.P. Duprex, Mumps Virus, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B978-012-801238-3.02623-4.

History The earliest description of mumps dates back to the fifth century BC by Hippocrates of a disease capable of spreading in humans with characteristic enlargement close to the ears and painful swelling of the testes common in young men. Infection in the central nervous system (CNS) and meninges in some cases of mumps was first noted by Hamilton in 1790. In 1934, Johnson and Goodpasture demonstrated the filterable nature of the causative agent and Koch’s postulates were fulfilled by infection of human volunteers with virus propagated in the salivary glands of monkeys. The first vaccine against mumps was developed in 1946, and it protected macaques against infection.

Taxonomy and Classification Mumps virion is a spherical or pleomorphic particle with glycoprotein projections 150-nm or more in diameter (Fig. 1). It is stable at 41C for several days but its lipid membrane is sensitive to ether and other membrane-destroying reagents, and has haemagglutination and neuraminidase (HN) activities. It contains a nonsegmented negative-strand RNA genome and is classified in the family Paramyxoviridae, in the genus Orthorubulavirus within the Rubulavirinae. MuV is the prototype species, and two other human Rubulavirinae, parainfluenza virus 2 (hPIV2) and hPIV4 have been identified. Other rubulaviruses infect a range of vertebrates – for example, Mapuera virus infects bats and porcine rubulavirus neonatal pigs. Simian virus 5 (SV5) was originally isolated from rhesus and cynomolgus monkey kidney-cell cultures and thus designated as a primate virus. SV5 was sometimes referred to as canine parainfluenza virus although it has a more extended host range and has now been isolated from dogs, pigs and human bone marrow. Consequently, the virus has now been renamed PIV5. MuV has only been isolated from humans, although other species including macaques, dogs, mice and ferrets can be experimentally infected. Recently, sequences of a closely related virus have been identified in samples from fruit bats.

Properties of the Virion Paramyxoviruses consist of an inner ribonucleoprotein (RNP) core surrounded by a lipid bilayer membrane from which the glycoproteins protrude (Fig. 1). Electron microscopy (Fig. 1(a)) shows that MuV has a typical paramyxovirus structure with a lipid

Fig. 1 The structure of the mumps virion. In electron micrograph (a) the arrow points to a nucleocapsid released from a virus particle showing the characteristic herringbone structure. Schematic representation (b) indicates the location of the major viral structural proteins (see Fig. 2 for an explanation of the shapes and abbreviations).

634

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00099-0

Mumps Virus (Paramyxoviridae)

635

Fig. 2 Transcription, replication, and translation of the MuV genome. The mumps gene order, the size and the position of encoded gene, sequence (positive strand) of the Ig sequences (a); the major transcripts derived from the MuV genome and their sizes (b); the proteins and their sizes in numbers of amino acid residues and their schematic representations (c), and the replicative intermediates (d).

bilayer membrane surrounding an internal RNP complex, termed the nucleocapsid. The nucleocapsid displays the herringbone structure characteristic of Paramyxoviridae (arrow, Fig. 1(a)) and is approximately 1 mm in length with a diameter of 17 nm and an internal central core of 5 nm. Some pleomorphic particles have been reported to contain more than one RNP structure. The biological significance of such polyploid particles has not been investigated. The RNP contains a negative (
) sense RNA genome covered with nucleocapsid (N) protein as well as a phospho- (P) protein and the large (L) protein. The RNP is surrounded by a lipid bilayer derived from the host cell in which the matrix (M) protein of the virus is embedded (Fig. 1(b)). This protein interacts with the internal core N protein and the cytoplasmic tails of the viral glycoproteins. Spikes (10–15 nm in length) protrude from the membrane and these contain the haemagglutinin–neuraminidase (HN) and the fusion (F) glycoproteins. The HN glycoprotein is probably a homo-tetramer and the F protein a homo-trimer. The ratio of HN and F glycoproteins in the spike complexes has not been elucidated. The spikes have haemagglutination and neuraminidase activity and will haemolyse red cells from a range of different species. Hemolysis reflects the ability of the virus to fuse with infected cells.

Properties of the Genome MuV has a single nonsegmented negative-strand RNA genome (Fig. 2(a)). The nucleotide sequences of the complete genome is 15,384 nucleotides in length. The MuV gene order and transcription of the genome is similar to other paramyxoviruses, with seven transcription units separated by intergenic (Ig) sequences. The basic unit of infectivity is the negative-stranded, encapsidated RNP (Fig. 2(d)). The virion is probably polyploidic containing several RNPs.

Properties of the MuV Proteins The properties of the MuV proteins are summarized in Table 1 and the number of amino acid residues in each viral protein is given in Fig. 2(c). Six structural proteins, namely, the N, P, M, and L proteins, as well as two glycosylated membrane-spanning proteins, the HN and F proteins, have been identified in MuV virions. At least two nonstructural proteins (V and W) are synthesized from transcripts of the V/W/P gene. The presence of a small hydrophobic (SH) protein has been demonstrated in MuV-infected cells using antisera to peptides derived from the deduced amino acid sequence. It is not clear whether the protein is incorporated into virions. At least one strain (Enders) expresses the SH gene as a tandem read? through transcript with the F gene in tissue culture. It is unlikely that the SH protein is translated from an F–SH bicistronic mRNA. The growth of Enders strain in tissue culture may not require the expression of SH. A recombinant virus lacking the SH protein has been shown to be capable of growing in several cell types, formally proving a nonessential nature of the protein for growth of the virus in cell culture.

636

Table 1

Mumps Virus (Paramyxoviridae)

Properties of the proteins of MuV

Protein

Size (kDa)

Function Phosphorylated structural protein of RNP; possible role in regulation of transcription and regulation Associated with RNP; possible role in RNA synthesis Polymerase activity; role in capping, methylation and polyadenylation Associated with inner side of membrane; interacts with N protein and HN and F glycoprotein F2-F1 heterodimer activated by a proteolytic cleavage of F0; fusion of virion membrane with plasma membrane (with HN) Glycoprotein with haemagglutination and neuraminidase activities; accessory role in fusion with plasma membrane Membrane protein of unknown function. Sequence used to determine genotype Role in STAT1 and STAT3 degradation Unknown role

Nucleocapsid protein

N

68–73

Phosphoprotein Large Matrix Fusion

P L M F

45–57 4200 39–42 65–74

Haemagglutinationneuraminidase Small hydrophobic Non-structural V Non-structural W

HN

74–80

SH V or NS1 W or NS2

6 23–28 17–19

Fig. 3 Consensus sequences of the seven gene start (GS) and gene end (GE) sequences. The various consensus sequences are shown as positive strand sequences. The specific intergenic sequences and their positions in the genome are shown in Fig. 2(a).

Physical Properties The MuV virion is sensitive to heat and treatment with ultraviolet (UV) light. The virus is inactivated by 0.2% formalin and the presence of the lipid bilayer confers sensitivity to both ether and chloroform. Treatment with 1.5 M guanidine hydrochloride leads to a selective inactivation of neuraminidase but not haemagglutination activity of the virus, indicating that separate domains of the HN molecule are responsible for these two functions.

Replication MuV infects a variety of cells in vitro but whether this is the case in vivo remains to be determined. The attachment of MuV is mediated primarily through the interaction of the HN glycoprotein with an a2,3-linked sialic acid. After introduction of the RNP into the cell, primary transcription of the negative-stranded genome occurs within the cellular cytoplasm. This is mediated by the viral L and P proteins (Fig. 2(b)). The transcription complex recognizes the 30 end of the genome and transcribes the genes sequentially (Fig. 2(c)), stopping at each Ig sequence to synthesize polyadenylate (An) tails of the various mRNAs by repeated transcription on a poly(U) stretch in the genome. The Ig sequences are between 1 and 7 nt-in length (Fig. 2(a)) and they are flanked by highly conserved gene end (GE) and gene start (GS) signal sequences (Fig. 3). Co-transcriptional editing is responsible for the generation of mRNAs encoding the P and W proteins of MuV. Some of the viral proteins are modified post-translationally (Table 1). The HN glycoprotein forms oligomers during transport through the Golgi complex whereas the F glycoprotein rapidly matures simultaneously with its glycosylation. During replication of the genome the RdRp ignores the GE, Ig, and GS signals to generate one single positive-strand RNA molecule. The role(s) of V and W nonstructural proteins in the processes of replication, transcription, translation, and assembly has yet to be elucidated. Neither is it clear what is the role of SH protein in the virus life cycle.

Serologic Relationships and Variability MuV is a monotypic virus, and tests with human sera indicate the existence of only a single serotype. Polyclonal sera from infected individuals show a low level of cross-reactivity with hPIV2 and PIV5. Variability between strains has been demonstrated using monoclonal antibodies against the HN and N glycoproteins showing the greatest diversity when single epitopes are examined.

Mumps Virus (Paramyxoviridae)

637

WHO currently recognizes 12 genotypes of MuV, assigned with letters from A to N (excluding E and M) which are based on the nucleotide sequence of the highly variable SH gene. These are routinely used in molecular epidemiological studies (see below).

Evolution and Genetics Human populations only became dense enough to sustain MuV from about 4000 years ago and, therefore, it has been suggested that the virus must have evolved from an animal reservoir. Recently closely related Rubulaviruses have been identified in a number of different bat species. Neither temperature-sensitive nor any other conditional lethal mutants of MuV have been reported nor has recombination been described in any nonsegmented negative-strand RNA viruses. Although host range mutants have not been isolated, adaptation of the virus to grow in embryonated eggs or in chicken embryo fibroblasts requires a number of blind passages. Strains adapted in this way do not readily grow and fail to generate syncytia in mammalian cells in culture. As MuV is a neuropathogenic virus some clinical isolates have been adapted to grow in the CNS of experimental animals to study their neuropathogenicity. These viruses have been used in reverse genetics approaches to identify molecular determinants of neuropathogenesis. These main determinants appear to reside in the viral envelope proteins. At present neutralizing monoclonal antibody escape mutants of the HN glycoprotein are the only type of MuV mutants which have been described.

Epidemiology Geographic and Seasonal Distribution Prior to vaccination the peak incidence of mumps was in children of 5–7 years of age. The overall risk of infection in unvaccinated populations has been observed to be equal between genders but males are at increased risk of complications. Epidemics of mumps were seen to occur at intervals of 2–5 years. Seasonal variation occurs in temperate climates with highest incidence in February and March. The overall annual incidence in Europe in the pre-vaccine era (1977–1985) was 290 per 100,000 of the population. The seroprevalence of mumps IgG in a nonimmunized population of adults can be greater than 90%. Clinically apparent mumps infection following a previous infection may occur though an accurate history of previous infection can be difficult to establish. It is generally considered that wild-type infection gives rise to a lifelong immunity against the disease.

Transmission and Tissue Tropism The virus replicates in the epithelium of the upper respiratory tract and the salivary glands and is transmitted in salivary droplets. Patients are infectious from 3 days before until approximately 4 days after the onset of clinical symptoms. Mumps can also cause viruria but this is not considered important in transmission. Transmission has been demonstrated by a direct contact with upper respiratory tract secretions and droplets and only occurs in the acute phase. It has been proposed that primary viraemia occurs following the replication of the virus in local lymphoid tissues. Viraemia may lead to subsequent replication in other target organs including the kidneys, thyroid glands and sexual organs. Salivary glands are commonly involved but not essential to pathogenesis.

Clinical Features of Primary Infection A prodromal phase is usually followed by clinical mumps symptoms within 24–48 h which include low-grade fever, headache, malaise, myalgia and anorexia. The median incubation period is 19 days and can vary from 14 to 25 days. Infectivity is usually present from 48 h prior to symptoms until several days after onset. Asymptomatic infection occurs more frequently in adults with an estimated subclinical infection rate of 15%–20%.

Systemic Manifestations Parotid Glands The predominance of parotitis (Fig. 4) has ensured that other clinical features are commonly described as complications rather than systemic manifestations. Recovery from parotitis is usually swift and complete. Parotid swelling can obscure the angle of the mandible and the orifice of Stensen’s duct can become erythematous and edematous. Lymphatic obstruction may rarely lead to presternal and supraglottic edema.

638

Mumps Virus (Paramyxoviridae)

Fig. 4 Pathogenesis and systemic manifestations of MuV infection Parotitis- Parotid swelling occurs in as many as 95% of individuals with symptoms; a further 75% of these subsequently develop swelling in the contra-lateral gland Neurological- Aseptic meningitis is the most frequent of these manifestations occurring in as many as 1%–10% of infections. Encephalitis occurred in approximately 1 in 6000 patients that presented in the prevaccine era. Transient sensorineural deafness has been reported in adult military personnel whilst permanent deafness may occur in up to 1 in 15,000 cases of mumps. Pancreatitis- Pancreatitis has been reported in up to 4% of cases presenting to hospital. Genitourinary- Orchitis can occur in as many as 30% of post-pubertal males presenting with symptomatic mumps and close to 40% of these can develop bilateral swelling. Epididymitis is associated with 85% of cases of orchitis. Oophoritis is present in comparatively fewer post-pubertal females at a rate up to 7% with no subsequent infertility reported. Transient nephritis is relatively frequent though few cases go on to develop severe nephritis and thrombocytopenia. Cardiac- Myocarditis has been reported in 4%–15% of patients presenting to hospital with mumps though ECG findings may reflect symptoms of an acute infection. Musculoskeletal- Young male adults make up the largest proportion to suffer from monoarticular or polyarticular mumps arthropathy.

Neurological Disease Aseptic meningitis has been reported as up to 3 times more frequently in men than in women and occurs in the presence or absence of parotitis. It is usually benign with no permanent neurological damage. Before the development of the currently used liveattenuated vaccine, MuV was the most common cause of viral meningitis and encephalitis in the USA. Encephalitis usually has a favorable prognosis, although minor neurological changes, such as learning and concentration impairments and sudden deafness are well-documented sequelae. Other neurological syndromes proposed to be associated with mumps included cerebellar ataxia, facial palsy, Guillain-Barré syndrome, hydrocephalus, poliomyelitis-like syndrome and transverse myelitis.

Pancreas The clinical course of pancreatitis due to mumps virus is typically benign though cases of operable pseudocysts have been noted.

Mumps Virus (Paramyxoviridae)

639

Genitourinary Tract Orchitis occurs more frequently in post-pubertal males than oophoritis in post-pubertal females (Fig. 4). Late atrophy of testicular tissue may lead to infertility in approximately 17% cases of epididymitis and rarely sterility ensues. The risk of miscarriage is considered to increase in pregnant mothers with mumps infection but there is no evidence of the disease causing congenital malformations.

Cardiac Symptoms The causative role of mumps virus in myocarditis is uncertain (Fig. 4). Fulminant fatal myocarditis with dilated cardiomyopathy has been described in rare instances.

Musculoskeletal System Monoarticular and polyarticular mumps arthropathies have been described (Fig. 4).

Other Symptoms Other organs or glands that may be affected include the eyes, gall bladder, lacrimal glands, liver and thyroid gland. Mumps virus has also been proposed to be associated with diabetes mellitus, haemophagocytic syndrome and thrombocytopenia.

Pathogenesis The virus replicates primarily in the nasal mucosa and epithelium of the upper respiratory tract. After penetrating the draining lymph nodes, a transient viremia occurs and thereafter various target organs such as the salivary glands, the kidneys, pancreas, and the CNS are infected. Salivary glands are commonly involved but not essential to pathogenesis. Infection of the salivary glands produces parotitis, the most predominant clinical feature of the disease. Viral replication leads to tissue damage and the subsequent immune response causes inflammation and swelling of the gland. Dissemination into the kidneys can lead to a prolonged infection of this organ and viruria. Virus can be isolated from throat swabs, blood, saliva and urine. Involvement of the CNS may be as high as 50% of cases and parotitis is not required for this to occur. MuV can be readily detected in the cerebrospinal fluid (CSF) in cases of meningoencephalitis. Pathological changes initiated by mumps virus are nonspecific. In the parotids, diffuse interstitial edema and serofibrinous exudate consisting predominately of polymorphonuclear leukocytes (PML) have been observed in the connective tissue and ducts. Degeneration of ductal epithelium can be seen as a result of accumulation of neutrophils and necrotic debris in the lumen. Involvement of the pancreas or testis may lead to a more marked interstitial hemorrhage and PML accumulation. This can lead to vascular compromise and eventual epithelial atropy, hyalinization and fibrosis. Mumps virus infection can induce the production of interferons and anti-mumps IgA, IgG and IgM antibodies as well as the activation of cell-mediated immunity. A viruria can frequently be detected though it is unclear whether this is due to a local replication of the virus in the kidneys or whether it is of haematogenous origin. Central nervous system (CNS) involvement occurs more frequently in the meninges with electroencephalograms revealing little deviation from normal. Although rare, mumps encephalitis with perivenous demyelination and microglial cell increase has been observed. Conversely, the death of neurons without demyelination has been demonstrated in an apparent case of primary mumps encephalitis. The contribution or extent of immune response-related damage to CNS in mumps encephalitis has been difficult to estimate.

Immune Response It is not known whether the humoral or cell-mediated immune (CMI) response is the most important factor in the clearance of MuV infection. The correlates of protection in terms of antibody titer or CMI responses have not been determined. Both play a role although neither seems to be required exclusively for a successful control of the infection. Eleven days after infection, the humoral immune response is well established (Fig. 5) and the presence of neutralizing anti-MuV antibodies probably terminates viremia. Similarly, the appearance of IgA in saliva stops excretion of infectious MuV. The precise time at which MuV reactive cytotoxic T lymphocytes appear is unclear, although these have been demonstrated in both the blood from patients with the natural infection and in vaccinated individuals. The magnitude and effectiveness of cell-mediated immune response may be related to the genetic human leukocyte antigen (HLA) background of the host. A complication in the development of the CMI response is the tropism of the virus for T and B cells. A reduction in CMI responses to antigens previously recognized has been observed, although the mechanism is unclear. This response is less severe and of shorter duration than the immunosuppression associated with a measles virus infection. The virus grows well in activated but not in resting T lymphocytes and infection of the CNS during MuV infection

640

Mumps Virus (Paramyxoviridae)

PRIMARY INFECTION OR PRIMARY VACCINE FAILURE

D E T E C T I O N

P C R

Oral Fluid

SECONDARY INFECTION OR SECONDARY VACCINE FAILURE

Oral Fluid

IgG

LABORATORY

A N T I B O D Y R E S P O N S E

PRIMARY INFECTION OR PRIMARY VACCINE FAILURE IgM

IgG SECONDARY INFECTION OR SECONDARY VACCINE FAILURE

IgM

Incubation (14 - 25 days) Parotid Swelling (4 - 7 days) CLINICAL Contralateral Parotid Enlargement Prodromal Symptoms (malaise, myalgia,headache, low-grade fever)

4 Exposure

3

2

1

1 Onset of Clinical Symptoms

2

3

4

8

(weeks)

Fig. 5 The laboratory diagnosis of mumps virus infection. The clinical course of mumps virus infection and laboratory evidence to support a diagnosis of mumps through detection of viral RNA by PCR (oral fluid) or antibody response in a single (positive anti-mumps IgM antibodies) serum specimen or an increase in anti-mumps IgG antibodies between paired serum specimens.

is thought to occur via transfer of infected monocytes into the choroid plexus and the meninges. Perinatal exposure of the fetus to MuV via the placenta does not appear to lead to infection of the fetus. However, congenital infection can give rise to anomalous immune responses to MuV in the newborn child. In these children, CMI but no humoral immune responses are detectable. Neutralizing B cell epitopes have been defined in the HN glycoprotein of MuV but no other B or T cell epitopes have been detected.

Differential Diagnosis The diagnosis of mumps is more readily suspected clinically with classical swelling of the parotid(s). Other infectious causes of parotitis comprise viral and bacterial infections including Staphylococcus aureus and atypical mycobacteria, though rarely described. A viral cause was identified by serologic testing in 14% (84/601) cases of mumps-like illness in children who were tested mumpsnegative. The main viral aetiologies were Epstein-Barr virus (7%), parainfluenza virus types 1, 2 or 3 (4%) and adenovirus (3%). Other viruses that have implicated in parotitis include coxsackieviruses, HHV6B and influenza A viruses. Non-infectious causes of enlarged parotids include trauma, calculi, tumors, Sjögren syndrome and sarcoidosis. Symptomatic treatment of virus-related parotitis include thiazide diuretics, phenylbutazone and phenothiazines. In the absence of parotitis, symptoms or signs of mumps systemic manifestations may predominate and specific diagnosis can be obtained with laboratory analyzes.

Laboratory Diagnosis The laboratory evidence to support a diagnosis of mumps commonly comprises detection of viral nucleic acids (RNA) and/or antiMuV antibody response (IgM or significant rise in IgG titers) (Fig. 5). Serum and oral fluid (OF) specimens are routinely used for the determination of mumps virus antibody response. The advantages of OF rather than blood collection include accessibility, reduced hazard, cost and convenience. Mumps virus can usually be detected in OF 2 days prior to 5 days after the onset of clinical

Mumps Virus (Paramyxoviridae)

641

symptoms by culture, immunofluorescence (IF) or polymerase chain reaction (PCR). Mumps virus may also be detected in urine during the first 2 weeks of mumps infection. Mumps virus is present in CSF in the first 3 days and up to the 6th day of clinically apparent mumps meningitis/ encephalitis. A lumbar puncture taken in patients with mumps meningo-encephalitis commonly reveals an elevated white blood cell count and normal or weakly elevated protein. A low glucose concentration in the CSF has been measured in up to 30% of patients which is more frequent than other viral meningitides. PML predominance in CSF has also been seen in approximately 20%–25% of patients. Increased CSF: serum anti-MuV IgG antibody ratios (e.g., 41:50) may be indicative of an active CNS infection.

Detection of Mumps Virus Virus culture has been considered the gold standard for mumps confirmation though it is time and resource intensive and presently it is not widely used any more. Mumps virus RNA can be detected by various nested reverse transcriptase PCR (RT-n-PCR) assays. In addition, real time ((RT)-PCR) has been used to detect MuV RNA in OF, CSF, respiratory and urine samples. The advantages of (RT)PCR include greater sensitivity and specificity as well a significant reduction in the likelihood of contamination. Furthermore, quantitative real-time (RT)-PCR is increasingly being used in investigations of mumps infection to quantify viral load. MuV RNA has been detected in patients with mumps infection and healthy children following immunization live mumps vaccines. A greater sensitivity of (RT)-PCR versus cell culture has been demonstrated in OF for mumps infection and in CSF for clinically apparent mumps CNS disease. However, a lower sensitivity of (RT)-PCR relative to cell culture has been observed in urine specimens and may be attributable to inhibition. SH gene sequencing has shown geographically distinct strains, and can distinguish between wild virus and vaccine strains.

Antibody Response Standard enzyme-labeled immunoassays (ELISAs) are the preferred method for the detection of mumps virus IgM and IgG antibodies. The efficiency and lower complexity of the enzyme immunoassay (EIA) method to measure and quantify mumps virus IgM and IgG has ensured its predominant use over of a variety of other techniques. There are capture or indirect assays to detect mumps virus-specific antibodies that use recombinant mumps nucleoprotein (rMuVN) as an antigen. Mumps virus IgM antibodies has been detected in CSF from patients with clinically apparent mumps meningitis but the sensitivity of this detection method has remained at a level of ca. 50%. A significant rise in IgG antibody levels between an acute and convalescent sample can diagnose primary infection and the level of significant and diagnostic rise depends on the assay. Mumps virus IgG detection by EIA is not an international standard for protective immunity following infection or vaccination. A residual low-level serum IgG antibody level in adulthood following immunization with MMR vaccine as a child does not correlate with protection against mumps infection. The measurement of mumps virus IgG avidity has been proposed to assist in the differentiation of primary and secondary mumps virus infection. The strength of binding of mumps virus IgG to antigen has been shown to increase with time following primary mumps infection. Commercially available assays have been used to evaluate primary and secondary vaccine failure during a mumps outbreak. Mumps virus IgG may be of low or borderline avidity in vaccinated individuals with mumps virus infection. There are currently no commercial mumps virus IgG avidity assays.

Neutralisation Tests The plaque reduction neutralisation test (PRNT) and focus reduction neutralisation test (FRNT) are the most specific assays for measuring neutralizing antibodies against mumps virus. PRNT has been shown to detect antibodies below the level of detection of IgG binding antibody assays. FRNT has been validated against PRNT and the method is more accessible, faster, utilizes lower serum volumes and consumes less reagents than PRNT. A protective antibody level with PRNT or FRNT has not been determined. A recombinant mumps virus GFP expression inhibition assay has been described with ca. 95% correlation with a conventional CPE-based neutralisation assay. Ultimately, it is not clear whether a detectable level of mumps neutralizing antibody confers protection from natural infection.

Utilization of Assays Primary Infection and Primary Vaccine Failure The presence of mumps virus IgM provides laboratory evidence to support a diagnosis of primary mumps infection or primary vaccine failure. A documented history of mumps virus vaccination is necessary to distinguish a primary vaccine failure from a primary infection. Mumps virus IgM is most reliably detected 1 week after symptom onset, declines within 1 month and persists at

642

Mumps Virus (Paramyxoviridae)

low levels for a minimum of 4 months. However, mumps virus IgM can also be detected with a secondary infection or a secondary vaccine failure. Moreover, the absence of mumps virus IgM in acute illness does not exclude an infection. Mumps virus IgG seroconversion is consistent with primary infection although dependent upon collection of acute and convalescent samples with no established standard rise in IgG titer. The detection of mumps virus RNA in high levels is consistent with primary infection or primary vaccine failure although the levels are dependent on sample timing.

Secondary Infection and Secondary Vaccine Failure Mumps virus IgM is usually present in low levels or absent in secondary infection or secondary vaccine failure. Mumps virus RNA detection is predominately detected with lower viral RNA amounts than in primary infection at an equivalent clinical time point. A documented history of mumps virus vaccination is necessary to distinguish secondary vaccine failure from secondary infection.

Prevention and Control Adaptation of MuV to growth in embryonated eggs and chicken embryo fibroblasts allowed the development of live-attenuated vaccines. In 1946, Enders observed that adaptation of MuV to chicken cells was associated with the loss of virulence in monkeys. In the past, killed virus preparations were used as vaccines in humans, although these vaccines did not lead to a life-long protective immunity and their use has been discontinued. Mumps virus vaccines are available as a monovalent vaccine or in combination with other vaccines such as the measles, mumps and rubella (MMR) with or without varicella (MMRV) vaccines. MMR vaccine is a live attenuated vaccine used commonly worldwide. The Jeryl-Lynne (JL) strain of MuV is most frequently used in the MMR vaccine and interestingly it has been shown to be comprised of two strains, the major (JL5) and the minor (JL2) components. JL vaccine has been in global use since the 1970s and an initial field-randomized controlled clinical trial reported an efficacy exceeding 95% with the monovalent vaccine. A low complication rate and subsequent eradication of mumps infection has been reported following its introduction in some countries. However, there is now evidence for the efficacy of JL mumps vaccine component to be as low as 62% during some epidemics. There are 4 other major vaccine strains (Urabe, Leningrad-3, Lenningrad-Zagreb and Rubini). There have been demonstrable increases in central nervous system (CNS) complications that have limited the use of the Urabe strain in the vaccine. However, the Urabe strain has, however, shown greater immunogenicity in inducing antibody responses (97%) compared to Jeryl Lynn strain (90%). The Lenningrad-Zagreb strain is an attenuated form of the Leningrad-3 strain. The use of this strain as a vaccine has been limited due to complications including aseptic meningitis although this complication has not been found in other studies. The Rubini strain, when introduced, was shown to not achieve sufficient levels of protection from wild type virus and it is not recommended by the World Health Organization. Mumps vaccination has substantially reduced the incidence of mumps infection worldwide. After successful licensing of the vaccine in 1967 in the USA, the incidence of mumps dropped from 76 to 2 cases per 100,000 population. A similar decrease in mumps incidence was found in many countries after introduction of the MMR vaccine. Clinically apparent mumps infection can occur following a previous immunization, often 10 or more years later, and outbreaks of mumps in teenagers and young adults are common in many countries with a childhood vaccination program. Mumps symptoms in these cases are milder, short-lived and rarely are complications other than transient parotitis reported.

Future Perspectives Over the last 10 years, the development of reverse genetics systems for all members of the Mononegavirales has had a significant impact on our understanding of the pathogenesis of these viruses. Reverse genetic systems have been generated for PIV5, hPIV2, and MuV, and to date they have been used to begin to define the structural and functional relationships and the roles that various proteins play in attenuating these viruses. The opportunity to examine the contribution of individual proteins from neurovirulent strains, such as Urabe, to neuropathogenesis in animal models should help to identify virulence determinants, something which is important if it becomes necessary to develop new and safer MuV vaccines. One of the key challenges for the future is to develop safe, non-neurovirulent vaccines that induce a long-lasting protective immune response. Correlates of protection are still not well understood, but the increasing number of mumps outbreaks in vaccinated populations show the need for such vaccines.

Further Reading Rubin, S.A., Sauder, C., Carbone, K.M., 2013. Mumps virus. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Wolters Kluwer/ Lippincott Williams and Wilkins Oxford University Press, pp. 1024–1041. Rima, B.K., 2010. Mumps: Epidemic parotitis. In: Warrall, D., et al. (Eds.), Oxford Textbook of Medicine, fifth ed. Oxford University Press, pp. 513–515.

Murine Leukemia and Sarcoma Viruses (Retroviridae) George R Young and Kate N Bishop, Francis Crick Institute, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary Endogenous virus A retroviral genome integrated within that of its host and inherited in a Mendelian fashion. Exogenous virus Infectious viral particles transmitted between individuals or causing spreading infection within an individual. Inbred mice Genetically invariant strains of mice developed through inbreeding programs.

Integration The process or action of a retrovirus inserting its reverse transcribed genome into the DNA of an infected cell. Provirus A viral genome integrated within the DNA of an infected cell (not synonymous with “endogenous virus” as infections of somatic cells will not be vertically inherited). Viral core The assembled structure in the center of the virion encompassing the capsid protein shell, viral nucleic acids, and enzymes.

Classification Murine Leukemia Viruses (MuLVs or MLVs) are members of genus Gammaretrovirus of the family Retroviridae, subfamily Orthoretrovirinae. As with other retroviruses, MLV virions contain two copies of a non-segmented positive-sense single-stranded RNA genome and fall into the larger Ortervirales order. As single-stranded RNA viruses replicating through a DNA intermediate, alternative classification places them within Baltimore Group VI. MLVs exist both as exogenous viruses and as endogenous retroviruses (ERVs) within the genome of their host.

History As judged by the fossil record of endogenous examples within species of Mus, MLVs have been infecting mice for at least 1.5 million years (my), although other gammaretroviruses are likely to have been circulating within their ancestors for considerably longer. The first breeding of fancy mice and of early inbred lines, primarily from stocks of Mus musculus domesticus wild-caught in China, revealed a variety of neoplastic and leukemic pathologies that, thanks to detailed record keeping, were observed to have familial associations. The search for the etiology of such heritable cancers led to the creation of numerous modern laboratory mouse lines, many bred for their specific resistance or susceptibility to disease. Using these inbred lines, a viral cause of mammary carcinoma, Mouse Mammary Tumor Virus (MMTV), was identified by John Bittner in 1936. Although MLVs were also quickly identified by the breeding of strains with high incidences of leukemia in the 1920s and 30s, it was not until 1957 that the first MLV was isolated by Ludwik Gross. MLVs with different properties – generally either T lymphoma-inducing (e.g., Gross- and Moloney-MLV) or erythroleukemia-inducing (e.g., Friend- and Rauscher-MLV) – were subsequently isolated, several of which remain actively used in research. The deliberate selection of inbred lines created great diversity in the complement of endogenous MLVs between strains and, depending on relatedness, only around 30% of proviral loci are shared between common inbred mouse lines. Selection for inbreds with altered characteristics or phenotypic differences in turn elucidated the various impacts of MLVs and other mobile elements in genomes; indeed, many of the first characteristics mapped to genomic loci were determined to result from disruption of gene function due to MLV insertions, including dilute (d) in Myo5a, hairless (hr) in Hr, which remains named for the phenotype, and rodless retina (rd1) in the Pde6b gene. Work with MLVs revealed the means of retroviral replication by reverse transcription and formed the basis for much of the field of Retrovirology, for example early serological classification of MLVs defined various “group specific antigens”, for which the gag gene is named.

Genome and Structure Mature MLV virions are spherical enveloped particles typically B100–120 nm in diameter with a classical C-type morphology defined by viral assembly at the plasma membrane and an electron-dense polyhedral core in electron micrographs (Fig. 1, top), although overall size and core structures are pleomorphic. Virions budding from the cell membrane are initially immature, formed from assembled Gag polyproteins, with an electron-lucent core (Fig. 1, bottom). Gag is then cleaved by the virally encoded protease to allow the multimerization of CA hexamers and pentamers into the mature CA lattice that surrounds the viral core. The MLV genome defines the prototypic genetic structure of all simple retroviruses with three genes (50 -gag-pol-env-30 ), along with the order of the proteins within the polyproteins encoded by these genes: gag encodes the Gag polyprotein, cleaved to release matrix, p12, capsid, and nucleocapsid (MA-p12-CA-NC), pol encodes the Pol polyprotein, cleaved to release protease, reverse transcriptase, and integrase (PR-RT-IN), and env encodes the Env polyprotein, cleaved into surface and transmembrane (SU-TM)

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00038-2

643

644

Murine Leukemia and Sarcoma Viruses (Retroviridae)

Fig. 1 Transmission electron micrographs of MLV particles within spleen and lymph node tumor sections following spontaneous disease development in immunodeficient C57BL/6 inbred mice. Top row, mature virions showing the electron-dense polyhedral core. Bottom row, immature virions showing the electron-lucent circular core. Images were acquired at 5000–12000  magnification using a JEOL 100E  microscope and ORIUS 1000 CCD camera. Presented fields are standardized to 300 nm and scale bars show 100 nm.

(Fig. 2). Thus, products of gag contain all elements necessary for assembly and release of virions, pol the enzymes required for the process of retroviral replication, and env the structural elements required for target cell entry. The specific roles of these proteins are discussed in greater detail elsewhere. Whilst env is expressed from a spliced RNA, the products of gag and pol are translated from an unspliced genomic transcript (Fig. 2). Initiation at the Gag N-terminus either gives rise to the Gag polyprotein or, due to read-through of the in-frame UAG (amber) stop codon, a Gag-Pol polyprotein. This differentiates MLVs from other retroviral genera, which typically produce the Pol polyprotein through ribosomal frameshifting. The efficiency of the read-through thus also dictates the ratio of Gag: Pol gene products, at around 20:1. Certain MLVs also produce a longer, glycosylated, form of the Gag polyprotein (glyco-Gag or gGag), initiated upstream of the canonical start codon, which is not cleaved to yield the structural Gag proteins (gPr80, Fig. 2). gGag is transported to the cell membrane and shed from infected cells and, whilst gGag deficient viruses replicate well in vitro, such mutants exhibit reduced in vivo fitness. The role of gGag has yet to be fully elucidated but it has been proposed to overcome restriction factors including murine Apobec3 and Serinc5 (see below). Although all retroviruses express MA, CA and NC, different viruses release different additional Gag-cleavage products. The functions of these proteins are less well characterized and they are usually named for their apparent molecular weight. MLV, like many other genera, releases a protein from between MA and CA, called p12 (Fig. 2). This protein contains a four amino-acid motif referred to as the Late (L)-domain which recruits components of the cellular endosomal sorting complexes required for transport (ESCRT) machinery required for immature particles to bud from the plasma membrane. It also contains two functional domains that are important for viral infectivity. The N-terminal functional domain binds to the CA protein and stabilizes the CA lattice whilst the C-terminal domain interacts directly with host chromatin facilitating subsequent integration. In the integrated, proviral form, the long terminal repeats (LTRs) flank the viral genome and contain the regulatory elements necessary for expression within the U3 region (Fig. 2). The LTR’s complement of transcription factor binding sites dictate spatial and temporal expression patterns and, hence, too the mode of disease in MLV infection. Exchanging the LTR of Moloney-MLV for that of Friend-MLV, for example, switches its mode from T lymphomagenic to erythroleukemogenic, and vice versa.

Replication The process of retroviral replication is discussed in detail elsewhere, yet MLVs differ from other retroviruses in certain key aspects. Significantly, where many retroviruses, such as HIV-1, possess mechanisms to gain access to the cell nucleus, allowing cell-cycleindependent infection, MLVs are incapable of infecting non-dividing cells. If target cells are arrested in S phase or at the G2/M boundary, although viral entry, reverse transcription, and formation of the pre-integration complex (PIC) can complete at normal rates, integration cannot occur, even after extended periods. Thus, MLV infection is believed to require dissolution of the nuclear envelope in order for the PIC to gain access to the host chromosomes and, accordingly, successful infection is highly dependent on the cycling state of target cells.

Murine Leukemia and Sarcoma Viruses (Retroviridae)

645

Fig. 2 To-scale schematic representation of the MLV genome detailing the protein products of the gag, pol, and env genes, their apparent molecular weights, and points of enzymatic cleavage. The proviral DNA genome includes long terminal repeats at either end (composed of U3 – gray, R – white, and U5 – black) that both result from and allow for reverse transcription. Genomic mRNAs may be translated to yield only the gag gene products (green) through termination at the first UAG stop codon or, alternatively, read-through of this codon allows the translation of pol gene products (orange) in the form of a Gag-Pol precursor. The env gene products (blue) are produced from a spliced mRNA with the indicated splice donor (SD) and splice acceptor (SA) points. All mRNAs have a 50 7mG cap (50 CAP) and are polyadenylated (p(A)) at the 30 end. See main text for protein name two-letter abbreviations.

Once inside the cell nucleus, mechanisms of integration differ between retroviruses, resulting in varying target site selection preferences clearly visible with high-throughput sequencing. MLVs display a strong preference for integration within regions of active transcription and, specifically, for areas surrounding promoter-proximal CpG islands and transcription start sites. This differs significantly from HIV-1, where integration within transcriptional units (regions spanning from points of transcription initiation to the corresponding points of termination) is favored, but no specific enrichment is seen near transcription start sites and small reverse-associations are visible for CpG islands. Integration site selection in MLVs is mediated through IN binding host bromodomain and extraterminal (BET) proteins.

Host-Virus Interactions Host-virus interactions can be divided into the innate and the adaptive arms. Methods of innate control or resistance to MLV infection were some of the first subjects to be studied with inbred mice and the identification of Friend Virus Susceptibility 1 (Fv1) in 1970 and its positional cloning in 1996 initiated the study of host “restriction factors”. Common laboratory inbred mice express either Fv1b or Fv1n, each allele conferring resistance to virus of the opposing N- or B-tropism, determined by differences at residue 110 of the MLV CA. The range of Fv1-based restriction is now known to extend beyond the gammaretroviruses and its evolution and continued positive selection has occurred in tandem with the speciation of the Myodonta, over at least 45 my. Fv1 derives from the gag gene of an ERV and likely blocks infection through a process involving its scaffolding over incoming retroviral cores. A more common co-option of ERVs is seen in the Fv4, Rmcf, and Rmcf2 genes, each representing an MLV envelope glycoprotein (of ecotropic, polytropic, and xenotropic origins, respectively, see below) whose expression acts to prevent superinfection through interaction with the cognate cellular receptors. As well as co-opting retroviral proteins in host defense, a number of other host genes play significant roles as restriction factors. The murine Apobec3 cytidine deaminase inhibits MLV infection through both G-to-A hypermutation of the viral genome during reverse transcription and deaminase-independent mechanisms. Similarly to Fv1, Apobec3 exhibits significant positive selection and different mouse lines possess different alleles. The allele of C57BL/6 has particularly strong antiviral activity due to the recent capture of an MLV LTR, acting as an enhancer element for increased expression, as well as loss of exon 5, which increases protein synthesis. Studies of the mutational profile of integrated proviruses reveals that hypermutation is a substantial burden to infecting viruses, both in vitro and in vivo. Another host factor, Tetherin (Bst2), acts to prevent virion release from the cell surface and can have significant impacts in in vivo infection settings. Adult mice typically mount robust humoral responses to immunogenic epitopes within env, which are capable of inhibiting cell-to-cell spread of virus by neutralization and elimination. Both CD4 þ and CD8 þ T cells additionally respond to both Env and

646

Murine Leukemia and Sarcoma Viruses (Retroviridae)

Gag determinants and mediate direct killing, which has been shown to be relevant in limiting viral spread before the humoral response is mounted. After an initial period of viremia, infections within adults are typically controlled within 14–21 days, as exemplified by the Friend-MLV model system. Conversely, infection of neonates results in immunotolerance to viral antigens, persistent infection, and high levels of viremia. This is mediated through thymic deletion of Env-reactive T cells, rather than through B cell tolerization, and experimental introduction of T cell help allows infected mice to mount effective humoral responses. Although not causing complete tolerance, there is also evidence that background expression of MLV ERVs comparably shapes the repertoire of T cells able to respond to exogenous virus.

Pathology: Murine Leukemia Viruses MLVs can be subdivided into different groups according to the host range of their envelopes. Ecotropic MLVs (eMLVs) exhibit tropism for murine cells but not for cells of other species and utilize the cationic amino-acid transporter mCat1 (Slc7a1) as a receptor. eMLVs split into three groupings phylogenetically: AKV-type, CasBrE-type, and HoMuLV-type, the latter two having only been isolated from wild mice. Xenotropic MLVs (xMLVs) exhibit tropism for non-murine cells, using the inorganic phosphate exporter Xpr1 (Xpr1) as a receptor. Polytropic and modified polytropic MLVs (p/mpMLVs), differentiated by variation within their LTRs, also utilize Xpr1, but can infect both murine and non-murine cells. Amphotropic MLV (aMLVs) exhibit the same wide host range as p/mpMLVs but utilize one or both of the phosphate transporter Pit1 (Slc20a1) and Pit2 (Slc20a2) paralogues as receptors. These, although having only been identified as pathogens of wild mice, bear strong phylogenetic similarities to several model MLVs, including both Friend- and Moloney-MLV. The apparent historic escape of mXpr1 from xMLV envelope underlines the likely extended periods of receptor-envelope coevolution and suggest a certain plasticity in receptor usage over time. Indeed, endogenous MLVs with ecotropic host ranges have been identified within wild mice that use Smit1 (Slc5a3) in place of mCat1, as well as polytropic viruses using Pllp in place of Xpr1. Nevertheless, the global prevalence of retroviral pathogens of wild mice, including of MLVs, is largely unstudied. Indeed, whilst MLVs may be considered the primary burden, the evolution of Fv1 would suggest that both MMTVs and as yet unidentified lentiviruses circulate within certain species of Mus. In wild mice, exogenous MLVs are of low pathogenicity and disease occurs after long latency periods (these viruses are frequently described as “slowly transforming”, therefore), allowing for efficient horizontal transmission. Lesser routes of infection may be venereal, perinatal, transplacental, or through saliva but, primarily, transmission occurs through milk, from infected dams to nursed pups and the fostering of pups born of infected dams eliminates transmission. Whereas infection of adults rarely results in leukemogenesis for most viruses, neonatal infection frequently results in disease after 2–18 months, commonly in B/pre-B cells and visible within the spleen. Subsequent to initial viremia, a general requirement for MLV leukemogenesis is the spontaneous generation of recombinants, historically named mink cell focus-inducing (MCF) viruses due to their acquisition of a polytropic host range. MCF viruses form through individual or sequential recombination events with endogenous MLVs and are typified by wholly replaced or partially-recombinant env genes. Replicating at high titers, MCF viruses cause oncogenesis through random insertional mutagenesis – insertion near to, and the resulting dysregulation of, a cellular proto-oncogene (c-onc) – and it is common that resulting tumors are infected solely by MCF viruses, rather than with the initially-transmitted MLV. In the absence of MCF recombinants, ongogenesis may still occur, albeit at a reduced rate and with extended latency periods.

Pathology: Murine Sarcoma Viruses Early passage experiments with MLVs gave rise to occasional instances of increased pathogenicity and of altered disease characteristics – neoplastic rather than leukemogenic modes of disease. Named Murine Sarcoma Viruses (MuSVs or MSVs), these viruses cause disease after far shorter latency periods than for MLVs and are hence described as “strongly/acutely transforming”. MSVs, while derived from MLVs, acquire cellular oncogenes (termed v-onc genes when carried by the virus) at the expense of viral sequence and are, hence, replication incompetent, requiring complementation from a helper virus for their spread. Exapted v-onc genes are capable of inducing rapid transformation. Examples include the HRas GTPase (Harvey-MSV v-ras, controlling cell proliferation, differentiation, adhesion, and migration), Abl1 tyrosine kinase (Abelson-MSV v-abl, controlling cell division, differentiation, and stress responses), and the transcription factor c-Fos (Finkel-Biskis-Jinkins-MSV v-fos, pairing with c-Jun to form the AP-1 complex, controlling genes influencing cell proliferation, survival, and polarity). All known MSVs have been derived from experimental laboratory processes including the repeat passage of viral stocks or tumor extracts through susceptible mice, repeated neonatal inoculation, passage through alternate species, and host irradiation. As such, MSVs are experimental phenomena and are not believed to be a characteristic of disease within wild mice.

MLV Vectors The study of MSVs showed that it was possible to complement defective particles in trans. This allowed for designed, defective, genomes to be packaged in the same way and for stable integration of vectored sequences. MLV-based retroviral vector systems are

Murine Leukemia and Sarcoma Viruses (Retroviridae)

647

now routinely used in research laboratories as a way of expressing introduced genes. These systems are single cycle, meaning that no new viral particles are produced following reverse transcription and integration of the encapsidated RNA. Such retroviral vectors have also been used in the clinic as gene therapy vectors for several immunodeficiency disorders. However, insertional mutagenesis resulted in malignancy in some cases and other systems may provide improved safety features.

Endogenous MLVs Electron micrographs of both healthy and neoplastic tissues frequently reveal C-type retroviral particles as incidental observations and, indeed, common inbred mouse lines harbor between 50 and 80 complete MLV proviruses within their genomes, with a further number of intact env-deficient integrations, fragmented proviruses, and solo LTRs. Xenotropic, polytropic, and modified polytropic MLV, termed Xmv, Pmv, and Mpmv as endogenous loci, constitute the majority of endogenous MLV, with ecotropic MLV (Emv loci) being found in low numbers and in comparatively few inbred mice by comparison, for example the single Emv2 locus of C57BL/6 mice. The majority of endogenous MLVs are replication incompetent through point mutation, insertion, or deletion, although certain high-leukemia-incidence inbred mice (AKR and C58) notably carry infectious Emv loci. MLV loci are specifically targeted for epigenetic silencing through Trim28/Setdb1 by KRAB zinc-finger proteins (for example by Zfp809, which targets MLVs utilizing a reverse transcriptase proline-tRNA primer binding site (PBSPro)) and, hence, suppression can be reversed by treatment with demethylating agents, including BrdU and 5-AzaC. Potentially as a result of gradual changes in the epigenetic landscape accumulated with time, expression of endogenous MLVs increases in aged mice, as well as varying between tissues as a result of differing milieus of transcription factors. Accumulation of MLV products can induce progressive autoimmune conditions, including spontaneous vasculitis and glomerulonephritis. Expression of select proviruses can also be induced by stimulation of cells with TLR agonizts, including LPS, Poly(I:C), and Pam3CSK4. In conditions of immunodeficiency, activation of endogenous MLVs in this manner by products of commensal flora can result in their expression and recombinational repair, formation of MCF recombinants, and subsequent oncogenesis. In 2006, a new gammaretrovirus was isolated from prostate cancer patient tissue. Named xenotropic murine leukemia virusrelated virus (XMRV), this was potentially the third class of retrovirus to be pathogenic in humans. In 2009 XMRV was also linked to chronic fatigue syndrome. However, subsequent studies failed to find an association of XMRV with disease and, in most cases, failed to find the virus in human samples. In 2011, work from multiple laboratories revealed that XMRV was derived from recombination between two murine ERVs during serial passage of a human prostate tumor in nude mice to make the 22Rv1 and CWR-R1 cell lines in the mid-1990s. Indeed, it is likely that MLVs contaminate other cell lines historically passaged through mice. These studies separately highlighted that certain MLV RT-utilizing commercial reverse transcription kits harbor low levels of MLV DNA contamination.

See also: Avian Leukosis and Sarcoma Viruses (Retroviridae). Fish Retroviruses (Retroviridae). Jaagsiekte Sheep Retrovirus (Retroviridae). Structure of Retrovirus Particles (Retroviridae). The Role of Retroviruses in Cellular Evolution

Further Reading Coffin, J.M., Hughes, S.H., Varmus, H.E. (Eds.), 1997. Retroviruses. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Groom, H.C., Bishop, K.N., 2012. The tale of xenotropic murine leukemia virus-related virus. Journal of General Virology 93, 915–924. Kozak, C.A., 2015. Origins of the endogenous and infectious laboratory mouse gammaretroviruses. Viruses 7, 1–26. Mukerjee, S., Thrasher, A.J., 2013. Gene therapy for PIDs: Progress, pitfalls and prospects. Gene 525, 174–181. Rein, A., 2011. Murine leukemia viruses: Objects and organisms. Advances in Virology. 403419. Skorski, M., Bamunusinghe, D., Liu, Q., Shaffer, E., Kozak, C.A., 2019. Distribution of endogenous gammaretroviruses and variants of the Fv1 restriction gene in individual mouse strains and strain subgroups. PLoS One 14, e0219576. Young, G.R., Yap, M.W., Michaux, J.R., Steppan, S.J., Stoye, J.P., 2018. Evolutionary journey of the retroviral restriction gene Fv1. Proceedings of the National Academy of Sciences of the United States of America 115, 10130–10135.

Newcastle Disease Virus (Paramyxoviridae) Ben Peeters and Guus Koch, Wageningen Bioveterinary Research, Lelystad, The Netherlands r 2021 Elsevier Ltd. All rights reserved.

Nomenclature AAvV-1

The suggested nomenclature for AAvV-1 isolates is derived from that used for avian influenza virus isolates. At first mention, the isolate should be fully listed using the format: AAvV-1/ Species/Country (state, city or more specific location)/isolate number, name, or other unique

Glossary ICPI Intracerebral pathogenicity index Lentogenic Avirulent Mesogenic Moderately virulent

identifier/year of isolation (e.g.: AAvV-1/Chicken/ USA/LaSota/1946). Subsequently, this may be appropriately abbreviated (LaSota/1946). Certain isolates, in particular those used for vaccine production, are so well known that they often are listed by one name (LaSota, B1, Ulster, Herts33, etc.).

OIE World Organisation for Animal Health (Office International des Epizooties) Velogenic Very virulent

Classification The taxonomic name for Newcastle disease virus (NDV) has recently changed from Avian paramyxovirus 1 (APMV-1) to Avian Avulavirus 1 (AAvV-1). Together with 18 other avian avulaviruses (AAvV-2 to –19) AAvV-1 forms the genus Avulavirus within the family Paramyxoviridae in the order Mononegavirales. Phylogenetic analysis of genome sequences is the standard procedure to characterize AAvV-1 strains. The current generally accepted phylogenetic classification is based on the complete nucleotide sequence of the fusion protein (F) gene. Over time, AAvV-1 has undergone considerable genetic and antigenic drift. This genetic diversity is represented by groups of genetically related viruses denoted as genotypes and sub-genotypes. A system to define genotypes based on a mean evolutionary distance of 10% between groups is used to define genotypes. An evolutionary distance of 3%–10% is used to define sub-genotypes. Furthermore, a (sub-) genotype must consist of at least 4 epidemiologically-independent isolates, and the bootstrap value at branch points must be at least 60 (using the Neighbor Joining and the Maximum likelihood methods, with standard errors being calculated based on 500 bootstrap replicates). Strains of AAvV-1 can be grouped into 2 main classes, class I and class II. Class I viruses are mostly low pathogenic (lentogenic) viruses found in wild birds and belong to a single genotype. Class II viruses are much more diverse and currently 18 genotypes (with some containing sub-genotypes) can be discriminated.

Virion Structure AAvV-1 virions consist of enveloped pleomorphic particles ranging in size from approximately 100 nm to more than 500 nm in diameter. The majority of virus particles contains one functional genome although particles containing two or three genomes have also been observed. The F protein and hemagglutinin-neuraminidase protein (HN) are anchored in the virion envelope which is derived from the membrane of the infected cell. The F and HN proteins participate in attachment and entry, while HN is also involved in the release of progeny virus from infected cells. The matrix protein (M) is located immediately beneath the viral envelope. It maintains the shape of the virus and assists in the packaging of newly assembled viruses. Three proteins are associated with the single-stranded viral RNA genome. The nucleoprotein (NP) covers the entire viral RNA to form the ribonucleoprotein (RNP), which is the template for transcription and replication by the viral RNA-dependent RNA polymerase (large protein; L) in conjunction with the phosphoprotein (P) which acts as cofactor of the polymerase (Fig. 1).

Genome The genome of AAvV-1 consists of a non-segmented single-stranded RNA of negative polarity with a size of 15,186, 15,192 or 15,198 nucleotides (nt). The variation in genome size is due to a 6 nt deletion/insertion in the intergenic region between the NP and P genes, or a 12 nt deletion/insertion in the P gene. The AAvV-1 genome contains six open reading frames (ORF) which encode for NP, P, M, F, HN and L. Two additional, non-structural proteins, V and W, are generated by RNA editing during P gene transcription. The 3′ and 5′ ends of the genome comprise the non-transcribed leader (55 nt) and

648

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20933-3

Newcastle Disease Virus (Paramyxoviridae)

649

Fig. 1 AAvV-1 genome.

Fig. 2 AAvV-1 life cycle.

trailer (114 nt) regions. These regions contain regulatory sequences involved in transcription and replication of the genome. The terminal 10–20 nucleotides at the 3′ and 5′ ends are highly conserved and show complementarity to each other, suggesting an important role in genome replication. Transcription is initiated at the leader region and takes place by a sequential ‘stop-start’ mechanism in which the polymerase terminates transcription of the proximal gene and starts transcription of the distal gene. To this end, each gene is flanked by conserved transcription-initiation and transcriptiontermination sequences, the so called ‘gene-start’ and ‘gene-end’ boxes, which are separated by intergenic regions that vary in length from 1 to 47 nucleotides. Since not every stop results in a start event, the sequential transcription process gives rise to an mRNA gradient from the 3′ to the 5′ end of the genome, resulting in higher amounts of the structural proteins compared to the polymerase protein (Fig. 2).

Life Cycle AAvV-1 infection is initiated by HN-mediated attachment of the virus particle to sialic acid-containing glycoproteins on the cell surface. Binding of HN to sialic acid triggers a conformational change in the F protein that leads to fusion of the viral envelope with the plasma membrane. It has been reported that NDV may also enter the host cell through receptor-mediated endocytosis. After entry, the RNP is released into the cytoplasm where the genomic RNA is transcribed by the L protein to produce capped and polyadenylated mRNAs for synthesis of the viral proteins. When sufficient amounts of the viral proteins have accumulated, the polymerase complex switches from transcription to replication of the genome. Newly formed genomic RNA is then covered by NP and associates with a few copies of the polymerase complex to form RNPs. The other components of the virus particle are transported to the cell membrane where they are assembled under the direction of the M protein, followed by encapsidation of the

650

Newcastle Disease Virus (Paramyxoviridae)

RNP and subsequent budding of mature virions. Finally, the neuraminidase activity of the HN protein removes sialic acid from progeny virus particles, which facilitates detachment from the cell and prevents virus self-aggregation. The V protein is rich in cysteine and binds zinc while its carboxy-terminal portion has an anti-interferon activity, allowing the virus to counteract the innate immune response of the host.

Epidemiology At least 241 species from 27 of the 50 orders of birds have been shown to be susceptible to AAvV-1 infection. However, clinical disease signs, if any, may vary considerably between different species. Virulent AAvV-1 strains have been isolated from all types of poultry, ranging from pigeons to ostriches. Chickens infected with virulent AAvV-1 show overt disease signs and often succumb to the infection, whereas ducks may not show clinical signs. In some poultry species the disease may be milder than in chickens and cause problems in diagnosis, e.g. in pheasants and ostriches. Furthermore, there is often a difference in the severity of disease between young and adult birds. AAvV-1 isolates have been frequently obtained from wild birds, especially migratory aquatic birds. Most of these isolates were of low virulence for chickens. Occasionally, virulent viruses have been detected in wild birds found dead near infected poultry, suggesting that spill-over from infected poultry to wild birds can occur. For example, in North America AAvV-1 causes periodic die-offs in cormorants, which may also represent a reservoir species for this virus. Wild birds, captive birds and racing pigeons have been implicated in the introduction of virulent NDV into poultry in a number of outbreaks over the last two or three decades. Psittacine birds such as parrots have been shown to excrete virulent AAvV-1 intermittently for extremely long periods of time without showing clinical signs, emphasising the potential role of these birds in dissemination of AAvV-1. In the late 1970s a genotype VI AAvV-1 strain that had adapted to pigeons (known as PPMV-1) arose in the Middle East. This virus spread in racing and show pigeons throughout the world, resulting in a true panzootic (see below). The virus still remains enzootic in racing pigeons in many countries, posing a continuing threat to poultry. AAvV-1 infected birds shed virus in oropharyngeal secretions and faeces. Susceptible birds may become infected by ingesting or inhaling contaminated material. Vaccinated chickens may shed velogenic AAvV-1 for 6–9 days after infection, while unvaccinated wild or captive exotic birds like parrots, cormorants, and pigeons may have prolonged shedding of virus without clinical signs. The emergence of virulent AAvV-1 is a serious concern to the global poultry industry. AAvV-1 strains circulating in wild bird reservoirs are probably the major risk for the introduction of virulent AAvV-1 in poultry. Through ecological contacts and trade, these viruses can be transmitted from wild birds to domesticated poultry, where they cause clinical disease. Also, continuous circulation of lentogenic AAvV-1 among poultry forms a potential risk factor for the emergence of virulent AAvV-1. Furthermore, vaccine-derived AAvV-1 strains used in domesticated poultry have been reported to occur in wild birds, which further provides evidence of virus exchange between wild and domesticated birds. Thus, the continuous circulation and maintenance of AAvV-1 between wild and domesticated birds constitute a considerable threat for the emergence of virulent AAvV-1 strains. Introduction of virulent AAvV-1 into poultry has huge economic consequences in particular for exporting countries. Since the emergence of velogenic AAvV-1, four major AAvV-1 panzootics have occurred. The first started around the mid-1920s in Asia and Europe and was initially caused by genotype II viruses and later also by genotype III and IV viruses. In this case, spreading to the rest of the world was relatively slow and took about twenty years. The second panzootic started in the late 1960s and was caused by genotype V viruses. This time the panzootic developed within four years presumably due to the global increase in large-scale commercial poultry production and the international trade of poultry and captive wild birds. The third panzootic was caused by genotype VI viruses and started in the early-1980s among racing pigeons but eventually affected several other bird species. The fourth panzootic was caused by genotype VII viruses and started in the mid-1980s, resulting in severe economic losses in a large number of countries in South East Asia, the Middle East, Europe, Africa, and America. Currently, genotype VII viruses still constitute the most rapidly evolving strains of the virus. The recent expansion in the geographic distribution and host range of sub-genotype VIIi strains may signify the start of the fifth panzootic.

Pathogenesis Velogenic viruses can be further divided into viscerotropic velogenic (with a tropism for intestinal organs and tissues) and neurotropic velogenic (causing predominantly neurological signs). All mesogenic and velogenic AAvV-1 isolates require immediate notification to the World Organisation for Animal Health (OIE). Hence, pathotype identification is necessary for diagnosis of Newcastle disease (ND) in poultry. A common method to discriminate between different pathotypes is to the establish the mean death time (MDT) in experimentally infected 9-10-day-old embryonated chicken eggs. Viruses are diagnosed as velogenic when the MDT is 40–60 hours, mesogenic when the MDT is 60–90 hours, and lentogenic when the MDT is 4 90 hours. In case of a suspected outbreak, the pathogenicity test prescribed by the OIE is the intracerebral pathogenicity index (ICPI) in ten 1-day old SPF chickens. Velogenic strains have ICPI values from 1.4 to 2.0, while the values for mesogenic strains range from 0.7 to 1.4. Lentogenic isolates generally have ICPI values of 0.0–0.7. The major determinant of AAvV-1 pathogenicity is the F protein. The capacity of the F protein to act as a fusion protein must be activated by cleavage of the precursor protein F0 into F1 and F2. This cleavage is required for entry of AAvV-1 into host cells. Cleavage of

Newcastle Disease Virus (Paramyxoviridae)

651

F0 is mediated by cellular proteases and the type of protease that is capable of cleavage is dependent on the amino acid sequence motif around the cleavage site. The cleavage site of F0 of lentogenic AAvV-1 is characterised by a monobasic amino acid sequence motif at the C-terminus of F2 and a leucine at the N-terminus of F1, 112G-R/K-Q-G-R↓L117. F0 of lentogenic strains are cleaved mainly only extracellularly by trypsin-like proteases present in the respiratory and intestinal tract. Virulent strains have a multibasic amino acid sequence motif at the C-terminus of F2 and a phenylalanine at the N-terminus of F1, 112R/G/K-R-Q/K-K/R-R↓F117, and are cleaved intracellularly by ubiquitous furin-like proteases found in most host tissues. This difference in protease activation is the major determinant of systemic replication associated with severe disease of velogenic AAvV-1. Apart from the F protein, also other viral proteins may have a role in determining the pathogenicity of AAvV-1. The most notable example is the V protein which is involved in counteracting the host immune response. In general, the contribution of other viral proteins to pathogenicity is only minor when compared to the F0 cleavage site. The OIE has defined a Newcastle disease outbreak as an infection with any AAvV-1 that has an ICPI equal to or greater than 0.7. Detection of multiple basic amino acids near the cleavage site of the F protein is also acceptable to diagnose Newcastle disease. Viruses with a polybasic F cleavage site and phenyl alanine at position 117 are considered velogenic or mesogenic, while those with a monobasic F cleavage site and leucine at position 117 are considered lentogenic. However, if the analysis of the cleavage site of a suspected case reveals a lentogenic sequence, the ICPI test is still required by the OIE for the in vivo determination of pathogenicity.

Clinical Features The clinical symptoms after an AAvV-1 infection can differ greatly, depending on the pathotype of the virus and/or vaccination or infection status of the birds. In fully susceptible birds, the most severe symptoms are observed with velogenic viscerotropic strains which can cause mortality rates up to 100%. Symptoms include conjunctivitis, nasal discharges, dyspnoea, diarrhoea, ruffled feathers, prostration, tremors, and paralysis. Throughout the digestive tract haemorrhages may be observed, especially in the proventriculus and the caecal tonsils. Necrosis may be observed in internal organs such as the spleen, liver, and gut associated lymphoid tissue. Infections with velogenic neurotropic viruses are characterised by a mortality rate between 50% in older and 90% in younger susceptible birds. Mainly neurological and some respiratory clinical signs with almost no gastrointestinal involvement are observed. Typically, the affected birds show tremors, head twisting, opisthotonus and paralysis. Gross lesions are often absent but histologically, necrosis of Purkinje fibres as well as perivascular cuffing are often observed. Infection with mesogenic strains is also associated with neurological and respiratory symptoms but with a low mortality rate. Clinical signs are associated with a drop in egg production and mild to moderate respiratory illness. Infection with lentogenic viruses are generally associated with mild respiratory signs or no evidence of clinical disease, in particular in young fully susceptible birds.

Diagnosis Virus isolation is still regarded as the gold standard method for the diagnosis of an AAvV-1 infection. The choice of samples in live birds include cloacal and oropharyngeal swabs. Samples from dead birds should include tissue samples in addition to cloacal and oronasal swabs. The samples are inoculated into the allantoic cavity of 9- to 11-day-old specific pathogen and antibody free embryonated chicken eggs, and after 4–7 days of incubation a hemagglutination test (HA) is used to detect the presence of the virus in the allantoic fluid. Since other viruses also possess HA activity, it is necessary to further confirm the identity of the virus by either a hemagglutination inhibition test (HI) or molecular tests such as the polymerase chain reaction (PCR) or nucleotide sequencing. Virus identification by quantitative polymerase chain reaction (qPCR) is faster and less cumbersome than conventional diagnostic techniques. In addition, it also provides equal or even greater sensitivity of virus detection than virus isolation. M-, L- and F-gene based qPCR assays have been described as standard methods for AAvV-1 screening. However only F-gene based PCRs can be used for pathotyping directly from clinical samples. AAvV-1 class I isolates may escape detection using F-gene based PCRs assays. Therefore, a multiplex M- and L-gene based PCR was proposed. For pathotyping, it is important to continuously monitor the genetic diversity of the evolving AAvV-1 isolates, so that the primers and probes can periodically be updated to identify all variants. In addition to disease identification and pathotyping, the qPCR assay can also be used for the quantification of viral load in different organs or swabs from vaccinated animals that have been challenged with a virulent AAvV-1 strain. Serology is not very useful for the diagnosis of ND because serologic methods cannot differentiate antibodies induced after infection with AAvV-1 from those induced by vaccination. Nevertheless, serological tests are valuable tools to assess the humoral immune responses following vaccination. In countries that do not vaccinate, serology can confirm exposure to AAvV-1. In case of a suspicion based on clinical signs, a rise in HI titre may be an indication that exposure has occurred. The widest used serological test for AAvV-1 is the HI test which measures the ability of AAvV-1 specific antibodies to inhibit the agglutination of erythrocytes by the AAvV-1 particles. Virus neutralisation test (VNT) is yet another serological test that is useful for measuring AAvV-1 specific neutralising antibodies. Cross-reactivity of antibodies directed against other AAvV

652

Newcastle Disease Virus (Paramyxoviridae)

serotypes in particular against AAvV-3 may complicate interpretation of HI results. Enzyme-linked immunosorbent assays (ELISA) are also used in AAvV-1 serology, and several commercially available ELISA kits based on viral proteins or whole virus antigen have been developed. A tool that has recently revolutionised the diagnosis of infectious diseases is next-generation sequencing (NGS). NGS is not only important in tracking disease epidemics, but it also facilities the rapid, sensitive, and specific detection and differentiation of mixed infections within a single host. Furthermore, it can also be used to detect low frequency variants within a mixed population which would escape detection using classical diagnostic tools. With sequencing services becoming cheaper and cheaper, in the near future, NGS is likely to become a routine tool for viral diagnosis.

Prevention Tight biosecurity at the farm level and of the entire poultry production chain is critical to prevent the introduction of diseases to domestic poultry. Water and feed quality and pest and litter management are areas that need to be tightly controlled. All-in-all-out practices and routine disinfection and cleaning procedures between production rounds should be applied to reduce the risk of virus introduction. Biosecurity measures also should be employed in backyard flocks. Even with a good biosecurity practice, vaccination is still required to optimally protect the birds against economically devastating diseases such as ND. Because vaccines in the field are not able to completely prevent vaccinated birds from being infected with velogenic AAvV-1, the role of vaccination in the control of ND is to prevent losses from morbidity and mortality. Vaccination increases the resistance to infection and decrease the number of birds that become infected and the quantity of virus being shed, thus reducing within- and between-flock transmission. The use of vaccines derived from naturally occurring highly immunogenic AAvV-1 strains of low or intermediate virulence with ICPI o0.4 has been quite successful and has limited the economic losses due to ND in many countries. To date, a number of lentogenic AAvV-1 strains such as LaSota, Ulster, B1, F, V4, and I2 are extensively used as live vaccines. Live vaccines derived from mesogenic viruses such as the Komarov and Mukteswar strains, have been used in the past to booster the immune response following priming with lentogenic viruses. However, for safety reasons and because of the new definition of ND adopted by the OIE, use of these vaccines is no longer allowed. Live attenuated AAvV-1 vaccines applied by aerosol stimulate both mucosal and systemic immune responses. When applied correctly, a single dose of live AAvV-1 vaccine should be sufficient to induce an immune response capable of protecting 100% of the animals against clinical disease after challenge. However, more vaccinations are required to attain such levels in practice. Thus, shedding of virulent challenge virus via the cloacal and oropharyngeal routes may still occur. Replication and shedding of virulent virus can be substantially reduced when much higher doses of live vaccine are administered. However, because of vaccination reactions that can cause growth retardation and high cost of vaccination per bird, this is not an economically viable option. Thus, new approaches are required to tackle the problem of virus shedding associated with the use of conventional ND vaccines. Live AAvV-1 vaccines are suitable for mass application via drinking water or spray. In addition, the vaccine virus may spread from vaccinated birds to suboptimally vaccinated ones, thereby contributing to overall herd immunity. In spite of all their benefits, live AAvV-1 vaccines have also shortcomings. Maternal antibodies in chickens up to the age of 3 weeks may interfere with the infection and replication of vaccine virus. After weaning of the maternal antibodies, vaccination may cause respiratory disease or cause growth retardation. These post vaccination reactions can predispose the birds to secondary infections by other pathogens. Furthermore, vaccines are produced using genotypes I or II seed strains which are phylogenetically divergent from currently prevalent genotypes. Hence, although the vaccines are still protective against clinical signs and mortality caused by any AAvV-1 isolate, their reduced ability to block shedding of contemporary viruses post challenge increases the risk of the continuous presence of the virulent virus in the environment. This is particularly more dangerous with genotype VII isolates, whose shedding from vaccinated birds is significantly higher than those of other genotypes. Therefore, considering the above limitations, live attenuated vaccines must be used with care and the state-of-the-art vaccines are urgently needed to address these weaknesses. Rationally designed vaccines targeting the prevailing genotypes, the so-called genotype-matched vaccines, are highly needed to overcome the shortcomings of classical vaccines. Reverse genetics-based live attenuated vaccines are probably the most promising candidates. Vector vaccines (see below) form another alternative approach having the advantage of in ovo application in the face of maternal immunity and claims of live-long protection. Inactivated vaccines are not suitable for mass application because they have to be applied via the parental route, and thus individually. Furthermore, they do not replicate, and thus cannot spread horizontally among the birds. Individual application is labour intensive and thus expensive. The same nonreplicating feature, however, makes them safe with no risk of reversion to virulence. Inactivated AAvV-1 vaccines are generally poor inducers of mucosal or cell-mediated immune responses but induce high HI titres. Therefore, for best results, these vaccines are administered after initial priming and/or boosting with live vaccines in layer flocks of 15–17 weeks or older. Inactivated vaccines require the use of adjuvants which may cause undesirable reactions in vaccinated birds such as a drop in egg production. Furthermore, a withdrawal period is required before birds immunised with inactivated adjuvanted vaccines can be processed for human consumption. Therefore inactivated vaccines cannot be applied in broilers. A promising strategy that can be used to combat pathogens is the use of recombinant viral vector vaccines. The most common vectors used in poultry are the vaccinia virus, fowl pox virus (FPV), herpes virus of turkeys (HVT), and Marek’s disease virus (MDV). Because of their large double-stranded DNA genome, these viruses have a very high capacity for the insertion of foreign

Newcastle Disease Virus (Paramyxoviridae)

653

genes. They are highly immunogenic, capable of inducing strong inflammatory innate immune responses, and they can be easily propagated on a large scale. Recombinant vaccinia virus expressing the F gene from AAvV-1 was shown to induce strong immunity that protected birds against virulent AAvV-1 challenge. Recombinant FPV-vectored ND vaccines generated by replacing the thymidine kinase gene with AAvV-1 F, HN or F and HN were shown to induce protective immunity against virulent AAvV-1 challenge in chickens. One of the advantages of an FPV-vectored vaccine is that it does not cause post vaccination respiratory reactions. However, the presence of anti-FPV antibodies in vaccinated chickens may dramatically interfere with the efficiency of this vector. Furthermore, this vaccine may not be suitable for young birds. This particular shortcoming can be overcome by the use of recombinant herpes virus vectored vaccines which can be used at the hatchery both in ovo in 18-day-old embryo’s or in 1-dayold chickens. It elicits a strong and long lasting cell mediated and humoral immunity due to its ability to persist as a latent infection in the vaccinated chicken.

Further Reading Alexander, D.J., 2009. Ecology and epidemiology of newcastle disease. In: Capua, I., Alexander, D.J. (Eds.), Avian Influenza and Newcastle Disease: A Field and Laboratory Manual. New York: Springer, pp. 19–26. Ayala, A.J., Dimitrov, K.M., Becker, C.R., et al., 2016. Presence of vaccine-derived newcastle disease viruses in wild birds. PLOS ONE 11 (9), e0162484. Cardenas-Garcia, S., Diel, D.G., Susta, L., et al., 2015. Development of an improved vaccine evaluation protocol to compare the efficacy of Newcastle disease vaccines. Biologicals 43 (2), 136–145. Cattoli, G., Susta, L., Terregino, C., Brown, C., 2011. Newcastle disease: A review of field recognition and current methods of laboratory detection. Journal of Veterinary Diagnostic Investigation 23, 637–656. Czegledi, A., Ujvari, D., Somogyi, E., et al., 2006. Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications. Virus Research 120, 36–48. Diel, D.G., da Silva, L.H., Liu, H., et al., 2012. Genetic diversity of avian paramyxovirus type 1: Proposal for a unified nomenclature and classification system of Newcastle disease virus genotypes. Infection, Genetics and Evolution 12, 1770–1779. Dimitrov, K.M., Afonso, C.L., Yu, Q., Miller, P.J., 2017. Newcastle disease vaccines – A solved problem or a continuous challenge? Veterinary Microbiology 206, 126–136. Dimitrov, K.M., Ramey, A.M., Qiu, X., Bahl, J., Afonso, C.L., 2016. Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus). Infection, Genetics and Evolution 39, 22–23. Dortmans, J.C., Koch, G., Rottier, P.J., Peeters, B.P., 2011. Virulence of newcastle disease virus: What is known so far? Veterinary Research 42, 122. Miller, P.J., King, D.J., Afonso, C.L., Suarez, D.L., 2007. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge. Vaccine 25, 7238–7246. Miller, P.J., Haddas, R., Simanov, L., et al., 2015. Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features. Infection, Genetics and Evolution 29, 216–229. Miller, P.J., Koch, G., Newcastle Disease, 2013. Diseases of Poultry. Swayne, D.E. (Ed.), thirteenth ed. John Wiley & Sons, Inc. Palya, V., Kiss, I., Tatar-Kis, T., et al., 2012. Advancement in vaccination against Newcastle disease: Recombinant HVT NDV provides high clinical protection and reduction challenge virus shedding, with the absence of vaccine reaction. Avian Diseases 56, 282–287. Peeters, B.P.H., De Leeuw, O.S., Koch, G., Gielkens, A.L.J., 1999. Rescue of Newcastle disease virus from cloned cDNA: Evidence that cleavability of the fusion protein is a major determinant for virulence. Journal of Virology 73, 5001–5009. Rauw, F., Gardin, Y., Palya, V., et al., 2010. Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine 28, 823–833.

Relevant Websites http://agriculture.vic.gov.au/agriculture/pests-diseases-and-weeds/animal-diseases/poultry/newcastle-disease Agriculture Victoria – Newcastle disease. https://ec.europa.eu/food/animals/animal-diseases/control-measures/newcastle-disease_en European Commission. Animal Diseases and control measures – Newcastle disease. http://www.efsa.europa.eu/en/efsajournal/pub/477 European Food Safety Authority – Opinion of the Scientific Panel on Animal Health and Welfare (AHAW) to review Newcastle disease focussing on vaccination worldwide in order to determine its optimal use for disease control purposes. https://www.agric.wa.gov.au/livestock-biosecurity/newcastle-disease Government of Western Australia. Department of Primary Industries and Regional Development – Newcastle disease. https://www.gov.uk/guidance/newcastle-disease Gov.uk – Newcastle disease: How to spot and report it. http://www.cfsph.iastate.edu/DiseaseInfo/disease.php?name=newcastle-disease&lang=en The Center for Food Security and Public Health – Newcastle Disease. https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/avian-influenza-disease/vnd United States Department of Agriculture – Virulent Newcastle Disease. https://www.wur.nl/en/Research-Results/Research-Institutes/Bioveterinary-Research/Animal-diseases/Virology/Newcastle-disease-3.htm Wageningen University and Research – Newcastle Disease. http://www.oie.int/en/animal-health-in-the-world/animal-diseases/Newcastle-disease/ World Organisation for Animal Health – Newcastle Disease. http://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_nd.htm World Organisation for Animal Health – Terrestrial Animal Health Code. Infection with Newcastle Disease.

Orthobunyaviruses (Peribunyaviridae) Alyssa B Evans, Clayton W Winkler, and Karin E Peterson, National Institutes of Health, Hamilton, MT, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of C.H. Calisher, Orthobunyaviruses, In Encyclopedia of Virology (Third Edition), Edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00623-3.

Glossary Arbovirus A virus that is transmitted to vertebrates through the bite of a hematophagous (blood-feeding) arthropod. Endemicity When a virus is found consistently in a region, generally determined by measuring neutralizing antibody responses in the primary animal reservoir for that particular virus. Orthobunyavirus Genus of viruses in the family Peribunyaviridae, order Bunyavirales. They are enveloped viruses with tri-segmented single stranded negative-sense

RNA genomes. These segments are L (large), M (medium) and S (small). Reassortment When one or more segments of one orthobunyavirus is combined with segments from another virus to form a new reassortant virus (e.g., the M segment from Batai virus reassorted with the L and S segments from Bunyamwera virus to generate Ngari virus). Transovarial The transmission of a virus or other pathogen from an organism, such as a mosquito, to its offspring by infection of the eggs in the ovary.

Classification Serological Classification The orthobunyaviruses are enveloped, negative-sense, single-stranded RNA viruses containing three genomic segments, the Large (L), Medium (M), and Small (S) segments (Fig. 1(A)). Traditionally, the orthobunyaviruses have been classified into serogroups via serological assays such as complement fixation, hemagglutination (HI), and neutralizing antibody assays. However, classification into serogroups is complicated by the tri-segmented nature of the genome of these viruses. Different serological assays target different genome segments, which can lead to contradicting results. Complement fixation assays interact with the Nucleocapsid protein encoded by the S segment, while HI and neutralization assays interact with the Gn and Gc envelope glycoproteins encoded by the M segment (Table 1). Although none of the serological tests interact with proteins generated by the L segment, differences between M and S segments can affect serological testing. Related segmented viruses can exchange genome segments in a process called reassortment (Fig. 1(B)). Reassortant viruses may be determined to fall into two different serogroups depending on the serological test used in classification. Even without the process of reassortment, viruses within a serogroup can group differently depending upon the serological test used. Despite these caveats, serogroups are a useful way of determining antigenic relatedness and are an important consideration in diagnostic testing. At least 20 orthobunyavirus serogroups have been identified or proposed (Table 2). The Bunyamwera, California, and Simbu serogroups contain many of the most significant human and livestock pathogens of the Orthobunyavirus genus. The orthobunyaviruses do not have any crossreactivity with other genera of bunyaviruses.

Phylogenetic Analysis by Sequencing With the increased capacity and decreased cost of sequencing technology, classification of viruses by phylogenetic relationships has become more common, and the orthobunyaviruses are no exception. Based on genetics, distinguishing characteristics of the Orthobunyavirus genus include specific consensus terminal nucleotide sequences and the M segment which encodes the envelope proteins and a nonstructural protein as a precursor polyprotein in the order of Gn-NSm-Gc. As with serology, different results can be obtained depending on the genome segment(s) used for the analysis. Additionally, the viruses/strains used in studies and methods of phylogenetic analysis can yield differing results between studies. However, in the phylogenetic analyses that have been performed, genetic relationships tend to group with serological classifications. The advantage of sequencing over serology is that all three segments can be compared. Despite advances in the technology, sequencing is still more difficult, time-consuming, and expensive than serology. However, sequencing information is an important tool for evaluating virus genetic relatedness and assessing reassortment between orthobunyaviruses, particularly in cases were serology analysis results in conflicting or inconclusive classification. According to the International Committee for the Taxonomy of Viruses 9th Report, when sequencing data is available, orthobunyaviruses are categorized as different species when there is more than a 10% difference in amino acid sequences of the nucleocapsid protein encoded by the S segment. However, this classification is limited because it does not account for the genetic diversity in the M and L segments.

654

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21542-2

Orthobunyaviruses (Peribunyaviridae)

655

Fig. 1 Structure, genomic makeup and reassortment potential of orthobunyaviruses. (A) Orthobunyaviruses are enveloped spherical viruses with two envelope proteins (Gn and Gc) in the membrane, which surrounds three negative strand RNA segments of genome (L, M, S), associated with the N and L proteins. The genome and encoded proteins are shown below. (B) Due to the tri-segmented nature of orthobunyaviruses, when two viruses infect the same cell the individual segments of each viral genome can be reassorted and produce eight possible different viruses. These include the original two parental strains as well as a multiple combinations of L, M, and S segments.

Table 1

Orthobunyavirus proteins of Bunyamwera virus prototype

Genome segment

Protein

Amino acids

Function

Large

L protein

2238

RNA-dependent RNA-polymerase: Mediates transcription and replication Endonuclease domain: involved in cap-snatching from host mRNAs

Medium

Glycoprotein(n) (Gn) Glycoprotein(c) (Gc) Nonstructural protein M (NSm)

296 146 956

Envelope glycoprotein; chaperone for Gc trafficking to the Golgi Envelope glycoprotein: Class II fusion protein May play a role in virion assembly and budding; not strictly required for replication in vitro

Small

Nucleocapsid (N)

233

Encapsidates genomic and anti-genomic RNAs to form ribonucleoproteins

Nonstructural protein S (NSs)a

101

Inhibits host RNA Polymerase II; Type I IFN-antagonist; not required for replication Inhibits apoptosis in BUNV, but increases apoptosis in LACV

a

Not all orthobunyaviruses encode NSs, including members of the Anopheles A, Anopheles B, and Tete serogroups.

Genome Genome and Encoded Proteins Orthobunyaviruses contain tri-segmented, single-stranded, negative-sense RNA genomes (Fig. 1(A)). The Large (L) segment is the largest at B6.9 kb and encodes the L protein, which functions as the viral RNA-dependent RNA-polymerase (RdRp, L protein) and contains an endonuclease domain (Table 1). The Medium (M) segment is B4.5 kb and encodes a polyprotein in the order Gn-NSm-Gc. This protein is proteolytically processed into envelope glycoproteins (Gn and Gc) and a nonstructural protein (NSm) by internal signal peptides and host signal peptide peptidase. The Small (S) segment is B1 kb and encodes the nucleocapsid (N) protein. Most orthobunyaviruses also encode a second nonstructural protein on the S segment (NSs) in an overlapping reading frame with N. However, some orthobunyaviruses, including members of the Anopheles A, Anopheles B, and Tete serogroups, do

656

Orthobunyaviruses (Peribunyaviridae)

Table 2

Orthobunyavirus family members ICTV recognized species; group virus not listed as ICTV species

Virus

Serogroup

Anopheles A virus Lukuni virus Tacaiuma virus Anopheles B virus Boraceia virus Bakau virus Ketapang virus Nola virus Tanjong Rabok virus Telok Forest virus Anadyr virus

Anopheles A Anopheles A Anopheles A Anopheles B Anopheles B Bakau Bakau Bakau Bakau Bakau Bunyamwera

mosquitoes

Anhembi virus Batai virus Birao virus Bozo virus Bunyamwera virus

Bunyamwera Bunyamwera Bunyamwera Bunyamwera Bunyamwera

mosquitoes mosquitoes mosquitoes mosquitoes mosquitoes

Cache Valley virus

Bunyamwera

Calovo virus Fort Sherman virus Germiston virus Ilesha virus

Bunyamwera Bunyamwera Bunyamwera Bunyamwera

Kairi virus Lokern virus Maguari virus Main Drain virus

Bunyamwera Bunyamwera Bunyamwera Bunyamwera

Ngari virus*

Bunyamwera

Northway virus Playas virus Potosi virus Tensaw virus Bwamba virus

Bunyamwera Bunyamwera Bunyamwera Bunyamwera Bwamba

Pongola virus California encephalitis virus Jamestown Canyon virus Keystone virus La Crosse virus Lumbo virus Melao virus San Angelo virus Serra do Navio virus Snowshoe hare virus Tahyna virus Trivittatus virus Inkoo virus South River virus Chatanga virus Morro Bay virus Infirmatus virus Achiote virus Jerry Slough virus

Bwamba California California California California California California California California California California California California California California California California California California

Disease

Vector

Ticks

livestock, avian, humans: mild; fever, joint pain, rash sheep: embryonic and fetal death, stillbirths, congenital defects; human disease (rare)

humans: mostly febrile, occassional association with meningoencephalitis and hemorrhagic fever not reported

experimental infection in sheep: severe musculoskeletal and nervous system malformations, death in fetuses livestock, avian, humans: fatal hemorrhagic fevers in humans and ruminants

human encephalitis humans: febrile illness, hemorrhagic complications

Host References Hughes et al. (2017) Hughes et al. (2017) Hughes et al. (2017) Mohamed et al. (2009) Mohamed et al. (2009) Zeller et al. (1989) Zeller et al. (1989) Zeller et al. (1989) Zeller et al. (1989) Zeller et al. (1989) Shchetinin et al. (2015) (in Russian) de Souza Lopez et al. (1975) Dutuze et al. (2018) Arbocat Arbocat Dutuze et al. (2018)

mosquitoes mosquitoes mosquitoes mosquitoes mosquitoes

Zeller et al. (1989) Arbocat Calisher (1983) Pachler et al. (2013)

mosquitoes mosquitoes mosquitoes mosquitoes

Soto et al. (2009) Meyers et al. (2015) Pollitt et al. (2006) Tangudu et al. (2018); Arbocat

mosquitoes

Briese et al. (2006)

mosquitoes mosquitoes mosquitoes mosquitoes

Meyers et al. (2015) Calisher et al. (1983) Harrison et al. (1995) Tangudu et al. (2018) Groseth et al. (2014)

neuroinvasive in humans

mosquito

Groseth et al. (2014) Evans and Peterson (2019)

neuroinvasive in humans Rash in 1 human neuroinvasive in humans

mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito mosquito

Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans Evans

neuroinvasive in humans mostly flu-like, neuroinvasive neuroinvasive in humans neuroinvasive in humans

and and and and and and and and and and and and and and and and and

Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson Peterson

(2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019) (2019)

Orthobunyaviruses (Peribunyaviridae)

Table 2

657

Continued

Virus

Serogroup

Acara virus Benevides virus Benfica virus Bushbush virus Capim virus Guajara virus Juan Diaz virus Moriche virus Enseada virus Alajuela virus Calchaqui virus Gamboa virus Pueblo Viejo San Juan virus Soberania virus Apeu virus Bruconha virus Caraparu virus Gumbo limbo virus Itaqui virus Madrid virus Marituba virus Murutuca virus Nepuyo virus Oriboca virus Ossa virus Restan virus Vinces virus Ananindeua virus Bertioga virus Bimiti virus Cananeia virus Catu virus Guama virus Guaratuba virus Itimirim virus Mahogany Hammock virus Mirim virus Moju virus Timboteua virus Guaroa virus Koongol virus Wongal virus Leanyer virus Buffalo Creek virus Gan Gan virus Mapputta virus Maprik virus Murrumbidgee virus Salt Ash virus Trubanaman virus Minatitlan virus Palestina virus Nyando virus Botambi virus Olifantsvlei virus

Capim Capim Capim Capim Capim Capim Capim Capim Enseada (proposed) Gamboa Gamboa Gamboa Gamboa Gamboa Gamboa Group C Group C Group C Group C Group C Group C Group C Group C Group C Group C Group C Group C Group C Guama Guama Guama Guama Guama Guama Guama Guama Guama Guama Guama Guama Guaroa (proposed) Koongol Koongol Leanyer (proposed) Mapputta Mapputta Mapputta Mapputta Mapputta Mapputta Mapputta Minatitlan Minatitlan Nyando Olifantsvlei Olifantsvlei

Abras virus Babahoyo virus Pahayokee virus

Patois Patois Patois

Disease

Not Not Not Not

reported reported reported reported

Not reported

humans: febrile illness

humans: febrile illness

Vector

mosquitoes mosquitoes mosquitoes mosquitoes mosquitoes mosquitoes

Host References

birds birds birds birds birds

(Arbocat) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) de Souza et al. (2016) Chiang et al. (2018) Chiang et al. (2018) Chiang et al. (2018) Chiang et al. (2018) Chiang et al. (2018) Chiang et al. (2018) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Nunes et al. (2005) Shchetinin et al. (2015) de Souza Lopez et al. (1975) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Aguilar et al. (2010) Shchetinin et al. (2015) Shchetinin et al. (2015) Savji et al. (2011) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Calisher et al. (1983) Calisher et al. (1983) Groseth et al. (2014) Arbocat Taxonomy of the order Bunyavirales Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) (Continued )

658

Orthobunyaviruses (Peribunyaviridae)

Table 2

Continued

Virus

Serogroup

Patois virus Shark River virus Zegla virus

Patois Patois Patois

Aino virus

Simbu

Akabane virus

Simbu

Buttonwillow virus Cat Que virus Douglas virus Faceys paddock virus Ingwavuma virus Inini virus Jatobal virus Kaikalur virus Manzanilla virus Mermet virus Oropouche virus Para virus Peaton virus Sabo virus Sango virus Sathuperi virus Shamonda virus Schmallenberg virus

Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu Simbu

Shuni virus Simbu virus Thimiri virus Tinaroo virus Utinga virus Yaba
7 Bahig virus Batama virus Matruh virus Tete virus Tsuruse virus Weldona virus Lednice virus MPoko virus Turlock virus Umbre virus Yaba
1 virus Anhembi virus Cachoeira Porteira virus Iaco virus Macaua virus Sororoca virus Taissui virus Wyeomyia virus Bellavista virus Kaeng Khoi virus Tataguine virus Witwatersrand virus Wolkberg virus

Simbu Simbu Simbu Simbu Simbu Simbu Tete Tete Tete Tete Tete Tete Turlock Turlock Turlock Turlock Turlock Wyeomyia Wyeomyia Wyeomyia Wyeomyia Wyeomyia Wyeomyia Wyeomyia Unclassified Unclassified Unclassified Unclassified Unclassified

Disease

ruminants: hydranencephaly/arthrogryposis, abortion, stillbirths, congenital abnormalities of CNS ruminants: hydranencephaly/arthrogryposis, abortion, stillbirths, congenital abnormalities of CNS

Vector

biting midges

biting midges

humans: febrile illness

ruminants: teratogenic, congenital malformations, CNS disease

biting midges

Ticks

Humans: fever, headache, rash, joint pain not reported

Host References Aguilar et al. (2018) Shchetinin et al. (2015) Zeller et al. (1989); Aguilar et al. (2018) Agerholm et al. (2015); Saeed et al. (2001) Agerholm et al. (2015); Saeed et al. (2001) Saeed et al. (2001) Zhang et al. (2015) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Aguilar et al. (2011) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Agerholm et al. (2015) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Saeed et al. (2001) Shchetinin et al. (2015) Mohamed et al. (2009) Shchetinin et al. (2015) Mohamed et al. (2009) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Shchetinin et al. (2015) Chowdhary et al. (2012) Chowdhary et al. (2012) Chowdhary et al. (2012) Chowdhary et al. (2012) Chowdhary et al. (2012) Chowdhary et al. (2012) Chowdhary et al. (2012) Hang et al. (2016) Groseth et al. (2014) Shchetinin et al. (2015) Shchetinin et al. (2015) Jansen van Vuren et al. (2017)

Orthobunyaviruses (Peribunyaviridae)

659

Fig. 2 Orthobunyaviruses enter host cells by receptor-mediated attachment of virus envelope (1) leading to clathrin-mediated endocytosis (2). The virus then uncoats (3) in endosomal vesicles and the RNA segments are released in the cytoplasm where they undergo primary transcription (4), translation of viral proteins (5) and genome replication (6). The components are transported to the Golgi complex (7) where virus assembly takes place. Virus particles then migrate to the membrane (8) where they fuse with the plasma membrane and are released via exocytosis.

not have NSs. In Bunyamwera virus (BUNV) and La Crosse virus (LACV), the NSs has been shown to antagonize the host antiviral response by targeting host Polymerase II, leading to impaired host transcription and interferon production.

Life Cycle Most studies of orthobunyavirus replication have focused on just a select few viruses, primarily LACV and BUNV, the latter of which is the orthobunyavirus prototype. There is likely a great deal of diversity in the mechanisms used by different orthobunyaviruses for replication. From studies of LACV and BUNV, it has been shown that orthobunyaviruses utilize receptor-mediated endocytosis to enter cells (Fig. 2). The entry receptor(s) are currently not known for any of the orthobunyaviruses. Upon entry, orthobunyaviruses uncoat at the endosomal membrane, likely through conformational changes in Gc, which is the predicted orthobunyavirus fusion protein. The negative-sense genome segments are used as the template for the L protein RdRp to generate viral mRNA. The L protein contains an endonuclease domain that steals the 50 7-methylguanylate capped ends of host mRNAs in order to prime transcription in a process called cap-snatching. Orthobunyaviruses require active host translation for viral transcription to occur. Genome replication uses full-length anti-sense ribonucleoproteins (RNPs) as templates for the RdRp in a primer-independent process to generate the full length negative-sense genome segments. The 50 and 30 ends of each genome segment are complementary and form a panhandle that circularizes each segment. The genome segments form RNP complexes through interactions with the N protein, and the panhandles are thought to associate with the L protein in the virion.

660

Orthobunyaviruses (Peribunyaviridae)

Several orthobunyaviruses have been shown to create Golgi-derived viral factories to facilitate virus replication through endosomal complex required for transport (ESCRT) machinery. Restructuring of the Golgi complex for viral replication likely helps facilitate efficient virus assembly, maturation, and budding, processes that occur at the membrane of the Golgi. Assembly and budding may be facilitated by NSm, however, in studies utilizing recombinant viruses, the importance of NSm for the production of infectious virus varies between orthobunyaviruses. The Golgi membranes express the viral glycoproteins. The Gn and Gc glycoproteins dimerize and form a tripod-like trimer of dimers on the envelope surface. Genomic RNPs are trafficked to these membranes where the UTRs mediate packaging. These interactions facilitate budding, then exocytosis releases virions from the cell. Once released from cells, the orthobunyavirus glycoproteins experience a final enzymatic cleavage maturation step that results in fully infectious virions. Virion size varies between orthobunyaviruses but is approximately 108 nm in diameter.

Transmission Vector Most isolations of orthobunyaviruses have been made from arthropods, and all orthobunyaviruses are thought to be carried and transmitted by hematophagous insects and/or arachnids. Orthobunyaviruses are found throughout the world, but the range of individual viruses is restricted by their arthropod vector and vertebrate host ranges. The majority of orthobunyaviruses utilize mosquito vectors. However, viruses in the Simbu serogroup are transmitted by biting midges, viruses in the Tete serogroup are transmitted by ticks, and one virus, Kaeng Khoi virus, has been found in bed bugs. Viruses from other serogroups have been isolated from non-mosquito insects, but their roles as vectors is unclear. For many orthobunyaviruses in the California serogroup, transovarial transmission in mosquito vectors has been well established as an over-wintering mechanism. However, evidence of transovarial transmission of other orthobunyaviruses is limited, so it is unclear if transovarial transmission is widely utilized as an overwintering mechanism for orthobunyaviruses.

Vertebrate Hosts Vertebrate hosts have not been identified for many of the orthobunyaviruses. Some vertebrate hosts have been characterized for orthobunyaviruses, and range in diversity from birds to mammals. Most viruses in the California serogroup are transmitted by small mammals like hares, chipmunks, and squirrels. Bunyamwera serogroup viruses utilize a wide variety of vertebrate hosts including rodents, humans, and ruminants. Gamboa serogroup members appear to be maintained in a mosquito to bird transmission cycle, and Simbu viruses use a variety of mammals and birds. Due to the lack of evidence of transovarial transmission for many orthobunyaviruses, and the wide range of vertebrate hosts that the viruses have been associated with, most orthobunyaviruses are likely maintained in a vector to vertebrate host to vector transmission cycle.

Reassortment Evidence of Reassortment Reassortment is the process where related segmented viruses exchange genome segments creating novel reassortant viruses (Fig. 1 (B)). Reassortment among the orthobunyaviruses has been well documented between viruses as well as viruses within a serogroup. Based on phylogenetic analyzes, there is evidence of reassortment within several orthobunyavirus serogroups including the California, Guamá, Bunyamwera, and Simbu serogroups. Interestingly, the M segment is the most commonly reassorted segment between orthobunyaviruses, possibly because it encodes the envelope proteins that control virus entry.

Effects of Reassortment Of particular interest and concern are reassortant viruses with increased pathogenicity compared to their parental viruses. In addition to the naturally occurring reassortant orthobunyaviruses, laboratory derived reassortant viruses have been generated between viruses in the California serogroup. Because many orthobunyaviruses share geographical ranges, vector species, and vertebrate hosts, the occurrence of novel reassortant orthobunyaviruses arising with enhanced pathogenicity is possible.

Epidemiology Viral Discovery The prototypical and namesake orthobunyavirus, BUNV, was first discovered in Uganda in 1943 as a result of Aedes mosquito surveillance studies looking for Yellow fever virus. This route of discovery is fairly typical as novel orthobunyaviruses have been discovered and isolated in surveys of known vector species, presumed vector species, host species and host parasite

Orthobunyaviruses (Peribunyaviridae)

661

species. Alternatively, novel viruses have also been identified as a result of human or animal disease. Because of the potential for reassortment of orthobunyaviruses, the expanding range of vector species, the ability of some orthobunyaviruses to colonize new vectors and the increasing numbers of novel human cases associated with orthobunyavirus infection, surveillance, identification, characterization and early detection of novel pathogenic viruses that could lead to an outbreak virus is paramount.

Surveying Viral Endemic Ranges and Tracking Viral Spread Areas of endemicity for most orthobunyaviruses have been determined by a combination of vector and host molecular identification surveys, and seroprevalence studies. However, most molecular identification studies are limited by being short in duration and only determining the presence of a virus rather than establishing if that virus has a replicative life cycle in a putative endemic area. To supplement these survey studies, seroprevalence studies in host species, including wildlife, domesticated livestock and humans have also been completed. While these types of studies can be complicated by viral misidentification due to antibody cross-reactivity within and between orthobunyavirus serogroups, they do utilize easily accessible samples that can be monitored longitudinally to determine endemicity. Seroprevalence sampling is also a convenient and effective tool to monitor viral spread particularly for human disease-causing orthobunyaviruses. This technique is commonly employed by government agencies to monitor health risks to their population and provide public awareness. Such information is becoming increasingly important in the modern era of human global travel and expanding vector ranges.

Orthobunyavirus Outbreaks and Tracking Due to the inherently high mutation rates of RNA viruses and the ability of orthobunyaviruses to reassort and acquire pathogenic features, periodic outbreaks and emergence of novel orthobunyaviruses occur. A recent example involves an outbreak of Schmallenberg virus, which is transmitted by biting midges, in sheep and cattle during 2011–2012 in Europe. The first cases reported were in December of 2011 in the Netherlands and Belgium, but shortly thereafter, cases were reported in France, Luxemburg, and Spain corresponding to the initial outbreak. In late May of 2012, new cases were reported in France, Germany and the UK indicating that the virus had reemerged following the winter. Early in 2012, a serological test was developed which allowed for the wide-spread tracking of the virus, which revealed the virus had spread to the entirety of northern Europe by early 2013. The speed with which this virus spread was surprising and was likely influenced by propagation by a broad-ranged, mobile vector, the shipping of livestock, and the availability of naïve hosts. Recent serological surveys have indicated that Schmallenberg virus continues to circulate at low levels in northern European livestock herds, but no further outbreaks have been reported. Outbreaks of reassortant orthobunyaviruses have also occurred in humans. One such virus, Ngari virus, emerged at the end of the last century in East Africa. In 1988 and again in 1997 and 1998, outbreaks of severe hemorrhagic fever occurred in eastern Africa. While at the time all febrile illness cases were diagnosed as malaria, random serological surveys of patient samples suggest that more than 5000 cases could be attributed to a Bunyamwera serogroup orthobunyavirus infection. Initial molecular analysis of patient samples determined the causative virus was a reassortant of BUNV, as the S and L segments, but not the M segment, matched this virus. Further studies showed Ngari virus to be a novel reassortant with the L and S segments from BUNV and the M segment from Batai virus that had in fact been first discovered in 1979. Both BUNV and Batai viruses cause mild febrile disease in humans, though Batai virus can cause severe congenital malformations in ruminants as well. However, neither parental virus is known to cause severe hemorrhagic fever demonstrating how reassortment can lead to a more pathogenic virus and more severe human infection. Another factor that influences outbreaks and the detection of disease is alterations in climate. For example, Ngari virus may have gone unrecognized as a human pathogen until much later had the outbreaks in the 1980s and 90s not occurred. Those years were associated with unusually high rainfall and increased mosquito breeding in the outbreak area which likely led to a large increase in reported human febrile cases. This increase in cases led researchers to perform retrospective molecular studies that identified Ngari virus and drew attention to its pathogenic potential in humans. It is possible that other orthobunyaviruses which possess pathogenic potential have yet to be discovered due to a low incidence of human cases. The life cycle of orthobunyaviruses within a vector may also affect outbreaks. For some orthobunyaviruses, such as those in the California and possibly the Simbu serogroups, the viruses are transovarially transmitted, allowing them to over-winter in vector species and reemerge each year in endemic regions. This process is also likely aided by viral genetic factors that sustain infection throughout the life of the vector which allows for more host encounters. For other serogroup viruses, it is unclear how they reemerge in subsequent years, which suggests an unrecognized means of overwintering or a persistent infection of host species. One California serogroup virus, Snowshoe hare virus, has been isolated from snowshoe hares during the winter months when mosquitos are absent, suggesting long-term infection of the host may contribute to overwintering of this virus. However, for most orthobunyaviruses, persistent infection seems unlikely as immunity and seroconversion within host populations is typically widespread and quite effective at clearing infection.

662

Orthobunyaviruses (Peribunyaviridae)

Host and Viral Determinants of Disease-Species Barriers Outbreaks can occur when a virus crosses a species barrier and infects a new, immunologically naïve species. Factors that control the crossing of this barrier include species-specific factors, such as cell receptors, elements of the immune response, and host factors required for viral replication. The orthobunyavirus NSs protein can specifically shut down mammalian, but not mosquito, host cell transcription by inhibition of the RNA polymerase II which allows the virus to suppress the innate immune response. This demonstrates species-specific antagonism that correlates with host disease as these viruses typically cause disease in mammalian hosts. Furthermore, studies of select ruminant-infecting versus human-infecting orthobunyaviruses have shown species-specific restriction by BST2/tetherin expression in the host, but it is unknown whether this is a major species determinant across all orthobunyaviruses. Laboratory evidence suggests that orthobunyaviruses that cause disease in humans (Oropouche) can reassort with those that cause disease in animals (Schmallenberg). Thus, depending upon the genetic factors of novel reassortant viruses, orthobunyaviruses could in principle jump species to cause an outbreak in a naïve host.

Clinical Features and Pathogenesis Orthobunyaviruses can causes disease in animals and in humans (Table 2). Disease in animals is most commonly reported in livestock, although zoonotic transmission is known to be important for some viruses suggesting wild populations may suffer from disease as well. Human disease ranges from mild febrile illness to hemorrhagic fever to acute encephalitis. In some cases, infection can be fatal. Here we will discuss by serogroup several of the more recent and consistently relevant orthobunyaviruses that cause disease. Not all serogroups will be represented as some do not contain known disease-causing viruses.

Anopheles A In humans, Tacaiuma virus is known to cause myalgia, arthralgia, headaches, chills and weakness that lasts from days to a week with a sudden onset. At least a transient viremia is associated with infection as virus has been isolated from the blood of an acute patient. Although the number of infections per year is unknown, seroprevalence studies show specific antibodies in 0.5%–1% of residents in endemic areas.

Bunyamwera Bunyamwera virus (BUNV) is present on both the African and South American continents and known to infected both humans and animals. High viremia is known to occur in some mammals. Infection in ruminants can include spontaneous abortion and teratogenic effects. In Brazil in 2013, two horses were found to be infected with BUNV and both developed neurological disease and died. Symptoms in humans are typically mild including fever, arthralgia and rash, but young or immunocompromised individuals can develop severe encephalitis. In endemic areas such as sub-Saharan Africa, seroprevalence has been recorded as high as 82% in some areas. Ngari virus, as discussed above, has caused several large hemorrhagic fever outbreaks in humans in East Africa. It has also been associated with hemorrhagic disease in small ruminants such as sheep and goats although to a lesser extent. Disease in humans is characterized by sudden onset of fever and headache followed by gastrointestinal and mucosal hemorrhage. Up to 0.3% of cases have been fatal.

Bwamba Bwamba virus causes clinically significant, although rarely fatal disease in humans. Symptoms are typically acute (4–5 days) and include general malaise associated with fever, headache, arthralgia and widespread or focal myalgia. Skin rash is also common. Intestinal tract involvement also commonly occurs, typically as diarrhea and can progress to intestinal and mucosal hemorrhagic complications in rare cases. Viremia is short-lived (1–2 days) which may be in part why this virus has not been associated with large outbreaks. However, seroprevalence in central and southern Africa can be quite high suggesting the virus is widespread. Neutralizing antibodies have also been found in birds, nonhuman primates and livestock although no associated disease has been described.

California Jamestown Canyon virus is endemic to North America and has been associated with human disease in both Canada and the USA. Since 2013, the number of reported human disease cases has been increasing dramatically, leading many to consider it an emerging pathogen. However, these numbers may appear inflated because in the same year, the Centers for Disease Control instituted testing for Jamestown Canyon virus as a standard practice for all suspected arbovirus cases. Symptoms of infection are commonly flu-like, involving fever and myalgia. Severe cases may involve meningitis or meningoencephalitis that can result in hospitalization and in rare instances death. In reported cases, neurological complications are more common than febrile illness

Orthobunyaviruses (Peribunyaviridae)

663

and typical occur in older adults (40–59 years). Dependent on region, seroprevalence ranges from B4%–40% in the human population and as high as B90% in deer in Nova Scotia. No disease has been described in animals. La Crosse virus (LACV) is also endemic to North America and is currently the leading cause of pediatric arboviral encephalitis on that continent. It is estimated that 300,000 infections occur annually with approximately 70 of those resulting in encephalitis. Encephalitic disease is far more common in younger individuals (16 years or younger), although immunocompromised individuals are also at risk. In less severe cases, symptoms include fever lasting 2–3 days, nausea, vomiting, headache, lethargy and fatigue. In severe disease cases, hospitalization is typically required, and patients can experience paralysis, seizures and coma. In these cases, CT scan and MRI can reveal areas of hyperintensity associated with diffuse edema and inflammation in the brain and spinal cord. Abnormal electroencephalographic readings are also common. LACV disease is lethal in up to 1.9% of cases, but most patients appear to recover completely. However, long-lasting neurological sequelae has been reported in as many as 15%–30% of cases upon post-discharge checkup more than a year later. While LACV viremia in humans has not been well studied, seroprevalence can range as high as B23% in some endemic areas. Viremia is known to occur in small mammals such as squirrels and chipmunks which are thought to be important zoonotic factors. Laboratory infections of young mice have demonstrated they are susceptible to viral-induced neurological disease, however, disease in wild-animals or livestock populations has not been reported.

Simbu Akabane virus is broadly distributed and has been shown to cause disease in ruminants in Australia, the Middle East, Africa, southeast Asia and Japan. Clinical disease involves congenital defects in the offspring of infected host species. Disease incidence is greatly influenced by the timing during pregnancy when the maternal host becomes infected. Infection early during pregnancy (80–150 days for cattle, 28–56 days for goats and sheep) can result in up to 80% of offspring having abnormalities. Infection later in pregnancy carries a much lower risk. Offspring which are infected late in pregnancy and do develop disease are typically born alive but are often uncoordinated, unable to stand, or have flaccid paralysis and are blind. These animals can also exhibit cavitation of the cerebrum that causes hydranencephaly. Offspring infected early in pregnancy commonly exhibit arthrogryposis and sometimes torticollis, kyphosis and scoliosis as a result of muscle atrophy resulting from motor neuron loss. These animals often are severely hydraencephalic which can cause midterm abortion. If the offspring is carried to term, they typically do not survive birth and may cause obstetric complications for the mother that results in infertility and potentially death. In adult animals, the virus typically does not cause disease except for a very small number of cases in Japan. Asymptomatic infection typically results in long-lasting immunity that can be verified serologically. No human or wild ruminant disease cases have been reported, however, mice and hamsters can be infected in the laboratory.

Diagnosis Serological Assays Most orthobunyavirus infections remain undetected as they are asymptomatic or do not induce sufficient disease to warrant diagnostic testing. Other than seroprevalence surveys, the presence of virus infection is only analyzed once clinical signs of disease are observed in livestock or in human cases. Clinical signs are generally the first preliminary diagnostic and are based on the relevant disease signs correlating with those known for that specific virus as well as the endemicity of the virus for that region. Once an orthobunyavirus is suspected as a causative agent, typically a virus-specific Enzyme-linked Immunoabsorbant Assay (ELISA) will be performed, followed by plaque reduction neutralization tests (PRNTs) on serum from individuals or animals to test for virus-specific antibodies against the suspected orthobunyavirus. Specific diagnosis of an individual virus can be complicated by the cross-reactivity of antibodies to orthobunyaviruses within a serogroup. Additionally, these assays usually require two tests; one taken when a patient or animal first presents with clinical signs (acute phase) to determine if an antibody response is present, which indicates an infection but not necessarily an active infection. A subsequent analysis is performed a few weeks later (convalescent phase) to determine if antibody titers have risen, which would be indicative of a recent or an ongoing infection. A confirmed case is determined based on the four-fold rule: if the suspected orthobunyavirus has a four-fold higher antibody titer than the other serogroup viruses tested, and there is at least a four-fold rise in antibody titer from the acute serum to the convalescent serum, then the case is confirmed. Therefore, this type of test often does not result in an immediate diagnosis, which may limit its usefulness for an acute disease case.

Polymerase Chain Reaction (PCR) Detection of Viral RNA Recent strides have been made in developing PCR assays that detect individual strains of viruses based on clear genetic differences. Although this assay is far more specific than sero-conversion assays, it also has limitations, as detection requires the virus to be present in the tested sample. For several viruses, including those that cause encephalitis, the presence of clinical signs occurs once the virus has been cleared from the blood and is present primarily in the central nervous system. A negative test in serum or in CSF

664

Orthobunyaviruses (Peribunyaviridae)

for virus cannot be taken as an indication that the patient does not have that infection. Thus, PCR analysis may work for positive confirmation for a virus, but not to rule out a specific orthobunyavirus.

Treatment and Prevention There is no known specific treatment for orthobunyavirus infections. In the cases of human encephalitis infection, palliative care is given to patients to aid in recovery. Instead, the primary focus is generally towards prevention. Vaccine studies, including the identification of T cell and B cell epitopes that are protective against different orthobunyaviruses, are being investigated. However, vaccine development can be lengthy and costly. Additionally, the low incidence of disease, anti-vaccine public sentiment and potential for a higher mutation rate for RNA viruses may be barriers for establishing effective vaccination against different orthobunyaviruses. Another avenue for prevention is limiting arthropod transmission of virus to the vertebrate host. This can be done by using aerial spray pesticides to control mosquitoes and ticks in outdoor environments, removal of stagnant water where mosquitoes like to lay eggs and using insect repellant when in areas that are frequented by arthropods.

Acknowledgments The authors would like to than Ryan Kissinger and Durbadal Ojha for the design of the figures. This work was supported by the National Institute of Allergy and Infectious Diseases, Division of Intramural Research.

References Agerholm, J.S., et al., 2015. Virus-induced congenital malformations in cattle. Acta Veterinaria Scandinavica 57, 54. Aguilar, P.V., et al., 2011. Iquitos virus: A novel reassortant Orthobunyavirus associated with human illness in Peru. PLOS Neglected Tropical Diseases 5 (9), e1315. Aguilar, P.V., et al., 2018. Genetic characterization of the patois serogroup (Genus Orthobunyavirus; Family Peribunyaviridae) and evidence that estero real virus is a member of the genus orthonairovirus. The American Journal of Tropical Medicine and Hygiene 99 (2), 451–457. Aguilar, P.V., et al., 2010. Guaroa virus infection among humans in Bolivia and Peru. The American Journal of Tropical Medicine and Hygiene 83 (3), 714–721. Briese, T., et al., 2006. Batai and Ngari viruses: M segment reassortment and association with severe febrile disease outbreaks in East Africa. Journal of Virology 80 (11), 5627–5630. Calisher, C.H., et al., 1983. Identification of hitherto unrecognized arboviruses from Ecuador: Members of serogroups B, C, Bunyamwera, Patois, and Minatitlan. The American Journal of Tropical Medicine and Hygiene 32 (4), 877–885. Chiang, J.O., et al., 2018. Characterization of the gamboa virus serogroup (Orthobunyavirus Genus, Peribunyaviridae Family). The American Journal of Tropical Medicine and Hygiene 98 (5), 1502–1511. Chowdhary, R., et al., 2012. Genetic characterization of the Wyeomyia group of orthobunyaviruses and their phylogenetic relationships. Journal of General Virology 93 (Pt 5), 1023–1034. de Souza Lopez, O., et al., 1975. Bertioga (Guama group) and Anhembi (Bunyamwera group), two new arboviruses isolated in Sao Paulo, Brazil. The American Journal of Tropical Medicine and Hygiene 24 (1), 131–134. de Souza, W.M., et al., 2016. Molecular characterization of Capim and Enseada orthobunyaviruses. Infection, Genetics and Evolution 40, 47–53. Dutuze, M.F., et al., 2018. A review of Bunyamwera, Batai, and Ngari Viruses: Understudied orthobunyaviruses with potential one health implications. Frontiers in Veterinary Science 5, 69. Evans, A.B., Peterson, K.E., 2019. Throw out the map: Neuropathogenesis of the globally expanding California serogroup of orthobunyaviruses. Viruses 11 (9). Groseth, A., et al., 2014. Molecular characterization of human pathogenic bunyaviruses of the Nyando and Bwamba/Pongola virus groups leads to the genetic identification of Mojui dos Campos and Kaeng Khoi virus. PLOS Neglected Tropical Diseases 8 (9), e3147. Hang, J., et al., 2016. Genome sequence of bellavista virus, a novel orthobunyavirus isolated from a pool of mosquitoes captured near iquitos, Peru. Genome Announcement 4 (6). Harrison, B.A., et al., 1995. Isolation of potosi virus from Aedes albopictus in North Carolina. Journal of the American Mosquito Control Association 11 (2 Pt 1), 225–229. Hughes, H.R., et al., 2017. Full genomic characterization of California serogroup viruses, genus Orthobunyavirus, family Peribunyaviridae including phylogenetic relationships. Virology 512, 201–210. Jansen van Vuren, P., et al., 2017. Isolation of a novel orthobunyavirus from bat flies (Eucampsipoda africana). Journal of General Virology 98 (5), 935–945. Meyers, M.T., et al., 2015. Management factors associated with operation-level prevalence of antibodies to cache valley virus and other bunyamwera serogroup viruses in sheep in the United States. Vector-Borne and Zoonotic Diseases 15 (11), 683–693. Mohamed, M., et al., 2009. Viruses in the Anopheles A, Anopheles B, and Tete serogroups in the Orthobunyavirus genus (family Bunyaviridae) do not encode an NSs protein. Journal of Virology 83 (15), 7612–7618. Nunes, M.R., et al., 2005. Molecular epidemiology of group C viruses (Bunyaviridae, Orthobunyavirus) isolated in the Americas. Journal of Virology 79 (16), 10561–10570. Pachler, K., et al., 2013. Molecular characterization of the African orthobunyavirus Ilesha virus. Infection, Genetics and Evolution 20, 124–130. Pollitt, E., et al., 2006. Characterization of Maguari orthobunyavirus mutants suggests the nonstructural protein NSm is not essential for growth in tissue culture. Virology 348 (1), 224–232. Saeed, M.F., et al., 2001. Phylogeny of the Simbu serogroup of the genus Bunyavirus. Journal of General Virology 82 (Pt 9), 2173–2181. Savji, N., et al., 2011. Genomic and phylogenetic characterization of Leanyer virus, a novel orthobunyavirus isolated in northern Australia. Journal of General Virology 92 (Pt 7), 1676–1687. Shchetinin, A.M., et al., 2015. Genetic and phylogenetic characterization of tataguine and witwatersrand viruses and other orthobunyaviruses of the anopheles A, Capim, Guama, Koongol, Mapputta, Tete, and Turlock serogroups. Viruses 7 (11), 5987–6008. Soto, V., et al., 2009. Complete nucleotide sequences of the small and medium RNA genome segments of Kairi virus (family Bunyaviridae). Archives of Virology 154 (9), 1555–1558.

Orthobunyaviruses (Peribunyaviridae)

665

Tangudu, C.S., et al., 2018. Evidence that Lokern virus (family Peribunyaviridae) is a reassortant that acquired its small and large genome segments from Main Drain virus and its medium genome segment from an undiscovered virus. Virology Journal 15 (1), 122. Zeller, H.G., et al., 1989. Electron microscopic and antigenic studies of uncharacterized viruses. II. Evidence suggesting the placement of viruses in the family Bunyaviridae. Archives of Virology 108 (3-4), 211–227. Zhang, J., et al., 2015. Molecular Characterization and Seroprevalence in Pigs of SC0806, a Cat Que Virus Isolated from Mosquitoes in Sichuan Province, China. Vector-Borne and Zoonotic Diseases 15 (7), 423–431.

Further Reading Collins, A.B., Doherty, M.L., Barrett, D.J., Mee, J.F., 2019. Schmallenberg virus: A systematic international literature review (2011–2019) from an Irish perspective. Iranian Journal of Veterinary Research 72, 9. Dutuze, M.F., Nzayirambaho, M., Mores, C.N., Christofferson, R.C., 2018. A review of Bunyamwera, Batai, and Ngari viruses: Understudied orthobunyaviruses with potential one health implications. Frontiers in Veterinary Science 12, 69. Elliott, R.M., 2014. Orthobunyaviruses: Recent genetic and structural insights. Nature Reviews Microbiology 12, 673–685. Romero-Alvarez, D., Escobar, L.E., 2018. Oropouche fever, an emergent disease from the Americas. Microbes and Infection 20, 135–146.

Relevant Websites https://www.cdc.gov/jamestown-canyon/index.html Jamestown Canyon virus. CDC. https://www.cdc.gov/lac/index.html La Crosse encephalitis. CDC. https://www.aphis.usda.gov/animal_health/animal_diseases/schmallenberg/downloads/SBV_EE_February%202014.pdf Schmallenberg. USDA APHIS.

Parapoxviruses (Poxviridae) Hanns-Joachim Rziha, Eberhard Karls University of Tübingen, Tübingen, Germany Mathias Büttner, Leipzig University, Leipzig, Germany r 2021 Elsevier Ltd. All rights reserved. This is an update of D. Haig, A.A. Mercer, Parapoxviruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00461-1.

Introduction Parapoxviruses (PPVs) are epitheliotropic viruses found worldwide. The individual viruses generally exhibit a narrow host range and infect via scarified or damaged skin and give rise to pustular lesions of the skin and occasionally the buccal mucosa. These lesions are associated with low mortality and high morbidity. In addition to a narrow host range, most of the PPVs can also infect humans. However, the zoonotic potency of PPV is mainly restricted to an occupational infection of localized self-limited manifestation without generalization and life threatening pathology. Therefore, PPV never attained the attention of some Orthopoxviruses (OPV) especially Vaccinia virus (VACV) and Variola virus. There are numerous historical references to diseases of domesticated animals such as sheep and cattle that we would now suspect to be the result of infection by PPVs. These references include Jenner’s ‘spurious’ cowpox which is likely to have been caused by the PPV, pseudocowpox virus (PCPV). In the latter part of last century, reports appeared in the scientific literature which recognized the distinct identities of the diseases caused by members of this genus. Following an extensive study of contagious pustular dermatitis of sheep, Aynaud produced a report in 1923, which included the observation that the disease could be transmitted by a ‘filterable’ agent. The isolation of PPV in cell culture was reported from 1957 to 1963. Detailed reports of the transmission of each disease to humans appeared in 1933 (ORFV), 1963 (PCPV), and 1967 (bovine papular stomatitis virus (BPSV)). The first molecular analyses of PPV genomes appeared in 1979 followed in 1989 by the first description of PPV DNA sequence and in 2004 the reporting of the full genome sequence for two strains of ORFV and one of BPSV.

Taxonomy and Classification Parapoxvirus (PPV) represents a genus of the subfamily Chordopoxvirinae of the family Poxviridae. Originally, classification of different PPV species was based on the host and/or the pathology of the disease and genomic restriction profiling, but now sequence homologies are particularly taken into account. Currently PPV encompasses four species including the type-species Orf virus (ORFV) with the synonyms contagious pustular dermatitis virus or contagious ecthyma virus, the Bovine papular stomatitis virus (BPSV), the Pseudocowpox virus (PCPV) synonymous with milker’s nodule virus or paravaccinia virus, and the Parapoxvirus of red deer (PVNZ) first described in New Zealand but later also found in Europe. Tentative species of Parapoxvirus are Auzduk disease or camel contagious ecthyma virus, chamois contagious ecthyma virus, and Sealpox virus. Morphological virus particle similarity and limited partial DNA sequence homologies led to speculations about the presence of novel PPV, for instance the Squirrel poxvirus (SQPV) that, however, turned out to represent a new genus of Chordopoxvirinae. Whole viral genome sequencing will clarify such questions.

Host Range, Epidemiology, and Virus Propagation Natural infection by ORFV has been reported in many wildlife and domestic small and larger ruminants such as bighorn, and thinhorn sheep, domestic and Rocky Mountain goat, Dall’s sheep, chamois, ibex, Himalayan thar, musk-ox, reindeer, caribou, steenbok, camelids from which all are transferable to humans. Experimental inoculations have shown that monkeys are susceptible to ORFV but a wide range of other animals including mouse, rabbit, dog, cat, and domesticated chicken are resistant. BPSV and PCPV both establish infection in cattle and humans but all other species tested, including sheep, are resistant. Both, ORFV and PCPV can affect reindeer where ORFV infection caused the more severe lesions. The PPV of red deer induces only very mild lesions in sheep and so far was not found in other species. The PPV of seals has been reported in a range of seals and sea lions. PPVs do not produce lesions on the chorioallantoic membrane of the developing chick embryo. The PPVs of cattle and sheep are widespread throughout the world, essentially wherever their host animals are kept. The viruses are maintained in populations by a combination of persistent (chronic) infection, frequent reinfection, and the environmentally resistant nature of the viruses. ORFV shed in scab material can remain infective under dry conditions for lengthy periods (at least 4 months and possibly years) and infection of naive animals by virus persisting in heavily contaminated areas such as barns, yards, and sheep camps is likely to play a major role in maintaining the disease. One study has shown that if the scab material is ground up so as to release the virus, then exposure to field conditions quickly results in inactivation of ORFV. Several studies have shown that a productive

666

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00058-8

Parapoxviruses (Poxviridae)

667

Fig. 1 Orf (Ecthyma contagiosum) in Ovis aries. Note the pustular lesions around the mouth and nares aggravated by bacterial super-infection.

infection can be established in animals which have recovered from a previous infection. Such reinfections result in lesions that are smaller and resolve more quickly than primary infections. This short-lived immunity is likely to contribute to the persistence of the disease. In the case of PCPV, it is apparent that infection can be spread within dairy herds by contamination of milking machinery and milkers’ hands. The introduction of procedures, which reduce damage to teats and improve general hygiene at milking, can control the spread of the disease. The most widely used permissive cell culture systems are primary ovine or bovine cells derived from sources such as testis, skin biopsy and embryonic kidney, lung, and muscle. Passaging PPV in cell cultures rapidly (after six and more passages) leads to genomic alterations as a result of adaptation to in vitro conditions. There have also been reports of ORFV isolates adapted to growth in few cell lines, e.g., Vero cells, accompanied by substantial genomic changes and attenuation. Yields of infectious ORFV from cell culture tend to be 10- to 100-fold lower than those achieved with VACV.

Serologic Relationships PPVs show extensive antigenic cross-reactivity, although monoclonal antibodies can be used to distinguish each of the species. There are also antigens shared with other poxvirus genera but there is no cross-protection between PPVs and either orthopoxviruses or capripoxviruses.

Clinical Features PPVs cause inflammatory and/or proliferative lesions that are confined to the skin and oral mucosa with no evidence of systemic spread. Infection is initiated in abrasions and generally proceeds through an afebrile, self-limiting lesion that resolves within 3–9 weeks without leaving a scar. ORFV lesions (Fig. 1) are most generally seen around the mouth and nares; hence, the infection is commonly referred to as scabby mouth or sore mouth. Lesions are also observed on other parts of the body, for example, the coronet, udder, or vulva. Following experimental inoculation of scarified skin, lesions progress through erythema, papule, vesicle, pustule, and scab before resolving. Large, proliferative, tumor-like lesions can be observed. It is likely that these are a result of an immune impairment of the host animal.

Orf, Scabby Mouth, Contagious Pustular Dermatitis (CPD), Ecthyma Contagiosum Lesions around the mouth can interfere with feeding or suckling and especially in young animals result in failure to thrive. Teat lesions can have similar effects through the inhibition of suckling. Lesions in sheep and goats can develop into tumor-like cauliflower proliferative erosions usually accompanied by secondary bacterial infection. Edema of the head and swelling of the regional lymph nodes are common but rather non-specific signs of severe progression. In severe cases, glossitis phlegmonosa and ulcerosa with secondary bacterial infections can lead to starvation especially in young animals. Most common is the labial form that is the basis for the name of the disease. Blisters and yellowish pustules that may reach pea size are formed on the lips and at the corners of the mouth and can extend up to the nose, ears and eyelids. The mild labial form heals within 3–6 weeks. Pustules can also develop on the udders of ewes shortly before the lambing period. Secondary bacterial infections cause complications. The podal form (scabby foot) can occur simultaneously with the labial manifestation or independently. Lesions develop at the

668

Parapoxviruses (Poxviridae)

Fig. 2 Bovine Papular Stomatitis (BPS). (A) Cockade-like inflammatory zones at the muzzle. (B) Circle of inflammation around protected zone.

corner edges of the hooves, at the pasterns and in the hoof gaps. The ends of the digits are painful and lead to lameness and the refusal to stand. The genital form is less common. Typical pustule and crust formation occurs on the udder mostly developing into mastitis. Skin lesions also can occur on the inner leg, the labia or the prepuce.

Milker’s Nodule, Paravaccinia, Pseudocowpox It is probable that all PPVs are able to infect humans, although a human case of the PPV of red deer is not reported. Transmission to humans occurs readily although there is little evidence of human to human transmission. Nowadays human infections with ORFV are less frequent since milking of cows by hand became uncommon. Usually, Orf of man is an occupational disease of veterinarians, farmers, butchers and abattoir workers. Highest risk of infection is linked to handling of sheep fleece or wool and ritual slaughter of affected sheep, e.g. at religious feasts. Progression of the lesions is essentially as seen in sheep and cattle such that the infection is benign and confined to localized pustular lesions on the skin mainly at the hand and fingers at the points of virus entry. Restitutio ad integrum without leaving a scar usually occurs after a few weeks post infection. More severe progressive disease can occur in immune-compromised individuals. Severe reactions and proliferation, called giant Orf, have also been recorded in otherwise normal individuals in cases of burns and in cases of atopic dermatitis. Erythema multiformae reactions in the form of rashes on the backs of the hands and on the legs and ankles are common.

Bovine Papular Stomatitis (BPS) Bovine popular stomatitis normally is a mild form of inflammation around the muzzle and mucous membrane of the mouth in large ruminants (Fig. 2). Horseshoe or coroner herds of inflammation are commonly seen on the muzzle often interrupted by zones of normal cells probably protected by interferon (ring zone phenomenon). However, more severe lesions can occur resulting in extension of confluent inflammation to the hard gum and far down to the esophagus. Signs of BPS may arise spontaneously in apparently healthy animals (especially young calves) without reports about skin damage or injury. Similar to infections with PPV of red deer subclinical persistence of the virus cannot be excluded. The trigger of clinical manifestation can be either unusual virulence of BPSV or immune suppression or both in synergy.

Parapox of Red Deer Initially described in red deer of New Zealand it became evident that red deer in Europe and probably worldwide can become infected with this unique species of PPV. In Italy, clinical manifestation in red deer was seen as inflammation of the mouth whereas in Germany subclinical infection was diagnosed by viral DNA presence and virus isolation in cell culture. For red deer in New Zealand it is reported that PPV infection in the growing deer antler can affect antler growth and severely affect marketability of the product.

Physical Properties Although exclusively tested with ORFV, PPVs seem to be resistant to desiccation and within scab material of infected animals; the dried viruses retain infectivity for at least 4 months and possibly years. Under laboratory conditions, infectivity can be maintained over many years. UV light, g-irradiation, or heating at 561C for more than 1 h can destroy infectivity of PPVs.

Parapoxviruses (Poxviridae)

669

Fig. 3 Electron micrograph of a negatively stained characteristic ORFV particle showing the crisscross arrangement of the tubular core filaments (‘ball-of-wool’ appearance).

Virion Properties The ovoid PPV virion has a length of 220–300 nm and a width of 147–170 nm and thereby are smaller than most of the brickshaped virions of Chordopoxvirinae. PPV are surrounded by a tubule-like spiral structure resulting in the characteristic and distinct ‘ball-of-wool’ appearance of PPV by electron microscopy (Fig. 3). Thin sections of virions reveal a lipoprotein bilayer surrounding a biconcave core and two associated lateral bodies. Further studies revealed ORFV to undergo morphogenesis similar to VACV. Thus, the intracellular mature virion (MV) is enveloped by a membrane derived from the endoplasmic reticulum. Subsequently two additional, probably Golgi-derived membranes form the wrapped virions (WV), which distribute by cell-to-cell infection. Eventually, the WV fuse with the cell membrane to release MVs as extracellular virions (EV). Under hostile conditions, e.g., non-permissive cells or adverse physical or chemical conditions, virus particles can lose their typical morphology showing unconventional shape such as disrupted arrangement of the spiral coil. Such particles may still be infectious.

PPV Genomes The PPV core harbors a linear, double-stranded DNA genome with sizes of approximately 130 to 140 kbp encoding 124–134 predicted genes (Fig. 4), and an unusually high average G þ C content of 63%–64%. The genome is terminally cross-linked by short, ca. 60 bases long single-stranded hairpin loops. Its genomic organization shows the typical poxviral structure: A central core region encoding essential genes conserved in position, spacing, and orientation, and variable genomic termini harboring genes dispensable for in vitro growth and involved in virus virulence and pathogenesis. Inverted terminal repeats (ITR) of varying sizes (2.6–3.5 kbp) embrace the genomic ends. During virus attenuation by cell culture passaging ITR have undergone genetic recombination between non-homologous segments, which resulted in transposition and/or duplication of DNA sequences. In addition, deletions of non-essential gene regions were found to create virus variants of reduced virulence. To date the complete genome sequences of 14 ORFV, 2 BPSV, and 2 PCPV strains have been published, and use of next generation genomic sequencing methods will increase their number in the near future (see also Diagnosis).

Viral Replication As with all poxviruses, the PPVs replicate in the cytoplasm of the infected cell leading to a shut-down of the cellular protein synthesis. The viral cores not only harbor the DNA genome but also are able to produce early poxviral transcripts upon cell entry.

670

Parapoxviruses (Poxviridae)

Fig. 4 Genomic map of ORFV strain BO15. Arrows indicate gene transcription, rightward orientation above the line and leftward below the line. Colored in red are the virulence genes introduced in the text and in green those involved in virion maturation and mentioned in the text.

Early viral gene transcription is controlled by early promoters and according to their definition are also expressed in the presence of inhibitors of DNA synthesis. Sequences around the start and the end of PPV early genes contain A þ T-rich control motifs very similar in structure and function to Orthopoxviruses. Analyses of vaccinia virus recombinants carrying large multigene DNA fragments of ORFV indicate the regulated cascade of early, intermediate, and late PPV gene expression is very comparable to Orthopoxviruses. PPV DNA replication begins approximately 4–8 h post infection (hpi) and viral particles are produced at 16–36 hpi continuing until at least 48 hpi.

Viral Proteins SDS-polyacrylamide gel electrophoresis analyses of ORFV and PCPV virions resolve 30–40 polypeptides ranging in size from 10 to 220 kDa including 10–13 surface polypeptides. Significant antigenic overlap among PPV has been reported. However, pronounced cell association of the virus and the lack of additional specific antibodies limit the knowledge of electrophoretically demonstrable PPV proteins. For ORFV the most prominent polypeptides are found in the mol. wt. range of 30 and 56 kDa. The 39 kDa major envelope protein of ORFV represents an immuno-dominant antigen recognized by several independently derived monoclonal antibodies. This protein, formerly also named F1L, is encoded by the open reading frame ORF 059, and is a major heparin-binding protein potentially involved in binding to cells during virus entry. Another major structural, 37/42 kDa protein of ORFV exhibits strong amino acid similarity with the F13L gene product of the extracellular enveloped form of VACV. This B2L named ORFV protein is encoded by ORF 011 and found to induce antibody as well as T cell responses during ORFV infection. The product of ORF 104 is the so-called 10-kDa protein of ORFV, an orthologue of the VACV A27L gene product (14-kDa fusion protein). Removal of ORF 104 leads to disruption of the spiral tubular structure of ORFV. Recently, two other late core proteins were identified, which are encoded by ORF 050 and ORF 086, respectively.

Known or Putative Genes Involved in Pathogenesis and Virulence Poxviruses, particularly Orthopoxviruses, contain extensive arrays of immunomodulatory genes to defend the virus against the host innate and acquired immune responses and to manipulate various intracellular signaling pathways, e.g., regulating apoptosis. Many of those immune evasion genes have been acquired during co-evolution of viruses with their hosts. During the last decade, those genes were increasingly identified also in PPV, especially in ORFV. Most of the factors influencing virulence, pathogenesis, or host range are dispensable for virus growth in vitro and are encoded in the genomic termini. In the following subsections, potential virulence factors of ORFV and their encoding open reading frame (ORF) numbers are briefly described.

Parapoxviruses (Poxviridae)

671

VEGF-E – ORF 132 The first of these factors reported in ORFV was the functional homolog of the mammalian angiogenic vascular endothelial growth factor (VEGF). This early expressed, unspliced viral VEGF is unique for PPV. In contrast to the other members of the VEGF family it interacts only with VEGF receptor 2 (VEGFR-2), but not with either VEGFR-1 or VEGFR-3, and was classified as VEGF-E. Deletion of the ORFV VEGF-E gene proved its major role as a virulence determinant. VEGF-E can induce epidermal hyperplasia, which might contribute to scab formation and thereby continuously provide permissive cells for productive virus replication. Recent reports also indicate the role of VEGF-E in wound healing. Expression of ORFV VEGF-E can promote epidermal regeneration and reduce wound inflammation, and together with enhancing IL-10, skin repair in mice is positively influenced. In different PPV strains two VEGF-E variants, designated VEGF-NZ2 and VEGF-NZ7, can be distinguished. Despite these variants sharing only 41% amino acid identity, the structure of the functional protein domains is highly conserved. Since the VEGF-E gene displays a remarkably low G þ C content as compared to the flanking gene regions, a more recent gene acquisition from the mammalian host is suspected.

Viral Interferon Resistance Protein – ORF 020 An interferon resistance protein (OVIFNR) is encoded by the early ORF 020 gene, a functional homolog of the VACV E3L gene. It inhibits the antiviral activity of interferon induced shutdown of cellular protein translation by binding to viral dsRNA and thus, preventing activation of the interferon-inducible protein kinase (PKR). In addition, OVIFNR acts as a PKR antagonist and prevents binding of PKR to the PKR activator. The gene product exists in 2 isoforms, the shorter, non-essential sh020, and the essential fulllength 020 product, which is not dispensable for ORFV replication.

Viral dUTPase – ORF 007 At the left genomic side gene ORF 007 encodes a functional dUTPAse. This virion-associated enzyme is suspected to be involved in ORFV virulence since it is missing in some attenuated ORFV strains.

Viral IL-10 – ORF127 This early gene of PPVs encodes a polypeptide with remarkable homology to mammalian, especially ovine IL-10. The mammalian IL-10 is a pleiotropic cytokine known to induce immune-stimulating or -suppressive effects in various cell types. IL-10 can prevent the synthesis of other cytokines, such as IFN-gamma in blood monocytes or TNFa, IL-1b and IL-8 in macrophages and keratinocytes. Additionally, T-helper 1 cell activation can be indirectly suppressed by decreased antigen processing and presentation or recruitment of dendritic cells (DCs). Several reports indicate that ORFV IL-10 displays a similar range of activities as its cellular counterpart. Immature DCs accumulate at the site of ORFV infection, which is possibly a result of ORFV IL-10-mediated blocking of DC maturation as well as decreasing interferon-gamma production. This might be one factor contributing to the short-term ORFV-specific immune response. The secreted ORFV IL-10 represents a virulence determinant, a recombinant ORFV lacking the gene is attenuated compared to wild-type or to ORFV IL-10 gene-reconstituted virus in sheep. As mentioned above, together with the ORFV VEGF-E, the ORFV IL-10 can regulate skin repair, accelerate wound healing, and limit local skin inflammation.

Viral GM-CSF Inhibitory Factor (GIF) – ORF 117 The gene ORF 117 encodes a viral protein, which is a secreted dual inhibitor both of granulocyte macrophage colony-stimulating factor (GM-CSF) and of interleukin-2 (IL-2), and is therefore termed GM-CSF/IL-2 inhibition factor (GIF). Due to its dimeric structure, GIF can bind two molecules of the ovine but not of human target cytokines. Among other properties, GM-CSF regulates the recruitment, differentiation, and activation of macrophages, neutrophils, and DCs. Activated T cells are an important source of GM-CSF during immune responses to pathogens. IL-2 is produced predominantly by T cells and stimulates the expansion and activation of T cells and natural killer cells. Consequently, interaction of GIF with these cytokines can help ORFV to escape the host cellular immune attack. The GIF protein is conserved in ORFV strains and also expressed in other PPVs, though there is only approx. 40% amino acid identity.

Chemokine Binding Protein (vCBP) – ORF112 The early gene ORF 112 encodes a viral chemokine-binding protein, structurally and functionally related to the type II CCchemokine binding proteins (CBP-II) of the Orthopoxvirus and Leporipoxvirus genera. CBP-II regulates recruitment of T cells, monocytes/macrophages, and DCs to sites of infection. In addition, vCBP binds to lymphotactin, a C-chemokine attracting T and B cells, and neutrophils. By disrupting chemokine gradients, the vCBP may inhibit immune cell trafficking to the site of infection and prevent key aspects of the anti-PPV immune response. After deletion of ORF 112 the knock-out mutants are attenuated in sheep showing its critical role in virulence and pathogenesis of ORFV. It seems reasonable to assume that the PPV encoded IL-10,

672

Parapoxviruses (Poxviridae)

GIF, and CBP act in concert to counteract innate immune responses of the host. In addition, BPSV express a vCBP sharing 40% sequence identity with that of ORFV and which exhibits comparable inhibition of chemokines.

NF-κB Inhibitors – ORF 002, 024, 073, 119, 121 Among others, the nuclear factor kappa B (NF-κB) signaling pathway plays a vital role in regulating innate immunity, inflammation, programmed cell death (apoptosis), and keratinocyte proliferation. Therefore, not only poxviruses evolved strategies to counteract this essential regulatory pathway. To date five ORFV encoded polypeptides have been identified in vitro to target NF-κB pathway signaling factors. The ORFV encoded inhibitors display no homology to those from other poxviruses. Gene ORF 002 encodes an early-late protein found in the nucleus of the infected cells and binds to NF-κB-p65. Knock-out of this non-essential gene has no effect on ORFV virulence. ORF 002 is present in different ORFV and PCPV strains, but seems to be lacking in BPSV. The non-essential, early gene ORF 024 has no influence on virulence or disease severity. This gene product interferes with the core element of NF-κB pathway, the IKK kinase complex. The product of the late gene ORF 073 was the first poxviral virion-associated NF-κB inhibitor. The virion component ORF 073 acts transiently very early in ORFV infection without the need of de novo protein synthesis. Infection of sheep with an ORFV 073 knock-out mutant demonstrated the role of this non-essential gene in pathogenesis. The ORF 119 late gene product represents another virion protein unique for PPV. Similar to the ORF 073 protein, it also acts very early in ORFV infection, but unlike 073 it is also found late in infection in the cell nucleus. Elimination of ORF 119 gene resulted in ORFV attenuation in sheep. Interestingly, the loss of ORF 119 is found in attenuated ORFV strains after cell culture passaging. Finally, ORF121 encodes an early, cytoplasmically expressed inhibitor of NF-κB signaling by counteracting the translocation of NF-κB-p65 into the nucleus. This dispensable ORFV gene contributes to reduction of epidermal pathology and decreased cellular inflammatory response in the sheep skin.

Poxvirus APC/C Regulator (PACR) – ORF 014 The multicomponent anaphase promoting complex or cyclosome (APC/C) is a dominant regulator of the cell cycle. The poxvirus APC/cyclosome regulator (PACR) protein is expressed from the ORFV gene ORF 014. PACR seems to be incorporated into APC/C leading to its inhibition. It is suggested that PACR promotes an S-phase-like state of the infected cells favoring ORFV DNA replication. Although not essential for in vitro replication, ORF 014 deletion substantially reduces ORFV growth and plaque size. Amino acid comparison revealed a PACR homolog encoded by BPSV gene 013.

Ankyrin Repeat (ANK)/F-box Genes – ORFs 008, 123, 126, 128, 129 Ankyrin repeats (ANK) are protein consensus motifs crucial to facilitate protein-protein interactions, and are present in a plethora of cell proteins involved in cell–cell signaling, cytoskeleton integrity, regulation of transcription and cell cycle, inflammatory response, or protein transport. Those proteins are absent in most viruses except Chordopoxviruses. The poxviral ANK proteins share a common molecular structure including a protein motif at their C-termini, called F-box, which is also present in cellular proteins. This PRANC (Pox protein Repeats of Ankyrin C-terminal) domain is responsible for recruiting specific substrates to SCF1 ubiquitin ligase. For ORFV five ANK proteins are known and coded by ORF 008, 123, 126 (ANK-1), 128 (ANK-2) and 129 (ANK-3). The ORF 126 encoded ANK-1 protein co-localizes with mitochondria. A recent study demonstrated for the first time an influence of the five ORFV ANK proteins on the Hypoxia-inducible factor (HIF) pathway. The HIF pathway is not only crucial in the regulation of cellular responses to hypoxia, but also in angiogenesis- and anti-apoptotic programs. Upon ORFV infection, HIF gene expression is upregulated due to binding and sequestration of the cellular HIF inhibiting factor (FIH) by ORFV ANK proteins.

Inhibitor of Apoptosis – ORF 125 Programmed cell death or apoptosis is a highly effective innate response to eliminate infected cells. Thus, especially large DNA viruses evolved various immune evasion strategies limiting or preventing apoptosis. In addition to the anti-apoptotic effect of ORF 020 (described above), an anti-apoptotic protein is coded by the ORFV early gene ORF 125, which is essential for ORFV replication. This Bcl2-like inhibitor of apoptosis is directed to mitochondria to inhibit cytochrome C release, caspase activation, and cellular DNA fragmentation. Unlike other described poxviral Bcl2-like factors, the ORF 125 product targets cellular Bak. Recently, additional host proteins were identified to interact with ORF 125 indicating further biological functions.

Pathogenesis and the Host Immune Response to Infection The histological sequence of events in the skin of sheep after ORFV infection is similar in primary and reinfection lesions, in spite of differences in the magnitude of the lesions and the time taken to resolve. Antibodies to ORFV envelope proteins have been used to detect ORFV antigen in epidermal keratinocytes, particularly those regenerating the damaged skin. Basal keratinocytes at the root of hair follicles can also contain virus. In vitro ORFV can persist in an infective state in keratinocytes for up to ten days. Some

Parapoxviruses (Poxviridae)

673

infected cells show evidence of a ballooning-like degeneration. There is no evidence that ORFV infects other, non-epithelial cell-types in vivo. ORFV lesions often exhibit epidermal down-growths (rete formation) into the dermis. This is particularly marked in primary lesions. Another characteristic feature is extensive capillary dilation and proliferation. A-type intra-plasmatic eosinophil inclusion bodies can be seen in infected cells. By using its unique gene coding for vascular epidermal growth factor (VEGF-E, see also above), ORFV is able to promote endothelial cell proliferation and such create permissive cells for virus spread. ORFV lesions contain a dense accumulation of immune and inflammatory cells underneath and adjacent to virus-infected cells. These include neutrophils, lymphocytes (T and B cells), and dendritic cells that express major histocompatibility complex class II antigens. A dense network of probably immature dendritic cells is characteristic of Orf lesions in sheep. The accumulating cells increase and decrease in number in parallel with the presence of virus in epidermal cells. The histology of human ORFV lesions is generally similar to that described in sheep. A comparison of ORFV and PCPV lesions in humans has not revealed any histopathological differences.

Immune Response to Infection Typical for poxviruses PPVs induce a vigorous immune and inflammatory response in their hosts and have evolved to replicate in the presence of this response. In sheep experimentally infected with ORFV, studies of the skin and lymph draining into (afferent lymph) and out of (efferent lymph) local lymph nodes have demonstrated that activated CD4 þ (helper) and CD8 þ (cytotoxic) T cells, B cells, and antibodies are generated as part of the sheep-acquired immune response to infection. The cytokines generated in lymph in response to virus reinfection are typical of type 1 antiviral cell-mediated immune responses and include interleukin (IL)  1b, IL-2, tumor necrosis factor (TNF)-a, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-a, and IFN-g. Studies of ORFV reinfection in sheep depleted of specific lymphocyte subsets or treated with the immunosuppressant drug cyclosporine-A indicated that at least CD4 þ T cells and interferons are important components of the host-protectiveresponse against infection. These studies also indicated that the cutaneous damage sustained during ORFV infection is due in large part to the virus rather than host immune-mediated responses. Sheep infected with ORFV mount detectable antibody responses to a small number of viral antigens but there is considerable individual qualitative and quantitative variation in the response. The lack of ORFV neutralizing antibody is exceptional. No apparent correlation between antibody titers and severity of viral lesions exists and passive antibody transfer does not confer protection against virus challenge.

Diagnosis After a long period of restricted diagnosis based on clinical observation and morphological rating in electron microscopy, molecular methods have opened the capability of rapid and exact diagnosis even for differentiation at the species level. Most rapid differentiation from Orthopoxviruses still is the negative staining method of virions for electron microscopy; however, its low sensitivity requires high numbers of intact virus particles in lesion samples (about 105 particles). Polymerase chain reaction (PCR), especially real time PCR, became a method of choice for PPV diagnosis because of the high sensitivity and specificity when appropriate primers are chosen. For PPV species resolution and molecular epidemiology less conserved genes or sequences at the terminal regions of the genomes can be targeted with a set of primers. Thus, even differentiation of ORFV isolates from sheep or goats is possible. Whole genome (WGS) or next generation sequencing (NGS) achieves most precise characterization and differentiation of PPV genomes. To avoid genome alterations caused by in vitro isolation of PPV, WGS is attempted to be applied directly out of lesion material as demonstrated with a case of PPV infection in seals. In general, differential diagnosis in affected domestic animals is very important since some diseases caused by notifiable infections such as Foot-and-mouth disease virus (FMDV), Bluetongue virus (BTV), BVDV /mucosal disease virus, Border disease virus, Malignant catarrhal fever virus, Lumpy skin and Sheep or Goat pox virus can cause similar lesions and must be excluded. Natural antibody formation after PPV infection is highly restricted to the major envelope 39 kDa protein (see above). These antibodies do not possess neutralizing potency and in general, antibody detection in animals for diagnostic purpose is not constructive since PPV infections are so widespread. In the case of isolated PPV-free flocks of sheep or goats, antibody detection for status surveillance and maintenance makes sense. After infection of humans or for retrospective resolution of suspected human infections antibody detection by ELISA can be applied but whenever possible molecular diagnosis with fresh biopsies or lesion material should be preferred.

ORFV Vector Vaccines ORFV has proven useful as a novel poxvirus vector platform for delivering heterologous antigens without the risk of a vigorous immune response against the vector backbone. The restricted host range, lack of systemic spread even in immuno-compromised animals, and induction of strong humoral and cellular immune responses to expressed antigens are excellent viral vector properties. Furthermore, even inactivated ORFV particles exhibit nonspecific immuno-modulatory and immune enhancing effects successfully applied in different pre-clinical disease models especially with non-permissive animals. Live ORFV recombinant

674

Parapoxviruses (Poxviridae)

vaccines are capable of mediating excellent and long-term protective immunity against diverse viral infections. Most of the recombinants were created with the highly attenuated ORFV strain D1701-V by replacing its VEGF-E virulence gene by foreign antigen insertion. Additional new insertion sites have been established in this ORFV strain, which enables expression of at least three more different transgenes. Because an ORFV early promoter regulates transgene expression in strain D1701-V, no ORFV replication or production is needed to elicit protective immunity, even under non permissive conditions. Moreover, transgene substitution of the virulence genes ORF 024 or ORF 121 (see above) of the non-attenuated ORFV strain IA82 also allowed generation of protective recombinant vaccines, and deletion of ORF 113 as well as 116 allowed marker gene expression.

Oncolytic Potency Besides the investigation of various other promising viruses, e.g., measles virus, among the poxviruses especially VACV, Myxoma virus, and ORFV are candidates for virotherapy of tumor diseases. The combined advantages of ORFV as a potent vector with immune stimulating efficacy and the capability to lyse tumor cells in vitro are favorable characteristics for future prospects in oncotherapy.

Prevention and Control Vaccines against ORFV have been available for many years and are widely used to protect lambs against the debilitating effects of natural infection. These vaccines consist of live and essentially non-attenuated virus that is applied to a scratch on the skin of a leg. The ensuing infection does not interfere with feeding and provides significant protection against infection for some months. However, the scab derived from vaccination lesions is likely to contaminate the environment and contribute to the perpetuation of the disease. A few attenuated vaccine virus strains for parenteral application have been developed but they also induce only short lasting protection (up to six months), however the severity of reinfections can be effectively reduced. By analogy with many Orthopoxviruses including vaccinia virus inactivated ORFV does not mediate protective immunity as learned from many vaccination attempts. New vaccines that induce protection against Orf but do not shed infectious virus are highly desirable. This might be achieved by deleting genes encoding viral virulence determinants or by delivering the protective antigens of the virus in an appropriate way. Acyclic nucleoside analogs, such as acyclovir ACV or cidofovir CDV selectively interacting with poxviral DNA polymerase are effective against human PPV infection. Topical treatment with CDV resulted in complete regression even of giant orf lesion in immunocompromised patients.

Further Reading Fleming, S.B., Wise, L.M., Mercer, A.A., 2015. Molecular genetic analysis of Orf virus: A poxvirus that has adapted to skin. Viruses 7, 1505–1539. Friederichs, S., Krebs, S., Blum, H., et al., 2014. Comparative and retrospective molecular analysis of Parapoxvirus (PPV) isolates. Virus Research 181, 11–21. Rziha, H.-J., Büttner, M., Müller, M., et al., 2019. Genomic characterization of Orf virus strain D1701-V (Parapoxvirus) and development of novel sites for multiple transgene expression. Viruses 11, 127. Weber, O., Mercer, A.A., Friebe, P., et al., 2012. Therapeutic immunomodulation using a virus – The potential of inactivated orf virus. European Journal of Clinical Microbiology and Infectious Diseases 32, 451–460.

Parechoviruses (Picornaviridae) Sisko Tauriainen, University of Turku, Turku, Finland Glyn Stanway, University of Essex, Colchester, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary cre Short RNA stem loop needed to generate the primer for PeV RNA replication. Encephalitis Inflammation of brain tissue. IRES (internal ribosome entry site) Region of the PeV genome, which is needed for initiation of translation. Meningitis Acute inflammation of the protective membranes covering the brain and spinal cord, the meninges. Meningoencephalitis An acute inflammation of the brain and meninges.

Myalgia Nonspecific pain or tenderness in muscles. Myositis Inflammation of muscle tissue. NPGP Motif associated with translation termination/ reinitiation in several PeV species. Orchiodynia Pain in the testicles. Sepsis A systemic multi-organ infection, usually caused by bacteria. When it is caused by viruses, the condition is often called Sepsis-like disease or viral sepsis.

The first two Parechoviruses (PeVs) were discovered in a diarrhea epidemic in the 1950s and were named Echovirus 22 and Echovirus 23. They were classified as enteroviruses (EVs), but were reclassified as Parechoviruses and first renamed Human Parechoviruses (HPeV1 and HPeV2), but recently they have been renamed again as Parechovirus-A1 and Parechovirus-A2 (PeV-A1 and PeV-A2), respectively. It took several decades before the third type, PeV-A3, was discovered in 1999. Thereafter a further 16 types have been identified, increasing the total identified PeV-A types up to 19. In addition to the PeVs found in humans, several PeVs infecting rodents have been detected. The PeV-Bs were first discovered in bank voles (Clethrionomys glareolus) in Sweden and were named as Ljungan virus. Another PeV was already detected in 1972 in an African wood mouse (Hylomyscus sp.), but the virus was later reclassified as an additional PeV species, PeV-C, based on the genome organization and sequence. The fourth species, PeV-D, contains a virus found in a pet ferret (Mustela putorius furo).

Classification Parechoviruses are classified within the Realm Riboviria, Order Picornavirales, Family Picornaviridae, Genus Parechovirus. Phylogenetically, Parechovirus clusters with several other Picornaviridae genera, together forming a group currently called supergroup 4 (SG4), which in the future may formally be designated as a Picornaviridae Subfamily. Currently, genus Parechovirus consists of four species, Parechovirus A (previously called Human parechovirus), Parechovirus B (previously called Ljungan virus), Parechovirus C and Parechovirus D (PeV-A to D). Originally on the basis of serology, but now defined by amino acid identities within the virus capsid proteins, parechoviruses are grouped into types (originally serotypes). 19 PeV-A types are currently recognized. Five PeV-B types are known, while only one type has been identified for each of PeV-C and PeV-D.

Virion Structure As in other picornaviruses, the parechovirus virion is composed of an icosahedral protein capsid enclosing the genomic RNA. The capsid is composed of 60 copies of each of three proteins, VP0, VP3, and VP1 (Fig. 1). These three proteins have the same core structure, an 8-stranded anti-parallel b-barrel. Copies of VP1 are clustered around the capsid 5-fold axis, while VP0 and VP3 alternate around the 3-fold and 2-fold axes. The average diameter of the virion is somewhat smaller than that of many picornaviruses and the surface is relatively flat, with no sign of the deep “canyon” that is the site of receptor binding in enteroviruses. However, in PeV-B five copies of a very large protrusion, made up of the C-terminal region of VP1, circle the 5-fold axis. Parechoviruses were the first picornaviruses found to lack the cleavage of VP0 to give VP4 and VP2, and are now known to share this property with several more recently described Picornaviridae genera. In the picornaviruses where VP0 cleavage occurs, this takes place after assembly, as the final step of virion maturation and is necessary for stability and infectivity, so its absence in parechoviruses is intriguing. Another interesting feature of PeV is that the VP3 protein has a longer N-terminal region than most other picornaviruses and it contains several basic amino acids. When the structures of parechoviruses were first analysed, contacts between the capsid proteins and the genomic RNA were more evident than in other picornaviruses and extensive amounts of the RNA could be visualized, indicating essentially identical contacts between the RNA and capsid proteins. Contact regions include the VP3 N-terminus, which was found to be on the inside of the virion, and this protein may also serve to help neutralize the charge of the RNA to facilitate packaging. A number of RNA stem-loops, arrayed along the genomic RNA, and with a conserved

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21509-4

675

676

Parechoviruses (Picornaviridae)

Fig. 1 Virion structure of PeV-A1. The icosahedral capsid is made up of 60 copies of 3 virus proteins VP0 (orange), VP1 (dark blue), and VP3 (pale blue). The image was generated from PDB ID: 4Z92 using NGL viewer and RCSB PDB. Reproduced from Kalynych, S., Pálková, L., Plevka, P., 2016. The structure of human parechovirus 1 reveals an association of the RNA genome with the capsid. Journal of Virology 90, 1377–1386, available from the RCSB PDB database, using NGL Viewer. Rose, A.S., et al., 2018. NGL viewer: Web-based molecular graphics for large complexes. Bioinformatics 34 (21), 3755–3758, doi:10.1093/bioinformatics/bty419.

apical sequence motif, have been identified and may be the observed points of contact and possible packaging signals. The unusually ordered protein/RNA contacts are also seen in other picornaviruses where VP0 cleavage does not occur. This possibly indicates that these non-VP0 cleaving picornaviruses may follow a distinct assembly pathway where multiple capsid subunits align themselves along the RNA and then interact with one another to give an intact virus particle. Alternatively, a similar mechanism may occur in other picornaviruses, but some RNA/protein interactions are broken as a consequence of the structural change to the virus particle caused by VP0 cleavage, giving a less ordered RNA structure. Parechoviruses are also unusual in lacking N-terminal myristoylation of VP0, thought to be important in interactions with membranes during early events in the infection of cells by many picornaviruses. This suggests that parechoviruses, and other picornaviruses lacking VP0 myristoylation, use fundamentally different pathways during entry and uncoating from picornaviruses with a myristoylated VP0/VP4.

Genome Parechoviruses have a typical picornavirus genome organization (Fig. 2). A long (about 700 nucleotides in PeV-A) 50 untranslated region (50 UTR) is followed by a single open-reading frame (around 2200 codons) and a short 30 UTR (70–120 nucleotides). The genome is polyadenylated at the 30 end and a small protein, VPg, is covalently attached by an O-tyrosyl link to the 50 end. The genome is analogous to a cellular mRNA and it is translated to give a polyprotein, which is cleaved by a virus-encoded protease to give precursors and the final virus proteins. The picornavirus 50 UTR contains several RNA secondary structures, particularly within the extensive IRES domain, involved in translation of the RNA. Five structurally-distinct IRES types (I-V) have been identified among picornaviruses. PeV-A, -B, and -C have an IRES of type II, while PeV-D has a type IV IRES, suggesting that recombination has played a role in the evolution of the different genera. Clear evidence of recombination has been found within the large number of PeV-A isolates that have been sequenced and this may be of significance in the generation of epidemic or pathogenic strains. The order of proteins encoded within the open reading frame is essentially conserved among picornaviruses. The parechovirus gene order is 1AB(VP0)-1C(VP3)-1D(VP1)-2A-2B-2Chel-3A-3B(VPg)-3Cpro-3Dpol (Fig. 2). The more conserved proteins (2Chel, 3Cpro-3Dpol) possess the typical motifs of a putative helicase, chymotrypsin-like protease and polymerase, respectively. 2B and 3A are more diverse among picornaviruses, but as in most picornaviruses both parechovirus proteins are highly hydrophobic. They are associated with intracellular membranes when over-expressed in mammalian cells. The PeV 2C is largely associated with lipid droplets. There are two variable loci within picornavirus genomes, where proteins of quite different types can occur, L, which is encoded before the capsid proteins in some viruses, and 2A. L does not occur in parechoviruses. The PeV-A 2A was found to be related to a group of cellular proteins and similar proteins are present at the 2A locus in several other picornaviruses. This type of

Parechoviruses (Picornaviridae)

677

Fig. 2 The PeV genome as seen in the species PeV-A. The single stranded positive sense RNA genome is around 7300 nucleotides long in PeV-A and it encodes a single polyprotein. The open-reading frame is flanked by 50 and 30 untranslated regions (UTRs) and there is a geneticallyencoded 30 poly A tail. A small protein, VPg, which is the product of the 3B region, is covalently attached to the 50 terminus (green circle). Following translation, the polyprotein is cleaved into several individual proteins. The structural proteins VP0, VP3, and VP1 are encoded by the 1AB, 1C, and 1D regions, respectively (yellow). The other non-structural proteins are encoded by the 2A-3D regions (red). The proteins encoded by the 2C, 3C, and 3D regions have helicase, protease and polymerase activities, respectively.

protein is termed Hbox/NC from two characteristic amino acid motifs, but its biochemical properties and function in virus replication remain largely obscure. The only property ascribed to the parechovirus 2A to date is binding to the 30 UTR of genomic and dsRNA, but its functional significance is not clear. An interesting difference of the other 3 Parechovirus species from PeV-A is that although these also encode an Hbox/NC type 2A protein, additionally they seem to possess a second type of 2A, a small protein characterized by an NPGP motif. Such a protein is associated with ribosome skipping, a translation termination/reinitiation event, leading to the polyprotein being produced in two pieces. These 3 species are said to encode 2A1 (NPGP type) and 2A2 (Hbox/NC type), although it is not clear whether 2A1 is a separate protein or if it remains part of the preceding protein, VP1, possibly accounting for part of the large 5-fold related protrusions seen in the PeV-B capsid structure.

Life Cycle The cell and molecular biology of parechoviruses has not been studied extensively, but some specific details are known. Some of the PeV-A types have an RGD (arginine-glycine-aspartic acid) motif close to the VP1 C-terminus and this interacts with cell surface integrins during the entry process. Several RGD-dependent integrins have been found to be potentially utilized as the PeV receptor, including avb1, avb3, and avb6, with most proof of avb1 being the main receptor for PeV-A1. Entry of the virus into the cell seems to be via endocytosis into endosomes and acidification of the endosome seems to be vital for infection, presumably to trigger uncoating of the RNA. The receptors used by the members of PeV-A which lack an RGD motif (including PeV-A3) are not known, as is the case for viruses in PeV-B to -D, which also lack RGD. Following the release of the genomic RNA, translation occurs and gives rise to the parechovirus polyprotein. Translation is initiated by a cap-independent, IRES-driven mechanism. This has been shown to depend on RNA structures within the type II IRES, particularly domains I and J/K. These probably serve as biding sites for proteins usually involved in cellular mRNA translation, as well as IRES-specific factors (ITAFS), but this has not been studied directly. It is assumed that all processing of the polyprotein in PeV-A is performed by 3Cpro, as 2A was shown not to have a protease activity, while PeV-B to -D have the additional “processing” activity of the NPGP-containing 2A1 protein. RNA replication depends on stem-loops and pseudoknots close to the 50 terminus of the RNA, but little is known about the details of the process. As in other picornaviruses, the genome harbors a small RNA element, the cre, which is needed to template the 3Dpol-mediated addition of two uridine residues to VPg so it can be used as a primer in RNA replication. The picornavirus cre occurs in diverse locations in different genera or species and in PeV-A it is found in the VP0-encoding region, while in PeV-B it is in the 3B-encoding region. During infection, picornaviruses manipulate intracellular membranes and generate replication complexes formed from components of these membranes to produce nascent RNA. In parechovirus-infected cells, it has been reported that the Golgi disintegrates and the endoplasmic reticulum is modified. Replication complexes seem to contain Golgi markers, as well as the virus 2C protein, while some of the 2C locates to other membrane structures. In contrast to enteroviruses, PeV replication complex formation does not depend on COP-I, indeed COP-I is dispersed in PeV-infected cells. These results emphasize that there are significant differences between picornaviruses in intracellular replication events. Later steps in infection, including assembly, whether there is some distinct pathway of maturation of the particle in the absence of VP0 cleavage and how the virus is released from the cell, have not been studied directly.

Epidemiology PeV-As are very common and they infect mainly young children. Their seasonality depends much on the geographic location and may differ between the PeV types. However, seasonality is best known for PeV-A1 and PeV-A3, but a clear picture for other types is lacking. Most studies showed the peak of infections to be either in summer or in fall. Nordic countries have mostly a peak of

678

Parechoviruses (Picornaviridae)

Fig. 3 PeV-A type distribution around the world. Pie charts show the type distribution of each PeV-A type, named in charts as PeV-A1 ¼ A1, PeV-A2 ¼ A2, etc. Studies from Malawi, Pakistan, and India are based on one or two large studies and represent background population. The Bolivian results are based on a small study population and thus does not likely represent real type distribution. The result from USA is based on clinical surveillance data and a small epidemiological series, thus PeV-A3 is overrepresented. In addition, the European data is partly based on a clinical data making PeV-A3 overrepresented. Distribution of viruses in charts: USA: 15% PeV-A1, 82% PeV-A3, 3% PeV-A4; Bolivia: 13% PeV-A1, 13% PeV-A2, 6% PeV-A3, 25% PeV-A4, 19% PeV-A7, 19% PeV-A9, 6% PeV-A12; Europe: 62% PeV-A1, 32% PeV-A3, 1% PeV-A4, 1% PeV-A5, 5% PeV-A6; Malawi: 27% PeV-A1, 14% PeV-A2, 10% PeV-A3, 9% PeV-A4, 8% PeV-A5, 2% PeV-A6, 1% PeV-A7, 8% PeV-A8, 2% PeV-A9, 3% PeV-A10, 1% PeV-A11, 3% PeV-A12, 2% PeV-A14, 6% PeV-A16, 3% PeV-A17; Pakistan: 27% PeV-A1, 2% PeV-A2, 12% PeV-A3, 8% PeV-A4, 5% PeV-A5, 2% PeV-A6, 2% PeV-A7, 3% PeV-A8, 8% PeV-A10, 2% PeV-A11, 8% PeV-A12, 7% PeV-A13, 12% PeV-A15, 2% PeV-A16; India: 40% PeV-A1, 5% PeV-A2, 12% PeV-A3, 11% PeV-A4, 11% PeV-A5, 3% PeV-A6, 2% PeV-A7, 4% PeV-A8, 3% PeV-A10, 1% PeV-A11, 3% PeVA13, 4% PeV-A14, 2% PeV-A16. Not typed viruses were left out of the results.

infections in fall and Central European countries have a peak in summer. Furthermore, PeV-A3 showed a biannual cycle in several countries, such as in the Netherlands, Scotland, USA and Australia. PeV-As have been detected in all continents. PeV-A1 is especially common and it mainly infects children under the age of 2 years. Most studies show PeV-A1 to be the dominating type, but there are big differences in its prevalence. European studies show up to 70%–90% of detected PeVs to belong to PeV-A1, in contrast to studies from Asia, South America, and Africa, which show PeV-A1 to represent 25%–60% of detected PeVs. This is the case in studies from either healthy population or samples from children with varying symptoms, such as diarrhea, respiratory symptoms or fever. However, in studies done on samples from neonates with serious infections such as sepsis-like disease, meningitis or encephalitis, the most prevalent type is PeV-A3. There are several reports of PeV-A3 outbreaks around the world. In some regions, PeV-A3 seems to be very common and the virus circulates in the population with infections peaking every 2–3 years. In Europe, USA, Australia, and Japan, the type distribution is limited to PeV-A1 to PeV-A6. Types PeV-A7 to PeV-A19 can be found in Asia, South America, and Africa (Fig. 3). However, there is no clear picture on the prevalence of these types. Although PeV-A types are mostly detected in humans, several types have also been isolated from non-human primates. PeV-A types 1, 4, 5, 12, 14, and 15 have been detected in Rhesus macaques, which were in close contact with humans. PeV-A types are seen as human viruses, but additional studies are needed to evaluate the possibility of animal reservoirs of PeV-A viruses.

Seroprevalence The seroprevalence of PeV-As differs very much between virus types and populations. However, serological studies have only been done in a few countries and they have mostly focused on PeV-A1. A study comparing the seroprevalence of PeV types 1–6 in Finnish and Dutch populations found PeV-A1 and PeV-A2 to be very common and the least common type was PeV-A3, with only minor differences between these populations for most types. Other studies do not support PeV-A2 to be common, and the virus is isolated only occasionally. In addition, PeV-A3 is found to be common in different populations, however, there might be a bias in the frequency of PeV-A3, since this type is the one seen in the clinics and therefore it is reported most often. Despite differences seen in studies done in different regions, PeV-A1 is the most common type found around the globe, reaching seroprevalence up to about 82%–99% in adults. Types 4, 5, and 6 are also fairly common, with seroprevalences of 35%–75%. Studies from Japan show seroprevalences against PeV-A3 of 80%–85% in children of 5 years of age, but lower seroprevalence in adults. A recent study compared PeV-A3 seropositivity in three populations, the Netherlands, Australia and USA,

Parechoviruses (Picornaviridae)

679

and the study showed prevalence in these populations ranging from 58% up to 83%, confirming the frequent findings of PeV-A3 in the clinics. Seroprevalences of types 7–19 are unknown.

Outbreaks The first reported hospital outbreak caused by PeV-A1 was in 1964–1965 in a nursery of premature infants. The babies presented with upper and lower respiratory symptoms. After this, several outbreaks have been reported from different continents. After the discovery of PeV-A3, most reported outbreaks have been found to be caused by this type. This is quite understandable, since this is the most pathogenic type and may cause severe symptoms in neonates. So far the largest reported outbreaks have been in Japan starting from 2006 and in Australia from 2013, which are described below.

Japan Japan has experienced several PeV-A3 outbreaks; these were detected in 2006, 2008, 2011, 2014, and 2016. The numbers of PeV-A3 positive cases have risen from the early epidemics (2006, 2008) with around 50 cases, to more than 300 in 2014 and 2016. This is either due to rising infection rates of PeV-A3 or just due to increased diagnostic activity in response to better awareness of the virus. Even in 2017, a year of lower PeV-A3 activity, there were about 100 cases detected. The outbreaks almost followed the biannual cycle seen elsewhere, except the outbreak in 2011, with a three-year gap on either side. In addition to the severe central nervous system (CNS) infections and sepsis-like disease in neonates, these outbreaks manifested with myalgia and myositis both in children and adults. The association to myalgia and myositis has not been described elsewhere. It could be due better awareness among Japanese scientists and doctors of PeV-A3 infections, since this virus was first detected in Japan. However, there could also be some genetic or other factor in the Japanese population making them more susceptible to PeV-A3 caused myalgia.

Australia So far, the biggest and best-documented epidemic is the one from Australia in 2013 to 2014. The succeeding epidemics occurred in 2015 to 2016 and 2017 to 2018. All epidemics had their peak in November or December, which are summer months in the southern hemisphere. This pattern fits the earlier finding from the Netherlands and UK, where PeV-A3 has a biannual seasonality and most cases were seen in summer months. The 2013/4 epidemic started in October and lasted until February 2014. More than 198 cases were identified and all typed samples were of PeV-A3 type. The epidemic was detected in New South Wales and it spread nationwide. The epidemic confirmed many earlier findings, such as most affected children were under the age of 3 months (93%) and both boys and girls were infected. The 2015/6 epidemic spread over a larger area from Western Australia to Sidney and the island of Tasmania. It had a slightly longer duration, from September 2015 to May 2016. The 2017/8 epidemic was first noted in Victoria, spreading thereafter to Queensland and subsequently nationwide. In both the 2015/6 and 2017/8 epidemics, more than 200 infected individuals were identified.

Parechovirus Species B-D The first characterized non-human PeV was Ljungan virus (a member of PeV-B) found in bank voles (Clethrionomys glareolus) in Sweden near the Ljungan River. Now five PeV-B types have been identified in several rodents including montane vole (Microtus montanus), southern red-backed vole (Myodes gapperi), yellow-necked mice (Apodemus flavicollis) and Eurasian red squirrels (Sciurus vulgaris), as well as in bird feces in Japan. PeV-Bs have so far been detected in several European countries, USA and Japan. Information from other regions is lacking. There has been speculation on whether PeV-B is able to infect humans and cause several different diseases, such as myocarditis, type 1 diabetes and intrauterine fetal death. However, no clear proof of human infections has been shown. PeV-B has never been detected from human stool samples, despite several large studies, with 2000–4000 screened samples. Seroprevalence studies have shown a significant proportion of positivity, but as there is no other evidence of PeV-B being able to infect humans, it is speculative whether this positivity is due to cross-reactive antibodies against antigenically related viruses. Thus, it still remains to be proven if PeV-Bs can infect humans and cause disease in humans. Other species (PeV-C, PeV-D) have so far only been detected in rodents.

Clinical Features The clinical picture of PeV-A1 and PeV-A3 is fairly well described in the literature. For other types the information is limited or lacking. One reason for this may be that these viruses mostly cause asymptomatic or very mild infections and therefore they are not seen in the clinics, this is probably true for types PeV-A2 and PeV-A4 to PeV-A6. For types PeV-A7 to PeV-A19, the reason could be that these types are mostly found in Asia, South America and Africa and studies from these continents, apart from Japan, are scarce. PeV-A1 was originally found to mostly cause fairly mild gastrointestinal and respiratory symptoms. However, in studies done after 1990 the proportion of PeV-As detected in these cohorts is quite low. Other symptoms that PeV-A1 has been connected to

680

Parechoviruses (Picornaviridae)

include fever, cough, rash, and otitis media. PeV-A1 has also been detected in serious infection cases throughout the decades. Severe infections include sepsis-like disease, meningitis, encephalitis, Guillain-Barré syndrome, paralysis, myocarditis, and hepatitis. Most of these findings are individual detections or case reports. A recent study from Iran found that all viruses of 12 positive CSF samples were PeV-A1, which is a rare observation since after its discovery, PeV-A3 has been reported to be almost exclusively the CNS infection-causing type. PeV-A3 is the type, which causes most severe infections and these are the best documented. Despite this, most PeV-A3 infections are mild or asymptomatic. Most common severe infections are sepsis-like disease, meningitis, encephalitis or meningoencephalitis. In Japan, PeV-A3 has caused myalgia in adults, but reports from other countries are lacking. In adults, clinical infection with any PeV-A is quite rare and most severe cases are seen in babies below the age of 3 months. In the 2013/4 Australian epidemic the most common symptoms in severe PeV-A3 infections were: fever, tachycardia, tachypnea and irritability/pain, which were present in more than 90% of patients. The next most common were rash and poor feeding (over 70%), followed by poor perfusion, diarrhea and lethargy, with each being present in more than 40% of patients. Hepatitis was seen in 33% of studied patients. Additionally, some patients (less than 15%) presented with upper respiratory symptoms, abdominal distention, vomiting, edema, seizures, apnea/preapnea or myoclonic jerks. The patients were described to be “red, hot, and angry”. Other studies have quite frequently reported seizures in PeV-A3 infected children, but definite numbers are hard to give with small patient numbers. In addition, to having quite severe symptoms at the acute phase, it has been shown that many children suffering from PeV-A3 caused encephalitis have long-term sequelae. After one year of follow-up 7 of 8 children showed at least some developmental concern. Earlier studies already suggested long-term sequelae, but with no follow-up and low patient numbers, this could not be confirmed. Now, after the Australian epidemic there are follow-up studies, which will provide more information on long-term consequences. More studies are needed to clarify the whole picture of PeV-A3 caused diseases. PeV-A3 has caused myalgia and myositis in Japanese children and adults. Symptoms are mostly muscle weakness and pain in arms and legs, sometimes even preventing patients from walking, as well as fever, loss of appetite, sore throat and diarrhea. Most adult male patients presented also with orchiodynia. Most of these cases had elevated creatine phosphokinase (CPK) levels, but no elevation of C-reactive protein (CRP). Most of the myalgia patients have been between ages 20–60 and are predominantly male. One report suggests the cause of myalgia to be an elevation of IL-6 by the PeV-A3 infection, since it is known that IL-6 causes myalgia, but this theory needs further studies. So far, the patients have recovered in about a week and no long-term sequelae have been reported. PeV-A types 2, 4–6 are rare in clinical cases. There was an outbreak of PeV-A4 in Helsinki in 2012 with a sepsis-like disease. This virus was discovered to be a recombinant, but it is not known if the recombination was the cause of more severe symptoms. Mostly PeV-A types 4–6 are seen in fever of unknown causes, upper respiratory infections and diarrhea. There are a few reported cases where PeV-A6 has been connected to severe CNS infections, but number of cases have been very low. In addition, the first detection of PeV-A6 was from a child with Reye’s syndrome, but the connection to this disease has not been confirmed. The clinical symptoms of PeV-A types 7–19 are mostly unknown and the types are rarely found. They have been detected in stool samples of children with or without diarrhea in Asia (except Japan), South America and Africa but the numbers are still low. It is possible that these types mostly cause asymptomatic infection and are thus seen rarely in the clinics. It is just as likely that these types are undiagnosed in developing countries.

Virus Genetics and Association to Disease The Australian epidemic-causing PeV-A3 strain was shown to be a recombinant virus. This was also the case for the sepsis-like disease-causing PeV-A4 strain in Finland. Despite genetic analyses comparing PeV-As involved in CNS-infections, sepsis-like disease or myalgia to those causing minor symptoms, no clear evidence of genetic differences explaining differences in the pathogenicity have been identified.

Pathogenesis Research on how PeV-As cause disease is scarce. It is not even known what cell types PeV-As infect in the CNS. White matter damage in the brain of encephalitis cases has been seen, but this does not necessarily mean that these cell types are directly infected. In cell culture settings it has been demonstrated that PeV-As can infect many different human cell lines, such as neuronal cells (SH-SY-5Y), astrocytes (T98G, U373-MG), lung (A549) and colon epithelial cells (HT-29). Neutralizing antibodies are important in preventing secondary PeV-A infections and in protecting new-born babies in their first months of life by maternal antibodies. As PeV-A1 is very prevalent, and according to some studies almost all mothers are positive for PeV-A1 neutralizing antibodies, it is not surprising that severe PeV-A1caused infections are not often seen in neonates. This is in contrast to what is found for PeV-A3, where a good proportion of mothers were negative for PeV-A3 antibodies (30%–90%). This difference in the presence of neutralizing antibodies could be one explanation why most severe infections seen in neonates are caused by PeV-A3. However, this does not explain why older children do not get severe infections. It is not known if the brains of an infant is more susceptible to PeV-A infections than the brains of an older child.

Parechoviruses (Picornaviridae)

681

Diagnosis Earlier, when PeVs were still typed as enteroviruses and the diagnosis was done in cell cultures, PeV-A1 and PeV-A2 were detected as part of enterovirus (EV) diagnostics. When molecular methods were introduced to enterovirus diagnosis, PeV diagnosis went from bad to worse, as EV RT-PCR does not detect PeVs. PeV-A diagnosis these days is mostly done by RT-PCR followed by sequencing to establish the PeV-A type. The diagnostic RT-PCR is based on primers amplifying a conserved region in the 50 UTR and the sequencing is based on either the VP1 region or the VP3/VP1 junction. Mostly those laboratories that have introduced PeV in their routine screening use in house RT-PCRs and there are numerous publications describing the methods. However, today there are also commercial PeV-A nucleic acid detection kits available, either as a PeV-A specific RT-PCR or PeV-A is included in multiplex PCR panels for the detection of meningitis or respiratory pathogens. Although viral culture and isolation is an important tool for further studies, the diagnosis should not be based on cultures. PeVA3, which is the most clinically important PeV-A, does not grow very well in the cells mostly used for diagnostic viral culture and even successful growth does not always produce a clear cytopathic effect. Furthermore, PeV-A types 7–19 might not be detected, since it is not known whether they grow in cell cultures, if they produce CPE or which cells should be used for virus culture. Diagnosing PeV-As has proven beneficial in several studies and the outcome of knowing the infective agent has reduced the length of stay in hospitals and unnecessary use of antibiotics.

Treatment and Prevention There are no specific antiviral medication or vaccines against PeV-As. There is an urgent need for medication since a great proportion of children suffering of severe PeV-A sepsis-like disease or CNS infection need intensive care. Recovered patients are often left with long-term problems and some infections even lead to death. Medication could help in reducing the number of children left with long-term sequelae. The problem with a preventive vaccine would be to decide when to vaccinate, since most children get the severe infection in their first weeks of life. The best option may be to vaccinate mothers against PeV-A3, which in the best case would lead to protective maternal antibodies. At the moment the only treatment option is intravenous administration of immunoglobulins (IVIG). This has been successfully used in clinical settings, but the success of treatment depends on the presence of specific antibodies, which could vary from batch to batch. Successful treatment was accomplished in a child suffering from PeV-A1 associated dilating cardiomyopathy. PeV-A1 is very common in the population and therefore IVIG is almost guaranteed to be positive for PeV-A1 antibodies. In contrast, according to several studies, seropositivity against PeV-A3 is not very high in European countries and therefore IVIG might lack PeV-A3 antibodies and thus not work. There is a clear need for antiviral medication against PeV-A3, but research to develop medication is not very active. Only a few in vitro-studies have been published. Studies have revealed potential compounds such as: 2-C-methylcytidine (20 CMC), a nucleoside analog, as well as itraconazole (ITZ) and posaconazole (PSZ), which are FDA-approved antifungal compounds. Of these 20 CMC reduced PeV-A1 and PeV-A3 growth in cell culture, but ITZ and PSZ only reduced the growth of PeV-A3. However, these substances could be clinically useful as the need for medication is the greatest for PeV-A3. Several compounds known to inhibit EV replication in vitro have been tested, but these compounds show no effect on PeVs. This leads to the conclusion that the PeV-As lifecycle differs greatly from the EV lifecycle and therefore more studies are needed for targeted drug design against PeV-As to be possible. Furthermore, the finding that ITZ and PSZ inhibited PeV-A3 growth but had no effect on PeV-A1 suggests that there may be significant differences in the lifecycle of different PeV-A types.

Further Reading Aizawa, Y., Izumita, R., Saitoh, A., 2017. Human parechovirus type 3 infection: An emerging infection in neonates and young infants. Journal of Infection and Chemotherapy 23, 419–426. Britton, P.N., Jones, C.A., Macartney, K., Cheng, A.C., 2018. Parechovirus: An important emerging infection in young infants. The Medical Journal of Australia 208, 365–369. Britton, P.N., Khandaker, G., Khatami, A., et al., 2018. High prevalence of developmental concern amongst infants at 12 months following hospitalised parechovirus infection. Journal of Paediatrics and Child Health 54, 289–295. Brouwer, L., Karelehto, E., Han, A.X., et al., 2019. High frequency and diversity of parechovirus A in a cohort of Malawian children. Archives of Virology 164 (3), 799–806. de Crom, S.C., Rossen, J.W., van Furth, A.M., Obihara, C.C., 2016. Enterovirus and parechovirus infection in children: A brief overview. European Journal of Pediatrics 175, 1023–1029. Kalynych, S., Pálková, L., Plevka, P., 2016. The structure of Human Parechovirus 1 reveals an association of the RNA genome with the capsid. Journal of Virology 90, 1377–1386. Kolehmainen, P., Siponen, A., Smura, T., et al., 2017. Intertypic recombination of human parechovirus 4 isolated from infants with sepsis-like disease. Journal of Clinical Virology 88, 1–7. Mizuta, K., Aoki, Y., Komabayashi, K., et al., 2018. Parechovirus A3 (PeV-A3)-associated myalgia/myositis occurs irrespective of its genetic cluster: A longitudinal molecular epidemiology of PeV-A3 in Yamagata, Japan between 2003 and 2016. Journal of Medical Microbiology 68 (3), 424–428. Olijve, L., Jennings, L., Walls, T., 2017. Human parechovirus: An increasingly recognized cause of sepsis-like illness in young infants. Clinical Microbiology Reviews 31, 1–17.

682

Parechoviruses (Picornaviridae)

Shakeel, S., Dykeman, E.C., White, S.J., et al., 2017. Genomic RNA folding mediates assembly of human parechovirus. Nature Communications 8, 5. van der Linden, L., Wolthers, K.C., van Kuppeveld, F.J., 2015. Replication and inhibitors of enteroviruses and parechoviruses. Viruses 7, 4529–4562. Zheng, L., Wang, F., Huang, J., Xin, H., 2015. Evaluation of the association of zoonotic Ljungan virus with perinatal deaths and fetal malformation. Birth Defects Research, Part C 105, 81–85.

Relevant Websites http://www.europic.org.uk/ Europic '79. http://www.picornastudygroup.com/ Picornaviridae Study Group. http://www.picornaviridae.com/ Picornavirus. https://talk.ictvonline.org/taxonomy/ Taxonomy International Committee on Taxonomy of Viruses. https://viralzone.expasy.org/ ViralZone root ExPASy.

Parvoviruses of Carnivores, and the Emergence of Canine Parvovirus (Parvoviridae) Colin R Parrish and Ian EH Voorhees, Cornell University, Ithaca, NY, United States Susan L Hafenstein, Pennsylvania State University, Hershey, PA, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Carnivora An order of eutherian mammals that share a common ancestor, which includes wolves, dogs, cats, raccoons, bears, weasels, hyaenas, seals, and walruses.

Translesional polymerases Specialized DNA polymerases that are expressed in cells that contain damaged DNA. This damage repair mechanism is error-prone.

Classification (Compact) Family – Parvoviridae Genus – Protoparvovirus Species – Carnivore protoparvovirus 1 Despite infecting a broad range of host species within the Order Carnivora, all Carnivore protoparvovirus 1 variants share over 97%–98% nucleotide sequence identity, forming a single distinct clade (Fig. 1). The most closely related viruses known outside this group are only about 60% or less identical (Ungulate PPV 1 and the Rodent PPV 1 and Rodent PPV 2). Within the Carnivore protoparvovirus 1 clade, the two major subclades comprise the viruses related to FPV, and those within the lineage of viruses that emerged in dogs in the 1970s which are identified as CPV-variants no matter which hosts they are isolated from (Fig. 2).

Virion Structure and Genome Like other members of the family Parvoviridae, the Carnivore protoparvoviruses 1 virion consists of a single linear strand of DNA of about 5000 nucleotides in length packaged within a non-enveloped icosahedral protein capsid. The capsid is initially assembled from 60 copies of a mixture of two different forms of a capsid protein (virus protein 1 and 2 (VP1 and VP2)). The two forms are produced by alternative splicing of the same message, sharing a common C-terminal sequence of 568 residues, with the VP1 form containing an additional 145 residue N-terminal domain. VP2 makes up a majority (about 90%) of the 60 capsid proteins, and in a full (DNA containing) capsid around 24 residues may be cleaved from the N-terminus by host proteinases to form VP3. The robust capsid is a compact T ¼ 1 icosahedron that is B26 nm in diameter with a prominent raised region or spike surrounding each threefold symmetrical axis, an elevated cylindrical pore-like structure around each fivefold axis formed by 5 antiparallel beta strands, and depressions around the fivefold axis and also spanning the two-fold axis. The genome of the virus is composed of two major coding regions, with the NS1 and NS2 proteins encoded within the lefthand end of the genome (the 30 -end of the minus sense DNA strand that is packaged into these viruses). The VP1 and VP2 proteins are encoded by right hand ORFs. The messenger RNAs that encode the NS and VP proteins are expressed from the P4 and P38 promoters, respectively. Like the VP forms, the NS1 and NS2 are produced by alternative splicing. Additionally, a small (58–68 residue) alternatively translated non-structural protein (SAT) is encoded within the VP1/2 mRNA, overlapping the sequence near the VP2 N-terminus.

Life Cycle Infection of host cells requires that the virus capsid bind to the transferrin receptor type-1 (TfR) on the surface of the cell, which mediates rapid uptake into the cell through clathrin-mediated endocytosis. The TfR is a large dimeric receptor that is expressed on the surface of all cells, where its normal function is to bind iron-loaded (holo) transferrin, and to transport that into the endosomal system of the cells, where the iron is released at the low pH of the early or recycling endosome, after which the TfR and iron-free (apo) transferrin recycle back to the cell surface. The TfR is assembled as a dimer of three domains, the apical domain, the protease-like domain, and the helical domain that forms the dimer interface (Fig. 3(a)). Once inside the cell the capsid enters the cytoplasm, and the ssDNA (likely without the capsid) enters the nucleus, where the 30 -end of genome in a palindromic hair-pinned form acts as a template for DNA synthesis to generate a dsDNA form. Genome replication occurs via a variation of the rolling-hairpin replication process, that involves nicking of the double stranded replicative form of the DNA by the viral NS1 protein. The 30 -end of the DNA generated is then extended by one or more of the host cell DNA polymerases to create a new double strand, displacing the second strand when replicating on double stranded

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00098-9

683

684

Parvoviruses of Carnivores, and the Emergence of Canine Parvovirus (Parvoviridae)

Fig. 1 Evolutionary relationships among species within the genus Protoparvovirus. A related parvovirus, Carnivore amdoparvovirus 1, within the genus amdoparvovirus is used as an evolutionary outgroup. For each species, the nearly full genome NCBI Ref sequences was used. In addition, multiple isolate sequences were included for Carnivore PPV 1 to display the key variants within the species. Numbers represent node support (out of 100). GTR PhyML tree. 100xBS replicates. Abbreviations: PPV ¼ protoparvovirus; FPV ¼ feline panleukopenia virus; RPV ¼ raccoon parvovirus; CPV ¼ canine parvovirus.

DNA. While host DNA polymerase delta appears to be the primary polymerase involved in the viral genome replication, other canonical DNA polymerases or translesional polymerases may also play roles in viral genome replication when they are present. Replication produces both positive and negative strand viral genomes; however, negative strands are preferentially packaged by these viruses (other members of the Parvoviridae may package either strand with similar efficiency). Packaging is mediated by the helicase domain of the viral NS1 protein, and for these viruses the ssDNA DNA is fed in a 30 -to-50 orientation into the preassembled capsid through one of the fivefold capsid pores.

Epidemiology Carnivore protoparvovirus 1 was first described as the etiological agent of severe enteric disease in cats as early as the 1920s. By the 1940s, the virus was recognized in number of wild and domestic non-canine carnivores around the world, and referred to as feline panleukopenia virus (FPV), mink enteritis virus (MEV), or raccoon parvovirus (RPV) depending on the host infected. In early 1978, a variant of those viruses (named canine parvovirus (CPV)) or canine parvovirus type-2 (CPV-2) emerged to cause disease in domestic and wild dogs and that reached pandemic proportions by the end of that year. Today, natural infections by various strains of these viruses are found in cats and dogs, as well as domestic or captive populations mink, foxes, and raccoon dogs, as well as in most major groups of wild terrestrial carnivores. It is unclear whether Pinnipeds (seals, sealions and relatives) or bears are susceptible to infection or to clinical disease by these viruses. Occasionally indications of infection of other hosts have been reported, including reports in captive rhesus macaques, and in insectivores such as hedgehogs. Testing for viral DNA or antibodies indicates that Carnivore protoparvovirus 1 is very widespread in nature among susceptible carnivore populations. Likewise, phylogenetic studies of the viruses and their transmission reveals widespread global dissemination of virus genotypes as there are close similarities between viruses that are derived from distant locations. Such rapid global dissemination was well documented during the original emergence and spread of CPV in dogs in 1978 and 1979, as dogs and related canids (coyotes and wolves) throughout the world became infected over a period of a few months. There are also significant DNA sequence similarities between the viruses found infecting different host species, suggesting frequent cross-species transmission (Fig. 2). Domestic dogs and cats remain the largest host populations for these viruses around the world by a wide margin, and are assumed to maintain the most common virus strains in wide circulation. It is likely that the viruses in other hosts mostly originate from spill-over infections of viruses from cats or dogs, perhaps followed by local transmission within that new host - there is evidence of viral lineages in wild raccoons and in farmed mink and arctic foxes, likely because those populations are large enough to reveal outbreaks and to sustain transmission for weeks or months. As mentioned above the viruses appear to be very widespread in nature due to the very high levels of shedding that occurs in the feces of infected animals, and the stable capsid which can persist in an infectious form in the environment for weeks or even months. Most puppies or kittens acquire maternal antibodies to the virus from their mother, mostly in the form of colostral IgG,

Parvoviruses of Carnivores, and the Emergence of Canine Parvovirus (Parvoviridae)

685

Fig. 2 Evolutionary relationships among viral variants within Carnivore protoparvirus 1 collected from various host species. Tree tips colored according to host species.

protecting them from infection by virus for the first 8–16 weeks of life (depending on the levels of maternal antibodies transferred). For domestic animals, those maternal antibodies also prevent infection by vaccines, such that the young become receptive to vaccine and to wild virus in the environment at about the same time. There has therefore continued to be relatively high levels of parvovirus infection in young animals even in the face of widespread application of vaccines, and the virus also infects most wild animals in the early months of life. Both FPV-like and CPV-like strains of virus continue to circulate in parallel, with most viruses in cats being FPV-like, and all strains in dogs being CPV-derived viruses.

686

Parvoviruses of Carnivores, and the Emergence of Canine Parvovirus (Parvoviridae)

Fig. 3 (A) Structure of the CPV capsid (colored blue) in complex with host TfR (colored red ¼ protease domain; yellow ¼ helical domain; and green ¼ apical domain) determined by cryo-electron microscopy. (B) Roadmap of CPV capsid with the TfR footprint indicated (bold outline). Changes that occurred at VP2 residues 301 and 93 (colored in yellow) expanded the host range of the virus, enabling binding and entry of host cells using the canine TfR.

Host Range Variation, Its Control, and the Emergence of CPV Host range variations are often associated with small numbers of amino acid differences in the viral capsid, which affect the structure and interaction with the host TfR. When viruses or viral DNA are recovered from various hosts (e.g., raccoons, foxes, and mink), they often show host range variation that appears to be related to differences in binding to the host receptor. The bestdocumented example of host range variation was the acquisition of the canine host range by the ancestor of the CPV lineage. When FPV viruses were compared to the earliest CPV for which sequences are known (from 1978), at least 2 key mutations were identified that alter residues in the viral capsid and together control the ability of the virus to bind to the canine TfR. Mutagenesis studies and the cryo-electron microscopy structures of the TfR from a host related to the dog – the black backed jackal – in complex with the CPV-2 capsid, have provided high resolution structural detail and context to this host-range acquisition. Those revealed that the TfR bound the capsid through a relatively small interface (Fig. 3), with parts of two loops of the TfR apical domain interacting with portions of three loops exposed on the surface of the CPV capsid. The canine TfR differs from the related forms found in other carnivore hosts in that it has an N-linked glycosylation in the apical domain, which blocks the binding of viruses that are similar to the FPV. The mutations in the virus that resulted in it gaining the canine host range occur within or adjacent to the TfR footprint (Fig. 3(b)), altering the flexibility of the capsid loops that interact with the TfR. These changes allowed the modified receptor to interact with the apical domain around the additional glycosylation site, mediating cell infection. The original strain of CPV, termed CPV type-2, spread world-wide during a period of around 1 year, when it was replaced by a variant form (identified as CPV- type-2a) which was essentially the CPV-2 genotype, but had acquired an additional group of 5 mutations which altered sites on the surface of the capsid, further expanding the virus’s host range.

Pathogenesis and Clinical Features The pathogenesis of the viruses are all similar, and they infect through the oral or oro-nasal routes, and likely establish their initial infections in the tonsils and retro-pharyngeal lymphoid tissues. Virus spreads free in the plasma, as well as likely by binding to or infecting lymphocytes, and therefore infects cells in many secondary tissues. A defining feature of the pathogenesis of these viruses is that they only replicate in cells that are undergoing mitosis, because the polymerases, nucleotides and other cellular components required for viral DNA replication are only expressed in cells that are dividing. This also results in a strong age-relationship to the disease due to the reduced proportions of dividing cells as animals age. Two diseases are observed only after the virus infects neonatal animals, due to the populations of cells that are replicating shortly after birth. Those diseases are cerebellar hypoplasia in cats due to virus replication in and killing of the germinal cells of the cerebellum, thereby resulting in permanent ataxia, and viral

Parvoviruses of Carnivores, and the Emergence of Canine Parvovirus (Parvoviridae)

687

myocarditis in dogs due to virus infection of myocardial cells, which results in a slowly developing myocarditis where host responses target the infected myocytes, resulting in cardiac failure a few weeks after infection. Both of those diseases are rare because most kittens or puppies are now protected during that age by maternal antibodies. In animals that are infected at two months of age or later (after maternal immunity has waned), many develop only mild or subclinical infections. However, a proportion of infected animals develop severe disease due to viral replication in the rapidly dividing cells that regenerate the epithelium of the small intestinal villi. The epithelial cells are continuously turned over, and loss of the regenerating cells results in the loss of epithelial integrity, so that infected animals develop severe diarrhea and vomiting, as well as loss of the intestinal barrier.

Diagnosis There are many diagnostic tests that can be applied to detect these viruses, or to provide evidence of their infection. The virus may be detected in feces using point-of-care ELISA tests that involve antibody capture of the viral particles from feces (or sometimes from pharyngeal swabs), followed by a second reagent that detects the bound virus. That test depends on the presence of high levels of free virus in the feces, so sensitivity is lost after host antibodies are produced that bind to the virion, or once viral shedding falls to low levels. Other tests include virus isolation into tissue cultures of feline cells (which are susceptible to all strains of virus), or PCR detection of the viral DNA. The PCR test is very sensitive and can detect the DNA of actively replicating virus, as well as DNA in the tissues that represents previous viral infection but where infectious virus is no longer present. Quantitative PCR may be used to distinguish between those circumstances, as false-positive DNA tests are otherwise very common since most animals have some residual DNA.

Treatment No specific treatment is known that effectively targets the virus or its replication. The cerebella hypoplasia and myocarditis that develop after neonatal infection are irreversible once infection and replication of the virus occurs. For animals with enteritis the treatment is mainly supportive, including replacing the fluid and electrolyte losses from the diarrhea and vomiting. Antibiotics may be given to forestall secondary infections, but their efficiency is marginal in most cases.

Prevention The most effective prevention involves the use of live attenuated vaccines that are given by parenteral routes, and which replicate systemically and induce the production of highly protective immunity that appears to last for the life of the animal. However, like the natural infection, the vaccine is blocked by the presence of maternal antibodies, so that it is necessary to wait until animals are between 8 and 16 weeks of age when the maternal immunity has waned sufficiently to allow virus infection and replication. Since animals may be exposed to wild type viruses from the environment during the same period, continuing disease is seen even when vaccines are used diligently.

Further Reading Barrs, V.R., 2019. Feline Panleukopenia: A re-emergent disease. Veterinary Clinics of North America: Small Animal Practice 49, 651–670. Carmichael, L.E., 2005. An annotated historical account of canine parvovirus. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health 52, 303–311. Lee, H., Callaway, H.M., Cifuente, J.O., et al., 2019. Transferrin receptor binds virus capsid with dynamic motion. Proceedings of the National Academy of Sciences of the United States of America 116, 20462–20471. Parker, J.S., Parrish, C.R., 1997. Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid. Journal of Virology 71 (12), 9214–9222. Parrish, C.R., 1995. Pathogenesis of feline panleukopenia virus and canine parvovirus. Baillière's Clinical Haematology 8, 57–71. Reed, A.P., Jones, E.V., Miller, T.J., 1988. Nucleotide sequence and genome organization of canine parvovirus. Journal of Virology 62 (1), 266–276. Voorhees, I.E.H., Lee, H., Allison, A.B., et al., 2019. Limited intra-host diversity and background evolution accompany 40 years of canine parvovirus host adaptation and spread. Journal of Virology 94, e01162.

Polioviruses (Picornaviridae) Philip D Minor, St. Albans, United Kingdom r 2021 Published by Elsevier Ltd.

Introduction A funerary stele from about 1300 BCE currently in the Carlsberg museum in Copenhagen shows the priest Rom with a withered single limb and down-flexed foot typical of motor neuron destruction caused by poliovirus infection. It is considered to be the first documentary evidence for an infectious disease of humans. Two highly effective vaccines were developed in the 1950s, and a major program of the World Health Organization (WHO) is underway which may make poliomyelitis the second human disease to be eradicated globally after smallpox.

The Virus Genome The virus contains a single strand of messenger sense RNA of about 7500 nt. A long highly structured 50 noncoding region of about 740 nt which serves as an internal ribosomal entry site (IRES) is followed by a single open reading frame and terminates in a 30 noncoding region of c. 70 nt followed by a polyadenylate tract. The 50 end of the genome is covalently linked to a virus-encoded protein termed VPg. The single open reading frame encodes the structural proteins which make up the capsid (collectively termed the P1 region) followed by regions encoding the nonstructural proteins P2 and P3. P1 is divided into VP1, VP3, and VP0 (VP2 plus VP4). P2 is cleaved into 2Apro, 2B, and 2C, and P3 into 3A, 3B (or VPg), 3Cpro, and 3Dpol by virus-encoded proteases. 3Dpol is the viral polymerase, but all proteins in the nonstructural part of the genome play a role in RNA replication, which also depends on RNA structural elements and host cell membranes which are extensively rearranged in the course of infection. A significant structural element involved in replication is the stem loop designated cre (cis acting replication element) which is essential to the initiation of genome replication but can be moved to any position by molecular manipulation to any site in the genome where it remains functional, as mentioned below.

The Virus Particle The infectious virus particle consists of 60 copies each of VP1, VP2, VP3, and the smaller protein VP4 surrounding the viral genome; VP2 and VP4 are generated by autocatalytic cleavage of VP0 in the last stage of the maturation of the virus particle. The proteins are arranged with icosahedral symmetry such that VP1 is found at the pentameric apex of the icosahedron and VP2 and VP3 alternate around the pseudo-sixfold axes of symmetry of the triangular faces of the icosahedron. VP4 is located internally about the pentameric apex and is myristylated. The pentameric apex of the particle is surrounded by a dip or canyon into which the cellular receptor for poliovirus fits. The atomic structure of the virus was solved in 1985.

The Poliovirus Receptor Although some strains of type 2 poliovirus are able to infect normal mice, only the higher primates and Old World monkeys are susceptible to infection by all serotypes as they possess a specific poliovirus receptor that is required for infection, and this species restriction is one of the factors that makes polio eradication theoretically possible. The receptor molecule was identified in 1986 and it is a three-domain membrane protein of the immunoglobulin superfamily termed CD155. It is necessary and sufficient for the infection of cells in vitro, and transgenic mice carrying the gene for the poliovirus receptor can be infected by all poliovirus types, developing paralysis when infected with wild-type virus. Such mice have been developed as alternatives to monkeys in the safety testing of live poliovirus vaccines. The tissue distribution of the receptor does not explain the targets of infection of the virus and it is possible that innate immunity plays a significant role in its highly specific tropism in normal individuals.

Classification of Picornaviridae The members of the family Picornaviridae to which poliovirus belongs are small nonenveloped viruses of about 30 nm in diameter when hydrated, essentially almost featureless when examined by electron microscopy and containing a single strand of an RNA molecule of messenger sense. The family is currently split into sixty three genera and one hundred and forty seven species on the basis of sequence similarities.

688

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00123-5

Polioviruses (Picornaviridae)

Table 1

689

Species within the genus Enterovirus

Species

Serotype

Human enterovirus A Human enterovirus B

Coxsackievirus A2, 3, 5, 7, 8, 10, 12, 14, 16 Enterovirus 71 Coxsackievirus B 1–6, coxsackievirus A 9 Human echovirus 1–7, 9, 11–21, 24–27, 29–33 Human enterovirus 69

Human enterovirus C Human enterovirus C Bovine enterovirus Porcine enterovirus A Porcine enterovirus B

Poliovirus 1, 2, 3 Coxsackie virus A 1, 11, 13, 15, 17–22, 24 Human enterovirus 68, 70 Bovine enterovirus 1, 2 Porcine enterovirus 8 Porcine enterovirus 9, 10

The genus Enterovirus, of which poliovirus is the archetypal species, is currently further subdivided into twelve species of which A to D and the three rhinovirus species infect humans. Poliovirus is classified as a Human enterovirus type C. The classification of the enterovirus species and serotypes is shown in Table 1. The sequences of the capsid region of the viruses within a species define a virus type while sequences of other regions of the genome do not. This suggests that viruses within a particular species can exchange genome segments more or less freely. The comparison of the sequence of field poliovirus isolates has proved a very valuable tool in the WHO program to eradicate polio. Viruses from the same geographical region tend to cluster in terms of their sequence largely independent of the year of isolation. Thus, it is possible to identify a virus from a case as indigenous to the region where it was isolated or whether the virus was an importation from another geographic region. Similarly, as virus circulation is restricted by effective vaccination programs, the variety of sequences found declines, indicating progress in the vaccination program long before the disease is eradicated. Finally, it is possible to identify strains as wild type or vaccine derived. The rate at which the sequence of a vaccine or wild-type virus lineage drifts in an epidemic or during chronic infection of immunodeficient individuals unable to clear the virus is amazingly constant at about 2%–3% silent substitutions per year, or 1% for all substitutions. Thus, the comparison of viral sequences can date the common ancestor of two polioviruses accurately and give an estimate how long they have been circulating.

Pathogenesis and Disease Poliomyelitis gets its name from the specificity of the virus for the motor neurons that form the gray matter (polios and myelos, Greek for gray and matter, respectively) of the anterior horn. Despite the obvious physical signs of muscle atrophy and motor neuron degeneration, few cases of poliomyelitis are identifiable in the literature before the end of the nineteenth century, and it is believed that it was extremely rare. However, at this time, the disease began to occur in large epidemics, initially in Scandinavia (particularly Sweden), and then in the USA, most commonly affecting young children (hence the alternative name of infantile paralysis). The legs are more commonly affected than the arms, and paralysis tends to occur in one limb rather than symmetrically. Poliovirus occurs in three antigenically distinct serotypes designated 1, 2, and 3, such that infection with one serotype confers solid protection only against other viruses of that serotype. Infections primarily occupy the gut, and most are entirely silent, but in a small number of cases they lead to a systemic infection (the minor disease) 3–7 days post exposure, characterized by fever, rash, or sore throat. Depending on the strain or type of virus, usually less than 1% of all infections lead to a more serious major disease or poliomyelitis which develops on average 7–30 days after infection. Spinal poliomyelitis resulting in lower limb paralysis or bulbar poliomyelitis in which the breathing centers are affected occurs when the lower or upper regions, respectively, of the spinal cord are affected. In the mid-twentieth century, about 10% of the polio cases lead to death, 10% of cases recovered without sequelae and 80% had permanent residual paralysis. Encephalitis may occur but it is a rather rare complication. Meningitis, also known as abortive or nonparalytic poliomyelitis, occurs at a rate of about 5% of poliomyelitis cases. Infection is mainly fecal–oral, although the virus can also be transmitted by the respiratory route from the throats of infected individuals. The primary site of infection in the gut is not known but may be the lymphoid tissues, specifically the Peyer’s patches and tonsils or the mucosal surfaces in the gut or throat; infectious virus can be found in the local lymph nodes and in more distal mesenteric lymph nodes but it is not clear whether the virus actually replicates there. The Peyer’s patch-associated M-cells are believed to be the major infected cell types in the gut. Viremia may occur about 7 days after infection, corresponding roughly to the appearance of the minor disease. It is believed to seed sites including the peripheral and central nervous systems resulting in the major disease. Thus, antibodies should prevent poliomyelitis by blocking viremia; the protective effect of passively administered antibodies was shown in the 1950s. This explains the change in the epidemiology of the disease in the beginning of the twentieth century as hygiene improved, and children were exposed to poliovirus infection later in life when maternal antibody levels had declined to levels no longer able to confine the infection to the gut.

690

Polioviruses (Picornaviridae)

Vaccines Inactivated polio vaccine (IPV) was developed by Dr. Jonas Salk. It consists of wild-type virus that has been treated with low concentrations of formalin sufficient to inactivate the virus without affecting its antigenic properties to a great extent. The vaccine was first licensed in 1955 in the USA and its use reduced the number of cases by over 99%. It has been shown to be capable of eradicating the disease in certain countries, notably Scandinavia and the Netherlands. However, there remained controversy over whether a nonreplicating vaccine could eradicate the disease and the virus altogether in low income countries where exposure is more intense, and it was possible that live-attenuated vaccines imitating natural infection might give better protection against infection by wild-type viruses. The live-attenuated vaccines given orally (oral polio vaccine, OPV) that formed the basis of the eradication campaign were developed by Albert Sabin and introduced in the early 1960s. The Sabin vaccines have been shown to be able to interrupt epidemics and to break transmission chains if used correctly, resulting in the eradication of the virus in entire regions and possibly eventually the world. It was also known that the Sabin vaccines altered in the vaccine recipients, becoming more neurovirulent particularly in the case of the type 2 and 3 components and that in rare instances, now estimated at about 1 case per 750,000 first-time vaccinees, the vaccine could cause poliomyelitis in recipients and their close contacts. Therefore, there is increasing usage of IPV in developed countries. WHO have recommended the inclusion of a dose of IPV in all immunization programs. Both IPV and OPV contained a single representative of each of the three serotypes.

Attenuation of the Sabin Vaccine Strains The genome of poliovirus is of positive (messenger) RNA sense and therefore the viral RNA is infectious. Complete genomes of the virulent precursors of the Sabin vaccine strains or isolates from vaccine-associated cases can be cloned, sequenced, and mutations introduced or segments exchanged. Genomes can be synthesized de novo. Virus recovered from RNA transcribed from the modified plasmids was tested in monkeys or in transgenic mice carrying the poliovirus receptor. It was possible to identify two differences between Leon, the virulent precursor of the Sabin type 3 vaccine strain which would attenuate Leon or would de-attenuate the Sabin strain, one in the 50 noncoding region, the other in capsid protein VP3. Similarly, there were two major attenuating mutations in the type 2 strain, one in the 50 noncoding region and another one in capsid protein VP1. Other attenuating mutations with weak effects may also exist. The situation with type 1 poliovirus was more complicated. Again, there was one mutation in the 50 noncoding region but several others throughout the capsid region. All three of the 50 noncoding mutations affect the highly structured region shown in Fig. 1. All 50 noncoding mutations have been shown to affect the initiation of protein synthesis, all involve allowed but altered base pairing, and all revert or are suppressed in vaccine recipients when sequential isolates are made. Excretion of virus following vaccination with OPV continues on an average for 4 or 5 weeks and the reversions observed occur early in the excretion period, usually within 1 or 2 days for the type 3 strain and within a week for the other two types. Other changes also take place, including the reversion or more usually direct or indirect suppression of the VP3 mutation in the type 3 strain. It is possible to alter the base pairing in domain 5, exchanging GC or GU base pairs for AU to stabilize the structure genetically while altering its thermodynamic stability. Thus, where a GU base pair is replaced by an AU base pair two mutations will be required to generate the more thermodynamically stable GC. The thermodynamic stability of domain 5 has been shown to correlate with the virulence of the virus so that in principle a virus of any desired degree of attenuation can be generated by changing appropriate base pairs; this forms the basis of some novel vaccines which are in development as described later. In addition, the viruses recombine with each other at high frequency. The type 3 strain in particular is usually excreted by vaccinees as a recombinant from about 11 days post immunization with the part of the genome encoding the nonstructural genes from the type 1 or type 2 strains. Complex recombinants with portions from different serotypes are also common, although their selective advantage is not known. The adaptation of the virus to the gut is therefore rapid and subtle and by a variety of mechanisms including reversion, second site suppression, and suppression of the phenotype of poor growth by enhancing fitness by an entirely unrelated route, and may involve mutation or recombination. Once adapted, virus excretion persists typically for several weeks. The drift in the viral sequence over prolonged periods of time referred to above occurs in addition to these selected changes, and appears to be a consequence of a purely stochastic process.

The Global Polio Eradication Program For many years, it was thought that OPV could not be effective in tropical countries, and despite large-scale use its impact was in fact minimal. The reasons put forward included interference by other enteric infections, and many other factors, including breastfeeding. The most plausible explanations are that the vaccine used in routine vaccination programs was probably poorly looked after and therefore inactive, and the epidemiology of the disease was different in tropical and temperate climates. In countries of Northern Europe, poliomyelitis is highly seasonal, occurring essentially only in summer. Thus, a routine vaccination campaign in which children are immunized at a specific age is able to reduce the number of susceptible individuals during the winter when the virus is not freely circulating, so that circulation of the wild-type virus in the summer is impaired. Eventually, the wild-type virus

Polioviruses (Picornaviridae)

691

Fig. 1 Structure of RNA domain in the 50 noncoding region of the poliovirus genome involved in attenuating the neurovirulence of the Sabin live-attenuated vaccine strains. Top: type 1; center: type 2; bottom: type 3. Differences from the type 3 sequence are shown in bold, illustrating the general conservation of the base-paired structures. Base changes involved in attenuation are shown by arrows for the three types. Note that for types 1 and 3, the changes result in weaker interactions in the secondary structure but allowed base pairing compared to the wild-type sequence.

692

Polioviruses (Picornaviridae)

dies out. In tropical climates, however, exposure is less seasonal, so that there is no respite during which immune populations can be built up by immunization; it remains a matter of chance whether a child is exposed first to the vaccine or wild-type virus, and the impact on reducing the disease is correspondingly weaker. The strategy required is to immunize large proportions of the population at once. This approach was successfully followed in the 1960s in the USA in the southern states, where the climate is more tropical and routine immunization was less effective. However, it was not until the 1980s that the strategy was used in developing countries in South America when vaccine was given in mass campaigns termed National Immunization Days (NIDs). The success of the program led to a resolution in 1988 by WHO to eradicate polio from the world by the year 2000. While this goal has still not been achieved at the time of writing, Pakistan and Afghanistan are currently the only two countries from which the wild type viruses have never been eliminated. Northern India proved extremely difficult and some children contracted poliomyelitis after 10 or more immunizations with OPV. One approach adopted was to use monovalent vaccine instead of the trivalent form containing all three serotypes. The rationale is that in the trivalent vaccine the different serotypes compete with each other so that the required serotype may not infect and induce immunity, whereas a monovalent type 1 vaccine used in an area where this is the problem serotype will be effective. There is evidence that this gives faster immune responses, and it was first used in Egypt where it eradicated the remaining type 1 strains. India was declared free of polio in 2014 after three years without a case. The whole of Africa was declared free of wild type poliovirus of all three serotypes in 2020. The type 2 component was removed from all OPVs in 2016 as no naturally occurring case of poliomyelitis caused by wild type 2 has been identified globally since 1999 and the type 2 component was the main source of circulating vaccine derived polioviruses (cVDPVs) which were a major cause of residual polio cases in the world. While there are only two countries in which poliomyelitis has not been eradicated, many countries have suffered reintroductions. This is shown in Fig. 2, which shows cases of the regions where polio has not been completely eliminated. The first examples of this phenomenon were in central Africa. A decision was taken because of shortage of funds to concentrate immunization efforts in Nigeria which had many cases (Fig. 2(a)). Thus, immunization in the surrounding countries suffered, and at the same time resistance to immunization grew in northern Nigeria because of local concerns about supposed contaminants in the vaccine. During the period when immunization ceased, there was a resurgence in polio in Nigeria, which spread to adjacent countries where the immunization activities had been reduced (Fig. 2(b)). As a result, polio spread from Nigeria across much of central Africa, being brought under control eventually by coordinated NIDs across most of the continent, an operation of unprecedented scale. Shortly after this, with polio still endemic in Nigeria, the annual pilgrimage to Mecca resulted in the introduction of polio from northern Nigeria into Yemen and Indonesia (Fig. 2(c)). In 2006, polio was introduced into several African countries from northern India (Fig. 2(d)). The difficulties of eradication are hard to overestimate, and are associated with the fact that most infections are entirely silent. Despite this, as stated above, there has been no case of poliomyelitis caused by a naturally occurring type 2 virus since 1999 and there have been no cases caused by wild type 3 virus since 2012. It is clear that as long as one country remains a source of the virus the world remains at risk. This complicates the strategies to be followed once there is some confidence that the virus has in fact been eradicated in the wild, which are further complicated by the fact that OPV is a live vaccine derived from wild-type virus that is able to change in infected vaccinees, from whom virus may be isolated for significant periods of time.

Vaccine-Derived Poliovirus Vaccine-associated paralytic poliomyelitis (VAPP) cases were recognized from the first use of the Sabin vaccine strains, although their unambiguous identification required molecular methods applied in the 1980s. VAPPs occur in primary vaccinees at a rate of about 1 per 750,000 but can also occur in contacts and those previously vaccinated. The occurrence of such cases, while rare, indicates that the vaccine strains can revert to virulence in recipients and that it is possible for the vaccine strains to spread from person to person. In 2001, it was recognized for the first time that the Sabin vaccine strains could be the cause of outbreaks of poliomyelitis when about 22 cases were identified in Hispaniola, comprising Haiti and the Dominican Republic. Sequencing of the strains showed that they were very closely related to the type 1 Sabin vaccine strains and not to the previous endemic strains of which the last had been isolated 20 years earlier. Moreover, the sequence diversity indicated that the outbreak strains had been circulating unnoticed for ca. 2 years, as they differed from the vaccine strain by about 2% overall. In addition, they were shown to be recombinant strains with a major portion of the nonstructural regions of the genome from viruses identified as species C enteroviruses unrelated to the vaccine strains. At least three different genomic structures were identified. Subsequently, it was reported that between 1988 and 1993 all supposed wild type 2 strains isolated from poliomyelitis cases in Egypt were in fact heavily drifted vaccine-related strains. Outbreaks in the Philippines in 2001, two in Madagascar in 2002 and 2005, one in China in 2004, and one in Indonesia in 2005 and many others have been recorded. The most likely explanation is that vaccination programs become less vigorous once polio is eliminated from a country and other health issues take priority, so that while administration of OPV continues, the coverage is less than 100% giving perfect conditions for the selection of transmissible strains. For reasons that are not understood, almost all such strains to date have been recombinants with unidentified species C enteroviruses and most are type 2, followed by type 1, with type 3 being rare. So far, the outbreaks have been limited and easy to control; this was particularly shown in Indonesia where a type 1 outbreak from a virus introduced from Nigeria occurred at the same time as an outbreak of circulating vaccine-derived

Polioviruses (Picornaviridae)

(a)

Wild poliovirus*, 15 Apr. 2002−14 Apr. 2003

Under investigation Importation Wild virus type 1 Wild virus type 3 Wild virus types 1 & 3 Endemic countries *Excludes viruses detected from environmental surveillance and vaccine-derived polio viruses. Data in WHO HQ as of 15 Apr. 2003.

(b)

693

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2003. All rights reserved

Wild poliovirus*, 12 Apr. 2004−12 Apr. 2005

Wild virus type 1 Wild virus type 3 Wild virus types 1 & 3 Endemic countries Reestablished transmission countries Case or outbreak following importation *Excludes viruses detected from environmental surveillance and vaccine-derived polio viruses. Data in WHO HQ as of 12 Apr. 2005.

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2005. All rights reserved

Fig. 2 Occurrence of cases of poliomyelitis in remaining areas of infection from 2002 to 2006. World Health Organization (WHO).

694

Polioviruses (Picornaviridae)

(c)

Wild poliovirus*, 25 Jan. 2005−24 Jan. 2006

Wild virus type 1 Wild virus type 3 Wild virus types 1 & 3 Endemic countries Reestablished transmission countries Case or outbreak following importation *Excludes viruses detected from environmental surveillance and vaccine-derived polio viruses. Data in WHO HQ as of 24 Jan. 2006.

(d)

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2005. All rights reserved

Wild poliovirus*, 30 Aug. 2005−29 Aug. 2006

Wild virus type 1 Wild virus type 3 Wild virus types 1 & 3 Endemic countries Case or outbreak following importation (6 months) Case or outbreak following importation (6−12 months) As of 1 Jan. 2006, Egypt and Niger were reclassified as nonendemic countries. *Excludes viruses detected from environmental surveillance and vaccine-derived polio viruses. Data in WHO HQ as of 29 Aug. 2006.

Fig. 2 continued.

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2006. All rights reserved

Polioviruses (Picornaviridae)

695

poliovirus (CVDPV). The wild-type outbreak persisted longer. Virologically, there is no real reason why vaccine-derived viruses should not become as aggressive as the wild-type viruses from which they were derived, although this does not seem to have happened at the time of writing. The occurrence of such viruses is clearly a problem when cessation of vaccination is considered and was a major factor in the decision to remove the type 2 component of OPV. A second issue is the occasional case of long-term excretion of polioviruses by individuals deficient in humoral immunity. The viruses are termed immunodeficient vaccine-derived polioviruses (IVDPVs). Probably only a few percent of these hypogammaglobulinemia individuals will become long-term excreters of virus even if given OPV, and most stop shedding virus spontaneously after a period, which may be 2 or 3 years. Occasionally virus excretion may continue for much longer; in one instance isolates were available for 19 years, and the sequence data and medical history of the patient suggest that virus has been shed for well over 30 years. The patient remains entirely healthy, although the virus is highly virulent in animal models. No treatment has been successfully applied to such individuals, although there are claims for a success with an antiviral compound in one case. It is of interest that the mechanism by which virus is cleared is still not known. An infected individual can be expected to excrete the virus for over 4 weeks and will continue to do so after an immune response is detectable. Immune-deficient patients can stop excreting virus spontaneously with no evidence of an immune response and while termination of excretion correlates in general with a rise in fecal anti-poliovirus IgA levels, this is neither necessary nor sufficient.

Cessation of Vaccination Wild type 1 poliovirus currently circulates only in Pakistan and Afghanistan and wild type 2 and 3 have been eliminated from the entire world. Once the wild virus has been eradicated vaccination in its current form is hard to justify. WHO have produced very stringent guidelines (the Global Action Plan, third edition, or GAP3) on containment of poliovirus as this becomes a major concern as eradication approaches. In 2016 the type 2 component of oral polio vaccine was withdrawn on the grounds that there had been no wild type 2 polio since 1999, the type 2 component interfered with immunization with the other two strains in OPV and the type 2 component was responsible for most cases of vaccine associated poliomyelitis and circulating vaccine derived strains. However, withdrawal was problematic in so far as there were still type 2 cVDPVs, so a fall back policy was needed which took the form of supplies of monovalent type 2 OPV intended to deal with future cVDPV outbreaks should they arise. This strategy failed in so far as outbreaks continued to occur and the programmatic response caused more type 2 cVDPV to be generated. A more radical approach is therefore being adopted in which a new live attenuated type 2 vaccine has been developed that is designed to be more genetically stable than the Sabin strain, based on the understanding gained of the molecular basis of attenuation. Two candidates were developed. Both contain modified domain 5 structures in which base pairs are modified to fine tune the thermodynamic stability to produce the desired degree of attenuation by substituting weaker AU base pairs for GC pairs as mentioned earlier. One candidate also includes a high fidelity RNA polymerase to reduce the generation of mutations and in this candidate the cre was moved to a region to the 50 side of domain 5. This last manipulation means that the attenuating domain 5 structure cannot be replaced by a single recombination event without losing cre and thus the ability to replicate. Overall, the virus should therefore be genetically more stable than the Sabin strain. The other candidate possessed the attenuated domain 5 but was produced by synthesis of the genome and deoptimizing codon usage so that virus production and replication is far less efficient. Both candidates have been evaluated in clinical trials and shown to be immunogenic and genetically stable over the time covered. Vaccine usage based on at least one of the strains will begin in 2021. In addition, OPV usage has stopped in many countries and been replaced by the use of Inactivated Polio Vaccine. This switch has taken place in Europe, the whole of North America and much of central and Southern America as well as in Australia and New Zealand. The production of IPV requires the growth and inactivation of live poliovirus which may be a problem if the virus escapes the manufacturing facility. There are stringent requirements for handling poliovirus but a safer strain would be advisable. A strain designated S19 in which domain 5 has been seriously weakened by base pair exchanges so that virus growth is seriously hampered has been developed. The strain is unable to infect animal models and is genetically very stable making it safe for use. Its use in production is being explored. These developments give hope that cessation of vaccination in its current form can occur safely and that both wild type and vaccine derived polioviruses can be eradicated. The progress will need to be monitored closely.

Further Reading Bodian, D., 1955. Emerging concept of poliomyelitis infection. Science 122, 105–108. Fine, P.E., Carneiro, I.A., 1999. Transmissibility and persistence of oral polio vaccine viruses: Implications for the global poliomyelitis eradication initiative. American Journal of Epidemiology 150, 1001–1021. Hammon, W.D., Coriell, L.L., Wehrle, P.F., et al., 1953. Evaluation of Red Cross gammaglobulin as a prophylactic agent for poliomyelitis. Part 4: Final report of results based on clinical diagnosis. JAMA 151, 1272–1285. Kew, O., Morris-Glasgow, V., Landarverde, M., et al., 2002. Outbreak of poliomyelitis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus. Science 296, 356–359. Minor, P.D., 1992. The molecular biology of poliovaccines. Journal of General Virology 73, 3065–3077.

696

Polioviruses (Picornaviridae)

Minor, P.D., John, A., Ferguson, M., Icenogle, J.P., 1986. Antigenic and molecular evolution of the vaccine strain of type 3 poliovirus during the period of excretion by a primary vaccinee. Journal of General Virology 67, 693–706. Minor, P.D., Nathanson, N., Ahmed, R., Gonzalez-Scarano, F., et al., 1997. Viral Pathogenesis. Philadelphia: Lippincott-Raven Publishers, pp. 555–574. Nkowane, B.U., Wassilak, S.G., Orenstein, W.A., 1987. Vaccine associated paralytic poliomyelitis in the United States: 1973 through 1984. JAMA 257, 1335–1340. Paul, J.R., 1971. A History of Poliomyelitis. New Haven, CT: Yale University Press. Sabin, A.B., 1956. Pathogenesis of poliomyelitis: Reappraisal in the light of new data. Science 123, 1151–1157. Sabin, A.B., Ramos-Alvarez, M., Alvarez-Amesquita, J., et al., 1960. Live orally given poliovirus vaccine: Effects of rapid mass immunization on population under conditions of massive enteric infection with other viruses. JAMA 173, 1521–1526.

Relevant Website http://www.polioeradication.org Global Polio Eradication Initiative: GPEI.

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae) Jianqiang Zhang, Iowa State University, Ames, IA, United States Alan T Loynachan, University of Kentucky, Lexington, KY, United States Gregory W Stevenson and Jeffrey J Zimmerman, Iowa State University, Ames, IA, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Chronic persistent infection An infection in which the virus continues to replicate in the host, despite an adaptive immune response. In contrast to “latent persistent infection”, chronic infection is eventually cleared by the immune system. Clinical signs Objective evidence of disease perceptible to an observer. In contrast, “symptoms” are subjective sensations described by the affected person. Half-life The time required to inactive one-half of a viral population. Half-life is situation-specific, i.e., dependent on the environment (temperature, humidity, pH, light, etc.). Pathogenicity The quality or state of being able to produce disease. Sometimes incorrectly used as a synonym for virulence.

Sensitivity, analytical For any assay, the relationship between the concentration of the target in the sample and the probability of detecting the target. It is sometimes (incorrectly) described as the limit of detection. Sensitivity, diagnostic For any assay, the probability of a positive test result when testing a true-positive specimen. Specificity, analytical The degree to which an assay is selective for the target of interest. Assays that lack analytical specificity will cross-react with other targets (false positive). Specificity, diagnostic For any assay, the probability of a negative test result when testing a true-negative specimen. Virulence A measure of pathogenicity. For example, if mortality is the criterion, virulence can be expressed as the “case-fatality rate.”

Classification Porcine reproductive and respiratory syndrome (PRRS) was first reported in the late 1980s in the U.S. and early 1990s in Europe. The etiologic agent, PRRS virus (PRRSV), was identified in Europe (Type 1, PRRSV-1) and North America (Type 2, PRRSV-2) in 1991. Equine arteritis virus (EAV) was first isolated from the lung tissue of an aborted fetus during an epidemic of abortions and respiratory disease in pregnant mares in Bucyrus, Ohio, USA, in 1953. The nomenclature is in transition, but the current taxonomic classifications of PRRSV and EAV are given in Table 1. Both are members of the family Arteriviridae in the suborder Arnidovirineae within the order Nidovirales. However, PRRSV-1 (species name Betaarterivirus suid 1) and PRRSV-2 (species name Betaarterivirus suid 2) respectively belong to the subgenera Eurpobartevirus and Ampobartevirus in the genus Betaarterivirus and the subfamily Variarterivirinae. In contrast, EAV (species name Alphaarterivirus equid) belongs to the genus Alphaarterivirus in the subfamily Equarterivirinae. PRRSV strains are highly genetically diverse, especially in open reading frame (ORF) 5 and non-structural protein (nsp) 2 regions. Based on ORF5, PRRSV-1 and PRRSV-2 vary by B44% in nucleotide sequence; within PRRSV-1 and PRRSV-2, nucleotide sequence variations are approximately 30% and 21%, respectively. Phylogenetic analyses of ORF5 sequences classified PRRSV-1 viruses into 4 subtypes and PRRSV-2 viruses into 9 lineages. EAV field strains are also diverse, although to a lesser extent than PRRSV. EAV field isolates from North America and Europe differ approximately 15% in nucleotide sequences, with the major differences located in the nsp2, ORF3, and ORF5 regions. Phylogenetic analyses of ORF5 sequences classified EAV isolates into North American and European clades, with the latter further divided into European subgroup-1 (EU-1) and European subgroup-2 (EU-2).

Table 1

Taxonomy of EAV and PRRSV

Order

Suborder

Family

Subfamily

Genus

Nidovirales Arnidovirineae Arteriviridae Equarterivirinae Alphaarterivirus Variarterivirinae Betaarterivirus

Subgenus

Species

Virus name

Alphaarterivirus Equine arteritis virus (EAV) equid Eurpobartevirus Betaarterivirus Porcine reproductive and respiratory suid 1 syndrome virus 1 (PRRSV-1) Ampobartevirus Betaarterivirus Porcine reproductive and respiratory suid 2 syndrome virus 2 (PRRSV-2)

Note: Based on the International Committee on Taxonomy of Viruses report released in 2018.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20926-6

697

698

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

Fig. 1 Electron micrograph of arterivirus particles and schematic diagram of arterivirus virion. (A) Transmission electron micrograph of extracellular porcine reproductive and respiratory syndrome virus (PRRSV) particles. Bar ¼ 25 nm. (B) Schematic diagram of equine arteritis virus (EAV) virion. Image adapted from (A) Snijder, E.J., Meulenberg, J.J.M., 1998. The molecular biology of arteriviruses. Journal of General Virology 79, 961–979 with permission. (B) Balasuriya, U.B.R., Go, Y.Y., MacLachlan, N.J., 2013. Equine arteritis virus. Veterinary Microbiology 167, 93–122 with permission.

Virion Structure Both PRRSV and EAV virions are spherical, with a diameter of 50–60 nm and inapparent surface projections (Fig. 1(A)). The virion includes a nucleocapsid core composed of an infectious RNA genome and nucleocapsid (N) protein, surrounded by a lipidcontaining envelope with 7 viral-encoded proteins (Fig. 1(B)). It has been assumed that the arteriviruses have an icosahedral nucleocapsid core, but recent cryo-electron tomography studies suggest that the PRRSV nucleocapsid core is pleomorphic. The N protein interacts with the viral RNA during virus assembly and is the most abundant structural protein in arterivirus virions. The non-glycosylated membrane (M) protein and the glycosylated GP5 protein are major envelope proteins that form disulfidelinked heterodimers. The small non-glycosylated envelope protein (E), glycosylated GP2, GP3, GP4 proteins, and the newly discovered ORF5a protein are minor envelope proteins. The GP2/GP3/GP4 glycoproteins are covalently associated and form a heterotrimeric complex on the virion surface. The buoyant density of infectious arterivirus particles is B1.18–1.22 g/cm3 in cesium chloride and B1.13–1.17 g/cm3 in sucrose. Based on a study of 4 PRRSV-2 isolates, virus half-life was found to be highly temperature dependent: 155 h at 4°C (39°F), 84.5 h at 10°C (50°F), 27.4 h at 20°C (68°F), and 1.6 h at 30°C (86°F). Half-life estimates are not available for EAV, but infectious virus was detected up to 75 days at 4°C (39°F), 48–72 h at 37°C (99°F), and 20–30 min at 56°C (133°F). Both PRRSV and EAV are stable at −70°C (−94°F) for years without significant loss of infectivity. PRRSV is stable at neutral pH but is inactivated by low and high pH (o6 or 47.5); this may apply to EAV as well, although it has not been experimentally investigated.

Genome Organization PRRSV and EAV have similar genomic organization, i.e., positive-sense, single-stranded, 3′-polyadenylated RNA (B15 kb for PRRSV and B12.7 kb for EAV), presumably with a cap structure at the 5′ end. The genome includes 5′ untranslated region (UTR), 10 ORFs (ORFs1a, 1b, 2a, 2b, 3, 4, 5a, 5b, 6 and 7), and 3′ UTR (Table 2; Fig. 2). Based on EAV studies, the 5′ UTR is involved in translation, replication and transcription. An RNA hairpin in EAV 3′ UTR is required for RNA synthesis and is essential for a pseudoknot interaction with a hairpin in the N protein gene. ORF1a and ORF1b occupy approximately 5′ three-quarters of the genome. ORF1a is translated into replicase polyprotein (pp) 1a, which is further processed by viral encoded proteinases (papain-like proteinases and serine proteinase) into nsps 1–8 (Table 2; Fig. 2). ORF1b translation involves a −1 programmed ribosomal frameshifting (PRF) which extends pp1a into pp1ab. The ORF1bencoded portion of pp1ab is further cleaved by viral-encoded serine proteinase into nsps 9–12 (Table 2 and Fig. 2). Some important functional domains conserved in arterivirus ORF1b-encoded protein include the RNA-dependent RNA polymerase in nsp9, putative multinuclear zinc-binding domain and RNA helicase in nsp10, and the NendoU endoribonuclease domain in nsp11 (Fig. 2). Recently, a conserved small ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame was identified in PRRSV, but not in EAV genome, and translation of this small ORF involves a −2 PRF mechanism. The 3′ one-quarter of the genome includes ORFs 2a, 2b, 3, 4, 5a, 5b, 6, and 7, which respectively encode structural proteins E (EAV) or GP2 (PRRSV), GP2 (EAV) or E (PRRSV), GP3, GP4, ORF5a, GP5, M, and N (Table 2; Fig. 2). These structural proteins are not directly translated from genomic RNA; rather, they are translated from subgenomic (sg) mRNAs (see Life Cycle).

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

Table 2

Nonstructural and structural proteins of EAV and PRRSV

ORF

Protein

PRRSV-1b

PRRSV-2c

1–224 225–5408

1–221 222–7412

1–189 190–7701

nsp1

5405–9751 225–5405, 5405–9751 255–1004

7406–11785 222–7406, 7406–11785 222–1376

7695–12071 190–7695, 7695–12071 190–1338

nsp2

1005–2717

1377–4610

1339–4926

nsp3

2718–3416

4611–5300

4927–5616

nsp4

3417–4028

5301–5909

5617–6228

nsp5

4029–4514

5910–6419

6229–6738

nsp6

4515–4580

6420–6467

6739–6786

nsp7

4581–5255

6468–7274

6787–7563

nsp8

5256–5405

7275–7409

7564–7698

nsp9 nsp10

5256–5405, 5405–7333 7334–8734

7275–7406, 7406–9328 9329–10654

7564–7695, 7695–9617 9618–10940

nsp11

8735–9391

10,655–11,326

10,941–11,609

nsp12

9392–9748

11,327–11,782

11,610–12,068

E (EAV); GP2 (PRRSV) GP2 (EAV); E (PRRSV) GP3 GP4 ORF5a GP5 M N

9751–9954

11,796–12,545

9824–10507 10,306–10,797 10,700–11,158 11,112–11,291 11,146–11,913 11,901–12,389 12,313–12,645 12,646–12,704

pp1a

ORF1b ORF1a/b

– pp1ab

ORF2b ORF3 ORF4 ORF5a ORF5b ORF6 ORF7 3′ UTR

Predicted protein

EAVa 5′ UTR ORF1a

ORF2a

Predicted nucleotide position

699

EAVa

PRRSV-1b

PRRSV-2c

Met1-Asn1727 (1727 aa)

Met1-Cys2396 (2396 aa)

Met1-Cys2503 (2503 aa)

12,073–12,843

Met1-Val3175 (3175 aa) Met1-Gly260 (260 aa) Gly261-Gly831 (571 aa) Gly832-Glu1064 (233 aa) Gly1065-Glu1268 (204 aa) Ser1269-Glu1430 (162 aa) Gly1431-Glu1452 (22 aa) Ser1453-Glu1677 (225 aa) Gly1678-Asn1727 (50 aa) Gly1678-Glu2370 (693 aa) Ser2371-Gln2837 (467 aa) Ser2838-Glu3056 (219 aa) Gly3057-Val3175 (119 aa) E (67 aa)

Met1-Pro3854 (3854 aa) Met1-Gly385 (385 aa) Ala386-Gly1463 (1078 aa) Ala1464-Glu1693 (230 aa) Gly1694-Glu1896 (203 aa) Gly1897-Glu2066 (170 aa) Gly2067-Glu2082 (16 aa) Ser2083-Glu2351 (269 aa) Ala2352-Cys2396 (45 aa) Ala2352-Glu3036 (685 aa) Gly3037-Glu3478 (442 aa) Gly3479-Glu3702 (224 aa) Gly3703-Pro3854 (152 aa) GP2 (249 aa)

Met1-Asn3960 (3960 aa) Met1-Gly383 (383 aa) Ala384-Gly1579 (1196 aa) Gly1580-Glu1809 (230 aa) Gly1810-Glu2013 (204 aa) Gly2014-Glu2183 (170 aa) Gly2184-Glu2199 (16 aa) Ser2200-Glu2458 (259 aa) Ala2459-Cys2503 (45 aa) Ala2459-Glu3143 (685 aa) Gly3144-Glu3584 (441 aa) Gly3585-Glu3807 (223 aa) Gly3808-Asn3960 (153 aa) GP2 (256 aa)

11,801–12,013

12,078–12,299

GP2 (227 aa)

E (70 aa)

E (73 aa)

12,404–13,201 12,946–13,497 13,499–13,630 13,494–14,099 14,087–14,608 14,598–14,984 14,985–15,098

12,696–13,460 13,241–13,777 13,778–13,933 13,788–14,390 14,375–14,899 14,889–15,260 15,261–15,411

163 aa 152 aa 59 aa 255 aa 162 aa 110 aa

265 aa 183 aa 43 aa 201 aa 173 aa 128 aa

254 aa 178 aa 51 aa 200 aa 174 aa 123 aa

a

Based on EAV VBS strain (ATCC VR-796) (GenBank accession number # DQ846750). Based on PRRSV-1 strain Lelystad (GenBank accession number # M96262). c Based on PRRSV-2 strain VR2332 (GenBank accession number # PRU87392). b

The PRRSV and EAV N proteins are phosphorylated and the N-terminal domain of the N protein is presumed to interact with the genomic RNA during nucleocapsid assembly. M and GP5 are two major envelope proteins. The nonglycosylated M protein lacks an N-terminal signal sequence and is presumed to span the membrane three times. M protein forms heterodimers with GP5 protein, probably by the formation of a disulfide bridge between a Cys residue in the M ectodomain and a Cys residue in the GP5 ectodomain. The glycosylated GP5 protein contains an N-terminal signal sequence, a hydrophobic region spanning the membrane three times, and a cytoplasmic domain. The PRRSV and EAV GP5 ectodomain is an important target for neutralizing antibodies. E, GP2, GP3, GP4, and ORF5a protein are minor envelope proteins. The disulfide-linked GP2-GP3-GP4 trimers have been identified in both PRRSV and EAV; in addition, the disulfide-linked GP2-GP4 dimers are found in EAV. All major (N, GP5 and M) and minor (E, GP2, GP3, and GP4) structural proteins are required for the production of infectious progeny virus, as demonstrated by individually knocking out the expression of each structural protein. Knocking out ORF5a protein expression led to the production of infectious progeny virus with a small plaque phenotype and significantly reduced virus

700

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

Fig. 2 Genome organization of equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV). In open reading frame (ORF) 1a, the papain-like proteinase domains (P) in the nsp1-nsp2 region and their predicted cleavage sites are indicated with blue triangles. In nsp4, the main viral protease (serine proteinase, S) and its predicted cleavage sites are indicated with red triangles. “TM” indicates the putative transmembrane domains in nsp2, nsp3, and nsp5 of the ORF1a-encoded polyprotein. In ORF1b, the four most conserved replicase domains are indicated: the RNA-dependent RNA polymerase (R) in nsp9, putative multinuclear zinc-binding domain (Z) and RNA helicase (H) in nsp10, and the NendoU endoribonuclease domain (N) in nsp11. Minor structural proteins E (encoded by ORF2a in EAV and ORF2b in PRRSV), GP2 (ORF2b in EAV and ORF2a in PRRSV), GP3 (ORF3), GP4 (ORF4) and ORF5a (encoded by ORF5a) and major structural proteins GP5 (ORF5), M (ORF6) and N (ORF7) are indicated. Modified with permission from Snijder, E.J., Kikkert, M., 2013. Arteriviruses. In: Knipe, D.M., Howley, P.M., (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Lippincott Williams and Wilkins, pp. 859–879.

titer. Knocking out minor structural proteins (E, GP2, GP3, and GP4) produced non-infectious subviral particles consisting of N, GP5, M, and the genomic RNA, suggesting that major structural proteins N, GP5, and M are essential and sufficient for forming virion scaffold (virus-like particle), although such particles lack infectivity.

Life Cycle The virus infection cycle starts with virus attachment and entry into host cells. This process involves the interaction between the viral proteins and receptor(s) on the host cells. In pigs, PRRSV primarily infects monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells. Continuous cell lines MARC-145 and CL2621, both derived from African green monkey kidney MA-104 cells, are also permissive to PRRSV infection. The scavenger receptor CD163, expressed on both porcine macrophages and MARC-145 cells, can alone mediate virus attachment and entry (including internalization and uncoating). Sialoadhesin (Sn, or CD169), which is expressed on macrophages, but absent on MARC-145 cells, can enhance virus internalization, but cannot mediate uncoating. There are reports that glycosaminoglycans (heparan sulfate) on the cell surface may interact with sialic acids on the PRRSV virion surface and are implicated in the initial binding step, but heparan sulfate is not absolutely required for PRRSV entry. In horses, EAV infects equine endothelial cells, monocytes, macrophages, and a small subpopulation of CD3+ T cells. In vitro, EAV can infect a broad range of continuous cell lines from various host species, including baby hamster kidney (BHK-21), African green monkey kidney (Vero, MARC-145), rabbit kidney (RK-13), and mouse C2C12 cells. Recent studies suggest that equine CXCL16 (EqCXCL16) protein, encoded by the CXCL16 gene located on equine chromosome 11, acts as a receptor for EAV entry into some cell populations in horses. However, EAV may also use other as-yet-unidentified molecule(s) for entry into equine cells. That is, EqCXCL16 protein is not expressed on non-equine host cell lines permissive to EAV infection, thus it remains to be determined if CXCL16 orthologue proteins are present on these cell lines and act as receptors for EAV or if EAV uses other as-yetunidentified receptors to infect these cell lines. The PRRSV and EAV GP5 and M proteins were initially hypothesized as viral ligands for interacting with cell receptors. However, studies with chimeric viruses indicated that GP5 and M ectodomains are not the determinants for PRRSV and EAV cell tropism. Rather, PRRSV/EAV chimeric virus studies suggested that the minor envelope proteins GP2, GP3, and GP4, in combination with the E protein, may be responsible for interacting with the cellular receptor(s). In fact, some studies suggested that PRRSV GP4 and GP2 proteins interact with the receptor CD163. As shown in Fig. 3, after binding to receptor(s) on host cells, PRRSV and EAV are postulated to enter through standard clathrin-dependent receptor-mediated endocytosis, with the viral nucleocapsid released into the cytosol following endosome acidification and membrane fusion. Positive-sense genomic RNA is directly translated into two large replicase polyproteins, pp1a and pp1ab. Posttranslational processing of pp1a and pp1ab involves a complex proteolytic cleavage cascade mediated by three (EAV) or four (PRRSV) ORF1a-encoded proteinases (EAV: papain-like proteinases in nsp1β, nsp2, and serine proteinase in nsp4; PRRSV: papain-like proteinases in nsp1α, nsp1β, nsp2, and serine proteinase in nsp4) (Fig. 2). Viral nonstructural proteins then assemble into a replication and transcription complex (RTC) associated with vesicular double-membrane (double membrane vesicles, DMV) derived from the endoplasmic reticulum. The RTC directs synthesis of both genome-length and subgenome

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

701

Fig. 3 Overview of arterivirus infection cycle. Following entry by receptor-mediated endocytosis and release of the genome into the cytosol, genome translation yields the replicase polyproteins pp1a and pp1ab which are cleaved by multiple internal proteases into nonstructural proteins. The viral nonstructural proteins assemble into a replication and transcription complex (RTC) on double membrane vesicles (DMV) to engage in minus-strand RNA synthesis. Both genome-length and subgenome (sg)-length minus strands are produced, with the latter serving as templates for the synthesis of sg mRNAs required to express the structural protein genes residing in the 3′-proximal quarter of the genome. Novel genomes are packaged into nucleocapsids that become enveloped by budding from smooth intracellular membranes, after which the new virions leave the cell using the exocytic pathway. Modified with permission from Snijder, E.J., Kikkert, M., 2013. Arteriviruses. In: Knipe, D.M., Howley, P.M., (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Lippincott Williams and Wilkins, pp. 859–879.

(sg)-length minus strands. The genome-length minus strand serves as a template for replication of genomic RNA. Nested sets of sglength minus-strand RNAs serve as templates for generating positive-strand sg mRNAs from which structural proteins are translated. Novel genomes are encapsidated by N protein to form nucleocapsids that become enveloped when budding into the lumen of the smooth endoplasmic reticulum and/or Golgi complex. The formation of GP5/M heterodimer is a primary determinant of virus budding. After budding, new virions accumulate in intracellular vesicles and are then transported to the plasma membrane where the progeny virus is released.

Epidemiology Pigs (Sus scrofa) and collared peccary (Pecari tajacu) are susceptible to PRRSV. The susceptibility of other species within the superfamily Suoidea (families Suidae and Tayassuidae) is likely, but it has not been determined. Members of family Equidae, i.e., horses, donkeys, zebras, as well as hybrid crosses (mules and hinnies), are susceptible to EAV. Neither virus is infectious for humans, i.e., zoonotic, and therefore do not affect public health. Both viruses have a global distribution, with some exceptions. PRRSV is not present in Oceania, parts of South America (Argentina, Brazil), parts of Scandinavia (Finland, Norway, Sweden), and Switzerland. EAV has not been reported in Japan, Iceland, and New Zealand. Both PRRSV- and EAV-infected animals shed virus in oronasal secretions, urine, semen, and perhaps briefly in feces. Shedding in boar (PRRSV) and stallion (EAV) semen presents the risk of long distance virus transmission to susceptible females through the use of artificial insemination, as has been documented in the literature. PRRSV transmission can occur by intranasal, intramuscular, oral, intrauterine, and vaginal routes of exposure. Indirect transmission occurs by exposure to contaminated fomites (equipment, instruments, clothing, medications) or via aerosols. Parenteral

702

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

exposure (breaks in the skin) is a particularly efficient mode of transmission. Because PRRSV is present in oropharyngeal fluids for weeks, the dominance behaviors associated with mixing pigs can result in rapid transmission via bites, scrapes, and abrasions. Aerosol transmission is dependent on the presence of meteorological conditions able to support infectious virus and compatible with the movement of virus in space, e.g., low temperature, moderate levels of relative humidity, a rising barometric pressure, low sunlight levels, and slow, directional winds. EAV transmission is primarily via respiratory and venereal routes. Aerosol transmission occurs via infectious droplet particles originating either from the respiratory tract or from the environment, e.g., urine splashes. Extensive EAV transmission can occur among groups of horses brought together for sales, racetracks, fairs, etc. Vertical transmission, i.e., transplacental transmission to fetuses, is a feature of both viruses when females become infected in late gestation. PRRSV-naïve females infected in late gestation may farrow virus-infected piglets and will shed virus in mammary secretions throughout lactation. Likewise, EAV-susceptible mares infected in late gestation may produce congenitally-infected foals. Both viruses can produce chronic persistent infections. Infectious PRRSV has been detected in individual animals at 175 days post exposure (DPE). The mechanism(s) by which the PRRSV persists in the face of an active immune response is unknown, but does not depend on the age of the pig at the time of exposure and does not involve evasion of immunity through in vivo viral mutation. Similarly, EAV carrier stallions may shed infectious levels of EAV in semen for weeks to years (see EAV Pathogenesis).

Clinical Features PRRSV Pigs of all ages are susceptible to PRRSV infection. Clinical signs are highly variable, but may include pyrexia (39–41°C), anorexia, lethargy, dyspnea, and variable hyperemia or cyanosis of skin on the extremities. Affected pigs may be leukopenic, anemic, and thrombocytopenic during the acute stage of the infection. The expression of clinical disease reflects the virulence of the PRRSV initiating the infection in combination with various co-factors, e.g., pig immune status, concurrent viral or bacterial co-infections, and herd management practices. Frequent recombination and mutation of PRRS viruses has led to the emergence of variants of atypically high virulence (ATV), including PRRSV-2 variants in North America, PRRSV-1 variants in Europe, and PRRSV-2 variants in Asia (“highly pathogenic” or HP-PRRSV). Some so-called “neurotropic” viruses may induce nervous signs, including head tilt, ataxia, lateral recumbency, and/or convulsions. Mortality ranges from 0% to 10% depending on variant virulence and the presence of co-factors, but may be higher with ATV viruses. Infection in breeding-age boars may be inapparent or result in reduced libido and semen quality. Shedding of virus in semen can result in PRRSV transmission to sows through natural service or artificial insemination. Infection in pregnant females may result in transmission of PRRSV to the developing fetuses. Due to individual fetal placentation, some or all of the fetuses will be infected. Abortion rates may be 10%–50% (“abortion storms”). Aborted litters contain a mix of normal pigs, weak and small pigs, stillborn pigs, and partially or completely mummified pigs. If brought to term, litters may include viable, but PRRSV-infected, piglets. Transplacental transmission is most efficient in the third trimester, but some ATV PRRS viruses are able to cross the placenta with moderate efficiency during the first and second trimesters, resulting in aborted litters composed of smaller mummified fetuses, or irregular returns to heat without observed pregnancy loss. In commercial herds, clinical signs range from subclinical to severe respiratory and reproductive diseases. In acute outbreaks in PRRSV-naïve herds (populations without prior immunity), clinical signs may be observed in all ages. More typically, swine herds are endemically infected with one or more PRRSV variants, in which case clinical signs are observed periodically in susceptible members of the population, i.e., weaned pigs (nursery-grower pigs) with declining levels of maternal immunity and replacement breeding stock (gilts or boars) that lack adequate immunity against the PRRS viruses circulating in the herd.

EAV Clinical signs associated with EAV infection vary by strain virulence, animal age, route of infection, host susceptibility, and environmental conditions. Only one EAV serotype is recognized, but field isolates can be separated into those that produce subclinical infection (EAV KY63, EAV PA76, EAV KY77, and EAV CA95), mild disease (EAV SWZ64, EAV AUT68, EAV IL94, and EAV CA97), or moderate-to-severe disease (EAV KY84, EAV AZ87, EAV IL93, EAV PA96, and Bucyrus strain). The vast majority of adult horses are subclinically infected, but highly virulent strains can rarely result in death of adult animals. Pyrexia (up to 41°C) and leucopenia are the most common clinical signs. Less commonly, horses will develop depression, anorexia, nasal congestion and discharge, lymphadenopathy, mild-to-severe dyspnea, edema of the limbs, ventrum, scrotum, mammary gland, periorbital regions, and intermandibular space, conjunctivitis, corneal opacity, diarrhea, stiffness, icterus, and oral and cutaneous papules. Infection in stallions can result in subfertility for 6–7 weeks post infection due to diminished libido and decreased semen quality. Genetically susceptible stallions can become persistently infected and continuously shed virus in semen. Acutely infected pregnant mares are at increased risk of abortion and 10%–71% will abort during the second to tenth month of gestation. Neonatal infection can present as severe dyspnea due to interstitial pneumonia and older foals are susceptible to progressive pneumoenteric syndrome. Most adult horses spontaneously recover, but young and debilitated animals may develop significant disease and die.

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

703

Pathogenesis and Immunity PRRSV Pathogenesis PRRSV infection consists of three stages: acute infection, persistence, and elimination. Acute infection is characterized by extensive viral replication in lung and lymphoid tissues and viremia (21–35 days). Persistence is marked by the resolution of viremia and progressively less PRRSV replication in lymph nodes and tonsils. Elimination begins when viral shedding ceases and is complete when virus is cleared from all tissues. The entire course of the infection can be ≥175 days and the detection of carrier animals is problematic in the latter stages of the infection. PRRSV replicates in a subset of monocyte-derived cells that display CD163 and sialoadhesin (Sn) receptors, including pulmonary alveolar macrophages and pulmonary intravascular macrophages in the lung, monocyte-derived macrophages in lymphoid tissues, and dendritic cells and monocyte-derived macrophages that reside in perivascular locations in most organs. CD163 is required for PRRSV binding, internalization, and replication; but co-expression of Sn augments virus internalization. Most PRRS viruses replicate in CD163+Sn+ cells, but ATV variants may also replicate in CD163+Sn- cells, a factor that likely contributes to the greater breadth and severity of clinical disease, lesions, and mortality. PRRSV-infected macrophages are killed by necrotic or apoptotic mechanisms, but the majority of cell death in PRRSV-infected tissues is due to indirect killing of uninfected cells (bystander apoptosis). In the lung, cells killed by bystander apoptosis are mostly macrophages, with fewer lymphocytes and alveolar pneumocytes. In lymph nodes and thymus, cells killed by bystander apoptosis are mostly lymphocytes, with fewer macrophages. ATV PRRSV variants induce higher levels of bystander apoptosis than less virulent variants. The reduction in the number, phagocytic capacity, and bactericidal capacity of pulmonary alveolar macrophages and pulmonary intravascular macrophages is thought to be responsible for an increased incidence of septicemia in PRRSV-infected pigs (Streptococcus suis and others) and to opportunistic bacterial bronchopneumonia. PRRSV infection alters innate immunity and inflammatory/immuno-regulatory cytokines. Secretion of pro-inflammatory cytokines from PRRSV-infected macrophages, including TNF-alpha, IL-1, and IL-6, promote the influx and activation of leukocytes, increased microvascular permeability, and induction of systemic effects, such as pyrexia, anorexia, and lethargy. Even higher levels of these and other pro-inflammatory cytokines in HP-PRRS viruses are suggested as a cause of death similar to “macrophage-activation-syndrome” or “cytokine-storms” in humans. Reproductive failure usually occurs in females infected during the third trimester of gestation. During the first two trimesters, CD163+Sn- macrophages predominate in the placenta, but in the third trimester the number of highly permissive CD163+Sn+ macrophages continually increases. Thus, in the third trimester, the virus is able to efficiently transit the maternal-fetal-interface and encounter PRRSV-permissive macrophages in the fetal placenta. In acutely-infected gestating females, PRRSV is present in the maternal endometrium, primarily in CD163+Sn+ macrophages. These infected cells undergo direct apoptosis and also induce bystander apoptosis in both the maternal endometrium and interfacing fetal placenta, including endometrial epithelial cells and placental trophoblasts. This results in the direct apposition of PRRSV-infected, macrophage-rich, endometrial lamina propria and fetal placental stroma. At the maternal-fetal-interface for a particular fetus, the degree of direct and bystander apoptosis is directly proportional to the number of PRRSV-infected macrophages in the maternal endometrium, the concentration of PRRSV in the fetus’ thymus, and the risk for fetal death. PRRSV replicates to its highest concentration in fetal thymus, tonsils, and lymph nodes, thereby contributing to fetal death, or if pregnancy continues to term, the birth of PRRSV-infected piglets. PRRSV ATV variants induce higher levels of apoptosis at the maternal-fetal-interface and in fetal tissues, and are able to replicate in CD163+Snmacrophages; features that explain, at least in part, the higher rates of abortion associated with ATVs across a broader range of gestational ages.

PRRSV Immunity PRRSV-induced alterations of immuno-regulatory cytokines and in the numbers of various lymphocyte subsets cause a prolonged delay in the implementation of an effective adaptive immune response. Regardless, PRRSV infection induces an immune response that slowly eliminates the virus and establishes memory that may (or may not) protect against other PRRS viruses. Non-neutralizing antibodies against PRRSV structural and nonstructural proteins appear 7–10 DPE. Neutralizing antibodies appear 21–28 DPE, i.e., concurrently with the resolution of viremia. Given their role in cell tropism and interactions with cell receptors, it has been hypothesized that neutralizing antibodies target glycoproteins GP2, GP3, and GP4, but data confirming this hypothesis have not yet been produced. Despite the fact that their role in controlling PRRSV is not clearly established, neutralizing antibodies are the best predictor of the level and duration of viremia. Interferon-γ ELISPOT has demonstrated a consistent T-cell response to PRRSV, but its significance is uncertain. Overall, the protracted development of neutralizing antibody is credited for the prolonged PRRS viremia and the delayed cell-mediated immune response for the persistence of PRRSV in lymphoid tissues.

EAV Pathogenesis Virus transmission can occur by either venereal or respiratory routes of exposure. Within 24 h of respiratory exposure, EAV replicates in alveolar macrophages and bronchiolar epithelial cells. The virus localizes in tonsils and regional lymph nodes within 48 h and then rapidly disseminates throughout the host by cell-associated viremia. Viremia lasts 3–19 DPE and nasal shedding

704

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

occurs for 2–21 DPE. Once viremic, the virus replicates in macrophages, endothelial cells, and vascular smooth muscle myocytes. The combination of direct virus-mediated injury to endothelial cells and leukocyte infiltration results in vascular necrosis and panvasculitis. The resultant vascular injury and increased permeability contribute to perivascular hemorrhage and edema that grossly manifests as the characteristically observed clinical sign. Virus clearance typically occurs by 28 DPE in concurrence with a rising serum neutralizing antibody response. Strain virulence and host genetic factors greatly influence the host proinflammatory immune response and disease severity. EAV virulence determinants are complex and involve both nonstructural and envelope proteins. EAV exhibits tropism for CD14+ monocytes through interactions with GP2, GP3, GP4, GP5, and M envelope proteins and CD3+ lymphocytes through interactions with GP2, GP4, GP5, and M envelope proteins. Host genetic factors can contribute to the clinical outcome. Horses can be separated into EAV-susceptible or -resistant phenotypes based on the in vitro susceptibility of their CD3+ lymphocytes. Based on genome-wide association studies, the susceptible phenotype was associated with a common, genetically dominant haplotype located on equine chromosome 11. Further investigation identified two allelic variants of chemokine ligand 16 (CXCL16) with strong associations to the susceptible and resistant phenotypes. The susceptible CXCL16 phenotype has EAV receptor activity and the resistant phenotype lacks receptor activity. Susceptible vs resistant phenotype horses differ in proinflammatory (tumor necrosis factor α, interleukin (IL)-1β, IL-6, and IL-8) and immunomodulatory cytokine mRNA expression. In horses possessing the resistant phenotype, clinical signs are more severe, but clearance of the virus is rapid. Stallions exhibiting the susceptible phenotype are more prone to develop persistent infection. The CXCL16 phenotypes vary by breed and correlate to observed variations in breed EAV seroprevalence. Persistently infected carrier stallions serve as the natural reservoir of EAV. Persistent infection is testosterone dependent and occurs in 10%–70% of infected stallions and sexually mature colts. The susceptible CXCL16 CD3+ T lymphocyte phenotype is at higher risk for developing the EAV carrier state. Carrier stallions may never eliminate infection or may eventually clear infection after weeks, months, or years. In persistently infected stallions, the virus localizes to the reproductive tract where it primarily resides in vimentin+ stromal cells and lymphocytes in the ampullae of the vas deferens and to a lesser degree in other accessory sex organs. Microscopic lesions in persistently infected stallions consist of mild-to-moderate lymphoplasmacytic inflammation in the accessory sex organs. Long-term carrier stallions show no clinical signs of infection and have no alterations in semen quality. Carrier animals retain high serum neutralizing antibody levels and have significant local mucosal antibodies, but EAV persists and is continually shed into semen. EAV immune evasion mechanisms are suspected to play a role. Pregnant mares vertically transmit EAV to the fetoplacental unit. Within the fetus and placenta, the virus replicates in a wide array of cell lineages, but rarely results in fetoplacental pathology. Fetal pathology, when present, consists of necrotizing vasculitis, tissue hemorrhage and edema, and effusion. Abortion is hypothesized to occur as a result of myometrial vasculitis and subsequent placental hypoxia and detachment, or by direct viral replication within the fetus resulting in fetal stress and expulsion.

EAV Immunity EAV infection induces protective immunity. Complement-fixing and neutralizing antibodies develop 1–2 weeks following infection. Complement-fixing antibodies remain for 8 months, but neutralizing antibodies remain for years. EAV-infected horses develop immune responses to GP5, N, and M structural proteins and non-structural proteins 2, 4, 5, and 12. Foals acquire passive immunity from immune mares, and colostrum-associated virus neutralizing antibodies remain for 2–7 months after birth.

Diagnosis PRRSV Acute PRRS is suggested by clinical signs and lesions, but a definitive diagnosis requires laboratory confirmation based on evaluation of specimens, e.g., lung, lymphoid tissues, bronchoalveolar lavage fluids, serum, semen, and/or oral fluids. Preferred specimens in cases of reproductive failure include fetal lung and thymus, serum collected from dams shortly after abortion, and “processing fluid” from live-born littermates or cohorts. Processing fluid is a composite sample consisting of the blood-serum exudate recovered from tissues collected at the time of castration and/or tail-docking of neonatal piglets. Lesions in lungs and lymph nodes include lymphoplasmacytic bronchointerstitial pneumonia with necrotic intra-alveolar macrophages and variable hyperplasia of type II pneumocytes. Lymph nodes are enlarged and edematous with variable necrosis of lymphoid follicles. In addition to lesions in lungs and lymph nodes, sows with reproductive signs often have lymphoplasmacytic perivascular endometritis. Lesions are inconsistent, mild, and observed in a minority of PRRSV-infected fetuses. PRRSV antigens can be visualized in frozen tissue sections by fluorescent antibody staining or in paraffin-embedded tissue sections prepared from tissues fixed in 10% neutral-buffered formalin by immunohistochemical staining. Monoclonal antibodies specific for the highly conserved nucleocapsid protein are typically used. Both fluorescent antibody and immunohistochemical staining are less analytically sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Virus isolation in cell culture is not routinely used for diagnosis, but may be performed when viable PRRSV is needed for further analyses. PRRSV is heat-liable, so samples should be immediately chilled (4°C) and maintained on ice until tested. The preferred antemortem sample is serum, but lung and lymphoid tissue samples collected at necropsy may also be used. PRRSV is fastidious and inoculation of several cell types maximizes the chances of isolation, e.g., primary porcine alveolar macrophages,

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

705

African monkey kidney cells (MA-104, CL-2621, MARC-145), and an immortalized pulmonary alveolar macrophage cell line, ZMAC-1. Most commonly, RT-PCR is used to confirm PRRSV infection. Reverse transcription loop-mediated isothermal amplification and in situ hybridization assays have been described for detection of PRRSV RNA, but are not widely used. Commercial PRRSV RTPCRs are analytically sensitive, analytically specific, quantitative, and compatible with high-throughput testing. PRRSV is detected in serum 6–48 hours post exposure, with peak virus concentration 4–14 DPE, reaching 1  102–105 TCID50 per ml of serum or gram of lung tissue, but as high as ≥1  108 TCID50 in ATV variants. Notably, RT-PCR testing of serum will not detect carrier animals in the persistence or elimination phases of infection. Individual specimens may be “pooled” by combining roughly equal volumes into one sample for PRRSV rRT-PCR testing. This approach reduces testing costs, but the inclusion of virus-negative specimens will dilute the concentration of virus and increase the probability of false-negative RT-PCR results. Interpretation of PRRSV RNA assays is complicated by the use of modified-live (MLV) PRRSV vaccines. Like field viruses, MLV vaccine viruses replicate in vaccinated animals, or in non-vaccinated animals from vaccinated populations, for a prolonged period. Sequencing of ORF5, the major envelope protein gene, or whole genome sequencing with phylogenetic analysis, are used to differentiate MLV viruses from wild-type viruses and, in epidemiological studies, to describe and compare viruses in farms, production systems, and geographic regions. A variety of tests have been described for the detection of PRRSV antibodies, e.g., virus neutralization, fluorescent microsphere immunoassay, immunoperoxidase monolayer assay, and indirect fluorescent assay. Commercial enzyme-linked immunosorbent assays (ELISAs) for the detection of antibody in serum and/or oral fluid specimens are commonly used because they can provide high diagnostic sensitivity and specificity and are compatible with high throughput testing. Most commercial ELISAs detect IgG antibodies against both PRRSV-1 and PRRSV-2 nucleocapsid antigens, but some are species-specific. A disadvantage of antibody testing is that current assays cannot differentiate between antibodies produced in response to wildtype infection vs MLV vaccination. An advantage of antibody testing is that antibody is detected for ≥6 months and positive results reveal prior PRRSV exposure, even in RT-PCR-negative animals. Periodic population sampling (serum or oral fluids) and antibody testing can provide a cost-effective approach for monitoring PRRSV circulation or vaccination.

EAV EAV infection may be subclinical, variable in clinical presentation, or resemble other infectious and non-infectious equine diseases. Therefore, a definitive diagnosis of EAV requires confirmation based on laboratory testing of clinical specimens. Appropriate specimens from acutely infected horses include nasopharyngeal swabs, nasal washings, conjunctival swabs, and whole blood collected in anticoagulant, e.g., ethylenediaminetetraacetic acid (EDTA) or sodium citrate. Swabs should be placed in a suitable viral transport medium. Fresh and formalin-fixed lung, liver, spleen, thymus, and lymph nodes should be collected from animals that have died and, in addition, both allantochorion and amnion should be collected from aborted fetuses. Fresh samples should be held at 4°C and submitted to the laboratory by overnight delivery. Freezing of samples should generally be avoided. Virus isolation and RT-PCR can be attempted on samples collected from acutely infected horses, aborted fetoplacental units, and semen from carrier stallions. Rabbit kidney (RK-13) cells are optimal for EAV isolation, but Vero cells and equine lung cells can also be used. Virus isolation on RK-13 cells is the World Organization for Animal Health (OIE) standard for the detection of EAV in semen and is the recommended test for international trade. Virus isolation has comparable sensitivity to RT-PCR, but in contrast to RT-PCR, cannot produce rapid results. Various RT-PCR assays (single step, nested, real-time, and insulated isothermal) have been developed to target specific genes (ORFs 1b, 3, 4, 5, 6, and 7), but have varying degrees of analytical sensitivity and specificity. Reagent kits can also differ in analytical sensitivity. A recently developed RT-insulated isothermal PCR assay was shown to have a diagnostic sensitivity of 100% and specificity of 98.33% and overall diagnostic accuracy similar to virus isolation. Frozen or formalin-fixed tissues from deceased animals and fetoplacental units can be used to visualize virus by immunohistochemistry and in situ hybridization. Immunohistochemistry should be conducted using monospecific polyclonal serum against EAV or monoclonal antibody derived from the highly conserved EAV nucleocapsid protein. Conventional RNA in situ hybridization was less sensitive than immunohistochemistry or an in situ hybridization signal amplified system (RNAscope®) using oligonucleotide probes targeting ORFs 5, 6, 7, and the 3′ untranslated region. Serum antibody assays have been developed to indirectly identify horses exposed to EAV and to detect acutely infected animals. The virus neutralization test is the World Organization for Animal Health (OIE) standard for the serologic diagnosis of EAV. Both diagnostically sensitive and highly specific, a diagnosis of acute infection requires the demonstration of a significant increase, e.g., ≥4-fold increase, in neutralizing antibody titers in serum samples taken 3–4 weeks apart from the animal of interest. Complement fixation, indirect fluorescent assay, agar gel immunodiffusion, various ELISAs, and a microsphere immunoassay have also been described, but variation in viral antigen and serum can affect test performance. In stallions, a diagnosis of persistent EAV infection is based on a combination of the virus neutralization test and the detection of EAV in semen. Stallions can be serologically screened for neutralizing antibody because all carrier stallions have virus neutralization titers ≥1:8. Persistence is then confirmed by virus isolation or detection of EAV nucleic acid in the sperm-rich fraction of semen or by test breeding 2 seronegative mares and assessing them for seroconversion within 4 weeks post-breeding.

706

Porcine Reproductive and Respiratory Syndrome Virus and Equine Arteritis Virus (Arteriviridae)

Prevention and Control There are no specific anti-viral treatments for EAV or PRRSV. In clinically-affected animals, supportive veterinary care and the use of anti-inflammatory agents and/or antimicrobials may be warranted. Overall, the focus must be on prevention and control, i.e., stopping the transmission of virus to susceptible animals using appropriate biosecurity measures and reducing the clinical effect of infection through the use of vaccines. Prior to introduction into herds, animals should be quarantined and tested to determine infection status. Thereafter, animals and/or population should be monitored routinely for changes in infection status. Knowledge of true infection status should be an important consideration in management, husbandry, and biosecurity decisions. Shedding of virus by infected animals in body secretions and excretions leads to contamination of the environment with infectious virus, i.e., housing, trucks, trailers, transport equipment, and other fomites. Both PRRSV and EAV can be inactivated by lipid solvents and by common disinfectants and detergents. Vaccines may be useful in managing EAV and PRRSV, but their limitations and recommendations for use should be fully appreciated.

Further Reading Balasuriya, U.B.R., Carossino, M., Timoney, P.J., 2018. Equine viral arteritis: A respiratory and reproductive disease of significant economic importance to the equine industry. Equine Veterinary Education 30, 497–512. Balasuriya, U.B.R., Go, Y.Y., MacLachlan, N.J., 2013. Equine arteritis virus. Veterinary Microbiology 167, 93–122. Sarkar, S., Chelvarajan, L., Go, Y.Y., et al., 2016. Equine arteritis virus uses equine CXCL16 as an entry receptor. Journal of Virology 90, 3366–3384. Snijder, E.J., Kikkert, M., 2013. Arteriviruses. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Lippincott Williams and Wilkins, pp. 859–879. Snijder, E.J., Kikkert, M., Fang, Y., 2013. Arterivirus molecular biology and pathogenesis. Journal of General Virology 94, 2141–2163. Zimmerman, J.J., Dee, S.A., Holtkamp, D., et al., 2019. Porcine reproductive and respiratory syndrome viruses (porcine arteriviruses). In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., et al. (Eds.), Diseases of Swine, eleventh ed. John Wiley and Sons, Inc, pp. 685–708.

Relevant Websites http://www.oie.int/en/standard-setting/terrestrial-manual/access-online/ OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. http://www.oie.int/international-standard-setting/terrestrial-code/access-online/ OIE Terrestrial Animal Health Code.

Prions of Vertebrates Jonathan DF Wadsworth and John Collinge, UCL Institute of Prion Diseases, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved. This is an update of J.D.F. Wadsworth, J. Collinge, Prions of Vertebrates, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B978-0-12-801238-3.02648-9.

Glossary Codon 129 polymorphism There are two common forms of PRNP encoding either methionine or valine at codon 129; a major determinant of genetic susceptibility to and phenotypic expression of prion disease. Conformational selection model A hypothetical model which explains transmission barriers on the basis of overlap of permissible conformations of PrPSc (prion strains) between mammalian species. Although a wide range of mammalian PrPSc conformations may be possible, only a subset will be compatible with each individual PrP primary structure. Molecular strain typing A means of rapidly differentiating prion strains by biochemical differences in PrPSc. Prion The infectious agent causing prion diseases. Prion incubation period The interval between exposure to prions and the development of neurological signs of prion disease; typically months even in rodent models and years to decades in humans. Prion protein (PrP) A glycoprotein encoded by the host genome and expressed in many tissues but especially on the surface of neurons. Prion strain Distinct isolates of prions originally identified and defined by biological characteristics which breed true in inbred mouse lines.

PRNP The human prion protein gene; mouse gene is designated Prnp. Protein-only hypothesis That prions lack a nucleic acid genome, are composed principally or solely of abnormal isomers of PrP (PrPSc), and replicate by recruitment of host PrPC. PrP Prion protein. PrPC The normal cellular isoform of PrP rich in a-helical structure. PrPSc The “scrapie” or disease-associated isoform of PrP which differs from PrPC in its conformation and is generally found as insoluble aggregated material rich in b-sheet structure. Subclinical infection A state where host prion propagation is occurring but which does not produce clinical disease during normal lifespan; essentially a carrier state of prion infection. Transmission barrier This describes the observation that transmission of prions from one species to another is generally inefficient when compared to subsequent passage in the same host species.

Introduction The prion diseases are a closely related group of neurodegenerative conditions which affect both humans and animals. They have previously been described as the subacute spongiform encephalopathies, slow virus diseases, and transmissible dementias, and include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in deer and elk, and the human prion diseases, Creutzfeldt–Jakob disease (CJD), variant CJD (vCJD), Gerstmann–Sträussler–Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru. While rare in humans, prion diseases are an area of intense research interest. This is first because of their unique biology, in that the transmissible agent appears to be devoid of nucleic acid and to consist of a post-translationally modified host protein. Secondly, because of the ability of these and related animal diseases to cross from one species to another, sometimes by dietary exposure, there has been widespread concern that the exposure to the epidemic of BSE poses a distinct threat to public health in the United Kingdom and other countries. The extremely prolonged and variable incubation periods of these diseases, particularly when crossing a transmission barrier, means that it will be some years before the full consequences of human exposure to bovine prions can be predicted with confidence. In the meantime, we are faced with the possibility that significant numbers in the population may be incubating this disease and that they might pass it on to others via blood transfusion, blood products, tissue and organ transplantation, and other iatrogenic routes.

Aberant Prion Protein Metabolism is the Central Feature of Prion Disease The nature of the transmissible agent in prion disease was a subject of heated debate for many years. The understandable initial assumption that the causative agent of “transmissible dementias” must be some form of virus was challenged by the failure to directly demonstrate such a virus (or indeed any immunological response to it) and by the remarkable resistance of the transmissible agent to treatments that inactivate nucleic acids. These findings led to suggestions that the transmissible agent may be devoid of nucleic acid and might be a protein. Subsequently in 1982, Prusiner and co-workers isolated a protease-resistant sialoglycoprotein, designated the prion

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20992-8

707

708

Prions of Vertebrates

protein (PrP), that was the major constituent of infective fractions and was found to accumulate in affected brain. The term prion (from proteinaceous infectious particle) was proposed by Prusiner to distinguish the infectious pathogen from viruses or viroids and was defined as “small proteinaceous infectious particles that resist inactivation by procedures which modify nucleic acids.” Initially, PrP was assumed to be encoded by a gene within the putative slow virus thought to be responsible for these diseases; however, amino acid sequencing of part of PrP and the subsequent recovery of cognate cDNA clones using an isocoding mixture of oligonucleotides led to the realization that PrP was encoded by a single-copy chromosomal gene rather than by a putative viral nucleic acid. Following these seminal discoveries, a wealth of data has now firmly established that the central and unifying hallmark of the prion diseases is the aberrant metabolism of PrP, which exists in distinct conformational states with different physicochemical properties. The normal cellular form of the protein, referred to as PrPC, is a highly conserved cell surface glycosylphosphatidylinositol (GPI)-anchored sialoglycoprotein that is sensitive to protease treatment and soluble in detergents. In contrast, disease-associated isoforms of PrP are found only in prion-infected tissue and composed of detergent-insoluble infectious polymeric PrP assemblies some of which acquire protease-resistance and are classically designated as PrPSc (the scrapie isoform). Due to its physicochemical properties, the precise atomic structure of the infectious particle or prion is still undetermined. The essential role of host PrP for prion propagation and pathogenesis is demonstrated by the fact that knockout mice lacking the PrP gene (Prnp% mice) are entirely resistant to prion infection, and that reintroduction of PrP transgenes restores susceptibility to prion infection in a species-specific manner.

Human Prion Diseases are Biologically Unique Human prion diseases are biologically unique and can be divided etiologically into inherited, sporadic, and acquired forms. Approximately 85% of cases of human prion disease occur sporadically as Creutzfeldt–Jakob disease (sporadic CJD) at a rate of roughly 1 case per million population per year across the world, with an equal incidence in men and women. The etiology of sporadic CJD is unknown, although hypotheses include somatic PRNP mutation, or the spontaneous conversion of PrPC into PrPSc as a rare stochastic event. Polymorphism at residue 129 of human PrP (encoding either methionine (M) or valine (V)) powerfully affects genetic susceptibility to human prion diseases. About 38% of Europeans are homozygous for the more frequent methionine allele, 51% are heterozygous, and 11% homozygous for valine. Homozygosity at PRNP codon 129 predisposes to the development of sporadic and acquired CJD and is most strikingly observed in vCJD. At the time of writing all but one pathologically proven cases of vCJD studied so far have been homozygous for codon 129 methionine of PRNP with one heterozygous patient confirmed in 2016. Approximately 15% of human prion diseases are associated with autosomal dominant pathogenic mutations in PRNP. How pathogenic mutations in PRNP cause prion disease is yet to be resolved, however, in most cases the mutation is thought to lead to an increased tendency of PrPC to form a pathogenic PrP isoform. However experimentally manipulated mutations of the prion gene can lead to spontaneous neurodegeneration without the formation of detectable protease-resistant PrP. These findings raise the question of whether all inherited forms of human prion disease invoke disease through the same mechanism, and in this regard it is currently unknown whether all are transmissible by inoculation. Because of the extensive phenotypic variability seen in inherited prion disease and its ability to mimic other neurodegenerative conditions, notably Alzheimer’s disease, PRNP analysis should be considered in all patients with undiagnosed dementing and ataxic disorders. Recently, a novel clinical and pathological phenotype associated with a Y163X mutation in PRNP was identified which generates a prion protein systemic amyloidosis, characterized by slow disease progression, diarrhea, autonomic failure and neuropathy. This newly recognized novel disease phenotype indicates that PRNP analysis should also be considered in the investigation of unexplained chronic diarrhea associated with a neuropathy or an unexplained syndrome similar to familial amyloid polyneuropathy. Although the human prion diseases are transmissible diseases, acquired forms have, until recently, been confined to rare and unusual situations. The two most frequent causes of iatrogenic CJD occurring through medical procedures have arisen as a result of implantation of dura mater grafts and treatment with human growth hormone derived from the pituitary glands of human cadavers. Less frequent incidences of human prion disease have resulted from iatrogenic transmission of CJD during corneal transplantation, contaminated electroencephalographic (EEG) electrode implantation, and surgical operations using contaminated instruments or apparatus. The most well-known incidences of acquired prion disease in humans resulting from a dietary origin have been kuru that was caused by cannibalism among the Fore linguistic group of the Eastern Highlands in Papua New Guinea, and more recently the occurrence of vCJD in the United Kingdom and some other countries that is causally related to human exposure to BSE prions from cattle. Incubation periods of acquired prion diseases in humans can be extremely prolonged, and it remains unclear how many people within the UK and elsewhere may have become infected and what proportion of these individuals may go on to develop clinical disease rather than remaining as asymptomatic carriers.

The Protein-Only Hypothesis of Prion Propagation Despite extensive investigation, no evidence for a specific prion-associated nucleic acid has been found. Instead, a wide body of data supports the idea that infectious prions consist principally or entirely of an abnormal isoform of PrP. PrPSc is derived from PrPC by a post-translational mechanism and neither amino acid sequencing nor systematic study of known covalent post-translational modifications have shown any consistent differences between PrPC and PrPSc. The protein-only hypothesis, in its current form, argues that prions propagate by means of seeded protein polymerization, a process that involves the recruitment of PrP monomers to fibrillar

Prions of Vertebrates

709

assemblies of PrP followed by fission of the polymer to produce more “seeds” and that this conversion involves only conformational change. However, the underlying molecular events during infection that lead to the conversion of PrPC to prototypical PrPSc and how prototypical PrPSc accumulation leads to neurodegeneration remain poorly defined. Notably, in multiple prion strain/host combinations it now appears that the majority of disease-related PrP and prion titer may be destroyed by proteases under conditions that are typically employed to detect prototypical PrPSc. These data suggest that the term PrPSc, often used synonymously with prion infectivity, should be restricted to material as classically biochemically defined (infectious detergent-insoluble, protease-resistant PrP assemblies). Such prototypical PrPSc constitutes a small (variable) fraction of total disease-related PrP isoforms and its proportional contribution to both infectivity and neurotoxicity remains unclear. Resolving the structural relationship between protease-sensitive and proteaseresistant disease-related infectious PrP assemblies and whether these simply comprise different-sized aggregates of essentially the same PrP conformer has yet to be established. It is now clear that a full understanding of prion propagation will require knowledge not only of the structure of PrPC and prototypical PrPSc assemblies but also the role of other protease-sensitive disease-related PrP isoforms that are generated during the course of prion disease pathogenesis.

Structure and Putative Function of PrPC PrP is highly conserved among mammals, has been identified in marsupials, birds, amphibians, and fish, and may be present in all vertebrates. It is expressed during early embryogenesis and is found in most tissues in the adult with the highest levels of expression in the central nervous system, in particular in association with synaptic membranes. PrP is also widely expressed in cells of the immune system. As a GPI-anchored cell surface glycoprotein, it has been speculated that PrP may have a role in cell adhesion or signaling processes, but its precise cellular function has remained obscure. Mice lacking PrP as a result of gene knockout (Prnp% mice) show no gross phenotype; however, these mice are completely resistant to prion disease following inoculation and do not replicate prions. Prnp% mice do however show subtle abnormalities in synaptic physiology, peripheral myelination and in circadian rhythms and sleep. While the relative normality of Prnp% mice was thought to result from effective adaptive changes during development, data from Prnp conditional knockout mice suggest this is not the case. These mice undergo ablation of neuronal PrP expression at 9 weeks of age and remain healthy without evidence of neurodegeneration or an overt clinical phenotype. Thus, acute loss of neuronal PrP in adulthood is tolerated and the pathophysiology of prion diseases appears to be unrelated to loss of normal PrP function in neurons. Nuclear magnetic resonance (NMR) measurements and crystallographic determination of normal PrP structures from numerous mammalian species, including human PrP, show that they have essentially the same conformation. Following cleavage of an N-terminal signal peptide, and removal of a C-terminal peptide on addition of a GPI anchor, the mature PrPC species consists of an N-terminal region of about 100 amino acids which is unstructured in the isolated molecule in solution and a C-terminal segment, also around 100 amino acids in length. The C-terminal domain is folded into a largely a-helical conformation (three a-helices and a short antiparallel b-sheet) and stabilized by a single disulfide bond linking helices 2 and 3. There are two asparagine-linked glycosylation sites. The N-terminal region contains a segment of five repeats of an eight-amino-acid sequence (the octapeptide-repeat region), expansion of which by insertional mutation leads to inherited prion disease. While unstructured in the isolated molecule, it seems likely that the N-terminal region of PrP may acquire coordinated structure in vivo through coordination of either Cu2 þ or Zn2 þ ions.

Structural Properties of PrPSc PrPSc is extracted from affected brains as highly aggregated, detergent insoluble material that is not amenable to high-resolution structural techniques. However, Fourier transform infrared spectroscopic methods show that PrPSc, in sharp contrast to PrPC, has a high b-sheet content. PrPSc is covalently indistinguishable from PrPC but can be distinguished from PrPC by its partial resistance to proteolysis and its marked insolubility in detergents. Under conditions in which PrPC exists as a detergent-soluble monomer and is completely degraded by the nonspecific protease, proteinase K, PrPSc exists in aggregated assemblies with the C-terminal two-thirds of the protein showing marked resistance to proteolytic degradation leading to the generation of N-terminally truncated fragments of di-, mono-, and nonglycosylated PrP (Fig. 1). Due to the difficulty in performing structural studies on native PrPSc assemblies considerable international effort has instead focused on attempting to produce infectious synthetic b-sheet-rich forms of recombinant PrP which may be amenable to NMR or crystallographic structure determination. Direct in vitro mixing experiments were first attempted to produce PrPSc in vitro. In such experiments PrPSc was used in excess as a seed to convert PrPC to a protease-resistant form, designated PrPRes. While there are now many historical examples in the literature of conditions that generated PrPRes, such reactions were not able to demonstrate de novo production of prion infectivity. However, the discovery of a protein misfolding cyclic amplification (PMCA) system, established that substantial amplification of PrPRes and prion infectivity can be achieved in the absence of living cells. Although the generation of infectious prions from recombinant PrP alone has been reported the low prion titer of such preparations has so far precluded meaningful structural analyzes. Consequently, the requirement to isolate and study ex-vivo prions continues. Progress in understanding prion structure has been severely hindered by the difficulty of isolating relatively homogeneous prion particles from infected tissue and unequivocally correlating infectivity with composition and structure. However new methods for isolating exceptionally pure, high-titer infectious prion preparations from mouse brain have recently been developed

710

Prions of Vertebrates

Fig. 1 PrP analysis by immunoblotting. Immunoblot analysis of normal human brain and vCJD brain homogenate before and after treatment with proteinase K (PK). PrPC in both normal and vCJD brain is completely degraded by proteinase K, whereas PrPSc present in vCJD brain shows resistance to proteolytic degradation leading to the generation of N-terminally truncated fragments of di-, mono-, and non-glycosylated PrP.

Fig. 2 Characterization of disease-related prion protein in human prion disease. (a) Immunoblots of proteinase K-digested tissue homogenate with monoclonal antibody 3F4 showing PrPSc types 1–4 in human brain and PrPSc type 4t in vCJD tonsil. Types 1–3 PrPSc are seen in the brain of classical forms of CJD (either sporadic or iatrogenic CJD), while type 4 PrPSc and type 4t PrPSc are uniquely seen vCJD brain or tonsil, respectively. (b) Brain from a patient with vCJD showing spongiform neurodegeneration following hematoxylin- and eosin staining (H&E), reactive proliferation of astroglial cells following staining with a monoclonal antibody recognizing glial-fibrillary acidic protein (GFAP), and abnormal PrP immunoreactivity following immunohistochemistry using anti-PrP monoclonal antibody ICSM 35 (ICSM 35). Scale (main panels) ¼ 100 mm. Inset, high-power magnification of a florid PrP plaque. Courtesy of Professor Sebastian Brandner.

which showed that PrPSc in these preparations is assembled into rod-like assemblies (PrP rods) that faithfully transmit prion strain-specific phenotypes when inoculated into mice. Sensitive cell culture infectivity assays demonstrated that the PrP rods are intrinsically infectious and that aggregates of the rods behave as discrete infectious particles in cell culture. Electron tomography of negatively-stained prion rods from multiple prion strains revealed a common three-dimensional architecture comprising a pair of short, intertwined fibers, each with a double helical repeating substructure, separated by a distinct gap of 8–10 nm in width. The architecture of the infectious PrP rods, which cause lethal neurodegeneration, readily differentiates them from all other protein assemblies so far characterized in other neurodegenerative diseases.

Prion Disease Pathogenesis Microscopic examination of the central nervous system of humans or animals with prion disease reveals typical characteristic histopathologic changes, consisting of neuronal vacuolation and degeneration, which gives the cerebral gray matter a microvacuolated or “spongiform” appearance, and a reactive proliferation of astroglial cells (Fig. 2). Demonstration of abnormal PrP immunoreactivity, or more specifically biochemical detection of PrPSc in brain material by immunoblotting techniques, is diagnostic of prion disease (Figs. 1 and 2) and some forms of prion disease are characterized by deposition of amyloid plaques composed of insoluble aggregates of PrP. The histopathological features of vCJD are remarkably consistent and distinguish it from other human prion diseases with large numbers of PrP-positive amyloid plaques that differ in morphology from the plaques seen in kuru and GSS in that the surrounding tissue takes on a microvacuolated appearance, giving the plaques a florid appearance (Fig. 2). Although the pathological consequences of prion infection occur in the central nervous system and experimental transmission of these diseases is most efficiently accomplished by intracerebral inoculation, most natural infections do not occur by these means. Indeed, administration to sites other than the central nervous system is known to be associated with much longer incubation periods, which in humans may extend to 50 years or more. Experimental evidence suggests that this latent period is associated with clinically silent prion replication in lymphoreticular tissue, whereas neuroinvasion takes place later. The M-cells in the intestinal epithelium appear to mediate prion entry from the gastrointestinal lumen into the body, and follicular dendritic cells

Prions of Vertebrates

711

(FDCs) are thought to be essential for prion replication and for accumulation of disease-related PrP assemblies within secondary lymphoid organs. B-cell-deficient mice are resistant to intraperitoneal inoculation with prions probably because of their involvement with FDC maturation and maintenance. However, neuroinvasion is possible without FDCs, indicating that other peripheral cell types can replicate prions. The interface between FDCs and sympathetic nerves represents a critical site for the transfer of lymphoid prions into the nervous system; however, the mechanism by which this is achieved remains unknown. Distinct forms of prion disease show differences in lymphoreticular involvement that may be related to the etiology of the disease or to divergent properties of distinct prion strains. For example, the tissue distribution of PrPSc in vCJD differs strikingly from that in classical CJD with uniform involvement of lymphoreticular tissues. Depending upon the density of lymphoid follicles, PrPSc concentrations in vCJD peripheral tissues can vary enormously, with levels relative to brain as high as 10% in tonsil or as low as 0.002% in rectum. In contrast, in sporadic CJD, PrPSc has only been irregularly detected by immunoblotting in noncentral nervous system tissues at very low levels. Tonsil biopsy is used for diagnosis of vCJD, and to date it has shown 100% sensitivity and specificity for diagnosis of vCJD at an early clinical stage, although some patients show scanty deposition of abnormal PrP and a large number of follicles may have to be examined by immunohistochemistry. Anonymous studies of discarded surgical lymphoreticular tissues (tonsil and appendix) have estimated that the overall prevalence of BSE-related prion infection within the UK may be 1 in 2000 of the population. The demonstration of extensive lymphoreticular involvement in the peripheral pathogenesis of vCJD raises concerns that iatrogenic transmission of vCJD prions through medical procedures may be a major public health issue. Disturbingly, cases of transfusion-associated vCJD prion infection have occurred. Prions resist many conventional sterilization procedures and surgical stainless steel-bound prions transmit disease with remarkable efficiency when implanted into mice. While effective methods for prion decontamination of surgical instruments and medical equipment have been reported these have yet to be effectively implemented. Notably, there is now considerable evidence for human transmission of amyloid-beta protein pathology and cerebral amyloid angiopathy resulting from discontinued medical practices involving treatment with human cadaveric pituitary-derived growth hormone or cadaveric dura mater grafting. These findings suggest that it is now important to systematically re-evaluate the potential risks for iatrogenic transmission of proteopathic seeds from individuals with other neurodegenerative diseases.

Prion Strains A major problem for the “protein-only” hypothesis of prion propagation has been to explain the existence of multiple isolates, or strains, of prions. Prion strains are distinguished by their biological properties: they produce distinct incubation periods and patterns of neuropathological targeting (so-called lesion profiles) in defined inbred mouse lines. As they can be serially propagated in inbred mice with the same Prnp genotype, they cannot be encoded by differences in PrP primary structure. Usually, distinct strains of conventional pathogen are explained by differences in their nucleic acid genome. However, in the absence of such a scrapie genome, alternative possibilities must be considered. Support for the contention that prion strain specificity may be encoded by PrP itself was provided by study of two distinct strains of transmissible mink encephalopathy prions which can be serially propagated in hamsters, designated “hyper” and “drowsy”. These strains can be distinguished by differing physicochemical properties of the accumulated PrPSc in the brains of affected hamsters. Following limited proteolysis, strain-specific migration patterns of PrPSc fragments on western blots are seen which relate to different N-terminal ends of PrPSc following protease treatment implying differing conformations of PrPSc. Distinct PrPSc conformations are now recognized to be associated with numerous other prion strains and, similarly, different human PrPSc isoforms propagate in the brain of patients with phenotypically distinct forms of CJD. The different fragment sizes seen on Western blots, following treatment with proteinase K, suggests that there are several different human PrPSc conformations, referred to as molecular strain types. These types can be further classified by the ratio of the three PrP bands seen after protease digestion, corresponding to N-terminally truncated cleavage products generated from di-, mono-, or nonglycosylated PrPSc. Four types of human PrPSc have now been commonly identified in sporadic and acquired CJD using molecular strain typing (Fig. 2), although much greater heterogeneity seems likely. Polymorphism at PRNP residue 129 dictates the propagation of distinct PrPSc types in humans. To date, the repertoire of PrPSc conformations that can be stably propagated by human PrP with 129 methionine or 129 valine remains unknown. In the inherited prion diseases specific pathogenic PrP point mutations dictate the propagation of prion strains that are distinct from those generated from wild-type human PrP. The identification of strain-specific PrPSc structural properties facilitates an etiology-based classification of human prion disease by typing of the infectious agent itself. Stratification of human prion disease cases by PrPSc type enables rapid recognition of any change in relative frequencies of particular PrPSc subtypes (for example in relation to either BSE exposure patterns or iatrogenic sources of vCJD prions). However, efforts to produce a unified international classification and nomenclature of all human PrPSc types has been complicated by the fact that the N-terminal conformation of some PrPSc subtypes seen in sporadic CJD can be altered in vitro via changes in metal-ion occupancy or solvent pH. Although agreement is yet to be reached on methodological differences, nomenclature and the biological importance of relatively subtle biochemical differences in PrPSc, there is strong agreement between laboratories that a significant proportion of the phenotypic diversity in human prion disease relates to the propagation of distinct human prion strains defined by distinct PrPSc conformations and assembly states. However because host genetic makeup and numerous other factors may also significantly influence prion disease phenotype, it is expected that the actual

712

Prions of Vertebrates

number of distinct human prion strains may be considerably less than the number of identified phenotypes. Detailed transgenic modeling therefore remains crucial to establishing how many human prion strains exist. The hypothesis that alternative conformations or assembly states of PrP provide the molecular substrate for clinicopathological heterogeneity seen in human prion diseases (and that this relates to the existence of multiple human prion strains) has been strongly supported by transmission experiments to conventional and transgenic mice. Transgenic mice expressing only human PrP with either valine or methionine at residue 129 have shown that this polymorphism dictates both the propagation of distinct human PrPSc conformers and the occurrence of associated patterns of neuropathology and these data provide the molecular basis for PRNP codon 129 as a major locus influencing both prion disease susceptibility and phenotype in humans. Biophysical measurements suggest that this powerful effect of residue 129 on prion strain selection is likely to be mediated via its effect on the conformation of PrPSc or its precursors or on the kinetics of their formation, as it has no measurable effect on the folding, dynamics, or stability of PrPC. These data are consistent with a conformational selection model of prion transmission barriers and strongly support the “protein only” hypothesis of infectivity by suggesting that prion strain variation is encoded by a combination of PrP conformation and assembly state. Heterozygosity at codon 129 is thought to confer resistance to prion disease by inhibiting homologous protein-protein interactions essential for efficient prion replication while the presence of methionine or valine at residue 129 controls the propagation of distinct human prion strains via conformational selection. Kuru imposed strong balancing selection on the Fore population essentially eliminating PRNP 129 homozygotes. Elderly survivors of the kuru epidemic, who had multiple exposures at mortuary feasts, are, in marked contrast to younger unexposed Fore, predominantly PRNP 129 heterozygotes. Recently a novel protective PrP variant with a coding change at residue 127 (glycine to valine) has been found to colocalize with kuru exposure. Variants at codons 127 and 129 of PRNP demonstrate the population genetic response to an epidemic of prion disease and represent a powerful episode of recent selection in humans. Remarkably, mice expressing only human PrP 127 valine appear to be as resistant to prion disease as PrP null mice and understanding the structural basis of this effect may provide critical insight into the molecular mechanism of mammalian prion propagation.

Neuronal Cell Death in Prion Disease Although various mechanisms have been proposed to explain neuronal death in prion disease, the precise structure of the infectious agent and the cause of neuronal cell death in prion disease remains unclear. While PrPC is absolutely required for prion propagation and neurotoxicity, knockout of PrPC in adult brain and in embryonic models has no overt phenotypic effect, effectively excluding loss of of PrPC function in neurons as a significant mechanism in prion neurodegeneration. Notably, however, there is also considerable evidence that argues that prototypical PrPSc and indeed prions may not themselves be highly neurotoxic. Instead, it is now hypothesized that the neurotoxic entity may comprise a distinct toxic monomeric or oligomeric PrP species designated PrPL (for lethal). The steady-state level of PrPL could determine the rate of neurodegeneration with subclinical prion infection states generating PrPL at levels below the threshold required for neurotoxicity. Direct support for this hypothesis has been demonstrated by depleting endogenous neuronal PrPC in mice with established neuroinvasive prion infection. This depletion of PrPC reverses early spongiform change and progression to clinical prion disease despite the slow accumulation of extraneuronal PrPSc to levels seen in terminally ill wild-type mice. These data establish that propagation of non-neuronal PrPSc is not pathogenic, but arresting the misfolding of PrPC within neurons during scrapie infection prevents prion neurotoxicity. Importantly, this model also validates PrPC as the key therapeutic target in prion disease. Anti-PrP monoclonal antibodies cure prion-infected cells readily and passive immunotherapy with monoclonal antibodies that bind PrPC has shown powerful therapeutic effects in several animal models. Based upon this research, and proceeding with extreme caution under very tightly controlled conditions, administration of a humanized anti-PrP monoclonal antibody to a limited number of CJD patients has now been approved. Although PrPSc has long been considered as a target for chemotherapy, drugs interacting with PrPSc are likely to be prion strain-specific and may only target a specific subset of PrPSc conformers resulting in propagation of drug resistant prions.

Future Perspectives Mammalian prions exist as multiple strains which produce characteristic phenotypes in defined hosts. How this strain diversity is encoded by an apparently protein-only agent remains one of the most interesting and challenging questions in biology, with significant evolutionary implications. The principles of protein conformation-based inheritance established from studying mammalian prions have now been formally demonstrated by elegant studies with analogous systems in yeast models indicating the very wide potential relevance of understanding prion biology. The novel pathogenic mechanisms involved in prion propagation appear highly relevant to other neurological and non-neurological illnesses. Indeed, advances in understanding prion neurodegeneration are already casting considerable light on related mechanisms in other, more common, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. While the protein-only hypothesis of prion propagation is supported by compelling experimental data, and is able to encompass the phenomenon of prion strain diversity, detailed structural studies remain problematic. However, recent advances made with prion isolation together with improved methods in cryo-electron microscopy and high-speed vertically-orientedprobe force microscopy suggests that obtaining a high resolution prion structure will be achievable.

Prions of Vertebrates

713

See also: Prions of Yeast and Fungi

Further Reading Asante, E.A., Smidak, M., Grimshaw, A., et al., 2015. A naturally occurring variant of the human prion protein completely prevents prion disease. Nature 522, 478–481. Collinge, J., 2016. Mammalian prions and their wider relevance in neurodegenerative diseases. Nature 539, 217–226. Collinge, J., Clarke, A.R., 2007. A general model of prion strains and their pathogenicity. Science 318, 930–936. Gill, O.N., Spencer, Y., Richard-Loendt, A., et al., 2013. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: Large scale survey. BMJ 347, f5675. Jaunmuktane, Z., Mead, S., Ellis, M., et al., 2015. Evidence for human transmission of amyloid b pathology and cerebral amyloid angiopathy. Nature 525, 247–250. Mead, S., Gandhi, S., Ayling, H., et al., 2013. A novel prion disease associated with diarrhoea and autonomic neuropathy. New England Journal of Medicine 369, 1904–1914. Mead, S., Stumpf, M.P., Whitfield, J., et al., 2003. Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics. Science 300, 640–643. Mead, S., Whitfield, J., Poulter, M., et al., 2009. A novel protective prion protein variant that colocalizes with kuru exposure. New England Journal of Medicine 361, 2056–2065. Prusiner, S.B., 1998. Prions. Proceedings of the National Academy of Sciences of the United States of America 95, 13363–13383. Purro, S.A., Farrow, M.A., Linehan, J., et al., 2018. Transmission of amyloid-b protein pathology from cadaveric pituitary growth hormone. Nature 564, 415–419. Sandberg, M.K., Al-Doujaily, H., Sharps, B., et al., 2014. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked. Nature Communications 5, 4347. doi:10.1038/ncomms5347. Terry, C., Harniman, R.L., Sells, J., et al., 2019. Structural features distinguishing infectious ex vivo mammalian prions from non-infectious fibrillar assemblies generated in vitro. Scientific Reports 9, 376. Terry, C., Wenborn, A., Gros, N., et al., 2016. Ex vivo mammalian prions are formed of paired double helical prion protein fibrils. Open Biology 6, 160035. doi:10.1098/rsob.160035. Wadsworth, J.D., Asante, E.A., Collinge, J., 2010. Review: Contribution of transgenic models to understanding human prion disease. Neuropathology and Applied Neurobiology 36, 576–597. Wenborn, A., Terry, C., Gros, N., et al., 2015. A novel and rapid method for obtaining high titre intact prion strains from mammalian brain. Scientific Reports 5, 10062. doi:10.1038/srep10062.

Pseudorabies Virus (Herpesviridae) Thomas C Mettenleiter and Barbara G Klupp, Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany r 2021 Elsevier Ltd. All rights reserved.

History The first description of symptoms due to pseudorabies virus (PrV) infection came from infected cattle (‘mad itch’), although pigs have later been found to represent the natural hosts of this virus. Hence, Suid Alphaherpesvirus 1 (SuHV-1) is the taxonomic name of PrV. Throughout this article, we will use the term PrV as this is the name most used in the scientific community. Symptoms of PrV infection in swine range from respiratory signs to severe central nervous system disorders. In other susceptible species productive infection is nearly invariably fatal and characterized by severe central nervous symptoms which prompted its designation as pseudorabies due to the rabies-like clinical picture. A typical symptom in species other than pigs is extensive pruritus which resulted in the name ‘mad itch’ to describe the disease present in cattle in the United States in the first half of the 19th century. In 1902, the Hungarian veterinary pathologist Aladár Aujeszky reported successful isolation of the infectious agent from a diseased ox, a dog and a cat, and differentiated it from rabies. The agent could be passaged in rabbits reproducing the typical symptoms. Guinea pigs and mice were also found to be susceptible, whereas chicken and doves were resistant. Thus, the illness has become widely known as Aujeszky’s disease (AD). However, it was not until 1931 that Richard Shope established the identity of the ‘mad itch’ agent with an infectious agent widely present in domestic pig holdings in the US. In Germany, Erich Traub was the first to cultivate PrV in vitro in porcine organ explants in 1933. One year later, Albert Sabin published his findings of a serological relationship between PrV and herpes simplex virus (HSV) resulting in the inclusion of PrV into the herpesvirus group.

Classification PrV belongs to the Alphaherpesvirinae subfamily of the Herpesviridae within the order Herpesvirales. Originally based on serological studies and later confirmed by molecular biological analyses and comparison of deduced amino acid sequences of homologous proteins, it was shown to be most closely related to bovine herpesvirus 1 (BoHV-1) and equine herpesvirus 1 (EHV-1), and also to varicella-zoster virus (VZV). This prompted its assignment to the Varicellovirus genus within the Alphaherpesvirinae. Its current correct taxonomic designation is Suid Alphaherpesvirus 1 following the binary nomenclature for viruses. Alphaherpesviruses are characterized by a rapid lytic replication, a pronounced neurotropism with establishment of latency in sensory ganglia, and a broad host range. All this applies especially to PrV.

Virion Structure PrV particles exhibit typical herpesvirion morphology with a diameter of ca. 180 nm. At the centre is the genomic DNA which is enclosed in an icosahedral capsid. The capsid is surrounded by a structure designated as tegument which equals the matrix of RNA viruses but is significantly more complex. Finally, the virion envelope which contains virally encoded glycosylated and nonglycosylated proteins anchored in the lipid bilayer encloses the nucleocapsid and tegument (Fig. 1).

Genome The PrV genome consists of a double-stranded linear DNA. Complete genomic sequences have been published which comprise between ca. 140 and 145 kbp encoding a minimum of 70 open reading frames which all exhibit homology to genes in related alphaherpesviruses (Fig. 2). The genome contains a long (UL) and short (US) unique region with the latter being bracketed by inverted repeats resulting in two possible isomeric forms of the genome with inverted US regions relative to the UL. This arrangement has been designated as a class D herpesvirus genome. So far, three functional origins of replication have been mapped, two in the inverted repeats and one in the middle of the unique long region. Compared to the genomes of other alphaherpesviruses, which generally are largely collinear in their gene arrangement, the PrV genome specifies an inversion of ca. 40 kbp which encompasses genes homologous to the UL27 to UL44 genes of herpes simplex virus (Fig. 2). Although a similar inversion is also present in the genome of infectious laryngotracheitis virus, a distantly related avian alphaherpesvirus, its biological significance is unclear. A list of the identified PrV genes and function of encoded proteins is shown in Table 1. Wherever possible, PrV genes have been named after their homologs in herpes simplex viruses (HSV). However, the UL3.5 gene of PrV which has homologs in other alphaherpesviruses such as varicella-zoster virus or bovine and equine herpesvirus 1 is absent from the HSV genome. In contrast, PrV does not specify homologs to the UL45, US5, US10, US11 and US12 genes of HSV. Approximately half of the PrV genes are considered as ‘nonessential’, which indicates that they are dispensable for viral replication, at least in cultured

714

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20929-1

Pseudorabies Virus (Herpesviridae)

715

Fig. 1 The PrV virion. A schematic diagram of the PrV virion is presented between an electron micrograph of a negatively stained PrV virion (left) and a thin-sectioned virus particle (right). The location of the virion subcomponents core, capsid, tegument and envelope is indicated. Spikes at the envelope represent viral glycoproteins.

cells. Also, it is estimated that about half of the protein products are located in the virion and can be considered structural components of the virus particle.

Capsid As a typical herpesviral capsid the icosahedral PrV capsid is composed of 161 capsomers consisting of 955 copies of the major capsid protein, the product of the UL19 gene (see below). Homologs of the HSV pUL18 and pUL38 which form triplexes connecting and stabilizing the capsomers as well as the pUL35 protein located at the tip of the hexons have been identified. Also present are homologs for the pUL6 portal protein which forms a dodecameric channel for package and release of the viral genome at one pentonal site. Intimately associated with the capsid is the capsid vertex specific complex (CVSC) consisting of the UL17 and UL25 gene products.

Tegument The tegument of herpesviruses is a complex structure which, in the case of PrV, contains in excess of 15 virally encoded proteins. It has become clear that tegument formation is an important step in the morphogenesis of the PrV virion requiring a network of partially redundant protein-protein interactions. The tegument can be divided into capsid-proximal and envelope-proximal parts. The capsid-proximal tegument is comprised of the UL36 gene product, the largest protein in PrV consisting of 3108 amino acids, which physically interacts with the UL37 gene product. From cryoelectron image reconstructions of herpesvirus particles, the UL36 gene product is thought to contact the capsid. On the other hand, the UL46, UL47, UL48 and UL49 gene products are easily stripped from the capsid together with the envelope and, thus, are located in the envelope-proximal tegument. Correlating with these findings, the PrV UL49 gene product has been shown to interact with the intracytoplasmic carboxytermini of the gE and gM envelope proteins. Both parts of the tegument may be connected by the UL48 gene product. Tegument proteins enter the cell after fusion of the virion envelope and the cellular plasma membrane during entry, and prime the cell for virus production. The alphaherpesvirus UL48 gene products are strong transactivators of viral immediate-early gene expression, whereas the UL41 proteins possess endoribonucleolytic activity to degrade preexisting cellular mRNAs. Interestingly, cellular proteins have also been detected in the PrV tegument including cellular actin, annexins and heat shock proteins. Their biological role is unclear.

Envelope Receptor binding proteins as well as major immunogens are located in the viral envelope. More than 10 envelope constituents have been identified in PrV. Most of them are modified by the addition of carbohydrates and, thus, represent glycoproteins. Several type I, type II and type III PrV membrane proteins have been described (Table 1). Since early nomenclature of PrV glycoproteins was somewhat confusing, it has been agreed to name them after their HSV counterparts. Several of these glycoproteins form complexes such as the homo-oligomeric gB, and the heterodimeric gE/gI, gH/gL, gK/pUL20 and gM/gN. Glycoprotein N and the gM/gN complex which is conserved throughout the Herpesviridae have for the first time been identified in PrV as has the gK/pUL20 interaction. Nonglycosylated membrane proteins include the pUs9, pUL43 and pUL56 gene products (see Table 1). The nonstructural gG is proteolytically cleaved and released from infected cells.

Replication Cycle PrV is amongst the most studied animal herpesviruses and has become a major model virus to understand fundamental herpesvirus biology. PrV infection of host cells starts with the interaction of the envelope glycoprotein C (gC) with cell-surface

716

Pseudorabies Virus (Herpesviridae)

Fig. 2 The PrV genome. The transcript and gene organization as deduced from the complete genomic sequence is shown. The linear form of the PrV genome comprises the unique long sequence, the internal repeat region, the unique short sequence and the terminal repeat. The predicted locations of PrV open reading frames (see Table 1), 5′- and 3′ nontranslated regions, DNA repeats, splice sites and origin of replication are shown. Reprinted with permission from the American Society for Microbiology from Klupp, B.G., Hengartner, C., Mettenleiter, T.C., Enquist, L.W., 2004. Journal of Virology 78, 424–440.

ORF locationa

1370-2254 1631-2254 3873-2788r 4890-3952r 7733-4845r 7720-8430 9390-8584r 9314-9610 9648-10,397 10,461-11,702 11,803-14,055 14,074-16,155 19,673-16,911r 21,700-19,544r 25,383-21,850r 25,699-28,845 29,581-28,766r 30,986-29,574r 30,985-31,332 31,491-32,279 32,334-32,645 42,472-33,146r 45,269-42,510r 45,326-46,432 46,763-49,135 491,45-50,056 51,655-50,558r 51,784-52,938 52,998-54,119 54,186-55,628 56,695-55,859r 57,433-55,859r 59,071-57,467r 59,679-59,164r 59,672-60,634 60,770-62,830 66,226-64,649r 66,333-66,818 66,905-70,897 71,063-71,953 73,285-72,149r

pORF1.2 pORF1 pUL54 pUL53 pUL52 pUL51 pUL50 pUL49.5 pUL49 pUL48 pUL47 pUL46 pUL27 pUL28 pUL29 pUL30 pUL31 pUL32 pUL33 pUL34 pUL35 pUL36 pUL37 pUL38 pUL39 pUL40 pUL41 pUL42 pUL43 pUL44 pUL26.5 pUL26 pUL25 pUL24 pUL23 pUL22 pUL21 pUL20 pUL19 pUL18 pUL15 (Ex2)

PrV ORFs

Protein

Table 1

294 207 361 312 962 236 268 98 249 413 750 693 920 718 1177 1048 271 470 115 262 103 3108 919 368 790 303 365 384 373 480 278 524 534 171 320 686 525 161 1330 296 735

31.0 21.8 40.4 33.8 103.3 25.0 28.6 10.1 25.9 45.1 80.4 75.5 100.2 78.5 125.1 115.3 30.4 51.6 12.7 28.1 11.5 326.7 98.2 40.0 86.5 34.4 40.1 40.3 38.1 51.3 28.2 54.6 57.4 19.1 35.0 71.9 55.2 16.7 146.0 31.7 79.1

Length (aa) MW (kD)

VP5 VP23

gH

gC VP22a VP24

VP19c RR1 RR2 VHS

VP26 VP1/2

dUTPase gN VP22 VP16/αTIF VP13/14 VP11/12 gB ICP18.5 ICP8

ICP27 gK

Alias

Membrane protein, pUL56 family; vesicle trafficking Membrane protein, pUL56 family; vesicle trafficking Early protein; gene regulation Type III membrane protein; glycoprotein K; complex with pUL20; viral egress Helicase-primase primase subunit; DNA replication Tegument protein; complexed with pUL7; viral egress (secondary envelopment) Deoxyuridine triphosphatase; nucleotide metabolism Type I membrane protein; glycoprotein N; complexed with gM Tegument protein; interacts with C-terminal domains of gE & gM Tegument protein; gene regulation (trans activator); viral egress (secondary envelopment) Tegument protein; viral egress (secondary envelopment) Tegument protein; signaling Type I membrane protein; glycoprotein B; viral entry (fusion); cell-cell spread DNA packaging terminase subunit 1; associated with pUL15, pUL33 & pUL6 Single-stranded DNA-binding protein; DNA replication DNA polymerase catalytic subunit; DNA replication; complexed with pUL42 Tegument protein; primary virion tegument; nuclear egress; interacts with pUL34 DNA encapsidation; efficient localization of capsids to replication compartments DNA Cleavage/encapsidation; associated with pUL28 & pUL15 Type II nuclear membrane protein; primary virion envelope protein; nuclear egress; interacts with pUL31 Capsid protein Tegument protein; interacts with pUL37 & pUL19; capsid transport Tegument protein; interacts with pUL36; capsid transport Capsid triplex subunit; complexed with pUL18 Large subunit of ribonucleotide reductase; nucleotide metabolism Small subunit of ribonucleotide reductase; nucleotide metabolism Gene regulation (inhibitor of gene expression); virion host cell shut off factor DNA replication; DNA polymerase processivity subunit; complexed with pUL30 Type III membrane protein; unknown; inhibits membrane fusion in transient assays Type I membrane protein; glycoprotein C; viral entry (virion attachment); binds to heparan sulfate Scaffold protein; substrate for pUL26; capsid morphogenesis Scaffold protein; proteinase; capsid morphogenesis Capsid associated protein; complexed with pUL17 (CVSC); DNA packaging and nuclear egress Unknown Thymidine kinase; nucleotide metabolism Type I membrane protein; glycoprotein H; viral entry (fusion); cell-cell spread; complexed with gL Capsid associated protein; complexed with pUL16 Type III membrane protein; viral egress; complexed with gK Major capsid protein; forms hexons und pentons Capsid triplex subunit 2; complexed with pUL38 DNA packaging terminase subunit; DNA cleavage/encapsidation; interacts with pUL33, pUL28 & pUL6

Function/propertyb

V (E) V (E) NS V (E) NS V (T) NS V (E) V (T) V (T) V (T) V (T) V (E) pC NS NS pV (T) V (?) NS pV (E) V (C) V (T) V (T) V (C) V (?) V (?) V (?) ? V (E) V (E) pC pC; V (?) V (C) NS NS V (E) V (T)? V (E) V (C) V (C) pC (Continued )

Virion subunitc

Pseudorabies Virus (Herpesviridae) 717

597 328 159 398 483 63 393

77,235-76,165r 73,336-75,129 75,156-76,142 77,234-77,713 77,683-78,879 78,845-80,296 80,254-80,445 82,105-80,924r

82,104-84,635 84,632-86,683 87,649-86,849r 89,471-87,540r 89,470-91,974 92,033-92,470 93,320-92,646r 94,030-93,317r 95,036-94,086r 95,484-95,014r 97,884-96,652r 107,695-103,343r 137,044-141,396 116,181-117,275 128,558-127,464r 118,239-119,405 118,401-119,405 119,465-120,964 121,145-122,347 122,368-123,465 123,569-125,302 125,360-125,656 125,879-126,649

pUL15 (Ex1) pUL17 pUL16 pUL14 pUL13 pUL12 pUL11 pUL10

pUL9 pUL8 pUL7 pUL6 pUL5 pUL4 pUL3.5 pUL3 pUL2 pUL1 EP0 IE180 (IRS) IE180 (TRS) US1 (IRS) US1 (TRS) US3 (minor) US3 (major) US4 US6 US7 US8 US9 US2 39.6 42.9 36.9 53.7 44.3 38.7 62.4 10.6 27.7

364

388 334 499 400 365 577 98 256

DNA-packaging tegument protein; DNA cleavage/encapsidation; complexed with pUL25 (CVSC) Tegument protein; complexed with pUL11 & pUL21 Unknown Serine/threonine protein kinase Deoxyribonuclease; DNA processing Myristylated tegument protein; viral egress (secondary envelopment); complexed with pUL16&pUL21 Type III membrane protein; glycoprotein M; viral egress (secondary envelopment); C-terminus interacts with pUL49; complexed with gN; inhibits membrane fusion in transient assays DNA replication origin-binding helicase; DNA replication Helicase-primase subunit; DNA replication; part of pUL5/pUL8/pUL52 complex Tegument protein; virion formation and egress; complexed with pUL51 Capsid portal protein; DNA packaging; docking site for terminase Helicase-primase helicase subunit; DNA replication; part of pUL5/pUL8/pUL52 complex Nuclear protein; unknown Tegument protein; viral egress (secondary envelopment) Nuclear protein; unknown Uracil-DNA glycosylase; DNA repair Glycoprotein L; membrane anchored via complex with gH; viral entry (fusion); cell-cell spread Gene regulation (trans activator of viral and cellular genes); early protein Gene regulation; immediate-early protein

Function/propertyb

PK PK gG gD gI gE 11K 28K

Minor form of the serine/threonine protein kinase Major form of the serine/threonine protein kinase; viral egress (nuclear egress) Type I membrane protein; glycoprotein G Type I membrane protein; glycoprotein D; viral entry (receptor binding protein) Type I membrane protein; glycoprotein I; cell-cell spread; complexed with gE Type I membrane protein; glycoprotein E; cell-cell spread; complexed with gI; C-terminus interacts with pUL49 Type II membrane protein; axonal transport Prenylated tegument protein; unknown

RSp40/ ICP22 Immediate-early protein; gene regulation

UNG gL ICP0 ICP4

V57

OBP

gM

VP18.8

Alias

NS V (T) secreted V (E) V (E) V (E) V (E) V (T)

V (?)

NS ? V (T) V (C) NS NS V (T) NS NS V (E) V (?) V (?)

V (C) V (T)? NS ? NS V (T) V (E)

Virion subunitc

b

a

Numbering starts at +1 on the UL end of the genome. r indicates ORF encoded on reverse strand; AccNo. JQ809328.1. Function/Property as demonstrated for the PrV and/or HSV-1 homolog. c V (C): virion capsid component; V (T): virion tegument component; V (E): virion envelope component; V (?): virion component of unknown subviral localization; pV: primary enveloped virion precursor component (not found in mature virion); NS: non structural protein; pC: present in intranuclear capsid precursor forms but not found in mature virion;?: unknown.

90.5 71.2 29.0 70.3 92.1 15.8 24.0 25.6 33.0 16.5 43.8 148.9

843 683 266 643 834 145 224 237 316 156 410 1450

64.2 34.8 17.9 41.1 51.3 7.0 41.5

Length (aa) MW (kD)

ORF locationa

Continued

Protein

Table 1

718 Pseudorabies Virus (Herpesviridae)

Pseudorabies Virus (Herpesviridae)

719

Fig. 3 The PrV replication cycle. A diagram of the replication cycle of PrV is shown together with electron micrographs showing the respective stages. After attachment (1) and penetration (2), capsids are transported to the nucleus (3) via interaction with microtubuli (4) docking at the nuclear pore (5) where the viral genome is released into the nucleus. Here, transcription of viral genes and viral genome replication occurs (6). Concatemeric replicated viral genomes are cleaved to unit-length molecules during encapsidation (7) into preformed capsids (8) which then leave the nucleus by budding at the inner nuclear membrane (9) followed by fusion of the envelope of these primary virions located in the perinuclear cleft (10) with the outer nuclear membrane (11). Final maturation then occurs in the cytoplasm by secondary envelopment of intracytosolic capsids via budding into trans-Golgi derived vesicles (12) containing viral glycoproteins (black spikes) resulting in an enveloped virion within a cellular vesicle. After transport to the cell surface (13), vesicle and plasma membranes fuse releasing a mature enveloped PrV particle from the cell (14).

heparansulfate-containing proteoglycans (Fig. 3). This interaction is beneficial but not essential for the second step which involves binding of the essential envelope gD to one of its cellular receptors, e.g., nectin. Interestingly, HSV and bovine herpesvirus 1 also use heparansulfate and nectin for attachment. However, gD-negative and even gC- and gD-negative infectious PrV mutants have been isolated which indicates that infection can also occur by other routes. Interestingly, these mutants harbour mutations in gB and gH. Penetration, i.e., fusion of viral envelope and cellular plasma membrane, requires the essential gB and gH/gL which represent glycoproteins conserved throughout the Herpesviridae indicating a common herpesvirus membrane fusion mechanism. The capsid with associated inner tegument is then transported to the nuclear pore via microtubules where it docks and releases the viral genome through one vertex via the nuclear pore into the nucleus of the cell. Empty capsids may remain bound to the nuclear pore for a considerable time. This cascade of events can be alleviated by transfection of naked genomic viral DNA. In the nucleus, transcription of viral genes is initiated by expression of the major immediate-early (IE) gene resulting in the translation of a 180 kDa immediate-early protein (IE180). Although IE180 has long been considered the only immediate-early protein of PrV, studies using specific inhibitors have identified that the US1 (RSp40) mRNA is also expressed with immediate-early kinetics. Like other herpesvirus IE proteins, PrV IE180 is a potent transcriptional activator that induces the expression of viral early (E) genes. These encompass genes encoding enzymes involved in nucleotide metabolism (e.g., UL23 ¼ thymidine kinase; UL2 ¼ UracilDNA-glycosylase; UL39/40 ¼ Ribonucleotide-Reductase; UL50 ¼ dUTPase), DNA replication (UL30/UL42 ¼ DNA-polymerase and associated factor; UL5 ¼ helicase; UL8/UL52 ¼ primase; UL29 ¼ ssDNA-bdg. protein; UL9 ¼ origin-binding protein) as well as two protein kinases (pUS3, pUL13). DNA replication, whether exclusively via rolling circle or involving intra- and intermolecular recombination and branching, results in the formation of head-to-tail fused concatemers of the genome. Finally, late genes encoding primarily virion structural proteins are expressed. After translation in the cytosol, capsid proteins are transported into the nucleus for capsid assembly and DNA packaging. Capsid assembly is morphologically similar in all herpesviruses: capsids containing the major capsid protein pUL19, triplex proteins pUL18 and pUL38, hexon-tip protein pUL35, and portal protein

720

Pseudorabies Virus (Herpesviridae)

pUL6 assemble autocatalytically with a protein scaffold consisting of the UL26.5 gene products. Packaging occurs via the unique portal at one vertex comprising 12 molecules of the pUL6 protein. Genome-length molecules are cleaved from concatemeric replication products during packaging which requires the terminase complex (pUL15, UL28, pUL33) as well as pUL32. The CVSC of pUL17 and pUL25 stabilizes the capsid and is essential for triggering primary envelopment. It is located at the outside of the capsid. Egress of herpesvirus capsids from the nucleus occurs by budding at the inner leaflet of the nuclear membrane followed by de-envelopment (fusion) at the outer leaflet. For primary envelopment, the conserved heterodimeric nuclear egress complex (NEC) consisting of pUL31 and pUL34 proteins has been shown to be crucial in all three subfamilies of herpesviruses. Crystal structures for the HSV-1, PrV and human cytomegalovirus (HCMV) NEC show a conserved arrangement with extensive interfaces between the complex partners. The NEC which is localized in the nuclear membrane recruits cellular protein kinase C which phosphorylates and, thereby, dissociates nuclear lamins allowing access of nascent capsids to the inner nuclear membrane. Primary enveloped virions in the perinuclear space also contain the NEC which constitutes part of the primary envelope (pUL34 is a type II transmembrane protein) and tegument (pUL31). The pUS3 protein kinase is also part of the primary PrV virion. pUS3 has been detected in primary and mature PrV virions, whereas pUL31 and pUL34 proteins are absent from mature virus particles. The mechanisms of de-envelopment are unclear. However, in the absence of the nonconserved and nonessential pUS3, primary enveloped virions accumulate in the perinuclear space demonstrating the participation of this protein in nuclear egress. Within the cytoplasm, virion morphogenesis is completed by tegumentation, final envelopment and transport of mature virus particles for release at the plasma membrane. Tegumentation apparently starts at two sites, the capsid and the future envelopment site. At the capsid, the conserved tegument protein pUL36, the largest gene product of PrV with 3108 amino acids, interacts with pUL37. At the future envelopment site, i.e., at vesicles derived from the Golgi apparatus, the carboxytermini of the type I membrane protein gE and the type III gM bind the pUL49 tegument protein. Presumably, pUL48 links the two parts of the tegument which drives budding of tegumented virions into these trans-Golgi vesicles containing viral glycoproteins to yield mature virions within a vesicle. PrV gM has been shown to relocate other viral and cellular proteins to the trans-Golgi and, thus, may be involved in assembling the envelope proteins. On the other hand, the conserved pUL11 is thought to be involved in directing tegument proteins to the envelopment site. Finally, virion-containing vesicles move to the cell surface, a process in which pUL20 is involved, where plasma and vesicle membranes fuse resulting in release of infectious particles. Apparently, the gK/pUL20 complex inhibits an immediate re-fusion of released virions with the cell they just left. Besides infection by free virions, infectivity can also be transmitted via direct cell-cell transmission. Although several virion proteins required for penetration (gB, gH/gL) are also required for direct cell-cell spread, its mechanism remains enigmatic. Interestingly, direct cell-cell spread of PrV does not require the presence of the receptor-binding gD nor does syncytium formation after transient expression of gB and gH/L which contrasts the situation in other alphaherpesviruses such as HSV.

Clinical Features and Pathogenesis PrV is able to productively infect most mammals with the exception of higher primates including humans. However, primate and human cells are infectable in cell culture and the reason for the natural resistance is not clear. Equids and goats are also rather resistant but may be infected experimentally. In addition, pseudorabies has been reported in many species of wild mammals, including wild boar, feral pigs, coyotes, raccoons, rats, mice, rabbits, deer, badgers, and coatimundi. It is so far unknown whether these animals play a role in farm-to-farm transmission of PrV. In susceptible species other than porcines, infection is invariably fatal and animals die from severe neuronal disorders. After PrV infection of the natural host, the clinical outcome varies depending on the age of the animal, the virulence of the virus and the route of infection. In nature infection occurs predominantly oro-nasally, although genital transmission may also take place especially in feral pigs. After replication in epithelial cells the virus gains access to neurons innervating the facial and oropharyngeal area, in particular the olfactory, trigeminal and glossopharyngeal nerves. By fast axonal retrograde transport the virus reaches the cell bodies of infected neurons where either lytic or latent infection ensues (see below). By a short viremia PrV may be disseminated to other organs where the virus replicates in epithelia, vascular endothelium, lymphocytes, and macrophages. Neonate piglets become prostrate and die quickly, often without nervous signs. In slightly older piglets, severe CNS disorders are characterized by incoordination, twitching, paddling, tremors, ataxia, convulsions and/or paralysis whereas itching is only rarely present (Fig. 4). Mortality in piglets until 2–3 weeks of age may be as high as 100% resulting in severe losses. Piglets between 3 and 6 weeks may still exhibit neurological signs and a high morbidity, but mortality is usually reduced. Infection in older pigs induces primarily respiratory symptoms like coughing, sneezing and heavy breathing resulting from viral replication in and destruction of pulmonary epithelium. Despite the absence of overt nervous signs, virus gains access to neurons and remains latently established in the olfactory bulb, trigeminal ganglia, and brain stem or, after venereal transmission, in the sacral ganglia. PrV infection of pregnant sows may result in abortion, or the delivery of stillborn or mummified fetuses due to endometritis and necrotizing placentitis with infection of trophoblasts. In non-porcine species, PrV infection is strictly neurotropic and invariably fatal, sometimes after a rapid, peracute course without any preceding overt clinical signs. Pruritus is a lead symptom of PrV infection in these species which, in particular in rabbits and rodents, may result in violent itching and automutilation. Often the death of mice, rats, cats or dogs on farms is a warning sign for the presence of PrV prior to symptoms in pigs.

Pseudorabies Virus (Herpesviridae)

721

Fig. 4 Neurological symptoms of PrV infection in piglets. The animals show ataxia (A), convulsions and paralysis (B) which ultimately lead to death.

Transmission occurs via virus-containing body fluids like nasal and genital secretions which gain access to unprotected epithelial surfaces within the respiratory or genital tract. Airborne transmission is efficient only at short range. Carnivores become infected by ingesting contaminated meat. After primary replication in epithelial cells the virus enters the endings of sympathetic, parasympathetic or sensory and motor neurons innervating the area of primary replication. Infection occurs most likely by the same mechanism as outlined above for cultured cells. De-enveloped and partially de-tegumented virus particles are retrogradely transported to the neuronal cell body, where DNA replication and formation of progeny virions ensues. Primarily complete enveloped virions within transport vesicles are then translocated to the synapse for transsynaptic transfer. Depending on the virulence of the virus and the age and immune status of the host, infection may not proceed beyond the first neuronal level, i.e., ganglia directly innervating the affected peripheral site. However, virus may also spread to the brain resulting in ganglioneuritis and encephalitis. Lymphocytes can also become infected by PrV and infection of these cells may help viral spread within the body playing an important role in infection of the fetus. However, the percentage of infected cells in the blood is rather low, even during acute infection, and difficult to detect. A major target organ for latency in swine are tonsils, and tonsil biopsies allow reliable detection of virus either by molecular biological techniques or virus isolation. There are no specific gross lesions of Aujeszky’s disease. Only in piglets there may be necrotizing tonsillitis, rhinotracheitis or proximal esophagitis. Other lesions commonly seen include pulmonary edema, necrotizing enteritis, and multifocal necrosis of the spleen, lung, liver, lymph nodes, and adrenal glands. Histologically, PrV causes a nonsuppurative meningoencephalitis and paravertebral ganglioneuritis. The grey matter is especially affected, and infected neurons or astrocytes may present acidophilic intranuclear inclusions. The presence of viral antigen can be visualized by immunostaining and viral genomes can be detected by in situ hybridization. PrV infected cells usually show more or less extensive degeneration and necrosis due to the lytic viral replication. Whether apoptosis induced by PrV infection also plays a role in vivo is unclear. Mainly a T-cell reaction of the immune system induces ganglioneuritis, polio- or panencephalitis with foci of gliosis contributing to the loss of neuronal function. The described extraneural lesions in pigs or acute myocarditis in carnivores might provide additional explanations for the fatal outcome of infections in which virus can not be recovered from the brain.

Immunity Live as well as inactivated vaccines induce efficient protective immunity against AD. Antibodies against a number of viral structural and nonstructural proteins have been detected in infected animals and virus-neutralizing murine monoclonal antibodies have been isolated. Antibody responses are primarily directed against the major surface glycoproteins including gB, gC, gD and gE as well as the secreted gG. The most potent complement-independent virus neutralizing antibodies are directed against gC, gD and, to a lesser extent, gB, and subunit vaccines consisting of gB, gC, gD, as well as anti-idiotypic anti-gD antibodies and heterologous vectors expressing gC or gD elicit protective immunity. In contrast, anti-gE antibodies require complement for neutralization, and anti-gG antibodies have no neutralizing ability at all. In diagnostic assays, antibodies against whole virus or specific for gB are being used to serologically detect PrV infection. Major targets for cell-mediated immunity in pigs are primarily gC and to a lesser extent gB. Although the numerous elaborate immune evasion mechanisms of beta- and gammaherpesviruses including expression of virokines and viroceptors have not been found in alphaherpesviruses, these viruses still interact with the immune system to evade its activity. Like other alphaherpesvirus gC proteins, PrV gC binds species-specifically to porcine complement component C3, and

722

Pseudorabies Virus (Herpesviridae)

the gE/gI complex binds the Fc portion of porcine IgG. The secreted gG may bind chemokines, thereby impairing intercellular signaling. Moreover, infection of cells by PrV results in downregulation of MHC-I antigen presentation, and envelope glycoproteins present at the plasma membrane of infected cells are internalized by as yet unknown factors resulting in a paucity of antigens presented to the immune system at the cell surface. PrV gN inactivates the transporter which translocates processed peptides for loading onto MHC-I molecules into the endoplasmic reticulum.The combined action of these mechanisms may give the virus an edge over the immune system facilitating establishment of latency and further virus spread.

Latency Like other herpesviruses, PrV has the capacity to become latent in neurons. During latency, the genome persists largely quiescent in a presumably circular form. Expression is restricted to one region, the LAT gene, which encompasses part of the inverted repeat and adjoining UL region. It is located antiparallel to the gene for the major immediate-early protein IE 180 and the EP0 gene (Fig. 2). The LAT gene is transcribed into three different RNAs of 8.4, 8.0 and 2.0 kb. The 8.4 and 2.0 kb species are derived from a larger precursor by splicing. During latency, only the 8.4 kb RNA is produced from a separate promotor which, apparently, is active only under latent conditions, whereas the 8.0 and 2.0 species are transcribed during lytic infection. Detailed RNA analyses revealed the existence of eleven micro-RNAs derived from the LAT which play a role in latency and virulence of PrV. Like other herpesviruses, PrV encodes proteins which are able to suppress apoptotic cell death which is a prerequisite for the establishment of latency. The PrV Us3 protein kinase has been demonstrated to mediate this function in porcine fetal trigeminal neurons. Like other alphaherpesviruses, PrV establishes latent infections predominantly in neuronal tissues such as the trigeminal or sacral ganglia. However, tonsils have also been identified as sites of PrV latency.

Epidemiology and Prevention In the 20th century PrV has become a pathogen distributed worldwide with the exception of Australia, Canada and the Scandinavian countries. In particular in major swine producing areas, PrV infection caused significant economic losses amounting to hundreds of million of dollars making it one of the most devastating pig diseases. Control and eradication of PrV infection in pigs relied on two strategies. In areas with a low prevalence of infection, serological identification and consequent elimination of seropositive animals resulted in the successful eradication of AD e.g., from the United Kingdom, Denmark and East Germany. PrV infection can be diagnosed by detecting either viral nucleic acid using polymerase chain reaction (PCR) or the infectious agent (antigen detection by immunofluorescence or virus isolation). PCR is also suitable for detecting latent viral genomes. A PrV specific immune response in live animals can be confirmed using various serological assays such as virus neutralisation test, latex agglutination test and ELISA systems based on either complete virus particles or distinct viral antigens, e.g., gB. To reduce disease in areas of high infection prevalence, vaccination using either live-attenuated or inactivated vaccines has also been used. However, vaccination does not result in sterile immunity, and vaccinated animals may still be infected with and carry the virus but these carriers were no longer identifiable by serological analysis. This problem has been solved by the advent of so-called ‘marker’ vaccines. This concept provided a break-through in animal disease control, and serves as a blueprint for control of other infectious diseases. It was based on the finding that several immunogenic envelope glycoproteins of PrV such as gC, gE and gG (see above) are not required for productive replication and, thus, can be deleted from the viral genome without abolishing virus replication. These gene-deleted strains can be produced easily in conventional cell systems and can be administered as inactivated as well as

Fig. 5 The principle of DIVA or marker vaccination. Whereas after wild-type infection antibodies are produced against all immunogenic viral proteins, after vaccination with a gene-deleted virus antibodies against the missing gene product (circled) will not be formed. Thus, presence or absence of these antibodies is used to differentiate between infected and vaccinated animals.

Pseudorabies Virus (Herpesviridae)

723

modified-live vaccines. In fact, gene-deleted PrV strains were the first genetically engineered live-virus vaccines ever licensed. Thus, PrV has pioneered modern vaccinology. Whereas animals vaccinated with these vaccines do not mount an immune response to the missing gene product, wild-type virus infection invariably results in seroconversion for the differentiating antigen. Serological assays (ELISA) have subsequently been developed that allow easy and sensitive detection of antibodies against these marker proteins resulting in the unambiguous identification of wild-type virus infected animals despite vaccination (Fig. 5). Thus, virus circulation can be reduced by vaccination, and, subsequently, infected animals which still harbour field virus can be identified and eliminated resulting in a cost-efficient eradication. This ‘differentiating infected from vaccinated animals’ (DIVA) approach used first in the eradication of PrV, has been a breakthrough in animal disease control and is now widely accepted and practiced on other infectious diseases such as bovine herpesvirus 1 infection, classical swine fever and foot-and-mouth disease. Its application resulted in the eradication of PrV from heavily infected West Germany within ten years, and also recently succeeded in eliminating PrV infection from USA and New Zealand pig herds. Although European wild boar and American feral pigs also harbour PrV, there is no epidemiological link between PrV in wild boar and domestic pigs in Europe since clearly different viral strains have been isolated from either. However, infected feral pigs may represent a source of infection of domestic pig holdings in the Southern part of the USA. Recently, a surge in PrV infections has been noticed in China despite extensive vaccination. It has been attributed to the emergence of hypervirulent PrV strains, although the molecular basis for this increased virulence remains unclear.

Pseudorabies Virus as a Tool in Neurobiology Like other alphaherpesviruses, PrV exhibits a distinct neurotropism invading the CNS via peripheral nerves. While wild-type strains of PrV may spread within the CNS both, laterally and transsynaptically, attenuated PrV mutants have been identified which, under proper assay conditions, travel more or less exclusively along nerves and are transported transsynaptically. This particular property has prompted an increasing use of PrV as a suitable transneuronal tracer to label neuronal connections in experimental animal models being useful in mice and rats for elucidating detailed neuroanatomical networks. The virus used most in these studies is the Bartha strain of PrV, a modified-live vaccine strain which had been attenuated by the Hungarian veterinarian Adorjan Bartha by multiple passages in embryonated chicken eggs and chicken embryo fibroblasts. Molecular biological analyses demonstrated that this strain carries several lesions compared to wild-type PrV: it lacks the gE, gI and Us9 genes, contains a mutation in the signal sequence for gC, specifies attenuating mutations in the UL21 gene, and expresses a UL10 gene product (gM) which is not glycosylated due to mutation of the N-glycosylation site. The glycoprotein deletions and the UL21 mutation have been shown to be most important for the observed attenuation. Recently, genetically engineered Bartha-derivatives expressing a range of chromogenic or fluorescent marker proteins have been used in tracing studies. Moreover, elegant mutants that express their markers only under specific conditions, e.g., in transgenic cells or animals expressing cre-lox recombinase under control of tissue-specific promotors have added another possibility of tissue-specific labelling.

Future Perspectives PrV is a most fascinating virus with several interesting properties. The availability of conventional and genetically engineered marker vaccines allows effective and cost-efficient disease control campaigns which have been shown to result in the eradication of virus and disease from animal populations. Although PrV infection is still widespread, in particular in certain areas in Eastern Europe and Asia, concerted efforts could result in the elimination of the disease at the global level. Beyond its importance as the causative agent of a relevant animal disease, it is an ideal tool to study basic mechanisms of herpesvirus biology with the enormous advantage of an experimentally accessible natural virus-host system by infection of pigs. Moreover, its broad host range allows the use of other well-defined animal models for neuroanatomical, immunological and molecular biological studies. Since PrV grows exceedingly well in tissue culture, it is also well suited for detailed analysis of the requirements for replication of other (alpha) herpesviruses. Thus, PrV will remain under intensive scrutiny.

Acknowledgments We thank Thomas Müller and Jens Teifke for help with the manuscript and Harald Granzow and Mandy Jörn for Figs. 1 and 3.

Further Reading Freuling, C.M., Müller, T.F., Mettenleiter, T.C., 2018. Veterinary Microbiology 206, 3–9. Klupp, B.G., Hengartner, C., Mettenleiter, T.C., Enquist, L.W., 2004. Journal of Virology 78, 424–440. Mettenleiter, T.C., 2016. Viruses 8, 266. doi:10.3390/v8100266. Mettenleiter, T.C., Ehlers, B., Müller, T., Yoon, K.-J., Teifke, P.T., 2012. In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.M., Stevenson, G.W. (Eds.), Diseases of Swine, tenth ed. John Wiley & Sons, pp. 421–446. Pomeranz, L., Reynolds, A.E., Hengartner, C.J., 2006. Microbiology and Molecular Biology Reviews 69, 462–500.

Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus (Caliciviridae) Lorenzo Capucci, Patrizia Cavadini, and Antonio Lavazza, The Lombardy and Emilia Romagna Experimental Zootechnic Institute, Brescia, Italy r 2021 Elsevier Ltd. All rights reserved.

Classification Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) belong to the Lagovirus genus within the Caliciviridae family. RHDV and EBHSV are among the most pathogenic and contagious animal viruses, causing an acute and almost always lethal hepatitis in their main hosts, respectively, RHD in the European rabbit (Oryctolagus cuniculus) and EBHS in brown hare (Lepus europaeus). These viruses cause disease only in adults while animals under 7–8 weeks of age are resistant, as the infection is subclinical. In 2010 a third lagovirus emerged, which is related to RHDV but shows significantly different characteristics, hence the proposed name of RHDV type 2 (RHDV2). Namely, RHDV2 (1) infects and causes RHD in rabbits but also an EBHS-like disease in hares, with a degree of susceptibility that is species dependent; (2) causes RHD also in very young rabbits (2–3 weeks of age onwards); and (3) has a specific antigenic profile that allows to overcome RHDV-induced immunity. Also, during its spread in Europe, RHDV2 became more virulent, approaching that of RHDV. The development and application of serological ELISA for RHD on farmed and wild rabbits led to suspect that under the tip of the viral iceberg (i.e., pathogenic lagoviruses) there were nonpathogenic RHDV-related viruses. Since mid-1990s the employment of genomic techniques allowed the discovery of two main lineages of nonpathogenic lagoviruses, the so-called rabbit caliciviruses (RCVs): RCV-E identified in Europe, and RCV A-1 first identified in Australia but present also in rabbit populations in Europe. RCVs cause subclinical infections, have a tissue tropism for intestinal epithelia cells and behave as enteric viruses. Similarly, a hare nonpathogenic lagovirus, called hare calicivirus (HaCV), was recently discovered in brown hares.

Virion Structure RHDV and EBHSV are spherical particles with an outer diameter of 40 nm, whose structure is defined by characteristic cup-shaped depressions (Fig. 1). The 180 VP60 subunits that comprise the capsid of lagoviruses are organized as 90 dimers, each appearing as an arch-like capsomer. Particles with a different morphology are detectable in liver and spleen of rabbits that died as a result of chronic RHD (see “Clinical features”). This is a smooth particle of around 28–30 nm without the cup-shaped depression and relative protrusions, corresponding to the S domain only. The RHDV capsid is resistant to low pH but it is disassembled to subunits at pH 410. The assembled VP60 is also resistant to trypsin. RHDV VP60 is assembled into three domains, the N-terminal arm, aa 1–65 (NTA), the shell, aa 66–229 (S), the protrusion, aa 238–579 (P), and a short hinge (aa 230–237) that connects S and P. The S domain shares high sequence and structural homology with those of other caliciviruses.

Fig. 1 Electron micrographs of purified RHDV particles. Micrographs show the typical calicivirus morphology (left) and smooth particles (right), constituted only by the S domain of VP60, purified from the liver of a rabbit died due to chronic RHD (right). Negative staining (NaPT 2%) Bar ¼ 100 nm.

724

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20998-9

Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus (Caliciviridae)

725

The P domain consists of subdomains P1 surmounted by the P2. The external part of P2 is formed by seven loops of various lengths. Indeed, the most exposed surface loop L1 (aa 300–314), which exhibits high sequence variations among RHDV isolates, stimulates production of antibodies that can protect rabbits from RHD. X-ray crystallography studies showed that loop 7 contains the key residues of the binding pocket of the histo-blood group antigens, considered anchors/receptors on lagomorph epithelial cells for all lagoviruses (see below). Part of these residues on loop 7 interact also with the protein nuclein, a nuclear protein present also on the surface of cell membranes, indicated as cell receptor for RHDV2.

Genome The genomes of Lagoviruses are composed of single positive stranded polyadenylated RNA B7.4 kb long (Fig. 2). An additional RNA species of 2.2 kb, collinear with the 30 end of the gRNA and designated subgenomic RNA (sgRNA), is also produced during replication. sgRNA usually contributes to the production of high levels of structural proteins required during the intermediate and late stages of infection (e.g., VP60 and VP10). The genomic structure is composed of two slightly overlapping open reading frames (ORFs): ORF1 encodes for a large polyprotein that is cleaved by a viral protease into seven nonstructural (NS) proteins and the major capsid protein VP60; ORF2 produces a minor structural protein, VP10, that might regulate virus replication and virion release from infected host cells. In addition, both genomic RNA and sgRNA are polyadenilated at the 30 end and covalently linked to a small virus-encoded protein (VPg) at the 50 , which is essential for the translation of viral RNA. The genetic similarity between RHDV and EBHSV is approximately 70% for the VP60 protein gene but it increases up to 80% at the amino acidic level. Based on phylogenetic analysis performed with the VP60 coding sequences, the lagoviruses could be divided in two genogroups (GI and GII) that can be subdivided into six genotypes (GI.1, GI.2, GI.3, GI.4, GII.1, and GII.2), further subdivided into variants (four variants for GI.1 and three variants for GII.1) (Fig. 3). Since the identification in 2010 of RHDV2 (GI.2), several complete genome sequences of the circulating strains were obtained and phylogenetic analysis showed frequent recombination events. In particular, two types of recombinants strains were detected in Europe and Australia, both with the structural proteins VP60 and VP10 originating from strains GI.2, and different NS proteins

Fig. 2 Genome organization of lagoviruses. The genome has a VPg protein attached to the 50 end of the gRNA and sgRNA and a 30 poly A tail. The positive sense RNA genome is organized into two ORFs. The ORF1 polyprotein undergoes to autocatalytic processing due to the activity of the 3C-like protease.

Fig. 3 Genetic relatedness of lagoviruses. Phylogenetic tree of lagoviruses including both pathogenic (RHDV, RHDV2, and EBHSV) and nonpathogenic viruses (RCV E1, RCV E2, RCV A1, and HaCV). The coding sequences of the genes encoding VP60 of representative lagoviruses were used to construct the tree. Bootstrap values greater than 60% are shown.

726

Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus (Caliciviridae)

originated respectively from the virulent variant GI.1b (no more circulating in Europe) or the nonpathogenic strain GI.4 identified in Australia. The evolutionary rate of VP60 gene for RHDV has been estimated to be 2.77  10–3 substitutions per site per year and the estimation of the evolutionary time scale places the date of origin of RHDV between 1970 and 1981 and hence only shortly before its first description in 1984. Similar studies performed for RHDV2 circulating in Europe identified a mean substitution rate of 3.93  10–4 substitutions per site per year, and place the RHDV2 origin on July 2008, just two years before its first identification in France. The mean rate of nucleotide substitution for EBHSV ranges from 2.6 to 3.6  10–3 subs/site/year and the estimated time scale places its origin between 1969 and 1980, which fits with its circulation a few years before the original diagnosis in 1980.

Epidemiology The pathogenic lagoviruses independently emerged on at least three distinct occasions. EBHSV and RHDV emerged in the early 1980s, but while EBHS in hares was first noticed in Scandinavian countries, RHD in rabbits was reported in China in 1984 and two years later in Europe. In a few years RHDV caused devastating epidemics worldwide where the European rabbit is present. Few RHD outbreaks occurred in the USA, South America, and Canada where rabbit populations are very scarce. In 1995 RHDV was released into Australia, as a new possible rabbit biological control agent, like the Myxoma virus in the 50s, starting an epizootic that continues to this day. A first antigenic subtype of RHDV, named RHDVa (GI.1a), was identified in Europe in 1996. However, VP60 sequence of RHDVa of China isolates deposited in Genebank, dating back to 1985, confirms that the emergence of RHDV occurred in that country. About 30 years later, the third emergency occurred in Europe, firstly noticed in France both in wild and farmed rabbits that succumbed to RHD although they were vaccinated against RHDV. The distinctive features of RHDV2 favored its rapid and wide diffusion in Europe and Australia, almost replacing RHDV. Particularly, RHDV2 diffused in an almost immunologically naïve rabbit population, with a high RHDV herd immunity largely ineffective against RHDV2. In addition, a broader usage of the HBAGs as anchors to the cells in comparison with RHDV enlarged the potential host susceptibility, further considering that RHDV2 kills also kittens. More recently, RHDV2 outbreaks were registered also in North America in colonies of European rabbits. EBHS is a disease of the brown hare (Lepus europaeus) that occurred in Europe some years before the appearance of RHD. In fact, the earliest confirmed case was from 1981 in Sweden. Thereafter it has been reported in many European countries but never outside Europe. Cases of EBHS are observed also in mountain hare (Lepus timidus) and Italian hare (Lepus corsicanus); however, they were limited to the areas were these species live in sympathy with brown hare. In relation to the presence of RCVs, several studies have been performed in Australia to understand the influence of RCV-A1 on the diffusion of the virulent lagoviruses. As RCV-A1 is antigenically distinct from RHDV and even more from RHDV2, rabbits infected by RCV-A1 are poorly protected against RHD. RHDV and EBHSV spread very quickly and the infection can occur mainly through the oral routes. Transmission could be direct due to exposure to infected or dead animals or indirectly by means of fomites, contaminated vegetation, food, bedding, water, equipment, tools, vehicles, etc. Indeed, considering the stability of lagoviruses in the environment, they could be easily passively transmitted by other animals, like carnivores, scavenger birds, insects (flies), and even humans. The virus is excreted in feces, urine, and respiratory secretions. The virus has been detected also in bone marrow, where it remains infectious for several months, as well as in rabbit meat, where the virus is able to survive freezing. Thus, importation of contaminated rabbit meat from subclinically infected, regularly slaughtered rabbits is considered a likely way of transmission of lagoviruses to new areas/continents.

Clinical Features RHD and EBHS usually appear abruptly and the clinical evolution can be peracute, characterized by sudden death, acute, or subacute/chronic. Peracute and acute forms are prevalent where animal populations are fully susceptible to the diseases. Animals with the acute form survive fairly longer (2–4 days), with signs of dullness/apathy, anorexia and prostration, neurological signs (e.g., incoordination, opisthotonos, convulsions, squeals, and paddling) and respiratory signs (e.g., dyspnea and a terminal, bloodstained, foamy nasal discharge). With respect to RHD, EBHS has a slightly longer course (death occurs at 3–5 days p.i.); the number of hares showing subclinical/chronic signs is higher (30%–50%) and mortality rate is lower (40%–60%) compared with RHD infected rabbits. In wild hares, changes in behavior could be observed (e.g., lack of fear, dullness, jumping into the air, circling, incoordination and convulsion excitement, respiratory distress) before death. Rabbit and hares with the subacute/chronic form of the disease survive longer (over 6–8 days p.i.) and develop severe generalized jaundice, accompanied by weight loss and lethargy. Death usually occurs due to liver dysfunction, but some animals may survive, readily recovering in few days.

Pathogenesis and Host Immunity RHD and EBHS can be experimentally reproduced by inoculation of seronegative adult animals with infected liver homogenates. The oro-nasal route permits to study the natural course of infection and its pathogenesis. In experimentally infected rabbits, RHDV

Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus (Caliciviridae)

727

Fig. 4 Typical pathological lesions associated with RHD. (a) Rabbit died as a result of an acute form of RHD showing frothy, bloody pollution of the nostrils area; (b) lung congestion and edema with multifocal hemorrhages; (c) enlarged, degenerated and discolored liver; (d) spleen congestion and enlargement.

is detected in the epithelial cells of the nasal cavity, nasopharynx, and in the salivary glands at 12–24 h p.i. At 24–48 h p.i. the virus is detected in the hepatocytes, where it progressively replicates, inducing liver necrosis and inflammatory changes until the death of the animal. The virus induces high-level of viremia. In the terminal stages of the disease, disseminated intravascular coagulation and ischemic necrosis in various organs (including the central nervous system), shock, and hemorrhages are observed. In rabbits with subacute/chronic disease (up to 7–10 days p.i.), the viral load is in general lower, and the liver necrosis is focal. Mononuclear phagocytic cells in blood and organs (e.g., liver Kupffer cells) contain viral antigen, according to their role in the viral clearance. At necropsy, both in case of RHD and EBHS, a frothy, bloody pollution of the nostrils area and, in case of subacute/chronic disease, an icteric discoloration in the pinnae of the ears and of external mucosae could be observed. Indeed, the most severe lesions are liver necrosis and degeneration (pale, swollen, friable, yellowish-brown in color, and marked lobular pattern) and spleen congestion and enlargement, accompanied by poor blood coagulation and petechial hemorrhages in almost all organs (Fig. 4). The histopathological changes observed in rabbits infected with RHD and hares with EBHS are similar. These include necrotic hepatitis with multifocal necrosis, fibrosis and calcification (mainly in hares), follicular karyorrhexis and red pulp necrosis of spleen and lymphoid tissue, edema, microthrombi, hyperemia, hemorrhages, leukocyte infiltration and calcification in lungs, trachea, kidneys, and SNC. Young animals infected with either RHDV or EBHSV show only a subclinical infection, with the innate immune system seemingly playing a key role for this distinct clinical outcome as opposed to the lethal hepatitis displayed in adult animals. In fact, rabbit kittens treated with a corticosteroid with pleiotropic actions and then challenged with RHDV developed RHD in 100% of cases. In addition, a recent genome-wide study of liver transcripts from adult and young rabbits showed that kittens infected with RHDV displayed an increased level of expression of multiple genes encoding components of the innate immune response (particularly those associated with MHCII genes) compared with adult rabbits. In contrast, the same genes were downregulated in kittens during RHDV2 infection. The authors proposed a model where the innate immune system of young rabbits is able to respond rapidly to pathogens, including RHDV, whose replication is hindered in liver (very few hepatocytes become infected). Conversely, RHDV2 prevents the innate immune response, replicating at high level also in the liver of kittens, so causing RHD. The final failure of the liver functions, resulting in animal death, occurs a few hours before the appearance in the blood of high levels of virus-specific IgM, which triggers the rapid removal of the virus from the blood stream. The huge quantity of IgM-virus

728

Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus (Caliciviridae)

immunocomplexes is mainly conveyed to the liver and spleen to be cleared, and this could contribute to cause the chronic form of the disease. In addition to specific IgM, also virus-specific IgA is detectable in the serum of rabbits at 3–4 days p.i. Conversely, virus-specific IgG appears in serum 7–8 days p.i., becomes the Ig dominant class in few weeks, and remains at detectable levels for over one year. In case of lagovirus infection in disease resistant animal (i.e., RHDV and EBHSV in young, RCVs in rabbits, HaCV in hares) the kinetic of immunoglobulin classes is similar, but with serum titers 20–40 times lower. Animals vaccinated with inactivated vaccine develop an IgM response in 6–8 days, followed by IgG at 10–14 days with titers similar to those found in rabbits infected by nonpathogenic virus. Specific IgG remain detectable for several months, while usually IgA are not found, presumably because parenteral vaccination of a killed virus does not involve the mucosal immune system, main producer of IgA. Conversely, seropositive rabbits, when infected with a wild type virus develop a transient peak of IgA, detectable in serum for 1–2 months, due to the replication of the virus at the mucosal level. Experimental infections demonstrated that even very low levels of specific IgG protect rabbits from RHD. Altogether, available data indicate that humoral immunity is the main defense mechanism in lagomorphs against lagoviruses-induced diseases.

Diagnosis Both RHD and EBHS are characterized by typical clinical signs as well as by characteristic gross lesions but confirmation by laboratory diagnosis is strongly recommended. Initially, the hemagglutination (HA) test using human red cell type “0” and immunoelectron microscopy were largely used for detection of RHDV but these tests were soon replaced by techniques such as reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Liver and spleen are the best organs to use for diagnosis as they contain very high levels of virus. Hence, ELISA methods using polyclonal or specific monoclonal antibodies are very reliable, being able to detect the agent at high dilutions of the tissue homogenate. For chronic cases of the disease, tests such as HA and ELISA are less reliable due to the presence of high levels of virus-IgM immnocomplexes. Since RT-PCR tests can be positive also in convalescent animals for over two months, in recently vaccinated rabbits and in animals with ongoing not pathogenic RCV infections, false positive RHD or EBHS results are possibly. Consequently, the use of ELISA or of quantitative real time RT-PCR is strongly recommended for a reliable diagnosis of the diseases. ELISA-based methods are commonly used as serology tests for seroepidemiological surveys investigating the prevalence of infection or vaccine efficacy. Detection of lagovirus-specific antibodies is based on three different ELISA-based assays: (1) Classical ELISA (spELISA): the purified virus (or virus-like particles) adsorbed directly to the solid phase. Since adsorption to the plastic causes capsid denaturation with exposure of the internal structure (i.e., the S domain high conserved among all lagoviruses), spELISA is the best method to detect the subset of cross-reactive antibodies. This ELISA is recommended to identify newly infected animals that have not been previously infected by any lagovirus (pathogenic or not). (2) Competition ELISA (cELISA): the virus–antibodies reaction occurs in the liquid phase where the virus is in a native conformation and exposes only external antigenic determinants (i.e., present on the VP60 P2 sub domain). Consequently, cELISA is optimal to detect the subset of specific and protective antibodies in the serum. Testing positive RHDV, RHDV2, and EBHSV sera with homologous and heterologous cELISAs provides a virus-specific antigenic profile. (3) Isotype ELISAs (isoELISA): distinct methods to detect virus-specific IgM, IgA, and IgG. These methods can be used in seroepidemiological studies on wild rabbit populations since they allow the distinction of seropositive rabbits in those who have been recently infected (high IgM titers), those recently reinfected (positive IgA titers) or those with an older infection (only IgG positive).

Prevention and Control Eradication of RHD is unfeasible in regions where wild European rabbits are present and where there are high numbers of small backyard farms, usually lacking biosecurity approaches. Effective measures to prevent RHD could be adopted only in farmed, domestic/pet, or laboratory animals through the application of vaccination programs. Several vaccines for RHDV2 as well as RHDV/RHDVa are commercially available, prepared from clarified inactivated liver suspension of experimentally infected rabbits (Lagovirus do not replicate in in vitro systems) used in conjunction with adjuvants (incomplete mineral oil or aluminum hydroxide). Vaccines prepared with a specific lagovirus (RHDV/RHDVa or RHDV2) are poorly cross-protective against infections by other lagoviruses. Combined vaccination with both antigenic virus strains is highly advisable unless there is a clear understanding of the epidemiological situation of the region where the vaccine is sought. There are no commercial vaccines for EBHS, but when EBHS outbreaks occurs, autogenous killed virus vaccines can be produced from livers of the hares infected at the beginning of the outbreak, to immunize the remaining apparently unaffected hares. Since protection induced by vaccination usually lasts for over 1 year, most vaccine manufacturers recommend a single vaccination, from 25 to 30 days of age onwards, with annual boosts. However, a booster vaccination after 3–4 weeks and then a

Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus (Caliciviridae)

729

further vaccination after 6–12 months depending on epidemiological situation is recommended to ensure a good level of protection. RHD is an OIE-listed disease, hence reporting the disease to veterinary authorities is compulsory in most countries. When an RHD outbreak in a farm is laboratory confirmed and the agent typed, all the surviving animals, including young rabbits, should be immediately vaccinated. In addition, direct prophylaxis actions should be adopted, including cleaning and disinfection of premises, equipment, plants, and instruments; safe removal of carcasses and fomites; and control of movement of people vehicles and animals. A quick response is vital for containing outbreaks in disease-free regions and preventive actions should be extended to rabbit farms surrounding the outbreak.

Treatment Given that immunity starts very quickly, vaccination can also be considered an effective post-exposure emergency measure, especially when a homologous vaccine is used and depending from the farm organization. Serotherapy through the parenteral administration of a homologous hyperimmune antiserum has been shown to be effective in limiting the spread of the disease and reducing economic losses. This short-lived, passively acquired immunity, can protect animals that have not yet developed clinical signs. Other treatments are currently limited to supportive care especially in pet rabbits.

Further Reading Abrantes, J., van der Loo, W., Le Pendu, J., Esteves, P.J., 2012. Rabbit haemorrhagic disease (RHD) and rabbit haemorrhagic disease virus (RHDV): A review. Veterinary Research 43, 12–31. OIE’s Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2012. Chapter 2.6.2. Rabbit Haemorrhagic Disease, seventh ed., version adopted in May 2016. Bárcena, J., Verdaguer, N., Roca, R., et al., 2004. The coat protein of rabbit hemorrhagic disease virus contains a molecular switch at the N-terminal region facing the inner surface of the capsid. Virology 322, 118–134. Cooke, B.D., 2014. Australia’s War Against Rabbits. The Story of Rabbit Hemorrhagic Disease. CSIRO Publishing. Eden, J.S., Read, A.J., Duckworth, J.A., Strive, T., Holmes, E.C., 2015. Resolving the origin of rabbit hemorrhagic disease virus: Insights from an investigation of the viral stocks released in Australia. Journal of Virology 89, 12217–12220. Frolich, K., Lavazza, A., 2007. European brown hare syndrome. In: Alves, P.C., Ferrand, N., Hacklaender, K. (Eds.), Lagomorph Biology. Berlin: Springer, pp. 253–262. Lavazza, A., Capucci, L., 2007. How many calicivirus are there in the rabbit? A review on RHDV and correlated viruses. In: Alves, P.C., Ferrand, N., Hacklaender, K. (Eds.), Lagomorph Biology. Berlin: Springer, pp. 263–278. Le Gall-Reculé, G., Lavazza, A., Marchandeau, S., et al., 2013. Emergence of a new lagovirus related to rabbit haemorrhagic disease virus. Veterinary Research 44, 81–94. Le Pendu, J., Abrantes, J., Bertagnoli, S., et al., 2017. Proposal for a unified classification system and nomenclature of lagoviruses. Journal of General Virology 98, 1658–1666. Lopes, A.M., Breiman, A., Lora, M., et al., 2017. Host specific glycans are correlated with susceptibility to infection by lagoviruses, but not with their virulence. Journal of Virology 92, e017559-17. Lopes, A.M., Capucci, L., Gavier-Widén, D., et al., 2014. Molecular evolution and antigenic variation of European brown hare syndrome virus (EBHSV). Virology 468–470, 104–112. Lopes, A.M., Dalton, K.P., Magalhães, M.J., et al., 2015. Full genomic analysis of new variant rabbit hemorrhagic disease virus revealed multiple recombination events. Journal of General Virology 96, 1309–1319. Neave, M.J., Hall, R.N., Huang, N., et al., 2018. Robust innate immunity of young rabbits mediates resistance to rabbit hemorrhagic disease caused by lagovirus europaeus GI.1 But Not GI.2. Viruses 10, 512–534. Neimanis, A., Larsson Pettersson, U., Huang, N., Gavier-Widén, D., Strive, T., 2018. Elucidation of the pathology and tissue distribution of lagovirus europaeus GI.2/RHDV2 (rabbit haemorrhagic disease virus 2) in young and adult rabbits (oryctolagus cuniculus). Veterinary Research 49, 46–61. Wang, X., Xu, F., Liu, J., et al., 2013. Atomic model of rabbit hemorrhagic disease virus by cryo-electron microscopy and crystallography. PLoS Pathogen 9, e1003132. Zhu, J., Miao, Q., Tang, J., et al., 2018. Nucleolin mediates the internalization of rabbit hemorrhagic disease virus through clathrin-dependent endocytosis. PLoS Pathogen 14, e1007383.

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae) Peter J Kerr, University of Sydney, Sydeny, NSW, Australia and CSIRO Health and Biosecurity, Black Mountain Laboratories, Canberra, ACT, Australia r 2021 Elsevier Ltd. All rights reserved.

Glossary European rabbits Oryctolagus cuniculus: this species evolved in the Iberian Peninsula and southern France. All the domestic and farmed breeds of rabbit including laboratory rabbits are derived from the original wild populations. European rabbits have been spread around the world and established in the wild in Australia and New Zealand as well as parts of the Americas and many islands.

Feral Feral populations are previously domestic animals that have established in the wild. Host-pathogen coevolution The dynamically opposed processes of natural selection acting on a virus to maximize its transmission and the host species to control virus replication. Species-jump The shift of a virus into a novel host-species in which it was not previously established.

Myxoma Virus and the Fibroma Viruses Classification Family: Poxviridae; subfamily: Chordopoxvirinae; Genus: Leporipoxvirus; Species: Myxoma virus (type species); Rabbit fibroma virus (Shope fibroma virus); Squirrel fibroma virus; Hare fibroma virus.

Virion Structure The virions are indistinguishable from those of other poxviruses such as Vaccinia virus: brick shaped approximately 300 × 250 × 75 nm with convex ends and a disordered surface tubular structure. As with other poxviruses, two infectious forms of virus are formed: intracellular mature virions, which in thin section electron micrographs have an external lipoprotein membrane surrounding a nucleoprotein core and two lateral bodies and are released on cell rupture and extracellular enveloped virions that are surrounded by an additional cell-derived lipid envelope containing virus-encoded proteins not present in the intracellular mature form.

Genome Like all poxviruses, the leporipoxvirus genome consists of double stranded DNA with terminal inverted repeats (TIR) and closed single strand hairpin termini. The Lausanne strain is the type sequence of myxoma virus with a genome of 161,777 base pairs (bp) including TIRs of 11,577 bp. There are 158 unique open reading frames defined; 11 of which are duplicated in the TIRs. Full genome sequences have also been obtained for the Kazza strain of rabbit fibroma virus and the MSW strain of Californian myxoma virus.

Life Cycle Myxoma virus replication is similar to that of other chordopoxviruses, the best characterized being the orthopoxvirus vaccinia virus. The key steps in replication are cell attachment and entry followed by early gene expression, uncoating of the viral core, DNA replication, intermediate and late gene expression and viral assembly all of which occur in the cytosol of the infected cell. No specific cell surface receptor has been identified for myxoma virus. Following attachment of the mature virion, uptake is triggered via macropinocytosis, which requires activation of multiple cell-signalling pathways and dynamic actin rearrangements. This delivers the virion into the endocytic pathway. Subsequent fusion of the viral membrane and the endosomal membrane releases the viral nucleoprotein core into the cytoplasm where it is transported to the perinuclear region. Enveloped virus presumably requires different attachment factors and must disrupt its extra envelope membrane to expose the fusion entry complex, 11 highly conserved viral proteins assembled on the mature viral membrane, before entry can occur. This disruption may possibly occur at the cell surface or within the endosome. Gene expression is temporally regulated by viral transcription factors and promoters. Early gene expression occurs within the viral core from packaged transcription machinery. This is followed by uncoating of the genome. Viral DNA replication, intermediate and late gene transcription and gene translation and viral assembly occur in membrane-bound, discrete viral factories that form around the unpackaged core. DNA replication involves formation of long concatemers that are resolved into unit length genomes by the viral Holliday junction resolvase. Intermediate and late gene transcription occurs from the newly synthesised DNA.

730

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20935-7

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae)

731

Viral assembly is a complex process involving membranes, probably derived from the endoplasmic reticulum, that complex with viral scaffold proteins and expand to form crescents that grow into spheroids containing the viral genome and core proteins and called immature virions. Subsequent proteolytic processing, disulphide bond formation and the assembly of surface membrane proteins forms the mature virion consisting of the nucleoprotein core and two lateral bodies enclosed within the viral surface lipoprotein membrane. Mature virions are infectious and released upon cell lysis. Some mature virion particles can acquire an extra double membrane, likely from Golgi membranes, together with additional viral envelope proteins, to form wrapped virions. These wrapped virions are transported to the cell plasma membrane where exocytosis occurs with the loss of one layer of the double membrane to form extracellular enveloped virions which are considered important in cell-cell spread and dissemination within the host.

Epidemiology The natural history of the leporipoxviruses can be considered in two parts. Firstly, in their “natural hosts” and geographic range (Table 1) and secondly, the species jump of the South American type of myxoma virus into a novel host, the European rabbit (Oryctolagus cuniculus), and expansion of its geographic range into Australia, Europe and new areas of South America. With the exception of hare fibroma virus, the known leporipoxviruses are native to the Americas. All appear to have a relatively narrow host range in which the viruses typically cause cutaneous fibromas. Transmission relies on biting arthropods, usually fleas or mosquitoes, which pick up virus on their mouthparts when they probe through the virus-rich epidermis overlying the fibroma. Virus is then passively transmitted from the mouthparts when the vector feeds on another animal. The virus does not replicate in the vector.

Myxoma Virus Myxoma virus causes a lethal disease of European rabbits termed myxomatosis because of the mucinous character of the skin lesions on incision. This disease was first described following an outbreak in rabbits housed in a laboratory in Montevideo, Uruguay in 1896. European rabbits are not native to the Americas but have been widely introduced and are present as both domestic and feral populations with widespread feral populations in Argentina and Chile. Subsequent studies in Brazil showed that myxoma virus in this region most likely circulates in the native forest rabbit or tapeti Sylvilagus minensis (also referred to as S. brasiliensis minensis and S. brasilienis). In this species, the virus induces a localized cutaneous fibroma 1–2 cm in diameter at the inoculation site. Infection appears to have little impact on the host although more generalized disease was reported in juvenile tapetis. The fibroma is cleared by the host immune response within a few weeks. Myxomatosis is predominantly a disease of European rabbits but it occasionally occurs in European brown hares (Lepus europaeus), Iberian hares (L. granatensis) and mountain hares (L. timidus). In addition, North American Sylvilagus species such as S. nuttallii (mountain cottontail) and S. audubonni (desert cottontail) are susceptible to experimental infection with South American myxoma virus and could potentially transmit virus. However, their natural geographic range does not overlap with the South American virus. Myxoma virus has been isolated from European rabbits in Central America and Colombia with some isolates antigenically related to Brazilian and others to Californian myxoma virus. The virus has been deliberately released in Chile and has also spread into Argentina.

Californian Myxoma Virus A virus related to South American myxoma virus circulates in the brush rabbit, Sylvilagus bachmani on the west coast of the United States and Mexico. Hairless, well delineated fibromas 1–2 cm in diameter occur at the base of the ears, on the legs or even the lip. Table 1

The Leporipoxviruses and probable natural hosts

Virus

Natural host

Geographic range

Disease

Myxoma virus

Brazil; Argentina West coast of USA and Mexico

Localized cutaneous fibroma

Rabbit fibroma virus

Forest rabbit (tapeti) (Sylvilagus menensis) in South America Brush rabbit (S. bachmani) in North America eastern cottontail (S. floridanus)

Localized cutaneous fibroma

Squirrel fibroma virus

Grey squirrel (Sciurus carolinensis)a

Eastern and Central North America Eastern North America

Hare fibroma virus

European brown hare (Lepus europaeus)b

Europe

Cutaneous fibroma; may be generalized in young squirrels and affect internal organs Localized cutaneous fibroma

A poxvirus that antigenically cross-reacted with Californian myxoma virus has been described from a fibroma on a Western grey squirrel (Sciurus griseus griseus). An uncharacterized poxvirus has been demonstrated by electron microscopy in fibromas on hares (Lepus capensis) from Kenya.

a

b

732

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae)

More than one fibroma may occur on an individual. Following experimental inoculation, fibromas take several weeks to develop and begin to scab 5 weeks or so after infection before being cleared by the host immune response. Occasionally, fibromas can persist for up to 3 months. Outbreaks of myxomatosis occur in European rabbits on the west coast of the USA and Mexico. Some strains of Californian myxoma virus cause a fulminant form of myxomatosis with death occurring before the typical clinical signs of myxomatosis can develop. The virus appears well adapted to the brush rabbit. Experimentally, other Sylvilagus species may be infected with Californian myxoma virus but titres of virus in the lesions are too low for vector transmission. European brown hares (Lepus europaeus) are susceptible to infection. Brush rabbits could be infected with South American myxoma virus but titres in the lesion were insufficient for mosquito transmission.

Myxomatosis in European Rabbits The lethality of myxomatosis in European rabbits, together with the species-specificity of the virus, suggested that myxoma virus might be used as a biological control for this introduced pest species in Australia. Experimental field trials in 1950 led to the unexpected spread of myxoma virus across a large rabbit-infested area of the south-eastern part of the continent causing enormous numbers of deaths. The virus has circulated in the Australian rabbit population ever since. Subsequently, in 1952, a landowner in France inoculated two wild rabbits with a separate strain of myxoma virus and from this introduction the virus spread and established throughout the wild and farmed rabbit populations of Europe and Britain. As in Australia, massive population crashes occurred. Thus from a relatively obscure origin in the Americas, myxomatosis had emerged as a major disease of domestic and wild rabbits. These separate introductions on two continents initiated one of the best documented examples of host-pathogen coevolution following a species jump by a pathogen. The initially released viruses had case fatality rates in excess of 99% with death typically occurring 8–14 days after infection. This meant that there was only a short time period between developing transmissible virus titres in the lesions and death of the rabbit. With no natural host reservoir, virus was rapidly selected for slightly lower virulence, which allowed the infected rabbit to survive for longer in an infectious state and so increased the likelihood of vector transmission. These moderately attenuated viruses had case fatality rates ranging from 50% to 99%. At the same time the very high population mortality rate meant that resistance to myxomatosis was strongly selected in the wild rabbit populations. Because of the short generation interval in rabbits this resistance could be measured in real-time. At one Australian study site, case fatality rates for a particular virus strain dropped from 90% to 26% over seven generations. So both the virus and the rabbit were undergoing rapid natural selection. Resistance was slower to emerge in Europe and Britain although strong resistance now exists in wild rabbits. The rabbits are not resistant to actual infection but control of virus replication by the innate immune system occurs distal to the inoculation site. Resistance can be overcome by viruses of higher virulence or manipulation of the innate immune response. It is likely that resistance in the wild rabbit population has selected for higher virulence (when measured in non-resistant rabbits) in the circulating virus strains to maintain transmissibility since, unlike in the natural host species, highly attenuated viruses are poorly transmitted from European rabbits.

Clinical Disease in European Rabbits Two clinical forms of myxomatosis are described. A nodular or myxomatous form characterized by a large elevated primary cutaneous lesion at the inoculation site and multiple secondary cutaneous lesions – this was the typical disease seen following the introduction of myxoma virus in Australia and Europe. Subsequently, a second form of disease with minimal cutaneous lesions, sometimes termed amyxomatous, has emerged. In the nodular form, the initial clinical sign is a slight redness and swelling at the inoculation site 2–3 days after inoculation. Elevated rectal temperature 4 40°C can occur from day 4 onwards. Depending on the virus strain, this primary lesion gradually enlarges and may become as much as 2 cm in height and 4–5 cm in diameter by 10–12 days after infection. It sits loosely within the skin and late in infection may erode and ulcerate at the surface with haemorrhage and scabbing. From 4–5 days after infection, the ano-genital area becomes increasingly red and swollen, the eyelids redden at the edges and start to thicken and the ears swell especially at the base. By 8–12 days after infection the head can be extremely swollen with drooping ears and swollen, closed eyelids often accompanied by a mucoid or mucopurulent conjunctival discharge (Fig. 1). Ano-genital swelling can be quite extreme at this stage. Secondary elevated cutaneous lesions up to 2 cm in diameter are present on the body, legs, ears and head. Swollen upper respiratory tract mucosa together with mucoid or mucopurulent discharge in the nasal passages can cause respiratory tract obstruction and dyspnoea. Death typically occurs around this point although the proximate cause of death is unclear. Viruses of lower virulence cause a more protracted although essentially similar clinical course; secondary bacterial infection can be a major cause of mortality in these cases. Inappetence or anorexia and significant weight loss occurs but some rabbits can mount a successful immune response and survive infection. Recovered rabbits have long-term immunity to reinfection. Vector transmission can occur from cutaneous lesions and other sites where high titres of virus occur in the epidermis such as eyelids and base of the ears. Virus is shed in most secretions and contact transmission can occur via conjunctival or respiratory tract inoculation but not orally. In the amyxomatous form, no or minimal cutaneous lesions are seen but otherwise a similar clinical course to the nodular form occurs with swollen head, eyelids and ano-genital region and death occurring 2–3 weeks after infection (Fig. 2). Secondary

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae)

733

Fig. 1 Domestic rabbit with severe nodular myxomatosis. Figure is from: Kerr, P.J., Cattadori, I.M., Rogers, M.B., et al., 2017. Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia. PLOS Pathogens 13, e1006252, under the terms of the Creative Commons Attribution License.

Fig. 2 Domestic rabbit with amyxomatous form of myxomatosis.

bacterial infection may be important in disease outcome. Transmission occurs between rabbits in direct contact but not via aerosol; the role of vectors is unclear. Viruses causing amyxomatous disease in laboratory rabbits have also been described from Australia and Britain. Some of these viruses cause a peracute syndrome manifesting as pulmonary oedema and haemorrhage in multiple tissues 9–16 days after infection. In these rabbits, rectal temperatures were elevated briefly early in infection before returning to the normal range and then often spiking to very high levels shortly before death. The Californian myxoma viruses can also cause a peracute form of myxomatosis with minimal cutaneous lesion development and death in 7–9 days after infection. In these forms of the disease, infected rabbits may simply be found dead and in those animals the early clinical signs may not be noticed.

Pathogenesis of Myxomatosis in European Rabbits Following intradermal inoculation, myxoma virus initially replicates in MHC-II positive cells in the dermis and the dermal-epidermal interface. From here it spreads to infect the cells of the epidermis and to the draining lymph node where it can be detected within connective tissue of the subcapsular sinus and subsequently within paracortical lymphocytes. From the draining lymph node, virus spreads throughout the body, probably within infected lymphocytes as there is no free virus in the blood, to distal lymphoid tissues and cutaneous and mucocutaneous sites such as eyelids. In males, high titres may occur in testis and epididymis. At the inoculation site, keratinocyte hypertrophy and hyperplasia is followed by ballooning degeneration of cells and vesicle formation within the thickened epidermis (Fig. 3). Oedema and the accumulation of large amounts of mucinous material in the dermis causes disruption of the collagen and hair follicles. Few inflammatory cells are seen in the epidermis where most of the virus is present. Deeper in the dermis there may be large numbers of neutrophils particularly around small blood vessels, which are frequently distorted by “myxoma cells,” large stellate cells, probably fibroblasts in origin, which appear to bud through and disrupt the endothelium allowing extravasation of red blood cells. Virus is present in the myxoma cells and within endothelial cells. Late in

734

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae)

Fig. 3 Histopathology of primary lesion. Proliferation and hypertrophy of cells in the epidermis (E) with ballooning degeneration and beginnings of vesicle formation (V). The underlying dermis (D) is disrupted by pale staining mucinous material.

infection, there may be complete loss of the epidermis with formation of a thick scab. Intracytoplasmic eosinophilic inclusions may sometimes be prominent in degenerating epidermal cells but are not always seen in haematoxylin and eosin stained sections. The histological appearance of secondary cutaneous lesions, eyelids and ears is similar to that at the primary lesion but usually less advanced at the time of death. Disruption of lymphoid tissue architecture with depletion of lymphocytes, destruction of follicles and proliferation of reticuloendothelial cells is a major feature of the acute phase of the disease particularly in lymph nodes; the spleen is less consistently affected and even some lymph nodes may have normal areas. Large numbers of neutrophils may be present in affected lymph nodes. In the amyxomatous form of myxomatosis, the lesion at the inoculation site can include minor hypertrophy and hyperplasia of epidermal cells with little deposition of mucin within the dermis and limited disruption of collagen and blood vessels. Despite the limited pathology the titres of virus at these sites can be very high. In the peracute form of the disease, neutrophils are rarely seen in any tissues. However, large numbers of bacteria may be present throughout the tissues, within blood vessels or macrophages but without an obvious inflammatory response. High titres of virus can occur in liver and lung. Myxoma virus is profoundly immunosuppressive. Over 40 genes encode proteins with demonstrated or probable roles in manipulating or suppressing host anti-viral responses. These include proteins that suppress type I and type II interferon responses, inhibit multiple steps in inflammatory pathways, inhibit tumour necrosis factor, inhibit activation of macrophages and T cells, competitively bind pro-inflammatory chemokines, downregulate MHC-I molecules from the surface of infected cells and inhibit cell death pathways. All of these proteins must have evolved in the Sylvilagus host species where the local innate and adaptive immune responses are sufficiently suppressed to allow continued replication at the inoculation site. This is despite the development of an adaptive immune response demonstrated by resistance to superinfection within a few days and neutralizing antibody in the serum. In the European rabbit these proteins facilitate dissemination and generalization of the virus with replication in multiple tissues and ultimately death of the rabbit.

Diagnosis Diagnosis of the acute or chronic nodular forms of myxomatosis in European rabbits is relatively straightforward since the clinical appearance is almost unique. More chronic forms of myxomatosis may be seen in wild rabbits where genetic resistance alters the outcome of infection. In these cases, large raised, grey, hairless swellings can be present over the head, ears and body and the affected rabbit often appears emaciated. At necropsy, in the nodular form the main findings are on the external body and have been described above. Internally, there may be little to see beyond swollen lymph nodes and testes. The spleen may or may not be enlarged. There is usually little or no gross pathology in the lung, liver and other internal tissues. In chronic infections or recovering animals, lymph nodes and spleen may be massively enlarged and significant fat loss and emaciation will be apparent. For the amyxomatous form, the external lesions may be relatively minor although head and ano-genital swelling and some swelling of the eyelids will usually be apparent. In the peracute form, there may be massive pulmonary oedema with swollen, wet lungs and fluid and froth filling the trachea and bronchi (Fig. 4). In some cases there will be visible haemorrhages over the lung surface and through the parenchyma. The liver may be enlarged and firm but this is less consistent. Haemorrhage may also be present in the wall of the appendix and caecum or in the hind legs. Subcutaneous oedema of hind legs and rump is common. Lymph nodes tend to be very small. Differential diagnosis includes Leporid herpesvirus 4, which can also cause swelling of the head, blepharoconjunctivitis and death. The peracute forms of myxomatosis must be distinguished from other causes of acute death such as rabbit haemorrhagic disease, bacterial septicaemias, coccidiosis and poisonings for pest control. Low virulence forms of amyxomatous virus may circulate undiagnosed in rabbit farms and be confused with bacterial respiratory disease.

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae)

735

Fig. 4 Pulmonary oedema in peracute death. Arrow indicates froth filling the trachea which has been opened along its length.

In the acute stage of infection high titres of virus are present in lymph nodes, eyelids, testis and cutaneous lesions. At later stages, cutaneous lesions and eyelids are a more reliable source of virus. Conjunctival swabs are a readily collected source of virus for diagnosis from the live rabbit. Polymerase chain reaction (PCR) using DNA extracted from tissues or conjunctival swabs is the simplest and fastest means to confirm diagnosis. Electron microscopy can demonstrate typical poxvirus particles. Myxoma virus can be readily cultured in cell lines such as RK-13. In these cells, virus replication causes raised proliferative foci of cells with formation of multinucleate syncytia followed by monolayer destruction. The virus remains predominantly cell associated. Myxoma virus can also be grown on the chorioallantoic membrane of embryonated chicken eggs. Virus identity can be confirmed by PCR or immunostaining. The histology of skin lesions is reasonably characteristic. Immunostaining with specific antibody can be used on impression smears or frozen or fixed histological sections to detect myxoma virus. Serum antibodies to myxoma virus can be detected as early as 8–10 days after infection by ELISA or virus neutralization assay.

Treatment There is no specific treatment for myxomatosis. Many wild rabbits survive infection; some domestic rabbits infected with attenuated viruses will recover although illness may be prolonged for several months. General supportive care with attention to hydration and nutrition may assist survival. Antibiotics may be needed to control secondary bacterial infection.

Prevention of Myxomatosis Vaccination and quarantine are the main means of preventing myxomatosis in domestic and farmed rabbits. Three types of live vaccine are available. No inactivated vaccines have been successfully developed. (1) Heterologous live vaccines. Infection with rabbit fibroma virus provides cross-protection from myxoma virus. Following the spread of myxoma virus in Europe, vaccines based on rabbit fibroma virus were widely used in farmed and domestic rabbits and attempts also made to vaccinate wild rabbits. (2) Attenuated live myxoma virus vaccines. These have been developed by serial passage of virulent myxoma viruses in cell culture. Examples include the Borghi strain based on the MSD strain of Californian myxoma virus and the SG-33 strain based on an attenuated field strain from France. (3) Recombinant live myxoma virus. This vaccine was released commercially in 2012 and expresses the Rabbit haemorrhagic disease virus type 1 (RHDV-1) capsid protein providing dual protection against both myxomatosis and rabbit haemorrhagic disease due to RHDV-1 (but not RHDV-2). Insertion of the RHDV capsid protein gene into the myxoma virus genome has been done such that critical virulence genes have been disrupted, producing an attenuated myxoma virus vaccine. Vaccination is not permitted in Australia. Introduction of myxoma virus into rabbit farms has occurred in new stock incubating the virus even though these were reportedly vaccinated. Vaccination and quarantine of any introductions should be routine. In countries where myxoma virus circulates in wild rabbits, protection from mosquitoes and fleas is critical to protect unvaccinated rabbits. Rabbits suspected of having myxomatosis should be culled or isolated until diagnosis is confirmed. Virus can be readily transmitted between in-contact rabbits and on contaminated water bottles or feeders. The virus is potentially transmitted in semen from infected males.

736

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae)

Rabbit Fibroma Virus Rabbit fibroma virus circulates in eastern cottontails, Sylvilagus floridanus, which are widely distributed in the eastern and central United States, north into Canada and south into Central America. In the eastern cottontail, rabbit fibroma virus causes cutaneous fibromas, 1.5–2 cm in diameter, appearing as a raised hairless dome or flat plaque, most often on the feet or legs and not firmly attached to underlying tissues. More than one fibroma may be present. It takes 3–4 weeks after inoculation for the fibroma to mature into a readily transmissible form with high titres of virus in the surface epidermis. In immunologically mature cottontails the fibroma is cleared by the immune response after 6–8 weeks. In very young suckling cottontails fibromas can grow extremely large and cause death with virus also present in internal tissues. When slightly older kittens are infected, the fibroma can persist in an infectious state for as long as nine to ten months, despite the presence of neutralizing antibody in the serum and resistance to superinfection within a few days of the initial infection. This persistence would allow overwintering in the absence of mosquito vectors. Histologically, in cottontails the fibroma consists of collagen, fibroblasts including stellate “fibroma cells” and fibrocytes. The overlying epidermis is thickened with hyperplasia, hypertrophy and degeneration of keratinocytes. Pegs of epidermis extend deep into the fibroma. Eosinophilic intracytoplasmic inclusions may be present in degenerating keratinocytes. Neutrophils and occasional lymphocytes are present within the fibroma while the underlying tissue can be massively infiltrated with lymphocytes. In European rabbits, inoculation of rabbit fibroma virus induces a cutaneous fibroma at the inoculation site. This persists for 2–3 weeks before clearance by the immune response. In immunosuppressed or immunologically immature suckling rabbits more generalized fibromatosis and death can occur. Rabbit fibroma virus is rarely able to be experimentally transmitted by vectors from European rabbits, despite reaching very high titres in the fibroma. Localized outbreaks of rabbit fibroma virus occur in domestic European rabbits housed outdoors in the United States presumably spread by mosquitoes from local cottontail populations. Rabbit fibroma virus can be cultured in cell-lines such as RK-13 where it causes similar cytopathic effect to myxoma virus.

Squirrel Fibroma Virus Squirrel fibroma virus affects juvenile grey squirrels, Sciurus carolinensis in the eastern United States. Affected squirrels may have a few well-separated cutaneous fibromas or generalized fibromatosis with dozens of coalescing fibromas covering the body and nodules in lung, liver, kidney, spleen and lymph nodes. Fibromas range from a few mm to 3 cm in diameter. This highly variable host response was confirmed experimentally with some suckling squirrels developing generalized disease following inoculation while others in the same litter only had a single fibroma at the inoculation site. Adult squirrels appear refractory to infection: experimentally only 50% developed short-lived nodules at the inoculation site and seroconverted. Histologically, the fibromas resemble those caused by rabbit fibroma virus in cottontails. Woodchucks (Marmota monax) can be infected experimentally although young animals are easier to infect than adults. Thickened nodules develop in the skin and subcutaneous tissues at the inoculation site. Squirrel fibroma virus replicates poorly in European rabbits and does not replicate in eastern cottontails. Squirrel fibroma virus can be cultured in primary squirrel kidney cells and rabbit embryo fibroblasts. It replicates poorly in primary rabbit kidney cells and does not replicate in chick embryo fibroblasts. Fibromas have also been described in fox squirrels (Sciurus niger) and porcupines (Erethizon dorsatum) from the eastern USA but these were not further characterized. A poxvirus, which cross-reacted antigenically with Californian myxoma virus, has been reported from a western grey squirrel (Sciurus griseus griseus). However, the virion morphology resembled a parapoxvirus. Squirrel fibroma virus is distinct from squirrel poxvirus, which occurs in grey and red squirrels (Sciurus vulgaris).

Hare Fibroma Virus Hare fibroma virus causes cutaneous fibromas in European brown hares (Lepus europaeus). Little is known of the natural history of the virus, although occasional large outbreaks are recorded. One or more fibromas 1–3 cm in diameter raised above the skin are seen, usually on feet or ears. In most cases these scab and resolve over 1–3 months. Outbreaks have been reported in Germany, France and Italy. Hare fibroma virus replicates poorly in European rabbits but will form subcutaneous nodules in new born animals. It forms pocks on the chorioallantoic membrane of chicken embryos and plaques on primary rabbit kidney cell monolayers but does not replicate in RK-13 cells. Fibromas containing poxvirus particles have also been described in cape hares (Lepus capensis) from Kenya. Unfortunately, further characterization of this virus was not possible.

Further Reading Cameron, C., Hota-Mitchell, S., Chen, L., et al., 1999. The complete DNA sequence of myxoma virus. Virology 264, 298–318. Fenner, F., Fantini, B., 1999. Biological control of vertebrate pests. The history of myxomatosis – An experiment in evolution. New York: CAB International.

Rabbit Myxoma Virus and the Fibroma Viruses (Poxviridae)

737

Fenner, F., Ratcliffe, F.N., 1965. Myxomatosis. Cambridge: Cambridge University Press. Grilli, G., Piccirillo, A., Pisoni, A.M., et al., 2003. Re-emergence of fibromatosis in farmed game hares (Lepus europaeus) in Italy. Veterinary Record 153, 152–153. Kerr, P.J., 2012. Myxomatosis in Australia and Europe: A model for emerging infectious diseases. Antiviral Research 93, 387–415. Kerr, P.J., Cattadori, I.M., Liu, J., et al., 2017. Next step in the ongoing arms race between myxoma virus and wild rabbits in Australia is a novel disease phenotype. Proceedings of the National Academy of Sciences of the United States of America 114, 9397–9402. Kilham, L., Herman, C.M., Fisher, E.R., 1953. Naturally occurring fibromas of grey squirrels related to Shope’s rabbit fibroma. Proceedings of the Society for Experimental Biology and Medicine 82, 298–301. Shope, R.E., 1932. A transmissible tumour like condition in rabbits. Journal of Experimental Medicine 56, 783–802. Spiesschaert, B., McFadden, G., Hermans, K., Nauwynck, H., Van de Walle, G.R., 2011. The current status and future directions of myxoma virus, a master in immune evasion. Veterinary Research 42, 76. Willer, D.O., McFadden, G., Evans, D.H., 1999. The complete genome sequence of Shope (rabbit) fibroma virus. Virology 264, 319–343.

Relevant Website http://www.oie.int/en/animal-health-in-the-world/animal-diseases/Myxomatosis/ Myxomatosis: OIE - World Organisation for Animal Health.

Rabies and Other Lyssaviruses (Rhabdoviridae) Ashley C Banyard, Animal and Plant Health Agency, Addlestone, United Kingdom; University of West Sussex, Falmer, United Kingdom; and St. George's Medical School, University of London, London, United Kingdom Anthony R Fooks, Animal and Plant Health Agency, Addlestone, United Kingdom; University of Liverpool, Liverpool, United Kingdom; and St. George's Medical School, University of London, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary Anteriograde Directed or moving forward. Apoptosis The death of cells which occurs as a normal and controlled part of an organism’s growth or development. Autophagy It is a normal physiological process in the body that deals with destruction of cells in the body. It maintains homeostasis or normal functioning by protein degradation and turnover of the destroyed cell organelles for new cell formation. During cellular stress the process of autophagy is upscaled and increased.

Paraesthesia An abnormal sensation, typically tingling or pricking (‘pins and needles’), caused chiefly by pressure on or damage to peripheral nerves. Pleomorphic A variable appearance or morphology. Pruritis An itching sensation. Retrograde Directed or moving backwards. Sylvatic rabies The sylvatic rabies disease is transmitted by wildlife, particularly foxes and wolves.

Classification (Compact) Lyssaviruses are classified within the family Rhabdoviridae within the order Mononegavirales. Within the lyssavirus genus, there are 16 different viral species, classified as separate entities according to divergence across genomic sequence. The genus currently includes: Aravan lyssavirus (ARAV), Australian bat lyssavirus (ABLV), Bokeloh bat lyssavirus (BBLV), Duvenhage lyssavirus (DUVV), European bat −1 lyssavirus (EBLV-1), European bat −2 lyssavirus (EBLV-2), Gannoruwa bat lyssavirus (GBLV), Ikoma lyssavirus (IKOV). Irkut lyssavirus (IRKV), Khujand lyssavirus (KHUV), Lagos bat lyssavirus (LBV), Lleida bat lyssavirus (LLEBV), Mokola lyssavirus (MOKV), Rabies lyssavirus (RABV), Shimoni lyssavirus (SHIBV) and West Caucasian bat lyssavirus (WCBV). These 16 viruses are accepted by the International Committee on Taxonomy of Viruses (Fig. 1). Two further novel lyssaviruses, Kotalahti bat lyssavirus (KBLV) and Taiwanese Bat Lyssavirus (TBLV), have been reported and group genetically within the lyssavirus genus but remain as tentative species until fully characterised.

Virion Structure All lyssaviruses exhibit a bullet-like virion morphology with an envelope derived from the plasma membrane of the infected host cell. One end of the bullet shape is conical, the other flat with approximate dimensions of B75 nm and a length of B180 nm, depending on the virus.

Genome The lyssavirus genome consists of a single negative stranded (−) RNA molecule, consisting of approximately 12,000 nucleotides that encode 5 viral proteins with a conserved gene order; nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and large (L) polymerase protein. The N, M and L proteins are conserved in structure and length across each of the lyssaviruses, whilst the P and G proteins vary in length. The conserved gene order has been proposed to have a role in the control of protein expression. The genomes of lyssaviruses are encapsidated in their entirety with the N protein and as such the viral RNA is protected from host cell based degradation. This encapsidated state also means that replicative mechanisms that might result in genetic recombination are considered to be unlikely although they have been reported in isolated studies.

Life Cycle In the host, lyssaviruses infect peripheral nerves, often via muscle cells, at the motor endplate of neuromuscular junctions. The virus binds to a cellular receptor and enters endosomal transport vesicles via receptor-mediated endocytosis. Several cellular receptors have been proposed including the nicotinic acetylcholine receptor (nAChR), neuronal cell adhesion molecule (NCAM) and the p75 neurotrophin receptor (p75NTR). Once in neurons the virus travels in a retrograde fashion utilising microtubules

738

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20936-9

Rabies and Other Lyssaviruses (Rhabdoviridae)

739

Fig. 1 Phylogenetic analysis of globally circulating lyssaviruses.

along axonal processes. Primary transcription occurs, leading to the production of viral proteins, after ribonucleoproteins (RNPs), a viral unit consisting of the viral RNA, N and P are released into the cytoplasm of the infected cell. Primary transcription initiates when the polymerase complex binds to the genome promoter at the 3′ proximal end of the infecting genome. N, P and L interact in as yet undefined ratios to initiate primary transcription. As the transcriptase complex processes nascent mRNA it stutters at distinct intergenic boundaries, with reinitiation of transcription only occurring through binding at the 3′ terminus of the negative sense genome. This results in a transcriptional gradient whereby the 3′ proximal genes are produced to the highest levels with downstream genes being expressed at consequtively lower levels as the genome is traversed by the transcriptse complex. Following round of primary transcription, at some point during infection, the viral transcriptase complex switches to a replicative mode that enables the viral machinery to transcribe through the intergenic control regions present at each gene boundary to generate a full length antigenome positive sense (+) RNA. This positive sense RNA is a replicative intermediate that is fully encapsidated by N and serves as the template for nascent negative sense genome RNA synthesis. This secondary transcription occurs where viral RNA synthesis occurs in inclusion bodies (IBs), sites where viral proteins accumulate. As new RNPs are formed within IBs, they are transported to post-synaptic membranes where the nascent virions are assembled and transmitted in a process that depends on the presence of viral G on the budding membrane. Moving transynaptically through the peripheral nervous system the virus eventually enters the central nervous system (CNS) and once in the brain, massive viral replication occurs causing clinical disease. From the brain the virus then spreads centifugally being excreted in saliva facilitating the onward transmission of the virus.

Epidemiology Human rabies is a neglected zoonosis, especially in countries where the virus is endemic such as across Africa and Asia. In endemic areas, it is estimated that a human fatality from rabies occurs every 10–20 min, with 40%–50% of the victims being children less than 15 years of age. Children are thought to be disproportionately affected because of their small size and frequent interactions with domestic or free-roaming dogs. In endemic countries, it has been proposed that rabies be considered a neglected paediatric disease. The estimated annual 59,000 deaths worldwide do not capture the true burden of this disease. A profound lack of surveillance due to limited health care services, including an absence of laboratory capabilities and both medical and veterinary

740

Rabies and Other Lyssaviruses (Rhabdoviridae)

infrastructures, results in under-reporting, which is further confounded by cultural, religious and social taboos. Furthermore, human rabies can present with symptoms that are common to other clinically indistinguishable conditions (for example, Guillain–Barré syndrome and cerebral malaria) and as such, in the absence of laboratory confirmation of disease, rabies is likely misdiagnosed. Consequent inaccuracies in reporting the burden of disease means that rabies is often not considered a high priority disease in endemic settings. Lyssaviruses are capable of infecting all mammalian species. The development of serological techniques and later genetic methods of virus characterisation led to the understanding that multiple lyssaviruses exist that have been characterised as 16 distinct viral species. Whilst the archetypal lyssavirus, RABV, is most commonly associated with dogs, its evolution is though to originate in bats and most lyssavirus species are now thought to be maintained by Chiropteran hosts with few exceptions. Where multiple isolates have been described, the chiropteran hosts and geographical range are relatively well documented. However a number of lyssaviruses have been characterised following only a single isolation and so their epidemiology and true association with different bat species remains speculative. It is likely that RABV evolved from a bat-associated progenitor and that numerous cross-species transmission events from bats to terrestrial carnivores resulted in the distribution of closely related but genetically distinct classical rabies viruses described. Across the globe, seven primary lineages of RABV have been described and within each lineage, numerous variants often described as causing clinical disease within a particular mammalian host across a certain geographical range, have emerged. The most widespread lineage has been described across several continents including Europe, the Americas, Africa and Asia, being maintained by both dog populations as well as by several terrestrial wildlife species including foxes, raccoon dogs, skunks and jackals. Classical rabies is found regularly in bat species across the Americas, with over 40 species of bat being reported as potential transmitters of disease to the human and terrestrial carnivore populations. From an economic perspective the vampire bat rabies lineage causes rabies within livestock, as well as the human population, and as such is considered of great economic significance in areas of vampire bat habitation. Genetic analyses of RABV across the globe have demonstrated that natural landscape features such as mountain ranges and waterways are frequent barriers to epizootic spread. Man-made roads and human mediated movement of animals, often accompanying large-scale human migrations can, however, facilitate disease dispersion. Habitat fragmentation may also affect the epidemiology of the disease and spread of the virus. Genetic characterisation has also demonstrated where human translocation of animals has introduced rabies lineages in to new communities of both human and animals. The latter is particularly evident where the virus has then established a cycle of infection within a new geographical location. The use of vaccination campaigns, parenteral for domestic dog populations, and oral for wildlife populations has eliminated dog rabies across Europe, the United States and Canada and substantially reduced its prevalence in Latin America, with a resulting decrease in incidence of human rabies in these areas. Within Europe there are six lyssaviruses, all are considered pathogenic and capable of causing the disease, rabies: the European Bat Lyssaviruses (EBLV-1 and EBLV-2) that circulate in Chiropteran hosts, the latter being endemic within UK bat populations; BBLV that has been detected in Natterer’s bats in Germany and France; Kotohlahti Bat lyssavirus for which only genetic evidence has been reported in a Brandt’s bat in Finland; WCBV isolated from a Bent Winged bat in the Caucasus mountains and LLEBV isolated in Bent winged bats in both Spain and France. WCBV and LLEBV are both genetically, highly divergent from all other European lyssaviruses. Alongside European viruses, three Eurasian viruses have been characterised, all isolated from bats; Aravan Virus (ARAV) in Kyrgystan, Khujand (KHUV) Virus isolated in Tajikistan and Irkut Virus (IRKV) was isolated in Eastern Siberia. Across the rest of the Old World, several other lyssaviruses have been described including within Africa: LBV isolated from a number of different bat species across sub-Saharan Africa; MOKV which is prevalent across Africa and has been associated with two recorded human cases; DUVV, that was initially isolated from a fatal human bat bite case in Kenya in 1971 and subsequently from a number of fruit bats in South Africa; and SHIBV that was first isolated in 2009 from a Commerson’s leaf-nosed bat in Kenya. Most recently, IKOV was isolated from an African Civet in Tanzania in 2010 and although discovered in a terrestrial carnivore is believed to be of bat origin. Within Australasia, only Australian Bat Lyssavirus (ABLV) has been reported and has been isolated from five different bat species since its initial isolation in 1996. In Asia only GBLV has been reported in Sri Lanka in Fruit bats in 2014 and 2015.

Clinical Features Human and animal rabies are often characterised into two classical forms: furious (often termed encephalitic) and paralytic. The factors determining the progression into either form are not understood, and whilst some specific symptoms are often reported, case definition can only generally be concluded during the neurological phase (Fig. 2). Incubation periods vary, although most patients develop symptoms between 20 and 90 days following exposure, however, there have been reports of extreme cases of incubation periods ranging between 14 and 19 years although the possibility of a secondary infection cannot be eliminated in such reports. Regardless, it is clear that the incubation period following infection can vary greatly depending on a range of factors. Due to the neurotropic nature of the virus, a bite/site of infection that is highly innervated (e.g., face and hands) can result in a reduced incubation period or faster progression of disease than following infection at poorly innervated sites. Alongside this, the presence/ absence of permissive receptors in the tissue surrounding the infection site can affect viral entry and spread, hence increasing the incubation period. The dose of infective virus also plays a role in determining incubation period as does the immune status and age of an individual infected. Certainly, incubation periods in paediatric cases are shorter than those in adults, presumably due to their

Rabies and Other Lyssaviruses (Rhabdoviridae)

741

Fig. 2 Clinical disease progression in rabies.

smaller stature. Incubation periods following exposure to other lyssavirus are very poorly defined as exposure times are often difficult to determine. In particular it is recognised that often bat bites may go totally unnoticed leading to cryptic cases of rabies. A case of EBLV-1 infection in a human recorded an incubation period of 45 days. Further to this, a case of EBLV-2 infection in a bat handler reported a 19 week incubation period, however, the patient had a documented history of bat bites which may have confounded determination of the incubation period. DUVV infection has been reported to have an incubation period of around 4 weeks whereas ABLV may have a much longer incubation period as a case in 1998 reported onset of clinical disease 20 months after exposure to a bat. Within the chiropteran host, even more protracted prodromes have been described with a bat in a rehabilitation center developing clinical disease 9 months after being received and testing positive for EBLV-2. From a human perspective, initial symptoms occur as the virus initiates replication in the dorsal root ganglia of neurons at the bite site. These symptoms can include pain, paraesthesia and pruritus which then develop to the neurological phase often characterised by more pronounced clinical signs. Where furious rabies develops clinical disease can include transient agitation, hypersalivation and hydrophobia whilst the paralytic form of disease can manifest with muscle weakness leading to paralysis. Regardless, both forms result in to coma and death with no treatments identified that can halt the progress of disease once clinical signs have developed.

Pathogenesis The most common route of infection with a lyssavirus is via the mechanistic action of a bite from an infected animal which contains live virus in its saliva. Historically, this mechanism was first described 200 years ago by experimental work in 1804 by Zinke. Alongside this early observation, it was later determined that these viruses are unable to cross an intact dermal barrier. However, if the skin is broken, the risk of infection is greatly increased. Lyssaviruses can also infect hosts via mucous membranes including the nasal lining, oral cavity, conjunctivae, external genital organs and the anus. Rare reports of airborne transmission have not been scientifically corroborated. Human-to-human transmission has been reported very rarely with transmission of bodily fluids being implicated as the source of virus. Alongside this, there have been occurrences of infection through transplantation of infected tissue. A case in 2004 resulted in three rabies fatalities when an infected donor supplied a liver section, a kidney and a section of artery to three separate recipients. Within 30 days of transplantation all three recipients had returned to the hospital with rapidly progressing neurological disease, which was diagnosed post mortem as rabies infection. A further case in Germany resulted in the deaths of three individuals following organ donation and corneal transplantation has also facilitated infection in the recipient following surgery. Whilst it is appreciated that lyssaviruses are highly neurotropic, the mechanism behind viral entry of neurons at peripheral inoculation sites remains largely undefined. The location of nicotinic acetylcholine receptors (nAChR) in the vicinity of post-synaptic membranes on muscle cells may support infection prior to neuronal invasion. It is then thought that virus replication in the musculature and consequent virus egress into synaptic clefts may facilitate neuroinvasion via receptors at pre-synaptic axonal membranes. Once inside a cell the virus encounters host immune effector mechanisms that attempt to

742

Rabies and Other Lyssaviruses (Rhabdoviridae)

clear the infection. Critically, RABV virulence resides in the viruses ability to evade the localised immune response alongside retaining the viability of the neurons it has invaded following infection. The demonstration of immune effector cells within the CNS have suggested that RABV replication in the CNS occurs in the face of a fully immunocompetent cellular environment although it has long been stated that the immunocompromised nature of the CNS enables ease of virus replication. From a host response perspective, experimental models have demonstrated that wildtype and fixed rabies strains induce different inflammatory markers both in the periphery and within the CNS. Critically, virus clearance from the brain, that predominantly correlates with an increase in B cell infiltration and antibody production in the CNS, is reduced during infection with pathogenic RABV isolates. This supports the concept that adaptive immune responses are impaired in natural RABV infections. Further, blood–brain barrier permeability to the infiltration of immune cells is important for successful virus clearance, with enhanced permeability being associated with an increase in viral clearance of attenuated RABV in experimental models. Canine models of infection have suggested that in line with the pathogenicity observed, the immune response in the CNS is limited with wildtype RABV infection having a minimal effect on the permeability of the blood brain barrier. Certainly, permeability of the blood–brain barrier is of vital importance for viral clearance. Interestingly, virusneutralizing antibodies (VNAs) have been reported in sera and cerebral spinal fluid during the clinical stages of human infection but appear unable to clear virus alone. Patients that develop neutralizing antibodies early during the disease course are considered potentially favorable candidates for aggressive treatment of the disease. Interestingly, almost all reported human rabies survivors were diagnosed on the basis of neutralizing antibodies in CSF or serum with no other viral products being detected in submitted samples. Immune effectors appear restricted by elements of the viral protein repertoire including poor induction of host type I IFN responses, apoptosis of induced antigen-activated T-cells and reduced T cell proliferation and apoptosis. A further mechanism by which RABV may replicate alongside minimal pathological changes has been proposed through M protein influenced autophagy. This may enable preservation of brain and spinal cord function despite abundant viral replication. Apoptotic responses appear to be common following experimental infection with attenuated viruses.

Diagnosis Ante-mortem laboratory tests can only confirm infection: a negative ante-mortem test result cannot be used to rule out infection. Notably, no clinical feature is pathognomonic for rabies. Hydrophobia or aerophobia (fear of drafts or fresh air) provide a strong indication of RABV infection but laboratory diagnosis is always required to confirm rabies. A number of diagnostic tests are available to diagnose RABV infection but the use of any given test depends on the laboratory capability of the local infrastructure to support diagnostic testing (Fig. 3). Typically, rabies diagnosis is performed on post mortem material. However, where available, clinical rabies can be diagnosed ante-mortem with testing of either nuchal (nape of the neck) skin biopsies or saliva enabling detection of viral RNA.

Fig. 3 Rabies diagnostic testing in laboratories with capacity to perform recommended tests. Samples are submitted from a variety of sources. The frontline test on post mortem material is the FAT. The RTCIT, and where unavailable the MIT, are used as secondary confirmatory tests. In many laboratories, molecular tools such as PCR are used as a more rapid secondary test platform.

Rabies and Other Lyssaviruses (Rhabdoviridae)

743

Live virus has been occasionally isolated from human saliva and salivary excretion can be detected before the onset of clinical disease in experimental canine models. Molecular testing using RT-PCR has proven to be a rapid, highly sensitive and specific tool for ante-mortem diagnosis. However, testing saliva samples should involve testing multiple samples as intermittent secretion of the virus into this fluid means that negative results does not prove absence of infection. The invariably fatal nature of rabies infection means that almost all laboratory confirmations of rabies are made post mortem. Samples of brain tissue are optimal for post mortem diagnosis with the fluorescent antibody test (FAT) detecting virus antigen in brain material as the diagnostic test recommended by both the WHO and the OIE. Efficient diagnosis by FAT depends on the quantity and quality of the sample in terms of autolysis and decomposition. In addition, the requirement of expensive equipment and secondary antibody conjugate to detect virus antigen does not allow to use FAT in resource limited settings where, in most cases, the virus is endemic. The development of the direct “rabies immunoperoxidase test” (dRIT) has overcome the problems associated with conducting FAT in resource limited settings. Other antigen detection tests, including lateral flow devices (LFDs) have been developed but require further development and validation before being useful as diagnostic for an invariably fatal disease. Molecular tools, such as reverse transcription-polymerase chain reaction (RT-PCR) have been developed that have many advantages over traditional tests and, through the highly sensitive and specific detection of viral nucleic acid, are slowly replacing classical antigen based assays. One-step closed tube RT-PCR systems have been developed that dramatically reduce the risk of contamination. RT-PCR assays that can detect RNA of many lyssaviruses are available and are replacing the exiting confirmatory tests for rabies diagnosis in animals. In comparison to the FAT, RT-PCR methods are less prone to subjective interpretation and have the added benefit that they can identify the virus species, following sequencing of the derived amplicon, a useful tool in cases with undefined history of potential exposure events. Molecular assays are now recognised by the OIE; however, these tests need to be performed in well controlled environments to avoid false positive results and, as a consequence, the standardization of the assays and utilization of appropriate controls are required before these tools can be used for routine post mortem diagnosis of rabies in animals in resource-limited settings (WHO, 2013). Most post mortem diagnoses are currently based on the previously recommended techniques including FAT, tissue culture isolation and mouse inoculation tests: adoption of new reliable and validated tools (dRIT, RT-PCR) have improved diagnostic options for rabies diagnosis in endemic areas where empirical data are lacking and the burden of the disease is disproportionately high. The use of serological assays for detection of a current rabies infection is generally limited to post-vaccinal assessment of serostatus. The OIE recommends the rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralisation (FAVN) test for monitoring of antibodies to allow both international travel in companion animals and evaluation of human vaccination. Both of these tests measure the ability of antibodies in suspect sera to neutralise live virus. Once the virus is applied to the sera, fluorescent antibody staining is used to detect any virus that has not been neutralised by the sera. In order to quantify the virus neutralizing antibody, standardised reference sera from the WHO and OIE are used as a comparison. These tests are widely considered as the gold standard in serological assays but they have some logistical restrictions as the use of live virus requires containment facilities which may not be available in resource limited settings. This has lead to efforts to develop serological assays that do not require the use of live virus. Novel enzyme-linked immunosorbent assay (ELISA) techniques are available that are capable of the detection of rabies specific antibodies in serum from both humans and animals. However, the current ELISAs have a major drawback in that they are unable to conclusively show the presence of neutralizing antibodies within a sample.

Treatment No tools exist to prevent the clinical phase of lyssavirus infection and so the development of antivirals for rabies remains an urgent priority. Prevention of rabies disease is best achieved through multiple activities including: preventing transmission from dogs to humans via dog vaccination campaigns; promoting awareness of rabies to avoid exposure; ensuring availability of post exposure rabies immunoglobulin and vaccine; and encouraging responsible dog ownership. It is anticipated that dog-mediated human rabies will be confined to history with the goal of eliminating transmission of the virus between dogs and humans targeted for 2030. For this target to be achieved improved diagnostic tools, improved veterinary and medical infrastructures, cheaper and efficacious biologicals (vaccines, new antivirals and HRIG), and increased education and awareness of rabies is required. Where disease has developed the outlook for treating clinical rabies remains poor. Despite reports of promising molecules from reports of in vitro studies, following attempts with the same molecules in vivo, no molecules have shown sufficient promise to warrant further investigation. However, developing novel antiviral treatments remains a major challenge as both viral factors, including the neurotropic nature of the virus, and host factors, such as variable stages of presentation, exposure history and immune response, preclude progress. Any candidate molecules need to be able to cross the blood–brain barrier, disrupt and potentially halt virus replication without negatively impacting on host neurons and also inhibit detrimental host responses to RABV infection. Strategies reported have included ultrasound and microbubbles, which have been used to try and increase permeability of the BBB to facilitate the entry of therapeutics to the optimal site for action. Other tools, such as small interfering RNA (siRNA) or microRNAs (miRNA) that bind to and target RNA species for degradation within the cells have demonstrated some suppressive activity on virus replication in vitro. Some broad spectrum antiviral molecules incliding Favipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) have demonstrated an in vitro effect although again in vivo studies have shown limited use of these molecules. Other

744

Rabies and Other Lyssaviruses (Rhabdoviridae)

delivery methods including stable nucleic acid lipid particles and nanoengineered particles with si-RNAs or miRNAs are being assessed for utility as rabies antiviral molecules. The urgent need for rabies antiviral treatments means that if any benefit can be demonstrated with drugs licensed for human use in experimental models, there is good rationale for assessing them in human rabies cases where consent is given.

Prevention Despite the high number of annual human deaths from rabies, the disease can be readily prevented with the use of vaccines. Rabies vaccines have been available since 1885, when Louis Pasteur established that immunisation with desiccated nerve tissue from an infected rabbit prevented disease in a young boy who had been attacked by a rabid dog. Since the dawn of vaccination, safe and efficacious rabies vaccines have been developed. Human rabies vaccines are administered intramuscularly or intradermally with the primary goal of vaccination being the induction of virus neutralizing antibodies directed against the viral G protein. This response provides protection against RABV infection although the location and mechanisms by which neutralizing antibodies prevent viral replication remain unknown. Human rabies vaccines are based on inactivated purified viruses and as such are poorly immunogenic and often require three doses given within a 4-week period to reliably induce protective titers of neutralizing antibodies. The development of inactivated, lyophilized, cell culture-based vaccines with increased antigenicity has led to a reduction in the number of doses needed to elicit a sufficient neutralizing antibody response. This in turn has reduced the number of clinic visits required and has improved complete vaccine administration through increased patient uptake. All licensed rabies vaccines have superb safety records and adverse post-vaccination events are rarely reported. As with most other vaccine formulations for infectious diseases, pre-exposure immunisation (PrEP) using standard vaccines is recommended for travellers to rabies endemic areas. The invariably fatal nature of the disease and the strong association in endemic areas with infection of children under 15 years of age has led some to suggest that PrEP should be considered as part of a recommended paediatric vaccination strategy. PrEP regimens consist of four vaccine doses being administered on days 0, 7, 14 and 21 or 28. In endemic areas, economic limitations restrict the availability of vaccines and as such extensive PrEP coverage is not generally considered possible. As such post exposure prophylaxis (PEP) is generally relied upon following a potential exposure event. Rabies is unusual in that, even following a potential exposure event, vaccination is effective and can prevent the development of disease as long as its administered in a timely manner. However, before vaccination is considered, primary wound care including the irrigation of wounds with soapy water; thorough scrubbing of affected areas to remove foreign bodies; and the use of iodine based antiseptics can significantly reduce the likelihood of establishing a productive infection. Suturing the wounds should be avoided. Then, where available, and individuals are aware of its utility, PEP can be sought. PEP administration is guided by WHO recommendations that define responsive activities depending on the degree of exposure with category I and II exposures warranting vaccination alone whilst severe exposures, termed category III, requiring rabies immunoglobulin (RIG) alongside vaccination. In exposed unvaccinated individuals, the severity of exposure dictates if the vaccine has to be combined with a rabies immunoglobulin (RIG). The application of RIG directly to the wound serves to neutralise any live virus present in the wound area and prevent any further live virus infection of exposed cells and prevents spread of the virus in the time before vaccination elicits sufficient immunity, generally considered to be up to 14 days. RIG is generated from both human vaccinees (HRIG) and vaccinated horses (Equine- ERIG). RIG administration is based on weight with 20 IU/kg for HRIG and 40 IU/kg for ERIG being recommended by the WHO. It is also suggested that all of the calculated dose of RIG should be infiltrated into wounds, with any residual RIG should be injected intramuscularly at a distant site to the wound. Certainly, whilst ERIG is safe, easier to produce than HRIG, effective and increasingly available in rabies endemic countries, the search for alternatives continues. To try and extend the use of available RIG in endemic regions studies have looked at their application to wounds alone, with any residual product being reserved for future patients rather than being injected at distant sites. Vaccination is performed after RIG administration at a site distant to the wound itself. Vaccination for PEP can be time-consuming and costly, requiring multiple clinic visits and studies have tried to reduce PEP vaccination regimens to one week, making PEP more affordable and accessible. In cases of licks or minor scratches, several vaccination regimens are approved for intramuscular PEP and a two site 4-dose intradermal regimen has been adapted in some regions. PEP should be administered rapidly following a potential exposure to increase the probability of preventing clinical disease. Although the clinical disease development is almost 100% preventable with timely PEP, failures most often due to alterations in PEP administration, have been reported. The majority of exposed individuals do not receive PEP when required or they are administered PEP without RIG. Both vaccines and RIG are costly and in some countries patients often are expected to pay for required biologicals, medical supplies and treatment alongside covering all costs associated with travel to health care facilities. The role of vaccines in protection against RABV is well defined with correlates of protection against the virus, in the form of neutralizing antibody titres of or above 0.5 International Units (IU), being defined as protective. Serological responses to lyssaviruses are targeted towards the trimeric lyssavirus G protein, the primary antigen on the surface of the virion that is responsible for cell receptor binding and entry. However the divergence of lyssaviruses has led to the categorisation of viruses within the genus into three phylogroups based on their reactivity against sera from vaccinated individuals (see below). Differences within G give an indication of the antigenic diversity within the genus although the lack of an atomic structure for this protein does not allow to identify the key antigenic determinants at the amino acid residue level. Human rabies vaccines

Rabies and Other Lyssaviruses (Rhabdoviridae)

745

are formulated from inactivated preparations of whole virus and it is widely accepted that a protective titer of 0.5 international units (IU)/ml is sufficient for protection against RABV. Serum from vaccinated individuals has been utilised to determine cross neutralisation of different lyssavirus species demonstrating that vaccine derived sera does not neutralise all of the lyssavirus species. Phylogroups broadly define the relationship between existing vaccines and neutralisation with phylogroup I viruses (RABV/ABLV/ARAV/BBLV/DUVV/EBLV-1/EBLV-2/GBLV/IRKV/KHUV) being neutralised whilst phylogroup II (LBV/MOK/SHIBV) and phylogroup III (LLEBV/IKOV/WBCV) viruses failing to be neutralised. Still, the epidemiology of these divergent lyssaviruses is ill defined and only 13 human fatalities have been directly attributed to infection with lyssaviruses other than classical rabies virus ad as such the burden is undefined. Attempts are being made to increase the valency of vaccine preparations to enable cross lyssavirus protective responses to be induced although exact requirements for any pan-lyssavirus vaccine remain to be defined.

Conclusions Aims to reduce the human burden from rabies are ongoing with the OIE targeting the elimination of dog-mediated rabies by 2030. A first steps toward human rabies elimination is to ensure a globally available diagnostic capability to accurately confirm infection with rabies or a lyssavirus. This requires laboratory networks capable of performing diagnosis across resource limited regions where the virus is endemic. In the future, approaches to rabies diagnosis should combine laboratory confirmation with point-ofcare testing in the field. Studies are ongoing to develop novel diagnostic tools. Alongside this, endemic regions need to ensure that rabies is a notifiable disease to ensure that laboratory networks and government bodies develop the infrastructure to accurately diagnose and report the disease. Several pathways to rabies prevention have been developed through the WHO, OIE and other international organisations. Elimination of RABV from dogs populations requires extensive in country veterinary capabilities to enable a sustained approach of vaccination of companion animals, and control and vaccination of free-roaming and community dogs. Estimated costs required to eliminate dog-mediated rabies in all endemic countries have been estimated at US$6.3 billion with most human rabies cases being prevented through control, licensing and vaccination of dog populations in endemic regions. Alongside parenteral vaccination of dogs, the oral vaccination of free-roaming dog populations has been proposed although assessment of vaccine coverage using oral formulations remains difficult. Lessons learned from extensive and highly successful oral vaccination campaigns to protect wildlife species has demonstrated difficulties in achieving adequate seroconversion even though significant successes have been made. From a prevention perspective, clearly adoption of rabies vaccination as part of a paediatric regimen in endemic areas would reduce the human burden of disease significantly. The existing recommendation is that PEP, in the form of vaccination alone, is still given to exposed individuals who received PrEP. Hence, there are still costs associated with exposure events but the requirement for RIG would reduce dramatically. RIG constitutes the highest cost in PEP and therefore a vaccine only approach would make a significant difference in the costs associated with rabies. The number of animal bites considered to be potential exposure events is often high in regions where rabies is endemic. It is estimated that PEP is given to 415 million individuals annually to prevent the establishment of disease. Optimization of awareness to ensure exposed individuals practice thorough wound cleaning and development of vaccines to reduce production costs and doses required could improve prospects for exposed individuals in endemic areas drastically. Despite all the challenges, the 2030 eradication goal will promote global initiatives to encourage international organisations, governments, charities, non-governmental bodies and potentially pharmaceutical companies to develop strategies to achieve global human rabies elimination.

Further Reading Abela-Ridder, B., Knopf, L., Martin, S., et al., 2016. The beginning of the end of rabies? The Lancet Global Health 4, e780–e781. Banyard, A.C., Fooks, A.R., 2017. The impact of novel lyssavirus discovery. Microbiology Australia 38, 18–21. Bourhy, H., Reynes, J.-M., Dunham, E.J., et al., 2008. The origin and phylogeography of dog rabies virus. Journal of General Virology 89, 2673–2681. Dodet, B., Korejwo, J., Briggs, D.J., 2013. Eliminating the scourge of dog-transmitted rabies. Vaccine 31, 1359. Fooks, A.R., Cliquet, F., Finke, S., et al., 2017. Rabies. Nature Reviews Disease Primers 3. Hampson, K., Coudeville, L., Lembo, T., et al., P., 2015. Estimating the global burden of endemic canine rabies. PLoS Neglected Tropical Diseases 9, e0003709. Hemachudha, T., Ugolini, G., Wacharapluesadee, S., et al., 2013. Human rabies: Neuropathogenesis, diagnosis, and management. The Lancet Neurology 12, 498–513. Johnson, N., Cunningham, A.F., Fooks, A.R., 2010. The immune response to rabies virus infection and vaccination. Vaccine 28, 3896–3901. Lafon, M., 2005. Rabies virus receptors. Journal of Neurovirology 11, 82–87. Mitrabhakdi, E., Shuangshoti, S., Wannakrairot, P., et al., 2005. Difference in neuropathogenetic mechanisms in human furious and paralytic rabies. Journal of the Neurological Sciences 238, 3–10. Rupprecht, C., Kuzmin, I., Meslin, F., 2017. Lyssaviruses and rabies: Current conundrums, concerns, contradictions and controversies. F1000Research 6. Rupprecht, C.E., Nagarajan, T., Ertl, H., 2016. Current status and development of vaccines and other biologics for human rabies prevention. Expert Review of Vaccines. 1–19. Schnell, M.J., Mcgettigan, J.P., Wirblich, C., Papaneri, A., 2010. The cell biology of rabies virus: Using stealth to reach the brain. Nature Reviews Microbiology 8, 51–61. Wallace, R.M., Undurraga, E.A., Blanton, J.D., Cleaton, J., Franka, R., 2017. Elimination of dog-mediated human rabies deaths by 2030: Needs assessment and alternatives for progress based on dog vaccination. Frontiers in Veterinary Science 4. WHO, 2013. WHO Expert Consultation on Rabies, Second Report. WHO Technical Report Series No. 982. Geneva: World Health Organization.

746

Rabies and Other Lyssaviruses (Rhabdoviridae)

Relevant Websites www.rabiesalliance.org Global Alliance for Rabies Control: Home. www.missionrabies.com Mission Rabies – Taking up the fight against rabies in India. http://web.oie.int/boutique/index.php?page=ficprod&id_produit=1641&lang=en OIE standards and guidelines related to trade and poultry diseases. www.oie.int/animal-health-in-the-world/rabies-portal Rabies Portal: OIE – World Organisation for Animal Health. http://www.who.int/rabies/resources/who-cds-ntd-nzd-2018.03/en/ WHO Laboratory Techniques in Rabies. www.who.int/rabies/en WHO – Rabies – World Health Organization.

Respiratory Syncytial Virus (Pneumoviridae) Tiffany King, Nationwide Children’s Hospital, Columbus, OH, United States and The Ohio State University College of Medicine, Columbus, OH, United States Tiffany Jenkins, Nationwide Children’s Hospital, Columbus, OH, United States and The Ohio State University, Columbus, OH, United States Supranee Chaiwatpongsakorn, Nationwide Children’s Hospital, Columbus, OH, United States Mark E Peeples, Nationwide Children’s Hospital, Columbus, OH, United States and The Ohio State University College of Medicine, Columbus, OH, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of Tracy J. Ruckwardt, Peter L. Collins, Barney S. Graham, Human Respiratory Syncytial Virus, In Reference Module in Biomedical Sciences, Elsevier Inc, 2018, doi:10.1016/B978-0-12-801238-3.02599-X.

Nomenclature

Paramyxoviridae

Metapneumovirus

Proper name for the human and avian viruses most closely related to the orthopneumoviruses Mononegavirales Order of negative strand virus Orthopneumovirus Proper name of human, bovine and ovine RSV and pneumonia virus of mice

Glossary Antigenome Positive sense single strand copy of the genome, used the template for producing genomes. F protein Fusion glycoprotein. G protein Attachment glycoprotein. Genome Negative single strand RNA encodes all the viral mRNAs and the positive strand antigenome. L protein Large RNA-dependent RNA polymerase protein. M2–1 protein Small protein that enables the polymerase to transcribe mRNA without releasing from the nucleocapsid. M2–2 protein Small protein that inhibits replication at the expense of replication. Minigenome System launched from plasmids that replaces all the viral genes with a marker gene that is used to study viral replication and mRNA transcription.

Pneumoviridae

Family that included the Pneumoviridae before 2016 Family that includes orthopneumoviruses and the metapneumoviruses

N protein Nucleocapsid protein. NS1 protein Inhibits the initial interferon response. NS2 protein Inhibits the amplification of the interferon response. Nucleocapsid Helical complex of the genome or antigenome covered by the nucleocapsid protein. P protein phosphoprotein interacts with the nucleocapsid and the L protein. Post-F Structure of the F protein after it is triggered to refold. Pre-F Structure of the F protein before it is triggered to refold, causing membrane fusion. Replicon Self-replicating RSV genome that lacks the RSV F and G genes and so cannot produce virions. RSV Respiratory syncytial virus SH protein Small hydrophobic glycoprotein.

History Chimpanzee coryza agent was first recognized in 1955 in chimpanzees with respiratory symptoms housed in an animal facility and in one of the animal handlers. Nasal samples transmitted the disease to other chimpanzees and caused cytopathology in cell culture. Human adolescents and young adults had serum antibodies to the agent. The virus was soon isolated and characterized. Human respiratory syncytial virus (RSV) is now recognized as the most important cause of lower respiratory tract infection in infants and young children and among the most frequent causes for hospitalizations in childhood. With the advent of highly sensitive RT-PCR assays, RSV has also been recognized as a major cause of “influenza-like illness” in the elderly, responsible for approximately one-third of the excess deaths each winter that had been traditionally attributed to influenza (Falsey et al., 1992; Walsh et al., 2004).

RSV Disease RSV spreads through aerosolized droplets and contaminated surfaces, and typically requiring close contact with an infected individual. On average, the incubation period lasts 3–5 days before clinical signs become apparent: upper respiratory tract infection include rhinorrhea, sneezing, coughing, and occasionally a low-grade fever. Some 25%–40% of RSV infections in young children progress to acute lower respiratory tract infections (ALRI), in most cases bronchiolitis and/or pneumonia. Children with

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00142-9

747

748

Respiratory Syncytial Virus (Pneumoviridae)

Fig. 1 Pneumoviridae genome organization.

ALRI may develop signs of tachypnea, inspiratory crackles, and expiratory wheezing. Recovery occurs 7–12 days after the onset of clinical symptoms except in severe cases, in which cough and wheeze may progress to dyspnea and hypoxia. The disease can be lethal when the infant experiences exhaustion from increased and persistent respiratory effort, resulting in respiratory failure. The decision to hospitalize a child is often based on the presence of a hypoxemic state. Treatments may involve oxygen supplementation and supportive care but severe cases may require mechanical ventilation. Fatal RSV infections are rare in the developed world. They involve peribronchiolar infiltrates consisting of lymphocytes, plasma cells, and macrophages, with neutrophils comprising a larger portion of the inflammation early in the course of infection. There is often necrosis and sloughing of bronchiolar epithelial cells into the bronchiolar lumen. Obstruction can occur in the small bronchioles caused by the combination of enhanced mucus production, sloughed epithelial cells and inflammation. Despite the middle name of the virus, syncytia are rarely observed histologically in cases of human RSV but are a prominent feature of bovine RSV (bRSV) infections. T cell deficient individuals may develop a “giant cell pneumonia”, where syncytia formation is more readily apparent, but this is not a feature in immunocompetent individuals. Infection with RSV and RSV-induced ALRI early in life has been associated with the development of asthma in early adolescence (Schwarze et al., 1997; Sigurs et al., 1995; Welliver and Duffy, 1993). Infections of infants and young children with RSV are the most frequent cause of hospitalization for children, responsible for over 570,000 annual hospitalizations and a major economic burden in the USA. Globally, fatal RSV infections are estimated to be 48,000–75,000 per year. However, the vast majority of these RSV-related deaths occur in developing countries, where ready access to medical care is not readily available. Worldwide, RSV is second only to malaria as a leading cause of death in the first year of life. Throughout life, older children and adults are infected multiple times with RSV, developing symptoms of a common cold (Hall et al., 1991). The inability to prevent subsequent infections is likely due to the inability of the virus to induce a strong memory immune response. In individuals who are immunosuppressed or 65 and older with weakening immune systems, RSV can be life threatening. RSV rivals influenza viruses each winter season as a cause of “excess deaths” with as many as 14,000 deaths in the USA, often in elderly individuals with underlying health problems.

Classification Nonsegmented negative sense viruses like RSV, with an RNA genome and envelope are included in the order Mononegavirales. The RSV genome is approximately 15.2 kb in length with 10 gene units separated by intergenic regions (IR) of variable lengths. The genome is protected in a helical nucleocapsid structure formed by the nucleocapsid (N) protein. The Pneumoviridae family (Fig. 1) includes the orthopneumovirus genus, human, bovine and ovine RSV, as well as the related pneumonia virus of mice. The metapneumovirus genus includes the more distantly related human and avian metapneumoviruses. The avian metapneumoviruses are divided into three types, A, B and C. A and B are primarily European and C is primarily North American. Human metapneumovirus is most highly related to avian metapneumovirus C, the North American strain, suggesting that the transition from birds to humans took place in America. All members of the Pneumoviridae infect the respiratory tract only, causing respiratory disease. They also share many of the same genes, including N, phosphoprotein (P), matrix (M), small hydrophobic (SH), attachment glycoprotein (G), fusion (F), the large (L) polymerase, and the M2 gene, encoding two proteins. The M2-1 and M2–2 proteins enable transcription and regulate replication, respectively. The nonstructural, NS1 and NS2, genes are found in the orthopneumoviruses, but not in the metapneumoviruses (Fig. 1). The source of these nonstructural genes is not clear as no other virus has similar genes and no obviously similar genes have been found in other organisms. Interestingly, the F-M2 gene block follows the SH/G gene block in pneumoviruses but precedes the SH/G gene in metapneumoviruses. It apparently switched position in the divergence between the two groups, a change that would require recombination. Unlike positive strand viruses that replicate their RNA genomes outside the context of a nucleocapsid and have high rates of recombination due to polymerase copy choice directed by hybridization, recombination is exceedingly rare among the Mononegavirales, likely because the nucleocapsid covers both the genome and antigenome RNAs preventing hybridization from nascent RNA/polymerase complexes. But despite the rarity of such a recombination event, the appearance of the heavily

Respiratory Syncytial Virus (Pneumoviridae)

749

Fig. 2 RSV virion organization. Modified from the dissertation of Sara Johnson, The Ohio State University, 2015.

glycosylated G protein in the Pneumovirinae likely represents another such rare recombination event. The G protein is related to the highly glycosylated mucins that decorate the surface of the respiratory epithelium, suggesting that an ancestral Pneumoviridae virus acquired this gene from the cells in which RSV replicates. The members of the Pneumoviridae have many similarities with the members of the Paramyxoviridae, with whom they were classified until recently. The two families share their N, P, M, F, and L proteins. A few paramyxoviruses have a small hydrophobic (SH) protein, but most do not, and none of them have homologs to the nonstructural proteins, NS1 and NS2, or M2 proteins. Furthermore, the Pneumoviridae attachment protein, G, is completely different from the hemagglutinin/neuraminidase (HN) attachment protein of most members of the Paramyxoviridae, which bind to sialic acid as its receptor and cleave sialic acid from its terminal position on glycans, releasing these viruses from their receptor.

Virus Entry The Virion The RSV virion incorporates 8 of the 11 viral proteins the virus produces. As shown in Fig. 2, the nucleocapsid structure containing the RSV genome is wrapped by N protein and associated with the L/P polymerase complex and M2–1 protein. That ribonucleoprotein complex is surrounded by a lipid membrane. The M protein underlies the membrane, and the three viral glycoproteins are incorporated into the membrane. The shape of the RSV virion can be spherical, but is more often rod-shaped. The RSV virion infects cells by binding to a receptor on the cell surface primarily with its G protein, and its F protein causes the virion membrane to fuse with the cell membrane. Once the membranes fuse, the nucleocapsid with its associated polymerase, is spilled into the cytoplasm where the L/P polymerase complex with help from the M2-1 protein produce 10 viral mRNAs that are translated into 11 viral proteins. The L/P polymerase complex replicates the RSV genome via an antigenome intermediate that is also encapsidated by the N protein is in a helical nucleocapsid. The three viral glycoproteins are produced in the ER where the F and G are decorated with Nlinked glycans that are processed as they move through the Golgi complex. In addition, the G protein is decorated with B35 O-linked glycans in the trans-Golgi. Both the G and F proteins arrive on the cell surface where they are joined by the M protein underlying the membrane, the nucleocapsid with its L/P/M2–1 polymerase complex. New virions are formed by budding through the plasma membrane. Most virions produced in cell lines (B90%) remain associated with the cells, whereas in primary well differentiated human bronchial epithelial (HBE) cultures grow at the air-liquid interface and form tight junctions, they are released efficiently and apically. Historically, most studies on RSV replication have been performed in HEp-2, HeLa or Vero cells, immortal cell lines that grow well in the laboratory as submerged cultures. However, RSV attachment to these cells, particularly the receptor recognized by the G protein, is different from its receptor in HBE cultures, an ideal model for the airway epithelium where RSV specifically infects the ciliated cells.

The F Glycoprotein RSV enters a target cell by fusing its membrane with the membrane of the cell, a process mediated by the fusion (F) protein. The F protein is a highly conserved, type I transmembrane glycoprotein trimer. It is produced as a precursor, F0, in the ER where it is also

750

Respiratory Syncytial Virus (Pneumoviridae)

modified by the addition of 5 N-linked glycans. During passage through the Golgi these glycans are matured, and the F0 protein is cleaved, activating its fusion function. The transmembrane part of the protein, F1, is linked by two disulfide bonds to F2. Like many other class I viral fusion proteins, the Pneumoviridae F protein is a homotrimer and each monomer is activated by proteolytic cleavage, orthomyxoviruses by a furin-like protease. However, unlike the other Pneumoviridae F proteins, the RSV F protein is cleaved at two sites releasing a 27 amino acid peptide known as pep27. Pep27 does not appear to remain associated with the F protein. Following cleavage, the F monomers assemble into functional trimers. The pep27 of human RSV has no known function, but the pep27 of the bovine RSV functions as a tachykinin-like peptide, promoting smooth muscle contraction and perhaps mediating other proinflammatory responses. The RSV F protein arrives at the cell surface as a fully function, metastable prefusion F (pre-F) trimer (Fig. 3, left) that undergoes major structural changes to accomplish membrane fusion. The process of refolding into the postfusion F (post-F) protein (Fig. 3, right) causes membrane fusion, thereby initiating virus infection. It is not clear what triggers refolding, but when pre-F is triggered, much of the top part of the molecule undergoes dramatic refolding, almost doubling the length of the F protein. The apical surface of the pre-F trimer contains 4 short a-helices and two b-sheets but during the refolding process the loops between these a-helices and b-sheets, as well as the b-sheets themselves, transform into one very long a-helix that is tipped with the highly hydrophobic fusion peptide at the N-terminus of F1 that had been revealed by cleavage in the trans-Golgi. The three long a-helices of the trimer form a coiled coil and their three fusion peptides insert into the target cell membrane thereby linking the viral membrane with the target cell membrane. The F protein trimer then folds in half, bringing its N-terminal and C-terminal regions together and allowing the three C-terminal a-helices to lock in place in the grooves of the long N-terminal helix to form the very stable “6-helix bundle” (6HB) of the post-F structure. In so doing, the virion and plasma membranes are brought together and their lipids begin to mix, initiating the membrane fusion process. In traditional laboratory cultured cell lines, the F protein on the infected cell surface enables that cell to fuse with neighboring cells, resulting in the classical multinucleated giant cell or ‘syncytial’ pathology that gives RSV its middle name. Six major antigenic sites have been identified on the surface of the pre-F protein by monoclonal antibody binding. Two of these sites at the top of pre-F are lost during the transition from pre-F to post-F (Fig. 3, red and orange) because these are the regions that refold dramatically following triggering. The remaining antigenic sites are present, to varying degrees on both pre-F and post-F. No antigenic site is uniquely post-F. All these observations are consistent with the rearrangement of the top part of the pre-F protein, while the lower portion of the pre-F protein remains largely intact. The potency of neutralizing monoclonal antibodies follows the same trend: antibodies to the epitopes unique to the pre-F form are, approximately 10 times more efficient at neutralizing the virus than the antibodies that bind both pre-F and post-F. This much higher potency likely reflects interference with triggering or refolding of this dynamic region of the pre-F protein, thereby blocking its ability to cause fusion and initiate infection. It is not clear what triggers the RSV pre-F protein to refold, leading to membrane fusion. The RSV pre-F protein does not require its attachment protein to trigger and cause fusion. All paramyxovirus pre-F proteins do. While the G protein is not essential for infection in vivo, the F protein is capable of mediating at least some attachment and infection in immortalized laboratory cells, B10-fold less efficiently than in the presence of G, likely because of the much more efficient cell binding ability of the G protein that would position the F protein close to the cell membrane. Several proteins, including nucleolin, EGFR, ICAM-1 and TLR4, interact with the F protein and could be receptors for pre-F. Binding to one of these receptors might be the trigger that initiates preF refolding. It is also possible that contact with a more common molecule or structure on the plasma membrane of a target cell such as a charged glycan or a lipid membrane triggers the F protein. While most evidence points to the plasma membrane as the point of RSV entry, there are reports that RSV is taken into a cell by micropinocytosis and fusing with intracellular membranes. Low ionic conditions have been shown to trigger the structural conversion of pre-F to post-F and initiate cell to cell fusion. Low ionic strength conditions might be provided in intracellular vesicles, enabling entry at that site.

The G Glycoprotein The G glycoprotein is the primary attachment protein of RSV. It is a type II transmembrane protein that is rich in Ser, Thr and Pro, and highly modified by the addition of glycans, similar to the mucins in the respiratory tract where RSV resides. The initial G protein molecular weight is 32 kDa but its final form in immortalized cells is 90–100 kDa. The first modifications, palmitylation and N-linked glycosylation, occur during translation in the endoplasmic reticulum resulting in a 45 kDa species. Palmitylation modifies cysteines within the cytoplasmic N-terminal domain while N-linked glycosylation occurs in the two hypervariable mucinlike regions of the ectodomain. Once in the Golgi, O-linked glycans are added to these mucin-like regions, resulting in a fully glycosylated 90–100 kDa glycoprotein. The range in its size is likely due to minor variations in the many attached glycan chains. Finally, the G protein reaches the plasma membrane where it is incorporated into budding virions. The N and O-linked glycosylation sites reside in two large mucin-like domains that flank a conserved central region and cysteine noose. Three to 5 N-linked glycans are added, depending on the RSV strain and B35 O-linked glycans modify the many Ser and Thr residues in the mucin-like domains. The G protein produced in HBE cultures is much larger, 180 kDa, glycoprotein, when evaluated under reducing conditions. It is not yet clear if this larger size is due to non-disulfide, covalent dimerization or to additional post-translational modifications.

Respiratory Syncytial Virus (Pneumoviridae)

751

Fig. 3 RSV F protein trimer with its antigenic sites indicated on both its pre-F and post-F forms. Pre-F is the metastable, functional F protein which is anchored in the virion membrane. Post-F is the post-triggered, refolded F protein that brought the virion and cell membranes together and caused them to fuse. Note that the blue, purple, yellow and green antigenic sites remain relatively intact following refolding while the orange and red sites disappear, refolding into long helices that form the core of the long stalk of post-F that trimerize and insert into the target cell membrane via their hydrophobic N terminus. The stalk of the pre-F protein then swings upwards to contribute its three helices to the post-F stalk, forming the extremely stable 6-helix bundle. Modified from Graham, B.S., 2017. Vaccine development for respiratory syncytial virus. Current Opinion in Virology 23, 107–112.

Unlike the F glycoprotein whose sequence is highly conserved (88%), the G glycoprotein is highly variable, with as little as 48% conservation between RSV subgroups. A portion of the C-terminal hypervariable region of the G glycoprotein has been used to define two antigenic subgroups, A and B, and further divisions into genotypes that are useful for epidemiology. The A and B subgroups have not diverged enough to be recognized as serotypes because antibodies raised against one subgroup are able to neutralize viruses from the others subgroup. However, cross-subgroup neutralization is not as potent as intra-subgroup neutralization, indicating that these differences could be important in protection. The only region that is completely conserved in the G protein is a 13 amino acid central conserved domain which is followed by a mostly conserved sequence containing 4 completely conserved cysteines that form a “cysteine noose” (Fig. 4). The C-terminal side of this noose includes a CXXXC (CX3C) motif. Only one other CX3C motif is found in Genbank and that belongs fractalkine, a chemokine which interacts with its receptor CX3CR1 on immune cells to induce chemotaxis. G protein has, likewise, been shown to induce chemotaxis in immune cells through its interaction with CX3CR1. CX3CR1 has also been detected on the ciliated cells in primary HBE cultures and may be the receptor for RSV on ciliated cells, its target cell. It is not clear how this interaction would occur because the disulfide bonding pattern of the cysteine noose of G differs from that of fractalkine resulting in a different structure for the CX3C in the G protein. A generally conserved region of the G glycoprotein, C-terminal to the cysteine noose, is a stretch of positively charged amino acids. In immortalized cell lines, these residues interact with the heparan sulfate attached to transmembrane proteoglycans (HSPG), which serve as the receptor for RSV infection in these cells. Heparan sulfate is an unbranched polysaccharide chain of a repeating disaccharide, hexuronic acid and hexosamine. The polysaccharide chain is modified by the addition of sulfate groups, giving the heparan sulfate a negative charge. It is the charge–charge interaction between the cluster of positively charged amino acids in the G protein with the negatively charged sulfate groups on the HSPG that enables RSV to attach to these cells. Many other viruses that infect immortalized cells also use this “non-specific” mechanism to attach and initiate infection. Some of the G glycoprotein produced during infection is released from infected cells in a soluble form. This “soluble G” is produced by initiating translation at the second AUG of the G protein, reaching the cell surface. The result is a G protein whose N-terminus includes part of the hydrophobic transmembrane domain and contains the full ectodomain of G. It appears to be released from the cell by cell surface proteases. Approximated 20% of the G glycoprotein expressed in cultured cells is in a soluble

752

Respiratory Syncytial Virus (Pneumoviridae)

Fig. 4 The RSV G protein and its roles in attachment. (A) G protein domains and predicted glycosylation sites (4 N-linked, green triangles; B35 O-linked, blue horizontal lines). (B) General 2D structure with two disulfide bonds creating a “cysteine noose”. (C) Attachment sites for heparan sulfate proteoglycans on immortalized cell lines and for CX3CR1 on primary well differentiated human bronchial epithelial cultures.

form. Soluble G may play an immunomodulatory role via its interaction with the CX3CR1 receptor on immune cells. Soluble G could also be a decoy for neutralizing antibodies, although most neutralizing antibodies (90%–99%) are against the F protein, not the G protein. However, it remains possible that non-neutralizing antibodies to the G protein may suppress RSV infection in another manner such as antibody dependent cellular cytotoxicity or complement mediated lysis. As described above, in immortalized cells G attaches to the negatively charged heparan sulfate proteoglycans (HSPG) to initiate infection via a stretch of positively charged residues C-terminal to the cysteine noose. Soluble HS interferes with this interaction, inhibiting RSV infection of immortalized cells but not infection of HBE cultures, suggesting that RSV uses a different receptor in vivo. HSGP is not detectable on the apical surface of the ciliated cells that RSV infects. CX3CR1, a chemokine receptor which interacts with the CX3C motif is found on the apical surface of ciliated cells in HBE cultures. A G-specific monoclonal antibody, 131-2G, which binds to G in the region of the CX3C motif does not prevent RSV from infecting immortalized cells, but it does prevent RSV from infecting HBE cultures. These findings confirm that RSV uses different receptors in standard laboratory cells compared to primary HBE cultures and are consistent with CX3CR1 being a receptor for RSV in vivo.

Genome Transcription and Replication The result of fusion between the virion membrane and the cell membrane is that the RSV nucleocapsid enters the cytoplasm of a target cell, its associated polymerase complex produces viral mRNAs, antigenomic and genomic RNAs in localized regions that grow into viral inclusion bodies in the cytoplasm. Viral genes are transcribed into mRNAs for each gene in order, from the 30 to 50 end of the genome. As the polymerase reaches a gene end (GE) sequence, it repeatedly copies the poly U tract in the GE, generating a poly A tail before releasing the mRNA. The polymerase then scans through the intergenic region (IR), 1–52 nt long. Some of the polymerase molecules release while crossing the IR, creating a gradient of reduced gene expression for each subsequent gene. Eventually a sufficient amount of N protein is produced to encapsidate antigenome transcripts, producing full-length antigenome nucleocapsids. Finally, encapsidated antigenomes are copied many times, beginning at the ‘trailer complement’ sequence at the extreme 30 end of the antigenome to produce full-length genomes that are encapsidated. These new encapsidated genomes either reenter the replication cycle or are incorporated into new virions during the budding process at the plasma membrane. Deletion of the M2–2 gene from the RSV genome results in enhanced mRNA transcription and reduced genome replication indicating that the M2-2 protein encourages the opposite, genome replication, but the mechanism is not known. The template for mRNA transcription and replication is the genome-nucleocapsid complex, not free genomic RNA. The nucleocapsid is composed of either the genomic or the antigenomic RNA and over 2000 N proteins in a long helical structure that looks like a string of vertebrae. One “vertebra” represents one revolution of the helix of 10 N proteins, each holding 7 nucleotides of genomic RNA in the groove between its N- and C-terminal domains. The viral L (large) protein is an RNA-dependent RNA polymerase that is complexed with a tetramer of the P (phosphoprotein). This L/P complex transcribes the 10 mRNAs and the antigenome from the genome template, and the genome from the antigenome template. The determination of whether the L/P complex will produce a full-length viral antigenome or the series of 10 viral mRNAs takes place in the leader sequence, the promoter at the extreme 30 end of the genome. The leader sequence contains two overlapping viral promoters, one for transcription and one for replication. To produce mRNA, the L protein, initiates transcription at the third and fourth nucleotides of the leader sequence. Starting at that position rather than the first and second nucleotides of the leader results in the transcription of a 25 nt “leader transcript” that terminates and is released, allowing the L/P complex to recognize the GS sequence and begin the process of transcribing the mRNAs. The L polymerase protein is a doughnut-shaped structure that complexes with a tetrameric P protein. To transcribe the genomic RNA into complementary mRNA, the C-terminal domains of the P proteins each pull an N protein off the RNA template, allowing the RNA to pass through the polymerase, followed by the N proteins being replaced. The L/P protein complex remains associated

Respiratory Syncytial Virus (Pneumoviridae)

753

with the nucleocapsid, scanning until it finds the first GS signal where it reinitiates transcription, this time producing the NS1 mRNA. The M2-1 protein associates with the L/P complex involved in mRNA transcription, functioning as an “anti-termination” factor that prevents premature termination of viral mRNA transcripts. The lengthening transcript passes through the polyribonucleotidyltransferase (capping enzyme) domain on the opposite side of the L protein, where a cap is added to its 50 end. The next step in mRNA processing is the addition of two methyl groups, one to the cap and one to the first nt of the mRNA by the methyltransferase domain of the L protein. This portion of the L protein has not yet been visualized. The addition of these methyl groups is important to make the viral mRNAs appear to be cellular mRNAs and avoid the induction of interferon. Finally, the L/P/M2–1 polymerase slips on the poly-U tract of the GE signal, repeatedly copying it to produce the poly A tail and eventually releases the NS1 mRNA. The polymerase continues along the nucleocapsid template by sliding through the IR and re-engaging at the next, NS2, GS sequence where it transcribes, caps, methylates, polyadenylates, and releases NS2 and each of the downstream viral mRNAs in sequence. During transit through each IR, some of the polymerase molecules fall off the template such that the level of transcription of each downstream gene is less than the one before it. Another less frequent occurrence is that the polymerase misses the GE sequence, continuing transcription through to the following GE. The result is a bicistronic “read-through” transcript. These read-through events may help more polymerase molecules reach the downstream genes. If instead of initiating transcription at the third nucleotide, the L/P complex initiates transcription at the first two nucleotides, the resulting full-length antigenome is produced. If the concentration of N protein in the local area is high enough, as the complementary “antigenome” RNA strand exits the polymerase, it is encapsidated by newly synthesized N proteins, guided into position by the P protein. Once antigenome nucleocapsids are produced, they are used as the template to produce copies of the genomic RNA that are also encapsidated. The trailer complement promoter on the antigenome appears to be stronger than the leader promoter because the number of genomes in an infected cell greatly outnumber the number of antigenomes. All but one of the RSV genes encode a single protein. The M2 gene encodes two proteins, M2-1 and M2-2, which are translated from the same mRNA by initiating translation at two different sites and in different, but partially overlapping reading frames. The M2-L gene junction is unique among the junctions in RSV. The GS for the L gene precedes the GE of the M2 gene. To transcribe the L gene, the polymerase must terminate the M2 mRNA at its GE and backtrack by retrograde scanning to find the GS site for the L gene to initiate its transcription. This type of GE and GS placement is used by other negative strand viruses, such as Ebola, at many of its gene junctions.

Non-Structural Viral Proteins The NS1 and NS2 proteins are not present in virions but are produced in the infected cell. The NS1 protein plays an important, but as yet undefined role in RSV replication. Both NS1 and NS2 proteins have roles in efficiently evading innate immune responses. NS1 interferes with the induction of interferon by preventing the interaction between RIG-I and MAVS. NS2 interferes with the amplification phase of interferon signaling by cleaving the STAT-2 protein that is involved in transmitting the signal from the interferon receptor to the nucleus to activate antiviral genes and enhance interferon production. The M2-2 protein is also non-structural. As discussed above, its expression enhances viral genome replication at the expense of mRNA production.

The Third Viral Glycoprotein The SH protein is the third viral glycoprotein, along with F and G, is expressed on the virus surface. Although SH is not required for RSV replication in vitro or in vivo, two distinct functions have been identified for SH. First, the SH protein appears to be a “viroporin”, a virus-produced membrane channel, that forms pentameric structures within membranes increasing permeability to ions. Second, SH has an immune modulatory role that protects infected cells from TNF-a mediated apoptosis. SH inhibits TNF-a by: (1) inhibiting of TNF-a production by infected cells; and (2) inhibiting of downstream TNF-a signaling that would have led to phosphorylation of p65 by NF-κB. It remains unclear if these functions are related and their significance to RSV pathogenesis.

Animal Models of RSV Human RSV is host-specific and replicates poorly in most other species, which is thought to be due to the inability of the virus to impede the interferon response in species other than humans. The paucity of animal models that accurately recapitulate human RSV infection and disease has been a hindrance in the understanding of RSV pathogenesis and immunity. Chimpanzees are the only non-human species in which RSV causes respiratory symptoms and have been used in the past to test vaccine candidates. However, chimpanzees are no longer available for such studies in the United States and many other parts of the world. Other non-human primates such as African green monkeys and Rhesus macaques can be infected with RSV but demonstrate few if any symptoms or pathology. The most common small animal model used to study RSV is the mouse, particularly BALB/c. A murine model lacking signal transducer and activator transcription 1 (STAT1), and thus an interferon-response, is more susceptible to RSV infection. Attempts to develop a “mouse-adapted” strain of RSV through serial passage have been unsuccessful, with the virus failing to gain the ability

754

Respiratory Syncytial Virus (Pneumoviridae)

to replicate to high titers even after numerous passages in vivo. Hamsters, guinea pigs, ferrets, and cotton rats have all been used as alternatives to mice for RSV studies because they are more susceptible to RSV. The cotton rat (Sigmodon hispidus) has become one of the most widely-used and accepted rodent models for RSV investigation, and is approximately 100-fold more permissive for RSV infection than the BALB/c mouse. The cotton rat was used as a model to study formalin-inactivated RSV (FI-RSV) vaccine-enhanced disease because it develops a pulmonary disease that more-closely reflects of the disease in humans. The predictive quality of the cotton rat is so high that the FDA-approved prophylaxis for RSV, palivizumab (Synagis), proceeded to clinical trials based on the safety and efficacy studies in the cotton rat model alone, bypassing non-human primate studies. Alternative animal models for RSV research include the highly related bovine RSV (bRSV) that naturally causes respiratory infection in calves, resulting in tachypnea, oculonasal discharge, hypoxemia, and fever. The bovine respiratory tract offers more parallels to the human respiratory system than the murine model, as both human and bovine respiratory tracts are lined predominately by ciliated epithelium, contain abundant submucosal glands, and have both palatine and nasopharyngeal tonsils, all of which are features not present in the mouse respiratory tract. Alternatively, pneumonia virus of mice (PVM) is a orthopneumovirus that naturally infects and induces severe respiratory disease in mice. However, PVM is not as closely-related to human RSV as is bRSV.

Protection From RSV Disease Vaccination – The First Attempt In the mid-1960s the first attempt to develop an RSV vaccine was launched. Using the “Salk” approach that had been very successful for poliovirus vaccination, RSV was grown in cell culture and inactivated with formalin. Children were vaccinated before RSV season with no ill effects. However, after natural RSV infection, 80% of the vaccinated children were hospitalized with enhanced RSV disease (ERD) and two children died. Only 5% of the children in the control group who had received parainfluenza virus vaccine were hospitalized and none experienced ERD (Kim et al., 1969). The cause has not been clearly identified but evidence for an enhanced Th2 response, the response to cell culture contaminants, poorly neutralizing antibodies that recognized the post-F rather than pre-F protein, and complement deposition in the airways have all been presented as potential explanations.

Passive Immunity and Prophylactic Therapy Maternal antibodies (Abs) are transmitted to the fetus in utero from approximately 26 weeks of gestation up until birth, and levels of these Abs in infants directly correlate to Ab titers in the mother. However, the half-life of antibodies is 21–26 days and maternal Abs usually become undetectable by 4 months of life. Infants born prematurely receive fewer transplacental Abs and are therefore at a higher risk for severe hRSV infection. Injection with a humanized monoclonal Ab, palivizumab (Synagis), has been used to supplement maternally derived Abs in infants born prematurely or with other conditions that make them more susceptible to RSV infection. However, palivizumab levels also wane with time and must be replenished once a month for 5 months to maintain an effective neutralizing titer. The cost of this treatment is high and so is not used for all at risk infants. Palivizumab binds to site 2 on both the pre-F and post-F protein (Fig. 3) and neutralizes RSV. Currently, a monoclonal Ab is being tested in infants that uniquely recognizes a pre-F site (Fig. 3), neutralizing RSV approximately 10 times more efficiently than palivizumab. In addition, this pre-F Ab contains several mutations in its heavy chain that triples its half-life. It may, therefore, be possible to protect infants during their most vulnerable first winter season with a single injection before the RSV season (November-March).

Maternal Vaccination Approximately 60% of severe cases of RSV occur in infants, less than 6 months old. Active vaccination during this time, especially during the first 4 months of life is not likely to be effective because the immune response of infants that young is poor. An alternative, immunization of pregnant women during their third semester, is currently being tested. Such a vaccine would likely protect the pregnant woman from RSV disease and allow RSV neutralizing antibodies to cross the placenta, supplement the mother’s own antibodies in protecting the infant after birth. Because antibodies that uniquely recognize pre-F are the most effective for neutralizing RSV, pre-F would seem to be an ideal RSV vaccine, but pre-F is metastable, and readily converts to post-F. However, solving the structure of the pre-F protein enabled the prediction of mutations to stabilize it. Testing, selection, combining and retesting has resulted in several stabilized versions of the RSV F protein that are currently in clinical trials as a vaccine for pregnant women.

Respiratory Syncytial Virus (Pneumoviridae)

755

Live Attenuated Vaccines RSV live attenuated vaccines (LAV) are replicating RSV viruses with mutations that weaken their ability to replicate and cause disease. This approach was very successful in developing the current vaccines against measles, mumps, rubella, chickenpox, and polio (Sabin) which are all given by intramuscular injection. Similarly, the live attenuated influenza virus vaccine that is given intranasally has been effective. Live attenuated RSV vaccines would also be given intranasally. It has been difficult to find live RSV vaccine candidates that do not cause symptoms but are still protective. The Laboratory of Infectious Diseases at the National Institute of Allergy and Infectious Diseases (NIAID) in the USA has been leading the development of RSV LAVs. They have generated many vaccine candidates and tested 14 of them in sero-positive adults, sero-positive children and sero-negative children. Two of the promising candidate vaccines in phase 1 trials have now proceeded to phase 2 trials. Live attenuated vaccines must be produced in one of three cell lines approved by the World Health Organization for that purpose. Of these, Vero, a cell line derived from African green monkey kidneys, produces the most RSV and is therefore the best choice for vaccine production. However, when RSV is produced in Vero cells, much of its B95 kDa G protein is cleaved by cathepsin L resulting in a truncated, 55 kDa protein. This Vero-grown virus is 5-fold less infectious in HBE which would reduce the efficiency of vaccine production by 5-fold. However, the location of this cleavage site has been identified and mutated to prevent cleavage, allowing enhanced vaccine virus yields which should enhance manufacturing efficiency.

Vectored RSV Vaccines Several RSV vaccine candidates are based on virus vectors that are non-replicating (adenovirus) or poorly replicating (Modified Vaccinia Ankara (MVA) and Sendai virus, a paramyxovirus of mice). All these vectors express the RSV F protein and in some cases a stabilized pre-F protein, and particularly to the pre-F protein, are much more neutralizing than antibodies to the G protein, at least by the standard neutralization assays. Some of these vectors also express the G protein or internal viral proteins such as N and M2 in order to induce T cell immunity capable of killing RSV-infected cells during an RSV infection.

Treatment of RSV: Antiviral Agents Once an individual is infected with RSV, a vaccine is not useful. RSV infections might be halted by small molecule antiviral agents capable of inhibiting RSV replication. Attempts to develop such drugs have been mounted over the past 40 years but have not yet resulted in an approved product. Blind screening of chemical libraries for small molecules that inhibit RSV infection or cell–cell fusion resulted in compounds that bind to the F protein and prevent it from refolding. Although these fusion inhibitors showed promising early results, most have not reached clinical trials due to safety concerns and poor pharmacokinetic properties. However, one of them, lumicitabine is currently being evaluated in hospitalized adults in clinical trials. Other efforts have targeted the RSV polymerase, or L protein, including nucleoside analogs and non-nucleoside inhibitors. Multiple companies now have effective candidate drugs that inhibit RSV replication and are bioavailable. As with all acute infections, the difficulty is in diagnosing an RSV infection early enough for an inhibitor to have a significant effect on symptoms.

References Falsey, A.R., Treanor, J.J., Betts, R.F., Walsh, E.E., 1992. Viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings. Journal of the American Geriatrics Society 40, 115–119. Hall, C.B., Walsh, E.E., Long, C.E., Schnabel, K.C., 1991. Immunity to and frequency of reinfection with respiratory syncytial virus. Journal of Infectious Diseases 163, 693–698. Kim, H.W., Canchola, J.G., Brandt, C.D., et al., 1969. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. American Journal of Epidemiology 89, 422–434. Schwarze, J., Hamelmann, E., Bradley, K.L., Takeda, K., Gelfand, E.W., 1997. Respiratory syncytial virus infection results in airway hyperresponsiveness and enhanced airway sensitization to allergen. Journal of Clinical Investigation 100, 226–233. Sigurs, N., Bjarnason, R., Sigurbergsson, F., Kjellman, B., Bjorksten, B., 1995. Asthma and immunoglobulin E antibodies after respiratory syncytial virus bronchiolitis: A prospective cohort study with matched controls. Pediatrics 95, 500–505. Walsh, E.E., Peterson, D.R., Falsey, A.R., 2004. Risk factors for severe respiratory syncytial virus infection in elderly persons. The Journal of infectious diseases 189, 233–238. Welliver, R.C, Duffy, L., 1993. The relationship of RSV-specific immunoglobulin E antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7–8 years. Pediatric Pulmonology 15, 19–27.

Further Reading Collins, P.L., Karron, R.A., 2013. Respiratory syncytial virus and metapneumovirus. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed. vol. 1. Lippincott Williams & Wilkins: Philadelphia, pp. 1086–1123.

756

Respiratory Syncytial Virus (Pneumoviridae)

Collins, P.L., Hill, M.G., Camargo, E., et al., 1995. Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proceedings of the National Academy of Sciences of the United States of America 92, 11563–11567. Johnson, S.M., McNally, B., Willette, M., et al., 2015. Respiratory syncytial virus uses CX3CR1 as a receptor on primary human airway epithelial cultures. PLOS Pathogens 11, e1005318. McLellan, J.S., Chen, M., Joyce, M.G., et al., 2013. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592–598. Shi, T., McAllister, D.A., O'Brien, K.L., et al., 2017. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: A systematic review and modelling study. Lancet 390 (10098), 946–958.

Relevant Websites https://path.azureedge.net/media/documents/RSV-snapshot-2020_03_26_High_Resolution_PDF_1.pdf RSV Vaccine and mAb Snapshot. https://www.cdc.gov/rsv/index.html RSV. Home. Respiratory Syncytial Virus. CDC.

Rhinoviruses (Picornaviridae) Matti Waris, University of Turku, Turku, Finland Olli Ruuskanen, Turku University Hospital, Turku, Finland r 2021 Elsevier Ltd. All rights reserved. This is an update of N.W. Bartlett, S.L. Johnston, Rhinoviruses, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B9780-12-801238-3.02658-1.

Nomenclature AOM Acute otitis media CDHR3 Cadherin related family member 3 CPE Cytopathic effect cre Cis-acting replication element Ct Cycle threshold EV Enterovirus ICAM-1 Intracellular adhesion molecule 1

Glossary AOM Acute otitis media, infection of the middle ear. CPE Cytopathic effect, microscopically detectable effect of viral infection in infected cell. Cre Cis-acting replication element; the action on a genetic element on the activity elsewhere in the same nucleic acid molecule. IRES Internal ribosomal entry sites, secondary structure on the 50 UTR of positive-sense RNA viruses for ribosome contact.

IFN Interferon IL Interleukin IRES Internal ribosomal entry sites LDLR Low-density lipoprotein receptor RV Rhinovirus Tm Melting temperature UTR Untranslated region VP Viral protein

Myristoylation Covalent attachment of a derivative of the fatty acid myristic acid. ORF Open reading frame, gene region that encodes a protein. SYBR Green Cyanine dye forming a highly fluorescent complex with dsDNA. Uridylation Post-translational addition of one or more uridines. UTR Untranslated region, gene region not encoding a protein.

Classification Rhinoviruses (RVs) form three species A, B, and C within the Enterovirus genus of Picornaviridae family. The current total number of types is 169 (Table 1). Initially, 100 RV serotypes were numbered as new serotypes were identified. Phylogenetic analysis divided them into two groups, RV-A and RV-B, and the types were assigned with their original serotype number, e.g., A1, A2, B3 … B6, A7, etc. New genetically defined RV-A and RV-B types from number 100 on are assigned the next available number for the species in question, i.e. A100–A109 and B100–106. There are currently 80 types in RV-A and 32 in RV-B. Type A87 is missing since it was reclassified as enterovirus D68. Types A44, A95, A98 have also been deleted, because they were reassigned as variants of A29, A8, and A54, respectively. Analysis of clinical specimens positive for RV by RT-PCR but unculturable by traditional methods revealed a third rhinovirus species, type C. RV-C types are numbered starting from C1, and their current total is presently 57. As agreed by the International Committee on Taxonomy of Viruses in 2012, the sequence identity is 470% within a species, and 487% in VP1 gene or 490% in VP2/VP4 gene region within a type. Current status of the picornavirus taxonomy including approved RV types can be found at “See Relevant Websites section”.

Virion Structure Picornaviruses are small, non-enveloped viruses with an icosahedral protein capsid shielding an RNA genome. The diameter of the virus particle is about 30 nm. The outer part of the capsid is formed from 60 copies each of three proteins VP1, VP2, and VP3 in pseudo T ¼ 3 symmetry (Fig. 1(A)). A further 60 copies of a fourth, internal capsid protein VP4 is connected with the externum. A mature virion is formed from 12 pentamers, each pentamer containing five protomers with one copy of each of the structural proteins. VP4 protein undergoes post-translational modification with a hydrophobic amino-terminal myristic acid. A micelle of 5 VP4 units, in contact with the internal N-terminus of VP1, reside under the vertex of the core pentamer. Between species, there are differences in the capsid surface topographies due to differences in VP tertiary structures (Fig. 1(B–C)).

Genome Picornaviruses are single-stranded RNA viruses with positive polarity and 7.1–7.2 kb long genome. A single open reading frame (ORF) is flanked by untranslated regions (UTRs) with a polyA tail at the 30 end and a small viral protein of the genome (VPg) at the

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00078-3

757

758

Rhinoviruses (Picornaviridae)

Table 1

Rhinovirus species and types. Group refers to receptor usage (see life cycle)

Species

Group

Types

RV-A

Major

RV-A RV-B

Minor –

RV-C

–

A7–13, A15–22, A24, A28, A32–34, A36, A38–41, A43, A45, A46, A50, A51, A53–61, A63–68, A71, A73–82, A85, A88–90, A94, A96, A100–109 A1, A2, A23, A25, A29–31, A47, A49, A62 B3–6, B14, B17, B26, B27, B35, B37, B48, B52, B69, B70, B72, B79, B83, B84, B86, B91–93, B7, B99, B100–106 C1–57

Fig. 1 Rhinovirus structure. (A) Schematic presentation of the RV capsid structure showing a protomer with three viral proteins VP1, VP2, and VP3 as a triangle and a pentamer of five protomers (thick blue lines). The capsid is formed from 12 pentamers. The triangular face of an icosahedron is indicated with dashed gray lines. (B–E) Capsid structure images of different RV species and major (RV-A16) and minor (RV-A2) receptor group RV-As. VP1 proteins around the pentamer vertex are discernable by the yellow coloring of heights. Blue color indicates depths. Predicted RV capsid structures were obtained under CC by 4.0 from McErlean et al. (2015): PLoS One, doi:10.1371/journal.pone.0001847.g005. Type names were updated according to the current classification.

50 -end (Fig. 2). The about 600 bases long 50 UTR contains secondary and tertiary structures acting as internal ribosomal entry sites (IRES). PolyA tail is required for infectivity and the 40–50 bases long 30 UTR for efficient replication, while VPg is dispensable in in vitro and in vivo experimental settings.

Life Cycle Receptor All RV-B types and the major group of RV-As use intracellular adhesion molecule 1 (ICAM-1) as their receptor, while the minor group of 10 RV-As use low-density lipoprotein receptor (LDLR) family proteins (Table 1). RV infection up-regulates the synthesis of membrane-bound ICAM-1 and down-regulates that of the soluble ICAM-1, thereby facilitating the epithelial cell infectivity. Cadherin-related family member 3 (CDHR3), mainly expressed in airway ciliated epithelial cells, has been identified as a putative receptor for RV-Cs, based on CDHR3 expression vector-transfected HeLa cell line, HeLa-E8. Experimental data on virus-receptor interactions involving CDHR3 has been described for types C2, C15, C40, C41, and C45. In primary human airway epithelial cells,

Rhinoviruses (Picornaviridae)

759

Fig. 2 Schematic illustration of rhinovirus genomic organization and protein formation. The genome is translated into a large polyprotein that is proteolytically processed through several precursor states to 11 proteins. First, protease activity of 2A co-translationally cleaves off the structural proteins’ precursor P1. Then, subsequent cleavages take place through the action of 3C protease, except the final autocatalytic cleavage of VP0 to VP4 and VP2, turning the provirion into a mature virion.

Table 2

Receptor usage of rhinoviruses

Species

Prototype

Receptora

Binding site on the virus capsid

RV-A (major) RV-A (minor) RV-B RV-C

A16 A2 B3 C15

ICAM-1 LDLR ICAM-1 CDHR3

Canyon at the 5-fold axis, VP1 and VP3 Dome at the 5-fold axis, VP1 Canyon at the 5-fold axis, VP1 and VP3 Surface groove at 3-fold axis, mainly VP2 and VP3

a

ICAM-1, intracellular adhesion molecule 1; LDLR, low-density lipoprotein receptor; CDHR3, cadherin -related family member 3.

knock-out of CDHR3 reduced RV-C15 replication by 80%, while binding of the virus on the cells was not affected. Most of the RVC/CDHR3 receptor-related work have been done with a growth adapted variant of the type C15 virus. Cryo-electron microscopy studies indicate different binding sites on the virion surface for each of the receptor molecules (Table 2).

Entry Receptor-mediated endocytosis of both RV-A receptor groups and RV-Bs occurs predominantly via clathrin-coated pits in HeLa cells, but for ICAM-1-mediated endocytosis in other cells alternative routes may be in use. Since, in contrast to LDLR, ICAM-1 has no known internalization signals, co-receptor(s) are likely to be involved with ICAM-1.

Uncoating All events of RV replication take place in the cytoplasm, where the viral RNA must be released from the endosome. The release is triggered by low endosomal pH inducing conformational changes in the capsid proteins. For minor receptor group viruses, the acidic trigger is direct and independent of dissociated LDLR. For major receptor group viruses, it is indirect and in synergy with the action of ICAM-1. VP4 and the N-terminus of VP1 induce membrane permeability through pore formation, resulting in RNA release. Empty virus capsids are transferred to lysosomes and degraded.

Translation Guided by IRES at the 50 UTR, the viral RNA is translated as mRNA by host cell ribosomes and other cellular translation factors into a single polyprotein of 250 kD. The polyprotein is proteolytically processed via precursors (Fig. 2). First, the protease activity of 2A cleaves the polyprotein P1 to release it from the entire polyprotein. Then, protease 3C sequentially cleaves all the remaining several intermediate precursors, except VP4 and VP2, that remain as the precursor protein V0 until autocatalytic cleavage in the newly formed capsid packaged with viral RNA.

760

Rhinoviruses (Picornaviridae)

Replication The RNA-dependent RNA polymerase protein 3D is mainly responsible for the replication of RV genome, facilitated by other nonstructural proteins. To initiate the replication, 3D polymerase uridylates the VPg protein using cis-acting replication element (cre) as the template. Cre has a hairpin RNA structure, and its location in the genome is species-specific: within 2A in RV-A, 1D (VP1) in RV-B, and 1B (VP2) in RV-C. Two adenosines in the loop of cre serve the uridylation. Uridylated VPg acts as the primer for RNA replication at the 30 polyA tail. The negative-sense RNA copy is then used as the template for positive-sense copies, resulting in an average ratio of 50 copies of ( þ )RNA to one copy of (  )RNA.

Assembly Structural proteins VP1, VP3 and VP0 form protomers, which organize to pentamers and subsequently form the capsid. When the newly synthesized genomic RNA of the virus enters, the final maturation of the capsid takes place by autocatalytic cleavage of VP0 into VP2 and VP4, rendering the particle infectious. VP4 is myristoylated, which, in analogy with other enteroviruses, likely has a role in the final proteolytic step of maturation. Release. In susceptible, cultured cell-lines, RV infection causes a strong cytopathic effect (CPE), indicating lytic release of nascent viruses. Ciliated epithelial cells are found in excretions of RV infected individuals regardless of symptom severity. On the other hand, studies of differentiated airway epithelial cells show only little CPE, also suggesting non-lytic RV release with an unknown mechanism.

Epidemiology RV is circulating in the populations all year round, with typical peaks of high prevalence in early autumn and late spring. In year-round surveys with multiplex PCR detection methods, RV is the most common virus in all age groups, constituting 30%–50% of the findings. The epidemic seasons mirror each other in temperate zones of northern and southern hemispheres but are less clear near the equator. The autumn increase of RV prevalence coincides with the start of a new school year. When other respiratory viruses become more common during winter, the number of RV infections decrease, or their share of all infections is reduced and vice versa in the spring (Fig. 3). Thus, RV seems to be highly transmissible but sensitive to interference by other virus infections. Compared to influenza, respiratory syncytial virus (RSV), parainfluenza viruses and metapneumovirus, RV is more often detected as a co-infection with other viruses. These epidemiological observations could be explained by RV being sensitive to but a relatively poor inducer of host interferon (IFN) system. RV-A is usually the most prevalent species, sometimes overtaken by RV-C, but RV-B is less common, representing rarely more than 10% of the findings. Some longitudinal studies have implicated RV-A12 and RV-A78 as more prevalent types. In addition to them, RV-C15 and RV-C2 are detected more frequently than others in general. Still, further studies over more extended periods of time must be performed to pinpoint these or any other types as more transmissible or more pathogenic.

Pathogenesis Symptomatic RV infection begins with coryza, rhinorrhea or nasal obstruction. The neutrophilic inflammatory response to the infection leads to increased vascular permeability and mucin secretion. An uncomplicated RV infection is limited to the upper

Fig. 3 Seasonality of rhinovirus infections. The graph shows the monthly incidence of RV as compared to selected other respiratory viruses, including influenza A and B virus, respiratory syncytial virus, parainfluenza virus types 1–4, and metapneumovirus. The incidence was calculated as findings per total number of monthly specimens in multiplex RT-PCR. Overall, 7436 specimens were tested; 1385 were positive for RV and 1440 for the other viruses. Data from the Clinical Microbiology laboratory of Turku University Hospital.

Rhinoviruses (Picornaviridae)

761

respiratory epithelium, but it is not effusively cytotoxic even in lower airways. However, there is a disruption in the epithelial barrier function, which may open the window for other opportunistic pathogens. Neutrophils are attracted to the infection site mainly by chemokines interleukin (IL)-8 and IP-10, which are hallmarks of a symptomatic RV infection. Other proinflammatory cytokines induced by the host innate immune response include IL-6 and RANTES. Induction of type I and III IFNs is detectable through an expression of IFN stimulated genes and proteins, such as myxovirus resistance protein A and viperin, both locally and systemically. However, RV does not induce particularly strong IFN response that is often associated with fever. IFNs, in turn, are effective against RV infection. Poor IFN response is associated with prolonged RV infections in patients with a common variable immunodeficiency. Reduced in vitro IFN production has been found in studies on RV infection in airway epithelial cells from patients with asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Asymptomatic RV infection differs in the host transcriptome analysis from the symptomatic infections, which are characterized by overexpression of IFN-, inflammation-, neutrophil- and monocyte-related genes. Asymptomatic infections generally have lower viral loads.

Clinical Features RV is the most commonly detected respiratory virus in all age groups, which makes it also the most common cause of any acute infection in humans. Children acquire their first RV infection early, and by two years of age, practically all have been infected at least once, with a mean annual rate of 3.5 infections. In adults, RV infections account for approximately 20%–50% of respiratory tract infections annually. In practice, over 90% of the subjects suffer at least one RV infection in a year. Species A and C are more common and may cause more severe illnesses early in life than RV-B.

Asymptomatic Infections Many studies have shown that RV may be more common in asymptomatic subjects than in cases with respiratory symptoms. In analogy with COVID-19, this may have significance for transmission of the infection, but this phenomenon has remained poorly studied. RV RNA can be detected before, during, and after symptomatic infection, in subclinical infection, or may rarely be innocent contamination. Different studies have identified RV RNA in 12%–50% of asymptomatic children. In adults, asymptomatic respiratory infections, mostly caused by RV, are detected in 4%–6% of the subjects. It is essential to recognize that in otherwise healthy subjects, RV is not known to induce chronic illness. For these reasons, it has been concluded that RV RNA finding signifies most probably actual infection with or without symptoms.

Common Cold Compared with other respiratory viruses, RVs have a dominant role as causative agents of upper respiratory tract infections in young children, adults and older people. The incubation period of RV common cold is two days. The most common symptoms of RV-induced common cold include rhinorrhea, nasal congestion, cough and sore throat. The symptoms are most severe during the first four days of illness and last, on an average, 8–10 days. In the older people, the median duration of the illness is 16 days. RV RNA positivity lasts up to four weeks after an acute RV infection, but the infectious period is likely to be closer to that of the most substantial symptoms.

Acute Otitis Media (AOM) RSV has earlier been considered as a major predisposing factor for the development of AOM. Studies with RT-PCR suggest a dominant role of also RVs. In large Finnish Otitis Media Cohort Studies, 32%–42% of AOM episodes were associated with RV infections. RV RNA has been detected in 17%–41% of the middle ear fluid specimens from children with AOM.

Bronchiolitis and Recurrent Wheezing RV is associated in up to one-quarter of bronchiolitis cases in infants, second only to RSV found in about two-thirds of them. RSV dominates in infants and RV in children older than 12 months. Interestingly, RV-infected children had a significantly shorter length of hospital stay as compared with children with RSV bronchiolitis. Innate immune responses inversely correlate with the RV disease severity. Several studies have shown that infants with a history of bronchiolitis will have significantly more often recurrent wheezing than controls without bronchiolitis. RV detection in the first wheezing episode is the most critical risk factor, followed by positive family history for asthma or atopy. Severe episodes of bronchiolitis increase the odds of early asthma. Asthma at seven years has been diagnosed in 52% or 15% of children, who have suffered RV or RSV bronchiolitis, respectively. There is evidence that early prednisolone treatment of RV-induced wheezing may reduce recurrent wheezing. This requires further confirmation, as prednisolone treatment does not achieve a similar benefit in bronchiolitis caused by RSV.

762

Rhinoviruses (Picornaviridae)

Asthma RV infections are responsible for the majority of asthma exacerbations in children and adults. Up to 90% of asthma exacerbations in children are associated with virologically defined natural respiratory virus infections. RV is the most common finding and is detected in approximately two-thirds of the cases. Children with asthma and IgE-mediated allergic sensitization are more prone to RV infections. Polymorphisms in CDHR3 and chromosome locus 17q12–21 are associated with increased risk of RV wheezing. There is evidence that asthmatic patients have more frequent RV, more severe, and longer-lasting lower respiratory tract infections than non-asthmatics. Many studies have unveiled the mechanisms of increased susceptibility to RV infections in asthmatic patients. They include changes in airway epithelium, defective RV-induced secretion of IFN-b, IFN-l, IL-10 and probably IFN-a, and impaired alveolar macrophage function. RV infection during the first weeks of life is a significant risk factor because it may permanently mold the response to subsequent infections and increase the risk of asthma. RV-A and RV-C are more likely to induce wheezing than RV-B.

Pneumonia Many different studies suggest that RVs are important causative agents of pneumonia both in children and adults, and in immunosuppressed subjects. RV has been identified from induced sputum, tracheal brushing, bronchoalveolar lavage and lung tissue samples, strongly suggesting that it can cause lower respiratory tract infections. In studies on childhood community-acquired pneumonia RV is detected in one-fifth of the cases. The clinical profile of RV infections has included pneumonia in 11%–53% children admitted to hospital. RVs are commonly detected in children with severe pneumonia also in low-income countries. In a cornerstone study among 2320 adults with radiographic evidence of pneumonia, viral pathogens were detected in 589 of the cases and out of those RV was detected in 194 cases. RV may also be the causative agent of severe pneumonia in adults.

Chronic Obstructive Pulmonary Disease (COPD) Respiratory viruses are involved in 40%–50% of acute exacerbations of COPD, which is the fourth leading cause of death worldwide. RV is the most common virus causing COPD exacerbations. Viral exacerbation is associated with a more severe illness than exacerbation with no virus. Experimental RV infections in patients with mild COPD have confirmed the causative role of RV in exacerbations. There is evidence that patients with COPD may have impairment in the immune response to RV and COPD patients may have chronic RV infections as also seen in patients with immunodeficiency diseases and lung and hematopoietic stem cell transplant recipients.

Diagnosis As with other respiratory viruses, there are no clinical features of RV infection, allowing a specific diagnosis without laboratory tests. Nucleic acid detection is the only method for universal RV detection. Virus culture is less sensitive in general and not available for wild type RV-C strains. The significant number of different strains has thwarted the development of antibody reagents for diagnostic antigen detection. For the same reason, antibody detection for serological RV diagnostics in clinical use is a futile task.

Sampling For the nucleic acid detection or culture, a nasopharyngeal swab is the specimen of choice, but any sample containing respiratory excretions, including nasal swab, nasopharyngeal of tracheal aspirate, (induced) sputum and bronchoalveolar lavage may be used. For culture, the specimen should be immediately placed into virus transport medium, for PCR, a swab can be transported even in a dry test tube for more flexible downstream processing in the laboratory. RV can be detected in respiratory tract tissue biopsies. Although RVs do not survive acidic pH of a normal stomach, they can be often detected in the feces of children.

Nucleic Acid Detection All members of Enterovirus genus, including human enterovirus (EV) and RV species, have highly conserved motifs at the 50 UTR. These enable universal amplification of EVs and RVs with a single primer pair in RT-PCR. The optimal primer site is at the distal part of the 50 UTR (Fig. 4). Using advanced probe design, it is also possible to simultaneously differentiate RVs from EVs in a realtime RT-PCR assay. Using only dsDNA dye, like SYBR Green and melting curve analysis, it is possible to make a differential diagnosis with 80% accuracy, due to lower average melting temperatures of RVs. Additional conserved sites closer to the proximal part of the 50 UTR are applicable but result in longer amplicons with less efficient amplifications or less specific primers and probes. Many commercial assays do not differentiate between RVs and EVs, which is suboptimal, since it may delay early recognition of EVs in respiratory samples or lead to overestimation of EVs in stool samples. The copy number or threshold cycle (Ct) determination may elucidate clinical relevance of RV detection in co-infections.

Rhinoviruses (Picornaviridae)

763

Fig. 4 Rhinovirus 50 UTR target site for RT-PCR. (A). 50 UTR with predicted secondary structure. The prediction was performed for RV-A1 genome (GenBank D00239.1) using mfold (unafold.rna.albany.edu). Base pairs: red line, C-G; blue line, A-U; green line, G-U. The coding sequence for 1A (VP4) starts at nucleotide 623. (B). Nucleotide homology at the optimal 50 UTR target site for RT-PCR. Frequency of the identical nucleotides of RVs (blue line) and EVs (gray line) as compared to the consensus RV sequence. The positions refer to RV-A1 genomic sequence (GenBank D00239.1). Sites for universal RV and EV primers are indicated by orange arrows and a target site allowing design of probes differentiating between RVs and EVs by a green line.

Culture Routine culture of RV-A and RV-B strains can be carried out in heteroploid HeLa cells and primary diploid fibroblast cell lines MRC-5 and WI-38. RV-C strains can only be cultured in respiratory organ culture. HeLa-E8 cells over-expressing CDHR3 seem not to be applicable for universal RV isolation in cell culture. Since 30%–50% of circulating strains are representatives of RV-C species, culture is not recommended as the primary diagnostic method for RV infections. RV isolation is usually performed in roller tube cultures at 33–351C for the best yield, but the multiplication of wild type viruses is not restricted at 371C. A blind passage of week-old cultures increases the yield. A specific diagnosis of a positive culture requires confirmation by RT-PCR. A high Ct value in RT-PCR, corresponding to low amounts of virus in a cell culture supernatant, may indicate traces of non-replicating seed virus, abortive infection, or slow adaptation of the virus. Former methods for differentiation between cultivable RV and EV, by acid lability test (RVs are sensitive to pHo5) or serological typing, are nowadays mostly not in use.

Typing Identification of the strain in individual infections has no clinical value. Still, it is of great interest from an epidemiological point of view, which may also reflect on drug development. While the whole genome sequencing is developing, single amplicon sequencing, by Sanger or NGS methodology, is used for RV typing. To cover the high number of different RV types, highly degenerate primers targeting gene regions for VP1 or VP4/2 are used. Typing directly from RNA of clinical specimens usually has up to 75% success rate depending on the amount of virus and primability of the strain in question. The resulting sequence is compared with those in the GenBank, and the type is revealed among the nearest matches, following the rules explained above in the Classification section. At the species level, the more sensitive 50 UTR sequencing can be used, but there is some ambiguity in 50 UTR between RV-A and C species. On the other hand, 50 UTR sequence may strengthen the VP gene sequencing results. High resolution melting temperature (Tm) analysis of 50 UTR amplicons can be used to discern whether RV in sequential samples from the same individual represents the same RV type (same Tm) or subsequent infections with multiple types (different Tms).

764

Rhinoviruses (Picornaviridae)

Treatment No antiviral drugs have been approved for clinical use in HRV infections. Interestingly, the monoclonal anti-IgE antibody, omalizumab, reduces RV infections and shedding of RV in asthmatic children. Azithromycin, a macrolide antibiotic, reduces in vitro replication of RV. Furthermore, azithromycin increases RV-induced IFN and IFN-stimulated gene expression. In a controlled clinical study, azithromycin reduced the duration of RV-induced episodes of asthma-like symptoms in young children.

Prevention There is no vaccine against RV infections. Avoidance of exposure to RV is the best way to prevent infection. A major route of transmission is probably from hands of infected subjects to an intermediary surface or directly to the fingers of the susceptible recipient, who is then infected by self-inoculation of the nose or eye. Handwashing with soap is considered better than an alcohol sanitizer because the effectiveness of the latter is questionable against the non-enveloped RV. Airborne RV has also been identified in exhaled breath and coughs of children and adults with an acute respiratory illness. Thus, aerosol transmission is a potential mode of transmission. Physical distancing, covering coughs and sneezes or using face mask may be recommended, as with any other respiratory infection, to prevent the spread of the infection.

Further Reading Altman, M.C., Beigelman, A., Ciaccio, C., et al., 2020. Evolving concepts in how viruses impact asthma: A work group report of the microbes in Allergy Committee of the American Academy of Allergy, Asthma & Immunology. Journal of Allergy and Clinical Immunology 145, 1332–1344. Bartlett, N., Wark, P., Knight, D. (Eds.), 2019. Rhinovirus infections: Rethinking the Impact on Human Health and Disease. Academic Press: Elsevier. Jacobs, S.A., Lamson, D.M., George, K.S., Wash, T.J., 2013. Human rhinoviruses. Clinical Microbiology Reviews 26, 135–162. Jain, S., Self, R.G., Wunderink, R.G., et al., 2015. Community-acquired pneumonia requiring hospitalization among U.S. adults. New England Journal of Medicine 373, 415–427. Leung, N.H.L., Chu, D.K.W., Shiu, E.Y.C., et al., 2020. Respiratory virus shedding in exhaled breath and efficacy of face masks. Nature Medicine 26, 676–680. McIntyre, C.L., Knowles, N.J., Simmonds, P., 2013. Proposals for the classification of human rhinovirus species A, B and C into genotypically assigned types. Journal of General Virology 94, 1791–1806. Miller, K.E., Linder, J., Kraft, D., et al., 2016. Hospitalization and outpatient visits for rhinovirus-associated acute respiratory illness in adults. Journal of Allergy and Clinical Immunology 137, 734–743. Oliver, M.E., Hinks, T.S.C., 2020. Azithromycin in viral infections. Reviews in Medical Virology. e2163. doi:10.1002/rvm.2163. Osterback, R., Tevaluoto, T., Ylinen, T., et al., 2013. Simultaneous detection and differentiation of human rhino- and enteroviruses in clinical specimens by real-time PCR with locked nucleic acid probes. Journal of Clinical Microbiology 51, 3960–3967. Paul, A.V., Wimmer, E., 2015. Initiation of protein-primed picornavirus RNA synthesis. Virus Research 206, 12–26. Peltola, V., Waris, M., Kainulainen, L., Kero, J., Ruuskanen, O., 2013. Virus shedding after human rhinovirus infection in children, adults and patients with hypogammaglobulinaemia. Clinical Microbiology and Infection 19, E322–E327. Ritchie, A.I., Wedzicha, J.A., 2020. Definition, causes, pathogenesis, and consequences of chronic obstructive pulmonary disease exacerbations. Clinics in Chest Medicine 41, 421–438. Ruuskanen, O., Waris, M., Ramilo, O., 2013. New aspects of human rhinovirus infections. The Pediatric Infectious Disease Journal 32, 553–555. Zlateva, K., van Rijn, A.L., Simmonds, P., et al., 2020. Molecular epidemiology and clinical impact of rhinovirus infections in adults during three epidemic seasons in 11 European countries (2007–2010). Thorax 75, 882–890.

Relevant Websites www.picornaviridae.com Picornavirus Home. https://virologydownunder.blogspot.com/2015/04/rhinoviruses-rvsa-primer.html Rhinoviruses (RVs)...a primer.

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae) Tetsuro Ikegami, The University of Texas Medical Branch at Galveston, Galveston, TX, United States r 2021 Elsevier Ltd. All rights reserved.

Nomenclature

NSm

ABIN2 A20-binding inhibitor of NF-kB 2 ALT Alanine aminotransferase AMTV Arumowot virus AST Aspartate aminotransferase BCX4430 Galidesivir CPK Creatinine phosphatase DC Dendritic cells DC-SIGN Human dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin DC-SIGNR Liver/lymph node-specific intercellular adhesion molecule 3 grabbing nonintegrin (L-SIGN) eIF2a Eukaryotic initiation factor 2a ELISA Enzyme-linked immunosorbent assay ENSO El Niño Southern Oscillation GTV Guertu virus HRTV Heartland virus IFN Interferon IFN-AR Interferon-a/b receptor IRF-3 Interferon regulatory factor 3 LAMP Loop-mediated isothermal amplification LDH Lactate dehydrogenase L-segment Large segment M-segment Medium segment NIH National Institutes of Health

Glossary 78-kD protein A protein encoded by the RVFV M-segment. It is expressed via cotranslational cleavage of a glycoprotein precursor from the first initiation codon. Ambi-sense A viral coding strategy encoding two different genes in negative- and positive-sense RNA, respectively. Antigenic complex A group of phleboviruses, which show high cross-reactivity via complement fixation test and hemagglutination inhibition test. Cap-snatching Use of 50 short fragments of capped cellular mRNAs to prime viral mRNA synthesis. Cleavage of cellular mRNA is mediated via an endonuclease activity of viral L proteins. Category A-C Priority pathogens The National Institutes of Health in the United States of America list and classify pathogens based on emerging threat to the national security and public health.

Nonstructural gene encoded by the bunyavirus M-segment NSs Nonstructural gene encoded by the bunyavirus S-segment ORF Open reading frame PKR Double-stranded RNA-dependent protein kinase PTV Punta Toro virus RIG-I Retinoic acid-inducible gene I RNP Ribonucleocapsid RVF Rift Valley fever RVFV Rift Valley fever virus SAP30 Sin3A associated protein 30 SFNV Sandfly fever Naples virus SFSV Sandfly fever Sicilian virus SFTSV Severe Fever with Thrombocytopenia Syndrome virus S-segment Small segment STAT Signal transducer and activator of transcription T-705 Favipiravir TBK1 TANK-binding protein 1 TFIIH Transcription factor II H TOSV Toscana virus TPL2 Tumor progression locus 2 TSI-GSD-200 A formalin-inactivated RVF vaccine under clinical trials for human use (U.S.) UUKV Uukuniemi virus VLP Virus-like particle

DC-SIGN A C-type lectin protein expressed on the surface of dendritic cells or macrophages. DC-SIGN can bind to specific types of carbohydrates and the interaction can lead to phagocytosis. Floodwater Aedes spp. Floodwater Aedes spp. mosquitoes in floodplain habitat lay eggs in moist or waterlogged soil. Eggs survive drought and hatch upon rainfall or flooding. Select agent Biological agents and toxins regulated under the Federal Select Agent Program in the United States of America, which potentially pose a severe threat to public health and safety. Transcription factor II H TFIIH play important roles in transcriptional initiation via RNA polymerase II and DNA nucleotide excision repair. TFIIH is comprised by ten subunits, XPB, XPD, p52, p44, p62, p34, p8, cdk7, cyclin H, and MAT1.

Introduction The family Phenuiviridae, within the order Bunyavirales, consists of 15 genera: Beidivirus, Goukovirus, Horwuvirus, Hudivirus, Hudovirus, Mobuvirus, Phasivirus, Pidchovirus, Tenuivirus, Wenrivirus, Wubeivirus, Laulavirus, Kabutovirus, Banyangvirus, and Phlebovirus.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21499-4

765

766

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

Viruses belonging to the former 12 genera are not known to cause any diseases in vertebrates. Many viruses belonging to the genus Phlebovirus are transmitted via arthropod vectors, such as sandflies, mosquitoes, or ticks, and they cause mild to severe diseases in humans and/or in animals. Some tick-borne phleboviruses were reclassified into the Banyangvirus or Kabutovirus genera, which were newly made in 2018–2019, are also described in this article. At least 100 distinct phleboviruses have been characterized and named, although only 10 species have been officially approved via the International Committee for Taxonomy of Viruses: Bujaru, Candiru, Chilibre, Frijoles, Mukawa, Punta Toro, Rift Valley fever, Salehabad, Sandfly fever Naples, and Uukuniemi phlebovirus. These 10 phleboviral species can be distinguished via four-fold differences in the two-way cross-neutralization test. The remaining phleboviruses have not been officially assigned to species. Such phleboviruses can be further grouped into antigenic complexes via serological cross-reactivity using a complement fixation test and a hemagglutination inhibition test. In addition to serological tests, phylogenetic analyzeses can be additionally applied to the grouping of phleboviruses. Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by Rift Valley fever virus (RVFV), which is endemic to sub-Saharan Africa, but has spread to Egypt, Madagascar, the Comoros, Yemen, and Saudi Arabia. RVF is one of the most pathogenic phleboviral diseases affecting ruminants and humans, and it has been classified as a Category A Priority Pathogen by the United States (U.S.) National Institutes of Health (NIH), and as a Blueprint priority disease by the World Health Organization. In response to an increasing fear of bioterrorism, RVFV is also designated as an overlap select agent by the U.S. Department of Health and Human Services and the U.S. Department of Agriculture, meaning that its possession and handling are strictly monitored in the U.S. Phlebotomus fever (three-day fever) is a disease caused by Sandfly fever Naples virus (SFNV) and Sandfly fever Sicilian virus (SFSV), which are transmitted via sandflies in Eurasia and Africa. Although the febrile disease is self-limiting, outbreaks may affect the health status of soldiers or travelers for up to two weeks before a complete recovery from the illness occurs. Moreover, Toscana virus (TOSV), which was first isolated from sandflies in Italy in 1971, has spread into Europe and North Africa. Aseptic meningitis or meningoencephalitis caused by TOSV are more severe than the typical phlebotomus fever. In the New World, phlebotomus fever can be caused by several different phleboviruses, such as the Punta Toro (PTV), Chagres, and Candiru viruses. Furthermore, febrile illness associated with a viral meningitis can be caused by a tick-borne Bhanja virus, which is widely distributed throughout Eurasia and Africa. Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is not pathogenic to humans. This virus has been used as a model for phleboviral virology studies. In the past decade, the Severe Fever with Thrombocytopenia Syndrome phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus, was identified in East Asia (China, Korea, Japan, Vietnam, Taiwan). A genetically similar pathogenic tick-borne phlebovirus was also identified in the U.S. Those pathogenic tick-borne phleboviruses were reclassified from the genus Phlebovirus into the genus Banyangvirus, which now includes Huaishangyang banyangvirus (formerly SFTSV), Heartland banyangvirus (HRTV), and Guertu banyangvirus (GTV). Due to public health concerns, both SFTSV and HRTV are classified as Category C Priority Pathogens in the U.S.

Virion Structure, Genome, and Strategy of Replication The phleboviral genome consists of three single-stranded RNA segments: negative-sense Large (L)- and Medium (M)-segments, and an ambi-sense Small (S)-segment (Fig. 1). The L-segment encodes a single open reading frame (ORF) for the RNA-dependent RNA polymerase and the M-segment encodes a single ORF for a precursor protein, which can be co-translationally cleaved into Gn and Gc envelope glycoproteins. Some phleboviruses also encode non-structural M (NSm) proteins upstream of the Gn proteins. The S-segment encodes an ORF for the nucleoprotein (N) in the negative-sense RNA, and an ORF for the non-structural S (NSs) protein in the positive-sense RNA. Phleboviral virions are spherical or pleomorphic. In virions, ribonucleocapsids (RNP), which consist of viral RNA encapsidated with N proteins, are surrounded by a viral envelope, which comprises a host-derived lipid bilayer and GnGc proteins. RVFV virions are 90–110 nm in diameter, with surface spikes 10–18 nm in length. Cryoelectron tomography has further characterized the surface arrangement of hollow-like capsomers composed of Gn/Gc heterodimers and/or homodimers arranged in T ¼ 12 icosahedral symmetry. The T ¼ 12 icosahedral symmetry of capsomers was also found in virions of a tick-borne UUKV, which is 125 nm in its mean diameter, with surface spikes 12–15 nm in length. Virions attached to cell surface receptors are internalized via endocytic pathways. One receptor molecule for phleboviruses is the human dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), which is a C-type lectin expressed on the surface of a specific subset of dendritic cells (DCs) and macrophages. Binding to specific types of mannose-rich or fucosylated glycans leads to an uptake of ligands via endocytosis. Other molecules, such as DC-SIGNR (L-SIGN), heparan sulfate proteoglycans, and the non-muscle myosin heavy chain IIA (NMMHC-IIA), are also important for the attachment of some phleboviruses or banyangviruses. Endocytosis involved in phleboviral entry is mediated via caveolae or clathrin, depending on the virus. In late endosomes, low pH leads to an exposure of hydrophobic fusion loops as a result of structural changes in the Gc protein (class II fusion protein). Fusion loops can then be inserted into cellular endosomal membranes, triggering the release of RNP into the cytoplasm. Once RNP is released into the cytoplasm, L proteins associated with incoming virions start the transcription of viral mRNAs (primary transcription). L proteins cleave host cytoplasmic mRNA, typically 10–20 nt from the 50 termini, and use those oligonucleotides as primers to synthesize their own transcripts (cap-snatching). Viral mRNA is not polyadenylated at the 30 terminus, and the lengths of transcripts are shorter than those of viral genomic RNA templates because transcriptional termination occurs

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

767

Fig. 1 Genome structure of phleboviruses. The schematics represent the genome structure of Rift Valley fever virus (RVFV). Viral mRNAs are transcribed via cap-snatching of host mRNA. N and NSs mRNA are transcribed from negative- and positive-sense S-segment, respectively. The RVFV M mRNA can be translated into two precursors, which generate (i) 78-kD þ Gc, and (ii) NSm þ Gn þ Gc. Tick-borne phleboviruses do not encode NSm gene, whereas glycoprotein precursors have not been characterized for most phleboviruses. Newly identified tick-borne phleboviruses (e.g., Blacklegged tick phleboviruses) encode a large N gene and a small NSs-like open reading frame in S-segment.

within untranslated regions in L-, M-, and S-segments. The positive-sense S-segment of RVFV or UUKV can also be packaged into virions, which allows the accumulation of primary transcripts of NSs mRNA from virion-associated positive-sense S-segments, together with N mRNA, during primary transcription. With an increase in cellular levels of N and L proteins, the replication of viral genomic RNA occurs. The sequences of the 30 and 50 termini of L-, M-, and S-segments are complementary, and thus the RNPs of L-, M-, and S-segments form panhandle structures. The panhandle structures are considered to support encapsidation of RNA with N proteins and promote the recruitment of L proteins for viral genomic RNA replication. RVFV N proteins can be assembled into a hexameric ring, structurally displaying both a RNA binding cleft and a N oligomerization groove, which allows the multimerization of N proteins along with genomic RNA. Viral RNA synthesis occurs via genomic RNA encapsidated with N proteins. Accumulation of genomic RNA during viral RNA replication can also amplify viral mRNA synthesis (secondary transcription), followed by further accumulation of viral proteins. The assembly of viral components occurs in the Golgi complex or in the ER-Golgi intermediate compartment. Although phleboviruses do not encode any matrix proteins, the cytoplasmic domain of Gn play a role in interactions with RNP in the Golgi. The Golgi retention signal is also encoded surrounding the Gn cytoplasmic tail. The Gc protein cytoplasmic tail at the C-terminus is very short, which encodes a presumable ER retrieval signal. Co-expression of Gn and Gc proteins leads to the co-localization of both Gn and Gc proteins in the Golgi. Mutagenesis of the Gc cytoplasmic tail could lead to mislocalization of Gc into the ER, even in the presence of Gn proteins, indicating that the Gc cytoplasmic tail plays a role in the dimerization of Gn and Gn proteins, or in the trafficking of Gc proteins to the Golgi. The formation of virus-like particles (VLPs) occurs with the expression of Gn and Gc proteins. In the case of RVFV, however, the presence of RNP facilitates the production of VLPs. In an artificial over-expression system of RVFV Gn and Gc proteins using insect Sf9 cells, VLPs consisting of either Gn, Gc, and N proteins, or Gc and N proteins could be produced without RNP.

Functions of Nonstructural Proteins NSs proteins, which are expressed from the S-segment, are the major virulence factors for pathogenic phleboviruses and banyangviruses, and they share low similarity in amino acid level. RVFV NSs proteins form filamentous structures in the nuclei of infected cells, sequestering p44 protein and promoting post-translational degradation of p62 protein, both of which are among ten subunit proteins of transcription factor (TF) IIH, which is essential for cellular transcription via RNA polymerase II. The expression of RVFV NSs proteins can thus induce general transcriptional suppression, including that of the IFN-b gene. RVFV NSs filaments also bind to Sin3A Associated Protein 30 (SAP30) and form a repressor complex on the IFN-b gene promoter, which can be another mechanism inhibiting IFN-b gene expression in RVFV-infected cells. RVFV NSs proteins also localize to the cytoplasm and promote post-translational degradation of dsRNA-dependent protein kinase (PKR), which is a cellular sensor of dsRNA and triphosphated ssRNA. PKR bound to RNA substrates exposes kinase-active domains, which can phosphorylate the eukaryotic initiation factor (eIF) 2a subunit and lead to the shutoff of host and viral protein syntheses. Because RVFV NSs proteins can promote the degradation of PKR, infected cells can maintain active viral protein synthesis due to a lack of PKR-mediated eIF2a phosphorylation. TOSV NSs proteins also inhibit the expression of IFN-b gene, while promoting the posttranslational degradation of PKR and the retinoic acid-inducible gene I (RIG-I), but not that of TFIIH p62. SFTSV NSs proteins form inclusion bodies in the cytoplasm, which sequester interferon regulatory factor 3 (IRF3) via binding to TANK-binding protein 1 (TBK1). These inclusion bodies of SFTSV NSs proteins also sequester cellular signal transducer and activator of transcription (STAT) 1 and STAT2. Accordingly, both

768

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

the induction and signaling pathways for the IFN-b gene can be inhibited by SFTSV NSs proteins. Moreover, the SFTSV NSs proteins bind to the A20-binding inhibitor of NF-kB 2 (ABIN2), which in turn inhibits the suppression of tumor progression locus 2 (TPL2) and p105 via ABIN2, and leads to the upregulation of anti-inflammatory genes, such as IL-10. HRTV NSs protein does not form inclusion bodies in the cytoplasm, but it binds to TBK-1, which hinders the association of TBK1 with IRF3. Other pathogenic phleboviruses, such as PTV and SFSV, also inhibit the expression of IFN-b gene, but these inhibitor mechanisms have so far remained unknown. The NSs protein of Arumowot virus (AMTV) inhibits the upregulation of IFN-b gene in murine cells, whereas the AMTV NSs proteins can be posttranslationally degraded via proteasomal pathway in human cells, indicating varied stability of NSs proteins in different host species. The NSm protein is encoded upstream of the GnGc coding region in the M-segment, although tick-borne phleboviruses or banyangviruses do not encode the NSm gene. The role of NSm proteins is largely unknown in most phleboviruses. The RVFV M-segment encodes five in-frame AUGs upstream of the GnGc coding region, and cellular ribosomes can initiate translation from the 1st and the 2nd AUGs via leaky scanning. The first AUG encodes a precursor protein of 78 kD, whereas the second AUG encodes a precursor that can be co-translationally cleaved into the NSm, Gn, and Gc proteins. RVFV lacking four of the five AUGs can still replicate in cells, indicating that 78 kD or NSm proteins are dispensable in the viral life cycle. In contrast, specific knockout of the 78 kD protein may result in poor viral transmission in mosquitoes. In one study, RVFV 78 kD proteins were incorporated into virions in mosquito C6/36 cells, but not in Vero cells, indicating an interesting biological difference between mosquito and mammalian cells. Furthermore, RVFV NSm proteins localize to the mitochondrial outer membrane via basic amino acids upstream of a putative transmembrane domain at the C-terminus and suppress apoptosis. Abolishing of both the 78kD and NSm proteins can partially attenuate RVFV.

The Viruses Rift Valley Fever Virus RVF is now endemic in sub-Saharan Africa, Madagascar, the Comoros, Egypt, Saudi Arabia, and Yemen, and it is primarily maintained via floodwater Aedes spp. mosquitoes (e.g., subgenera Neomelaniconion, Aedimorphus) (Table 1). RVFV can be vertically transmitted into offspring via transovarial transmission, and viruses are considered to maintain their infectivity in drought-resistant eggs for several years. Other mosquito species also play a role in the amplification of RVFV via susceptible animals in nature (e.g., mosquitoes of the genera Culex, Coquillettidia, Anopheles, Eretmapodites, Mansonia). In endemic areas, animals such as African buffalos, domestic ruminants, bats, rodents, and other wild mammals are likely involved in the maintenance of RVFV. It has been suggested that increased biting of susceptible animals by RVFV-infected mosquitoes can trigger RVFV amplification and epidemics. In terms of increasing mosquito populations, the establishment of habitats favorable for the hatching of infected mosquitoes (e.g., heavy rain, irrigation) is important. The prediction of RVF outbreaks linking to heavy rainfall in climates during the El Niño Southern Oscillation (ENSO) condition is one of the important efforts for allowing early preparation for RVF outbreaks in eastern Africa. RVFV isolates can be grouped into lineages A – G (alternatively, lineages A – N). The maximum genetic diversity of L-, M-, and S-segments among RVFV strains is 4%, 5%, and 4%, respectively, at the nucleotide level; and 1%, 2%, and 1%, respectively, at the deduced amino acid level. High similarity among RVFV isolates allows cross-neutralization using antisera raised against one RVFV strain. An RVF outbreak is a major public health threat, and can also cause devastating economic damage by affecting animal industry. For example, a single RVF outbreak in South Africa in 1951 resulted in an estimated 500,000 abortions and 100,000 deaths among sheep. During RVF outbreaks, high rates of abortions occur in sheep (up to 100%), goats (up to 100%), and cattle (up to 40%). Newborn lambs and goat kids less than one week old are highly susceptible to RVFV and, due to an acute or peracute liver necrosis, case fatality rates are typically higher than 90%, whereas those of newborn calves range from 20% to 70%. Adult ruminants are more resistant to RVFV infection than are newborn animals, and the disease is associated with hepatic necrosis, resulting in case fatality rates of 5%–30% for sheep and goats, and 10% for cattle. Camels are also susceptible to RVFV, although the clinical signs vary a lot according to different reports (e.g., asymptomatic, icterus, ocular discharge, blindness, hemorrhage, nervous symptoms, abortion). RVFV does not usually cause diseases in pigs, horses, dogs, cats, or birds. Humans can be infected with RVFV via the bite of an infected mosquito, or through an exposure to the bodily fluids of infected animals. Infected humans exhibit a biphasic febrile illnesses. An estimated 18,000–200,000 people were infected during the first RVF outbreak in Egypt in 1977–1978, and 598 patients died. Although the overall case fatality rate of humans is 1%–2%, the case fatality rate among hospitalized patients was estimated to be 34% during the RVF outbreak in Saudi Arabia in 2000–2001. The incubation period is typically 4–6 days, and clinical signs start abruptly. These include malaise, severe chills, severe headache, nausea, weakness, and fever (38.8–39.51C). Clinical signs can be reduced in 3–4 days, although severe clinical signs can recur thereafter. In the convalescence phase, most patients recover without sequela in two weeks or more, although some patients experience neurological disorders, thrombosis, or vision loss weeks or months later. Some patients suffer from an acute hemorrhagic syndrome, including macular rash, ecchymosis, bleeding, or disseminated intravascular coagulation. RVF can be diagnosed based on the detection of elevated IgM and IgG specific to RVFV via an enzyme-linked immunosorbent assay (ELISA), using paired serological samples. The detection of viral neutralizing antibody can improve the specificity of

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

Table 1

Phleboviruses in the old world (mosquito-, sandfly-borne, and other non tick-borne viruses)

Antigenic complex

Virus

Isolations

Sandfly Fever Naples

Sandfly Fever Naples Toscana

South Europe, North Africa, Middle East, South and Central Asia Sandflies (P. perfiliewi and P. perniciosus, Italy, Spain, Portugal, France, Malta, Croatia, Bosniaand others) Herzegovina, Greece, Turkey, Cyprus, Morocco, Algeria, Tunisia Portugal, France Sandflies (P. perniciosus) Spain Sandflies (P. perniciosus) Portugal Sandflies (Phlebotomus spp.) Italy Sandflies (P. perfiliewi) Iran Sandflies (Phlebotomus spp.) Turkey Sandflies (Phlebotomus spp.) Sandflies (P. perniciosus, P. longicuspis) Tunisia Striped grass mice (Lemniscomys striatus) Central African Republic Gerbil (Tatera kempii) Central African Republic Gerbil (Tatera kempii)

Massilia Granada Arrabida Fermo Tehran Zerdali Punique Gordil Saint Floris Sandfly Fever Sicilian

Geographic distribution

Sandflies (Phlebotomus papatasii, P. perniciosus, P. perfiliewi and others)

Sandfly Fever Sicilian Sandfly Fever Turkey Sandfly Fever Cyprus Corfou

Sandflies (P. papatasii and others)

Toros Dashli

Sandflies (Phlebotomus spp.)

769

Diseases

(Human) Phlebotomus fever

(Human) Phlebotomus fever, aseptic meningitis, meninoencephalitis

Unknown (Human) Seroplevalence in Spain Unknown Unknown Unknown Unknown Unknown Unknown Unknown

South Europe, North Africa, (Human) Phlebotomus fever Middle East, South and Central Asia Turkey (Human) Phlebotomus fever, aseptic meningitis, meninoencephalitis

Human

Cyprus

(Human) Phlebotomus fever

Sandflies (P. neglectus)

Greece

Sandflies (Phlebotomus spp.) Sandflies (Sergentomyia spp.)

Turkey Iran

Similar viral RNA was detected from a patient with encephalitis Unknown Unknown

Sandflies (Phlebotomus spp.) Sandflies (P. perfiliewi, P. perniciosus) Sandflies (Phlebotomus spp.) Sandflies (Phlebotomus spp.) Sandflies (Phlebotomus spp.) Sandflies (Phlebotomus spp.) Mosquitoes (Culex, Mansonia spp.) Gerbils (Tatera kempii) Wild rats (Arvicanthis niloticus, Thamnomys macmillan) Shrew (Crocidura sp.) Striped grass mice (Lemnyscomys striatus) Birds (Turdus libonyanus) Odrenisrou Mosquitoes (C. albiventris)

Iran Italy Tunisia Turkey Portugal Italy Kenya, Sudan, Uganda, Ethiopia, Nigeria, Senegal, Central African Republic, Rhodesia, Zimbabwe, South Africa

Unknown Unknown Unknown Unknown Unknown Unknown (Human) Seroplevalence in Somalia, Sudan, and Egypt

Ivory Coast

Unknown

Rift Valley fever

Rift Valley Mosquitoes (Aedes, Culex, Coquillettidia, fever Anopheles, Eretmapoidites, Mansonia spp.)

Sub-Saharan Africa, Egypt, Madagascar, Saudi Arabia, Yemen

(Human) Febrile illness, hemorrhagic fever, encephalitis, blindness (Ruminant) Febrile illness, abortion (Newborn lambs, goat kids) Acute hepatitis

Karimabad

Karimabad Sandflies (Phlebotomus spp.)

Iran

(Human) Seroplevalence in Iran, Azerbaijan, Uzbekistan, Tajikistan, Pakistan

Salehabad

Salehabad Arbia Medjerda Adana Alcube Ponticelli Arumowot

(Continued )

770

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

Table 1

Continued

Antigenic complex

Virus

Isolations

Gabek Forest

Spiny mouse (Acomys cahirinus) Hedgehog Sudan, Nigeria, Benin, Senegal, Central African Republic (Atelerix albiventrix) Bushbaby (Galago senegalensis) Gerbil (Tatera kempii) Wild rat (Arvicanthis niloticus) Striped grass mice (Lemniscomys barbarus)

Unclassified Salanga virus

Wild rodent (Aethomys medicatus)

Geographic distribution

Central African Republic

Diseases

Unknown

Unknown

serological diagnosis. To confirm the presence of pathogenic RVFV strains, it is important to isolate RVFV in cultured cells (e.g., Vero or BHK cells), or detect viral RNA via quantitative reverse transcriptase (RT)-PCR, RT-Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification, or next-generation sequencing, although the period of viremia is short in ruminants and humans. Post-mortem diagnosis can be made via the detection of RVFV-specific antigens or viral RNA, associated with histopathological lesions. Vaccination is likely the most effective way of preventing the outbreak of RVF. There are several licensed RVF vaccines for veterinary use in endemic countries, but no licensed RVF vaccines for humans are currently available. The live-attenuated Smithburn vaccine was developed via serial intracerebral passages of the pathogenic Entebbe strain in the 1950s. This vaccine is manufactured in South Africa and Kenya, and more than 46 million doses have been sold. Although long-term protective immunity can be induced via a single dose of the Smithburn vaccine, drawbacks include potential abortion or fetal malformation in pregnant animals. The live-attenuated Clone 13 vaccine originated from a plaque variant of the 74HB59 strain in the Central African Republic and the vaccine virus has an in-frame 69% truncation of the NSs gene, which is a major virulence factor for RVF. Accordingly, this strain induces host type I IFN responses and it is avirulent in most immunocompetent animals. The Clone 13 vaccine has been licensed in several African countries, and more than 28 million doses have been given in South Africa. Three different inactivated RVF vaccines are also available in Africa: the formalin-inactivated RVFV strain (South Africa), the formalininactivated RVFV Menya/Sheep/258 strain (Egypt), and the Binary Ethylenimine-inactivated RVFV ZH501 strain (Egypt). In the U.S., the live-attenuated MP-12 vaccine was generated via serial passaging of the pathogenic ZH548 strain in human lung diploid MRC-5 cells in the presence of a chemical mutagen, 5-fluorouracil. The MP-12 vaccine shows attenuation via point mutations in the L-, M-, and S-segments, and it exhibits protective efficacy in livestock ruminants. Vaccination of ewes in the second or third trimester can confer protective immunity not only for vaccinated ewes, but also for newborn lambs via colostrum containing the neutralizing antibody. The MP-12 vaccine has been conditionally licensed for veterinary use upon RVFV introduction in the U.S. As mentioned above, there are currently no licensed human RVF vaccines. In the U.S., the formalin-inactivated Entebbe strain was developed as a human RVF vaccine candidate (TSI-GSD-200), and its safety and immunogenicity has been tested in clinical trials. This vaccine elicits protective immunity via three primary doses, and the maintenance of immunity requires one or more booster doses. A live-attenuated MP-12 vaccine has also been studied in clinical trials for potential human use. In comparison to the formalin-inactivated vaccine, this vaccine can induce long-term protective immunity via a single dose, with no major adverse effects reported in clinical trials. Several other vaccine candidates are being characterized for safety, immunogenicity, and efficacy at the research level. There are no effective treatment regimens for RVF patients. Ribavirin, a guanosine analog, has been shown to effectively limit RVFV replication in animal models. In mice, ribavirin can limit acute liver disease, but not viral encephalitis, in a postexposure treatment regimen, whereas favipiravir (T-705), another guanidine nucleoside analog, showed protective efficacy against late-onset RVFV encephalitis in mice. The combined use of ribavirin and favipiravir could thus improve the protective efficacy of RVF in the mouse model. Galidesivir (BCX4430), an adenosine nucleoside analog, also showed protective efficacy in mice. In Japan, favipiravir has been approved for the treatment of influenza, whereas it is under a phase 3 clinical trials for influenza in the U.S. BCX4430 has also been studied for safe dosage in phase 1 clinical trials. A number of antiviral candidates have been studied in animal models, but most hits have not been further tested in clinical trials.

Sandfly Fever Naples and Sandfly Fever Sicilian viruses During the World War II, phlebotomus fever (three-day fever, Pappataci fever) was caused by either SFNV or SFSV among soldiers who were deployed and stayed in the Mediterranean region. SFNV and SFSV are widely distributed in southern Europe, North Africa, the Middle East, and southern and central Asia, and are transmitted via sandflies (Phlebotomus papatasii, P. perniciosus, P. perfiliewi, and others). Phlebotomus fever typically occurs between May and October, when female sandflies feed on human blood. The incubation period for this disease is typically 3–5 days. Phlebotomus fever is characterized by an abrupt onset of fever, headache, myalgia, nausea, retro-orbital pain, conjunctival injection, rash, or leukopenia, which typically lasts for three days. The

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

771

febrile illness is self-limiting and patients can fully recover without sequelae. Since the 1980s, few reports of SFNV detection or isolation have been made from human or sandfly samples, with no known reason. The diagnosis of phlebotomus fever can be made via serological tests for IgM, IgG, and neutralizing antibodies, using sera from the acute and convalescent phases. Although the detection of viral RNA or viral isolation can better specify the genotype of the phlebovirus, the short viremic phase may hamper sensitive detection of viruses. Most cases of phlebotomus fever do not require specific treatment.

Toscana Virus TOSV was first isolated in Italy in 1971, and its presence has been further confirmed in many European and African countries (Table 1). Phylogenetically, TOSV genome sequences can be clustered into three lineages: Lineage A (Italy, France, Algeria, Tunisia, Turkey), lineage B (France, Spain, Portugal, Morocco, Turkey), and lineage C (Croatia, Greece). While the vectors of sandfly viruses are P. perfiliewi and P. perniciosus, TOSV has also been isolated from other species, including Sergentomyia minuta, P. longicuspis, P. sergenti, and P. neglectus. The incubation period of TOSV infection is 3–7 days, and the disease is characterized by an abrupt onset of fever, headache, myalgia, nausea, and due to viral aseptic meningitis, neurological signs such as neck rigidity, Kernig sign, reduced Glasgow Coma Scale score, tremors, paresis, and nystagmus. Most cases are self-limiting, and the patients usually fully recover within 7 days without sequelae. In rare cases, TOSV infection leads to a severe meningoencephalitis, with clinical signs such as seizure, paresis, aphasia, hearing loss, deep coma, hydrocephalus, orchitis, or intravascular coagulation. Severely affected patients may exhibit long-term sequelae, such as aphasia, blurred vision, deafness, severe headache, or depression. In Tuscany, the seroprevalence of TOSV increases in an age-dependent manner, and 20–30 cases of TOSV meningitis are reported annually, particularly during the summer season. The diagnosis of TOSV infection can be made serologically, as well as via detection of TOSV RNA in blood or cerebrospinal fluid samples. Patients with meningitis or meningoencephalitis may require supportive care in a hospital.

Punta Toro and Cocle Viruses PTV is transmitted by sandflies (Lutzomyia spp.) in Panama (Table 2). The Adames strain of PTV was isolated from a febrile entomologist in Darien Province in 1972, and the Balliet strain was isolated from a febrile soldier undergoing a jungle warfare training in Colon Province in 1966. The genetically-related Cocle virus can also cause febrile illness in humans. In an investigation of acute dengue in Panama in 2009, 13.4% of suspected acute dengue patients, who were negative for dengue virus, were positive for PTV RNA, indicating that PTV infection is an important cause of non-dengue febrile illnesses in Panama. Clinical signs of PTV and Cocle virus infection have been characterized as fever, chills, myalgia, severe headache, retro-orbital pain, and arthralgia, with full recovery without sequelae. Syrian hamsters infected with the Adames strain of PTV show anorexia, ruffled fur, and lethargy, with pathological findings including hepatic and splenic necrosis, and hemorrhage in the duodenum. In contrast, inbred mice exhibit age-dependent and strain-dependent susceptibility to the Adames strain. In rodent models, the Balliet strain did not show notable pathogenicity. The PTV infection model has been used as a surrogate experimental model for the pathogenesis of RVF, as well as for antiviral screening.

Phleboviruses in the Candiru Antigenic Complex The Candiru antigenic complex consists of at least 13 viruses (Candiru, Morumbi, Serra Norte, Alenquer, Itaituba, Jacunba, Ariquemes, Oriximina, Turuna, Mucura, Maldonado, Echarte, and Nique viruses) found in Brazil, Peru, or Panama (Table 2). Six viruses (Candiru, Morumbi, Serra Norte, Alenquer, Maldonado, and Echarte) have been isolated from febrile patients. Clinical signs of illnesses are considered similar to those of phlebotomus fever.

Chagres Virus The Chagres virus has been isolated from febrile humans and sandflies in Panama (Table 2). Clinical signs of Chagres virus infection include fever, severe headache, myalgia, arthralgia, and retro-orbital pain lasting for a couple of days. Seroprevalence studies were exhibited in populations in Panama in 1965, with especially high seroprevalence among residents of San Miguel Island.

Huaiyangshan Banyangvirus (Formerly Severe Fever With Thrombocytopenia Syndrome Virus: SFTSV) SFTS is endemic in northeast to southeast Asia, including China, the Korean Peninsula, and Japan, whereas it was also found in Taiwan, and Vietnam (Table 3). SFTSV was first isolated in 2009, during an investigation of an etiological agent causing unusually high fatality rates in humans via thrombocytopenia, fever, gastrointestinal symptoms, or leukocytopenia in China’s Hubei and Henan provinces. Transmission of SFTSV occurs via a bite of an infected tick (e.g., Haemaphysalis longicornis, Amblyomma testudinarium, Rhipicephalus microplus, Ixodes nipponensis). In China, SFTS cases occur from March to November, with a peak between May and July. After hatching, larvae of H. longicornis attach to hosts and feed on blood for several days (between June and October).

772

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

Table 2

Phleboviruses in the new world (mosquito-, sandfly-borne, and other non tick-borne viruses)

Antigenic complex Virus

Isolations

Geographic distribution

Human diseases

Candiru

Candiru Morumbi Serra Norte Alenquer Maldonardo Echarte Itaituba Jacunba Ariquemes Oriximina Turuna Mucura Nique

Human Human Human Human Human Human Opossum (Didelphis marsupialis) Rodent (Myoprocta acouchy) Sandflies (Lutzomyia spp.) Sandflies (Lutzomyia spp.) Sandflies (Lutzomyia spp.) Mosquito (Anopheles triannulatus) Sandflies (Lutzomyia spp.)

Brazil Brazil Brazil Brazil Peru Peru Brazil Brazil Brazil Brazil Brazil Brazil Panama

Phlebotomus Phlebotomus Phlebotomus Phlebotomus Phlebotomus Phlebotomus Unknown Unknown Unknown Unknown Unknown Unknown Unknown

Punta Toro

Punta Toro (Adames strain) Punta Toro (Balliet strain) Cocle Capira Campana Letica Buenaventura

Human

Panama

Phlebotomus fever

Human Human Sandflies Sandflies Sandflies Sandflies

Panama Panama Panama Panama Columbia Columbia

Phlebotomus fever Phlebotomus fever Unknown Unknown Unknown Unknown

Bujaru

Bujaru Munguba

Rodent (Proechimys guyannensis oris) Sandflies (Lutzomyia umbratilis)

Brazil Brazil

Unknown Unknown

Tapara

Tapara Urinara

Sandflies Sandflies

Brazil Brazil

Unknown Unknown

Frijoles

Frijoles Joa

Sandflies (Lutzomyia spp.) Sandflies (Lutzomyia spp.)

Panama Brazil

Unknown Unknown

Aguacate

Aguacate Armero Durania Ixcanal

Sandflies Sandflies Sandflies Sandflies

Panama Columbia Columbia Guatemala

Unknown Unknown Unknown Unknown

Chilibre

Chilibre Cacao

Sandflies (Lutzomyia spp.) Sandflies (Lutzomyia spp.)

Panama Panama

Unknown Unknown

Rift Valley fever

Belterra

Sandflies (Lutzomyia spp.), Mosquitoes (Sabethes chloropterus)

Brazil

Unknown

Brazil Brazil

Unknown Unknown

Panama

Phlebotomus fever

Columbia French Guiana, Trinidad, Brazil United States (Texas) Brazil Brazil Brazil

Unknown Unknown

Salobo Icoaraci

Others

Chagres Mariquita Itaporanga Rio Grande Ambe Anhanga Urucuri

(Lutzomyia (Lutzomyia (Lutzomyia (Lutzomyia

(Lutzomyia (Lutzomyia (Lutzomyia (Lutzomyia

spp.) spp.) spp.) spp.)

spp.) spp.) spp.) spp.)

Mosquitoes (Aedes, Anopheles, Culex, Sabethini spp.), Sandflies (L. flaviscutellata) Sandflies (Lutzomyia spp.), Mosquitoes (Sabethes chloropterus) Sandflies (Lutzomyia spp.) Mosquitoes (Culex spp.), Opossum (Caluromys spp.), Birds (Thamnophilus aethiops) Southern Plains Woodrat (Neotoma micropus) Drain fly (Psychodidae sp.) Sloth (Choloepus brasiliensis) Guyenne spiny rat (Proechimys guyannensis) Long-tailed spiny rat (Proechimys longicaudatus)

fever fever fever fever fever fever

Unknown Unknown Unknown Unknown

Larvae then molt and become nymphs, which then feed on the blood of hosts for another several days (between March and September) after surviving the winter without feeding. Nymphs become adults after dropping off of the host and molting. Adults feed on host blood for 7–14 days (between April and September) and then lay up to 2500 eggs. One study showed that microinjection of SFTSV into H. longicornis resulted in transovarial transmission of SFTSV into larvae, nymphs, and adults; ticks in

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

Table 3

773

Tick-borne phleboviruses/Banyangviruses

Genetic group

Virus

Isolations

Geographic distribution Diseases

SFTS/HRTV (Banyangvirus)

SFTS

Ticks Haemaphysalis longicornis Amblyomma testudinarium Rhipicephalus microplus Ixodes nipponensis

China, Korean Peninsula, Japan, Vietnam, Taiwan

Guertu Heartland

Ticks (Dermacentor nuttalli) Ticks (Amblyomma americanum)

China (Xinjiang) United States

SFTS/HRTV (unclassified)

Bhanja (Phlebovirus)

Hunter Island group Ticks (Ixodes eudyptidis)

Unknown

Malsoor

Bats (Rousettus leschenaultii)

Bhanja

Ticks (Haemaphysalis, Amblyomma, Rhipicephalus, Hyalomma, Dermacentor spp.) Hedgehog (Atelerix albiventris), Squirrel (Xerus erythropus) Ticks (Haemaphysalis punctata) Ticks (Rhipicephalus geigy) Ticks (Rhipicephalus pulchellus) Ticks (Dermacentor marginatus) Ticks (Amblyomma americanum)

Central and southern (Humans) Febrile illness, Europe, southern and meningoencephalitis (Young central Asia, Africa ruminants): CNS diseases

Portugal Guinea Somalia Armenia United States (Kentucky)

Unknown Unknown Unknown Unknown Unknown

Ticks (Haemaphysalis spinigera) Bird (Zoothera citrina) Ticks (Dermacentor auratus, Haemaphysalis spp., Ixodes granulatus) Ticks (Haemaphysalis Leporispalustris), Snowshoe hare (Lepus americanus) Ticks (Haemaphysalis longicornis)

India

Unknown

Malaysia

Unknown

Canada (Ontario), United States (Wisconsin, Alaska) Russia

Unknown

Northern and eastern Europe (Finland, Norway, Poland, Czechoslovak, Hungary), Russia Egypt France Russsia Norway United States (Alaska) United States (Alaska) United States (Texas) Pakistan France Tajikistan Russia (Far East) Russia (Far East) Australia (Macquarie Island) Australia (Macquarie Island) United States (Oregon) Scotland, UK France Tunisia

Unknown

Palma Forecariah Kismayo Razdan Lone Star

Kaisodi (Phlebovirus) Kaisodi Lanjan

Silverwater

Khasan Uukuniemi (Phlebovirus)

Australia (Albatross Island) India

(Humans) Febrile illness with thrombocytopenia, leukopenia, multiorgan dysfunctions (Cheetahs, cats) Hemorrhage in digestive tract, thrombocytopenia, decreased leukocyte counts Unknown (Humans) Febrile illness with thrombocytopenia, leukopenia

Uukuniemi (S23 strain)

Ticks (Ixodes ricinus), Rodent (Apodemus flavicollis), Birds (Turdus merula and others)

EgAN1825–61 Chizé Zaliv Terpeniya Fin V707 Murre RML-105355 Sunday Canyon Manawa Grand Arbaud Gissar Rukutama Komandory Precarious Point

Bird (Phylloscopus trochilus) Ticks (Ixodes frontalis) Ticks (Ixodes putus) Ticks (Ixodes uriae) Bird (Uria aalge) Ticks (Ixodes uriae) Ticks (Argas cooley) Ticks (Argas abdussalami) Ticks (Argas reflexus) Ticks (Argas reflexus) Ticks (Ixodes signatus) Ticks (Ixodes uriae) Ticks (Ixoides uriae)

Catch-me-cave

Ticks (Ixoides uriae)

Oceanside St. Abb’s Head Ponteves Tunis

Ticks Ticks Ticks Ticks

(Ixoides uriae) (Ixoides uriae) (Argas reflexus) (Argas reflexus)

Unknown

Unknown

Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown (Continued )

774

Table 3

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

Continued

Genetic group

Virus

Isolations

Geographic distribution Diseases

Mukawavirus (Phlebovirus) Kabutovirus (Kabutovirus)

Mukawa

Ticks (Ixodes persulcatus)

Japan

Unknown

Kabuto Mountain

Ticks (Haemaphysalis flava)

Japan

Unknown

all three developmental stages can transmit SFTSV into mice via saliva. Moreover, tick colonies derived from SFTSV-infected H. longicornis collected in China also showed evidence of transovarial and transstadial transmission. In addition to the reservoir role of ticks, other animals may serve as amplifying hosts for SFTSV. High seroprevalence of SFTSV antibodies has been shown in sheep, goats, cattle, dogs, and chickens in China. Moreover, viral RNA has been detected in cattle, deer, wild boars, hedgehogs, common shrews, fulvous harvest mice, house mice, and cats. It is therefore likely that those animals may be involved in viral amplification or in the life cycle of H. longicornis. It has also been suggested that the geographical distribution of H. longicornis is similar to the flyway route of migratory birds, which can harbor this tick, across China, Korea, and Japan. In Japan, a 50-year-old woman who was bitten by a stray cat died from SFTS without any evidence of a tick bite, which suggested cat-to-human transmission of SFTSV. Human-to-human transmission of SFTSV can also occur via exposure to the bodily fluids of an infected patient. Typically, people who have had direct contact with the blood of an index case (e.g., cleaning blood from the face or body, removing tubes or needles), including that of a cadaver, without using personal protective equipment (e.g., gloves, eye protection, mask, gown) could contract SFTS. SFTSV strains can be phylogenetically clustered into multiple clades, with the L-, M-, and S-segments grouped into genotypes A – F. The majority of SFTSV strains belong to genotypes A, D, and F, which are commonly found in the mainland China. Genotype B is found in Japan, South Korea, and the Zhoushan islands in China, indicating transmission of this genotype among those three countries. Due to the co-circulation of several genotypes, reassortant strains among the A, C, D, and F genotypes have also been isolated. The amino acid similarity among the GnGc proteins of those genotypes is higher than 97%, and SFTS patient sera can cross-neutralize different SFTSV genotypes. The case fatality rate of SFTS patients is typically 6–33%, with 343 deaths among 5360 laboratory confirmed cases in China from 2011 to 2016; 56 deaths among 172 cases in South Korea from 2013 to 2015; and 394 SFTS cases with estimated 27% case fatality rate in Japan from 2013 to 2019. SFTSV was also found in blood samples from two patients (27- and 28-year-old) in Vietnam and a patient (70-year-old) in Taiwan. Elderly individuals are more susceptible to SFTS, and deaths occurs more frequently in individuals older than 50 years of age. In China, nearly 97% of the abovementioned SFTS patients were farmers, and up to 21% of them could recall receiving a tick bite within the previous two weeks. The incubation period of SFTSV is typically 6–14 days, followed by an initial fever stage, which lasts for 5–11 days and involves high fever (381C – 411C), myalgia, headache, fatigue, anorexia, nausea, vomiting, and/or diarrhea. During this period, thrombocytopenia, leukocytopenia, and lymphadenopathy can accompany the clinical signs. At 7–13 days after the disease onset, surviving patients start showing signs of recovery based on increasing platelet counts or a reduction in viral loads. Conversely, disseminated intravascular coagulation and multiple organ dysfunction/failure may occur in severely ill SFTS patients in this stage of the disease. Moreover, hemorrhagic manifestations (e.g., ecchymosis, pulmonary or gastrointestinal bleeding, infarctions), necrotizing lymphadenitis, elevation of liver enzymes (associated with secondary hepatocyte injury other than viral infection), elevation of muscle enzymes, or neurological manifestations (e.g., apathy, lethargy, muscular tremor, convulsions, coma) may also occur in severely ill patients. Bone marrow hematopoietic cells in SFTS patients do not show apparent cytological changes, indicating that thrombocytopenia and leukocytopenia may not occur via direct damage to those cells. The median time from the onset to death is typically 8–9 days. The convalescence phase typically starts 11–19 days after the onset of the disease, and recovery occurs within 3–4 weeks after the onset. Viremia in SFTS patient blood can be detected for 2–3 weeks after the onset of the disease, and it peaks around 6 days after onset. SFTS can be diagnosed based on the detection of viral L-, M-, or S-segment RNA via quantitative RT-PCR in serum samples. Alternatively, a RT-LAMP assay can be used to detect SFTSV RNA. Serum IgM or IgG antibodies specific to SFTSV proteins can be used to evaluate seroconversion during an SFTSV infection. The presence of neutralizing antibodies against SFTSV also strengthens the serological diagnosis of SFTSV infection. Little is known about animal diseases caused by natural SFTSV infections. In a zoo in Japan, two cheetahs exhibited anorexia, vomiting, hemorrhage, thrombocytopenia, decreased leukocyte counts, and elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatinine phosphatase (CPK), and total bilirubin. One of these animals died on day 4 and the other one died on day 7. Post-mortem examination revealed bleeding in the esophagus and ulcers in the gastrointestinal tract. SFTSV was isolated, and viral genome sequences were clustered into a clade with those of SFTSV previously isolated from humans in the same region. Animal models of SFTS are critical for understanding pathogenesis and allowing the development of vaccines and antivirals. Rhesus macaques experimentally infected with SFTSV developed mild fever, decreased counts of platelets and white blood cells, and elevations of ALT, AST, or LDH, mimicking clinical signs exhibited by patients with nonlethal, mild SFTS. Viral RNA in blood peaked at days 3–5, and it was undetectable by 7 days post infection (dpi). In contrast, cynomolgus macaques inoculated with SFTSV did not show notable clinical signs or viremia, except for a temporary decrease in platelets. Immunocompetent ferrets are uniquely susceptible to

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

775

SFTSV infection in an age-dependent manner. Upon SFTSV infection, older ferrets (Z 4 years old) showed an elevated body temperature, thrombocytopenia, reduced white blood cell counts, and elevated AST and ALT, finally succumbing to infection by 8 dpi. Young ferrets (r 2 years old), however, survived the infection without notable clinical signs. Experimental infection of 0.5–2 years old cats with SFTSV via intravenous route resulted in a lethal SFTS-like disease with marked leukopenia and thrombocytopenia in four out of six cats, in which viral antigens were detected in immunoblast-like cells and macrophages in lymphoid tissues. Immunocompetent adult Balb/c mice, Syrian hamsters, and goats have not shown notable clinical signs in response to experimental SFTSV infection. In contrast, adult C57BL/6 mice inoculated with SFTSV exhibit transiently decreased platelet and white blood cell counts, and elevations of AST, ALT, and blood urea nitrogen, although all infected mice survived the infection without changes in body weight. Interferon a/b receptor (IFN-AR) knockout C57BL/6 or 129/Sv mice are highly susceptible to SFTSV, and typically succumb to infection within 7 dpi. The disease can vary to some extent depending on the virus strains. Pathological findings include gastric and intestinal distensions, lymphoid depletion, and necrosis of lymph nodes or spleen. Viral antigens or RNA are found in reticular cells in the spleen, epithelial cells in the stomach or intestines, or mononuclear cells in various organs. Similarly, STAT2 knockout Syrian hamster strain is highly susceptible to SFTSV infection; death often occurring within 6 dpi when using 10 PFU of SFTSV. The disease in hamsters was characterized by thrombocytopenia, elevations of serum AST and ALT levels associated with multifocal neutrophilic hepatitis, and lymphoid necrosis in the spleen, but leukopenia was less evident in this hamster model. No licensed SFTS vaccines are currently available for humans. SFTSV GnGc proteins are the targets of neutralizing antibodies, and passive transfer of neutralizing antibodies may protect IFN-AR knockout mice from lethal SFTSV challenge. Further research is required to identify safe and immunogenic vaccine candidates that can induce protective immunity in target populations. Presently, there are no approved treatment regimens for SFTS patients. IFN-AR knockout mice have been used to evaluate potentially effective antivirals. Ribavirin has been shown to partially protect mice from lethal SFTSV infection. In contrast, favipiravir (T-705, per os or intraperitoneal inoculation) could fully protect IFN-AR knockout mice or STAT2 knockout hamsters from lethal SFTSV infection. Further preclinical and clinical trials will potentially lead to the approval of antivirals against SFTS infections.

Heartland Banyangvirus (HRTV) Another tick-borne pathogenic banyangvirus, HRTV, was first identified in 2009 in two patients in Missouri in the U.S.As of 2018, over 40 cases have been identified in the midwestern and southern U.S., in the states of Missouri, Kansas, Oklahoma, Arkansas, Illinois, Indiana, Kentucky, Tennessee, Georgia, and North Carolina (Table 3). In northern Missouri, serological prevalence of HRTV infection was 0.9%, indicating that some infected patients do not seek for medical care. Among the ten documented clinical cases (patients were 50–80 years of age), eight patients were hospitalized, three of whom died. Nine patients had recently been exposed to tick bites. HRTV has been isolated from the lone star tick (Amblyomma americanum), which is thus considered to be the vector for HRTV. Lone star ticks prefer their second developmental phase in the forest, and altogether they require three hosts in their life cycle. In Missouri, the seasonal peak of each stage is as follows: larvae from July to September, nymphs from May to August, and adults from May to July. HRTV RNA has been detected in different pools of depleted, host-seeking nymphs, but not in adults or larvae, indicating that transstadial transmission may occur from infected larvae to nymphs, but it is likely less frequent from nymphs to adults, or from adults to eggs. A serological survey of wild and domestic animals in Missouri in 2012–2013 revealed that neutralizing antibodies specific to HRTV were detected in northern raccoon, horse, white-tailed deer, dogs, and Virginia opossums, indicating that larval or nymphal lone star ticks may be newly infected via viremic blood from those animals. In a large serological survey across the U.S., samples seropositive for HRTV were identified in raccoons (Missouri, Tennessee, Kentucky, Indiana, Texas), white-tailed deer (Florida, Georgia, North Carolina, Vermont, New Hampshire, Maine), coyotes (Illinois, Kansas), and moose (New Hampshire). The incubation period of HRTV infection is not precisely known, but clinical signs of illness typically start within two weeks after a tick bite. Clinical signs of non-lethal HRTV infections are characterized by fever, fatigue, headache, anorexia, nausea, myalgia, non-bloody diarrhea, leukopenia, thrombocytopenia, and mild to moderate elevations of serum AST and ALT levels. Patients exhibited persistent fatigue or impaired short-term memory, yet they tended to be fully recovered 4–6 weeks after the onset of symptoms. In two lethal cases, erythrophagocytosis with histiocytic hyperplasia was noted in the bone marrow, spleen, and lymph nodes, indicating the occurrence of hemophagocytic lymphohistidiocytosis. HRTV antigens were detected in mononuclear phagocytic cells in those patients, showing erythrophagocytosis in the bone marrow. Moreover, one of the two patients also exhibited viral antigens in multiple tissues, including thalamus, liver, gallbladder, pancreas, heart, lung, intestine, kidney, and testes. Acute gastritis, gastric and duodenal ulcers, hemorrhage in the colon, acute pyelonephritis, atherosclerosis in cardiac arteries and aorta, and systemic amyloidosis were also observed in a different HRTV-infected patient. HRTV infection can be diagnosed by detection of viral RNA in blood or serological tests, including a plaque reduction neutralizing test and an IgM or IgG detection assay (e.g., microsphere immunoassay). There are no licensed vaccines or antivirals against HRTV infection. To model HRTV infection, several animal species have been tested for susceptibility to viral infection. No detectable viremia occurs in wild raccoons, goats, New Zealand white rabbits, Syrian hamsters, C57BL/6 mice, cynomolgus macaques, or chickens inoculated with HRTV. In contrast, IFN-a/b/g receptor knockout Ag129 mice succumbed to infection in a dose-dependent manner. Clinical signs of infected Ag129 mice include ruffled fur, hunched back, hematochezia, and ocular discharge, with viremia progressively increasing until the death. Post-mortem examination showed enlarged pale spleen, hepatic hemorrhage, enlarged

776

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

gall bladder, and/or excess peritoneal fluid, with HRTV antigens detected in mononuclear cells in the spleen, hepatic sinusoid, or renal interstitium. STAT2 knockout Syrian hamsters infected with HRTV exhibited viremia and a transient reduction of body weight, and mild elevations of alkaline phosphatase, but most animals survived the challenge. Leukocytopenia and thrombocytopenia are not evident in this model. Histopathological examination of one moribund hamster showed neutrophilic inflammation in the spleen, liver, lung interstitium, heart, and lymph nodes.

Bhanja Virus Bhanja virus was first isolated from Haemaphysalis intermedia ticks feeding on a paralyzed goat in India in 1954. Later, this virus was also isolated from African hedgehogs and African ground squirrels. Bhanja virus is widely distributed throughout central and southern Europe, southern and central Asia, and Africa, with samples isolated in India, Italy, Slovakia, Armenia, the Balkan region including Bulgaria, Romania and Croatia, and Africa including the Central African Republic, Senegal, and Nigeria (Table 3). Accidental laboratory infections with Bhanja virus have been characterized by a sudden onset of myalgia, arthralgia, frontal headache, and photophobia. Clinical signs are then cleared in 2–10 days. Clinical signs exhibited by a patient in Zagreb, who was naturally infected with Bhanja virus, were characterized by an initial phase of fever, back pain, photophobia, and vomiting, followed by an unconscious phase with fever, meningitis, and muscle hypertonia, with gradual recovery over 45 days. Seropositivity has also been observed in man, sheep, goat, and cattle in Europe.

Other Phleboviruses There are at least 100 distinct phleboviruses, and evidence of related human or animal disease has not been identified for many of them. Most phleboviruses have been isolated from sandflies, mosquitoes, ticks, or mammals. Transmission patterns and associations with human or animal illnesses are largely unknown for these phleboviruses. The current classification, determined via antigenic cross-reactivity using the complement fixation test and plaque reduction neutralization test, involves the following antigenic complexes: Sandfly Fever Naples, Sandfly Fever Sicilian, Salehabad, Rift Valley fever, Karimabad, Candiru, Punta Toro, Bujaru, Tapara, Frijoles, Aguacate, Chilibre, SFTS/Heartland, Bhanja, Kaisodi, and Uukuniemi. Genetic grouping had been alternatively applied to those that could not be readily analyzed by antigenic characterization. The Sandfly Fever Naples antigenic complex consists of SFNV and TOSV, as well as the Massilia, Granada, Arrabida, Fermo, Tehran, Zerdali, and Punique viruses, among others. The Massilia and Granada viruses share highly similar L- and S-segments, but the M-segment sequences of these two viruses are distinct, indicating that genetic reassortment may have created these two phleboviruses. Seropositivity for the Granada virus was found in healthy humans in the Granada area. The Sandfly Fever Sicilian antigenic complex consists of SFSV, as well as the Sandfly Fever Turkey (SFTV), Sandfly Fever Cyprus (SFCV), Corfou, Toros, and Dashli viruses. SFCV and SFTV were isolated during outbreaks of phlebotomus fever in Cyprus in 2002 and in Turkey in 2008, respectively. The SFTV infection also caused meningitis or encephalitis in some patients. In Greece, viral RNA similar to that of Corfou virus was detected in cerebrospinal fluid of a patient with an acute encephalitis, and this unisolated viral strain was provisionally named Chios virus. Toros and Dashli viruses were isolated from sandflies, whereas human illnesses associated with these viruses have not been reported. The Salehabad phlebovirus antigenic complex consists of at least Salehabad phlebovirus, Arbia virus, Adria virus, Medjerda virus, Adana virus, Alcube virus, Ponticelli virus, Arumowot virus, and Odrenisrou virus. There is no clear evidence of a human disease associated with these phleboviruses. A partial L-segment RNA of Adria virus was, however, detected in Greece in the blood of a hospitalized febrile 2.5-year-old child who exhibited fever, vomiting, and tonic seizures. Adria virus has never been isolated, yet its RNA has been detected in two pools of sandflies in Albania. Arumowot virus (AMTV), which is proposed to be a member of the Salehabad antigenic complex, has shown seropositivity in humans in Somalia, Sudan, and Egypt, as well as in sheep in Burkina Faso. AMTV is transmitted by mosquitoes and distributed throughout several African countries that overlap with the RVF endemic area. In contrast to RVFV, AMTV has not been experimentally found to cause any lethal diseases in adult mice, hamsters, or sheep. The functional incompatibility of respective N, L, and GnGc proteins also hampers the formation of reassortant strains between RVFV and AMTV. Guertu banyangvirus (GTV), in the SFTS/Heartland antigenic group, was isolated from Dermacentor nuttalli ticks in Guertu county in Xinjiang, China in 2014, and it shows a distinct but close phylogenetic relationship with other SFTSV genotypes. GTV and SFTSV can be cross-neutralized by each other’s antisera due to conserved antigenic epitopes. Seropositivity has been shown in populations in Xinjiang, although associated clinical diseases have not been characterized in humans. In contrast, Hunter Island Group virus, which was isolated from Ixodes eudyptidis ticks parasiting in shy albatrosses on Albatross Island, Australia, and Malsoor virus, which was isolated from wild bats in India, are phylogenetically close to SFTSV/HRTV, yet they have not been shown to cause human or animal diseases. The Bhanja antigenic group consists of at least Bhanja virus, Forecariah virus, Kismayo virus, Razdan virus, Palma virus, and Lone Star virus. Little is known about the pathogenicity of Palma, Forecariah, Kismayo, and Razdan viruses in humans and animals. Lone Star virus was isolated in 1967 from lone star ticks (Amblyomma americanum) collected from woodchucks in Kentucky, but no association with a human disease has been identified. The Kaisodi and Uukuniemi antigenic complexes consist of tick-borne phleboviruses distributed worldwide, with no clear association with human or animal diseases. Antigenically, the Uukuniemi group has at least two complexes – the Uukuniemi

Rift Valley Fever Virus and Other Phleboviruses (Phenuiviridae)

777

antigenic complex and the Murre antigenic complex. The former includes at least Uukuniemi virus (S23 strain), EgAN1825–61 virus, Chizé virus, Zaliv Terpeniya virus, and Fin V707 virus, and the latter includes at least Murre virus, RML-105355 virus, and Sunday Canyon virus. The Manawa, Grand Arbaud, and Precarious Point viruses are antigenically distinct from the others. Other Uukuniemi-like phleboviruses, such as the Catch-me-cave, Oceanside, Ponteves, and St. Abbd Head viruses, have not been sufficiently characterized to allow their antigenic grouping. The Kabuto Mountain virus and Mukawa virus, which were isolated from ticks in Japan, are classified as independent species within the Kabutovirus and Phlebovirus genera, respectively. A recent study showed further genetic diversity of tick-borne phleboviruses. Many novel phleboviruses (e.g., Blacklegged tick phleboviruses) have not been successfully grown in culture cells, although viral RNA (L- and/or S-segments) has been amplified from total RNA collected from ticks. Interestingly, M-segment RNA could not be detected in those studies. Moreover, N genes appear to be longer, and NSs genes much shorter, than those of other phleboviruses. There are a number of phleboviruses for which there is only limited information regarding their pathogenicity or transmission to humans or animals. Further studies are needed to better understand the life cycle of each phlebovirus and their potential impact on public health.

Further Reading Abudurexiti, A., Adkins, S., Alioto, D., et al., 2019. Taxonomy of the order Bunyavirales: Update 2019. Archives of Virology 164, 1949–1965. Alkan, C., Bichaud, L., de Lamballerie, X., et al., 2013. Sandfly-borne phleboviruses of Eurasia and Africa: Epidemiology, genetic diversity, geographic range, control measures. Antiviral Research 100, 54–74. Brault, A.C., Savage, H.M., Duggal, N.K., et al., 2018. Heartland virus epidemiology, vector association, and disease potential. Viruses 10, 498. Gai, Z., Liang, M., Zhang, Y., et al., 2012. Person-to-person transmission of severe fever with thrombocytopenia syndrome bunyavirus through blood contact. Clinical Infectious Diseases 54, 249–252. Matsuno, K., Weisend, C., Travassos da Rosa, A.P., et al., 2013. Characterization of the Bhanja serogroup viruses (Bunyaviridae): A novel species of the genus Phlebovirus and its relationship with other emerging tick-borne phleboviruses. Journal of Virology 87, 3719–3728. Mendoza, C.A., Ebihara, H., Yamaoka, S., 2019. Immune modulation and immune-mediated pathogenesis of emerging tickborne banyangviruses. Vaccines 7, 125. Nunes-Neto, J.P., Souza, W.M., Acrani, G.O., et al., 2017. Characterization of the Bujaru, frijoles and Tapara antigenic complexes into the sandfly fever group and two unclassified phleboviruses from Brazil. Journal of General Virology 98, 585–594. Palacios, G., da Rosa, A.T., Savji, N., et al., 2011a. Aguacate virus, a new antigenic complex of the genus Phlebovirus (family Bunyaviridae). Journal of General Virology 92, 1445–1453. Palacios, G., Tesh, R., Travassos da Rosa, A., et al., 2011b. Characterization of the Candiru antigenic complex (Bunyaviridae: Phlebovirus), a highly diverse and reassorting group of viruses affecting humans in tropical America. Journal of Virology 85, 3811–3820. Palacios, G., Savji, N., Travassos da Rosa, A., et al., 2013a. Characterization of the Salehabad virus species complex of the genus Phlebovirus (Bunyaviridae). Journal of General Virology 94, 837–842. Palacios, G., Savji, N., Travassos da Rosa, A., et al., 2013b. Characterization of the Uukuniemi virus group (Phlebovirus: Bunyaviridae): Evidence for seven distinct species. Journal of Virology 87, 3187–3195. Palacios, G., Tesh, R.B., Savji, N., et al., 2014. Characterization of the Sandfly fever Naples species complex and description of a new Karimabad species complex (genus Phlebovirus, family Bunyaviridae). Journal of General Virology 95, 292–300. Palacios, G., Wiley, M.R., Travassos da Rosa, A.P., et al., 2015. Characterization of the Punta Toro species complex (genus Phlebovirus, family Bunyaviridae). Journal of General Virology 96, 2079–2085. Swanepoel, R., Coetzer, J.A.W., 2004. Rift valley fever. In: Coetzer, J.A.W., Tustin, R.C. (Eds.), Infectious Diseases of Livestock with Special Reference to Southern Africa, second ed. Cape Town: Oxford University Press, pp. 1037–1070. Tokarz, R., Williams, S.H., Sameroff, S., et al., 2014. Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses. Journal of Virology 88, 11480–11492.

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae) Katherine N Ward, University College London, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary Allogeneic Refers to the genetic differences between individuals of the same species. Antibody avidity The binding strength between antibody and antigen. In primary infection the first IgG antibodies produced are of low avidity but with time the immune response matures, specific B cell clones undergo somatic mutation and IgG antibody of higher avidity is produced. Astrocytes Brain cells that form a supporting framework for neurons. Other properties include proliferation after damage. Atherosclerosis A disease characterized by thickening and loss of elasticity of arterial walls. CD4, CD134, CD46 and CD8 molecules These are some of the cell surface antigens of leukocytes also known as CD (cluster of differentiation) antigens. As lymphocytes differentiate, they express different patterns of CD molecules on their cell surface, which can aid in determining the type and maturation stage of the cells being examined. Chromosomal integration of HHV–6 (CIHHV–6) A condition in which the complete HHV–6 genome is integrated into the host cell chromosome in every nucleated cell in the body and transmitted from mother or father to child in the germ line. Encephalitis Inflammation of the brain causing a reduced level of consciousness together with other symptoms such as fever and fits. Usually caused by an infection. Episome An episome is an extrachromosomal replicating genetic element that is not necessary for cell survival. Viral episomes can persist latently in the host cell but have the potential for virus replication. Febrile fits or convulsions An epileptic seizure occurring in childhood associated with a febrile illness. The peak age of occurrence is in the second year of life. Febrile status epilepticus A serious complication of febrile fits, convulsions lasting more than 30 min – a medical emergency. Founder effect The reduced genetic diversity, which results when a population is descended from a small number of colonizing ancestors.

Graft-versus-host disease Reaction occurring when T-lymphocytes present in a graft recognize and attack host cells. Hematopoietic stem cell transplant (HSCT) Colloquially referred to as bone marrow transplant. A transplant of selfrenewing stem cells that are capable of giving rise to all of the formed elements of the blood (i.e., leukocytes, erythrocytes and platelets). Horizontal transmission The spread of an infectious agent from one individual to another. Limbic encephalitis Encephalitis with characteristic signs and symptoms originating in the hippocampus in the temporal lobe of the brain. MHC (major histocompatibility complex) A genetic region encoding molecules involved in antigen presentation to T lymphocytes. Monoclonal antibodies Homogeneous antibody derived from a single B lymphocyte and therefore each antibody molecule is identical to all others. Peripheral blood mononuclear cells (PBMC) Any peripheral blood cell having a round nucleus, i.e., lymphocytes and monocytes. Peripheral Occurring outside of the primary lymphoid organs. Telomeres Caps at the ends of human chromosomes, which are composed of repeats of a DNA sequence of 6 base pairs. The telomere is maintained (copied) by a specialized enzyme that protects against loss of genetic information. Temporal lobe epilepsy A chronic disorder of the nervous system characterized by seizures that originate in the temporal lobe of the brain. Temporal lobe The lower lateral lobe of the cerebral hemisphere; contains the hippocampus. Vertical transmission Genetic transmission from one generation to another. Also extended by others to include transmission of infection from one generation to the next i.e., through the placenta. Virus reactivation A term usually applied to herpesviruses which describes a switch from latent infection to productive also known as active infection, i.e., virus replication.

Introduction In 1986 human herpesvirus 6 (HHV–6) was discovered by accident in lymphocyte cultures designed for the isolation of HIV. The virus was initially named human B lymphotropic virus but was later found to infect and replicate in T lymphocytes (also known as T cells), at which point the name was changed to its present form. Identification of another closely related T lymphotropic virus, HHV–7, followed in 1990. Soon after HHV–6 was discovered, it was realised that viral isolates fell into two readily distinguishable groups, variants A and B, which differed in their molecular, biological and epidemiological properties. The two groups showed different abilities to grow in tissue culture of different T cell lines, specific immunological reactivity with monoclonal antibodies, distinct patterns of restriction endonuclease sites, and specific and conserved interstrain variations in their DNA sequences. In 2011, in the face of accumulating evidence, the International Committee on Taxonomy of Viruses declared that the two variants met the formal definition of separate herpesvirus species, namely HHV–6A and HHV–6B. In this article, wherever possible, the two

778

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00089-8

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

779

Fig. 1 Overview of HHV–6 genome. (a) Schematic representation of the HHV–6 genome (not to scale). U unique region, DRL direct repeat left, DRR direct repeat right. (b) Schematic representation of the DR (not to scale). TMR telomeric repeat, imp TMR imperfect telomeric repeat, pac1 and pac2 packaging sequences.

species of HHV–6 (A and B) are distinguished and the more general term HHV–6 is reserved for studies where either the distinction was not made or there is no clear point to be elucidated. No disease is causally linked to HHV–6A, and its natural history is unknown. In contrast, primary or first infections with HHV–6B and HHV–7 are ubiquitous in early childhood sometimes causing exanthem subitum (roseola infantum); subsequent viral latency gives the potential for reactivation, transmission and disease. HHV–6B reactivation is well established as a cause of encephalitis principally after hematopoietic stem cell transplant (HSCT). A unique feature of HHV–6 amongst the human herpesviruses is the persistence of the viral genome in human chromosomal telomeres in a minority of individuals with the capacity for transmission from one generation to the next. Telomeres are the essential capping structure of chromosomes that play roles in cancer and ageing raising the possibility that disruption of telomeres by the acquisition of HHV–6 nucleic acid may cause consequential disease. Importantly, the phenomenon of viral integration with its accompanying very high levels of HHV–6 DNA throughout the body has resulted in inappropriate association with disease. It also confounds laboratory diagnosis of active HHV–6 infection potentially resulting in the unnecessary use of antiviral therapy. It was originally considered that chromosomally integrated virus remained latent in the host without reactivation but there is now increasing evidence for viral reactivation and consequent disease. Much remains to be discovered about this fascinating phenomenon.

Biology of the Viruses Viral Genomics The virus species, HHV–6A, HHV–6B and HHV–7, belong to the roseolovirus genus of the betaherpesvirus subfamily, so named because of the propensity of HHV–6B and HHV–7 to cause the childhood illness, roseola infantum (exanthem subitum). The only other human betaherpesvirus is the more distantly related human cytomegalovirus (HCMV). Many roseolovirus genes are homologs of core genes shared by all herpesviruses, most of which encode structural proteins or proteins involved in virus replication. The viruses also contain genes unique to betaherpesviruses and those specific to roseoloviruses; encoded proteins include transcription factors controlling the gene expression cascade (see below “Virus Structure and Replication”). Open reading frames (ORF) are named with a prefix of U or DR (Fig. 1 and see below “HHV–6A and HHV–6B”). Table 1 gives a selected list of HHV–6 and HHV–7 genes and gene products. Notably major functions of many roseolovirus genes are known only by inference from that of their homologs in HCMV or other herpesviruses. Only a few genes unique to roseoloviruses have been studied functionally including an immunoevasin encoded by U21, the gQ1 and gQ2 glycoproteins encoded by U100 and lastly the U94 parvovirus rep gene homolog, which is specific to HHV–6A and HHV–6B.

HHV–6A and HHV–6B HHV–6A (strains U1102 and GS) and HHV–6B (strains Z29 and HST) are 160–162 kilobases (kb) in size. Their complete nucleotide sequences have been published; both genomes are co-linear consisting of a long unique (U) region, containing the majority of genes, bracketed by terminal direct repeats on the left and right (DRL and DRR) yielding the arrangement DRL-U-DRR (Fig. 1(a)). The few open reading frames (ORFs) in the DR are prefixed ‘DR’ and those in the unique region U1-U100 from the left to the right hand of the sequence. The DRs are bounded by multiple copies of short sequences that are either identical to the 6 base pair repeat sequences found in human telomeric DNA or imperfect copies of the telomere repeat sequence (TMR or impTMR). At the extreme ends of each DR are “pac” sequences for cleavage and packaging of unit length genomes during virus replication (Fig. 1(b)). As to the molecular differences between HHV–6A and HHV–6B underlying their recent classification as separate viral species, HHV–6A is predicted to have 102 genes and HHV–6B 97. Not all HHV–6A genes have counterparts in HHV–6B and vice versa. The two genomes have an overall nucleotide sequence identity of 90%. Divergence of specific sequences (e.g., the immediate early, IE, region) is higher than 30% and there are clear functional differences between the IE1 (U90) genes of HHV–6A and HHV–6B. Moreover, even highly conserved sequences with homology higher than 95%, e.g., U94, as well as divergent genes such as U83, are

780

Table 1

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

Selected HHV-6 and HHV-7 genes and gene products

Gene/Gene producta

HHV–6A

HHV–6B

HHV–7

HCMVb

Core herpesvirus

Nucleic acid metabolism U69c/protein kinase confers ganciclovir sensitivity U86/ immediate early protein – IE-A (IE2) U90/IE-A (IE1) U94d/rep protein

þ þ þ þ

þ þ þ þ

þ þ þ –

þ þ – –

þ – – –

Structural proteins U11/tegument phosphoprotein U14/tegument phosphoprotein U57/major capsid protein

þ (p100)e,f þ þ

þ (101K)e,f þ þ

þ (pp89)f þ (pp85)f þ

þ þ þ

– – þ

Virus attachment/membrane fusion proteins U39/glycoprotein B (gB) U48/glycoprotein H (gH) U82/glycoprotein L (gL) U100c/glycoproteins Qg

þ þ þ þ

þ þ þ þ

þ þ þ þ

þ þ þ –

þ þ þ –

Immunomodulatory molecules U12/chemokine receptor U21/down regulator of MHC class 1 U51/chemokine receptor U83/potential viral chemokine

þ þ þ þ

þ þ þ þ

þ þ þ –

þ – þ –

– – – –

a

Known function by experimental validation or by analogy with homologous gene. Human cytomegalovirus. c Homolog of CMV U97. d Homolog of adeno-associated virus type 2 (AAV–2) rep gene. e Only 80% amino acid identity between HHV–6A and HHV–6B. f Major antigenic target for human antibody response. g Spliced envelope glycoproteins: gQ1 and gQ2 for HHV–6A and HHV–6B and gQ for HHV–7. Likely target for neutralizing antibodies. b

characterized by specific amino acid signatures, which permit distinction between HHV–6A and HHV–6B. At present, the full implications of genomic and proteomic differences between the two species are unknown although it is clear that such differences may have profound biological effects. In contrast, there is very little variability within strains of HHV–6A or HHV–6B. For instance, the IE1 gene is highly conserved (495%) within clinical isolates of each species.

HHV–7 The full sequences of three isolates of HHV–7 (strains JI, RK and UCL-1) have been published. The genome is arranged in the same way as HHV–6 with a U region flanked at either end by DR regions bounded by telomeric repeats but is smaller than that of HHV–6A/B, namely 144–153 kb with fewer genes, namely 86. Over 90% of the ORFs of HHV–7 are similar in amino acid sequence to those of HHV–6A and HHV–6B. There are only a small number of HHV–6 genes without a counterpart in HHV–7 and vice versa.

Virus Structure and Replication In common with all herpesviruses, HHV–6 and HHV–7 have a linear double-stranded DNA genome surrounded by an icosahedral capsid separated by amorphous material, the tegument, from an outer envelope containing viral glycoproteins. HHV–6 and HHV–7 are characterized by growth in T lymphocytes, although as described below (Roseolovirus tropism) they can infect other cell types. Replication is slow in vitro with the production of swollen (cytomegalic) cells and results in cell death. The entry of roseoloviruses into the cell requires attachment of viral glycoproteins to the target cell receptor, which is CD46 for HHV–6A, CD134 for HHV–6B and CD4 for HHV–7. In the case of HHV–6A and HHV–6B, virus attachment involves a complex of glycoproteins gH/gL/gQ1/gQ2 whereas the virus attachment protein or complex for HHV–7 likely involves gH, gL and gQ. Following attachment, fusion between the viral envelope and the cell membrane occurs by a poorly understood mechanism, which may involve gB and gH. The viral nucleocapsid is then transported through the cytoplasm to the nucleus, likely using the microtubule network. As for other herpesviruses, once viral DNA is released in the nucleoplasm, the gene expression cascade commences. Roseolovirus genes are expressed in a temporally ordered manner starting with immediate early (IE) genes from the IE-A locus, which

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

781

comprises two genetic units, IE1 and IE2, with transcription occurring in the absence of de novo protein synthesis. Dependent on prior synthesis of IE proteins, regulation of gene expression follows starting with early (E) genes (encoding enzymes dedicated to nucleotide metabolism and DNA synthesis) and followed by late (L) genes (encoding virus proteins for structure, e.g., viral capsid proteins, and virus attachment). During active infection, HHV–6 and HHV–7 genomes are predicted to change from linear to a circular, episomal form with replication of the genome through a rolling circle mechanism. A long continuous DNA molecule known as a concatemer is generated, which contains multiple copies of the viral genome joined end to end. This molecule is cleaved into single viral genomes, which is packaged into nucleocapsids thanks to specific cleavage packaging signals present in the DR regions of the genome. The capsids exit the nucleus, acquiring an intermediate envelope by budding through the inner part of the nuclear membrane, are de-enveloped by fusion with the external part of this membrane and appear coated with tegument in the cytoplasm. This is followed by acquisition of the final envelope carrying viral glycoproteins and transport to the cell membrane in a Golgi-derived vesicle. Mature viruses are released from the cell by exocytosis.

Roseolovirus Cell Tropism Tropism is the predilection of a virus to invade and reproduce in a particular cell type and is a determinant of its propensity to cause disease.

HHV–6A and HHV–6B The major IE genes of HHV–6A and HHV–6B have promoters that are highly active in T cells reflecting their principal tropism for activated CD4 þ T lymphocytes. Primary isolation of either species from a human sample requires co-cultivation with T lymphocytes derived from umbilical cord blood mononuclear cells or peripheral blood mononuclear cells (PBMC). Subsequently some isolates have been adapted to replicate in CD4 þ T cell lines; HHV–6A strains replicate in HSB-2 and JJhan cells whereas HHV–6B grows in the less differentiated MOLT-3 T cell line. Both HHV–6A and HHV–6B can also infect cultured glial cells of central nervous system origin such as astrocytes, although HHV–6A has the greater capacity being capable of productive replication whereas HHV–6B merely persists at low level. These differences in cell tropism are at least partially explicable because the cellular receptors for the two virus species are different. The HHV–6A cell receptor, CD46, is a ubiquitous molecule (also known as membrane cofactor protein), which is a regulator of the complement cascade. This molecule is expressed on all nucleated cells but not all cell types are permissive for HHV–6A replication indicating a lack of specific cellular factors important for the viral life cycle. The HHV–6B cell receptor, CD134, is expressed on activated but not resting naïve T lymphocytes and a secondary co-stimulatory molecule for T cell differentiation.

HHV–7 Tropism is more restricted for this virus, which infects T cells expressing its receptor CD4 and replicates efficiently in the T cell line, Sup-T1.

Chromosomal Integration of HHV–6A and HHV–6B HHV–6 chromosomal integration was first described in 1993 in 3 individuals with unusual HHV–6 DNA levels so high in PBMC that they could be detected by Southern blotting rather than the very much more sensitive technique of PCR. Fluorescent in situ hybridization (FISH) using HHV–6 specific probes identified integrated viral sequences close to, or in, the telomeres of the short arm of chromosome 17 in each case. Subsequently, the telomeric location was confirmed by direct sequencing of junctions between viral and host cell DNA recovered by PCR from patient’s cells. Study of a number of Japanese and other families throughout the world indicated vertical transmission from one generation to the next. Such integration is unique amongst the human herpesviruses in that it occurs naturally in vivo. It is now clear that chromosomally integrated HHV–6 (CIHHV–6), formerly regarded as an oddity, is in fact relatively common, being found inherited in about 1% of humans. CIHHV–6 is alternatively termed inherited CIHHV–6 (iCIHHV–6). In each case of CIHHV-6, one copy of HHV–6A or HHV–6B is integrated into a chromosomal telomere in every nucleated cell in the body because of Mendelian inheritance. Viral integration is not limited to chromosome 17 as telomeric integration sites have been identified on several different chromosomes by FISH (see Fig. 2 for an example). Integration is normally restricted to a particular chromosome per individual but very rarely two sites, if inherited from both parents. Studies of a few dozen CIHHV–6 genomes have identified a founder effect suggesting that chromosomal integration into the germline is an extraordinarily rare event i.e., most CIHHV–6 individuals acquired their virus in the remote past thousands of years ago. This hypothesis needs further confirmation by nucleic acid sequencing of many more HHV–6 genomes.

Mechanism of chromosomal integration Exactly how HHV–6 integrates into the ends of chromosomes is unknown but this region is notoriously unstable. Proteins associated with the telomeric DNA sequence at human chromosome ends carry out and regulate the maintenance and protection

782

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

Fig. 2 Fluorescent in situ hybridization (FISH) mapping of HHV–6 chromosomal integration site 9q. Example of dividing cells showing the green signal from the HHV–6 specific probe on one homolog of chromosome 9 (arrow) and the red signal from the 9q telomeric probe on both homologs. Inset right: two enlarged images of the homologs of chromosome 9; the upper image shows superimposed signals from the HHV–6 and 9q telomeric probes and the lower the other homolog with signals from the 9q telomeric probe. Inset lower left: Probes as above; interphase nucleus with uncondensed nucleic acid. CIHHV–6 chromosomally integrated HHV–6.

of telomeres by means of DNA repair and homologous recombination. It is therefore presumed that homologous recombination between the telomere repeat sequences in the viral genome and those present at the ends of human chromosomes is responsible for HHV–6 integration. Another herpesvirus, Marek’s disease virus of chickens, has telomeric repeat sequences similar to HHV–6. Marek’s virus is found integrated into the telomeres of avian chromosomes in newly harvested lymphoma cells. Being closely related to HHV–6, HHV–7 also has telomeric repeats at the extremities of the genome but there is no evidence for vertical transmission of this virus suggesting that although such repeats might be necessary they may not be sufficient for integration. It is therefore hypothesized that the HHV–6 U94 rep gene, which is unique amongst the herpesviruses, might be the additional missing factor. This gene codes for a protein, which shares homology with the REP68/78 protein of the adeno-associated parvovirus type 2 (AAV–2) and which is known to be essential for AAV–2 chromosomal integration. Despite this promising hypothesis, U94 is not necessary for the chromosomal integration of HHV–6A into several different cell lines, suggesting that this gene is dispensable for HHV–6A/B integration at least under these conditions.

Viral Latency HHV–6 and HHV–7 persist in the host throughout life following primary infection with the possibility of subsequent reactivation. Like all herpesviruses, their life cycle has two phases. One is the lytic phase, in which infection of a permissive cell results in coordinated expression of most genes, DNA replication, viral assembly, and release of progeny viruses. Alternatively, the virus infects a cell and becomes latent. Latency is a reversible state in which viral DNA (without viral replication) remains in the cell’s nucleus, with limited gene expression. HHV–6 and HHV–7 can be detected in a small proportion of the PBMC of healthy individuals by PCR when sufficient quantities of DNA are tested. The site of latency for HHV–6A is unknown. Candidates for HHV–6B include monocytes and early bone marrow progenitor cells. For HHV–7, CD4 þ T helper cells are the proposed site of latency. HHV–6 and HHV–7 persistence probably includes not only a latent state in cells, such as PBMC, with virions only produced during episodes of reactivation, and chronic replication with continuous or frequent but intermittent production of infectious virus. Salivary glands are a candidate site for chronic replication of HHV–6B and HHV–7 (see “Transmission” below).

State of viral genome during latency Roseolovirus latency is poorly defined in molecular terms. The assumption was that similar to other human herpesviruses, such as herpes simplex virus, the roseolovirus genomes would be in the form of covalently closed circular episomes. However, the presence of roseolovirus episomes has never been documented. Our understanding of HHV–6A and HHV–6B latency and persistence in the host has undergone significant revision in the light of studies characterising CIHHV–6. It is not formally proven, but entirely possible such integration is an important property of HHV–6 that is the basis of latency after post-natal infection.

Reactivation from latency In the case of HHV–6B, differentiation of bone marrow progenitor cells might lead to viral reactivation as occurs with the related betaherpesvirus, HCMV. Virus reactivation is common after immunosuppression accompanying organ transplant indicating the

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

783

importance of T lymphocytes in controlling HHV–6B virus infection. HHV–6B reactivation occurs after allogeneic HSCT and the disease syndrome, DRESS (see below “HHV–6 and DRESS”), both conditions in which CD134, the receptor for HHV–6B, is upregulated facilitating virus replication.

CIHHV–6 and the possibility of reactivation The majority of CIHHV–6 genomes contain a full set of intact viral genes and therefore might have the capacity for viral gene expression and full reactivation. There is limited evidence for this possibility in tissue culture after stimulation with drugs such as histone deacetylase inhibitors. Importantly, symptomatic reactivation has been reported in a few cases of immunocompromised patients (see below “CIHHV–6 and Potential for Disease in the Immunocompromised”). It is presumed that for the virus to reactivate from its chromosomal latent state, the genome must revert from linear to circular form so that virus replication can take place. The molecular mechanism for this is currently unknown, although extrachromosomal circular HHV–6 molecules have been reported in a study of human telomeres that carry an integrated form of HHV–6.

Epidemiology Occurrence HHV–6B and HHV–7 primary infections First infection with HHV–6B occurs in almost all children worldwide by the time they are two years old, the peak age of acquisition being between 9 and 21 months. Primary HHV–7 infection is similarly ubiquitous also occurring early in life but usually somewhat later than the latter over the first 5 or 6 years of life.

HHV–6A In contrast to HHV–6B and HHV–7, natural primary infection with HHV–6A has never been identified and the virus itself is only rarely detected.

Chromosomal integration In contrast to the almost universal postnatal acquisition of HHV–6B, both HHV–6A and HHV–6B are found chromosomally integrated in a small proportion of the human population (as described above). Although HHV–6A is rare in the general population, it is commonly found chromosomally integrated; one third of HHV–6A cases have CIHHV–6A.

Tissue Distribution The distribution of HHV–6A in the body differs from that of HHV–6B. Thus, HHV–6B is commonly found in saliva and blood whereas HHV-6A has only rarely been found in these sites. HHV–6B is also the predominant species found in brain tissue. In vivo HHV–7 is commonly found in blood and saliva but is rarely been found in brain tissue.

Transmission Horizontal transmission of HHV–6 and HHV–7 It is presumed that HHV–6B and HHV–7 are transmitted between individuals via the saliva of infected individuals. As with the other human herpesviruses, close contact is required i.e., with older siblings and parents rather than casual contacts.

Vertical transmission of HHV–6A and HHV–6B Two possibilities for the route of congenital infection with these viruses were investigated by Hall and colleagues in Rochester, USA, one being germline, vertical transmission, and the other a conventional infectious process whereby virus crosses the placenta. The results showed that the majority if not all congenital infections are due to Mendelian inheritance of CIHHV–6. One-third of the positives were HHV–6A as opposed to the control post-natal infections all of which were with HHV–6B. The suggested possibility that a small proportion of cases were due to reactivation of virus from its latent chromosomally integrated state and crossing the placenta to infect the fetus remains to be formally proven.

Transmission of HHV–6 by organ donation Since HHV–6 replicates in T lymphocytes and is likely latent in monocytes and bone marrow progenitor cells, it is to be expected that the virus can be transmitted via blood transfusion and organ transplant. HHV–6B transmission has been well documented in an infant who received an HSCT from his non-identical twin brother, who had exanthem subitum at the time of marrow donation. There are also reports of primary HHV–6 infection acquired by infants after liver transplant from their HHV–6 seropositive mothers.

784

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

If an organ donor has CIHHV–6, then integrated virus will be transmitted to the recipient. In the case of allogeneic HSCT from a donor with CIHHV–6A or CIHHV–6B the result is an extremely high HHV–6 DNA load in the blood of the engrafted recipient, which must always be distinguished from active infection (see below ‘CIHHV–6 and misdiagnosis of active infection’).

Immune Responses For HHV–6, the tegument proteins, p100 for HHV–6A and 101K for HHV–6B, encoded by U11, are a major target of the antibody response. However, the HHV–6A and HHV–6B proteins have an 80% amino acid identity and substantial antibody cross-reactivity exists. Antibody responses to HHV–7 are directed predominantly against the tegument proteins pp85 and pp89, encoded by U14 and U11 respectively. There is a low level of cross-reactivity between the antibody response to HHV–6 and HHV–7 (see below “HHV–6 and HHV–7 Laboratory Tests and Diagnosis of Infection”). As with other viruses, roseoloviruses are susceptible to neutralisation by antibodies able to block binding to their receptors on host cells. Neutralizing antibodies have been described for a number of HHV–6 glycoproteins including gB, the gH/gL complex and gQ. For HHV–7, neutralizing antibodies to the HHV–7 gQ homolog have been reported. Whilst inside the cell the virus is effectively hidden from the immune response especially whilst latent. However, viral proteins produced during active infection are at risk of being processed for presentation by major histocompatibility complex (MHC) class I molecules to CD8 þ cytotoxic T cells with subsequent destruction of the infected cell.

HHV–6, HHV–7 and Immunomodulation HCMV, the prototypical human betaherpesvirus, encodes many gene products that function to subvert host defense mechanisms. This virus is particularly adept at interfering with antigen processing and presentation by MHC class 1 to cytotoxic T cells. Both HHV–6 and HHV–7 infect CD4 þ T helper lymphocytes, the cornerstone of the adaptive immune response, and it seems likely that in common with HCMV, HHV–6 and HHV–7 have genes that function to facilitate the avoidance of immune surveillance during primary infection, latency and reactivation. So far, this topic remains largely unexplored but a few genes have been identified that encode immunomodulatory molecules (see Table 1 for some examples). In particular, HHV–6A and HHV–6B encode a down regulator of MHC Class 1. Based on the above limited evidence, it has been suggested that infection with either HHV–6 or HHV–7 (but primarily the former) interferes with the functioning of the host immune response, and therefore contributes to overall mortality by enhancing the pathogenic effects of agents such as HIV and HCMV or exacerbates graft-versus-host-disease. Further studies are required to establish the clinical impact, if any, of immunomodulation due to HHV–6 and HHV–7.

HHV–6A, HHV–6B and HHV–7 Disease Associations The association of HHV–6A, HHV–6B and HHV–7 with disease is fraught with difficulty since HHV–6B and HHV–7 are universal in the human population from an early age and both primary infection and subsequent reactivation/reinfection may be asymptomatic. The problem is to distinguish infection from infection that has induced disease. In the case of HHV–6 A and HHV–6B, matters are further complicated by the need to differentiate HHV–6A from HHV–6B and to determine whether CIHHV–6 is present. HHV–6 in particular has been alleged to cause many different diseases in both immunocompetent persons and those with cellular immunodeficiency but without compelling evidence for an etiological role. No disease has been causally linked to HHV–6A. The only examples of proven disease are HHV–6B and HHV–7 as the cause of exanthem subitum, and HHV–6B as the cause of encephalitis after organ transplant, particularly after HSCT.

CIHHV–6 and Disease HHV–6 integration can occur in several distinct chromosomes but is invariably telomeric. It is well established that the selfrenewal potential of cells is directly proportional to telomere length and telomerase activity. The shortest chromosomal telomere, not average telomere length, is critical for cell viability and chromosome stability. Several diseases are linked with telomere dysfunction and/or telomerase mutations such as cancer, hematopoietic dysfunction, pulmonary fibrosis, liver and cardiovascular disease. Telomeric integration of HHV–6 therefore has the potential to disrupt normal telomere function leading to disease.

CIHHV–6 and potential for disease in the immunocompetent Neurodevelopment is known to be adversely affected by some viral infections. HHV–6 is closely related to HCMV, which is a major cause of central nervous system disease in the newborn child. A prospective study of children born with CIHHV–6, and possible neurodevelopmental consequences suggested such infection may be associated with cognitive impairment at 12 months of age. Because CIHHV–6 is present in only 1% of births, large population studies are needed.

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

785

In a groundbreaking study of almost 20,000 persons in a large general population in Canada, CIHHV–6 was identified as a risk factor for angina pectoris. It was suggested that coronary artery disease might be the result of viral integration leading to telomeric instability, namely deletions and shortening. Alternatively, endothelial cells with integrated HHV–6 might express viral proteins and/or produce virions leading to chronic inflammation and atherosclerosis. Other much smaller studies have not identified a significantly increased frequency of CIHHV–6 in a range of hematological malignancies. Notably no study so far has taken account of the likelihood that integration into different chromosomal sites might have different pathological consequences.

CIHHV–6 and potential for disease in the immunocompromised There is limited evidence of symptomatic reactivation of CIHHV–6 in three different reports. One in a child with X-linked severe combined immunodeficiency after HSCT, a second in a patient treated with a histone deacetylase inhibitor and a third in a patient who received a liver transplant from a donor with CIHHV–6. Despite the above case of reactivation with accompanying morbidity post-HSCT, this has not been reported in the few other cases where CIHHV–6 was identified in the donor or recipient and the frequency and type of diseases caused by CIHHV–6 in HSCT recipients remain unknown.

HHV–6B and HHV–7 Primary Infection and Disease Exanthem subitum This usually benign rash illness of early childhood begins with a high fever followed by a rose pink, non-pruritic, macular rash; the rash is commonly mistaken for measles, rubella or an allergic reaction to antibiotics. The disease was given its name because of the sudden appearance of the rash (subitum means sudden in Latin). Primary infection does not cause exanthem subitum in all affected children but most have some symptoms ranging from fever, to febrile convulsions, to febrile status epilepticus (FSE). The severity of disease depends on the population studied. Notably, 20% of cases of fever in children aged 6–8 months that come to medical attention are due to primary HHV–6B infection.

Encephalitis In the USA and the United Kingdom, primary HHV–6B and to a lesser extent primary HHV–7 infections are an important cause of complex febrile convulsions and FSE. In Japan HHV–6B is also reported as a cause of encephalitis with an incidence estimated as 5.5/100,000 infants. This difference may be due to a genetic susceptibility of the Japanese. One possibility is that the thermolabile phenotype of carnitine palmitoyltransferase 2, which occurs only in Japanese, is a predisposing factor for encephalitis, with it being inactivated during the high fever characteristic of primary HHV–6B infection; the enzyme is involved in the production of ATP during mitochondrial fatty acid oxidation and its dysfunction would produce an energy crisis.

FSE and temporal lobe epilepsy Primary HHV–6B and HHV–7 infections are a common cause of FSE. Although the short-term outcome of FSE is good, it has long been debated whether this condition predisposes in the long-term to temporal lobe epilepsy (TLE), also known as mesial temporal lobe epilepsy. TLE is one of the most common and intractable form of seizure disorders. FSE may cause hippocampal injury and HHV–6B DNA has been identified in samples of resected temporal lobe in astrocytes from patients with TLE. Whilst the role, if any, of HHV–6B and HHV–7 in epilepsy remains unresolved, it is noteworthy that after HSCT HHV–6B limbic encephalitis (see below “HHV–6B Encephalitis After HSCT”) predisposes to TLE.

HHV–6 and HHV–7 Disease in Older Children and Adults Delayed primary infection Almost all children are infected with HHV–6B by their second birthday and primary infection delayed until after this point must be extremely rare. On the other hand. HHV–7 encephalitis due to delayed primary HHV–7 infection, defined as at or beyond 6 years of age, proven by antibody testing and exclusion of other causes has been reported in one adult and two adolescents. It is tempting to hypothesize that, as when primary Epstein-Barr virus (EBV) infection is delayed beyond early childhood, the consequences of infection are more severe, i.e., infectious mononucleosis in the case of EBV.

HHV–6 and HHV–7 and the possibility of encephalitis After primary infection, HHV–6 and HHV–7 persist in the host and, when reactivated, there is the potential to cause disease. Encephalitis is a rare but serious disease in immunocompetent older children and adults. The most common cause is herpes simplex virus and the key diagnostic test is detection of specific viral DNA in cerebrospinal fluid (CSF). Similarly, both HHV–6A and HHV–6B DNA have been concluded to be a cause of encephalitis based on the finding of viral DNA in the CSF. However, the diagnosis is not straightforward due to the phenomenon of CIHHV–6; in this condition, inevitably HHV–6 DNA will be present in amounts equal to the number of leukocytes present in CSF but it is not necessarily an indicator of viral replication. Formal proof of suspected HHV–6 encephalitis requires evidence of active virus infection and cell destruction in the brain and this, to date, is lacking in immunocompetent older children and adults.

786

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

HHV–6B and DRESS (drug rash with eosinophilia and systemic symptoms) This disorder is also known as drug-induced hypersensitivity syndrome (DHIS) or drug hypersensitivity syndrome (DHS). DRESS is characterized by a severe multiorgan hypersensitivity reaction that appears 3–6 weeks after exposure to certain therapeutic agents. Rising HHV–6B DNA levels in blood occur after rash onset together with increasing amounts of HHV–6-specific IgG antibody. However, it is unclear whether the virus plays a causal role in this syndrome rather than being a consequence of immune activation.

HHV–6 and multiple sclerosis Multiple sclerosis is a progressive demyelinating disease characterized by an immune-mediated focal breakdown of myelin sheaths around neuronal axons, causing impaired conduction of nerve impulses and symptoms ranging from blurred vision to paralysis. Viral infections are likely candidates given their associations with encephalitis and demyelination. HHV–6B antigen, a marker of active viral protein expression was first localized to myelin-producing oligodendrocytes in multiple sclerosis lesions in 1995. Since then large numbers of conflicting reports have explored the role of HHV–6 in multiple sclerosis. These studies have utilized a myriad of diagnostic techniques in different sample populations rendering data interpretation challenging.

Alzheimer’s disease There is growing evidence that inflammation plays an important role in this disease, with chronic brain infection by neurotropic agents being one plausible cause of neuroinflammation. There is provocative new evidence that HHV–6A and HHV–7 RNA levels are high and correlate with amyloid plaque and neurofibrillary tangle densities as well as clinical dementia ratings in brain samples from patients with Alzheimer’s disease. Although this suggests that a relationship might exist between these viruses in the brain and Alzheimer’s disease, direct involvement in causing the disease is not supported and must remain speculative.

HHV–6B and Disease After Organ Transplant Since HHV–6B infection is almost universal in older children and adults, it is not surprising that HHV–6B reactivation is common after solid organ and HSCT. Such reactivations occur in about half of all patients within 2–4 weeks of transplant. Hepatitis, pneumonitis, bone marrow suppression and encephalitis have been reported for liver, lung and heart transplant patients but a synergistic pathological role for HCMV could not be ruled out in all cases. As discussed below, a causative relationship for HHV–6B is most convincing for HSCT patients.

HHV–6B encephalitis after HSCT Not surprisingly, HHV–6B reactivation is common early after allogeneic HSCT because of the accompanying immunosuppression. Causal association with disease is fraught with difficulty as the described cases are often complex involving several different possible pathological mechanisms or infections. Proof remains elusive for proposed links of HHV–6B to hepatitis and pneumonitis. There is some support for a connection to acute graft-versus-host disease and bone marrow suppression but only fever with rash and encephalitis are agreed to be caused by HHV–6B. Encephalitis is rare after allogeneic HSCT but the most common cause is HHV–6B, which carries significant morbidity and mortality; individuals at highest risk are those recipients with poor T cell function, i.e., the donor was mismatched as regards histocompatibility, the presence of acute graft-versus-host disease and an unrelated cord blood transplant; the latter is a particularly high risk. In the first described case of HHV–6B encephalitis there was evidence of infection, namely viral protein in astrocytes and neurons, with necrosis and demyelination in the diseased area of the brain. The virus shows a predilection for the hippocampus and patients characteristically present with post-transplant limbic encephalitis (PALE) – the clinical picture includes depressed consciousness, convulsions, confusion and disorientation, often with short-term memory loss. In cerebrospinal fluid, as well as HHV–6 DNA, there may be evidence of inflammation – leukocytes and elevated protein levels. Mortality is high but the virus may also have more subtle effects; a recent prospective study has reported a correlation between HHV–6 reactivation and impaired cognitive function after allogeneic HSCT in the absence of overt encephalitis.

HHV–7 and Disease after Organ Transplant Although HHV–7 is capable of reactivation in immunosuppressed patients there is little if any evidence of consequent disease.

HHV–6 and HIV HHV–6 is also frequently detected in patients with HIV but unlike in transplant patients HHV–6 is not considered as a significant cause of morbidity and mortality in this patient population.

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

787

HHV–6 and HHV–7 Laboratory Tests and Diagnosis of Infection Unfortunately, antibody tests cannot distinguish between HHV–6A and HHV–6B due to antigenic cross-reaction between these two closely related species. Virus detection is therefore the most useful test but distinction must be made between viral chromosomal integration versus the very much more usual acquisition of active infection by the classical process of primary infection.

Antibody Detection – HHV–6 and HHV–7 The first tests to be developed used indirect immunofluorescence and these remain the gold standard. Such tests allow evaluation of amount of HHV–6 or HHV–7 IgG antibody and in sequential serum samples, and when modified to detect IgG antibody avidity can distinguish primary (low avidity) from long-standing infection (high avidity). Although different less labor-intensive tests now exist, in particular enzyme immunoassay, systematic comparisons have rarely been carried out. Testing for roseolovirus antibodies is rarely available in diagnostic laboratories.

Primary HHV–6B IgM antibodies can be detected 5 days after the onset of infection and IgG antibodies by day 7 the latter increasing for up to 3 weeks and persisting thereafter. HHV–6B DNA appears transiently in the blood but subsides with the appearance of antibodies.

Primary HHV–7 Although less well studied the immune response is similar to that of HHV–6; some cases are accompanied by a rise in HHV–6 antibodies due to limited antigen cross reaction between the two viruses.

Virus detection – HHV–6A, HHV–6B and HHV–7 Roseoloviruses can be grown by coculture of a sample of an individual’s PBMC with phytohemagglutinin-stimulated cord blood lymphocytes. Infected cells can be individually identified by the use of specific monoclonal antibodies or nucleic acid tests. Nowadays virus isolation tests are not used diagnostically and real time quantitative PCR that distinguishes between HHV–6A and HHV–6B DNA is the preferred assay, although no quantitative threshold has been formally established to differentiate latent infection from active viral replication.

CIHHV–6 and Misdiagnosis of Active Infection In chromosomal integration, HHV–6 DNA can be found not only in whole blood but also in “cell-free” samples such as serum or plasma and CSF, which contain human chromosomal DNA released from damaged cells during sample preparation. For most viruses detection of significant levels of viral DNA in the blood, especially in serum or plasma, can be taken as evidence of active infection but HHV–6 is an exception as such high amounts may instead be due to viral integration into host leukocyte chromosomes. For example, after HSCT from a donor with CIHHV–6 there will be asymptomatic elevation of HHV–6 DNA load in the recipient rising to characteristically and strikingly high levels that correlate with leukocyte engraftment. In the past, misunderstanding of this has led to the inappropriate use of antiviral therapy but in CIHHV–6 even prolonged exposure will be ineffective in reducing viral DNA levels. On the other hand, if the stem cell recipient has viral chromosomal integration rather than the donor, the converse occurs as the recipient’s leukocytes are replaced by donor leukocytes and the level of HHV–6 DNA in blood drops as donor leukocytes engraft.

Diagnosis of CIHHV–6 CIHHV–6 should always be suspected if there is an abnormally high HHV–6 DNA load in peripheral blood. CIHHV–6 can be confirmed by evidence of one copy of viral DNA/cellular genome. Comparison of two quantitative real-time PCR results (one for HHV–6 and one for a human gene present in all nucleated cells) is acceptable albeit with a significant margin of error due to inherent assay imprecision. Droplet digital PCR is the most accurate method as it gives an absolute number. Other possibilities for confirmation are HHV–6 DNA in either hair follicles or nails; sites where the virus is only found in CIHHV–6, or by FISH demonstrating HHV–6 integrated into a human chromosome.

HHV–6 and HHV–7 and Antiviral Therapy HHV–6 and HHV–7 are not susceptible to acyclovir as they lack a gene for thymidine kinase but they are sensitive to ganciclovir as they encode a kinase (gene U69) able to phosphorylate this drug. In vitro studies show that all human betaherpesviruses are susceptible to ganciclovir, foscarnet and cidofovir but all have significant side effects. Despite this, these antiviral agents are

788

Roseoloviruses: Human Herpesviruses 6A, 6B and 7 (Herpesviridae)

commonly and effectively used to treat HCMV infections in the immunocompromised, especially organ transplant patients. Of the three roseoloviruses, it is only HHV–6B that is proven to cause serious disease worthy of antiviral therapy. To date there have been no controlled trials of ganciclovir, foscarnet or cidofovir therapy for HHV–6B. However, there is enough evidence in the literature of reduction of amount of HHV–6B DNA in blood or simply a clinical response to treatment to support recommendation of ganciclovir or foscarnet for treatment of HHV–6B encephalitis; no recommendation can be made for cidofovir. It is also important to remember that antiviral therapy will not reduce the HHV–6 DNA integrated into host chromosomes.

Concluding Remarks Whereas the natural history of HHV–6A remains unknown, the pathogenic potential of HHV–6B and HHV–7 is well understood for both viruses as a cause of febrile fits in young children and for HHV–6B as the major cause of encephalitis after HSCT. Issues still to be resolved are a proposed link of FSE to TLE, and effective prevention and treatment strategies, such as new antiviral drugs and immunotherapy, to improve the dismal outcome of HHV–6B encephalitis after HSCT. HHV–6 reactivation has been claimed as a cause of many different diseases although for most there is no compelling evidence for an etiological role. In some cases, HHV–6 may play an immunomodulatory role but alternatively may merely be a bystander reactivated by inflammation or immune reactivation. In other cases, association with active infection and disease may have been wrongly suggested by the phenomenon of HHV–6 chromosomal integration. It is also important any study of disease associations distinguishes HHV–6A from HHV–6B by the use of appropriate tests so that the role of HHV–6A in particular can be more fully explored. Studies to determine whether CIHHV–6 is significantly associated with disease are at an early stage. Although relatively rare in the human population, it is estimated that 40–70 million individuals worldwide carry a chromosomally integrated copy of HHV–6. Understanding the full pathogenic potential of CIHHV–6 at the level of different chromosomes and distinguishing HHV–6A from HHV–6B will necessitate very large prospective studies.

Further Reading Aimola, G., Beythien, G., Aswad, A., Kaufer, B.B., 2020. Current understanding of human herpesvirus 6 (HHV–6) chromosomal integration. Antiviral Research 176, 104720. doi:10.1016/j.antiviral.2020.104720. Clark, D.A., 2016. Clinical and laboratory features of human herpesvirus 6 chromosomal integration. Clinical Microbiology and Infection 22, 333–339. Collin, V., Flamand, L., 2017. HHV–6A/B integration and the pathogenesis associated with the reactivation of chromosomally integrated HHV–6A/B. Viruses 9, 160. doi:10.3390/v9070160. Gravel, A., Dubuc, I., Morissette, G., et al., 2015. Inherited chromosomally integrated human herpesvirus 6 as a predisposing risk factor for the development of angina pectoris. Proceedings of the National Academy of Sciences of the United States of America 112, 8058–8063. Hall, C.B., Caserta, M.T., Schnabel, K., et al., 2008. Chromosomal integration of human herpesvirus 6 is the major mode of congenital human herpesvirus 6 infection. Pediatrics 122, 513–520. Hall, C.B., Long, C.E., Schnabel, K.C., et al., 1994. Human herpesvirus-6 infection in children. A prospective study of complications and reactivation. New England Journal of Medicine 331, 432–438. Huang, Y., Hidalgo-Bravo, A., Zhang, E., et al., 2014. Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome. Nucleic Acids Research 42, 315–327. Krug, L.T., Pellett, P.E., 2014. Roseolovirus molecular biology: recent advances. Current Opinion in Virology 9, 170–177. Pellett, P.E., Ablashi, D.V., Ambros, P.F., et al., 2012. Chromosomally integrated human herpesvirus 6: Questions and answers. Reviews in Medical Virology 22, 144–155. Schwartz, K.L., Richardson, S.E., Ward, K.N., et al., 2014. Delayed primary HHV–7 infection and neurologic disease. Pediatrics 133, e1541–e1547. Ward, K.N., 2005. The natural history and laboratory diagnosis of human herpesviruses-6 and -7 infections in the immunocompetent. Journal of Clinical Virology 32, 183–193. Ward, K.N., 2014. Child and adult forms of human herpesvirus 6 encephalitis: Looking back, looking forward. Current Opinion in Neurology 27, 349–355. Ward, K.N., Hill, J.A., Hubacek, P., et al., 2019. Guidelines from the 2017 European conference on infections in leukaemia for management of HHV–6 infection in patients with hematologic malignancies and after hematopoietic stem cell transplantation. Haematologica 104, 2155–2163. Ward, K.N., 2013. Human herpesviruses-6 and -7 (HHV–6A, HHV–6B and HHV–7). Encyclopaedia of Life Sciences. Chichester: John Wiley & Sons, Ltd, doi:10.1002/ 9780470015902.a0023616. http://www.els.net. Yamanishi, K., Mori, Y., Pellett, P.E., 2013. Human herpesviruses 6 and 7. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed., 2. Wolters Philadelphia PA: Lippincott Williams & Wilkins, pp. 2058–2079.

Rotaviruses (Reoviridae) Juana Angel and Manuel A Franco, Pontifical Javeriana University, Bogota, Colombia r 2021 Elsevier Ltd. All rights reserved. This is an update of J. Angel, M.A. Franco, H.B. Greenberg, Rotaviruses, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B978-0-12-801238-3.02662-3.

Glossary Antigenemia Presence of viral antigen in blood. CFTR Cystic fibrosis transmembrane conductance regulator, a chloride channel localized in the apical membrane of epithelial cells implicated in secretory diarrhea. Genotype Specific genetic makeup of one or more viral genes determined by sequence comparison.

Intussusception Pathological event in which the intestine acutely invaginates upon itself and becomes obstructed, followed by local necrosis of gut tissue. Serotype Significant differences in the antigenic composition of the neutralizing antigens, VP4 and VP7 in the case of rotavirus. Transcytosis Active transport by which polymeric IgA and IgM antibodies are transported from the basolateral to the lumen of the intestine by the polymeric Ig receptor.

Classification Using electron microscopy, rotaviruses were discovered as the etiological agents of epizootic diarrhea of infant mice (EDIM) in 1963 and of calf scours in 1969. Employing this same technique, Ruth Bishop identified a rotavirus (RV) in intestinal biopsies of children with diarrhea in 1973. Rotaviruses are classified in the genus Rotavirus of the family Reoviridae that comprises icosahedral, nonenveloped viruses with segmented, double-stranded RNA (dsRNA) genomes. Based primarily on sequence and antigenic differences of VP6, rotaviruses are classified into ten groups (A–J). Most human pathogens fall into groups A, B, C, and H. The information presented in this article is mostly limited to group A rotaviruses which are, by far, the most prevalent in humans. Serotypes within each group are defined by epitopes in VP7 (glycoprotein, G types) and VP4 (protease, P types) that induce neutralizing antibodies. As the genes encoding these two proteins segregate independently during genome segment reassortment, a binary serotyping system has been developed to identify isolates. Genotypes, determined by nucleic acid sequence similarity of genes encoding VP7 and VP4, and serotypes, determined by antigenic similarity in VP4 or VP7, as tested by neutralization assays, are generally equivalent for VP7. Currently, 36 G genotypes have been described among group A rotaviruses. G1, G2, G3, G4, G9, and G12 constitute the most prevalent G serotypes of humans detected globally and appear to be equally virulent. For VP4, direct relationship between genotypes and serotypes is unclear and, therefore, a dual system for P classification is in use. The RV classification-working group endorsed the following nomenclature: first, the G serotype/genotype (“X” if it is unknown), second, the P serotype, and lastly, the P genotype denoted in brackets. At least 51 P genotypes, that have been further grouped into 5 serogroups (P[I]–P[V]), and 14 serotypes have been described. The predominant circulating human RV strains encode the P[4], P[6], and P[8] genotypes. P[4] and P[8] genotypes correspond to two subtypes of the P1 serotype (P1A and P1B, respectively) that share some cross-reactive epitopes.

Virion Structure Rotaviruses were given their name because, when examined by classical electron microscopy, they appear as wheel (rota)-shaped, 70 nm particles. However, by cryoelectron microscopy (a method that permits visualization of the viral spikes), the viral diameter is 100 nm, with an icosahedral structure T ¼ 13l (Fig. 1), and formed by three concentric layers of proteins. The core comprises three viral structural proteins (VP) and the 11 segments of dsRNA in the viral genome: 120 copies of the scaffolding protein (VP2), and anchored to each segment of dsRNA are the RNA-dependent RNA polymerase (VP1) and the guanyl tranferase and methyltransferase (VP3) (Fig. 1). The intermediate layer is made up of 260 trimers of the structural protein VP6, the most abundant and most antigenic viral protein. Particles with these protein layers are non-infectious but transcriptionally active and called double layered particles (DLP). Their surface has 132 channels of three types that penetrate into the interior of the capsid. These channels are important during viral replication, allowing exchange of compounds in aqueous solution to the inside of the capsid and the export of nascent RNA transcripts. The external layer comprises 260 trimers of the glycoprotein VP7, which depend on bound calcium ions for stability, and 60 trimeric spikes formed by VP4. Several RV structural proteins and nonstructural proteins (NSPs) have been crystallized, permitting detailed molecular studies of viral physiology. Using this method, the rearrangement of the viral spike protein (VP4) upon trypsin cleavage (a process that greatly enhances viral infection) has been characterized: The cleavage of VP4 by trypsin into VP8* and VP5* (the proximal portion), which remain non-covalently associated with each other on the virion surface, generates a “rigidification” of the spike.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21279-X

789

790

Rotaviruses (Reoviridae)

VP7

VP4 VP6 VP2

VP1 and VP3

Viral RNA

Fig. 1 An artist's reconstruction based on cryoelectron microscopy studies of an RV particle. Shown are the seven structural proteins, and the viral RNA. Reproduced with permission from Andrew Swift, Swift Illustration.

These changes resemble the conformational transitions of membrane fusion proteins of enveloped viruses. Structure-based epitopes for neutralizing antibodies have been identified on the two RV outer layer proteins: for VP7 two unique areas are targeted by neutralizing antibodies, some of which stabilize the capsid and prevent viral uncoating. The viral hemaglutinin VP8* contains at least four virus-neutralizing epitopes and antibodies directed against these epitopes probably block virus attachment. Antibodies directed at VP5* appear to neutralize the virus by inhibiting cell entry during capsid uncoating.

Genome The RV genome comprises 11 dsRNA segments that code for six structural proteins and six nonstructural proteins. The size of segments varies from 0.6 to 3.3 kbp. Each segment contains a single long open reading frame (ORF), with the exception of segments 9 and 11, each of which may contain two ORFs. The 50 and 30 ends of the RNA segments have noncoding regions that differ between rotaviruses from groups A, B, and C. These sequences are important in transcription, replication, and reassortment of the virus genome. The viral RNA itself is not infectious. For this reason, the engineering of recombinant rotaviruses has been very difficult, and this has impeded functional analysis of the viral RNA noncoding sequences and the viral proteins. This problem was partially solved by the development of a cell-free system that supports the synthesis of dsRNA from exogenous mRNA, and by gene silencing using small interfering RNA (siRNA) to specifically inhibit translation of viral proteins. In addition, an entirely plasmid- based reverse genetics system for introduction of site-specific mutations into the dsRNA genome of infectious RV has been very recently developed. Rotaviruses are highly variable. Three mechanisms for generating genetic diversity have been identified: point mutation, genome segment reassortment, and recombination. Rotaviruses have a high rate of mutation and it has been estimated that, on average, at least one mutation occurs during each genome replication. Animal and human group A rotaviruses can undergo genome segment reassortment in vitro and in vivo. Reassortment originates in cells simultaneously infected with two different rotaviruses strains and results in progeny viruses that have a combination of genes from each parental strain. Natural reassortment in vivo appears to influence the serotypic diversity in humans, especially in less developed countries (see below). Recombination and related rearrangements of viral RNA segments (e.g., partial gene duplication or deletions) probably play a minor role in generating viral diversity but may be important in longer-term viral evolution.

Life Cycle RV entry into host cells is a multistep process and several molecules have been identified as RV receptors or co-receptors. However, the process has been shown to vary in different viral strains. For example, for several animal RV strains, the first binding step involves the interaction of VP8* with sialic acid, while some human RV strains appear to bind initially to GM1 ganglioside. As a second step, RV binds to the integrin a2b1 in an interaction mediated by the integrin-binding motif DGE in VP5*. In addition to these two interactions, integrins avb3 and axb2, and the heat shock protein hsp70 have also been shown to be involved at a later step of RV cell entry. These interactions probably induce conformational changes in the two viral surface proteins, allowing viral

Rotaviruses (Reoviridae)

791

entry. It seems that the association of some of these molecules with rafts is important for viral entry. A specific interaction between A-type histo-blood group antigen (HBGA) and human RV VP8* to promote RV cell attachment was observed. This interaction seems important since rotaviruses of the five described P genogroups (P[I]–P[V]), differentially infect humans or animals: P[II] rotaviruses infect humans, while P[III] rotaviruses infect both humans and animals, and viruses of both genogroups have different binding patterns to the type A antigen. Although the clinical significance of this discovery is incompletely understood, persons with Lewis-negative secretor-positive HBGA phenotypes have an augmented susceptibility to P[8] RV strains, compared with nonsecretor individuals. Also, genetic differences in HBGA expression may explain the susceptibility of neonates to some RV genotypes and the high prevalence of P[6] RV infections in Africa. Furthermore, research on the importance of HBGA in RV vaccine take and efficacy is ongoing. Although it was initially proposed that rotaviruses enter by direct penetration, current models favor the hypothesis that virus entry is by endocytosis. Viral particles then traffic to the early endosome, where disassembly of VP4 and VP7 – or viral decapsidation – is postulated to occur, due to low Ca2 þ concentrations. This event functions as a signal for VP5* rearrangement that allows permeabilization of the membrane of the endosome. Then, the viral transcriptase (DLPs) gains access to the cytoplasm and begins to synthesize viral mRNAs. The mRNAs produced by the transcriptase are exact copies of each genome segment, with a 50 -terminal type 1 methylated cap structure and without a 30 -terminal poly(A) sequences. The viral mRNAs are translated, giving rise to the structural and nonstructural proteins necessary to complete the viral replication cycle. NSP3 is reported to shut off the synthesis of cellular proteins and induces the preferential translation of viral proteins. These proteins accumulate in the cytoplasm in an electron dense region called the viroplasm (constructed mainly by NSP2 and NSP5), where the viral genome is replicated and the assembly of progeny double-layered particles takes place. The mechanism by which one viral particle assembles with each and only one of the 11 RNA segments is unknown. Synthesis of the dsRNAs occurs following the packaging of viral mRNAs into intermediate precursors of double-layered particles. Assembled double-layered particles and probably VP4 interact with NSP4, which is synthesized by ribosomes associated with the endoplasmic reticulum (ER), and bud into the ER lumen. In this organelle, the double-layered particles acquire a transient lipid membrane. Then, in a very poorly understood process (probably related to the high Ca2 þ levels of the ER), the viral particles acquire VP7, locking VP4 into place, and lose the transient enveloping membrane, giving rise to the triple-layered particle. The physiological mechanism of exit of the mature triple layer viral particle from the cell is unknown. In polarized Caco-2 cells (intestinal epithelial cells derived from a human colon adenocarcinoma), RV is released, at least in part, before cell death using a vesicle-associated vectorial transport system, which bypasses the Golgi apparatus and lysosomes, to the apical pole. However, rotaviruses are lytic viruses, and also exit the cell after cell lysis. The mechanism of cell death induced by RV is incompletely understood. Results in polarized Caco-2 cells and in vivo studies in mice suggest that it is by viral induced cell apoptosis. Up until recently, most in vitro cellular models of RV infection comprised transformed cell lines and animal RV. Novel insights in human virus-cell interactions are being achieved using human intestinal enteroids produced from self-organizing and selfrenewing stem cells, isolated from crypts of intestinal samples, that differentiate to enterocytes, goblet cells, enteroendocrine cells, and Paneth cells, modeling human gastrointestinal epithelium.

Epidemiology Before the introduction of RV vaccines, a meta-analysis presented in 2008 estimated that RV-induced GE caused around 453,000 deaths of children under 5 years old, accounting for 5% of children's deaths of all causes and 37% of those attributable to GE worldwide. More than 90% of these deaths occur in developing countries of sub-Saharan Africa, and South Asia. Although deaths due to RV are rare in developed countries, the incidence of viral infection is the same as in developing countries and health costs associated with RV disease are considerable. In the United States, for example, prior to the introduction of vaccine, an estimated of 58,000–70,000 rotavirus-associated hospitalizations had occurred each year and cost-effectiveness studies clearly justified the use of RV vaccines. After introduction of RV vaccines, norovirus has become the principal cause of medically consulted visits due to GE in the United States. The multisite MAL-ED cohort study of children aged 0–2 years, performed at eight low-resource settings in different continents, assessed the etiology of diarrhea of any severity using quantitative PCR for an extended range of enteropathogens. The results published in 2017 showed that in the first and second year of life the incidence of diarrhea was 304.9 and 242.7 episodes per 100 child-years, respectively. RV was the principal pathogen identified in the first year of age and the fourth in the second year of age (23.9 and 17.7 episodes per 100 child-years, respectively). In two of eight settings RV vaccine had been introduced to the national vaccine programs, and the total incidence of RV disease was less than half, compared with that of settings where vaccination was absent (4.2% versus 8.9%). Overall, strain variability is less than would be expected from a random assortment of G and P genotypes since human RV strains belonging to G1, G3, and G4 serotypes are preferentially associated with P[8] while G2 serotype strains are most frequently associated with P[4] genotypes. Besides, six strains of RV represent more than 90% of worldwide circulating species A RV in children necessitating medical attention: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8]. In addition to these more prevalent strains, rotaviruses with unusual serotypes circulate and can be identified sporadically in developed and particularly in developing countries. Several serotypes may coexist within a community, but, especially in temperate climates, each season is usually dominated by a single serotype that may change from season to season. The global distribution of

792

Rotaviruses (Reoviridae)

rotaviruses varies over continents. Prior to the introduction of RV vaccines G1P[8] represented over 70% of RV infections in North America, Europe, and Australia, but only about 30% of the infections in South America and Asia, and 23% in Africa. These differences in geographic distribution can probably be explained by variations in sanitary and climatic conditions and/or closer contact of individuals with animal rotaviruses in areas with more RV diversity. From 2000–2007 the most prevalent RV strain in India was G12, and during this period other strains such as G2P[11] and G3P[11] were infrequently identified. G and P associations of a probable zoonotic origin have also been detected in Africa. From 2000–2010 G12 strains emerged and G2P[4] reemerged in Latin America and the Caribbean countries. In several cases, human rotaviruses have been found that have genes of animal RV origin (natural reassortment), adding a further dimension to strain diversity. Even though rotaviruses have an important host range restriction, there is rising evidence of zoonotic transmission to humans, although animal origin strains rarely, if ever, persist in a human population. Notably, host range restriction has been exploited for the development of several viral vaccines. In the temperate zones of the world (mostly developed countries), prior to vaccine introduction, RV infection occurred primarily during epidemic peaks in the cooler months of the year. A yearly wave of rotaviral illness spread over the United States; beginning in the southwest in November and terminating in the northeast in March. A similar phenomenon has been reported to occur in Europe. This pattern has been markedly attenuated in the US since the introduction of vaccine and is unseen in countries within 101 of latitude from the Equator (mostly developing countries), where epidemics occur year-round. Use of mathematical models of RV transmission dynamics suggest that in the United States spatiotemporal variations in birth rate may have explained RV seasonality, and that for countries localized in the tropics, high birth rates and RV transmission, instead of environmental factors, may explain this lack of seasonality. There has been substantial disruption of the RV circulation patterns after the introduction of RV vaccines. Rotaviruses are highly contagious and spread easily but, unlike certain human bacterial infections that occur disproportionately in developing countries, the prevalence of RV infection is the same in developed and developing countries, implying that sanitation and hygiene are ineffective measures for disease control.

Clinical Features Rotaviruses are highly contagious since approximately 1011 particles per gram of feces are excreted, only 10 or less particles are necessary to infect a new host, and they are very resistant to ambient conditions. In addition, viral excretion in most children lasts for up to 10 days and, in some children, may extend for 2 months after onset of infection. Rotaviruses are mainly transmitted by an oral–fecal route although, in some cases, a respiratory route has been suggested. In developing countries, the peak incidence of RV disease occurs in children between 6 and 11 months of age. In contrast, in developed countries, the highest incidence is observed in older children (2 years old). This difference is probably related to differences in sanitation in the different settings, but also influence by the patterns of annual viral circulation. Notwithstanding this variation, RV incidence is similar in both developing and developed countries but mortality is mainly observed in developing countries, presumably due to limited access to appropriate health care. The relative protection of infants younger than 2 months of age, which occurs worldwide, could be related to the presence of protective maternal antibodies. Up to 50% of adults caring for children with RV diarrhea can become infected and, of these, 50% develop disease symptoms, which are generally mild. The primary clinical syndrome caused by RV infection is acute gastroenteritis. After a short incubation period of 48 h or less, children frequently present with vomiting that lasts for 1–2 days. The vomiting is often accompanied with fever (37.91C or greater). Subsequently, or at the same time, a watery diarrhea appears, (commonly more than six loose stools in 24 h) and high severity, lasting 5–6 days and, if left untreated, frequently induces dehydration. It is estimated that up to 50% of RV infections in children are asymptomatic but some of these may represent second or third exposures. Children attending day care institutions are at high risk of developing RV-induced diarrhea. Although RV infections in neonatal care units have been classically described as asymptomatic, probably due to the presence of maternal antibodies, severe symptomatic outbreaks have been described. RV strains that induce nosocomial infections in neonatal nurseries are generally different from those circulating in the community. RV infection can cause severe and prolonged disease in children with primary immunodeficiencies, some of whom develop systemically disseminated infections. Acquired immunodeficiency also predisposes to severe RV disease in bone marrow- and liver-transplanted children. The role of RV-induced disease in immunosuppressed adults with HIV seems, at present, less important.

Pathogenesis Rotaviruses are very well adapted to their host: they replicate very efficiently, sterilizing immunity is not developed and, despite an important host range restriction, many animal hosts exist (Table 1). These characteristics help to explain the high viral prevalence, and suggest that the prevention of severe disease is an appropriate goal for vaccination. Important viral antigenemia and some level of viremia are observed in the initial phase of RV-induced diarrhea in children and animals. In mice, extraintestinal viral replication occurs commonly during homologous and some heterologous RV infections. Also in this model, the level and location of extra intestinal replication varies between RV strains and low-level replication can occur in several leukocytes subsets. Elevation of hepatic transaminases has been detected in 20% of children with RV gastroenteritis. However, the clinical relevance of these findings is unclear and, in children and in animals, the bulk of RV replication occurs in the mature villus tip cells of the small bowel.

Rotaviruses (Reoviridae)

Table 1

793

Virus-associated factors that contribute to high viral prevalence and reinfections

Characteristic

Comments

Natural infection does not generate sterilizing immunity Multiple animal hosts exist Short incubation period (1–2 days) The entry cell is the same as the cell used for viral replication Virus is excreted in high quantities (up to 1011 pfu g1 of feces) Up to 30% of children excrete antigen up to 57 days after onset of diarrhea High rate of viral mutation and gene reassortment Over 50% of infections are asymptomatic

Goals of vaccination are to decrease severe disease but not to prevent infection Eradication does not seem feasible Does not allow time for the recall of high levels of immune effector mechanisms Does not allow time for the recall of high levels of immune effector mechanisms High levels of viral dissemination in the environment High level of viral dissemination May permit the virus to evade the immune system. Currently unproven RV well adapted to the human host

Note: Reprinted from Franco, M.A., Angel, J., Greenberg, H.B., 2006. Immunity and correlates of protection for rotavirus vaccines. Vaccine 24 (15), 2718–2731, with permission from Elsevier.

RV-induced diarrhea probably occurs by multiple mechanisms that vary, depending on the animal species analyzed. Pathological findings in the small intestine of children with RV diarrhea include: shortening and atrophy of the intestinal villi, enterocyte vacuolization with distended cisternae of the endoplasmic reticulum, and mononuclear infiltration in the intestinal lamina propria. However, in children a direct relationship between the extent of histopathology and disease has not been demonstrated. This finding suggests that RV diarrhea can occur without important enterocyte death. In pigs, RV infection induces an intestinal lactase deficiency and increased lactose in feces induces an osmotic diarrhea. Elevated levels of lactose are also observed in the feces of some RV infected children. Lactase deficiency could be due to RV-induced enterocyte destruction or to alteration in the synthesis or metabolism of disaccharidases. In mice, two mechanisms of diarrhea, independently of enterocyte destruction, have been identified. NSP4, and a derived peptide of NSP4, induce diarrhea in mouse pups but not adult animals, making it the first viral enterotoxin described. However, its role in RV-induced diarrhea in other species remains uncharacterized. Also in mice, RV activates the intestinal autonomous neural system and increases the secretion of water and electrolytes, as well as the intestinal motility. In fact, RV infection and NSP4 can induce secretion of 5-hydroxytryptamine (serotonin) from enteroendocrine cells in humans and mice, which can activate enteric nerves of the small intestine leading to increased intestinal motility. Racecadotril, an inhibitor of the enteric nervous system, is somewhat efficacious in treating RV-induced diarrhea in children, suggesting that this mechanism plays at least some role in human diarrhea.

Diagnosis Initial efforts to isolate wild-type rotaviruses in tissue culture were unsuccessful and even today it is a complex procedure. For this reason, diagnosis is more commonly made using commercial ELISAs that detect viral antigen (mostly VP6) in the feces. Rotavirus is shed in very large amounts making the ELISA a highly effective, inexpensive, simple and accurate diagnostic assay. For epidemiological studies, human RV strains present in feces can be grown in cell culture in MA104 (African green monkey kidney) and Caco-2 cells using trypsin for enhancement of viral growth during the culture. Trypsin cleaves VP4 and increases infectivity in culture. Genotype characterization of RV strains is mainly by reverse transcription-polymerase chain reaction (RT-PCR). RV detection by electron microscopy is only conducted in research laboratories, especially when other viral pathogens are also being investigated.

Treatment RV diarrhea is self-limited and treatment is aimed at reducing symptoms until the immune response resolves the infection. Children with mild diarrhea are treated by oral rehydration. Those presenting moderate to severe dehydration may require intravenous rehydration. In cases of severe disease, treatment with probiotics, preparations that contain antibodies against RV, Racecadotril (an inhibitor of the enteric nervous system), and Nitasoxanide (a drug of unknown mechanism of action) have been shown to accelerate resolution of the disease. However, it is unclear whether these interventions truly provide significant advantages to ill children.

Immune Response and Prevention Immunity to RV is incompletely understood and animal models have been important in the acquisition of our current knowledge. Pepsin and gastric acid seem to be important host defense factors against RV infection, since these factors inactivate rotaviruses in adult mice but not in suckling mice. In addition, the innate immune response and interferons (IFN), in particular, seem to mediate an antiviral effect, and viral mechanisms for evading this response involving NSP1 have been described. However, mice lacking T and B cells become chronically infected with RV, suggesting that the adaptive immune system is essential for viral elimination. Also, children lacking T and B cells, or only B cells have been shown to shed virus chronically. Several recent studies in mice have addressed the role of IFNs in the immune response against rotaviruses: RV infection of intestinal cells of mouse pups induces expression of mRNA of type I and III IFNs, treatment of mice with these IFNs reduced RV

794

Rotaviruses (Reoviridae)

replication, and in parallel, enhanced RV replication was detected in mice lacking receptors for these IFNs. In agreement with these findings, it has been shown that intracellular RV replication is recognized by RIG-I and/or MDA5, which activate the transcription factors IRF3 and NF-κB, that in turn induce expression of type I IFN and interferon-secreted genes (ISGs). After RV induced production of IFN, it signals, in an autocrine fashion, through STAT1/STAT2/IRF9 complexes to further induce type I IFNR expression, more IFN and ISG expression, and an antiviral state. Moreover, RV has developed numerous mechanisms to evade the type I IFN response: RV NSP1 triggers degradation of IRF3 and inhibition of NF-κB activity; RV inhibits the function of STAT1 activation in vitro and in vivo in mice, by an unknown mechanism, possibly inhibiting all types of IFN responses; it significantly depletes IFN-type I, II, and III receptors in vitro and in vivo in mice. When human intestinal enteroids were used, type III IFNregulated response was the predominant transcriptional pathway stimulated by human RV infection, which is, however, unable to restrict viral replication. A recent study showed that RV activates another component of the innate immune response, the inflammasome, and Nlrp9b, specifically expressed in murine intestinal epithelial cells, was recognized as the NOD-like receptor (NLR) that restricts murine RV infection. Intestinal antibodies seem to be the principal mechanism that mediates protection against viral reinfection. In agreement with the fact that most viral replication occurs in the intestine, the localization of virus specific B cells in the intestine seems important for their capacity to mediate protection. It is postulated that local IgA antibodies can mediate expulsion of rotaviruses inside the enterocytes and exclusion (to avoid de novo infection of enterocytes) of rotaviruses in the gut lumen. Neutralizing antibodies to VP4 and/or VP7 can block enterocyte infection directly when present in the gut lumen (exclusion). In mice, anti-VP6 non-neutralizing polymeric IgA may bind virus VP6 during transcytosis from the basolateral membrane of enterocytes to the gut lumen and “expulse it”. These antibodies may also inhibit RV transcription intracellularly. In addition, antibodies to NSP4 may block diarrhea, but not infection, by blocking the enterotoxic property of the molecule. However, the antiviral effects of antibodies against VP6 and NSP4 have only been shown to be protective in mice. In piglets, a model that is probably closer to humans than mice, the presence of antibodies against VP4 and/or VP7 seems necessary for protection. Although local antibodies appear to be the principal mechanism that protects against viral infection, T cells also directly mediate antiviral immunity, at least in mice. CD4 þ T cells are also essential for the development of more than 90% of the RV-specific intestinal IgA, and thus their presence seems critical for the establishment of protective long-term memory responses. Moreover, murine RV-specific CD8 þ T cells are involved in the timely resolution of primary RV infection and can mediate shortterm partial protection against reinfection. In children from middle- to high-income countries, primary RV infections are generally the most severe, with severity decreasing as the number of reinfections increases. Complete protection against moderate-to-severe illness is achieved after two natural RV infections, whether symptomatic or asymptomatic. However, in a cohort study in India, where close to 22% of deaths due to RV worldwide occur and half of the children were infected by 6 month of age, the severity of diseases decreased between the second and third infection, and 79% of protection against moderate-to-severe illness was observed only after the third infection. In agreement with the results of animal models mentioned previously, total serum RV IgA, induced by a primary infection, seems to be the best but imperfect correlate of protection against subsequent reinfections. Primary RV infection induces homotypic-neutralizing antibodies to VP7 and VP4 and some level of heterotypic immunity. The lack of understanding of RV immunity limits our capacity to develop new RV vaccines. Due to the relative inaccessibility for study of the human intestinal immune tissue, cells involved in the RV immune response have mainly been studied in peripheral blood. It is hypothesized that at least a fraction of these lymphocytes are recirculating to and from the intestine, and thus may reflect intestinal immunity. In blood of children with acute RV infection, plasmablasts and plasma cells (secreting mostly RV specific IgM) are found, and these cells express intestinal homing receptors. In the convalescence phase, RV-specific B cells are mainly memory cells, some of which express intestinal homing receptors. In children and adults with acute infection, RV-specific CD4 and CD8 T that secrete gamma interferon circulate, but in unexpectedly low frequencies. It is speculated that the limited induction of RV-specific T cells can be related to the occurrence of delayed viral clearance and symptomatic reinfections in a subset of individuals. Class II T cell tetramers with RV specific epitopes have been described and used to show that a subset of circulating RV-specific CD4 are probably originated in the intestine. The first commercially available RV vaccine was a simian-human reassortant tetravalent vaccine (RotaShield). Although this vaccine was highly effective, it was withdrawn from the market 1 year after its introduction due to its temporal association with low levels of intussusception (1 in 10,000 children). Two new RV vaccines received worldwide recommendation by the World Health Organization and have now been licensed for use in more than 100 countries. The three dose vaccine produced by Merck (RotaTeq™, RV5) contains mono-reassortants of a bovine virus with G1, G2, G3, G4, and P1A[8] human RV genes. The two dose vaccine produced by GlaxoSmithKline (GSK, Rotarix™, RV1) is a human-attenuated G1P1A[8] virus. In trials that involved over 60,000 infants from middle- to high-income countries, both of these vaccines showed to be safe and to provide over 70% protection against any RV diarrhea and over 98% protection against severe RV infection. As observed for other oral vaccines, both RV vaccines are less efficacious, with 44% protection, against severe RV gastroenteritis in settings with high under-5 mortality rates. Importantly, protection against severe disease generally decreased between the first and second year after vaccination. A systematic review of the effectiveness of RV1 and RV5 in the first decade of global postlicensure confirmed that vaccine effectiveness in routine programmatic use depends on under-5 mortality rates. The median vaccine effectiveness against RV disease

Rotaviruses (Reoviridae)

795

were: RV1 84% (range, 19%–97%) and RV5 90% (range, 63%–100%) in low-mortality countries; RV1 75% (range, –2% to 94%) in medium-mortality countries; and RV1 57% (range, 18%–69%) and RV5 45% (range, 43%–92%) in high-mortality countries. The median RV1 and RV5 effectiveness against RV infections requiring hospitalizations were higher: 88% (range, 70%–95%) and 94% (range, 83%–100%), respectively. A decline of 35% in RV induced mortality has been observed from 2008 to 2011 in Mexico, and other countries, after introduction of RV vaccination nationwide. The lower efficacy of oral vaccines in low-income settings remains incompletely understood. To increase RV vaccine effectiveness in these settings, investigators have performed clinical trials with variations in the age of administration, timing of breastfeeding, improvement of nutrition status, and supplementation with zinc, which have, unfortunately, shown none or modest improvement. Even though concomitant administration of live oral polio and RV vaccination is recommended, it interferes with RV vaccines immunogenicity, and consequently, some improvement may arise in settings transitioning from oral polio formulations to parental vaccines. Rotavirus vaccines postlicensure studies revealed: (a) herd immunity appears to be generated following RV vaccination in highand middle-income countries; (b) changes in the age of individuals infected with RV (increase in unvaccinated children 6–16 years of age or individuals 4 70 years of age) (c) changes in the seasonal pattern of RV disease. Also, in some settings, there is evidence that RV vaccines deploy different immunological pressures that impact, to some extent, the diversity of circulating RV strains. However, the fact that RV1 and RV5 induce wide protection against homotypic (strains with genotypes similar to those included in the vaccine formulation) and heterotypic (strains with genotypes different to those included in the vaccine formulation) strains has been corroborated in a great number of different settings. More studies are necessary to establish if waning of vaccine effectiveness after the first year of life is occurring in the different settings, which may have important consequences for vaccination programs, since a booster dose of rotavirus vaccine may become necessary. In post-vaccination surveillance programs, a very low (1–6 per 100,000) increased risk of intussusception has been observed in RV1 or RV5 vaccinated children, primarily in the first week after the first dose of vaccines in several high- and middle-income countries. However, health authorities have generally concluded that the benefits of RV vaccination outweigh these small potential risks. Some evidence indicates that the increased risk of intussusception after RV vaccination may be lower in low-income countries. In China, a lamb RV strain based vaccine (LLR, Lanzhou Institute of Biological Produces) was licensed in 2000, and in Vietnam a human G1P[8] strain based vaccine (RotavinTM, PolyVac) was licensed in 2012. RotavacTM (Bharat Biotech), a three-dose vaccine containing a natural human-bovine reassortant 116E neonatal strain (G9P[11]) was prequalified by WHO in 2018, allowing its procurement by UNICEF for global use. RotasiilTM (Serum Institute of India), a three-dose pentavalent vaccine composed of bovinehuman VP7 reassortants of the UK strain developed at The NIH, was also recently prequalified by WHO. A neonatal RV3-BB strain based vaccine, generated in Australia, is being evaluated for neonatal vaccination. In addition, current studies in animal models and people are also focused on the development of third generation nonreplicating RV vaccines, such as recombinant RV proteins or virus-like particles.

Further Reading Angel, J., Steele, A.D., Franco, M.A., 2014. Correlates of protection for rotavirus vaccines: Possible alternative trial endpoints, opportunities, and challenges. Human Vaccines & Immunotherapeutics 10 (12), 3659–3671. Bines, J.E., At Thobari, J., Satria, C.D., et al., 2018. Human neonatal rotavirus vaccine (RV3-BB) to target rotavirus from birth. The New England Journal of Medicine 378 (8), 719–730. Burnett, E., Jonesteller, C.L., Tate, J.E., Yen, C., Parashar, U.D., 2017. Global impact of rotavirus vaccination on childhood hospitalizations and mortality from diarrhea. The Journal of Infectious Diseases 215 (11), 1666–1672. Crawford, S.E., Ramani, S., Tate, J.E., et al., 2017. Rotavirus infection. Nature Reviews Disease Primers 3, 17083. Kanai, Y., Komoto, S., Kawagishi, T., et al., 2017. Entirely plasmid-based reverse genetics system for rotaviruses. Proceedings of the National Academy of Sciences of the United States of America 114 (9), 2349–2354. Kotloff, K.L., Platts-Mills, J.A., Nasrin, D., et al., 2017. Global burden of diarrheal diseases among children in developing countries: Incidence, etiology, and insights from new molecular diagnostic techniques. Vaccine 35 (49 Pt A), 6783–6789. Lee, B., Dickson, D.M., deCamp, A.C., et al., 2018. Histo-blood group antigen phenotype determines susceptibility to genotype-specific rotavirus infections and impacts measures of rotavirus vaccine efficacy. The Journal of Infectious Diseases 217 (9), 1399–1407. Lopez, S., Sanchez-Tacuba, L., Moreno, J., Arias, C.F., 2016. Rotavirus strategies against the innate antiviral system. Annual Review of Virology 3 (1), 591–609. Nair, N., Feng, N., Blum, L.K., et al., 2017. VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans. Science Translational Medicine 9 (395). Platts-Mills, J.A., Liu, J., Rogawski, E.T., et al., 2018. Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: A reanalysis of the MAL-ED cohort study. The Lancet Global Health 6 (12), e1309–e1318. Saxena, K., Simon, L.M., Zeng, X.L., et al., 2017. A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection. Proceedings of the National Academy of Sciences of the United States of America 114 (4), E570–E579. Shah, M.P., Dahl, R.M., Parashar, U.D., Lopman, B.A., 2018. Annual changes in rotavirus hospitalization rates before and after rotavirus vaccine implementation in the United States. PLoS One 13 (2), e0191429. Tate, J.E., Burton, A.H., Boschi-Pinto, C., Parashar, U.D., N. World Health Organization-Coordinated Global Rotavirus Surveillance, 2016. Global, regional, and national estimates of rotavirus mortality in children o5 Years of Age, 2000–2013. Clinical Infectious Diseases 62 (Suppl 2), S96–S105. Tate, J.E., Yen, C., Steiner, C.A., Cortese, M.M., Parashar, U.D., 2016. Intussusception rates before and after the introduction of rotavirus vaccine. Pediatrics 138 (3). Zhu, S., Ding, S., Wang, P., et al., 2017. Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells. Nature 546 (7660), 667–670.

796

Rotaviruses (Reoviridae)

Relevant Websites https://www.cdc.gov/rotavirus/about/symptoms.html CDC. https://www.path.org/diarrheal-disease/ Diarrheal Disease PATH. http://rotacouncil.org/ Rotacouncil. https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg Rotavirus classification working group: RCWG. https://www.who.int/immunization/diseases/rotavirus/en/ WHO Rotavirus.

Rubella Virus (Picornaviridae) Annette Mankertz, Robert Koch-Institute, Berlin, Germany r 2021 Elsevier Ltd. All rights reserved. This is an update of T.K. Frey, Rubella Virus, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00499-4.

Glossary Birth defects Malformations or abnormalities developed during gestation that are apparent at, or soon after, delivery. Congenital infection Infection of the fetus during gestation as a result of virus passage through the placenta. Elimination program Vaccination program designed to decrease the number of susceptible individuals in order to eliminate transmission of a pathogen in a geographic region and limit circulation after imports from other regions. Enveloped virion Virus particle with a lipid bilayer membrane, or envelope, surrounding the capsid or core. The envelope is usually derived from host cell membranes and contains virus-specific glycoproteins decorating its outer surface. Icosahedral capsid In the virus particle, the proteinaceous shell surrounding the virus genome – in this case, with symmetry of an icosahedron (appears quasi-spherical or round in electron micrographs). Live, attenuated vaccine A vaccine formulation using an infectious variant of the virus that has been attenuated or

weakened by repeated passage in cell culture or in alternate hosts. Such vaccines recapitulate the infection, including induction of a complete adaptive immune response, but do not cause disease due to a loss of pathogenic features. Persistent infection Infection characterized by continued presence of the virus, often despite the induction of an adaptive immune response. Plus-strand RNA virus Virus with a single-stranded RNA genome that is of messenger RNA sense and can be directly translated to produce virus-specified proteins. Serodiagnosis Detection of virus-specific antibodies for diagnosis of infection or verification of the immune status. Briefly the presence of IgM antibodies can indicate an acute infection, while presence IgG can be taken as a token of previous infection or immunity. Systemic infection Infection that spreads from local site entry via the lymphatic system and blood stream to one or more target internal organs or tissues.

Introduction Rubella virus (RV) infection leads to a benign disease with rash and fever in children and adults. In contrast, infection during the first trimester of pregnancy can result in a miscarriage, fetal death, stillbirth, or infants with malformations, known as a congenital rubella syndrome (CRS). Norman Gregg, an Australian ophthalmologist, first reported the association of congenital cataracts as a consequence of gestational rubella in 1941, establishing RV as a major teratogen. Vaccination programs administering live-attenuated vaccines have been successful in greatly reducing the incidence of both rubella and CRS, particularly in the Americas, where rubella elimination was verified in 2015. In the end of 2014, 140 countries had introduced national rubella vaccination programs (54 had not) and a global coverage with rubella vaccine had increased to 47%. This does not prevent the birth of approximately 110,000 children with CRS annually, as estimated by WHO.

Classification The classification of RV is currently under reassessment by the ICTV: Until 2019 RV was the only member of the genus Rubivirus (family Togaviridae) which comprises mainly arthropod-borne alphaviruses. This classification was based on similarities in virion structure, gene expression strategy, and some sequence similarity between conserved regions of the alphavirus genome and their counterparts in the rubivirus genome. However, RV differs from alphaviruses in its mode of transmission, genome organization, virion structure and also in sequence relationship. Thus, RV has been moved to the family of Matonaviridae. The rubella virion consists of a single-stranded, plus-sense genomic RNA with a variable length of approximately 9760 to 9778 nucleotides. Phylogenetic analysis of RV is based on a reference sequence within the E1 gene (nt 8731–9469). The World Health Organization (WHO) has proposed a standardized taxonomy for RVs which consists of two clades 1 and 2, and 13 genotypes within these two clades. The sequences in these two clades differ by approximately 8%. Clade 1 includes ten genotypes and is distributed worldwide. Clade 2 contains three genotypes and appears to be restricted mostly to Asia. Genotypes 1E, 1G, 1J and 2B currently dominate global circulation.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21546-X

797

798

Rubella Virus (Picornaviridae)

Fig. 1 Cryoelectron micrograph of rubella virions. Unlike conventional negative staining, the virions have a uniform structure when visualized by this technique; however, pleomorphism of particle diameter and the gap between the core and envelope remains apparent. Courtesy of Teryl Frey (unpublished data).

Host Range and Virus Propagation RV has no known natural host other than humans. No reliable animal model exists for the study of RV pathogenesis. RV replicates in a number of laboratory cell culture lines; however, in most of these no cytopathic effects (CPEs) are usually observed. Continuous cell lines commonly used to propagate RV include Vero (African green monkey kidney), RK-13 (rabbit kidney), BHK21 (baby hamster kidney), HaCaT cells (human keratinocytes). Also HUVEC (primary fetal endothelial cells derived from human umbilical vein) can be used. In all cell lines in which RV replicates, persistent infections are readily established and maintained, whether or not the cell line exhibits a functional interferon system. Infectious virus is released into the media for several subcultures of infected cells. Persistent RV infection in cell culture cannot be cured by the inclusion of neutralizing antibodies in the culture medium because virus budding occurs at intracytoplasmic locations. Thus, RV is highly adapted for persisting infection.

Properties of the Virion Rubella virions are 60–70 nm spherical particles composed of an electron-dense core separated from the lipid envelope by an electron-lucent zone. Rubella virions exhibit a marked degree of pleomorphism (Fig. 1). The virion has a density of 1.18–1.19 g/ml, whereas isolated capsids have a density of 1.44 g/ml. The genome is contained within a quasi-spherical core or capsid, composed of a single virus-encoded structural protein, C. The C protein (B34 kDa) is present as disulfide-linked homodimers. Although presumed to be icosahedral with a T ¼ 4 symmetry, the precise structure of the RV capsid has not been reported. The capsid is surrounded by a lipid envelope in which two virus-specified glycoproteins, E1 and E2, are embedded, which form the virion spikes. E1 has a molecular weight of 59 kDa whereas E2 is a heterogeneous species ranging from 44 to 50 kDa due to differential glycosylation. Both E1 and E2 appear to be primarily in the form of heterodimers which are easily disrupted by routine preparation techniques. The higher-order architecture of the virion spikes is unclear. E1 is more exposed on the virion surface than is E2, contains both the viral hemagglutinin and receptor site, and is also immunodominant in terms of humoral immunity. Rubella virions are stable at physiological pH values and can be frozen at temperatures below –201C for years, without a significant loss of infectivity. Live, attenuated vaccine virus is stored in lyophilized form and must be reconstituted before use. Rubella virions are susceptible to most commonly used inactivating agents, such as formaldehyde, UV light, and lipid solvents.

Genomic Organization The RV genomic RNA is approximately 9762 nucleotides in length. It contains a 50 terminal cap structure and a 30 terminal poly(A) tract. A distinctive feature of the genomic RNA is that it contains 30% guanine residues and 39% cytosine residues, the highest G þ C content of all RNA viruses. The genome encodes two long, nonoverlapping open-reading frames (ORFs) plus untranslated regions (UTRs) at the 50 and 30 ends and between the ORF's (Fig. 2). The 50 proximal ORF (NS) encodes nonstructural proteins which are translated into a 2116-amino-acid precursor that is proteolytically cleaved into two proteins, 150 kDa (P150) and 90 kDa (P90), which are at the N- and C-termini of the ORF, respectively. The cleavage is mediated by a papain-like cysteine protease located at the C-terminus of P150. P150 and P90 are responsible for virus RNA replication: P150 contains a domain predicted to have methyl/guanylyltransferase activity (responsible for forming the cap structure at the 50 end of the genomic and

Rubella Virus (Picornaviridae)

799

Fig. 2 Coding strategy of the RV genome. Shown is a schematic representation of the RV genomic RNA with untranslated regions (UTRs) drawn as solid black lines and coding regions (ORFs) as open boxes (NS-ORF, nonstructural protein ORF; S-ORF, structural protein ORF). Within each ORF, the coding sequences for the proteins processed from the translation product of the ORF are delineated and, in addition, within the NS-ORF, the locations of motifs associated with the following activities are indicated: methyl/guanylyltransferase (MT), protease (P), helicase (H), and RNA-dependent RNA polymerase (R). The sequences encompassed by the subgenomic RNA (SG RNA) are also shown. The scale at the top of the diagram is in kilobases.

subgenomic RNAs) in addition to the protease, whereas P90 contains both a helicase domain and an RNA-dependent RNA polymerase (or replicase) domain. The 30 located ORF (S) encodes the structural proteins. The 1063-amino-acid product is cleaved by a cellular protease into the virion proteins C, E1 and E2. The order of the virion protein genes within the ORF is 50 C-E2-E1 30 . The S-ORF is translated from a subgenomic RNA synthesized in infected cells and containing the sequences from the start site through the 30 end of the genome. An infectious clone for RV has been developed, which is beneficial for studies based on sitedirected mutagenesis. It is a cDNA copy of the viral RNA under the control of promoter contained in a plasmid. Since RV has a plus-sense genome, in vitro transcripts from the plasmid will initiate virus replication following transfection into susceptible cells.

Intracellular Replication Cycle Myelin oligodendrocyte glycoprotein (MOG) has been identified as a cellular receptor for RV. Since this molecule is expressed mainly on nervous tissue and MOG-independent infection has also been demonstrated, other not yet identified receptors are thought to be responsible for virus entry. Following attachment to the receptor, the virus is taken into the cell by receptor-mediated endocytosis. In the reduced pH environment of the endocytic vesicle, fusion between the viral envelope and the vesicular membrane occurs, releasing the capsid and genomic RNA into the cytoplasm of the cell. The genomic RNA is translated to produce the NS protein precursor which is cleaved into P150 and P90 (Fig. 3). These proteins then use the genomic RNA as a template for the synthesis of a genome-length, minus-sense RNA, which is subsequently used as the template for the synthesis of both the genomic and the subgenomic RNAs. Synthesis of the subgenomic RNA is initiated by internal recognition of sequences on the genome-length, minus-sense RNA template. Host-cell proteins are likely involved in the replication process and, interestingly C protein seems to be as well. RNA synthesis is asymmetric in infected cells in that more of the plus-sense species than the minus-sense RNA is produced. The uncleaved NS protein precursor is active in minus-strand RNA synthesis, while the cleaved P150/P90 complex appears to be active in only plus-strand RNA synthesis. Thus the activity of the NS protease through mediating this cleavage is important in regulating plus- and minus-sense RNA synthesis. RV RNA synthesis occurs in cytopathic vacuoles of lysosomal origin, in infected cells. In the structural protein ORF S, E2 and E1 are immediately preceded by hydrophobic signal sequences that direct translation of secreted and membrane-associated proteins into the lumen of the endoplasmic reticulum (ER). Therefore, following translation of the C sequences within the ORF, the E2 signal sequence mediates association of the translation complex with the ER. C–E2 cleavage is mediated by signal endopeptidase, which functions in the lumen of the ER to remove signal sequences from secreted and membrane-associated proteins. Unlike the proteins of the alphaviruses, RV C protein does not have autocatalytic protease activity. Following cleavage, the E2 signal sequence remains associated with C. Similarly, the E1 signal sequence maintains the association of the translational complex with the ER, signalase mediates the E2–E1 cleavage, and the E1 signal sequence remains attached to E2. Soon after synthesis, heterodimerization of E2 and E1 occurs in the lumen of the ER. The three-dimensional folding of E1 appears to be a complicated process requiring intramolecular disulfide-bond formation by all 20-Cys residues in the ectodomain of the protein. Both E1 and E2 acquire high-mannose glycans in the ER; E1 contains three potential glycosylation sites and E2 contains four and all appear to be utilized. The sites of O-glycosylation of the E2, which accounts for the size heterogeneity of E2, are not known. E1 contains an ER retention signal that is only overridden once conformational folding is complete, after which the E1–E2 heterodimer migrates to the Golgi. In the Golgi the N-glycans of both E2 and E1 are modified to complex form, although modification is not complete and the extent of modification on both proteins is heterogeneous. Modifications of the O-glycans on E2 also occur and E2 contains a Golgi retention signal, indicating that the Golgi is the preferred site of viral budding in infected cells. However, late in infection E1 and E2 migrate to the cell surface and budding can also occur on the plasma membrane in some cell lines.

800

Rubella Virus (Picornaviridae)

Fig. 3 Replication strategy of RV. The plus-sense genome and subgenomic RNA are represented by solid black arrows indicating plus polarity; beneath each of them, the ORFs that they contain are shown as open boxes. The minus-sense genome RNA complement, represented as a dotted arrow, is used solely as a template for the two plus-sense RNA species. Putative cis-acting sequences on each RNA, which are recognized by the virus RNA-dependent RNA polymerase to initiate synthesis of complementary RNAs, are marked with stars. The general functions of the virus proteins are indicated by arrows (e.g., P150 and P90 functioning as the RNA-dependent RNA polymerase by interacting with cis-acting sequences on the viral RNA species and synthesizing complementary strands).

RV capsid morphogenesis occurs in association with cell membranes. The association of RV capsid protein with membranes is probably mediated by the E2 signal sequence, which is retained at the COOH terminus of C. In fact, C may associate with the E2–E1 heterodimer and migrate as a passenger on the cytoplasmic side of vesicles transporting E2–E1 from the ER to the Golgi and among the Golgi stacks. Unlike the alphaviruses, whose capsids accumulate in infected cells, RV capsids only become visible in association with deformed, thickened membranes that appear to be in the early process of budding. A putative encapsidation signal has been localized near the 50 terminus of the genomic RNA. The C protein is phosphorylated and phosphorylation/ dephosphorylation by cell enzymes is proposed as the regulator of the process by which the genome is unpacked following entry (phosphorylation) and encapsidated later in infection following replication (dephosphorylation). Interestingly, in cells in which the complete S-ORF is expressed in the absence of genomic RNA, virus-like particles form and are secreted and these have the same morphology and isopycnic density as the virions do. RV replicates in the cytoplasm of the infected cell. None of the virus proteins exhibit any involvement with the nucleus during infection and RV infection does not appear to inhibit cell macromolecular synthesis in any grossly detectable manner; however, perturbations of specific macromolecular products and induction of specific genes occurs. Microscopically, RV-infected cells appear similar to uninfected cells; however, rearrangements of cellular cytoskeletal elements and organelles such as mitochondria have been reported. Moreover, RV infection induces a shift of the bioenergetic state of epithelial cells (Vero and A549) and human umbilical vein endothelial cells to a higher oxidative and glycolytic level. RV reportedly inhibits growth in primary human cell cultures in part due to an inhibition of mitosis; however, the virus has no reproducible effect on the growth of stable cell lines. In those cell lines which exhibit CPE (Vero and RK-13 cells), cell death is due to apoptosis, although the extent of apoptosis seems to vary according to virus strain and cell line infected.

Genetics and Evolution The RV genome is extremely stable. Currently, more than 80 genomes of independent strains of RV have been fully sequenced and all are nearly identical in terms of the size of the genome as well as the coding and noncoding regions within the genome. The only

Rubella Virus (Picornaviridae)

801

exceptions are short deletions occasionally encountered in the junction region. A unique feature of RV evolution is that changes to G and C are selected for, indicating an adaptive advantage of the high G þ C content of the genome. There are no known close relatives of RV, and the origin of the virus prior to its introduction into the human population is unknown. Except for short stretches at the 50 end of the genome and at the subgenomic promoter site, RV and the alphaviruses share no nucleotide homology. Accordingly, these two genera are only distantly related and RV has been recently regrouped to the Matonaviridae family. Phylogenetic analysis of the NS proteins indicates that RV is more closely related to hepatitis E virus than to alphaviruses and this dissimilarity is borne out by differences in the order of motifs in the NS-ORF. Thus, it is hypothesized that the evolution of the togaviruses may have been more complicated than simple divergence from a common ancestor and probably involved recombination events between progenitors of the current alphaviruses, RV, hepatitis E virus, and, possibly, certain plant viruses.

Serologic Relationships and Variability RV is monotypic and immunological characterization of diverse strains, including both clade 1 and 2 viruses, has only revealed subtle antigenic differences which map to C or E2. As might be anticipated from the lack of serologic cross-reaction with other viruses like e.g., alphaviruses, there is no homology at the amino acid level between RV and alphaviruses within the virion proteins.

Epidemiology Historically, RV was endemic worldwide. Over the past 50 years, vaccination programs have curtailed this distribution. In 2016, 22,361 rubella cases were reported to WHO, a 97% decrease from 670,894 cases reported in 2000. Before the advent of vaccination programs, rubella was considered a childhood disease in temperate zones, with seasonal peak occurrence in the spring and epidemics at 5–9-year intervals. However, in tropical zones the highest infection rates were in children under 5 years of age. RV is not as transmissible as measles virus and even during the epidemics, many susceptible individuals are spared. Thus, infection of adolescents and especially young females in childbearing age, a group of the population that should be protected against rubella infection, is not uncommon. RV elimination has been verified in the Americas in 2015 but since the vaccination coverage decreases, it remains doubtful, whether this great success can be sustained. Other parts of the world experience large rubella outbreaks due to inadequate vaccination programs, groups that do not take vaccination e.g., for religious communities or ethnic minorities which are underserved by national health programs.

Transmission and Tissue Tropism RV is transmitted between individuals by aerosol. The epithelium of the buccal mucosa provides the initial site for virus replication and the mucosa of the upper respiratory tract and nasopharyngeal lymphoid tissue serve as portals of virus entry. The virus is then spread locally by the lymphatic system, seeding regional lymph nodes where further virus replication occurs. After an incubation period of 7–9 days, virus appears in the blood. Viremia ceases with the onset of detectable rubella-specific antibody, shortly after the rash appears 2–3 weeks post infection. Patients are most infectious immediately before and during the rash phase. RV generally disappears from nasopharyngeal secretions within 4 days of the appearance of rash. Congenitally infected infants shed high loads of virus for 3–6 months following birth and are a source of transmission during that period. Reinfection with RV occurs, usually without clinical illness or virus shedding. There are very few described cases in which RV reinfection of pregnant women with welldocumented immunity has resulted in CRS as the result of a two rather rare events: the mother’s lack of immunity after vaccination and her exposure to rubella in a highly immune population during early pregnancy. During pregnancy, placental tissues are very susceptible to infection. Placental infection results in scattered foci of necrotic syncytiotrophoblast and cytotrophoblast cells and evidence of damage to vascular endothelium. Following placental infection, virus can spread to the fetus but this does not always occur and RV is more often recovered from placental tissue than from fetuses. Once fetal infection occurs, virus spreads throughout the fetus and almost any organ may be infected. Severe fetal damage is only associated with infection during the first trimester of pregnancy; the rate of CRS is 485%, 25%, and 10% when infection occurs during the first, second, and third months, respectively. This is due to a combination of an apparent decline in the efficiency of placental transfer after the first trimester and a reduction in the ability of the virus to inflict damage after this time of gestational development.

Clinical Features of Infection Rubella acquired in childhood or early adulthood is usually mild and it is estimated that up to 50% of rubella infections are clinically inapparent, making rubella surveillance for elimination programs very difficult. Symptomatic rubella encompasses

802

Rubella Virus (Picornaviridae)

maculopapular rash, lymphadenopathy, low-grade fever, conjunctivitis, sore throat, and arthralgia. The rash is the most prominent feature and appears following an incubation period of 16–20 days. The rash begins as distinct pink maculopapules on the face that then spread over the trunk and distally onto the extremities. The maculopapules coalesce and the rash fades over several days. An associated posterior cervical and suboccipital lymphadenopathy is also characteristic. Infrequent complications include thrombocytopenia and post-infectious encephalitis. Acute polyarthralgia and arthritis following natural RV infections of adults are common and occur more frequently and with a greater severity in women than in men. Joint involvement is usually transient; however, chronic arthritis, persisting or recurring over several years, has been reported. The clinical manifestations of CRS apparent at birth vary widely, most frequently including thrombocytopenic purpura (“blueberry muffin syndrome”), intrauterine growth retardation, congenital heart disease (patent ductus arteriosus or pulmonary artery or valvular stenosis), psychomotoric retardation, birth defects in the eye (cataract, glaucoma, retinopathy), hearing loss, and hepatomegaly and/or splenomegaly. Nearly 80% of CRS children show some type of neural involvement, particularly neurosensory hearing loss. Most clinical manifestations of congenital rubella syndrome are evident at or shortly following birth. Some of these defects are transient. However, recognition of retinopathy, hearing loss, and mental retardation may be delayed in some cases. Progressive consequences of congenital rubella have become increasingly visible as CRS children from the 1964 epidemic have been followed longitudinally. These predominantly involve endocrine dysfunction (diabetes mellitus, which ultimately affects 40% of CRS patients, and thyroid dysfunction). A rare, fatal neurodegenerative disease, progressive rubella panencephalitis (PRP), described in CRS patients, bears superficial resemblance to subacute sclerosing panencephalitis associated with measles virus. Subsequently, PRP cases have been reported in individuals infected postnatally.

Pathogenesis, Pathology, and Histopathology There is limited information on the pathogenesis of uncomplicated rubella because of the benign nature of the illness. With respect to the complications that can accompany acute rubella, the postinfectious encephalitis is thought to be autoimmune in nature since RV cannot be isolated from the cerebrospinal fluid or brain at autopsy. Interestingly, however, extensive inflammation and demyelination are not observed. In a few cases of rubella arthritis, the presence of RV in synovial fluid and/or cells has been demonstrated and therefore it is assumed that virus persistence is involved. However, considering the age and sex factors in the incidence of arthritis, it seems likely that immunopathological mechanisms also play a role. Human leukocyte antigen (HLA) class II haplotypes were associated with differences in predisposition of adult women to arthritis and arthralgia following rubella vaccination to experience arthritis and arthralgia and generally with responses to vaccination and viral infection. Following fetal infection, virus can be isolated from practically every organ of aborted fetuses or infants who die soon after birth. However, only 1 in 103 to 1 in 105 cells are infected and it is not known how such a low infection rate leads to the profound birth defects exhibited in CRS. RV antigen was found in interstitial fibroblasts in the heart, adventitial fibroblasts of large blood vessels, alveolar macrophages, progenitor cells of the outer granular layers of the brain, and in capillary endothelium and basal plate in the placenta. Affected organs are usually small for gestational age and contain reduced numbers of cells. Considering the inhibitory effect of RV on primary cells, it is thought that virus infection early in organogenesis inhibits cell division, leading to both retardation and alteration in organ development. Moreover, RV infection has shown virus persistence continuing after birth, as evidenced by virus shedding, which generally ceases within 6 months of age. Whether virus persistence continues beyond cessation of shedding and plays a role in the delayed and progressive manifestations of CRS is not known. Transcriptome analysis have shown genes involved in cytokine production and cytokine regulation were differentially regulated in wild type RV and vaccine virus-infected cells. Increased synthesis of inflammatory cytokines following RV infection was verified as well as down-regulation of chemokine CCL14, which is implicated in supporting embryo implantation at the fetal-maternal interface. GO term-based cluster analysis of the down-regulated genes of HUVEC revealed an enrichment of the GO terms “sensory organ development”, “ear development” and “eye development”. When human induced pluripotent stem cells (iPSCs) were infected with RV followed by differentiation into cells of the three embryonic lineages (ecto-, meso-, and endoderm) as a cell culture model for blastocyst- and gastrulation-like stages, the RV-infected endodermal cells showed profound alterations of the epigenetic landscape including the expression level of components of the chromatin remodeling complexes and an induction of type III interferons. Histologically, affected organs of CRS patients show a limited number of well-recognized malformations, with noninflammatory histopathology predominating. Particularly apparent are vascular lesions and focal destruction in tissues bordering these lesions. These lesions are likely to be due to virus replication in the vascular endothelium and damage to neighboring tissue may play a role in the pathogenesis of CRS. The neuropathology of CRS is of interest not only because of the defects manifest shortly after birth, but also because some CRS patients develop schizophrenia-like symptoms later in life. CRS brains are generally free of gross morphological malformations, with a common tendency toward microcephaly. Vascular damage, leptomeningitis, decreased number of oligodendroglial cells, and alteration of white matter are observed. Magnetic resonance imaging of a group of CRS adults with schizophrenia-like symptoms revealed specifically reduced cortical gray matter and enlargement of the ventricles, which were not previously observed characteristics of CRS-induced neuropathology. Interestingly, the comparative finding that non-CRS schizophrenia patients exhibit a pattern of brain dysmorphosis similar to that found in CRS patients with schizophrenia-like symptoms supports the hypothesis that schizophrenia is developmental in nature (there is some evidence for a viral trigger to schizophrenia).

Rubella Virus (Picornaviridae)

803

Immune Response and Serodiagnosis Following an acute infection, anti-RV IgM antibodies are detectable a few days after rash onset and for four to six weeks afterwards, so that detection of IgM using ELISA is important for the diagnosis of an acute RV infection; in the succeeding weeks anti-RV antibodies in all immunoglobulin classes appear. The dominant early and persistent IgG response is in the IgG1 subclass and antibodies of this class persist indefinitely after natural infection of otherwise healthy individuals. The majority of the antibody response is directed to the E1 glycoprotein, with proportionally lesser amounts of the response directed against E2 or C; neutralizing and complement-fixing antibodies are induced as well. When investigating relationships between rubella vaccine-specific humoral and cellular immunity, a significant correlation between neutralizing antibodies and IFN-g secretion was observed. Because of the importance of serology testing for rubella, many rubella tests are on the market but standardization between the tests is missing. This leads often to an apparent change in titer values (and the respective concern) but the phenomenon may be based solely on the use of two discordant ELISA tests. RV-specific cellular immune responses are measurable within 1–2 weeks of onset of illness. Major histocompatibility complex (MHC)-restricted CD4 þ epitopes have been mapped to all three of the virus structural proteins; however, CD8 þ epitopes have thus far only been mapped to the C protein. HLA class I and II alleles both positively and negatively associated with antibody and lymphoproliferative responses following rubella vaccination have been determined. Following fetal infection, the fetus produces IgM antibodies, detectable at 18–20 weeks of gestation, and maternal IgG antibodies crosses the placenta. Both types of antibodies exhibit virus-neutralizing activity in vitro; however, these antibodies are not sufficient to resolve virus infection during gestation. As discussed above, the intracellular maturation of virus probably shields it from antibody recognition. After birth, the presence of IgM or a lack of decline of IgG antibodies are both considered indicative of a fetal infection as well as the shedding of virus that can be detected be RT-PCR. CRS infants exhibit various degrees of impairment in the cellular immune response to RV and it is thus a deficiency in this arm of the immune response that allows the virus to persist. Considering the cellular immune deficiency in CRS infants, it is curious that detectable virus persistence ends a few months after birth. The way by which virus persistence is cleared under these conditions is not understood.

Diagnosis The common symptoms of acute rubella, maculopapular rash, low-grade fever, and lymphadenopathy are nonspecific and easily confused with other illnesses like e.g., measles, scarlatina, or infection with parvovirus B19. In compliance to WHO elimination programs every suspected rubella case should be confirmed by a laboratory diagnosis. Laboratory confirmation of a suspected case of rubella requires PCR detection of the genome in a throat swab taken within 5 days after onset of symptoms and the detection of IgM antibodies in an acute-phase serum specimen. The diagnosis of acute rubella is of utmost importance when an infection during early pregnancy is suspected. In these cases, the detection of IgM alone is not a reliable basis to decide whether an RV infection has occurred in early pregnancy; a specialized laboratory should be consulted for verification. Laboratory diagnosis will usually employ IgG avidity, and detection of the E2-reactive IgG antibodies in a Western blot to exclude an earlier infection. The risk of congenital infection is related to the gestational age at the time of maternal infection. When pregnant women are infected with rubella during the first 11 weeks of pregnancy, up to 90% of liveborn infants may suffer from CRS. There is no other intervention for congenital rubella than abortion. Diagnosis of CRS in a newborn is initially made on the basis of symptoms and confirmed in the laboratory by serological testing for the presence of IgM antibodies, by lack of decrease of IgG antibodies in serum specimens, and by detection of virus RNA. CRS infants shed high amounts of virus and therefore are a source of contagion. Comparison of serologic markers revealed that compared with healthy children, children with CRS have significantly higher RVspecific IgG and stronger C and E2 protein-specific antibody responses. ELISA-based detection of IgG as a surrogate marker for immunity by occupational physicists is routine practice as well as for pre-pregnancy planning. IgG detection can also be used in lieu of proof of vaccination, which is required in many countries for the enrollment in school or university. Individuals found to be seronegative should subsequently be vaccinated. In case of women who are pregnant at the time of testing, vaccination should be offered directly post-partum.

Prevention and Control of Rubella There is no specific treatment of RV infection, but since the infection is vaccine-preventable and humans are the only host, it is possible to eliminate rubella. Several live attenuated vaccines were developed and placed in use by 1970; the vaccine RA 27/3 is used in most countries. Most national immunization programs recommend two doses of the MMR or MMRV vaccine during childhood. If not already covered by a routine vaccination in childhood, women in reproductive age and health care workers should be protected against rubella. Attenuated RV vaccines cause subclinical infection with transient viremia in susceptible patients. Natural transmission of vaccine virus has not been reported. The RA27/3 vaccine strain produces seroconversion in greater than 95% of recipients. Vaccineinduced titers are lower than those induced by a natural infection but a life-long immunity is mounted in most cases. Reinfections can occur but in contrast to primary infections, they do normally not endanger the unborn child. The RV vaccine is generally

804

Rubella Virus (Picornaviridae)

administered to children in a trivalent form with the measles- and mumps-attenuated vaccines (MMR) or as a tetravalent vaccine with varicella virus vaccine (MMRV). The RV vaccines have been most successful in terms of induction of immunity with the absence of side effects. However, two issues have arisen concerning rubella vaccination. The first is that the vaccine virus can cross the placenta and infect the fetus. However, registries of deliveries to women inadvertently vaccinated during early pregnancy revealed no reported congenital abnormalities. Similar findings were reported in subsequent studies conducted in other countries in conjunction with mass vaccination campaigns. Nevertheless, due to a theoretical risk vaccination during pregnancy is contraindicated and deferred until postpartum. Second, is the occurrence of arthralgia and arthritis following vaccination. Joint complications are nonexistent in children with the currently used rubella vaccines; however, transient arthralgia and arthritis is reasonably common among adult female vaccinees (or vaccine recipients). There have also been reports of chronic arthritis and related neurological involvement following vaccination of adult women. Although these complications are consistent with complications that can accompany natural rubella infection in adult females, recent studies have shown that the incidence of such vaccine-related complications is low and cannot be statistically differentiated from the incidence of similar symptoms in control, unvaccinated populations. Since the inception of rubella vaccination in 1969 in the US, many countries have implemented rubella vaccination for children, health care workers and individuals working in community facilities as well as women in childbearing age. The cost of the vaccine, the general mildness of the disease compared to other infections like tuberculosis, malaria and HIV, and the nature of national public health infrastructures are factors impeding vaccination programs in developing countries. However, efforts among developing countries have accelerated, particularly in conjunction with ongoing measles elimination programs, and as of December 2016, 152 (78%) of 194 countries had introduced RV vaccine into the national immunization schedule. In addition to its efforts to initiate rubella vaccination programs in developing regions, the WHO established a global Measles and Rubella Laboratory Network that is indispensable in surveying the way to elimination. The WHO Regions of the Americas have eliminated rubella and CRS in 2010 and verified this success in 2015, while other regions like e.g., Europe and the Western Pacific have set goals for elimination of rubella and CRS by 2020.

Future RV is a viral pathogen, that infects only humans. Due to the presence of a safe and efficient vaccine, global implementation of rubella vaccination in accordance with the WHO elimination goal is of vital importance to eliminate rubella and prevent CRS in the future. Moreover, RV and the attenuated vaccine are a fascinating model to unravel teratogenic effects and compare it with other agents causing congenital diseases like e.g., Zika virus, cytomegalovirus or toxoplasmosis. The follow-up of CRS patients can further teach us of a very special condition, e.g., the high incidence of diabetes in CRS patients which is statistically the strongest direct association between a specific human virus and an autoimmune disease. The mechanism of viral involvement, if any, in each of these diseases is not fully understood. Unfortunately, our present understanding of the disease mechanisms in the RV-related syndromes is hindered by the lack of a suitable animal model system that fully mimics the infection seen in humans, making the development of an animal model a research priority. RV is taxonomically unique and appears to have evolved as a recombinational hybrid of distantly related ancestor viruses. Thus, investigation of the molecular biology of RV likely will reveal novel replication strategies and yield insight into virus evolution.

Further Reading Best, J.M., Castillo-Solorzano, C., Spika, J.S., et al., 2005. Reducing the global burden of congenital rubella syndrome: Report of the World Health Organization steering committee on research related to measles and rubella vaccines and vaccination, June 2004. International Journal of Infectious Diseases 192, 1890–1897. Bilz, N.C., Willscher, E., Binder, H., et al., 2019. Teratogenic rubella virus alters the endodermal differentiation capacity of human induced pluripotent stem cells. Cells 8 (8), 870. Dimech, W., Grangeot-Keros, L., Vauloup-Fellous, C., 2016. Standardization of assays that detect anti-rubella virus IgG antibodies. Clinical Microbiology Reviews 29 (1), 163–174. Frey, T.K., 1994. Molecular biology of RV. Advances in Virus Research 44, 69–160. Geyer, H., Bauer, M., Neumann, J., et al., 2016. Gene expression profiling of rubella virus infected primary endothelial cells of fetal and adult origin. Virology Journal 13, 21. Grant, G.B., Desai, S., Dumolard, L., Kretsinger, K., Reef, S.E., 2019. Progress toward rubella and congenital rubella syndrome control and elimination – Worldwide, 2000–2018. Morbidity and Mortality Weekly Report 68 (39), 855–859. Hobman, T.C., 2013. Rubella virus. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, sixth ed. Philadelphia: Lippincott Williams and Wilkins, pp. 1069–1100. Hyde, T.B., Sato, H.K., Hao, L.J., et al., 2015. Identification of serologic markers for school-aged children with congenital rubella syndrome. The Journal of Infectious Diseases 212 (1), 57–66. Lambert, N., Strebel, P., Orenstein, W., Icenogle, J., Poland, G.A., 2015 6. Rubella. Lancet 385 (9984), 2297–2307. Lazar, M., Perelygina, L., Martines, R., et al., 2016. Immunolocalization and distribution of rubella antigen in fatal congenital rubella syndrome. EBioMedicine. 3, 86–92. Ovsyannikova, I.G., Salk, H.M., Larrabee, B.R., Pankratz, V.S., Poland, G.A., 2015. Single nucleotide polymorphisms/haplotypes associated with multiple rubella-specific immune response outcomes post-MMR immunization in healthy children. Immunogenetics 67 (10), 547–561. Pitts, S.I., Wallace, G.S., Montana, B., et al., 2014. Congenital rubella syndrome in child of woman without known risk factors, New Jersey, USA. Emerging Infectious Diseases 20 (2), 307–309. Tzeng, W.P., Matthews, J.D., Frey, T.K., 2006. Analysis of RV capsid protein-mediated enhancement of replicon replication and mutant rescue. Journal of Virology 80, 3966–3974. Vynnycky, E., Adams, E.J., Cutts, F.T., et al., 2016. Using seroprevalence and immunisation coverage data to estimate the global burden of congenital rubella syndrome, 1996–2010: A systematic review. PLoS One 11 (3).

Saint Louis Encephalitis Virus (Flaviviridae) William K Reisen, Lark L Coffey, and Daniele M Swetnam, University of California, Davis, CA, United States Aaron C Brault, Centers for Disease Control and Prevention, Fort Collins, CO, United States r 2021 Elsevier Ltd. All rights reserved.

Flaviviridae Viral family Flavivirus Viral genus

Nomenclature Culex

Mosquito genus

Glossary Bridge vector Arthropod capable of moving an infectious agent from an enzootic cycle to hosts typically not involved in the enzootic cycle. Diapause Period of hormonally induced reproductive inactivity for insects typically triggered by shortening day length and cool temperatures (akin to hibernation of mammals). Extrinsic incubation period Time period within the arthropod vector between the ingestion of an infectious agent and when the vector is capable of transmitting following biological amplification.

Gonotrophic cycle The period between blood feeding and the oviposition of eggs by an insect. Vector competence The capacity of an arthropod to become infected with an infectious agent (typically through oral ingestion), disseminate the agent to other tissues and subsequently transmit through blood feeding, typically through salivary secretions. Viremia Presence of virus in the peripheral circulation of a vertebrate host.

History Saint Louis encephalitis virus (SLEV) probably has been present in the New World within enzootic cycles for thousands of years, although recent molecular genetic studies indicate the current lineages may have diverged as recently as 200 years ago from a common flaviviral ancestor within present day Mexico. The arrival of European settlers in the 1600s and the extensive agricultural development that followed greatly altered the landscape by clearing and irrigating vast areas of North America and establishing extensive urban centers. These changes likely increased the abundance of Culex mosquito species and avian hosts such as house finches (Haemorhous mexicanus) and mourning doves (Zenaida macroura). Furthermore, the introduction of new avian hosts such as house sparrows (Passer domesticus) and vectors such as Culex pipiens complex mosquitoes, intensified human-vector mosquito contact, and likely increased the incidence of human infection; moreover, clinical diagnosis of disease caused by SLEV assuredly had been confused with other infections causing fever and/or central nervous system (CNS) disease during summer months. During the summer of 1933, a major encephalitis epidemic with more than 1,000 clinical cases occurred in St. Louis, Missouri. These cases occurred during the middle of an exceptionally hot, dry summer and were concentrated within areas of the city adjacent to open storm water and sewage channels that produced a large number of Culex mosquitoes. A virus, later named “Saint Louis encephalitis virus” (SLEV), was isolated from a human brain specimen during an autopsy during this outbreak. The epidemiological features of this epidemic included the late summer occurrence of cases (especially in persons 450 years of age), exceptionally warm temperatures during the summer months preceded by a wet spring, and elevated Culex mosquito abundance associated with poorly draining waste water systems. These features have remained the hallmarks of SLEV epidemics through the 21st century. A multidisciplinary team of entomologists, vertebrate ecologists, epidemiologists and microbiologists from the University of California at Berkeley subsequently investigated an SLEV epidemic in the Yakima Valley of Washington State 1941–1942 and established the components of the summer transmission cycle, including the identification of wild birds as the primary vertebrate hosts and Culex mosquitoes as principal vectors. The isolations of SLEV from Culex tarsalis and Cx. pipiens mosquitoes were among the first of any virus from mosquitoes and stimulated the redirection of mosquito control in North America from Anopheles malaria vectors and pestiferous Aedes to Culex vectors. A more thorough understanding the basic transmission cycle, an appreciation of the wide range of clinical symptoms, and the development of laboratory diagnostic procedures subsequently has provided an expanded view of the public health significance of SLEV, with epidemics or clusters of cases recognized annually throughout the United States. Wide geographic distribution and transmission since 1933 has resulted in 41000 deaths, 410,000 cases of severe illness and an estimated 41,000,000 mild or subclinical infections. The largest documented SLEV epidemic occurred during 1975 in the Ohio River drainage, with 42000 human cases documented. Other substantial human epidemics, involving up to hundreds of cases, have occurred in Missouri (1933, 1937), Texas (1954, 1956, 1964, 1966), Mississippi (1975), Florida (1977, 1990) and Louisiana (2001). Smaller outbreaks have been recognized in California (1952, 1989), New Jersey (1962), Arizona (2015) and several other states plus Ontario (1975), Canada, and Cordoba, Argentina (2005) (Fig. 1). Cases reported annually to the Centers for Disease Control and Prevention

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00002-3

805

806

Saint Louis Encephalitis Virus (Flaviviridae)

Fig. 1 Geographic distribution of historical Saint Louis encephalitis human cases reported in the Americas through November 2017. Dot size represents the number of human cases reported in each episode. Colors represent year of detection. Reproduced with permission from Diaz, A., Coffey, L.L., Burkett-Cadena, N., Day, J.F., 2018. Reemergence of St. Louis encephalitis virus in the Americas. Emerging Infectious Diseases 24 (12).

(CDC) from 1964 to 2018 are shown in Fig. 2. Geographical distribution of cases can be seen throughout the continental United States with recurrent disease transmission centers existing along the Ohio River basin, Florida, Texas and California (Fig. 3).

Virion Structure/Genome SLEV virions are approximately 50 nanometers in diameter and are comprised of a lipid envelope in which viral structural proteins, (pre)membrane (prM/M) and envelope (E) glycoproteins, are embedded. The structural proteins associate as heterodimers within the lipid envelope with T ¼ 3 symmetry. The lipid envelope scaffolds an internal icosahedral capsid (C) protein constellation encompassing a single-stranded (monopartite), positive-sense viral genome of approximately 11 kb in length. The RNA genome is flanked by 50 and 30 untranslated regions (Fig. 4). A 50 m7GpppAmpN2 cap is present that protects the virus from endoribonuclease degradation. No polyadenylation is present at the 30 end of the genome; however, a non-coding sequence of variable length (B300 nucleotides) is present and single-stranded RNA structures are formed that block 50  30 endoribonucleases degradation forming subgenomic RNAs that are believed to serve as decoys for siRNA antiviral pathways in both vertebrate and

Saint Louis Encephalitis Virus (Flaviviridae)

807

Fig. 2 Number of human Saint Louis encephalitis virus cases reported to the Centers for Disease Control and Prevention ArboNet system from 1964 to 2018 by year.

Fig. 3 Map of United States indicating the cumulative number of Saint Louis encephalitis virus cases per state between 1964 and 2018 (data from ArboNet).

invertebrate hosts. The 50 and 30 ends of the genome have conserved cyclization sequences that are involved in transcriptional and translational initiation. The coding region of the virus is expressed as one long translated polyprotein that is cleaved by host and viral proteases. The three structural proteins (C, prM and E) are coded at the 50 end of the genomes and the seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) are found in the 30 portion of the genome (Fig. 4). The nonstructural proteins principally serve to replicate viral RNA through RNA-dependent RNA polymerase (NS5) and helicase (NS3), process

808

Saint Louis Encephalitis Virus (Flaviviridae)

Fig. 4 Depiction of the genomic structure of the Saint Louis encephalitis virus. The 11 kb RNA genome is flanked by 50 and 30 untranslated sequences. The 50 structural proteins are delineated as w ell as the 30 non-structural protein genes.

translated viral proteins with a protease (NS2B/NS3) and cap viral RNA with methyltransferase (NS5). Nonstructural proteins are also involved in the antagonization of host-induced antiviral responses.

Classification/Genetic Characterization Saint Louis encephalitis virus (SLEV) is a mosquito-borne flavivirus within the family Flaviviridae. Serologically, SLEV has been classified as a member of the Japanese encephalitis serocomplex with the most closely related viruses (West Nile, Japanese encephalitis, Usutu and Murray Valley encephalitis viruses). Subsequent to its initial isolation from a brain sample during the human encephalitis outbreak in St. Louis, Missouri in 1933, serological assays, which included mouse protection assessments with viral preparations incubated with convalescent serum dilutions, were performed that demonstrated that the isolated virus was antigenically distinctive from other viruses typically associated with human encephalitis cases during summer months, such as the equine encephalitis viruses, poliomyelitis and vesicular stomatitis viruses. Marked differences in the severity of SLEV epidemics stimulated interest in possible differences among isolates made over time and space. Detailed studies during the 1980s clearly demonstrated geographic variation among 43 different SLEV isolates using oligonucleotide fingerprinting and virulence in model vertebrate hosts. These strains were grouped into 6 clusters: (1) east central and Atlantic USA, (2) Florida epidemic, (3) Florida enzootic, (4) eastern USA, (5) Central and South America with mixed virulence, and (6) South America with low virulence. Changes in virulence were attributed, in part, to differences in mosquito vector competence and were supported by the historical presence or absence of human cases. Subsequent sequencing studies extended the understanding of SLEV genetics and provided further insight into patterns of geographical variation. Sequences of the envelope gene from SLEV strains isolated in California from 1952 through 1995 varied temporally and spatially, but indicated regional persistence in the Central Valley for at least 25 years as well as sporadic introduction and extinction. Studies in Texas using a single-strand conformation polymorphism technique showed that multiple SLEV strains circulate concurrently and remain highly focal, whereas other strains amplify and disseminate aggressively during some summers, but then disappear. Further analysis of sequences from 62 isolates made throughout the known geographical range of SLEV indicated that there have been 7 lineages that overlapped somewhat with the 6 groups previously defined using oligonucleotide fingerprinting: (I) western USA, (II) central and eastern USA and 3 isolates from Mexico and Central America, (III) one mosquito isolate from Argentina, (IV) 5 isolates from Panama mosquitoes, (V) South American strains plus an isolate from Trinidad, (VI) one Panama isolate from a chicken, and (VII) two isolates from Argentinian rodents. Subsequent re-analysis of 73 envelope gene sequences from GenBank basically supported these groupings, but had low support for separation of the Panama isolates. This analysis also indicated limited exchange between North and South American clades, especially California, but indicated repeated circulation among the Caribbean Islands, Mexico and the southern USA. Collectively, these data indicated that SLEV strains vary markedly in virulence and that the frequency and intensity of epidemics in the USA may be related to genetic selection by different host systems. Interestingly, transmission within the Neotropics appears to have given rise and/or allowed the persistence of less virulent strains that rarely amplify to produce epidemic level transmission, a scenario duplicated by West Nile virus in the Americas. Several changes have been proposed since the initial designation, including joining lineages IV and VI, and the addition of lineage VIII and the Palenque lineage (Fig. 5). Lineage I is geographically restricted to the western US, comprises genomes from 1950 to 1998 and can be divided into Clades IA and IB. Clade IA contains only genomes collected from Kern County, California between 1950 and 1970. Sequences belonging to Clade IB have been detected in the greater western US, including California from 1978 to 98, as well as New Mexico and Colorado in 1972, and Texas from 1966 to 1989 (Hale County, El Paso, and Dallas). Whereas lineage I genomes have only been detected in a small geographic area, lineage II is more widely distributed throughout North, South and Central America. Kramer and Chandler (2001) initially divided lineage II into six clades (IIA-IIF). Lineage I clade IIA consists of genomes from Jamaica in 1962, Brazil in 1968, and the central and eastern US from 1933 to 1996, including Florida, Kentucky, Mississippi, Missouri and Texas. Lineage II clade IIB contains sequences from Central America, specifically Guatemala in 1969 and 1978, and several genomes from the southeast portion of the US, including, Florida in 1962, Tennessee in 1974 and the Gulf Coast region of Texas (Harris, Jefferson and Nueces Counties) between 1983 and 2003. The sequences of lineage I clade IIC, although restricted to the US, are split between the eastern (Tennessee, Florida and Maryland) and western (California) states and were from genomes detected between 1975–1977 and 1988–1992, respectively. Genomes belonging to lineage I clade IID were from 1962 to 1973 from Florida, Mexico and Panama. In its initial description by Kramer and Chandler (2001), lineage I clade IIE contained the two earliest SLEV sequences, Parton and Hubbard, from Missouri in 1933 and 1937, respectively. Later studies indicated that a third genome from the US (state unknown) during 1966, Laderle, also belonged to this

Saint Louis Encephalitis Virus (Flaviviridae)

809

Fig. 5 Phylogenetic tree based on the envelope coding sequences of Saint Louis encephalitis viruses indicating the relationship of different geographic lineages (designated by roman numerals). Isolate locations are color-coded according to the legend.

clade. Finally, clade IIF was defined using one genome from Guatemala collected in 1969. However, several studies found evidence of sequencing inconsistencies in the Parton genome that altered the phylogenetic clustering pattern of clade IIE so that the three genomes clustered within clade IIA. Similarly, a revised Guatemala 1969 genome clustered within clade IIB. Finally, one additional clade, clade IIG, was identified in 2008 and contains only a single genome collected in Florida during 1962. Both lineages I and II have been associated with epidemics in North America. Lineage III was responsible for epidemic disease in Argentina and more recently, in the western US, and also was detected in Brazil during 2004. Like lineages II and III, lineage V also has been detected throughout the Americas. In a pattern very similar to lineage III, lineage V historically was restricted to South America (Argentina in 1978, Brazil from 1960 to 1973, Peru in 1975, Trinidad in 1955) and subsequently expanded into the western US, including western Texas (El Paso) in 2002 and California between 1998 and 2003. The remaining lineages: IV, VI, VII, VIII and Palenque, are all geographically restricted. Lineage IV originally was defined by a single sequence collected in Panama during 1983; however, support for the separation of lineages IV and VI was very weak leading investigators to suggest combining the two lineages. Lineage VII contains two genomes from Argentina during 1966 and 1967 and the distantly related Palenque lineage that has only been detected in Palenque National Park in southern Mexico. Finally, lineage VIII appears to be restricted to the Para state in the northern Amazon region in Brazil and was detected between 1964 and 1984. Taken together, several trends have been reported for SLEV across multiple lineages. Overall, there appears to be a consistent pattern of northward SLEV dispersal. In particular, the Gulf of Mexico region (bounded by latitudes 151N and 301N) appears to act as a major source for SLEV expanding into North America. South America, specifically Brazil and Argentina, also appears to be a source of SLEV moving into California (lineages III and V). A degree of isolation separating the eastern and western US has been reported in several lineages. For instance, lineage IIB in the Gulf Coast region of Texas clusters more closely with SLEV collected in the Eastern or Central US, whereas lineages IB, III and V from west and north Texas are related more closely to SLEV in California. It is important to note that our current understanding of SLEV lineages is restricted by limited and inconsistent virus surveillance and sequencing. Additional SLEV sequences, especially from new locations, will likely refine our understanding of endemic SLEV circulation and dispersal events. Furthermore, while multiple studies have described the geographic distribution of each SLEV lineage, only one has expanded on their specific nucleotide or amino acid differences. That study defined 17 amino acid changes within the E protein associated with different lineages or clades. However, several of the amino acid changes were shared across multiple lineages and no defining amino acid changes were described for clade IID. Additional studies are needed to identify the genotypic differences among the SLEV lineages, especially outside of the E protein.

Life Cycle St. Louis encephalitis virus is transmitted in enzootic transmission cycles between viremic avian hosts and Culex spp. mosquito vectors. Birds develop viremias of sufficient magnitude to infect mosquitoes with virus following bloodfeeding. Experimental data also suggests that nestlings develop elevated viremias and could be instrumental for seasonal enzootic amplification of the virus.

810

Saint Louis Encephalitis Virus (Flaviviridae)

Although humans can show symptoms of viral infection, they serve as tangential or “dead end” hosts, not manifesting viremias of sufficient magnitude to infect additional mosquito vectors. Once exposed to virus through the ingestion of a viremic blood meal, Culex vectors' midguts are infected and the virus disseminates through the open circulatory system (hemocoel) of the mosquito, infecting the salivary glands. Subsequent feeding by the mosquitoes and its associated salivation inject viral particles into another avian host. The time period in which the virus traverses from the midgut to the salivary glands is defined as the extrinsic incubation period, the duration of which is directly related to temperature.

Host Range Although a wide variety of mosquitoes occasionally have been found infected in nature, three avian-feeding species within the genus Culex appear to be the most frequently infected and important arthropod hosts: Culex pipiens (including the subspecies Cx. p. quinquefasciatus at southern latitudes, Cx. p. pipiens at northern latitudes, and intergrades) in urban and periurban environments throughout North and South America, Culex tarsalis in irrigated agricultural settings in western North America including northern Mexico, and Culex nigripalpus in the southeastern U.S., the Caribbean and parts of the Neotropics. Although these species feed predominantly on birds, they also feed on mammals including humans, and therefore function as both maintenance and bridge vectors. Other Culex species such as Culex stigmatosoma in the west, Culex restuans and Culex salinarius in the east, and perhaps species in the subgenus Melanoconion in the Neotropics also may be important in local transmission. Ticks have been found naturally infected, but their role in virus epidemiology most likely is minimal. The importance of avian host species appears to be related to vector Culex host selection patterns as well as to avian susceptibility to the virus. Species can be separated into those frequently, sporadically and never found infected in nature, and these groupings are related directly to their nocturnal roosting/nesting behavior and the questing behavior of Culex vectors. Wild birds do not develop apparent illness following experimental infection, but their viremia response varies markedly, depending upon virus strain, bird species and bird age. Viremia titers sufficient to infect mosquitoes typically are limited to 1–5 days post-infection. Based on serological surveys during or after epidemics, peridomestic passeriforms, including house finches, house sparrows, cardinals (Cardinalis cardinalis) and blue jays (Cyanocitta cristata), columbiforms, including mourning doves and rock doves (Columba livia) or domestic pigeons (Columba livia domestica), and galliforms, especially domestic chickens (Gallus gallus), seem to be infected most frequently. SLEV strains isolated from Cx. pipiens complex mosquitoes from the central and eastern U.S. produced elevated viremias in house sparrows, whereas strains isolated from Cx. tarsalis from the western U.S. were weakly viremogenic. Although host competence studies have been limited, the adults of few bird species seem to develop elevated viremias. However, nestling house finches, house sparrows and mourning doves produce high viremias that readily infect mosquitoes. Therefore, the nesting periods of multibrooded species may be critical for virus amplification. Regardless of their viremia response, most experimentally infected birds produce antibody and, although titers typically decay rapidly, these birds remain protected from reinfection for life. Although frequently antibody positive during serosurveys, SLEV infection does not produce elevated viremias or cause clinical illness in domestic animals, including equines, porcines, bovines or felines. Similar to wild birds, immature fowl o1 month old (including chickens and ducks) consistently develop sufficient viremia to infect mosquitoes, but do not develop clinical illness. Adult chickens (422 wks old) usually fail to develop a detectable viremia, and along with immature birds, develop long-lasting antibodies. The response of wild mammals to natural or experimental infection varies. Serosurveys occasionally have shown higher SLEV prevalence in mammals than in birds, but these data could be confounded because mammalian hosts typically live longer than avian hosts and therefore have a longer history of exposure. Overall, the role of mammalian infection in SLEV epidemiology is complex and difficult to interpret. All reputed Culex vectors feed most frequently on avian hosts, occasionally on large mammals and lagomorphs, rarely on rodents, and almost never on bats.

Epidemiology Human SLEV disease cases typically peak in late summer/early fall and have historically occurred during periods of warm and dry conditions that serve for the optimal development of Culex vectors. Furthermore, hot conditions shorten the extrinsic incubation period due to the faster rate of viral replication and reduce the duration of the gonotrophic cycle, increasing the rate of blood feeding and host contact. Transmission of SLEV is complex and requires that the virus replicate in and avoid the immune responses of alternating insect and vertebrate hosts under temperatures ranging from those found in diapausing mosquitoes to more than 431C in febrile avian hosts. Annual transmission activity may be divided into overwintering, vernal and/or summer amplification, and autumnal subsidence periods. Three possible mechanisms may explain the persistence of SLEV at temperate latitudes; however, few supportive field data are available. First, low level vertical passage of SLEV from infected females to their progeny has been demonstrated repeatedly in laboratory experiments. Although not detected for SLEV in nature, vertical transmission has been documented for other viruses in the Japanese encephalitis (JEV) complex, including JEV and West Nile virus (WNV). Second, Cx. p. pipiens females destined for diapause have been shown to take small blood meals during late summer and early fall without ovarian development. Two isolations of SLEV made from diapausing Cx. p. pipiens females collected resting during winter in Maryland were considered to have been infected by

Saint Louis Encephalitis Virus (Flaviviridae)

811

this mechanism, although infection by vertical transmission also was possible. Third, Culex p. quinquefasciatus and Cx. nigripalpus do not enter reproductive diapause, remain reproductively active or quiescent during winter at southern latitudes and, depending upon ambient temperature, could maintain SLEV by continued, infrequent transmission among resident birds. Experimentally infected, reproductively active Cx. p. quinquefasciatus females have been shown to survive winter as gravid females and to then transmit SLEV to recipient birds throughout the following spring. SLEV also may persist over-winter within vertebrate host populations. Passeriform birds infrequently develop chronic infections that persist as long as a year following experimental infection. However, attempts to demonstrate natural relapse or to trigger recrudescence experimentally have not been successful. Flaviviruses, including SLEV, also have been isolated repeatedly from bats, and experimental infections in bats destined for hibernation have been maintained for 20 days at 101C. When returned to room temperature, SLEV was detected in the brown fat and at low levels in the blood. These data indicated that bats could function as an over-wintering host. However, studies of mosquito host selection patterns indicated that bats rarely, if ever, are fed upon by host-seeking mosquitoes, raising the unanswered question of why so many field-collected bats have neutralizing antibodies. An alternative hypothesis to local persistence involves annual or periodic re-introduction of virus into northern latitudes from southern refugia. Long-distance movement of SLEV has been indicated indirectly from genetic evidence as well as by the re-appearance of SLEV after years of absence. For instance, genotype III SLEV that was identified in California and Arizona in 2015 is most closely related genetically to genotype III SLEV associated with the 2005 Cordoba, Argentina outbreak, indicating long-distance introduction of this virus to North America. Two possible hypotheses address re-introduction, but neither are well supported by field evidence. Many species of birds and some bats have long distance annual migrations that could allow the transport of virus from foci active during winter in southern latitudes or south of the equator to receptive areas north of the equator during spring. These vertebrate migrations typically are very consistent in their summer and winter destinations, and this would allow the same or similar genetic strains to re-appear each summer at the same locality. However, molecular genetic studies of North, Central and South American isolates indicate that they are relatively distinct, thereby implying infrequent genetic exchange. In addition, north-bound migratory birds do not seem to be frequently involved in transmission because they infrequently are found positive for virus or antibody. Regardless of the persistence mechanism, summer enzootic amplification transmission in North America involves Culex mosquitoes and primarily birds in the orders Passeriformes and Columbiformes. Humans become infected tangentially to the primary cycle, do not develop viremias sufficient to infect mosquitoes, and are considered to be “dead end” hosts. Transmission appears to be initiated after Culex spp. vectors resume blood-feeding and reproductive activity, and ambient temperatures warm sufficiently to allow the replication of virus in the mosquito vector. Infection is acquired when a female Culex mosquito blood feeds on a viremic avian host. Virus imbibed within infectious blood meals taken in early in spring when ambient temperatures average o151C may lay dormant until warm conditions or changes in mosquito physiology stimulate replication. Under warm temperatures, virus replicates rapidly, disseminates within the mosquito during the ensuing extrinsic incubation period, and then may be transmitted by bite after the female oviposits and attempts to imbibe a subsequent blood meal. The duration of the extrinsic incubation period is temperature-dependent and requires 410 days and perhaps 2 mosquito gonotrophic cycles when temperatures average 221C. In contrast, the viremia response in susceptible avian hosts typically is of short duration, lasting 2–4 days. Four distinct transmission cycles of SLEV are defined by differences in the biology of the primary vector mosquito species and their distribution, and include: (1) rural North America, west of the Mississippi River transmitted by Cx. tarsalis, Cx quinquefasciatus in southern California and Cx. pipiens in Washington; (2) rural and urban central and eastern North America transmitted by members of the Cx. pipiens complex; (3) Florida, Caribbean and parts of Central America transmitted by Cx. nigripalpus; and (4) urban and rural South America transmitted by Cx. pipiens complex and mosquitoes of other taxa. Intensity of enzootic transmission and occurrence of new human cases always subsides rapidly during autumn. Cool evening temperatures slow the replication of SLEV within infected mosquito hosts, decreasing the efficiency of transmission and, concurrently, the combination of cool water temperature and shortening days during larval development initiates reproductive diapause (Cx. tarsalis, Cx. p. pipiens) or quiescence (Cx. p. quinquefasciatus, Cx. nigripalpus) in vector females emerging during fall. The fall mosquito population declines in abundance and divides into newly emerged females that do not routinely blood feed and survive the winter, and remnants of the summer population that continue reproductive activity, but usually fail to survive winter. The critical day length that triggers the onset of diapause in Cx. p. pipiens may occur in late summer at northern latitudes, markedly shortening the SLEV transmission season. During warm days, however, females may become infected when taking partial blood meals from viremic birds, survive winter and then transmit the virus after diapause is terminated by warm spring temperatures. Cx. p. quinquefasciatus does not undergo diapause, so that reproductive activity may continue through winter, albeit at a rate slowed by winter temperatures. Populations exploiting underground storm water systems for resting or for larval development may be exposed to relatively warm temperatures throughout winter. Five factors have been associated with human risk of SLEV infection: (1) place of residence, (2) age, (3) occupation, (4) socioeconomic status and (5) climate. Clearly, the place of residence markedly affects the risk of infection, with geographic regions in the southern U.S. historically having the greatest numbers of human cases and greatest incidence of disease (Fig. 3). Based on experimental infection patterns in laboratory mice, virus strains from this geographical area also exhibit greater neurovirulence than strains from the western U.S. or South America. Because of greater mosquito abundance relative to humans and host selection patterns, urban residents seem to be at greater risk for SLEV infection than are rural residents; however, these conclusions may be confounded by protective immunity acquired early in life that may be greater among rural residents and by low apparent: inapparent case ratios that require a substantially large population to produce recognizable clusters of human cases.

812

Saint Louis Encephalitis Virus (Flaviviridae)

In the absence of acquired immunity, clinical illness and fatality rates, but not necessarily infection rates, increase dramatically with age. Infection seems to occur equally among different age classes as indicated by the increase in antibody as a function of age in endemic areas and by cohort seroconversion rates determined after epidemics in previously unexposed populations. For example, using data following the 1964 Houston, Texas epidemic, seroprevalence rates remained similar among age cohorts, whereas the case-incidence rates increased from 8.2 per 100,000 for the 0 - 9 year old cohort to 13.5–27.6 for the 10–59 year old cohort and to 78.0 for the 460 year old group; apparent to inapparent ratios decreased concomitantly from 1:806 to 1:490–1:239 and to 1:85, respectively. Case-fatality rates among 2288 cases reported to the CDC from 1971 to 1983 increased from o6.7% for 0–64 year old age groups to 9.5% for the 65–74 year class to 18% for the 475 year groups. In the western United States, where SLE historically has been a rural disease, infection risk was found to be greatest among male agricultural workers who frequently lived in suboptimal housing and worked at night. However, infection patterns during urban outbreaks indicated that attack rates were highest among elderly women. These data indicated that there may be differences in risk related to vector species, with elderly women infected most readily during urban outbreaks associated with the Cx. pipiens complex, and men working outdoors at greatest risk during rural outbreaks associated with Cx. tarsalis. Historically, socioeconomic status has been related closely to the distribution of cases during urban epidemics. Homes and municipal drainage systems frequently were not well maintained in low income neighborhoods, and this was related to the distribution of human cases, but not necessarily the occurrence of virus within the enzootic transmission cycle. Television and air conditioning ownership that brought people indoors during the evening Culex hostseeking period was found to reduce risk. Climate variability affects temperature and precipitation patterns, mosquito abundance and survival, virus replication in the vector, and therefore SLEV transmission. Annual changes based on the El Niño/southern oscillation in the Pacific sea surface temperature markedly alter precipitation and temperature patterns over the Americas and cycle with varying intensity at 3–5 year intervals. These cycles alter storm tracks that affect mosquito and avian abundance, the intensity and frequency of rainfall events, and ground water depth; all related to SLEV risk. Above normal temperatures have been especially necessary for northern latitude SLEV epidemics, because elevated temperatures are required for effective SLEV replication within the mosquito vector.

Pathogenesis/Clinical Features Like many other flaviviruses within the Japanese encephalitis virus serocomplex that can elicit encephalitic syndromes such as WNV and Murray Valley encephalitis virus, human disease is typically recognized in more serious cases that manifest as aseptic meningitis, encephalitis and/or meningoencephalitis. However, the majority of human exposures result in asymptomatic infection. Clinical severity is more notable in individuals 465 years of age. Humans serve as incidental hosts and do not produce viremias sufficient to infect mosquitoes. Similar to most arboviruses that cause CNS disease, infection with SLEV does not result in a clear clinical picture in humans and most infections remain unrecognized, unless associated with an epidemic. When presented with such diverse symptoms, few physicians initially suspect SLEV, even in endemic areas. Most SLEV infections, especially in young or middle age groups, fail to produce clinical disease, and infected individuals rarely experience more than a mild malaise of short duration with spontaneous recovery. In humans, clinical disease due to SLEV infection may be divided into 3 syndromes in increasing order of severity: (1) febrile headache with fever, headache possibly associated with nausea or vomiting, but no CNS illness; (2) aseptic meningitis with high fever and stiff neck; and (3) encephalitis (including meningoencephalitis and encephalomyelitis) with high fever, altered consciousness and/or neurological dysfunction. The onset of illness may be sudden (o4 days after infection) and acute, leading rapidly to encephalitis, or progressing gradually through all 3 syndromes. Symptoms may resolve spontaneously during any stage of the illness, with full recovery. Acute illness may be followed by “convalescent fatigue syndrome” in o50% of patients, with complaints of general weakness, depression, and the inability to concentrate that generally resolve within 3 years. Other sequelae include headache, disturbances in gait, and memory loss. Transmission of SLEV from an infected mosquito into the epidermis results in initial infection of either Langerhans cells, epidermal cells, melanocytes or tissue macrophages as initial sites of viral replication based on studies with SLEV as well as other flaviviruses. Viral infected myeloid cells are trafficked to the draining lymph nodes where viral replication is associated with subsequent serum viremia and viral dissemination to alternative tissues including the peripheral and central nervous system. The mechanism(s) of neuroinvasion have not been conclusively demonstrated; however, axonal retrograde transport, introduction into the CNS through the olfactory bulb, a Trojan horse movement of SLEV through infected circulating monocytes and direct hematogenous passage through compromised point of the blood brain barrier have all be suspected as potential routes. CNS pathology consists of necrosis of neurons and glia cells and inflammatory changes. Inflammatory changes typically are most important in slowly progressing or sublethal CNS disease and sequelae. Viral clearance is dependent upon a functional immune system and the rapid production of neutralizing antibody, which typically appears within 7 days of infection. Viral pathogenesis associated with SLEV infection is principally the result of a combination of viral replication within neural tissue and the subsequent necrosis associated directly with viral infection or the subsequent inflammatory process that neuronal infection elicits.

Diagnosis The identification of human SLEV cases is primarily based on serological laboratory confirmation of patients exhibiting aseptic encephalitis. Detection of SLEV-specific reactive immunoglobulin is typically made by ELISA with subsequent confirmation by

Saint Louis Encephalitis Virus (Flaviviridae)

813

plaque reduction neutralization testing. Testing of patient sera and/or cerebral spinal fluid for viral RNA by reverse transcription polymerase chain reaction (RT-PCR) within the first five days post-symptom onset also serves as a direct confirmation of SLEV infection.

Prevention There are no approved antiviral or therapeutic treatment regimens for SLEV human infection and all medical care is supportive. As such, the principal preventive action is to reduce human exposure to mosquitoes. Reducing human/mosquito contact is principally managed by mosquito control activities focused on larviciding, with the implementation of adult mosquito control in situations in which active transmission necessitates targeting infected adult mosquitoes. Best results have been achieved using an integrated management approach that focuses on mosquito vector population suppression through habitat inspection and larviciding. Failure of larval management can be followed by emergency adult control focusing on eliminating infected adult mosquitoes and thereby reducing the force of transmission and preventing human infection. Even if a vaccine was available, protection of the human population by vaccination does not seem cost effective or prudent, because there is no human-to-human transmission, few human infections produce disease, and infection rates remain relatively low, even during epidemics. However, if regional infection rates were to become high, thereby placing selected cohorts at high risk for disease, then selective vaccination may be warranted. There currently is no approved commercial vaccine for SLEV, although vaccination against other flaviviruses such as JEV may impart some protection. Control of avian hosts such as house sparrows and pigeons in urban situations could be done, but this approach is not generally acceptable to the public. Notification of the public of infection risk through the media and the wide-scale use of personal protection through changes in behavior (staying indoors after sunset) and/or repellent application have been credited with reducing the number of infections during the 1990 epidemic in Florida.

Further Reading Auguste, A.J., Pybus, O.G., Carrington, C.V.F., 2009. Evolution and dispersal of St. Louis encephalitis virus in the Americas. Infection, Genetics and Evolution 9, 709–715. Curren, E.J., Hills, S.L., Fischer, M., Lindsey, N.P., 2018. St. Louis encephalitis virus disease in the United States, 2003–2017. The American Journal of Tropical Medicine and Hygiene 99 (4), 1074–1079. Day, J.F., 2001. Predicting St. Louis encephalitis virus epidemics: Lessons from recent, and not so recent, outbreaks. Annual Review of Entomology 46, 111–138. Diaz, A., Coffey, L.L., Burkett-Cadena, N., Day, J.F., 2018. Reemergence of St. Louis encephalitis virus in the Americas. Emerging Infectious Diseases 24 (12). Kopp, A., Gillespie, T.R., Hobelsberger, D., et al., 2013. Provenance and geographic spread of St. Louis encephalitis virus. mBio 4 (3). Kramer, L.D., Chandler, L.J., 2001. Phylogenetic analysis of the envelope gene of St. Louis encephalitis virus. Archives of Virology 146, 2341–2355. Monath, T.P., 1980. St. Louis Encephalitis. Washington, D.C: American Public Health Association. Monath, T.P., Tsai, T.F., 1987. St. Louis encephalitis: Lessons from the last decade. The American Journal of Tropical Medicine and Hygiene 37, 40s–59s. Ortiz-Martinez, Y., Vega-Useche, L., Villamil-Gomez, W.E., Rodriguez-Morales, A.J., 2017. Saint Louis encephalitis virus, another re-emerging arbovirus: A literature review of worldwide research. Le Infezioni in Medicina 25 (1), 77–79.

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae) Malik Peiris and Leo LM Poon, The University of Hong Kong, Pok Fu Lam, Hong Kong r 2021 Published by Elsevier Ltd.

Glossary Lymphopenia Reduction in the lymphocytes in the circulating blood below the normal range for age. Pathognomonic Characteristic and diagnostic of a particular disease.

PEGylated A molecule conjugated with polyethylene glycol (PEG). Radiological abnormalities Pathological findings observed by medical imaging procedures (e.g., chest X-ray). Rhinorrhea Runny nose.

History Coronaviruses OC43 and 229E were first discovered in the 1960 as causes of mild upper respiratory disease. The discovery that a novel coronavirus was the cause of severe acute respiratory syndrome (SARS) in 2003 led to increased activity of research on human coronaviruses, resulting in the discovery of NL63, HKU1 and, in 2012 the identification of Middle East respiratory syndrome (MERS)-coronavirus as a novel zoonotic disease with epidemic potential. Both SARS and MERS coronaviruses are identified by WHO as pathogens of greatest concern for global public health for which countermeasures are urgently needed.

Emergence of SARS From November 2002 to January 2003, clusters of cases of an unusually severe atypical pneumonia were observed in Guangdong Province, China. The disease was characterized by the lack of response to conventional antibiotic therapy and the occurrence of clusters of cases within a family or health care setting. In retrospect, these were the first known cases of the disease that was later called severe acute respiratory syndrome (SARS). In January, the numbers of cases “atypical pneumonia” continued to increase with examples of “super-spreading incidents” that were to punctuate the course of the subsequent SARS epidemic. On February 21, 2003, a health care worker in a hospital in the city of Guangzhou, the provincial capital of Guangdong, arrived in Hong Kong and checked in at Hotel M. He had treated patients with “atypical pneumonia” in Guangzhou and had been ill himself since 15 February. His one-day stay on the ninth floor at this hotel led to the infection of at least 17 other guests or visitors, some of whom traveled later to Hanoi, Toronto, Vancouver, Singapore, USA, Philippines, Guangzhou, and Australia. Five of these secondary cases initiated clusters of infection in Hanoi, Singapore, Toronto and two clusters of infection in Hong Kong. This was the most significant single event in the global spread of SARS, and arguably the most dramatic single event in the global spread of any infectious disease. In response to the outbreaks in Hanoi and Hong Kong, on 12 March WHO issued a Global Health Alert warning of a pneumonia that was a particular risk to health care workers. Subsequently, Singapore and Toronto also reported clusters of cases. On 15 March, WHO issued a Travel Advisory. The new disease was named Severe Acute Respiratory Syndrome (SARS) and a preliminary case definition was provided. Within weeks, SARS had spread to affect 8096 patients in 29 countries across five continents with 744 fatalities, an overall case-fatality rate of 9.6%. Health care facilities served as a major amplifier of the infection, constituting 21% of all reported cases. By 21–24 March, the etiological agent of SARS was identified to be a novel coronavirus, subsequently named as SARS coronavirus (SARS-CoV). Early case detection and isolation of infected individuals reduced and interrupted SARS-CoV transmission throughout the world. By July 5, 2003, WHO announced that all chains of human transmission of SARS were broken and the outbreak was at an end. Although SARS was subsequently to re-emerge to cause limited human disease (and in one instance, limited human-to-human transmission) as a result of laboratory escapes and zoonotic transmission from live game animal markets of Guangdong in December 2003–January 2004, the human outbreak of SARS had been efficiently controlled. Emergence of MERS: Almost a decade later, a virus was identified from a patient with a severe pneumonia in Saudi Arabia and later identified to be a novel betacoronavirus, initially named EMC, but later renamed as Middle East Respiratory Syndrome coronavirus (MERS-CoV). The report of this discovery via Promed on September 20, 2012 led to recognition of other cases, and to a retrospective recognition that a previously undiagnosed cluster of patients with pneumonia in Jordan in 2012 was linked to the same virus. As of October 2020, ca. 2600 confirmed cases of MERS with 880 deaths (34%) have been reported to WHO from 27 countries.

814

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00138-7

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

815

Fig. 1 Schematic drawing of a SARS or MERS viral particle.

Virology SARS- and MERS-CoVs Coronaviruses are currently genetically classified into 4 genera (a, b, g, and δ) and a putative one (e). SARS- and MERS-CoVs are now classified within the sub-genus Sarbecovirus and Merbecovirus, respectively, in the genus Betacoronavirus within the family Coronaviridae, under the order Nidovirales. Coronaviruses are enveloped positive-sense, single-stranded RNA viruses with a genome size of approximately 30 kb. Their virus particles are about 100–140 nm in diameter with a distinctive corona of petal-shaped spikes on the surface which is comprised of the spike glycoprotein (S) (Fig. 1). The S protein is in a trimeric form on the viral surface and it has an N-terminal subdomain (S1) which contains the motifs responsible for receptor binding. A more conserved C-terminal subdomain (S2), which contains heptad repeats and a coil-coil structure, is important in the membrane fusion process. The S1–S2 subdomains remain in a non-cleaved form in newly synthesized progeny viral particles and cleavage of S into S1 and S2 subunits is essential for the viral entry process. The envelope also contains a transmembrane glycoprotein M and in much smaller amounts, an envelope (E) protein. The M protein is a triple-spanning membrane protein and it has a key role in coronavirus assembly. The hemagglutinin-esterase (HE) glycoprotein, found in some betacoronaviruses, are absent in both SARS- and MERSCoVs. The nucleocapsid protein (N) interacts with the viral genomic RNA to form the viral nucleocapsid. Within the cell, viral replication complexes are located at double-membraned vesicles or autophagosomes within the cell for viral RNA synthesis.

SARS- and MERS-CoV Genomes The genome organization of SARS- or MARS-CoV is that of typical coronaviruses. The viral genomes of SARS- and MERS-CoVs have, respectively, at least 14 and 11 open reading frames (ORFs) (Fig. 2(A)). Both genomes code for 16 nonstructural proteins (nsp1–16) in their replicase genes (Fig. 2(B)). The capped genomic RNA encoding the replicase gene functions as mRNA to generate polyproteins 1a and 1ab. The translation of ORF1b is directed by a –1 ribosomal frameshift (RFS) signal that contains a nucleotide slippery sequence and an RNA pseudoknot. By contrast, the structural and accessory proteins are products derived from capped subgenomic RNA (sgRNA) which are synthesized by discontinuous RNA transcription. The polyproteins 1a and 1ab generated from the replicase gene of SARS- or MERS-CoV are cleaved by a papain-like proteinase (part of nsp3) and a 3C-like proteinase (nsp5 or 3CLpro) to generate 16 nsps (Fig. 1(B)). As SARS-CoV is relatively well characterized, the biological functions of these nsps described below are primarily based on the findings of studies on SARS-CoV. Nsp12 is a primer-dependent RNA-dependent RNA polymerase (RdRp), whereas nsp8 is a noncanonical RdRp (nsp8) synthesizing primers utilized by nsp12. The N-terminus of Nsp12 has a domain (nidovirus RdRp-associated nucleotidyltransferase, niRNA) which is unique to nidoviruses. In addition, eight nsp7 and eight nsp8 subunits are able to form a hexadecamer with a hollow, cylinder-like structure. RNA-binding studies and the overall architecture of this nsp7–nsp8 complex suggest that it may encircle RNA and confer processivity of nsp12. The nsp9 is a single-stranded RNA-binding protein and is able to interact with nsp8. The dimerization of nsp9 is essential for virus replication. The nsp10 contains two zinc finger motifs and is suggested to be a regulator of vRNA synthesis. The nsp13 is a helicase and unwinds duplex RNA (and DNA) in a 50 -to-30 direction. The nsp14, nsp15, and nsp16 have been shown to have 50 -to-30 exonuclease, endoribonuclease, and 20 -O-ribose methyltransferase activities, respectively. These three proteins, together with nsp3 (see below) are distantly related to cellular enzymes involved in RNA metabolism. These observations may be relevant to viral RNA processing. The exonuclease activity of nsp14 is known to be essential for replication fidelity. The nsp10, 13, 14 and 16 are the subunits essential for forming the cap structure of viral RNA. The nsp3 contains multiple domains and it has 8 domains which are conserved in all coronaviruses (ubiquitin-like domain 1, hypervariable region, X domain, ubiquitin-like domain 2, papain-like protease 2 domain, zinc-finger domain and Y1 and CoV-Y domain). Apart from facilitating viral RNA synthesis and releasing nsp1-nsp3 from the polyprotein 1a and 1ab, nsp3 is also involved in various post-translation modifications (de-ADP-ribosylation, deubiquitination, and de-ISGylation) and evasion of host innate immunity. The biological functions of nsp1, nsp2, nsp4, nsp6, and nsp11 are not entirely clear. The nsp1 is reported to induce chemokine dysregulation and

816

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

Fig. 2 SARS and MERS CoV genomes. (A) Genomic organization of SARS and MERS CoVs. The ORFs are expressed from the genomic RNA and a nested set of subgenomic mRNA (not shown) that all have a common leader sequence derived from the 50 end of the genome will be expressed. The genomic RNA and all sgRNA contain a 50 cap and a polyadenylated tail at the 30 end. (B) Domain organization of the proteins for ORF1ab. Black and white arrow heads represent the sites cleaved by papain-like and 3C-like proteinases, respectively. The ribosomal frameshift (RFS) site is highlighted by a broken line.

host mRNA degradation, and regulate host gene expression. The nsp2 is reported to be involved in mitochondrial biogenesis and intracellular signaling, but it is dispensable for virus replication. Both nsp4 and nsp6 contain a putative transmembrane domain and they are suggested to be essential for membrane rearrangement. The majority of nsps of MERS-CoV (nsp3, 5, 7–10, 12–16) are assumed to have similar biological functions, whereas the functions of other MERS-CoV nsps are not clear. Both SARS- and MERS-CoVs have 5 structural proteins (S, E, M, ORF3a and N) encoded by the corresponding ORFs as indicated (Fig. 2(A)). The proteins are the basic protein components of the viral particles. Some studies suggest that ORF3a of SARS-CoV is also a structural protein. This protein can induce membrane rearrangement and activate NF-κB and the NLRP3 inflammasome in infected cells. However, the precise function of this protein in these virions is not entirely clear. Apart from the ORFs encoding the replicase and structural proteins, SARS-CoV and MERS-CoV genomes contain additional ORFs that code for accessory proteins (SARS-CoV: 3b, 6, 7a, 7b, 8a, 8b, and 9b and MERS-CoV: 3, 4a, 4b 5 and 8b). The accessory proteins of SARS- and MERS-CoV have very different sequences, suggesting that the proteins may have very different biological functions. Genetically modified recombinant SARS- and MERS-CoVs without these accessory ORFs have been shown to be replication competent in cell cultures, indicating that the accessory ORFs may not be essential for virus replication in vitro. However, recombinant viruses with deletions in these regions are attenuated in vivo, suggesting that these proteins may have functions that are important for viral replication, immune evasion and pathogenesis in vivo. The accessory proteins of ORF3b and ORF7a of SARS-CoV induce apoptosis in transfected cells. ORF3b is reported to act as an interferon antagonist during the infection. There is also evidence suggesting that the 7a protein is incorporated into virions. The protein encoded by ORF6 has been shown to inhibit the nuclear import of STAT1 and function as an interferon antagonist in SARS-CoV-infected cells. In addition, this protein is also reported to interact with the viral RNA transcription complex. These properties may be related to virus virulence. Interestingly, comparative sequence analysis of SARS-CoV isolated from palm civets (see below) and humans showed that all animal strains contained a 29-nucleotide (nt) sequence which is absent from most human strains obtained in the later phase of the SARS outbreak. The ORF8 in human SARS-CoVs encodes 8a and 8b proteins, whereas the corresponding ORF in the animal isolates encodes a single protein, known as the 8ab protein. These proteins from the animal and human ORF8 have differential binding affinities to various SARS-CoV structural proteins. Furthermore, the expression of E can be downregulated by 8b but not 8a or 8ab in infected cells. These observations may suggest that the 29-nt deletion modulates the replication or pathogenesis of the human SARS-CoV. The crystal structure of the 9b protein suggests that it may be a lipid binding protein and that it may be a virion-associated accessory protein. But its function is yet to be identified. Overall, these accessory proteins may have a role in viral replication and pathogenesis. The biological functions of MERS-CoV accessory proteins, except ORF4a and ORF4b, are not clear. All the accessory proteins are dispensable for in vitro virus cultures. ORF4a and ORF4b of MERS-CoV are known to suppress innate sensing triggered by dsRNA. Thus, these proteins may have essential roles in virus pathogenesis. Interestingly, deletions in ORF3 and ORF4b sequence can be found in MERS-CoV circulating in West and North African dromedary camels, but not in those animals found in East Africa and the Arabian Peninsula (see below).

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

817

Fig. 3 Phylogenetic analysis of representative RNA-dependent RNA polymerase sequences from a, b, g and δ coronaviruses. The phylogenetic tree was constructed by the neighbor-joining method and bootstrap values were determined with 500 replicates. Bootstrap values are shown as indicated. Scale bar indicates an estimated genetic distance.

Phylogeny The phylogeny of nidoviruses is based on the concatenated 5 highly conserved domains in 3 nsps (nsp5: 3CLpro; nsp12: NiRAN and RdRp; nsp13: ZBD and HEL1). Currently, viruses under the same subgenus have patristic pairwise distances less than 0.22. As formal virus taxonomy analyses primarily focuses on (almost) full genome sequences, partial virus sequence fragments derived from potentially novel virus species are not considered in these analyses. Thus, virus diversity within each subgenus is expected to be underestimated. SARS-CoV, MERS-CoV and their related animal viruses are grouped into subgenera Sasbecovirus and Merbecovirus, respectively (Fig. 3). The search for the precursor of SARS-CoV led to the discovery of a number of novel coronaviruses in both wild and domesticated animals. In particular, a vast number of alphacoronaviruses and betacoronaviruses were identified in bats. Interestingly, these recently discovered bat coronaviruses appear to be in evolutionary stable while many other mammalian coronaviruses still appear to be under evolutionary selection pressure, raising an intriguing possibility that bats may in fact be the precursors, not only of SARS-CoV, but also for many other mammalian coronaviruses. It is suggested that some other human coronaviruses, such as NL63 and 229E primarily originated from bat coronaviruses. Genetic and phylogenetic analysis indicates that the viruses associated with the early phase of the human SARS outbreak are more closely related to the viruses found in palm civets and other small mammals in the live game animal markets in Guangdong. The genomes of viruses in the early phase of the human outbreak in 2003 were observed to be under strong positive selective pressure, suggesting that the virus was rapidly adapting to the new host, humans. Furthermore, SARS-CoV in civets was also found to be under strong positive selection pressure, supporting the view that civets were not the natural host of the precursor SARS-like coronavirus. MERS-CoV found in humans and camels can be divided into 3 clades (A–C). Both clades A and B viruses were found in humans and camels from the Arabian Peninsula, whereas clade C viruses found in African camels are very diverse. The genomes of these viruses are genetically relatively stable, but some studies suggest that the S genes of these camel and human viruses are under positive selection pressure. This may be due to adaptive evolution of these viruses to achieve better attachment to the cells of their new hosts. Deletions in accessory genes of MERS-CoV were found in camel and human viruses. In addition, recombination

818

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

between MERS-CoV in camels or humans has been reported, for example, giving rise to the lineage 5 clade B viruses currently causing outbreaks of human disease in the Arabian Peninsula.

Virus Receptors The S1 subunit of coronavirus spike proteins contains two distinctive structural domains, the N-terminal domain (NTD) and the receptor binding domain (RBD). The RBD binds to the major functional receptor to achieve viral attachment and entry. However, work on other coronaviruses indicate that the NTD may also have strain-specific binding affinity to other host proteins or sugar residues. Both SARS- and MERS-CoVs can bind to multiple non-functional receptors and it is generally assumed that the NTDs may also facilitate virus-host interactions. The functional receptor for SARS-CoV in human cells is the angiotensin-converting enzyme 2 (ACE-2) which binds the RBD (amino acid residues 424–494) of the SARS-CoV S protein. While the human SARS-CoV S protein binds efficiently to both human and civet ACE-2, the civet-like SARS-CoV S protein binds efficiently to ACE-2 from civets but poorly to human ACE-2. These findings explain the increased human transmissibility of SARS-CoV in the later stages of the SARS outbreak. An observation that human SARS-CoV efficiently infects civets under experimental conditions, the poor virulence and transmissibility of re-emergent SARS in December 2003–January 2004 when humans are believed to have been infected with a civet-like SARS-CoV and the observation that there was approximately 20% sero-prevalence to SARS-CoV in those working in Guangdong live game animal markets although none of them had SARS-like disease. The spike protein of some bat SARS-like coronavirus binds to human ACE-2. Other cell-surface molecules such as L-SIGN, DC-SIGNR, DC-SIGN (CD209), and LSECtin may serve as binding receptors but they do not appear to be functional viral receptors in the absence of ACE-2. They may, however, promote cell-mediated transfer of the virus to other susceptible target cells. The RBD of MERS-CoV S protein (AA residues 484–567) can bind to its human functional receptor, dipeptidyl peptidase-4 (DPP4). The RDB of human MERS-CoV is identical to that of dromedary camel virus. MERS-CoV can experimentally infect animals having DPP4 with conserved critical binding residues to those of human DPP4 (e.g., camelids, primates, and rabbits). By contrast, animals with critical DPP4 residues that are different to those of human DPP4 are not susceptible to MERS-CoV infection (e.g., mice, rats and ferrets). MERS-CoV is also known to bind to sialic acid, CEACAM5 and GRP78.

Epidemiology Ecology, Animal Reservoir and Zoonotic Transmission Both SARS- and MERS-CoVs are of animal origin, but their host origins and modes of zoonotic transmission differ (see below).

SARS In the early phase of SARS epidemic (before the end of January 2003), 39% of patients with SARS in Guangdong had handled, killed, or sold wild animals or prepared and served them as food. However, such risk factors were found in only 2%–10% of cases from February to April 2003 when the virus had adapted to efficient human-to-human transmission. Thus, the early epidemiological evidence pointed to the live game animal trade as an interface for the emergence of the SARS-CoV. SARS-like coronaviruses were identified in a number of small mammalian species sold in the live game animal markets in Guangdong, including the palm civet (Paguma larvata), raccoon dog (Nyctereutes procyonides), and the Chinese ferret badger (Melogale moschata). A high proportion of individuals working in these markets had developed antibodies to SARS-CoV, although none of them had a history of the disease. Viruses isolated from the re-emergent SARS cases in Guangdong in December 2003–January 2004 were more similar to those found in civets in these markets, rather than to viruses causing the global outbreak in early 2003. These observations strongly implicated that the live game animal trade is the interface for interspecies transmission of a precursor animal SARS-like coronavirus to humans. SARS-CoV is shed for weeks in experimentally infected palm civets but many of the other species appear to clear the virus more rapidly. While civets in live-animal markets tested positive for SARS-like coronavirus RNA and antibody, civets tested in the farms that supply these markets and those caught in the wild rarely had evidence of infection. Thus, although palm civets and other small mammals sold in these markets were likely the intermediate host that amplified and maintained the virus in these markets and provided the source of virus for repeated human exposure to zoonotic infection, they were not the natural reservoir of the precursor SARS-CoV. Several sarcoviruses related to SARS-CoV have now been identified in Rhinolophus bats in China. Such bats were also sold live in these game animal markets. So far, none of the reported bat CoV is genetically identical to SARS-CoV. It is now believed that SARS-CoV is a recombinant virus of bat sarbecoviruses. Studies in Yunnan, China, has revealed several bat coronaviruses containing all genetic elements needed for generating SARS-CoV in a single geographical location.

MERS-CoV Is enzootic in dromedary camels which is the main source of zoonotic infection. Bactrian camels have no evidence of infection. The majority of dromedary camels in the Middle East, Africa and Central Asia have serological evidence of MERS-CoV infection. Infection in dromedary camels is characterized as a mild upper respiratory infection. Virus shedding in camel herds is highly seasonal (winter months and the calving season) and is most commonly seen in calves although adults can get re-infected. Settings

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

819

where camels from different sources intermingle (camel markets, abattoirs, racing meets) are associated with higher rates of virus detection and may show less pronounced seasonality. Zoonotic transmission is stochastic and uncommon and the routes by which such transmission occurs are unclear. Direct contact with infected camel upper respiratory secretions is one presumed route of infection to humans. A gastro-intestinal route of infection has also been proposed, based on studies of virus tropism in the human gastrointestinal tract and experimental animal models. While virus RNA has been detected in camel milk, presumably derived by the suckling calf contaminating the udder and thence the milk. Viable virus has not been isolated from milk and it is likely that passive maternal antibody found in camel milk would neutralize infectious viruses. Most zoonotic transmission appears to be asymptomatic or mild, while those individuals with underlying co-morbidities are more likely to manifest severe disease. Over 50% of patients with laboratory confirmed “primary” MERS infection (not explainable by potential contact with other MERS patients) had recent direct or indirect contact with camels. Case-control studies have confirmed direct exposure to dromedaries within two weeks of disease onset as a significant risk factor. MERS-CoV transmission between humans can lead to outbreaks, especially within health care facilities. Transmission may also occur within households or other community settings but this is less frequent. MERS patients have been diagnosed in 27 countries to date, some associated with index cases acquiring infection in the Arabian Peninsula. Zoonotic MERS has only been reported in the Arabian Peninsula, not in Africa where the majority (470%) of infected camels are found. The reason for this is at present unclear. MERS-CoV found in African camels is genetically highly similar but phylogenetically distinct from those found in camels in the Arabian Peninsula. MERS-CoV neutralizing antibody was detected in archived serum samples collected in 1983, indicating that the virus has been circulating in camels for over 30 years. It is unclear whether dromedary camels are the primary reservoir of MERS-CoV. There are several merbecoviruses found in Tyloncteris and Pipistrellus bats, suggesting that the precursor(s) of MERS-CoV derives from bats but a closely related ancestor is yet to be found.

Human-to-Human Transmission Both SARS and MERS-CoV appear to be more stable and remain viable for longer on surfaces and in aerosols than other enveloped respiratory viruses. Both viruses are more stable at low humidity and low temperature environment such as those found in airconditioned spaces. This may explain why the major outbreaks of both have occurred in air conditioned environments such as modern hospitals. The findings are also compatible with epidemiological observations that transmission may occur via fomites, large droplet and aerosol routes. Aerosol generating procedures such as tracheal intubation, manual ventilation before intubation, non-invasive ventilation and tracheostomy have contributed to the transmission in health care settings. SARS-CoV is present in high titers in feces and this may well be a source of infection. The largest single outbreak of SARS in an apartment block in Hong Kong where over 300 individuals were infected from a single index case, is believed to have been caused by aerosols generated from infected feces via a faulty sewage system. The incubation period for both SARS and MERS ranges from 2 to 14 days. While a asymptomatic infection is rare in SARS it appears to be more common in MERS. The majority of cases did not transmit disease at all and only a few patients accounted for a disproportionately large number of secondary cases, referred as “super-spreading events”. While some host factors may play a role in these super-spreading events, in many cases there was a unique combination of host factors and environmental circumstances that facilitated transmission. In contrast to the high transmission rates in these super-spreading events and within hospitals, there was less evidence of secondary transmission within the family or households. SARS-CoV viral load in the upper respiratory tract was lower in first few days of illness and it peaked in the beginning of the second week of the disease. This correlated with minimal transmission occurring in the first five days of illness and explains why health careassociated outbreaks was more common because hospitalization and use of aerosol generating procedures are more common towards the end of the first week of illness. Taken together, with the lack of asymptomatic infections the public health measures of aggressive case detection and isolation lead to efficient interruption of transmission of the SARS-CoV outbreak in 2003. The characteristics of the epidemiology of MERS is generally comparable to that of SARS, except that asymptomatic infections are more common in MERS. The basic reproduction number (Ro) of SARS is estimated to range from 2 to 4 while that for MERS over the course of an outbreak is o1. However, there is considerable heterogeneity of effective reproduction number (Rt) during the course of an outbreak, being 1.3–5.4 in the early stages of a hospital outbreak followed by a decrease to o1 within 2–6 weeks.

Clinical Features The clinical features of SARS and MERS are not pathognomonic. Fever, cough, headache, myalgia, nausea, diarrhea and malaise are commonly reported at early stages of the disease followed by progression to shortness of breath, acute respiratory distress syndrome (ARDS) and multiple organ failure in more severe cases. Rhinorrhea was uncommon in both diseases (Table 1). Lymphopenia was common in both diseases. Differences seen in MERS compared to SARS include a higher proportion of patients who are older and/or with co-morbidities and the presence of hypotension that lead to the need for vasopressors. In both diseases, symptoms may be atypical in immune-compromised patients. A contact history with SARS-CoV positive individuals was an important epidemiological risk factor for SARS. A travel history to the Arabian Peninsula, direct contact with dromedary camels or visiting health care facilities in these affected areas are important factors relevant to initiating specific diagnostic tests for MERS.

820

Table 1

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

Comparison of virological, clinical and epidemiological features of SARS and MERS SARS

MERS

Virus taxonomy Genus betacoronavirus; subgenus Sarbecovirus Genus betacoronavirus; subgenus Merbecovirus Receptor ACE2 DPP4 Animal reservoir and Natural reservoir Rhinolophus bats, Zoonotic Natural reservoir: unknown. Natural host and source of zoonotic infection: zoonotic source interface – live game animal markets Dromedary camels Clinical and epidemiological aspects (see notes below) Health care workers 21% Males 47% Fever ++++ Headache ++ Myalgia +++ Cough +++ Shortness of breath ++ Sore throat + Nausea/vomiting + Diarrhoea + Rhinorrhoea (+)

Approx. 17% Approx 63% ++++ + ++ +++ ++ + + + (+)

Co-morbidities Diabetes Hypertension Chronic lung disease Chronic renal failure Chronic heart disease Malignancy

++ ++ + + + +

+ Not noted + o1% + +

Approximate frequency of occurrence: + 1%–25%; ++ 26%–50%; +++ 51%–75%; ++++ 76%–100%.

Radiological abnormalities were observed in 460% of cases at initial stages of the disease and preceded lower respiratory tract symptoms in approximately 41% of patients with SARS. In MERS, chest radiographs or computed tomography scans showed multilobar airspace disease, ground glass opacities, and sometimes, pleural effusions, but some patients with mild or asymptomatic infection may show minimal changes in lungs. Liver dysfunction and lymphopenia are seen in both infections. High serum levels of chemokines (IL-8, CCL2, and CCl10) and pro-inflammatory cytokines have been seen in patients with SARS. In MERS, higher serum levels of IL-6 was associated with a severe disease. The overall case fatality ratio for confirmed SARS and MERS cases has been 9.6% and 35%, respectively. Since MERS-CoV infections, unlike SARS, can often be mild and unrecognized by the health care system, this apparently higher fatality rate may be misleading. In both diseases, infections of children and young adults have been mild. Case-fatality rate increased progressively with increasing age in SARS and with the presence of co-morbidities such as diabetes, cardiac disease, obesity, chronic respiratory disease, end stage renal disease in the case of MERS. Autopsy findings of those who died in the first 10 days of illness with SARS were diffuse alveolar damage, desquamation of pneumocytes, and hyaline membrane formation. Viral RNA was detected by quantitative polymerase chain reaction (PCR) at high copy numbers in the lungs, intestine and lymph nodes, and at lower levels in the spleen, liver and the kidneys. In the lungs of the patients who died within the first 10 days after disease onset, viral antigen and viral nucleic acid were detectable by immunohistochemistry and in situ hybridization in alveolar epithelial cells and to lesser extent in macrophages. Some studies also reported virus particles or viral RNA in other organs but these findings require independent confirmation. There are limited autopsy studies on MERS. A diffuse alveolar damage was seen in a man who died approximately 12 days after onset of illness. MERS-CoV antigen was detected in pneumocytes and syncytial epithelial cells. No evidence of infection in the kidneys was observed. In an immunocompromised patient with a T cell lymphoma who died of MERS around 3 weeks after onset, histopathological examination showed necrotizing pneumonia, pulmonary diffuse alveolar damage, acute kidney injury, portal and lobular hepatitis and myositis with atrophic changes in the muscles. The brains and the heart were histologically normal. Virus infection was demonstrated in pneumocytes, pulmonary macrophages and renal proximal tubular epithelial cells.

Laboratory Diagnosis Specimens from the lower respiratory tract such as sputum or endotracheal aspirates have higher viral load and give a higher diagnostic sensitivity than those from the upper respiratory tract for both SARS or MERS. Highly sensitive and specific real-time PCR assays for the detection of viral RNA remain the best choice for early diagnosis. For diagnosis of MERS, initial screening by

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

821

RT-PCR targeting the E gene followed by confirmatory tests with RT-PCR for the ORF1 gene region is recommended and only patients positive in both assays are diagnosed as confirmed cases of MERS. While SARS-CoV RNA remains detectable in the respiratory secretions and feces for many weeks after the onset of illness, specimens rarely yield a virus isolate after the third week of illness, likely because of the emerging antibody responses. Viral RNA can be detected in feces, serum, and urine in patients with SARS, later in the illness. MERS-CoV RNA may also be detected in serum approximately in one-third of patients, especially those with a more severe disease; and in the stool of around 15% of patients, less often that was seen in SARS. In both SARS and MERS, a negative PCR result in an individual specimen does not exclude the disease. Testing multiple specimens improves the diagnostic accuracy. Both viruses can be cultured from clinical specimens in Vero or Vero-E6 cells but the sensitivity of virus culture is much lower as compared to RT-PCR. Immunofluorescence or neutralization tests have been used for serological diagnosis or sero-epidemiology of SARS-CoV. A MERS-CoV S1 ELISA is available for the detection of MERS-CoV antibody. Positive results are best confirmed by neutralization tests with live MERS-CoV or spike-pseudotyped virus. Seroconversion occurs during the second week of illness in both diseases and can provide reliable retrospective diagnosis. While a seroconversion is invariable in patients with a severe MERS disease, antibody responses may be marginal or absent in those with a mild MERS infection. Waning antibody responses need to be considered in sero-epidemiologial studies. MERS-CoV-specific T cell response has also been used as an indicator of past infection that may complement conventional serology.

Animal Infection Models Experimental animal models are important in the studies related to pathogenesis, transmission and the effectiveness of therapeutics and vaccines. Experimental animal models for both SARS and MERS are sub-optimal, especially in relation to pathogenesis and there are only few that serve to investigate transmission.

SARS Experimental infection with SARS-CoV leads to virus replication in Cynomolgous and Rhesus macaques, African green monkeys and marmoset monkeys, with variable manifestations of the disease. Part of the variation in the development of the disease may relate to age (older animals develop more severe disease) or virus strains used. Mice (BALB/c, C57/BL6, 129SvEV-lineage), Syrian golden and Chinese hamsters, ferrets, and cats are also susceptible to SARS-CoV. Only some of these animals develop pathological lesions in the lungs (ferrets, hamsters, marmosets, aged BALB/ C mice). Interestingly, while SARS-CoV replicates in the lung of both young and aged (12–14 months) BALB/c mice, only aged mice manifest clinical symptoms and histological evidence of lung pathology. This is reminiscent of the disease in humans where children suffer only a mild form of the disease. Serial passages in mice has led to the development of virus strains (MA15) with increased pathogenicity in mice. Ferrets are susceptible to SARS-CoV infection and can transmit the virus to other ferrets. They develop fever and show variable histological changes in the lungs but there is no mortality. In most experimental models, viral titers peak early (within first 4 days) and the virus is cleared by the end of the first week, which is not comparable to the human disease. Furthermore, few animal models reproduce the gastrointestinal manifestations of the illness. While an ideal animal model for SARS pathogenesis or transmission is lacking, those that support viral replication (with or without clinical disease) are adequate ones for evaluating the efficacy of vaccines.

MERS Mice, ferrets, guinea pigs, ferrets and hamsters are not suitable experimental models to study MERS-CoV because the DPP4 receptors of these species do not support virus attachment. Human DPP4 has been transduced into mouse lungs by adenovirus vectors and these mice are susceptible to infection and they develop lung pathology. Human DPP4 (hDPP4) transgenic mice are susceptible to MERS-CoV infection but because the expression of hDPP4 was not physiological in its tissue distribution, infection lead to unusual manifestations such as central nervous system disease, which is not comparable to the one seen in humans. On the other hand, hDPP4 knock-in mice have key regions of the mouse DPP4 replaced with amino acid residues of the hDPP4 and these mice are susceptible to infection. Adapting virus strains by passage in such mice led to virus strains that caused a disease. Rabbits have DPP4 similar to that of humans and they are, therefore, susceptible to MERS-CoV infection. In contrast to humans, the distribution of DPP4 is predominantly in the upper respiratory tract and consequently, infected rabbits do not develop a lung disease. Marmosets are also susceptible to an infection and a disease although these findings have not been reproduced by different researchers. Rhesus macaques are susceptible to an infection and they manifest a mild form of disease. Dromedary camels, alpacas and llamas are all susceptible to experimental MERS-CoV infection and they can be used as experimental models for infection and transmission. Camels have been used to study the effectiveness of vaccines.

822

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

Pathogenesis SARS The primary mechanism of lung damage appears to be due to infection of type 1 and type 2 pneumocytes which are the key target cells of the virus. Type 2 pneumocytes are important in the repair of lung injury and infection of these cells can potentially impair the regenerative processes of the lung and aggravate the respiratory impairment. While mice deficient in NK, T or B lymphocytes display similar kinetics of viral replication to that seen in normal mice, infection of mice with defects in the STAT1 signaling pathway results in a more prolonged viral replication and a more severe disease. These findings indicate the importance of innate immune responses in the control of the infection, at least in the mouse. Infection of epithelial cells, macrophages, and myeloid dendritic cells fails to induce a type 1 interferon response although IFN-inducible genes are activated. Multiple accessory proteins mediate innate immune evasion leading to innate immune dysregulation. Patients with a severe SARS had higher plasma levels of IFN-g, IL-1, IL-6. IL2, TGBb, CCL2, CXCL10, CXCL9, and IL8 compared to those with an uncomplicated disease. There is evidence of viral replication within intestinal epithelial cells but there is minimal cellular infiltrate or disruption of intestinal architecture and the pathogenesis of diarrhea in SARS remains unclear.

MERS-CoV DPP4, the receptor for MERS-CoV is less abundant in upper respiratory tract epithelium but it is expressed in the epithelial cells of the distal airways and alveoli as well as in the kidneys, intestine, liver, thymus and the bone marrow. Patients with a chronic lung disease have an increased DPP4 expression. Limited autopsy studies and in ex vivo cultures of human lung specimens infected with MERS-CoV have identified evidence of virus infection in ciliated epithelial cells of the bronchioles, type 1 and 2 alveolar epithelial cells in the lung, alveolar macrophages, lung microvascular endothelial cells, renal proximal tubular epithelial cells and human primary intestinal epithelial cells or small intestine explants. The DPP4 distribution and tropism of the virus explains why MERS-CoV causes severe lower respiratory tract infections with minimal upper respiratory symptoms and also explains the mild renal dysfunction observed in patients with MERS and virus detection in the stool and urine in some patients. Unlike SARS-CoV, MERS-CoV can infect human dendritic cells and can infect activated T cells leading to apoptosis. Thus, MERS-CoV may impair adaptive immune responses. As with SARS, MERS-CoV has potent mechanisms for innate immune evasion which contribute to innate immune dysregulation and pathogenesis. Patients with a severe MERS had higher plasma levels of IL-6, IL8, CXCL10 and CCL5 compared those with a mild disease.

Treatment In the absence of specific antiviral therapies of proven efficacy, good supportive care remains the major form of therapy for both SARS and MERS.

SARS SARS emerged as a disease of unknown etiology and empirical therapeutic options were initially tested including broad spectrum antivirals and immunomodulators such as ribavirin, intravenous immune globulin, type 1 interferon, SARS convalescent plasma and corticosteroids. However, in the absence of controlled clinical trials, no conclusions can be drawn on the efficacy or adverse effects of these interventions. A clinical trial of 400 mg lopinavir with 100 mg ritonavir orally every 12 h (added to an existing regimen of ribavirin and corticosteroid therapy) appeared to provide clinical benefit compared to historical controls. However, the lack of concurrent controls makes it difficult to draw conclusions. Similarly, a limited clinical trial of 13 patients using interferon alfacon-1 treatment showed a trend toward improved radiological and clinical outcomes, without, however, reaching a statistical significance. A meta-analysis of observational studies of passive immunotherapy for SARS suggested a decrease in mortality with therapy with convalescent plasma from patients with SARS, particularly if there was evidence of the presence of neutralizing antibodies to SARS-CoV. Studies in primate models demonstrated prophylactic and therapeutic benefit for PEGylated recombinant interferon a-2b and from small interfering RNA therapies. Anti-SARS-CoV activity has been demonstrated in vitro, and sometimes in experimental animal models, for several therapeutic substances already in clinical use for other diseases, including lopinavir–nelfinavir, glycyrrhizin, baicalin, reserpine and niclosamide. Screening of combinatorial chemical libraries in vitro has lead to identification of potential inhibitors of the viral protease, helicase and spike protein-mediated entry.

MERS Given the promising observational data with the use of convalescent antibody therapy with SARS, a similar approach was attempted for MERS. However, the poor convalescent antibody levels and waning immunity in patients with MERS and because

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

823

many patients are elderly or have co-morbid conditions, it turned out very difficult to collect adequate amounts of convalescent plasma for good clinical trials. A human polyclonal antibody to MERS-CoV produced in transchromosomic cattle has been shown to be safe in phase 2 clinical trials (AIMS). Similarly, several anti-S monoclonal antibodies with neutralizing activity against MERS-CoV are under development and they have shown in vivo efficacy in transgenic mice and Rhesus macaque models. Some of these antibodies are proceeding to phase 1 clinical trials. Corticosteroid treatment of MERS-CoV infected patients was not significantly associated with a difference in mortality but it was associated with a delay in MERS-CoV RNA clearance suggesting a lack of efficacy of corticosteroids against SARS-CoV infection. Based on the data from in-vitro and Rhesus macaque challenge studies where high doses of ribavirin and IFN-a2b administered within 8 h post challenge showed partial efficacy in reducing clinical symptoms and viral loads, varying combinations of ribavirin with several interferons (IFN-a2b, IFN-a2a, IFN-b1a) have been used to treat patients with MERS. A retrospective cohort study showed no effect on mortality or viral load, after adjusting for confounding factors. It is now recognized that ribavirin concentrations required to inhibit MERS-CoV need to be far higher than what is clinically acceptable for humans. A double-blind randomized trial comparing a combination of lopinavir/ritonavir and recombinant IFN-b1b to placebo is presently ongoing in Saudi Arabia.

Vaccines and Immunity SARS A wide range of strategies have been explored for the development of SARS vaccines, including inactivated whole virus vaccines, subunit vaccines (baculovirus expressed S1 subdomain or the complete trimeric spike protein of the virus expressed in mammalian cells), DNA vaccines expressing S (full-length and fragments), N, M, or E proteins; and vectored vaccines based on modified vaccinia Ankara (MVA) virus, vesicular stomatitis virus, adenoviral vectors carrying S, M, or N proteins and attenuated parainfluenza virus type 3 vectored vaccines carrying S, E, M, and N proteins. Neutralizing antibody responses and where appropriate, cell-mediated immune responses have been measured as correlates of immunity. Some of these vaccines have been evaluated in experimental models by challenging with infectious SARS-CoV. It has been shown that the S protein is the principal immunogen that induces antibody response with neutralization-mediated protection. Passive immunization with human monoclonal antibodies against the S protein has been successful at protecting mice and ferrets from an experimental challenge by reducing viral load in the lungs but not in the nasopharynx. However, S proteinbased vaccines have also been shown to elicit Th2 immunopathology following experimental live virus challenge. A newly emerged SARS outbreak will probably arise from an animal reservoir and it is, therefore, important to investigate crossprotection against animal SARS-like CoV. While human SARS-CoV S protein was neutralized by antibodies to the civet SARS-like virus, civet-like S protein was not neutralized by antibodies against the human SARS-CoV. On the other hand, antibodies against human SARS-CoV appeared to enhance the infectivity of the GD03 and SZ3 pseudo-typed viruses. The development of vaccines that can prevent re-emergence of SARV-CoV from its zoonotic reservoir remains a challenge.

MERS There are no licensed vaccines against MERS so far, although there are some in clinical trials. Pre-clinical studies have shown that both antibody responses to the receptor binding domain of the spike protein and cellular immune responses are required for full protection against MERS-CoV infection. In spite of genetic heterogeneity of MERS-CoV in Africa, virus neutralization epitopes remain conserved and a protective neutralizing antibody-based vaccine to any MERS-CoV strain is likely to cross-protect against most MERS-CoV strains currently circulating in camels. MERS vaccines based on the virus spike protein, live-attenuated, viral vectored vaccines, DNA vaccines and virus-like particle-based vaccines are in the development and they have undergone initial dose-response tolerability and immunogenicity studies. T cell responses have been consistently observed in MERS survivors and they are more long-lasting than antibody responses. In pre-clinical models, intranasal vaccination with the N protein was shown to generate airway memory CD4 T cells responses that protected against a challenge against multiple CoV. As with SARS, the possibility of disease enhancement by vaccination needs to be considered. Pulmonary Th2-immunopathology, associated with eosinophilic infiltration and increased pro-inflammatory cytokines in the lungs has been observed after immunization with an inactivated MERS vaccine followed by a wild-type MERS-CoV challenge in a transgenic mice model. Vaccines have also been developed for vaccinating dromedary camels with an idea of reducing zoonotic infections. A vaccinia (MVA) vectored vaccine administered by an intranasal route was shown to protect camels from an experimental challenge. However, an experimental challenge was carried out within a few weeks of vaccination and it remains unclear whether a vaccine will provide protection for several years in field-settings. It is known that a natural infection does not protect seropositive camels against mucosal re-infection of the nasopharynx. It may be that mucosal immunity is more important than systemic serum antibody-mediated. On the other hand, it is possible that even if the vaccine is not providing sterilizing immunity, vaccination may lead to lower peak viral titers, shorter duration of virus shedding, reduced transmission within camel herds and reduce zoonotic transmission. An important aspect of acceptance of such a vaccine by camel owners is the fact that it also protects against camel pox, which is considered as a more important disease of camels than MERS-CoV infection which is a very mild illness in camels.

824

Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Coronaviridae)

Conclusion Given that the precursor viruses of SARS remain in bats, it is conceivable that SARS may return as a public health threat. However, the wild game animal markets which allowed the virus to transmit to humans are now prohibited in mainland China, thus reducing the possibility of potentially new SARS-like viruses to infect humans. However, the situation is very complex, which is evidenced by the emergence of COVID-19 pandemic caused by a SARS-CoV-2 which likely originated from wild game. MERS-CoV continues to cause zoonotic infections, followed by outbreaks in health care facilities associated with transmission between humans. Such repeated outbreaks may provide the virus an opportunity to further adapt to humans in order to become a virus with a higher potential to infect humans. In addition, we do not know how extensive MERS-CoV infection is in camelexposed humans in Africa where 470% of infected dromedary camels are found. It is important to understand the extent of zoonotic transmission and disease, if any, in Africa.

Further Reading Arabi, Y.M., Balkhy, H.H., Hayden, F.G., et al., 2019. Middle East respiratory syndrome. New England Journal of Medicine 376, 584–594. Channappanavar, R., Perlman, S., 2017. Pathogenic human coronavirus infections: Causes and consequences of cytokine storm and immunopathology. Seminars in Immunopathology 39, 529–539. Fehr, A.R., Channappanavar, R., Perlman, S., 2017. Middle East respiratory syndrome: Emergence of a pathogenic human coronavirus. Annual Review of Medicine 68, 387–399. Peiris, J.S.M., Lai, S.T., Poon, L.L., et al., 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 1319–1325. Peiris, J.S.M., Guan, Y., Yuen, K.Y., 2004. Severe acute respiratory syndrome. Nature Medicine 10 (12 Suppl), S88–S97. Song, Z., Xu, Y., Bao, L., et al., 2019. From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses 11, 59. Sikkema, R.S., Farag, E.A.B.A., Islam, M., et al., 2019. Global status of Middle East respiratory syndrome coronavirus in dromedary camels: A systematic review. Epidemiology and Infection 147 (e84), 1–13. [2019]. Zaki, A.M., van Boheemen, S., Bestebroer, T.M., Osterhaus, A.D.M.E., Fouchier, R.A., 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. New England Journal of Medicine 367, 1814–1820.

Relevant Website https://www.who.int/emergencies/mers-cov/en/ WHO. Middle East respiratory syndrome coronavirus (MERS-Cov).

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Coronaviridae) Malik Peiris, The University of Hong Kong, Pok Fu Lam, Hong Kong r 2021 Published by Elsevier Ltd.

Glossary Lymphopenia Reduction in the lymphocytes in the circulating blood below the normal range for age. Reproduction number The number of secondary infections, on average, generated by one infected person. RNA Ribonucleic acid. Non-pharmaceutical interventions Public health measures other than drugs or vaccines that are used to contain an infectious disease outbreak. Acute Respiratory Distress Syndrome (ARDS) Occurs when fluid builds up in the tiny, elastic air sacs (alveoli) in the lungs leading to reduced oxygenation of the blood.

CD4 T cells T cells bearing the cluster differentiation marker 4. These T cells have a helper function in both cell-mediated and antibody-mediated immune responses. CD8 T cells T cells bearing the culture differentiation marker 8. These T cells have a cytotoxic function and recognize and kill other cells that express “foreign” antigens which may be of viral or malignant origin. JAK inhibitors inhibitors of the Janus kinase (JAK)/signal transducers which are activators of the transcription (STAT) pathway are linked to various cytokines and are involved in a variety of immune-mediated and inflammatory diseases.

An unusual cluster of severe pneumonia was noted in Wuhan, China, in December 2019. The etiological agent was identified to be a novel coronavirus closely related, but not identical to, the virus that caused SARS in 2003. The virus was named SARS coronavirus 2 (SARS-CoV-2) and the disease coronavirus virus disease 2019 (COVID-19). Within months, the virus spread to cause a pandemic which has had catastrophic impacts on global health, economy and society. SARS-CoV-2, SARS-CoV-1 (causing SARS epidemic in 2003) and related viruses found in Rhinolophid bats are classified within the sub-genus Sarbecovirus, genus Betacoronavirus, Family Coronaviridae. Closely related viruses have been found in Rhinolophus genus bats, notably RaTG13 and RMYN02, which respectively, share 96.3% and 93% nucleotide identity with SARS-CoV-2. RMYN02 and SARS-CoV-2 both have furin cleavage sites within the spike protein while RaTG13 does not. The natural reservoir from which SARS-CoV-2 emerged is likely to be bats of the Rhinolophus genus. What remains unclear is whether there were intermediate hosts that facilitated transfer and adaptation of the precursor virus to humans. The receptor used by SARS-CoV-2 to gain entry to cells is angiotensin-converting enzyme 2 (ACE-2). The median incubation period of SARS-CoV-2 infection is around 5 days (range 2–14 days) and the reproduction number (Ro) is estimated to be 2.5. Infected persons may transmit infection from 1 to 2 days prior to onset of symptoms to around 7–10 days after symptom onset. However, severely ill patients and immunocompromised individuals may be infectious for longer periods of time. Pre-symptomatic as well as asymptomatic infections may lead to transmission. The virus is transmitted via large respiratory droplets or respiratory aerosols, predominantly over close range (a few meters) although there are occasional instances of transmission over greater distances. Crowded indoor environments are more conductive to transmission and singing or loud speaking by infected individuals increases the risk of transmission. The virus remains viable for many hours on smooth surfaces (stainless steel, glass, plastic) but survival is much shorter on non-porous surfaces such as cloth or paper. Therefore, indirect transmission from contaminated surfaces via hands to eyes, nose or mouth may potentially contribute to transmission. While the virus RNA can be detected in feces for prolonged periods, infectious virus has infrequently been detected and the degree of infectiousness of feces remains unclear. Super-spreading events are prominent drivers of transmission. In the early stages of the pandemic, non-pharmaceutical interventions including case detection, isolation, contact tracing, quarantine, physical distancing, reduction of mobility and travel related measures were successfully used to reduce transmission. Symptoms include fever or chills, cough, shortness of breath or difficulty in breathing, fatigue, muscle or body aches, headache, sore throat, congestion or runny nose, nausea, vomiting or diarrhea. Loss of smell or changed sense of taste is frequently reported and is associated with infection and damage of olfactory neurones in the nasopharynx. Progression of clinical disease may lead to hypoxia and acute respiratory distress syndrome (ARDS). Radiological changes include bilateral ground glass opacities and alveolar exudation. Lymphopenia with increased serum transaminase, C-reactive protein and d-dimer levels are commonly seen in severe cases. Progression of disease is associated with difficulty in breathing, leading to ARDS, sometimes leading to a fatal outcome. The overall infection fatality risk increases progressively with age; those aged 15–44, 65–74 and 475 years having infection fatality risks of 0.03%, 3.1% and 11.6%, respectively; males roughly having twice the risk of females across age spectrums. COVID19 infection in children and young adults is often mild or asymptomatic. The presence of co-morbidities including heart, respiratory, renal and liver diseases, cancer, diabetes and obesity increase the risk of severe infection and fatal outcome. Molecular detection of SARS-CoV-2 RNA is the mainstay of diagnosis. Detection of viral protein (usually nucleoprotein) by rapid antigen detection tests give more rapid results and are sensitive in detecting specimens with high viral load who have highest transmissibility of infection. Antibody responses to multiple viral proteins (spike, nucleoprotein, ORF8) and virus neutralizing antibodies are progressively detectable towards the end of the first week after onset of symptoms, and are detectable in most

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00155-7

825

826

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Coronaviridae)

patients by the end of the third week of infection. Neutralizing antibodies target the spike protein and is protective. CD4 and CD8 T cell responses are also elicited following infection but their role in protection remains to be elucidated. Direct viral damage as well as immunopathology contribute to pathogenesis, a hyper-inflammatory state being observed in severely ill patients. An intravascular coagulopathy also contributes to pathogenesis, often involving the microvasculature but sometimes leading to thrombosis of large blood vessels with poor prognosis. Supportive care in the management of patients include provision of supplemental oxygen or mechanical ventilation as and when required. Randomised clinical trials are beginning to identify specific therapies with proven clinical efficacy and this is a fast-moving area of knowledge. There is emerging consensus for the beneficial use of corticosteroids in those patients who require supplemental oxygen or mechanical ventilation. The antiviral drug remdesivir improves time to recovery but does not appear to provide survival benefit when used by itself. However, a combination of remdesivir with immunomodulators (e.g., JAK inhibitors such as barcitinib) may provide improved benefit. There has been a rapid progress in developing and evaluating COVID-19 vaccines. These have included protein subunit, viral vectored (e.g., adenoviral vectors) and RNA vaccines targeting the viral spike protein; and inactivated whole virus vaccines which elicit immune responses against the structural proteins of the virus. By the end of the year 2020 phase 3 trial data show acceptable levels of efficacy and safety with RNA and adenoviral vectored vaccines targeting the virus spike protein providing evidence that the viral spike is a protective antigen. In December 2020 some countries have started to vaccinate their populations. The duration of vaccine induced protection remains unknown. Most clinical trials evaluate protection from virologically confirmed symptomatic clinical disease and it is unclear whether there will be comparable impact in reducing transmission, a question of key relevance in disease control and population immunity.

Further Reading Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nature Microbiology 5, 536–544. Chin, A.W.H., Chu, J.T.S., Perera, M.R.A., et al., 2020. Stability of SARS-CoV-2 in different environmental conditions. Lancet Microbe 1 (1), e10. doi:10.1016/S2666-5247(20)30003-3. Hu, B., Guo, H., Zhou, P., Shi, Z.L., 2020. Characteristics of SARS-CoV-2 and COVID-19. Nature Reviews Microbiology 6, 1–14. Krammer, F., 2020. SARS-CoV-2 vaccines in development. Nature 586, 516–527. Leung, G.M., Cowling, B.J., Wu, J.T., 2020. From a sprint to a marathon in Hong Kong. The New England Journal of Medicine 382 (18), e45. Mann, R., Perisetti, A., Gajendran, M., et al., 2020. Clinical characteristics, diagnosis, and treatment of major coronavirus outbreaks. Frontiers of Medicine 7, 581521. doi:10.3389/fmed.2020.581521. Vabret, N., Britton, G.J., Gruber, C., et al., 2020. Immunology of COVID-19: current State of the Science. Immunity 52, 910–941.

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae) Phyllis J Kanki, Harvard T.H. Chan School of Public Health, Boston, MA, United States r 2021 Elsevier Ltd. All rights reserved.

Glossary Beta chemokines A family of cellular proteins produced in response to acute or chronic inflammation to attract a variety of white blood cells. They bind to cellular targets by specific beta chemokine receptors, that are one of the members of the 7-transmembrane chemokine receptors. Beta chemokines include: CCR5, MIP1a and MIP 1b. CCR5 A cell membrane protein expressed on several cell types including peripheral blood-derived dendritic cells, CD34 þ hematopoietic progenitor cells and certain activated/memory Th1 lymphocytes. This receptor is well defined as a major co-receptor for primate lentiviruses, in conjunction with CD4 þ , the major cellular receptor. CCR5 is implicated in susceptibility to HIV-1, HIV-2 and some SIV infections. CD4 þ T cell An immune cell, lymphocyte (white blood cell) characterized by the CD4 þ antigen (protein) on its surface. This is a T lymphocyte considered to have a “helper” function to enhance the cellular immune response. The CD4 þ is the primary receptor for SIV and HIV, and upon infection the virus can destroy the CD4 þ cell. The drop in CD4 þ T lymphocyte cells is a major determinant of the progression of SIV/HIV infection to AIDS. CD4 þ A large glycoprotein that is found on the surface of helper T lymphocyte cells, regulatory T cells, monocytes, and dendritic cells. Its natural function is as a co–receptor that assists the T cell receptor (TCR) to activate its T cell following an interaction with an antigen-presenting cell. CD4 þ is the primary receptor used by SIV and HIV viruses to enter and infect host T cells. Chemokine receptor A member of the family of seventransmembrane G-protein-coupled protein molecules on the surface of certain cells that interact with specific chemokines to activate various cellular functions. Chemokine receptors on lymphocytes or monocytes bind to the envelope protein of SIV or HIV and serve as a coreceptor with CD4 (the primary cellular receptor for SIV and HIVs), allowing fusion and entry of the virus into the susceptible cell. HIV-2 The second HIV virus discovered in registered sex workers in Dakar, Senegal in 1984, the virus is more closely related to the simian immunodeficiency virus (SIV). There are 9 lineages of HIV-2 described, although only A and B are

thought to transmit in human populations. Although HIV-2 can cause AIDS, it has a distinct epidemiology, lower rates of transmission and slower progression to disease. Long-term non progressors HIV-1 infection and rate of disease progression is variable but the majority of infected patients in the absence of therapy develop AIDS. HIV-1 infected patients with greater than 8–10 years of infection with high CD4 þ cell counts have been considered longterm non progressors. Long-term non progressors can be further characterized based on the stability and level of their plasma viral load. Old World primates The first monkey species of the infraorder Anthropoidea are thought to have arisen B34 million years ago (Oligocene), New World monkeys appeared 30 million years ago most probably originating in Africa but moving to South and North America. The technical distinction between New World platyrrhines (flat sideways facing nostrils) and Old World catarrhines (downward facing nostrils) is the shape of their noses. The first apes lived in African forest B20 million years ago evolving as a division within the Catarrhine suborder. Two ape genera are endemic in Africa: Pan (chimpanzee and bonobo) and Gorilla (gorilla). The monkey superfamily of ancestral catarrhine primates or Old World monkeys (family Cercopithecidae) in current day are represented by 71 species. Most Old World monkeys are limited to sub-Saharan Africa, although macaques (Macaca sp.) are found in Asia. SHIV Recombinant SIV/HIV chimeric viruses generated in the laboratory, in which subsets of viral genes from one parent virus (SIV) were replaced with the homologous regions of the other (HIV). This has allowed researchers to study particular aspects of virus infection or immunity. SHIVs have been useful in HIV-1 vaccine development research. SIV A primate lentivirus, first discovered in captive macaques with a simian AIDS syndrome. SIV is known to naturally infect B40 African Old World primate species without apparent disease. Cross-species transmission events of ancestral SIV gave rise to the origin of HIV-1 and HIV-2. TRIM5a TRIM5a belongs to the large family of tripartite motif (TRIM) proteins with many cellular functions including antiviral defense.

Introduction The virus responsible for human acquired immunodeficiency syndrome (AIDS) was discovered in the early 1980s. This new virus was a member of the Lentivirus genus of the Retroviridae family of RNA viruses and later designated as Human immunodeficiency virus type 1 (HIV-1). The first simian lentivirus, Simian Immunodeficiency Virus (SIV) was identified in 1984 from immunodeficient captive rhesus macaques (Macaca mulatta), its relatedness to HIV was demonstrated by serologic cross-reactivity to HIV-1 viral antigens. In 1985, serum samples from registered female sex workers in Dakar, Senegal, demonstrated unusual immunoblot

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21277-6

827

828

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

reactivity to HIV-1 with strong reactivity to gag and pol antigens but minimal reactivity to the envelope proteins, suggesting infection with a related virus. The samples were subsequently tested for antibody reactivity to the recently described SIV and they reacted strongly with the envelope proteins, indicating that this new human virus was more closely related to SIV than to HIV-1. The close antigenic relatedness of both SIV and HIV-2 to the prototype HIV-1 virus prompted both the discovery and further classification of these related viruses.

SIV and HIV-2-Taxonomy and Classification SIV and HIV are viruses belonging to the Lentivirus genus in the family of Retroviridae. The term lentivirus refers to the long incubation period that has been a hallmark of this group of viruses. There are 45 known primate lentiviruses which naturally infect various African Old World monkeys and apes. Phylogenetic analyzes have revealed that HIV-1 and HIV-2 are human viruses that resulted from multiple cross species transmissions from monkey and ape SIVs. HIV-1 resulted from four independent cross-species transmissions from chimpanzees (SIVcpz) and gorillas (SIVgor) as well as recombination events. SIV cpz in humans gave rise to the HIV-1 groups M and N. The HIV-1 group O viruses are thought to have arisen from SIVgor but may also have been independently transmitted from a related SIVcpz. Within the SIVs, there is phylogenetic evidence that at least five of them arose from recombination events between two or more parental SIV strains. HIV-2 in humans resulted from multiple transmissions of SIVsmm (simian immunodeficiency virus, sooty mangabey), this SIV grouping also includes the SIV viruses originally found in captive macaques with an immunodeficiency syndrome similar to human AIDS. Molecular clock studies have estimated the most recent common ancestor of HIV-2 was introduced into humans between 1920 and 1940. Since HIV-20 s initial discovery, nine distinct lineages have been identified, only groups A and B are endemic, while the other lineages are represented by 1–2 reports. Individuals harboring non-A and non-B HIV-2 were asymptomatic at the time of the virus description, while the lack of secondary transmission suggests that not all cross-species transmission from SIVsmm monkeys have been successful from the virus’ standpoint. Phylodynamic analysis of HIV-2 sequences indicate Guinea Bissau as the geographic origin of Group A and Ghana as the geographic origin for Group B. The global epidemiology of HIV-2 is supported by social-political events such as wars, colonialism, immigration patterns and other population movements.

SIV and HIV-2-Virion Structure Primate lentiviruses have a common morphology with viral particles 80–110 nm long and 25–50 nm wide, consisting of an electron dense conical or wedge-shaped nucleoid composed of the viral capsid that houses a pair of single-stranded RNA genomes and viral enzymes. The virion is covered by a host cell derived membrane with viral envelope glycoprotein spikes. The envelope spike proteins are more prominent on SIV and HIV-2 compared with HIV-1.

SIV and HIV-2-Genome The primate lentiviruses have a similar organization of open reading frames within the B9 kb genome. The genome is flanked by palindromic 50 and 30 long terminal repeat sequences important to viral replication. There are three structural genes, gag (group specific antigen), pol gene encoding the polymerase including the protease, reverse transcriptase and integrase enzymes and env (envelope). There are five more accessory genes, vif (viral infectivity factor), tat (trans activator), and rev that are involved with viral transcription, while vpr, nef, vpx and vpu are variability represented depending on the SIV/HIV grouping (Fig. 1). The gag and pol messages are unspliced, the env singly spliced, and the major regulatory genes doubly spliced and derived from multiple messages. SIV and HIV-2 target the CD4 bearing cells including helper lymphocytes, monocytes, macrophages and microglia cells in the brain. Several members of the seven-transmembrane G-protein-coupled receptor family function as coreceptors in association with CD4 to permit viral entry and infection. Different from HIV-1, both HIV-2 and SIVs demonstrate more promiscuous utilization of coreceptors including CCR1–5, CCR8–10, CXCR4–6, GPR1 and GPR15 and many others, some of them associated with specific SIV species. In the case of HIV-2, use of coreceptors other than CCR5 would impact the efficacy of antiretroviral therapy (ART) drugs whose mechanism of action is to block viral infection via CCR5 coreceptor usage.

SIV and HIV-2 Lifecycle The replication cycle of SIV and HIV-2 include the following steps: (1) adsorption to the major receptor, CD4 and relevant coreceptor with subsequent fusion to the host cellular membrane, (2) penetration and uncoating; (3) reverse transcription is carried out by the viral reverse transcriptase, resulting in a DNA copy of the viral RNA genome; (4) transport of viral DNA to the nucleus; (5) integration of the viral DNA into the host cell DNA which is facilitated by the viral integrase; (6) transcription of the

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

SIVagm, SIVmnd-1

5’LTR

5’LTR

5’LTR

vpx

3’LTR

tat

rev

vif

pol gag

rev

tat

pol

rev

tat

tat

rev

rev

vpr

nef 3’LTR

env

vpr

vif

nef

env

vpr

gag

HIV-1, SIVcpz

tat

rev

vif

gag

HIV-2, SIVmac, SIVsmm

tat

pol

829

vpu env

nef

3’LTR

Fig. 1 Genomic structure of representative primate lentiviruses.

viral mRNA and splicing; (7) translation of viral structural and accessory proteins; (8) transport of the viral structural proteins to the cytoplasmic membrane and virus assembly; and (9) viral budding, virion maturation and release.

SIV Epidemiology SIV was discovered in captive Asian rhesus macaques dying with a syndrome similar to human AIDS. Subsequent studies could not identify SIV in Asian macaques in the wild, whereas numerous African monkeys were found to have high prevalence rates of SIV. In 1986, studies to transmit leprosy from a captive sooty mangabey to a rhesus macaque resulted in simian AIDS, this solidified the evidence that SIV was not a natural infection in the Asian macaque and the artificial infection with the African SIVsmm was pathogenic. Natural SIV has now been described in 40 different African Old World monkeys and anthropoid apes but not Asian Old World monkeys, such as macaques, in the wild. This suggests that the SIV emerged after the Old World primates began speciation within Africa. Although not extensively studied in the wild, it is considered that SIV in the African monkey and ape is typically nonpathogenic. This is supported by the high prevalence rates founds in various serologic surveys, and high levels of viremia despite robust cellular and humoral immune responses to the virus. It has been postulated that a number of host immune mechanisms may distinguish natural SIV infection from the pathogenic HIV/SIV. In at least some African Old World monkeys like the African Green monkey and sooty mangabey, there appears to be host cell restriction to SIV virus entry, mediated by both CD4 and CCR5. Natural SIV infections also appear to resolve immune activation by the virus which thereby preserves central memory CD4 þ T cells, the major target in development of immunodeficiency. The nef gene of certain natural SIVs have the ability to downmodulate CD3-TCR from the surface of CD4 þ T cells, rendering them resistant to activation. This function has been lost in the SIVs from chimpanzees or HIV-1, apparently linked to the high levels of chronic immune activation and pathogenic outcomes.

SIV Clinical Features SIV in the natural African primate host is generally non-pathogenic. However, SIV in the Asian macaque host causes an immunodeficiency syndrome marked by profound destruction of CD4 þ T cells in the periphery and lymphoid organs throughout the body. The end-stage disease can include opportunistic infections, SIV meditated inflammatory disease, and cancer. The clinical course is dependent on the SIV strain and host macaque species with development of AIDS-like disease between 6 and 24 months. Similar to HIV-1 infection in humans, SIV replicates to high levels in the macaque host with viremia as high as 109 viral RNA copies per ml of plasma. The level of SIV RNA in the plasma is an indicator of clinical prognosis in the Asian macaque. The gastrointestinal tract and other mucosal sites are a major site for SIV induced CD4 þ T cell destruction with accompanying immune activation and lymphocyte proliferation. Polyclonal B-cell activation results in hypergammaglobulinemia similar to the human condition. Anemia, weight loss and diarrhea are common clinical features of the disease. Lymphomas are the most common neoplasia seen in SIV infected macaques. Unlike the pathogenesis of infection in the natural African primate, chronic immune activation and inflammation appear to contribute to disease pathogenesis. SIV crosses the blood-brain barrier and infects

830

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

cells of the macrophage lineage resulting in viral encephalitis. In addition, SIV antigen and antibody complexes in the kidney can result in immune complex glomerulonephritis. SIV in the Asian macaque has been studied intensively as a model of HIV-1 infection and pathogenesis. Notably, SIV disease pathogenesis in the Asian macaque demonstrated the importance of lymphoid organ and gut-associated lymphocyte infection and destruction in disease pathogenesis. This model system has also been used to better understand viral and host parameters involved in HIV sexual transmission, including oral, anal and vaginal routes of infection. Anatomic and physiologic similarities of the macaque with humans has increased the relevance of the system to understand the genotypic and phenotypic viral characteristics involved in sexual transmission. Transmission of genetically diverse SIV populations has been shown in the macaque model similar to HIV infection in humans and recombination of viral variants has also been demonstrated after mucosal SIV exposure. SIV has also been useful to better understand HIV perinatal transmission particularly breast milk transmission. Female macaques can be SIV infected after delivery and the infant allowed to breast feed, this approach reduces the variables involved in fetal exposure and transplacental maternal antibody transfer. This has allowed researchers to better understand the dynamics of SIV and HIV breastmilk transmission. There is significant heterogeneity in the disease progression dependent on the specific SIV strain, molecular clone or Asian macaque species host. In some instances, this diversity has been useful, such as the discovery of SIV variants with specific tropism for monocyte/macrophages that result in SIV meningoencephalitis. However, the differences between HIV-1 and SIV cellular tropism mediated by the CD4 þ and coreceptors resulted in an imperfect model to study potential HIV vaccine candidates at the interface of the HIV-1 envelope and vaccine induced immune response. As a result, researchers developed the chimeric SHIV virus consisting of the SIV backbone with an HIV envelope in order to test for the generation of HIV neutralizing antibodies and its ability to protect from a SHIV challenge virus in the macaque host.

Immunity to SIV SIV in the Asian macaque has also been extensively studied to better understand HIV immunity. HIV-1 could not infect Old World primate cells which led to the discovery of intrinsic host factors such as TRIM5a with retrovirus-specific antiviral activity. During viral entry into a susceptible host, TRIM5a binds the viral capsid blocking its function prior to viral integration into the host DNA. Other intrinsic antiviral factors including APOBEC3G, BEST-2/tetherin and SAMHD1 were discovered through SIV studies and likely play a role in the viral host range and transmission of these viruses in nature. In the Asian macaque SIV model there is little evidence for innate immune responses with only slight increases in type 1 interferon and interferon-stimulated gene levels at the site of infection. At the peak of viremia however there is a dramatic increase in innate antiviral immune response in all lymphoid tissues and SIV-specific CD8 þ T cells are seen shortly after the peak viremia. The control of SIV replication by CD8 þ lymphocytes has been demonstrated in depletion studies using antibodies to the a-chain of the CD8 complex, which is accompanied by corresponding increases in SIV viremia. Similar to HIV infection in humans, detailed studies of well characterized MHC-I-restricted T-cell epitopes in SIV infected macaques has shown targeting of specific SIV peptide sequences with the evolution of viral variants, some of which are capable of escaping the response and increasing the resultant viral population. The B cell or humoral immune response also plays a role in controlling SIV replication as demonstrated by the transfer of high titer SIV specific-gamma globulin to inhibit SIV replication and slow the progression to disease. B-cell depletion studies have resulted in rapid uncontrolled SIV replication and a shortened course to SIV disease.

Epidemiology of HIV-2 In 1985 when HIV-2 was discovered, the number of AIDS cases reported in West Africa was quite low. The original Senegalese sex workers that had evidence of HIV-2 were healthy, and testing of hospitalized patients that might have AIDS or an AIDS-like disease revealed HIV-1 infection and often a connection to Central Africa but not HIV-2. In 1986, the isolation of HIV-2 (LAV-2) from an AIDS patient originating from Cape Verde, and subsequent AIDS case reports prompted the research community to fear a second global AIDS epidemic. Large scale serologic surveys conducted in healthy and hospitalized patients in West Africa showed varying rates of HIV-2 but the highest prevalence was in healthy adults rather than hospitalized patients. At that time, AIDS cases were reported at higher levels in Central Africa, yet there was no evidence of HIV-2 in AIDS or hospitalized patients from Zaire (DRC), Burundi, Tanzania, Kenya, Zambia and Cameroon. Although HIV-2 infection in sub-Saharan Africa was concentrated in West Africa, Mozambique and Angola were notable exceptions. These countries although distant from West Africa, were often on the same Portuguese trade routes as Guinea Bissau and Cape Verde, both West African countries with some of the highest rates of infection. In the early 1990s, there were reports of HIV-2 in southwestern India and Goa, a former Portuguese colony. These disparate geographic sites shared economic and political ties with Portugal and some of the first HIV-2 cases in Europe were Portuguese military veterans who had served in the war of independence in Guinea Bissau (1963–1974). In Europe, other former West African colonial powers such as France and England began to detect cases of HIV-2

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

831

infection, often with a connection to West Africa. In the late 1980s, France was one of the first countries to screen for HIV-1 and HIV-2 in their blood banks; in current times HIV-2 represents 1%–2% of new HIV diagnoses in France. Portugal is another country with significant HIV-2, likely due to their strong political and economic ties to West African countries such as Guinea-Bissau and Cape Verde. In 2009, over 1800 HIV-2 infections had been identified, one third of which were native borne Portuguese without a known West African connection; suggesting local transmission consistent with an endemic infection. Currently, longitudinal studies of HIV-2 infection in Guinea-Bissau, Senegal and the Gambia document the decline in HIV-2. In Guinea Bissau, the HIV-2 prevalence rate in the adult populations was 8.9% in 1987, but this was halved by 2007, with the highest rates of infection in the elderly. Modeling data from rural Caió in Guinea Bissau (1990–2007) predicts continued decline in HIV-2 prevalence and incidence with eventual extinction of the virus in this locale by the second half of the century.

Risk and Transmission of HIV-2 Similar to HIV-1, many of the risk factors for HIV-2 heterosexual transmission would include: unprotected sex with an HIV-2 infected partner, genital ulcer disease and lack of male circumcision. The prospective study of registered sex workers in Dakar began in 1985 and analysis of risk determinants of HIV-2 versus HIV-1 infection demonstrated virus-specific differences. The approximate log-linear relation of HIV-2 infection with increasing years of sexual activity was consistent with the hypothesis that the virus had been in the population for at least several decades. The most common modes of transmission in HIV-2-endemic areas are heterosexual and perinatal transmission; since Senegal like most West African countries had evidence of both HIV-1 and HIV-2 infections, measurement and comparison of incidence rates for both viruses was possible. In this high-risk group, the heterosexual transmission of HIV-2 was significantly slower than that of HIV-1, with HIV-1 increasing 12-fold over an 8-year period, while HIV-2 remained stable. This strongly suggested differences in the heterosexual transmission of these two related immunodeficiency viruses. In different mathematical modeling studies the male-to-female transmission rates of HIV-2 and HIV-1 were estimated with a 3.5–8.9 difference between HIV-1 and HIV-2 in infectivity per sexual act with an infected partner. The mathematical model of the concomitant transmission of the two viruses within the same sexually active population suggested a positive association between pathogenicity and reproductive success, indicating that HIV-1 would competitively displace HIV-2 in the long term. In the study of both viruses in Dakar, Senegal, over more than 25 years, the decrease of HIV-2 prevalence and incidence was accompanied by the increase in HIV-1 prevalence and incidence. Perinatal transmission of HIV-2 and HIV-1 has been studied in Guinea Bissau, Ivory Coast, France, and Senegal, with all demonstrating extremely low rates of HIV-2 perinatal transmission (0%–4% transmission) in contrast to that of HIV-1 (15%–45% transmission) in the absence of ART prophylaxis. In studies that measured perinatal transmission of both viruses, the rate of HIV-1 transmission was 10- to 20-fold higher than that of HIV-2 (Table 1).

Diagnosis of HIV-2 The close relatedness of HIV-2 to HIV-1 also meant that methods to diagnose the virus, through antibody, antigen, and even genetic material required specific assays. Blood bank screening for HIV-2 was instituted in the United States in 1990, following the lead of many European nations. As a result, the vast majority of commercial HIV ELISA assays utilize a combined antigen source that incorporates HIV-2 specific antigens along with HIV-1 antigens. However, confirmation of HIV-2 serostatus requires HIV-2 specific assays such as peptide specific assays or HIV-2 immunoblots. Immunoblots demonstrating a profile of antibody recognition of HIV-2 envelope proteins are typically used to confirm HIV-2 diagnosis. Early on it was recognized that HIV-2 was less prevalent globally and therefore commercial companies were slow to develop HIV-2 specific diagnostics. This became even more pertinent, when the description of HIV-2 and HIV-1 dually infected individuals was first noted in West Africa; relying heavily on unequivocal type-specific diagnosis of both viruses with type-specific peptide assays or nucleic acid diagnosis. In recent times, most national serologic surveillance surveys have not distinguished these virus types, and it is likely that in some regions of the world such as West Africa, the serologic surveys are detecting both HIV-2 and HIV-1 without distinguishing them. As a result, much of what is known about the current status of HIV-2 infection even in West Africa is based on research reports. Table 1

Key Characteristics of SIV, HIV-2 and HIV-1

Host Geographic distribution Plasma viral load Perinatal transmission Progression to AIDS

Natural SIV

HIV-2

HIV-1

African Old World monkeys and apes Sub-Saharan Africa species specific High Low Absent

Humans West Africa-historical ties to West Africa Low 0%–4% without ART Rare – more slowly

Humans Worldwide High B25% without ART High morbidity

832

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

Although numerous genetic tests for HIV-1 are approved and widely used in diagnosis and monitoring of therapy, this is lacking for HIV-2. Babies exposed to HIV-2 from their mothers would require an HIV-2 specific DNA test, which are not available except through a few research laboratories in the US and France. This has also compromised treatment efforts for HIV-2, where quantitation of HIV-2 viral load is required to monitor the success of ART therapy.

HIV-2 Clinical Features Early case reports described HIV-2-infected people with disease consistent with an AIDS diagnosis. The disease characteristics, including tuberculosis, chronic diarrhea, and Candida infections, which were similar to diseases seen in HIV-1-associated AIDS in the same settings. Central nervous system involvement has also been described in HIV-2 AIDS cases. However, classical African AIDS comorbidities, such as tuberculosis, often have had only a weak epidemiological association with HIV-2, even in HIV-2endemic areas. In addition, large serological surveys of healthy and AIDS patients failed to demonstrate a strong association of HIV-2 with AIDS and a prospective study was warranted. The prospective cohort study of Dakar sex workers initiated in 1985 compared disease progression in women with known time of infection with HIV-2 or HIV-1, with follow-up exceeding 22 years, representing one of the longest HIV natural-history studies in the literature to date. The majority of HIV-2-infected women (85%) were AIDS-free after 8 years of HIV-2 infection. Using a definition of long-term non-progression employed for HIV-1 infection this would’ve classified 95% of HIV-2 infected women as long-term non-progressors. In the absence of ART therapy, HIV-2 infected individuals studied in France showed a slower rate of CD4 þ cell decline (9 cells/ml/year) compared to HIV-1 infected individuals (49 cells/ml/year). In Guinea Bissau, the policeman cohort study has recently described a median time to AIDS for HIV-2 as 14 years as compared to a median time to AIDS of 6.2 years in HIV-1. Thus, it may be that HIV-2 infected individuals may follow a similar progression to AIDS as HIV-1 although much slower. Since this cohort is dominated by men in distinction from other HIV-2 natural history studies, the role of gender in disease progression warrants further study.

Pathogenesis of HIV-2 As studies continued to document HIV-20 s distinct differences in epidemiology, transmission and pathogenicity, other pertinent research questions emerged to identify and characterize the viral and host immune mechanisms responsible. Evidence for a lower viral burden in HIV-2-infected individuals was first reported from the decreased ability to isolate virus and later on quantitative PCR based DNA and RNA studies. While levels of HIV-2 proviral DNA were similar to those of HIV-1 proviral levels, they failed to correlate with levels of viral RNA. Thus, it appears that significant differences occur upon expression, release, and/or maintenance of HIV-2 virions in the bloodstream. Plasma viremia comparisons revealed a 30-fold lower median HIV-2 viral load compared to HIV-1 infected women, irrespective of the length of time infected. This reinforced the concept that plasma viremia was linked to the differences in the pathogenicity of the two related HIVs. HIV-1 viral integration mapping studies had demonstrated a major effect on viral transcription, however, mapping of HIV-2 integration sites during in vitro infection failed to identify a major difference between HIV-2 and HIV-1. However, accumulation of viral mRNA was attenuated in HIV-2 infection, relative to HIV-1. The differences in viral mRNA were consistent with the differences in plasma viral loads between HIV-1 and HIV-2, and suggested that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, could be explained by lower rates of viral replication in vivo. HIV diversity is considered a major determinant in the virus’ pathobiology. In studies of sequence diversity in the central HIV-2 envelope the rates of viral divergence and diversification were slower in individuals infected with HIV-2 compared to individuals with HIV-1. Viral evolution occurs slowly in HIV-2 infection, which is consistent with the slow disease progression, and supports the notion that viral evolution may be a relevant correlate for disease progression. In detailed analysis of the HIV-2 envelope, while the C2 and C3 regions appear to be well-exposed, similar to HIV-1, they appear to be structurally constrained and consequently mutations are thought to have a negative effect on virus fitness, this could represent a fundamental difference between the biology of the two HIVs and their interaction with the host immune response. Since slower disease course appeared to be common in HIV-2 infection, it was reasonable to consider that certain subsets of the population would possess host characteristics that might predispose them to a more rapid disease course. Some human leukocyte antigen (HLA) class I alleles have been associated with slower or faster rates of HIV-1 disease progression, suggesting that the presented epitopes or the restricting HLA class I molecule may play a central role in determining the ability to control viral replication. A case-control study investigated possible associations between HLA and the risk of disease progression in HIV-2. The HLA class I status was molecularly typed in female sex workers from the Dakar cohort; HLA B35 was associated with lack of p26 antibodies and higher risk of disease progression. The same association was found for the class I haplotypes B35-Cw4 and A23-Cw7, similar to the association with HIV-1. The data showed that certain HLA molecules are associated with risk of disease progression in HIV-2; some of the alleles and haplotypes involved in susceptibility to disease are similar for both HIV-1 and HIV-2. Therefore, certain genetic factors may be shared by HIV-1 and HIV-2 with respect to susceptibility to enhanced disease progression.

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

833

Immunity to HIV-2 HIV-2 Humoral Immunity The attenuated phenotype of HIV-2 infection in vivo has sparked considerable interest in understanding the immunopathogenesis of this particular HIV infection. Although certain viral determinants appear to be central to the lower replicative capacity in vivo, the virus appears uniquely immunogenic, which may be central to the explanation of the weaker, more attenuated phenotype of HIV-2 compared to HIV-1. The antibody response to most HIV-2 structural proteins occurs shortly after infection and is long lived. Antibodies to gag, pol, and env encoded antigens are routinely demonstrated with cross-reactivity in the gag and pol to HIV-1 analogous related proteins. IgG1 and IgG2 are the predominant antibody subclasses. Similar to HIV-1, the lack of antibodies to the p26 gag antigen was correlated with disease progression, but unlike the situation with HIV-1, this does not appear to be related to circulating p26 antigen. Also similar to the observations in HIV-1, antibody response to the tat viral protein appeared shortly after seroconversion and were stably maintained, while the majority of HIV-2 infected individuals have antibodies to HIV-2 tat, those that lack the antibodies are more likely to progress to clinical disease. The low viremia, reduced transmission and slow disease progression of HIV-2, led to the interest in HIV-2 as a model of lentivirus control. The identification of potent and broadly neutralizing antibodies has been considered critical components for an effective HIV-1 vaccine. Autologous and heterologous neutralizing antibodies (NAb) responses in HIV-2 infection have been studied, where heterologous refers to an HIV-2 individual’s sera to neutralize HIV-2 viruses other than their own. NAb to autologous HIV-2 was considered as a potential mechanism for HIV-2 viral control, however, studies failed to demonstrate a significant inverse relationship between NAb titers and plasma viremia. In multiple studies of the heterologous HIV-2 NAb responses, potent Nab responses were found with a significant positive association between these titers and HIV-2 plasma viral load. In individuals with higher viral loads suggestive of disease progression, the higher heterologous NAb production may reflect abnormal polyclonal B- cell activation, similar to what is seen in HIV-1. The development of NAb responses was analyzed in HIV-1 infected Dakar sex workers assessed longitudinally for up to 14 years with heterologous responses developing fully after 2.5 years of infection, this is similar to what is seen in the development of HIV-1 NAb responses. There was no correlation between titers of either heterologous or autologous NAbs and clinical progression in the cohort and HIV-2 envelopes failed to demonstrate any evidence of escape from NAb selection pressure. Thus, HIV-2 appears to induce superior NAb titers which may result from preserved CD4 þ T cell help that is maintained in HIV-2 infection. This is coupled with the lack of HIV-2 variability which allows for persistence of NAb epitopes that would promote higher affinity maturation by B cells. Antibody-dependent complement-mediated inactivation by HIV-2 has not been well studied. One study compared the effect of complement on HIV-1 and HIV-2 and found a higher magnitude of anti-viral activity in HIV-2 compared to HIV-1 via activation of the classical complement pathway via IgG. Complement increased the HIV-2 antiviral activity 32-fold whereas the increase in HIV1 antiviral effect was modest. This may result from intrinsic differences in the envelope spike proteins of HIV-2 versus HIV-1, where the HIV-2 envelope is more accessible with functional and stable envelope spike proteins.

HIV-2 Cellular Immunity T cell activation in HIV-2 infection has been studied where CD4 þ and CD8 þ T cell populations were independently assessed with the hypothesis that distinct factors mediate immune activation in specific T cell subsets. CD8 þ T cell immune activation has previously been strongly associated with HIV-1 disease progression. CD8 þ T cell activation was significantly different between HIV-2 and HIV-1 infected individuals although both groups expressed significantly higher activation level compared to seronegative individuals. Unlike HIV-1, there was no association of CD8 þ or CD4 þ activation with HIV-2 viral load or CD4 þ cell count. Numerous studies have described robust gag specific CTL responses in HIV-2 infection, in one study, 88% of patients demonstrated CD8 þ mediated responses. These were notable for their presence even in patients with undetectable HIV-2 viremia. The responses were found to be more polyfunctional in HIV-2 compared to HIV-1 as measured by secretion of IFN-g, IL-2, TNF-a, MIP-1b and CD107a mobilization. HIV-2 CD8 þ responses to HIV-1 was also demonstrated, HLA-B5801 positive subjects showed broad cross-recognition of various HIV-1 subtypes suggesting a response that tolerated extensive amino acid substitutions within the HLA-B5801-restricted HIV-1 and HIV-2 epitopes.

Treatment of HIV-2 There has been tremendous progress and success in the science and clinical testing of antiretroviral therapies (ART) for HIV-1 infection. Previous and even current guidelines for ART treatment of HIV-2 have often been based on HIV-1 without significant scientific evidence or clinical testing. This is at least partially due to the overall lower global prevalence of HIV-2, its African geographic localization and the paucity of AIDS-related HIV-2 cases in resource rich settings. There are also diagnostic challenges to the clinical monitoring of HIV-2 therapy. The lack of approved nucleic acid tests to confirm HIV-2 infection, monitor viral loads and characterize host-cell tropism are lacking. There has been no formal regulatory approval of ART drugs for HIV-2 treatment and

834

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

only in 2018 have results from small clinical trials been reported. Several other trials are ongoing which will help to determine the optimal first-line therapy regimen for HIV-2. HIV-2 is considered susceptible to the class of nucleoside reverse transcriptase inhibitors (NRTIs) which are typically combined with non-nucleoside reverse transcriptase inhibitors (NNRTI) in most first-line ART regimens in Africa. Early in vitro studies indicated high level resistance of HIV-2 to NNRTI class of drugs. This is due to the known mechanism of action of this class of ART drugs where the drug binds to a specific subunit of the HIV-1 reverse transcriptase enzyme, that is not present in HIV-2. There is active research on protease inhibitors that may be effective in HIV-2 ART. There are currently 10 approved protease inhibitors for HIV-1 treatment, only four of these (ritonavir boosted -lopinavir, -darunavir and -saquinavir) are considered active for HIV-2 treatment due to naturally occurring secondary mutations or polymorphisms in the HIV-2 protease. Upon selection of primary resistance mutations to protease inhibitors, pre-existing secondary changes may explain the acquisition of multi-PI resistance phenotype. HIV-2 is also intrinsically resistant to the fusion inhibitor enfuvirtide. Maraviroc is an entry inhibitor that blocks HIV entry via the chemokine receptor, CCR5. While there is limited in vitro data suggesting that HIV-2 may be susceptible to this drug, it is anticipated that the virus’ ability to utilize other non-CCR5 coreceptors may compromise in vivo efficacy. HIV-2 treatment in West Africa has relied on the availability of drugs available for standard HIV-1 treatment. Historically, the typical first line ART regimens in Africa were NRTI and NNRTI based regimens. Since HIV-2 patients would not be susceptible to NNRTI drugs, they were initiated on therapy with NRTIs and available protease inhibitors. Reports from those HIV-2 infected West African patients indicated high clinical and virologic failure, mortality and loss to follow-up. Upon genotypic analysis, a significant proportion (30%–65%) of failing patients had developed multiclass NRTI and protease inhibitor resistance. There are no current guidelines for second-line ART for HIV-2 infected patients. There are ongoing clinical trials to determine if drug resistance testing can provide clinical benefit in HIV treatment. In recent years the WHO guidelines for first line ART have included integrase inhibitors. Clinical studies support their use as first-line ART in HIV-2 and this has been bolstered by in vitro selection studies, biochemical analyzes and studies with site-directed HIV-2 mutants.

Prevention of HIV-2 Although HIV-2 transmission by sexual and perinatal transmission appears to be reduced compared to HIV-1, risk factor analysis confirms that methods of prevention for both viruses should be effective. Prevention of HIV-2 by sexual transmission would include protected sexual intercourse, reducing concomitant sexual transmitted diseases and male circumcision. In addition to sexual and perinatal transmission, blood borne transmission should also be considered. Screening of blood banks for HIV-2 has been instituted since the 1990s in most countries. While HIV-2 transmission through intravenous drug use is biologically possible, current epidemiology has not documented significant HIV-2 infection in intravenous drug users. Although these interventions have been extensively studied with HIV-1, they have not been studied with HIV-2 because the transmission rate has been relatively low and geographically isolated to West Africa. Antiretroviral prophylaxis has been an effective means of preventing perinatal transmission of HIV-1 and would be considered a means of preventing HIV-2 perinatal transmission. Since the majority of HIV-2 infected pregnant women would have low HIV-2 plasma viral loads and reduced cervical shedding, their risk of perinatal transmission is considered to be significantly lower that HIV-1 infected woman, 0%–4% with or without prophylaxis. As discussed in the HIV-2 therapy section, there are certain classes of ART that are ineffective in blocking HIV-2 infection, so drugs for HIV-2 prophylaxis must take this into consideration. In the US, a regimen with two NRTIs and a ritonavir-boosted protease inhibitor or integrase strand inhibitor are recommended; NNRTIs should not be used since they are not active against HIV-2. It is recommended that all infants borne to an HIV-2 infected woman receive 4 weeks of zidovudine prophylaxis. Since breastmilk transmission is also potential, breast-feeding is not recommended in HIV-2 infection. Again, there are no clinical trials that have addressed the efficacy of these interventions to prevent HIV-2 perinatal transmission.

Interactions of HIV-2 and HIV-1 in Vivo Early in the description of HIV-2 in West Africa, serologic surveys for both HIV-2 and HIV-1, found samples that reacted with both viruses. This created a diagnostic challenge and required the development of type specific antibody assays or nucleic acid tests to confirm the HIV-dual infection status of the individual. Isolation of both HIV-1 and HIV-2 viruses has been reported from selected HIV-dual cases and nucleic acid evidence has been demonstrated in most of these with the improvement of these assays. HIV dual in regions of the world that lacked evidence of HIV-2 infection alone, were considered to result from misinterpretation of laboratory data. However, in West Africa, where HIV-2 and HIV-1 infection co-exist, HIV-dual infection has been well documented.

HIV-2 Protection From HIV-1 Demonstrated differences in the infectivity and disease potential of HIV-2 compared to HIV-1 supported the notion that upon interaction, HIV-2 might protect from HIV-1 infection or disease, analogous to an attenuated virus vaccine model. In 1995, an

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

835

8-year study of the Dakar female sex worker cohort, the hypothesis that the attenuated phenotype of HIV-2 infection might protect from subsequent HIV-1 infection was addressed. HIV-1 infection in previous HIV negatives along with super-infection of HIV-2 infected was documented over the study period with both serology and nucleic acid assays. A Poisson regression model was used to estimate the independent effect of demographic, behavioral, and biologic variables on the risk of HIV-1 infection. Despite higher incidence of other sexually transmitted diseases, HIV-2 infected women had lower incidence of HIV-1 than seronegatives, with a statistically significant incidence rate ratio (IRR) of 0.32. When immunosuppression by reduced CD4 þ cell count was accounted for, the IRR associated with HIV-2 was reduced further to 0.23. This analysis led to the conclusion that HIV-2 infection conferred a significant reduction (32%) in the subsequent risk of HIV-1 infection. The generalizability of these findings was questioned by studies from other West African sites. In Ivory Coast, Guinea Bissau and the Gambia, studies originally designed as cross-sectional surveys were analyzed for short periods of longitudinal observation, as a result of their design they did not possess sufficient statistical power, capable only of detecting an extremely high protected fraction (499%) of HIV-1 infection due to HIV-2 infection. Continued analysis of the Dakar cohort extended the observation period from the first published report to over 13 years, HIV-2 protection ranged from 52% to 74% depending on the method of analysis. In 2012, results from a cohort of 233 HIV-dual and HIV-1 infected in Guinea Bissau policemen were reported, the cohort had been followed for B20 years. They reported 53% slower progression to AIDS in individuals with dual infection compared to those with HIV-1 infection, as reflected by higher CD4 þ cell counts. Dual infected individuals where HIV-2 infection had preceded HIV-1 had the longest progression time to AIDS, median time to AIDS estimated at 129 months (93–165) compared to singly HIV-1 infected individuals with a median time to AIDS of 68 month (60–76). This was accompanied by significantly higher CD4 þ cell counts. This study was extended with further analysis to show that HIV dual infected individuals, where the HIV-2 infection had preceded HIV-1, had longer survival times compared to individuals with HIV-1 alone, measuring all-cause mortality as the outcome measure. This suggested that HIV-1 disease progression was inhibited by prior HIV-2 infection and again demonstrated the importance of in vivo studies to understand the pathogenesis of these related viruses. These results also seemed to confirm the 1995 findings from the Dakar cohort, and further strengthen the view that prior HIV-2 infection could modulate the pathogenicity of HIV-1 in vivo.

In vitro evidence for HIV-2 protection from HIV-1 Studies have described in vitro interactions of HIV-1 and HIV-2 that support the in vivo observations, these range from virus-virus interactions to potential immune mediated mechanisms for HIV-2 protection. Prior studies had reported that HIV-2 inhibits the replication of HIV-1 at the molecular level. This inhibition was selective, dose-dependent, and nonreciprocal. Though the exact mechanism remains to be defined, the inhibition appeared to be mainly due to an intracellular molecular event because it could not be explained solely on the basis of cell surface receptor mediated interference. The results supported the notion that the inhibition likely occurred at the level of viral RNA, possibly involving competition between viral RNAs for some transcriptional factor essential for virus replication. Using an in vitro HIV-1 challenge system, PBMCs from HIV-2 infected women could not support replication of a CCR5-dependent HIV-1 virus compared with CXCR4-dependent virus. Resistance was transferable, CD8 dependent and strongly correlated with beta chemokine production in the media. All resistant cultures were rendered susceptible by addition of a cocktail of antibodies to beta chemokines. HIV-2 infection might dramatically influence beta chemokine production by enhancing it in magnitude and/or duration, thus enabling HIV-2 infected individuals to cope favorably with subsequent exposure to HIV-1. This is supported by studies demonstrating that binding of the HIV-2 envelope to the alpha chain of CD8 stimulates dramatic levels of beta-chemokine production in comparison to HIV-1 envelope stimulation activity. Further, unstimulated HIV-2 PBMCs had diminished surface CCR5 receptor expression on CD4 þ T cells. In vitro up-regulation of the CCR5 receptor was readily demonstrated indicating that the receptor was functional. The in vivo down-regulation of the receptor was not correlated with activation markers (HLA-DR), beta chemokine levels or plasma viral load. It was postulated that the down regulation of the CCR5 receptor in HIV-2 infection contributed to slower disease course, by decreasing susceptibility of target cells for infection in conjunction with HIV-2 specific cellular immune responses. It is recognized that CTLs are able to lyse infected cells before progeny virions are produced and the production of granzyme particles will elicit beta chemokines therefore providing two mechanisms by which viral replication could be limited or prevented. In the Guinea Bissau policeman cohort, HIV-1 disease progression was most attenuated in HIV-2-infected subjects who had stable asymptomatic disease. This suggested that HIV-1 was inhibited by immune responses mounted to HIV-2, where CD8 þ gagspecific T cells had already been documented to be present in the majority of individuals with asymptomatic HIV-2 infection. Extensive cross-reactivity has been demonstrated between HIV-1 and HIV-2 epitopes, particular for CD4 þ and CD8 þ T-cells directed against the most conserved regions of the gag proteins between HIV-2 and HIV-1. It is therefore plausible that the potent and unusually high avidity T-cell responses elicited by HIV-2 contribute to the control of HIV-1 replication in vivo. Although HIV-2 infection is also characterized by very potent neutralizing antibody response that sometimes cross-neutralize HIV-1, the neutralization of HIV-1 is much less potent. HIV-2 infection results in a low level persistent viral infection, which elicits robust humoral, innate, and adaptive cellular immunity; cross-reactivity likely inhibits the replication of HIV-1 and ensuing disease progression. The mounting of the immune response to HIV-2 would be critical to the protective effect for both HIV-1 immunopathology and the development of disease.

836

Simian Immunodeficiency Virus (SIV) and HIV-2 (Retroviridae)

Since its initial discovery, studies of HIV-2 in infected individuals in West Africa have provided compelling evidence for potential mechanisms that may be critical to the pathogenicity of HIV viruses in general. Observational and in vitro studies suggest that the attenuated HIV-2 virus infection may provide cross-protective immunity for HIV-1. Suggesting that one plausible explanation for the relatively low rates of HIV-1 infection may be due to the co-existence of HIV-2, a unique feature of West Africa. Unbiased, powerful studies, using sensitive and specific classification methods, will effectively address the generalizability of the observation of HIV-20 s protective efficacy against subsequent HIV-1 infection described in West African studies over multiple decades. The investigation of virologic and immunologic mechanisms responsible for HIV-2 attenuated phenotype and protection from HIV-1 disease has significant implications for HIV pathogenesis studies and vaccine development.

Further Reading Bell, S.M., Bedford, T., 2017. Modern-day SIV viral diversity generated by extensive recombination and cross-species transmission. PLoS Pathogens 13 (7), e100646. Chahroudi, A., Bosinger, S.E., Vanderford, T.H., Paiardini, M., Silvestri, G., 2012. Natural SIV hosts: Showing AIDS the door. Science 335 (6073), 1188–1193. Esbjornsson, J., Mansson, F., Anders, K., et al., 2012. Inhibition of HIV-1 disease progression by contemporaneous HIV-2 infection. New England Journal of Medicine 367, 224–232. Gottlieb, G.S., Raugi, D.N., Smith, R.A., 2017. 90–90–90 for HIV-2? Ending the HIV-2 epidemic by enhancing care and clinical management of patients infected with HIV-2. Lancet HIV 5, e390–e399. Kanki, P.J., Mboup, S., 2015. HIV-2: Lessons from the Dakar cohort. In: Hope, T., Richman, D.D., Stevenson, M. (Eds.), Encyclopedia of AIDS. New York, NY: Springer, pp. 1–17. Kirmaier, A., Krupp, A., Johnson, W.E., 2014. Understanding restriction factors and intrinsic immunity: Insights and lessons from the primate lentiviruses. Future Virology 9 (5), 483–497. Marlink, R., Kanki, P., Thior, I., et al., 1994. Reduced rate of disease development with HIV-2 compared to HIV-1. Science 265, 1587–1590. Nyamweya, S., Hegedus, A., Jaye, A., et al., 2013. Comparing HIV-1 and HIV-2 infection: Lessons for viral immunopathogenesis. Reviews in Medical Virology 23, 221–240. Travers, K., MBoup, S., Marlink, R., et al., 1995. Natural protection against HIV-1 infection provided by HIV-2. Science 268, 1612–1615. VandeWoude, S., Apetri, C., 2006. Going wild: Lessons from naturally occurring T-lymphotropic lentiviruses. Clinical Microbiological Reviews 19, 728–762. Visseaux, B., Damond, F., Matheron, S., Descamps, D., Charpentier, C., 2016. HIV-2 molecular epidemiology. Infection, Genetics and Evolution 46, 233–240. Zhou, Y., Bao, R., Haigwood, N.L., Persidsky, Y., Ho, W.Z., 2013. SIV infection of rhesus macaques of Chinese origin: A suitable model for HIV infection in humans. Retrovirology 10, 89.

Relevant Websites https://www.health.ny.gov/diseases/aids/providers/testing/docs/guidelines.pdf 2018 Guidelines for use of the HIV Diagnostic Testing Algorithm for Laboratories. https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-hiv-2-infection Infection. https://www.sciencedirect.com/topics/medicine-and-dentistry/simian-immunodeficiency-virus Simian Immunodeficiency Virus.

Sindbis Virus (Togaviridae) Satu Kurkela, University of Helsinki and Helsinki University Hospital, Helsinki, Finland r 2021 Elsevier Ltd. All rights reserved.

Nomenclature arbovirus Arthropod-borne virus BFV Barmah Forest virus CHIKV Chikungunya virus E1 Envelope glycoprotein 1 E2 Envelope glycoprotein 2 EEEV Eastern equine encephalitis virus

MAYV Mayaro virus ONNV O0 nyong-nyong virus ORF Open reading frame RRV Ross River virus SINV Sindbis virus VEEV Venezuelan equine encephalitis virus WEEV Western equine encephalitis virus

Classification Sindbis virus (SINV) is an arbovirus, which belongs to Western equine encephalitis (WEE) complex of the genus Alphavirus in the family Togaviridae. The phylogenetic relationships of alphaviruses resemble those of the suggested serocomplex division (SFV, WEE, EEE, VEEE). A phylogenetic division of SINV into six genotypes has been suggested, with African and European virus strains forming genotype 1, Australian, East Asian and Central Asian strains genotypes 2–4, the closely related Whataroa virus genotype 5, and the South-West Australian strains genotype 6 (Fig. 1). Alphaviruses that are pathogenic to humans can also be divided into arthropathic and neuropathogenic viruses. SINV is the only arthropathic alphavirus belonging to the WEE complex, while the others (Chikungunya, Ross River, Barmah forest, O0 nyong-nyong, and Mayaro) belong to the SFV complex. SINV, alongside with e.g., SFV, is widely used as a model for viral encephalitis in mice, as an expression vector in cell biology, as well as in versatile gene therapy applications.

Virion Structure SINV is a spherical virus of 70 nm in diameter with T ¼ 4 icosahedral symmetry. SINV particle consists of a nucleocapsid core, a host cell derived lipid membrane, and a glycoprotein shell. The nucleocapsid core includes an RNA genome and 240 copies of the

Fig. 1 Geographical distribution of SINV. Regions with serological, virological or clinical evidence of human infections are marked in green. Regions with signs of SINV in some specific animal species such as mosquitoes, birds, etc., are marked in blue. Black stars indicate regions with known SINV infection outbreaks. Colored triangles indicate virus isolates belonging to genotypes 1–6 shown in red, orange, cyan, green, black and blue, respectively. Base map from: http://d-maps.com. From Adouchief, S., Smura, T., Sane, J., Vapalahti, O., Kurkela, S., 2016. Sindbis virus as a human pathogen-epidemiology, clinical picture and pathogenesis. Reviews in Medical Virology 26, 221–241. The image is reproduced with the permission of the rights holder, John, Wiley & Sons.

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00048-5

837

838

Sindbis Virus (Togaviridae)

Table 1

SINV proteins and their key functions

Protein

Length (aa), size (kDa)

Functions

nsP1 nsP2

540 aa, E60 kDa 807 aa, E90 kDa

Methylation and capping of newly synthesized viral genomic and subgenomic RNA Protease, RNA helicase and RNA triphosphatase activity required for viral RNA replication and transcription Host translation shut-off

nsP3

556 aa, E60 kDa

Macrodomain with phosphatase activity and nucleic acid binding ability Interference of the innate antiviral mechanisms

nsP4 C E3

610 aa, E70 kDa 264 aa, E30 kDa 64 aa, E7 kDa

RNA-dependent RNA Polymerase Formation of nucleocapsid with viral RNA Proper folding of pE2 pE2/E1 complex formation Transport of structural components to the site of budding Spike folding and spike activation for viral entry

E2

423 aa, E46 kDa

Receptor binding Receptor-mediated endocytosis Antigenic and neutralization epitopes

6K

55 aa, E6 kDa

Glycoprotein trafficking Virion assembly Virus particle formation Membrane permeability

E1

439 aa, E47 kDa

Fusion of the viral envelope with the host endosomal membrane during virus entry Antigenic and neutralization epitopes

capsid protein. The glycoprotein shell features 80 trimeric spikes consisting of three heterodimers of E1 and E2 transmembrane glycoproteins, projecting from the membrane. The ectodomain of E2 forms the outer portions of the spike, while the cytoplasmic tail interacts with the nucleocapsid. The ectodomain of E1 has a more tangential orientation within the viral membrane. E1 is responsible for membrane fusion, while E2 is responsible for the host cell receptor recognition (Table 1).

Genome SINV has a linear single-stranded 11.7 kilobase RNA genome of positive polarity. The coding region consists of two open reading frames (ORF). The N-terminal ORF is translated directly from the 50 two-thirds of the genomic RNA, and encodes the nonstructural polyproteins P123 and P1234, from which the nonstructural proteins nsP1–4 are cleaved. The C-terminal ORF is expressed through a subgenomic mRNA, and encodes a structural polyprotein p130, from which the structural proteins C (capsid), E3, E2, 6K, and E1 are cleaved by cellular and virus-encoded proteases (Fig. 2).

Life Cycle The host cell entry of SINV occurs by receptor-binding, followed by clathrin-meditated endocytosis. The low pH of the endosome triggers viral fusion, and nucleocapsid is released into the cytoplasm. A conformational rearrangement of E1 and E2 occurs, during which E2 dissociates from E1. Subsequently, E1 forms homotrimers and expose their fusion peptides, which drives the fusion of the viral membrane with the endosomal membrane. This results in the release of viral genome into the cytoplasm. The nonstructural proteins are translated from the full-length genomic viral RNA to polyprotein P123, and specifically in SINV, by translational readthrough of an opal termination codon to polyprotein P1234 (Fig. 2). The polyproteins are processed by the protease located within nsP2 protein. nsP1 and nsP2 are involved in several enzymatic activities essential for RNA replication (Table 1). nsP4 functions as RNA-dependent RNA polymerase. The structural proteins are translated from a subgenomic RNA to a C-pE2-6K-E1 polyprotein, from which C protein is released by autoproteolysis. pE2 (precursor of E3 and E2) and E1 are translocated across the ER membrane and undergo post-translational modifications (Fig. 2). pE2 and E1 form heterodimers, which are processed and transported to plasma membrane. pE2 is cleaved by a furin-like protease to yield E3 and E2 proteins. Viral RNA replication takes place in cytoplasmic vacuoles. The minus-strand synthesis predominates early in infection, and it is dependent on a replication complex composed of P123 and nsP4. The plus-strand synthesis requires a replication complex composed of individual nonstructural proteins, and it takes place later in infection. In the virus particle assembly, the nucleocapsid core is formed by the autoproteolytic process of the capsid protein. The trimers consisting of three heterodimers of E1 and E2 are transported to the plasma membrane, and interact with the nucleocapsid cores in the cytoplasm, forming progeny viruses. During the release from cells, virions acquire a membrane bilayer derived from the host cell plasma membrane.

Sindbis Virus (Togaviridae)

839

Fig. 2 Schematic representation of SINV life cycle, genome and proteins.

Epidemiology SINV was first isolated in 1952 from a pool of Culex pipiens and Cx. univittatus mosquitoes in the village of Sindbis located in the Nile river delta near Cairo in Egypt. SINV was later isolated from humans in Uganda in 1961, however, without a reported link to a clinical illness. The virus was recovered for the first time from a clinically ill patient in South Africa in 1963 from skin lesions, and subsequently associated with a rash-arthritis syndrome. In 1960s, SINV disease outbreaks were reported in South Africa. At the same time, antibodies to SINV were first detected in Northern Europe, several Mediterranean countries, and the Volga delta region in humans and/or birds. In 1967, first clinical patients were noted in Sweden, while in 1974, a disease outbreak took place both in South Africa and in Northern Europe (Finland and Sweden). Fig. 3 illustrates some of the key events in the history of SINV emergence. Clinical SINV disease only occurs in geographically restricted areas with an unpredictable temporal appearance. Between 1974–2002, disease outbreaks in Finland took place in a seven year interval, during which the largest outbreak took place in 1995 with 1310 laboratory confirmed cases. Finland has the highest incidence of SINV disease (in epidemic years 425/100,000/year), and approximately 5% of the Finnish population has been estimated to be seropositive for SINV. Data from Northern Europe indicates a higher SINV antibody prevalence in men than in women, while the incidence rates are higher in women than in men. Incidence rates in Northern Europe are highest in middle-aged adults. Fig. 1 shows the geographical regions in which SINV has been detected in humans and some animal species, as well as areas of SINV outbreaks. SINV or antibodies to SINV have been detected in several parts of Europe, Africa, and Oceania, as well as sporadically in different parts of Asia. SINV is not present in the Americas. Constant reports of clinical disease appear primarily from Finland and Sweden, as well as from South Africa. In recent years, the presence of SINV has been reported in new geographical regions, such as Germany, Czech Republic, the United Kingdom, and Israel, where, however, clinical reports are rare or nonexistent. The restricted geographical occurrence of clinical SINV disease may be due to differences in clinical alert, virus ecology, as well as genetic factors involving both human populations and virus strains. In endemic areas, human infections coincide with activity of enzootic vector mosquitoes. In Northern Europe, enzootic vector mosquito species include at least Cx. torrentium, Cx. morsitans and Cx. pipiens. In South Africa, Cx. univittatus appears to play an important role as an enzootic vector for SINV. Bridge vectors to man have been suggested to include e.g., Aedes cinereus and Ochlerotatus annupiles species. Birds have been identified as the natural hosts of SINV. Experimental SINV infection of different species in the Passeriformes, Galliformes and Anseriformes orders have shown that viremia reaches a level and duration which is sufficient to infect both the

840

Sindbis Virus (Togaviridae)

Fig. 3 Timeline of selected events in the history of Sindbis virus as a human pathogen. Arrows indicate disease outbreaks in South Africa (blue) and in Northern Europe (green). From Adouchief, S., Smura, T., Sane, J., Vapalahti, O., Kurkela, S., 2016. Sindbis virus as a human pathogenepidemiology, clinical picture and pathogenesis. Reviews in Medical Virology 26, 221–241. The image is reproduced with the permission of the rights holder, John, Wiley & Sons.

enzootic vectors and the bridge vector mosquitoes. There is also a wide variety of bird species within these three orders in which SINV antibodies have been found in nature. In Sweden, the highest SINV seroprevalence has been detected in Fieldfare, Redwing, and Song thrush of the order Passeriformes, which have been suggested as the main amplifying hosts in that region. In Finland, also the role of Galliformes in SINV ecology has been proposed. Migratory birds have been suggested to play a role in the spread of SINV to non-endemic areas. Field investigations have provided only limited information, but phylogeographic studies have provided further evidence on their role. SINV genotype 1 is the only genotype that has been associated with human outbreaks. Recent evidence suggests that genotype 1 has originated from central Africa. Genotype 1 was introduced to Sweden in 1920s as a single introduction, probably via migratory birds, and it subsequently dispersed to other parts of northern, eastern and central Europe in 1960s and 1970s. This data fits well with the epidemiological information available as to emergence of SINV disease and outbreaks.

Clinical Features Human SINV disease is also known as Pogosta disease (Finland), Ockelbo disease (Sweden), and Karelian fever (Russia). In many respects, clinical SINV infection resembles that of CHIKV infection, but in a much milder form. SINV infection is frequently subclinical or even asymptomatic. Particularly in children, the disease is usually mild, and often presents without joint symptoms. There are no reports of fatal infections. In Northern Europe, 3%–6% of the clinically ill patients become hospitalized over the course of illness. Human SINV reinfections have not been reported, and it appears likely that a lifelong immunity follows the infection. The incubation period of SINV infection is typically less than seven days. The hallmarks of an acute SINV infection are itchy, maculopapular exanthema (papules approximately 3 mm in diameter) over the trunk and limbs and mild fever, followed by muscle pain and joint symptoms, particularly in several large joints. Inflamed joints typically include ankles, fingers, wrists and knees, but often also hips, shoulders, and elbows. The joint pain, which often manifests as tenderness in movement and aching in rest can be fluctuating, but resides in the same articulations. Other manifestations include general malaise, headache, dizziness, nausea, and lymphadenopathy. Basic blood chemistry tests are typically within normal range. Extra-articular symptoms alleviate within a few weeks.

Sindbis Virus (Togaviridae)

841

Most patients with a symptomatic SINV infection have a relatively mild and self-limiting course of illness. Yet, a considerable proportion will experience long term sequelae. Over that past few decades, several clinical investigations have been conducted in Northern Europe to better understand the frequency and severity of persistent joint manifestations after SINV infection. Prospective studies from Finland show that approximately 50% of patients have a varying degree of joint manifestations attributable to SINV infection 12 months post infection, and 25% for three years post infection. In a small proportion (o5%) of patients, the long term joint manifestation can be defined as arthritis in an examination by a professional rheumatologist. Female gender and higher age are risk factors for prolonged symptoms. Of note, pre-existing rheumatic and autoimmune diseases are more frequent among those patients who experience persistent manisfestations. The estimated public health burden from persistent joint manifestations after SINV infection is considerable in areas with high SINV seroprevalence. In Finland, for instance, there are specific geographical areas in which the seroprevalence of SINV can reach 410%. In such areas, differential diagnosis of patients manifesting with unspecified joint symptoms should include consideration of long term sequeale of SINV infection. Although SINV antibodies are found even in high frequency in various animals in many regions in the Old World, no clear evidence exists as to SINV pathogenicity in animals.

Pathogenesis SINV has been shown to infect bone-associated connective tissue and to replicate in the skin in mouse models. SINV has also been isolated from human skin lesion biopsies. Pathological examination of patients’ skin lesions reveals edema in the upper dermis with inactive T-lymphocytes and lymphoblasts, as well as macrophages present in the area. Primary human myoblasts and myotubes are susceptible to SINV infection in vitro, and signs of tissue regeneration following necrosis has been observed in a patient with chronic myalgia after SINV infection. Major human antibody responses are targeted to the SINV structural proteins E1 and E2. Patients show a strong antibody response with particularly IgG3 subclass antibodies predominating. In the related CHIKV infection, IgG3 response protects patients from a chronic disease and mouse models suggest therapeutic effect from a passive transfer of patient anti-CHIKV antibodies. Elevated levels of type 1 interferons are seen in SINV mouse models, and also in patients infected with a related virus, namely CHIKV. Macrophages and macrophage induced cytokine response have been shown to play an important role in the immune response of SINV and other alphaviruses. Replicaton of SINV in human macrophages has been demonstrated, and SINV infection promotes macrophage activation, subsequently inducing the secretion of TNF-alpha, IL-1beta and IL6, as well as increased expression of matrix metalloproteinases 1 and 3, which may be associated with joint tissue damage. Evidence suggests an association between alphavirus arthralgias and autoimmune diseases or their markers. Elevated antinuclear and anti-mitochondrial autoantibodies and rheumatoid factor have been observed in patients examined three years after SINV infection. Symptomatic SINV infection is also associated with HLA DRB1*01 allele, which is linked to e.g., rheumatoid arthritis.

Diagnosis Even in endemic countries, the clinical diagnosis of SINV infection is not straightforward, and therefore a clinically suspected case should be confirmed in a laboratory. This is of importance not only to establish the correct diagnosis, but also to avoid unnecessary and ineffective therapies, and to provide information of the prognosis. Laboratory diagnostics are based on the detection of SINV IgM antibodies or seroconversion of IgG antibodies. IgM antibodies can be detected within 8 days and IgG antibodies within 11 days after the onset of disease. IgM antibodies commonly persist for several months post infection (430% after six months). IgG antibodies remain positive, likely for whole life as a sign of immunity. Immunoassays for SINV are only available in specialized laboratories, and they are based on in-house enzyme immunoassays and immunofluorescence techniques. Molecular diagnostic techniques are not readily applicable for laboratory diagnostics of human SINV disease due to the typically low level of viremia and a short viremic period in the acute phase. Virus isolation from blood or skin lesions has been successful only in rare occasions.

Treatment Specific antiviral treatment is not available for SINV infection. Symptomatic treatment can be considered, including antihistamins for itching rash, and non-steroidal inflammatory drugs for joint pain. Intra-articular corticosteroids are sometimes used for persisting joint symptoms.

Prevention Persons without immunity to SINV and who are exposed to bridge vector mosquitoes may become infected. Prevention is based on protective clothing and mosquito repellents in endemic areas with a known SINV circulation. Vaccines or prophylactic medication are not available.

842

Sindbis Virus (Togaviridae)

Further Reading Adouchief, S., Smura, T., Sane, J., Vapalahti, O., Kurkela, S., 2016. Sindbis virus as a human pathogen-epidemiology, clinical picture and pathogenesis. Reviews in Medical Virology 26, 221–241. Hesson, J.C., Verner-Carlsson, J., Larsson, A., et al., 2015. Culex torrentium mosquito role as major enzootic vector defined by rate of Sindbis virus infection, Sweden, 2009. Emerging Infectious Diseases 21, 875–878. Jalava, K., Sane, J., Ollgren, J., et al., 2012. Climatic, ecological and socioeconomic factors as predictors of Sindbis virus infections in Finland. Epidemiology and Infection 141, 1857–1866. Kurkela, S., Manni, T., Myllynen, J., Vaheri, A., Vapalahti, O., 2005. Clinical and laboratory manifestations of Sindbis virus infection: prospective study, Finland, 2002–2003. The Journal of Infectious Diseases 191, 1820–1829. Ling, J., Smura, T., Lundström, J.O., et al., 2019. Introduction and dispersal of Sindbis virus from Central Africa to Europe. Journal of Virology 93, e00620. Pietilä, M.K., Hellström, K., Ahola, T., 2017. Alphavirus polymerase and RNA replication. Virus Research 234, 44–57. Rupp, J.C., Sokoloski, K.J., Gebhart, N.N., Hardy, R.W., 2015. Alphavirus RNA synthesis and non-structural protein functions. Journal of General Virology 96, 2483–2500. Suhrbier, A., Jaffar-Bandjee, M.C., Gasque, P., 2012. Arthritogenic alphaviruses – An overview. Nature Reviews Rheumatology 8, 420–429.

Relevant Website https://www.ecdc.europa.eu/en/sindbis-fever/facts Facts about Sindbis fever.

Tick-Borne Encephalitis Virus (Flaviviridae) Teemu Smura, Helsinki University Hospital and University of Helsinki, Helsinki, Finland Suvi Kuivanen, University of Helsinki, Helsinki, Finland Olli Vapalahti, Helsinki University Hospital and University of Helsinki, Helsinki, Finland r 2021 Elsevier Ltd. All rights reserved. This is an update of T.S. Gritsun, E.A. Gould, Tick-Borne Encephalitis Viruses, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B978-0-12-801238-3.02668-4.

Classification Tick-borne encephalitis virus (TBEV) is a flavivirus (genus Flavivirus, family Flaviviridae). Within the genus Flavivirus, TBEV is a member of a monophyletic tick-borne flavivirus group. The genus also contains three other ecological groups: the insect-specific flaviviruses (ISFV), the mosquito-borne flaviviruses (MBFV) and the flaviviruses with no known arthropod vectors (NKV). There are three well-established subtypes of TBEV: European (Eur), Siberian (Sib) and Far-Eastern (FE) subtypes that reflect phylogenetic and geographic relationships. TBEV-Eur is carried mainly by Ixodes ricinus ticks in central and north-eastern Europe, whereas TBEV-Sib and -FE are found mainly in Ixodes persulcatus ticks in an area reaching from north-eastern Europe to the Russian Far East, China and Japan. Louping ill virus and Spanish sheep encephalitis viruses form a sister clade to the European TBEV subtype. These are further related to Greek goat encephalitis virus and Turkish sheep encephalitis virus. Recently, divergent lineages of TBEV have been characterized from Eastern Siberia (Irkutsk and Transbaikal), Mongolia, Western Siberia (river Ob region) and Tibetan highlands. These have been proposed to form new subtypes of TBEV (Baikalian, Obskaya and Himalayan subtypes) (Fig. 1). Other tick-borne flaviviruses outside the TBEV clade described above include Omsk hemorrhagic fever, Langat virus, Alkhurma hemorrhagic fever virus and Powassan virus, the latter representing ecoepidemiologically and clinically parallel pathogen to TBEV in North America.

Virion Structure TBEV particles are approximately 50 nm in diameter. The virion consists of three structural proteins: capsid (C), precursor of membrane (prM) and envelope (E), as well as a positive-sense RNA genome and a lipid membrane derived from the host cell. (Fig. 2) Seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) are expressed in infected cells. The envelope is formed by E and M heterodimers that form tetramers in a head-to-tail manner giving the virion an icosahedral shape. (Fig. 2(D)) The capsid protein C encloses the genome that is approximately 11 kB long single-stranded positive-sense RNA. The genome has a single open reading frame of approx. 11,000 nucleotides flanked by 50 and 30 untranslated regions (UTRs).

TBEV Life Cycle TBEV virions are internalized mainly via receptor-mediated endocytosis. In mammalian cells, laminin-binding protein (LBP) and the aVb3 integrin are major candidates for TBEV receptors, but less is known of the receptors in tick cells. The virions are stable in slightly basic conditions (BpH 8), and the gradual acidification of the endosome causes the E homodimers to rearrange into homotrimers. Viral and endosomal membranes fuse and release the nucleocapsid into the cytoplasm. After nucleocapsid uncoating, the genome is translated into one long polyprotein of approximately 3400 amino acids at the ER membrane. The viral genome is replicated at membrane-associated replication complexes (RC) in the perinuclear region of the cell. The amino-terminal part of the polyprotein consists of three structural proteins followed by seven nonstructural proteins that are co- and post-translationally cleaved by several proteases, most importantly by the host signal peptidase and viral serine protease (NS2B/NS3). The individual proteins are released either on the luminal (prM, E, NS1 and NS4B) or the cytoplasmic side (NS2A, NS2B, NS3, NS4A and NS5) of the ER. (Fig. 3).

Viral Proteins The capsid protein C (11 kDa) forms homodimers that enclose the viral genome into an electron dense core of ca. 30 nm. The precursor of membrane protein (prM) is cleaved by furin into ‘pr’ and membrane M (26 kDa) proteins prior to virion budding. The prM protein enables E protein to fold correctly into its native conformation. The envelope protein E (53 kDa) is a membrane protein that typically has 1 or 2 glycosylation sites, and functions in receptor binding and membrane fusion. E monomer consists of four domains (dI, dII, dIII and dIV). The putative receptor-binding sites reside in domain III, and the fusion peptide is located at the tip of domain II. NS1 (46 kDa) is a multifunctional glycoprotein that exists as a dimer and a hexamer. It translocates into the ER by a signal sequence at the carboxy-terminus of E, and is cleaved by a host signal peptidase. Shortly after synthesis, the downstream cleavage

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00141-7

843

844

Tick-Borne Encephalitis Virus (Flaviviridae)

Fig. 1 A phylogenetic tree based on the complete coding regions of TBEV. Omsk hemorrhagic fever virus (OHFV) that is the closest relative to TBEV was included as an outgroup. The three well-established subtypes; European, Far Eastern and Siberian are shown in orange, green and blue colors, respectively. Of the proposed new subtypes, the two Baikalian subtypes form sister clades to the Far Eastern subtype while the proposed “Obskaya subtype” forms a sister clade to the Siberian subtype. Louping ill virus (LIV), Spanish sheep encephalitis virus (SSEV), Greek goat encephalitis virus (GGEV) and Turkish sheep encephalitis virus (TSEV) form sister clades to the European subtype of TBEV.

of NS1 from NS2A is catalyzed by an unknown ER-resident host protease. NS1 functions in replication, forming the replication complex (RC) together with NS4A and NS4B. Dimeric NS1 forms soluble hexamers in the Golgi network that are released outside of the infected cell, and possess immunomodulatory activities via interactions with the host complement system. NS1 shares 90% amino acid homology with different TBEV strains and as high as 44% homology with other flaviviruses. The extracellular form of NS1 elicits a strong immune response in the host in natural flavivirus infections. NS2A is a small hydrophobic protein (22 kDa) that functions in virus replication and assembly. The downstream cleavage from NS2B occurs via the viral serine protease. NS2B is a membrane-associated protein (14 kDa) that functions as a key cofactor of the viral serine protease complex with NS3. NS2A and NS2B function in immunomodulation and together with NS3, NS2B has been shown to play a role in TBEV neuroinvasiveness. NS3 is a multifunctional cytoplasmic protein associated with membranes via its interaction with NS2B. NS3/NS2B catalyzes the autocleavage of NS2A/NS2B and NS2B/NS3 and the trans cleavage of NS3/NS4A and NS4B/NS5. NS3 possesses RNA helicase, nucleotide 50 -triphosphatase (NTPase) and RNA 50 -triphosphatase (RTPase) activities, and it associates with viral protein NS5 in replication. NS4A and NS4B are small integral membrane proteins (16 kDa and 27 kDa, respectively) connected via a conserved signal peptide 2k that serves as a signal sequence for NS4B. NS4A and NS4B function in RC formation and immunomodulation. NS5 (104 kDa) contains two distinct domains: the N-terminal S-adenosyl-methionine (SAM) transferase domain associated with the 50 -cap formation of newly synthesized viral RNA, and the C-terminal RNA-dependent RNA polymerase domain (RdRp). The interdomain region of NS5 is involved in RC formation. The genome and viral proteins are schematically described in Fig. 3. Immature virus particles are assembled in the ER from the structural proteins and the genome. The lipid envelope is gained by budding into the ER. pH and protease-dependent maturation of the virion occurs in the trans-Golgi network (TGN), where prM and E undergo major structural reorganization. Mature particles are formed once pr is cleaved from M. Three types of virions are released from infected cells by exocytosis: mature, partly mature and immature (Fig. 2(A)). Flavivirus-infected cells also release particles containing only the envelope proteins E and M and the lipid envelope. These virus-like particles (VLPs) are noninfectious and approximately 30 nm in diameter. The maturation and assembly of VLPs seem very similar to those of the virus: VLPs undergo low-pH induced rearrangement of envelope proteins and are able to undergo membrane fusion.

Epidemiology TBEV is maintained in nature by ticks and their rodent hosts, typically in geographically restricted “TBEV foci” (Fig. 4). Although the virus has been detected from at least 22 tick species, Ixodes ricinus and I. persulcatus are considered to play the main role in virus transmission. Humans do not contribute to the circulation of TBEV, but can be infected as accidental (dead end) hosts. Ticks serve both as a reservoir and a vector for the virus. Ticks can obtain TBEV either by feeding on a viremic animal or by non-viremic

Tick-Borne Encephalitis Virus (Flaviviridae)

845

Fig. 2 Structure of the TBEV virion. (A) Electron cryo-micrograph of smooth mature particles (black arrowheads), immature (white arrows), partially mature (white arrowhead), and damaged (black arrows) TBEV particles. The scale bar is 100 nm. The image is courtesy of Dr. T. Füzik et al. Nat Commun. 2018 Jan 30;9(1):436. Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody and is reproduced under a Creative Commons Attribution 4.0 International License. (B) Schematic representation of the TBEV virion. Multiple copies of the C protein (green) encapsulate the viral genome (lilac). The nucleocapsid is surrounded by a lipid membrane (light blue), in which E and M proteins (yellow and gray, respectively) are embedded; (C) Surface representation of the TBEV virion (wwPDB: 5O6A). An icosahedral asymmetric unit is outlined in black. The three E proteins within each asymmetric unit are shown in blue, red, and yellow. Symmetry axes are indicated by the black pentagon (five-fold), the triangles (three-fold), and the ellipse (two-fold); (D) Three E-M-M-E heterotetramers on the TBEV surface. Three domains of E are highlighted in red (I), yellow (II), and blue (III), and the fusion loop is highlighted in turquoise. E protein domain IV and M protein are not visible on the virion surface. Figure reproduced from Pulkkinen, L., Butcher, S.J., Anastasina, M., 2018. Tick-borne encephalitis virus: a structural view. Viruses 10 (7), 350. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

transmission between co-feeding ticks. During host infestation, Ixodes ticks tend to aggregate to certain preferred feeding sites. During such co-feeding events, the virus can transmit from one tick to another without a need for host viremia. Thereby, also animals with antibodies against TBEV can support TBEV circulation. Co-feeding transmission, especially between tick larvae and nymphs, is considered to amplify the spread of TBEV in the tick population. Among ticks, TBEV may also be transmitted transovarially from infected adult females to their offspring and transstadially, i.e., the tick stays infected during molting and can therefore carry the virus through all of its life span. These two mechanisms are likely to support the persistence of TBEV in the tick population. The prevalence of TBEV among the ticks of a given TBEV focus is usually low; typically only a few percent or less of ticks in a given TBEV focus are TBEV RNA positive (Fig. 4). Since the efficient co-feeding transmission requires overlapping activity periods for tick larvae and nymphs, microclimatic conditions favoring such activity patterns are considered to play a major role in the susceptibility of a tick population for TBEV circulation. The immature life stages (larvae and nymphs) of Ixodes ticks feed mainly on small mammals, whereas the adult ticks mainly feed on larger mammals such as cervids. Considering the TBEV circulation, the most important mammalian species are considered to be the abundant rodent species of Apodemus and Myodes genera. Due to its dependence on the population dynamics of ticks and various mammalian species, as well as climatic factors suitable for co-feeding transmission, TBEV is typically found focally within the distribution area of the host ticks.

846

Tick-Borne Encephalitis Virus (Flaviviridae)

Genomic RNA ~11 kbp Translation

(a)

Polyprotein 3400 amino acids vs C

s

f s

pr M

12 10 8

(b)

NS1

53

47

*

E

prM

s

M

v

25

27

?

vs

v

NS4A NS4B 14

69

NS5

27

103

Lumen of ER

NS2B

NS2A

s

NS4B

s v

v

NS4A v

v

C NH2

v NS3

*

NS1

s

f

v NS2A NS2B

Co-translational processing and translocation

*

* *

?

E

Cytosol

NS3

v COOH

v

NS5

(c)

Fig. 3 Genome strategy of TBEV. (a) Genomic RNA is presented as a solid line on the top; the 50 - and 30 -UTR are depicted in the predicted conformations according to Gritsun, T.S., Venugopal, K., Zanotto, P.M., et al., 1997. Complete sequence of two TBFV isolated from Siberia and the UK: Analysis and significance of the 50 - and 30 -UTRs. Virus Research 49, 27–39. (b) Translation and co-translational processing of flavivirus polyprotein. The flavivirus polyprotein is depicted as a bar, with specified individual proteins and their molecular masses (numbers below the bar). (c) Membrane topology of viral proteins in relation to the lumen of the ER and cytoplasm after the completion of co-translational processing and translocation. Adapted from Westaway, E.G., Mackenzie, J.M., Khromykh, A.A., 2003. Kunjin RNA replication and applications of Kunjin replicons. Advances in Virus Research 59, 99–140. Transmembrane domains are shown as cylinders. Glycosylation is indicated as (*). The polyprotein is processed by ER signalases (s) and viral-specific protease NS2B-NS3 (V). The cleavage of M from prM is carried out by the Golgi protease, furin (f) and cleavage between NS1 and NS2A is by an unknown (labeled as?) ER protease.

It has been estimated that over 10,000 TBEV cases occur every year in northern Eurasia. Ixodes ticks are active in temperatures above 5 degrees, and in Central Europe, there is typically a two-peak distribution of cases in early and late summer following the activity pattern of ticks. Human behavior (e.g., outdoor recreational activities), socio-economic factors, awareness of the disease and vaccine coverage are considered to affect the incidence of the disease significantly. In Europe, the disease incidence is highest in Central and Eastern Europe. Recently, previously unknown TBEV foci have been detected in Western Europe (e.g., The Netherlands and England) and Northern Europe (e.g., Finland, Sweden, Norway and Denmark), suggesting that TBEV endemic areas may be spreading toward these directions. During the years 2000–2016, the number of TBE cases reported in Europe (excluding Russia) fluctuated between 2000 and 3500 cases per year. Over one third of these cases were reported in Czech Republic and Lithuania. The incidence of the disease is highest in Baltic countries; Estonia, Latvia and Lithuania. The majority of the notified cases occur among individuals of age 40–69, with increasing incidence towards the older (60  69) age group. The disease is more prevalent in males than females in all age groups. In Russia, more than 27,000 TBE cases were registered between the years 2007–2016, with highest incidences in the Western Siberian part of the country: Altai, Krasnoyarsk, Tomsk, Khakassia and Tuva. Compared to the 1990s, the incidence of TBE has decreased in Russia during the last two decades. The disease occurs also in China, mainly in the north-eastern regions including Inner Mongolia Autonomous Region, Heilongjiang province and Jilin province. While the virus and antibodies against it have been detected in ticks and reservoir animals in Korean peninsula and Japan, only a few cases have been reported from Japan and none from the Republic of Korea.

Clinical Features TBEV is transmitted from the saliva of an infected tick within minutes after the tick bite. In addition, TBEV infection may occur via the gastrointestinal tract by unpasteurized milk from an infected animal. The route of infection as well as the infecting TBEV subtype may affect the clinical picture of the disease. European subtype TBEV infection with involvement of CNS is typically a biphasic illness. After an incubation period of on average 8 days (range 4–28 days), the first phase may present with general malaise, fatigue, headache, myalgia,

Tick-Borne Encephalitis Virus (Flaviviridae)

847

Fig. 4 TBEV ecological cycle. Dashed arrows depict the different stages of the Ixodes ricinus or I. persulcatus tick (top: eggs, right: larva, below: nymph, left: adult). Black arrows depict the presence and transtadial transmission of TBEV. The co-feeding transmission from infected nymphs to uninfected larvae, both of which typically feed on rodents, is supported by suitable environmental conditions, and enhances the circulation of TBEV. The virus may also transmit transovarially. Larger mammals such as cervids support larger tick populations and birds may transmit TBEV to establish geographically remote foci. Vertical transmission may also occur among rodents. (Picture by Marjukka Törmi).

anorexia, nausea, vomiting and low grade fever. This phase usually lasts for 2–4 days. For the majority of the patients, the disease is resolved after the first phase. However, after a lag period of approximately a week (1–20 days), about one third of the patients enter a second phase of the disease that manifests with high fever and headache. The severity and occurrence of neurological symptoms and manifestations vary and may include nuchal rigidity, sensitivity to light, diplopia, dizziness, cerebellar ataxia, disorientation, hallucinations, epileptic seizures, dysphasia and pareses, and in extreme cases, tetraparesis. The severity of the disease increases with age; the CNS manifestations are meningeal in the majority of those under 40, and above this age meningoencephalitis and myelitis myeloradiculitis occur more often. Recovery from more severe forms of the disease may take months. 13%–36% of patients can have neuropsychiatric sequelae such as pareses, hearing and balance disturbances, headache, difficulties to concentrate, and depression. The severity and case fatality rate of the disease is considered to vary between the different TBEV subtypes. The case fatality rate in TBEV-Eur infections is o2%, whereas for TBEV-Sib infections the case fatality rate is 2%–8%. For the Far Eastern TBEV subtype, the case fatality rate has been reported to be up to 40% in some years, but it is unclear how many milder TBEV-FE cases may go unnoticed. The disease caused by the Siberian subtype of TBEV is often monophasic, may proceed more rapidly and may present with a chronic form. In fatal Siberian subtype infections, death usually occurs 3–7 days after onset of the disease. For the Far Eastern subtype, the typical first-phase symptoms are followed by a stiff neck, sensorial changes, visual disturbances and variable neurological dysfunctions. In fatal cases, death occurs within the first week after onset of illness.

Pathogenesis Langerhans dendritic cells are the primary targets of vector-mediated flavivirus infections. Tick saliva facilitates the infection by modulating the innate immune responses of the first infected cells. Infected dendritic cells then migrate to draining lymph nodes, from which the infection spreads to other organs such as the spleen, liver and the kidneys. The hallmark of TBEV pathogenesis is the entry of the virus into the CNS and brain, characterized by breakdown of the blood-brain-barrier (BBB) and neuroinflammation. The BBB is a semi-permeable physical barrier between the blood circulation and the brain, consisting of epithelial cells, glial cells, neurons and pericytes. The inflammatory responses to TBEV may lead to increased permeability of the BBB, and the virus has been suggested to cross the BBB via either direct infection of the BBB cells, trans- and paracellular transport, or within migrating leukocytes. In addition, retrograde axonal transport may serve as a potential route for TBEV to enter the CNS.

848

Tick-Borne Encephalitis Virus (Flaviviridae)

Diagnosis As the clinical picture of TBE is often similar to other viral CNS infections (caused by e.g., enteroviruses or herpes simplex virus 1) a laboratory confirmation is required to diagnose the disease. Anamnestic information of tick bites or potential exposure to ticks or unpasteurized milk during the tick season in TBEV endemic areas is the basis for suspicion of TBE. Notably, tick bites may often be unnoticed by the patient. It is also essential to know the TBEV vaccination history and possible exposure to other flaviviruses or flavivirus vaccines, particularly as a TBEV vaccinee may nevertheless have an acute TBE and the vaccination or other flavivirus infections affect the serological results and their interpretation. The diagnostics is primarily based on detection of IgM and IgG antibodies from serum during the 2nd phase of TBE. Additional confirmation of CNS infection can be acquired through detection of these antibodies in cerebrospinal fluid. Typically, the antibodies are measured by EIA or IFA and the strongest reactivity can be detected against the viral E protein. Also reliable TBEV IgM immunochromatographic tests are available. Usually, viral RNA can no longer be detected during the 2nd phase of the disease, although it has been occasionally detected in early blood samples. On the other hand, during the first phase the patient rarely seeks medical care, and therefore, serum samples from the early stage of the infection are rarely available. However, from such samples, TBEV can be readily isolated, or detected by RT-PCR while antibodies are not yet present. In fatal cases, viral RNA is found and the virus can often be isolated from autopsy brain samples. Although the diagnostics is in principle straightforward, since a single serum sample IgM and IgG test can usually provide the diagnosis, there are some caveats: (1) in TBEV Siberian subtype infections, a first sample from a patient with CNS symptoms may be antibody negative. This is possibly due to the faster invasion of the CNS by the Siberian TBEV subtype, without the first, systemic phase. In addition, the antibody positivity in CSF has preceded that of the serum in some cases. Therefore, in the TBEV endemic areas, where Siberian and/or Far Eastern subtypes are circulating, it is particularly important to have a second sample 1–2 weeks after the onset of illness. (2) The positive TBEV antibody reactivity can, in principle, be due to cross-reactive antibodies raised by infection with other flaviviruses. This is especially relevant in the areas where e.g., both West Nile virus (WNV) and TBEV circulate. In such circumstances a comparison of serum IgM levels or the neutralizing antibody titers for both viruses may be needed (3) In case of vaccine failure infections, IgM is seldom found in the first serum sample, while IgG levels may already be very high. However, in a second sample anti-TBEV IgM antibodies are usually positive. In all of these cases, reviewing anamnestic and clinical information, studying the CSF, neutralization tests, or testing for TBEV NS1 antibodies, as well as having consecutive samples are helpful.

Treatment The treatment is supportive as currently there is no specific antiviral therapy for TBE. In Russia and Kazakhstan, post-exposure treatment with hyperimmune serum is provided, but this is no longer used elsewhere. Long term rehabilitation may be needed.

Prevention TBE can be prevented by vaccination. Commercially available vaccines contain formalin-inactivated TBEV and are widely used across the TBE-endemic areas of Eurasia. Three doses are typically required for effective protection. For the vaccines used in Europe, the second dose is given after an interval of 1–3 months, and the third dose after an interval of 5–12 months, although accelerated protocols are also in use. The first booster vaccination is recommended 3 years later, and further boosters are recommended with e.g., 3–10 year intervals, depending on the age of the vaccinee. At population level, the vaccinations have shown to be effective: The highest vaccine coverage is in Austria, where nearly 90% of the population is vaccinated, and the case numbers have diminished despite increasing in neighboring countries and 98% of the remaining cases occur in the unvaccinated population. Despite full vaccination series, individual vaccine failure infections may occur. WHO recommends population-wide vaccination, if the pre-vaccination incidence is more than 5/100,000. It should be noted, however, that the risk to acquire TBEV infection may vary considerably even in a small geographical scale, due to the focal occurrence of the virus. In endemic areas, measures to minimize tick bites such as protective clothing and repellents as well as avoiding the consumption of unpasteurized milk products are important. Tick removal is an uncertain preventive measure, since the virus is transmitted soon after a tick bite. Updated surveillance of disease occurrence through diagnosed cases and One Health surveys, and increasing people’s knowledge of TBE and vaccination are of key importance.

Further Reading Füzik, T., Formanová, P., Ru˚ˇzek, D., et al., 2018. Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody. Nature Communications 9 (1), 436. doi:10.1038/s41467-018-02882-0. Lindquist, L., Vapalahti, O., 2008. Tick-borne encephalitis. Lancet 371 (9627), 1861–1871. Pulkkinen, L., Butcher, S.J., Anastasina, M., 2018. Tick-borne encephalitis virus: A structural view. Viruses 10 (7), 350.

Tick-Borne Encephalitis Virus (Flaviviridae)

849

Ruzek, D., Avšicˇ Županc, T., Borde, J., et al., 2019. Tick-borne encephalitis in Europe and Russia: Review of pathogenesis, clinical features, therapy, and vaccines. Antiviral Research 164, 23–51. doi:10.1016/j.antiviral.2019.01.014. World health Organization, 2011. Vaccines Against Tick-Borne Encephalitis: WHO Position Paper. vol. 86. World health Organization. pp. 241–256.

Relevant Website https://www.ecdc.europa.eu/en/tick-borne-encephalitis Tick-borne encephalitis. ECDC - Europa EU.

Transmissible Gastroenteritis Virus of Pigs and Porcine Epidemic Diarrhea Virus (Coronaviridae) Qiang Liu and Volker Gerdts, University of Saskatchewan, Saskatoon, SK, Canada r 2021 Elsevier Ltd. All rights reserved.

Classification Transmissible gastroenteritis virus (TGEV) of pigs and porcine epidemic diarrhea virus (PEDV) are two porcine coronaviruses in the genus Alphacoronavirus of the Coronavirinae subfamily in the Coronaviridae family within the order Nidovirales. Based on sequence heterogeneity, PED viruses are tentatively categorized as “classical” and “emerging” strains. The classical strains include PED viruses identified between 1970s to 2010, whereas PED viruses isolated after 2010 are referred to as emerging strains. The emerging strains are further divided into “non-S INDEL (insertions and deletions)” and “S INDEL” strains on the basis of the spike (S) protein sequences and virulence in piglets. Another proposal classifies PED viruses into up to five genotypes.

Virion Structure The enveloped virions of coronaviruses are spherical and/or pleomorphic with diameters of 144.8 ± 7.2 nm for TGEV (PUR46MAD strain) and 95–190 nm for PEDV (CV777 strain). Viral envelope contains the spike (S), membrane (M), and envelope (E) proteins. Homotrimeric S protein complexes form the distinctive “corona-like” structure on the surface of the virions. Within the envelope, there exists a nucleocapsid consisting of the nucleocapsid (N) protein and viral genomic RNA.

Genome Both TGEV and PEDV have a typical genomic organization of coronaviruses. The positive-sense, single-stranded RNA genome is approximately 28 kb in length with a 5′ cap structure and a 3′ polyadenylated tail. The coding sequence is flanked by untranslated regions (UTRs) at 5′ and 3′ ends. The N-terminal two-thirds of the genome contain one major open reading frame ORF1a encoding replicase polyprotein pp1a. A -1 frameshift just 5′ to the stop codon of ORF1a gives rise to ORF1b encoding a much longer replicase polyprotein pp1ab. These polyproteins are cleaved into 16 nonstructural proteins (nsps) mostly involved in viral RNA replication. The rest 10-kb genome codes for structural proteins S, M, E, and N, as well as accessory proteins with various functions (Fig. 1). TGEV encodes three accessory proteins, whereas PEDV encodes one.

Life Cycle The life cycles of TGEV and PEDV consist of virion attachment and entry, viral RNA translation, viral RNA replication and transcription, virion assembly and release (Fig. 2). Virion attachment to a host cell requires the interaction between viral S protein with cellular receptors. Aminopeptidase N (APN) is a major receptor for TGEV. However, whether APN is an essential receptor for PEDV is debatable. PEDV S protein can bind to sialic acid on the cell surface that may mediate virion attachment. After receptor binding, the S protein undergoes proteolytic cleavage that in turn induces membrane fusion and virion entry. Viral RNA with a 5′ cap structure and a 3′ polyadenylated tail serves as an mRNA that is translated by cellular translation machinery to generate viral replicase proteins pp1a and pp1ab. Replication and transcription complexes formed by these proteins produce both genomic and subgenomic progeny RNA species. Structural and accessory proteins are translated from subgenomic RNAs. Nucleocapsids formed by the N protein and progeny viral genomic RNA are enveloped in the endoplasmic reticulum – Golgi intermediate compartment with the involvement of S, E, and M proteins. The assembled virions are transported to the cell surface and released.

Epidemiology TGEV, first described in 1946 in USA, has been detected all over the world. PEDV was first isolated in 1978 in Belgium and is widespread in Europe and Asia ever since. The appearance of PEDV in North America was reported in 2013 where the virus continues to circulate in swine herds and there is a potential for new PEDV strains to emerge.

850

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20928-X

Transmissible Gastroenteritis Virus of Pigs and Porcine Epidemic Diarrhea Virus (Coronaviridae)

851

Fig. 1 Genome organization of TGEV and PEDV. Genes encoding structural proteins are presented in yellow. Putative accessory genes are shown in green. Nonstructural proteins encoded by ORF1a/b are presented in blue. Abbreviations: TGEV, transmissible gastroenteritis coronavirus; PEDV, porcine epidemic diarrhea virus; S, spike; E, envelope; M, membrane; N, nucleocapsid. Genomes have 5′ cap and 3′ poly A tail. Reproduced from Gerdts, V., Zakhartchouk, A., 2017. Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. Veterinary Microbiology 206, 45–51, with permission.

Fig. 2 PEDV replication cycle. PEDV binds a cellular receptor such as pAPN via the spike (S) protein. Penetration and uncoating occur after the S protein-mediated fusion of the viral envelope with the plasma membrane. Following disassembly, the viral genome is released into the cytoplasm and immediately translated to yield replicases ppla and pp1ab. These polyproteins are proteolytically cleaved into 16 nsps comprising the replication and transcription complex (RTC) that first engages in the minus-strand RNA synthesis using genomic RNA. Both full- and sub genomic (sg)-length minus strands are produced and used to synthesize full-length genomic RNA and sg mRNAs. Each sg mRNA is translated to yield only the protein encoded by the 5’-most ORF of the sg mRNA. The envelope S, E, and M proteins are inserted in the ER and anchored in the Golgi apparatus. The N protein interacts with newly synthesized genomic RNA to form helical RNP complexes. The progeny virus is assembled by budding of the preformed RNP at the ER-Golgi intermediate compartment (ERGIC) and then released by the exocytosis-like fusion of smooth-walled, virion-containing vesicles with the plasma membrane. Reproduced from Lee, C., 2015. Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virology Journal, 12, 193, under BioMed Central license agreement.

852

Transmissible Gastroenteritis Virus of Pigs and Porcine Epidemic Diarrhea Virus (Coronaviridae)

Table 1

Vaccines for TGEV and PEDV

Virus Region/country Vaccines in development

Commercial vaccines

TGEV North America

Live attenuated vaccines (mono, bi-, and trivalent for TGEV, rotavirus, and E. coli )

Europe

Asia

PEDV North America

Europe Asia

Recombinant proteins expressed in baculovirus, yeast, and plants; live attenuated vaccine; DNA vaccine Recombinant proteins expressed in baculovirus, yeast, and plants; live attenuated vaccine; DNA vaccine Recombinant proteins expressed in baculovirus, yeast, and plants; live attenuated vaccine

Live attenuated vaccines (mono, bi-, and trivalent for TGEV, rotavirus, and E. coli ) Inactivated vaccines (mono, bi-, and trivalent for TGEV, rotavirus, PEDV and/or E. coli ); live attenuated trivalent for TGEV, PEDV, and porcine (China) Inactivated vaccine; recombinant alphavirus-based vaccine

Recombinant proteins expressed in yeast and baculovirus; DNA vaccine; infectious clone for live attenuated vaccine; DNA vaccine Inactivated vaccine Recombinant vaccines expressed in baculovirus, Inactivated bivalent TGEV and PEDV vaccine (China, PEDV strain CV777); yeast, plants, Lactobaccilus casei, Salmonella live attenuated trivalent TGEV, PEDV, and porcine rotavirus (China, typhimurium and others PEDV strain CV777); live attenuated vaccines (Japan, PEDV strain 83P5; South Korea, PEDV strains SM98-1 and DR-13; Philippines, PEDV strain DR-13); inactivated vaccine (South Korea, PEDV strain SM98-1)

Note: Gerdts, V., Zakhartchouk, A., 2017. Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. Veterinary Microbiology 206, 45–51, with permission.

Clinical Features Both TGEV and PEDV cause enteritis in pigs with very similar clinical symptoms. Major clinical signs include vomiting, watery diarrhea, dehydration, and weight loss. The mortality rate can reach greater than 90% and is inversely related to the age of pigs.

Pathogenesis Villous enterocytes in small and large intestine are the major target cells of TGEV and PEDV infections. Viral infection causes cell death that results in villous atrophy followed by malabsorption, diarrhea, dehydration, anorexia, and eventually animal death. The molecular mechanisms for pathogenesis are not very well understood. There is evidence to suggest that viral proteins, such as spike and ORF3 proteins may affect viral virulence. At the molecular level, TGEV and PEDV have been shown to modulate multiple cellular processes/pathways including endoplasmic reticulum stress, cell cycle, and mitogen-activated protein kinase signaling.

Diagnosis Because clinical signs cannot distinguish between TGEV and PEDV infections, additional assays are required for diagnosis. Common diagnostic assays include viral antigen detection by histoimmunochemistry and enzyme-linked immuosorbent assay (ELISA), viral RNA detection and genotyping by PCR and sequencing, virus isolation by cell culture, as well as antibody detection by serology.

Treatment There is no specific treatment for TGEV and PEDV infections.

Prevention Enhanced biosecurity procedures are a general means to contain the spread and prevent the entrance of viral infection in pig farms, but vaccination represents the most effective way in preventing TGEV and PEDV outbreaks. Because of high mortality in piglets, it has become a common practice to vaccinate sows in order to transfer lactogenic immunity to protect piglets from TGEV and PEDV infections. Live attenuated and inactivated virus vaccines have been developed for both TGEV and PEDV. Since the spike protein is the major immunogen, numerous technologies have been employed to express the spike protein. These include DNA vaccine, vectored vaccine, subunit vaccine, and dendritic cell-based vaccine. Experimental and commercial TGEV and PEDV vaccines are listed in Table 1.

Transmissible Gastroenteritis Virus of Pigs and Porcine Epidemic Diarrhea Virus (Coronaviridae)

853

Further Reading Carman, S., Josephson, G., Mcewen, B., et al., 2002. Field validation of a commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds. Journal of Veterinary Diagnostic Investigation 14, 97–105. Choudhury, B., Dastjerdi, A., Doyle, N., Frossard, J.P., Steinbach, F., 2016. From the field to the lab – An European view on the global spread of PEDV. Virus Research 226, 40–49. Crawford, K., Lager, K.M., Kulshreshtha, V., Miller, L.C., Faaberg, K.S., 2016. Status of vaccines for porcine epidemic diarrhea virus in the United States and Canada. Virus Research 226, 108–116. Cubero, M.J., Leon, L., Contreras, A., et al., 1993. Transmissible gastroenteritis in pigs in south east Spain: Prevalence and factors associated with infection. Veterinary Record 132, 238–241. Delmas, B., Gelfi, J., L’haridon, R., et al., 1992. Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357, 417–420. Diel, D.G., Lawson, S., Okda, F., et al., 2016. Porcine epidemic diarrhea virus: An overview of current virological and serological diagnostic methods. Virus Research 226, 60–70. Eleouet, J.F., Rasschaert, D., Lambert, P., et al., 1995. Complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus. Virology 206, 817–822. Fehr, A.R., Perlman, S., 2015. Coronaviruses: An overview of their replication and pathogenesis. Methods in Molecular Biology 1282, 1–23. Gerdts, V., Zakhartchouk, A., 2017. Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. Veterinary Microbiology 206, 45–51. Hooper, B.E., Haelterman, E.O., 1969. Lesions of the gastrointestinal tract of pigs infected with transmissible gastroenteritis. Canadian Journal of Comparative Medicine 33, 29–36. Hsu, T.H., Liu, H.P., Chin, C.Y., et al., 2018. Detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in Taiwan. Archives of Virology 163, 3113–3117. Hu Jr., X., Li Jr., N., Tian Jr., Z., et al., 2015. Molecular characterization and phylogenetic analysis of transmissible gastroenteritis virus HX strain isolated from China. BMC Veterinary Research 11, 72. Ji, C.M., Wang, B., Zhou, J., Huang, Y.W., 2018. Aminopeptidase-N-independent entry of porcine epidemic diarrhea virus into Vero or porcine small intestine epithelial cells. Virology 517, 16–23. Jung, K., Saif, L.J., 2015. Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. The Veterinary Journal 204, 134–143. Kocherhans, R., Bridgen, A., Ackermann, M., Tobler, K., 2001. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes 23, 137–144. Langel, S.N., Paim, F.C., Lager, K.M., Vlasova, A.N., Saif, L.J., 2016. Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV): Historical and current concepts. Virus Research 226, 93–107. Lee, C., 2015. Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virology Journal 12, 193. Li, B.X., Ge, J.W., Li, Y.J., 2007. Porcine aminopeptidase N is a functional receptor for the PEDV coronavirus. Virology 365, 166–172. Li, W., Luo, R., He, Q., et al., 2017. Aminopeptidase N is not required for porcine epidemic diarrhea virus cell entry. Virus Research 235, 6–13. Li, W., Van Kuppeveld, F.J.M., He, Q., Rottier, P.J.M., Bosch, B.J., 2016. Cellular entry of the porcine epidemic diarrhea virus. Virus Research 226, 117–127. Lin, C.M., Saif, L.J., Marthaler, D., Wang, Q., 2016. Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains. Virus Research 226, 20–39. Masters, P.S.P., Stanley, 2013. Coronaviridae, Fields Virology, sixth ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins. Miyazaki, A., Fukuda, M., Kuga, K., Takagi, M., Tsunemitsu, H., 2010. Prevalence of antibodies against transmissible gastroenteritis virus and porcine respiratory coronavirus among pigs in six regions in Japan. The Journal of Veterinary Medical Science 72, 943–946. Nakagawa, K., Lokugamage, K.G., Makino, S., 2016. Viral and cellular mRNA translation in coronavirus-infected cells. Advances in Virus Research 96, 165–192. Niederwerder, M.C., Hesse, R.A., 2018. Swine enteric coronavirus disease: A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada. Transboundary and Emerging Diseases 65, 660–675. Pensaert, M.B., Bouck, P.D.E., 1978. A new coronavirus-like particle associated with diarrhea in swine. Archives of Virology 58, 243–247. Pineyro, P.E., Lozada, M.I., Alarcon, L.V., et al., 2018. First retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in Argentina. BMC Veterinary Research 14, 292. Rasmussen, T.B., Boniotti, M.B., Papetti, A., et al., 2018. Full-length genome sequences of porcine epidemic diarrhoea virus strain CV777; Use of NGS to analyse genomic and sub-genomic RNAs. PLoS One 13, e0193682. Risco, C., Anton, I.M., Enjuanes, L., Carrascosa, J.L., 1996. The transmissible gastroenteritis coronavirus contains a spherical core shell consisting of M and N proteins. Journal of Virology 70, 4773–4777. Saif, L.J., Van Cott, J.L., Brim, T.A., 1994. Immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine. Veterinary Immunology and Immunopathology 43, 89–97. Shirato, K., Maejima, M., Islam, M.T., et al., 2016. Porcine aminopeptidase N is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity. Journal of General Virology 97, 2528–2539. Song, D., Moon, H., Kang, B., 2015. Porcine epidemic diarrhea: A review of current epidemiology and available vaccines. Clinical and Experimental Vaccine Research 4, 166–176. Stevenson, G.W., Hoang, H., Schwartz, K.J., et al., 2013. Emergence of Porcine epidemic diarrhea virus in the United States: Clinical signs, lesions, and viral genomic sequences. Journal of Veterinary Diagnostic Investigation 25, 649–654. Subramaniam, S., Yugo, D.M., Heffron, C.L., et al., 2018. Vaccination of sows with a dendritic cell-targeted porcine epidemic diarrhea virus S1 protein-based candidate vaccine reduced viral shedding but exacerbated gross pathological lesions in suckling neonatal piglets. Journal of General Virology 99, 230–239. Wesley, R.D., Woods, R.D., Mckean, J.D., Senn, M.K., Elazhary, Y., 1997. Prevalence of coronavirus antibodies in Iowa swine. Canadian Journal of Veterinary Research 61, 305–308. Zuniga, S., Pascual-Iglesias, A., Sanchez, C.M., Sola, I., Enjuanes, L., 2016. Virulence factors in porcine coronaviruses and vaccine design. Virus Research 226, 142–151.

Vaccinia Virus (Poxviridae) Yan Xiang and Rebecca K Lane, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of G.L. Smith, Vaccinia Virus, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd, 2008, doi:10.1016/B978-012374410-4.00525-2.

The Origin of Vaccinia Virus Vaccinia is the name given to the smallpox vaccine that has been in use since the 19th century, long before the nature of the vaccine was defined and the virus bearing its name was isolated from it. The name (vacca, Latin for cow) reflects an age-old misconception that the vaccine was derived from cowpox. While the original smallpox vaccine used by Jenner in the 1790s is assumed to be cowpox virus (CPXV), the identity and source of the vaccines utilized during much of the history of smallpox vaccination were not well-characterized. Jenner and other early European vaccinators derived vaccination material from both cowpox and horsepox, and the vaccine stocks were propagated by serial passage in humans and animals. In 1939, it was discovered that the vaccine strains in use at that time were serologically different from CPXV, and subsequent studies confirmed that vaccinia virus (VACV) and CPXV are distinct virus species. The natural host and origin of VACV remain unknown, but the most probable ancestor of VACV is the horsepox virus (HSPV), considered to be a rodent virus that sporadically caused the now-extinct disease in horses. VACV is phylogenetically closer to HSPV than any other virus species. A commercial smallpox vaccine produced in the USA in 1902 was recently found to be 99.7% identical to HSPV with respect to the genome sequence.

Classification VACV belongs to the genus Orthopoxvirus (OPXV) of the family Poxviridae. Orthopoxvirus consists of more than ten genetically and antigenically closely related species, including CPXV, HSPV, monkeypox virus and variola virus, the causative agent for smallpox. Infection by one orthopoxvirus species induces an immune response that can cross-protect against all orthopoxvirus species, making VACV an effective vaccine against both smallpox and monkeypox. As the prototype poxvirus, VACV has been studied most intensively. The genome of more than 100 different VACVs has been completely sequenced. The Copenhagen vaccine strain was the first one sequenced and hence its genome has been used as the reference. The genes in Copenhagen were named with the convention that uses a letter (from A to O) to denote the genome fragment that is produced from digestion with HindIII restriction endonuclease, a number (from 1 to 57) for the open reading frame (ORF) number within the fragment, and L or R to represent the direction of transcription. The Copenhagen gene names are also used for the corresponding genes in other VACV strains. The letter L or R of the gene name is dropped when referring to the encoded protein. The VACV strain that has been the standard for laboratory research is the Western Reserve (WR), which originated from passaging the New York City Board of Health strain (NYCBH) in mice and is neurovirulent in that host.

Virion Structure Two distinct forms of infectious virus are produced from infected cells. The more abundant and stable form is the intracellular mature virion (IMV), now simply referred to as the mature virion (MV). The less numerous and more fragile form is the cellassociated and extracellular enveloped virion (CEV and EEV), now collectively referred to as the extracellular virion (EV). The virions are complex and barrel shaped, lacking icosahedral or helical symmetry. The virion dimensions are approximately 360
270
250 nm, amongst the largest of animal viruses. The ultrastructure as well as molecular architecture of the virions have been studied extensively with electron microscopy and mass spectrometry. The MV form has an outer membrane layer embedded with B30 different viral envelope proteins, many of which are involved in virus attachment or entry. Some of the MV envelope proteins are known targets of neutralizing antibodies. Unlike envelope proteins from many other viruses, none of the MV membrane proteins is modified by glycosylation. This may be due to an unusual process of MV envelope biogenesis that is unlike the budding process used by other viruses (see the section titled “Assembly”). Lying within the envelope is a dumbbell-shaped virus core, flanked by two amorphous “lateral bodies” in the concavities of the core. The surface of the core contains a palisade of protein spikes as well as pores for transport of molecules. The core encapsulates the virus genome along with all the enzymes and factors required for transcribing the early subset of viral genes (see the section titled “Transcription and translation”). Additional core proteins play roles in virus structure or morphogenesis. In total there are approximately 50 core proteins. The EV form is essentially an MV wrapped with an extra envelope embedded with at least six EV-specific proteins. MV and EV are subjected to neutralization by different sets of antibodies, due to their difference in antigen composition in the outer envelope.

854

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00047-3

Vaccinia Virus (Poxviridae)

855

The EV envelope proteins are mostly glycosylated, and two of those are known targets of neutralizing antibodies. Although produced in lower amount than MV in infected cells, EVs are critical for viral dissemination in infected hosts, as they are specialized in long-range spread.

Genome The VACV genome consists of a linear, double-stranded DNA (dsDNA) of 177–200 kilobase pairs (kbp), tightly packed with approximately 200 genes. The two DNA strands are covalently linked together at the ends by hairpin loops into one polynucleotide chain. The regions at the two termini are inverted terminal repeats (ITR), whose sequences are reverse complement of each other. ITRs contain a few genes and a variable number of tandem repeats. A core set of B90 VACV genes are conserved among nearly all vertebrate poxviruses, encoding essential proteins that function at various stages of virus replication, including entry, gene transcription, genome replication and virus assembly. Most of these genes are located within the central B100 kbp of the genome. Outside this conserved central region, the rest of the genome is more variable between different VACV strains, encoding proteins that are usually nonessential for virus replication but often participate in some aspect of host interactions, such as inhibition of cell death or evasion of host immunity. The naked VACV genome by itself is not infectious, as a virus-encoded and virion-packaged transcription system is required for initiating transcription from the genome. However, the VACV genome, which has been cloned in a bacterial artificial chromosome, can be initiated to produce infectious virus by using a related, replication-defective poxvirus as the helper. A chemically synthesized genome has been used to resurrect the horsepox virus, which appears to have become extinct in nature.

Life Cycle Entry VACV is capable of entering all mammalian and even insect cells that have been tested, with the exception of perhaps some primary human lymphocytes. It is thus presumed that either the entry requires no specific cellular receptors or that the receptors are ubiquitous molecules present on nearly all animal cells. The entry of MVs has been most extensively studied and will be described first. Four VACV proteins on the MV envelope are known to mediate MV attachment to cells. Three bind glycosaminoglycans (chondroitin or heparin) on the cell surface, while one binds laminins of the extracellular matrix. However, none of these viral proteins is individually required for entry, as loss of any one of the proteins has no deleterious effect on entry. VACV can enter cells either by direct fusion at the plasma membrane or by endocytosis followed by fusion with the endosomal membrane (Fig. 1), preferring one over the other depending on the virus strain or cell type. For entry through the plasma membrane, MV uses a virus-encoded entry fusion complex (EFC) to fuse directly with the plasma membrane in a pH-independent manner. The EFC consists of eleven proteins, including nine that form the core complex and two that peripherally associate with the complex. All EFC members are essential for entry, as loss of any one of the EFC proteins causes an entry defect. For entry through the endosomal path, MV particles are first internalized by macropinocytosis or fluid phase endocytosis. The interaction of MV with the cell surface triggers a signaling cascade that induces membrane rearrangements and the formation of actin- and ezrin-enriched membrane protrusions that engulf the virus particles. The internalized virus particles escape the endosomes by fusing the viral envelope with endosomal membranes. This process is activated by endosomal acidification and also requires the EFC. A viral protein, A26, regulates this process by interacting with the EFC and suppressing fusion at neutral pH. The low pH environment of the endosome results in a conformational change of A26 that de-represses the fusion machinery. Strain-specific differences in A26 expression may influence the preferred entry path used by different VACV strains. Similar to MV, EV can enter through both the plasma membrane and the endocytic pathway in an EFC-dependent manner. The binding of EV to cells triggers the disruption of the EV outer envelope via a ligand-induced, non-fusogenic mechanism. This reaction occurs at neutral pH and involves cellular polyanionic molecules and two EV glycoproteins. Once devoid of the outer wrapper, the underlying MV particles can enter cells essentially as free MV particles as described above.

Transcription and Translation The transcription of VACV genes occurs in the cytoplasm and depends on a completely virus-encoded transcription system, including transcription factors, a multi-subunit DNA-dependent RNA polymerase, capping and methylating enzymes, and a poly (A) polymerase. The transcriptome and translatome of VACV have been extensively studied with RNA-seq and Ribo-seq. The B200 genes of VACV are classified into three temporal classes: early, intermediate, and late, with some genes belonging to more than one temporal class. Each gene class is defined by class-specific transcription factors recognizing promoters with characteristics of that class. The sequential transcription of different classes of genes in a “cascade” fashion is enforced by the staged expression of these different class-specific transcription factors. The transcription factors for early genes are products of late genes and packaged in the virion. In turn, early and intermediate genes code for the necessary transcription factors for intermediate and late gene class, respectively (Fig. 1). The switch of the transcription program from cellular to viral and from one temporal class to another is also

856

Vaccinia Virus (Poxviridae)

Fig. 1 Vaccinia virus life cycle. Mature virus (MV) virions enter cells by either fusion of the viral envelope with the plasma membrane or by endocytosis. Endocytosed virions escape to the cytoplasm by low pH-dependent fusion with the endosomal membrane. Once inside the cell, transcription of early mRNAs begins within the viral core and transcripts are exported into the cytoplasm for translation. The uncoating process then disassembles the core to release the viral genome, allowing for replication to proceed. Genome replication and transcription of intermediate and late genes take place in cytoplasmic virus factories. Transcription occurs in a temporally-regulated manner; early genes produce transcription factors for intermediate genes, and likewise, intermediate genes produce transcription factors for late genes. New virions begin to form as membrane crescents and assembling cores grow into a spherical immature virion (IV). Newly-replicated viral DNA is packaged into the IV. Condensation of the viral core, enzymatic cleavage of several proteins, and structural changes result in an intracellular mature virion (IMV). The IMV either remains in the cell until lysis or is wrapped with a double membrane from the trans-Golgi or endosomal vesicles to form the wrapped virion (WV). WV can exit cells by fusion of its outer envelope with the cell’s plasma membrane, releasing double-enveloped extracellular virus (EV) by exocytosis.

facilitated by an increase in mRNA turnover mediated by two virus-encoded decapping enzymes, which remove the 50 -cap of mRNAs that protects them from a cellular 5–30 mRNA exonuclease. As described above, the virion core is packaged with the complete transcription apparatus for early genes, so synthesis of early mRNAs does not require de novo transcription or translation and starts inside the virion cores immediately after cell entry. Nearly half of the VACV genes are transcribed early, and the mRNAs are extruded through pores in the virion core into the cytoplasm for translation. Early proteins include modulators for various cellular antiviral responses, enzymes and factors for genome replication, and transcription factors for intermediate genes. Some early viral proteins and the cellular ubiquitin proteasome system are required for a deliberate process of core uncoating, which simultaneously disrupts the transcription apparatus for early genes and releases the genome for replication. The replicated genome serves as the template for the transcription of intermediate and late genes (collectively referred to as post-replicative genes), which appears to be coupled with the translation at sites of genome replication. Post-replicative gene expression requires genome replication, probably because the genome becomes accessible to the newly synthesized transcription factors only after core uncoating and genome replication. The products of post-replicative genes include all virion components as well as regulatory proteins for virion morphogenesis. The viral mRNAs do not undergo splicing but possess all the characteristic features of eukaryotic mRNA with a methylated cap at the 50 end and a poly(A) tail at the 30 end. Early VACV mRNAs have defined 30 -ends owing to the recognition of a specific transcription termination signal by the vaccinia termination factor. In contrast, no specific termination signal exists for postreplicative genes. Consequently, the post-replicative mRNAs are long with heterogeneous 30 -ends, leading to the formation of dsRNA by overlapping transcripts. The dsRNA represents a pathogen-associated molecular pattern that activates several innate antiviral pathways. As will be described later, VACV encodes proteins to specifically counteract the responses to dsRNA. Another unusual feature of post-replicative mRNAs is the presence of a poly(A) leader of heterogeneous lengths at the 50 -untranslated region. These poly(A) leaders are not derived from the DNA template, but are formed instead by RNA polymerase slippage. It has been shown that the 50 poly(A) leader confers translational advantage for the mRNAs.

Vaccinia Virus (Poxviridae)

857

Genome Replication VACV early gene products generate a complete DNA replication apparatus in the cytoplasm, including a DNA polymerase, a helicase-primase, a single-stranded DNA-binding protein, a processivity factor, and a DNA ligase. All except for the DNA ligase are required for genome replication, as a cellular DNA ligase can also fulfill the apparently essential function of DNA ligation. In addition, a viral serine/threonine protein kinase and a viral uracil DNA glycosylase are required. The former phosphorylates a cellular protein that, in its unphosphorylated state, binds DNA and inhibits viral DNA replication. The latter is required for its interaction with the processivity factor but not for its enzymatic activity. Genome replication starts within 2 h after infection and takes place in discrete juxtanuclear areas of cytoplasm called virus “factories”, which are thought to be derived from individual infecting virions and devoid of most cellular organelles. Several models of DNA replication have been advanced, with a selfpriming model being the more favored by the existing data. In this model, a nick is first introduced into a DNA strand near the terminal hairpin loop, creating an open 30 end which is extended by the polymerase to the end of the hairpin. The newly synthesized DNA segment then folds back to form a hairpin, essentially creating a primer for continuous DNA replication by strand displacement, until both strands of the DNA genome are traversed and the template is exhausted. An alternative model dictates that the genome replicates in a semi-conservative manner with leading and lagging strand synthesis, like eukaryotic DNA replication. This model is supported by reports of VACV DNA covalently linked to RNA, putative Okazaki fragments, and the essential role of the DNA ligase and primase. Regardless of the exact mechanism, the product of replication is a concatemer of multiple unit-length genomes connected by junctions that resemble Holliday junctions (HJ). The resolution of the concatemers into unit-length genomes is carried out by a viral HJ resolvase with similarity to the bacterial RuvC HJ resolvase.

Assembly Virus assembly commences in the cytoplasmic factories following genome replication and post-replicative gene expression. The first viral structures discernible by electron microscopy are crescent-shaped membranes, which are highly unusual, open-ended membranes. The origin and biogenesis of the crescents are still debated, but the available evidence favors a model that involves the modification and scission of the ER membranes by viral proteins. Two MV envelope proteins and six regulatory viral membrane assembly proteins (VMAPs) are known to be required. One envelope protein, A17, is inserted into the ER membranes and functions similarly to cellular reticulons, which promote membrane curvature. VMAPs are thought to participate in the rupture of the ER and the stabilization of the resulting open membranes. A17 also interacts with a capsid-like protein, D13, which assembles a honeycomb-like scaffold on top of the membranes to give them a spherical shape. The crescents grow in length while engulfing the core proteins, eventually closing off to form the spherical immature virions (IVs). A complex of seven core proteins is required for the association of the core with the crescents. Before the membrane is completely sealed, the newly synthesized virus genome is also packaged into the IV, facilitated by a viral ATPase and a viral telomere-binding protein. Proteolytic cleavage of several virion proteins accompanies the maturation of the spherical IVs into the brick-shaped MVs. Particularly, the cleavage of A17 by a viral protease removes the capsid-like scaffold from the surface of MV. MV envelope proteins are nonglycosylated, probably because they are synthesized on viral membranes that are already well separated from the ER. A unique cytoplasmic redox system formed by three viral proteins catalyzes the unusual cytoplasmic disulfide bond formation in some MV envelope proteins, including the EFC proteins. The majority of MV particles remain within the cell until it lyses, but a small fraction of them are transported on microtubules to near the microtubule organizing center where they are wrapped by trans-Golgi or endosomal vesicles that have been modified by the inclusion of several viral proteins. The wrapping of MV with two additional membranes produces the wrapped virion (WV) with a total of three membranes. WVs are transported to the cell surface on microtubules, facilitated by the interaction between microtubule motor proteins and two viral proteins on the surface of WV. The outer membrane of the WV fuses with the plasma membrane to externalize the virion via exocytosis, producing extracellular virions (EV) with two membranes (Fig. 1). Most of the EVs remain attached to the cell (CEV), while some are released (EEV), with the relative ratio influenced by both the cell type and some EV proteins. The interaction of EV with the surface of a VACV-infected cell triggers a signaling cascade that induces actin polymerization and the formation of finger-like actin tails right beneath the plasma membrane. These motile actin tails propel EVs away from the cell and into surrounding cells, preventing superinfection and facilitating efficient cell-to-cell spread of EVs.

Host Range Although its natural hosts are unknown, VACV is capable of infecting a wide range of wild and experimental animals, including primates, ungulates and rodents. Cultured cells from an even broader range of animal species are permissive for VACV replication, with the exception of a few cell types, such as Chinese hamster ovary (CHO) cells. In rare cases when VACV does fail to infect, the block in replication occurs after cell entry at an intracellular step, an example being the block of intermediate protein synthesis in CHO cells. A variety of viral “host-range” genes that are essential for VACV replication only in some cell types have been identified. Of them, two pairs of functionally redundant genes, E3L/K3L and K1L/C7L, affect VACV replication in the widest range of mammalian cells. Many host range genes function by antagonizing host antiviral factors that would otherwise block VACV replication. The E3L/K3L pair mainly inhibits dsRNA-activated protein kinase R (PKR), while the K1L/C7L pair inhibits two interferon-inducible antiviral factors SAMD9 and SAMD9L. Host species-specific differences in PKR and SAMD9L are known to

858

Vaccinia Virus (Poxviridae)

affect VACV replication in cells of different species. For example, a species-specific difference in Chinese hamster SAMD9L results in resistance to inhibition by both K1L and C7L, preventing VACV from replicating in CHO cells.

Virulence Naturally circulating VACV or its close relatives have been reported to cause infections in bovines and humans. In the Indian subcontinent, buffalopox virus (BPXV), a VACV sub-lineage, has sporadically caused pox lesions in Asian buffalos and their direct human contacts since the 1930s. Similarly, in south America, VACV-like viruses have repeatedly caused disease outbreaks in cattle and their human handlers since the late 1990s, having a significant impact on the dairy industry and public health in Brazil. VACV circulation has been observed in several species of marsupials and wild rodents in Brazil, suggesting that wild rodents are reservoirs or intermediate hosts for VACV transmission. Phylogenetic analyses suggest that the feral BPXV and Brazilian VACV-like viruses may derive from escaped smallpox vaccine viruses. VACV pathogenesis has been studied extensively with mouse models. The intranasal or intradermal routes of infection are commonly used, as respiratory tract and direct skin contact represent the main transmission routes for natural OPXV infection. Intranasal infection usually causes a systemic illness in mice, manifesting in body weight loss and the spread of virus to many tissues. Intradermal infection causes a localized disease in mice, characterized by relatively healthy animals with skin lesions. The viruses do not spread from the skin and the lesions heal in about 2 weeks. Many VACV proteins have been found to contribute to virulence in the mouse models, including enzymes that are involved in nucleotide metabolism, proteins that affect EV production, and proteins that modulate the host antiviral response. Among them, the loss of proteins that are involved in EV production results in the greatest virus attenuation.

Immunomodulators A growing list of VACV proteins have been found to modulate various aspects of the host immune system, particularly the innate immune system. These so-called immunomodulators are divided into the secreted proteins that act extracellularly and the intracellular factors that act within infected cells. The extracellular proteins include binding proteins for complement factors and various cytokines such as type I and type II interferons, IL-1b, IL-18, TNF-a, and CC chemokines. The interaction of the specific viral binding protein prevents the corresponding immune molecule from binding to its cellular receptor and from triggering the downstream signaling event. The intracellular immunomodulators include inhibitors of apoptosis and the signal transduction pathways initiated by cell pattern recognition receptors and interferons. They also include inhibitors of some interferon-induced antiviral factors, such as PKR and SAMD9 (see the section titled “Host range”). The signaling pathways and antiviral factors are often modulated by multiple VACV proteins that target different points of the pathway or through different mechanisms. For example, PKR is inhibited by two VACV proteins, with one sequestering dsRNA that is needed for PKR activation and the other acting as a pseudo-substrate inhibitor of PKR.

VACV: The Smallpox Vaccine Jenner’s inquiry in the 1790s heralded a new era of infectious disease prevention. In honor of Jenner’s discovery, “vaccination” is now used generally as the term for the preventive immunization procedure against any infectious disease. As the vaccine for smallpox, VACV is one of the most successful vaccines in human history and remains the only vaccine that successfully eradicated a human disease. During the global smallpox eradication campaign, several replication-competent VACV strains were used as the vaccine by different countries. They include the NYCBH strain (Americas), the Copenhagen strain (Denmark), the Lister strain (UK), the Ankara strain (Turkey), and the Tian Tan strain (China). However, vaccination with these strains was associated with a relatively high rate of adverse reactions, including generalized vaccinia, progressive vaccinia, eczema vaccinatum, and postvaccinial encephalitis. Some of these adverse reactions could be fatal and were mainly associated with individuals having an underlying immune deficiency. The vaccination of the general public ceased globally after the eradication of smallpox in the late 1970s. For many years afterwards, the only smallpox vaccine available for limited use in the US was the Dryvax vaccine, manufactured from the lymph fluid of calves’ skin infected with the NYCBH strain. The heightened concern over smallpox bioterrorism after 2001 led to a concerted effort by the public and private sectors in developing vaccines and antivirals against smallpox. The first new smallpox vaccine approved in the US is ACAM2000, which was derived from a clone of the Dryvax vaccine and manufactured on the Vero monkey cell line. ACAM2000 has a similar safety profile as the Dryvax vaccine and is contraindicated in individuals with atopic dermatitis or immune deficiencies. The vaccine is administered by scarification of the skin using a bifurcated needle containing the vaccine, and a successful vaccination results in VACV replication at the inoculation site producing a pustule or “take”. A safer vaccine, derived from the highly attenuated Modified vaccinia Ankara strain (MVA), was recently approved in the US for use against both smallpox and monkeypox. MVA was developed in the 1970s through passaging the Ankara strain in chick embryo fibroblast cells for more than 500 generations, resulting in the loss of nearly 30 kb of genomic information. MVA is replication-defective in most mammalian cells, and the MVA-based smallpox vaccine is approved for use even in people with atopic dermatitis or immune deficiencies. The vaccine is administered by two subcutaneous injections 4 weeks apart.

Vaccinia Virus (Poxviridae)

859

Smallpox was eradicated before the correlates of protective immunity to smallpox could be studied with modern immunology, but recent studies with animal models and proteome-wide immune profiling have provided a clear understanding of the immune responses to VACV immunization. A single immunization with live VACV elicits robust antibody and cytotoxic T cell responses to a wide array of viral proteins, which could last for decades in humans. Neutralizing antibodies play an essential role in protection against OPXV infection, while the T-cell response is essential for recovery from infection in the absence of antibodies. The antibody responses predominantly recognize structural, late VACV proteins, while the cytotoxic T cell responses predominantly recognize epitopes present in nonstructural, early VACV proteins. For optimal protection against OPXV infection, antibodies against both MV and EV are required. One protein (B5) is the major target of neutralizing antibodies against EV, while multiple proteins are the targets of MV neutralizing antibodies. None of the MV neutralizing antibodies are individually required for MV neutralization or dominant in all vaccinated individuals. Instead, the highly redundant neutralizing antibody responses may be a feature of the smallpox vaccine that ensures protection in very different human populations.

VACV: The Vaccine Vectors VACV has been used as a vector for developing vaccine candidates for infectious diseases and cancer, and as an oncolytic virus to target human cancers. As a vector, VACV has the advantage of the relative ease of generating recombinant virus, a large capacity for accommodating exogenous DNA, a high level of expression of biologically active protein and a wide host range. For human vaccine development, VACV strains that are replication defective in mammalian cells, such as MVA and NYVAC, are often used. NYVAC derived from VACV Copenhagen by the specific deletion of 18 genes including the host range genes K1L and C7L (see the section titled “Host range”), which are essential for replication in mammalian cells. Consequently, NYVAC is highly attenuated, but remains capable of inducing strong immune responses. These vectors have been used for candidate vaccines against AIDS and malaria with some success in human clinical trials. A replication-competent VACV that expresses the rabies glycoprotein has been used as an oral based rabies vaccine for wildlife, which is responsible for significantly reducing the incidence of rabies in the US and Europe.

Antiviral Drugs ST-246 (Tecovirimat) is the first antiviral drug that has been approved in the US for treating smallpox. It is a potent inhibitor of a highly conserved OPXV envelope protein essential for wrapping of MVs (see the section titled “Assembly”). ST-246 is effective against VACV in animal models and presumably also in humans. Another class of antiviral that has been in clinical trials for treating OPXV infection is Cidofovir and its derivatives. Cidofovir is an acyclic nucleoside analog licensed for treating human cytomegalovirus infection that also inhibits the poxvirus DNA polymerase.

Further Reading Condit, R.C., Moussatche, N., Traktman, P., 2006. In a nutshell: structure and assembly of the vaccinia virion. Advances in Virus Research 66, 31–124. Esparza, J., Schrick, L., Damaso, C.R., Nitsche, A., 2017. Equination (inoculation of horsepox): An early alternative to vaccination (inoculation of cowpox) and the potential role of horsepox virus in the origin of the smallpox vaccine. Vaccine 35, 7222–7230. Lane, J.M., Ruben, F.L., Neff, J.M., Millar, J.D., 1969. Complications of smallpox vaccination, 1968: National surveillance in the United States. New England Journal of Medicine 281, 1201–1208. McFadden, G., 2005. Poxvirus tropism. Nature Review Microbiology 3, 201–213. Moss Poxvirus, B., 2013. DNA Replication. Cold Spring Harbor Perspectives in Biology 5, a010199. Moss, B., 2013. Poxviridae. In: Knipe, D.M. (Ed.), Fields Virology, sixth ed., 2. Philadelphia, PA: Lippincott Williams and Wilkins, pp. 2129–2159. Moss, B., 2015. Poxvirus membrane biogenesis. Virology. 479–480, 619–626. Moss, B., 2016. Membrane fusion during poxvirus entry. Seminars in Cell & Developmental Biology 60, 89–96. Sánchez-Sampedro, L., Perdiguero, B., Mejías-Pérez, E., et al., 2015. The evolution of poxvirus vaccines. Viruses 7, 1726–1803. Schmidt, F.L., Bleck, C.K., Mercer, J., 2012. Poxvirus host cell entry. Current Opinion in Virology 2, 20–27. Smith, G.L., Vanderplasschen, A., Law, M., 2002. The formation and function of extracellular enveloped vaccinia virus. Journal of General Virology 83, 2915–2931. Smith, G.L., Benfield, C.T., Maluquer de Motes, C., Mazzon, M., 2013. Vaccinia virus immune evasion: Mechanisms, virulence and immunogenicity. Journal of General Virology 94, 2367–2392. Veyer, D.L., Carrara, G., Maluquer de Motes, C., Smith, G.L., 2017. Vaccinia virus evasion of regulated cell death. Immunology Letters 186, 68–80.

Relevant Website https://www.viprbrc.org/brc/home.spg?decorator=pox (ViPR). Poxviridae. Virus Pathogenh Database.

Varicella-Zoster Virus (Herpesviridae) Jeffrey I Cohen, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Published by Elsevier Ltd.

Glossary Dermatome The area on the skin the is innervated by a single cranial or spinal nerve. Varicella A disease also known as chickenpox that presents with a disseminated vesicular rash.

VariZIG Varicella immune globulin obtained from plasma of persons with antibodies to varicella-zoster virus. Vesicle A small fluid filled blister on the skin. Zoster A disease also known as shingles that usually presents with a dermatomal, vesicular rash.

Classification Varicella-zoster virus (VZV) is a member of the Alphaherpesvirus subfamily of the family Herpesviridae. Other alphaherpesviruses that infect humans include herpes simplex viruses (HSV) 1 and 2, and very rarely macacine herpesvirus 1 (B virus). All of these agents exhibit relatively short replicative cycles, destroy the infected cell, and establish latent infection in sensory ganglia. Simian varicella virus (SVV) is the most closely related, well characterized virus to VZV. Natural infection with SVV can cause a varicella-like illness in Old World monkeys; the virus establishes latency in trigeminal ganglia and can spontaneously reactivate to cause a rash which can transmit the virus to naïve animals. SVV shares a nearly identical set of genes with VZV; the virus is not known to infect humans.

Virion Structure It is not possible to obtain high titers of cell free VZV, so high resolution structural analysis of the virus has not been performed. Nonetheless, using electron microscopy the virus has been shown like other herpesviruses to contain DNA within a nucleocapsid that is surrounded by a tegument and viral envelope (Fig. 1). The nucleocapsid is derived from the major nucleocapsid protein (encoded by ORF 40) and, largely by analogy with other herpesviruses, proteins encoded by VZV ORFs 20, 21, 23, 33, 33.5, and 41. Like other alphaherpesviruses the nucleocapsid consists of 162 capsomeres arranged in an icosahedron. Several viral proteins have demonstrated to be localized in the tegument including proteins encoded by ORFs 4, 9, 10, 11, 47, 62, 63, and 66. Viral glycoproteins stud the membrane and include gB, gC, gE, gH, gI, gK, gL, gM, and gN.

Genome The VZV genome consists of double-stranded DNA and like other herpesviruses is usually linear in the virion and circularizes in latently infected cells. It contains about 125,000 base pairs with a single unpaired nucleotide at both ends of the genome. The VZV genome consists of a unique long region (UL, which includes ORFs 1–61 and a portion of ORF0 [sometimes referred to as ORF S/L]), an internal repeat short region (IRS, which includes ORFs 62–64), a unique short region (US, which includes ORFs 65–68), a terminal repeat short region (TRS, which includes ORFs 69–71, and a portion of ORF 0), as well as very short inverted repeat regions (terminal and internal repeat long; TRL and IRL) (Fig. 2). The viral genome encodes at least 20 small noncoding RNAs as well as the viral latency associated transcript (VLT). The genome exists predominantly as two different isomers depending on the orientation of the unique short region. It has five repeat (R) regions. R1 is located in ORF11, R2 in ORF 14, R3 in ORF 22, R4 between ORF62 and ORF63, and R5 between ORF60 and ORF61. R3 is the largest of the repeats, approximately 1000 base pairs in length. VZV encodes at least 70 genes, 5 of which are not conserved with HSV. These latter genes are ORF 0, 1, 2, 13, 32, and 57; ORF13 which encodes the thymidylate synthetase has a homolog in human herpesvirus 8, while the other four genes have homologs in equine alpha-herpesviruses. VZV has the most stable genome of all the human herpesviruses. Comparison of over 1300 whole VZV genomes from throughout the world has resulted in classification of viruses into 7 clades (1 6, 9) and 2 provisional clades (VII, VIII). At least one whole genome sequence is required for classification into a clade, while a partial genomic sequence defines a provisional clade. Clade 1 contains European isolates including VZV Dumas (the first completed VZV genome), clade 2 contains Asian isolates including the parental VZV Oka strain and the Oka vaccine derived from the parental strain, clade 3 contains European isolates

860

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21315-0

Varicella-Zoster Virus (Herpesviridae)

861

Fig. 1 Electron micrograph of varicella-zoster infected cell. Nucleocapsids in the nucleus (bottom) and virions budding from the cell membrane (top). Reproduced with permission from Cohen, J. I., Straus, S. E., 2001. Varicella-zoster virus and its replication. In: Knipe, D. M., Howley, P. M., Cohen, J. I., et al. (Eds.), Fields Virology. Philadelphia, Lippincott-Williams & Wilkins, pp. 2707–2730.

Fig. 2 Map of the varicella-zoster virus (VZV) genome (top) and the herpes simplex virus (HSV) genomes. Unique short (US), unique long (UL), terminal repeat (TR), internal repeat (IR), long (L), and short (S) regions are shown. Genes unique to varicella-zoster or herpes simplex are circled. Reproduced with permission from Cohen, J.I., Straus, S.E., Arvin, A.M., 2007. Varicella-zoster virus: Replication, pathogenesis, and management. In: Knipe, D.M., Howley, P.M., Cohen, J.I., et al. (Eds.), Fields Virology. Philadelphia, Lippincott-Williams & Wilkins, pp. 2773–2818.

including VZV HJO, clade 4 contains African isolates including VZV DR, clade 5 contains European isolates including VZV CA123, and clade 9 contain a United States isolate VZV KY037798.

Life Cycle VZV infects epithelial cells in the oropharynx and infects T cells that circulate throughout the body. Virus spreads to the skin, resulting in the rash of varicella, but also to other tissues including sensory, autonomic, and cranial nerve ganglia where the virus establishes latency. In the skin, virus in present in vesicles which become pustular and break down. VZV is released from the skin to infect other persons. In vitro, the virus has been shown to infect CD4 and CD8 T cells, NK and NKT cells, B cells, and monocytes. During initial infection, the virus enters host cells. The inability to obtain high titers of cell-free virus, has prevented researchers from identifying important steps in VZV entry and many steps are inferred from studies of the well characterized other human

862

Varicella-Zoster Virus (Herpesviridae)

alpha-herpesvirus HSV. Initially, the virus attaches to host cells by electrostatic interactions between viral glycoproteins and host cell glycosaminoglycans. Heparan sulfate on the surface of cells binds to VZV gB. The virus is thought to undergo endocytosis followed by fusion of the virus membrane to the host cell membrane. Three entry mediators, mannose-6 phosphate (cation independent), myelin-associated glycoprotein, and insulin degrading enzyme have been proposed as entry mediators, but these have not been confirmed in other laboratories. Glycoproteins gB, gE, gH, gL are all essential and as such are likely to have key roles in VZV infection. VZV gE is the most abundant glycoprotein on the virion and by analogy to the most prevalent glycoproteins in other herpesviruses is likely important for attachment to cells and binding to a receptor. By analogy with other herpesviruses, gH, and its chaperone gL, likely trigger activation of gB and all three glycoproteins are required for fusion of the virus membrane to the cell. The viral nucleocapsid is then uncoated and transported to the nucleus, where tegument proteins are released in the nucleus and the linear viral DNA circularizes and DNA is likely replicated through a rolling circle model. Initially the immediate-early (IE) genes are transcribed and translated which include IE4, IE61, IE62, and IE63. Since it is difficult to obtain high titer cell-free VZV, the kinetics (IE, early, or late) of most viral genes have not been formally determined, but have been putatively assigned based on homology with HSV. VZV IE62 is the major viral transactivator and upregulates expression of IE4 and IE61 as well as putative early and late VZV genes. IE62 binds to cellular transcription factors USF, TFIIB, and SP1 as well as to TATA binding protein. VZV IE4 transactivates some viral IE, early, and late genes and binds to TFIIB and to the NF-κB subunits p50 and p65. IE61 can upregulate expression of putative IE, early, and late VZV genes and can modulate expression of viral genes that are transactivated by IE4 or IE62. IE63 may down-regulate expression of some viral genes and binds to anti-silencing function-1 protein, RNA polymerase 2, and IE62. VZV putative early genes encode proteins important for viral DNA replication. These include the large (ORF28) and putative small (ORF16) subunits of the viral DNA polymerase, the single-stranded DNA binding protein (ORF29), the origin binding protein (ORF51), the viral dUTPase (ORF8), the viral uracil DNA glycosylase (ORF 59), the large (ORF19) and small (ORF18) subunits of the viral ribonucleotide reductase, and the viral thymidine kinase (ORF36). The latter protein phosphorylates acyclovir resulting in inhibition of virus replication. VZV putative late proteins include protein kinases, a protease (OR33), nucleocapsid and tegument proteins, and glycoproteins. The viral protein kinases (ORF47 and ORF66) phosphorylate both cellular and viral proteins. Nucleocapsid proteins include the major nucleocapsid protein (ORF40), a 115 kDa protein (ORF21), a 30 kDa protein (ORF23), and the viral assembly protein (ORF33.5) which is thought to form a scaffold for assembly of the nucleocapsid. Tegument proteins include ORF10 which transactivates IE62 and the ORF11 RNA binding protein. VZV encodes multiple glycoproteins important for virus entry and egress. VZV gE (ORF68) forms a heterodimer with gI (ORF67); the latter is important for trafficking of gE to the cell surface. gE is essential for virus infection and binds the Fc receptor of immunoglobulin. gH (ORF37) forms a heterodimer with gL (ORF60); the latter is important for maturation of gH and trafficking of gH to the plasma membrane. gH/gL are thought to activate gB (ORF 31) to allow the virus to fuse to the cell membrane. Other glycoproteins include gC (ORF14), gK (ORF5), and gM (ORF50) which forms a heterodimer with gN (ORF9A); of these three glycoproteins, only gK is essential for virus growth in vitro. Virus egress from cells is not well understood. In cell culture no cell-free infectious virus is released, instead the virus spreads by cell-cell fusion. In vitro, virus is directed to pre-lysosomes where it is degraded. In contrast, in vivo virus is released through an alternative pathway, likely in cells not expressing the cation-independent mannose-6 phosphate receptor which otherwise binds to mannose-6 phosphate on the viral envelope and directs the virus for degradation in pre-lysosomes. VZV establishes latency in neurons of the dorsal root ganglia (alongside the spine), cranial nerve ganglia, enteric ganglia (in the intestine), and autonomic (e.g., celiac and vagus nerve) ganglia. The virus enters dorsal root and cranial nerve ganglia either by the bloodstream or by retrograde transport from cutaneous nerves. Analysis of transcripts expressed in latently infected human ganglia obtained less than 9 h after death indicates that two transcripts are usually expressed in both the nucleus and cytoplasm of neurons- VZV ORF63 and VLT (VZV latency associated transcript). VLT RNA is expressed more abundantly than ORF63 RNA in latently infected human ganglia. ORF63 encodes IE63, an immediate-early protein in lytically infected cells, which is the homolog of HSV US1.5. In lytically infected cells VLT is spliced in different forms than in latently infected cells, and in these lytic cells VLT encodes a late protein; VLT protein was not detected in latently infected cells. In latently infected cells VLT is a multi-spliced unique transcript that is expressed anti-sense to ORF61. Somewhat surprisingly, not all neurons latently infected with VZV DNA express ORF63 or VLT transcripts. Expression of VLT inhibits ORF61 (a potent viral transactivator) expression in vitro which suggests that VLT could help the virus to maintain latency. Additional transcripts have been identified in latently infected ganglia including ORF4, ORF21, ORF29, ORF62, and ORF66. VZV microRNAs have not been detected in human ganglia. While numerous prior studies reported VZV proteins in latently infected human ganglia, more recent reports indicate that detection of some of these proteins may have been due to cross-reactivity of the antibodies with human proteins. Thus, at present VZV proteins are generally not thought to be expressed in latent infection. The viral genome is thought to be a circular episome exclusively in neurons.

Epidemiology Infection with VZV occurs worldwide and prior to the development of the varicella vaccine over 95% of children in temperate countries were infected with the virus before age 15. Varicella occurs more commonly in the winter and spring, often occurring at a later age in tropical climates. Varicella may occur after exposure of susceptible persons to chickenpox or to herpes zoster. Over 95% of primary infections result in symptomatic chickenpox. VZV is transmitted by the respiratory route and virus has been detected by

Varicella-Zoster Virus (Herpesviridae)

863

PCR in room air from patients with varicella or zoster. Intimate, rather than casual contact is important for transmission. Chickenpox is highly contagious; about 60%–90% of susceptible household contacts become infected. In contrast, only 20%–30% of susceptible contacts of persons with zoster develop varicella. Patients with varicella are infectious from two days before the onset of the rash until all the lesions have crusted. Primary infection with VZV results in viral replication in epithelial cells of the upper respiratory tract and oropharynx with lesions present on the respiratory mucosa. T cells subsequently become infected and transmit virus throughout the body including to the skin and to the dorsal root and cranial nerve ganglia where the virus establishes latency. Virus has been detected by PCR in the oropharynx and virus has been cultured from the blood early during varicella. Virus infection of epidermal cells in the skin is thought to be initially limited by interferon; later T cells trafficking in the skin may become infected and disseminate the virus to various organs throughout the body including the nervous system. Zoster is due to reactivation of VZV in patients who have had prior chickenpox; some of these patients may not recall the primary infection. The virus usually reactivates from dorsal root or cranial nerve ganglia, travels down the axon, and replicates in the skin resulting in a rash in the dermatome innervated by the nerve. Zoster does not result from exposure to chickenpox or to other cases of zoster. In contrast to varicella, zoster occurs throughout the year. The risk of reactivating VZV due to varicella vaccine virus is lower than due to wild-type virus. The risk of zoster rises steadily with age, and persons who live to age 85 have a 50% risk of reactivating the virus and developing zoster, if they do not receive the zoster vaccine. Immunocompromised patients, such as those with the acquired immunodeficiency syndrome (AIDS), hematologic malignancies, as well as persons who have receive hematopoietic stem cell or organ transplants, or those on immunosuppressive medications that reduce cellular immunity have a high incidence of zoster. Viremia and subsequent cutaneous or visceral dissemination of lesions may occur in zoster, especially in immunocompromised patients. Injury to a nerve is also a risk factor for reactivation of the virus from the ganglia innervated by the nerve. The rate of zoster has been increasing in the last 60 years perhaps because of longer lifespans or better recognition of the disease. Recurrent zoster is uncommon; less than 4% of patients experience a second episode. Asymptomatic viremia has been detected in bone marrow transplant recipients and has been followed by recovery of cell mediated immunity. Severe VZV infections have been reported in children and adults with heterozygous mutations in subunits of RNA polymerase III (POLR3A, POLR3C, POLR3E, or POLR3F). These patients have reduced induction of interferon after stimulation of cells with poly dA: dT or after VZV infection. Unlike HSV, the VZV genome is AT-rich, which may explain the severe CNS or pulmonary disease seen with VZV, but not HSV in persons with these mutations.

Clinical Features The incubation period for chickenpox is 2 weeks, with a range of 10–21 days. The disease begins with fever and malaise, followed by a generalized vesicular rash (Fig. 3) which evolves into pustules before crusting. Lesions tend to appear first on the head and trunk and then spread to the extremities. New lesions usually follow viremic waves for three to five days and, in the normal host, most lesions are crusted and healed by two weeks. Lesions in different stages coexist in an individual. The disease is usually self-limited in the normal host. Complications of varicella are more common in neonates whose mothers were first infected 5 days before to 2 days after the infant was born, in children with malnutrition, in immunocompromised patients (e.g., malignancy or immunosuppressive therapy), in pregnant women, and in older adults. These complications include bacterial superinfection of the skin, cerebellar ataxia, pneumonia, hepatitis, thrombocytopenia, and less commonly encephalitis, meningitis, purpura fulminans, and central nervous system vasculopathy (vasculitis of the cerebral arteries) which often presents as a stroke. Group A streptococcus or staphylococcus is the most common serious superinfection and can result in cellulitis, fasciitis, or bacteremia. Reye syndrome occurs in rare children who take aspirin to treat varicella fever. Severe complications occur more often in children with impaired T cell immunity such as those with leukemia, HIV, or congenital immune deficiencies or those receiving corticosteroids. Prior to the widespread use of the varicella vaccine in the United States, there were about 3–4 million cases of varicella each year with about 100 deaths; however, the number of cases has dropped sharply and death from varicella in the United States is very rare. Pregnant women with primary infection during the first trimester can give birth to infants with the congenital varicella syndrome, manifested by limb hypoplasia, scarring of the skin, central nervous system abnormalities, and cataracts. Pregnant women with primary infection during the third trimester are more likely to have complications of varicella, especially pneumonia. Zoster usually presents with pain and dysesthesias 1–4 days before the onset of the vesicular rash. The rash is usually painful and confined to a single dermatome (Fig. 4) but may involve several adjacent dermatomes. The trigeminal or thoracic dermatomes are most often affected. Fever and malaise often accompany the rash. Vesicles often are pustular by day 4 and become crusted by day 10 in the normal host. Some patients have pain, but do not develop a rash which is referred to as zoster sine herpete. Postherpetic neuralgia, manifested by pain that persists at least one month after the initial rash resolves can last for months to several years, and is the most common and disconcerting complication of zoster in the normal host. Postherpetic neuralgia is more common in the elderly. Ocular disease manifested by keratitis, episcleritis, iritis or glaucoma is not uncommon. Other less common ocular complications include acute retinal necrosis which can occur in apparently healthy persons, or progressive outer retinal necrosis which occurs in very immunocompromised persons. Less common complications include encephalitis, myelitis, the Ramsay-Hunt syndrome (lesions in the ear canal, with auditory and facial nerve involvement), facial weakness, bacterial superinfection, and pneumonitis. Patients can present with strokes concurrent with or within a few months after presenting with

864

Varicella-Zoster Virus (Herpesviridae)

Fig. 3 Child with disseminated rash of varicella. Courtesy of the Centers for Disease Control and Prevention.

Fig. 4 Adult with zoster in the ophthalmic branch of the trigeminal nerve (A) and vesicles and pustules with the rash of zoster (B). Reproduced with permission from Cohen, J. I., 2013. Herpes zoster. The New England Journal of Medicine 369, 255–263.

zoster due to central nervous system vasculopathy. Patients with impaired T cell immunity are more likely to develop disseminated disease with neurologic, ocular, or visceral involvement. Patients with AIDS have a high frequency of zoster and may develop recurrent or chronic disease with verrucous, hyperkeratotic skin lesions. Immunocompromised transplant recipients can present with viral hepatitis, pancreatitis, or disease involving other organs, sometimes in the absence of, or before development of a rash.

Pathogenesis Varicella lesions are readily recognized in the skin and mucous membranes. However, similar lesions also occur in the mucosa of the respiratory and gastrointestinal tracts, liver, spleen, and any tissue, and remain unrecognized except in severe cases. With severe disease there is inflammatory infiltration of the small vessels of most organs. Zoster causes inflammation and necrosis of the sensory ganglia and its nerves, and skin lesions which are histopathologically identical to those seen with varicella. Cutaneous lesions due to VZV begin with infection of capillary endothelial cells followed by direct spread to epidermal epithelial cells. The epidermis becomes edematous with acantholysis and vesicle formation. Mononuclear cells infiltrate the small

Varicella-Zoster Virus (Herpesviridae)

865

vessels of the dermis. Initially, vesicles contain clear fluid with cell-free virus, but later the vesicles become cloudy and contain neutrophils, macrophages, interferon, and other cellular and humoral components of the inflammatory response pathways. Subsequently, the vesicles dry leaving a crust that heals, usually without scarring. Cells infected with VZV show eosinophilic intranuclear inclusions with multinucleate giant cell formation. These changes are not specific for VZV, as they are seen with herpes simplex and cytomegalovirus infections. Infection with VZV elicits both a humoral and cellular immune response. The ability of VZV immune globulin (VariZIG) to attenuate or prevent infection in exposed children (see Prevention and Control, below) indicates that virus-specific antibody is important for protection from primary infection. The presence of VZV-specific IgG does not correlate, however, with protection from zoster. Antibody to VZV is often present by the time the rash of varicella first appears. Virus-specific IgM, IgG, and IgA are present within five days of symptomatic disease; however, only IgG persists for life. Antibodies to viral glycoproteins gE, gB, gH, and the immediate-early 62 protein have been detected during acute infection, and the titers of antibodies to these proteins are boosted during recurrent infection. The mere presence of antibody to VZV glycoproteins in children with leukemia who had received live varicella vaccine is not adequate to prevent breakthrough varicella or zoster. Cellular immune responses to VZV are more important in recovery from acute varicella infection and for prevention of, and recovery from, zoster. The level of cellular immunity correlates with disease severity during acute varicella. Cytotoxic T cells that lyse virus-infected cells are present by two to three days after the onset of the rash of varicella. Cell-mediated immunity, as measured by lymphocyte proliferative response, is directed against cells expressing glycoproteins gE, gB, gH, gI, and gC, IE62, IE63, and other viral proteins. Interferon is present in VZV vesicles. Most varicella infections result in lifelong immunity to reinfection. Second episodes of varicella are rare; these individuals tend to have reduced humoral and cellular immunity to VZV at the time of the second infection. Zoster is associated with a reduction in cellular immunity to VZV that, in the normal host, is partially restored in response to this recurrent infection. Recurrent zoster is uncommon, except in severely immune deficient patients, such as those with AIDS.

Diagnosis Varicella and zoster are usually diagnosed clinically. Varicella typically presents with a disseminated rash with lesions present in various stages including vesicles, pustules, and crusts. Zoster usually presents with a similar rash, but with pain and skin lesions that are localized to one or two dermatomes and does not cross the midline. In cases where the diagnosis is less certain, scraping of skin lesions to unroof the vesicles or pustules followed by PCR for VZV DNA is diagnostic. PCR for VZV DNA is much more sensitive than staining the fluid for viral antigen or trying to culture the virus from the fluid. Detection of multinucleated giant cells in the specimen is consistent with a herpesvirus infection; however, it is not specific for VZV. In rare cases of zoster which present with visceral disease before, or in the absence of a rash, PCR of blood for VZV is used to make a diagnosis. Patients who present with stroke and are suspected of having VZV vasculopathy are usually diagnosed by PCR for VZV DNA in the cerebrospinal fluid or by demonstrating a significantly higher level of VZV antibody in the cerebrospinal fluid relative to the level of antibody in the blood. Testing for VZV antibody in the blood is indicative of prior infection but is not useful for diagnosis of acute disease. Testing for antibody can be helpful in immunocompromised persons exposed to varicella or zoster, to determine if they have never been infected in the past, and thereby determining whether post-exposure prophylaxis is needed (see below).

Treatment Treatment of varicella includes acetaminophen for fever and anti-pruritics when itching is problematic. While acyclovir is licensed for treatment of varicella, it is not recommended by the American Academy of Pediatrics for otherwise healthy children under age 13 since it shortens the duration of disease by only 1 day. Nonetheless, some physicians recommend acyclovir since it may reduce rare complications in children. Acyclovir is recommended for persons at higher risk of complications of varicella including persons 13 years or older, infants born to mothers with varicella at the time of delivery, immunocompromised children (including those on steroid therapy) regardless of their age, children with chronic pulmonary or skin disease, those taking aspirin (at higher risk of Reye’s syndrome), and persons already with complications associated with varicella. It is often given to pregnant women with varicella in their third trimester who have an increased risk of severe disease. Acyclovir decreases visceral dissemination of varicella in the immunocompromised host. Oral valacyclovir has better bioavailability than oral acyclovir and is approved for treatment of varicella in persons aged 2 years or older; it is not recommended for pregnant women. Acyclovir also reduces the risk of VZV spread of trigeminal zoster to the eye and modestly shortens the duration of zoster symptoms in the normal host. Analogs of acyclovir, such as famciclovir and valacyclovir, result in higher levels of antiviral activity than oral acyclovir and have been licensed for oral therapy of zoster in the United States. Treatment is given within 3 days of the onset of the rash, as well as if new lesions are continuing to occur. Antiviral therapy is recommended for persons with zoster who are age 50 or above, who are immune compromised, who have disease involving the eye or face, or have other complications associated with zoster, and those with moderate to severe pain. Acyclovir shortens the duration of lesions and reduces dissemination of the virus in immunocompromised patients; acyclovir reduces zoster-associated pain but does not reduce the rate of post herpetic neuralgia. Corticosteroids, when used early during zoster, reduce acute pain. Herpes zoster, particularly in elderly patients may lead to prolonged and severe postherpetic neuralgia.

866

Varicella-Zoster Virus (Herpesviridae)

Treatment of postherpetic neuralgia is difficult and often unsatisfactory, but many patients experience improvement with gabapentin, pregabalin, tricyclic antidepressant drugs like amitriptyline, or lidocaine patches. Acyclovir-resistant strains of VZV have been reported in patients with AIDS and transplant recipients; these infections are best treated with foscarnet.

Prevention Passage of wild-type VZV in cell culture by Takahashi in 1974 led to attenuation of the virus and changes in its temperature sensitivity and infectivity for certain cell lines. Comparison of the complete nucleotide sequence of the Oka vaccine strain with its parental virus shows 42 nucleotide and 20 amino acid differences. Multiple nucleotide changes in several genes are responsible for attenuation of the vaccine for growth in skin. The Oka vaccine strain can be distinguished from wild-type strains by differences in restriction endonuclease patterns. The live, attenuated varicella vaccine (Oka strain) was licensed in the United States in 1995 and is recommended for vaccination of healthy children age 1–12 and for older persons who have not been infected with the virus. Two doses of vaccine are recommended, the first at one year of age and the second at 4–6 years of age. The vaccine has also been combined as a single injection with the measles, mumps, and rubella vaccines. Varicella vaccine is about 90% effective in preventing varicella and more than 95% effective in preventing moderate or severe varicella. A mild papular rash, usually near the injection site, may occur during the first two weeks after vaccination. It is usually mild but can be severe if the vaccine is given to patients experiencing periods of profound cellular immune impairment. The live vaccine virus establishes neural latency and can reactivate. Zoster has been reported in vaccine recipients, especially those who are immunocompromised, but the rate appears to be lower than that following natural infection. The varicella vaccine virus has very rarely been transmitted to other persons, and only when the vaccine recipient has developed a rash. The vaccine is not given to persons on high dose immunosuppression, persons with hematologic malignancies, HIV patients with CD4 counts below 200 cell/ul, or pregnant women. Two vaccines are available to prevent zoster. A high titer formulation of the live attenuated Oka vaccine virus is given as a single dose and is approved for persons age 50 or older. The vaccine reduces the frequency of zoster and postherpetic neuralgia by 51% and 67%, respectively, in healthy persons Z60 years old. The vaccine retains its efficacy to prevent postherpetic neuralgia in persons older than age 70 but is less effective to prevent zoster in these older persons. The most common side effects are mild injection site reactions; no significant severe adverse reactions have been attributed to the vaccine. The vaccine is not given to persons on high dose immunosuppression, persons with hematologic malignancies, or HIV patients with CD4 counts below 200 cell/ul. In 2017 a two-dose adjuvanted subunit vaccine was approved for prevention of zoster. The vaccine consists of VZV glycoprotein E and AS01B adjuvant (saponin and monophosphoryl lipid A) in a liposomal formation. The subunit vaccine is 97% effective to prevent zoster in persons age 50–69, and 91% effective for preventing zoster and 89% effective for preventing postherpetic neuralgia in persons age 70 and older. The adjuvanted subunit has more side effects than the live attenuated zoster vaccine; 17% of persons receiving the subunit vaccine had systemic or injection site reactions sufficiently severe to temporarily prevent normal activities. These vaccine reactions included pain, redness, or swelling at the vaccination site, or generalized muscle pains, fever, headache, fatigue, or gastrointestinal symptoms. In 2018 the Advisory Committee for Immunization Practices at the Centers for Disease Control and Prevention gave a preferential recommendation for the zoster subunit vaccine over the live attenuated zoster vaccine for prevention of zoster and its complications. Prevention of varicella can be achieved by restricting exposure to persons with varicella or zoster until all lesions have crusted. This is particularly important for hospital workers and immune deficient patients. For persons exposed to varicella, three options are available- vaccination with the live, attenuated vaccine, giving immunoglobulin prophylaxis, or providing acyclovir to persons soon after exposure. Vaccination is preferred for postexposure prophylaxis of non-immunocompromised persons who were exposed to varicella within the prior three days. Vaccination is 70%–90% effective in this situation. Varicella-zoster immune globulin (VariZIG), prepared from human plasma, prevents or attenuates varicella in 90% of seronegative persons if given within four days of exposure to the virus. Recent recommendations indicate that it can be used up 10 days after exposure to varicella or zoster. VariZIG has no effect in modifying zoster. VariZIG is recommended for individuals at high risk for developing severe varicella and is indicated in those (a) with recent, close contact to patients with varicella or zoster, (b) who are susceptible to varicella, and (c) who fall in a high-risk category. The latter include premature or certain newborn infants, pregnant women, and patients with congenital or acquired cellular immunodeficiencies. Oral acyclovir is an alternative to VariZIG in high risk VZV seronegative persons exposed to varicella or zoster. Acyclovir is estimated to be about 85% effective in preventing varicella when given for one week beginning 7–9 days after exposure.

Further Reading Abendroth, A., Arvin, A.M., Moflat, J.F., 2000. Varicella-Zoster Virus. Current Topics in Microbiology and Immunology. 342. New York: Springer. Arvin, A.M., Gilden, D., 2013. Varicella-zoster virus. In: Knipe, D.M., Howley, P.M., Cohen, J.I. et al. (Eds.), Fields Virology, sixth ed. Philadelphia: Lippincott Williams & Wilkins, pp. 2015–2057. Cohen, J.I., 2013. Clinical practice: Herpes zoster. The New England Journal of Medicine 369, 255–263. Cunningham, A.L., Lal, H., Kovac, M., et al., 2016. Efficacy of the herpes zoster subunit vaccine in adults 70 years of age or older. The New England Journal of Medicine 375, 1019–1032.

Varicella-Zoster Virus (Herpesviridae)

867

Depledge, D.P., Ouwendijk, W.J.D., Sadaoka, T., et al., 2018. A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene 61. Nature Communications 9, 1167. Dooling, K.L., Guo, A., Patel, M., et al., 2018. Recommendations of the advisory committee on immunization practices for use of herpes zoster vaccines. Morbidity and Mortality Weekly Report 67, 103–108. Gershon, A.A., Breuer, J., Cohen, J.I., et al., 2015. Varicella zoster virus infection. Nature Reviews Disease Primers 1, 15016. Jensen, N.J., Rivailler, P., Tseng, H.F., et al., 2017. Revisiting the genotyping scheme for varicella-zoster viruses based on whole-genome comparisons. Journal of General Virology 98, 1434–1438. Leung, J., Harpaz, R., 2016. Impact of the maturing varicella vaccination program on varicella and related outcomes in the United States: 1994–2012. Journal of the Pediatric Infectious Diseases Society 5, 395–402. Markus, A., Golani, L., Ojha, N.K., et al., 2017. Varicella-Zoster virus expresses multiple small noncoding RNAs. Journal of Virology 91, e01710–e01717. (9). Ogunjimi, B., Zhang, S.Y., Sørensen, K.B., et al., 2017. Inborn errors in RNA polymerase III underlie severe varicella zoster virus infections. Journal of Clinical Investigation 127, 3543–3556. Oliver, S.L., Yang, E., Arvin, A.M., 2016. Varicella-Zoster virus glycoproteins: Entry, replication, and pathogenesis. Current Clinical Microbiology Reports 3, 204–215. Sadaoka, T., Depledge, D.P., Rajbhandari, L., et al., 2016. In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency. Proceedings of the National Academy of Sciences of the United States of America 113, E2403–E2412. Warren-Gash, C., Forbes, H., Breuer, J., 2017. Varicella and herpes zoster vaccine development: Lessons learned. Expert Review of Vaccines 16, 1191–1201. Xing, Y., Oliver, S.L., Nguyen, T., et al., 2015. A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL. Proceedings of the National Academy of Sciences of the United States of America 112, 6056–6061. Zerboni, L., Sen, N., Oliver, S.L., Arvin, A.M., 2014. Molecular mechanisms of varicella zoster virus pathogenesis. Nature Reviews Microbiology 12, 197–210.

Variola and Monkeypox Viruses (Poxviridae) Lalita Priyamvada and Panayampalli S Satheshkumar, Centers for Disease Control and Prevention, Atlanta, GA, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of S. Parker, D.A. Schultz, H. Meyer, R.M. Buller, Smallpox and Monkeypox Viruses, In Reference Module in Biomedical Sciences, Elsevier Inc., 2014, doi:10.1016/B978-0-12-801238-3.02665-9.

Glossary Exanthem Eruptive skin rash. Papule Small, solid, elevated lesion of the skin that is often inflammatory. Pock Pitted scar or mark caused by a pustule or pimple. Prodrome Early sign or symptom of illness.

Pustule Small elevated pus-containing lesion of skin. Sequelae Pathological condition occurring as a result of disease. Vesicle Small cavity within the epidermis containing serous fluid.

History Variola virus (VARV), the causative agent of smallpox, is perhaps the singular most devastating human pathogen in history. Since the earliest evidence of smallpox infections preserved in Egyptian mummies dating back to 1200 BCE, through thousands of years later, human beings have battled this highly virulent and contagious virus in numerous outbreaks worldwide. Entire communities have perished during smallpox epidemics, including an estimated 300–500 million individuals in the 20th century alone. Those who survived infection suffered disfiguring residual scarring or blindness. Between the 10th and 18th centuries, efforts to control the spread of smallpox mainly involved quarantine, and intranasal and intradermal variolation using powdered or pulverized smallpox lesions. Thereafter, following Jenner’s inoculation studies using cowpox virus, vaccination emerged as a safer, more effective alternative in the fight against smallpox. In the 20th century, the generation of freeze-dried formulations extended the shelf life of vaccines, which allowed for greater distribution and reach worldwide. Widespread public health campaigns that involved ring vaccination and mass vaccination strategies were deployed around the globe. As a result of these extensive efforts, the number of new smallpox cases worldwide dwindled. The last naturally-occurring cases of smallpox were recorded in the Indian subcontinent in 1975 and in the Horn of Africa in 1977. In 1980, the WHO declared that smallpox had been eradicated worldwide. In contrast to the ancient descriptions of VARV, monkeypox (MPXV) was discovered relatively recently in 1958. MPXV was first discovered when monkeys that were part of a polio research study developed pox-like vesiculopapular skin lesions. Although the clinical symptoms observed were virtually identical to smallpox, serial passage of the isolated virus in mice and rabbit skin distinguished the virus from VARV. The first human case of monkeypox was recorded a decade later in 1970 when a 9-year old boy from Zaire (now Democratic Republic of Congo, DRC) presented with pox lesions in a region that had previously been cleared of smallpox. It is highly probable that human MPXV cases occurred prior to 1970, but were previously misidentified as VARV infections due to the clinical similarities and geographical overlap between the two viruses. Although smallpox was eradicated in 1980, isolated MPXV cases and larger outbreaks have been reported over the past few decades. These have occurred mainly in Africa, but exported cases have also reached North America, Europe and Asia. A zoonotic disease, a majority of monkeypox cases to date have been caused by direct or indirect contact with infected rodents and primates. Today, MPXV remains endemic in the Congo Basin, and has taken the place of VARV as the most critical orthopoxvirus for human health.

Classification and Structural Morphology VARV and MPXV are both members of the Poxviridae family of large double-stranded DNA viruses. Within the poxvirus family, they belong to the sub-family Chordopoxvirinae, which constitutes poxviruses that infect vertebrates, and are further classified into the Orthopoxvirus (OPXV) genus based on serological cross-reactivity with other species within the genus. Like other OPXVs, VARV and MPXV produce large brick-shaped particles that are approximately 140–260 nm in diameter and 220–450 nm long, containing a linear DNA genome approximately 200 kb in length. The genome, enclosed within an enveloped capsid, is monopartite and encodes around 200 genes. As is a classic feature of OPXVs, both VARV and MPXV exist in two distinct forms during viral replication; these forms are named intracellular mature virus (MV) and enveloped virus (EV). Although the same viral genome is shared between the two forms, the particles differ in the number of membranes surrounding the core. While MVs possess a singular viral membrane, EVs undergo an additional double-membrane wrapping step during the viral life cycle resulting in the presence of two viral membranes encapsulating the genome. As a consequence, MVs and EVs express different viral surface proteins, which further results in unique antigenic signatures differentiating the two forms of virions.

868

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21545-8

Variola and Monkeypox Viruses (Poxviridae)

869

Fig. 1 Highly magnified images of variola and monkeypox viruses obtained using transmission electron microscopy. Image on left shows a variola virion with an internal dumbbell-shaped core. Image on right shows the “M” (mulberry type) monkeypox virion in human vesicular fluid. Photographs obtained from the CDC Public Health Image library, courtesy of Dr. Fred Murphy and Sylvia Whitfield (VARV), and Charles D. Humphrey, Tiara Morehead, and Russell Regnery (MPXV).

Electron microscopy (EM) images have revealed morphological details of the exterior and interior surfaces of the VARV and MPXV virions (Fig. 1). The outer surface of VARV and MPXV MV particles consists of a lipoprotein membrane with a knobby, mulberry-like appearance due to the presence of irregularly-shaped surface tubules. Enclosed within is a core wall surrounding the dumbbell shaped viral core, as well as two amorphous structures named lateral bodies. The viral core contains tightly compressed nucleoprotein and a DNA genome. The characteristic morphology of VARV and MPXV particles has made EM-based visualization a valuable confirmatory technique for VARV and MPXV infection diagnosis, both historically during the smallpox eradication campaign, as well as more contemporarily in recent monkeypox outbreaks.

Viral Life Cycle Much of what is understood about the viral life cycle of OPXVs like VARV and MPXV stems from studies of the prototypic OPXV, vaccinia virus (VACV). The steps involved in viral entry and replication of OPXVs have been described in extensive detail elsewhere. Briefly, particles can enter cells either by fusion directly at the plasma membrane or through macropinocytosis. Proteins on the surface of EVs bind to host attachment receptors resulting in the rupture of the EV membrane. This exposes the MV membrane which expresses the entry-fusion complex required for virus-host cell fusion. Viral fusion releases the internal core into the cytoplasm where viral enzymes packaged within the core initiate early transcription and translation. Early proteins include immunomodulatory factors, as well as viral enzymes that facilitate DNA replication. As DNA replication occurs, intermediate and subsequently late genes are transcribed and expressed under regulation by stage-specific viral transcription factors. Late translation products include structural proteins required for virion assembly. Following infection, all the steps involved in viral replication cycles of VARV and MPXV occur entirely within the cytoplasm. Particle assembly occurs within electron dense regions termed “virus factories” and begins with the formation of immature virions consisting of a newly generated DNA core and a singular viral membrane. The immature virions undergo maturation through proteolytic processing of core proteins mediated by viral encoded protease to generate MVs. Although entirely infectious, most of the MVs remain in the cytoplasm, and only a proportion undergo an additional wrapping event resulting in a double-membrane enveloped wrapped virus (WV). The WV is an intermediate between the MV and the cell-associated EV (CEV) or extracellular EV (EEV) forms, both of which are responsible for a majority of viral dissemination within a host during infection. Egress of the enveloped viral forms occurs through membrane fusion. Viral egress triggers the formation of actin tails which can propel viral particles from an infected cell towards neighboring cells. CEVs and EEVs are launched from the tips of microvilli protruding out of cell surfaces toward other cells within the microenvironment, at which point another round of infection can occur. MVs largely remain trapped within infected cells until cell lysis, following which they are released and are able to infect other cells. A simplified illustration of the OPXV life cycle is provided in Fig. 2.

Pathogenesis The pathogenesis of VARV and MPXV are virtually identical with one important exception at the point of entry. While VARV was a solely human pathogen, monkeypox is a zoonotic disease. Therefore, the point of entry for most VARV infections during historic smallpox outbreaks occurred within the respiratory mucosa during person-to-person viral transmission. In contrast, a majority of human MPXV cases are caused by animal exposure and likely require contact with lesions on the skin or oral mucous membranes. After infection has occurred, both viruses travel to the closest lymph nodes through the lymphatic system and replicate in lymphoid tissue.

870

Variola and Monkeypox Viruses (Poxviridae)

Fig. 2 Simplified illustration of the OPXV life cycle.

Fig. 3 Illustration showing the timeline of clinical stages and symptoms in smallpox and monkeypox disease. Days abbreviated as “d”.

Primary viremia is initiated as the virus enters the blood stream and migrates to other sites within the body. Between days 3–14 after infection, the virus spreads to other lymph nodes, the bone marrow, spleen, kidneys and liver. Secondary viremia occurs as the virus disseminates to the oral mucosa and skin. Viral replication in the blood vessels of the dermis causes the formation of lesions that eventually spread to the epidermis. This results in the appearance of pus-filled pocks that are characteristic of smallpox and monkeypox disease. With virus rapidly spreading throughout the body, a systemic inflammatory response is generated causing toxemia, which can lead to death.

Clinical Features As with viral pathogenesis, the clinical features of the two orthopoxviral diseases are nearly indistinguishable but for a few differences. As shown in Fig. 3, human MPXV and VARV infection begin with an incubation period lasting approximately 7–17 days during which no clinical symptoms are observed. This is followed by a prodromal period characterized by high fever, malaise, fatigue and headaches, among other symptoms. In case of smallpox, individuals often suffer very severe fevers with body temperatures exceeding 401C. During MPXV infection, fevers typically range between 38.5–40.51C. Another notable difference during this period is the lack of severe lymphadenopathy in smallpox cases, which is often observed in human MPXV infections.

Variola and Monkeypox Viruses (Poxviridae)

871

The prodromal period is followed by a decline in fever and the onset of rash lesions. Rashes usually appear centrifugally, starting on the throat and oral cavity which can cause difficulties in eating and drinking. Rashes then appear on the face, arms and legs, and finally on the trunk, palms and soles. Over the first week of the rash period, lesions progress from macules, to papules, to vesicles to pustules. The lesions in MPXV and VARV infections are hard and deep, progress slowly and can cause significant pain to infected individuals until they begin to crust. The rash period ends as pustules form scabs, and the scabs crust and fall off. Depending upon the severity of symptoms, an infected individual may succumb to disease during or after the rash period, or survive with sequelae including but not limited to scarring, vision loss or blindness and respiratory complications. The clinical course and severity of disease in MPXV and VARV infections can depend on several factors such as age, immune status, infecting dose, route of infection and the infecting virus strain. The differences in disease severity after VARV infection are illustrated in Fig. 4.

Epidemiology Smallpox For thousands of years until its eradication in 1980, smallpox outbreaks caused widespread mortality and devastation. After the earliest descriptions of pox-like rashes on Egyptian mummies, independent records of human disease resembling smallpox appeared in China, India and Western Asia between the 4th and 10th centuries AD. Through colonization, the virus was brought to Europe and northern Africa, and later to the Americas and southern Africa in the 16th and 17th centuries. With the exception of Australasia, smallpox had become endemic globally by the turn of the 18th century. Through the next two centuries, vaccination efforts strengthened and brought about a large decline in the number of outbreaks worldwide. Nonetheless, smallpox remained a persistent problem for densely populated regions in Africa and Asia through the second half of the 20th century. Through the introduction of the Intensified Smallpox Eradication Program in 1967, and active engagement by global, national and local leaders in widespread immunization campaigns, additional countries eliminated smallpox over the next decade. In 1977, the last known natural case of smallpox occurred in Somalia. In 1980, the WHO declared that smallpox had been eradicated worldwide, and no new VARV infections have been recorded since.

Monkeypox The first known case of human monkeypox was detected in Zaire (now DRC) in 1970. Over the next decade, an additional 54 cases were recorded in Western and Central Africa, 44 of which occurred in DRC. Interestingly, while no deaths were reported in West

Fig. 4 Clinical and epidemiological features of disease following VARV infection. Images showing Malignant, Ordinary and Modified smallpox, and Alastrim obtained from the CDC Public Health Image Library. Photographs by Dr. Dean, Dr. Robinson and Dr. Noble. Source of Hemorrhagic smallpox image: Herrlich, A., Mayr, A., Munz, E., Rodenwaldt, E., 1967. Die Poken; Erreger, Epidemiologie und klinisches Bild, second ed., Stuttgart: Thieme.

872

Variola and Monkeypox Viruses (Poxviridae)

Africa due to monkeypox during this time, the case mortality rate in DRC was 21%. Later studies demonstrated two distinct clades of MPXV based on genomic alignments and orthologous gene clusters, which were subsequently named for the regions in which they were isolated: Congo Basin and West Africa. Despite the eradication of smallpox in 1980, monkeypox persisted in the Congo Basin. Over three hundred cases were reported between 1981–1986, partly due to increased interest in monkeypox surveillance in the region. Sero-surveillance data indicated that prior smallpox vaccination conferred 485% protection from MPXV during this period, and case-control studies revealed a large majority of infections occurred due to contact with wildlife. As smallpox vaccine-mediated immunity waned, several human monkeypox outbreaks occurred in DRC over the subsequent decades. The years 1996–1997 witnessed a prolonged outbreak of 88 possible cases, following which 42500 cases of suspected human MPXV infection were reported during 1998–2004. The first human monkeypox outbreak outside Africa occurred in 2003 in the United States, as a result of the importation of infected animals from Ghana. It is hypothesized that West African rodents carrying MPXV transmitted the virus to indigenous rodents including prairie dogs, which eventually spread monkeypox to humans. In all, 37 confirmed human infections were identified, and no deaths were reported. In contrast, an MPXV outbreak occurring the same year in the Republic of Congo (ROC) involved one death and one case of severe sequelae out of a case total of 11 infected persons. Other significant outbreaks have occurred since, the first in DRC involving over 750 cases in 2006–2007, and more recently in Nigeria in 2017. The 2017 Nigerian monkeypox outbreak has been the largest to date in the West African nation, occurring nearly 40 years after its last confirmed human case. To date, over 200 confirmed and suspected monkeypox cases have been identified, although the total disease burden remains unknown. In 2018 and 2019, a total of six MPXV cases were reported in the United Kingdom (4), Israel (1) and Singapore (1), all involving travelers from Nigeria who fell ill with monkeypox upon arrival. While five of these cases originated in Nigeria, the sixth was a nosocomial infection of a healthcare worker in the United Kingdom. In conjunction, ROC reported over 40 suspected human monkeypox infections in 2017–2018. The recent re-emergence of monkeypox in Western and Central Africa and increase in travel-related cases have elevated the importance of monkeypox as a growing public health threat to humans.

Diagnosis The accurate diagnosis of OPXV infections, including VARV and MPXV, is complicated by a few factors. First, poxviral and nonpoxviral exanthema can appear very similar, making it exceptionally challenging to identify the causative pathogen purely by clinical evaluation. This is illustrated in Fig. 5, which shows a side-by-side comparison of pustular and maculopapular lesions caused by VARV and varicella zoster virus (causative agent of chickenpox and shingles). Second, prior vaccination with the smallpox vaccine may confound the serology-based diagnosis of an ongoing OPXV infection. Third, antigenic and structural similarities between VARV, MPXV and other OPXVs requires the use of advanced laboratory techniques and exclusive resources to differentiate between these viruses. As such, a combination of detailed case history including prior vaccination records, clinical examination and laboratory assays is critical for the accurate and efficient diagnosis of VARV and MPXV. Due to the eradication of smallpox, clinical and laboratory testing to confirm VARV infection do not routinely occur in the present day. In case of MPXV, the symptoms associated with the prodromal and rash phases listed in Fig. 3, as well as the presence of swollen lymph nodes are typical clinical features of monkeypox disease. Importantly, the physical characteristics of the lesions (monomorphy versus pleomorphy) and their distribution on the body (centrifugal versus centripetal) can help to distinguish between OPXVs and other non-poxviral exanthematous infections. Laboratory-based tests to distinguish between OPXV and nonOPXV infections include electron microscopy negative staining, immunohistochemistry, serological assays, and viral isolation, culture and amplification. Of the aforementioned techniques, only virus isolation and DNA-based detection can parse MPXV apart from other OPXVs at this time. As a result, the current laboratory criteria for confirmation of monkeypox require the PCR-based detection of MPXV DNA in clinical samples or a positive MPXV culture from virus isolated from a clinical specimen.

Fig. 5 Image showing lesions caused by VARV (left panel) and varicella zoster virus (right panel). The lesions have similar morphologies that appear undistinguishable to an untrained eye. Images obtained from the CDC Public Health Image Library. Photographs by Dr. Noble and Dr. K.L. Herrmann.

Variola and Monkeypox Viruses (Poxviridae)

873

Prevention The earliest efforts in smallpox prevention can be dated back to 10th century China, where dried pustular lesions from infected persons were applied to the nostrils of uninfected persons. This practice was observed to sometimes protect the recipients from smallpox. In the 17th and 18th centuries, “pox-laden” blankets were wrapped around uninfected children in India, with the intent of transmitting mild disease and conferring protective immunity. The instillation of smallpox pustular matter into the skin of uninfected persons, also known as variolation, was also practiced during this time in China, India and Turkey. Variolation was introduced to England in the 18th century and subsequently to multiple English colonies. With the introduction of variolation in new areas, many communities experienced a decline in smallpox incidence. However, safer alternatives were imperative, as variolation itself caused a case mortality rate of 0.5%–2%. In 1796, Dr. Edward Jenner injected material from the vesicle of a milkmaid with cowpox into the arm of a smallpox nonimmune boy. He then infected the boy with VARV and observed that the boy did not develop smallpox disease. This initial challenge experiment, an egregious violation of ethics in human research by today’s standard, was the foundation of modern-day vaccination. Far safer than variolation, vaccination quickly became widespread in the 19th century. By the onset of the 20th century, a number of Western nations had entirely eliminated smallpox from their population through mass and ring vaccinations. Interestingly, the smallpox vaccines used during the 20th century WHO smallpox programs, also known as first generation smallpox vaccines, were formulated using VACV, not cowpox virus. The origin of VACV is unclear, and the cause for a switch from cowpox virus to VACV, if one even occurred is not well understood. The mostly widely used and studied first generation smallpox vaccine was Dryvax, based on the NYCBH (New York City Board of Health) strain of VACV. Others include Lister strain-based vaccines Pourquier and Lancy-Vaxina Berna. First generation vaccines were largely propagated in animal skin and lymph and although efficacious in controlling smallpox incidence, were associated with a wide range of serious post-vaccinial complications. Second generation smallpox vaccines, including ACAM2000, Elstree-BN and VV Lister/CE, were manufactured using modern cell culture in accordance with Good Manufacturing Practices (GMP). These vaccines were formulated with either the same vaccine strains as first generation vaccines or their purified clonal variants. ACAM2000 was licensed in the United States in 2007, available for use under limited circumstances and part of the Strategic National Stockpile (SNS). Given that smallpox is no longer an imminent threat, the vaccine is only administered to individuals with a high risk of exposure, such as certain military, healthcare and laboratory personnel. ACAM2000 is a live replication-competent vaccine derived from a less virulent subpopulation of Dryvax vaccine NYCBH strain. The vaccination schedule requires a singular dose of vaccine administered percutaneously using a bifurcated needle as pictured in Fig. 6. Individuals with certain health conditions, including immune deficiency disorders, cardiac disease, chronic skin conditions such as eczema, as well as infants and pregnant women cannot receive ACAM2000 as a pre-exposure vaccine as its effectiveness relies on the local replication of VACV at the site of vaccination. Third generation vaccines were developed as a response to the need for safer vaccines with fewer side effects. Towards the end of the smallpox eradication era, the Lister strain based LC16m8 and Modified Vaccinia Ankara (MVA) were evaluated for their safety and immunogenicity in several clinical trials. Both vaccines showed significantly safer post-vaccination health outcomes in their respective studies compared to first and second generation vaccines. The most recent smallpox vaccine to have received licensure and have been added to the SNS is the MVA-based third generation vaccine named Jynneos (also known as Imvanex, Imvamune or MVA-BN). Jynneos is a replication-deficient (in mammalian cells) vaccinia virus-based vaccine that was approved by the FDA for smallpox and monkeypox prevention in 2019. The approval of Jynneos is noteworthy for the following two reasons: first, Jynneos was shown to cause no adverse events even in recipients with immune deficiencies or exfoliative skin conditions. Since ACAM2000 is contraindicated for these groups of individuals, Jynneos would be a safe way to provide these susceptible groups with protective immunity in the event of a future smallpox outbreak. Second, Jynneos is the first and only FDA-approved monkeypox vaccine. Although the cross-protection and safety testing of Jynneos is currently ongoing in MPXV endemic regions, Jynneos is the only vaccine that is presently approved for prevention of monkeypox disease in the United States.

Fig. 6 Image showing the correct technique for smallpox vaccine administration using a bifurcated needle. Image obtained from the CDC Public Health Image Library. Photograph by James Gathany, 2002.

874

Variola and Monkeypox Viruses (Poxviridae)

Treatment At present, the only FDA-approved treatment for smallpox is the small molecule inhibitor tecovirimat (also known as ST-246, TPOXX). Approved in 2018 by the FDA, tecovirimat targets a conserved VARV gene involved in EV formation. A decrease in the production of EV particles, as caused by tecovirimat, reduces overall viral spread and associated pathology. The anti-VARV potency of tecovirimat has been demonstrated in several in vitro studies. Since VARV is a human pathogen that has been eradicated, other OPXV animal models involving smallpox-like illness have been used to evaluate the antiviral efficacy of tecovirimat, such as ectromelia virus in mice, rabbitpox virus in rabbits and MPXV in primates in accordance with FDA recommendations. Tecovirimat is also well tolerated with few side effects in healthy individuals. As it has not been evaluated in humans experiencing VARV infection, it is unknown whether tecovirimat will be effective in the event of a smallpox outbreak. There are no approved treatments for monkeypox at this time. If an exposure occurs in the absence of prior smallpox or monkeypox vaccination, a clinical regimen involving vaccine immune globulin (VIG), tecovirimat and other investigational drugs currently in development, including Cidofovir and Brincidofovir (CMX001) may be considered.

Disclaimer The conclusions, findings, and opinions expressed by authors contributing to this article do not necessarily reflect the official position of the U.S. Department of Health and Human Services or the Centers for Disease Control and Prevention.

Further Reading Beer, E.M., Rao, V.B., 2019. A systematic review of the epidemiology of human monkeypox outbreaks and implications for outbreak strategy. PLOS Neglected Tropical Diseases 13, e0007791. Chan-Tack, K.M., Harrington, P.R., Choi, S.Y., et al., 2019. Assessing a drug for an eradicated human disease: US Food and Drug Administration review of tecovirimat for the treatment of smallpox. The Lancet Infectious Diseases 19, e221–e224. Damon, I.K., 2013. Poxviruses. In: Knipe, D.M., Howley, P. (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Lippincott Williams & Wilkins. Durski, K.N., Mccollum, A.M., Nakazawa, Y., et al., 2018. Emergence of monkeypox - West and Central Africa, 1970–2017. Morbidity and Mortality Weekly Report 67, 306–310. Fenner, F., Henderson, D.A., Arita, I., Jezek, Z., Ladnyi, I.D., 1988. Smallpox and its Eradication. Geneva: World Health Organization. Jezek, Z., Fenner, F., 1988. Human monkeypox. In: Melnick, J.L. (Ed.), Monographs in Virology, vol. 17. Basel: Karger. Mccollum, A.M., Damon, I.K., 2014. Human monkeypox. Clinical Infectious Diseases 58, 260–267. Merchlinsky, M., Albright, A., Olson, V., et al., 2019. The development and approval of tecoviromat (TPOXX(R)), the first antiviral against smallpox. Antiviral Research 168, 168–174. Moss, B., 2013. Poxviridae: The viruses and their replication. In: Knipe, D.M., Howley, P. (Eds.), Fields Virology, sixth ed. Philadelphia, PA: Lippincott Williams & Wilkins. Pittman, P.R., Hahn, M., Lee, H.S., et al., 2019. Phase 3 efficacy trial of modified vaccinia ankara as a vaccine against smallpox. The New England Journal of Medicine 381, 1897–1908.

Relevant Websites https://www.cdc.gov/smallpox/clinicians/algorithm-protocol.html Evaluating Patients for Smallpox: Acute, Generalized Vesicular or Pustular Rash Illness Protocol. https://www.who.int/csr/disease/smallpox/en/-WHO | Smallpox. World Health Organization. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/smallpox-variola-virus-infection-developing-drugs-treatment-or-prevention-guidance-industry-Smallpox (Variola Virus) Infection: Developing Drugs for Treatment or Prevention Guidance for Industry.

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae) Peter J Walker, The University of Queensland, St. Lucia, QLD, Australia Robert B Tesh, The University of Texas Medical Branch, Galveston, TX, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of H. Bourhy, A.J. Gubala, R.P. Weir, D.B. Boyle, Animal Rhabdoviruses, In Encyclopedia of Virology (Third Edition), edited by Brian W.J. Mahy and Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00783-4

Glossary Akt A serine/threonine-specific protein kinase that plays important roles in cellular signaling pathways (also known as protein kinase B). AP1 A transcription factor (activation protein 1) that regulates cellular gene expression. Epicardium The outer layer of heart tissue. Fas A member of the tumor necrosis factor (TNF) receptor superfamily which has a central role in the regulation of apoptosis (also known as Apo1 or CD95). Hypocalcemia A low level of calcium in the circulating blood. JNK c-Jun N-terminal kinases are serine/threonine kinases and members of the mitogen-activated protein (MAP) kinase family involved in signal transduction. Leucopenia A decreased total number of white blood cells in the circulating blood. NF-κB Transcription factor that plays a key role in regulation of cellular gene expression during viral infection.

Pericardial fluid Fluid within a double-walled sac that contains the heart and the roots of the great blood vessels. PI3K Phosphoinositide 3-kinases involved in signal transduction by phosphorylating the inositol ring of phosphatidylinositol. Src Non-receptor tyrosine kinases (src is short for sarcoma) that play key roles in signal transduction from transmembrane receptor tyrosine kinases. Sternal recumbency Reclined in a position of comfort on the chest bone. Synovial membranes Connective tissue membranes lining the cavities of the freely movable joints. Thoracic fluid Fluid in the chest cavity. TURBS A ‘termination upstream ribosome-binding site’ that allows stop-start expression from consecutive or partlyoverlapping open reading frames.

Classification Apart from rabies, the animal rhabdoviruses that can cause disease in livestock fall into two genera of the family Rhabdoviridae, order Mononegavirales. Four viruses assigned to the genus Vesiculovirus cause vesicular stomatitis in cattle, horses and swine, and occasionally spill over to cause disease in humans: vesicular stomatitis Indiana virus (VSIV; species Indiana vesiculovirus), vesicular stomatitis New Jersey virus (VSNJV; species New Jersey vesiculovirus), Cocal virus (COCV; species Cocal vesiculovirus) and vesicular stomatitis Alagoas virus (VSAV; species Alagoas vesiculovirus). Antigenically, these represent two major serotypes, Indiana and New Jersey. The Indiana serotype is subdivided into four subtypes represented by VSIV (Indiana 1), COCV (Indiana 2), VSAV (Indiana 3) and Morreton virus (MORV; Indiana 4; species Morreton vesiculovirus). Antibodies to MORV has been detected in animals and humans but to date the virus has not been associated with disease. The term vesicular stomatitis virus (VSV), as commonly used in the literature, usually refers to vesicular stomatitis Indiana virus and/or vesicular stomatitis New Jersey virus. Other vesiculoviruses that infect mammals, including humans, are Piry virus (PIRYV; species Piry vesiculovirus) and Isfahan virus (ISFV; species Isfahan vesiculovirus), as well as Chandipura virus (CHPV; species Chandipura vesiculovirus) which has been associated with major outbreaks of an influenza-like illness and encephalitis in India. Eight other vesiculoviruses have been isolated from mammals or hematophagous insects but have not been associated with disease. These include American bat vesiculovirus (ABVV; species American bat vesiculovirus), Carajas virus (CARV; species Carajas vesiculovirus), Jurona virus (JURV; species Jurona vesiculovirus), Malpais Spring virus (MSPV; species Malpais Spring vesiculovirus), Maraba virus (MARV; species Maraba vesiculovirus), Perinet virus (PERV; species Perinet vesiculovirus), Radi virus (RADV; species Radi vesiculovirus) and Yug Bogdanovac virus (YBV; species Yug Bogdanovac vesiculovirus). In the genus Ephemerovirus, bovine ephemeral fever virus (BEFV; species Bovine fever ephemerovirus) causes ephemeral fever in cattle and water buffalo. Kotonkan virus (KOTV; species Kotonkan ephemerovirus) is the only other member of the genus Ephemerovirus to have been associated with clinical disease, both naturally and in experimentally infected cattle. There is no evidence of human infection with either BEFV or KOTV. Ephemeroviruses are distinguishable antigenically in virus neutralization tests using polyclonal hyperimmune sera or ascites fluids. Six other ephemeroviruses have been isolated from healthy cattle and/or hematophagous insects but have not been associated with disease in animals or humans. These include Berrimah virus (BRMV; species Berrimah ephemerovirus), Kimberley virus (KIMV; species Kimberley ephemerovirus), Adelaide River virus (ARV; species Adelaide River ephemerovirus), Obodhiang virus (OBOV; species Obodhiang ephemerovirus), Koolpinyah virus (KOOLV; species Koolpinyah ephemerovirus) and Yata virus (YATV; species Yata ephemerovirus).

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.20979-5

875

876

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

Virus Structure VSV virions are enveloped, bullet-shaped particles, approximately 190 nm in length and 80 nm in diameter (Fig. 1). One end is conical and comprises approximately 25% of the length of virions; the cylindrical trunk comprises 75% of the length. Projections, 5–10 nm long and about 3 nm in diameter, form a honeycomb pattern on the surface. Internally, the nucleocapsid is 45 nm in diameter with cross-striations spaced at 4.5–5 nm. Defective-interfering particles with incomplete genomes are formed under some conditions in cell culture and appear as proportionally truncated virions. VSV particles are composed of approximately 74% protein, 20% lipid, 3% RNA and 3% carbohydrate. Cryo-electron microscopy and crystallographic studies have revealed the fine structure of purified VSV virions. The viral RNA is precisely complexed with the nucleoprotein (N) to form an inner helical nucleocapsid with the 30 end located in the conical tip of the bullet and the 50 end is at the base of the trunk. The inner nucleocapsid is encased in a second helical layer formed from the matrix protein (M) which then interacts with the glycoprotein (G) which projects through the lipid envelope to form trimeric spikes on the virion surface. Two other VSV proteins (P and L) are also components of the nucleocapsid but their precise structural locations have not yet been determined.

A

B

C M G N

RNA L

P

Fig. 1 (A) Negative stain, transmission electron micrograph of VSV virions. (B) Negative stain, transmission electron micrograph of a BEFV virion, illustrating the prominent axial channel. Bar = 50 nm. (C) Schematic illustration of a rhabdovirus virion. Structural proteins N, P, M, G and L, and the (−) ssRNA genome are indicated. The size, relative quantities and precise location of the proteins do not accurately reflect the content in virions. (A) Courtesy F.A. Murphy, CDC Public Health Image Library

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

877

BEFV virions are also bullet-shaped (Fig. 1). The virions (140–200 nm  60–80 nm) display a prominent axial channel intruding from the base, a precisely coiled, helical nucleocapsid with 35 cross-striations at intervals of 4.8 nm, and an envelope with projections extending 8 nm from the surface. Truncated defective-interfering particles are frequently observed following passage of the BEFV in cell culture.

Genomes and Proteins Like most other rhabdoviruses, vesiculoviruses and ephemeroviruses have non-segmented, negative sense (  ), single-stranded (ss) RNA genomes (Fig. 2). Vesiculovirus genomes range in size from approximately 10.7 to 11.3 kb. The genomes feature 30 leader and 50 trailer regions in which the three terminal nucleotides (nt) are complementary (i.e., 30 -UGC...ACG-50 , in negative sense). In the VSIV genome, the 30 leader is 47 nt in length and the 50 trailer is 57–59 nt in length. The 30 leader region is followed consecutively by five genes (N, P, M, G and L), each bounded by conserved transcription initiation (30 -UUGUC….) and transcription termination/polyadenylation (….ACUUUUUUU-50 ) sequences. Each gene contains a long open reading frame (ORF) encoding a protein that forms a structural component of virions. In VSIV, the P gene also contains an alternative ORF that encodes two small proteins (C0 and C00 ) from different initiation codons. Other vesiculoviruses have very similar genome organizations to VSIV. Ephemerovirus genomes are significantly larger than those of vesiculoviruses, ranging in size from approximately 14.5 to 16.1 kb. They are similar in organization to the genomes of vesiculoviruses, including the same conserved complementary terminal trinucleotides, the same conserved transcription initiation and transcription termination sequences, and the five structural protein genes (N, P, M, G and L). However, ephemeroviruses have 3–5 additional genes located between the G gene and L gene, including the GNS gene and the a gene. In the BEFV genome (14,900 nt), the a gene is followed by the b and g genes. The a gene contains two consecutive long ORFs, a1 and a2 separated by a single nucleotide. Although not yet demonstrated experimentally, expression of the a2 ORF appears to be possible by a stop-start mechanism involving a conserved ‘termination upstream ribosome-binding site’ (TURBS) in the mRNA (i.e., 50 -UGGGA-30 ). In KOTV, the a gene also contains two consecutive long ORFs (a1 and a2) separated by a single nucleotide and a TURBS sequence upstream of the ORF junction. The KOTV a gene is followed by b, g and δ genes. The five vesiculovirus structural proteins (N, P, M, G and L) are encoded in long ORFs in the corresponding genes. The N protein (B47 kDa) is a major component of virions. It associates with genomic RNA to form a ribonucleoprotein (RNP) which serves as the template for replication and transcription. The N protein also associates with anti-genomic RNA during replication but does not associate with the viral mRNAs. The P protein (B29 kDa) is a phosphoprotein that complexes with N to prevent both self-aggregation and binding of N to cellular RNA, and then acts as a chaperone to deliver N to complex with genomic or anti-genomic RNA. P is also a cofactor of the viral polymerase, interacting with the L protein to position it on the N-RNA template. The L protein (B241 kDa) is an enzyme that is responsible for RNA transcription and replication. It performs the functions of the RNA-dependent RNA polymerase (RdRp) as well as 30 -polyadenylation and 50 -capping of mRNA, and protein kinase activities. The N, P and L proteins, together with the viral RNA, are components of the inner helical nucleocapsid in virions. The M protein (B26 kDa) is also a major component of virions. It binds to N to form an outer helix layer, providing rigidity by clamping adjacent turns of the RNP. In infected cells, M is also 0

1

2

3

4

5

6

7

8

VSIV

9

10

11

12

13

14

15 kb

11,161 nt

mRNAs

3’

5 N

P

M

G

L

ORFs C

50 nt leader

59 nt trailer

BEFV

14,900 nt

mRNAs

3’

5’ N

P

M

G

GNS

α1α2 β γ

L

ORFs

50 nt leader

70 nt trailer

Fig. 2 Schematic illustration of the genome organizations and transcription strategies of VSIV and BEFV. Solid arrows indicate the major transcriptional products. Open boxes indicate long open reading frames (ORFs).

878

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

responsible for several cytotoxic effects associated with VSIV infection including cell rounding, regulation of apoptosis, inhibition of host cell transcription and inhibition of nuclear export of host RNAs. The G protein (B67 kDa) is a class I transmembrane glycoprotein which forms trimers that project through the lipid envelope. The C-terminal cytoplasmic domain of G interacts with the nucleocapsidbound M protein layer to promote budding of virions at cellular membranes. The N-terminal ectodomain of G forms the outer layer of the virion where it mediates binding to cellular receptors, membrane fusion and entry into cells. The G protein also induces virusneutralizing antibodies. Two small basic protein (C0 and C00 ) encoded in vesiculovirus P genes are expressed in infected cells (B8 kDa and B6.5 kDa, respectively) but they are not required for normal replication in mammalian cell cultures. Their roles are currently not known but they may be involved in pathogenesis of transmission by insect vectors. Evidence of their expression indicates that alternative ORFs as small as 165 nt (encoding proteins of 55 amino acids) are potentially functional in rhabdoviruses. Ephemeroviruses also contains five structural proteins (N, P, M, G and L) corresponding approximately in size and basic functions to those of vesiculoviruses and most other rhabdoviruses. The ephemerovirus GNS gene, which is located immediately following G gene, also encodes a class I transmembrane glycoprotein (GNS) that shares moderate levels of sequence identity with the BEFV G protein, including a sub-set of the 12 cysteine residues that are variously conserved in animal rhabdovirus G proteins and form disulfide bridges that stabilize their folded structure. BEFV GNS (B90 kDa) is expressed in infected cells but is not present in virions produced either in mammalian cells or insect cells and does not induce virus-neutralizing antibodies or pH-activated cell fusion. The function of GNS is currently unknown. Ephemerovirus a1 proteins have the structural characteristics of class IA viroporins, featuring an N-terminal ectodomain containing multiple large aromatic residues (tyrosine, tryptophan and phenylalanine), a central transmembrane domain, and a C-terminal cytoplasmic domain that is rich in basic residues (lysine, arginine and histidine). BEFV a1 (B10.5 kDa) has been shown to localize in the Golgi complex and increase cellular permeability. The C-terminal cytoplasmic domain of BEFV a1 also interacts specifically with importin b1 and importin 7, suggesting that it may also modulate components of nuclear trafficking pathways. Other potential accessory proteins (a2, b, g and δ) encoded in the ephemerovirus G–L intergenic region have not yet been identified or characterized.

Life Cycle Attachment, Entry and Uncoating Most studies of rhabdovirus replication have been conducted by using VSV as a model (Fig. 3). Virions enter cells by endocytosis via clathrin-coated pits in a dynamin-2 dependent manner. Adsorption to cells is mediated by the G protein which binds to its

Fig. 3 A schematic illustration of the replication cycle of VSV. The replication cycle (described in the text) is depicted showing attachment of virus to the cell, internalization, release of the viral core into the cytoplasm, primary viral mRNA synthesis, mRNA translation, genomic replication, secondary viral mRNA synthesis, assembly and budding of infectious particles. Illustration courtesy of S.P. Whelan.

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

879

receptor on the cell surface. The low-density lipoprotein receptor (LDLR) serves as the primary receptor for VSV but other LDLR family members containing structurally homologous class A cysteine-rich repeats can serve as alternative receptors. Low pH within endocytic vesicles induces a conformational rearrangement of the G protein that exposes the fusion domain and triggers fusion between the endosomal and viral membranes, releasing viral nucleocapsids into the cytosol, either directly or via a secondary fusion involving an intraluminal vesicle. During this process, the M protein is shed from the surface of nucleocapsids and migrates to the nucleus to inhibit host cell transcription. Primary viral transcription occurs directly from the RNP complex comprising the L protein, P protein and the N-RNA template. BEFV also enters cells via the clathrin-mediated, dynamin-2-dependent endocytic pathway and has been shown to require microtubules as well as Rab 5 and Rab 7, regulatory guanosine triphosphatases that associate with the sorting endosome and are involved in endosomal membrane fusion. To enhance virus entry, BEFV activates the Src-JNK-AP1 and PI3K-Akt-NF-κB signaling pathways to upregulate clathrin and dynamin 2 expression. Although mediated through the viral G protein, the cellular receptor for BEFV entry is not yet known.

Gene Expression and Replication VSV transcription occurs exclusively in the cytosol from the RNP complex, generating 50 -capped and 30 -polyadenylated mRNAs. Transcription from the (  ) RNA genome is initiated by a sequence-specific promoter at the 30 end, first generating a short (47 nt) leader RNA (Le þ ) and then commencing mRNA synthesis at a conserved initiation sequence (30 -UUGUC-50 ) at the start of the N gene. N gene transcription terminates with polyadenylation of the mRNA at another conserved sequence (30 -AUACUUUUUUU-50 ) at the end of the N gene. The polymerase then recommences transcription at the next conserved initiation sequence and terminates at the next conserved termination/polyadenylation sequence, moving progressively in this fashion to the 50 end of the genome to produce 5 viral mRNAs. There is a decrease in mRNA copy numbers as the polymerase moves along the genome (N > P > M > G > L), apparently due to decreasing efficiency of re-initiation of transcription at each gene junction. The mechanisms of mRNA capping and polyadenylation differ from those employed for host mRNAs. Methylated caps are formed on the nascent 50 -triphosphorylated mRNAs (50 -pppApApCpApG-) in a multi-step process coordinated and catalysed by several active sites in the L protein: (1) although not formally demonstrated, a GTPase appears to produce GDP from GTP; (2) a GDP polyribonucleotidyl transferase links the L protein covalently via a histidine residue (H1227) to the RNA to form an intermediate structure (50 -LpApApCpApG-) and then transfers the 50 -monophosphorylated RNA to GDP (50 -GpppApApCpApG-); and (3) a methyl transferase then sequentially methylates the ribose 20 -O and guanosine N-7 positions in the cap structure (7mGpppAmpApCpApG-). Similarly, polyadenylation of VSV mRNAs is catalysed by the L protein in an unconventional pseudo-templated reaction in which the polymerase reiterates transcription in the conserved tract of U residues at the end of each gene. The capped and polyadenylated mRNAs are translated by cytoplasmic polysomes, except for the G mRNA which is translated by membrane-bound polysomes. Following translation, RNA replication occurs in the cytoplasm, generating full-length ( þ ) RNA and then full-length (  ) RNA. Like transcription, replication is conducted by RNP complexes using the N-RNA template. However, during replication the RdRp initiates at the 30 end of the template, reads through transcriptional control signals and continues to terminate at the 50 end. Newly synthesized N, P and L proteins are required to simultaneously encapsidate the nascent antigenomic RNA and then genomic RNAs, ensuring that they are never naked in infected cells. Encapsidation is thought to protect the genome and antigenome from degradation and detection defensive responses by host cells. Nascent RNP complexes containing (  ) sense genomic RNA undergo rounds of secondary transcription. Switching between transcription and replication is not well understood but involves the increasing accumulation of N protein to encapsulate nascent genomic and antigenomic RNA. According to a proposed ‘single initiation stop-start model’, interaction between N-P complexes and the 47 nt Le þ RNA modifies of the transcriptase, promoting read-through of the initiation and termination signals. However, several observations have raised issues with this model, leading to a proposed ‘two polymerase entry model’ in which the polymerase can initiate either at the genome 30 terminus for replication or internally at the start of the N gene for transcription. To enter full replication mode, this model requires suppression of Le ( þ ) synthesis. In support of this model, it has been reported that there are indeed two distinct forms of RNP complex in infected cells, with the replication complex comprising only the P, L and the N-RNA template and the transcription complex sequestering several additional host proteins including elongation factor 1a, heat shock protein 60 and RNA guanidinyl transferase.

Assembly and Budding Assembly and budding of VSV from infected cells occur at the plasma membrane and involve interaction of the viral RNP complex with the M protein. M is synthesized as a soluble cytoplasmic protein. Three forms of M are generated from alternative in-frame initiation codons but only the full-length protein is associated with budding and assembly. A proportion of full-length M is transported to the inner leaflet of plasma membrane where it forms microdomains. The G protein is synthesized at membranes of the rough endoplasmic reticulum where the signal peptide is removed and the step-wise process of N-linked glycosylation commences. G is then trafficked via the Golgi complex to the plasma membrane where it forms separate microdomains from the M protein. The M and G microdomains appear subsequently to coalesce to establish the sites of virion assembly and budding from infected cells. By interacting with M, the RNP complex is condensed into a tightly packed helix. Late domain motifs (24PPPY27 and 37 PSAP40) in the flexible N-terminal region of M then mediate the recruitment of host cell factors that are required for the late step

880

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

of virus egress. The 24PPPY27 domain interacts with the WW domains of E3 ubiquitin ligase Nedd4 and 37PSAP40 recruits Tsg101/ vps23, a component of the ESCRT (endosomal sorting complex required for transport) complex. Recruitment of ESCRT to the site of viral budding facilitates multivesicular body (MVB) biogenesis as well as the constriction and severing the neck of the bud, allowing the release of mature virions.

Inhibition and Modification of Host Cell Functions VSV deploy a range of strategies to enhance viral replication processes and inhibit host defensive responses, many of which are mediated through the three forms of the M protein (M1, M2 and M3). The M protein targets several cellular proteins to inhibit host gene expression. At one level, M shuts off transcription by host RNA polymerases I and II. In the case of RNA polymerase II, inhibition has been shown to be due to inactivation of transcription factor TFIID. The M protein also blocks nuclear-cytoplasmic export by interacting with the nucleoporin Nup98 and the mRNA export factor Rae1. Although not attributed directly to the M protein, host protein synthesis is also blocked directly during VSIV infection by inhibition of host translation initiation factors eIF2 and eIF4E. In addition to favouring viral replication, inhibition of host gene expression results in cell rounding due to disruption to the cytoskeleton, apoptosis and inhibition of the interferon response. The M protein also appears to directly target the host defensive response by interaction with LMP2, a chymotrypsin-like catalytic subunit of the immunoproteasome which is involved in the generation of MHC class I-compatible peptides. BEFV activates the Src-JNK-AP1 and PI3K-Akt-NF-κB signaling pathways to enhance virus replication (see above). BEFV has also been shown to induce apoptosis in infected cells through the activation of the Fas-mediated and mitochondria-mediated caspasedependent pathways. Specific viral proteins involved in activation of these signaling pathways have not yet been identified. However, the C-terminal domain of the BEFV a1 has been shown to bind importin b1 and importin 7, suggesting that it may modulate components of nuclear trafficking pathways.

Epidemiology Based on antigenic and genetic differences, vesicular stomatitis viruses are divided into two distinct serotypes, Indiana and New Jersey. The Indiana serotype is further divided into four subtypes: classical Indiana or Indiana 1 virus (VSIV), Cocal or Indiana 2 virus (COCV), Alagoas or Indiana 3 virus (VSAV) and Morreton or Indiana 4 virus (MORV). At present, all of these viruses are restricted to the Americas, although the known geographic distribution of each within the continents varies. Vesicular stomatitis New Jersey virus (VSNJV) and VSIV occur occurs throughout both continents from Canada to Argentina. In temperate regions of the Americas, their occurrence is sporadic with seasonal activity during warmer months of the year. In tropical and subtropical regions of Mexico, Central and South America, VSNJV and VSIV are endemic. In contrast, COCV has only occasionally been reported from Trinidad and Brazil, VSAV from north-eastern Brazil, and MORV from eastern Colombia. Each of these viruses, except MORV, have been associated with vesicular disease in livestock. Despite many years of research, the epidemiology of vesicular stomatitis is not well understood. Serologic studies indicate that in regions where these viruses occur, a wide variety of mammals (wild and domestic) as well as humans are naturally infected, although the precise mode of their infection is uncertain. Vesicular stomatitis viruses have been isolated from a variety of field-collected biting insects (sandflies, mosquitoes, black flies, midges and eye gnats) and they are presumed to be arthropod-borne. Experimental studies have demonstrated infection, replication, bite transmission and even vertical (transovarial) transmission of VSV in some of these insects, although how the insects acquire the virus in nature is puzzling. Some insects, such as black flies (Simulium), biting midges (Culicoides) and eye gnats (Hippolates) are probably infected during outbreaks by feeding on vesicular lesions on livestock; fluid and exudate from such lesions contain high titers of infectious virus. However, results of field and experimental studies indicate that equines, bovines, swine and a variety of wild mammals have low levels of viremia during acute infection with VSV. In experimental studies, the duration of VSV viremia in livestock and wild animals is quite transient and often undetectable, with the development of high titers of specific neutralizing antibodies in the serum within 5–7 days. This suggests that a hematophagous insect, such as a sandfly or a mosquito, that fed directly on the blood of an infected mammal would not ingest sufficient virus to become infected. Birds are relatively refractory to VSV infection and there is no good evidence for persistent or congenital VSV infection in mammals. VSNJV, VSIV, COCV and MORV have all been isolated from biting arthropods collected at localities where no livestock cases of vesicular disease were occurring. This observation raises the question: how is VSV maintained in endemic regions, during interepidemic periods or at times when the presumed insect vectors are absent or in low numbers? The most likely explanation is vertical transmission (VT) in phlebotomine sandflies. Experimental VT has been demonstrated in sandflies with both VSNJV and VSIV. VT occurs for a number of arboviruses in their arthropod vectors. In the case of VSV, VT would explain how the virus survives in nature between epizootics or when most of the local animal population is immune. During vesicular stomatitis epizootics in livestock, the virus can also be transmitted by direct contact (i.e., among animals feeding from the same container, by infected calves feeding on mother, virus-contaminated milking machines, or to animal handlers and veterinarians working with infected animals). This probably occurs by introduction of virus though cuts or scratches in the skin or buccal mucosa, since there is not good evidence of aerosol transmission in nature or excretion of virus in milk or excrement of infected animals.

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

881

In contrast to VSV, BEFV occurs across a very large geographic range encompassing tropical and sub-tropical regions from Africa, the Middle-East, Asia and Australia. The virus does not occur in the Americas, the Pacific Islands or Antarctica, or in Europe excluding eastern Thrace. The vertebrate host range includes cattle, water buffalo and yak, as well as a wide range of wild African ungulates. The disease in livestock occurs seasonally, principally in the summer and early autumn, and with the onset of the monsoon season in Asia. Outbreaks are usually associated with periods of high rainfall which precipitate the emergence of insect vectors in large numbers. BEFV can also spread in epizootics that follow the pattern of prevailing winds with a general southward movement in the southern hemisphere and northward movement above the Equator. Wind-borne movement of insect vectors is the likely mechanism of spread. Vectors include biting midges (Culicoides spp.) and mosquitoes. BEFV has been isolated from Anopheles bancroftii, Culicoides brevitarsis, Culicoides coarctatus, a pool of mosquitos of mixed species in Australia, and from a mixed pool of biting midges in Africa. The virus has also been recovered from biting midges and mosquitoes of several other species following experimental infection. The abundance and distribution of insects from which BEFV has been isolated suggests that several major vectors may be involved in transmission. However, several lines of evidence indicate that, in Australia, mosquitoes rather than biting midges serve as the principal vectors. There is no evidence of direct transmission of BEFV between cattle, even when encouraged by smearing nasal or ocular discharges on mucosal surfaces. Molecular epidemiological studies, primarily using the deduced amino acid sequences of the BEFV G protein ectodomain, have identified three largely independent epidemiological systems (episystems) in East Asia, Australia and the Middle-East. However, the absence of BEFV sequence data Africa and most of southern and western Asia represents a large gap in our understanding of the phylogeography. In each of the major episystems, there is evidence of relatively rapid spread across vast distances and the continual evolution of new lineages. Recent evidence of the presence of viruses of the East Asian lineage in the Middle East suggests that trade in livestock can contribute to translocation of the virus globally.

Pathogenesis and Clinical Features The disease vesicular stomatitis primarily affects livestock (horses, cattle and swine), although humans can also develop the illness when handling affected animals or working with the virus in the laboratory. Vesicular stomatitis is of economic importance to farmers because it causes weight loss, reduced milk production and mastitis in cows, and lameness in affected animals. In addition, there are national and international restrictions on shipment of animals and semen from affected regions to VSV-free areas. Another major reason for its importance is that the clinical signs of vesicular stomatitis are similar to those of foot-and-mouth disease. In bovines and swine, the two diseases can only be differentiated by laboratory tests. The incubation period for vesicular stomatitis ranges from 2 to 8 days. In affected livestock, excessive salivation (drooling) may be the first sign of illness. Subsequently, vesicular lesions (blisters) develop on the lips, around nostrils, tongue, corners of the mouth and buccal mucosa. The vesicles rupture with grazing or eating, leaving painful ulcers, so the affected animals often stop eating, resulting in weight loss. Affected cows may also develop lesions on their teats; mastitis and reduced milk production often result. In addition, livestock also can develop vesicular lesions in the coronary band of their feet, causing swelling and sometimes loss of the hoof. Lameness may result, which in some animals such as thoroughbred horses can be a major loss for owners. Histopathologic examination of skin lesions shows intercellular edema in the malpighian layer and epithelial cell necrosis extending down to the basal cells with neutrophilic and monocytic inflammation. The virus destroys cells above the basal layer forming large vesicles which rupture leaving cutaneous ulcers and necrotic tissue (Fig. 4). Without complications, the ulcers heal and most livestock recover in about two weeks. Serological surveys indicate that many livestock as well as wild mammals and humans residing in rural areas where VSV is endemic have neutralizing antibodies to one or more of the five VSV serotypes or subtypes. This suggests that many VSV infections are probably subclinical or unrecognized. The explanation for this apparent paradox may be the route of infection. Intracerebral or intranasal inoculation of VSNJV or VSIV into adult mammals usually results in encephalitis and death. In contrast, when livestock and most other adult animals are inoculated intramuscularly or intravenously, they show little sign of illness and rapidly develop

Fig. 4 Tongue of a horse infected with VSNJV, showing extensive ulceration and loss of epithelium. Photo courtesy of R.A. Bowen.

882

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

neutralizing antibodies to the virus. However, in bovines, equine or swine, if these viruses are inoculated intradermally or intralingually or are rubbed onto abraded skin or buccal mucosa, the animal usually develops vesicular lesions on and around the site of introduction. Experimental transmission studies of VSNJV by infected black flies to cattle have given similar results. When the infected insects fed around the mouth, nostrils or coronary band of cattle, vesicular lesions developed at the bite sites; however, when the infected flies were allowed to feed on the flank or neck of the animals, infection occurred but vesicles were not observed. Most VSV human infections are also probably asymptomatic or unrecognized. Serologic surveys among people living in rural communities in Central and South America in regions where VSNJV, VSIV, and MORV are endemic have detected specific neutralizing antibody prevalence up to 90%. Furthermore, the prevalence of VSV antibody increased with age, suggesting continued exposure and infection. In contrast, reported VSV infections in persons who handle sick livestock or in laboratory personnel working with the virus have developed an acute febrile illness with headache, myalgia, chills, anorexia and weakness lasting 3–6 days. In these individuals, the time and method of exposure were known (a hand laceration contaminated with a sick animal’s saliva; a needle stick; or possibly an aerosol with a high dose of virus). It is uncertain whether humans naturally infected in endemic areas have a similar but unrecognized febrile illness or have an asymptomatic infection. The route of infection and the virus dose could also influence the outcome of infection in humans. Bovine ephemeral fever is a disease affecting cattle and water buffalo. There is also a high prevalence of BEFV neutralizing antibodies in yaks in Tibet. Clinical signs of BEFV infection can include a bi‐phasic fever, salivation, ocular and nasal discharge, recumbency, muscle stiffness, lameness and anorexia. As suggested by the name, ephemeral fever usually manifests with rapid onset and rapid recovery, lasting only 1–3 days, but prolonged paralysis and ataxia can sometimes occur. During epizootics in Australia, infection and morbidity rates are commonly very high (sometimes approaching 100% of susceptible livestock) but mortality rates are low, rarely exceeding 1%. However, reports from East Asia suggest somewhat higher mortality rates (5%–10%) have occurred historically and, in recent years, there have been reports from Taiwan (2002), mainland China (2011) and Turkey (2012) of case‐fatality rates ranging from 20% to 50%, suggesting there may have been a shift in virulence of some BEFV strains of Asian origin. The economic impacts of BEFV can be high due to cessation of lactation in dairy cattle, loss of condition in beef cattle and immobilization of water buffalo used for draft power. Bovine ephemeral fever is principally an inflammatory disease. The incubation period is normally 2–4 days. Viremia usually persists for 1–3 days and peaks approximately 24 h before the onset of fever. The initial sites of infection are not known but the virus has been isolated from neutrophils and reticulo-endothelial cells of the lungs, spleen and lymph nodes. There is also evidence of infection in synovial membranes, epicardium and aorta, and in cells derived from synovial, pericardial, thoracic and abdominal fluids. There is not widespread tissue damage. The primary lesion is a vasculitis affecting the endothelium of small vessels of synovial membranes, tendon sheaths, muscles, facia and skin. The onset of fever and other clinical signs is accompanied by marked leucopenia, relative neutrophilia, elevated plasma fibrinogen and elevated levels of cytokines including interferon a, interleukin 1 and tissue necrosis factor. There is also a significant hypocalcemia that is thought to be responsible for sternal recumbency.

Control and Treatment Control of vesicular stomatitis is difficult, since knowledge of the ecology, mode of transmission and maintenance mechanisms of the viruses causing the disease is limited. If sandflies or some other arthropod are the natural reservoir of the virus, then VSV probably cannot be eradicated. However, control measures can be taken during outbreaks of vesicular stomatitis to stop or to reduce spread of the virus. While VSV does not appear in urine or feces of infected animals, it is present in high titers in their saliva and in exudate from the vesicles and ulcerative lesions. Thus, cleaning water pails and feeding troughs with a viricidal disinfectant (i.e., 10% solution of household bleach) will decrease virus transmission. Many outbreaks in dairy cows have been traced to contaminated milking equipment, so cows with vesicular lesions should be milked last, and the equipment disinfected between uses. Infected livestock also should be quarantined and separated from other animals in the herd. Likewise, animals from an infected farm should not be transported to other premises. Generous application of insecticides in barns and corrals with infected livestock will reduce flies and other insect pests that might feed on lesions of affected animals and mechanically transmit the virus to others. Persons handling sick animals should also wear disposable gloves to avoid transmitting the virus to other animals or infecting themselves. Dairy farmers sometimes don’t report vesicular stomatitis in their herds; if quarantined, the mill can't be sold until the farm is declared free of the disease. However, even if not reported, owners can initiate the above control measures. At present, there are no effective VSV vaccines available for livestock. Various inactivated VSV vaccines have been tested but immunity generally wanes after a few years and reinfection with the same serotype can occur. Natural BEFV infection induces a strong neutralizing antibody response and apparently durable immunity. Following experimental infection, neutralizing IgG antibody appears 4–5 days after the onset of clinical signs and peaks within 1–4 weeks. Although there are some reports that cattle with high levels of neutralizing antibody can be susceptible to experimental challenge, other evidence suggests a good correlation between protection and neutralizing antibody. Colostral antibody has also been shown to protect calves against experimental challenge. High levels of cytokines circulate during the acute phase of infection but little is known of the role of innate or adaptive cell mediated immunity in recovery from infection or protection against natural or experimental challenge. Several forms of live-attenuated, inactivated, subunit and recombinant BEFV vaccines have been reported and vaccines of varying format are produced for commercial use. Live-attenuated vaccines have been produced in mice and in cell cultures. In

Vesicular Stomatitis Virus and Bovine Ephemeral Fever Virus (Rhabdoviridae)

883

general, live vaccines are relatively effective in inducing protection but require at least two doses in adjuvant to generate durable immunity. Inactivated vaccines have been produced by treatment of BEFV with formalin or b-propiolactone but they have generally poor efficacy. Consecutive vaccinations with live-attenuated and killed preparations have also been used with some success. A purified G protein subunit vaccine delivered in Quil A adjuvant has been shown experimentally to provide reliable protection following a two-dose treatment at an interval for 21 days. Recombinant BEFV vaccines employing the BEFV G protein delivered in vaccinia and capripox viral vectors have also been trialed. The major clinical signs of bovine ephemeral fever can be treated very effectively with non-steroidal anti-inflammatory drugs, such as phenylbutazone, which can reduce the duration of the disease. Calcium injections given early in the course of disease may also be used to treat animals that are recumbent. Where possible, affected cattle should be provided with shelter, water and food, particularly in hot weather.

Further Reading Albertini, A.A.V., Baquero, E., Ferlin, A., Gaudin, Y., 2012. Molecular and cellular aspects of rhabdovirus entry. Viruses 4, 117–139. Gaudier, M., Gaudin, Y., Knossow, M., 2002. Crystal structure of the vesicular stomatitis virus matrix protein. Proceedings of the National Academy of Science of the United States of America 21, 2886–2892. Ge, P., Tsao, J., Schein, S., et al., 2010. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science 327, 689–693. Joubert, D.A., Blasdell, K.R., Audsley, M.D., et al., 2014. Bovine ephemeral fever rhabdovirus a1 protein has viroporin-like properties and binds importin b1 and importin 7. Journal of Virology 88, 1591–1603. Letchworth, G.J., Rodriguez, L.L., Del Cbarrera, J., 1999. Vesicular stomatitis. The Veterinary Journal 157, 239–260. Liang, B., Li, Z., Jenni, S., et al., 2015. Structure of the L protein of vesicular stomatitis virus from electron cryomicroscopy. Cell 162, 314–327. Lyles, D.S., 2013. Assembly and budding of negative-strand RNA viruses. Advances in Virus Research 85, 57–90. Lyles, D.S., Kuzmin, I.V., Rupprecht, C.E., 2013. Rhabdoviridae. In: Knipe, D.M., Howley, P.M., Cohen, J.I., et al. (Eds.), Fields Fields Virology, sixth ed. Philadelphia: Lippincott Williams and Wilkins, pp. 885–924. Roche, S., Rey, F.A., Gaudin, Y., Bressanelli, S., 2007. Structure of the prefusion form of the vesicular stomatitis virus G protein. Science 315, 843–848. Rodriguez, L.L., Pauszek, S.J., 2012. Genus vesiculovirus. In: Dietzgen, R.F., Kuzmin, I.V. (Eds.), Rhabdoviruses: Molecular Taxonomy, Evolution, Genomics, Ecology, HostVector Interactions, Cytopathology and Control. Norfolk: Caister Academic Press, pp. 23–35. Tesh, R.B., Bolling, B.G., Beaty, B.J., 2016. Role of vertical transmission in mosquito-borne arbovirus maintenance and evolution. In: Vasilakis, N., Gubler, D.J. (Eds.), Arboviruses: Molecular biology, Evolution and Control. Norfolk: Caister Academic Press, pp. 191–217. Trinidad, L., Blasdell, K.R., Joubert, D.A., et al., 2014. Evolution of bovine ephemeral fever virus in the Australian episystem. Journal of Virology 88, 1525–1535. Walker, P.J., 2005. Bovine ephemeral fever in Australia and the world. Current Topics in Microbiology and Immunology 292, 57–80. Walker, P.J., Klement, E., 2015. Epidemiology and control of bovine ephemeral fever. Veterinary Research 46, e124. Whelan, S.P.J., Barr, J.N., Wertz, G.W., 2004. Transcription and replication of non-segmented negative-strand RNA viruses. Current Topics in Microbiology and Immunology 283, 61–119.

Relevant Websites https://talk.ictvonline.org/ictv-reports/ictv_online_report/negative-sense-rna-viruses/mononegavirales/w/rhabdoviridae Rhabdoviridae.

West Nile Virus (Flaviviridae) Fengwei Bai and Elizabeth Ashley Thompson, The University of Southern Mississippi, Hattiesburg, MS, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of Laura D. Kramer, Elizabeth Kauffman, West Nile Virus (Flaviviridae), In Reference Module in Biomedical Sciences, Elsevier Inc., 2017, doi:10.1016/B978-0-12-801238-3.02696-9.

Glossary Autoimmune disease A disease caused by attacks from the own immune system. Blood-Brain Barrier A highly selective semipermeable border in the capillaries that blocks the passage of certain substances in the blood into the brain and spinal cord tissue. Encephalitis Inflammation of the brain. Epidemiology The study deals with the incidence, distribution, and possible control of diseases and other factors relating to health.

Meningitis Inflammation of the protective membranes covering the brain and spinal cord. Neuroinvasive disease Disease caused by a pathogen that infects nerve cells. Pathogenesis The mechanism of how a disease develops. Viremia The presence of viruses in the blood. Virion The complete, infectious viral particle.

Classification West Nile virus (WNV) is a member of the genus Flavivirus within the family of Flaviviridae, which also includes three other genera, i.e., Pestivirus (including bovine viral diarrhea virus), Hepacivirus (including hepatitis C virus, HCV), and a recently established genus Pegivirus (including human pegivirus-1). The family Flaviviridae is named after the Latin word flavus (yellow) referring to jaundice caused by yellow fever virus (YFV). Flavivirus genus is composed of more than 90 viruses, many of which are important mosquito-transmitted human pathogens, including WNV, dengue virus (DENV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), Zika virus (ZIKV) and YFV. While most of these viruses are transmitted by mosquitoes and ticks, some transmit without known transmission vectors, such as HCV. Despite many flaviviruses can cause human diseases varying from fever to life-threatening hemorrhagic fever and encephalitis, some do not infect humans and other vertebrate animals, such as mosquito-specific flaviviruses.

Virion Structure WNV has a relatively smooth protein surface (no projections or spikes), enveloped, spherical structure with icosahedral symmetry approximately 50 nM in diameter. Cryoelectron microscopy (cryo-EM) shows a concentric multilayer organization of the virus. The outermost layer contains Envelope (E) and Membrane (M) transmembrane proteins. A host-derived lipid bilayer appears non-spherical with the transmembrane components of the M and E proteins. WNV and other flaviviruses are icosahedral enveloped viruses composed of a lipid bilayer surrounding a nucleocapsid containing its genomic RNA complexed with multiple copies of C proteins (Fig. 1).

Genome and Viral Proteins WNV has a positive-sense, linear, single-stranded (ss) RNA genome, which is approximately 11 kb long and is flanked by 50 and 30 non-coding stem-loop structures with a 50 cap (m7GpppAmp) and no poly-A tail, respectively. The coding region of the genome has a single open reading frame (ORF), which is translated into a polyprotein that is cleaved by viral and host proteases into three structural proteins (capsid [C], pre-membrane [PrM/M], and envelope [E]), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Fig. 2). However, certain studies suggest that additional proteins may also be produced through ribosomal frameshifting. While non-structural proteins play essential roles in the replication of viral genomic RNA, the structural proteins assemble the virion and mediate host receptor binding and viral entry during the virus life cycle (Table 1).

C Protein The capsid (C) protein is composed of a large number of positively charged amino acid residues and multiple copies dimerize and tetramerize into a nucleocapsid that is encapsulated by a lipid bilayer derived from the host cell membranes. The N- and

884

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-809633-8.21504-5

West Nile Virus (Flaviviridae)

885

Fig. 1 WNV virion structure. A 17 Å structure of WNV virion determined by cryo-EM. (A) surface shaded view of the virus with one asymmetric unit of the icosahedron is shown as a triangle. The 5-fold and 3-fold icosahedral symmetry axes are shown. (B) A central cross section showing the concentric layers of density. Reproduced with permission from Mukhopadhyay, S., Kim, B.S., Chipman, P.R., Rossmann, M.G., Kuhn, R.J., 2003. Structure of West Nile virus. Science 302, 248.

C-terminal parts of C protein have been shown to facilitate RNA folding during viral replication through RNA chaperon activity to the capsid, and the central part of the C protein structure shows the presence of four a-helices likely involved in direct interaction with the viral RNA. In addition, it has also been associated with claudin protein degradation, which leads to the epithelial barrier damage and virus dissemination in the human host. While the C protein has a role in the induction of apoptosis, it is also suggested to block apoptosis through a phosphatidylinositol (PI) 3-kinase-dependent pathway.

prM/M The prM protein of WNV is a short, glycosylated protein with a molecular weight of 20–26 kDa, which plays an essential role in viral particle formation and secretion. PrM chaperones E protein in the formation of immature virions and prevents a premature fusion during virus egress. The viral particles mature during virus egress through the secretory pathway, where cleavage of prM by a furin-like protease generates infectious particles, albeit the efficiency of the cleavage may be fairly low at approximately 30%. The immature prM-containing flavivirus particles are non-infectious, which may due to the requirement of the cleavage of prM to M to activate the membrane-fusion machinery of the virus.

E Protein The envelope (E) protein is a transmembrane glycoprotein embedded into the lipid bilayer by a C-terminal a-helicase. One hundred and eighty copies of E proteins, together with the same numbers of M proteins span through the lipid bilayer and cover the outer surface of the virion. Thus, E and M are the main antigens triggering neutralizing antibody responses in WNV infection and mediating the host receptor binding. Flavivirus E ectodomain is composed of three structural domains. i.e., DI, DII, and DIII that share a variable degree of homology among different flaviviruses. DI is a b-barrel structure containing a single glycosylation motif, while DII forms an elongated finger-like structure that contains an internal fusion loop, which is conserved among flaviviruses. DIII is an immunoglobulin (Ig)-like domain that has a major role in cellular receptor binding and viral entry. Although neutralizing epitopes have been described in all three domains, antibodies against DIII are generally considered to show higher neutralizing capacity compared to DI and DII-specific antibodies.

NS1 NS1 is a highly conserved protein sharing ca. 20%–40% identity and 60%–80% similarity among flaviviruses. The flavivirus NS1 protein is a multifunctional glycoprotein that can be found intracellularly, on the cell surface of infected cells, or secreted by infected cells as a hexamer and it circulates in the bloodstream. NS1 is a diagnostic target as it is secreted and circulates in patients’ blood at an early stage of viral infection. The intracellular dimer form of NS1 facilitates viral replication and assembly, while the secreted hexameric NS1 downregulates viral immune responses. WNV NS1 has been shown to inhibit complement activation by binding the regulatory protein factor H, attenuate classical and lectin-dependent complement activation by directly interacting with C4, and antagonization of production of type I interferon. WNV NS1 has also been shown to induce hyperpermeability of brain endothelial cells and vascular leakage in the mouse brain, and induce hyperpermeability of human brain endothelial cells resulting in neurological complications and encephalitis. Besides NS1, WNV also produces an additional non-structural protein, NS10 , a C-terminally extended product of NS1 generated as the result of a  1 programmed ribosomal frameshift, which leads to an addition of 52 amino acid (aa) residues at the C terminal of NS1 protein. The full-length NS10 may not be essential for virus replication in vitro or virulence in mice, but the sequence of the last 20 aa of NS10 appears to be responsible for its cellular retention.

886

West Nile Virus (Flaviviridae)

NS2A and NS2B NS2A and NS2B are small hydrophobic proteins. NS2A is a transmembrane protein involved in the production of virus-induced membrane structures and virion assembly, while NS2B is a cofactor of NS3 protease. WNV NS2A plays a role in the inhibition of type I interferon (IFN-a/b) production and it has been suggested to play a role in virus-induced cell apoptosis. NS2B binds to NS3 to function as a membrane anchor for the viral protease and is essential to NS3 proteolytic activity.

NS3 NS3 is a highly conserved protein across all flaviviruses and its N-terminal end contains a trypsin-like serine protease. When activated by binding to its cofactor NS2B, NS3 forms an NS2B-NS3 protease that cleaves the viral polyprotein into structural and nonstructural proteins. The NS2B-NS3 complex localizes to convoluted membranes, paracrystalline structures, and in the proliferating endoplasmic reticulum (ER), suggesting that these membranes are also involved in the proteolytic process. Besides its N-terminal protease functions, the C-terminus of NS3 possesses other important functions such as helicase, nucleoside triphosphatase, and RNA triphosphatase activities that are essential for viral replication. During viral replication, NS3 helicase separates the double-stranded (ds) RNA intermediates or disrupt secondary structures formed by the single-stranded (ss) RNA template. NS3 helicase has been shown to promote WNV resistance to antiviral action of 20 ,50 -oligoadenylate synthetase 1b (OAS1b). Recently, it was shown that the WNV NS3 helicase domain inhibits type I IFN signaling. The NS2B-NS3 protease of WNV, DENV, ZIKV, and JEV has been shown to interrupt human but not mouse, cGAS-STING pathway thus inhibiting IFN-mediated antiviral responses in humans.

NS4A and NS4B NS4A and NS4B are small hydrophobic proteins that are linked by a 2K peptide composed of 23 amino acid residues. The complete cleavage of the polyprotein generates the 2K fragment that directs NS4B into the ER. Mutations in the 2K fragment resulted in resistance against the antiviral action of the IFN-inducible OAS1b during WNV infection. The NS4A and NS4B proteins have been related to membrane rearrangement in infected cells and to inhibit IFN signaling during WNV infections. NS4A and NS4B seems to be inducers of an unfolded protein response, which displays a high correlation in inhibiting IFN-a stimulated JAKSTAT signaling during WNV infection. Similar to HCV, WNV NS4A functions as a cofactor of NS3 to regulate the ATPase activity of the S3 helicase. The flaviviral NS4B appears to play an important role in viral replication by facilitating the formation of the viral replication complexes and in counteracting innate immune responses.

NS5 Flavivirus NS5 is the largest non-structural protein encoded by the viral genome. It contains an N-terminal methyltransferase (MTase) activity and a C-terminal RNA-dependent RNA-polymerase activity for viral genome replication. The NS5 MTase activity is responsible for the viral genome 50 capping, which mimics eukaryotic mRNA and allows to use of host protein synthesis machinery for efficient viral gene translation. Specifically, NS5 has been shown to sequentially catalyze methylation of a guanine N-7 residue followed by a ribose 20 O site on the 50 end of viral RNA. Like other RNA viruses, WNV NS5 RNA-dependent RNA-polymerase lacks a proof-reading capability; thus, the viral populations display a variable level of sequence diversity which may favor the selection of variants in response to selective immunological pressures. Besides the functions in virus replication, NS5 protein has been shown to function as a potent antagonist of type I IFN-mediated JAK-STAT signaling. Like in most flaviviruses, WNV NS5 is located in the cell cytoplasm at sites of virus replication. However, several studies have also shown that a significant proportion of NS5 of WNV (Kunjin strain), DENV, JEV, YFV, and ZIKV is found in the nucleus suggesting its important role during flavivirus infection. In DENV infection nuclear NS5 seems to downregulate interleukin-8 (IL-8) production and interfere with the host cellular mRNA splicing.

Life Cycle WNV enters a host cell through receptor-mediated endocytosis after the E protein attachment to cell surface receptor(s). It appears that receptor recognition and attachment is mediated by multiple molecules in a combinatory or consecutive manner. WNV and other flaviviruses make initial contact with the host cell by binding to glycosaminoglycans (GAGs), such as heparan-sulfate proteoglycans or syndecans. GAGs are long, anionic, unbranched polysaccharides located on the surface of eukaryotic cells and in the extracellular matrix. GAGs are prominently exposed on the cell surfaces of all tissues, providing an easily accessible primary receptor for viral adhesion by electrostatic interactions. Several molecules have been implicated as cellular receptors that mediate WNV entry and infection, including mammalian avß3 integrin, DC-SIGN/DC-SIGNR, mannose receptor, and mosquito mosGCTL-1. However, like other flaviviruses, WNV entry process involves multi-protein interactions with a high degree of redundancy and a single, specific entry receptor might not exist. After attachment to the host cell surface receptors, WNV will be endocytosed into early endosomes with the pH dropping from neutral to slightly acidic and becoming more acidic during maturation to the late endosomes. The acidic pH in the late endosome triggers the conformation change of E protein leading to the

West Nile Virus (Flaviviridae)

887

fusion of the viral lipid membrane with the endocytic membrane and the release of the viral RNA genome into the cell cytoplasm. Using the cellular translational machinery, the viral RNA is translated into a single polyprotein which is cleaved into ten viral proteins. Genomic positive-sense RNA is transcribed into complementary negative-sense RNA on the ER membranes, which serves as templates for the synthesis of 10- to 100-fold positive-sense RNA molecules. The newly synthesized positive-sense RNA is packaged in viral C protein to form the nucleocapsid, and bud into the cytoplasm via the Golgi complex. Immature virus particles are transported to the cell surface in exocytic vesicles acquiring an envelope consisting of a host-derived lipid bilayer and viral prM/ M and E proteins. The viral particles mature as cellular enzymes cleave the prM, resulting in the release of mature virus from the cell surface. Mature virions are released from the infected cell by exocytosis beginning at 10–12 h after the infection. (Fig. 2).

Fig. 2 WNV life cycle. 1. WNV particle binds to a cellular receptor on the cell surface; 2. The viral particle is enclosed in an endosome via a receptor-mediated endocytosis; 3. The acidic pH in the late endosome triggers the membrane fusion between the viral lipid membrane with the endocytic membrane and release nucleocapsid into the cell cytoplasm; 4. Uncoating of the nucleocapsid releases positive sense, single-stranded viral RNA genome ( þ ssRNA); 5. Using the cellular translational machinery, the viral RNA is translated into a single polyprotein; 6. The polyprotein is cleaved into ten viral proteins; 7. þ ssRNA is transcribed into complementary negative-sense RNA (-ssRNA); 8. Synthesis of þ ssRNA using -ssRNA as a template; 9. The newly synthesized þ ssRNA is packaged in viral C protein to form the nucleocapsid, and bud into the cytoplasm via the Golgi complex; 10. The immature virus is transported in an exocytic vesicle and acquires an envelope consisting of a host-derived lipid bilayer and viral prM/M and E proteins; 11. The viral particle matures as cellular enzymes cleave the prM, resulting in the release of mature virus from the cell surface.

Table 1

WNV proteins and their functions

Proteins

Functions

C prM/M E NS1 NS2A NS2B NS3 NS4A NS4B NS5

Nucleocapsid formation, viral RNA folding, regulation of cell apoptosis Viral particle formation and maturation Viral surface protein, receptor attachment, entry Viral replication and assembly, regulation of immune response Virion assembly, inhibition of IFN production, regulation of cell apoptosis Cofactor of NS2B-NS3 protease, polyprotein cleavage Polyprotein cleavage, helicase, inhibition of IFN production, inhibition of antiviral response Membrane rearrangement, inhibition of IFN production, regulation of immune response, cofactor of NS3 helicase Membrane rearrangement, inhibition of IFN production, regulation of immune response, formation of the viral replication complexes Methyltransferase, RNA polymerase, inhibition of IFN production, regulation of immune response

888

West Nile Virus (Flaviviridae)

Fig. 3 WNV transmission cycle. WNV maintains its transmission cycle between Culex mosquitos and birds in nature. Infected mosquito bites can also transmit WNV to accidental hosts, such as humans and horses. Other than mosquito bites, WNV can also be transmitted to humans through other routes, such as blood transfusion, organ transplantation, laboratory or intrauterine exposure.

Epidemiology WNV was first discovered in 1937 in West Nile Province Uganda Africa. In 1999, WNV gained entry into North America in New York City and within 3 years, the virus had spread to most of the continental states of the U.S. as well as the neighboring countries of Canada, Mexico and the Caribbean. WNV has now spread throughout the rest of the world except Antarctica. WNV is now considered as the most important causative agent of human viral encephalitis worldwide. In the last 20 years, WNV has been estimated to cause more than 6 million human infections, with over 24,000 neurological disease cases and 2300 deaths in the U.S. alone. In addition, a large outbreak occurred in Europe in 2018 involving over 2000 human cases in 15 countries. WNV transmission in nature is maintained in a cycle between mosquitos and a variety of bird species. The ability of different mosquito and bird species to acquire and transmit WNV is highly variable. In the U.S., the American robins (Turdus migratorius) and a few Culex mosquito species are thought to be the main host responsible for the maintenance and transmission of WNV. Humans, horses and other vertebrate animals can be infected with WNV through the bite of an infected mosquito but mammals are considered as “dead-end hosts” because the infection does not produce a viremia in a magnitude that can infect subsequently biting mosquitos. Besides mosquito bites, WNV transmission may also occur through blood transfusion, organ transplantation, breast-feeding, or intrauterine exposure, or accidental laboratory exposure (Fig. 3).

Clinical Manifestations WNV infection in humans is predominantly asymptomatic (80%), but it may result in a spectrum of diseases in about 20% infected people, ranging from a febrile illness known as WNV fever to severe neuroinvasive diseases, including acute flaccid paralysis, meningitis, and encephalitis. In those individuals who develop symptoms, most have WNV fever, chills, malaise, headache, backache, myalgias, arthralgias, gastrointestinal symptoms (nausea, vomiting, or diarrhea) and maculopapular rash. Most WNV fever patients recover completely, but fatigue and weakness can persist for weeks or months. About 1 in 150 people who are infected with WNV develop a severe neuroinvasive disease affecting the central nervous system (CNS), such as flaccid paralysis, encephalitis, and meningitis. Symptoms of neuroinvasive disease include high fever, worsening headache, neck stiffness (nuchal rigidity), confusion, stupor, tremors, seizures, muscle weakness or paralysis, and focal neurological deficits. About 10% of people who develop a severe neuroinvasive disease die. Risk factors for severe encephalitis and death include advanced age, history of cardiovascular diseases, hypertension or a chronic renal disease, HCV infection, and immunosuppression. Elderly individuals are more susceptible to neurological diseases that may result in death. Those elderly individuals who survive, as many as 50% may develop post-illness morbidity. Although WNV can be cleared from the body within a few weeks, many WNV neuroinvasive disease survivors suffer a long-term post-infectious CNS symptom. Post-infectious CNS symptoms include post-exertion fatigue,

West Nile Virus (Flaviviridae)

889

dizziness, altered sensation, arthralgias, impaired memory, difficulties in concentrating, depression, anxiety, sleep disruption, and recurrent headaches. Moreover, many WNV patients have developed autoinflammatory or autoimmune-related diseases, including classic autoimmune diseases, such as myasthenia gravis (MG). In recent years, the reported frequency of severe neurological illness associated with neuromuscular manifestations, post-infectious CNS symptoms, and autoimmune diseases has been increasing.

Pathogenesis Mosquito transmitted WNV infection begins in the skin where the virus replicates initially in keratinocytes and Langerhans cells (LCs), the skin resident dendritic cells (DCs). After being activated by the WNV antigens, LCs can migrate to the draining lymph node, leading to T cell priming and activation. WNV‐infected DCs produce large amounts of type I IFNs, however, IFN signaling is decreased in DCs of elderly individuals infected with WNV, which could partially explain why the elderly people are more susceptible to a clinical WNV infection. WNV enters the blood stream through the lymphatic drainage or directly by mosquito probing into blood vessels, resulting in a primary viremia that carries the virus to the peripheral organs, such as the spleen, the liver, and the kidneys, where the virus further replicates and re-enters into bloodstream creating a secondary viremia. WNV has also been shown to replicate efficiently in blood leukocytes, such as in neutrophils that may serve as a reservoir for WNV replication and dissemination. In mouse models, a sufficient magnitude of viremia is required for WNV to cross the blood-brain barrier (BBB) to infect the CNS. Although the detailed mechanisms are not completely understood, WNV has been suggested to enter the CNS through multiple pathways, including flow through the BBB tight junctions, direct infection of endothelial cells in the cerebral microvasculature, infection of olfactory neurons, infected leukocytes that “Trojan horse” WNV to the CNS, and/or direct axonal retrograde transport from infected peripheral neurons. Once in the brain, WNV can infect and replicate in various types of CNS residential cells, including neurons, astrocytes, and microglial cells. WNV infection results in necrosis or apoptosis of neuronal cells, which may be the consequence of WNV replication or activation of immune cells such as cytotoxic T cells, leading to neuroinvasive diseases.

Diagnosis Currently, the diagnosis of WNV infection is mainly based on typical clinical symptoms and WNV-specific immunoglobulin M (IgM) response. The incubation period for WNV infection ranges from 2 to 14 days and the presence of specific anti-WNV IgM in serum or cerebrospinal fluid (CSF) using an IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA) is typically used for the diagnosis. If necessary, plaque reduction neutralization test (PRNT) can be used to exclude the possibility of cross-reactivity with related flaviviruses. WNV-specific IgG ELISA is sensitive and can determine past exposure; however, the results need to be interpreted cautiously since false positives are possible due to cross-reaction with other closely related flaviviruses. Other laboratory techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) or RT-quantitative PCR (RT-qPCR) can be used to detect viral nucleic acid in serum, cerebrospinal fluid (CSF) and tissue specimens that are collected early in the course of illness; however, they are not commonly used for the diagnosis of WNV infection since the likelihood of detection of WNV RNA is low. Since WNV can be transmitted through blood transfusion, highly sensitive nucleic acid-amplification testing (NAT) assays have been developed for rapid screening of donated blood, which may also be used to complement IgM testing in the diagnosis of acute WNV infection within the first few days of clinical illness.

Treatment and Prevention Although the licensing of several WNV vaccines for veterinary use have succeeded, there are still no approved human vaccines or specific antiviral treatment available against WNV. Despite of several clinical trials with multiple vaccine candidates, none have proceeded to the final phase of clinical testing yet. The main reasons for the lack of a human vaccine include lack of evidence of sufficient protective immunity; safety concerns in causing potential antibody-dependent enhancement responses to other flavivirus infections; difficulties in recruiting enough WNV cases for a phase III vaccine efficacy study, and economic considerations. Thus, the most effective way to prevent WNV infection is to avoid exposure to mosquitos by applying insect repellents, wear long-sleeved shirts and pants, and taking measures to control mosquitoes in indoors and outdoors. While the specific treatments are not currently available, patients with severe illness often need to be hospitalized to receive supportive care, such as intravenous fluids and pain medication. In addition, clinical researches have documented that administration of intravenous high dose steroids may result in significant clinical improvement in WNV patients who suffer from a severe neuroinflammatory disease. However, the therapeutic benefit of the immunosuppressive drugs in the treatment of WNV disease is still a question of debate.

Acknowledgment This project was supported in part by the American Heart Association (AHA) under Award Number 17AIREA33671273 and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R15AI135893 to F.B.

890

West Nile Virus (Flaviviridae)

Further Reading Bai, F., Thompson, E.A., Vig, P.J.S., Leis, A.A., 2019. Current understanding of West Nile virus clinical manifestations, immune responses, neuroinvasion, and immunotherapeutic implications. Pathogens 8. Campbell, G.L., Marfin, A.A., Lanciotti, R.S., Gubler, D.J., 2002. West Nile virus. The Lancet Infectious Diseases 2, 519–529. Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012. West Nile virus: Biology, transmission, and human infection. Clinical Microbiology Reviews 25, 635–648. Diamond, M.S., Pierson, T.C., Fremont, D.H., 2008. The structural immunology of antibody protection against West Nile virus. Immunological Reviews 225, 212–225. Hawkes, M.A., Hocker, S.E., Leis, A.A., 2018. West Nile virus induces a post-infectious pro-inflammatory state that explains transformation of stable ocular myasthenia gravis to myasthenic crises. Journal of the Neurological Sciences 395, 1–3. Kaiser, J.A., Barrett, A.D.T., 2019. Twenty years of progress toward West Nile virus vaccine development. Viruses 11. Kaiser, J.A., Wang, T., Barrett, A.D., 2017. Virulence determinants of West Nile virus: How can these be used for vaccine design? Future Virology 12, 283–295. Londono-Renteria, B., Colpitts, T.M., 2016. A brief review of West Nile virus biology. Methods in Molecular Biology 1435, 1–13. Mackenzie, J.S., Gubler, D.J., Petersen, L.R., 2004. Emerging flaviviruses: The spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nature Medicine 10, S98–S109. Martin-Acebes, M.A., Saiz, J.C., 2012. West Nile virus: A re-emerging pathogen revisited. World Journal of Virology 1, 51–70. Mazeaud, C., Freppel, W., Chatel-Chaix, L., 2018. The multiples fates of the flavivirus RNA genome during pathogenesis. Frontiers in Genetics 9, 595. Murray, C.L., Jones, C.T., Rice, C.M., 2008. Architects of assembly: Roles of flaviviridae non-structural proteins in virion morphogenesis. Nature Reviews Microbiology 6, 699–708. Simmonds, P., Becher, P., Bukh, J., et al., 2017. ICTV virus taxonomy profile: Flaviviridae. Journal of General Virology 98, 2–3. Ulbert, S., 2019. West Nile virus vaccines – Current situation and future directions. Human Vaccines & Immunotherapeutics. 1–6. doi:10.1080/21645515.2019.1621149.

Yellow Fever Virus (Flaviviridae) Ashley E Strother and Alan DT Barrett, The University of Texas Medical Branch, Galveston, TX, United States r 2021 Elsevier Ltd. All rights reserved. This is an update of A.A. Marfin, T.P. Monath, Yellow Fever Virus, in Encyclopedia of Virology (Third Edition), Edited by Brian W.J. Mahy, Marc H.V. Van Regenmortel, Elsevier Ltd., 2008, doi:10.1016/B978-012374410-4.00535-5.

Nomenclature 30 UTR 30 untranslated region ALT Alanine aminotransferase AST Aspartate aminotransferase C Capsid E Envelope ELISA Enzyme linked immunosorbent assay FNV French neurotropic vaccine

Glossary Biosafety Level The level of safety measures implemented when working with a pathogen. Levels range from 1 to 4 with Level 1 requiring the least precautions and Level 4 requiring the most.

FVV French viscerotropic virus M Membrane NS Nonstructural prM pre Membrane YEL-AND Yellow fever vaccine associated neurotropic disease YF Yellow fever YFV Yellow fever virus

Neurotropic disease Disease affecting the brain. New World Refers to the Americas. Old World Refers to Africa. Viscerotropic disease Disease affecting kidneys, liver and heart.

Classification (Compact) Yellow fever virus (YFV) is the prototype virus of the family Flaviviridae that takes its name from the Latin for yellow (flavus). The virus is a member of the genus Flavivirus, which presently contains 67 human and animal viruses. In 1901 the virus was the first to be shown to be transmitted by a mosquito. In 1927 YFV was isolated by monkey to monkey passage of blood from Mr. Asibi, a Ghanaian man who had a mild case of YF. Wild-type strain Asibi is the prototype strain of YFV. On the basis of their ecology, flaviviruses have been termed arthropod-borne viruses, or “arboviruses”, because many are transmitted between vertebrate hosts by either mosquitoes or ticks. YFV was the first virus to be shown to be transmitted by mosquitoes. Carlos Finlay, a Cuban physician and scientist, proposed in 1881 that YF may be transmitted by mosquitoes rather than by a direct human contact. Subsequently, in 1900–1901 Major Walter Reed, a United States physician, led a team in Cuba composed of James Carroll, Aristides Agramonte and Jesse William Lazear that postulated and confirmed the theory that the virus was transmitted by a mosquito. Thus, YFV was the first virus to be shown transmitted by mosquitoes. Neutralization tests based on epitopes on the envelope protein have been used to show that flaviviruses consist of eight serological subgroups together with 17 viruses that were not assigned to any serogroup due to weak or no serologic cross-reactivity with other members of the Flavivirus genus. Interestingly, although YFV is considered the prototype flavivirus, it is not assigned to a serogroup and is serologically distinct from all other flaviviruses identified to date. YFV 17D vaccine strain was the first flavivirus genome to be sequenced. Genomic sequencing of flaviviruses has resulted in identification of genetic relationships that closely resemble those of serological relationships. On the basis of genetic relationships, YFV is closely related to nine other flaviviruses (Banzi, Bouboui, Edge Hill, Jugra, Saboya, Potiskum, Sepik, Uganda S and Wesselsbron viruses). YF virus is classified as biosafety/containment level/risk group- 3 (i.e., indigenous or exotic agents with a potential to cause serious and potentially lethal infection).

Virion Structure Virus particles are spherical with a lipid envelope and approximately 50 nm in diameter. The lipid envelope is derived from host cell membranes and constitutes approximately 20% of the total weight of the virus particle. Carbohydrates represent approximately 10% of the weight of virus particles and are found as glycolipids and glycoproteins; the envelope (E) protein is glycosylated at none, one or two sites, depending on the strain. The small 12 kd capsid (C) protein surrounds the genome of the virus and the envelope contains two proteins known as E and membrane (M). The E glycoprotein has a molecular weight of 53–59 kDa (depending on strain) and the M protein is 8 kDa. The M protein is thought to reside just below the E protein in virions. Two types

Encyclopedia of Virology, 4th Edition, Volume 2

doi:10.1016/B978-0-12-814515-9.00003-5

891

892

Yellow Fever Virus (Flaviviridae)

of virions are recognized; mature extracellular virions contain M protein while immature intracellular virions contain precursor M (prM)(27 kDa), which is glycosylated at one site in the “pr” region, that is proteolytically cleaved during maturation to yield M protein. The flavivirus virion is known to “breathe” between these two types of virions while mixtures of the two types are possible too. Cryo-electron microscopy has been used to determine the structure of a number of flavivirus virions but none have been solved for YFV. Recently, in 2019, the first structure of YFV E protein (vaccine strain 17D-204) has been determined and has been found to be similar to that of other flaviviruses.

Genome The genome consists of a single-stranded, positive-sense (i.e., infectious) RNA of approximately 10,500–11,000 nucleotides with a single open reading frame of 10,233 nucleotides. The genome of the 17D-204 vaccine virus is 10,862 nucleotides in length but some strains have shorter or longer sequences due to variability in the length of the 30 untranslated region (30 UTR). The 30 UTR is 511 nucleotides in length for 17D-204 vaccine virus and contains approximately 40 nucleotide repeats (RYF) and strains have either none, 1, 2 or 3 copies. In addition, some strains have a duplication of 126 nucleotides in the 30 UTR; thus, the variability in length of the genome. Although the genome is positive-sense RNA and functions as an mRNA there is no terminal poly (A) tract at the 30 terminus. The 50 terminus of the genome possesses a type I cap (m-7GpppAmp) followed by the conserved dinucleotide AG. The 50 UTR is 118 nucleotides in length and there is very little sequence variation between any YFV strain sequenced to date. The genome encodes one single open reading frame that is co-and post-translationally processed by viral and cellular proteases to generate three structural proteins (capsid [C], membrane [M] and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) in the gene order 50 NTR-C-M-E-NS1-NS2A-NS3-NS4A-NS4B-NS5–30 NTR (Fig. 1). The

Fig. 1 Genome structure of yellow fever virus. Yellow fever has a genome length of 10,862 nucleotides with some variability between strains due to differences in length of the 30 untranslated region (30 UTR). The 50 UTR has a length of 118 nucleotides and the 30 UTR has a length of 511 nucleotides for 17D vaccine. Cleavage sites are denoted by arrows corresponding to the enzyme responsible. Golgi protease cleavage is denoted by a black arrow, signal peptidase by a pink arrow, NS3 protease by a green arrow, and unknown enzyme cleavage is denoted by blue triangles. Glycosylation sites are denoted by blue tabs.

Table 1

Structural and Non-structural proteins. Columns show protein length, function, and immune response involvement of the proteins

Protein

Length Function (AA)

Immune response

Capsid

121

Inhibits Dicer to disrupt RNA silencing

prM

89

NS1

352

Viral budding by gathering RNA into nucleocapsid Chaperone for E protein to prevent premature maturation Part of viral envelope along with E Part of viral envelope along with M; receptor binding; virion assembly Virus particle morphogenesis

NS2A

224

Virus particle morphogenesis

NS2B

130

NS3 NS4A

623 126

NS4B NS5

250 905

Co-factor for NS3 protease activity; induces membrane rearrangement Serine protease and helicase Co-factor for NS3 helicase activity; induces membrane rearrangement Scaffold for replication complex RNA-dependent RNA polymerase; methyltransferase; RNA capping

Membrane 75 Envelope 493

Uncleaved prM-E may contribute to immune evasion; weak neutralizing epitopes Induces mitochondrial- mediated apoptosis Determines neurovirulent phenotype of virus; cytotoxic T-cell epitope; neutralizing epitopes Attenuation of TLR3 signaling; suppression of antibody dependent cellular toxicity and complement lysis of infected cells; cytotoxic T-cell epitope Reduction of IFN-b gene expression; inhibition STAT1 and STAT2 phosphorylation; cytotoxic T-cell epitopes; viroporin Inhibition STAT2 phosphorylation; cytotoxic T-cell epitopes; viroporin Inhibition STAT2 phosphorylation; cytotoxic T-cell epitopes Inhibition STAT1 and STAT2 phosphorylation; viroporin Inhibition STAT1 and STAT2 phosphorylation; viroporin Inhibition of Tyk2 and Jak1 phosphorylation; reduction in STAT2 gene transcription; cytotoxic T-cell epitopes

Yellow Fever Virus (Flaviviridae)

893

Fig. 2 Wild-Type Yellow Fever Virus Phylogeny. The yellow fever virus genomes are shown in their relation to one another in a Neighbor Joining tree. Nt ¼ nucleotides.

seven NS proteins form the replication complex and have functions related to replication of the virus and interactions with the host immune response. The functions of the NS proteins have been determined and are shown in Table 1. Phylogenetic studies have identified seven genotypes of YFV (Fig. 2). There are five YFV genotypes in circulation within Africa: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Within South America, there are two genotypes: South America I and South America II. While genotypes differ up to 30% in nucleotide sequence, there is very little information on phenotypic differences, including virulence, between genotypes other than South America II genotype is associated with an enzootic transmission cycle whereas the other genotypes are associated with an epizootic transmission cycle.

Life Cycle YFV has a life cycle that involves both vertebrate and arthropod hosts, with the mosquito acting as the invertebrate host and primates as the vertebrate hosts. Both vertebrate and arthropod hosts are necessary for maintaining the virus in nature. Mosquitoes are infected when they bite and feed on the blood of a viremic primate. The blood contains virus such that when the mosquito feeds, the virus is taken up into the mosquito midgut. The virus multiplies in the mosquito midgut epithelium and crosses the midgut into the mosquito body into the haemolymph and subsequently disseminate other tissues, including the salivary glands and reproductive tract. When the virus-infected mosquito takes its next blood meal, virus present in the salivary glands is injected into a noninfected primate. The mosquito uses saliva to stop the blood clotting and so virus-infected saliva enters the primate and infection is initiated. It takes between 7 and 14 days for virus to be taken up during one feeding and spread to the salivary glands for transmission to a new primate (a process termed the extrinsic incubation period). If a non-virus-infected mosquito bites the primate during the viremic phase it becomes infected and the transmission cycle continues. The virus-infected primate has a short viremic phase, which is usually during the incubation phase in the primate where there are no clinical signs of infection. In addition to the mosquito-primate transmission cycle, due to virus-infection of the mosquito reproductive tract, vertical (transovarial) transmission enables the virus to survive in mosquito eggs during the dry season. This is termed overwintering. The initial events of the virus replication cycle involve the binding of virus to cell receptor(s) that is mediated by the E protein. The cellular receptor molecules have so far not been identified for YFV. Uptake of the virus particle into cells occurs via receptormediated endocytosis followed by pH-dependent membrane fusion activity to release the virus nucleocapsid into the cytoplasm.

894

Yellow Fever Virus (Flaviviridae)

Table 2

Mosquito and non-human primate species infected with yellow fever virus in Africa and South America

Country

Mosquitoes

Non-human Primates

South America

Haemagogus sp. Sabethes chloropterus Aedes aegypti

Alouatta sp. Ateles sp. Cebus sp. Saimiri sp. Logothrix sp. Saguinus sp. Callithrix sp. Cercocebus sp. Erythrocebus sp. Cercopithecus sp.

Africa

Aedes africanus Ae. furcifer Ae. vittatus Ae. luteocephalus Ae. simpsoni

Family Cercopithecidae Pan troglodytes Colobus sp. All other African monkey species

The virus replicates in a variety of vertebrate and mosquito cell cultures. Host-cell macromolecular synthesis is not shut-off during YFV replication and is not decreased until cytopathic effect is evident late in the infection process. The major animal hosts are many species of both New World and Old World primates (see Table 2). Note that although YFV is described as a mosquito-borne virus, there have been reports of isolation of the virus from Amblyomma variegatum ticks in central Africa indicating that the virus replicates in both mosquito and tick cells; however, there is no evidence of human disease caused by tick-borne infection.t

Epidemiology YFV is found in 46 countries in tropical South America and sub-Saharan Africa (see Fig. 3); the virus is not found in Asia. Using phylogenetic analyses, it has been inferred that YFV originated in central or east Africa and then moved westwards across Africa. It was introduced from Africa into the Americas between 400 and 500 years ago, consistent with the theory that the virus was introduced into the New World with the slave trade. Genetic studies have shown that South American viruses evolved from those originated from west Africa. YFV exists in nature in tropical regions of Africa and South America and is transmitted between non-human primates by diurnally active tree hole-breeding mosquitoes in what is termed the sylvatic cycle. Humans infected as part of this sylvatic cycle have what is termed “jungle yellow fever” (Fig. 4). The monkey and mosquito species carrying YFV differ by geographic location (Table 2). The main vector in Africa is Aedes africanus while in South America it is Haemagogus species. Other mosquito species involved in transmission include Ae. furcifer, Ae. vittatu, Ae. luteocephalus and Ae. simpsoni in Africa, and Sabethes chloropterus in South America. Primate species involved as vertebrate hosts in Africa do not tend to get fatal disease while in South America human outbreaks are preceded by epizootics where monkeys succumb to fatal infections. However, not all South America nonhuman primate species succumb to YFV infection. Urban yellow fever is the term given to a transmission cycle involving Ae. aegypti and humans, and takes place in cities as Ae. aegypti is a peri-domestic mosquito species (Fig. 3). Although urban YF is periodically reported in Africa, there have been very small numbers of urban YF cases in the Americas since 1942, even though Ae. aegypti has reinfested the majority of tropical South America during the last 60 years. Countries in South America that have experienced outbreaks of YFV include Brazil, Paraguay, Argentina, Columbia, Bolivia, and Peru with annual activity reported in Brazil, Peru and Bolivia; YF activity is rare in Argentina and Paraguay. The majority of YF cases in the Americas are reported from Peru where the virus is endemic, although outbreaks in Brazil tend to involve higher numbers of cases with large outbreaks identified every 8–10 years. Since the year 2000, in Africa, YF outbreaks have occurred in Guinea, Liberia, Mali, Senegal, southern Sudan, Togo, Uganda, Burkina Faso, Côte d0 Ivoire, Ethiopia, the Democratic Republic of the Congo, Angola, and Ghana. While YF activity is most often seen in west Africa, there are increasing numbers of outbreaks in east Africa. For example, the first outbreak in Ethiopia was in 1959, with another epidemic following in 1960 to 1962 followed by a gap of 50 years before an outbreak in 2013. Not only does the monkey and mosquito species vary by geographic location but the genotype of YFV also varies by geographic location. West African genotype viruses are genetically distinct from those found in east and central Africa; the Angola genotype is the oldest genotype but has only been found in Angola with spillover into the Central African Republic in 2016. The two South American genotypes are geographically separated with South American genotype II found only in Bolivia and Peru as well as a small region of Brazil while South American genotype I found in the remainder of tropical South America. YFV is currently not found in central America and the last cases were in Panama in 1974. Nonetheless, the virus is still found in Trinidad.

Yellow Fever Virus (Flaviviridae)

895

Fig. 3 Yellow fever virus distribution in South America and Africa. Countries in yellow denote where yellow fever virus has been previously isolated.

Fig. 4 Transmission cycles. The sylvatic cycle for yellow fever virus is shown on the left where virus circulates between no-human primates and mosquitoes. The middle arrow denotes a spill-over event where YFV can enter a human population and establish an urban transmission cycle between humans and mosquitoes, shown on the right.

The reason for the lack of YFV in Asia is not understood. The mosquito vector, Aedes aegypti, is found in Asia and laboratory studies indicate that Asian monkey species succumb to YFV infection and Asian strains of Ae. aegypti can transmit YFV. A number of hypotheses have been proposed to explain this phenomenon but none have been proven. In 2016 the first cases (n ¼ 11) on YF were reported in China following infection of Chinese workers in Angola