Encyclopedia of Virology, Volume 5 Diagnosis, Treatment and Prevention of Virus Infections [4 ed.] 0128234091, 9780128234099

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ENCYCLOPEDIA OF VIROLOGY FOURTH EDITION

Volume 5

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 5

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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.

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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.

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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.

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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

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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.

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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

DIAGNOSIS

Introduction to Virus Diagnosis and Treatment Maija Lappalainen, HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Hubert GM Niesters, Department of Medical Microbiology and Infection Prevention, Division of Clinical Virology, University Medical Center Groningen, Groningen, The Netherlands r 2021 Elsevier Ltd. All rights reserved.

There are more than 320,000 mammalian viruses, of which a little over 200 are known to infect humans (Anthony et al., 2013; Woolhouse et al., 2012). The number, however, is steadily increasing. During recent years, we have witnessed the emergence of new viral diseases, such as MERS (Memish et al., 2020), infection caused by Zika virus (Rather et al., 2017), and the COVID-19 pandemic caused by SARS-CoV-2 in 2020 (Hu et al., 2020). Viral diagnostics is increasingly important in clinical medicine. The growing number of new diseases, the expansion of specific antiviral drugs and the need for effective vaccines poses a challenge for the diagnostic tools available. This volume focuses on clinical virology. It leads us through the presentation of traditional, modern and future diagnostic tools and provides views on how to implement these tools in various clinical settings. Electron microscopy is a highly valued method allowing visualization of virus particles and combined with antibody-based labeling approaches. It is still the method of choice for identifying ultrastructural changes associated with virus-host cell invasion and replication. Molecular methods, in particular the PCR, have replaced viral culture in many laboratories, limiting the use of this traditional method of virus detection or replacing it altogether. We have to realize that most viruses cannot be grown easily or cannot be grown in vitro at all. In special circumstances, however, viral culture may still be needed. Traditional serological assays have largely been replaced by enzyme immunoassays and serodiagnostics are at their best in identifying and pinpointing the time of primary infection, in assessing immune status vs. susceptibility, and in pre-transplant donor-recipient matching. Challenges with conventional diagnostic methods have been stated in several studies. Only 62% of viral respiratory infections among children can be confidently attributed to known pathogens (Arnold et al., 2008) and in another study, it was demonstrated that in 42.7% of cases of viral gastroenteritis, the pathogen could not be identified with conventional methods (Vu et al., 2019). It has been estimated that up to 40% of viral encephalitis infections remain undiagnosed with modern clinical tests (Kennedy et al., 2017). Therefore, new approaches, like microarrays, sensitive multianalyte methods on microfluidic platforms, and high throughput sequencing (HTS) are needed. In addition, we have to also realize that pathogens can follow a hit-and-run procedure before a clinical syndrome is detected or diagnosed. The data provided with new technology could not only augment diagnostics and help to choose the correct therapy, but also facilitates virological research, since more precise, low-cost and accessible solutions are necessary. The ongoing COVID-19 outbreak emphasizes the importance of biosafety and biosecurity procedures as well as quality assurance and standardization of diagnostic assays to ensure the appropriate performance of the assays used. Many laboratories already have accreditation according to ISO15189:2015, but as more and more hospitals undergo comprehensive on-site surveys conducted by Joint Commission International (JCI) in order to achieve accreditation, this also highlights the accreditation status of the laboratories they use. Viruses have a quite simple structure with an encapsidated nucleic acid. They borrow molecular equipment from host cells to complete their replication cycle, thus having only a few targets for antiviral agents. In addition to high costs, the existing antiviral treatments still have safety and efficacy challenges. Fortunately, success stories have also been seen with, for instance, treatment of HIV-1 and hepatitis C, encouraging the development of therapies targeting different steps in the viral replication cycle. Updated reviews about management and treatment of clinically relevant viral diseases will be presented. Vaccine development normally consists of a series of steps that can take many years. However, urgent need, as noted for the development of a SARS-CoV-2 vaccine, may change the development process. Some of the steps in the research and development process are happening in parallel, while still maintaining strict clinical and safety standards. As an example of triumph of vaccine efficacy, will be the permanent cessation of poliovirus. The number of potentially pathogenic viruses is very large, while the resources for disease research and development (R&D) are limited. To ensure efforts under WHO’s R&D Blueprint are focused and productive, a list of diseases and pathogens are prioritized for R&D in public health emergency contexts. The priority 10 viral diseases are discussed. In the future, with the developing techniques, the clinical virology laboratories are able to produce an enormous amount of detailed data about viruses, pathogenesis and recovery from the infection. The leading challenge is to fully utilize this data in the patient’s clinical management and treatment. The prerequisite for this is good communication and collaboration between laboratory personnel, researchers and clinicians. The improved exchange of information and growing information and knowledge, in turn, significantly aids clinicians in battling viral infections.

References Anthony, S.J., Epstein, J.H., Murray, K.A., et al., 2013. A strategy to estimate unknown viral diversity in mammals. mBio 4. doi:10.1128/mBio.00598-13. Arnold, J.C., Singh, K.K., Spector, S.A., Sawyer, M.H., 2008. Undiagnosed respiratory viruses in children. Pediatrics 121, e631–e637. doi:10.1542/peds.2006–3073.

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Hu, B., Guo, H., Zhou, P., Shi, Z.-L., 2020. Characteristics of SARS-CoV-2 and COVID-19. Nature Reviews Microbiology. doi:10.1038/s41579-020-00459-7. Kennedy, P., Quan, P.-L., Lipkin, W., 2017. Viral encephalitis of unknown cause: Current perspective and recent advances. Viruses 9, 138. doi:10.3390/v9060138. Memish, Z.A., Perlman, S., van Kerkhove, M.D., Zumla, A., 2020. Middle East respiratory syndrome. Lancet 395, 1063–1077. Rather, I.A., Lone, J.B., Bajpai, V.K., Paek, W.K., Lim, J., 2017. Zika virus: An emerging worldwide threat. Frontiers in Microbiology 8, 1417. doi:10.3389/fmicb.2017.01417. Vu, D.-L., Sabrià, A., Aregall, N., et al., 2019. Novel human astroviruses: Prevalence and association with common enteric viruses in undiagnosed gastroenteritis cases in Spain. Viruses 11, 585. doi:10.3390/v11070585. Woolhouse, M., Scott, F., Hudson, Z., Howey, R., Chase-Topping, M., 2012. Human viruses: Discovery and emergence. Philosophical Transactions of the Royal Society B: Biological Sciences 367, 2864–2871. doi:10.1098/rstb.2011.0354.

Electron Microscopy for Viral Diagnosis Roland A Fleck, King’s College London, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary

Å Å ngström a unit of length used to define very small distances. One ångström is equal to 10
10 m (one ten-billionth of a meter or 0.1 nanometers). Bacteriophage (Phage) a virus which infects only bacteria. DNA See Nucleic acid. Cryo From the Ancient Greek κrύος (krúos, “icy cold, chill, frost”) and used as a prefix cryo- for the study of biological materials at very low temperatures by electron microscopy. Diffraction Limit The resolution of a microscope is principally limited by the physics of diffraction. An optical system with performance at the instrument's theoretical resolution limit is said to be diffraction-limited. Ernst Karl Abbe, described the diffraction limit of a microscope as d ¼ l/ 2n.sina, where d is the resolvable feature size (resolution), l is the wavelength of the illumination source, n is the index of refraction of the medium the sample is imaged in, and a is the half-angle subtended by the objective lens. The electron wavelength l is B100,000 times shorter than that of visible light and this difference is key to the greater resolving power of the electron microscope. ELISA (Enzyme-linked immunosorbent assay) or enzyme immunoassay (EIA) is a technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. In an ELISA, detection is by a highly specific antibody-antigen interaction. Freeze Etching Is the sublimation of surface ice under vacuum to reveal details of the freeze fractured face that were originally hidden. The fracture face of the sample can be imaged directly in a SEM or as a metal replica of the facture face in a TEM. Freeze Fracture Is a technique where a frozen specimen is broken to reveal internal structures. Leidenfrost Effect A physical phenomenon in which a liquid, close to a surface that is significantly hotter than the liquid's boiling point, produces an insulating vapor layer that prevents the liquid making physical contact with the hot surface. When a warm sample is placed in a liquid cryogen the film of insulating vapor slows down cooling. Due to the small temperature difference between solid and boiling point for nitrogen even when a very small sample is placed in liquid nitrogen a film of vaporized liquid nitrogen forms around the sample and impedes heat transfer. This limits the cooling rate achieved and prevents vitrification. Lyophilization Also referred to as freeze-drying, is a technique which utilizes low pressure and low temperature to dry a material by the sublimation of water. Sublimation converts water directly from a solid to a gas without the effect of a change in surface tension (the tendency of fluid

Encyclopedia of Virology, 4th Edition, Volume 5

surfaces to shrink into the minimum surface area possible) which increases as liquid water evaporates causing changes to virus morphology. Negative Stain Contrasting a thin specimen with an optically opaque electron dense fluid. Once dry, the background is stained, leaving the actual specimen untouched, and thus visible in the transmission electron microscope. Nucleic acid The genetic information of a virus is carried on nucleic acid Viruses may be divided into those of ribonucleic (RNA) or deoxyribonucleic (DNA) forms. RNA See Nucleic acid. SEM Scanning Electron Microscope, a microscopy technique in which a focused beam of electrons is scanned across a surface in a raster scan pattern, and the position of the beam is combined with the intensity of the detected signal to produce an image. Single Particle Analysis An image processing technique which is applied to TEM images. The approach digitally combines many images of similar small particles (e.g., a virus) to create an image with stronger and more easily interpretable features. Commonly many individual virus particles will be captured in a single photomicrograph and in many photomicrographs thousands of individual randomly orientated viruses exist. Each individually reveal the different unique faces of the virus. Each individual virus may be “boxed out” or digitally cut out and then processed to build up a three-dimensional reconstruction of the particle. Using cryo-electron microscopy it has become possible to generate reconstructions with near-atomic resolution. TEM Transmission Electron Microscope, a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. Tomography A technique for obtaining detailed 3D structures of sub-cellular macro-molecular objects. Electron tomography is an extension of traditional TEM where single 2D images are collected. Instead, the sample is tilted in the TEM at incremental degrees of rotation around the center of the target sample. At each tilt angle the TEM electron beam is passed through the sample and a 2D image collected. Each image is collected and digitally processed using a weighted back projection into a 3D volume of the sample. Vitrification The transformation of a water by rapid cooling into a non-crystalline amorphous solid, “glass” thus avoiding the crystalline ice states of water and change in volume associated with freezing. ll A unit of volume used to define a very small volume. 1 ml is equal to one millionth of a litre.

doi:10.1016/B978-0-12-814515-9.00129-6

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Electron Microscopy for Viral Diagnosis

Introduction to Virus Diagnosis by Electron Microscopy Organisms, causative of disease, commonly occupy a length scale below that visible by the naked eye. Light microscopes readily aid the observation of amoeba and bacteria but are unable to directly observe viruses, which are also a major cause of disease. A simple change to host phenotype may imply the presence of a pathogen further analysis, using specialist techniques and equipment is needed to confirm and identify a virus infection. The electron microscope (EM) developed by Ernst Ruska and Max Knoll in 1931, to exploit the shorter wavelength of electrons, created an instrument with the potential to overcome the diffraction limits of the light microscope (restricted by the longer wavelengths of visible light). Eight years after the initial EM development, Ruska with Kausche and Pfankuch were able to visualize a virus for the first time. This first view of a tobacco mosaic virus (TMV) heralded the start of modern virology. The initial proof of concept was rapidly followed by images of bacteriophage. The first EM studies on pathogenic viruses were published shortly after, in 1938 by von Borries et al., detailing the size and fine structure of smallpox, vaccinia virus and Ectromelia mouse pox. Soon after, the EM became the de facto tool of choice for the virologist. Providing a simple and almost direct way to observe nanometer (nm) scale biological structures. Today, the latest cryo transmission EMs (cryoEM) reveal near atomic level structural information of the virus and its assembly. Initially, positive staining by the application of phosphotungstic acid and osmium tetroxide to bacteriophage and proteins was tried, however, disruption to structures was observed due to the low pH of the staining solutions. Early studies of fine virus ultrastructure and morphology used the shadow casting techniques devised by Williams and Wyckoff in 1945, whereby a fine metal (e.g., palladium or platinum) was evaporated under vacuum and applied to a dried preparation of virus from a fixed low angle. Shadowed with B8 Å thickness of metal, at an angle of about 121 from the horizontal plane of the mount, the low angle and unidirectional coating created a shadow of the virus as the metal collected on the side of the virus facing the metal evaporation source. When viewed in the transmission electron microscope (TEM) the enhanced contrast between metal shadowed and shaded areas revealed the morphology and fine ultrastructural details of the virus. Although shadow-cast specimens provided useful details from both air-dried and freeze-dried material, the protocol itself is challenging and requires specialist vacuum metal evaporation technology and high quality “near pure” samples. The method of choice for direct virus detection is negative staining (NS) (Fig. 1). Established by Brenner and Horne as a fast, robust and universal EM technique NS has been used by virologists for over 60 years. The EM NS preparation technique is effective for fresh, aged, partly degenerated, or dried specimens (e.g., vesicle fluid, fecal samples) and can be applied readily to biological tissues; homogenized or mortar ground organs, biopsy (including wart and molluscum contagiosum skin lesions), autopsy specimens and cell cultures (both supernatant and lysed cell pellets). The versatility of NS also allows samples collected from soil and aquatic sources, e.g., from streams, urban irrigation systems, sewage outlets, standing water, lakes etc to be studied and can be used to detect agricultural or human sewage contamination of a eutrophicated water body. The potential of EM in the laboratory diagnosis of infectious diseases was first demonstrated in the control of smallpox outbreaks and developed into a routine procedure for the detection and diagnosis of common viral diseases by skilled operators. These virus diagnostic EM (DEM) laboratories became ubiquitous in hospitals and public health laboratories globally, providing a routine service for circulating gastrointestinal and respiratory infections. Routinely applied to human pathogens, e.g., viruses of medical importance, with around 21 viral families and genera being deemed medically significant (Table 1), DEM also found wide acceptance in veterinary virology. Due to the potentially serious consequences of viral outbreaks, rigorous, and universally recognized training of personnel dealing with virus identification was required. Today training in Diagnostic Electron Microscopy (DEM) of human and veterinary infectious diseases can be obtained via an annual External Quality Assurance (EQA) scheme prepared and distributed by the Robert Koch Institute (RKI), Berlin.

Diagnostic Transmission Electron Microscopy The transmission electron microscopy (TEM) is a powerful initial step in virus diagnosis for several reasons. The TEM provides an unbiased direct and rapid overview of the sample. The viral burden (amount), size and shape (critical for initial identification) of virus or viruses present is immediately apparent. The observed morphology, even for unexpected or emerging pathogens normally allows preliminary classification to family level based on particle morphology (Table 1). This “catch all” diagnostic facility is due to TEM sample preparation protocols targeting proteins, the viral capsid or ribonucleoprotein (RNP) complexes rather than the virus RNA or DNA genomes and makes TEM uniquely unbiased as a detection method. Initial identification of a virus requires only very small volumes (B1 ml) of samples carrying high virus (4106 particles ml
1) loads and can then direct the virologist to further methods (e.g., bioassays, serological, or molecular biology approaches) to identify virus genus and species. As a technique where identification is by morphology, DEM developed rapidly. During the early 1950s until the mid-1960s many new viruses were discovered and names were commonly assigned on Latinized abbreviations attributed to their morphology or niche and -viridae for virus families and -virus for viral genera (Table 1). Viral morphology and niche provided the basis for grouping viruses into families, be they a virus family able to replicate only in vertebrates, only in invertebrates, only in plants, only in bacteria or less commonly viruses able to replicate in more than one of these hosts. Viruses were then often further grouped or classified on the basis of size and shape, i.e., helical morphology is seen in nucleocapsids of many filamentous and pleomorphic viruses; helical nucleocapsids consist of a helical array of capsid proteins wrapped around a helical filament of nucleic acid.

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Fig. 1 Virus particles as revealed by different electron microscopy methods. (A) bacteriophage collected using a transmitted electron detector in a field emission scanning electron microscope (FEGSEM). B-C Influenza virus particles by different electron microscopy techniques (B) prepared by high pressure freezing, freeze fracture and direct imaging in the FEGSEM, (C) prepared by plunge freezing into liquid ethane and imaging at 200 kV by low dose cryo transmission electron microscopy, (D) conventional negative staining of air dried particles with 2% sodium silicotungstate (SST). (E-J) virus particles by negative staining, imaged at 120 kV on a TEM E, (H) parapoxvirus, (F) adenovirus, (G) reoviruses virus particles with monomorphic morphology (I) paramyxovirus (J) orthopoxvirus.

Icosahedral morphology is characteristic of the nucleocapsids of many “spherical or cubic” viruses. Some viruses (e.g., herpesvirus) also have an outer envelope which can be readily observed in the EM and can help in identifying the virus. Despite the role of EM as the first line diagnostic tool for gastrointestinal and influenza infections being superseded by genetic screening approaches (e.g., PCR). EM remains an important diagnostic tool in several clinical settings. Indeed, guidance issued by the UK's Royal College of Pathologists cites the Association of Clinical Pathologists Best Practice No. 160, which states that ‘Many respondents [to a review of laboratory practice in renal pathology] expressed the opinion that to carry out evaluation of renal biopsy specimens without at least having the availability of electron microscopy is negligent’. Other specialized areas include the diagnosis of primary ciliary dyskinesia; certain skin/connective tissue disorders, e.g., inherited bullous lesions and Ehlers–Danlos syndrome; and specialized areas of ophthalmic pathology. The versatility of DEM and the power to directly observe the morphology of virus particles, particularly for unknown or emerging diseases and complex co-infections remains its unique value. This is a specialist niché role and with the reduction in the number of DEM facilities globally one whose essential skilled operator base is increasingly difficult to maintain. Specialist DEM units are now largely maintained at a national level in specialist disease surveillance and control centers and continue to play a critical role as demonstrated by their contribution to the investigations into recent unexplained diseases, potential bioterrorism incidents and zoonoses. The identification of the Severe Acute Respiratory Syndrome (SARS) agent, the unexpected diagnosis of monkeypox virus infections in the USA, Buffalopox infections of humans, arboviruses and viral hemorrhagic fevers in India and Hendra paramyxovirus infections in Australia were by these specialist centers (e.g., the Center for Disease Control and Prevention, Georgia, USA and the National Institute of Virology, Pune, India). Based on its ability to recognize contaminations, rapid application of TEM has an important role for quality control of reference material or of virus preparations used for antibody production or directly for vaccination.

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Electron Microscopy for Viral Diagnosis

Table 1 Viruses families known to cause disease in humans, the origin of their name, nucleic acid relationship, symmetry of the capsomere as described by classical negative stain diagnostic electron microscopy and commonly observed disease Virus family

Nucleic Symmetry acid

Explanation of the origin of family name

Picornaviridae

RNA

Icosahedral pico (small) and RNA

Reoviridae

RNA

Icosahedral

Papovaviridae

DNA

Icosahedral

Retrovirus

RNA

Complex

Hepadnaviridae

DNA

Icosahedral

Adenoviridae

DNA

Icosahedral

Astroviridae Arenaviridae

RNA RNA

Icosahedral Complex

Bunyaviridae

RNA

Helical

Ebolavirus

RNA

Helical

Calicivirus

RNA

Icosahedral

Coronaviridae

RNA

Helical

Filoviridae

RNA

Helical

Herpesviridae

DNA

Icosahedral

e.g., Coxsackieviruses associated with neurological disease, had foot and mouth disease and respiratory tract illness in man respiratory, enteric, and orphan viruses because the Widespread in domestic animals and man, first agents were found in both respiratory and enteric isolated from respiratory and stool samples specimens papilloma, polyoma, and vacuolating agent, now split e.g., Human Papilloma Virus (HPV) causing cervical into Papillomaviridae and Polyomaviridae families cancers and genital warts reverse transcriptase e.g., causing Acquired Immune Deficient Syndrome (AIDS) following infection with the Human Immunodeficiency Virus (HIV) replication of the virus in hepatocytes and their DNA Including the Hepatitis B virus genomes, as seen in hepatitis B virus. adeno, “gland”; refers to the adenoid tissue from Infections are commonly associated with diarrhea in which the viruses were first isolated children and adults astron “star” small viruses associated with childhood diarrhea arena “sand” describes the sandy appearance of the The causative agent of Lassa fever in man virion. Bunyamwera, the place in Africa where the type strain Highly pathogenic in man causing Crimean-Congo was isolated hemorrhagic fever, Rift Valley Fever and Nephropathia epidemica Ebola River, close to the two simultaneous 1976 Morphologically similar to Marburg virus causing outbreaks: one in Nzara (a town in South Sudan) hemorrhagic fever in man with high levels of and the other in Yambuku village (Democratic mortality Republic of the Congo) where the virus was first identified. calix, “cup” or “goblet” from the cup-shaped Commonly infecting cats causing respiratory tract depressions on the viral surfaces infections in adult cats and fatal pneumonia in kittens, pigs and sealions, also infections in man corona, “crown” describes the appearance of the often reported as causing “common cold” symptoms peplomers protruding from the viral surface in man, human respiratory infections and “gasping disease” in chickens from the Latin filum, “thread” or “filament” describes including the Marburg virus which is highly infectious the morphology and pathogenic in man herpes, “creeping” describes the nature of the lesions Including the Epstein Barr virus responsible for Glandular fever (mononucleosis) and cold sores in man ortho, “true,” plus myxo “mucus,” a substance for the viruses responsible for Influenza which the viruses have an affinity. combining ortho with pox (pustule) descriptive of the the vaccinia, cowpox, and variola (smallpox) viruses infection para, “closely resembling” myxo including the viruses causing measles, mumps, respiratory syncytial infections and Newcastle disease parvus means, “small” one of smallest DNA viruses and including human parvovirus B19 which was initially discovered in human sera being screens for hepatitis B antigen. A canine variant causes bloody diarrhea in puppies and can lead to death pock means, “pustule” Including the pseudopoxvirus which causes udder infections in cows and is commonly named cow pox rhabdo, “rod” describes the shape of the viruses causing Rabies, an infection almost always fatal in man toga, “cloak” refers to the tight viral envelope. Including the virus responsible for Rubella “German Measles”

Orthomyxoviridae RNA

Helical

Orthopoxviruses

RNA

Complex

Paramyxoviridae

RNA

Helical

Parvoviridae

DNA

Icosahedral

Poxviridae

DNA

Complex

Rhabdoviridae

RNA

Helical

Togaviridae

RNA

Icosahedral

Symptoms and associated diseases attributed to the virus family

To partially address the combined challenges of fewer specialist viral DEM units and the essential requirement for experienced virus DEM specialists, Laue and Möller at the RKI have generated a publicly available database of EM images. Expansion of such archives will facilitate recognition of newly emerging as well as unexpected viruses. For instance, an increase in the local prevalence

Electron Microscopy for Viral Diagnosis

9

of plant viruses can serve as indicators of environmental stress and predict underlying contamination of irrigation water with pathogenic human viruses. Thus, TEM remains an integral part of the surveillance role of national reference laboratories, where prompt, reliable and comparable results are essential for efficient diagnostics during unexpected regional virus outbreaks or epidemics (e.g., rare zoonoses, such as Buffalopox). Despite this longstanding and continued value and capacity to inform, DEM as a tool in the clinical setting has been largely stagnant with little change in the technical procedures or their application for decades. In marked contrast, TEM and particularly cryoEM in the research setting has experienced an explosion in capacity and capability, heralding the “resolution revolution”.

Biological EM Challenges and Sample Preparation Biological EM is not without its challenges. The electron beam is generated under vacuum at pressures and temperatures that are nominally incompatible with liquid water, yet water is the most abundant cellular constituent. Biological tissues (carbon-based life forms) have poor contrast in the electron microscope because they are composed mainly of light elements. In fact, carbon is so “transparent” in the electron microscope that it is often employed as a film to support biological samples. To overcome these challenges, hydrated “live” tissue is converted to an EM stable state; conventional protocols employ a series of steps, including chemical fixation, alcohol dehydration and resin infiltration. Heavy metal salts are added for positive staining of fixed and embedded specimens or as negative stains of whole structures that have been deposited on a support film. More recently, cryofixation has been adopted and allows the “solidification of a biological specimen by cooling with minimal displacement of its components”. By using low temperature as a physical fixation strategy, the morphology and dimensions of the living material are retained, and soluble cellular components are not displaced, which means that processing artefacts commonly encountered in more conventional room-temperature EM techniques are either reduced or removed. CryoEM often allows direct observation of specimens that have not been stained or chemically fixed. However, due to the requirement for the cryo fixed sample to be maintained at cryogenic temperatures and the challenge of avoiding warming and/ or contamination during transfer into the TEM the approach is not regarded as suitable for DEM. Instead, cryoEM is reserved for high resolution research studies. Small particles like bacteria, viruses and proteins readily lend themselves to a simple protocol able to preserve their ultrastructure and introduce sufficient contrast to make their observation easy. Referred to a negative staining (NS) it is an easy, rapid, qualitative method for examining the structure of isolated organelles, individual macromolecules and viruses at the EM level (Fig. 2). This is the opposite of the stain binding to the sample and excess being removed, in which case a particle would appear dark against bright background (positive staining). Despite providing stunningly beautiful images of viruses, NS does not allow the highest resolution examination of samples. Negative staining involves the deposition of heavy atom stains, e.g., uranyl acetate, around a small particle to make it visible in the electron beam. Structural artefacts such as flattening of spherical or cylindrical structures are common. Nevertheless, negative staining is a very useful technique because of its ease and speed (taking only a few minutes for a skilled operator), and also because it requires no

Fig. 2 Representation of a negatively stained virus particle on a TEM grid. Negative staining requires very little specialized equipment and virus suspensions can be mounted directly on TEM support grids (e.g., carbon formvar) and examined after air-drying. However, due to their small size and the low atomic numbers of the elements forming the virus particle they provide little electron scattering on interaction with the electron beam (blue dotted lines). This means that they have very low contrast and do not stand out from the background. To visualize the virus, heavy metal (high atomic number, high scattering) stain such as uranyl acetate or phosphotungstic acid is used. The virus particles (smiley face) are surrounded by the stain, which scatters electrons strongly creating negative contrast, whereby the particle appears bright against the stain. As the stain has a small grain size, as it covers the virus particle it is also trapped in surface features. Differences in z “thickness” of stain which the electron beam passes through en route to the detector is responsible for variations in the level of signal detected and thus contrast, to reveal gross morphological features and details of subunits forming the protein structures.

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Electron Microscopy for Viral Diagnosis

Table 2 Common negative stains used for diagnostic electron microscopy. Other less common stains include; aluminum formate, uranyl oxalate, and uranyl sulfate and gold thioglucose Common negative stains:

Normal range for use (pH)

Normal concentration (w/v)

Phosphotungstic acid (PTA) Uranyl acetate (UA) Uranyl formate Sodium silicotungstate (SST) Ammonium molybdate Methylamine tungstate

5–8 4.2–4.5 4–4.5 5–7 6–7 5–8

1%–3% aqueous solution 1%–3% aqueous solution 0.5%–1% aqueous solution 1%–5% aqueous solution 1%–2% aqueous solution 2% aqueous solution

specialized equipment other than that found in a regular EM laboratory. The NS should not react with the specimen in a ‘positive staining’ manner (i.e., it should not bind to the specimen). However, uranyl based stains will bind to proteins and sialic acid carboxyl groups and to lipid and nucleic acid phosphate groups. One effect of this is to induce aggregation of the material.

General Protocol for the Study of Viruses by Negative Staining The method of surrounding or embedding specimens in opaque dyes is commonly used in light microscopy and dates from about 1884 (e.g., microbiologists use India ink to stain micro-organisms heat fixed to a slide. The background is stained with the ink while the organisms remain clear, creating a negative stain of the organism). A common clinical application of this procedure is to confirm the morphology of the encapsulated yeast Cryptococcus spp. which cause cryptococcal meningitis. The equivalent preparative technique applied to electron microscopy was derived from experiments on a bacteriophage by high resolution EM in the Cavendish Laboratory, Cambridge, during 1954. When mixed with 1% potassium phosphotungstate and airdried, gross morphological features of a human adenovirus and details of subunits forming the protein structures were revealed. The approach was first described by Brenner & Horne in 1959 and confirmed the earlier observation of Hall in 1955 working at the Massachusetts Institute of Technology that “bushy stunt virus stained with 5% phosphotungstic acid at pH 4.6 and insufficiently washed is the opposite to what I usually sought by the use of electron stains the viability of particles of low scattering power can be enhanced as well, if not better, by surrounding them with dense material rather than impregnating them with dense material” (sic). Negative staining is as diverse in how it is performed as it is ubiquitous as a rapid technique able to reveal the structure of small biological particles (e.g., viruses and proteins) in the EM. Many heavy metal contrasting stains are available (Table 2). Each has a “fine grain structure” suitable to reveal the fine structure of the virus particle without obscuring virus morphology, is electron dense to create strong contrast between the unstained specimen (the virus) and the surrounding (negative) stain and in almost all cases has the capacity to stabilize or otherwise fix the sample to preserve its structure in the EM. Fixed or unfixed samples may be used. With unfixed specimens there is the potential problem of changes occurring due to osmotic shock (or to changes in ionic composition) since most negative stains are made up in distilled water. Also, there may be risk of operator infection when examining unfixed bacterial or viral samples. Samples should be concentrated and purified to allow the operator the best chance of identifying and imaging relevant particles. Cell debris can be removed by centrifugation before loading samples onto grids. Due to the small volumes used in the preparation methods detailed below, the number of biological particles in the sample must exceed 106 particles/ml. There are a number of methods for preparing samples for examination by NS; the method used depends on either the sample to be stained or the method of choice in the laboratory. Below are outlined some of the simpler methods. Method A Negative Staining Single-droplet method. (1) Glow discharge carbon-formvar coated grids just before use to increase their hydrophilicity. This is essential to allow even wetting of the grid with the sample and stain and ensures even NS of the sample. (2) Place 1–3 ml of the sample on the grid (sufficient to cover the grid surface). (3) After B10 s slowly pipette 20 ml of stain on to sample, while gently absorbing the stain from the opposite side using a wedge of filter paper. The staining procedure should take B30–60 s. (4) After absorbing as much stain as possible allow the grid to dry and examine the grid as soon as possible, preferably the same day in the TEM. Some methods include a wash in distilled water after drying which can be helpful if the buffer becomes crystalline when dried or when the sample is too thick on the grid. Method B. Sequential two-droplet method. (1) (2) (3) (4) (5)

A suspension of particles/organelles is made in a suitable buffer or in distilled water. Place a drop (B5–10 ml) of the suspension on to a carbon-formvar glow discharged grid. When the suspension has partly dried the grid is washed by touching it three times to the surface of a drop of distilled water. Remove excess water by touching the grid to a filter paper. A small drop of stain is then applied to the grid.

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(6) After 10 s the excess stain is removed by touching the edge to a filter paper. The sample is wicked to dryness, dried at room temperature and viewed in the TEM. Alternatively, sequential droplets of; sample, stain, wash can be laid out on a hydrophobic surface (dental wax or parafilm). The carbon formvar TEM grid can be moved between each droplet with short timed exposures to complete the staining procedure.

Negative Staining for Diagnosis of Fecal Samples Make a 10%–20% suspension of feces in PBS. Centrifuge at 3000 rpm for 30 min to clarify. Collect the supernatant (i.e., the fecal extract) and store at 41C until EM examination has been completed. Original fecal specimens and extracts may be stored long term at
401C. Fecal extracts are examined by one of the following methods. (1) Ultracentrifugation Centrifuge 2 ml fecal extract at 20,000 rpm for one hour. Tip off the supernatant and drain the tube well. Reconstitute the deposit in a few drops of distilled water. Mix a drop of this suspension with an equal volume of 3% phosphotungstic acid pH 6.3. Place a drop of mixture on a formvar-carbon coated grid and then touch a piece of filter paper to the edge of the grid to blot dry. Examine the grid in the electron microscope at an instrument magnification around  30,000 to  60,000. If fecal specimens are very fatty and do not adhere to the grid well, mix one-part resuspended pellet with one part 0.05% bovine plasma albumin. Add one-part stain, place a drop on the grid and blot dry. (2) Lyphogel method Weigh out 0.1 g lyphogel into a small screw capped bottle. Add 0.55 ml fecal extract. Leave at room temperature for four hours or at 41C overnight. Place a drop of the residual fluid on a formvar-carbon-coated grid. Blot off the excess with a piece of filter paper. Wash the grid with 1–2 drops of distilled water and blot. Apply a drop of negative stain to the grid and again remove excess stain with filter paper. Examine grids in the microscope. (3) Sucrose method Place 4 ml sucrose solution (30% w/v in distilled water) in a centrifuge tube. Layer 1 ml fecal extract onto the sucrose. Centrifuge at 40,000 rpm for two hours. Drain the tubes well – use a swab or tissue if necessary, to ensure that all excess sucrose solution has been removed. Reconstitute the pellet in a few drops of distilled water and stain as in method 1. These methods are suitable for the detection of most of the usual gastroenteritis viruses eg rotavirus, adenovirus, astrovirus, etc., and are usually all that are required for examining fecal specimens from babies. Further concentration on cesium chloride gradients may be required to detect small round viruses and in particular the small, round, featureless, parvovirus like particles which are more usually associated with gastroenteritis in older children and adults. This method is used in addition to one of the three methods already described. (4) Cesium chloride gradients Place 2 ml fecal extract in a centrifuge tube and top up with phosphate buffered saline (PBS). Centrifuge at 40,000 rpm for one hour. Resuspend the deposit in 0.3 ml distilled water. Place 4.5 ml cesium chloride solution in a centrifuge tube (Cesium chloride is made up in distilled water to give a starting density of 1.38g/cc. Cesium chloride solution can be obtained from BDH as a 60% w/w solution for ultracentrifuge work. This solution diluted to 28% in distilled water gives a suitable starting concentration). Lift the grid up with a pair of forceps and stain by placing a drop of phosphotungstic acid on the grid and blotting dry as before. Examine the grids in the microscope.

Preparation of Agarose Slides Place 2 ml agarose (0.9% in distilled water) on a microscope slide. Allow the agarose to set and leave in a damp petri dish at 41C for at least one hour before use. One slide will accommodate three grids satisfactorily. Cesium chloride diffuses into the agarose leaving virus particles attached to the grid (Fig. 3). A general working assumption is that the threshold of visibility for virus particles is approximately 106 particles per ml. Further direct concentration of samples directly onto the TEM grid is possible. The Airfuge by Beckman Colter equipped with an EM-90 Electron Microscopy Particle Counting Rotor will concentrate up to 6 separate samples (100 ml) directly onto the EM grid and significantly improves the detection sensitivity of DEM.

Limitations of Traditional Processing Techniques and Advanced Protocols Traditional EM processing strategies introduce artefacts (perturbations to structure) that affect tissue structure, e.g., by shrinkage or swelling of the tissue under examination, the shrinkage of cellular organelles, and/or the extraction or redistribution of cellular constituents such as lipids, proteins and DNA. In addition, most chemical fixatives react with proteins and cross-link peptide chains as part of their action. These reactions can be highly deleterious to epitopes, significantly compromising immunohistochemical studies.

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Electron Microscopy for Viral Diagnosis

Fig. 3 Preparation of fecal samples for negative staining using an agarose slide method. 2 ml of agarose is added to a glass microscope slide and allowed to spread to form a uniform film. Once the agarose has set the sample collected from a cesium chloride gradient can be pipetted onto the agarose film and a TEM grid with previously glow discharged carbon-formvar film placed on top of the sample droplet. The cesium chloride will diffuse into the agarose leaving virus particles attached to the grid.

Specialist EM sample processing strategies have emerged to minimize or avoid these limitations. The capacity of EM can be enhanced by correlative light and electron microscopy (CLEM) workflows, where light microscopy (LM) events are co-localized with underlying ultrastructure. In its simplest form, LM generates a “Google Earth”-like map to identify areas of interest, which are then studied at the ultrastructural level by EM, so overcoming both the physical size limitations imposed on samples by EM and the diffraction limits of resolution imposed by LM. Pioneering groups now employ correlation in workflows which allow single particle analysis cryoEM techniques to be applied directly to structures in cells and tissues. Electron tomography is required to first determine the 3D structures of irregular samples such as cells and organelles. Once good tomograms are available, it is possible to extract multiple copies of a structure of interest and then apply single particle methods to obtain averages with greatly improved signal to noise ratios. By minimizing the use of chemical fixatives, immuno-electron microscopy (IEM) is also possible. IEM is based on the serological principles of the ELISA and can be used for specific virus identification during routine TEM diagnosis. IEM has the advantage that it works directly with raw serum, so no purification of immunoglobulins or conjugation steps are necessary and only small reaction volumes are required. If cell or tissue samples are prepared following the protocol as described by Tokuyasu in 1973, sections of tissue can be labeled with gold conjugated antibodies and ultrastructural details of virus replication processes revealed. A TEM DEM laboratory can maintain, refrigerated, active stable collections of antisera specific to a broad spectrum of virus species and isolates. Depending on the composition of antigens, as well as the epitopes present in the original virus purification, polyclonal antisera can be suitable for capturing multiple viruses within a genus. This versatility can be valuable in DEM, where serological relationships are revealed in the EM through strength of antibody attachment in the labeling step. A “strong” homologous reaction will be seen as a virion tightly packed with antibodies. For emerging isolates or a “less specific” heterogonous virus only weak antibody labeling will be seen. It is also possible to use monoclonal antibodies which targeting single epitopes to provide high specificity and reproducibility for distinguishing different virus isolates. Immuno-electron microscopy may be performed on fecal extract by; centrifugation of 1 ml fecal extract at 20,000 rpm for one hour. Remove the supernatant and resuspend the deposit in 0.2 ml PBS. Add 0.1 ml serum at the required dilution (dilute in PBS). Cover the tube with parafilm and leave at room temperature for one hour and at 41C overnight. Add cold PBS to fill the tube. Centrifuge at 18,000 rpm for one hour. Drain the tube well, resuspend the deposit in a few drops of distilled water and stain as in the ultracentrifugation method described above (method 1).

Cryo Electron Microscopy Recently, cryo-fixation has been adopted as a physical fixation strategy. The morphology and dimensions of the living material are retained, and soluble cellular components are not displaced, which means that processing artefacts commonly encountered in more conventional room-temperature EM techniques are either reduced or removed. Cryo-EM often allows direct observation of specimens that have not been stained or chemically fixed. Cryo-fixation has two distinct advantages over chemical fixation. It is rapid (measured in milliseconds), which means that the sample is preserved, hydrated and in a “close-to-life” state at the point of initiating fixation, and ensures simultaneous immobilization of all macromolecular components. Many protein networks are labile and prone to disruption with even a slight osmotic or temperature change; cryo-fixation minimizes these effects. Cryo-techniques also allow the study of biological samples with improved ultrastructural preservation and may facilitate the study of dynamic processes. Rapid freezing prevents artefactual aggregations of proteins because tissue fixation and processing can be performed with no or minimal use of cross-linking fixatives, which means that samples retain higher antigenicity. This level of preservation relies on vitrification (the transformation of water from a liquid to an amorphous state without inducing the nucleation of ice crystals). The nucleation of ice crystals is temperature- and pressure-dependent. Crystallization depends on the cooling rate, itself dependent on the thermal properties of water, the sample thickness, and the heat extraction flow at the surface of the specimen. Freezing is a time-dependent process. Vitrification sits at the beginning of a workflow where the sample is either subsequently processed to a stable room-temperature state for imaging or maintained in its vitreous state throughout. Due to the poor thermal conductivity of water, cooling of virus samples by simply plunging into liquid nitrogen (LN) would be too slow to allow for vitrification and samples would thus be badly damaged by ice crystals. Water was vitrified by vapor deposition on a cold surface as early as 1935 and by fast cooling of micro-droplets in 1980, however, these were either not useful or not amenable for direct observation at the TEM. It was Jacques Dubochet’s group which first successfully vitrified thin films of water containing small particles supported by the surface tension of water itself between the bars of

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Fig. 4 Cryo electron microscopy for near atomic resolution structural studies. The workflow is presented from left to right. The first step is the blotting of a small volume of pure virus particles (B4 ml) on a TEM grid with a suitable support film containing small regular holes. The blotting step produces a thin film of virus particles with many supported by surface tension across the holes in the film. The sample is then rapidly frozen by plunging into a cryogenic liquid to create a vitrified film of virus in water. The vitrified film will contain many individual particles which are imaged in the transmission electron microscope. These particles are randomly arranged and can be categorized as “ideal” (green boxed) when single isolated particles are held in the vitrified solution or “non-ideal” (red boxed) when particles are in contact with the air-water interface which can cause denaturing of structure. I In “thick” areas of the film, resolution can be limited by the volume or overlapping of particles. The resulting TEM images are relatively low contrast as no heavy metal stains are added to the sample. Individual particles are subsequently “boxed out” of many individual micrographs and classified on a basis of similarity before finally being processed to generate a high resolution 3D structure.

EM grids. The water films could be vitrified because they were plunged into liquid ethane rather than into liquid nitrogen or helium, which are not good cryogens due to the influence of the Leidenfrost effect (a film of insulating vapor which slows down cooling). The frozen samples could then be observed directly at their frozen hydrated state in the cryo-electron microscope and vitrification could be verified by electron diffraction. The plunging technique developed by this group is the basis of sample preparation used for modern single particle analysis and reconstruction, reaching near atomic resolution (Fig. 4). The success of the method lies not only in choosing the right cryogen but also in the high cooling rates that can be achieved during the freezing of extremely thin aqueous films (B100 nm), which are in the range of 107 C s
1. As Jacques Dubochet wrote: “It is the good fortune of the electron microscopist that, reducing sample size in order to increase the cooling speed, vitrification becomes easy just when the dimensions of the specimen are those suitable for electron microscopy.” This technique as the preparation step for single particle analysis was, together with transformative advances in understanding of EM image formation and image processing, rewarded by the award of the Noble Prize for Chemistry in 2017 for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution to Jacques Dubochet, Richard Henderson and Joachim Frank. There are a variety of methods for rapid freeze fixation of larger samples e.g., plant or animal tissue or even cell monolayers that are few mm in thickness. The depth of vitrification is often limited (e.g., a few microns) for samples plunged into liquid cryogen. The simplest add cryoprotectants to prevent ice crystal growth and achieve vitrification by plunging the sample into liquid nitrogen. An alternative technology is high-pressure freezing (HPF). First introduced by Moor and Riehle in 1968, HPF exploits the physical benefit of high pressure (210 MPa) to reduce the cooling rate required for the vitrification of water from several 100,000K s
1 to a few 1000 K s
1, making vitrification of relatively thick samples practicable. HPF machines synchronize pressurization and cooling of the sample (to below the glass transition temperature (Tg)) within 20 ms, in a highly reproducible manner, extending the depth of vitrification to as much as 200 mm. These HPF machines are now incorporating added functionality to achieve a high degree of temporal resolution. For example, light-sensitive cation channels can be expressed in cells and tissues, stimulated in the HPF pressure chamber at different time intervals (ranging from ms to s) and immediately cryo immobilized. Electron micrographs acquired from many time points reveal in minute detail sequences of morphological changes at ms precision. Using this approach, the process of ultrafast endocytosis has been characterized during neurotransmission. Recently, electrical stimulation has also been adapted to the HPF allowing electron microscopy to reveal complex events previously identified by electrophysiology. Tissue samples prepared this way require further modification to make them compatible with the transmission electron microscope. They must either be converted to a room-temperature stable state (e.g., by freeze substitution into a resin) or be sectioned to make them thin enough for the TEM electron beam to pass through the sample. This sectioning must be carried out whilst remaining below the glass transition temperature (Tg); when this is done, images close to the native structure of biological specimens can be achieved. The significant technical challenges of these techniques preclude them from use for DEM.

Advances in Scanning Electron Microscopy Cryo-FEGSEM has become well established as a research tool. The stability and emitter life of a FEG source, coupled with the stability and low keV sensitivity of a modern scanning electron microscope, is a powerful tool. Unlike TEM, in which high accelerating voltages are used to transmit the electrons through the specimen, SEM benefits from low accelerating voltages that

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limit beam interaction with the specimen. This is key to successful cryo-SEM but creates a challenge around generating sufficient signal for high-resolution imaging. Freeze fracture followed by cryo-FEGSEM can reveal ultrastructural details of viruses and permits high resolution direct visualization of cell membranes and the budding of virus particles (Fig. 1). Modern FEGSEM systems (with improved detector sensitivity and signal-to-noise ratios) are now achieving sub-nm resolution at 1 kV, making them highly capable tools for life science research, particularly when combined with high-resolution coating and fracture techniques. When equipped with a STEM detector it is possible to collect images of NS samples using the SEM similar to those generated by the TEM (Fig. 1). However, the use of a rasterizing scan to generate the image makes rapid screening of NS samples for DEM challenging. The operator must wait until the scan is complete before seeing the full scanned area. This prevents rapid DEM screening of samples. RKI have developed a field-based bioterrorism SEM protocol for the rapid detection of bacterial spores using portable “table-top” SEM’s. This approach provides a rapid direct test to determine if an unidentified powder is biological (i.e., anthrax spore) or chemical (i.e., talcum powder).

Conclusion Electron microscopy of viruses is largely determined by the quality of the sample preparation technique used. By optimizing the sample preparation steps, remarkable structural details of viruses, their organization and replication strategies can be revealed. Cell culture can be used to study the mode of replication and when combined with EM and protocols which allow thin sections of cells and tissues to be studies, direct observation of virus replication achieved. Selection of the most suitable technique is important. The diagnostic role of EM is principally by negative staining. Rapid sample preparation readily and flexibly applied to a wide range of samples facilitates rapid detection and identification of a viral pathogen. Higher resolution cryoEM approaches are better adapted for research applications where sufficient time is available for optimization of all conditions necessary to achieve near atomic resolution structures. The versatility of EM for virus detection maintains its critical role as a tool for diagnosis of viral diseases.

Further Reading Danev, R., Yanagisawa, H., Kikkawa, M., 2019. Cryo-electron microscopy methodology: Current aspects and future directions. Trends in Biochemical Sciences 44 (10), 837–848. Dubochet, J., McDowall, A.W., 1981. Vitrification of pure water for electron microscopy. Journal of Microscopy 124, RP3–RP4. Fleck, R.A., Humbel, B.M., 2019. Biological Field Emission Scanning Electron Microscopy. London: Wiley, p. 752. Gelderblom, H.R., 1996. Chapter – 41 Structure and classification of viruses. In: Baron, S. (Ed.), Medical Microbiology, fourth ed. Boston, USA: Addison-Wesley Publishing Company, Incorporated, Health Sciences Division. Gentile, M., Gelderblom, H.R., 2005. Rapid viral diagnosis: Role of electron microscopy. New Microbiologica 28 (1), 1–12. Gentile, M., Gelderblom, H.R., 2014. Electron microscopy in rapid viral diagnosis: An update. New Microbiologica 37 (4), 403–422. Glauert, A.M., Lewis, P.R., 1998. Biological Specimen Preparation for Transmission Electron Microscopy. London: Portland Press, p. 326. Griffiths, G., Lucocq, 2014. Antibodies for immunolabeling by light and electron microscopy: Not for the faint hearted. Histochemistry and Cell Biology 142, 347–360. Horne, R.W., Wildy, P., 1979. An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses. Journal of Microscopy 117 (1), 103–122. Laue, M., Möller, L., 2016. The VirusExplorer DEM - A Database for Diagnostic Electron Microscopy of Viruses, Zenodo. doi:10.5281/zenodo.221574. Madeley, C.R., Field, A.M., 1988. Virus Morphology, second ed. Edinburgh, London: Churchill Livingston. Nermut, M.V., Frank, H., 1971. Fine structures of influenza A2 (Singapore) as revealed by negative staining, freeze-drying and freeze-etching. Journal of General Virology 10, 37–51. Robards, A.W., Sleytr, U.B., 1995. Low temperature methods in biological electron microscopy. In: Glauert, A.M. (Ed.), Practical Methods in Electron Microscopy 10. Amsterdam: Elsevier, pp. 5–146. Stirling, J., Eyden, B., Curry, A., 2012. Diagnostic Electron Microscopy: A Practical Guide to Tissue Preparation and Interpretation, first ed. John Wiley & Sons. Tokuyasu, K.T., 1973. A technique for ultracryotomy of cell suspensions and tissues. Journal of Cell Biology 57 (2), 551–565.

Relevant Websites http://www.era.rothamsted.ac.uk/eradoc/OCR/ResReport1964-128-144 Report for 1964. e-RA. https://www.pirbright.ac.uk/cross-cutting-capabilities/bioimaging Specialists in imaging viruses and virus-host interactions. https://www.gu.se/core-facilities/centre-for-cellular-imaging-cci? Standard protocols for negative staining and chemical fixation of biological tissues with downloadable and printable pdf protocols. https://zenodo.org/record/221574 The VirusExplorer DEM - A database for diagnostic electron microscopy of viruses. https://zenodo.org/record/221524 The VirusExplorer DEM - A database for diagnostic electron microscopy of viruses. https://www.rki.de/EN/Content/infections/Diagnostics/SpecialLab/EM_content.html The virus diagnostic unit at the Robert Koch Institute.

Serological Approaches for Viral Diagnosis Klaus Hedman, University of Helsinki, Helsinki, Finland and Helsinki University Hospital, Helsinki, Finland Visa Nurmi, University of Helsinki, Helsinki, Finland r 2021 Elsevier Ltd. All rights reserved. This is an update of R. Vainionpää, M. Waris, P. Leinikki, 2015. Diagnostic Techniques: Serological and Molecular Approaches, In Reference Module in Biomedical Sciences, Elsevier Inc., doi:10.1016/B978-0-12-801238-3.02558-7.

Introduction Virus serodiagnostics (serology) can be employed for a number of clinical indications, including determination of the etiology and respective time(s) of primary infections or of secondary infections or recurrences, the latter comprising endogenous reactivations and exogenous reinfections. Based on several characteristics of virus-specific B-cell immune response, serology firstly aims at identifying and dating a recent active (i.e., replicative) infection (when infected?), and secondly at determining an individual’s lifelong infection history (ever infected?). The former question serves the clinician in diagnosis of diseases associated temporally with the corresponding infection types (primary; other). The latter question can serve two wide (and diverse) purposes: (1) Assessment of an individual’s status of immunity versus susceptibility; and (2) Uncovering the possibilities of infection chronicity or reactivation (via latency), or of other longer-term manifestations linked with lifelong history of contagion by some of the many human viruses that exist. Serology, in a nutshell, suits best for diagnostic assessment of such viruses that tend to infect the human body systemically (widely; deeply) rather than superficially (mucosa; skin). Understandably, for the immune response to ensue, the individual ought to be immunocompetent. This article briefly describes the principles of serological approaches and their utilities in essential (exemplary) clinical settings. Excluded is virus antigen detection (using antibodies as a tool), despite its ongoing or growing importance in management of e.g., some “heavyweight” pathogens such as HIV or hepatitis B. Following transmission, the virus begins to multiply, and after an ostensibly silent incubation period, the production of infectious virus begins, and the clinical symptoms appear simultaneously or subsequently, pending virus species, patient and clinical context. I.e., the appearance of virus-specific antibodies follows the primary contagion by a “window” of initial seronegativity lasting days to weeks, and a subsequent phase of antibody maturation lasting months. Having reached the detection level, the first antiviral immunoglobulin (Ig) class is IgM, followed by IgG. Around this time the infectious (or antigenic) virus in blood, termed viraemia, as a rule (pending methods sensitivity) disappears. In the first encounter with a given virus – termed a primary infection, or immune response – the IgG level tends to rise relatively slowly; as opposed to a subsequent encounter with the same virus/antigen – a secondary infection/response – in which the IgG level rises and peaks more rapidly; whereas the corresponding IgM may remain undetectable. Typical course of virus infection and antiviral antibody response are summarized in Fig. 1. The antiviral antibodies may be searched in two (or even more) successive serum samples taken at the acute and convalescent phases. In selected cases, other body fluids such as cerebrospinal fluid, saliva or potentially even urine can be analyzed for antiviral antibodies.

Principles of Serological Assays Serological diagnosis is usually based on either the demonstration of the presence of specific IgM or a significant increase in the levels of specific IgG between two consecutive samples taken 1–4 weeks apart. The antigen for the test can be either viable or inactivated virus or some of its components (proteins; epitopes) prepared by virological or molecular methods. Specific markers or physical separation are used to demonstrate the isotype of the antiviral antibody. In some cases, even IgG subclass specificities are determined although they have limited value in diagnostic work. In primary infection the antiviral IgM usually peaks at 7–10 days after onset of symptoms, and typically disappears during the subsequent weeks or months. However, it is not exceptional for the circulating IgMs to remain detectable even for years (IgM persistence). By contrast, in secondary infections – endogenous reactivation or exogenous reinfection – an IgM response often remains absent altogether. After primary infection the antiviral IgG usually becomes detectable a week or two later than the IgM, and often persists through life. This characteristic renders IgG an important hallmark of an individual’s lifelong infection history (with regard to a given immunogen/pathogen) – and thereby (pending assay calibration) an indicator of antiviral immunity vs. susceptibility – and (pending taxonomy and crossreactivity versus specificity) of infection latency, linked with the potentials for secondary infections, chronicity or other sequelae. In the early phase of primary infection, the avidity (functional affinity; average binding force) of antiviral IgG tends to be low, followed by increase during several months along with maturation of the immunoglobulin response. Diagnostic applications of IgG-avidity assays have been developed to help distinguish serological responses due to acute infections from those of chronic or past infections, and for a number of indications addressed below.

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Fig. 1 The course of virus infection, including shedding of infectious virus after incubation period and typical antiviral antibody response. Some typical diagnostic approaches have been marked.

Serological assays in general provide information of many types and perspectives, and for a number of clinical contexts/ conditions. By dating primary infection they can tell the etiology of an acute illness even when infectious virus or its components no longer are detectable. The assays are used widely for determination of the immune status, in screening of blood products for the risk of infectious-agent transmission, and for the need of prophylactic procedures. They are also widely used for epidemiological studies, determination of vaccine-induced immunity, and other similar public health purposes.

IgG Avidity IgG avidity, or functional affinity, is the average binding strength of an antibody population towards an antigen. Low-avidity IgG antibodies usually appear within 2–3 weeks after primary infection or immunization. Reflecting B-cell affinity maturation, IgG avidity increases during the following months. Subsequently, including re-infections and re-activations, the avidity remains elevated. On top of IgM and IgG, IgG avidity measurement provides an additional tool for sorting out the different stages of infection (low avidity during primary infection – increasing avidity during convalescence – high avidity as a marker of past infection, including chronic or recurrent infections). Indeed, valuable information is achieved when traditional IgM and IgG assays are combined with those of IgG avidity. During the past 3 decades, IgG avidity measurement has in the literature been adopted into use with numerous viral pathogens. During pregnancy, adverse clinical outcomes with certain pathogens (e.g., cytomegalovirus, parvovirus B19 and rubella virus) are mainly due to primary infection, moreover with the clinical picture and severity depending on the stage of pregnancy of the maternal infection. This highlights the importance of IgG avidity measurement in timing (dating) of obstetric infections. In addition to viral diagnostics, IgG avidity is increasingly utilized in other branches of clinical microbiology. More recently, the clinical relevance of antibody avidity is being investigated in many autoimmune diseases as well. In epidemiology, IgG avidity (along with IgM) measurement in a single sample has been proposed to be a cost-effective alternative to consecutive sampling, for assessment of new cases (primary infections). Most IgG avidity assays employ a chaotropic protein denaturant (e.g., urea) to disrupt the antigen-antibody bond. As high-affinity bonds tend to be more resistant to denaturation than those of low affinity, the result can be expressed by comparison of residual (continuously antigen-bound) antibody levels obtained with and without the denaturant. An approach more recently adopted from basic immunology into clinical use employs a soluble antigen to capture the high-avidity antibodies, while those of low avidity remain to bind selectively to the solid phase antigen (displaying higher epitope density required for low-affinity interaction). In analogy with the chaotrope approach, the avidity result can be expressed by comparing the results obtained with versus without the antigen in solution. Indeed, this approach lacks the chaotrope that can crystallize and hamper assay automation. Unlike the unequivocal affinity of a monoclonal antibody, the avidity of a polyclonal antibody population represents an approximation of the average of affinities, and tends to be expressed in arbitrary/relative values. The simplest is a ratio of signals obtained from two parallel measurements, often referred to as an avidity index (AI ¼ (signal with denaturation)/(signal without denaturation) ¼ relative proportion of denaturant resistant high avidity antibodies). As the AI is, to some extent, affected by the concentration of the corresponding IgG, the working dilution of the measurement may be chosen according to the IgG level. Due to its simplicity and acceptable sensitivity and specificity, the AI has gained notable popularity. Another approach employs serial dilutions of sample, whereupon avidity is calculated by the ratio of IgG end-point titers generated from titration curves. This latter approach has a broader dynamic range than the former and permits avidity measurement from samples the IgG concentration of which is too low for the AI approach. The sample titration though, understandably is more laborious and costly.

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Clinical specimens other than serum/plasma are useful in some situations. Whole blood e.g., dried on filter paper (Guthrie card) facilitates transport over or storage in challenging conditions (climatic; financial). Cerebrospinal fluid can identify immune response (reflecting infection) within the central nervous system (CNS), even if nucleic acid testing (NAT) is overtaking large parts of CNS diagnostics. Increasing attention in serodiagnostics is being paid to the use of noninvasive sample materials such as saliva – and recently, urine. The former has found use e.g., in epidemiology, while the latter’s value in clinical virology calls for in-depth assessment!.

General Considerations and Sources of Error The antigens used in serological assays play a central role in their clinical performance. Some epitopes are more immunogenic than others; and the immunoreactivity towards certain antigens/epitopes even correlates with the stage of infection (or duration/time from immunization). e.g., the antibodies against hepatitis B virus s-antigen indicate past infection or vaccination and are generally undetectable during acute or chronic infection. Low purity of antigen conceivably leads to increased background via nonspecific binding and some of the serological methods are more prone to this than others. Antibody responses elicited against certain viruses can also react against other, usually closely related, viral species through shared or similar epitopes. These cross-reacting antibodies can thus cause false positives unless: only unique epitopes are assayed; or cross-reacting antibodies are blocked and depleted with other viral antigens (e.g., anti-dengue virus antibodies with anti-zika virus gross-reactivity and anti-zika antibodies with anti-dengue cross-reactivity can be blocked with dengue antigen, leaving only zika specific antibodies unaffected). Not only serodiagnostics, but the entire antibody response against a virus can be influenced by cross-reacting antibodies of a previous infection. In the phenomenon called original antigenic sin, immune response against a virus can profoundly affect subsequent responses elicited against other viruses. e.g., infection by human bocavirus 1 produces a classical primary immune response. Subsequent infection by human bocavirus 2 can lead to activation of cross-reacting anti-bocavirus 1 memory B cells instead of primary immune response against bocavirus 2. Original antigenic sin not only hampers serodiagnostics but can also lead to weaker immune protection and more severe disease. Rheumatoid factor is an autoantibody and a diagnostic marker for autoimmune diseases such as rheumatoid arthritis and Sjögren’s syndrome. It binds the Fc portion of IgG and is usually of IgM glass. Rheumatoid factor can thus cause false positives in indirect IgM assays by binding to antigen-specific IgG, if the sample contains said antiviral IgG. In current IgM assays this is rarely an issue since they either are based on methodology not affected by rheumatoid factor, include removal of the bridging IgG or the sample is assayed for rheumatoid factor. A rare but extremely elusive source of false positives are serum antibodies against assay components that are generally considered immunogenically inert. Human antibodies against both streptavidin and its ligand biotin, a popular tool in molecular biology, have been reported in the literature and shown to cause false positives in several assays utilizing the streptavidin-biotin bond.

Restrictions and Limitations Serological assays have their restrictions. In some infections, e.g. superficially confined to the skin or mucosal membranes, the antibody response is not strong enough. Immunocompromised patients are often unable to mount a B-cell response. In newborns or infants the presence of (residual) maternal IgG can hamper identification of the baby’s own antibodies (hallmarking intrauterine infection). The converse also holds; functional comparison with the mother’s antibodies especially combined with follow-up can reveal significant qualitative differences, e.g., in antibody isotype, antigen-recognition or avidity, to indicate the emergence of the infant’s endogenous immunoreactivity.

Principles of Widely Used Serological Tests Immunoassays Immunoassays by definition take advantage of the highly specific antigen-antibody bond to recognize analytes (which in the context of this review are antibodies) in the sample. The majority of immunoassays used in serodiagnostics of viruses are based on sequential binding reactions that, step by step, build a molecular complex onto a solid surface, often beginning with antibodies in the sample targeting the antigen. Each reaction is separated by a washing step to remove unbound reagents. Provided that antiviral antibodies are present in the sample, the process ends at binding of a label generating a measurable signal that is proportional to the amount of those antiviral antibodies. Although many of the assays are rather time consuming and labor intensive, they also are robust, in general have high sensitivity and specificity, and are available in many optional formats. Commercial kits are available for numerous viruses and the assays can be run either manually or in automated systems, up to fully automated high-throughput pipelines used with many clinically relevant viruses. The different immunoassay approaches can also be adapted into multiplexing (i.e., simultaneous detection of antibodies against different viruses or epitopes).

Solid-phase microwell immunoassays Indirect immunoassays: The by far most popular format in antibody detection is indirect solid-phase enzyme immunoassay (EIA, Fig. 2(A)) alternatively termed enzyme-linked immunosorbent assay (ELISA). Antigens are first immobilized onto the surface of a

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Fig. 2 The most popular immunoassay formats: indirect (A), competitive (B) and capture (C) immunoassay.

microwell and then incubated with the sample. Alternatively, immobilization onto soluble magnetic microspheres can provide increased total surface area leading to higher sensitivity as well as to faster reaction times, as diffusion from the solution phase onto the surface of the microwell is no longer the limiting factor. The specific antibodies bind to the solid-phase antigen and are then detected with anti-immunoglobulin secondary antibodies. Commonly either anti-IgG or anti-IgM are used to assay IgG and IgM antibodies, respectively. With appropriate secondary antibodies, other immunoglobulin classes or subclasses can be assayed. The secondary antibody is coupled with a label that allows its detection. The signal furthermore is amplified, as several secondary antibodies can bind a primary antibody. Depending on the label, the assay nomenclature can vary, although the principle remains the same. In EIAs the label is an enzyme (e.g., horseradish peroxidase or alkaline phosphatase) that can activate multiple substrate molecules thus amplifying the signal (e.g., color reaction measured via absorbance). If fluorescent label is used the method may be called fluoro- (or fluorescence) immunoassay (FIA). A method utilizing chemiluminescence can be called chemiluminescence immunoassay (CLIA). The label can even be a strand of DNA that is amplified and quantified in a subsequent PCR-step. Nonspecific binding of antibodies can cause problems since they are equally recognized by the secondary antibody. This can be largely prevented by blocking the solid-phase with an inert protein or detergent. The indirect immunoassays remain, however, susceptible to cross reacting antibodies if additional measures are not taken to block them. Competitive immunoassays: In competitive immunoassays (Fig. 2(B)) labeled mono- or polyclonal antibody generates the signal by binding to its antigen. If a sample contains antibodies against the same epitope(s) they compete with the labeled antibody preventing it from binding. This results in a lower signal and the level of this inhibition is proportional to the amount of specific antibodies in the sample. Competitive immunoassays are highly specific since nonspecific or cross-reacting antibodies binding to other epitopes are not detected, unless they are bound close enough to cause steric hindrance. Provided that suitable monoclonal antibodies exist, competitive immunoassays can measure antibodies against single epitopes (e.g., a neutralizing epitope) without the need to separate said epitope from the rest of the antigen (which might not even be feasible). Competitive assays as such cannot distinguish between immunoglobulin glasses, but the IgG can be removed or precipitated from the sample to reveal possible underlying IgM reactivity. Capture immunoassays: Instead of antigens, the solid-phase of capture immunoassays (Fig. 2(C)) consists of anti-IgM or antiIgG capture-antibodies (or IgG-binding proteins such as protein A or G) that bind and “capture” the total IgM or IgG populations in the sample. Labeled antigen is then added and bound only by antibodies specific to it if present in the captured immunoglobulin population. This makes the method highly specific, although cross-reacting antibodies can still cause false positives. With IgG only a small fraction of the captured total antibody population is specific to a given antigen which can cause problems with sensitivity if signal is not sufficiently amplified. With IgM this is less of an issue since it is primarily produced during acute infections and generally does not persist afterwards. Capture immunoassays are also not affected by rheumatoid factor which makes them especially well suited for IgM diagnostics.

Immunoblotting Immunoblotting is a classical method for measuring antibody responses against multiple different antigens (i.e., a multiplex assay, actually). The separated antigen molecules are adsorbed as discrete bands on a solid strip and then incubated with the clinical sample. Antibodies present in the sample bind each to their respective antigens and are detected with a labeled secondary antibody visualizing or otherwise identifying the antibody-tagged bands. The latter are then compared to controls on another strip. Immunoblotting is useful (1) for control of antibody specificity, and (2) when the labeling pattern towards several molecules is required for diagnosis; although improved multiplex immunoassays of other types have decreased the popularity. Immunoblotting is still commonly used to ascertain the results of sensitive first-line serodiagnostics for e.g., HIV and hepatitis C virus (HCV).

Immunofluorescence microscopy Immunofluorescence assay (IFA) follows the same basic principle as the other indirect solid-phase assays. Infected cells immobilized on a microscope slide act as the antigen, and bound antibodies of a sample are detected by a fluorophore-labeled secondary (anti-immunoglobulin) antibody. The method is specific and sensitive, but quite labor intensive, and reading of the

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result with a microscope calls for experience. IFA has been a popular approach in the past but has now, to a large extent, been replaced by immunoassays of EIA-type.

Lateral-flow immunoassays In lateral-flow immunoassays the sample is applied on one end of a porous capillary strip and then diffuses along it, passing through several sections containing different reagents (analogous to distinct reaction steps of other immunoassays). Labeled secondary antibodies can bind those of the sample and move along with them. A band of immobilized antigen captures the specific antiviral antibodies whereby the labeled secondary antibody makes the band detectable. A control band further downstream captures the labeled secondary antibody indicating that excess reagents and nonspecific antibodies have successfully diffused through the system. One strip can hold several antigenic bands, and many of the assays are compatible with whole blood as sample. Performance of the test is often simple and the result is available in a few minutes to hours, making the approach, together with latex agglutination, the most popular form of POC serodiagnostics. While the sensitivity and specificity of these tests can be good, they tend to fall behind those of the more laborious immunoassays such as EIA.

Homogenous wash-free immunoassays Unlike the other, homogenous assays lack the laborious washing steps, whereby the results are obtained simply by combining the reagents with the sample. The assay can rely on proximity sensing techniques such as Förster resonance energy transfer, twophoton excitation energy transfer or their fluoro-chemical analogs. Briefly, included are two indicator molecules (or complexes) that generate a distinct signal only when in close proximity of each other. When coupled to antigens and/or antibodies, their binding brings the two indicators sufficiently close to generate the signal. In the absence of binding the indicator remains quiescent. Although such techniques have been successfully employed with PCR and microscopic imaging, they still are in their infancy in antibody detection. Commercial kits are available for antigen detection, but presently are more research than diagnostics oriented. Since the homogenous assay format is faster and simpler than the conventional immunoassays, it has great potential in POC diagnostics. Compared with lateral flow and latex agglutination it does require particular instrumentation, but with ongoing development of inexpensive portable technology this is becoming less of an issue.

Neutralizing Antibody Assay All antiviral antibodies by definition bind viral antigens. Neutralizing antibodies are such that, through this binding and without other components of the immune system, reduce or “neutralize” the infectious capacity of a virus (e.g., blocking the interaction between the virus and its receptor). They can be of IgG, IgM or IgA class and generally persist during lifetime. In neutralizing antibody assay (Fig. 3(A)), known amounts of infectious virus are mixed with the sample and incubated for a short period after which the residual infectivity is measured by infecting cultured cells and observing the outcome. This infectivity is then compared with the infectivity obtained without the sample and the neutralizing capacity is calculated by comparing the two results. Neutralizing antibody assay is specific and sensitive, but time-consuming and laborious, and therefore not widely used in routine diagnostic services. It measures, however, something the other serological methods do not: the function and efficacy of antiviral immunity, not its mere presence. For this reason neutralizing antibody assays are useful in more in-depth studies of the humoral immune response and especially with vaccines, as successful vaccination (i.e., immunity against the pathogen) generally both requires and can be demonstrated by the presence of neutralizing antibodies. Neutralizing antibodies can also be measured, to some extent, with conventional immunoassays with regard to neutralizing epitopes, e.g., using neutralizing antibodies in a competitive assay. This requires that, for a given virus: such epitopes have been discovered; a combination of all the relevant ones can be assayed; and they can be assayed independently from the nonneutralizing epitopes.

Fig. 3 Neutralizing antibody (A) and latex agglutination (B) assays.

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Latex Agglutination Latex agglutination (or fixation) assays, Fig. 3(B) are based on aggregation of antigen-coated beads (e.g., of latex) mixed with a sample containing antibodies against the antigen. When the bi- or multivalent antigen-binding sites of an antibody (IgG or IgM, respectively) attach to different latex beads, the latter are cross-linked and aggregate. The aggregation (or agglutination) may be observed visually or by increased light-scattering spectrophotometrically. The agglutination tests tend to be rapid (within minutes) and easy to perform. Their popularity as POC assays also is due to independence of complex/expensive equipment. The reaction, however, is sensitive to the prozone effect. Paradoxically, excessive concentrations of specific antibodies can bring about a negative result, when the cross-linking fails due to bead coverage. This in turn can be overcome by assaying at serial dilutions of the sample.

Hemagglutination Inhibition Many viruses bind to hemagglutinins, molecules at the surface of red blood cells (RBC) of various animal species, and causing in suitable conditions RBC aggregation leading to (hem)agglutination. Inhibition of the phenomenon by antiviral antibodies in patients’ sera has historically been used widely for diagnostic purposes. In the test, a virus preparation of predetermined hemagglutinating capacity, and standardized amount of red blood cells as well as the sample are mixed, and after incubation the residual hemagglutination is recorded. Both IgG and IgM antibodies are functional. Presently the hemagglutination inhibition test is used in seroepidemiological studies and as a reference test, most notably with influenza viruses. As the test utilizes complete virions it is well suited for assaying antibodies even on serotype level. The hemagglutination inhibition test is seldom used for diagnostic purposes nowadays.

Complement Fixation Complement fixation test is a classical diagnostic approach based on the capacity of complement, a group of heat-labile plasma proteins of the innate immune system, to bind antibodies in antigen-antibody complexes. When such complexes are present on the surfaces of red blood cells, the complement brings about their lysis which can be observed visually or measured. The sample’s endogenous complement is first deactivated by heating; the sample is then mixed with the viral antigen and a fixed amount of exogenous complement; and RBCs coated with anti-erythrocyte antibodies are finally added as an indicator. If the sample contains antibodies against the antigen, the exogenous complement gets depleted (fixed) by the immunocomplexes leaving the RBCs intact. If the antiviral antibodies are absent, the complement lyses the cells. The assay measures both IgG and IgM. Since the assay is laborious, calls for standardization of several biological variables and less sensitive than many other immunoassays it is rarely used nowadays.

Sequencing Analysis of Antibody Repertoire Advances in high-throughput DNA and RNA sequencing (HTS) and bioinformatics have made it possible to sequence, reconstruct and analyze the entire repertoire of B cell immunoglobulin genes (Ig-Seq). These data include all the different B cell clones of an individual and thus contain sequences for all the antibodies they produce. Compared with conventional PCR assays, HTS requires more extensive sample preparation, and the raw data typically comprise around 100 base pair fragments (reads) totaling up to 1012 base pairs per sample. Extensive data processing is thus required for comprehensible results. At present the process of sample preparation, sequencing and data analysis is too resource-demanding for most diagnostic purposes. The vast complexity and constant microevolution of the B cell population is also a challenge; and clear-cut, standardized and diagnostic-grade protocols are yet to be established. Ig-Seq has, however, already become an important and popular tool in research, including in-depth study of humoral immune responses elicited by viral infections or vaccinations. With further advancement in sequencing technology and above all in sequence analysis, clinical applications can be expected in the future. The unresolved challenge for HTS-based serodiagnosis is to link the amino acid sequence of an antigen-binding-site to its specific epitope (i.e. to deduce antibody function based on its sequence). Databases of confirmed antibody sequence-epitope pairs and predictive algorithms are being constructed to resolve the issue but are still at their early stages. The potential of such assays is, however, vast since a single test could reveal the entire infection history (all the pathogens against which antibodies are being produced) or susceptibility (absence of protective antibodies) of the patient.

Point-of-Care Serodiagnostics POC tests are becoming increasingly common in clinical practice. In proper use, they provide cost-efficient diagnostic guidance for quick clinical decision making. Their performance is rarely as good as that of conventional laboratory tests; their use should be quality-controlled, and a back up should be arranged with a virology lab. Most of them are based on easy-to-use lateral-flow or latex particle technology and yield the result in a few minutes. To curtail the diagnostic window, antibody and antigen detection can be combined into the same POC cassette (e.g., HIV Ag/Ab assay). While nucleic acid detection has largely replaced antigen detection and is now increasingly available in POC format, it does not provide equally straightforward synergy with antibody detection. POC tests are nowadays available for antibody screening of an increasing number of virus infections (HIV, HCV, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV)).

Serological Approaches for Viral Diagnosis

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Future Perspectives Driven by public health, scientific and commercial interests, new diagnostic tests for laboratory diagnosis of viral infections are continuously designed. The development of new molecular detection methods continues on two lines. On the one hand, it includes compact, fully integrated automates requiring minimal training to run and suitable for POC use. On the other hand, separate instruments for specimen handling, serological assays, nucleic acid extraction, PCR set-up, and amplification are integrated into high throughput flow systems, which allow efficient use of both commercial and laboratory designed assays. The use of multianalyte methods is becoming practical reality in antibody, antigen and nucleic acid detection and might significantly improve infectious disease diagnosis in the future. The current multiplex approaches, however, still lack a comprehensive library of compatible virus specific reagents (from which any combination of pathogens could be assayed). In antibody detection, multiplex approaches have so far been used mainly for research purposes. Multiplex PCR in a single tube or miniature format, on the other hand, still is to reach the sensitivity of standard PCRs targeting single or few analytes. Next-generation sequencing has paved the way to identification of new or emerging viruses. Simple microarrays, readable without bioinformatics, would reduce the cost of serological screening and allow the use of control measures to improved quality of results. Beyond classical virus diagnostics, analysis of virus-induced interferon gene response is an emerging scope in diagnostics. Inspired by single-molecule sequencing, state of the art EIAs also can detect individual antibodies or immunocomplexes and could significantly improve sensitivity. On the other hand, homogenous wash-free immunoassays can omit most hands-on steps that require expensive labor or instrumentation; their results can be read directly after adding a droplet of sample into a premade reagent mix. Use of noninvasive sample materials (urine or saliva) instead of whole blood, serum or plasma furthermore can allow for self-sampling, for convenience of the patient and cost-benefit of the society.

Further Reading Arnold, K.B., Chung, A.W., 2018. Prospects from systems serology research. Immunology 153, 279–289. Barzon, L., Percivalle, E., Pacenti, M., et al., 2018. Virus and antibody dynamics in travelers with acute zika virus infection. Clinical Infectious Diseases 66, 1173–1180. Chaudhary, N., Wesemann, D.R., 2018. Analyzing immunoglobulin repertoires. Frontiers in Immunology 9, 462. Chevaliez, S., Pawlotsky, J.M., 2018. New virological tools for screening, diagnosis and monitoring of hepatitis B and C in resource-limited settings. Journal of Hepatology 69, 916–926. Dubot-Pérès, A., Sengvilaipaseuth, O., Chanthongthip, A., Newton, P.N., de Lamballerie, X., 2015. How many patients with anti-JEV IgM in cerebrospinal fluid really have Japanese encephalitis? Lancet Infectious Diseases 15, 1376–1377. Enders, M., Schalasta, G., Baisch, C., et al., 2006. Human parvovirus B19 infection during pregnancy – Value of modern molecular and serological diagnostics. Journal of Clinical Virology 35, 400–406. Hedman, K., Seppälä, I., 1988. Recent rubella virus infection indicated by a low avidity of specific IgG. Journal of Clinical Immunology 8, 214–221. Landry, M.L., 2016. Immunoglobulin M for acute infection: True or false? Clinical and Vaccine Immunology 23, 540–545. Nurmi, V., Hedman, L., Weseslindtner, L., Hedman, K., 2020. Comparison of Approaches for IgG Avidity Calculation and a New Highly Sensitive and Specific Method with Broad Dynamic Range. Viruses, submitted. Parekh, B.S., Ou, C.Y., Fonjungo, P.N., et al., 2018. Diagnosis of human immunodeficiency virus infection. Clinical Microbiology Reviews 32, e00064. Picone, O., Bouthry, E., Bejaoui-Olhmann, Y., et al., 2019. Determination of rubella virus-specific humoral and cell-mediated immunity in pregnant women with negative or equivocal rubella-specific IgG in routine screening. Journal of Clinical Virology 112, 27–33.

A Brief History of the Development of Diagnostic Molecular-Based Assays Hubert GM Niesters, Department of Medical Microbiology and Infection Prevention, Division of Clinical Virology, University Medical Center Groningen, Groningen, The Netherlands r 2021 Elsevier Ltd. All rights reserved.

Isothermal Amplification Companies have developed isothermal amplification technologies to conquer the market without having to deal with the PCR patent rights. These technologies are not suitable for the development of lab-developed assays. Quantitation is possible and the branched-DNA technology (bDNA) (Urdea et al., 1993) (Fig. 1) and the NASBA or Nucleic Acid Sequence Based Amplification (Vandamme et al., 1995) (Fig. 2) have been commercially introduced for the detection of HIV-1; the bDNA has also been introduced for the detection of HBV and HCV. Isothermal amplification technologies have been successful in the early days of molecular-based assays; however, with the rapid development of PCR-based technologies in a number of research laboratories, these isothermal amplification technologies cannot currently compete with the clinical diagnostic market anymore. Only one isothermal amplification has been able to penetrate the diagnostic market and has retained its position successfully. This is the TMA or Transcription-Mediated Amplification (Stevens et al., 2009) technology, currently owned by Hologic. This is a variant of the NASBA technology and can amplify RNA as well as DNA by using an RNA polymerase and a reverse transcriptase. This latter enzyme also exhibits DNA polymerase activity for single-stranded DNA or single-stranded RNA. Finally, the so-called LAMP or loop-mediated isothermal amplification method (Notomi et al., 2015) was developed by Notomi and coworkers. This method can be used for RNA a well as DNA targets and amplifies long loop-like structures containing the target sequences. Although it is very rapid and specific, its use in clinical virology is rather limited.

Non-Isothermal PCR Based Technologies The first paper describing the PCR reaction (Fig. 3) was written by Saiki et al. in 1985 (Saiki et al., 1985). With the introduction of commercially available heat-stable enzymes like the Taq-DNA polymerase and the subsequent recombinant made enzymes in 1989, the technology was made available for the scientific community, as well as the basis for the introduction of commercial assays. The first one made available was for the detection of HIV-1 in 1992. Detection of any virus did become possible, and with the implementation of a new enzyme, the recombinant Tth DNA polymerase, even the reverse transcriptase step needed to transcribe viral RNA into the first DNA step needed for the PCR reaction became possible. The steps needed to implement these technologies into clinical practice were subsequently developed and introduced. The most important steps were to automate isolation of nucleic acids to ensure that larger quantities of patient materials could be processed. The methodology mostly used is based on the so-called Boom technology (Boom et al., 2002) (Fig. 4), which involves extracting nucleic acids using silica particles in a high salt chaotropic solution. Also, automation in PCR cyclers was further developed to ensure the repeated cycling of the PCR reaction. But most importantly, the development of the 50 -nuclease technology in 1991 that opened the road to the introduction of so-called TaqMan probes enabled the detection of the amplification products generated by the PCR method in real-time (Lee et al., 1993; Higuchi et al., 1993).

Fig. 1 Procedure of the bDNA assay (https://www.youtube.com/watch?v=yi-QZvhSCws).

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Fig. 2 Principles of the NASBA method.

Fig. 3 Principle of the PCR method.

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Fig. 4 The Boom method to isolate nucleic acids from clinical samples.

Fig. 5 Relation between Ct value and nucleic acid concentration. The higher the concentration, less cycles are needed to reach a plateau value. So low Ct value with high concentration. High Ct values indicate low concentration and more cycles are needed to reach the plateau value.

This real-time PCR method finally led to the introduction of the quantification of viral RNA or DNA in clinical samples. It opened up the ability to follow the effect of antiviral treatment in patients with, for instance, HIV-1, HBV, or HCV. In principle, every target could be quantified more accurately. To ensure standardization, reference materials were developed, and WHO international standards were slowly introduced. External quality control programs were set up, like from National Institute for Biological Standards and Control (NIBSC) (Shyamala et al., 2004), Quality control for molecular diagnostics (QCMD) (Matheeussen et al., 2020), or Institut für Standardisierung und Dokumentation (INSTAND), to ensure that data generated in different laboratories or by the assays developed by different commercial companies could be compared with one another. Also, with more strict accreditation guidelines in several countries like the ISO 15189:2015, more accurate and standardized data are being produced by clinical laboratories all over the world. The possibility of detecting any viral target in any clinical sample has given us a lot of new options and imposes new challenges. We have the technologies to detect new viruses very quickly, as has been done with SARS-CoV-2. We can also quantify the amount of virus in clinical specimens based on the so-called cycle threshold (Ct) of the PCR (Fig. 5); a low Ct value of 12–20 implies a very high viral load, while a high Ct value, 35 or higher, implies a low viral load. In clinical follow-up based on consecutive samples over time, the first patient samples usually show low Ct values indicating high viral load and the follow-up samples show low and

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Table 1 Overview of technologies used in clinical virology. Some examples of assays are mentioned, as well as the companies who have initially developed the technology. Some of them have been bought by other companies Molecular Based Technologies

Isothermal technologies

Signal Amplification Target Amplification

Used common name

Targets detected

Developed by/Current company (not complete)

Branched DNA

HIV-1, HBV, HCV

Chiron/Bayer

Hybrid Capture NASBA

HPV HIV-1

Digene/Qiagen Organon-Teknika/bioMerieux

TMA

HIV-1, HBV, HCV, Chlamydia trachomatis

GenProbe/Hologic

LAMP Thermo Cycling Conditions

Target Polymerase Chain Amplification Reaction Syndromic Testing Polymerase Chain Reaction

Point-of-Care testing Single target

Polymerase Chain Reaction

Eiken Chemical Any, also lab-developed Respiratory

Mayor diagnostic companies (Roche, Abbott, bioMerieux, Hologic and many more) BioFire/bioMerieux

Gastro-enteric Neurological targets HIV-1, HBV, HCV, M. tuberculosis

GenMark Qiagen Cepheid

subsequently not detectable viral load (high Ct value). The question still waiting to be answered is what the viral load level is that means that an individual is still infectious to others. The question of clinical relevance is important with respect to the new CE-IVD guidelines, which come into effect in 2022. It is clear that for viruses like HIV, HBV or SARS-CoV-2, a patient should not have an infection with these viruses at all Bert, I’m not sure I understand what you are saying here? Every positive test value, therefore, has clinical implications. However, for viruses that are latent in our body, like the herpesviruses CMV or EBV, the viral dynamic of the amount of target DNA is more relevant in relation to disease. But a rule of thumb is that low viral loads are better than higher loads. And there are always exceptions, such as HSV type 1, where detection has implications in specific clinical compartments. For example, a patient with HSV DNA in cerebrospinal fluid or in blood will be more severely ill than an individual with a vesicular eruption around the side of the mouth. There are, however, still open questions that need to be answered. PCR technologies are currently one of the most used amplification technologies and further developments in automation and multiplexing of targets have resulted in systems that can detect around 15–20 targets simultaneously. These so-called syndromic testing solutions are currently used to detect many viral and non-viral targets in respiratory, gastroenteric, or neurological samples simultaneously. Companies have introduced cartridgebased systems to detect multiple targets on a single sample for a short period of time, mostly between 45 and 90 min. Although viral targets are often detected, the clinical interpretation can remain a challenge if more than a single target is detected. A relative quantitation number is not always provided because an internationally accepted standard is not made available yet (Dien Bard and Alby, 2018; Leber et al., 2018; Babady et al., 2018). Topics related to the use of amplification technologies will be presented in several articles of this volume (Table 1).

References Babady, N.E., England, M.R., Jurcic Smith, K.L., et al., 2018. Multicenter evaluation of the ePlex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs. Journal of Clinical Microbiology 56 (2), e01658. doi:10.1128/JCM.01658-17. Boom, R., Sol, C.J., Schuurman, T., et al., 2002. Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented. Journal of Clinical Microbiology 40 (11), 4105–4113. doi:10.1128/jcm.40.11.4105-4113.2002. Dien Bard, J., Alby, K., 2018. Point-counterpoint: Meningitis/encephalitis syndromic testing in the clinical laboratory. Journal of Clinical Microbiology 56 (4), e00018. doi:10.1128/JCM.00018-18. Higuchi, R., Fockler, C., Dollinger, G., Watson, R., 1993. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Biotechnology 11 (9), 1026–1030. doi:10.1038/nbt0993-1026. Leber, A.L., Everhart, K., Daly, J.A., et al., 2018. Multicenter evaluation of biofire filmarray respiratory panel 2 for detection of viruses and bacteria in nasopharyngeal swab samples. Journal of Clinical Microbiology 56 (6), e01945. doi:10.1128/JCM.01945-17. Lee, L.G., Connell, C.R., Bloch, W., 1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Research 21 (16), 3761–3766. doi:10.1093/nar/ 21.16.3761. Matheeussen, V., Corman, V.M., Donoso Mantke, O., et al., 2020. International external quality assessment for SARS-CoV-2 molecular detection and survey on clinical laboratory preparedness during the COVID-19 pandemic, April/May 2020. Eurosurveillance 25 (27), 2001223. doi:10.2807/1560-7917.ES.2020.25.27.2001223.

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Notomi, T., Mori, Y., Tomita, N., Kanda, H., 2015. Loop-mediated isothermal amplification (LAMP): Principle, features, and future prospects. Journal of Microbiology 53 (1), 1–5. doi:10.1007/s12275-015-4656-9. Saiki, R.K., Scharf, S., Faloona, F., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230 (4732), 1350–1354. doi:10.1126/science.2999980. Shyamala, V., Cottrell, J., Arcangel, P., et al., 2004. Detection and quantitation of HBV DNA in the WHO international standard for HIV-1 RNA (NIBSC code: 97/656). Journal of Virological Methods 118 (1), 69–72. doi:10.1016/j.jviromet.2004.01.021. Stevens, W.S., Noble, L., Berrie, L., Sarang, S., Scott, L.E., 2009. Ultra-high-throughput, automated nucleic acid detection of human immunodeficiency virus (HIV) for infant infection diagnosis using the Gen-Probe Aptima HIV-1 screening assay. Journal of Clinical Microbiology 47 (8), 2465–2469. doi:10.1128/JCM.00317-09. Urdea, M.S., Wilber, J.C., Yeghiazarian, T., et al., 1993. Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay. AIDS 7 (Suppl 2), S11–S14. doi:10.1097/00002030-199311002-00004. Vandamme, A.M., Van Dooren, S., Kok, W., et al., 1995. Detection of HIV-1 RNA in plasma and serum samples using the NASBA amplification system compared to RNA-PCR. Journal of Virological Methods 52 (1–2), 121–132. doi:10.1016/0166-0934(94)00151-6.

Sequencing Strategies Sibnarayan Datta, Defence Research Laboratory, Defence Research and Development Organisation (DRDO), Tezpur, Assam, India r 2021 Elsevier Ltd. All rights reserved.

Nomenclature

SARS-CoV

Severe acute respiratory syndrome-related coronavirus SV40 Simian vacuolating virus 40 Zeptoliter 10–21 l

Gigabase (Gb) 1,000,000,000 bases Kilobase (Kb) 1000 bases MCPyV Merkel cell polyomavirus Nanometer 10–9 m

Glossary Base-calling The computational process of translating the raw output (chemiluminescence, fluorescence, change in pH or current flow, etc.) of the sequencing platforms into nucleotides. Contig (Contiguous sequences) are long DNA segments, reconstructed from the assembly of short sequences, based on the degrees of overlap among them. De novo sequencing Denotes sequencing a novel genome for which no reference sequence is available for alignment. Deep sequencing Deep sequencing is a next-generation sequencing approach denoting sequencing a particular genetic region multiple times, often hundreds or thousands of times. This approach allows detection of rare viruses or viral quasispecies that comprise less than1% of the sample. Degenerate primers Degenerate primers are a pool of similar but non-identical oligonucleotide primers, designed from an alignment of closely related sequences that have certain variabilities at the primer binding sites. These primers are useful and amplify similar genetic regions from closely related viral genomes. High-throughput sequencing This term is associated with the next-generation sequencing approach, which allows sequencing of hundreds of millions of DNA molecules in parallel, resulting in large volumes of data output.

Oligonucleotide primer Oligonucleotides are short DNA or RNA molecules (mostly 10–50 bases) which bind to specific regions of target nucleic acids and act as an initiation point for amplification or sequencing reactions. Polymerase chain reaction PCR is one of the most widely used molecular techniques for nucleic acid amplification (generating several million copies of the target DNA segment), which uses a thermostable DNA polymerase to mimic DNA replication, in vitro. Reverse transcription A reaction to generate complementary DNA (cDNA) from RNA using reverse transcriptase (RT) enzymes. Shotgun sequencing This is a rapid technique for sequencing large genomes. This technique involves fragmenting large pieces of nucleic acids into smaller ones, which are then randomly sequenced on NGS platforms. Resulting short sequences are assembled to reconstruct larger contiguous pieces or contigs. Viral quasispecies Evolution of viruses results in populations of closely related mutants that are beneficial to virus persistence in hosts or help the virus by immune escape. Zero-Mode Waveguide (ZMW) It is a photonic nanostructure that allows observation of fluorescent reactions occurring at a single-molecule level.

General Overview During the last few decades, risk of emerging and re-emerging viral diseases has significantly increased globally. Due to diverse human activities – including rapid and unplanned deforestation for agriculture and urbanization purposes, humans, animals, and plants are increasingly being exposed to wildlife, resulting in transmission and outbreaks of variant as well as new viruses. Moreover, improved facilities for global trade and transportation have hastened the transfer of viruses emerging in one part of the globe to other parts. Recent episodes of emergence/re-emergence and spread of Ebola, Nipah, Zika, Hantavirus, SARS (Severe acute respiratory syndrome), virulent forms of Influenza viruses, MERS (Middle east respiratory syndrome coronavirus), and SARS-CoV2 viruses attest to this changing epidemiology and predict future outbreaks with still unknown viruses. Additionally, detection and monitoring of viruses is also challenging due to their wide variability and rapid evolution. Apart from these natural consequences, a potential hazard of use of microbes, especially deadly human/animal viruses or highly damaging agricultural viruses, for acts of bioterrorism/agroterrorism, has also increased in recent years. Irrespective of the source, exact identification of the etiological agent is of prime importance to understand transmission routes and to formulate management strategies including quarantine, development of diagnostic tools, and therapeutic/prophylactic possibilities. Since most of the viruses are uncultivable in vitro, exact identification is primarily based on molecular virology methods such as PCR (Polymerase chain reaction) and/or nucleic acid sequencing. Due to their high evolutionary rates and wide diversity, amplification and sequencing of viral genomes is a big challenge to researchers. Before the availability of molecular biology tools, viral identification was mostly based on infection of vulnerable cell cultures, followed by morphological analyses of cytopathic changes in the cells and visualization of virus particles by

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electron microscopy. These classic methods were labor-intensive, time-consuming, and mostly unsuccessful as most of the viruses are uncultivable. During the last four decades, enormous development in science and technology has revolutionized the field of virology by obviating culture-based methods, significantly reducing the turnaround time, and allowing identification of non-cultivable viruses. Among the molecular tools, discoveries of rapid nucleic acid amplification and sequencing technologies have played a crucial role in understanding viral genome and evolution, resulting in development of diagnostics, therapeutics, and prophylactic agents. Sequencing has now become a “gold standard” technique in virology and with the introduction of next-generation technologies, speed of virus genome sequencing has exponentially increased the number of virus taxa and viral genomes in databases by permitting identification and taxonomic classification of numerous novel viruses. As a result of gradual evolution of sequencing methods and enormous technological refinements, three distinct generations of sequencing technologies are presently available. Each of these technologies has its own features and has distinct applications in virology. This article is intended to introduce the reader to available sequencing technologies and their specific applications in the field of virology so that the readers can decide the sequencing strategy best suited for their research.

First Generation Sequencing Technology Efforts to sequence nucleic acids started just after description of the double-helical model of DNA by Watson and Crick. Interestingly, it was an RNA molecule that was first sequenced. For the first time, nucleotide sequence of one of the smallest biologically active nucleic acid molecules (alanine transfer RNA, tRNAAla) was reported by Robert Holley in 1965, for which he was awarded the Nobel Prize in Physiology or Medicine, in 1968. However, it took quite a long time to come across suitable techniques to routinely sequence larger nucleic acids. During the 1970s, two brilliant methods for sequencing DNA strands were discovered and published almost simultaneously: The “dideoxy chain termination” method developed by Frederick Sanger and colleagues and the “chemical modification” based method developed by Allan Maxam and Walter Gilbert. These two techniques established the firstgeneration sequencing technologies and were revolutionary discoveries in the field of molecular biology, for which Frederick Sanger and Walter Gilbert were honored with the Nobel Prize in Chemistry in 1980. Initially, Maxam–Gilbert’s chemical modification-based sequencing method became instantly popular due to its simplicity in sample preparation, as compared to the Sanger’s dideoxy chain termination method. However, introduction of fluorescent-tagged dideoxy chain terminators made Sanger’s method more suitable for automation. The discovery of PCR further simplified sample preparation for sequencing by Sanger’s technique, significantly shortening turnaround time. Gradual refinement in instrumentation made this technique ready for high-throughput automated sequencing of comparatively large genomes. Due to its accuracy and robustness, Sanger’s sequencers are still the method of choice for validation of sequences obtained from technologically even more advanced next-generation sequencing (NGS) platforms. On the other hand, due to its limited prospect for automation, use of hazardous chemicals, and difficulties with sequencing long strands, use of Maxam–Gilbert’s technique gradually declined. Nevertheless, during the initial stages of development and testing of different sequencing techniques, viruses, primarily bacteriophages – served as a source for high-quality DNA in sufficient quantities, since they were easy to culture in the laboratory and had relatively smaller genome sizes. Bacteriophage phiX174 and MS2 DNA were the first to be sequenced, while the SV40 genome was the first animal virus genome to be sequenced. The Sanger sequencing technique, also known as “chain-termination sequencing” or “dye-terminator sequencing”, is principally based on the incorporation of chain-terminating dideoxynucleotides. In its present format, in a PCR-like reaction, template DNA is copied into single strands of DNA complementary to the target template using a DNA polymerase, a specific oligonucleotide primer, normal deoxynucleotides (dNTPs), and a lower molar concentration of four different dideoxynucleotides (ddNTPs, each being tagged with a particular fluorescent dye). While polymerization of nucleotides occurs by chance, whenever a particular ddNTP is incorporated into the growing chain, further polymerization is inhibited due to the absence of the 30 - hydroxyl group in the ddNTPs. Through repeat cycling of this reaction, n numbers (n being the number of bases in the template DNA) of DNA fragments of variable lengths (each different by a single nucleotide) are generated, having a fluorescently labeled dideoxynucleotide at the 30 end. These tagged amplicons are then electrophoretically separated through a capillary filled with an optimized polymer matrix, capable of resolving single-stranded fragments differing by only one base. As a result, a continuous flow of fragments is generated, with shorter fragments moving faster, while the longer ones move slower. At the capillary terminal, fluorescent tags on the fragments sequentially passing through a laser are excited, while a photoreceptor placed opposite registers the wavelength of the light emitted by the excited tags. Since each of the four ddNTPs are tagged with four distinct fluorescent dyes having precise emission spectra, a chromatogram of fluorescent peaks is generated by the continuous flow of tagged fragments through the laser-detector couple. Concurrently, depending upon the wavelength and intensity of a particular fluorescent tag, corresponding base is identified (technically termed “base-calling”) and this continuous “base-calling” represents the nucleotide sequence in the target sequence. Years of refinement in technologies and chemistries associated with this sequencing technique has resulted in highly reliable Sanger sequencing platforms that can analyze accurate sequence information of up to 900–1000 bases. Due to its unmatched reliability, this technology has dominated for almost four decades and is still considered the “gold standard” for DNA sequencing. Readers are encouraged to visit the website with the link provided at the end of this article. These online resources present sequencing technologies in the form of animated videos for better understanding.

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Owing to the need for pure, homogenous DNA, prior to the discovery of PCR, application of Sanger sequencing in virology was initially limited to cultivable viruses, including bacteriophages and certain other animal viruses for which cell culture was available. This requirement limited its wide application in virus sequencing, directly from human, animal, and plant samples, as viruses present in these are mostly heterogeneous and are present with large quantities of DNA from the host and other contaminating microorganisms. The discovery of PCR in the late 1980s by Kary Mullis (honored with the Nobel Prize in Chemistry in 1993) and coupling of reverse transcription with PCR (RT-PCR) entirely transformed the state of virus sequencing by facilitating rapid and targeted amplification of minute amounts of viral nucleic acids (DNA/RNA) from complex biological samples, hallmarking the era of culture-independent targeted sequencing of viruses.

Second Generation or Parallel Sequencing Technologies While the Sanger technology contributed enormously and significantly towards virus detection and discovery projects, evolution and commercial development of parallel sequencing technologies (aka next-generation sequencing, NGS) since 2000 has entirely changed the application and magnitude of sequencing in almost every field of biological research, including virology. In fact, the necessity to expand the scale of sequencing by several orders of magnitude to accomplish large-scale genome projects, like the human genome project, led to the invention of parallel or second-generation sequencing technologies. The glory of parallel sequencing technologies resides in its ability to simultaneously sequence millions of strands of heterogeneous nucleic acids with high sensitivity. Furthermore, unlike Sanger sequencing, parallel sequencing technology has enabled sequencing of samples in a sequence-independent manner, obviating the need for prior knowledge of the nucleic acid sequences. These secondgeneration sequencing technologies were built on newer sequencing chemistries, several innovations in microfluidics, amplification methodologies, and detection technologies. Since these platforms sequence nucleic acids in a massively parallel fashion, the volume of data generated by these platforms is huge, which prompted rapid advancement in the field of computation and bioinformatics. Unlike the Sanger method where a single large stretch of DNA is sequenced in each run, NGS platforms require larger DNA molecules to be fragmented into smaller sizes, which are then sequenced in parallel, and millions of short “sequence reads” are generated in each run. These short reads are subsequently assembled into larger contigs through complex computer programs. Due to their extremely large dynamic range of detection and sensitivity, presently available bench-top NGS platforms can sequence complete or near-complete genomes of viruses (known, unknown, or even unexpected) in a single run and within a few hours. NGS has the power to resolve sequences of minor target genomes from a highly complex mixture of non-target nucleic acids, where a computational subtraction method is used to remove non-target sequences (such as that of host sequences) while retaining the target virus sequences. Although, in such circumstances, yield and coverage of target viral sequences are often low, as the predominant non-target nucleic acids fiercely compete with the low abundant target viral nucleic acids for resources during sequencing. As an alternative to this approach, sensitivity, specificity, and turnaround time for NGS-based viral genome sequencing applications can be enhanced dramatically by enrichment of virus particles/genomes or depletion of host/non-target nucleic acids through ultracentrifugation/filtration and nuclease treatment-based methods. Nucleic acids can then be extracted from the resulting enriched viral particles, sequenced directly on NGS platforms or can be amplified through non-specific application techniques such as SISPA (Sequence-independent, single-primer amplification), VIDISCA (Virus Discovery based on cDNA Amplification), RCA (Rolling Circle amplification) and others, followed by running on NGS. Unlike Sanger sequencing, NGS does not require target-specific primers or any other sample-specific components, thereby allowing a uniform sequencing methodology that is similar for virtually all sample types. These features make NGS systems an indispensable tool in modern viral outbreak investigation. Once the sequence of a potential causative agent is determined, specific diagnostic assays could then be developed easily to monitor and contain further spread of the agent. Apart from the discovery of new viruses, NGS has numerous applications in virology, which include the study of virus integration sites, evolution, quasispecies, and emergence of antiviral/vaccine-escape mutations. Virus particle enrichment and nucleic acid amplification protocols coupled with NGS technologies have further enabled the efficient metagenomic study of viromes associated with humans, plants, and the environment. Over the years, NGS has become economically affordable, smaller in size (bench-top), while speed, quality, and quantity of data output have dramatically increased. Available benchtop models offer incredible NGS capabilities at very affordable costs. NGS technologies were commercially popularized through the 454 FLX pyrosequencer (Roche) and the Illumina Genome Analyzer (Illumina) and later, by the SOLiD platform (Life Technologies). Despite its huge success, Roche has discontinued marketing of its pyrosequencing based NGS platforms, although many of these instruments are still used in several laboratories. Eventually, rapid research and development has resulted in newer NGS platforms like the Ion Torrent PGM (Life Technologies). Although Roche 454 and Illumina are the most extensively represented technologies in virology related literature, in recent years, SOLiD (Sequencing by Oligonucleotide Ligation and Detection) has also gradually emerged as a robust and reliable platform. Even though each of these platforms exploit different sequencing chemistries and detection technologies, basic procedure is similar in principle and based on the “shotgun” sequencing technique. Initially, nucleic acid samples are fragmented by mechanical (ultrasonic based) or enzymatic (transposon-based) methods to produce nearly uniform length fragments between 100 and 1000 bases, depending on the sequencing platform being used. Mechanical fragmentation is preferred for clinical and environmental samples since it can withstand possible impurities that may inhibit enzymatic fragmentation methods. Enzymatic

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methods are suitable for relatively pure samples since fragmentation and adapter ligation can be done in a single reaction, thereby reducing time and handling steps for library preparation. Subsequently, a fragment library is prepared by ligation of adapters (for attachment to the sequencing matrix) and sample-specific barcodes (to identify the sample source after sequencing) to each of the fragments, attachment of each fragment on the sequencing matrix, amplification of the attached fragments and, finally, sequencing takes place. Different NGS platforms use their proprietary adapters and barcodes to fragment libraries for amplification (e.g., emulsion PCR in 454 & SOLiD platforms, and bridge amplification in Illumina platforms). Using distinct barcodes for each sample, samples from different sources can be prepared and sequenced collectively, making the maximum use of high throughput NGS sequencers, thereby cutting-down the per-sample sequencing costs. Different NGS platforms use distinct sequencing chemistries and detection technologies. The 454 platforms use sequencing-bysynthesis (SBS) chemistry where chemiluminescence, generated during incorporation of a nucleotide, is detected. Illumina platforms also use the SBS technology with reversible fluorescent dye-terminator chemistry where fluorescence signals generated during incorporation of nucleotides are detected. Similarly, Ion-Torrent also uses SBS technology with a novel semiconductorbased detection technology, which registers the drop in pH that occurs during incorporation of each specific nucleotide. Simply based on detection of voltage change, semiconductor-based signal capture technology is much faster, low-cost, and smaller than fluorescence or chemiluminescence detection-based technologies that require complex and sophisticated optics for fluorescence/ chemiluminescence image capture and advanced computational tools for image processing and data analyses. On the other hand, the SOLiD platform uses a rather complex sequencing-by-ligation (SBL) technology that uses a mixture of fluorescent-tagged oligonucleotide probes of various lengths and the mismatch sensitivity of DNA ligase to sequence the target DNA. Irrespective of the sequencing chemistry (SBS or SBL) used in these platforms, chemiluminescence/fluorescence image or pH changes occurring on the sequencing chip is collected periodically, integrated, and quality-assessed through computer-intensive complex image/ voltage processing algorithms to infer actual nucleotide sequence data from each fragment, which typically results in several gigabases (Gb) of sequence data. Despite being extensively used, different NGS platforms have certain limitations including base calling bias, reproducibility, errors associated with certain sequence motifs, or homopolymers containing regions. Therefore, it is recommended to get samples analyzed on two different NGS platforms simultaneously to reduce errors, albeit depending on the availability of funds. Nevertheless, with consistently improving chemistries, hardware, and software, many of the issues have either been resolved or will be resolved in newer platforms. For applications in virology, especially virus discovery and metagenomics, NGS platforms that can yield longer sequence reads are preferred since long reads are more suitable for de novo assembly of unknown genomes. On the contrary, for analysis of virus evolution and quasispecies, platforms that generate shorter but high-quality reads with increased depth should be preferred. For virus sequence detection, at certain levels of the target template, Illumina has been shown to have analytical sensitivity comparable to that of optimized qPCR but, at low target levels, reads generated by the Illumina platform are insufficient for de novo assembly of the genomes. Despite massive refinement in computational procedures for analysis of huge volumes of NGS data, de novo assembly and identification of short viral reads is still challenging, particularly for new viruses or highly variable viruses for which reference complete genome sequences are not available in the relevant databases. Therefore, development of a standardized universal bioinformatics pipeline for analysis of NGS data is required before these technologies can be adopted for routine viral diagnostics.

Application of NGS in Virology NGS has been extensively used by researchers for several interesting applications in virology. One of them is vector enabled metagenomics (VEM), where NGS analysis of nucleic acids of insect vector specimens collected in the field could be done to monitor viruses present in a given environment. Using this method, new plant and zoonotic viruses have been identified in different insects, such as whiteflies and mosquitoes, providing a powerful tool for vector-borne virus discovery and surveillance programs. Similarly, using NGS, clinically important novel viruses were detected in gastrointestinal tissue samples obtained from bats that are believed to be reservoirs for several emerging zoonotic viruses (Paramyxoviruses, SARS-CoV, Lyssaviruses, Filoviruses). The first complete genome sequence of the SARS-CoV2, etiological agent of the ongoing COVID-19 global pandemic was also determined by performing direct NGS of bronchoalveolar lavage fluid RNA extract from a patient suffering from severe pneumonia. Apart from directly detecting viruses in samples, an alternate strategy of virus discovery through NGS based sequencing of total small RNAs (sRNAs) from invertebrates is gaining importance. RNA silencing-based antiviral defense mechanisms in eukaryotes respond to viral infection by processing the invading viral RNA genomes into sRNAs of distinct lengths (B21–24 nucleotides). NGS-based deep sequencing of these sRNAs rich in processed viral genome, followed by assembly, could reconstruct the sequence of the original viral genome. This strategy has been successfully demonstrated in describing known and unknown viruses from diverse plants and animals, showing the universality of the mechanism. However, this strategy is challenging in cases of multiple infections with novel viruses, where small sequence reads from different viruses may interfere with the de novo assembly of each other, resulting in incorrect identification. Very recently, studies have successfully reconstructed complete or nearly complete ancient viral genomes, such as the hepatitis B virus, from archeological human genome NGS datasets through computational methods of screening for specific viruses. This has opened a new field for analyzing ancient viral sequences and the study of viral evolution over long periods of time. A similar approach of transcriptome subtraction has been used in the discovery of new viruses such as MCPyV associated with Merkel cell carcinoma. Additionally, deep sequencing of amplicons generated by primers that target highly conserved genetic regions within a virus family has led to the discovery of several new polyomavirus and papillomavirus genotypes. Deep sequencing NGS technology

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has also been used to determine retrovirus integration sites in natural infections, as well as those resulting from retroviral vectorbased gene therapy experiments. Choosing an NGS technology among the platforms available today is very important for specific applications, especially in virology. Sequencing viruses where a reference sequence is not available involves post-sequencing de novo (reference-independent) assembly of sequence reads. For such projects, datasets containing relatively long reads are preferable. On the other hand, when a reference genome is available, short-read technologies could be used to obtain high coverage of the genomes. Earlier, due to relatively long reads, pyrosequencing technology was the preferred NGS technology for metagenomic applications in virology. Following the discontinuation of pyrosequencing platforms in 2016, the consistent improvement in the read length of other NGS platforms has now enabled these alternative NGS technologies for wider applications in virology.

Third Generation Sequencing Technologies Subsequent to the second generation of sequencers, another class of parallel sequencers have arrived, the third-generation sequencers that are capable of sequencing extremely long stretches of DNA, generating sequence reads in kilobases. These technologies are, therefore, popularly termed “long-read sequencing” parallel technologies to distinguish them from “short-read sequencing” second-generation technologies. In comparison to the second-generation technologies that break long strands of nucleic acids into smaller fragments before amplification and sequencing, third-generation technologies neither need fragmentation nor amplification (thereby reducing time, cost, and amplification bias) and directly read long stretches of nucleic acids at the single-molecule level, providing nucleotide sequence in real-time. Although wide applications of the third-generation technologies are largely restricted due to their relatively high running costs and limited throughput capabilities. Since these technologies are still under active development, these limitations are expected to be addressed in the near future. Two of the third-generation sequencing technologies are commercially available, namely, the single-molecule real-time (SMRT) technology (Pacific Biosciences) and the Nanopore sequencing technology (Oxford Nanopore Technologies). In SMRT technology, sequencing takes place inside nano-photonic structures having zero-mode waveguide (ZMW) properties, which enables illumination of extremely small observation volumes (as small as zeptoliter), small enough to detect optical reactions occurring at the single DNA molecule level. Each SMRT chip contains many ZMWs and within each of these, one single-stranded DNA template with an active DNA polymerase is immobilized at the bottom of the well. Every time the polymerase incorporates a nucleotide into the growing complementary DNA strand, the dye linked to the incorporated nucleotide is cleaved off, causing it to illuminate the bottom of the wells and decay exponentially, which is detected by the optical sensors at the bottom of each ZMW. Optical signals from each of the ZMWs are detected continuously, resulting in real-time, parallelized single-molecule DNA sequencing. With presently available SMRT technology, extremely long reads ranging from 4190 kb with 499.999% consensus accuracy can be achieved. On the other hand, nanopore sequencing uses biological nanopores (nanometer diameter) formed by transmembrane proteins (porins), such as the heptameric transmembrane a-hemolysin (a-HL) from Staphylococcus aureus. Like SMRT, this technology also does not need amplification or any other modification to the nucleic acids. Using the principles of electrophoresis, nucleic acid strands are transported through the nanopores under a steady flow of ions. As the density of current flowing across the nanopore surfaces depends on the dimensions of the pores, there is a change in current flow across the nanopore depending on the dimension of the nucleotides occupying the pore. This modulation in current flow is detected by highly sensitive sensors and compared with previously optimized data to interpret the nucleotide sequence from current flow change data, in real-time. Presently, nanopore sequencers as small as USB drives (weighing only B100 g) are commercially available, which offer sufficiently long-read sequence data amounting up to 30 Gb of DNA sequences or 12 million reads of RNA. Being simple, requiring no DNA pre-treatment or amplification, operable at a wide range of conditions, this technology has allowed researchers to sequence samples collected at locations in the field. This technology has been successfully used in a number of field applications such as detection and monitoring of viral pathogens, such as during Ebola outbreaks, genotyping, antibiotic resistance, food safety to humans, and plant genome sequencing.

Sample Preparation Strategies for Sequencing Viruses Most of the time, virus samples are present with abundant non-target nucleic acids. Therefore, successful sequencing of virus nucleic acids needs specific strategies for sample preparation to achieve desired results.

Nucleic Acid Sequence Dependent Strategies for Known Viruses Sequencing of viruses, whose partial or complete genome is known, can be easily done by PCR/RT-PCR mediated amplification of the genome using specific primers, followed by sequencing on a Sanger platform. Owing to its high sensitivity, specificity, and capability to amplify non-cultivable viral nucleic acids from a mix of nucleic acids, introduction of PCR and RT-PCR based techniques have almost replaced classical methods, enabling rapid sequencing of viruses. However, unlike the highly conserved prokaryotic rRNA or the eukaryotic ITS genetic regions, there are no ubiquitously conserved genetic regions across all the virus families to design universal viral amplification PCR primers that can broadly amplify all virus families for sequencing-based identification. Therefore, for diagnostic purposes, primers can be designed targeting highly conserved regions in a particular virus

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family/genus for broad identification of variant or novel viruses belonging to the same family/genus, while type-specific primers can be designed by including type-specific variation at the 30 end. Amplicons generated can then be directly sequenced on a Sanger platform for confirmation. This strategy can be used for sequencing pure virus preparations (culture supernatant/ultracentrifuge purified samples) as well as field samples (from infected plants, clinics, or environment) where the target virus type is present in a mix of abundant contaminating nucleic acids. Generally, primers are designed to amplify 600–800 bp fragments (which is the limit of Sanger sequencing per run), having an overlap of at least 100–150 bp (for post-sequencing assembly of the fragments) with the neighboring fragment. Alternatively, virus genomes can be PCR-amplified to generate large amplicons (1–2 Kb) that can then be sequenced using nested overlapping primers covering 500–600 bases. Due to high genetic variability, it is often difficult to design virus family-specific PCR primers or even genus-specific PCR primers, especially for RNA viruses. So, for sequencing a particular virus, primers can be designed based on the type specimen sequence directly. However, for sequencing related viruses (different strains/types belonging to the same group), PCR primers must be designed to target highly conserved regions. For this, if available, complete genome sequences can be obtained from the database (e.g., GenBank RefSeq database) for the target virus and its related viruses, align them using a standard nucleic acid alignment algorithm, and manually inspect the alignment for highly conserved regions that flank a variable region within PCR amplifiable length. The conserved regions allow the design of broadly reacting primers, while the intervening variable regions allow the identification of the specific virus after sequencing. Designing broadly acting virus family/genus consensus primers is technically challenging and may require the introduction of a certain degree of degeneracy in the primers. However, degeneracy should be kept at a minimum, since increasing degeneracy compromises the specificity and sensitivity of the amplification. Since the Sanger method sequences a single strand of DNA at a time, it is essential to ensure that amplicons processed for direct sequencing are homogenous (i.e., no sequence variation among the amplicons). If related viruses are present, variants and quasispecies are expected in samples, which is very usual in field samples; cloning based library preparation of the PCR/RT-PCR amplicons should be carried out before Sanger sequencing. This can be done by simply using commercially available T-A/U-A based cloning vectors for amplicons generated by conventional Taq DNA polymerases. Alternatively, cloning can be carried out through engineering specific restriction endonuclease (RE) digestion sites at the 50 ends of the amplifying primers while designing, subsequently PCR/RT-PCR generated amplicons that can be digested by the respective RE, ligated into a suitable cloning vector and clonal DNA amplified, and isolated through routine laboratory methods. This ensures that an individual amplicon is present in each clone. Finally, random sequencing of DNA from individual clones is done to resolve the variants and quasispecies. In general, the more clones sequenced, the better the chances are for detecting minor populations of variant viruses in the sample.

Nucleic Acid Sequence Independent Strategies for Sequencing In contrast, sequence-dependent amplification strategies are not applicable for viruses where absolutely no sequence information is available in the relevant databases. Such unknown viruses can also be sequenced on the Sanger platform by adopting sequence-independent metagenomic sequencing approaches. This does not require any prior knowledge of sequences present in a given sample. The first step involves physical enrichment of virus particles from the sample and depletion of host genetic material or nontarget genetic material through nuclease digestion. Enrichment of virus particles starts with homogenization of the samples and suitable dilution for removal of particulate matter (coarse particles, bacteria, host cells) through centrifugation. Following centrifugation, samples can then be ultra-centrifuged through a suitable medium such as a cesium chloride density gradient to obtain pure virus particles. However, due to the requirement of expensive instrumentation, chemicals, and technical knowledge for ultracentrifugation dependent methods, alternative methods based on simple membrane ultra-filtration – through an appropriate pore size (0.45 m and 0.22 m), or PEG (Polyethylene glycol)-based virus particle precipitation are frequently used. Filtered or PEG precipitated intact virus particles are then treated with nucleases to remove non-viral or host-associated nucleic acids. Subsequently, nuclease resistant, capsid-protected viral nucleic acids are extracted and purified for downstream enzymatic reactions for cDNA preparation, conversion of single-stranded DNA/cDNA into double-stranded DNA, amplification of the viral genetic material using sequence-independent priming methods such as SISPA, virus discovery based on cDNA-AFLP (VIDISCA), or RCA to produce sufficient amounts of genetic material for sequence analyses. The Phi29 DNA polymerase-based RCA does not need prior virus particle separation, since this enzyme is capable of selectively amplifying circular DNA templates in a complex mix or non-target genetic material, thus very useful for enrichment of a number of human, animal, and plant viruses that have circular DNA genomes. These amplified viral genetic materials can then either be directly sequenced on NGS platforms or cloned to generate vector libraries for Sanger sequencing using vector-specific primers. These pre-sequencing enrichment methods reduce huge amounts of background non-target sequence data, thereby increasing the sensitivity of the sequencing methods and sequence coverage of the target nucleic acids. Nevertheless, owing to its parallel sequencing capabilities, application of target enrichment methods followed by NGS in viral metagenomics can immensely improve the chances of discovery of highly divergent or novel viruses compared to PCR-Sanger sequencing techniques. Although alleged to be associated with PCR bias, these methods have been extensively used for the discovery of a number of important viruses, especially in clinical, veterinary, and plant samples. Being based on non-specific amplification methods, extreme care should be exercised to prevent cross-contamination from the laboratory environment while performing sequence-independent amplification experiments.

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Virus Databases Successful completion of any sequencing project leads to the beginning of another involving analyses. Traditionally, after inspection and manual editing of the sequence data from a Sanger sequence, data are submitted to BLAST (Basic local alignment search tool) for a homology search (based on similarity) to determine if the sequence is the same as expected or is a potentially new sequence (no significant match in the database). The latter may warrant further analysis. On the other hand, analyses of large volumes of NGS data need specialized hardware and software solutions for storage and analyses. There are also several data analysis servers that can do the basic analyses as required. Primarily, short-read NGS data requires assembly of the short sequence stretches to create contigs (larger fragments, assembled on the similarity of the overlapping regions in short reads), which are then subjected to homology searches. Nevertheless, irrespective of the source of sequence data, its organization, analysis, and identification require searching for homology in the reference sequence data archived in the international databases. Since sequence databases play an important role in primer designing and sequence analysis, a certain amount of knowledge about databases is essential before embarking on a sequencing project. Instantly after the discovery of the first-generation sequencer, the volume of sequence data started to increase in molecular biology laboratories and there was a high requirement for data storage and retrieval management systems. Initially, nucleic acid sequence databases were largely localized to laboratories. In 1979, the Los Alamos Sequence Database was created at the Los Alamos National Laboratory, New Mexico, USA, which was later renamed as GenBank in 1982 and made available to the public. Simultaneously, in 1980, the EMBL Nucleotide Sequence Data Library (now EMBL-Bank, part of European Nucleotide Archive, ENA) was created as a centralized nucleotide repository at EMBL (European Molecular Biology Laboratory), Heidelberg, Germany. On the other hand, another database, the DNA Data Bank of Japan (DDBJ), was created in 1986 at the National Institute of Genetics at Mishima, Japan. Later, with the establishment of the International Nucleotide Sequence Database Collaboration (INSDC), these three primary international sequence databases, GenBank, EMBL-Bank, and the DDBJ – were linked to share data submissions and retrieval tools and to provide unrestricted access to publicly available data to everyone. By the end of 1981, the LANL database contained 263 sequences, including 50 sequences from eukaryotic viruses and 12 bacteriophage sequences, which, after the wide availability of automated Sanger sequencers, increased by doubling every 18 months. Following the commercialization of NGS platforms and the commencement of large-scale genome and metagenome projects, the volume of sequence data generation increased as never before, which required specialized virus databases for storage and retrieval. The Viral Genomes Resource hosted by the NCBI is one specialized virus reference sequence database, where annotated viral genome sequences cataloged at species level (RefSeq and genome neighbors) are available, which is very helpful in viral sequence identification and taxonomy. Similarly, the ViralZone database, hosted by the Swiss Institute of Bioinformatics, is another useful reference resource for viral sequence analyses. These primary and reference sequence databases have enabled rapid archival, assembly, annotation, taxonomy, discovery, and study of viral evolution, helping enormously at times of crisis. It is estimated that since 2000, there was almost a nine-fold increase in viral sequence data submitted to the primary databases, as well as for novel viruses, as seen by the proportionate increase in the RefSeq database. Considering the clinical significance of certain viruses, seven different modules have been created for influenza virus, Dengue virus, West Nile virus, Ebolavirus, MERS coronavirus, Rotavirus A, and Zika virus at the NCBI Viral Genomes Resource. These specialized modules have additionally improved analyses of new sequences related to these viruses. It is worth mentioning that the first virus genome of the SARS-CoV2 was shared on January 10th, 2020 via the GISAID (Global initiative on sharing all influenza data) platform, which is an international database for sharing information from all influenza viruses. With the COVID-19 pandemic and exponential increase in submission of virus sequences, a separate EpiCovTM database was created to accommodate these sequences. This database is freely accessible and features a number of analysis options including highly customised sequence search, sequence alignment phylogenetic analysis and clade identification, coordinates for diagnostic primers/probes, 3D structures of various virus proteins and drug targets etc., which has helped the scientists enormously in designing improved diagnostic approaches for tracing and tracking the virus, studying evolution and dynamics and also in identifying potential management strategies. Nevertheless, since January 10th, 2020 a total of 2,39600 SARS-CoV2 complete genome sequences have been submitted by December 4th 2020, nearly all of which were obtained by NGS. This gives an excellent idea of how modern sequencing technologies coupled with highly advanced bioinformatics and a curated database can help in management of a pandemic. Most of the time, homology-based search approaches (nucleotide BLAST) are useful in determining the identity of the query sequence by searching highly similar sequences deposited in the non-redundant (nr) database (megablast). However, sequences, especially arising from metagenomic studies, have very little or no similarity to sequences belonging to known viruses. In such cases, the search can be performed by low stringency searches – discontiguous megablast (searches more dissimilar sequences) and BLASTn (searches somewhat similar sequences). If the searches still do not return any similar sequences, a blastx search (searches protein databases using a translated nucleotide sequence query) can be done to identify sequences based on protein conserved domains that the query-sequence might encode. Apart from these, a proportion of sequences from metagenomic datasets may not be classified through any of the above-mentioned search strategies. These unclassified sequences are supposed to originate from truly unknown viruses or other “biological dark matter”, and detection of such coding sequences with no homologs, also termed as “ORFans”, may help in the discovery of novel viral species. The unknown protein sequences encoded in these sequences can be used for isolation and purification of the viruses from original samples, followed by sequencing of their complete genomes. During the last four decades, the field of molecular biology has grown vastly due to a number of discoveries and huge technological leaps. The discovery of nucleic acid sequencing and amplification technologies (primarily PCR) and the subsequent

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innovations in the sequencing chemistries and incorporation of various signal detection technologies has now enabled us to completely sequence complex genomes from higher organisms within days. For smaller genomes, especially from most of the pathogenic viruses, entire genome of an unknown virus can be sequenced and analyzed within a few hours. This can keep us a step ahead during viral outbreaks, where early detection, identification, and monitoring of the causative agent makes a great difference. Nowadays, third-generation sequencers that can be easily taken to the field for on-site investigations are available. Nevertheless, in addition to its increasing applications in virus discovery and viral genome sequencing, currently available parallel sequencing technologies are being widely used in monitoring of antiviral drug resistance, viral evolution, and quasispecies diversity. However, the application of sequencing for viruses has certain major challenges, which include low abundance of the target virus nucleic acids in a complex mix of non-target nucleic acids. Therefore, additional steps for enrichment of viral sequences are often needed. At present, three different generations of sequencing technologies are available; the basic technology, features, and specific applications of which have been discussed. Although, as with any other technology, these sequencing technologies have certain advantages as well as certain limitations. Therefore, the choice of sequencing platform is an important prerequisite for the success of a virus-related project and must be done carefully considering the specific objectives of the project.

Further Reading Barrientos-Somarribas, M., Messina, D.N., Pou, C., et al., 2018. Discovering viral genomes in human metagenomic data by predicting unknown protein families. Scientific Reports 8, 28. Barzon, L., Lavezzo, E., Militello, V., Toppo, S., Palù, G., 2011. Applications of next-generation sequencing technologies to diagnostic virology. International Journal of Molecular Sciences 12, 7861–7884. Chiu, C.Y., 2013. Viral pathogen discovery. Current Opinion in Microbiology 16, 468–478. Datta, S., Budhauliya, R., Das, B., et al., 2015. Next-generation sequencing in clinical virology: Discovery of new viruses. World Journal of Virology 4, 265–276. Delwart, E.L., 2007. Viral metagenomics. Reveiws in Medical Virology 17, 115–131. Deurenberg, R.H., Bathoorn, E., Chlebowicz, M.A., et al., 2017. Application of next generation sequencing in clinical microbiology and infection prevention. Journal of Biotechnology 243, 16–24. Edwards, R., Rohwer, F., 2005. Viral metagenomics. Nature Review in Microbiology 3, 504–510. Hall, R.J., Wang, J., Todd, A.K., et al., 2014. Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery. Journal of Virological Methods 195, 194–204. Hatcher, E.L., Zhdanov, S.A., Bao, Y., et al., 2017. Virus variation resource – Improved response to emergent viral outbreaks. Nucleic Acids Research 45 (D1), D482–D490. Levy, S.E., Boone, B.E., 2019. Next-generation sequencing strategies. Cold Spring Harbor Perspectives in Medicine 9. Nooij, S., Schmitz, D., Vennema, H., Kroneman, A., Koopmans, M.P.G., 2018. Overview of virus metagenomic classification methods and their biological applications. Frontiers in Microbiology 9, 749. Reuter, J.A., Spacek, D., Snyder, M.P., 2015. High-throughput sequencing technologies. Molecular Cell 58, 586–597. Shendure, J.A., Porreca, G.J., Church, G.M., et al., 2011. Current Protocols in Molecular Biology: Overview of DNA Sequencing Strategies. Hoboken: Wiley Press. Zheng, T., Li, J., Ni, Y., et al., 2019. Mining, analyzing, and integrating viral signals from metagenomic data. Microbiome 7, 42.

Relevant Websites https://nanoporetech.com/how-it-works How it works. Oxford Nanopore Technologies. https://www.thermofisher.com/blog/behindthebench/ How Does Sanger Sequencing Work?. https://www.youtube.com/channel/UCvJHVo5xGSKejBbBj0A5AyQ NCBI YouTube Channel. https://www.youtube.com/playlist?list=PLTt9kKfqE_0Gem8hIcJEn7YcesuuKdt_n Next Generation Sequencing (NGS) - YouTube. https://sapac.illumina.com/science/technology/next-generation-sequencing/beginners.html Next-Generation Sequencing Basics. https://www.youtube.com/watch?v=WdTX1yykLks Pyrosequencing. The Basic Principle and Steps ... - YouTube. https://www.pacb.com/videos/video-introduction-to-smrt-sequencing/ Sequencing 101: Video Introduction to PacBio Sequencing and the Sequel II System. https://www.youtube.com/watch?v=nlvyF8bFDwM SOLiD DNA Sequencing - YouTube. https://viralzone.expasy.org/ ViralZone root. https://www.ncbi.nlm.nih.gov/genome/viruses/ Viral Genomes - NCBI - NIH.

Validating Real-Time Polymerase Chain Reaction (PCR) Assays Melvyn Smith, Viapath Analytics, Specialist Virology Centre, King’s College NHS Foundation Trust, London, United Kingdom r 2021 Published by Elsevier Ltd.

IVD

Abbreviations CLIA

United States clinical laboratory improvement amendments. This is a regulatory body or program to ensure quality laboratory testing. Ct Crossing threshold, the cycle number at which the PCR enters the exponential phase. FDA United States food and drug administration. The FDA is a Federal agency responsible for protecting the public health through the control and supervision of medical devices, the FDA are one of the agencies responsible for developing and implementing CLIA.

In-vitro diagnostic products. These are the reagents or instruments used to diagnose a disease, in this case the PCR assay. LDT Laboratory developed test. LOD Limit of detection, the lowest amount of analyte detected in an assay. rt PCR Real-time polymerase chain reaction. RT-PCR Reverse transcription PCR. ΔRn The maximum difference in fluorescence between the start and end of the PCR.

Introduction For many years, the development of assays took place in the laboratories where the test was required, the so-called in-house or LDT. Although more and more commercially-developed tests have become available, novel assays continue to be developed in academic hospital laboratories. These laboratories are more able to respond quickly to new and re-emerging infections and crucially have the samples necessary to develop the test. This was the case with the new human coronavirus causing clusters of pneumonia epidemiologically linked to a seafood market in Wuhan, China in December 2019. Researchers from Shanghai, Wuhan, Beijing and Sydney sequenced a sample from a patient who had worked at the market; the sequence was deposited in the publicly available GenBank database on 10th January. Further sequencing showed the virus shared 85% identity with a SARS-like coronavirus found in bats. The first LDT was published on 23rd January 2020, followed a month later by the first of the commercial tests. Although often more expensive than LDTs, commercial kits enable the rapid introduction of new tests, with the further advantage of being CE marked or FDA approved, providing some form of reassurance to the laboratory. However, CE marking is only a declaration of compliance with European legislative requirements; it does not necessarily guarantee the rigorous validation of the assay, or give any details of how and when a test should be used. Furthermore, commercial assays, by definition, have to be commercially attractive. Therefore, specialist, small-scale tests for rarely occurring infectious pathogens will not be cost-effective. Thus, there will continue to be a need for laboratory-developed assays.

The Need for Validation In-House Assays Over the years many laboratories have established methodologies for validating their assays. However, the literature continues to show a lack of detail in some critical areas, e.g., optimization of extraction techniques, methods used in primer and probe design, no evidence of amplicon sequencing to confirm specificity, imprecise estimates of sensitivity and specificity and assays that do not include internal or extraction controls. Such lack of detailed experimental information makes assessing the clinical utility of the assay difficult. Some of these problems lie with the scientific literatures’ approach to publishing, where space limitations restrict the amount of detail permitted in a paper. These difficulties have led to a number of papers calling for an improvement in the standards of reporting diagnostic assays, including the STARD initiative (standards for reporting of diagnostic accuracy) and the MIQE guidelines (the minimum information for publication of quantitative real-time PCR experiments; Bustin et al., 2009). In addition to the fundamental requirements of good scientific practice, there are a number of regulatory bodies that require assays to be validated to certain standards, such as the FDA and CLIA requirements in the USA and the IVD Regulations (EU) 2017/7462017 in Europe. There is also an obligation on health institutions to be accredited according to the ISO 15189 standard. There has been an ongoing debate in Europe regarding CE marking of LDTs, similarly in the USA the FDA announced its intention to shift from a policy of enforcement discretion to exercising regulatory oversight at a future date on LDTs. However, the imposition of rigorous controls on the development of assays must not stifle innovation and the ability of front-line laboratories to respond quickly to new and emerging threats. Under these circumstances it makes sense for laboratories developing and publishing assays to ensure

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Validating Real-Time Polymerase Chain Reaction (PCR) Assays

Fig. 1 Assay validation: Key concepts.

that they produce rigorously documented experimental proof to support any potential CE-marking/IVDD requirements that may be necessary in the future.

Commercial Assays Although commercial assays are typically CE marked or FDA approved this does not necessarily mean the assay has been validated for all applications. Furthermore, although the assay may perform acceptably in the commercial developer’s laboratory, a number of factors can affect the assay’s performance elsewhere, e.g., staff competences and workflow systems, where not all laboratories may have separate working areas recommended to reduce contamination. There may also be differences in equipment maintenance schedules, including anything from freezers and pipettes to thermal cyclers, which can fundamentally affect the assay. Therefore, it is necessary for laboratories to verify the stated criteria. Although there are no formal requirements in the UK for laboratories to evaluate newly-introduced commercial assays, in the USA, FDA-approved, FDA-modified (i.e., tests modified from the manufacturer’s

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instructions but with performance characteristics determined by the user laboratory, consistent with CLIA requirements) and LDTs require verification before introducing the test into the clinical laboratory. CLIA stipulates that prior to implementation of an FDA-cleared test laboratories must verify the manufacturer’s performance specifications. Where a FDA-modified test or LDT is to be introduced, CLIA stipulates that, additionally, analytical sensitivity and specificity must be established. In an attempt to clarify the situation, it is the intention in this article to provide a set of basic working guidelines for the validation process. These guidelines can be applied throughout the continuous process of maintaining the validated status of an assay. The key concepts in the process of validating an assay are illustrated in Fig. 1.

The Validation Process: Consultation Stage The process begins with the development of a validation plan and involves decisions based on the clinical need for the assay, e.g., epidemiological studies, infection control or screening. As discussed previously, there are differences between the levels of validation laboratories perform when introducing commercial or LDTs. As a minimum, the laboratory introducing a commercial test must establish that the manufacturer’s performance claims can be reproduced. Once the performance characteristics of an assay have been met, whether commercial or LDT, the validation exercise must continue on a daily basis (see Fig. 1). This involves continually monitoring the levels of internal and external positive controls to ensure the validation status of the assay is maintained.

Preliminary Considerations The initial step is to define the purpose of the assay; all the subsequent steps in the validation process are guided by this decision. The following three variables that can affect an assay’s performance must be considered at this stage: (1) The sample-type and the host/pathogen interactions that determine whether a qualitative or quantitative assay is required. (2) The assay system: the biological, technical and operator-related factors that affect the assay’s ability to detect the target in the specific sample-type. All assays are considered multiplex, since they must include a co-amplified extraction control. (3) The result: will the result accurately predict the status of an individual or population in regard to the analyte detected? The sample-type (e.g., tissue, whole blood, CSF) may contain inhibitors that affect the activity of the polymerase in the PCR. Therefore, the extraction process needs to be evaluated and necessary alternatives considered. Quantification assays are useful in the management of patients, where pathogen load can be monitored during therapy. Some viral pathogens, such as EBV and CMV can establish a primary low-level latent infection and subsequently become reactivated, switching to a productive lytic infection. Is more than one pathogen to be identified in a multiplex assay? Another significant factor is the availability of sufficient numbers of well-characterized positive control samples to enable the validation. In order to maintain objectivity, the method chosen to resolve discrepant results must be established as part of the validation plan before testing begins. A quality assurance plan must be established for the assay. Consideration must also be given to the availability of external QA reagents. This can be a problem with new assays targeting rare pathogens, where QA panels are unlikely to be available; the laboratory may need to consider working with the providers to produce suitable reagents. Once the requirement for a test has been established, the next decision is whether to use a commercial assay or develop a LDT. In the USA the CLIA specify that laboratories using FDA-cleared assays must verify that the manufacturer’s performance specifications for accuracy, precision, reportable range and reference intervals can be replicated. For modified commercial assays the following should also be tested: (1) Analytical Sensitivity (LOD). (2) Analytical Specificity to include inhibitory substances. (3) Any other performance characteristics required for test performance. When assessing a commercial assay, the extraction process also needs to be verified. In most cases the manufacturer’s protocol includes details on the extraction process to use. However, the assay may need to be validated with a number of different extraction methods, depending on the type of equipment available.

The Validation plan Verification: the process of establishing whether the individual components of an assay meet the analytical performance requirements established at the start of the development process. Validation: the process of ensuring that the completed assay conforms to the users’ needs, requirements, and/or specifications under defined operating conditions. There are numerous aspects of an assay that need to be continuously monitored throughout its use in the laboratory. Micro-organisms, particularly viruses, often mutate. This means that the efficiency of the PCR needs to be monitored for potential false-negative results, as this may be the first sign that the primers and/or probe need to be updated and revalidated. Manufacturers continually develop new

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Table 1 Theoretical number of samples from subjects of known infection status required for establishing diagnostic specificity and sensitivity estimates using likely estimated specificity/sensitivity value and desired error margin and confidence Estimated specificity/sensitivity value

90% 92% 94% 95% 96% 97% 98% 99%

2% error in estimated specificity/sensitivity

5% error in estimated specificity/sensitivity

Confidence

Confidence

90%

95%

99%

90%

95%

99%

610 466 382 372 260 197 133 67

864 707 542 456 369 279 188 95

1493 1221 935 788 637 483 325 164

98 75 61 60 42 32 21 11

138 113 87 73 59 45 30 15

239 195 150 126 102 77 52 26

buffers, enzymes and extraction kits, these must be assessed and if adopted, re-verified, and the assay revalidated. New types of probe chemistry are constantly being introduced and these, too, need testing to maintain or improve the assay’s performance. Although each new assay may present its own particular challenges, the basic criteria for validation, such as specificity, sensitivity and reproducibility apply and any specific differences can be incorporated without difficulty. The first steps concern decisions based on the clinical need for the test, i.e., the scope, purpose and application of the assay and whether a commercial or a LDT will be required. For all in-house developments it is strongly recommended that the comprehensive MIQE guidelines are followed.

Analytical Verification Stage Reference Materials and Sample Numbers The first question that arises when developing a LDT, particularly for rare and emerging infectious disease pathogens, is the availability of samples. If sufficient samples are not available, are they obtainable elsewhere? If not, it may be necessary to construct test samples by spiking various concentrations of the analyte into a suitable matrix. Other sources of suitable samples may be other clinical/research laboratories or commercial standards, quality control materials or proficiency panels. Typically, 100 samples of 50–80 positive and 20–50 negative specimens are used. Consideration also needs to be given to potential inhibitory substances likely to be found in the specimens tested. Therefore, paired control specimens should be prepared by adding low concentrations of the analyte, with and without the known inhibitors, to suitable negative samples. However, such artificially constructed samples are unlikely to have the same properties as clinical samples. Therefore, when sufficient numbers of genuine samples become available, the assay and extraction methods will need to be re-assessed. Where samples are available, more than one specimen type may be required, e.g., respiratory pathogens can be extracted from nasopharyngeal aspirates, bronchoalveolar lavages or nose and throat swabs. A literature search should always be carried out to determine the type of specimens to be assessed and the most efficient extraction method for each of the sample type determined. When sufficient samples numbers are available, the numbers need to be calculated. Ideally this should be determined by statistical analyzes, where the sample size required to detect a significant difference is determined by the standard deviation from the difference of the means of paired samples used in the test under development and the gold standard comparator. Alternatively, they can be estimated from Table 1 below. This method allows for either a 2% or a 5% calculated error in diagnostic sensitivity and specificity. So, for example, if a 2% error is assumed, the number of samples required for an assay with 99% confidence and 99% estimated sensitivity/specificity is 164. However, if the same assay achieved a 95% estimated sensitivity/specificity then the samples required to achieve a high confidence (99%) would be 788.

Template, Primers and Probes: In-Silico Design A thorough literature review is first carried out to identify a suitable locus to be amplified. It may be necessary to consider using degenerate primers and possibly probes to cover sequence variants that occur in different strains of the pathogen. Thorough sequence searches with BLAST (Basic Local Alignment Search Tool; See “Relevant Websites section”) are required to ensure as many variants as possible are included in the design. A local database can be constructed and aligned using one (or more) of the sequence alignment program available, such as CLUSTALW (See “Relevant Websites section”). Optimal primer and probe function is critical to successful PCR. There a number of software packages available to assist in primer and probe design, such as Primer Express (Applied Biosystems) and OLIGO (See “Relevant Websites section”). If the assay is a multiplex, the sets of primers and probes must be checked for cross-reactivity, to eliminate any inhibition due to co-amplification issues. Multiplexing is a useful method of syndromic testing and reducing the cost diagnoses by targeting more than one pathogen in a sample. Furthermore, most, if not all assays will be multiplexed, due to the co-amplified EC.

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Any matches with other sequences, particularly from other pathogens likely to be encountered in the sample types under investigation will necessitate redesign. It must be stressed that this preliminary specificity check against the databases is not conclusive evidence. The assay itself must be tested against the range of pathogens likely to be encountered in the sample types under investigation.

Choice of the Quantification Standards There are two main options for quantifying by a real time PCR, either absolute or relative. In most diagnostic assays absolute quantification is chosen. A set of separately amplified, log-diluted previously quantified templates are used to form a standard curve, derived from Ct values plotted against the log concentrations of each standard. The copy number of any sample is estimated from its Ct intercept on the standard curve. The accuracy of the standards can be confirmed by testing against external QA controls. The National Institute for Biological Standards and Control in the UK produce over 300 WHO International standards. Several standards designed for nucleic acid based assay testing are also commercially available, including HIV, hepatitis, CMV, EBV, and B19 parvovirus and are reportable in international units/milliliter (IU/ml).

Reaction Controls The LDT must include positive controls (PC), negative, no template controls (NTC) and extraction controls (EC). The Ct values of the positive control must be recorded as part of the routine quality assurance. Typically, a single positive control for each specific pathogen, a NTC after every eight samples, and a further NTC as the last sample on the run is used to monitor the assay. Placing a NTC at the start of the set-up could give a false impression of any contamination problems. The PC can be produced from extracted clinical samples or from commercial sources and should be diluted to be reproducibly amplified at the lowest detectable level (typically a Ct of 30). An internal PCR or amplification control (IC) is an absolute requirement for any diagnostic assay. Nucleic acid from a suitable non-target pathogen can be spiked into each extracted specimen prior to the PCR and amplified and detected with a separate set of primers and probe. Preferably, an EC, using a non-related virus or bacterium is used to monitor extraction efficiency. Using this approach, the EC is spiked into the sample before extraction. This has the advantage over using naked nucleic acid as an EC, because it controls for the all the steps in the extraction process. Phocine herpesvirus is a useful EC for assays detecting viral DNA pathogens and for RNA viruses, MS2 phage or mengovirus can be used. The use of an RNA template in RT-PCR controls for both the critical reverse transcription step and the PCR. Inhibition can be assessed by comparing the Ct values of the amplified EC in samples with the Ct value of the control extracted in a negative matrix, e.g., water or elution buffer. A difference, typically greater than three Cts (approximately equivalent to one log) indicates inhibition and a potentially false negative result. It is necessary to establish that the primers and probes and the amount of control added to the reaction does not interfere with the amplification of the pathogen target.

Reverse Transcription PCR Reverse transcription PCR is used to detect RNA viruses, but the use of differential gene-expression assays is becoming more widespread to distinguish between infection and colonization in other infectious micro-organisms. Reverse transcriptionPCR can be carried out as a two-step reaction, where the RT step is carried out separately and an aliquot of the reaction transferred to the PCR. More usually, in infectious disease assays, a one-step reaction is employed, where the reverse transcription and PCR occur in the same tube on the thermocycler. The one-step is quicker and more suited to highthroughput diagnostic laboratories, since the potential for contamination is lessened by number of steps that require opening tubes. The choice of priming the RT step needs to be considered; typically, the downstream, anti-sense primer is used. However, random hexamers may be more efficient. Often the choice is down to a sensitivity/efficiency balance between the hexamers ability to bind and hence copy all the RNA species in the sample and the sensitivity provided by the sequence-specific binding of the downstream primer. Table 2

The Basic Westgard Rules

A 12 SD If one control measure exceeds the mean 72 SD, the control values from the previous run should be considered to rule out a trend. B 22 SD This rule detects systematic error. The rule is violated when two consecutive control values exceed the same mean 72 SD limit. C 41 SD This rule detects systematic error. The rule is violated when 4 consecutive control values exceed the same (mean þ 1 SD or mean  1 SD) limit. The run need not be rejected if this rule is violated but should trigger recalibration or equipment maintenance. D 13 SD This rule detects random error. Violation of this rule may also point to systematic error. The assay run is rejected if one control value exceeds the mean 73 SD. E R4 SD This is a range rule and it detects random error. The rule is violated when the difference in the SD between 2 control values exceeds 4 SD (i.e.,  2 SD and þ 2 SD). F 10X This rule detects systematic error. The rule is violated when the last 10 consecutive values are on the same side of the mean. Its violation often indicates the deterioration of assay reagents.

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Validating Real-Time Polymerase Chain Reaction (PCR) Assays

Westgard Rules For each run the Ct values of controls are plotted on Levy-Jennings or Shewhart charts to monitor the performance of the assay; visual presentation of the control data is helpful in spotting variations in the operation of the assay (see Table 2). Westgard rules can also be applied to determine when a corrective action should be taken and give some indication of the nature of the analytical error. The use of Westgard rules and Shewhart charts must form part of the ongoing monitoring of the assay’s validated status.

Experimental Optimization Optimal primer and probe concentrations must be verified, together with the PCR program itself. A well characterized, stable positive control must be used throughout this verification process to provide an invariant baseline from which the effects of changes to the various components can be measured. The formation of primer dimers and the degree of non-specific amplification can be assessed by analytical agarose gel electrophoresis which also provides evidence of the size of the amplicon. To optimize primer and probe concentrations, ideally two log-dilutions of the positive control are used in a checkerboard layout of varying primer/probe combinations. The optimal concentrations and thermal cycling conditions are those that provide the lowest Ct values, the greatest DRn and a consistent Ct difference of approximately three cycles between the two log-diluted samples. Once the basic parameters of the assay have been established it will be necessary to confirm the amplicon product by sequencing and BLAST analysis. Due to the short amplicon lengths in real-time PCR, the fragment may need to be cloned into a suitable plasmid vector.

Normalization Expression analyzes with reverse transcription PCRs is not normally used in diagnostic PCR. However, the range of diseases and their associated pathogens being diagnosed by RT-PCR is increasing. In some cases, e.g., with fungal species, there is a need to distinguish between carriage, environmental contamination and infection. Therefore, assays capable of detecting and quantifying differentially expressed genes indicative of the disease condition are needed. This requires carefully selected reference genes to control for variations in extraction, reverse-transcription and amplification efficiencies, so that comparisons across different mRNA concentrations can be made and fold changes in expression levels determined. A detailed literature search for suitable reference gene mRNA targets must be made and the selected candidates experimentally tested for stable expression in both diseased and non-diseased samples.

Analytical Specificity and Sensitivity Analytical specificity: the LDT’s ability to detect the target it was designed for and not cross-react with other analytes in the sample. Specificity is demonstrated either by spiking samples with a range of different pathogens prior to extraction, or adding the extracted nucleic acid from these pathogens to the extracted sample under investigation. All reactions should be analyzed by agarose gel electrophoresis to ensure that amplification has not occurred in any of the samples expected to be negative. In some cases, primers may amplify non-target templates, which may not be detected by the probe. This will compromise the sensitivity of the assay and must be eliminated, either by re-evaluating the PCR conditions (typically raising the annealing temperature), or preferably by redesigning the primers. Analytical sensitivity: The LDT’s ability to detect very low levels of a given analyte in a biological specimen. This is synonymous with the assay’s LOD, i.e., the lowest concentration of the analyte consistently detected in Z95% of samples tested with acceptable precision. The LOD is usually determined for diagnostic assays by using a set of log-diluted controls, such as patient samples, a suitable cell line, or proficiency panels. It is important to include a NTC in the LOD study to ensure that the PCR does not generate a signal that could interfere with true low-level positive signals from a sample. There is no hard and fast rule as to how many samples to use, The Clinical and Laboratory Standards Institute suggests a minimum of 60 data-points (12 separate measurements from each of 5 samples) are required from a manufacturer to establish the LOD. This seems a reasonable figure to adopt as a standard approach for both LDTs and commercially produced assays and will provide the necessary intra-assay variation measurements. This measurement should also be carried out on a second thermocycler to establish machine-to-machine variation. Estimation of the LOD is usually carried out by probit analysis, a type of regression used to analyze binomial response variables, where a sigmoid dose-response curve is transformed into a straight line that can be analyzed by regression either through least squares or maximum likelihood. For PCR assays the lowest concentration of analyte that can be detected with a stated probability can be determined by plotting the data from positive replicate results versus the analyte concentration.

Inhibition Study The factors that inhibit or prevent the amplification of nucleic acids by PCR can be present in the extracts from a number of sources. Inhibitors typically act by: (1) interference with the cell lysis necessary for extraction of nucleic acids, (2) interference by degrading the

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nucleic acid or inhibiting its capture, (3) inhibiting polymerase activity during amplification of the target DNA, resulting in falsenegative results or inaccurate quantification. Different sample types present different problems due to the range of endogenous inhibitors found, for example in fecal samples, complex polysaccharides, breakdown products from hemoglobin, and bile acids can all be present and careful thought needs to be given to the method of extraction. Other potential inhibitors include metabolites resulting from pathological conditions such as diabetes mellitus and homeostatic hepatitis, or from medications used in treatment. Manufacturers continue to develop their PCR reagents specifically to overcome many of the common endogenous inhibitors found in clinical specimens and it is recommended that a number of different commercial amplification kits are evaluated for their performance in this respect. As discussed previously, inhibitors can be detected using an IC. If the IC fails to amplify or exhibits suppression below the acceptable threshold, the amplification of the intended target sequence may also be inhibited. To avoid reducing the sensitivity of the assays, the IC should be used at the lowest concentrations that can be reproducibly amplified to minimize any competition between its amplification and that of the target pathogen. The IC must demonstrate inhibition by the substances expected to be found in the sample, for which an inhibition study needs to be carried out. The study must first identify what types of inhibitors are likely to be present in the sample types used in the assay. The IC can be tested in negative samples with and without the interfering substance(s) in parallel and the effect on the Ct values monitored. This test must be repeated with the full assay (including the pathogen detection primers and probes) using paired samples, with and without the target analyte to fully validate the assay. Once interference is found, of either or both the IC and target, samples can be serially diluted to test the limit at which the target will amplify. In most cases dilution of inhibited samples provides a straightforward method of enabling amplification. The assay must then be tested against clinical samples; this is because artificially constructed inhibition control samples may not accurately reflect the assays performance in routine use.

PCR Efficiency The efficiency of the PCR can have a significant impact on the robustness and precision of the assay. The efficiency of the reaction is determined by a number of factors, including primer design, cycling conditions and the reagents used in the reaction mixture. PCR efficiency is particularly important in assays reporting fold changes of mRNA for target genes relative to those of reference genes, where both templates must be amplified with equal efficiencies. PCR amplification efficiency is typically established using calibration curves and it makes sense to use the dilution series (covering 5–6 orders of magnitude) from the LOD analyzes for this purpose. The equation of the linear regression line, together with Pearson’s correlation coefficient (r) and the coefficient of determination (r2) are used to determine amplification efficiency. Amplification efficiency itself is determined from the slope of the log-linear portion of the calibration curve and is given by Eq. (1): E ¼ 10ð1=slopeÞ :

ð1Þ

Ideally the amount of template will double during each round of exponential amplification, this translates to a reaction efficiency of 2, therefore, using equation 1: 2 ¼ 10(1/slope) which gives a slope for the standard curve of  3.32. This ideal figure of 3.32 also represents the difference in cycle number for each log dilution in the series. The efficiency can also be expressed as a percentage of the template amplified in each cycle using Eq. (2): % Efficiency ¼ ðE  1Þ  100%

ð2Þ

In the ideal example given above; % Efficiency ¼ (2  1)  100% ¼ 100%. The experimental measurement should be carried out in triplicate on at least two thermal cyclers and the information included in the validation documentation. In practice acceptable assays should achieve efficiencies of 90%–100%. Reaction efficiencies lower than this may be caused by suboptimal reaction conditions or poor primer design. Reaction efficiencies of more than 100% could be due to measurement errors e.g., in preparation of the dilution series, or co-amplification of primer-dimers. Improving specificity will improve the sensitivity and increase the dynamic range of the assay, by reducing competition between the specific and non-specific amplification products.

Linear Dynamic Range or Reportable Range The linear dynamic range can be determined during the LOD analysis by extending the range of the dilution series. The dilution series is often set at the range one would expect the analyte to be found in clinical specimens, although this may not always be known, especially for a novel pathogen and so initial testing can be carried out from 1 up to 107 or higher copies/ml of target. If using a generic assay to detect a range of pathogens, e.g., a pan-fungal PCR, each of the species the assay is designed to detect must be tested. Similarly, for multiplex assays, the LOD must be determined individually for each target analyte in the assay and must include the individual targets in the multiplex at high and low concentrations to ensure competitive amplification does not prevent all targets being identified. The reportable range is defined as the lowest and highest results (in suitable units of concentration) reliably detected in the assay. The linearity of the range can be established by calibration curves, where an r2 value (coefficient of determination) of 1.00 indicates a perfect fit of the data points. Generally, in qPCR assays an r2 of not less than 0.99 is considered acceptable. The linearity and reportable range should be carried out on at least 5 log-dilutions of the target nucleic

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Validating Real-Time Polymerase Chain Reaction (PCR) Assays

acid extracted from an appropriate sample type (serum, urine, NPA etc.,) in triplicate, ideally on two different thermocyclers. Quantitative results from the test can only be reported when they fall within the linear range of the assay. A reportable range is not applicable to qualitative assays, since the result is simply positive or negative (below the LOD). However, a log-diluted panel should be tested, because the information derived is useful in assessing both the efficiency and clinical utility of the test and the results (as Ct values) reported as part of the supporting documentation for the assay.

Clinical Validation Stage Precision, Accuracy and Trueness Precision: a measure of the closeness of agreement between independent test results obtained under defined conditions. Accuracy: the level of agreement between the true value of an analyte and the value obtained by the new test. Trueness: defines the level of agreement between the average value obtained from a large series, the test and the accepted reference value. Precision experiments are designed to measure the random error of an assay over a pre-determined period of time by multiple measurements of an aliquot derived from a homogeneous sample. For qualitative assays random error can be established by testing the PC and NC material in triplicate over a period of 10 days on two different thermocyclers. For qPCR assays estimates can be determined from the quantification standards and two positive controls at the lower and upper ends of the assay’s reportable range. Accuracy refers to the closeness in agreement between a single measurement and the true value of the analyte under investigation. Trueness is determined by analyzing the average value obtained from a series of measurements with the new assay and the true value of the analyte (if an international standard is available) or a reference method (if a standard is not available). There are two approaches to evaluating trueness (Burd, 2010): (1) A comparison of methods study, where split samples are tested in parallel with an appropriate gold standard method. (2) A recovery study, where proficiency samples, or other verified sample types are compared with the assay under development. Although either of these methods is acceptable, a split sample, comparative study using clinical samples is preferred. However, in some cases, e.g., when developing an assay for a novel pathogen, a suitable gold standard assay may not available and a recovery study will have to suffice.

The Gold Standard Comparative Study The comparative study is the cornerstone of the validation exercise. Estimates of sensitivity and specificity are derived from comparisons between the LDT and an established gold standard, which, ideally, is the same type of test and has an assumed sensitivity and specificity of 100%. By definition the gold standard assay is an error-free diagnostic method, which, rarely (if ever) exists. Consequently, an earlier PCR assay, or a culture-based method is often used. Samples for the comparative study should be representative of the disease and population being investigated and also suitably distributed across both age range and gender. Where only limited samples are available, it may be necessary to use archived positive and negative specimens, or cultured material spiked into negative clinical samples. Other sources may be commercial standards, quality control materials or proficiency panels. Sample numbers can be determined as described previously. However, the sample numbers are more often selected for more pragmatic reasons, such as cost and feasibility and therefore, less than the statistically optimal number will be tested. This is often the case for a new or re-emerging infectious disease. The difficulty comes in interpreting the results of a study where the comparative test is not a “perfect” gold standard, (i.e., the alloyed standard). This is particularly so if the comparative method is cell culture, which is thought to be 100% specific, but provides less than optimal sensitivity. The strength of the PCR lies in its sensitivity (theoretically a single DNA copy). Thus a newly developed PCR assay may produce many more positive results than the reference method, leading to a misleading, lower estimate of specificity. The researcher is then left with the problem of deciding whether these are true or false positives. A thorough and detailed assessment of the sample derivation, clinical history and sequencing can help to determine the true status of any sample, together with discrepant analysis. In discrepant analysis the discordant results are resolved by a third test, such as another PCR directed to a different gene-target and the results from this resolver test are used to definitively assign the final results. It is important to include a number of randomly selected samples with concordant results for discrepant analysis so as to reduce bias. Further statistical analysis can be carried out to improve confidence in the results from the new LDT, particularly when the true disease status of the samples is difficult to evaluate. The agreement between the new test and comparator test can be expressed as the

Fig. 2 Fig. 2 2  2 Contingency Table.

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kappa value. The kappa statistic is a generic term for a number of similar measures of agreement applied to categorical data and is a measure of the proportion of agreement between results beyond chance. It is used in assessing the degree to which two or more raters, (i.e., tests) examining the same data, (i.e., specimens) agree in assigning the data to categories (positive or negative results). A kappa value of 1.00 indicates perfect agreement; a value of 0.00 indicates no agreement above that expected by chance, and a kappa value of  1.00 indicates complete disagreement. Generally, kappa statistics above 0.80 are considered almost perfect. The kappa value is calculated from the results in the standard 2  2 contingency table, used to record the results from the comparative test (see Fig. 2). OP ¼

TP þ TN n 

EP ¼ K¼

       TP þ FP TP þ FN FN þ TN FP þ TN  þ  n n n n

OP  EP 1  EP

where: K ¼ Kappa value. OP ¼ observed proportion of agreement. EP ¼ expected proportion of agreement. TP ¼ true positive. FP ¼ false positive. TN ¼ true negative. FN ¼ false negative. n ¼ total number of samples. Diagnostic sensitivity and specificity (DSe and DSp) are also calculated from the results in the contingency table. DSe ¼

TP TP þ FN

DSp ¼

TN FP þ TN

Two further important probabilities can be calculated from the data derived from the trueness study, the positive and negative predictive values (PPV and NPV). The PPV is a measure of a positive result from the test to truly predict the presence of disease/ infection, while the NPV is the probability that a negative result accurately indicate a non-diseased/uninfected status. PPV ¼

TP TP þ FP

NPV ¼

TN FN þ TN

The Study Without a Gold Standard In many cases, particularly for novel pathogens, a comparative method will not be available. Under these circumstances the gold standard may be a diagnosis determined by accepted clinical methods. It may also be necessary to incorporate results from more than one method to provide the comparative results. Whatever the approach chosen, estimating the performance characteristics of a new assay without a true comparative test is a challenging task. Typically, agreement is measured as the overall percentage or fraction of samples that have the same result (i.e., both either positive or negative). The difficulty with these simple agreement measures is that they do not take agreement by chance into account. A number of models have been proposed, based on latent class analysis, log-linear modeling and other techniques. Latent class analysis involves multiple imperfect tests that are used to construct a gold standard. Such models are based on the concept that the observed results of different, imperfect, tests for the same disease are influenced by a common but unobserved (latent) variable i.e., the true disease status.

Inter-Laboratory Testing The ultimate evidence of an assay’s fitness for purpose is its successful integration into other laboratories. The LDT reagents and samples used for the comparative study should be sent to a minimum of three laboratories willing to participate in testing. This will also provide additional data on the assay’s reproducibility and robustness.

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Validating Real-Time Polymerase Chain Reaction (PCR) Assays

Maintaining the Validated Status and Re-Validation The validated assay must be monitored consistently for repeatability through the performance of the run controls to evaluate any potential changes in the assay’s precision and accuracy. All reagents should be assigned batch numbers and new batches tested with the controls and a suitable number of positive and negative samples from a previous run to ensure consistent efficiencies between batches. If more than one thermal cycler is used, each machine should be referenced and each test carried out on it identified. The results of the PCR controls and quantification standards must be plotted daily on Shewhart charts and assessed by Westgard rules to ensure that reaction efficiencies are maintained. Participation in regular IQA and EQA schemes is fundamental to maintaining an assay’s validated status. The purpose of EQA testing is not solely to monitor the performance of the assay, but asses all aspects of the test procedure, including the extraction method and reporting accuracy. If an external panel is not available, alternatives can include blind sample testing, sample exchange with other laboratories, or clinical note reviews. Technical modifications continually arise as manufacturers develop and improve their reagents for extracting nucleic acids, the PCR and instrumentation. Assessment of new probe chemistry or the transfer of an assay to full or semi-automated instrumentation, normally only requires a methods comparison study. In this way any changes to the diagnostic sensitivity and specificity can be assessed quickly and accurately and the updated assay introduced with the minimum delay. Over time it is likely that an assay will require some degree of revalidation, either for purely technical reasons, or because of changes in the nature of the analyte detected. Regular monitoring of amplification efficiency together with clinical information will give an early warning of any changes in the sequence of the amplicon detected in an assay. Point mutations are common in the genomes of many infectious microorganisms, particularly RNA viruses. New viral lineages may also be introduced into the population being tested due to travel from abroad. Under such circumstances, significant modifications to the primers and probe may be required, necessitating reverification and revalidation of the assay. Amplicon sequencing should be part of the validation plan and an integral part of routine trouble-shooting algorithms, since it is capable of resolving errors due to non-specific binding of primers and probes.

Acknowledgment This article, including tables and figure, is based on author’s article “The Validation of Real-time PCR Assays for Infectious Diseases”, published in Real-Time PCR: Advanced Technologies and Applications (2013), edited by: Nick A. Saunders and Martin A. Lee, Health Protection Agency, Colindale, UK and Porton Consulting Research Ltd, Salisbury, UK (respectively), Caister Academic Press.

References Burd, E.M., 2010. Validation of laboratory-developed molecular assays forinfectious diseases. Clinical Microbiology Reviews 32, 550–576. Bustin, S.A., Benes, V., Garson, J.A., et al., 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55 (4), 611–622.

Further Reading Corman, V.M., Landt, O., Kaiser, M., et al., 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance 25 (3), 23–30. Hadgu, A., Qu, Y., 1998. A biomedical application of latent class models with random effects. Journal of the Royal Statistical Society Series C 47 (4), 603–616. Hadgu, A., Dendukuri, N., Hilden, J., 2005. Evaluation of nucleic acid amplification tests in the absence of a perfect gold-standard test – A review of the statistical and epidemiologic issues. Epidemiology 16, 604–612. Rådström, P., Knutsson, R., Wolffs, P., Lövenklev, M., Löfström, C., 2004. Pre-PCR processing: Strategies to generate PCR-compatible samples. Molecular Biotechnology 26, 133–146. Raymaekers, M., Smets, R., Maes, B., Cartuyvels, R., 2009. Checklist for optimization and validation of real-time PCR assays. Journal of Clinical Laboratory Analysis 23, 145–151. Rutjes, A.W.S., Reitsma, J.B., Coomarasamy, A., Khan, K.S., Bossuyt, P.M.M., 2007. Evaluation of diagnostic tests when there is no gold standard. A review of methods. Health Technology Assessment 11, 1–69. Saunders, N.A., Martin, A.L. (Eds.), 2013. Real-Time PCR: Advanced Technologies and Applications. UK: Caister Academic Press. Sloan, L.M., 2007. Real-time PCR in clinical microbiology: Verification, validation, and contamination control. Clinical Microbiology Newsletter 29, 87–95. Westgard, J.O., Barry, P.L., Hunt, M.R., 1981. A multi-rule Shewhart chart for quality control in clinical chemistry. Clinical Chemistry 3, 493–501. Wilson, I.G., 1997. Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology 63, 3741–3751.

Relevant Websites https://www.ebi.ac.uk/Tools/msa/clustalo/ Clustal Omega. http://www.ncbi.nlm.nih.gov National Center for Biotechnology Information. http://www.oligo.net/ oligo.net.

Rapid Point-of-Care Assays Jan G Lisby and Uffe V Schenider, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark r 2021 Elsevier Ltd. All rights reserved.

Introduction The first report of viruses as pathogenic agents was published in 1892, when the transmission of the tobacco mosaic disease by liquid filtered through porcelain filters capable of retaining bacteria was reported (Iwanowski, 1892). Subsequently, Loeffler and Frosch using similar techniques, reported the transmission of the causative agent of foot-and-mouth disease in 1898. The first human viral pathogen was reported in 1902, when it was reported that a filterable agent was the causative agent of yellow fever (Reed, 1902). Virology as a clinical diagnostic enterprise emerged with the development by Enders et al. in 1949 of the monolayer cell culture technology, allowing for growth of viruses outside the host or laboratory animals (Weller et al., 1949). For the subsequent decades, clinical diagnostic virology applied viral propagation in cell cultures, such as plaque assays, first described in 1952 (Dulbecco, 1952) and in the 1960s, detection of viral antigens or human antibodies raised against viral antigens was established. The first solid-phase assays, termed Enzyme Immunoassays (EIA) were made possible by the discovery in 1966 that antibodies (initially polyclonal) could be labeled with enzymes providing a detectable staining result (Nakane and Pierce Jr, 1966). The substitution of enzymes with a radioactive label in 1967 further enhanced the signal (Catt and Tregar, 1967). The description of the polymerase chain reaction (PCR) in 1985 (Saiki et al., 1985) and the subsequent upgrade of the technology by application of a heat-stable DNA polymerase (Saiki et al., 1988) resulted in a paradigm shift in clinical virology diagnostics and within the next decade, PCR almost completely replaced culture and serology methods as the preferred diagnostic intervention for acute viral infections (Mackay et al., 2002; Niesters, 2002). The primary purpose of human clinical viral diagnostics is to prove or disprove a definite viral diagnosis in the case of a clinical infection. The secondary purpose of the diagnostic efforts is to impact on clinical patient management, which may be in the form of cessation of unnecessary antibacterial treatment (if no other indication of a bacterial infection is present), initiation of specific antiviral treatment (if possible/available) and decision on contact isolation if the patient is admitted to a hospital or other infection control measures. Besides operational knowledge of specific immune and vaccination status, impact on clinical patient management requires availability of diagnostic results within a time window where impact is possible and patient outcome may be altered, which most often will be limited to the first few hours or days after patient symptom debut. For the first several decades of clinical diagnostic virology, this was problematic. Diagnostic methods based upon serology may deliver rapid results, especially methods based upon lateral flow technology (Koczula and Galotta, 2016), but while detection of a matured immune response to a previous infection with a viral pathogen – or a previous vaccination – by detection of IgG against the viral target is sensitive and specific, especially when matured IgG antibodies are detected (Prince and Lapé-Nixon, 2014), rapid detection of viral antigens in an acute infection by lateral flow is specific but insensitive (Chartrand et al., 2015) and detection of an acute viral infection by detection of IgM is sensitive but unspecific (Prince and Lapé-Nixon, 2014). With the emergence of the PCR technology, sensitive and specific detection of an acute viral infection became possible. Initially in the 1990s, diagnostic PCR in general was time consuming and cumbersome (Lisby, 1998; Smith et al., 1992), but eventually, fully automated PCR platforms were developed in the mid to late 2010s for the clinical diagnostic laboratory, bringing the turn-around-time of the diagnostic process in the laboratory down to a few hours (Wirden et al., 2017; Cobb et al., 2017). However, for most patients, a considerable time must be added for logistic delays, such as patient sample transportation to the laboratory, receipt and registration within the laboratory, reporting of test results and clinical action upon the test result. Often, the total turn-around-time from patient sample to clinical action upon the diagnostic result is more than 6–8 h. For acute viral infections, such as viral meningitis/encephalitis, viral respiratory infections and viral gastrointestinal infections, a diagnostic delay of 6–8 h or more may compromise the diagnostic impact on e.g., differential diagnostics (e.g., Influenza vs meningitis in an adult with high fever and muscle/head ache), antimicrobial use (at least the first 1–2 doses) and use of contact isolation facilities.

PoC Technologies The choice of technology for detection of viruses depends greatly upon the specific pathogen. In general, urine or throat swabs are well-suited specimens for virus detection, since virus often can be detected for a long time and specimens are easy to obtain. But, depending on the specific virus, blood, fecal or other specimens may be more adequate sampling sites (Paz-Bailey et al., 2018). Likewise, greater sampling volume and the processing of a greater fraction of the sample volume increases the sensitivity of detection. Some viruses such as the genus of flaviviruses can only be isolated from the blood stream in humans for a short time after infection. The preferred tests for such viruses such as yellow fever, dengue-, zika- or west nile virus are therefore lateral flow tests that are testing for virus specific antigens in combination with IgM and IgG human antibodies. The direct detection of virus is hence combined with the detection of host response by seroconversion. Other viruses, such as viruses infecting the respiratory tract or the gastro-intestinal channel of humans are well-detected by PCR based assays. PCR based amplification is especially beneficial when detection of multiple viruses (multiplex assays) is requested

Encyclopedia of Virology, 4th Edition, Volume 5

doi:10.1016/B978-0-12-814515-9.00130-2

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Rapid Point-of-Care Assays

(e.g., respiratory tract infections) or viruses with high mutations rates such as RNA viruses are being detected. Detection by isothermal amplification have been shown to work well with virus detection. Still, PCR is often preferred over isothermal amplification, as additional conserved nucleic acid regions are requested for design of isothermal amplifications assays compared to PCR based assays. To detect multiple viruses, current Point-of-care multiplex platforms are utilizing different strategies. In the Biofire Filmarray from BioMérieux, nested PCR amplification by a multiplex first round PCR is combined with second round nested monoplex pathogen specific PCR on microarray with fluorescence detection (Poritz et al., 2011). The ePlex platform from Genmark Dx is also based on microarray detection but with electrochemical detection in combination with multiplex PCR (Babady et al., 2018). Another strategy is applied by the QIAstat Dx platform from Qiagen in which the extracted nucleic acids are fractioned into several separate PCR chambers. Each chamber allows for a five-plex multiplex PCR with an internal control and the detection of fluorescence signal from hydrolysis probes (Parcina et al., 2020). A similar approach was applied by the Idylla Platform from Biocartis which initially was launched with a multiplex respiratory panel for detection of viral pathogens (Wouters et al., 2019). Most platforms that only detect a few pathogens simultaneously are based on the hydrolysis probe PCR technology, e.g., the Cobas Liat from Roche (Tanriverdi et al., 2010), the GeneXpert from Cepheid (Jenny et al., 2010), the Aries instrument from Luminex Corp (Voermans et al., 2016), the BD max from BD (Cárdenas et al., 2014), the Accula system from Mesa Biotech and the Simplexa from Focus Diagnostics (Woodberry et al., 2013). In contrast to these platforms, the IDnow instrument from Abbott is based on the isothermal nicking enzyme amplification reaction (NEAR) technology, which allows for faster amplification, but at the risk of non-specific amplification products (Nie et al., 2014; Tan et al., 2008). As the landscape of instruments in the PoC field increases, other technologies and combinations hereof are expected to be introduced. Hopefully, the choice of technology by the end-user will reflect the strategy and the implementation hereof, as envisioned by the manufacturer.

PoC Strategy and Implementation In general, any assay selected for use at a PoC setting should bring value to clinical patient management that cannot be obtained by testing the patient sample for the same microbial targets in the central clinical microbiology laboratory. The patient management value of any test will depend upon the actual patient population and the actual clinical setting, e.g., in an adult Emergency Room, Influenza A/B may be the only respiratory assay providing enough patient management value to be able to show a satisfactory benefit/cost ratio, but in a pediatric ED, a highly multiplexed respiratory panel may be preferable. Ideally, for each Point-of-Care setting (Fig. 1), for each diagnostic question (e.g., contact isolation yes/no, upper respiratory tract infection yes/ no) and for each patient population (e.g., adult/pediatric), a specific business case should be established, detailing the impact on clinical patient management obtained by PoC testing as well as the cost of PoC testing. PoC infectious disease testing performed outside the geography of the central clinical microbiology laboratory may in the near future be performed by individuals with different educations and skillsets, from highly trained biotechnicians to patients themselves. Depending upon the actual geographical location (Fig. 1), PoC testing may be classified as:

Point-of-Care Laboratory Grade 1A In this setting, PoC for infectious disease testing is performed “in hospital – in lab” in a 24/7 laboratory staffed with dedicated and trained biotechnicians. This may be considered as a satellite clinical microbiology laboratory and may be operated by trained clinical microbiology technicians but may also be operated by trained biotechnicians from other clinical specialties, such as clinical biochemistry or clinical pathology. If available 24/7, any assay performed within the geography of the clinical microbiology laboratory is considered as PoC Grade 1A.

Point-of-Care Laboratory Grade 1B In this setting, PoC for infectious disease testing is performed “in hospital – outside lab” by local healthcare professionals in e.g., the Emergency Department (ED), the Intensive Care Unit (ICU) or the Pediatrics Department. As the healthcare professionals performing the assays in this setting are not dedicated laboratory professionals, but most likely special trained nurses or nurse assistants, assays must be low complexity, easy to perform and fail-safe.

Point-of-Care Laboratory Grade 2A In this setting, PoC for infectious disease testing is performed “outside hospital – in healthcare facility” by healthcare professionals in e.g., a General Practitioners office or laboratory. In this setting, the assays may be performed in the actual office or in a side room facility. The healthcare professionals performing the assays will most likely not be professional laboratory workers but may be physicians, nurses or other trained staff. Thus, assays performed in this setting should be low complexity, easy to perform and fail-safe.

Rapid Point-of-Care Assays

47

Fig. 1 Different Point-of-Care settings. Point-of-Care Laboratory Grade 1A – in hospital – in lab; Point-of-Care Laboratory Grade 1B – in hospital – outside lab; Point-of-Care Laboratory Grade 2A – outside hospital – in healthcare facility; Point-of-Care Laboratory Grade 2B - outside hospital – outside healthcare facility; Point-of-Care Laboratory Grade 3 – home setting.

Point-of-Care Laboratory Grade 2B In this setting, PoC for infectious disease testing is performed “outside hospital – outside healthcare facility” by healthcare or other professionals in e.g., a third world field use or a military setting. Thus, assays performed in this setting should be low complexity, CLIA-waived or similar. The Clinical Laboratory Improvement Amendments of 1988 (CLIA) provides federal standards applicable to all US facilities performing diagnostic testing on human specimens. Waived tests include tests that have been cleared by The United States Food and Drug Administration (FDA) for home use (“see Relevant Websites section”).

Point-of-Care Laboratory Grade 3 In this setting, PoC for infectious disease testing is performed in a “home setting” by laymen individuals without any formal or informal previous training. Assays performed in this setting must be low complexity, CLIA-waived or similar. The basic requirement for any infectious disease PoC diagnostic assay should be equality regarding performance when compared to assays performed within the central laboratory. For Point-of-Care Laboratory Grades 1A and 1B, it is strongly recommended to establish local institutional or regional committees with participation of the local stakeholders and may include establishing local, regional or national guidelines. According to guidelines published by The American Society for Microbiology (Dolen et al., 2017) and The Danish Society for Clinical Microbiology (Lisby et al., 2017), it is recommended to maintain clinical microbiology laboratory oversight and expertize when PoC testing for infectious diseases is implemented. This may ensure selection of optimal assays and platform, verification of assays locally before implementation, test reporting and interpretation, quality control procedures and participation in quality control programs and – especially – electronic communication between the local PoC platform and the institutional Electronic Patient Record and/or the institutional Laboratory Information System. For Point-of-Care Laboratory Grade 2A, in some countries or regions, these recommendations may be implemented, in other countries/regions, this may not be feasible due to various factors such as organization of the local healthcare system. Analyzing more patient sample at the PoC setting and sending fewer samples to a central clinical microbiology service provider will increase the necessity to focus on “the full infectious disease picture” for each PoC geographical location. Without an established electronic communication between the individual PoC location and the central clinical microbiology

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service provider, expert guidance regarding diagnostic workup, test indication and interpretation, and, especially, surveillance of microbial identification and resistance patterns may be compromised. For Point-of-Care Laboratory Grade 2B settings, the same considerations as for Grade 2A may be applied. However, as this setting will likely be without readily accessible central clinical microbiology service providers (e.g., by military use or use in low-resource settings), electronic communication of individual patient test results and clinical microbiology oversight in general may not be relevant. The biggest challenge to clinical microbiology oversight and surveillance of microbial prevalence may arise from infectious disease testing performed in future Point-ofCare Laboratory Grade 3 settings (home use). Unless regulatory requirements regarding test result management, e.g., mandatory upload to cloud-based databases, are established, most likely test results based upon PoC testing in this setting will not be available for clinical microbiology guidance or local/national surveillance. The potential negative impact on local and national surveillance and antimicrobial use, especially in regions with unrestricted access to antimicrobials, may override the potential positive impact by use of infectious disease diagnostics in this PoC setting, e.g., patient/user satisfaction.

PoC Menus A variety of companies are marketing assays for immunofluorescence detection of antigens or antibodies on serum or feces e.g., Biocan or Abbott (“see Relevant Websites section”). Such test portfolios include viruses like adenovirus, Barmah Forest virus, Chikungunya virus, CMV, dengue virus, Ebolavirus, EBV, Hantaan virus, HAV, HBV, HCV, HIV, HSV 1 or 2, Influenza A and B virus, norovirus, ross river virus, rotavirus, RSV, SARS-CoV-2, rubellavirus and Zika virus. The current amplification based PoC tests are currently marketing panels targeting respiratory infections, gastrointestinal infections, CNS infections, HIV and HSV (Table 1). As such assays are being implemented in the clinical setting, it is anticipated that several companies will market returning travelers panels and post transplantation panels in the future. A pragmatic approach to selecting PoC menus for current and future PoC platforms could be to select “low hanging fruits” according to business cases based upon the designated geographic location of the PoC instrument in question (Fig. 1). E.g., for PoC Grade 1a and 1b, assays targeting pathogens requiring contact isolation of the patient (such as SARS-CoV-2, Influenza A&B, RSV, MRSA, VRE, CPO, norovirus) would most likely emerge as the most attractive assays seen from a benefit-cost perspective. In general, there is currently a lack of rapid open access amplification based PoC platforms that would allow end-users to apply laboratory developed assays allowing for fast detection of viruses according to local requirements for special diagnostics.

PoC Clinical Impact The clinical impact of PoC testing can be evaluated in several ways including (1) intervention studies in which the change in patient management of a positive test is compared to negative test results, (2) prospective or retrospective case-control studies comparing the effect on patient management of the introduction of a PoC compared to a previous standard of care and (3) randomized studies comparing current standard of care with PoC and the effect on patient management. Such studies have primarily been published on respiratory illness evaluating influenza PoC testing. As the results of these studies have been conflicting, further studies are needed to understand the clinical impact of PoC testing. Modeling and intervention studies in which the clinicians are blinded for PoC results have suggested that the introduction of PoC may result in better use of side room contact isolation (Pedersen et al., 2018), reduced prescription of antibiotics (Pedersen et al., 2018; Kaku et al., 2018), targeted use of antiviral treatment (Pedersen et al., 2018) and cost savings due deferred admissions and shortened hospital stay (Rathamat-Langendoen et al., 2019). The potential for cost savings has been supported by one study investigating the use of PoC in adults in the emergency room, in which a positive test lead to deferred admission and reduction in antiviral prescribing and resulted in overall cost savings of approximately $200 per emergency room visit (Hansen et al., 2018). A study on pediatric patients could not demonstrate an impact on admission or antibiotic prescription with exception of lower admission rate for children positive for influenza B but supported more appropriate prescription of antiviral treatment (Busson et al., 2019). Studies before and after introduction of PoC for influenza testing support a more targeted use of antiviral treatment for children and adults (Vos et al., 2019; Vecino-Ortiz et al., 2018; Benirschke et al., 2019) and better use of side room contact isolation facilities for immunocompromised adults (Vos et al., 2019), but the use of PoC testing did not change length of hospital stay for children (Vecino-Ortiz et al., 2018) or immunocompromised adults (Vos et al., 2019). Similar, no positive effect on antibiotic prescription could be established for adults in urgent care settings or for immunocompromised adults (Vos et al., 2019; Benirschke et al., 2019). Randomized studies on adults agrees on more appropriate use of antiviral treatment by use of PoC testing (Brendish et al., 2017; Andrews et al., 2017), but the studies are conflicting regarding whether PoC testing may shorten duration of antibiotics (Brendish et al., 2017; Shengchen et al., 2019) or stay in hospital (Brendish et al., 2017; Andrews et al., 2017; Shengchen et al.2019). In one study, the duration of antibiotics was shortened in the intervention group (Shengchen et al., 2019), but in another study no difference in initiation or mean duration of antibiotics could be established (Brendish et al., 2017). The two studies agree that stay in hospital was shortened in the PoC test group (Brendish et al., 2017; Shengchen et al., 2019), but a third study, having length of stay as the primary outcome, could only demonstrate a reduction in time-to-test-result, whereas the length of stay in hospital was not associated with PoC testing (Andrews et al., 2017). The cost of hospitalization was compared between

X X X

Aries Luminex Corp BDmax BD Vivalytic Bosch Cobas Liat Roche GeneXpert Cepheid Idnow Abbott Meridian Revogene Mesa Biotech Simplexa Focus Dx

X X

X X

SARS-CoV-2

Targeted testing

X (X)

Biofire Biomérieux ePlex GenMark NATlab Ador Dx QIAstat Qiagen Vivalytic Bosch (X) assays in pipeline

X

SARS-CoV-2 þ Influenza

X (X)

Gastrointestinal panel

Commercially available Point-of-Care assays

Syndromic testing

Table 1

X

X X X

Influenza A/B

(X)

X (X) (X)

X

X X

X

Influenza þ RSV

Meningitis/Encephalitis

X

RSV

X

Norovirus

X (X)

X X

X

X

GBS

Respiratory panel

X X

GAS

X

Enteric viral panel

(X)

X

HIV 1 þ 2

X

HSV 1 þ 2

Tropical diseases – Returning travelers panel

Rapid Point-of-Care Assays 49

50

Rapid Point-of-Care Assays

the PoC group and the standard of care group in one study, finding an average reduction in cost of approximately $200 (Shengchen et al., 2019). Most studies agree on PoC testing resulting in a more appropriate use of antiviral treatment, whereas the data are conflicting on the impact of PoC testing on use of antibiotics and the effect on length of stay in hospital, whereas the data the impact of PoC testing on use of side room contact isolation facilities are sparse. Based on current studies, it seems that PoC testing may impact patient management of adults seen in the emergency room more than children and immunocompromised adults, but more studies are needed to understand the true impact of PoC testing on patient management.

Conclusion Clinical virology diagnostics is currently undergoing a paradigm shift. The diagnostic procedure is no longer confined within the geography of a central medical/clinical microbiology/virology laboratory but may be performed closer to the patient by nonlaboratory trained personnel. The different versions of “Point-of-Care” testing may present challenges to platform and assay selection, verification of assay performance, training of non-laboratory personnel performing the tests as well as quality control. Clinical microbiology/virology oversight is highly recommended in order to maintain the same level of diagnostic quality compared to diagnostics by gold standard assays performed by a central laboratory. Furthermore, clinical microbiology laboratory oversight may ensure capture of the PoC assay result in the patient’s electronic medical record and in the laboratory information system. Thus, the likely loss of data caused by manual recording of test results in the electronic patient records may be avoided, maintaining local, regional and national surveillance of select infections, such as Influenza Virus infections. With limited resources available to the healthcare sector, benefit-cost assessments of new diagnostic interventions should be mandatory. The impact of PoC testing on clinical patient management and healthcare costs, such as use of antimicrobials, use of antivirals, use of side room contact isolation facilities and length of stay in hospital/ICU should be documented, as limited data is yet available.

References Andrews, D., Chetty, Y., Cooper, B.S., et al., 2017. Multiplex PCR point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: A quasi-randomised study assessing impact on length of stay and antimicrobial use. BMC Infectious Diseases 17, 671. Babady, N.E., England, M.R., Smith, K.L.J., et al., 2018. Multicenter evaluation of the ePlex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs. Journal of Clinical Microbiology 56, e01658-17. Benirschke, R.C., McElvania, E., Thomson, R.B., Kaul, K.L., Das, S., 2019. Clinical impact of rapid point-of-care PCR influenza testing in an urgent care setting: A single-center study. Journal of Clinical Microbiology 57, e01281-18. Brendish, N.J., Malachira, A.K., Armstrong, L., et al., 2017. Routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (ResPOC): A pragmatic, open-label, randomized controlled trial. The Lancet Respiratory Medicine 5, 401–411. Busson, L., Bartiaux, M., Brahim, S., et al., 2019. Contribution of the FilmArray respiratory panel in the management of adult and pediatric patients attending the emergency room during 2015–2016 influenza epidemics: An interventional study. International Journal of Infectious Diseases 83, 32–39. Cárdenas, A.M., Edelstein, P.H., Alby, K., 2014. Development and optimization of a real-time PCR assay for detection of herpes simplex and varicella-zoster viruses in skin and mucosal lesions by use of the BD Max open system. Journal of Clinical Microbiology 52, 4375–4376. Catt, K.J., Tregar, G.W., 1967. Solid phase radioimmunoassay in antibody-coated tubes. Science 158, 1570–1572. Chartrand, C., Tremblay, N., Renaud, C., Papenburg, J., 2015. Diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: Systematic review and meta-analysis. Journal of Clinical Microbiology 53, 3738–3749. Cobb, B., Simon, C.O., Stramer, S.L., et al., 2017. The COBAS 6800/8800 system: A new era of automation in molecular diagnostics. Expert Review of Molecular Diagnostics 17, 167–180. Dolen, V., Bahk, K., Carroll, K.C., et al., 2017. Changing Diagnostic Paradigms for Microbiology: Report on an American Academy of Microbiology Colloquium held in Washington, DC, from 17 to 18 October 2016. Washington, DC: American Society for Microbiology, doi:10.1128/AAMCol.17–18Oct.2016.https://www.ncbi.nlm.nih.gov/ books/NBK447255/. Dulbecco, R., 1952. Production of plaques in monolayer tissue cultures by single particles of an animal virus. Proceedings of the National Academy of Sciences of the United States of America 38, 747–752. Hansen, G.T., Moore, J., Herding, E., et al., 2018. Clinical decision making in the emergency department setting using rapid PCR: Results of the CLADE study group. Journal of Clinical Virology 102, 42–49. Iwanowski, D., 1892. Über die mosaikkrankenheit der tabakspflanze. Bulletin Scientific/Académie Impériale des Sciences de Saint Petersburg 35, 67–70. Jenny, S.L., Hu, Y., Overduin, P., Meijer, A., 2010. Evaluation of the xpert flu a panel nucleic acid amplification-based point-of-care test for influenza A virus detection and pandemic H1 subtyping. Journal of Clinical Virology 49, 85–89. Kaku, N., Hashiguchi, K., Iwanaga, Y., et al., 2018. Evaluation of FilmArray respiratory panel multiplex polymerase chain reaction assay for detection of pathogens in adult outpatients with acute respiratory tract infection. Journal of Infection and Chemotherapy 24, 734–738. Koczula, K.M., Gallotta, A., 2016. Lateral flow assays. Essays in Biochemistry 60, 111–120. Lisby, G., 1998. Application of nucleic acid amplification in clinical microbiology. In: Meltzer, S. (Ed.), PCR in Bioanalysis, series: Methods in Molecular Biology. New Jersey: The Humana Press Inc, pp. 1–29. Lisby, J.G., Andersen, D.T., Chen, M., et al., 2017. Anbefalinger vedrørende implementering og anvendelse af Point-of-Care teknologi til diagnostik af infektionssygdomme. Available at: https://dskm.dk/wp-content/uploads/2016/08/POC-Rapport-fra-DSKMs-Udvalg.pdf (accessed 5.10.2020). Mackay, I.M., Arden, K.E., Nitsche, A., 2002. Real-time PCR in virology. Nucleic Acids Research 30, 1292–1305. Nakane, P.K., Pierce Jr, G.B., 1966. Enzyme-labelled antibodies: Preparation and application for the localization of antigens. Journal of Histochemistry and Cytochemistry 14, 929–931. Nie, S., Roth, R.B., Stiles, J., et al., 2014. Evaluation of Alere i Influenza A&B for rapid detection of influenza viruses A and B. Journal of Clinical Microbiology 52, 3339–3344. Niesters, H.G., 2002. Clinical virology in real time. Journal of Clinical Virology 25 (Suppl 3), S3–s12.

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Parcina, M.P., Schneider, U.V., Visseaux, B., et al., 2020. Multicenter evaluation of the QIAstat respiratory panel – A new rapid highly multiplexed PCR based assay for diagnosis of acute respiratory tract infections. PLOS One 15, e023018. Paz-Bailey, G., Rosenberg, E.S., Doyle, K., et al., 2018. Persistence of zika virus in body fluids – Final report. New England Journal of Medicine 379, 1234–1243. Pedersen, C.J., Rogan, D.T., Yang, S., Quinn, J.V., 2018. Using a novel rapid viral test to improve triage of emergency department patients with acute respiratory illness during flu season. Journal of Clinical Virology 108, 72–76. Poritz, M.A., Blaschke, A.J., Byington, C.L., et al., 2011. FilmArray, an automated nested multiplex PCR system for mulit-pathogen detection. Development and application to respiratory tract infection. PLOS One 6, e26047. Prince, H.E., Lapé-Nixon, M., 2014. Role of cytomegalovirus (CMV) IgG avidity testing in diagnosing primary CMV infection during pregnancy. Clinical and Vaccine Immunology 21, 1377–1384. Rathamat-Langendoen, J., Groenewoud, H., Kuijpers, J., Melchers, W.J.G., van der Wilt, G.J., 2019. Impact of molecular point-of-care testing on clinical management and inhospital costs of patients suspected of influenza or RSV infection: A modeling study. Journal of Medical Virology 91, 1408–1414. Reed, W., 1902. Recent researches concerning the etiology, propagation and prevention of yellow fever, by the United States Army Commission. Journal of Hygiene 11, 101–119. Saiki, R.K., Gelfand, D.H., Stoffel, S., et al., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491. Saiki, R.K., Scharf, S., Faloona, F., et al., 1985. Enzymatic amplification of b-Globin sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354. Shengchen, D., Gu, X., Fan, G., et al., 2019. Evaluation of a molecular point of care testing for viral and atypical pathogens on intravenous antibiotic duration in hospitalized adults with lower respiratory tract infection: A randomized clinical trial. Clinical Microbiology and Infection 25, 141. Smith, T.F., Wold, A.D., Epsy, M.J., 1992. Diagnostic virology – Then and now. Advances in Experimental Medicine and Biology 312, 191–199. Tan, E., Erwin, B., Dames, S., et al., 2008. Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities. Biochemistry 47, 9987–9999. Tanriverdi, S., Chen, L., Chen, S., 2010. A rapid and automated sample-to-result HIV load test for near-patient application. Journal of Infectious Disease 201 (Suppl), S52–S58. Vecino-Ortiz, A.I., Goldenberg, S.D., Douthwaite, S.T., et al., 2018. Impact of a multiplex PCR point-of-care test for influenza A/B and respiratory syncytial virus on an acute pediatric hospital ward. Diagnostic Microbiology and Infectious Disease 91, 331–335. Voermans, J.J., Seven-Deniz, S., Fraaij, P.L., et al., 2016. Performance evaluation of a rapid molecular diagnostic, MultiCode based, sample-to-answer assay for the simultaneous detection of Influenza A, B and respiratory syncytial viruses. Journal of Clinical Virology 85, 65–70. Vos, L.M., Weehuizen, J.M., Hoepelman, A.I.M., et al., 2019. More targeted use of oseltamivir and in-hospital isolation facilities after implementation of a multifaceted strategy including a rapid molecular diagnostic panel for respiratory viruses in immunocompromised adult patients. Journal of Clinical Virology 116, 11–17. Weller, T.H., Robbins, F.C., Enders, J.F., 1949. Cultivation of poliomyelitis virus in cultures of human foreskin and embryonic tissues. Proceedings of the Society for Experimental Biology and Medicine 72, 153–155. Wirden, M., Larrouy, L., Mahjoub, N., et al., 2017. Multicenter comparison of the new COBAS 6800 system with COBAS Ampliprep/COBAS Taqman and Abbott RealTime for the quantification of HIV, HBV and HCV viral load. Journal of Clinical Virology 96, 49–53. Woodberry, M.W., Shankar, R., Cent, A., Jerome, K.R., Kuypers, J., 2013. Comparison of the Simplexa FluA/B & RSV direct assay and laboratory-developed real-time PCR assays for detection of respiratory virus. Journal of Clinical Microbiology 51, 3883–3885. Wouters, Y., Keyaerts, E., Rector, A., et al., 2019. Comparison of the Idylla™ respiratory (IFV-RSV) panel with the GeneXpert Xperts Flu/RSV assay: A retrospective study with nasopharyngeal and midturbinate samples. Diagnostic Microbiology and Infectious Disease 94, 33–37.

Relevant Websites https://www.globalpointofcare.abbott/en/product-details/id-now.html Abbott Global Points of Care. ID NOW™. http://www.rapidtest.ca/products Biocan Diagnostics Inc. https://www.cdc.gov/labquality/waived-tests.html Centers for Disease Control and Prevention. Waived Tests.

Standardization of Diagnostic Assays Sally A Baylis and C Micha Nübling, Paul-Ehrlich-Institute, Langen, Germany Wayne Dimech, National Serology Reference Laboratory, Fitzroy, VIC, Australia r 2021 Elsevier Ltd. All rights reserved.

Glossary International Standard Internationally recognized measurement standard established by the WHO.

International Unit Value given to WHO International Standards. WHO World Health Organization.

Introduction In clinical virology laboratories, diagnostic testing is performed to confirm the cause of viral infections, whether they are acute or chronic or represent reactivation of latent infections. The diagnostic test results help inform physicians about the best course of patient management, for example, whether to prescribe antiviral drugs or treat patients with specific immunoglobulins or administer vaccines prophylactically or therapeutically. Assay standardization is essential in order to compare diagnostic testing results produced by different laboratories, ensure robust testing procedures as well as to develop clinical practice guidelines where diagnostic test results such as viral loads, for example, are used in clinical decision making. In this article, we describe different aspects of standardization including the development and use of reference materials, the role of written standards as well as principles of quality assurance. Standardization has been applied extensively to the detection of viral DNA and RNA by nucleic acid amplification technology (nucleic acid testing (NAT) or nucleic acid amplification testing (NAAT)). In the case of serological assays used to detect viral antigens or specific antibodies induced following viral infections, standardization has been useful for a more limited range of assays. Testing of an immune response to an organism is not straightforward. When testing for antibodies against human immunodeficiency virus (HIV), for example, the different antigens derived from particular viral subtypes and mutations, the immune response to distinct antigens and the unique development of an individual’s immune response, makes the application of standardization difficult. In contrast, for serological assays that detect and quantify viral antigens, such as HIV p24, conventional approaches to standardization are more applicable.

Physical Standards (Reference Materials) Because of the complex, biological nature of diagnostic specimens tested in clinical virology laboratories, they cannot be evaluated by physicochemical means. Instead bioassays, measuring a functional biological activity are necessary. Reference materials, also termed standards, are essential for the control of such bioassays. These reference materials may be measurement standards, applied to quantitative assays. They can also be controls used qualitatively or quantitatively to monitor day-to-day performance of a test. The most important measurement standards used in virology laboratories are World Health Organization (WHO) International Standards (ISs) which have defined concentrations of specific analytes. The WHO ISs are considered “higher order standards” for biological assays and are critical for calibration of different assays using a common material and for ensuring calibration traceability. Concentrations of ISs are expressed in International Units (IUs) per ml. Because of the biological nature of WHO ISs results cannot simply be reported in International System of Units (SI)-related units such as moles, hence the adoption of the IU. The ISs are used to directly calibrate secondary standards, including ones that used at a regional or national level, calibrators used for commercially available assays, as well as control materials (e.g., working standards/reagents and run controls, independent of controls supplied by manufacturers of diagnostic kits) used to monitor assay performance. In turn, tertiary standards are directly traceable to secondary standards (Fig. 1). Collectively, these standards enable the comparison of results among different assays and different laboratories. Uncertainty values are not assigned to WHO ISs, since IU are arbitrary units and variance is associated the contents in the vial of standard. For secondary and tertiary standards there will be a measurement uncertainty when calibration is performed (Fig. 1). The WHO also produces reference materials termed International Reference Panels (IRPs) and no unitages are assigned to these materials. IRPs designed for NAT assays or antigen assays include different genotypes or strains of a virus that might be encountered globally and may challenge current assays. By analogy, IRPs designed for antibody assays reflect sera of patients who developed antibodies against different virus genotypes or strains or a different times post-infection. The WHO reference reagents (RRs), are usually interim standards with a unitage defined in units rather than IU. The types of reference material and their role in the clinical virology laboratory are summarized in Table 1.

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Fig. 1 Hierarchy of standards. The relationship between ISs and secondary and tertiary standards is shown, together with their uses. Reproduced, in modified form, with the permission of the Journal of Clinical Microbiology.

Table 1

Types of standard and their role in the clinical virology laboratory

Type of standard

Role

WHO IS

Highest order reference material. Used for calibration of secondary standards and evaluation of critical assay parameters such as limits of detection.

WHO IRP

Ensuring adequate detection of genetically and antigenically distinct strains of important viral pathogens and associated antibodies.

Secondary standards

National and regional standards traceable to the WHO IS with value assigned in IU. Manufacturer’s calibrators used for control of commercial assays with value assigned in IU. Working standards/working reagents used as routine external run controls for assays, assigned a value or range in IU.

Tertiary standards

Calibrators traceable to a secondary standard calibrated in IU. Working standards/working reagents used as routine external run controls for assays, assigned a value or range in IU traceable to a secondary standard.

Working standards/working reagents

Used as routine external run controls for assays, no unitage assigned or given an arbitrary value if no WHO IS exists.

Preparation and Evaluation of Standards The decision to develop a specific standard is based upon public health need and the prioritization of WHO standards is determined through discussion with the medical scientific communities worldwide and in consultation with the WHO Expert Committee on Biological Standardization (ECBS). The process for the preparation and evaluation of WHO standards, including their replacement is shown in Fig. 2. The starting materials for the preparation of any kind of standard are carefully pre-qualified to ensure they are fit for purpose. In the case of ISs used for NAT, they should contain the specific virus in sufficiently high titer, the strain should be detected in commonly used assays and the final formulation should not affect NAT assay performance. Similarly, starting materials for viral antigens or antibodies for IS are prepared at a titer that will enable the calibration of secondary standards. The ISs are usually lyophilized in order to maintain stability of the reference material, allowing worldwide distribution at ambient temperatures therefore avoiding cold chain difficulties. Batches of IS are tested to ensure that volume dispensed during filling is consistent and that the homogeneity and potency of the analyte after filling and lyophilization is adequate. After lyophilization, vials are filled with nitrogen and residual moisture in the freeze-dried residue (cake) is determined together with

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Fig. 2 Process for the development of WHO ISs, RRs and IRPs. The procedure is shown from the identification of a scientific need to develop a standard to the establishment of the standard and ultimately to its replacement. cIS, candidate IS. Reproduced, in modified form, with the permission of the Journal of Clinical Microbiology.

the levels of oxygen present in the final vial to ensure that levels do not exceed predefined specifications that may impact on the stability. Stability of IS must be evaluated to ensure that there is no degradation of the material during storage and shipment that would affect the unitage assigned to the analyte and, in turn, the value assigned to any secondary or tertiary standards. Occasionally, it has been necessary to add stabilizing agents to certain NAT standard preparations, especially for some RNA viruses that are more susceptible to degradation by nucleases. Long-term stability is predicted by accelerated degradation studies with incubation of the candidate material at higher temperatures, for defined time periods, together with continuous monitoring by testing of the IS after its establishment at defined time intervals. Details of the specifications are described in the WHO Recommendations for the preparation, characterization and establishment of international and other biological reference standards (2004). The WHO ISs are prepared using material that reflects the sample type being tested in an assay, and materials must be assessed for their “commutability” to ensure that their performance in different assays is equivalent to the usual test samples such as patient specimens tested routinely. The property of commutability is particularly affected by the composition of the sample matrix which may vary between the reference material and the routine specimens. The source materials for ISs for NAT are from viraemic plasma from infected donors or patients or by using virus stocks produced in cell culture, which may be heat-inactivated, and spiked into human plasma. For NAT standards, the use of intact virus particles is important because such preparations undergo nucleic acid extraction as well as the amplification and detection steps in a NAT assay. Materials made from in vitro RNA transcripts or plasmids do not control for the extraction step or reflect the complexities of viral genome structures and have not proved to be helpful in assay harmonization. Antigen standards have also been prepared from plasma from infected patients or from virus propagated in cell culture or from recombinant sources.

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Developing ISs for antibody detection, is more complex as a single reference preparation is not necessarily suitable for all purposes. An infected individual’s antibody response develops over time, with low avidity antibodies being replaced by higher avidity antibodies during the infection course. Furthermore, antibody features (antibody classes; epitopes and antigens targeted by antibodies) depend not only on strain or genotype of the pathogen, but also on parameters like the site and route of infection, inoculum size or infectious dose. Last but not least, the individual antibody response is unpredictable and often highly variable between infected individuals, dependent on different components of the individual immune system. Diagnostic assays may have different designs, e.g., in respect to detect “functional” antibodies (e.g., neutralizing antibodies) or “binding” antibodies, often with different antigens and epitopes used in the individual tests. With all these different aspects to consider, standardization of antibody tests is more difficult. Standardization of antibody assays restricted to a specific viral antigen, for example in the case of hepatitis B virus (HBV), anti-HBV core (anti-HBc), appears more feasible compared to assays detecting total antibodies to HBV: the designs of different anti-HBV assays may differ in the HBV antigens used with the consequence that each measures different subsets of anti-HBV antibodies. The IRPs for antibodies are often comprised of different specimens to reflect the diversity of antibody responses between individuals. Some reference materials have been prepared from therapeutic immunoglobulins, raising potential matrix (commutability) issues. The WHO reference materials i.e., ISs, IRPs and RRs are evaluated in international studies typically involving reference laboratories, clinical laboratories, regulatory organizations as well as manufacturers of diagnostic kits and reflecting different globally available assays. Such studies allow the head-to-head comparison of different assays used globally, including both commercially available tests as well as laboratory-developed tests (LDTs) or in-house assays. Potencies of the candidate reference materials are determined by the participants. Fig. 3 shows an example of the range of potencies reported by laboratories for the IS for hepatitis E virus (HEV) RNA. Potencies were determined by a mixture of qualitative and quantitative assays with a wide range of reported values. However, expressing results of other study samples against the candidate IS greatly reduced the variation in the measured potencies reported by participants. This reduction in variation is one of the main aims of the collaborative study because it demonstrates that the use of a reference material can improve harmonization of the results of diagnostic assays. The outcome of the studies are published on the WHO website to allow time for comments before it is presented to the WHO ECBS which endorses the proposed unitage and confirms whether materials are established as ISs, IRPs etc. Several thousand vials of each IS are prepared, once the stocks near exhaustion the IS must be replaced. The continuity of the IU assignment is important and replacement material must be carefully selected to ensure they are similar to the previous IS or have been demonstrated to perform in a similar way. In some cases, where large volumes of the original source material are available this process is easier. Preparation of the replacement IS follows the same manufacturing procedures as the establishment of a new IS. In the collaborative study the replacement material is evaluated alongside the existing IS in order to determine the potency of the new standard.

Written Standards and Guidelines In order to standardize diagnostic testing, the availability and correct implementation of reference materials is essential; underpinning this are documentary, written standards and guidelines relating to development of tests and their routine use in the diagnostic laboratory. With respect to the reference materials themselves, the WHO ECBS publishes a Technical Report Series (see “Relevant Websites section”) which records opinions of the committee with respect to the establishment of new reference materials and reviews strategic matters in the field of standardization including guidelines outlining the procedures used for the development and establishment of WHO ISs and IRPs together with guidance on the calibration of secondary and tertiary standards. The WHO also publishes biological standardization study reports (BS documents) which document each standardization project, including an analysis of the data generated during the collaborative study, and serve as a basis for the decision of the WHO ECBS to establish new and replacement standards. The International Organization for Standardization (ISO) is a non-governmental, independent standard setting body that aims to improve quality, reliability and safety of products and services by providing common standards around the world. Several ISO standards are important in the standardization of diagnostics. In particular, ISO 17511:2003 which comprehensively deals with metrological traceability of values assigned to calibrators and control materials. Supporting standardization is ISO 15189:2012 which sets out the requirements for quality management and competence in medical laboratories and is now mandatory in many countries. External quality assurance (EQA) schemes are important in independently demonstrating adequate quality of testing in laboratories. The materials used in EQA samples should be commutable and traceable to established standards and reference materials as defined in ISO 17043:2010 which provides guidance on the development and operation of EQA programmes. The Clinical and Laboratory Standards Institute (CLSI) is a voluntary organization developing clinical and laboratory practices globally, including, for example, guidance concerning commutability of reference materials. The International Laboratory Accreditation Cooperation (ILAC) is an international organization for accreditation bodies for testing and calibration laboratories and produces documents on measurement traceability and selection of reference materials. There are other guidelines and regulations relating to in vitro diagnostics either at a national or a regional level worldwide. The guidelines described above are not an exhaustive list of written standards, but provide some of the most important sources of information and are summarized in Table 2.

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Fig. 3 Harmonization of data by expressing potencies of a sample against a WHO IS. The top two panels show absolute mean values of the WHO IS for HEV RNA (left) and a blinded replicate sample S2 (right). By expressing the potency of S2 against the IS, the agreement is much improved between laboratories (bottom left). White indicates data reported from quantitative assays and orange indicates qualitative assays. The number of laboratories is indicated on the vertical axis. Laboratory code numbers are indicated in the respective boxes. Reproduced, in modified form, with the permission of Emerging Infectious Diseases.

Quality Assurance In the diagnostic virology laboratory, quality assurance (QA) is a planned and systematic set of activities designed to ensure confidence in the results of different diagnostic assays. The QA process includes the establishment of a quality management system (QMS) setting out the scope of activities, policies, procedures, documented information and resources needed for implementation

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Table 2

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Examples of written standards

World Health Organization WHO Technical Report Series 932 WHO Technical Report Series 1004

World Health Organization recommendations for the preparation, characterization and establishment of international and other biological reference standards Manual for the preparation of secondary reference materials for in vitro diagnostic assays designed for infectious disease nucleic acid or antigen detection: calibration to WHO international standards

International Organization for Standardization ISO 15189:2012 Medical laboratories – Requirements for quality and competence ISO/IEC 17025:2017 General requirements for the competence of testing and calibration laboratories ISO 17511:2003 In vitro diagnostic medical devices – Measurement of quantities in biological samples – Metrological traceability of values assigned to calibrators and control materials ISO/IEC 17043:2010 Conformity assessment – General requirements for proficiency testing CLSI Standard: EP30, 1st Characterization and Qualification of Commutable Reference Materials for Laboratory Medicine Edition European Commission Regulation (EU) 2017/746 of the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical devices Commission Decision of 3 February 2009 amending Decision 2002/364/EC on common technical specifications for in vitro-diagnostic medical devices (2009/108/EC) US Food and Drug Administration

https://www.fda.gov/medical-devices/ivd-regulatory-assistance/overview-ivd-regulation#9 https://www.fda.gov/medical-devices/device-advice-comprehensive-regulatory-assistance

and maintenance of the testing service. QA measures should include the use of standard operating procedures (SOPs) and associated documentation; correct handling of samples to ensure their appropriate storage and traceability, calibration and preventive maintenance of equipment, the use of designated laboratories, and the use of pre-qualified and in-date reagents. Operators should be suitably trained in technical procedures as well as specific assays, be competent in data interpretation, and able to undertake remedial/corrective actions where necessary. Audits and reviews are performed to ensure adequate performance and identify any issues and areas for improvement.

The Use of Controls Assessing an assay’s performance over time is achieved by testing of run controls and monitoring the test results. Controls for monitoring assays include ones provided in diagnostics kits supplied by the kit manufacturers – these typically include positive and negative controls or controls for quantification. Other controls, termed external run controls are provided by other organizations and represent an independent means of monitoring performance. All assays experience variation. The sources of variation are derived from the equipment and consumables used, operators, changes in calibration of instruments, transport and storage of reagents and general environmental conditions such as temperature and humidity. For serological assays, changes in reagent lot numbers are the greatest source of normal variation. The run controls are tested frequently, usually on a daily or per test run basis, and the results plotted on a Levey-Jennings (LJ) chart (Fig. 4). In a well-designed run control program, samples known to be reactive at medical decision points are used. Preferably they are of known homogeneity and stability, as are controls registered as in vitro diagnostic devices. Ideally, QC samples are calibrated against an IS (where one is available). This is usually the case in nucleic acid testing QCs. If this is not possible, the manufacturer should ideally reserve sufficient stock material to allow minimal QC lot-to-lot variation, releasing each new batch against in-house QC reference materials. By achieving consistency of QC reactivity from lot-to-lot, it is possible to monitor the performance of the assay over a long period of time. If the QC sample is calibrated against the corresponding IS and is proven to be commutable with patients samples, the results of QC testing can measure not only the imprecision of the test system (i.e., the standard deviation (SD) or coefficient of variation) but it can also measure the bias of the assay (i.e., how far removed the mean of the QC results are from the QC assigned value). These metrics are valuable in estimating the measurement of uncertainty of the assay. Critical to a run control program is determining the acceptance range of test QC test result. Acceptance limits set too tight will falsely reject test runs leading to unnecessary investigations, whereas limits set too broadly may result in incorrect results being reported and defects in the test systems not being identified. Traditionally, the mean and SD of QC results are used to determine acceptance limits. International guidelines suggest using 20 QC test results and set the acceptance limits at mean 7 2 SDs; and they apply a set of QC “rules”, called Westgard Rules, to detect “out-of-control” results. Although this approach works well for clinical chemistry, it is not applicable to infectious disease testing, especially serology, due to changes in reactivity of the QC sample due to reagent lots (Figs. 5 and 6). An alternative method for calculating acceptance limits has been published. QConnect Limits (see “Relevant Websites section”) use historical data from many laboratories using the same QC sample and assay

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Fig. 4 Results of a QC sample tested repeatedly over time plotted on a LJ chart. The vertical axis represents the signal to cut-off values of the QC sample. The horizontal axis represents the date of testing. The results reported over the period are all within the mean 7 2 standard deviations (indicated by upper and lower horizontal lines) of the QC test results displayed. The mean value is represented by the horizontal line in the middle of the plot.

combination (i.e., peer group). By using data from multiple laboratories over a long period of time, all sources of “normal” variation can be accounted for and more robust acceptance criteria established.

Batch Testing Commercial assays undergo testing by manufacturers to ensure adequate performance before they are released onto the market. In some regions of the world, regulatory authorities perform independent testing of assays before they are marketed as well as evaluating different batches during the lifetime of a particular product. In Europe, IVDs deemed to be high risk i.e., those where the consequences of a false diagnosis could cause serious harm both to the patient and to the community include tests for HIV and hepatitis B and hepatitis C. Independent batch testing is done for these high risk IVDs in some European countries using panels of well-characterized samples from patients or blood donors. Assays must show consistent performance, for example adequate sensitivity should be maintained, before new batches are released on the common market. In addition, technical files documenting the design, development, performance and manufacture of these high risk IVDs are independently reviewed by Notified Bodies who are involved in the conformity assessment of IVDs. With the introduction of new regulations (laws) concerning IVDs in Europe, verification of the performance of high risk IVDs by specialised reference laboratories will be a legal requirement before kits receive a EC declaration of conformity (i.e., CE-mark). Similar procedures are in place in the USA (see “Relevant Websites section”) and elsewhere.

External Quality Assessment/Proficiency Testing EQA schemes, also referred to as proficiency testing (PT) or ring trials, are an important part of the overall standardization process. The EQA organizers distribute the same materials (which are usually blinded) to participating laboratories. The laboratories test

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Fig. 5 Results of an anti-hepatitis C virus antibody QC sample tested repeatedly over time. A significant decrease in reactivity of the QC sample was associated with the introduction of a specific reagent lot (orange arrow). The results (signal to cut-off value on the vertical axis) of the QC sample tested on the affected reagent lot were outside the mean 7 2 SDs (upper and lower bold lines) of the QC test results displayed. Reagent lots are indicated at the top of the figure. The horizontal axis represents the date of testing.

the samples using routine methods and return results to the organizer. In some schemes associated metadata, such as reagent lot numbers and expiry dates are also collected by the organizer for analysis. The participation in EQA schemes provides laboratories with an objective means of assessing and demonstrating the reliability of the results they are producing. The EQA scheme covers the overall performance of a laboratory, including the entire process from receipt and storage of the samples, the laboratory testing, the interpretation and transcription of the data generated, to the reporting of results. Failure at any stage of this process affects the proficiency of the laboratory. Participation in EQA schemes is mandatory for laboratories accredited to ISO 15189:2012 and is important for laboratories seeking accreditation.

Laboratory Accreditation Accreditation of laboratories to ISO 15189:2012 or ISO/IEC 17025:2017 provides objective and internationally recognized evidence of a level of quality. ISO accreditation ensures that the laboratories have robust and well-documented procedures and that test systems employed are fit for purpose, validated and under control. When a previously validated assay (including commercial assays used in strict accordance with the manufacturer’s instructions for use) is introduced into use, verification is required before bringing the assay into routine use to demonstrate their utility. Validation protocols should be prospective i.e., they should set acceptance limits for assay performance which should be verified by the validation experiments and demonstrate that a particular procedure is suitable for its intended purpose. Once an analytical method has been established, a protocol for validation of the analytical procedure can be prepared. The analytical method should yield consistent results under standard operating conditions, critical points should be known, and robustness studies should be performed as part of the validation exercise. Upon completion of the validation experiments, the results are formally documented. Only if an analytical procedure meets the acceptance criteria set out in the validation protocol is the

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Fig. 6 Changes in QC sample reactivity for a combined serological assay detecting both anti-HIV antibodies and HIV p24 antigen. The changes are a consequence of changes in reagent lots. Reagent lots are indicated at the top of the figure. The vertical axis represents the signal to cut-off values of the QC sample. The horizontal axis represents the date of testing.

method considered validated. Validation includes analysis of performance characteristics such as analytical sensitivities (e.g., limit of detection, limit and range of quantification determined using reference materials where available), specificities as well as accuracy (closeness to an accepted value) and precision (how repeatable/reproducible a measurement is). Examples of accuracy and precision of measurements are shown in Fig. 7 demonstrating good to more variable and poor performance. Documented evidence of staff training and competence, instrumentation commissioning, maintenance and monitoring, and standardised procedures need to be in place. Accredited laboratories are required to identify, record and investigate any deficiencies detected and undertake a root cause analysis of the issues and implement corrective actions, and demonstrate the effectiveness of the action taken. All suppliers and subcontractors are managed under the QMS. All accredited laboratories undertake selfassessment in the form of internal audits and are subjected to external audits by their accrediting body and other stakeholders periodically. Similar regulatory requirements are in place in other jurisdictions such as the USA Clinical Laboratory Improvement Amendments (CLIA) regulations or local State-based regulations. In some parts of the world, for example Africa, there are frameworks in place to enable laboratories to work towards accreditation, these include Strengthening Laboratory Management Toward Accreditation (SLMTA) and Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA).

Commercial and Laboratory-Developed tests The availability of commercial assays which have been granted regulatory approval, such as clearance by the US Food and Drug Administration or EC declaration of conformity (i.e., have a CE-mark) in Europe, attesting to a certain level of quality have helped improve standardization in the diagnostic virology laboratory. This can be seen in the results from EQA studies where more

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Fig. 7 Precision and accuracy of measurement procedures. The upper left panel demonstrates the ideal situation where a measurement is both accurate and precise – hitting the target directly in the middle. In the case of the upper right panel, although the measurements are very precise they are off target i.e., they are not accurate. In the bottom left panel, the measurements are accurate, but they are not precise. Finally, in the lower right panel, the measurements are scattered being neither precise nor accurate.

consistent results are reported following the introduction of commercial assays over time. This is especially true once assays are calibrated in IU. Laboratory developed tests (LDTs) need to undergo extensive verification and validation before they are brought into routine use. IS are important for the calibration of LDTs and in the preparation of calibrated run controls, traceable to the IS, used to ensure consistent assay performance. Fig. 8 illustrates the steps involved in the introduction of an assay from initial evaluation and verification, through validation and routine clinical testing. The interplay between QC and EQA procedures ensure adequate test performance and identify out of specification test results which lead to quality improvements.

Virus Isolation Although used less frequently nowadays, virus isolation or culture is still in use in some diagnostic virology laboratories. The origins of the cell lines and tissue cultures used in isolation procedures should be defined to ensure identity and traceability as well as passage number. For example, continuous cell lines can be obtained from organizations such as the American Type Culture Collection and the European Collection of Authenticated Cell Cultures. The virus detection limit is an important validation parameter; however, this is likely to be specific for each indicator cell line and virus and may well vary for different strains. It is important to verify that the test matrix does not interfere with virus detection, and pre-dilution of samples may be necessary e.g., for stool or urine specimens; appropriate interference and negative controls should be included during isolation procedures. Virus isolation exercises may be offered by some EQA providers.

The Use of International Standards in Clinical Practice The standardization of NAT testing is important in clinical practice, not only for disease diagnosis, but also in decisions concerning when to implement treatment with antiviral drugs and how long treatment should continue. The first WHO IS developed for NAT testing was for hepatitis C virus (HCV). While this standard was established in 1997 to enable harmonized regulations for screening of plasma donations for blood product manufacturing, it soon found utility in clinical virology laboratories. In the European Association for the Study of the Liver (EASL) Recommendations on Treatment of Hepatitis C 2018, it is stated that HCV RNA assessment (in both acute and chronic cases) should be made by reliable, sensitive assays, and HCV RNA levels should be expressed in IU/ml using assays with a lower limit of detection r15 IU/ml. However, the vast majority of patients with an indication for anti-HCV therapy have an HCV RNA level above 50,000 IU/ml. For the treatment of acute hepatitis C in low- or middle-income countries and in specific settings in high-income countries, a qualitative HCV RNA assay with a lower limit of detection r1,000 IU/ml can be used to provide broad affordable access to HCV diagnosis and care. The

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Fig. 8 Assay implementation in a diagnostic laboratory, from initial evaluation and verification, through validation and routine clinical testing. QC and EQA procedures ensure adequate test performance and identify out of specification test results which lead to quality improvements. Reproduced in modified form with permission by Professor H. Niesters.

EASL recommendations include further parameters including lower detection limits (r15 IU/ml) indicating patient recovery following antiviral therapy, for example. Patients with chronic HBV infection are at increased risk of progression to cirrhosis and hepatocellular carcinoma (HCC). In the EASL 2017 Clinical Practice Guidelines on the management of HBV infection, HBV DNA levels exceeding 2,000 IU/ml, elevated alanine transaminase (ALT) and/or at least moderate histological lesions are an indication for treatment together with all cirrhotic patients with detectable HBV DNA. In patients where the levels of HBV DNA exceed 20,000 IU/ml, and ALT levels exceed 2  the upper limit of normal, treatment should commence regardless of the degree of fibrosis. Human cytomegalovirus (HCMV) is a major pathogen for immunocompromised patients, particular transplant recipients. The use of HCMV NAT is important for surveillance to guide antiviral treatment, and for therapeutic monitoring. In the “Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation”, reporting levels of HCMV DNA by NAT testing in IU/ml is recommended. In the future, it is hoped that it will be possible to define agreed thresholds to initiate pre-emptive therapy. There are several ISs for infectious disease antigens, such as HBV surface antigen (HBsAg), HIV-1 p24 as well as HCV core antigen. These have been used to calibrate immunoassays and they represent a cost-effective way of monitoring in infected patients undergoing therapy compared to NAT. However, quantitative results of antigen tests are used less frequently nowadays in clinical decision making, having been superseded by viral load testing by NAT. However, HBsAg testing is very widely used in screening blood donors to test and exclude donors with HBV infection. The level of antibody in a patient is not often used in the clinical setting. Qualitative testing for antibodies uses various processes to detect the presence of the immune reaction; however, a rise in the concentration of antibodies may be useful in confirming a recent infection. Furthermore, detection of antibody classes may provide insight into infection course (e.g., IgM in recent infection). Determination of antibody function may provide relevant information on immunity (e.g., neutralisation assays and protection against potential re-infection). Sometimes levels of immunity in patients are determined by assays calibrated with a WHO IS and the immunity threshold defined in IU/ml. However, this approach is sometimes questionable and standardization using a single reference material has proved challenging in certain circumstances, for example, with assays designed to measure antibodies against a single antigen or neutralization of virus infectivity. Several of the antibody standards that are available have been prepared to set a specification for therapeutic immunoglobulins and may not be the most suitable materials for standardization in the clinical laboratory setting.

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Conclusions The standardization of diagnostics in the clinical virology laboratory is driven by a combined approach using physical reference materials as well as written standards and adherence to quality assurance procedures. The WHO ECBS ensures that standards suitable for their purpose and will be replaced in a timely manner with the assurance of the continuity of the unitage. Discussions with the clinical virology community are essential to identify where there is a diagnostic need for new standards.

Further Reading Baylis, S.A., Wallace, P., McCulloch, E., Niesters, H.G.M., Nübling, C.M., 2019. Standardization of nucleic acid tests: The approach of the World Health Organization. Journal of Clinical Microbiology 57. (pii: e01056-18). Holden, M.J., Madej, R.M., Minor, P., Kalman, L.V., 2011. Molecular diagnostics: Harmonization through reference materials, documentary standards and proficiency testing. Expert Review of Molecular Diagnostics 11 (7), 741–755. doi:10.1586/erm.11.50. International Conference on Harmonization. 1994. Validation of Analytical Procedures: Text and Methodology (CPMP/ICH/381/95, ICHQ2[R1]). Miller, G.W., Myers, G.L., Lou Gantzer, M., et al., 2011. Roadmap for harmonization of clinical laboratory measurement procedures. Clinical Chemistry 57, 1108–1117. Wallace, P., 2021. Quality assurance in the clinical virology laboratory. In: Encyclopedia of Virology, 4th ed. World Health Organization, 2011. Laboratory Quality Management System. Available at: https://www.who.int/ihr/publications/lqms_en.pdf. World Health Organization, 2006. Recommendations for the Preparation, Characterization and Establishment of International and Other Biological Reference Standards (Revised 2004). Technical W.H.O. Report Series no. 932, pp. 73–131. Available at: http://www.who.int/immunization_standards/vaccine_reference_preparations/TRS932Annex %202_Inter%20_biol%20ef%20standards%20rev2004.pdf. World Health Organization. 2017. WHO Manual for the Preparation of Secondary Reference Materials for In Vitro diagnostic Assays Designed for Infectious Disease Nucleic Acid or Antigen Detection: Calibration to WHO International Standards. World Health Organisation Technical Report Series no. 1004, pp. 389–455. Available at: http://www. who.int/bloodproducts/norms/SecStandManWHO_TRS_1004_web_Annex_6.pdf?ua_1.

Relevant Websites https://www.fda.gov/medical-devices/ivd-regulatory-assistance/overview-ivd-regulation Overview of IVD Regulation. https://www.nrlquality.org.au/qc-limits QC Limits. https://www.who.int/biologicals/technical_report_series/en/ Technical Report Series (TRS).

Quality Assurance in the Clinical Virology Laboratory Paul Wallace and Elaine McCulloch, Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary AMR Analytical measurement range. Assay Drift. LoD Limit of detection.

LoQ Limit of quantitation. Probit Analysis. Reportable Range.

The Principles of Quality Assurance and Quality Management The principles of quality assurance (QA) and quality management (QM) have their origins in manufacturing and in particular the automotive industry. They were primarily used to support the stream-lining of processes and procedures, with the aim of increasing productivity, while reducing costs but maintaining the quality of the product delivered to the customer. Within the clinical laboratory, the concepts of QA and QM where first introduced in hematology and clinical chemistry by Belk and Sunderman in the late 1940s/early 1950s in an effort help reduce the high number of diagnostic errors observed in relation to the handling and testing of patient specimens which ultimately could have an impact on patient care. These concepts and general principles formed the QA and QM foundation commonly used within the clinical virology laboratory which continue to evolve in order to meet present regulatory demands and assure the safety and effectiveness of the virology testing service. The key components of quality assurance within a laboratory’s quality management system (QMS) are shown diagrammatically in Fig. 1. As well as supporting the whole diagnostic process (pre- analytical to post analytical) to delivery of patient result, the QMS also support the verification, validation and implementation phases of the diagnostic assays used within the virology laboratory. With the implementation of quality principles from other industry sectors comes the adoption of the associated terms and definitions used and one such quality term which has found its way into clinical virology in recent years is Total Quality Management (TQM). In the context of the clinical virology laboratory, TQM provides an alternative description for the integration of all quality parameters and the nurturing of a quality culture followed by all laboratory staff. So that the aim of the laboratory is not only to provide correct results, they also aim to ensure that the correct test is performed on the appropriate specimen, the correct result is obtained and interpreted, and provided to the right patient within a meaningful time frame and using the correct procedures. The goal of the laboratory is, to ensure confidentiality, the safety of the patient, as well as provide a mechanism for continuously monitoring and improving of the diagnostic service. In many countries, clinical virology laboratories and the testing services they provide are required to be accredited and in some regions accreditation is a pre-requisite to regional reimbursement policy. Accreditation also helps the clinical laboratory demonstrate the competence and reliability of its services to its immediate peers within the healthcare environment, the Clinicians who routinely utilize the testing service, and those external to the healthcare environment such as the legal profession. Increasingly, clinical virology laboratories are gaining accreditation to the Internationally recognized standard ISO15189 which was developed specifically for the medical laboratories involved in laboratory testing and examination, although in some country’s laboratories are accredited to the national regulatory framework. The quality management requirements of ISO15189 are also aligned to those in ISO 9001 which provides a basic standard and quality language across diverse industry sectors.

Quality Management System (QMS) In the context of the clinical virology laboratory, the QMS covers all the policies, documented processes, procedures, and records used in order to deliver the diagnostic testing service to the patient under its defined scope of accreditation. This also incorporates the operational environment in which the testing is performed, the equipment, specialist reagents and essential supplies required, as well as the qualification and competence of the staff tasked with conducting the testing service. The complexity of the quality management system depends on the scope of the service and in many larger Hospitals the QMS is managed centrally and covers the whole of the pathology services, Hematology, Blood Transfusion, Biochemistry, Microbiology including virology.

Quality Management Documentation The documentation used within the QMS is usually organized and managed through a clear document hierarchy. The top-level documents include the Quality Manual or Services Manual which stipulate the laboratory’s quality policy and objectives. These are monitored against defined quality performance indicators, such as the monitoring of the time taken to report a test result from receipt of specimen, and ensure that the service is achieving its stated quality intentions. The Quality Manual also sets out the

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Fig. 1 QMS/QA component.

laboratory organization, management roles and responsibilities in relation to the scope of the services provided. This will include the range and type of tests performed, the specimen types handled, the quality control measures and internal quality assurance procedures the laboratory undertakes, as well as the external quality assessment (EQA) schemes it subscribes to. Practical information on how to order the test, referral of tests and contact details for additional clinical and technical advice are also included within the quality manual or service manual. The procedural documents including standard operating procedures (SOPs), equipment operating procedures (EOPs), describe in detail how a specific laboratory activity is to be conducted or how a piece of equipment is to be used, who is authorized to do so, as well as when and where that activity is to be performed or equipment used. The procedural documents also describe the inputs and outputs of the procedure and where this information should be recorded. In addition to operating procedures some laboratories may also have separate specific work instruction documents. These detail the exact steps and methods to be employed when conducting a laboratory activity such as the consecutive tasks required in order to set up for the detection of a particular viral pathogen or the operation of a specific piece of equipment, for example the calibration and maintenance of a real-time Thermal Cycler. In some clinical virology laboratories, having separate work instructions means that they can be held directly within the laboratory for easy access and reference where the activity is taking place. The inputs and outputs of a procedure or specific work instruction are recorded within a Standard Operation Record (SOR). These include paper-based records as well as electronic outputs from equipment or devices within the laboratory such as a thermal cycler or serological autoanalyzer. In these cases, the devices are interfaced with the laboratory and the records are managed through a middleware software such as FlowG (See Relevant Websites section) which collects, collates, and stores the data which can include calibration and control performance data across a range of laboratory platforms. The middleware is also linked to the general Laboratory Information Management System (LIMS) which provides secure storage and management of all the laboratory data and patient results.

Document Control All documents within the clinical virology laboratory QMS need to undergo regular review at the specified intervals on when a particular policy, process, or procedure is changed in order to ensure that they remain fit for purpose and compliant. This is managed through a document control policy which also includes an index of all the documents within the laboratories QMS, who is responsible for each one, and when it is scheduled for review.

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Many clinical laboratories have moved away from a paper-based Quality Management and document control systems towards having a commercially available QMS software management solution such as Q-pulse (See Relevant Websites section) which is able to integrate directly into LIMS and makes the management of laboratory quality assurance information more efficient and compliant with regulatory standards such as ISO15189.

Specimen Management The ability of the clinical virology laboratory to carry out a test and to support a diagnosis depends extensively on the quality of the specimen the laboratory receives and the time it takes to get to the laboratory. Pre-analytical errors such as wrong labeling of the specimen, the incorrect specimen collection device, incorrect specimen type are the most frequently reported sources of error (between 48% and 62%) within the clinical laboratory. In some hospitals or institutes, it is the responsibility of specific clinical virology laboratory staff to provide training on appropriate sample collection, handling, transportation and storage of the clinical specimens to be used. As a result, the clinical virology laboratory details the quality requirements of the specimen types (i.e., serum, plasma, urine, stool, etc) the laboratory is capable of processing in relation to the test repertoire offered under the services it provides. This includes specific instructions to clinicians, general practitioners, and nursing staff on how and when to take the patient specimen as well as the collection device to be used and the minimum volumes required for the test requested. For example, most common serological tests are performed on either serum or plasma and are best tested within 48–72 h after collection in order to prevent degradation of the analytes, unless they are stored and shipped to the laboratory at between 2–81C. In comparison, for the molecular viral load determination of blood borne viruses such as HIV, HCV, and HBV, either EDTA plasma or sodium citrated plasma is preferable to serum as the degradation of nucleic acids has been observed within serum/clotted specimens and may result in the under-reporting of the viral load result. Heparinised specimens are also not recommended for molecular testing as the heparin has been show to inhibit technics such as PCR, although in most modern molecular technologies this does not appear to be a problem. Although all clinical specimens must be regarded as potentially infectious, most laboratories will also have a separate policy for the handling of potentially high-risk samples, such as those suspected of having a viral hemorrhagic fever (VHF). The policy covers the way that these specimens are handled and the labeling to be used. This will depend on risk factors identified and any regional regulatory and health and safety requirements the laboratory must adhere to. However, the sample selection process has been improved significant through the implementation of electronic test requisitions which allow authorized healthcare professionals such as general practitioners to request a test directly through an internet-based portal. This has helped make the process more efficient and standardized, as well as reduces both transcription and translation errors associated with manual processing. Many of the electronic test requisition systems also enable the generation of unique specimen-Patient barcodes which also help reduce the incidences of under labeling (i.e., not providing sufficient patient information in order to support a test requisition) or mislabelling as the additional information is a required field when completing the ‘on-line’ form and in many case is incorporated within the barcode generated.

Transportation and Storage of Specimens Transportation and storage can have a significant impact on the quality of the clinical specimen and hence the accuracy and outcome of the laboratory result. For example, sputum specimens should ideally be tested within 2 h of collection and stool samples within 12 h in order to prevent background bacterial flora growth masking the virologic examination. If this is not possible then the specimens can be stored at 4–81C but only for 48 h. So, it is essential that staff are appropriately trained and follow the correct procedures which are usually defined within the laboratory’s policy on the handling and delivery of laboratory specimens and in line with the requirements of any national Health and Safety at work legislation. The transportation of samples within the hospital or institute where the clinical virology laboratory is located is usually conducted through a trained internal porter service using containers dedicated to the movement of potentially biohazardous specimens. An approved specialist courier service will usually be contracted in order to transport specimens from external sites such as clinics, General Practitioner sites to the clinical virology laboratory. Transportation of specimens by road must comply with the carriage of dangerous goods regulations within that region (See Relevant Websites section), which state the specific packaging conditions that need to be followed dependant on the risk the viral pathogen poses. In general, most specimens such as blood, excreta, secreta, for diagnostic purposes will be transported under the category B classification unless they contain live virus cultures that are known to be life threatening to humans, under this circumstances the specimens should be transported under category A. Exempt patient specimens are those with a minimal likelihood that a viral pathogen is present and include blood transfusion products, and most dried blood spot applications. Where specimens need to be transported by air, the regulations of the International Air Transport Association (IATA) regulations are followed. In addition, where viral specimens are being transported from one country to another for reference testing, assay verification, or quality assurance purposes, etc it is important to verify whether the pathogen is regulated under any regional Human Pathogens and Toxins Regulations which could restrict its exportation and importation. Regulations vary from

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country to country dependant the apparent risk associated with the activities performed and also take into consideration whether the virus is a security sensitive biological agent.

Laboratory Specimen Retention Times Specimen retention defines the length of time clinical specimens will be retained by a clinical virology laboratory. Retained specimens are extremely important to the laboratory for confirmatory testing where required, infection control, and public health investigations, as well as for quality control and new assay evaluation. The specimen retention time will depend on the type and origin of the specimen, the needs of the patient, and also the storage capacity and capability of the laboratory. Monitoring retained specimens with regards to events such as the number of freeze/thaw cycles, viral load over time in storage, is important in assuring the quality of the specimens, particularly if they are going to be used for quality control purposes. Many clinical virology laboratories will utilize software options in order to create an inventory to track the use of important retained clinical specimens.

Personnel Training, Competency, and Continuous Professional Development (CPD) The clinical virology laboratory operates within a highly regulated environment and in many countries the laboratories can only perform their testing service under the appropriate legal licenses. An important aspect of human resource management particularly within the clinical virology laboratory is to ensure that all staff have defined roles and responsibilities, and they are clear on what is expected of them within the daily operation of the clinical laboratory. The structure and organization of the clinical virology human resource is dependant on the size of the laboratory, the range and complexity of the testing service provided, the volume of annual clinical specimens, and whether it is a separate independent entity or it is affiliated to a hospital or institute as part of the total pathology services provided. An organogram for the clinical virology laboratory with supporting narrative outlining the structure and reporting relationships is usually detailed within the Quality Manual and is an essential quality assurance requirement. The head of the laboratory (laboratory Director) will usually be a licensed Medical Virologist either with a Medical Doctorate (MD) or a Senior Clinical Scientist with a MSc, PhD, or equivalent professional qualification specialising in virology. They must ensure that the laboratory has sufficiently trained and competent staff in order to provide the scope of virology testing services specified to the appropriate regulatory standards within their jurisdiction, and where required, in line with their accreditation for example to ISO15189. The laboratory director is usually supported by a consultant clinical medical microbiologist and/or virologist responsible for the overall clinical practice and decision-making process. In line with the requirements of International Standards for Laboratory Accreditation, most laboratories will have a dedicated quality manager and nominated deputy this helps avoid potential conflicts of interest between roles as the quality manager does not have any laboratory operational responsibilities and can therefore assess and monitor the quality aspects of the laboratory impartially. Larger organizations may have a separate quality manager responsible for the whole of the pathology services provided by the hospital. The advantage of this is that they are independent of the clinical virology laboratory and can inspect and advise on quality issues. The disadvantage is they may not be familiar with the technicalities of the virology testing service offered which can potentially lead to misunderstandings. Under the direction of the laboratory director, the clinical virology laboratory will consist of technical supervisors (or technical section leaders) who lead specific laboratory sections such as the molecular, serology, or viral isolation & culture section and are responsible for the daily management of scientific and technical staff within their respective sections. The scientific and technical staff will be responsible for specimen processing, carry out the various testing procedures and other laboratory activities such equipment maintenance and calibration. The laboratory will also have general non-laboratory based clerical support staff performing administrative tasks including medical record keeping. Other laboratory support activities such as stock management, the procurement of laboratory supplies, and diagnostic kits would may be the responsibility of a dedicated laboratory manager or may be shared responsibilities in smaller organizations or hospitals. The human resource (HR) files for each staff member usually contains the most up to date job description, curriculum vitae, qualifications including copies of any certificates/diplomas, and laboratory licenses in regions where this is required. In addition to this training records, and details of any continuous education programs/events the staff member has undertaken are also included within the HR or personnel file, along with any relevant publications. Each laboratory employee should be provided with a clear training plan. This should cover the preliminary laboratory training requirements, initial duties, and responsibilities, for when the individual joints the laboratory. This should be assessed regularly and if necessary, amended in order to accurately reflect the current status of the individual followed by a further personal training and development plan which supports the employee’s continued professional development (CPD) requirements in line with the objectives of the clinical virology laboratory. The effectiveness of training and the competency of employees must also be reviewed regularly in accordance with the duties the employee is expected to perform. This can be achieved through practical laboratory assessments where the test results obtained by the employee on known QC samples or residual material from an external quality assessment cycle, are introduced into the routine workload and used as a way of checking and monitoring the employee’s ability over a defined period of time. Other aspects include monitoring the adherence to laboratory policies and procedures, observing performance when carrying out instrument maintenance and calibration checks, as well as the evaluation of problem-solving and data analysis skills through set

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technical questionnaires. The outcome of any competency assessment exercises are recorded within the employees training matrix and competency assessment records. If a performance issue is identified further training may be required followed by a reassessment of the employees’ competency. Staff training and competency assessment are key elements within a laboratory’s accreditation and some regulatory organizations such as the College of American Pathologists (CAP) in the United States specify the frequency that competency assessments must take place in relation to retraining requirements and test complexity. For example, for employees within the first year of their laboratory duties, competency must be assessed at least twice a year when engaged in moderate to high complexity testing (i.e., molecular testing). Most clinical laboratories will also have an employee handbook which is provided to each staff member when they commence work within the clinical laboratory, this usually includes guidance to the employee on human resource policies such as performance reviews, absence and holiday policies, information technology and security, as well as health and safety aspects related to the clinical virology laboratory working environment.

Laboratory Facilities and Equipment The quality of the test results a clinical virology laboratory provides are largely dependant on its facilities, organizational set-up, and equipment used. In general, the laboratory will be organized into functional areas designed to provide a safe and effective environment that ensures the delivery of the testing services to the appropriate quality and regulatory standards. For example, most clinical virology laboratories will have separate functional areas along the following lines:

• • • • • •

specimen collection and preparation area, general laboratory area, stores, plant room, etc., molecular virology, serology, virus culture and isolation unit (Note: in some specialist virology laboratories this could be further split dependant on the type and risk level of the viral pathogen), non-laboratory office area.

Each area will have its own set of operational specifications which may include temperature and cleanroom air-handling conditions. These are monitored as part of the laboratories QA measures against the specifications and defined control limits. In many laboratories the monitoring is 24/7 by way of an integrated electronic system which raises an alarm directly to laboratory management 24/7 in “real-time” should a control limit be broken, who can then take immediate action. In addition, the separation of functional activities and equipment helps prevent cross-contamination which could eventually lead to erroneous test results. Particular attention is paid to the set-up and management of the molecular virology area as molecular amplification technics such as PCR are extremely sensitive as well as pose a significant risk of introducing amplicon-based contamination if they are not properly controlled. In order to minimize the risk, most clinical virology laboratories will use rooms with a unidirectional workflow with relative air pressures with which to separate and manage the Pre-molecular amplification activities from the Postmolecular amplification activities. The number of separate rooms or areas the laboratory implement largely depends on the facilities and space available. Four designated areas or rooms provide the laboratory with the ability to separate the molecular activities into the reagent preparation area, the specimen extraction/processing area, the assay set-up room (template addition stage), and the molecular amplification room. Each room has its own equipment such as biological safety hoods, pipettes, centrifuges, fridge and freezers, etc as well as a dedicated consumable/reagent store which is restricted for use within that room. Staff movement follows the direction of the workflow, and returning to previous rooms in the workflow is not allowed. Personal protective equipment (PPE) such as glove and laboratory coats are required for different rooms and is often color coded for easier visual awareness. The management of waste from the molecular facilities should be properly segregated and disposed according to the institute’s disposal and infection control procedures. Amplicon contamination has been a serious problem in the past and has resulted in the shutdown of some laboratory facilities. The use of periodic wipe tests helps to monitor the laboratory environment for potential amplicon contamination. The frequency can be adjusted if any contamination is identified and thorough decontamination measures can then be put in place to rapidly address any issues found. It is also standard practice to include no template control (NTC)/negative controls in order check for potential contamination in the reagents, consumables, and general laboratory environment. Monitoring the positivity rate of the NTC over time can act as an indicator of potential contamination issues.

Equipment Qualification, Calibration, Maintenance and Monitoring From a quality assurance perspective equipment covers instruments and software including laboratory information management systems (LIMS). Regulatory agencies (ISO15189 or equivalent) place a great deal of emphasis on the quality assurance of

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equipment from initial purchase through qualification, calibration, maintenance and monitoring, to its eventual disposal at the end of its intended use and live cycle. Not only is equipment QA an important regulatory requirement, as specialist laboratory equipment is expensive the QA procedures employed can also help to increase the equipment lifespan and therefore provide an additional cost benefit to the laboratory.

Equipment Qualification The clinical virology laboratory will have an equipment validation and/or verification plan and the initial phase of this will be equipment qualification. The laboratory will evaluate the item of equipment in consultation with the equipment manufacturer in order to define both the functional and operational specifications of the equipment as well as the cost and any preliminary installation, calibration, and training requirements. This step is often called design qualification (DQ) and is followed by installation qualification (IQ) in which the laboratory establishes that the equipment is suitable for operation and meets the manufacturers specifications as well as any regulatory claims. Operational qualification (OQ) is the third step in equipment qualification and it involves proving the equipment’s performance within the clinical setting usually against a “gold standard” or predicate device. The final phase is performance qualification (PQ) where the performance of the equipment is monitored over time in the clinical setting against a defined set of performance criteria. Within industry this four-step approach has become known as the 4Qs and within clinical virology the steps are generally covered through assay verification and validation. For the most generic equipment used within the virology laboratory such as pipettes, vortex’s and microfuges the recommendations provided by the equipment manufacturer are usually sufficient for qualification for routine use. However, specialist virology equipment used for molecular and serological testing (e.g., thermal cyclers and autoanalyzer’s) will require specific qualification for the equipment’s intended use within the clinical context. These instruments can be fully integrated platform or cartridge based commercial assay systems which are essentially ‘sample in and result out’. Alternatively, they can be componentbased workflows where a combination of equipment is required to create a virology workflow which can be used to generate a test result. This is largely the case in molecular virology, particularly for laboratory developed assays, where the extraction platform, kit, molecular amplification platform, and detection kit are combined in a molecular workflow. The virology laboratory will define the equipment qualification criterion in accordance with the manufacturers’ equipment recommendations and the laboratories intended clinical use. The aim being at qualifying the laboratory equipment and monitoring performance to ensure the validity of data/result generated for the individual items of equipment as well as the whole test workflow.

Equipment Calibration When using laboratory equipment for measurement purposes whether monitoring viral load or the temperature of the laboratory environment maintaining the quality assurance of the equipment through calibration is essential. Calibration minimizes measurement uncertainty. It also helps establish operational performance criteria which is used to monitor and control errors by ensuring that measurements remain both appropriate and acceptable. Within the clinical virology laboratory, equipment calibration covers both the fixed plant and machinery used to control the different working environments within the clinical virology laboratory as well as the specialist instrumentation used within each of the designated laboratory areas. Fixed plant and machinery includes the laboratory’s heating, ventilation, and air conditioning (HVAC) systems which are designed to contain any potentially infectious material dependant on the biosafety classification level (1–4, with 4 being the highest risk based on the type of virus and work to be undertaken) as well as any fitted walk-in cold-rooms and specialist culture facilities. The calibration of specialist instrumentation including biosafety hoods,
801C freezers, thermal cyclers, autopipettes, etc. assures the user that the instrument is working within the manufacturers defined specifications. The calibration of pipettes can readily be performed by the laboratory themselves using gravimetric calibration which is an accepted standard for pipette calibration. However, for most accredited laboratories the calibration of fixed plant and machinery and some specialist instruments (such as thermal cyclers) is usually conducted by the equipment manufacturer or an approved external contractor to an international standard such as ISO17025 or equivalent. If a laboratory decides to change the manufacturers calibration requirements this could result in the manufacturer’s warrant for use being invalidated. It is therefore important that the laboratory discusses and agrees any proposed changes with the manufacturer before implementing them. The frequency of calibration depends on the specific requirements of the equipment, maintenance and safety record, annual service plan, as well as the extent of its use. Calibration events are recorded within a “equipment/calibration log” which identifies the equipment by its assigned asset number.

Equipment Maintenance and Monitoring The ongoing calibration, service, and preventive maintenance of laboratory equipment throughout its lifespan supports reliability and confidence in the performance of the equipment. Maintenance and monitoring should include a register of all equipment indicating serial numbers, assigned asset numbers as well a location within the laboratory. This is particularly important for

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equipment that is designated for use within a single laboratory location. Within the equipment log, details of the validation and outcome of any safety inspections are recorded along with any equipment failures and servicing events. Most laboratories will assign the responsibility for specific laboratory equipment to an operator or group of operators. It is their job to keep the equipment in good working order and to ensure that calibration and maintenance events are up to date as well as report any reoccurring problems to laboratory management. Routine preventive maintenance ensures the equipment warranty in line with the manufacturer’s recommendations and specified annual schedule. For example, the air filters within the HVAC system should be monitored and cleaned regularly and failure to do this could lead to potential laboratory contamination issues or safety issues is the filters become blocked and cause overheating. In addition, this can also lead to mechanical issues which shortened the equipment life span and become an additional cost burden to the laboratory. Most of the environmental monitoring within the laboratory (such as temperature and pressure) is managed through wireless monitoring systems. These systems consist of operating software, specific probes/sensors, and receivers/transmitters for picking up the data and enable real-time performance monitoring throughout the different laboratory environments. If the central power fails, the equipment is covered by a back-up power system which ensures the integrity of the laboratory environments is maintained and monitored 24/7. The metrics from the wireless monitoring system usually have operational tolerances set which provide an automatic alarm which is set electronically to the designated operator when the metric is broken. This enables the laboratory to take immediate action as well as helps with the identification of trends over time.

Internal and External Audits Internal and external audits are an essential part of the clinical virology laboratory’s quality assurance armory. The purpose of the internal audit is to provide an objective overview of the laboratory’s control and functional effectiveness over its policies, processes, and procedures in relation to the testing service it provides and the regulatory standard and environment it operates within. The quality manager in consultation with the laboratory management team will prepare an annual internal audit plan which sets out the areas to audited throughout the year in relation to the relevant clauses of the standard to which the laboratory is accredited, the date and time of the audit, and those responsible for each audit area. The depth, breadth and scope of each internal audit will vary depending on the technical complexity of the area, any findings from previous audit reports, as well as any open quality activities such as corrective actions and preventative actions (CAPA). Internal audits should only be conducted by those staff members who are trained and shown to be competent auditors. In addition, those staff members being audited need to ensure that they are fully prepared prior to the audit with a full understanding of the policies and procedures they are responsible for and a list of items that could be reviewed during the audit. This helps improve the efficiency of the audit and helps ensure that internal audits are completed within the allotted timeframe. The audit should be non-directive and allow the auditees responsible for their designated areas within the laboratory to explain their processes and procedures. The auditor is then able to gain an understanding of the staff competence, identify areas that may require improvements such as additional training, as well as potential risks or non-conformances within the written policy and procedures that could ultimately lead to a non-compliance with the regulatory standard. A documented plan can then be agreed with management outlining how any findings are to be addressed before any scheduled external regulatory audit takes place. The external audit is an independently conducted inspection usually by a national accreditation body (NAB) which assesses the laboratory’s technical competence and conformity to applicable international standard such as ISO15189 or ISO17025. The audits typically follow a four-year cycle with an annual surveillance audit over a three-year period and a re-accreditation audit every fourth year. Many countries have officially appointed National Accreditation Bodies that carry out conformity assessment and accreditation activities in particular for calibration and testing. The NAB’s are subject to oversight through regional co-operative bodies such as the EA in Europe, APAC in the Asia-Pacific, IAAC in the Americas, AFRAC in Africa, SADCA in Southern Africa, and ARAC in the Arabian region, all under the umbrella of the International Accreditation Forum (IAF) which aims to promote multilateral recognition arrangements (MLA) and common recognition and harmonization of the accreditation standards and practice across geographical regions. The International Laboratory Accreditation Cooperation (ILAC) supports the interests of those accreditation bodies that deal specifically with laboratory testing (ISO17025, ISO15189), measurement and calibration (ISO17511) as well as EQA or PT provider accreditation (ISO17043).

Assay Verification and Validation The implementation of a new assay method or the amendment of an existing assay method needs to be supported by an appropriate verification and/or validation study prior to routine use within the clinical virology laboratory. Assay verification is the process of testing and reviewing an assay’s performance in relation to its known or reported performance such as against the assay manufacturers’ defined performance specifications as they are stated within the package insert provided. Whereas, assay validation is the process of evaluating the assay method, establishing its performance characteristics and determining its fitness for use within the clinical virology laboratory. The verification and validation requirements depend on the type of assay, its intended use, and the amount of performance data available such as from previous clinical studies. There are numerous national and International regulatory guidelines which outline the verification, validation process as in many countries this is governed by the regulatory

The reportable range (also Analytical Measurement Range – AMR) is the interval between the upper and lower concentrations of an analyte for which the assay has acceptable levels of precision, accuracy and linearity

This is the ability of the assay to obtain test results which are directly proportional to the concentration of the analyte within a given range Paired/samples

Duplicate sample/lab peer review

Specificity (analytical and clinical)

Sensitivity (analytical and clinical)

Consider any potential interfering or cross-reacting substances that may be present in the specimen or matrix

The ability to return a “true negative” result when the target nucleic acid is not present

Qualitative assays: The number of true positive samples correctly identified by the assay divided by the total number of true positive samples Quantitative assays: Defined from the limit of detection and where appropriate confidence interval (CI) can be calculated in order to support the chance of obtaining a true positive result

The lowest amount of target nucleic acid that still returns a true positive result.

Limit of quantification The lowest concentration at which the analyte can be reliably detected within established imprecision (and where appropriate bias) criterion (LOQ)

Clinically relevant/ educational samples

Clinically relevant/ educational samples

“Educational” samples

The limit of detection The lowest quantity of a substance that can be distinguished from the absence of that substance (a blank value) within a stated confidence limit. The limit of detection can also be determined using a serial dilution of prequalified standard or reference material spiked into an (LOD) “Educational” samples appropriate clinical matrix. The dilutions are replicate tested and the results analyzed using PROBIT analysis

Linear/ Reportable range

Repeatability: Repeat testing under unchanged conditions for example within a single assay run (inter-assay) Reproducibility: Repeat testing of the same test sample in different runs across a time period, possibly using different operators (intra-assay)

Precision/imprecision The ability to consistently produce the same result for a given test sample

The closeness between values obtained from a series of test results. Where possible within the assay verification phase the test results are compared to “known” and/or an accepted reference value Based on consensus

Accuracy

EQA monitoring

Description/Definition

Key verification and validation parameters

Assay parameter

Table 1

If applicable

95% CI of þ ve result

Only if more than 1 control used

Inter-assay (Intra-assay)

“run to run”

IQC monitoring

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requirement within that country and the regulatory category of the assay method. However, the general parameters covered are the same and include accuracy, precision, reportable range, linearity (for quantitative assays), reference interval, analytical sensitivity, and analytical specificity as outlined in Table 1. Where a clinical virology laboratory uses a commercially developed assay such as one regulated and approved by the Food Drug Administration (FDA) in the US or a CE marked assay in Europe, the manufacturer is responsible for providing the clinical laboratory with the analytical performance characteristics as well as its expected clinical performance and supporting clinical data. The clinical laboratory verifies the assay performance against the manufacturer’s performance claims for the its intended use as defined within the instruction manual provided with the assay. Importantly, if the laboratory modifies the commercial assay and/or instructions for intended use, the assay is no longer considered a regulatory approved assay and the laboratory would be expected to fully validate the impact of these changes on the assay’s performance. It is important to remember that any changes to the manufacturer’s assay or protocol without prior approval could result in the manufacturer withdrawing its support for the assay. In comparison, where the assay has been developed in house by the clinical virology laboratory often referred to as a Laboratory Developed Test (LDT), the process of validating and establishing the performance of a laboratory developed assay would be more extensive and will also include comparative testing of the new test against a previous assay for the same targets as well as reference to clinical performance within the current literature if available. In both cases, commercial assay or LDT there is a requirement for well characterized reference material and, where available, International standards in order to support the verification and validation process as well as on-going Quality Control (see also section on Internal Quality Control Procedures and Monitoring Assay Performance Over Time). The patient samples, controls, or reference material used for verification and validation should be in the appropriate matrix and at clinically appropriate levels (if known). Where there is limited reference material available or a lack of suitably characterized patient material, the laboratory can use residual material from proficiency testing (PT) or external quality assessment (EQA) schemes. The EQA materials are generally highly characterized as well as have the advantage of comparative peer group data which can help facilitate the verification and validation process. It is also good practice to use the same assay reagent lots when conducting the verification and validation exercise in order to reduce the potential impact of variation introduced through different lots/batches of assay reagents. Finally, before undertaking any verification or validation study it is important that the laboratory ensures that the assay method is calibrated to the manufacturers requirements and there are no recalibration/maintenance events scheduled for the duration of the study. For the verification of a regulatory approved qualitative assay, the following approach and principles would apply.

Accuracy In order to verify accuracy a comparison of methods study can be carried out. Known patient samples which cover the assay range, and have previously been tested on the current assay method the clinical virology laboratory uses, are tested on the proposed new method over a defined period of time and the results compared. If insufficient in-house patient samples are available, the laboratory may obtain specimens from other laboratories. However, when using patient samples, it is important to ensure that the specimens have been treated the same prior to testing (i.e., storage conditions, number of freeze-thaw cycles etc). An alternative is to use commercially available control or characterized reference material containing the target of interest across the assay range. The number of samples to be tested and the frequency of testing depends on the type, complexity, and intended use of the assay method. Although some regulatory authorities do provide minimum requirements or recommendations. For example, 20 patient specimens over 2–5 testing events. The qualitative data (positive, negative) obtained is analyzed statistically using Cohen’s κ analysis.

Precision The precision of the assay method would be determined by testing one or two controls at clinically relevant levels (if known) repeatedly over a specified period of time in order to establish the between run precision. The recommended minimum time period for testing is 20 days in many guidelines. However, the testing interval should be sufficient to cover the routine operation within the laboratory, such as rotation of operators. The within-run precision can also be established by running duplicate control samples over a specified time period, usually 10 days. The results are analyzed using standard deviation and coefficient of variance and compared to the claims of the assay manufacturer or the results obtained for the current laboratory method being used.

Reportable range and limit of detection (LoD) The Reportable range of the assay is verified using known characterized positive clinical samples at clinically relevant levels or by using dilutions of commercially available standards or reference materials tested over a period of time in comparison to the current laboratory method. The LoD of the assay is verified by testing replicate samples at known levels above, at and below the determined LoD (e.g., LoD 7 20%) or as specified within the manufacturers Instruction manual in duplicate over a defined period of time (usually 20 days). The percentage detected at each level is then calculated and Probit analysis used to estimate the LoD, which can then be defined as a titer of the target analyte detected at a 95% Confidence Interval (CI). For the verification of a regulatory approved quantitative assay, such as a molecular assay used for the determination of viral load, the same basic principles used for verifying a qualitative assay apply for accuracy and precision although they need to verified across the analytical measurement range (AMR) of the assay and in particular at known clinically relevant or clinical decision making concentrations.

Linearity/analytical measurement range (AMR) In the context of a molecular viral load assay, the AMR or reportable range can be defined as the linear range of test values over which the laboratory can accurately detect the target viral nucleic acid within acceptable degrees of variation. Beyond the manufacturers stated limit of

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Fig. 2 Graph showing the linear relationship of test results across multiple clinical laboratories for a dilution series of SARS-CoV2.

quantitation (LoQ) the relationship between the measured and actual viral load concentration may not be linear and test results become unreliable. It is therefore important that the laboratory verify the AMR in the clinical setting they intend to use the assay method and in line with the manufacturer’s instructions and claims. In some cases, the manufacturer may only have performed an analytical assessment of AMR on contrived or synthesized control materials such as target viral nucleic acid in a plasmid construct and not true clinical samples at relevant titers. As a result, it may not be possible to establish the full analytical AMR equivalence with the manufacturer’s specifications. In these circumstances the laboratory should ensure that they assess the AMR within the known clinical context and in line with the laboratory’s pre-defined AMR acceptance criteria. At a minimum this should include samples at the low, medium, and high points of the AMR. Polynomial Regression analysis is used to assess the linearity of the assay across all relevant concentrations and if the relationship is found to be non-linear (not first order) those concentrations are removed from the analysis at the high and/or low end and the regression analysis repeated. The testing is usually performed in replicates (2
4) at each concentration and precision assessed across the linear range. The highest and lowest concentrations that produce a linear relationship are defined as the upper and lower limits of quantification. (Fig. 2) Where the AMR results are compared against the manufacturer’s claims or against the AMR of the comparative method the laboratory is currently using the proportional bias, as measured by the slope of the regression curve, and the constant or systemic bias, as measured by the y-intercept, are determined. A Bland-Altman difference plot is used to measure bias between assays. Most regulatory standards (such as ISO15189) require the laboratory to have a written policy on verification and validation. This defined the methods, frequency and limits of verification for an assay method. In general, the AMR should be verified in line with the following criteria:

• • • •

at changes of reagent lots, unless the laboratory can demonstrate that the use of different lots does not affect the accuracy of patient test results and the range used to report patient test data, if QC materials reflect an unusual trend or shift or are outside of the laboratory's acceptable limits, and other means of assessing and correcting unacceptable control values fail to identify and correct the problem, after major equipment maintenance or service, when recommended by the assay manufacturer.

In addition to this, some regulatory organizations may also specify the frequency of AMR verification, such as every 6 months (e.g., CAP). In most cases the laboratory will only test a smaller sub-set of linearity samples (3
4) at the low, mid, and high points within the AMR and only conduct further more extensive testing if these samples fall outside the acceptable criteria.

Sensitivity, Specificity, and Clinical Utility Key factors that can impact on the accuracy of a test to deliver a result are sensitivity and specificity as well as the prevalence of the disease in the population targeted by the assay method. In the context of a serology assay either targeted at detecting the viral antigen directly or through the indirect detection of antibodies to the viral pathogen, the diagnostic sensitivity and specificity are derived from test results on clinical samples obtained from selected reference patient groups within the population. The degree to

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which the reference groups represent all of the host and environmental factors within the total population targeted by the assay method can have a major impact on the accuracy of test result and its interpretation in the clinical setting. Therefore, the clinical virology laboratory also needs to consider this during the verification and validation phase, which is an ongoing process which continues for as long as the assay method is being used within the laboratory and requires constant vigilance, review, and where required reassessment. Specificity also includes any potential interfering or cross-reacting substances that may be present in the specimen or matrix. Interference is basically any non-target organism or substance that can cause a false negative result. Whereas, cross reactivity is a non-target organism or substance that can cause a false positive result. With molecular based assays this can be a problem, particularly when the assays are aimed at differentiating related viral strains. The assay manufacturer or laboratory who developed the assay should have chosen their primers and probes cautiously as well as made an initial “in silico” assessment of the available databases before testing in practice with known samples that are related taxonomically and also epidemiologically. The list of potential cross-reacting and interfering substances is usually documented within the manufacturer’s instruction manual. An assessment of analytical specificity should include testing the potential cross-reacting or interfering substance at the known highest concentration that could be expected to be observed within the patient sample. A common approach, in the absence of sufficiently available clinical samples, is to use spike samples in negative matrix and also weakly positive samples (e.g., LoD þ 10%) for each of the analytes of interest. From this the minimum inhibitory concentrations can be determined. For quantitative assays any potential cross-reacting targets should be assessed near the Lower Limit of Quantitation (LLoQ) and the quantified target values from spiked and unspiked samples compared and determined to be within the defined precision limits of assay. Many variables can influence the performance of the assay method, such as the prevalence of the viral disease, the clinical setting, and type of test being performed. Therefore during the clinical evaluation of the assay it is important to consider the testing purpose such as whether it is for diagnosis, screening, or therapeutic monitoring purposes; the environment the testing is likely to take place in such as the central clinical laboratory, out-reach clinic, or point of care setting; the specimen type (e.g., whole blood, plasma, sputum etc); type of result such as quantitative or qualitative result. Two other important measures of assay performance are positive predictive value (PPV), the probability that those testing positive by the test have the viral disease, and the negative predictive value (NPV), the probability that those tested negative by the test are do not have the viral disease. Both PPV and NPV depend on the sensitivity and specificity of the test and also the prevalence of the viral disease within the population which in turn gives the assay its clinical utility. In terms of the number of specimens that should be tested during clinical evaluation in order to assess PPV and NPV, some guidelines recommend 100 negative clinical samples for the evaluation of false positive rates, and 50 clinical samples known to be positive for the target viral pathogen are considered appropriate for determining the false positive rates. The CLSI guidelines (CLSI EP12-A2) recommend a minimum 50 positive and 50 negative clinical specimens. In low prevalent diseases, it can be difficult to obtain sufficient clinical samples to conduct a full clinical evaluation. In these circumstances the clinical laboratory has to consider alternative approaches such as including materials used in previous proficiency testing/EQA scheme challenges.

Internal Quality Assessment (IQA) “Split sampling” is an effective additional IQA measure for monitoring the whole laboratory testing process. This is where between 0.5%–1.0% of a laboratories clinical patient sample workload are subjected to internal quality assessment. The randomly selected patient samples are spilt and a new internal testing request generated with a unique laboratory IQA code. This IQA sample is processed through the laboratory in parallel to the original clinical patient sample. At the end of the testing process, results are generated for both original clinical patient sample as well as the IQA sample. These can then be compared internally by the laboratory quality team and any inconsistencies within the results and the report are investigated and corrective action taken through the laboratories quality system. The split sample approach is not always practice or appropriate particularly when the volume of the patient sample is small.

Internal Quality Control Procedures and Monitoring Assay Performance Over Time Assay variation is the result of systematic and random error which is inherent to the assay method. Common sources of error or variation can be attributed to operator, instrument, reagent lot, calibrator and calibration cycle. The amount and type of variation associated with an assay method is determined during assay verification and/or validation. Having established the extent and where known the clinical relevance of the variation it is important that the laboratory continues to monitor the variation to ensure that it remains within acceptable levels. This is done through Internal Quality Control on a daily run to run basis and is often a requirement of many regulatory bodies (including CLIA and ISO). In general, a commercial assay will be supplied with a set of controls. The intended use of these controls is to monitor the assay in line with the manufacturers defined performance criteria. As such, the manufacturers kit controls are intended for use only with the manufacturers assay and in addition are often specific to a particular kit batch or reagent lot. This can mean that the manufacturers kit controls do not support the monitoring of different reagent lots over time and would not necessarily identify a drift in the overall assay performance over time (assay drift). So, it is important that the operator is aware of the potential limitation of the manufacturers kit controls. For example, many of the controls provided with the manufacturers kit controls only monitor as specific part of the process such as with molecular tests where the manufacturers kit controls may just be free nucleic acid and would therefore only be suitable for controlling the

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amplification/detection steps of the assay and not the whole testing process from sample pre-treatment through the extraction phase, amplification and detection, to end result. Consequently, many regulatory and standards organizations either recommend as “best practice” or in some cases insist on the use of independent third-party controls either instead of, or in addition to those supplied by the assay manufacturer in order to demonstrate compliance. Such recommendations are made at the International level (ISO15189, CLSI) as well as the national level (e.g., RiliBak in Germany). Independent third-party controls are also often referred to as external quality controls or run controls. They aim to monitor the complete testing process and provide an accurate, unbiased measure of quality performance independent of the manufacturers specific control or calibrator. In general, for qualitative tests a minimum of a positive and negative control is included in each assay as specified in the laboratory procedure. This should also take into consideration the assay manufacturer’s instructions. For multiplex assays covering several different viral targets in a single assay, the laboratory may choose to rotate different positive controls for different targets within the multiplex assay over a given time period at a frequency defined within their laboratory procedure. For quantitative tests including molecular viral load assays, the laboratory should ensure that the controls it uses are at or near clinically relevant decision levels, where known and/or as specified within the current clinical guidelines. For viral load assays it is common practice to determine performance characteristics for the control under routine laboratory conditions by testing the control over a period of time (usually a minimum of 20 times) in order to obtain sufficient data points to determine the mean and standard deviation from which suitable control limits can be established. Some laboratories calculate a rolling mean based on the last set of observations reported in order to reduce the initial amount of testing and re-evaluate the mean and control limits after sufficient datasets are obtained. Where a Laboratory is using more than one of the same instruments/platforms, they would generally perform the study on one instrument and then perform a comparison testing to confirm performance on the other instruments. Alternatively, the laboratory could opt to run alternate QC runs across platform instruments. Alternative approaches have also been suggested for establishing and monitoring the performance of serology assays based on extended historical data and peer group assessment. This is covered within article by Sally Baylis et al. The most frequently used control rules applied within the clinical laboratory are those defined by Westgard (See Relevant Websites section). The performance can be monitored graphically by plotting the measurements in Levey-Jennings control charts and any deviations from the expected performance can be investigated and the appropriate corrective action taken if required. There are numerous web-based software packages available for the reporting and monitoring quality control data. Some such as Acusera 24.7 (See Relevant Websites section) offer LIMS connectivity, interlaboratory peer group assessment, as well as the ability for the laboratory to further interrogate parameters such as specific operator, and instrument associated with a particular such set of QC data. This enables the laboratory to proactively monitor QC performance and identify important trends or shifts in expected results over time which may impact on the assay performance and ultimately the patient test results.

Suitable Standards, Reference Materials and Controls to Support Quality Assurance in Virology International standards are essential to quality assurance in virology as they provide the means for the calibration of quality control and EQA materials which in turn help support the comparability of results across assay methods and provide confidence in the ability of the laboratory to deliver accurate and reproducible patient test results. In particular, the utility of viral load assays has improved significantly through the calibration of assays to the available International Standards particularly where patient management depends on the ability to relate patient results to prior results or to defined viral load values which support clinical decision making in line clinical practice, such as in the monitoring of post-transplant CMV infections. The development of WHO International standards is covered in chapter (Sally Baylis), however in the context of clinical virology, and in particular the quantitation of viral nucleic acid, the concepts of metrological traceability as defined in ISO17511 (In vitro diagnostic medical devices – Requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples) which are widely used in other areas of laboratory medicine such as clinical chemistry can generally be applied to molecular viral load assays. Metrological traceability describes the steps through which a patient test result can be traced back to a relevant higher order standard and the uncertainty of measurement in relation to each step estimated in a common unit of measurement. The highest order of standard would be one which is ultimately traceable to the metric system/international system of units (SI). WHO International standards are derived by consensus and are not ultimately traceable back to an SI based value and do not have an assigned uncertainty of measurement nevertheless their utility and importance in driving standardization in molecular virology continues to be demonstrated. Additional to the WHO initiatives, other international organization such as NIST (National Institute of Standards and Technologies) and LGC (Laboratory of the Government Chemist) have supported the development of methods such as Digital PCR (dPCR) which enables the counting of individual target viral nucleic acid molecules. Recent interlaboratory studies have demonstrated the accuracy and reproducibility of dPCR for the absolute quantification of DNA copy number concentration in comparison to techniques such as flow cytometry and isotope dilution-mass spectrometry. Thus, the inclusion of dPCR within the latest version of ISO17511 means it has the potential to become a recognized SI-traceable primary reference measurement procedure. However, dPCR still has limitation in the same context as any other PCR based methodology such as assay bias. Therefore, understanding the relationship between International Units (IU) and digital copies in a clinical context and across different non-PCR based molecular technologies such as Transcription Mediated Activation (TMA) used by Hologic, Strand

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Fig. 3 Schematic of the EQA cycle.

Displacement Amplification (SDA) employed by BD ProbeTec, etc is essential in further supporting standardization. In addition, the commutability of quality control or reference materials needs to be demonstrated relative to the patient specimen and assay technology it is tested on in order to further support comparability of results. The development of an International standard is primarily based on public health need. However relative to the number of viral pathogens routinely tested in the clinical laboratory there are very few International standards and certified reference materials (CRM) available with which to calibrate secondary standards and commercially available controls. Therefore, technologies such as dPCR and Next Generation Sequencing (NGS) enable commercial control manufacturers to molecularly characterized the materials in the absence of an International standard. When an International standard becomes available these materials can then be back calibrated to the standard to achieve further traceability.

External Quality Assessment and Performance Criteria External quality assessment (EQA) or proficiency testing (PT) is integral to quality assurance within the clinical virology laboratory. It provides assurance and confidence in the laboratory’s procedures and service provision. It also enables the laboratory to compare its performance to other laboratories carrying out similar tests, as well as provides an indication of areas for quality improvements. The participation in EQA is often an accreditation requirement as defined in ISO15189 and more recently ISO22870 which covers the specific use of point of care testing within the clinical laboratory setting. In many countries it can be a mandatory requirement with laboratories required to report their EQA performance to the national body within that region. In these circumstances, EQA is often linked to the test reimbursement policy within specific countries. The EQA provider, is also required to ensure that the EQA schemes it provides fulfill the requirements of ISO17043 (Conformity assessment – General requirements for proficiency testing) (Fig. 3). The EQA cycle starts with the EQA provider, supplying registered laboratories with “blinded” clinically represented samples at predetermined intervals throughout the year. The laboratory is required to test the EQA samples using its routine laboratory method and return their results through a web-based portal to the EQA provider within a defined period of time. Data analysis is carried out by the EQA provider and an EQA report which details the laboratories individual results and peer group performance is provided to the clinical laboratory along with a certificate of participation. In regions where there are defined national EQA performance criteria for specific target analytes, such as in Germany for the viral load determination of HIV, HBV, and HCV, the EQA provider will also report whether the laboratory has achieved the required level of proficiency.

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Fig. 4 Example of an HIV viral load EQA panel composition.

EQA Design and Objectives As molecular diagnostics account for the majority of the test output of the clinical virology laboratory, EQA design has progressed in order to meet increasing demand. Specialist EQA providers such as QCMD (See “Relevant Websites section”) which focus specifically on the provision of EQA within the area of molecular infectious disease offer a growing range of schemes covering viral quantitation, as well as the genotypic analysis for the detection and typing of drug resistance variants based on nucleic acid sequencing using technics such as NGS. The design and objectives of a clinical virology EQA scheme is dependant on many factors such as the type of method (e.g., qualitative, quantitative, sequencing, typing), its intended use, the matrix. In addition to this, in some countries where there are mandatory EQA schemes the regional regulatory bodies will often define the number and type of EQA samples that can be included within the EQA panel as well as the frequency of distribution to the laboratory (e.g., once every 6 months in the US and in Germany). Within the example shown in Fig. 4, the EQA panel consisted of two different viral strains of HIV, at a range of different clinically appropriate viral loads with the lower titer sample included to assess the sensitivity of the participating laboratory tests. In addition to this the panel also contained a negative matrix only sample in order to assess absolute specificity/false positivity. The inclusion of duplicate samples within an EQA challenge and across EQA distributions also supports the evaluation of homogeneity and reproducibility of the EQA panel members. For many quantitative EQA schemes, the assessment of performance is based on the consensus mean of results returned by all laboratories which tested the EQA sample, once any outlying values have been identified statistically and removed from the initial analysis. Alternatively, the consensus mean of the assigned method or technology peer group can also be used, such as in the example in Fig. 5(a) and (b). In both approaches the mean and standard deviation are calculated accordingly and a sample performance score based on the distance the laboratories result is from the consensus mean is established. In the example provided, zero points if the result is within one standard deviation from the mean. One penalty point if the result is between one and two standard deviations, two penalty points within two and three standard deviations, and three penalty points if the result is more than three standard deviations from the mean. The regional regulatory bodies have oversight of viral diagnostic testing for regulated analytes such as the blood borne viruses HIV, HBV, and HCV in some countries and define the minimum performance criteria for laboratories participating in these EQA schemes. For qualitative tests, failure of the laboratory to obtain results within 80% of the overall consensus is considered unsatisfactory and laboratories which fall outside this range require investigation, the outcome of which may involve escalation to the regulatory body.

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Fig. 5 Example of performance scoring within the HIV viral load EQA scheme. (a) Qualitative and quantitative panel score. (b) Individual sample analysis (quantitative).

It is important that clinical virology laboratory management and operational staff evaluate the outcome of the EQA schemes through regular quality meetings. A preliminary step is ensuring there are no administrative transcription errors which could inadvertently affect performance. The laboratory should also compare their results to previous EQA distributions and in line with known performance characteristics for the assay as defined through verification/validation and IQC monitoring. Where the IQC indicates that the performance of the assay is acceptable but the EQA indicates a possible quality issue, the laboratory should investigate further and if required obtain further information from the EQA provider as well as the assay manufacturer (if the assay used is commercially available). As well as meeting regional regulatory requirements, EQA schemes also provide the laboratory with educational feedback in relation to the test coverage for current clinically relevant viral strains, the incidence of false positive results, and differences in laboratory performance with a specific assay or technology. This also includes the monitoring of observed trends in laboratory EQA reporting over time, such as the move from reporting results in copies/ml and the gradual increase in the reporting of EQA results in IU/ml following the introduction of the first WHO International Standard for CMV viral quantitation and more recently the range of gene targets in assays targeting SARS-CoV-2 (Fig. 6(a) and (b)). The aim is to ensure optimal quality performance and compliance with the accreditation standard and regulatory authority. In addition, participation in EQA schemes also helps identify possible areas for improvement and ensure that the assay is clinically fit for purpose.

Summary and Future Aspects of QA to Virology Laboratory Viral tests are fundamental for patient management in diagnosis, infection control, and disease outbreaks where the rapid assessments of disease burden and progression is essential for evaluating the effectiveness of interventions and the verification of disease status. Technologies such as Next Generation Sequencing (NGS) and metagenomics allow the whole genome characterization at the nucleotide

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Fig. 5 Continue.

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Fig. 6 The range of different target gene loci reportedly used by laboratories for the detection of SARS-CoV2 within the QCMD EQA distribution (n¼ 1011 laboratories). (a) Major gene targets/gene combinations used. (b) Less commonly reported gene targets.

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level which as well as enhancing diagnostics also aids rapid epidemiology and infection control which is particularly important in relation to the recent Coronavirus pandemic. These technologies require advanced analytical and artificial intelligence software algorithms which support the extraction of diagnostic information. This poses additional challenges in quality assurance which will need to be addressed before such technologies can reach full routine potential and further drive diagnostic improvements as they become more accurate, simple, and affordable for use across a wide range of intended clinical settings.

Further Reading Baylis, S., 2020. Quality assurance and laboratory accreditation. In: Encyclopaedia of Virology, fourth ed. Elsevier Science. Baylis, S.A., Wallace, P., McCulloch, E., et al., 2019. Standardization of nucleic acid tests: The approach of the World Health Organization. Journal of Clinical Microbiology 57. Bustin, S.A., Benes, V., Garson, J.A., et al., 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55 (4). EP05-A3E. Evaluation of Precision of Quantitative Measurement Procedures, third ed. Clinical Laboratory Standard Institute (CLSI). Huggett, J.F., Foy, C.A., Benes, V., et al., 2020. The digital MIQE guidelines update: Minimum information for publication of quantitative digital PCR experiments for 2020. Clinical Chemistry 66 (8), 1012–1029. Hayden, R., Sun, Y., Tang, L., et al., 2017. Progress in quantitative viral load testing: Variability and impact of the WHO Quantitative International Standards. Journal of Clinical Microbiology 55 (2). ISO17511, 2020. In vitro diagnostic medical devices – Requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples. ISO17043, 2010. Conformity assessment – General requirements for proficiency testing. Miller, M.B., Atrzadeh, F., Burnham, C.D., et al., 2019. Clinical utility of advanced microbiology testing tools. Journal of Clinical Microbiology 57 (9). doi:10.1128/JCM.00495-19. Whale, A.S., Jones, G.M., Pavšicˇ, J., et al., 2018. Assessment of digital PCR as a primary reference measurement procedure to support advances in precision medicine. Clinical Chemistry 64 (9), 1296–1307.

Relevant Websites https://www.bipm.org/en/committees/cc/wg/jctlm-rt-nucleic-acids.html Bureau International des Poids et Measures. https://doi.org/10.1093/clinchem/hvaa125 Clinical Chemistry, https://www.cap.org/ College of American Pathologists. https://clsi.org/ Global Laboratory Standards for a Healthier World. www.Ideagen.com Ideagen. https://www.iso.org/ International Organization for Standardization. https://www.qcmd.org/ Quality Control for Molecular Diagnostics. www.randox.com/acusera-24-7-interlaboratory-data-management/ RANDOX. www.umcg.nl UMCG. www.unece.org United Nations Economic Commission for Europe. www.westgard.com WESTGARD. https://www.who.int/ World Health Organization.

Biosafety and Biosecurity in Diagnostic Laboratories Hannimari Kallio-Kokko, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Susanna Sissonen, Finnish Institute for Health and Welfare, Helsinki, Finland r 2021 Elsevier Ltd. All rights reserved.

Introduction Biosafety and Biosecurity Biosafety and biosecurity are not unambiguous concepts but have different meanings in different contexts. In laboratory context, biosafety usually refers to “the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release” (WHO Laboratory Biosafety Manual, 3rd edition). Biosafety aims to protect the workers, the community and environment. Many common laboratory practices such as good microbiological practices and procedures, use of personal protective equipment (PPE), decontamination and disinfection procedures or the use of safety equipment such as biosafety cabinets are part of laboratory biosafety. Laboratory biosecurity refers to “institutional and personal security measures designed to prevent the loss, theft, misuse, diversion or intentional release of pathogens and toxins” (WHO Laboratory Biosafety Manual, 3rd ed.). Laboratory biosecurity involves responsibility for the protection, control and accountability of biological materials. Measures that prevent the malicious use of pathogens and toxins such as permits and licenses for the handling, import and export of biological materials, physical structure of the laboratory facilities, restricted access or even ethical codes for life scientists can be regarded as biosecurity measures.

Diagnostic Laboratories Diagnostic laboratories handle human/animal samples that potentially contain hazardous microbes, and to ensure safe working environment to all personnel, the working practices maintained should follow thorough risk assessment n at all stages. The safe working practices in any laboratory contain codes of conduct, competent and appropriately trained staff, good microbial practices and procedures at all stages of handling potentially infective material. In addition, laboratory facility and equipment in use should be appropriate. Regardless of the focus of a laboratory, the possibility of highly contagious viruses, bacteria, fungi, parasites or prions being present in same sample should be taken into consideration, and be included in the risk evaluation to cover safe handling of samples and to ensure choice of proper and effective disinfectants. The selection and types of diagnostic assays performed in a laboratory affect the probability of risks as do e.g., the level of need for manual handling of samples. Working procedures that contain vigorous shaking (vortexing) or centrifugation of patient samples demand special biosafety precautions. Also assays that aim to concentration or multiplication of infectious agents (e.g., virus cultivation) cause special requirements for both the biosafety level of the facilities and utilization of other safety measures including PPE. The use of automated laboratory assay systems reduce the risk caused by manual sample handling, but problems in the function of machines rise another type of challenges, and handling and cleaning of possibly contaminated laboratory equipment is an important part of risk evaluation, the suitability of effective disinfectants should be ensured also with the equipment manufacturer. Even though potential to the presence of hazardous microbes is a reality in all diagnostic laboratories, and good basic laboratory practices, e.g., no eating or drinking in the laboratory are the ground for assuring laboratory biosafety, differences in the probability of highly contagious pathogens do exist. The epidemiological situations vary in different countries, leading also to differences in the level of immunity and susceptibility of individuals in the community. The type of a laboratory affect the sample material handled and assays performed, and laboratories providing advanced diagnostics and diagnostics for high threat pathogens need more control measures and enhanced biosafety precautions to be obtained.

Laboratory Biosafety Biological Agents Microorganisms are classified into four risk groups (1
4) based on the severity of the risk caused by the microbe to both an individual and the community. The severity of the disease, the transmissibility to other individuals as well as the availability of effective treatment and preventive measures affect the classification. Risk group 1 contains microbes that are unlikely to cause a disease in healthy humans. Risk group 2 contains microbes that are able to cause disease in humans, but are unlikely to cause serious threat to the environment or community. In case of an infection, effective treatment and preventive measures are available. Risk group 3 contains microbes that usually cause serious disease in humans, but pose a limited risk of spreading to the community, and effective treatment and preventive measures do exist.

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Risk group 4 contains microbes that usually cause serious disease in humans, and can easily be transmitted form one individual to another with no effective treatment or preventive measures usually available. For a single microbe, the classification may vary between different countries due to e.g., epidemiological situations or existing levels of immunity. Risk groups 2–3 contain viruses, bacteria, fungi and prions whereas all agents belonging to risk group 4 are viruses. For occupational evaluation of risks caused by handling microorganisms, it should be noted that the list of classified agents is based on the effect of those agents on healthy individuals only. Pre-existing disease, ongoing medication, compromised immunity, pregnancy or breast-feeding may affect the susceptibility to a pathogen. Therefore, special care should be taken when evaluating the risks of even microbes with lower classification for personnel on whose susceptibility may be affected for one or other reason.

Laboratory Biosafety Levels To ensure the safe working environment in laboratories, there are traditionally also four laboratory biosafety levels, which describe the level of the biocontainment structures and precautions required to handle biological agents in an enclosed facility. The classification is designed to provide protection to personnel, the environment, and the community. Biosafety level designations are a composite of the laboratory facilities, equipment, practices, operational procedures and PPE required for working with biological agents from the various risk groups (1
4). Differences between laboratory biosafety levels include requirements for ventilation systems, waste management, access control and safety practices such as work practices and the use of PPE. The lowest biosafety level with least specific requirements is level 1 (BSL1) precautions getting stricter step by step to the highest level 4 (BSL4). In BSL3 and BSL4 laboratories, special engineering and design features are required to prevent the accidental release of the pathogens from the laboratory. Also special training in handling the pathogens is required for the workers in high containment level laboratories. The risk groups of biological agents relate but do not equate to the biosafety level of laboratories. The assignment of handling an agent to a biosafety level for laboratory work must be based on a risk assessment covering all working procedures. Currently not only the classifications of agents and facilities but especially the risk based and the evidence-based approach to biosafety are emphasized more and more. WHO is updating the Laboratory Biosafety Manual, and when evaluating the risks, the main principles will be the same with the consequence of exposure and release, and likelihood of exposure varying from low to high. The response to the likelihood of the risks will contain core requirements (equivalent to BSL2 without BSC) for handling lower levels of risks, and as the risks elevate, heightened control measures are needed (equivalent to BSL2 þ and BSL3), ending to maximum containment procedures (equivalent to BSL4) if the highest risks are being present. Types of samples handled and diagnostic assays performed affect the needs for biosafety precautions, e.g., the propagation of risk group 4 viruses demand the highest level of biosafety practices. Procedures on samples that might contain agents with potentially severe consequences or on samples with known pathogens of high consequences need the highest level of biosafety practices obtained.

Risk Assessment Risk assessment is the process in which the risk(s) of working with hazards are assessed and evaluated. Based on the risk assessment the necessary control measures are determined to reduce the risk to an acceptable level. A hazard is something that can cause harm. A risk is the combination of the probability of the hazard to occur and the severity of the harm. Strategies for risk reduction in microbiological work include also the possible elimination of the work, substitution with an alternative organism/ activity, isolation of the hazard, use of engineering controls, use of administrative controls and use of PPE. As the risk assessment is the basis for the work safety, laboratories should have a routine for assessing risks, laboratory directors and principal investigators having the responsibility for follow up of proper evaluation and optimal practices. Risk assessment should take into account the pathogenicity of biological agents handled, mode of transmission of these agents, volume and concentration of infectious material handled. The presence of other risks such as use of needles and sharp items, high risk of creation of aerosols during working procedures, or use of hazardous chemicals and inflammables are included into evaluation. Also the availability of effective treatment or prophylaxis in case of accidental exposure is to be considered. The types of patient samples handled in the laboratory, the types of analyzes performed on the samples (e.g., the potential of aerosol formation), and the facilities available affect whether the risk can be safely handled. The route of transmission and the transmissibility of the agents affect the safety procedures selected for each work stage. The risk assessment should also cover the evaluation of effective decontamination methods and safe waste handling procedures as well as biosafety precautions. Training of the personnel is a key factor for ensuring the proper biosafety cultures in all laboratories, and the role of maintenance personnel (e.g., cleaners, technical personnel, maintenance personnel of laboratory equipment) should be included.

Routes of Transmission and Infective Dose In laboratory setting, the route of transmission may be different as compared to naturally acquired infections. Intact skin protects well against most microbes, but e.g., wounds or atopic eczema can break down this barrier. Mucous membranes and eyes are

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vulnerable, and serve a possible infection route for several microbes. In laboratory settings the most probable transmission routes for infective agents are through blood (needle stick accidents or cuts with sharp objects), mucosa and airways (spills or splashes into mouth, nose or eyes, through generation of aerosols), or gastrointestinal route (spills, by touching mouth with contaminated fingers or while consuming food in laboratory). All patient samples are considered potential to transmit infection to a laboratory worker. High risk of transmission is associated with contagious blood (blood borne pathogens e.g. HIV), nasopharyngeal specimens and other respiratory samples (air borne pathogens e.g., influenza viruses), feces (e.g., norovirus), brain biopsies and central nervous system specimens including cerebrospinal fluid (e.g. rabies virus, prions), tissue specimens, and skin biopsies and blisters (e.g., poxviruses and herpes simplex viruses). The most often reported ways of acquiring laboratory associated infections include splashes or sprays of infective material, needle stick injuries, accidents with sharp objects, and earlier also mouth pipetting. Working procedures such as pipetting and pipette spills on surface, taking blood samples or handling blood tubes as well as the usage of centrifuges, blenders, shakers, mixing instruments, and sonicators pose elevated risk for aerosol generation. The infective dose is the number of microbial particles needed to cause an infection, and it has an important role while evaluating the level of risk caused by handling the patient samples or enriched cultivations. For e.g., norovirus the infective dose is estimated be as low as only 10–20 virus particles.

Personal Protective Equipment (PPE) PPE is meant to block the direct transmission of an infective agent to a worker, and should be worn in circumstances where a risk assessment has shown the continuing needs for personal protection. PPE is usually regarded as the last measure to prevent the exposure to infectious agents; all other control measures such as the use of engineering controls should be applied before using PPE. PPE must be suitable for the purpose, suitable for the person and used correctly. The selection on the PPE is based on the risk assessment. Protective clothing, gloves for hand protection, facial masks for respiratory protection, and goggles and/or face shield for eye protection are examples of personal protective equipment available. It is recommended to avoid wearing protective laboratory clothing outside the laboratory, e.g., in canteens, coffee rooms, offices, libraries, staff rooms and toilets. While working in higher containment facilities, selected PPE is always put on prior to enter to, and removed before exit from the facilities. Protective clothing is considered the basic PPE used in all clinical laboratory settings. The selection of the material of protective clothing is based on the type of work performed. In case of a constant risk for heavy spills, the material should be water resistant, if no risk for heavy spills also water repellent material can be a choice. In case of contamination of the clothing during work, the decontamination of material by e.g., autoclaving before sending to laundry unit is obligatory. Especially in higher containment facilities, also disposable clothes, or positive pressure suits ventilated with motor unit are used. As the material of protective gloves vary, the selection is on the potential hazard (microbial and/or chemical). Nitrile has good shield against viruses and high or medium protection against some chemicals (high against e.g., glutaraldehyde, sodium hydroxide and hydrogen peroxide, medium against e.g., 70% ethanol, but not recommended against e.g. acetone or methanol). Latex is good against microbes, but pose a risk of allergies and does not protect against organic solvents. Vinyl is not suitable for work with highly infectious material. Standards, such as EU standard EN374-5:2016 “Protective gloves against dangerous chemicals and microorganisms” help in selecting the proper material for intended use. Glove packages should be marked with pictograms, which indicate the suitability of the glove against chemical and biological risks. After selecting the correct material, also proper way of using gloves is important. Appropriate gloves are to be worn for all procedures that may involve direct or accidental contact with potentially infective patient samples such as blood or body fluids, other potentially infectious materials or infected animals. Regular change of gloves while working, especially in case of a potential spill to hands, and removing the gloves aseptically is crucial to prevent contamination. Also cleaning the hands after removal of gloves ensure transmission prevention. For respiratory protection, several different types of masks are available, mask types varying in the level of protection provided. When working with material posing a high risk for airborne transmission, surgical masks offer the lowest protection for the worker, and FFP3/N-95 masks provide a better protection, assuming the person have passed the fit test with the FFP3/N-95 mask selected. Respirator with motor unit using filters against chemicals and/or microbes are a good choice when needed. To ensure best practices, the training of personnel should include the correct selection and use of PPE as well as the correct dressing and undressing procedures, keeping in mind that after each working period the outside of the PPE is considered potentially contaminated.

Biosafety Cabinets In diagnostic laboratories, biosafety cabinets are in use to protect the worker as well as to protect the sample from contamination. Biosafety cabinets use laminar airflow for cabinet air, and e.g., High-Efficiency particulate air (HEPA)-filters for incoming and/or exhaust air to capture potentially hazardous particles. Class I cabinet does not filter the incoming air, so while working in these only the worker is protected from contaminating agents. Class II cabinets drive both incoming and exhaust air through filters, and thus protect both the worker and the sample (Figs. 1 and 2). Class III cabinets completely isolate the sample from the worker and are the safest choice for work with highest threat pathogens (Fig. 3). In class III cabinets both incoming and exhaust air is filtered, and the cabinet connect to ventilation system through hard duct connection.

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Fig. 1 The air flow system in biosafety class II cabinet. Green arrows ¼ Room air or filtered air to exhaust; blue arrows ¼ Filtered air inside the cabinet; red arrows ¼ Contaminated air before filtering.

With class I and II cabinets, working practices affect the safety of the cabinet, vigorous moving of hands or blocking of the cabinet’s air circulation system by paper or material causing loss of safety. After each working session the cabinets are cleaned and treated with disinfectants chosen according to risk assessment, special care is taken when handling samples for nucleic acid detection assays to reduce the possibility of nucleic acid contamination, e.g., by using disinfectants that also break down nucleic acid chain residues. Proper training of personnel is crucial to ensure the optimal protection provided by biosafety cabinets.

Decontamination and Waste Management Very important is to carefully plan and instruct the disinfection practices of all laboratory work spaces as well as laboratory equipment, which have been in contact with potentially infectious agents. Selection of decontamination method is based on the evaluation of the properties of microbial agents handled in the laboratory. Use of disinfectants should follow the instructions provided by the manufacturer, and the suitability on special laboratory equipment confirmed from the equipment manufacturer. To ensure proper effect, the concentration of disinfectants used, the contact time of disinfectant on table surfaces or equipment, and the storage of the disinfectant should always be as instructed by the manufacturer. Several disinfecting chemical agents are available: aldehydes (e.g., glutaraldehyde and formaldehyde), alcohols (e.g., ethanol and propanol), biguanides (e.g., chlorhexidine), phenol compounds, halogens (e.g., chlorine compounds and iodine), quaternary ammonium compounds, and peroxygens (e.g., hydrogen peroxide and peracetic acids). Of these, peroxygens have the widest effective range against viruses covering both non-enveloped and enveloped virus groups. In some facilities, also UV-light (germicidal UV-light, 254 nm radiation) and ozone are in use for decontamination. Special attention to selecting the decontamination method is crucial when specimens handled in a laboratory have potential to contain highly resistant agents such as bacterial spores or prions.

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Fig. 2 An example of a class II biosafety cabinet.

Safe collection of all waste including sharp material (e.g., needles and pipette tips) prevent the risk of laboratory accidents and assure safe waste management at all stages. All waste is to be collected to specific waste buckets, and the content marked to assure safe disposal of the buckets. Special attention is required on the selection of waste buckets used for highly contagious waste, these should be leakage proof and sealed for safe transportation. When found necessary waste management should include decontamination of waste before disposal by chemical ways, heat inactivation or autoclaving depending on the potential infectivity of the waste (e.g., primary samples or virus cultivations). Incineration of special waste and safe transportation to disposal unit are also important. In higher containment laboratories, decontamination of sewage water is also required either by heating or by chemical ways before release to sewage system. In higher containment laboratories, fumigation of the facilities with e.g., hydrogen peroxide or formaldehyde is required on regular basis to ensure the safe maintenance of the facilities and laboratory equipment. Emergency plan including the choice of methods and disinfectants is important to ensure the quick and effective response in case of unexpected events.

Laboratory Accidents Exhaustive reports on laboratory-acquired infections are scanty. According to the studies, many laboratory accidents and laboratory-acquired infections originate from human factors such as insufficient training, lack of PPE, lack of risk assessments or inadequate risk assessments, lack of standard operating procedures or negligence of following the existing instructions. The best way to prevent laboratory accidents is by thorough education of the staff and maintenance of good laboratory practices on daily basis. Programmed action points and emergency plans in case of an accident prevent/mitigate effectively serious consequences. In laboratory settings, accidental routes of pathogen transmission include ingestion, injection, cuts and abrasions, and inhalation of infectious aerosols. According to published studies, in many cases the route of exposure remains unknown. Of the identified cases, it is usually by inhaling infections aerosols. An aerosol is a colloid of fine solid particles or liquid droplets, in air or another gas. An aerosol with a diameter of 5 mm or less can remain airborne for a long period of time, spread wide distances, and is easily inhaled. Proper PPE such as masks or respirators are to prevent primary contact. To mitigate the risks connected to aerosol generating procedures also the use of sealable buckets for centrifugation and opening the buckets only in biosafety cabinet offer best protection in case of e.g., broken tubes during centrifugation.

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Fig. 3 An example of a Class III biosafety cabinet.

In case of a spill of infectious material to mucosa or eye, thorough rinsing with clean water is usable first aid, and proper first aid, e.g., eye shower, should be available. In case of wounds to skin gentle cleaning with soap and water, and thorough rinsing with clean water are usable first aid. To prevent the serious consequences of accidents with certain known and preventable microbes, also post exposure prophylaxis is available (e.g., HIV drug administration after a needle accident while handling HIV-positive patient blood sample or rabies-immunoglobulin administration and/or vaccination after exposure to potentially infective sample from a rabies patient or animal). Prophylactic possibilities are included in the risk assessment.

Occupational Health and Special Groups Occupational health has an important role in providing guidance and in evaluating the workers ability to work in laboratory settings. Management of the facilities assure that both physical and mental health requirements are covered. Vaccines offered by occupational health for vaccine-preventable infectious diseases, such as influenza and hepatitis B are widely used for additional preventive measures, and can offer a protective method in addition to following good laboratory practices and using PPE. Use of vaccinations should be included in risk assessment especially if work contains virus cultivation or handling high virus concentrations of certain pathogens, e.g., rabies virus and TBE virus. Planning of working profile is important especially with special groups, which are more vulnerable to infections due to their health status or ongoing medication. Special planning is important e.g., with pregnant women, immunocompromised workers, or workers suffering from atopic eczema or allergies against e.g., disinfectants or other material used in PPE (e.g., chlorine or rubber). The need for regular, medical examinations should be included in the risk assessment.

Shipment of Clinical Specimens As samples often need to be transported from the medical unit taking the samples to diagnostic laboratory doing the microbiological analysis, laboratory has an important role also in instructing the clients to send the specimens. Instructing covers the selection of appropriate tubes for different specimens and assays, proper ways of packaging including the usage of

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Fig. 4 An example of a package for transportation of Category B substances.

leakage proof tubes, adsorbent and secondary package, and transportation systems available. International and local regulations guide the transportation of potentially infective samples. International Air Transport Association (IATA) supports aviation with global standards for e.g., airline safety and security, and offers instructions and guidelines for air transportation of highly infectious material. Category A samples listed by IATA demand more extensive safety procedures than Category B samples with lower infective potential (Figs. 4 and 5). Shipment of samples containing potentially highly infectious material demands careful planning and instructions in laboratories, special training in packaging and shipment, and well-organized contacts to couriers.

Laboratory Biosecurity During the last decades, the concerns for the deliberate malicious use of pathogens and toxins have arisen. Many pathogens and toxins can be used intentionally to cause harm for humans, animals or the environment. Laboratory biosecurity measures prevent the malicious use of micro-organisms. Also the information and knowledge related to pathogens as well as materials like laboratory equipment can be controlled by biosecurity measures. Microbial agents have a dual use nature: they can be used in benign diagnostics, research or commercial purposes but they can be also used in malicious purposes. It can be difficult to prevent the unauthorized use of biological agents since the same agents can be found in nature. Even the diagnostic laboratories should take the possibility of malicious use of micro-organism into account, even though the probability of malicious use is limited. Biosafety and biosecurity practices usually comply, and often biosafety and biosecurity measures must be integrated to have an effective biosecurity system. In some cases, biosafety procedures and biosecurity procedures may be in conflict. For example, the list of biological agents handled in the laboratory can be available for safety purposes but at the same time this information should be limited for security purposes. The key components of laboratory biosecurity are physical security and access control, personnel reliability, information security, transport security and accountability for materials. In addition, biosecurity awareness and emergency response planning are integral parts of an effective biosecurity system. Physical security control measures are used to prevent unauthorized access to sensitive biological materials or information. Physical security measures also improve laboratory biosafety since they restrict the access to the laboratory to only those personnel who are trained to work with the pathogens. Also personnel reliability measures prevent the pathogens and toxins out of the hands of individuals who might have malicious purposes. Personnel who handle or transport dangerous pathogens should be trustworthy, reliable and competent to perform their duties.

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Fig. 5 An example of a package for transportation of Category A substances.

Some information related to the working with biological agents can be sensitive and must be kept secure. Sensitive information can include, for example, the list of infectious agents and samples handled and stored in the laboratory, list of key personnel or laboratory security plans. Accountability for materials and inventory control mean that the laboratory should have an up-to-date overview of high-risk materials that they are handling and storing. Transport control ensures that the national and international rules for packaging, transport, import and export are being followed when biological agents are being transported. The biological material should only be sent to trustworthy and legitimate receivers, and the transport should be performed by approved couriers. The biosecurity measures needed are defined in the risk assessment. Basis of biosecurity in diagnostic laboratory consists of proper training of the personnel including also keeping up the knowledge of restricted sharing of information with third parties and restricted access to classified information. Restricted access to the facilities, including access control and follow up are also crucial. Thorough records and documentation of storages are also important to facilitate good follow up of all material that can potentially cause risk to the environment or that could be misused. Restricted access to delicate information of the facilities and the security systems ensure biosecurity.

Biorisk Management System and Legislation Biorisk management system describes the management of both biosafety and biosecurity risks. National legislation, regulation and policies as well as international regulation, policies and guidelines often set requirements that are related to laboratory biosafety and biosecurity. Some countries have strictly regulated biosafety and biosecurity systems. There are several international guidances and standards that are related to biosafety and biosecurity, such as WHO Laboratory Biosafety Manual, Biosafety in Microbiological and Biomedical Laboratories (BMBL) or the ISO 35001 Biorisk Management for Laboratories standard. Laboratory quality standards such as ISO 17025 General requirements for the competence of testing and calibration laboratories and ISO 15189 Medical laboratories – particular requirements for quality and competence comply with the implementation of laboratory biosafety and biosecurity. National and international Biosafety Networks aim to promote biosecurity and biosafety aspect in different laboratories by e.g., sharing best practices and by organizing training on biosafety and biosecurity for laboratory professionals. In many organizations, Biosafety Officer or Biorisk management advisor is the professional with expertize on biosafety and biosecurity regulations and safe practices, and responsible to supervise and guide personnel in matters connected to biosafety and biosecurity, e.g., in evaluating the risk assessments.

Further Reading Buchan, B.W., Mahlen, S.D., Relich, R.F., 2019. Interim Clinical Laboratory Guideline for Biological Safety. The American Society for Microbiology. Available at: https://www.asm. org/ASM/media/Policy-and-Advocacy/Biosafety-white-paper-2019.pdf. Centers for Disease Control and Prevention, 2009. Biosafety in Microbiological and Biomedical Laboratories, fifth ed. HHS publication no. (CDC) 21–1112. Available at: https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf. (accessed 2.01.16).

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Laboratory-acquired infections in Belgium, 2007–2012. Available at: https://www.biosafety.be/sites/default/files/2015_willemarck_lai_report_belgium_2007_2012_final.pdf. Peter Clevestig, 2009. Handbook of applied biosecurity for life science laboratories. Stockholm International Peace Research Institute (SIPRI). Available at: https://www.sipri.org/ sites/default/files/files/misc/SIPRI09HAB.pdf. WHO, 2006. Biorisk Management: Laboratory Biosecurity Guidance. Available at: https://www.who.int/ihr/publications/WHO_CDS_EPR_2006_6/en/. World Health Organization, 2004. Laboratory Biosafety Manual, third ed. Geneva. Available at: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf?Ua=1. (accessed 2.01.16).

Relevant Websites https://www.bureaubiosecurity.nl/en Biosecurity Office | Bureau Biosecurity. https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines.html Canadian Biosafety Standards and Guidelines. https://www.cdc.gov Centers for Disease Control and Prevention. https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection-methods/chemical.html Chemical Disinfectants. https://www.epa.gov/environmental-topics/chemicals-and-toxics-topics Chemicals and Toxics Topics. https://www.ecdc.europa.eu/en/home European Center for Disease Control and Prevention. https://www.iata.org IATA. https://www.epa.gov/pesticide-registration/selected-epa-registered-disinfectants Selected EPA-registered Disinfectants. https://www.who.int/ World Health Organization.

Screening for Viral Infections Walter Ian Lipkin, Nischay Mishra, and Thomas Briese, Columbia University, New York, NY, United States r 2021 Elsevier Ltd. All rights reserved.

Introduction Viral diagnostics are increasingly significant for public health and clinical medicine. Recognition of an infectious disease as viral was once important only as an insight that allowed practitioners to exclude the need for antibiotics. Tools for virus diagnosis, surveillance and discovery have become more urgent with the expansion of the inventory of effective and specific antiviral drugs, and of the emergence of new zoonotic agents that require assays to facilitate containment and to direct investments in vaccines. An additional incentive is the evidence to support new models of diseases wherein viral infections result in autoimmunity, neurodevelopmental damage, or neoplasia, that manifest months to years after the infection has resolved. In this article we will review the status of histopathology, culture, and molecular and serological assays in clinical and environmental microbiology.

Pathology Before the advent of high throughput genetic methods for detection and characterization of viral sequences the majority of viruses were detected using electron microscopy. Prominent examples of viral discovery enabled by electron microscopy include tobacco mosaic virus, ebolavirus, variola major virus, norovirus, polyomavirus, and SARS coronavirus (Kausche et al., 1939; Breman et al., 2016; Van Rooyen and Illingworth, 1944; Long et al., 1970; Kapikian et al., 1972; Haycox et al., 1999; Nicholls et al., 2003). Electron microscopy continues to be an important tool in virology where it is increasingly employed for confirmation of a molecular finding rather than as the first step in investigating outbreaks of infectious disease. Readers may wish to see Goldsmith and Miller for an excellent review of the history and use of electron microscopy in virology (Goldsmith and Miller, 2009). Histopathology and immunohistochemistry, too, have shifted from primary roles in viral identification to applications for viral biology and pathogenesis. In the earliest applications of polymerase chain reaction (PCR) in viral discovery, primer selection was guided by findings in analyses of clinical materials using panels of antisera to a wide range of viruses or hyperimmune sera from patients with specific diseases. In our own work with Sherif Zaki for example, immunological cross reactivity facilitated the discovery of West Nile virus NY99 (Briese et al., 1999). The finding of a viral nucleic acid sequence in an extract from an individual with disease is insufficient to demonstrate a causal relationship. An argument for a causal relationship is strengthened if footprints of the agent in an affected organ can be demonstrated through immunohistochemistry or in situ hybridization for viral proteins or nucleic acids, respectively (Lipkin, 2010).

Culture The focus of clinical diagnostic virology has shifted from culture to molecular methods. This largely reflects the introduction of commercial assays that allow rapid, inexpensive detection of viral pathogens using multiplex PCR panels, and quantitative PCR assays that can be used to monitor the efficiency of interventions by tracking viral burden. Culture nonetheless remains critical to viral discovery, and basic and translational viral research. Virus amplification through culture enhances the efficiency of detection through molecular methods. SARS coronavirus, for example, was readily characterized using sequencing, microarrays decorated with oligonucleotide probes representing viral sequences, and consensus PCR. Proof-of-causation through fulfillment of Koch’s Postulate requires propagation of an agent isolated from an individual with a disease, and recapitulation of the disease following introduction of the agent into a naïve host. Although alternative strategies for proving a causative relationship have been established, Koch’s Postulate remains the most persuasive. Virus isolation is also essential for understanding how it enters a cell, replicates itself, evades innate immune responses, and causes disease in animal models. Finally, virus isolation is imperative for developing and testing the efficacy of antiviral drugs and vaccines. Viruses can be isolated and propagated in cell culture systems or in live animals. The choice of an in vitro versus an in vivo strategy can have a substantive impact even for closely related viruses. Whereas inoculation of suckling mice is useful in detecting human enterovirus A, tissue culture is better for detection of human enterovirus B. Success depends on the presence of cell surface receptors that enable entry; intracellular factors required for trafficking of viral nucleic acids and proteins to appropriate sites for transcription, replication, translation, assembly and egress; and the ability of the virus to evade innate immune responses that inhibit viral replication. Accordingly, many clinical microbiology laboratories maintain multiple continuous cell lines for virus isolation. Some viruses cannot be propagated in continuous cell lines. Cytomegaloviruses, for example, require immature cells and are typically grown in primary fibroblast cultures. Other viruses require specific receptors that can be introduced through transfection, or more complex systems comprising multiple cell types that resemble organs (organotypic cultures). Still others are even more fastidious and require live animals such as suckling or transgenic mice. Culture in primary cells, transfected cells, organotypic cultures, and live animal systems is typically restricted to public health reference and research laboratories due to the need for substantial investment in resources and expertize.

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Nucleic Acid Tests Polymerase Chain Reaction Assays The discovery of PCR by Mullis in 1983 transformed clinical microbiology and public health (Saiki et al., 1985) by enabling rapid, inexpensive, tools for microbial differential diagnosis, surveillance, and discovery, as well cDNA cloning. In viral diagnostics, PCR can be broadly divided into assays that target single agents versus those that target more than one agent (multiplex PCR assays), assays that are designed to detect only specific viral species versus those that also detect related viruses (consensus PCR assays), and assays that are quantitative (qPCR assays). The amplification products of single agent PCR can be visualized after size fractionation through gel electrophoresis using dyes like ethidium bromide or the newer and safer stains (e.g., SYBRsafe or GelGreen among others) that also intercalate with nucleic acids. Alternatively, products can be quantitated using complementary oligonucleotide probes that contain both a fluorescent reporter dye and quenching molecule. In Taqman assays, probe bound to the template is degraded by the 5’ to 3’ exonuclease activity of the polymerase, thereby freeing the reporter molecules for detection. In molecular beacon assays, the quencher and reporter molecule are in close proximity to one another in a hairpin probe structure and become separated when PCR results in a product that is complementary to the hairpin loop sequence between them. Through use of a standard curve and known concentrations of template one can measure the amount of target in an experimental sample. Taqman and molecular beacon assays are exquisitely sensitive and typically can detect as few as 10 target molecules per reaction.

Multiplex PCR The majority of multiplex PCR assays are based on the Taqman system; however, gel based, mass spectrometric, and Luminex-bead based assays have also been established. At the time of writing, only five fluorescence reporter dyes can be unequivocally separated; thus, multiplex Taqman assays are limited to five targets (typically four viral and 1 host gene target control). The sensitivity is similar to single agent Taqman PCR. Two platforms combine PCR with mass spectroscopy to detect either the product itself or tags attached to the primers. The Ibis system, adopted by Abbott, uses matrix-assisted laser desorption–ionization (MALDI) MS to directly measure the molecular weights of PCR products in a sample and to compare them with a database of known or predicted product weights (Van Ert et al., 2004; Ecker et al., 2005; Sampath et al., 2007; Ecker et al., 2008). The Ibis system discriminates products of PCR amplification using universal bacterial 16S rRNA primers. There is no correlate universal viral primer set; thus, the platform is better suited to bacteriology than virology. MassTag PCR can accommodate up to 50 different primer sets. It uses atmospheric pressure chemical ionization (APCI) MS to detect small molecular weight reporter tags attached to PCR primers (Briese et al., 2005). Syndrome-specific MassTag PCR panels have been established for the detection of viruses, bacteria, fungi, and parasites associated with acute respiratory diseases, diarrheas, encephalitides/meningitides, and hemorrhagic fevers (Briese et al., 2005; Lamson et al., 2006; Palacios et al., 2006; Renwick et al., 2007; Dominguez et al., 2008; Tokarz et al., 2009; Palacios et al., 2009; Tokarz et al., 2010; Tokarz et al., 2011; Tokarz et al., 2012; Al-Samarrai et al., 2013; Hsu et al., 2013). Another highly multiplexed platform, established by Luminex Corporation uses flow cytometry to detect PCR amplification products bound to matching oligonucleotides on fluorescent beads (Brunstein and Thomas, 2006; Han et al., 2006; Li et al., 2007). Signal in multiplex PCR and Luminex assays requires the presence of three independent genetic targets (the forward primer, the reverse primer, and the probe). This provides high confidence in the fidelity of a finding. In contrast, mass spectrometric assays require the presence of only two genetic targets. Accordingly, we use them as screening tools and follow-up presumptive findings with single agent PCR or Sanger sequencing.

DNA Microarrays Microarrays are typically 750  250  1 mm glass slides that have been modified to bind oligonucleotides or peptides for detection of host or microbial nucleic acids or antibodies, respectively. This section of the article will focus on nucleic acid detection. Arrays for serology will be discussed below. Arrays comprise hundreds to millions of probes and can be designed to detect virtually any DNA or RNA target sequence. By varying the length of probes one can differentiate between truly complementary or less closely related sequences. Whereas probes less than 25 nt are specific, those more than 60 nt in length tend to be more promiscuous. Two longer probe array platforms were established for viral surveillance and discovery: the GreeneChip and the Virochip (Wang et al., 2002; Wang et al., 2003; Palacios et al., 2007; Quan et al., 2007). Both employ random amplification strategies prior to hybridization. Because host and viral sequences are amplified with similar efficiencies, the sensitivity for viral detection in tissues is lower than for targeted PCR assays. Host nucleic acid can be reduced by enzymatic digestion (DNA) or ribosomal RNA (rRNA) subtraction; nonetheless, these platforms are most successful with acellular template sources, such as virus cell culture supernatant, serum, plasma, cerebrospinal fluid, or urine. Viral nucleic acid that is hybridized to oligonucleotides on arrays can also be eluted from microarrays to enrich for relevant template prior to sequencing (SARS) (Wang et al., 2003).

High Throughput Sequencing The introduction in 2005 of automated pyrosequencing by 454 Corporation provided the first insights in the potential power of what has become known as ‘Next Generation Sequencing’ (NGS). Before the platform was replaced in 2013 by the more efficient Illumina system, pyrosequencing resulted in the discovery and implication of novel viruses in outbreaks of disease in humans,

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domestic animals and wildlife including an astrovirus associated with fatal encephalitis in X-linked agammaglobulinemia, disseminated arenavirus infection after organ transplantation, a polyomavirus causing vasculitis with myositis and retinal blindness, LuJo and Bundigbuyo virus associated hemorrhagic fever, and a reovirus that decimated wild and domestic salmon worldwide (Palacios et al., 2010). Whereas 454 pyrosequencing required three days to yield 50,000 sequence reads, hundreds of millions of reads can now be obtained in less than 24 h. The pace of viral discovery has accelerated accordingly, and sequencers are moving into clinical microbiology and public health laboratories worldwide. In the past several years, improvements in single molecule sequencing platforms have begun to address the potential drawback of the Illumina sequencing system concerning its limited read length (B350–700 bp). Recent sequencing technologies aim at fewer but longer reads, such as the single molecule real-time (SMRT) sequencing from PacBio (Eid et al., 2009), or the MinION from Oxford Nanopore (Lu et al., 2016). The latest Sequel sequencer by PacBio generates B2 million reads in a mean length range of 15–50 kb (up to B200 kb). However, it is quite a sizable instrument and takes B30 h to run. In contrast is the MinION by Oxford Nanopore, a tiny device of B100 g that is connected to a common USB port of a laptop computer. Data can be assessed in real time after a few minutes of run-time and downloaded once sufficient data for the application purpose are collected, commonly after 12–20 h. The device generates B10 million reads ranging in length from approx. 10 kb to several hundred kb. The long-read platforms are currently used mostly in combination with short read data to generate the most accurate assemblies with regard to sequence duplications or repetitive elements (Wick et al., 2017; Giddins et al., 2018).

VirCapSeq-VERT Host and viral nucleic acids compete as template in unbiased high throughput sequencing. In complex samples, such as tissue, blood, nasopharyngeal secretions, and feces, host sequences outnumber viral sequences by orders of magnitude. The relative sparsity of viral sequences in complex samples can lead to detection failure where the number of sequence reads is insufficient to include rarer templates. Thus, to enhance the probability for recovery of viral sequences, investigators typically obtain a minimum of 200 million reads per sample. This approach is resource intensive. It requires not only a substantial investment in sequencing but also in bioinformatic analysis. One strategy to focus and increase sensitivity for viral diagnosis and discovery has focused on enrichment of viral template through subtraction of host nucleic acid via nuclease digestion and depletion of rRNA. Another strategy is positive selection. VirCapSeq-VERT is a positive selection system designed to increase the sensitivity of NGS for detection of vertebrate viral sequences based on viral sequence capture (Briese et al., 2015). The platform comprises a library of biotinylated oligonucleotide probes that tile the coding sequences of all known vertebrate viruses with an inter-oligonucleotide distance of 100–150 nucleotides. cDNA libraries prepared from samples containing viral genetic material are hybridized with the oligonucleotides. The resulting viral nucleic acid/viral probe hybrids are collected using avidin coated magnetic beads and taken forward for sequencing. When compared with unbiased NGS, VirCapSeq-VERT enables a 100 to 1000-fold increase in sensitivity and improved depth of coverage. We find that 2.5 million reads with VirCapSeq-VERT provides similar sensitivity and genome coverage to that obtained with 200 million reads after subtraction (Williams et al., 2018). Bioinformatic complexity and computational resources are reduced accordingly.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPER) Platforms Bacteria and archaebacteria have evolved a response to invading bacteriophages that entails specific degradation of viral nucleic acid. Infection results in the integration of DNA fragments of bacteriophages into the bacterial genome as clustered repeats known as CRISPR arrays. Genes encoding CRISPER-associated (Cas) proteins with endonuclease (and in some instances integrase) activity are located in close proximity to CRISPR arrays. Subsequent infection with a similar bacteriophage results in expression from CRISPER arrays and Cas genes of RNA transcripts that target and degrade viral nucleic acid. CRISPER arrays can be genetically engineered to represent virtually any sequence. Thus, when joined with the appropriate Cas enzyme the system can be used to degrade vertebrate viral sequences in addition to bacteriophage sequences. CRISPER Cas systems can also facilitate gene editing by including Cas proteins with both endonuclease and integrase activities. For diagnostic purposes CRISPER Cas systems have been employed in conjunction with isothermal amplification in lateral flow assays and in CMOS arrays for multiplex detection of viruses in environmental and clinical samples (Gootenberg et al., 2018; Freije et al., 2019).

Serology Serology complements direct detection methods by providing a history of exposure to a viral agent. To date most serology assays have been performed using single agent enzyme-linked immunosorbent assays (ELISA), neutralization assays (NT), western blots (WB), immunofluoroscence assays (IFA), haemagglutination assay (HA), or hemagglutination inhibition assay (HAI).

Hemagglutination Inhibition Assays Serum agglutination, perhaps the first routine diagnostic test, was introduced in the 1800s for diagnosis of thypoid fever (Widal, 1896). Later, agglutination of blood cells was introduced by Landsteiner to determine ABO blood groups (Landsteiner, 1901).

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In virology, the hemagglutination (HA) assay is based on the capacity of some viruses responsible for prominent human and animal diseases to bind and cross-link red blood cell surface receptors. The resulting lattice formation deposits as a diffuse reddish layer on the surface of the reaction container. The presence of virus-specific antibodies (Abs) bind the virus and thus inhibit agglutination of red blood cells. Antibodies are quantitated by incubating fixed numbers of red blood cells and virus stock with dilutions of a test serum. Hemagglutination inhibition (HAI) assay are commonly used in typing and subtyping of influenza viruses (Hirst, 1942). Other agglutination assays include latex agglutination (LA) systems where microbeads are coated with Abs or viral antigens.

Plaque Reduction Neutralization (PRNT) Assays Antibodies can be detected and quantitated based on their capacity to prevent viruses from binding to and infecting their target cells (Pedersen and Spackman, 2014). The readout is a reduction in the number of lytic plaques in a culture of susceptible cells inoculated with the virus of interest. In PRNT as in hemagglutination assays, the requirement for infectious virus poses challenges for many clinical microbiology laboratories, particularly with assays for highly pathogenic viruses. Accordingly, in some instances investigators used less pathogenic viruses such as vesicular stomatitis virus (VSV) where the VSV glycoprotein gene has been exchanged for the glycoprotein of a more pathogenic virus such as ebola (Lee et al., 2017). Because of its native Ag presentation and biological signal readout PRNT is the still the method of choice for measuring functional (protective) immunity. Since even minor variation in protein structure result in changed neutralization behavior, it is also superior to the other methods in distinguishing closely related viruses via cross-neutralization studies. The specificity of PRNT assays is frequently exploited in differentiating immunological reactivity to related viruses. For example, PRNT is considered the gold standard in the differential serodiagnosis of infection with flaviviruses including Zika and dengue viruses (Kuno, 2003; Musso and Gubler, 2016). Only recently, Serochip analyses enabled the characterization of specific peptides that include distinguishing epitopes that now can be transferred other platforms such a peptide ELISA or lateral flow format with the promise of specific identification (Mishra et al., 2018). PRNT assays can also be employed to identify virus isolates through use of panels of hyperimmune sera generated in experimentally infected animals, and to group viruses taxonomically (Casals, 1957; The Enteroviruses, 1957; Schmidt et al., 1961; Muir et al., 1998).

Immunofluorescent Assays (IFA), Enzyme-Linked Immunosorbent Assays (ELISA) and Immunoblot Assays Each of these methods build on the specific binding between Ab and its matching Ag. In IFA, cultured cells are infected with virus, fixed on glass slides or in multi-well plates with acetone or formaldehyde, and incubated with the test serum, plasma or CSF. A reporter dye linked to a secondary Ab directed against the primary (test) Ab is then introduced prior to fluorescence microscopy. Secondary antibodies can also be conjugated to enzymes that yield products that can be visualized by light microscopy. Enzyme-based dye formation is also the basis for the ELISA (Engvall and Perlmann, 1971; Van Weemen and Schuurs, 1971), the most common assays used for antibody detection. Amongst the various platforms, the ELISA is the most versatile and popular. This is in part because of the multitude of ways to present Ags or Abs and the comparatively high throughput in 96-well format. Another advantage is the ability to present targets in native form supporting the detection of a wide variety of epitopes. The ELISA is typically performed in 96-well plates wherein wells coated with a viral antigen are incubated with serially diluted patient body fluid, a reporter enzyme (usually peroxidase or alkaline phosphatase) directly conjugated to the serum immunoglobulins (direct ELISA) or a secondary antibody (indirect ELISA), and finally a dye system that generates upon enzymatic catalysis a soluble colored product that can be read spectrophotometrically. A common variation is the sandwich ELISA wherein the well-surface is first coated with Abs specific for the Ag (capture Ab) before the antigen is applied. Next, the detection Ab is applied, either in direct or in indirect format. The advantage of the sandwich ELISA is that it increases the sensitivity and specificity of the assay. Western blot assays (also known and immunoblots) are typically performed including complex Ag preparations such as crude infected cell extracts that are size fractionated by electrophoresis in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels, transferred onto nitrocellulose or polyvinylidene difluoride membranes, and then incubated with sera. Immunoreactivity of antibodies in sera are detected using a species-specific secondary antibody that is linked to an enzyme that results in either a dye or chemiluminescence signal. Proteins subjected to SDS-PAGE are denatured. Thus, they are not designed to detect conformational epitopes that may be critical in viral neutralization.

Lateral Flow Assays (LFA) These assays are designed for point of care serodiagnosis and field applications. In LFAs. the liquid sample (plasma, serum, urine or CSF) flows by capillary action through a membrane where the antigen or antibody in the sample binds to a dye-labeled cognate marker (antigen or antibody). The antigen-antibody-dye marker complex continues to migrate through the membrane until further migration is blocked by binding of the complex to an antibody to a component of the complex that is fixed in position, the appearance of a line of dye indicates that the antigen or antibody was present in the sample.

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Multiplex Platforms for High Throughput Serology In contrast to molecular diagnostics where advances in technology such as PCR and HTS have dramatically improved sensitivity, specificity, and capacity for multiplex analyses, serologic methods remained largely unchanged. This lag is important given the role of serology in establishing the distribution and frequency of infection, testing the significance of association between the finding of an agent and disease, focusing efforts in pathogen discovery and surveillance, and for monitoring humoral responses to vaccines and immunomodulatory drugs. Most serological tests target only one agent; however, Luminex-based systems have been employed that can address up to 100 targets simultaneously. Phage display systems and arrays that comprise spotted recombinant proteins expressed in vitro in E. coli, S. cerevesiae, baculoviruses, and cell-free, coupled transcription-translation, have also been established. The VirScan platform comprehensively tests for the presence of antiviral antibodies through immunoprecipitation of viral peptides in bacteriophage followed by DNA sequencing. The current version screens 93,904 200-mer oligonucleotides, encoding 56-residue peptide that tile the proteome of 206 viral species with 28 residue overlap (Xu et al., 2015). The phage library is incubated with serum, plasma, or CSF. Bound antibodies are recovered by using a mixture of protein A and G coated magnetic beads. The bound phage DNA is sequenced and quantitated to identify immunoreactive epitopes. These assays are powerful but require expertize and resources for cDNA cloning, expression, sequencing, and bioinformatic analysis. An additional challenge is that VirScan may not have the resolution required to differentiate immune responses to closely related viruses. Peptide arrays (Serochips) are an alternative high throughput platform for unbiased serology. Serochips comprise up to 6 million distinct linear peptide sequences attached to a solid support. Like the VirScan platform, Serochips display only linear peptides; thus, neither platform will detect conformational or glycosylated epitopes. To ensure our ability to detect immune responses to discriminant as well as common viral epitopes we tile the entire proteome of the relevant viruses using 12 aa peptides with 11 aa overlap. After incubation with serum, plasma or CSF, arrays are incubated with fluorescence-labeled secondary antibodies (IgG or IgM). Finally, high-resolution image scans are obtained and relative fluorescence signal intensity is used to map immunoreactivity to specific viruses and identify peptides that can be transferred to less expensive and simpler methods for serodiagnosis such as ELISA, western blot, Luminex assays, or smaller peptide arrays. The detection of an IgG versus an IgM response is important to certain aspects of functional serology. During the challenge of the immune system with a new Ag, stimulated B-cells produce their membrane-presented Ab first as pentameric immunoglobulin class M (IgM) isotype antibodies, that usually exhibit limited specificity. During the next 2–4 weeks plasma B-cells transition after isotype switching and hypermutation to the production of highly specific monomeric immunoglobulin G (IgG) isotype antibodies (Nossal, 2007; Tonegawa, 1983). The ratio of IgM to IgG response may therefore be helpful in determining the timing of the infection. IgM vs IgG determination is easily achieved in all indirect assay systems through the choice of the secondary Ab, applying either an anti-human IgM or an anti-human IgG-specific Ab. There are many other considerations directing the choice of a particular serologic platform. This often includes the classical paradigm of confirming a positive result in one assay in a second technically independent assay, as it is still common practice in Lyme diagnosis; after initial screening by ELISA positive results are confirmed by immunoblot reaction patterns (Centers for Disease Control and Prevention, 1995; Tokarz et al., 2018).

Discussion/Summary Viral diagnostics is no longer a discipline that requires resources and expertize available only in reference laboratories. Although culture and electron microscopy remain important tools for basic and translational research, molecular methods have moved to the forefront of diagnostics in clinical microbiology and public health. This reflects not only lower costs and ease of use but also the capacity of molecular methods to yield actionable information in hours rather than days. Serology too is becoming more robust and convenient with the advent of multiplexed assays that provide specific information without virus cultivation and neutralization assays. Advances in molecular and serological methods allow clinicians and public health practitioners to simultaneously consider a wide range of probable candidate viral pathogens, and to detect the presence of pathogens that are not typically anticipated in a differential diagnosis. These include viruses known to cause disease in other geographic regions or in other species, or viruses not previously known to medicine or science. We have not addressed in this article the potential for host genetic, proteomic, metabolomic or microbiome analyses to provide diagnostic and prognostic information independent of virus identification that will impact interventions. This is an area of active research that we anticipate will be featured in future editions of this encyclopedia. We also anticipate that insights from wider application of molecular and serological tools will encourage the development of drugs and vaccines that reduce the burden of viral infections in both acute and chronic disease.

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Tokarz, R., Kapoor, V., Samuel, J.E., et al., 2009. Detection of tick-borne pathogens by MassTag polymerase chain reaction. Vector-Borne and Zoonotic Diseases 9, 147–152. Tokarz, R., Kapoor, V., Wu, W., et al., 2011. Longitudinal molecular microbial analysis of influenza-like illness in New York City, May 2009 through May 2010. Virology Journal 8, 288. Tokarz, R., Mishra, N., Tagliafierro, T., et al., 2018. A multiplex serologic platform for diagnosis of tick-borne diseases. Scientific Reports 8 (1), 3158. Tonegawa, S., 1983. Somatic generation of antibody diversity. Nature. 302 (5909), 575–581. Wang, D., Coscoy, L., Zylberberg, M., et al., 2002. Microarray-based detection and genotyping of viral pathogens. Proceedings of the National Academy of Sciences of the United States of America 99 (24), 15687–15692. Wang, D., Urisman, A., Liu, Y.-T., et al., 2003. Viral discovery and sequence recovery using DNA microarrays. PLOS Biology 1 (2), e2. Van Ert, M.S., Hofstadler, Y., Jiang, J., et al., 2004. Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis. BioTechniques 37 (4), 642. Van Rooyen, C.E., Illingworth, R.S., 1944. A laboratory test for diagnosis of smallpox. British Medical Journal 2 (4372), 526–529. doi:10.1136/bmj.2.4372.526. Van Weemen, B.K., Schuurs, A.H.W.M., 1971. Immunoassay using antigen-enzyme conjugates. FEBS Letters 15 (3), 232–236. Wick, R.R., Judd, L.M., Gorrie, C.L., Holt, K.E., 2017. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLOS Computational Biology 13 (6), e1005595. Widal, F.M., 1896. Serodiagnostic de la fiev́ re typhoide a-propos d’une modification par MMC Nicolle et al. Halipie. Bulletins et mémoires de la Société Médicale des Hôpitaux de Paris 13, 561–566. Williams, S.H., Cordey, S., Bhuva, N., et al., 2018. Investigation of the plasma virome from cases of unexplained febrile illness in Tanzania from 2013–2014: A comparative analysis between unbiased and VirCapSeq-VERT high-throughput sequencing approaches. mSphere 3 (4), e00311–e00318. Xu, G.J., Kula, T., Xu, Q., et al., 2015. Viral immunology. Comprehensive serological profiling of human populations using a synthetic human virome. Science. 348 (6239), aaa0698.

Clinical Diagnostic Virology Marcus Panning, Institute of Virology, Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany r 2021 Elsevier Ltd. All rights reserved.

Glossary DNA Deoxyribonucleic acid. Evidenced based medicine Uses the currently available best evidence in making decisions about the clinical management of individual patients. Multiplex PCR An assay to detect different pathogens/ targets simultaneously in a single or limited number of reaction tubes. Nucleic acid testing (NAT) The direct amplification and detection of DNA or RNA (after reverse transcription prior to PCR).

Point-of-Care-tests Rapid tests that can be performed close the patient with minimal hands-on time and ease of use. Polymerase chain reaction (PCR) Enzymatic thermocyclic amplification procedure for DNA. Randomized controlled trial A study that allocates enrolled patients randomly to either one of two arms: one intervention arm and one control arm. It thus eliminates bias. Reverse-transcription PCR For RNA, nucleic acids are transcribed into DNA using reverse transcriptase enzymes. RNA Ribonucleic acid.

Introduction Traditionally, clinical diagnostic virology methods included virus isolation using cell culture, antigen detection, and antibody detection methods. Until recently, these conventional methods were rather slow, labor-intensive and only available for a limited number of viral infections. In most instances diagnosis was retrospective. Thus, clinical diagnostic virology had little impact on patient management and clinical decision-making. However, clinical diagnostic virology has been revolutionized with the advent of nucleic acid testing (NAT) more than three decades ago. Molecular methods allow the detection of minute amounts of viral genomes and rapidly became indispensable tools for the clinical diagnostic laboratory. Target-specific amplification of (parts) of the viral DNA or RNA genome using polymerase chain reaction (PCR or reverse-transcription PCR for RNA viruses) is the most common technique used for NAT, but other technologies such as nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), or loop-mediated isothermal amplification (LAMP) have also found their niche. Beyond the pure detection of viral pathogens, NAT also provides a means to quantitate the amount of viral DNA/RNA (“viral load”). This was primarily made possible with the introduction of real-time quantitative PCR. Moreover, PCR combined with sequencing capabilities provides a tool to genotype and characterize a virus, i.e., to look for resistance-associated mutations in the viral genome. Technically, next-generation sequencing technologies are rapidly evolving and characterize viral genomes with unprecedented depth and accuracy. In combination with the increasing availability of antiviral therapeutics clinical diagnostic virology thus became essential for optimized patient care, e.g., in the context of viral load monitoring of HIV-, HBV- or HCV-infected patients or monitoring of CMV, EBV and other relevant viruses in immunosuppressed patients as well as for resistance testing. Consequently, with the advent of NAT many traditional “reference” methods became obsolete. It should be noted, however, that even in the era of NAT a mix of different techniques remains essential for the clinical virology laboratory to come to a valid and reliable diagnosis. Compared to conventional virological techniques NAT is faster, more sensitive and available for a wider range of pathogens. However, the first NAT assays were laboratory-based and still had turn-around times of up to 48 h. If testing for multiple viruses was required, this was mainly done in a sequential and time-consuming manner. In the next step, laboratories developed in-house assays that used the same PCR reaction components and the same thermal cycling profile. This allowed parallel testing for multiple targets but still required separate nucleic acid extraction and PCR set-up steps (Bonzel et al., 2008; Gunson et al., 2008). With constant technical improvement, multiplex panels for the simultaneous detection of a variety of pathogens in a single reaction have been introduced into routine diagnostics recently. This approach is also called “syndromic testing approach” and is evolving rapidly. Here, a simultaneous search for multiple pathogens causing a similar clinical picture is performed. Some of these assays require minimal hands-on time by using single-use cartridges and allow for near-patient testing (also termed “point-of-care testing” ¼ POCT). Time to result may be as low as o2 h on these integrated platforms. A number of different syndromic panels are now commercially available, e.g., for respiratory tract infections, gastrointestinal infections, and meningitis/encephalitis. Complementary to this, fully automated random-access platforms combining nucleic acid extraction and amplification/detection are now on the horizon for numerous pathogens, e.g., CMV, EBV, HIV, HCV, HBV and will certainly impact clinical diagnostic virology. Therefore, it is anticipated that rapid and near-patient testing will again constitute a dramatic change for the field of clinical diagnostic virology and may directly impact patient management. This includes but is not limited to the administration of (new) antiviral drugs (if available), appropriate use of antibiotics, and rapid implementation of infection control measures. This is accompanied by a variety of novel next-generation sequencing approaches entering diagnostic laboratories allowing “personalized medicine”. However, although many of these novel

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Fig. 1 Interdisciplinary network of clinical diagnostic virology.

techniques demonstrated very good diagnostic performance and accuracy the clinical impact remains unclear to date. It is mandatory that beyond many already published studies high-quality and randomized controlled trials will shed more light on the question if these new technologies will provide a benefit for the patient and will improve outcomes without doing harm. Another challenge remains the currently high costs of these novel assays, the optimal ordering strategy, rapid communication and interpretation of test results in the context of clinical disease and lastly quality control issues. Clinical diagnostic virology is truly interdisciplinary and requires a close partnership of infectious diseases clinicians, clinical virologists, microbiologists, laboratory/bioinformatics specialists, epidemiologists, infection control specialists, and public health members (Fig. 1). Foremost, close interaction with clinicians is essential to serve their needs and in turn for clinicians to understand test characteristics, result in interpretations and their utilization. This is especially true when new test panels will be introduced, which should be accompanied by performance evaluation and ideally cost-effectiveness calculation by the laboratory. Thus, clinical diagnostic virology should be an integrated part of antimicrobial and diagnostic stewardship programs (Dik et al., 2017). In this respect, clinical diagnostic virology should interact will all stakeholders, e.g., administration and purchasing department and develop solutions to the best of our patients. This may differ from country to country and even within countries with a very dynamic landscape of university-affiliated laboratories, large non-academic laboratories and those serving for basic and regular care. It is obvious that laboratories may serve different patient populations (e.g., number of immunosuppressed and immunocompetent patients, intensive care units, high or low throughput) and have different requirements (e.g., proximity to a hospital). In addition, large healthcare group laboratories may have their own needs and develop different approaches. Beyond primary patient care, clinical diagnostic virology also contributes to translational research and the implementation/ evaluation of emerging technologies and their transition into clinical care. Having this in mind data sharing to assess the burden of diseases and to better appreciate the epidemiology of viral infections seems mandatory on a national as well as international level. This article will briefly describe examples of syndromic testing approaches currently used in clinical diagnostic virology and how they might impact clinical decision making and the currently available evidence supporting this. Further, the impact of clinical diagnostic virology on current knowledge of hepatitis E virus infection will be briefly discussed as an example of an emerging infection and translational research.

Respiratory Tract Infections Background Acute respiratory tract infections (RTI) have a high disease burden worldwide and contribute significantly to mortality. In general, these acute RTI have a viral etiology and less common a bacterial one. Of note, acute RTI can be caused by a variety of respiratory viruses from different virus families including, e.g., influenza virus, respiratory syncytial virus (RSV) A/B, coronavirus, human metapneumovirus, and rhino-/enterovirus. Seasonal influenza alone causes up to 650,000 deaths worldwide and enormous socioeconomic losses each year. Clinical presentation can range from mild upper RTI to severe lower RTI and pneumonia. Of note, specific populations are at increased risk for severe clinical courses, e.g., immunosuppressed patients, HIV-infected patients, pregnant women, and the elderly. Except for viruses, atypical bacteria like Bordetella pertussis, Mycoplasma pneumonia and Legionella

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Table 1

Selection of currently available commercial multiplex panels from major companies

Manufacturer

Respiratory panel

Gastrointestinal panel

Luminex

BioMérieux

xTAG GPPa xTAG RVPa xTAG RVP Fast/FASTv2a NxTAG RPPa Verigene respiratory pathogens flex Verigene enteric pathogens test FilmArray Gastrointestinal (GI) Panel FilmArray Respiratory (RP) Panel

Fast track diagnostics Qiagen Genmark Seegene Hologic

FTD respiratory pathogens 21a, 21plusa, 33a QIAstat-Dx Respiratory Panel ePlex Respiratory Pathogen Panel Allplex respiratory panel 1–4a Panther fusion respiratory assays

FTD Viral Gastroenteritisa, Bacterial gastroenteritisa, Stool parasitesa QIAstat-Dx Gastrointestinal Panel – Allplex GI-Virus assaya –

Meningitis/encephalitis panel – – FilmArray Meningitis/Encephalitis (ME) Panel FTD Viral meningitisa, Bacterial meningitisa – – Allplex meningitis-V1/V2 assaya –

a

Non-cartridge/pouch-based systems.

pneumophila can also cause acute lower RTI/pneumonia. These bacteria are not very well detected by standard culture-based microbiological techniques and are therefore frequently included in modern molecular panels. Thus, testing for atypical bacteria should complement the diagnosis of acute RTI. Suitable clinical samples include nasopharyngeal swabs and aspirates/washings, pharyngeal swabs, high-quality sputum, and brochoalveolar lavage. Limitations apply as per the recommendation of the manufacturer of commercial assays as some assays have been only validated for a specific specimen type. The vast majority of (pediatric) RTI cases are seen by primary care physicians. However, it is currently impossible for primary care physicians to distinguish viral etiologies of an acute RTI and even more important to distinguish a viral from a bacterial etiology on clinical grounds only. This makes a laboratory-based diagnosis mandatory for a definite diagnosis. Critically, antibiotics are prescribed in >65% of consultations due to RTI although viruses cause the vast majority of RTI. Rapid identification of the etiology may lead to optimized patient care and treatment and may also reduce overall costs. From a public health perspective antibiotic resistance is an emerging problem worldwide and overuse/not appropriate use of antibiotics is therefore of great concern. In addition, viral infections like influenza can be treated with specific antiviral drugs, e.g., oseltamivir, but these drugs need to be given quite early in the course of the disease. Interestingly, the use of broadly reactive multiplex panels already allowed new insights into the epidemiology of previously underestimated viral infections like coronaviruses. Finally, a rapid diagnosis may also prove useful for better infection control and prevention within the hospital setting.

Assays In recent years, a paradigm shift has been observed for the detection of pathogens causing acute RTI. The first multiplex PCR assays were mostly laboratory-developed tests (LDT) and were mainly based on real-time PCR technology. One of the first commercial assays for the comprehensive detection of RTI pathogens was developed by Luminex Corporation. However, these assays still required separate nucleic acid extraction steps and the time to result was somehow in the range of around 8 h. Technological advancements led to the development of cartridge-based systems which combined nucleic acid extraction and PCR amplification/ detection allowing to test for a battery of different pathogens with minimal hands-on time within less than two hours. Different systems are now on the market and commercially available, e.g., FilmArray, ePlex or Verigene (Table 1). Popowitch et al. compared four different multiplex panels and determined overall sensitivity and specificity, ranging between 84.5% and 100%, respectively (Popowitch et al., 2013). It is important to note that sensitivity for different targets can vary and that laboratories are aware of the performance data and possible limitations of the assays they perform. Of note, selected pathogens yielded considerably lower sensitivities e.g., adenovirus and influenza virus in some studies, which is an issue of concern (Popowitch et al., 2013). Another study compared the Genmark ePlex RPP assay with LDT and showed a high overall agreement of 97.4% rendering them suitable for routine diagnostics (Nijhuis et al., 2017). Some of these assays already received FDA/CE marks and a recent study showed a high diagnostic accuracy of these multiplex panels (Huang et al., CMI 2018). Limiting their widespread implementation are considerable higher costs and presently limited availability of different panels on these platforms. However, recent experience has shown that the costs of multiplex panels are declining as more and more companies enter the market with newly developed panels. In addition, pending issues concerning quality control, reimbursement, and notifiable diseases have to be discussed in some countries. Critically, molecular assays need constant re-evaluation and close monitoring of new or mutated viral strains, which might affect assay performance. In 2009, a new influenza A virus emerged and challenged many established detection systems. In addition, e.g., influenza drift variants may go undetected if primer and/or probe binding regions are affected. It is a well-known phenomenon that especially RNA viruses are prone to genetic changes. Similarly, for enteroviruses and adenoviruses, a diversity of different types/ genotypes is known, and comprehensive detection of all members is challenging. It should also be noted that these panels include fixed combinations of pathogens and usually do not allow them to be customized. Prevalence of RTI pathogens vary in time and

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geographically and thus affect positive and negative predictive values. It is mandatory to record positivity rates for all detected pathogens for quality control purposes. The likelihood of false-positive results must be taken into account and might require alternative testing with individual assays. Thus, the laboratory must be aware of possible pitfalls of the assays in use (van Zyl et al., 2019). Strict operating procedures must be followed to minimize the risk of contamination. This holds especially true when nearpatient testing is done by non-laboratory personnel outside dedicated molecular diagnostic facilities. Further, NAT detects nucleic acids only and at present, it is impossible to assess infectivity/viability. Prolonged shedding of viral RNA has been described especially for entero-/rhinoviruses in the immunocompromised host. Finally, the role of co-detections is unclear to date. Some studies suggest that co-detections might be more severe compared to mono-infections whereas other studies did not show any differences.

Clinical Impact Although multiplex panels have been introduced by many laboratories worldwide within the last years the clinical impact of these rapid multiplex panels remains to some extent unclear to date. The first studies mostly used a before/after approach to analyze the impact after the introduction of multiplex testing approaches. Not surprisingly, these studies demonstrated a significant decrease in time to diagnosis (Rappo et al., 2016). Another study reported a significant increase in the number of detected pathogens. However, limitations apply as viral culture or other rather slow conventional methods were used in the prior season. Proper administration of antiviral therapies is another important issue in acute RTI. Vos et al. analyzed the impact of rapid viral testing in adult immunosuppressed patients and were able to show a more adequate use of oseltamivir (Vos et al., 2019b). Another recent systematic review and meta-analysis also yielded inconclusive results due to a very heterogenous study landscape (Vos et al., 2019a). In theory, the rapid identification of a viral etiology may help reduce unnecessary antibiotic treatment and initiate specific antiviral therapy, e.g., in influenza. Moreover, new antiviral therapies are in the pipeline of pharmaceutical companies, e.g., for RSV. The implementation of rapid molecular testing in the emergency department resulted in a more targeted approach to use oseltamivir treatment in immunocompromised patients. However, in the same study, no reduction in antibiotic prescriptions or its duration, admission and length of stay was seen. It is anticipated that new studies will shed more light on the clinical impact of rapid near-patient testing and will help to better appreciate their usefulness for patient care. Ideally, randomized controlled trials will be initiated to answer these urgent questions. It may also influence the decision to admit patients, reduce the length of stay, and avoid antimicrobials. Costs are also an important factor requiring careful consideration. As an example, multiplex panels showed to be cost-effective in the pediatric setting when children with influenza-like illness in the emergency department were analyzed. However, the estimation of costs and possible savings remains complex and varies from country to country. Beyond laboratory costs (which might increase due to higher test costs and extended availability of personnel) costs might decrease overall for the hospital. Recently, the €hr concept was introduced to analyze this in more detail (Dik et al., 2016). To summarize, the clinical benefit of rapid syndromic testing for the patient is to some extent unclear to date. A differentiated approach might be necessary taking into account e.g., the severity of the disease and the immune status of the patient. Syndromic testing might be beneficial for the immunocompromised host, but a more targeted approach might be necessary for the otherwise healthy outpatient and for children. It might be necessary to establish local testing algorithms for specific patient groups to use these promising new technologies properly. This may also include different testing approaches over the year to account for seasonal variations in the prevalence of RTI pathogens.

Gastrointestinal Infections Background Similar to the situation with acute RTI, gastrointestinal infections (GI) are also associated with a high disease burden worldwide. An estimated 2 billion cases occur each year and they constitute a major cause of emergency department visits and hospitalizations worldwide (Farthing et al., 2013). In particular children in resource-limited countries are affected and mortality in these settings can be very high. It is estimated that around 2 million deaths each year occur in developing countries. On the contrary, in industrialized countries, GI infections also have a great impact on individual and public health but to a lesser extent on mortality. However, although most infections are usually mild and self-limited in industrialized settings, severe courses and sometimes fatal outcomes are seen in vulnerable populations like the elderly and immunocompromised persons. Of note, outbreaks have been described, e.g., in the hospital setting, in nursing homes or on cruise ships. Transmission occurs via contaminated food or water or close contact with an acutely infected person. In addition, an aerosol transmission has been described for noroviruses. Relevant pathogens for GI are of viral, bacterial or parasitic origin. Traditional methods for the detection of GI pathogens include laborious and time-consuming microscopy, culture and antigen detection methods. The time to final diagnosis can take days. In addition, all of these techniques are less sensitive and specific compared to NAT. Timely diagnosis may prevent further spread and adverse patient outcomes and selected patients

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may benefit from rapid and appropriate antibiotic treatment. Isolation and/or cohorting of patients with norovirus associated GI is important in the health care setting.

Assays Numerous LDT, as well as commercial assays, are available for single pathogens but similar to RTI multiplex panels are now entering the market. Some of these platforms allow for rapid and near-patient testing using single-use cartridges or pouches (Table 1). In analogy to RTI panels different viral, bacterial and parasitic targets are included and allow for a “syndromic” testing approach. Most assays include more bacterial/parasitic targets than viral targets. Comparison studies have demonstrated equivalent or even superior performance to reference assays. In these studies, Z2 pathogens were detected significantly more frequently throughout different studies compared to conventional methods. Major viral targets included in these multiplex panels are adenovirus 40/41, astrovirus, norovirus genotype 1/2, rotavirus A, and in some assays sapovirus.

Clinical Impact The Luminex GPP assay was one of the first syndromic assays for GI pathogens on the market. However, it still required separate nucleic acid extraction procedures and multiple (post-PCR) processing steps. It was shown that costs for the clinical laboratory increased but overall, there were savings for the hospital (Goldenberg et al., 2015). Another study evaluated the impact of the FilmArray GIP on length of stay and patient isolation and concluded that patients spent considerable time without appropriate isolation methods in the hospital whereas others were placed under contact precautions without cause (Rand et al., 2015). One important observation was reported by Claas et al. in that 65% of positive results were from pathogens not specifically ordered by the treating physician (Claas et al., 2013). The clinical impact is much harder to assess in resource-limited settings where it became apparent that in children numerous pathogens can be detected in cases with diarrhea as well as in asymptomatic controls (Leva et al., 2016). This requires careful analysis of the local setting (i.e., patient population: adult versus pediatric, ambulatory versus in-hospital, immune status/previous antibiotic treatment, travel associated) and to evaluate the implementation of new methods in the respective laboratory. For some patients, a more tailored approach would be more effective but not possible with the fixed combinations delivered by the manufacturers. It seems advisable to develop algorithms, which patient should be tested with which method. This apparently needs a cross-sectional and collaborative effort of all involved partners within a hospital (Fig. 1). However, there is still limited evidence based data available to show a clear benefit for the patient without doing harm in industrialized settings. Of note, for bacterial pathogens culture and susceptibility testing is usually not recommended by many current guidelines but may prove useful in the context of outbreak investigations. It is also vital for public health surveillance and epidemiological purposes.

Central Nervous System Infections Background Infections of the central nervous system are usually caused by viruses or bacteria or to a lesser extent fungi. They can lead to encephalitis, which is an inflammation of the brain. It is frequently accompanied by meningitis, which is an inflammation of the meninges. Meningitis/encephalitis (ME) is associated with significant morbidity and mortality worldwide. Clinically, signs and symptoms of ME or often unspecific. Headaches and altered mental status are frequently reported. For diagnosis of microbial pathogens, Gram staining, microscopy and bacterial culture are standard laboratory methods. This is complemented by analysis of cerebrospinal fluid (CSF) for protein levels, white blood cell count and other clinical chemistry parameters. Molecular testing for viruses, which include herpes simplex viruses 1 and 2, varicella-zoster virus, and enterovirus as the most common viral pathogens causing ME has been well established by many laboratories for a long time. In addition, serological testing for flaviviruses (tickborne encephalitis virus, West Nile virus) can be performed depending on the local epidemiology.

Assays Compared to the situation for respiratory and gastrointestinal infections the availability of multiplex panels is rather limited. However, a number of different assays are in the pipeline of commercial companies and it is expected that they will soon enter the market. However, their performance has to be evaluated in comparison studies first. One of the first assays on the market was the Biofire FilmArray Meningitis/Encephalitis panel, which includes 14 targets including viral as well as bacterial/parasitic pathogens. Analytical performance studies showed somehow contrasting results when the assay was compared to culture and molecular methods (Leber et al., 2016). Of importance, false-positive results have been reported and the results of the FilmArray need to be interpreted cautiously and in combination with clinical signs and other laboratory markers. This again highlights the importance of strict contamination precautions, dedicated laboratory space and trained personnel for molecular testing. In addition, the pretest probability for ME has to be taken into account. Critical points of the multiplex panels for ME include e.g., the abovementioned sensitivity/specificity issues, the composition of the panels and the relevance of detecting bacteria in populations with

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successful immunization programs.

Clinical Impact Similar to the presently limited number of assays information on the clinical impact remains scarce. With respect to the FilmArray, no reduction in antimicrobial use or length of stay was reported in a study with rather small sample size. Thus, as soon as more assays are available and have proven their analytical performance ideally randomized controlled trials are necessary to assess the clinical impact of these assays. Ultimately, evidence based guidelines should provide reliable information on the use of these ME panels.

Hepatitis E Virus Infection Hepatitis E virus (HEV) infection is another example of clinical diagnostic virology leading to a fundamental change in current knowledge and clinical practice. Until recently, HEV was thought to be restricted to countries in Africa and Asia. In these countries, HEV genotypes 1 and 2 are causing HEV infections and transmission occurs through fecally contaminated water. Illness is usually short-lived mild hepatitis only. Exceptions are severe courses with a high fatality rate in pregnant women. Sporadically, outbreaks can occur with thousands of cases. In industrialized countries, HEV infections were occasionally seen in travelers who acquired infections abroad. Due to imperfect serological methods, this led to a gross underestimation of HEV seroprevalence rates (Kmush et al., 2015). However, interest and progress in HEV understanding dramatically changed when it became clear that HEV genotypes 3 and 4 are prevalent in industrialized countries and are transmitted zoonotically from infected animals (mostly swine, deer and others). Importantly, clinical diagnostic virology could demonstrate locally acquired HEV infection in chronic liver disease (Dalton et al., 2007). In brief, diagnosis is based on the detection of HEV-specific antibodies and more recently quantitative real-time PCR became available to detect HEV RNA in serum and stool samples. Of note, serological assays differ in their performance partially questioning the results of older seroepidemiological studies (Hartl et al., 2016). The first comparison studies were conducted showing significant differences among assays. However, in the absence of a true gold standard assay performance is still under evaluation. At the same time, the industry developed new test kits with improved performance characteristics. In addition, quantitative real-time PCR and newly established WHO standards for quantitation of viral HEV RNA were introduced. These are now widely used to monitor patients with chronic HEV infection and to control the success of antiviral treatment (Kamar et al., 2014). Using these novel tools, clinical diagnostic virology provided new data on seroprevalence in many European regions and elsewhere. Interestingly, using state-of-the-art diagnostic tools it soon became clear that beyond hepatitis other extrahepatic manifestations are associated with acute HEV infection including various neurological injuries (Dalton et al., 2016). Thus, clinical diagnostic virology led to new knowledge and changed what was previously thought to be accepted knowledge. To summarize, clinical diagnostic virology is an interdisciplinary subject requiring close collaboration foremost with clinicians and other related disciplines. Recently, multiplex panels proved to be valuable and powerful tools and are anticipated that fully automated random-access machines for almost all relevant viral targets will enter the market soon. However, critical issues remain, and we need evidenced-based data from high-quality trials on whether to implement these multiplex panels into routine diagnostics. Beyond technical issues, this also requires cost-effectiveness analysis and the development of locally adopted diagnostic testing algorithms to benefit the patient.

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Kmush, B.L., Labrique, A.B., Dalton, H.R., et al., 2015. Two generations of “gold standards”: The impact of a decade in hepatitis E virus testing innovation on population seroprevalence. The American Journal of Tropical Medicine and Hygiene 93, 714–717. Leber, A.L., Everhart, K., Balada-Llasat, J.M., et al., 2016. Multicenter evaluation of biofire filmarray meningitis/encephalitis panel for detection of bacteria, viruses, and yeast in cerebrospinal fluid specimens. Journal of Clinical Microbiology 54, 2251–2261. Leva, A., Eibach, D., Krumkamp, R., et al., 2016. Diagnostic performance of the Luminex xTAG gastrointestinal pathogens panel to detect rotavirus in Ghanaian children with and without diarrhoea. Virology Journal 13, 132. Nijhuis, R.H.T., Guerendiain, D., Claas, E.C.J., Templeton, K.E., 2017. Comparison of ePlex respiratory pathogen panel with laboratory-developed real-time PCR assays for detection of respiratory pathogens. Journal of Clinical Microbiology 55, 1938–1945. Popowitch, E.B., O'neill, S.S., Miller, M.B., 2013. Comparison of the biofire filmarray RP, Genmark eSensor RVP, luminex xTAG RVPv1, and luminex xTAG RVP fast multiplex assays for detection of respiratory viruses. Journal of Clinical Microbiology 51, 1528–1533. Rand, K.H., Tremblay, E.E., Hoidal, M., et al., 2015. Multiplex gastrointestinal pathogen panels: Implications for infection control. Diagnostic Microbiology and Infectious Disease 82, 154–157. Rappo, U., Schuetz, A.N., Jenkins, S.G., et al., 2016. Impact of early detection of respiratory viruses by multiplex PCR assay on clinical outcomes in adult patients. Journal of Clinical Microbiology 54, 2096–3103. van Zyl, G., Maritz, J., Newman, H., Preiser, W., 2019. Lessons in diagnostic virology: Expected and unexpected sources of error. Reviews in Medical Virology 29, e2052. Vos, L.M., Bruning, A.H.L., Reitsma, J.B., et al., 2019a. Rapid molecular tests for influenza, respiratory syncytial virus, and other respiratory viruses: A systematic review of diagnostic accuracy and clinical impact studies. Clinical Infectious Diseases 69 (7), 1243–1253. Vos, L.M., Weehuizen, J.M., Hoepelman, A.I.M., et al., 2019b. More targeted use of oseltamivir and in-hospital isolation facilities after implementation of a multifaceted strategy including a rapid molecular diagnostic panel for respiratory viruses in immunocompromised adult patients. Journal of Clinical Virology 116, 11–17.

Further Reading Binnicker, M.J., 2015. Multiplex molecular panels for diagnosis of gastrointestinal infection: Performance, result interpretation, and cost-effectiveness. Journal of Clinical Microbiology 53, 3723–3728. Ramanan, P., Bryson, A.L., Binnicker, M.J., Pritt, B.S., Patel, R., 2018. Syndromic panel-based testing in clinical microbiology. Clinical Microbiology Reviews 31. Schreckenberger, P.C., Mcadam, A.J., 2015. Point-counterpoint: Large multiplex PCR panels should be first-line tests for detection of respiratory and intestinal pathogens. Journal of Clinical Microbiology 53, 3110–3115. Webb, G.W., Dalton, H.R., 2019. Hepatitis E: An underestimated emerging threat. Therapeutic Advances in Infectious Disease 6.

Virus Diagnosis in Immunosuppressed Individuals Elisabeth Puchhammer-Stöckl, Medical University of Vienna, Vienna, Austria Fausto Baldanti, University of Pavia, Pavia, Italy and Scientific Institute for Research, Hospitalization and Healthcare, San Matteo Polyclinic Foundation, Pavia, Italy r 2021 Elsevier Ltd. All rights reserved.

Introduction The rapid and sensitive diagnosis of virus infections in the immunocompromised human host is of utmost importance to avoid the development of severe and possibly lethal virus diseases. In contrast to the immunocompetent host, the main virus infections leading to life-threatening infections in immunocompromised patients are often caused by viruses of overall low pathogenicity, such as herpesviruses, polyomaviruses and adenoviruses. Primary infection with these viruses may cause only mild disease or even asymptomatic infections in healthy individuals. However, they become dangerous pathogens in the absence of appropriate development of antiviral immune responses and may proceed to a high level or even unlimited virus replication, leading to highly increased morbidity and mortality. Most of these viruses are often latently present in the patients already before immunosuppression starts due to past infections and frequently reactivate and start extensive replication after the onset of immunosuppression. The major group of immunosuppressed patients, in whom an intensive and rapid virological diagnosis is required and close virological follow-up is needed, are recipients of solid organ transplantation (SOT) or hematopoietic stem cell transplantation (HSCT) recipients. These patients receive severe artificially induced immune suppression, mostly over their lifetime. The administration of immunosuppressive medication is urgently needed to protect the transplanted organs from the recipient’s immune response, directed against foreign antigens. A decrease of the host’s immune response can be caused by a number of drugs that influence different immune defense mechanisms, and thereby may avoid acute or chronic rejection of the transplant by the recipient. However, as a problematic and potentially dangerous side effect, this impairment of distinct immune mechanisms also leads to a decreased ability of the host to defend against viral infections and against infections with bacterial and fungal pathogens. Also, patients with genetic defects that impair different parts of the immune response are at high risk for virus infections. Individuals with Human immunodeficiency virus (HIV)-associated immunosuppression and AIDS may also suffer from severe virus infections, and rapid virological diagnosis is needed. Since the successful introduction and application of highly active antiretroviral therapy (HAART), severe virus infections have become quite rare in numerous HIV positive populations. However, especially patients diagnosed at a late time point after infection with HIV and those with limited access to antiretroviral drugs are still at high risk of developing opportunistic infections with other viruses.

Viral Diagnostic Tests Diagnosis of virus infections in immunosuppressed persons, especially in transplant recipients, encounters a number of specific problems. One main challenge is that virus screening must be performed preemptively in most cases, which means that virus replication must be detected already before the viruses start to cause clinical disease. This means a routine diagnostic follow-up schedule after transplantation is required. Patients must be screened for defined viral parameters at regular intervals and for a long time. Another aspect complicating the routine diagnosis of viruses with latency phases is that the presence of these viruses does not “per se” prove the patient’s risk to develop a virus-associated disease on follow-up. Indeed, a deep understanding of viral pathogenesis is required to select a better indicator of whether a specific virus disease is to be expected or not. In some cases, low-level virus replication is a physiological state and may be also observed in the immunocompetent host, while increasing to a certain level of virus replication may indicate a high risk for the development of virus diseases and may require immediate intervention. This level, deciding whether antiviral interventions are required, needs to be defined for each cohort and each virus separately. It must be confirmed that antiviral interventions are started at the optimal time point, avoiding unnecessary antiviral treatment, but starting early enough to avoid high-level virus replication and disease. Also, the virus emerging after transplantation may have been derived from the virus population of the transplant recipient as well as from the donor virus population transmitted by the transplant. The main analysis applied in the virological follow-up of transplant recipients is the quantitative analysis of the nucleic acid-load of different viruses as Cytomegalovirus (CMV), Epstein–Barr Virus (EBV), BK-Polyomavirus (BKPyV) or Adenovirus (ADV). This may be performed in blood but also in other compartments, as bronchoalveolar lavage (BAL), urine or stool, depending on the specific virus infection and transplant population. The extent of the recipient’s preexisting immune response against different viruses has a substantial impact on the course of the disease. Transplant recipients, who have no prior immunity for these viruses, show a particularly high risk of developing a severe primary infection by the donor virus after transplantation. To assess the individual risk of a patient developing severe virus infections, testing for the presence of virus-specific antibodies (ABs) in the donor and recipient prior to transplantation is routine. Blood samples collected from the patients are tested by Enzyme-linked immunosorbent assay (ELISA) test systems, determining in particular virus-specific IgG. The absence of virus-specific IgG in the recipient indicates that the patient is susceptible and could

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Table 1 Virus

Virus Diagnosis in Immunosuppressed Individuals

Clinical manifestations and virus diagnostics of the most important virus infections in the immunocompromised patient after transplantation Clinical manifestation

Cytomegalovirus End-organ diseases (Pneumonitis, Colitis, encephalitis etc.) disseminated CMV syndrome Epstein–Barr Posttransplant lymphoproliferative disease Virus BK Polyoma Polyomavirus associated nephritis, loss of Virus kidney transplant Hemorrhagic cystitis after HSCT Adenoviruses Generalized infection, especially in children after HSCT Herpes simplex Extended cutaneous lesions, rare virus generalized infections Varicella-Zoster Extended cutaneous lesions, rare Virus generalized infections

Diagnosis

Pre Tx serology tested Preemptive testing by quantitative PCR

Quantitative PCR from blood (also BAL, CSF other body fluids) Quantitative PCR from blood

Always, from donor and recipient

In all transplant recipients

Sometimes especially in children Not routinely done

In high-risk groups

Quantitative PCR from urine and blood Quantitative PCR from blood and stool PCR from vesicles, ev blood, CSF PCR from vesicles, ev blood, CSF

No

Especially in kidney transplant recipients, eventually in HSCT Especially in pediatric HSCT

No

No

Yes, vaccination possible

No

develop a primary virus infection. If the transplant derives from an IgG seropositive donor, this is a particularly high-risk situation, as it is most likely that a primary infection from the donor will occur. The determination of the recipient’s antiviral T-cell response is gaining growing interest to assess the individual risk for severe infections. In particular, the reconstitution kinetics of neutrophils, CD4 þ , CD8 þ T-cells and NK cells are of paramount importance in the HSCT setting to determine transplant success but are also actively investigated in SOT to determine the recovery of immune function following induction treatments. CD4 þ and CD8 þ T-cell responses to individual viruses can be investigated to determine the risk of specific virus-related diseases. However, this approach is so far not widespread and no consensus on protective responses has been reached yet. It was also shown that the level of Torque Teno Virus (TTV), a small commensal and non-pathogenic DNA virus present in the majority of the human population in blood, is associated with the level of drug-induced immunosuppression after SOT. TTV-DNA loads are also affected by the patient’s immune response and have been proposed as a potential indicator of overall immune impairment in SOT. This was reported in lung transplant and also kidney transplant recipients. An association was established between high immunosuppression, a high TTV load exceeding a specific cut off level and the occurrence of opportunistic infections. In contrast, low TTV-load was reflected low immunosuppression decreasing under a defined lower cut off level and was significantly associated with organ rejection processes after lung and kidney transplantation. The virus diagnosis procedures and follow-up recommendations are different and specific for each virus, which potentially causes a high risk to the immunocompromised host. Therefore, the most important virus pathogens and their follow-up will be separately reviewed in the following and are summarized in Table 1.

Human Cytomegalovirus (HCMV) Human Cytomegalovirus is a member of the herpesvirus family and belongs to the beta-herpesvirus subfamily. HCMV proved to be the major opportunistic agent in immunocompromised patients. Before the HAART era, HCMV was associated with most morbidity and mortality in HIV- infected individuals. Indeed, HCMV end-organ diseases were primary AIDS-defining clinical conditions, typically appearing in the presence of CD4 þ T-cell counts o200. Similarly, HCMV represents the main opportunistic pathogen in both HSCT and SOT recipients. Like all herpesviruses, HCMV is a widespread agent acquired early in life. In immunocompetent individuals, the majority of primary HCMV infections are asymptomatic, while in a minority of cases is a selfresolving mononucleosis syndrome. Following primary infection HCMV can establish lifelong latency in many cell types (myeloid cells precursors, endothelial cells, kidney and salivary glands epithelial cells). In immunocompetent individuals, reactivations are asymptomatic. The peculiar ability of HCMV to infect and become latent in many cell types may explain why this virus is so aggressive in immumocompromised patients. Indeed, in patients with impaired T-cell and NK immunity primary and reactivated infection can present as disseminated infections associated with end-organ diseases (retinitis, gastritis, small and large bowel ulcers, pneumonia, encephalitis, hepatitis). This wide range of end-organ diseases is due to HCMV establishing latency in endothelial cells, thus reactivation(s) can potentially involve any organ. From a diagnostic standpoint, it is then important to: (1) determine the donor (D)/recipient (R) serostatus and (2) periodically monitor the presence and the level of HCMV DNA in blood as a marker of reactivation and dissemination. The highest risk for HCMV disease in HSCT is in D/R þ combination, while in SOT is in the D þ /R group. In both cases, an HCMV infection could develop in the absence of specific memory B- and T-cell responses, with a high risk of dissemination and organ involvement. For

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this reason, high-risk transplant populations are often given antiviral prophylaxis. On the other hand, patients already HCMV experienced are at risk of symptomatic HCMV reactivations due to impaired immunity consequent to immunosuppressive antirejection treatments. To minimize the toxic effects of current antivirals, medium to low-risk patients are given pre-emptive treatment protocols based on the administration of antiviral treatment in the presence of documented HCMV replication when still asymptomatic. Periodic HCMV DNA quantitative determination in the blood is then required in the post-transplant period to detect the presence and the extent of virus replication (reactivation). A typical monitoring schedule involves HCMV DNA quantification once a week in the first 3 months (when immunosuppression is greater in SOT and engraftment is ongoing in HSCT) followed by once every 2 weeks in the second 3-month period and once a month in the following 6 months. Detection followed by the rising of HCMV DNA levels in the blood is indicative of asymptomatic HCMV replication and the potential for dissemination. For pre-emptive treatment initiation, different centers use different HCMV DNA cut-off levels due to a lack of standardization between real-time techniques. Recently, a national consensus conference indicated HCMV DNA cut-off levels for high, medium and low-risk SOT as well as HSCT recipients. The diagnosis of HCMV end-organ diseases requires the identification of viable virus (isolation) and virus antigens (immunohistochemistry) in organ tissues or a high level of CMV DNA in relevant fluids. Sequential determination of HCMV DNAemia is essential to identify treatment failures. Indeed, unchanged or increasing HCMV DNA levels following several days of treatment may indicate the “in vivo” selection of a mutant and drug-resistant HCMV strain. A number of drug-resistant mutants have been described and occur especially in the UL97 and UL54 genes. UL97 codes for a kinase required for the phosphorylation of ganciclovir and different mutations at this gene have been identified which confer resistance against this drug. The UL54 gene codes for the virus polymerase and distinct mutations in this gene may confer resistance against ganciclovir, foscarnet or cidofovir. To identify the development and extent of drug resistance, sequencing of the UL97 and UL54 genes is required. This can confirm the presence of ganciclovir, foscarnet and cidofovir resistant strains and is important to help direct a change of abtiviral agents. Finally, assessment of specific T-cell immunity has proven helpful in predicting patients at risk of HCMV reactivation and disease, as well as designing tailored preventive and treatment approaches.

Epstein–Barr Virus (EBV) EBV is a member of the herpesvirus family and belongs to the gamma-herpesvirus subfamily and has oncogenic potential. In fact, a peculiar characteristic of the two members of the gamma-herpesviruses (EBV and HHV-8) is their correlation with many lymphomas, sarcomas and carcinomas. Strikingly, EBV-induced malignancies are associated with different virus latency phases (Table 2). Thus, EBV is pathogenic both during replication and latency. EBV primary infection is acquired early in life and is mostly asymptomatic, justifying the impressive biologic success of this virus. Indeed, EBV seroprevalence is virtually 100% at the age of 20. Mononucleosis is the clinical manifestation of primary EBV infection in a small (but significant) fraction of individuals. Immunocompromised patients, especially transplant recipients, are at risk of complications in both the replicative phase (chronic active EBV infection) and latency (post-transplant lymphoproliferative disorders or PTLD, lymphomas and leiomyosarcoma). Among SOT recipients, D þ /R are at a higher risk of complications. Given the high EBV seroprevalence in young adults and adults, pediatric transplant recipients are at the highest risk of short-term complications. In addition, pediatric SOT recipients are also at the highest risk of late EBV-associated neoplasm due to the longer life expectancy and the greater cumulative effect of immunosuppression. EBV serology might be tricky to interpret due to the response to multiple antigens and the relevant antibody maturation (Table 3). Table 2 EBV associated tumors and expression of EBV latency genes (modified from J.L.Hsu, S.L. Glaser, Critical Reviews in Oncology/ Hematology 34; 27–53, 2000) EBV latency

Pattern of EBV latency gene expression

Malignancy

Type I

EBNA-1 EBERs

Burkitt’s lymphoma Gastric carcinoma

Type II

EBNA-1 LMP-1, -2A, -2B EBERs

Nasopharyngeal carcinoma Hodgkin’s disease (HD) Nasal T/NK lymphoma

Type III

EBNA-1, -2, -3A, -3B, -3C, -LP LMP-1, -2A, -2B, EBERs

AIDS-associated non-HD lymphoma PTLD (post-transplant lymphoproliferative disorders)

Other

EBNA-1, -2 EBERs

Leiomyosarcoma

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Table 3

Virus Diagnosis in Immunosuppressed Individuals Typicala EBV-serological profiles using the most frequently employed antigens and Ig isotypes

Antigen/Ig isotype condition

VCA/IgG

VCA/IgM

VCA/IgA

EA/DIgG

EA/R/IgG

EBNA/Ig

Susceptible/no previous exposure Primary infection Past infection Inconclusive: Primary or past infection Inconclusive: Primary or past infection Chronic active EBV infection

 þþ þ þ þ þþþ

 þþþ  þ  þ

 þ þ/ þ/ þ/ þþ

 þ  þ/ þ/ þþ

 þ/ þ/ þ/ þ/ þ

  þ þ  þ/

þ /  , Antibodies absent or present; þ , Antibodies present; þ þ , Antibodies present in elevated titers; þ þ þ , Antibodies present in strongly elevated titers. Abbreviations: EBV: Epstein–Barr virus. VCA: Virus capsid antigens. EBNA: EBV nuclear antigen. EA/D: Early antigen/diffuse. EA/R: Early antigen/restricted. Atypical patterns include VCA IgM positive only; EBNA positive only; or VCA IgM and EBNA positive, but VCA IgG negative. Atypical patterns merit repeat testing, testing of a follow-up sample, or testing by alternate methods.

a

Primary EBV infection (mononucleosis) and chronic active EBV infection (often a complication of initial mononucleosis) are observed almost exclusively in pediatric transplant recipients, while PTLD is an uncommon complication of HSCT and SOT in both pediatric and adult settings. The risk of PTLD is directly correlated with the level and duration of immune suppression. Thus, transplants (or rejection episodes) requiring aggressive immune suppressive treatments bear an elevated risk of EBV-PTLD. From a treatment standpoint, it must be underlined that PTLD, as well as all EBV-associated tumors, are induced during a replicationinactive phase of the virus life cycle. Currently available anti-herpetic drugs (inhibiting viral DNA polymerases) are ineffective. Indeed, during PTLD, EBV DNA replicates in its latent form via cellular DNA polymerases in proliferating cells. Then, measurement of EBV DNA in peripheral blood of transplant patients (although pivotal in the monitoring of PTLD development) has to take into account these pathogenetic peculiarities: (1) during mononucleosis or chronic active EBV infection EBV DNA is the expression of active virus replication and it can be used to monitor the efficacy of antiviral treatment, while (2) during PTLD EBV DNA is the expression of EBV-induced cell proliferation in the absence of active virus replication and it can be used to monitor the efficacy of immune suppression tapering as well as lymphocyte B lytic treatments. Cut-off levels to start treatment for PTLD are still debated. Monitoring EBV-specific T-cell responses to replicative phase antigens as well as to latency phase antigens might be helpful to define patients at risk of PTLD better.

Polyomaviruses (PyV) Polyomaviruses comprise a group of non-enveloped DNA-viruses that remain after primary infection and persist in the human host. PyVs are highly prevalent worldwide in human populations and include among others BK-PyV, JC-PyV, Merkel cell Carcinoma PyV and trichospinulosa PyV. The most important of these viruses in the transplant setting is BK-PyV, which is of clinical significance nearly exclusively after renal transplantation or HSCT. BK-virus is latently resident in kidney cells and may replicate to high levels after renal transplantation. In the worst case, it may lead to Polyomavirus associated nephritis (PVAN) and to loss of the transplant. Recent data provide evidence that BK-PyV associated disease is caused especially by the donor BK-PyV transmitted by the transplanted kidney. The key element in avoiding the development of PVAN is preemptive routine diagnostic testing for BK-PyV DNA by quantitative PCR assays in urine and blood. While low-level BK-PyV DNA is sometimes detectable in immunocompetent persons and is of no significant clinical value, urine BK-PyV DNA levels increasing over 4 7–8 log10 copies/mL signify substantial virus replication and reflect an increased risk for progression to BK-PyV DNAemia and further PVAN. Moreover, the detection of decoy cells in urine, which have enlarged cell nuclei and intranuclear viral inclusions, may be of diagnostic value. Either from the beginning, but at the latest as soon as the virus load in urine increases over 47–8 log10 copies/mL urine, quantitative PCR testing in blood is required to control the replication kinetics further. An increase of the BK-PyV DNAemia over 4 3–4 log10 copies/mL plasma indicates a high risk of developing PVAN. Renal histology is then required to help with management decisions to counteract further BK-PyV replication. These include reducing the individual immunosuppressive drugs to re-establish the recipient’s antiviral response and avoid progression and transplant loss. Without intervention, about 50% of highly viremic patients progress to PVAN. The quantitative PCR test cut-offs mentioned above are recommendations based on specific in-house PCR assays. An international BK-PyV calibrator approved by the World Health Organization (WHO) is available and this allows comparison of different quantitative PCR-assays used in different centers. Serology plays no role in routine testing and risk assessment of kidney transplant recipients. This is in part due to the fact that BK-PyV AB tests are not commercially available and only established in single laboratories in the course of scientific projects. However, an increasing amount of data suggest that a high level of BK-PyV specific ABs in the donor pre-transplantation is associated with a higher risk of the recipient developing PVAN, probably as it reflects a higher virus load in the donor kidney.

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BK-PyV may also cause hemorrhagic cystitis in patients after HSCT. Hemorrhagic cystitis may occur in up to 25% of pediatric and up to 54% of adult allogeneic HSCT patients. Diagnosis of BK-PyV associated cystitis is made using clinical parameters as well as by virus detection in urine by PCR. High-level viruria (47 log10 BK-PyV DNA copies/mL urine) as well as DNAemia seem to predict hemorrhagic cystitis in these patients. Other polyomaviruses cause infrequent complications in immunosuppressed patients after transplantation and are therefore not subject to routine follow up virus load testing by quantitative PCR. JC-PyV is latently present in the kidney and in the brain neurones of most people and may cause progressive multifocal leukoencephalopathy (PML) in rare cases of highly immune depressed AIDS patients or in HSCT patients. This is caused by reactivation of JC-PyV in severely T-cell deficient patients and due to progressive demyelination in the brain tissue which is mostly lethal. JC-PyV PCR tests are carried out in cerebrospinal fluid samples and also from blood to diagnose PML.

Adenoviruses (AdVs) Adenoviruses (AdVs) are non-enveloped DNA viruses that are widely distributed. AdV contains seven species, termed A to G. Most infections with AdVs occur at a young age and in the immunocompetent host are mostly associated with mild infections, such as respiratory infections, ocular infections or gastroenteritis. However, in the immunocompromised host, and especially in children after allogeneic HSCT AdVs are important pathogens, as they may extensively replicate and may cause life-threatening infections. In these patients, AdVs may cause hemorrhagic enteritis or cystitis, or affect other organs including lung, liver, kidney or heart tissue, and finally, AdV infections may cause multiorgan failure. The lethal outcome of generalized AdV infections in children after allo-HSCT is high. It is thought that the virus derives from extensive local replication especially in the gastrointestinal tract of severely immunosuppressed children and is further spread extensively to different organs by AdV viremia. Therefore, close routine diagnostic follow up for AdV is strongly recommended in this pediatric cohort. Also, adults may develop systemic AdV infections after allogenic HSCT, however, this is observed to a much lower extent. Preemptive diagnosis for AdV infections is based on quantitative AdV DNA PCR from stool and blood, using a pan-AdV PCR, detecting all AdV strains in one assay. Current PCR tests commonly target the AdV hexon gene which is a conserved region within AdV strains. Detection of virus DNA in blood is considered a marker of ongoing systemic disease. Therefore, most centers test for DNAemia routinely and on a weekly basis after transplantation. A level of 43 log10 copies/mL plasma is considered critical for the progression of infection to generalized disease and requires therapeutic interaction. This involves systemic treatment with cidofovir or brincidofovir. Also, a viral load of 46 log10 AdV DNA copies/g stool is considered critical. Before HSCT, intestinal AdV shedding is associated with an increased risk of later development of disseminated AdV disease post-transplantation. Therefore, AdV PCR testing of stool prior to transplantation may be a useful tool to assess the individual risk of patients for generalized AdV disease. AdV infections after solid organ transplantation are rare.

Other Viruses There are also other viruses that may cause significant complications in immunosuppressed patients. But as these occur less frequently, routine follow up testing is not initiated in these post-transplant risk groups.

Herpes Simplex Virus (HSV) Herpes simplex virus, especially HSV type 1, is highly prevalent in the human population. It usually causes a symptomatic or asymptomatic primary infection during early childhood and is thereafter latently present in ganglia cells. HSV may reactivate from time to time also in healthy individuals due to stressful situations or after extended sun exposure. It may then lead classically to vesicular lesions on the lips, herpes labialis, which is quite common. Under severe immunosuppression, HSV may also reactivate and may cause severe cutaneous manifestations with extended dermal lesions, which may also become necrotic. In rare cases, HSV may also affect different organs and even cases of HSV encephalitis may be observed. HSV infection is usually identified by clinically evident dermal manifestations. In difficult clinical situations, the diagnosis may be confirmed by HSV-specific PCR carried out on vesicle fluid. In generalized infection, blood or in suspected cases of encephalitis or meningitis also cerebrospinal fluid (CSF) samples will be tested by PCR. Early treatment with high dose intravenous aciclovir has to be administered as soon as possible to avoid or limit further virus dissemination. Frequently, HSV is also detected in patients in intensive care units, especially in the lower respiratory tract. Whether this has any clinical importance or whether this is due to subclinical replication under extremely stressing conditions, and eventually also due to intubation and artificial ventilation is still unclear.

Varicella-Zoster Virus (VZV) Varicella-Zoster Virus is a herpesvirus that is highly prevalent in the population, except in younger people if they have been vaccinated already against VZV.

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Primary infection with VZV manifests as chickenpox, classically associated with a generalized vesicular rash, and is highly contagious. Diagnosis is mostly made clinically on seeing the typical dermal lesions. The virus lies in a latent state in the dorsal root ganglia cells of the host and remains there for life. Reactivation of VZV is observed most frequently in older individuals and is associated with decreasing cellular immune response against VZV. Reactivation manifests typically as herpes zoster (shingles), manifesting as a vesicular rash that covers usually one dermatome. Herpes zoster may be accompanied by severe pain, or by complications affecting especially the central nervous system (CNS) and appear as meningitis, meningoencephalitis or transverse myelitis. Herpes Zoster infection is usually identified by clinically evident dermal manifestations. In difficult cases, the diagnosis may be confirmed by VZV-specific PCR performed from vesicle fluid. In generalized infection, VZV-PCR may be carried out in blood or CSF in suspected cases of encephalitis or meningitis. VZV may cause severe complications in the immunocompromised host. Reactivation of VZV latently present in the patient may start after immunosuppression and may lead to severe cases of herpes zoster, affecting more than one dermatome. In especially severe cases not only vesicles but severe and extended necrotizing dermal lesions may be observed. The clinical picture is also clear from the dermal manifestations, but in difficult cases, VZV-specific PCR from vesicle lesions is performed. VZV infection after immunosuppression may also cause meningoencephalitis and diagnosis by VZV PCR from CSF is needed. Rarely systemic infections occur. These have a dramatic course associated with the development of multiple ulcers in the gastrointestinal tract and with multi-organ failure. Rapid detection of VZV DNA by PCR is then required, also from blood. As the clinical appearance of generalized infection is often unclear at the beginning and the course of the disease is fulminant, mortality is high. Treatment with high dose intravenous antiviral drugs such as aciclovir is mandatory in all severe cases of VZV infection in the immunocompromised host. Children who have neither undergone primary VZV infection nor vaccination against VZV prior to immunosuppression, are at a particularly high risk of developing a primary infection with VZV after transplantation and the start of immunosuppression. Such primary varicella in the immunocompromised host is associated with especially severe clinical complications. Therefore, determination of the VZV ABs prior to transplantation is mandatory, especially in children, to assess the patients’ VZV serostatus. If no VZV specific ABs are detectable pre-transplantation, VZV vaccination has to be administered before the start of immunosuppression.

Hepatitis E Virus Hepatitis E virus is an RNA virus. HEV genotype 3 infections occur especially in pigs and boar in many countries and may be transmitted to humans mostly by eating undercooked food. Acute infection with HEV genotype 3 infection is self-limiting and asymptomatic in the immunocompetent host. However, when patients develop an HEV infection after transplantation, they may develop chronic hepatitis during immunosuppression. Undiagnosed this may lead to the rapid development of liver cirrhosis within a few years and death of the patient. Therefore, screening for HEV infection should be performed in all transplant patients with unclear elevation of liver enzymes. HEV-PCR can detect viremia from blood, and a positive result indicates ongoing acute or chronic infection. Also, HEV can be detected in the stool of the patients. HEV specific AB testing is not suitable in the immunocompromised host, as AB development may be reduced over a long time after infection in these patients. A decrease of immunosuppression and/or administration of ribavirin may lead to the termination of viral replication.

Conclusion A number of different viruses are dangerous pathogens for the immunosuppressed host. These may not only affect the patient as a single virus infection but may also appear in combination with other virus infections or with bacterial or fungal infections under immunosuppression. The diagnostics of virus infections in immunocompromised patients is of utmost importance for the patients’ short and long-time survival. But high virological expertize is needed to correctly perform appropriate tests at the optimal time points and establish exact quantification. Participation in quantitative quality control panels is needed, to allow interlaboratory comparisons and to assess quantitative cut-off levels, which are comparable between different centers. The diagnosis of virus infections especially after transplantation and the interpretation of the test results requires good knowledge of the particular viruses, their pathogenesis and life cycle, and the mechanisms of virus latency and replication. The differentiation between non-pathogenic and potentially pathogenic virus load levels is still difficult in most virus infections, and the identification of the turning point to highly dangerous levels of virus replication is challenging and needs highly experienced experts.

Further Reading Calarota, S.A., Aberle, J.H., Puchhammer-Stöckl, E., Baldanti, F., 2015. Approaches for monitoring of non virus-specific and virus-specific T-cell response in solid organ transplantation and their clinical applications. Journal of Clinical Virology 70, 109–119. Calarota, S.A., Chiesa, A., Zelini, P., et al., 2013. Detection of Epstein-Barr virus-specific memory CD4 þ T cells using a peptide-based cultured enzyme-linked immunospot assay. Immunology 139 (4), 533–544. Cesaro, S., Dalianis, T., Hanssen Rinaldo, C., et al., 2018. ECIL guidelines for the prevention, diagnosis and treatment of BK polyomavirus-associated haemorrhagic cystitis in haematopoietic stem cell transplant recipients. Journal of Antimicrobial Chemotherapy 73 (1), 12–21.

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Girmenia, C., Lazzarotto, T., Bonifazi, F., et al., 2019. Assessment and prevention of cytomegalovirus infection in allogeneic hematopoietic stem cell transplant and in solid organ transplant: a multidisciplinary consensus conference by the Italian GITMO, SITO, and AMCLI societies. Clinical Transplantation 16, e13666. González-Vicent, M., Verna, M., Pochon, C., et al., 2019. Current practices in the management of adenovirus infection in allogeneic hematopoietic stem cell transplant recipients in Europe: The advance study. European Journal of Haematology 102 (3), 210–217. Görzer, I., Haloschan, M., Jaksch, P., Klepetko, W., Puchhammer-Stöckl, E., 2014. Plasma DNA levels of torque teno virus and immunosuppression after lung transplantation. The Journal of Heart and Lung Transplantation 33 (3), 320–323. Hirsch, H.H., Randhawa, P.S., 2019. BK polyomavirus in solid organ transplantation – Guidelines from the American society of transplantation infectious diseases community of practice. Clinical Transplantation 33 (9), e13528. Jaksch, P., Kundi, M., Görzer, I., et al., 2018. Torque teno virus as a novel biomarker targeting the efficacy of immunosuppression after lung transplantation. The Journal of Infectious Diseases 218 (12), 1922–1928. Kotton, C.N., Kumar, D., Caliendo, A.M., et al., 2018. The third international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 102 (6), 900–931. Lee, D.H., Zuckerman, R.A., 2019. Herpes simplex virus infections in solid organ transplantation: Guidelines from the American society of transplantation infectious diseases community of practice. Clinical Transplantation 33 (9), e13526. Lhomme, S., Legrand-Abravanel, F., Kamar, N., Izopet, J., 2019. Screening, diagnosis and risks associated with Hepatitis E virus infection. Expert Review of Anti-infective Therapy 17 (6), 403–418. Lion, T., 2014. Adenovirus Infections in Immunocompetent and Immunocompromised patients. Clinical Microbiology Reviews 27 (3), 441–462. Navarro, D., San-Juan, R., Manuel, O., et al., 2017. Cytomegalovirus infection management in solid organ transplant recipients across European centers in the time of molecular diagnostics: An ESGICH survey. Transplant Infectious Disease 19 (6). Pergam, S.A., Limaye, A.P., 2019. Varicella zoster virus (VZV) in solid organtransplantation: Guidelines from the American society of transplantation infectious diseases community of practice. Clinical Transplantation 4, e13622. Rezahosseini, O., Drabe, C.H., Sørensen, S.S., et al., 2019. Torque-Teno virus viral load as a potential endogenous marker of immune function in solid organ transplantation. Transplantation Reviews 33 (3), 137–144. Thorley-Lawson, D.A., 2015. EBV persistence – Introducing the Virus. Current Topics in Microbiology and Immunology 390 (Pt 1), 151–209.

Diagnosis; Future Prospects on Direct Diagnosis Marianna Calabretto and Daniele Di Carlo, Sapienza University of Rome, Rome, Italy Fabrizio Maggi, University of Pisa, Pisa, Italy and University of Insubria, Varese, Italy Guido Antonelli, Sapienza University of Rome, Rome, Italy r 2021 Elsevier Ltd. All rights reserved.

The diagnosis of viral infection, especially the direct one (i.e., demonstration of viral particle - as a whole or through detection of viral antigens or nucleic acids), has been and is going to be, affected by several breakthroughs. Indeed, until a few decades ago, the direct diagnosis was made by performing viral isolation by using cell culture. This method, which today only in some instances still represents the “gold standard”, takes a long time to be carried out and requires highly skilled and experienced staff able to correctly interpret the viral cytopathic effect and to manage cell lines and highly pathogenic viruses. These characteristics do not fit with the current need for low-cost, accurate, and rapid virological diagnosis. Indeed, it is generally accepted that a rapid and more reliable diagnosis may lead to better management of the patient, including personalized therapy, reduced use of unnecessary drugs (for instance antibiotics), and shorter hospitalization. For the above reasons and considering the constant improvement of diagnostic assays, most clinical virology laboratories became essential in the hospital to make clinical decisions. Although the impressive achievement in terms of technical advances, it is important to note that today there is still no diagnostic approach (i.e., molecular detection, antigen identification, virus isolation, etc.) that: meets all needs of diagnostic virology laboratories; is suitable for all clinical situations; and applies to all virus types. This indirectly means that virologists, who are aware of these limitations, use a different assay depending on any specific clinical situation. This document provides an overview of the main technologies that, to our opinion, have been used in recent years, and will be used in the future, in the routine activity of a clinical laboratory of a big Hospital. In the framework of these technologies, we will try to outline possible future scenarios of their application in the context of the virological diagnostic.

PCR and Its Evolution Polymerase chain reaction (PCR) is the assay that allows the amplification of nucleic acid fragments of which initial and terminal sequences are known. Its use for the quantification of viral nucleic acids in body fluids has profoundly changed virological diagnosis. Up to now, the amplification of nucleic acids through PCR represents the gold standard for the screening, diagnosis, and follow-up of several viral infections, such as HIV, HBV, HCV, CMV, respiratory viruses. The most used tests in the laboratory are real-time PCR and reverse transcription-PCR (RT-PCR). The real-time PCR allows detection and quantification of a viral product during the run of the assay; “real-time”, as opposed to the standard PCR which quantified it only at the end of the session. This leads to: a reduction of run assay time; the lack of need for post PCR processing; an increase in sensitivity. RT-PCR, on the other hand, allows amplifying cDNA (complementary DNA) obtained by a previous reverse transcriptase reaction on mRNA and other RNA virus genomes. The introduction of viral load measurement in biological fluids through PCR has led to the reduction of morbidity, mortality, and disease transmission, especially in chronic infections such as HIV, HBV, and HCV. It found application also in monitoring viral infections in transplanted patients such as CMV. qPCR is based on the quantification of nucleic acids by comparing the number of amplification cycles, which is believed to be exponential, and the quantity of the final product of PCR with those of a reference sample. qPCR measures the intensity of fluorescence at specific times (generally at the end of each amplification cycle) to quantify the amount of target molecule. It gives the threshold per cycle (CT) and the difference in CT is used to calculate the amount of initial nucleic acid. However, this comparison is subject to factors that could produce inaccurate data. For example, the amplification cycles may not be exponential, or the target nucleic acid quantity may not be sufficient (Pekin et al., 2011; Quan et al., 2018). The future of quantitative PCR is digital PCR (dPCR). dPCR, indeed, thanks to the advances in nanofabrication and microfluidics that have now led to systems that produce hundreds to millions of nanoliter- or even picoliter-scale partitions, breaks down the body fluid by quantifying the presence of target nucleic acid in every single droplet and the measurement of the DNA quantity takes place after the amplification is completed. This allows having a more precise and accurate measurement of the number of amplified molecules. Furthermore, while qPCR is unable to distinguish differences in gene expression or variations in copy numbers that are smaller than twofold (Kuang et al., 2018), dPCR has shown to distinguish between copy number variations of that differ by only one copy (Baker, 2012). Its greater sensitivity, accuracy, and precision make dPCR a potential evolution in virological laboratory diagnostics to test viral load in a different compartment.

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Next-Generation Sequencing (NGS) The next-generation sequencing (NGS) (also called massively parallel or deep sequencing) represents a series of technologies that allow sequencing an entire genome within a few days. The peculiarity of NGS is to include together the three main steps of sequencing: sample preparation, sequencing, and data analysis. This saves significant time and reduces the number of errors in practice. There are three ways to sequence genetic material through NGS. The first method is similar to the classic Sanger method with the amplification and sequencing of a specific target. The second involves the amplification of all the genetic material present in the sample followed by NGS. Finally, in the third method, the viral genetic material is first enriched by various methods and then sequenced by NGS (Maggi et al., 2019). The applications of NGS in virology and clinical virology lab are various. First of all, it allows the identification of new pathogenic and non-pathogenic commensal viruses. For example, the Schmallemberg virus, a new Orthobunyavirus that causes malformations in cattle and sheep, was discovered through NGS (Hoffmann et al., 2012; Rosseel et al., 2012). Through a metagenomic approach, it is also possible to perform an etiological diagnosis of viral infection, as in the case of the recent identification of viruses responsible for diseases with severe fever but of unknown etiology (Yozwiak et al., 2012; McMullan et al., 2012). Furthermore, whole – viral genome sequencing may allow: the study of genetic mutations that can lead to drug resistance; the identification of variants that can overcome the immune response of the host; the identification of diversity in the viral variants/ genomes within the same host (useful in the case of viruses able to give chronic infection, such as HIV, HCV, HBV, or Herpesviruses) (Clevenbergh, 2000; Kim et al., 2014; Houldcroft, 2017) and, in this framework, study of the diversity of the viral genome and its evolution; to address molecular epidemiology and viral phylogeny. As far as HCV variants resistant to antiviral drugs are concerned, it is known that the resistance to some HCV antiviral drugs is due to mutations present in some non-structural proteins of the virus, NS3/4A, NS5A, and NS5B. Although now the issue is overcome by the introduction of very effective drugs, NGS would be possible to characterize the virus to administer the most effective antiviral drug. Besides, NGS was used to simultaneously differentiate HCV and HGV (Hepatitis G Virus) in the patient’s serum. The two viruses share the same transmission mode that can cause co-infection. The simultaneous diagnosis of the two viruses, therefore, improves the management of infection and therapy (Parker and Chen, 2017). NGS has the merit also to have demonstrated: the high variability of viruses that were believed to be genetically stable; that many anatomical sites considered sterile have their flora; that in biological samples it is possible to find viral elements not associated with diseases that are more numerous than pathogenic viruses. This aspect is important for the concept of virome, i.e., the presence of a commensal viral flora that is part of the human microbiome, the community of microorganisms present within the human body (Vu and Kaiser, 2017; Rascovan et al., 2016; Freer et al., 2018). NGS produces a high value of data that must be collected, evaluated, stored, and used correctly so that the potential of this technology can be exploited to the full. To reach this goal a synergistic activity between virology and “bioinformatics” which combines biology and data processing should be planned (Barzon et al., 2013).

CRISPR-Cas The microbial clustered regularly interspaced short palindromic repeats (CRISPR) represents one of the new technologies that is going to make a revolution also in the molecular diagnostics market, and some example are already available (Joung et al., 2020). In particular, such a technology has changed the perspectives of gene therapy and manipulation. CRISPR for editing cellular genes must be associated with the enzyme Cas (CRISPR-Cas) and an RNA that guides the complex to the target site of nucleic acid; the enzyme cuts the nucleic acid, triggering a cellular DNA repair mechanism that activates or deactivates the target gene or removes pieces of nucleic acid. The best example in virology is the use of technology to handle the integrated viral genome (Deng et al., 2018; Ford et al., 2019). Many viruses have indeed the ability to establish a persistent infection by integrating their genome into the host’s DNA. CRISPR technology has been used in vitro and in animal models to achieve integrated sequences, managing to eliminate chronic viral infections. For example, it was used in vitro to inactivate HIV gene expression in microglia and T cells. Besides, targeting the HIV LTR, gag, and pol genes it was possible to eliminate HIV proviral DNA from animal cells of the spleen, lungs, colon, heart, and brain (Hu et al., 2014; Wang et al., 2018; Yin et al., 2017). Also, CRISPR technology delivered by a lentivirus vector has been used to eliminate HIV proviral DNA from infected human peripheral blood mononuclear cells in a transgenic mouse model (Bella et al., 2018). The same results have also been achieved with other viruses, such as Herpesviruses. In particular, using CRISPR, 95% of the EBV genome in latently infected Burkitt lymphoma cells was achieved (van Diemen et al., 2016). These results suggest that in the future CRISPR technologies can be used also for therapeutic purposes like the eradication of chronic infections in the tissues and the oncological therapy of malignancies with viral etiology (Strich and Chertow, 2019; Myhrvold et al., 2018). One of the side effects of the combination of CRISPR/Cas13 is promiscuous ribonuclease activity upon target recognition. This has been exploited, combined with isothermal amplification, to quickly search for RNA or DNA with attomolar sensitivity and single-base mismatch specificity. This Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), has been used to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA (Roy et al., 2018; Gootenberg et al., 2017).

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Moreover, this method seems useful because SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications. Combined with HUDSON (heating unextracted diagnostic samples to obliterate nucleases), SHERLOCK can work directly on biological fluids without having been previously extracted and in less than two hours (Gootenberg et al., 2017). Another recent method, similar to SHERLOCK, is DETECTR (DNA endonuclease targeted CRISPR trans reporter). This uses Cas12a, similar to Cas13a but working on DNA, which produces single-stranded DNA cleavage activity after recognizing the target sequence. DETECTR has been used successfully to detect human papillomaviruses 16 and 18, two of the most important genotypes that cause neoplastic transformation. This method has proved to be simple and fast compared to other diagnostic methods for the diagnosis of human papillomavirus making it suitable for its use for rapid molecular diagnosis (Chen et al., 2018).

MALDI-TOF Mass Spectrometry Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) allows identifying with certainty and precision the type and strain of microorganism responsible for a specific pathology, through the analysis of a sample of the patient’s biological fluid (blood, urine, or liquor) subjected to spectrophotometric analysis, without the need of preliminary treatments. The sample to be analyzed is hit by a laser beam which first causes the fragmentation of its molecules and, subsequently, their ionization. Entering an electric field, these generate a signal that is read by software that produces a particular path. This will then be compared to the spectra already present in a database, allowing the recognition of a specific microorganism through this molecular “fingerprint”. In bacteriology and mycology, this technology found large application and then is widespread. In virological diagnostics is going to be used but still requires consolidation. It finds use to detect viral components in a wide variety of biological specimens, to spot genome mutations associated with drug resistance, and to identify the genotype of viruses (Cobo, 2013). MALDI-TOF MS can be used for rapid and accurate diagnosis of influenza viruses, being able to detect the different subtypes, and for the diagnosis of the various enteroviruses that cause pediatric gastroenteritis, especially if associated with Multiplex PCR (Chou et al., 2011; Piao et al., 2012). Furthermore, this method has found application in the identification of the viral genotypes of HPV, HBV, and HCV, improving the management of the infection and the response to the therapy (Gray and Coupland, 2014). Finally, through MALDI-TOF MS it is possible to identify drug resistance against antivirals, for example, resistance to ganciclovir (Zürcher et al., 2012). The main advantages of using this method derive from its rapid and reliable identification of a large variety of viruses, its simplicity of use, its sensitivity and accuracy. On the other hand, its diagnostic use is limited due to the limited coverage of virus species in the database and MALDI-TOF devices are at the moment still expensive.

Multiplex and POCT The need to optimize laboratory diagnosis in clinical practice has also led to the development of multiplex platforms over the years, both as regards bacteriological, parasitological, and virological diagnosis. In particular, multiplex PCRs allow detection or quantitation of sequences of multiple viruses in a single session using a set of specific primers (McCulloh et al., 2014). The advantages of multiplexing tests include reducing turn-around-time and improving patient management. The most recent multi-analytical tests allow analyzing a pattern of viruses and germs whose infections are associated with the same clinical manifestations, which possess the same tissue tropism and the same mode of infection. The manufacturing companies are dedicated to the development of syndromic panels specific for the body district, for example respiratory, gastrointestinal, central nervous system, and sexually transmitted diseases panels (Diaz-Decaro et al., 2018; Huang et al., 2018; Scagnolari et al., 2017; Zhang et al., 2015). The application of these panels is still ongoing but we have already obtained some results. For instance, multiplex PCR proves useful in the diagnosis of meningitis and encephalitis. Despite the suboptimal sensibility observed for a certain target, multiplex molecular panels allow simultaneous detection of multiple pathogens. Thus, in a situation that requires rapid and reliable diagnosis with small quantities of the sample – cerebrospinal fluid (CSF) – their application could improve a patient’s outcome (Dien Bard and Alby, 2018). The panels dedicated to sexually transmitted diseases that simultaneously diagnose HIV and Syphilis have led to increased compliance for monitoring patients with risky behaviors, such as drug addicts (Elnifro et al., 2000). In some circumstances, molecular assays have been made so simple to use that they are often marketed as point of care tests (POCT) which are characterized as simple and rapid diagnostic methods that can be used also by healthcare professionals with little or no laboratory experience. Over the last few years, the need for POCT has emerged considering the following advantages: POCT may be performed close to the patient thus reducing the time of diagnosis, especially in case of serious and potentially lethal

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pathologies. These features allow a rapid diagnosis made at the patient’s bed and also be made in complex situations where access to laboratory methods is difficult if not impossible (Briedigkeit et al., 1998; Johannessen, 2015; Haleyur Giri Setty and Hewlett, 2014). In particular, this approach has found an important development in the diagnosis of HIV-1, influenza viruses, viral hepatitis, and emerging viruses such as the Ebola virus. Especially when potentially dangerous viruses are involved, it is important to adequately train health professionals in the safe collection and handling of biological samples and the correct storage and use of tests for a reliable diagnosis (Li-Kim-Moy et al., 2016; Khuroo et al., 2015; Kaushik et al., 2016). Multiplex and POC tests can improve the management of the patients when it needs a viral diagnosis. Through the search for multiple microorganisms simultaneously the time-around-time is reduced leading to fast clinical management of the patient. This is especially important in intensive care or emergency room, with pediatric and geriatric patients, and in situations where resources are scarce and a rapid, inexpensive, and reliable test can save many lives, such as in developing countries (Baba et al., 2014).

Microfluidics Microfluidics is a technology based on the analysis of small quantities of fluids (10–9 to 10–18 liters) through a system of channels ranging from 10 to 100 micrometers. The material used for the construction of the canals is an optically transparent, soft elastomer: the poly(dimethylsiloxane) or PDMS. The peculiarity of this system lies in the use of very small quantities of samples and reagents, in the low cost, in its high resolution and sensitivity, in the run-time extremely short (Whitesides, 2006). Microfluidics can be an important tool in situations where, as in developing countries, access to other diagnostic methods for viral diseases is not feasible. Microfluidics can be an excellent tool for POCT, too. First, the diagnosis of viral infection can occur within 15–30 min. The ability to correctly diagnose even low-level infections, combined with high specificity and sensitivity, allows clinicians to set appropriate therapy in a short time with the result of a better therapeutic success (Simpson et al., 2018; Na et al., 2018). Furthermore, the microfluidic platforms have the unique ability to control the microenvironment through the precise manipulation of very small sample volumes for the assay, finding application in the discovery of new antiviral drugs and the development of vaccines (Chi et al., 2016; van der Borg et al., 2018; Chaipan, 2017). A recent study has demonstrated the ability to screen microfluidic chips and sorting HIV-1 particles according to epitope expression (Chaipan, 2017). Microfluidic technologies reduce reaction times while providing an increased number of information on the virus compared to the techniques currently used for the measurement of Viremia, such as qPCR. With this method, up to 12 influenza A subtypes can be found simultaneously in less than 100 min (Shen et al., 2019). Besides, the simultaneous identification of sensitive and resistant viruses makes it possible to assess the viral resistance to antiviral drugs before being administered to the patient. Microfluidic platforms also find space in biomedical research with the possibility of carrying out multiple experiments in a single device even with small quantities of a biological sample, reducing time and costs (Na et al., 2018). Such technology did not find so far an extensive application but it is our opinion that shortly the progress made by lab-on-a-chip microtechnologies will help this technology live up to its potential.

Conclusions and Future Perspectives Laboratory diagnostics of viral diseases are gaining increasing interest from companies, researchers, and clinicians. The new technologies will replace or improve the current ones, allowing them to reach increasingly optimal management of the patient with viral diseases. Furthermore, thanks to them it will be possible to give impetus to the world of virology through the discovery of new pathogenic and non-pathogenic viral species and their interactions with the host. Over recent years, there has been growing interest in using Artificial Intelligence (AI) in different areas of medicine. Following the recent events related to the COVID-19 pandemic, AI has shown how their application could improve the prediction, detection, and management of infectious diseases by the combination of laboratory results, medical history, symptoms, and instrumental tests, such as chest CT. Furthermore, the AI can be a useful support to the processing of laboratory results by eliminating operatordependent interpretation errors in diagnostic tests (Mei et al., 2020; Bansal et al., 2020). However, not all methods are equal and can lead to different results with the same biological sample. It is therefore important that research continues to develop ever more precise, low-cost, and accessible solutions.

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Whitesides, G.M., 2006. The origins and the future of microfluidics. Nature 442 (7101), 368–373. Yin, C., Zhang, T., Qu, X., et al., 2017. In vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in animal models. Molecular Therapy 25, 1168–1186. Yozwiak, M.L., Skewes-Cox, P., Stenglein, M.D., et al., 2012. Virus identification in unknown tropical febrile illness cases using deep sequencing. PLOS Neglected Tropical Diseases 6 (2), e1485. Zhang, H., Morrison, S., Tang, Y.W., 2015. Multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis. Clinics in Laboratory Medicine 35 (2), 461–486. Zürcher, S., Mooser, C., Lüthi, A.U., 2012. Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis. Journal of Clinical Virology 54, 359–363.

Further Reading Behjati, S., Tarpey, P.S., 2013. What is next-generation sequencing? Archives of Disease in Childhood: Education and Practice Edition 98 (6), 236–238.

TREATMENT

Antiviral Classification Guangdi Li, Xixi Jing, and Pan Zhang, Central South University, Changsha, China Erik De Clercq, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium r 2021 Elsevier Ltd. All rights reserved.

Nomenclature EC50 The concentration of a drug inducing its halfmaximal effective response.

Glossary Allosteric inhibitors Inhibitors block the enzymatic activity by targeting outside the active site of viral enzymes. Antiviral drug resistance The reduction in the effectiveness of an antiviral agent to treat an infectious disease, probably caused by amino acid mutations within or outside the drug-binding site.

IC50 The concentration of an inhibitor at which 50% of inhibition in its activity is achieved.

Drug susceptibility The sensitivity of viruses to one or more drugs. If a virus is susceptible, it can be treated with the drug. Genetic barrier to resistance The threshold above which drug resistance develops to a drug or a drug class. HAART Highly active antiretroviral therapy which contains a combination of Z3 antiretroviral drugs.

Introduction During the past decades, more than 100 antiviral agents or their combinations have been approved to treat 9 human infection diseases: HIV, HCV, influenza virus, RSV, HSV, HCMV, VZV, HBV, and variola virus (human smallpox). Antiviral agents can be possibly classified based on their chemical structures, drug targets, or mechanisms of action. For instance, most antiviral agents target either viral enzymes to block the viral replication or viral surface proteins to prevent the viral entry. Regarding the mechanisms of action, nucleoside analogs are effective viral polymerase inhibitors that resemble naturally occurring nucleosides to cause the termination of the nascent viral DNA chain, while protease inhibitors block the proteolytic processing by competing with protease substrate peptides. Based on the chemical structures, aciclovir and valaciclovir are classified as acyclic guanosine analogs to treat DNA viruses such as HSV and VZV, while cidofovir, adefovir, tenofovir alafenamide are acyclic nucleoside phosphonate analogs to treat HCMV, HBV, and HIV, respectively. Due to the variable nature of antiviral agents, this book article attempts to characterize the antiviral classification based on the drug targets in 9 human infectious diseases.

Human Immunodeficiency Virus (HIV) HIV Reverse Transcriptase HIV reverse transcriptase (RT) is an asymmetric heterodimer which harbors two enzymatic domains: the RNA- and DNAdependent DNA polymerase and the ribonuclease H (Li and De Clercq, 2016). After the viral entry of HIV particles, HIV RT produces the viral DNA genome based on the template of the single-stranded viral RNA genome released from HIV particles (Das et al., 2019). To inhibit HIV genome replication, nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) have been successfully developed to target HIV RT – an indispensable enzyme for HIV replication. On the one hand, the 50 triphosphate of the NRTIs act as chain terminators. Because NRTIs lack a 30 -OH group, the incorporation of NRTIs into the newly synthesized viral dsDNA terminates the elongation of DNA primer, thereby blocking HIV reverse transcription (Das and Arnold, 2013). On the other hand, NNRTIs are allosteric inhibitors that allosterically target a hydrophobic pocket located approximately 10–15 Å from the catalytic site of HIV-1 RT (Sluis-Cremer and Tachedjian, 2008). This causes conformation changes of the HIV-1 RT catalytic site, thus interrupting the viral dsDNA replication (De Clercq and Li, 2016). As of September 2020, fifteen HIV RT inhibitors have been approved by the US FDA for clinical use, including (1) seven NRTIs: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine; two NtRTIs (nucleotide RT inhibitors): tenofovir disoproxil fumarate and tenofovir alafenamide; and (2) six NNRTIs: nevirapine, delavirdine, efavirenz, etravirine, rilpivirine, and doravirine. Elsulfavirine has only been approved in Russia. For more than two decades, NRTIs have been clinically used as the backbone in the highly active antiretroviral therapy (HAART, a combination of Z3 antiretroviral drugs), while NNRTIs often serve as the third agent. Currently, tenofovir alafenamide and doravirine are widely administered in clinical practice because of a high genetic barrier to resistance, high safety profile, and low frequency of administration (Deeks, 2018; De Clercq, 2018). Novel RT inhibitors such as islatravir and dapivirine are currently under development (Baeten et al., 2016; Nel et al., 2016; Nakata et al., 2007).

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HIV Protease HIV protease plays an indispensable role in the proteolytic processing of Gag and Gagpol polyproteins to release key structural proteins (matrix, capsid, nucleocapsid, p6) and viral enzymes (reverse transcriptase, integrase, protease) (Li and De Clercq, 2016). HIV protease is a homodimer of two subunits and the active site of HIV protease is located at the center of the substrate-binding tunnel. This substrate-binding tunnel has been recognized as a conserved drug-binding pocket for the development of HIV protease inhibitors to prevent proteolytic processing (Ko et al., 2010). As of September 2020, 10 protease inhibitors have been approved by the US FDA. The era of protease inhibitors began in 1995 when saquinavir was the first protease inhibitor approved for clinical use. Subsequently, the US FDA approved ritonavir (a protease inhibitor and pharmacokinetic booster) in 1996, indinavir in 1996, nelfinavir in 1997, amprenavir in 1999, lopinavir and atazanavir in 2000, fosamprenavir in 2003, tipranavir in 2005, and darunavir in 2006. Over the past decades, HIV protease inhibitors have become an important ingredient of HAART (Hammer et al., 1997; Gulick et al., 1997). Nevertheless, the rapid emergence of resistance and poor bioavailability pose a challenge to the clinical use of protease inhibitors (Subbaiah et al., 2017). Currently, darunavir is the most popular protease inhibitor (Deeks, 2014), while saquinavir and amprenavir have been virtually discontinued (De Clercq and Li, 2016).

HIV Integrase HIV integrase, encoded by the HIV Pol gene, is an indispensable enzyme for integrating viral DNA genomes into human chromosomes by a series of DNA cutting and joining reactions (Asante-Appiah and Skalka, 1999; Esposito and Craigie, 1999). Since the viral DNA synthesized by HIV reverse transcriptase is initially blunt-ended, there are two critical steps of viral integration: (1) the 30 end processing reaction removes two nucleotides from each of the 30 ends of the viral dsDNA before viral integration; and (2) the DNA strand transfer reaction that breaks the human chromosome and 30 ends of viral DNA are joined to the 50 ends of human chromosome at the integration sites (Li et al., 2015). Antiviral compounds have been screened to inhibit either the 30 end processing reaction or the DNA strand transfer reaction (Delelis et al., 2008). Compounds against the 30 end processing reaction are inactive, but integrase strand transfer inhibitors (INSTIs) have shown potent antiviral activities in both in vitro and in vivo studies (Delelis et al., 2008). INSTIs selectively bind near the 30 end of the viral DNA in the structural complex of viral DNA and HIV integrase, thereby blocking the DNA strand transfer reaction (Delelis et al., 2008). As of September 2020, four INSTIs (raltegravir, elvitegravir, dolutegravir, bictegravir) have been approved by the US FDA. The first-generation INSTIs such as raltegravir and elvitegravir share a low genetic barrier to resistance, causing the loss of virologic activity in clinical studies (Brooks et al., 2019). In contrast, dolutegravir and bictegravir – two second-generation INSTIs – exhibit a higher genetic barrier to resistance, limited cross-resistance, and less drug-drug interactions (Oliveira et al., 2018; Podany et al., 2017). Currently, the WHO, IAS-USA, and EACS guidelines recommend the use of INSTIs in the first-line HIV regimens (Brooks et al., 2019). Novel INSTIs such as cabotegravir are undergoing clinical development (Orkin et al., 2020).

HIV GP41 GP41 is a transmembrane protein encoded by the env gene. As a key structure protein, HIV GP41 binds with GP120 to form HIV spike trimers on the surface of HIV particles (Mao et al., 2012). During the viral entry, the N-heptad repeat (NHR) and C-heptad repeat (CHR) of GP41 switch to a six-helix bundle (6-HB) structure which binds to the human cell membrane and drives the viral fusion into the human cells. Many antiviral agents have been developed to target the hydrophobic pocket within the NHR trimer, therefore preventing viral entry (Mostashari Rad et al., 2018). As the only GP41 inhibitor approved by the US FDA, enfuvirtide is a fusion peptide inhibitor that mimics the N-heptad repeat and prevents the formation of the six-helix bundle structure of GP41. Clinical use of enfuvirtide requires twice-daily subcutaneous injection (90 mg/Kg for adults, 2 mg/Kg for children aged 6–16 years) (Kitchen et al., 2008). Enfuvirtide is not commonly used in clinical practice because of its side effects (eosinophilia, neutropenia, increased risk of bacterial pneumonia), its short half-life, and lack of oral availability (Reust, 2011). Although many attempts have been made, the development of GP41 inhibitors remains difficult due to the emerging mutations and structural dynamics of HIV GP41.

HIV GP120 GP120, encoded by the env gene, is an envelope glycoprotein on the surface of HIV particles. During the viral entry, HIV GP120 binds to the CD4 receptor and the CCR5/CXCR4 co-receptor on the surface of human CD4 þ T cells (Falkenhagen and Joshi, 2018; Shaik et al., 2019). The Phe43 pocket and other conserved regions on the GP120 are considered as the real targets for developing anti-HIV drugs (Mostashari Rad et al., 2018). The drugs targeting GP120 can be called adhesion inhibitors, mainly blocking the binding of GP120 with CD4 (Pu et al., 2019). Fostemsavir (BMS-663068) is the first GP120-directed attachment inhibitor approved by the US FDA on July 3rd, 2020. Fostemsavir tromethamine is the prodrug of temsavir – the active moiety that binds to the conserved outer domain of GP120 which is adjacent to the CD4 binding loop (Langley et al., 2015). This binding inhibits the exposure of the chemokine co-receptor binding site and prevents the initial interaction of HIV GP120 with the CD4 receptor on human T cells and other immune cells

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(Langley et al., 2015). Fostemsavir is recommended for heavily treatment-experienced adults with multidrug-resistant HIV-1 infections. As the only compound in the class of GP120 drugs, fostemsavir harbors a unique resistance profile and exhibits no cross-resistance with other entry inhibitors (Lataillade et al., 2018).

Hepatitis C Virus (HCV) HCV NS3/4A Protease The nonstructural protein 3 (NS3) contains a serine protease domain and a helicase domain at the N-terminal and C-terminal regions, respectively (McGivern et al., 2015). The NS3 protein acts as a serine protease to cleave HCV polyprotein precursor at four junctions (NS3-NS4A, NS4A-NS4B, NS4B-NS5A, NS5A-NS5B) to release 5 viral proteins. As a short 54-amino-acid polypeptide, the nonstructural protein 4A (NS4A) serves as an essential co-factor for the NS3 serine protease in a non-covalent complex. NS3 protease is an attractive drug target because its inhibition can terminate the polyprotein processing and restore interferon gene expression (McGivern et al., 2015). NS3/4A protease inhibitors are a key element of direct-acting antivirals that effectively cure HCV infections by targeting one or more viral proteins (Li and De Clercq, 2017). As of today, ten NS3/4A protease inhibitors have been approved, including glecaprevir, grazoprevir, paritaprevir, simeprevir, vaniprevir, voxilaprevir, asunaprevir, danoprevir, boceprevir (discontinued), and telaprevir (discontinued) (Li and De Clercq, 2017). Moreover, danoprevir was approved in China to treat patients infected with HCV genotype 1b (Miao et al., 2020). Novel NS3/4A inhibitors such as seraprevir are currently under development. For instance, seraprevir is now evaluated in a phase 3 trial (NCT04001608).

HCV NS5A Phosphoprotein The nonstructural protein 5A (NS5A) is a zinc-binding and proline-rich hydrophilic phosphoprotein that executes versatile functions in genome replication, viral particle assembly, and human-virus interactions (Ross-Thriepland and Harris, 2015). The NS5A protein in the form of a dimer or multimer localizes to the ER-derived membranes via its amphipathic helix domain. NS5A inhibitors can target the amphipathic helix domain of NS5A dimer to inhibit the conformation of double-membrane vesicles and impair HCV RNA replication factories (Shanmugam et al., 2018). As of today, six NS5A inhibitors (ombitasvir, elbasvir, velpatasvir, daclatasvir, ledipasvir, pibrentasvir) have been approved by the US FDA. These NS5A inhibitors efficiently block in vitro and in vivo viral replication, though their exact models of drug actions are yet to be elucidated (Ross-Thriepland and Harris, 2015). Moreover, the clinical efficacy of NS5A inhibitors plus other directacting antivirals offers more than 90% of sustained virologic responses (Li and De Clercq, 2017). Many experimental NS5A inhibitors such as ravidasvir are still under development. Ravidasvir plus sofosbuvir offered promising virologic response rates in 298 patients infected with HCV genotype 4 (Esmat et al., 2017).

HCV NS5B Polymerase The nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase that is indispensable for HCV RNA synthesis and genome replication. The structure of NS5B consists of three subdomains: “palm”, “finger”, “thumb”, and the hydrophobic membrane anchoring C-terminus. As a promising drug target, the catalytic site of NS5B is encircled by the finger and thumb domains. NS5B inhibitors that alter structural conformation or interfere with viral RNA binding exhibit promising potency to inhibit HCV RNA replication (Kirby et al., 2015). Two NS5B inhibitors have been approved by the US FDA, including sofosbuvir (GS-7977; formerly PSI-7977) in December 2013 and dasabuvir (ABT-333) in December 2014. The NS5B inhibitors can be mainly divided into either nucleoside inhibitors (e.g., sofosbuvir) or non-nucleoside inhibitors (e.g., dasabuvir) (Li and De Clercq, 2017). On the one hand, nucleoside inhibitors block the viral RNA synthesis by mimicking natural substrates and competing with incoming nucleoside triphosphates at the catalytic site of NS5B (Li and De Clercq, 2017). On the other hand, non-nucleotide inhibitors noncompetitively block the allosteric pockets outside the catalytic site to prevent viral RNA synthesis (Li and De Clercq, 2017). For instance, dasabuvir (Kati et al., 2015), GSK5852 (Voitenleitner et al., 2013), beclabuvir (Gentles et al., 2014), and filibuvir (Fenaux et al., 2013) target the allosteric drug pockets in the palm I, palm II, thumb I, and thumb II domains, respectively. Although only two NS5B inhibitors have been approved, many novel NS5B inhibitors (e.g., beclabuvir) are still under development.

Respiratory Syncytial Virus (RSV) RSV RNA Polymerase RSV RNA-dependent RNA polymerase – a complex comprising the viral large polymerase subunit, the phosphoprotein, and the transcription elongation factor M2-1 – plays a critical role in viral mRNA transcription, mRNA capping/methylation, and genome

124

Antiviral Classification

replication (Fearns and Deval, 2016). The large polymerase subunit harbors the enzymatic domains that produce subgenomic mRNAs, RNA replication, and an antigenome RNA for genome RNA synthesis, while the other proteins act as essential cofactors (Fearns and Deval, 2016). Due to its unique features and essential nature, the large polymerase protein has proved to be a promising drug target for the development of nucleoside and non-nucleoside inhibitors. As of today, ribavirin is an FDA-approved nucleoside inhibitor that inhibits the activity of RSV RdRp, whereas it is largely discontinued to treat RSV infections due to limited efficacy and risk of serious side effects (Shook and Lin, 2017). Experimental inhibitors such as ALS-8176 and PC786 are still under clinical development. ALS-8176 (lumicitabine) is a nucleoside inhibitor with promising anti-RSV efficacy and safety in phase 2a study (NCT02094365) (DeVincenzo et al., 2015; Wang et al., 2015). PC786 is a potent non-nucleoside inhibitor that blocks RSV type A strains (IC50: o0.09–0.71 nM) and RSV type B strains (IC50: 1.3–50.6 nM) in cell cultures (Coates et al., 2017).

RSV Fusion Glycoprotein RSV fusion glycoprotein is a class I fusion protein that anchors in the membrane of viral particles via a transmembrane domain (Gilman et al., 2019). During the viral entry, RSV fusion glycoprotein in the trimeric form is undergoing dramatic changes from the metastable prefusion confirmation to the highly stable post-fusion conformation, thereby driving the fusion of viral membrane with the human cell membranes (Gilman et al., 2019). RSV fusion glycoprotein that induces RSV-neutralizing antibody responses is a key antigen for protective immunity (Tang et al., 2019). As a leading target of neutralizing antibodies and vaccines, RSV fusion glycoproteins have conserved sequences across different isolates of RSV type A and B strains (Tang et al., 2019). As of September 2020, palivizumab that binds to RSV fusion glycoprotein remains the only monoclonal antibody approved for the prevention of RSV infection in high-risk infants. Nevertheless, the clinical application of palivizumab is uncommon because of its low stability and its limited efficacy of 50% (Soto et al., 2020). Ongoing studies are currently evaluating many experimental inhibitors of RSV fusion glycoprotein, including preatovir, ziresovir, sisunatovir, MDT-637, JNJ-53718678, and ALX-0171. For instance, preatovir (GS-5806) efficiently inhibits the fusion glycoprotein of RSV-A (EC50: 0.51 7 0.25 nM) and RSV-B (EC50: 0.35 7 0.15 nM) strains in cell cultures (Perron et al., 2015), and it has completed clinical phase 2 trials (NCT02135614). Ziresovir is another selective and orally bioavailable inhibitor in phase 3 clinical trial (NCT04231968) (Zheng et al., 2019).

Herpes Simplex Virus (HSV) HSV DNA Polymerase UL30 HSV DNA replication requires seven viral proteins: (1) an origin binding protein UL9, (2) a single-strand binding protein ICP8; (3) a viral polymerase with two subunits UL30 and UL42; (iv) a helicase-primase complex with three subunits UL5, UL8, and UL52 (Chen et al., 2011). HSV DNA polymerase is a heterodimeric complex with two subunits: (1) the catalytic UL30 subunit which offers both polymerase and proofreading exonuclease activities; (2) the UL42 subunit that promotes processivity by tethering UL30 to viral DNA via its direct DNA binding (Vashishtha and Kuchta, 2015). The UL30 subunit belongs to the B-family of polymerases and forms a typical hand-shaped structure with the catalytic residues (D717, D888) located in the palm domain for dNTP polymerization (Vashishtha and Kuchta, 2015). The UL30 subunit can replicate viral DNA with high fidelity, even in the absence of the UL42 subunit. Due to its indispensable role, the UL30 subunit has been considered as a promising drug target to block viral DNA replication (Zarrouk et al., 2017). As of September 2020, many UL30 inhibitors such as idoxuridine, brivudine, trifluridine foscarnet, aciclovir, famciclovir, valaciclovir, and penciclovir have been approved for HSV treatment. Because current compounds face the challenge of drug resistance and adverse reactions (Vollmer et al., 2019), many experimental inhibitors such as synguanol (Vollmer et al., 2019), mitoxantrone dihydrochloride (Huang et al., 2019), and PHA767491 (Hou et al., 2017) are under development. For instance, synguanol is a methylenecyclopropane nucleoside analog that competitively inhibits HSV-1 UL30 (IC50: 0.33 7 0.16 mM) (Vollmer et al., 2019). Mitoxantrone dihydrochloride inhibits viral DNA synthesis by blocking the transcription of UL30 (IC50: 1.21 mM) (Huang et al., 2019). PHA767491 also inhibits UL30 to reduce the expression of HSV viral genes (UL5, UL8, UL29, UL30, UL42, UL52) (IC50: 1.86 mM) (Hou et al., 2017). Antiviral agents such as pritelivir and amenamevir that target HSV helicaseprimase complex are still under development (Poole and James, 2018).

HSV Envelope Protein Although HSV particles harbor at least 15 envelope proteins, four envelope glycoproteins (gD, gB, gH, gL) play an indispensable role in viral entry into all permissive cell types (Agelidis and Shukla, 2015). The viral entry begins with the binding of gD to a human receptor (nectin-1, herpesvirus entry mediator, or 3-O-sulfated heparan sulfate) (Atanasiu et al., 2018). This gD-receptor binding drives the conformation changes in gD to activate the regulatory proteins gH and gL, leading to the activation of gB into a fusogenic state for membrane fusion (Agelidis and Shukla, 2015). Docosanol (n-docosanol; behenyl alcohol) is a naturally occurring antiherpetic agent approved by the US FDA as a topical treatment for herpes labialis, as well as the over-the-counter medication for cold sores and fever blisters (De Clercq and Li, 2016).

Antiviral Classification

125

Although its exact mechanism of action remains unclear, docosanol may inhibit the interactions between HSV envelope proteins and human receptors (De Clercq and Li, 2016). Experimental inhibitors such as C19 and NGI-1 are still under development (Rinis et al., 2018; Lu et al., 2019).

Human Cytomegalovirus (HCMV) HCMV DNA Polymerase UL54 HCMV DNA polymerase is a multiprotein complex that contains a catalytic subunit UL54 and a processivity factor UL44 to synthesize long stretches of viral DNA during viral replication (Appleton et al., 2004). The homodimer UL44 binds to viral DNA to prevent dissociation from the template, thereby promoting the long-chain DNA synthesis within the catalytic subunit of UL54 (Appleton et al., 2004). Due to its essential role in viral DNA synthesis, UL54 has been recognized as an important drug target against HCMV. As of today, cidofovir, ganciclovir, foscarnet, valganciclovir, and fomivirsen (discontinued) have been approved for HCMV treatment. To inhibit viral DNA synthesis, cidofovir and ganciclovir are converted to DNA polymerase substrate analogs by the thymidine kinase of CMV. UL54 gene is prone to mutations that limit the efficiency of antiviral agents (Chou et al., 2016). Furthermore, cidofovir may induce nephrotoxicity especially in bone marrow transplant patients (Piret et al., 2017), which limits its clinical use. Novel compounds such as CMX001 (brincidofovir) are currently under development. CMX001 can be converted to cidofovir. The drug potency is much higher for CMX001 than for cidofovir. Cidofovir from CMX001 does not accumulate in the kidneys, therefore nephrotoxicity could be greatly reduced during the treatment (Marty et al., 2013).

HCMV Terminase UL56 HCMV DNA terminase complex is a hetero-oligomer composed of UL56, UL89, and additional viral subunits (UL51, UL52, UL77, UL93) (Ligat et al., 2018). This terminase complex cleaves HCMV DNA concatemers and packages the genome into the capsid for the DNA-packing process during the viral maturation and packaging (Ligat et al., 2018). The large terminase subunit UL56 plays an essential role in the viral DNA cleavage and packaging (Ligat et al., 2018). UL56 not only has the ATPase activity that hydrolyzes ATP to provide energy for the genome cutting and transfer activities but also has ATP-independent endonuclease activity driven by UL89 (Ligat et al., 2018). As an indispensable viral protein, UL65 has been considered to be a promising drug target. As of September 2020, letermovir remains the only UL56 inhibitor approved by the US FDA for preventing HCMV in adult HCMVseropositive recipients of an allogeneic hematopoietic stem cell transplant. Based on a phase 3 clinical trial (NCT02137772), letermovir prophylaxis significantly reduced the risk of HCMV infection, and its adverse effects were mild (Marty et al., 2017). Moreover, letermovir is a viral DNA-packaging inhibitor, remarkably specific for HCMV, but not other herpesviruses (Ligat et al., 2018).

Human Influenza Virus Viral RNA Polymerase The influenza polymerase is comprised of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), plus either polymerase acidic protein (PA) in influenza A/B or polymerase 3 protein (P3) in influenza C/D viruses (Takashita, 2020). The PB1 subunit encodes the RNA-dependent RNA polymerase, while the PA and PB2 subunits offer the endonuclease and cap-binding activities, respectively (Wandzik et al., 2020). During the transcription of viral mRNA, the cap-snatching of capped oligomers from human Pol II transcripts is mediated by the PB2 cap-binding and PA endonuclease activities. Subsequently, the PB1 active site undertakes the prime template-directed RNA synthesis, followed by the initiation, elongation, termination, and recycling of typical polymerase activities (Wandzik et al., 2020). Due to their importance, PA, PB1, and PB2 have been considered as promising antiviral targets. As of September 2020, the PA inhibitor baloxavir marboxil (S-033188) and the PB1 inhibitor favipiravir (T-705) have been approved for clinical use, while the PB2 inhibitor pimodivir (JNJ-63623872, VX-787) is currently evaluated in phase 3 trials (NCT03381196, NCT03376321). As a pyrazine derivative, favipiravir received conditional marketing approval only for patients with a novel or reemerging influenza when other antivirals are ineffective because favipiravir increases the risk for teratogenicity and embryotoxicity (Takashita, 2020). Baloxavir marboxil was approved for treating influenza A and B viruses, while its pattern of drug susceptibility follows the order: influenza A > B > C > D (Takashita, 2020). As a cyclohexyl carboxylic acid analog, pimodivir inhibits viral RNA binding by occupying the cap-binding domain of PB2, and it shows strong activity against influenza A but not B viruses (Takashita, 2020). Other novel polymerase inhibitors are still under development.

Neuraminidase Influenza surface glycoprotein neuraminidase is known for its multifunctional roles in viral entry and viral release. During the viral entry, the glycoprotein neuraminidase contributes to the viral binding to the sialic acid receptors of human cell glycoproteins,

Pimodivir (VX-787) Isocorilagin

Ribavirin Palivizumab

Aciclovir, brivudine, famciclovir, foscarnet, idoxuridine, penciclovir trifluridine, valaciclovir Docosanol Cidofovir, fomivirsen, foscarnet, ganciclovir, valganciclovir, Letermovir Aciclovir, brivudine, famciclovir, valaciclovir, vidarabine Adefovir, besifovirb, clevudineb entecavir, telbivudine, tenofovir alafenamide, tenofovir Tecovirimat

RNA polymerase

Fusion glycoprotein

DNA polymerase UL30 Envelope proteins

DNA polymerase UL54 Terminase UL56

DNA polymerase

DNA polymerase VP37 envelope wrapping protein

Respiratory syncytial virus (RSV)

Herpes simplex virus (HSV)

Human cytomegalovirus (HCMV)

Varicella-zoster virus (VZV) Hepatitis B virus (HBV) Human smallpox

Tenofovir exalidex

Filociclovir

Synguanol, filociclovir, MBX-2168; mitoxantrone dihydrochloride; PHA767491 NGI-1, C19

Presatovir (GS-5806), ziresovir (RO-0529, AK0529), MDT-637, JNJ53718678, sisunatovir, ALX-0171

b

Discontinued. Elsulfavirine was approved in Russia, albuvirtide was approved in China; favipiravir and laninamivir were approved in Japan; danoprevir was approved in China; clevudine was approved in South Korea and the Philippines; besifovir was approved in South Korea.

a

Baloxavir marboxil, favipiravirb Laninamivirb, oseltamivir, peramivir, zanamivir, Amantadinea, rimantadine

RNA polymerase Neuraminidase Matrix protein 2

Lumicitabine (ALS-8176)

Asunaprevir, faldaprevir furaprevir, narlaprevir, Seraprevir, vaniprevir, vedroprevir Ravidasvir, ruzasvir, odalasvir Adafosbuvir, deleobuvir, Lomibuvir, mericitabine, Radalbuvir, radalbuvir,

MK-8504, MK-8583, racivir, islatravir, rovafovir etalafenamide, censavudine, amdoxovir, elvucitabine, KM-023 Cabotegravir GSK373239

Danoprevirb, glecaprevir, grazoprevir, paritaprevir, paritaprevir, Simeprevir,

NS5A phosphoprotein Daclatasvir, ledipasvir, ombitasvir, elbasvir, velpatasvir, pibrentasvir NS5B polymerase Sofosbuvir, dasabuvir

NS3/4A protease

Human influenza virus

Hepatitis C virus (HCV)

Reverse transcriptase NRTIs: abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir alafenamide, tenofovir disoproxil fumarate, zidovudine, NNRTIs: delavirdinea, doravirine, efavirenz, elsulfavirineb, etravirine, nevirapine, rilpivirine, Integrase Bictegravir, dolutegravir, elvitegravir, raltegravir, gp41 Albuvirtideb, enfuvirtide gp120 Fostemsavir

Amprenavir, atazanavir, darunavir fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, TMB-607, TMC-310911, GRL-09510 saquinavir, tipranavir,

Protease

Human immunodeficiency virus (HIV)

Novel inhibitors

Approved drugs

Viral targets

Summary of viral proteins targeted by approved and novel inhibitors

Human viruses

Table 1

126 Antiviral Classification

Antiviral Classification

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leading to the enhancement of hemagglutinin receptor binding (Wen and Wan, 2019). During the viral release, neuraminidase cleaves sialic acids from cellular receptors and neuraminidase/hemagglutinin on nascent influenza virions so as to prevent virion aggregation and release new particles (McAuley et al., 2019). Due to its importance, neuraminidase has been recognized as a promising antiviral target (Kumar et al., 2020). As of September 2020, neuraminidase inhibitors such as oseltamivir, zanamivir, peramivir, and laninamivir have been approved for clinical use (Table 1). Oseltamivir is used orally as the first-line therapy, but its effectiveness is compromised by the development of drug resistance mutations in influenza A genotypes such as H3N2 and H5N1 (Kumar et al., 2020). Due to the emergence of drug resistance, it remains an urgent need to develop anti-influenza agents. Novel antivirals such as A-192558 and A315675 are still under development (Gubareva and Mohan, 2020).

Matrix Protein 2 The influenza matrix protein 2 (M2) is a 97-residue single-pass membrane protein that forms pH-gated proton channels in the viral lipid envelope (Schnell and Chou, 2008). By shuttling protons inwards and outwards through the viral membrane, matrix protein 2 equilibrates pH across the viral membrane during the viral entry and across the trans-Golgi membrane of host cells during the viral maturation (Mandala et al., 2020). Because the channel pore is essential for the proton shuttling, the pore of the M2 channel is a promising drug-binding pocket (Gu et al., 2013). As of September 2020, rimantadine and amantadine that target the M2 channel pore have been approved for anti-influenza treatment. For instance, amantadine targets the M2 channel by hydrophobic interactions between the adamantane group and the N-terminal gate of the channel (Gu et al., 2013). Mutations (e.g., V27A, L26F) within the channel pore weaken the hydrophobic interactions of rimantadine and amantadine with the M2 channel, thereby causing drug resistance and treatment failure (Gu et al., 2013). Novel inhibitors that are less prone to mutations may lead to better antiviral efficacy.

Variola Virus (Human Smallpox) VP37 Envelope Wrapping Protein The extinction of variola virus, the etiological agent of human smallpox was declared by the WHO in 1980 (Jordan et al., 2010). The F13L gene of variola virus encodes a highly conserved 37 kDa peripheral membrane protein called VP37. Before the viral budding, the wrapping complex requires VP37 and other viral proteins to interact with human membrane proteins; subsequently, it catalyzes the maturation of intracellular viral particles into the egress-competent form of the variola virus particles (Jordan et al., 2010). VP37 interacts with human proteins Rab9 and TIP47 (a Rab9-specific effector) in membrane fractions from infected cells to facilitate assembly of extracellular virus (Chen et al., 2009). As of September 2020, tecovirimat (ST-246) remains the only VP37 inhibitor approved for treating human smallpox, although its effectiveness has only been shown in animal models but not humans due to the extinction of the virus in human populations. As an 4-trifluoromethyl phenol derivative, tecovirimat blocks the interactions of VP37 with Rab8 and TIP47, thereby inhibiting the maturation of egress-competent enveloped virions for viral budding (Jordan et al., 2010). Novel compounds such as NIOCH-14 are still under development (Delaune and Iseni, 2020).

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) SARS-CoV-2 Polymerase SARS-CoV-2 RNA-dependent RNA polymerase, encoded by the NSP12 gene, forms a polymerase complex with its viral cofactors NSP7 and NSP8 to catalyze the viral RNA synthesis during the viral replication and transcription (Gao et al., 2020). Similar to SARS-CoV polymerase, the structure of SARS-CoV-2 polymerase contains three subdomains: fingers, a thumb, and a palm, while the catalytic site (S759-D760-D761) is located within the conserved motifs of the palm domain (Gao et al., 2020). The cofactors NSP7 and NSP8 interact with the thumb, while another NSP8 binds to the fingers, thereby conferring processivity to NSP12 (Hillen et al., 2020). Due to its indispensable role in viral RNA replication, SARS-CoV-2 polymerase is an important target for antiviral drug development (Li and De Clercq, 2020; Zhou et al., 2020; Jiang et al., 2020). As of October 2020, remdesivir (GS-5734) has been authorized in the US for clinical use as a polymerase inhibitor to treat patients infected with SARS-CoV-2. Remdesivir acts as a delayed chain terminator to interfere with the RNA synthesis of viral polymerase and evades the proofreading of viral exoribonuclease (Eastman et al., 2020). The intravenous use of remdesivir could shorten the recovery time and reduce the mortality rate of SARS-CoV-2 based on a randomized, double-blinded, placebocontrolled trial (NCT04280705) (Olalla, 2020). Other novel experimental inhibitors such as GS-441524 (Yan and Muller, 2020) and favipiravir (Arab-Zozani et al., 2020) might offer antiviral activities against SARS-COV-2, but their clinical use requires further investigation.

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Antiviral Classification

Varicella Zoster Virus (VZV) VZV DNA Polymerase VZV polymerase belongs to the DNA polymerase type-B family. As the essential component of VZV replication complex, DNA polymerase is encoded by the open reading frame 28 (ORF28). VZV DNA polymerase is an ideal target for the development of nucleoside analogs because it plays a critical role in viral DNA replication during the life cycle. As of September 2020, nucleoside analogs such as aciclovir, famciclovir, valaciclovir, brivudine, and vidarabine have been approved for VZV treatment (De Clercq and Li, 2016). These compounds could effectively target VZV polymerase and block viral DNA synthesis. As a prodrug of penciclovir, famciclovir effectively prevents the recurrence of VZV, and it has only mild adverse events (Wang et al., 2020). Furthermore, both aciclovir and famciclovir interventions offer high rates of benefit and showed a similar time to full crusting of lesions (Pott Junior et al., 2018).

Hepatitis B Virus (HBV) HBV DNA Polymerase HBV RNA-dependent DNA-dependent polymerase, encoded by the open reading frame P, acts with multifaceted functions such as (1) HBV reverse transcription that synthesizes the (  ) DNA strand from the viral RNA template within the catalytic site of HBV polymerase; (2) degradation of the viral RNA template by the RNase H activity of HBV polymerase (Menendez-Arias et al., 2014). HBV polymerase is mainly comprised of three functional domains: the template domain (AA positions: 1–183), the polymerase domain (349  691), and the RNase H domain (692  845) (Menendez-Arias et al., 2014). The catalytic site of the polymerase domain is considered as a promising drug target, whilst nucleos(t)ide inhibitors have been developed to compete with the incorporation of natural nucleotide substrates into the elongating DNA chain, thereby blocking viral DNA synthesis (De Clercq and Li, 2016). As of September 2020, many nucleos(t)ide inhibitors (entecavir, telbivudine, adefovir dipivoxil, tenofovir disoproxil fumarate, tenofovir alafenamide, clevudine, and besifovir) have been approved for clinical use. Note that clevudine was only approved in South Korea and the Philippines, while besifovir was approved in South Korea. Despite the success of nucleos(t)ide inhibitors to suppress the viral replication, they do not eliminate the virus from the hepatocytes, and a cure of HBV infection remains yet to be discovered. Novel HBV polymerase inhibitors such as tenofovir exalidex are still under clinical development (Martinez et al., 2020).

Conclusion Based on the classification of approved antiviral agents, several features could be summarized. First, antiviral agents mostly target the viral proteins with high specificity, leading to less toxicity compared with human protein targets. Second, among 9 infectious diseases, the most popular drug targets could be listed as follows: viral polymerase, viral envelope glycoproteins, and viral protease. These three viral proteins play a dispensable role in viral replication, making them promising viral targets for most human viruses. Third. antiviral agents are mostly small molecules that can be taken orally in clinical settings, while few antibodies and peptide inhibitors have been approved for antiviral use. Compared with monoclonal antibodies and peptides, small molecules are typically cheaper, chemically stabler, structurally simpler and better permeable. Although this article only describes 10 infectious diseases with approved drugs, future antiviral development should also focus on emerging infectious diseases such as coronaviruses, dengue, Zika, and Ebola. On October 14, 2020, the US FDA approved a mixture of three monoclonal antibodies (atoltivimab, maftivimab, odesivimab-ebgn), which marks the first FDA approval for the treatment of Ebola virus infection. Furthermore, it remains critical improving antivirals to combat emerging drug resistance mutations because drug resistance mutations have been observed in many viruses (e.g., HIV, HBV, influenza).

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Antiretroviral Therapy – Nucleoside/Nucleotide and Non-Nucleoside Reverse Transcriptase Inhibitors Timothy D Appleby and Killian J Quinn, King’s College Hospital, London, United Kingdom r 2021 Published by Elsevier Ltd.

Retroviruses and Reverse Transcription Retroviruses are a group of related viruses that depend on reverse transcription for their replication. They are subdivided into beta-, spuma-, gamma-, epsilon-, delta- and the lenti-retroviruses, to which HIV-1 and HIV-2 belong. The term retrovirus derives from the process of converting viral RNA to proviral DNA by use of the endogenous retroviral enzyme, reverse transcriptase (RT). Transcription is the process of copying DNA code into RNA – thus reverse transcription refers to copying an RNA code into DNA. Both DNA and RNA are composed of long chains of polysaccharide residues held together with phosphates, and each polysaccharide having a base attached to it. The two ends of a DNA and RNA chain are referred to the 50 end and the 30 ends, denoting the position at which the carbon chains link the polysaccharide, i.e., 5th position on their carbon chain, and at the other end the 3rd position. The genetic information of retroviruses is stored within two identical copies of positive sense RNA strands which exist as a dimeric molecule within the viral capsid. The genetic information is stored within nine reading frames, between 7 and 10 kilobases in length (9.7 kilobases in the case of HIV-1). Genes can be broadly divided into three main functional groups: (1) structural (2) regulatory and (3) accessory genes. Three genes (gag, pol and env) encode the structural polyproteins which undergo proteolytic cleavage to give rise to a total of 15 proteins involved in the viral lifecycle and structure. The pol gene encodes the three enzymes, RT, protease and integrase that are essential for the virus lifecycle and which are not available via the host machinery. The RT enzyme enables production of a complementary strand of viral DNA from single-stranded RNA (ssRNA). It is this process of reverse transcription that enables the integration of the resulting proviral DNA into the host genome and in turn effecting the persistence of the retroviral genome within the host DNA. Owing to its clinical importance, HIV-1 is by far the most studied retrovirus. Although there are multiple targets within the HIV-1 lifecycle, antiretroviral therapy targets 4 main steps namely: (1) viral entry (2) reverse transcription of viral RNA to doublestranded proviral DNA (dsDNA) (3) integration of dsDNA into the host genome and (4) proteolytic cleavage of viral polypeptides by the enzyme protease, enabling production of new viral proteins and ultimately new virus particles.

Reverse Transcriptase Structure and Functions RT is an asymmetric heterodimer of two related but distinct subunits, p66 and p51. It has two catalytic domains: the DNA polymerase active site and RNAse H both of which are necessary to carry out reverse transcription. These are a DNA polymerase that can copy either an RNA or a DNA template, and an RNase H that degrades RNA if it is part of an RNA–DNA duplex. Viral RT harnesses host nucleotides and initiates the process of reverse transcription of RNA into RNA-DNA duplex and subsequently double stranded DNA (Ren et al., 1995). With the activity of the RNAse H active site, the RNA component of the RNA-DNA double helix is digested, while the polymerase active site can then complete the synthesis of the double-stranded DNA to create a double-stranded helix which can then be integrated as proviral DNA into the host genome. RT was the first enzyme target in the development of ARV drugs and is the target for two distinct classes of ARV drug: the NRTIs and NNRTIs.

The Process of Reverse Transcription The HIV-1 life cycle begins with an interaction between the gp120 polypeptide on the viral surface and the CD4 subunits on the target cell membrane. This interaction leads to conformational change within gp120, in turn leading to release of gp41 which unfolds in a spring-loaded like mechanism piercing the target cell membrane. The gp41 subunit itself comprises two subdomains (heptad repeat, HR-1 and HR-2) which fold backwards upon one another. This mechanism results in the HIV-1 virion being pulled into close apposition to the target cell, a mixing of the viral and cellular lipid bilayers ensues with the creation of a “fusion pore” and the viral capsid is emptied inside the target cell. Following cytoplasmic penetration of the viral core, partial capsid dissolution and digestion of the matrix proteins releases viral RNA coupled with nucleoprotein into the host cells (“uncoating”) (Ren et al., 1995). It has been estimated that there are perhaps 50 RTs within each viral capsid which are released.

A tRNA primer binds to the primer binding site on the HIV-1 RNA Genomic viral RNA is plus ( þ ) sense. Reverse transcription of ( þ ) sense RNA into an RNA-DNA double helix is mediated by the RT polymerase active site. The process begins as the cellular tRNA3Lys molecule, an 18-nucleotide primer intrinsic to the host cell, hybridizes to a complementary region of the ( þ )RNA genome forming the primer binding site (PBS). The cellular tRNA3Lys primer is (  )sense whose 30 end is based-paired with the 50 prime end of the complementary ( þ )sense RNA. Although various

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NRTIs, with corresponding nucleosides, and year of licensing

Name

Abbreviation

Nucleoside

Year licensed

Abacavir Didanosine Emtricitabine Lamivudine Stavudine Tenofovir-DFa Tenofovir-AFb Zalcitabine Zidovudine

ABC ddI FTC 3TC d4T TDF TAF ddC AZT

Guanosine Adenosine Cytidine Cytidine Thymidine Adenosine Adenosine Pyrimidine Thymidine

1998 1991 2003 1995 1994 2001 2014 1992 1987

a

tenofovir disoproxil fumarate. tenofovir alafenamide.

b

primer models can be used to initiate reverse transcription in vitro, tRNA is used by all retroviruses. Host cellular tRNA3Lys is exclusively used by HIV-1 (Hu and Hughes, 2012; Hughes, 2015).

Reverse transcriptase (RT) starts at this binding site and copies RNA into a single strand of complementary DNA As the tRNA3Lys molecule anneals its 30 end to a complementary 50 end of the ( þ ) sense RNA, the RT polymerase active site binds to the viral RNA-tRNA complex at the PBS. Reverse transcription is activated by a signaling motif within the U5 region (non-coding region) of the HIV-1 genome, or primer activation signal. In doing this, RT navigates the 50 end, copying the ( þ ) RNA into a complementary strand of viral DNA, or cDNA and termed (  )strand strong-stop DNA or (  )ssDNA. To do this, viral RT harnesses host cell nucleotides and the process of reverse transcription of RNA is activated: DNA nucleotides are added onto the 30 end of the primer, synthesizing DNA complementary to the U5 and R region (a direct repeat found at both ends of the RNA molecule) of the viral RNA. At this point it only copies from the primer binding site back into the Long Terminal Repeat (LTR), so all that has been copied so far is the LTR plus a little extra (Hu and Hughes, 2012; Hughes, 2015) see “Relevant Websites section”. In vitro experiments indicate that the addition of the first few nucleotides is a slow process which speeds up after around 10 nucleotides are added. As the (  )ssDNA is released, it anneals to the 30 terminus of the vRNA, and primes further (  ) strand DNA synthesis and generates a full-length (  )strand DNA that is used as a template for ( þ )strand DNA synthesis.

RNase H degrades the section of the RNA which has been copied DNA synthesis initially creates an RNA-DNA duplex which is substrate for the RNAse H active site, resulting in simultaneous digestion of the copied ssRNA as polymerization progresses. RNAse H degradation removes the 50 end of the viral RNA thus exposing the newly formed (  )strand DNA. It is not known if the same RT molecule which synthesizes the viral DNA is responsible for digestion of the RNA template. This allows the tRNA/RT/ssDNA to dissociate from the HIV-1 RNA, and then reattach at the other end of the stretch of RNA, a process known as (  ) strand transfer. The ends of the ( þ ) sense viral RNA are direct repeats which allow transfer of the newly synthesized DNA to the 30 end of the RNA whereupon (  ) strand synthesis of viral single stranded DNA can continue. As DNA synthesis continues, so too does digestion of the viral RNA by the RNAse H enzymatic domain of RT. As the RNAse H reaches a region within the viral RNA which is purine rich, digestion is halted. As a result, this portion of ( þ ) sense viral RNA persists and acts as a primer to begin the synthesis of the ( þ ) sense viral DNA. Synthesis continues until RT encounters the tRNA3Lys primer which was initially incorporated and then digests this via RNAse H activity of RT. As the two ends of the DNA are complementary and easily stick together, the DNA can then circularize. RT completes the second strand of the DNA including the Long Terminal Repeat at each end. In the process of this the DNA loop breaks, leaving a doublestranded DNA fragment with a Long Terminal Repeat at each end thus leaving linear viral DNA (Hu and Hughes, 2012; Hughes, 2015).

Nucleoside/nucleotide Reverse-Transcriptase Inhibitors (NRTIs) NRTIs and Their Mechanism of Action NRTIs were the first active medicinal products used against HIV-1. The first to be licensed was 30 -azido-30 -deoxythymidine (AZT) in 1987, subsequently known as zidovudine (Cihlar and Ray, 2010). Since then, eight further pharmacologically useful NRTIs (abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir-DF*, tenofovir-AF**, zalcitabine) have been developed for the treatment of HIV-1 (De Clercq, 2009) (Table 1). NRTIs are pro-drugs that need to be phosphorylated intracellularly to become the active NRTI-triphosphate nucleoside analogs, a process catalyzed by cellular kinases (Anderson et al., 2004; Furman et al., 1986). They are structurally similar to the host nucleoside bases normally utilized by the RT active site (DNA polymerase) in the transcription of the ssRNA of the invading HIV-1 genome into dsDNA. The analogs bind competitively to proviral DNA, displacing the host nucleosides from DNA polymerase. However, the analogs lack the 30 -hydroxyl group: as a result, the native nucleosides (adenosine, thymidine, pyrimidine, guanosine) cannot bind to the dsDNA

Antiretroviral Therapy – Nucleoside/Nucleotide and Non-Nucleoside Reverse Transcriptase Inhibitors Table 2

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summary of NRTI backbone recommendations in national and international adult HIV-1 treatment guidelines Backbone

Alternative

BHIVA (British HIV Association, 2016)

TDF & FTC TDF & FTC

ABC & 3TC

WHO (World Health Organization, 2018)

TDF & 3TC TDF & FTC

AZT & 3TC

EACS (European AIDS Clinical Society, 2019)

ABC & 3TC TAF & FTC TDF & FTC TDF & 3TC 3TCa

with dolutegravir, if VL o 500,000 and CD4 4 200.

a

thus terminating the strand elongation, and thereby the process of reverse transcription. This is known as immediate chain termination (Sluis-Cremer et al., 2000). Tenofovir-DF (TDF) and tenofovir-AF (TAF) are nucleoside analogs that are phosphorylated at the 50 site, and are more commonly known as nucleotide analogs. This is an artificial distinction, as tenofovir acts pharmacologically by displacing host nucleosides, as with other NRTIs.

Clinical Uses of NRTIs NRTIs were the first class of antiretroviral drugs to be used in the treatment of HIV. Although first used alone as single agents (monotherapy) with modest effect, treatment failure as a result of emergent drug resistance occurred within 3–4 months of initiation (Larder et al., 1989). Further trials examined combinations of two NRTIs used concurrently, which showed improved outcomes within treatment-naïve patients with virological control sustained for longer periods and improved clinical outcomes. Trials examined two approaches: (1) The addition of a second NRTI to NRTI monotherapy where treatment failure had already occurred. (2) Head-to-head comparisons of NRTI monotherapy and dual therapy in treatment-naïve patients (Schooley et al., 1996; Hammer et al., 1996; Darbyshire and Aboulker, 1996). The improvement in outcomes with dual NRTI therapy was again short-lived, with resistance developing in the dual combinations. In 1996, triple-therapy, or highly active antiretroviral therapy (HAART), was introduced leading to great improvements in viral suppression, with higher barriers to resistance (Lange and Ananworanich, 2014). NRTIs are now primarily used in dual combinations as the “backbone” of antiretroviral therapy for the treatment of HIV-1, with the addition of a third agent, i.e., an NNRTI, a boosted protease inhibitor or an integrase inhibitor. National and international guidelines on the treatment of HIV-1 differ slightly in their preferred backbone and third agent (Table 2). However, dual NRTI is the preferred backbone in all British HIV Association (BHIVA) and World Health Organization (WHO) recommendations (British HIV Association, 2016; World Health Organization, 2018). The European AIDS Clinical Society (EACS) guidelines on the treatment of HIV-1 recommend dual NRTI as the backbone in all but one of their first line recommendations; EACS also recommends that a single NRTI (lamivudine) can be used with dolutegravir as first line treatment for HIV-1 (European AIDS Clinical Society, 2019). The most used NRTI combinations are available as fixed dose combinations, in single tablets. Tenofovir-DF and emtricitabine as Truvada, and abacavir and lamivudine as Kivexa or Epzicom. Both of these combinations are now available off patent in generic formulations. More recently, tenofovir alafenamide and emtricitabine have been combined into a fixed dose combination. NRTIs are also included in a number of other single table regimens, containing other classes of drugs.

NRTI Toxicities NRTIs are pro-drugs that are phosphorylated intracellularly into their active form. Some of the long-term toxicities with NRTIs are attributable to overactivation of this intracellular phosphorylation. NRTI-triphosphates may inhibit mitochondrial DNA polymerase g, leading to mitochondrial failure, and a pattern of toxicities resembling mitochondrial disease, especially in the older and now, less used drugs. These include lactic acidosis/hyperlactaemia and hepatic steatosis (didanosine, stavudine, zidovudine), pancreatitis (didanosine), peripheral neuropathy (stavudine, didanosine, zalcitabine), lipodystrophy (stavudine, all NRTIs) and myopathy (zidovudine) (Anderson et al., 2004; Lewis et al., 2001; Carr and Cooper, 2000). Zidovudine causes anemia and bone marrow suppression in about 5%–10% of patients. This is more common in patients with advanced HIV, and is also thought also to be as a result of mitochondrial toxicity (Carr and Cooper, 2000). Hypersensitivity reactions occur in approximately 5% of patients taking abacavir. These reactions usually happen within the first 6 weeks of commencing treatment, but can also develop after many months. Typically, hypersensitivity reactions affect the skin and

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cause fever and rash, but can affect multiple organs, including the gastrointestinal and respiratory systems. Severe reactions can sometimes develop, which can be fatal (Dean, 2012). People who have the HLA-B*5701 allele are more susceptible to abacavir hypersensitivity, and in practice all patients are tested for the allele before treatment is started. If the patient tests positive for the HLAB*5701 allele, they should not be started on abacavir. However, hypersensitivity reactions may still occur even in the absence of the HLA-B*5701 allele. If HLA-B*5701 testing is not available, abacavir could still be starting with caution and close monitoring (Dean, 2012; Mallal et al., 2008). TDF is usually tolerated well by patients, but can cause acute and chronic renal failure, interstitial nephritis, proximal tubular dysfunction, and rarely Fanconi’s syndrome. Stopping TDF due to renal complications is thought to account for fewer than 1% of discontinuations, but close monitoring of renal toxicity is suggested by most HIV treatment guidelines, especially in those most at risk of developing renal disease. These include older patients, including those with other risk factors for renal impairment, such as uncontrolled hypertension, diabetes mellitus, pre-existing low creatinine clearance and those taking nephrotoxic medications (World Health Organization, 2018). TDF has also been shown to cause a loss of bone mineral density (BMD) and an increased risk of fractures. Studies show 1%–3% greater loss in BMD with tenofovir-DF compared with TDF sparing regimens. The mechanism by which this occurs is not absolutely clear, but it may be linked to TDF’s effect on the proximal renal tubule. TDF may cause a sub-clinical tubulopathy and phosphate wasting, which may in turn drive reductions in BMD (Grant and Cotter, 2016). Tenofovir may also have a direct action on bone turnover, by acting directly on the osteoclasts and osteoblasts (Grigsby et al., 2010). Tenofovir alafenamide (TAF) is a newer formulation of tenofovir which was licensed in 2016. Plasma concentrations of tenofovir are significantly lower after oral administration of TAF, when compared to TDF. This means decreased exposure of the renal proximal tubule to the active drug, thereby decreasing the toxic effects on the kidney and bone. TAF is now recommended as a first-line treatment in BHIVA and EACS guidelines, and is an alternative to TDF for patients at risk of renal impairment patient at risk of low BMD. Some cohort studies have shown an increased risk of cardiovascular disease with use of abacavir, including events such as acute myocardial infarction within the first 12 months of use (but not stroke) (Sabin et al., 2008). This seems to be especially true in patients with pre-existing history of cardiac disease. However, this increased risk is not seen in all studies, and the link between abacavir and cardiovascular risk remains a topic of debate with concerns that such studies are confounded by selection bias of patients with a pre-existing increased cardiovascular risk (Llibre and Hill, 2016). See Table 3 for a summary of common NRTI toxicities.

Drug-Drug Interactions With NRTIs As NRTIs are not metabolized by the CYP450 enzyme system, there are fewer potentially significant drug-drug interactions compared with other anti-retroviral drug classes (Pau and George, 2014). TAF is a substrate of P-glycoprotein (p-gp), and therefore will interact with P-gp inducers such as rifampicin, resulting in a much lower concentration of TAF, potentially leading to treatment failure (British HIV Association, 2016).

Mechanisms of NRTI Resistance The mechanisms of viral resistance to NRTIs are complex. Mutations in the pol gene that codes for viral RT enzyme occurs under the selective pressure of NRTI drugs. One mechanism is known as discrimination, where the mutated RT loses its ability to bind to specific NRTIs, but keeps its affinity for the host nucleoside bases. This allows reverse transcription to continue without the NRTIs Table 3 Skin ABC Rash

Common NRTI toxicities, taken from European AIDS Clinical Society Guidelines, Version 10.0 GI

Liver

Nausea Diarrhea

AZT Nail pigmen-tation Nausea

Cardiac MSK

Renal

Metabolic/lipids Other

IHD

Systemic hypersensitivity (HLA B*5701 dependent)

Hepatic steatosis

Myopathy Rhabdo-myolysis

Hepatitis

↓ BMD Osteo-malacia ↑ fracture risk

Lipodystrophy Anemia Dyslipidemia Hyperlactaemia

3TC FTC TDF

TAF

↓ eGFR ↓ plasma lipids Fanconi syndrome ↑ weight

Note: European AIDS Clinical Society, 2019. EACS Guideline 10.0. [cited 2020 Jun 7]. Available from: http://www.eacsociety.org.

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causing chain termination. These are usually point mutations that confer resistance to specific NRTIs, e.g., M184V, K65R, L74V and Q151M (Marcelin, 2006). Some mutations enhance the ability of RT to remove NRTIs that have already bound to the 30 end of the expanding pro-viral DNA. This is known as primer unblocking, and is enhanced by specific mutations known as TAMs (thymidine analog mutations), examples of which are M41L, D67N, K70R, L210W, T215Y/F and K219Q/E. These mutations are selected by zidovudine and stavudine, but they can cause cross-resistance to most NRTIs. The extent of cross-resistance depends on the number of TAMs. For example, the combination of M41, L210W and T215Y causes high-level resistance to zidovudine and stavudine, and intermediate to high-level resistance to didanosine, abacavir and tenofovir (Marcelin, 2006).

New Drugs Islatravir is a novel agent, known as a nucleoside reverse transcriptase translocation inhibitor (NRTTI). It is an analog of deoxyadenosine and, like NRTIs, it is converted intracellularly to its active triphosphate form. Its main mode of action in though immediate chain termination. However, occasionally the proviral-DNA continues to be propagated despite incorporation of the active deoxyadenosine analog. In these cases, islatravir acts as a delayed chain terminator by causing structural changes in the proviral-DNA molecule which prevents nucleosides attaching further down the line. Islatravir has been shown to be active against HIV-1 and HIV-2, including virus with pre-existing NRTI-resistance. The active form of islatravir has a long intracellular half-life, and has the potential to be developed as a subdermal implant (Markowitz and Sarafianos, 2018).

Non-Nucleoside Reverse-Transcriptase Inhibitors (NNRTIs) NNRTIs and Their Mechanism of Action NNRTIs act by binding to a hydrophobic site that is remote from the active DNA polymerase site, known as the binding pocket, on the p66 subunit of the RT heterodimer. Binding causes a change in confirmation of the RT heterodimer and its active enzyme site, thereby inhibiting DNA polymerase activity and blocking the process of reverse transcription of viral ssRNA to pro-viral dSDNA (Weller and Williams, 2001). NNRTI drugs are structurally diverse (Fig. 1) and, unlike NRTIs, are not analogs of nucleosides. Among the pharmacologically useful NNRTIs are efavirenz, nevirapine, etravirine, rilpivirine, and more recently doravirine.

Clinical Uses of NNRTIs NNRTIs are used for the treatment of HIV-1 as part of combination antiretroviral therapy, and have demonstrated good efficacy in viral suppression. They are often used as the “third agent”, with the treatment “backbone” of two NRTI drug (see section above), as

Fig. 1 Chemical structures of NNRTIs (Scientific Figure on ResearchGate).

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an alternative to a boosted protease inhibitor or an integrase inhibitors. There is some variation in the recommendations for NNRTI use across international guidelines. The BHIVA guidelines for the treatment of HIV-1 suggest rilpivirine as a first line third agent, with efavirenz as an alternative (British HIV Association, 2016). The EACS guidelines suggest rilpivirine, or the newer NNRTI doravirine, as a suitable first line third agent with a dual NRTI backbone, with efavirenz as an alternative (European AIDS Clinical Society, 2019). The consolidated WHO guidelines suggest efavirenz as the first line NNRTI third agent, with nevirapine as an alternative (World Health Organization, 2018), and the American guidelines favor efavirenz and rilpivirine (Department of Health and Human Services, 2019). The HIV-2 virus is intrinsically resistant to all NNRTIs (Gilleece et al., 2010).

NNRTI Toxicities Older NNRTIs, and especially nevirapine, are associated with hepatotoxicity, including rarely, fatal drug induced liver injury. Although the risk with nevirapine hepatotoxicity is increased in female patients with CD4 cell counts 4 250 cells/mm3, and male patients with CD4 cell counts 4 400 cells/mm3, all patients require close monitoring of liver enzymes in the weeks after starting. Nevirapine and other NNRTIs can also cause hypersensitivity reactions, with rash commonly occurring within a few weeks of starting treatment. Hypersensitivity rash can happen in approximately 16% of patients starting nevirapine, but is also common in patients starting efavirenz and etravirine. If hypersensitivity and deranged liver enzymes occur concurrently after starting nevirapine, the medication should be stopped immediately. Central nervous system side effects and toxicities are common with efavirenz. These usually occur early on in treatment, in approximately 40% of patients, and can include sleep disturbance and vivid dreams, depression and suicidal ideation. Most of these side effects resolve with time, and are only severe enough to need discontinuation in about 3% of patients (Carr and Cooper, 2000). See Table 4 for a summary of common NNRTI toxicities. Doravirine is a newer NNRTI which has been shown to be as effective as a boosted protease inhibitor (darunavir/ritonavir) or efavirenz-based therapy, in combination with dual NRTI backbone. It also has fewer toxicities, including fewer central nervous system side effects when compared to efavirenz, and has fewer potential drug-drug interactions. It also appears to remain effective against HIV-1 with pre-existing NNRTI resistance mutations (Colombier and Molina, 2018).

Drug-Drug Interactions With NNRTIs All NNRTIs are metabolized by the CYP3A liver enzymes. Nevirapine and efavirenz are also metabolized by CYP2B6, and etravirine by CYP2C9 and CYP2C19 liver enzymes. Drugs that induced these enzymes can lead to reduced levels of NNRTI, and virological failure. Conversely, inhibition of these enzymes leads to increased levels of NNRTIs and potentially more side effects and toxicity. NNRTIs also act as inducers and/or inhibitors of CYP enzymes. For example, efavirenz is a mixed inducer and inhibitor of CYP enzymes, mainly inducing CYP3A and CYP2B6. The University of Liverpool provides a comprehensive and regularly updated resource on potential drug-drug interactions: See “Relevant Websites section”.

Mechanisms of NNRTI Resistance A disadvantage of NNRTI drugs is their low barrier to developing resistance, and the prevalence of NNRTI-resistant HIV-1 virus in ART-naïve patients (Snedecor et al., 2013). Mutations on the pol gene that codes for viral RT reduce the ability of NNRTIs to bind to the binding pocket on the RT p66 subunit, thereby reducing their activity. Just one mutation can mean complete resistance to a specific NNRTI. Single mutations can also cause resistance that extends across the more than one drug in the class. For example, a K103N mutation causes high-level resistance to both nevirapine and efavirenz (HIV Drug Resistance Database, 2020). For this reason, NNRTIs are not recommended second line, after failure of a first line NNRTI Table 4

Doravirine Efavirenz

Etravirine Nevirapine Rilpivirine

Common NNRTI toxicities, taken from European AIDS Clinical Society Guidelines, Version 10.0 Skin

Liver

Rash

Hepatitis

Rash Rash Hypersensitivity Rash

Renal

CNS

Metabolic

Depression Sleep disturbance Headache Suicidal ideation

Dislipidemia Gynecomastia

Hepatitis Hepatitis

↓ eGFR

Note: European AIDS Clinical Society, 2019. EACS Guideline 10.0. [cited 2020 Jun 7]. Available from: http://www.eacsociety.org.

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containing regimen (British HIV Association, 2016). However, the newer NNRTI doravirine maintains activity against HIV-1 with K103N and Y181C mutations (Feng et al., 2016).

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The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: Lessons for inhibitor design. Structure 3 (9), 915–926. [cited 2020 Oct 24]. Available from: https://pubmed.ncbi.nlm.nih.gov/8535785/. Sabin, C.A., Worm, S.W., Weber, R., et al., 2008. Use of nucleoside reverse transcriptase inhibitors and risk of myocardial infarction in HIV-infected patients enrolled in the D: a:d study: A multi-cohort collaboration. Lancet 371 (9622), 1417–1426. Schooley, R.T., Ramirez-Ronda, C., Lange, J.M.A., et al., 1996. Virologic and immunologic benefits of initial combination therapy with zidovudine and zalcitabine or didanosine compared with zidovudine monotherapy. The Journal of Infectious Diseases 173 (6), 1354–1366. https://academic.oup.com/jid/article-abstract/173/6/1354/1010046. Sluis-Cremer, N., Arion, D., Parniak, M.A., 2000. Molecular mechanisms of HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Cellular and Molecular Life Sciences 57 (10), 1408–1422. Snedecor, S.J., Khachatryan, A., Nedrow, K., et al., 2013. Prevalence of transmitted resistance to first-generation non-nucleoside reverse transcriptase inhibitors and its potential economic impact in HIV-infected patients. PLoS One 8 (8), e72784. Available at: https://dx.plos.org/10.1371/journal.pone.0072784. Weller, I.V.D., Williams, I.G., 2001. ABC of AIDS: Antiretroviral drugs. British Medical Journal. 1410–1412. [cited 2020 May 23]. Available from: www.bmjbooks.com. World Health Organisation, 2018. Consolidated Guidelines on HIV Prevention, Diagnosis, Treatment and Care for Key Populations. World Health Organisation. http://www.who. int/hiv/pub/guidelines/keypopulations-2016/en/.

Relevant Websites https://www.bhiva.org/guidelines Current Guidelines. Bhiva.

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http://www.eacsociety.org EACSociety. https://hivdb.stanford.edu/ HIV Drug Resistance Database. Stanford University. https://www.hiv-druginteractions.org/checker Interaction Checker. Liverpool HIV Interactions. http://www.mcld.co.uk/hiv/. The Molecules of HIV. https://www.who.int/health-topics/hiv-aids World Health Organization: HIV/AIDS.

Protease Inhibitors Vanesa Anton-Vazquez, King’s College Hospital, London, United Kingdom Frank A Post, King’s College Hospital NHS Foundation Trust, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary Bioavailability The proportion of a drug which enters the circulation when introduced into the body and so is able to have an active effect. Boosting Using an antiretroviral (ARV) drug or other drug to increase the effectiveness of another ARV drug. CD4 T lymphocyte A type of T lymphocyte that helps to coordinate the immune response by stimulating other immune cells, including B lymphocytes, CD8 T lymphocytes and macrophages. Cytochrome P450 A group of enzymes involved in the breakdown of drugs in the liver and play a key role in the metabolism of many drugs. Cytochrome P450 (CYP450) enzymes metabolize all protease inhibitors. Drug–drug Interaction A reaction between two or more drugs or between a drug and a food or supplement.

A drug interaction can decrease or increase the action of the drug. Protease A type of enzyme that breaks down proteins into smaller protein units. These smaller proteins combine with HIV’s genetic material to form a new HIV virus. Protease inhibitors (PIs) prevent HIV from replicating by blocking protease. Resistance When a virus, bacteria or fungus becomes non susceptible to a drug that was previously effective. Viral load Number of HIV RNA copies per milliliter of blood. Viral suppression When antiretroviral therapy (ART) reduces a person’s viral load (HIV RNA) to an undetectable level.

Protease Inhibitors Introduction The introduction of HIV protease inhibitors into clinical practice in 1995 changed the course of HIV infection, with an increase in survival in people living with HIV/AIDS (PLWHA) (Palella et al., 1998). Combination therapy consisting of two nucleoside analogs and a protease inhibitor reduced HIV viral load and increased CD4 cell counts rapidly for the very first time, stopping disease progression (Ho et al., 1995), and became a cornerstone of antiretroviral treatment. Ritonavir- or cobicistat-boosted protease inhibitors represent an important antiretroviral class due to their low potential to select resistance. However, they also introduced a variety of new challenges such as undesirable side effects, dosing and absorption difficulties and interaction with concomitant drugs. These characteristics should be fully considered and understood when using protease inhibitors.

Mechanism of Action Protease inhibitors (PIs) are one class of antiretroviral drug that target the viral enzyme, HIV-1 protease (Ali et al., 2010). Protease is an essential element for viral maturation in the HIV life cycle. PIs act by binding to the catalytic site of the HIV protease inhibiting cleavage of Pol polyprotein viral precursors into mature proteins that are essential for viral replication. As a result, noninfectious immature virions are released from the cell surface (Konvalinka et al., 2015). PIs are active against HIV-1 and HIV-2. To date, ten PIs have been approved by the FDA: ritonavir, followed by indinavir, nelfinavir, saquinavir, amprenavir, lopinavir/ritonavir, atazanavir, fosamprenavir, tipranavir, and darunavir (Table 1).

Pharmacodynamics and Pharmacokinetics in PIs Protease inhibitor concentrations in blood plasma must be high enough to inhibit HIV viral replication. The key is to maintain drug concentrations above the minimum effective concentration to ensure inhibition of viral replication and prevent emergence of resistance, and below the maximum safe concentration to avoid toxicity. PIs have poor oral bioavailability. This is mainly due to their susceptibility to oxidation by cytochrome P450 3A4 (CYP3A4) enzymes in the enterocytes and hepatocytes as well as the presence of P-glycoprotein (p-gp) efflux transporters that decreases gut absorption. In addition, there is substantial variability in the concentration of plasma PI levels due to inter-individual differences in CYP3A4 (Von Richter et al., 2004). To overcome the reduced bioavailability of PIs, co-administration of potent CYP3A4 and P-gp inhibitors, known as boosting, increases PI plasma exposure thereby allowing once or twice daily dosing, resulting in enhanced anti-viral efficacy and reduced

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Table 1

Classes of protease inhibitors (PIs)

HIV protease inhibitor

PI drug

FDA approval

Ritonavir Indinavir Nelfinavir Saquinavir Amprenavir Lopinavir/ritonavir Atazanavir Fosamprenavir Tipranavir Darunavir

RTV IDV NFV SQV APV LPV/r ATV FPV TPV DRV

1996 1996 1997 1997 1999 2000 2003 2003 2005 2006

toxicity (Larson et al., 2014). Ritonavir and cobicistat are the two main booster agents available in clinical practice (Boffito and Moyle, 2004). Ritonavir boosts intestinal absorption of the co-administered PI by inhibiting a number of CYP450 enzymes (2B6, 2C8, 2C9, 2D6 and 3A4) and the intestinal efflux transporter P-gp. In contrast, cobicistat inhibits CYP2B6 and 3A4 (with a lesser effect on 2C8 and 2D6 and no effect on 1A2, 2C9 and 2C19) and the efflux transporter P-gp (Nathan et al., 2013). As a boosting agent, ritonavir is dosed as 100 or 200 mg per day (well below the minimum effective concentration for inhibition of viral replication). Cobicistat is chemically similar to ritonavir, but has no antiviral activity. At a dose of 150 mg per day, it is used to boost atazanavir and darunavir and also the integrase inhibitor elvitegravir.

PIs in Combination With Other ART Classes PIs are typically administered in combination with one or two nucleoside reverse transcriptase inhibitors (NRTI). In ART naïve and ART experienced HIV patients, ritonavir-boosted darunavir or ritonavir-boosted atazanavir in combination with an NRTI backbone are the most widely used PIs. Boosted darunavir has been shown to be better tolerated with fewer side effects (Molina et al., 2008; Mills et al., 2009) and a lower incidence of treatment discontinuation when compared with boosted atazanavir (Lennox et al., 2014).

Protease Inhibitor-Based Regimens Triple therapy: Boosted PI þ 2 NRTI Darunavir/ritonavir (or cobicistat) OR Atazanavir/ritonavir (or cobicistat) Darunavir/ritonavir (or cobicistat) OR Atazanavir/ritonavir (or cobicistat)

Tenofovir (TDF or TAF)/emtricitabine

Abacavir/lamivudine

Dual therapy: boosted PI þ 1 NRTI or 1 INSTI

If ABC, TAF or TDF cannot be used, dual therapy could be considered with one NRTI (lamivudine) (Cahn et al., 2014) or, in those with CD4 cell counts >200 cells/mm3, one integrase-inhibitor (raltegravir) as an alternative to triple therapy for selected patients (Raffi et al., 2014). Darunavir/ritonavir Lopinavir/ritonavir

Raltegravir Lamivudine

Darunavir Darunavir is available in two single co-formulated tablets, darunavir-cobicistat and darunavir-cobicistat-emtricitabine-tenofovir alafenamide. Darunavir in combination with ritonavir or cobicistat, should not be used in patients with severe liver disease and should be avoided in individuals with sulfonamide allergy. In individuals with wild-type virus, the recommended dose is darunavir 800 mg/ritonavir 100 mg or cobicistat 150 mg once daily (Madruga et al., 2007; Clotet et al., 2007) The dose of darunavir 600 mg/ritonavir 100 mg twice daily is recommended if the following resistance mutations are present: V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, and L89V. Darunavir often

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retains activity against HIV virus with protease resistance-associated mutations (typically the result of prior exposure to unboosted PIs) and is the PI of choice for this group of individuals.

Atazanavir Atazanavir is available in 300 mg capsules and as co-formulated atazanavir 300 mg/cobicistat 150 mg. Atazanavir has better gastrointestinal tolerance than other PIs. It requires an acid pH for optimal drug absorption. Atazanavir has limited activity against HIV virus with protease resistance-associated mutations. Although more “lipid friendly” than lopinavir/ritonavir (Monforte et al., 2013; Squires et al., 2010), ATV/r and DRV/r have similar effects on lipids, glucose, insulin resistance (Ofotokun et al., 2015).

Lopinavir Lopinavir in fixed combination with ritonavir is available as tablets of 100 mg/25 mg and 200 mg/50 mg and as an oral solution (80/20 mg per mL) for pediatric use. The recommended dose of lopinavir in adults is 800 mg daily in combination with 200 mg of ritonavir, usually in two divided doses.

New PIs A new class of HIV protease inhibitor (GS-PI1) with high potency, high resistance barrier and long half-life has been developed to achieve metabolic stability without pharmacokinetic boosting. However, no new data have been presented since 2017 (Link et al., 2017).

Tolerability of PIs Common adverse events associated with protease inhibitors include hepatotoxicity and gastrointestinal side effects, dyslipidemia, lipodystrophy, insulin resistance and cardiovascular/renal toxicity.

Side effects Hepatotoxicity All the approved HIV protease inhibitors are metabolized by the liver and can inhibit the CYP 3A4 drug metabolizing enzymes. Among all PIs, ritonavir is the most potent CYP 3A4 inhibitor. Atazanavir may result in an increase in the indirect serum bilirubin concentration.

Gastrointestinal Protease inhibitors may cause diarrhea, nausea and vomiting. Lopinavir/ritonavir and fosamprenavir/ritonavir tend to show the highest rates of drug-related diarrhea, compared with atazanavir/ritonavir, darunavir/ritonavir, or saquinavir/ritonavir (Hill et al., 2009).

Dyslipidemia, lipodystrophy and insulin resistance The effect of HIV protease inhibitor on the lipid pathway is associated with the inhibition of SREBP-1, a transcription factor involved in the gene expression of adipocytes, leading to the deficiency of adiponectin, a protein hormone that plays a key role in the development of insulin resistance and lipodystrophy (Bastard et al., 2002; Rahmouni et al., 2008). In addition, PIs can inhibit glucose transporter-4, having a direct effect on glucose uptake and this leads to insulin resistance (Murata et al., 2000, 2002).

Cardiovascular PIs including lopinavir and darunavir (boosted with ritonavir) but not atazanavir have been associated with excess risk of cardiovascular disease (Ryom et al., 2018). ATV, possibly through its effects on bilirubin, has been associated with reduced progression of atherosclerosis (carotid artery intima media thickness) (Stein et al., 2015).

Kidney stones Atazanavir has been associated with the development of renal stones and interstitial nephritis (Hamada et al., 2012).

Drug–Drug Interactions Due to increased levels of other metabolized drugs when boosted-PIs are co administered, it is important to check and consider any potential drug interaction between the PI-booster and other concomitant medication (See “Relevant Websites section”). PIs can affect other drugs but also PIs can be affected by other drugs. Main drug interactions are shown in Tables 2 and 3.

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Table 2 Example of drugs whose plasma exposure are altered by boosted-PI coadministration Drug

Exposure of the non-ARV drug

Mechanism of drug-drug interaction

Warfarin Omeprazole Pravastatin Rosuvastatin Atorvastatin Simvastatin Amlodipine Diltiazem Verapamil Metoprolol Digoxin Citalopram Diazepam Mirtazapine Midazolam Paroxetine Sertraline Fluticasone Budesonide Salmeterol Sildenafil Etthinylestradiol

Variable Variable Increase

CYP2C19 inhibition/induction CYP2C19 inhibition/induction OATP1B1/B3 inhibition

Increase

CYP3A/5 inhibition

Increase

CYP-2D6 inhibition

Table 3

Variable Increase

CYP3A/5 inhibition

Decrease

CYP3A4 induction

Example of drugs whose co-administration alters plasma PI exposure

Drugs

Exposure of the PIs

Mechanism of drug-drug interaction

Clarithromycin Erythromycin Fluconazole Itraconazole Ketoconazole Fluoxetine Efavirenz Carbamazepine Phenytoin Phenobarbital Rifampin Rifabutin Efavirenz

Increase

Inhibition of CYP3A/5

Decrease

Induction of CYP3A/5

Resistance to PIs PIs have a high genetic barrier to resistance compared with other classes of ART such as (first-generation) integrase strand transfer inhibitors (INSTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), making them attractive agents for those with suboptimal adherence (Gardner et al., 2010). However, suboptimal plasma concentrations are highly undesirable as they can lead to drug resistance and subsequent virologic failure. In selected individuals, therapeutic drug monitoring may be a useful tool to assess adequate drug exposure (Calcagno et al., 2017).

Protease Inhibitors in Low Resource Settings Sub-Saharan Africa In sub-Saharan Africa, where 25.5 million people are living with HIV, access to HIV drugs and treatment monitoring is compromised by a variety of factors. The challenges to adequately respond to the HIV crisis in Africa and achieve the 90/90/90 strategy include financial constrain, distance to health centers, inadequate diagnostic capacity (e.g., CD4 count, viral load), recurrent stock

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out of ARVs and concurrent disease burdens. As a result, an increasing number of people in sub-Saharan Africa are at risk of treatment failure and drug resistance (Kanters et al., 2017; Boyd et al., 2015), and dolutegravir, a second generation INSTI with a high barrier to resistance, is increasingly replacing NNRTI in first-line therapy. Boosted protease-inhibitor remain important drugs in second-line regimens. Boosted PI therapy plus two NRTIs, administered without genotypic resistance testing or regular viral-load monitoring, had high rate of success, showing effective viral suppression (Paton et al., 2014). However, the use of protease inhibitor monotherapy has been shown to be inferior and should not be considered as an acceptable treatment alternative (Ciaffi et al., 2017; Paton et al., 2014). Therefore, the World Health Organization (WHO) recommends as second-line regimens a boosted protease inhibitor (atazanavir/ritonavir, lopinavir/ritonavir, or darunavir/ ritonavir) with 2 NRTIs such as zidovudine with lamivudine (3TC) (WHO, 2019; SECOND-LINE Study Group, 2013). Late presentation of HIV-positive adult patients into care is common in sub-Saharan Africa, with a high incidence of tuberculosis (TB). Several pharmacokinetic studies evaluating the effects of rifampicin on boosted PI exposure have reported high rates of liver toxicity (La Porte et al., 2004; Ribera et al., 2005; Haas et al., 2009). Hence, for HIV/TB patients requiring PI-containing ART, rifampicin should be replaced with rifabutin (administered at a dose of 150 mg/day) where available (WHO, 2010). Unfortunately, access to rifabutin in low- and middle-income countries, is very limited (Grinsztejn et al., 2014).

Protease Inhibitors in Clinical Practice Protease inhibitors remain widely used in clinical practice despite a large number of antiretrovirals available for the treatment of HIV infection. Most guidelines recommend the use of NNRTI and INSTI (with a NRTI backbone) as first line therapy. In special populations, including those with anticipated adherence issues, confirmed or suspected HIV resistance or intolerance to other classes of ART, boosted PI remain widely used. In clinical trials of first or second line therapy, virologic failure on boosted PI (when administered with at least one NRTI) is rarely associated with emergence of PI (or NRTI) resistance-associated mutations. Hence, boosted PI are particularly attractive for people with adherence issues and the preferred agents for those who have developed resistance to NNRTI and/or INSTI.

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Haas, D.W., Koletar, S.L., Laughlin, L., et al., 2009. Hepatotoxicity and gastrointestinal intolerance when healthy volunteers taking rifampin add twice-daily atazanavir and ritonavir. Journal of Acquired Immune Deficiency Syndromes 50 (3), 290–293. doi:10.1097/qai.0b013e318189a7df. Hill, A., Balkin, A., 2009. Risk factors for gastrointestinal adverse events in HIV treated and untreated patients. AIDS Reviews 11 (1), 30–38. Ho, D.D., Neumann, A.U., Perelson, A.S., et al., 1995. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373, 123–126. Kanters, S., Socias, M.E., Paton, N.I., et al., 2017. Comparative efficacy and safety of second-line antiretroviral therapy for treatment of HIV/AIDS: A systematic review and network meta-analysis. Lancet HIV 4, e433–e441. Konvalinka, J., Kräusslich, H.-G., Müller, B., 2015. Retroviral proteases and their roles in virion maturation. Virology 479–480, 403–417. La Porte, C.J.L., Colbers, E.P.H., Bertz, R., et al., 2004. Pharmacokinetics of adjusted-dose lopinavir-ritonavir combined with rifampin in healthy volunteers. Antimicrobial Agents and Chemotherapy 48 (5), 1553–1560. doi:10.1128/aac.48.5.1553–1560.2004. Larson, K.B., Wang, K., Delille, C., Otofokun, I., Acosta, E.P., 2014. Pharmacokinetic enhancers in HIV therapeutics. Clinical Pharmacokinetics 53, 865–872. Lennox, J.L., Landovitz, R.J., Ribaudo, H.J., et al., 2014. Efficacy and tolerability of 3 nonnucleoside reverse transcriptase inhibitor-sparing antiretroviral regimens for treatmentnaive volunteers infected with HIV-1: A randomized, controlled equivalence trial. Annals of Internal Medicine 161, 461. Link, J.O., Kato, D., Moore, M., et al., 2017. Novel HIV PI with high resistance barrier and potential for unboosted QD oral dosing. In: CROI. Seattle, WA: Gilead Sciences. Madruga, J.V., Berger, D., McMurchie, M., et al., 2007. Efficacy and safety of darunavir-ritonavir compared with that of lopinavir-ritonavir at 48 weeks in treatment-experienced, HIV-infected patients in TITAN: A randomised controlled phase III trial. Lancet 370 (9581), 49–58. Mills, A.M., Nelson, M., Jayaweera, D., et al., 2009. Once-daily darunavir/ritonavir vs. iopinavir/ritonavir in treatment-naive, HIV-1-infected patients: 96-week analysis. AIDS 23, 1679.

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Molina, J.M., Andrade-Villanueva, J., Echevarria, J., et al., 2008. Once-daily atazanavir/ritonavir versus twice-daily lopinavir/ritonavir, each in combination with tenofovir and emtricitabine, for management of antiretroviral-naive HIV-1-infected patients: 48 week efficacy and safety results of the CASTLE study. Lancet 372, 646. Monforte, A., Reiss, P., Ryom, L., et al., 2013. Atazanavir is not associated with an increased risk of cardio- or cerebrovascular disease events. AIDS 27, 407. Murata, H., Hruz, P.W., Mueckler, M., 2000. The mechanism of insulin resistance caused by HIV protease inhibitor therapy. Journal of Biological Chemistry 275 (27), 20251–20254. Murata, H., Hruz, P.W., Mueckler, M., 2002. Indinavir inhibits the glucose trans- porter isoform Glut4 at physiologic concentrations. AIDS 16 (6), 859–863. Nathan, B., Bayley, J., Waters, L., Post, F.A., 2013. Cobicistat: A novel pharmacoenhancer for co-formulation with HIV protease and integrase inhibitors. Infectious Diseases and Therapy 2 (2), 111–122. doi:10.1007/s40121-013-0013-7. Ofotokun, I., Na, L.H., Landovitz, R.J., et al., 2015. Comparison of the metabolic effects of ritonavir-boosted darunavir or atazanavir versus raltegravir, and the impact of ritonavir plasma exposure: ACTG 5257. Clinical Infectious Diseases 60 (12), 1842–1851. doi:10.1093/cid/civ193. Palella, F.J., Delaney, K.M., Moorman, A.C., et al., 1998. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. New England Journal of Medicine 338, 853–860. Paton, N.I., Kityo, C., Hoppe, A., et al., 2014. Assessment of second-line antiretroviral regimens for HIV therapy in Africa. New England Journal of Medicine 371 (3), 234–247. doi:10.1056/nejmoa1311274. Rahmouni, K., Sigmund, C.D., 2008. Id3, E47, and SREBP-1c: Fat factors con-trolling adiponectin expression. Circulation Research 103 (6), 565–567. Raffi, F., Babiker, A.G., Richert, L., et al., 2014. Ritonavir-boosted darunavir combined with raltegravir or tenofovir–emtricitabine in antiretroviral-naive adults infected with HIV1: 96 week results from the NEAT001/ANRS143 randomised non-inferiority trial. The Lancet 384 (9958), 1942–1951. doi:10.1016/s0140-6736(14)61170-3. Ribera, E., Azuaje, C., Lopez, R.M., et al., 2005. Once-daily regimen of saquinavir, ritonavir, didanosine, and lamivudine in HIV-infected patients with standard tuberculosis therapy (TBQD study). Journal of Acquired Immune Deficiency Syndromes 40 (3), 317–323. doi:10.1097/01.qai.0000182629.74336.4d. Ryom, L., Lundgren, J.D., El-Sadr, W., et al., 2018. Cardiovascular disease and use of contemporary protease inhibitors: The D:A:D international prospective multicohort study. The Lancet HIV 5 (6), e291–e300. doi:10.1016/s2352-3018(18)30043-2. SECOND-LINE Study Group, 2013. Ritonavir-boosted lopinavir plus nucleoside or nucleotide reverse transcriptase inhibitors versus ritonavir-boosted lopinavir plus raltegravir for treatment of HIV-1 infection in adults with virological failure of a standard first-line ART regimen (SECOND-LINE): A randomised, open-label, non-inferiority study. The Lancet 381 (9883), 2091–2099. doi:10.1016/s0140-6736(13)61164-2. Squires, K.E., Young, B., Dejesus, E., et al., 2010. Similar efficacy and tolerability of atazanavir compared with atazanavir/ritonavir, each with abacavir/lamivudine after initial suppression with abacavir/lamivudine plus ritonavir-boosted atazanavir in HIV-infected patients. AIDS 24, 2019. Stein, J.H., Ribaudo, H.J., Hodis, H.N., et al., 2015. A prospective, randomized clinical trial of antiretroviral therapies on carotid wall thickness. AIDS 29 (14), 1775–1783. doi:10.1097/qad.0000000000000762. Von Richter, O., Burk, O., Fromm, M.F., Eichelbaum, M., et al., 2004. Cytochrome P450 3a4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: A comparative analysis in paired tissue specimens. Clinical Pharmacology & Therapeutics 75, 172–183. WHO, 2010. Priority Research Questions for Tuberculosis/Human Immunodeficiency Virus (TB/HIV) in HIV-Prevalent and Resource-Limited Settings. Geneva: World Health Organization. World Health Organization, 2019. Consolidated Guidelines on the Use of Antiretroviral Drugs for Treating and Preventing HIV Infection. Geneva, Switzerland: WHO.

Further Reading Manosuthi, W., Wiboonchutikul, S., Sungkanuparph, S., 2016. Integrated therapy for HIV and tuberculosis. AIDS Research and Therapy 13, 22. Moyle, G.J., Back, D., 2001. Principles and practice of HIV-protease inhibitor pharma- coenhancement. HIV Medicine 2, 105–113. Renjifo, B., van Wyk, J., Salem, A.H., et al., 2015. Pharmacokinetic enhancement in HIV antiretroviral therapy: A comparison of ritonavir and cobicistat. Aids Reviews 17, 37–46. Tseng, A., Hughes, C.A., Wu, J., Seet, J., Phillips, E.J., 2017. Cobicistat versus ritonavir: Similar pharmacokinetic enhancers but some important differences. Annals of Pharmacotherapy 51, 1008–1022. WHO, 2014. Global Tuberculosis Report. World Health Organization.

Relevant Websites https://www.hiv-druginteractions.org/checker University of Liverpool.

HIV Integrase Inhibitors and Entry Inhibitors Daniel Bradshaw, Public Health England, London, United Kingdom Ranjababu Kulasegaram, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Glossary BCRP Breast cancer resistance protein. BIC/TAF/FTC Bictegravir/tenofovir alafenamide/ emtricitabine. CAB Cabotegravir. cART Combination antiretroviral therapy. CCR5 C-C chemokine receptor type 5. CD4 Cluster of differentiation 4. CXCR4 C-X-C chemokine receptor type 4. DTG Dolutegravir. E/c/TAF/F Elvitegravir/cobicistat/tenofovir alafenamide/ emtricitabine. E/c/TDF/F Elvitegravir/cobicistat/tenofovir disoproxil fumarate/emtricitabine. eGFR Estimated glomerular filtration rate. FDA Food and Drug Administration. FTC Emtricitabine. HIV-1 Human immunodeficiency virus type 1.

HIV-2 Human immunodeficiency virus type 2. HR1 First hepat repeat. INSTI Integrase strand transfer inhibitors. LEDGF Lens epithelium-derived growth factor. LTR Long terminal repeat. MATE 1 Multidrug and toxin extrusion transporter 1. NNRTI Non nucleoside reverse transcriptase inhibitor. NRTI Nucleos(t)ide reverse transcriptase inhibitor. OATP Organic anion transporting polypeptide. OBT Optimized background therapy. OCT2 Organic cation transporter 2. PEP Post-exposure prophylaxis. PI Protease inhibitor. PIC Pre-integration complex. PNGS Potential N-linked glycosylation sites. PrEP Pre-exposure prophylaxis. TDF Tenofovir disoproxil fumarate. UGT Uridine diphosphate glucuronosyltransferase.

Integrase Strand Transfer Inhibitors Integrase strand transfer inhibitors (INSTI) are one class of antiretrovirals effective against both HIV-1 and HIV-2. Integrase represents an attractive target for drug design as it has no human counterpart and INSTI are now considered preferred options in treatment guidelines owing to their virological efficacy, safety, tolerability and, in most cases, minimal potential for interactions with co-medications. The first in class INSTI, raltegravir, was approved by the United States Food and Drug Administration (FDA) in 2007, with three further agents, cobicistat-boosted elvitegravir, dolutegravir and bictegravir subsequently approved. A fifth drug, cabotegravir, is undergoing clinical trials. See the Table 1 for a summary of the key properties of approved INSTI and the investigational agent cabotegravir.

Virology HIV-1 integrase is a 32 kDa protein produced from the C-terminal portion of the Pol gene product and catalyses the integration of reverse transcribed viral cDNA into the host chromosome through a two step process, 30 processing and strand transfer. First, integrase self-associates into tetramers on the cDNA within the cytoplasm and mediates the hydrolysis of the final two nucleotides on long terminal repeats (LTR) of the 30 ends of both strands to generate reactive nucleophilic hydroxyl groups. Integrase remains bound to cDNA as a multimeric complex which bridges both ends of the cDNA within intracellular particles called pre-integration complexes (PIC). After nuclear translocation, integrase associates with lens epithelium-derived growth factor (LEDGF)/p75 and is directed to sites of open chromatin, where it mediates the nucleophilic attack of the viral 30 -hydroxyl cDNA across the major groove of the host DNA, resulting in insertion sites which are five base pairs apart on opposite chromosomal strands. The integration process is completed by DNA gap repair including a series of DNA polymerization, dinucleotide excision and ligation reactions. INSTI inhibit the second step catalysed by integrase, strand transfer, by competitive binding to the enzyme’s active site, resulting in displacement of the 30 end of the cDNA. In addition, INSTI chelate the divalent cation (Mg2 þ or Mn2 þ ), which is needed for integrase enzymatic activity. The genetic barrier to resistance of the first generation INSTI, raltegravir and elvitegravir, is relatively low, such that a single nucleotide substitution may cause a major reduction in susceptibility, as well as cross resistance. Most primary resistance mutations occur at the enzyme’s active site in response to drug pressure and are therefore associated with reduced viral replicative capacity (fitness). On continued drug exposure, primary resistance mutations are often followed by secondary (accessory) mutations, which either increase resistance or restore viral fitness, or both. The genetic barrier to resistance of the second generation agents, dolutegravir and bictegravir, is higher than first generation INSTI, probably due to structural differences. Second generation agents enter further into the pocket within integrase which is vacated by the displaced viral DNA base, making more

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intimate contact with viral DNA, and they may also have the ability to re-adjust their conformation in response to structural changes in the active site of raltegravir-resistant integrase. Multiple amino acid substitutions are therefore usually required before antiviral susceptibility is reduced and resistance mutations are rarely selected in individuals failing dolutegravir- or bictegravircontaining cART. In general, epidemiological studies have found negligible (o1%) levels of INSTI resistance in therapy-naïve individuals, and therefore routine pre-therapy integrase sequencing is not currently recommended.

First Generation INSTI Raltegravir Raltegravir is a highly potent antiretroviral compound, with a minimal inhibitor concentration in the nanomolar range. Following 10 days of monotherapy, plasma HIV-1 RNA levels reduce by a mean 2.0 log10 copies/ml. Dosing is with one 400 mg tablet twice daily, or two 600 mg tablets once daily. Raltegravir is generally well tolerated although the commonest reported side effects are headache, nausea and abdominal pain. Neuropsychiatric side effects have also been described, including insomnia and low mood. An uncommon but important toxicity is elevation in serum creatinine kinase levels, which may rarely be associated with rhabdomyolysis. Raltegravir has a low potential for interaction with co-medications. However, as metabolism is through glucuronidation by uridine diphosphate glucuronosyltransferase (UGT) 1A1, strong inducers of UGT1A1, such as rifampicin, reduce raltegravir exposure. Raltegravir should therefore be avoided with strong UGT1A1 inducers where possible, or administered at a dose of 800 mg twice daily where co-administration is unavoidable. The site which binds Mg2 þ or Mg2 þ can be chelated by orally ingested polyvalent cations, such as Al3 þ þ or Mg2 þ , which may reduce absorption. Raltegravir should therefore not be co-administered with aluminum or magnesium containing antacids.

Resistance Most resistance mutations involve amino acid substitutions in the vicinity of the integrase inhibitor binding pocket, either directly or indirectly altering the conformation of the active site. The signature mutations, observed both in vitro and in vivo, are at residues Q148 (to H, R or K), N155 (to H) and less frequently Y143 (to C, H or R). These mutations reduce enzymatic activity and therefore fitness and are likely to disappear on discontinuation of raltegravir. Accessory mutations, some of which are natural polymorphisms, are also mostly located close to the active site. The Y143 pathway is specific to RAL and relatively uncommon; residue 143 interacts directly with the oxadiazole ring of raltegravir, forming a stacking interaction which is abrogated when this position is mutated. By contrast, changes at residues 148 and 155 disturb the geometry of the integrase active site and thereby disrupt raltegravir binding. Q148 mutations impart a severe fitness cost and therefore are rapidly compensated for by secondary mutations including L74M plus E138A, E138K or G140S. The most common mutational pattern in this pathway is Q148H plus G140S, which also confers the greatest loss of drug susceptibility. N155 mutations tend to predominate early in the course of raltegravir failure, probably as changes at this position have less impact on fitness, but in ongoing drug exposure, are replaced by viruses with higher resistance, often bearing Q148H/R/K plus G140S mutations.

Clinical trials in cART naïve individuals In a randomised controlled phase 3 clinical trial conducted in therapy-naïve patients, the virological efficacy of raltegravir was noninferior to that of efavirenz, with fewer drug-related adverse events and smaller elevations in lipid levels (The STARTMRK study). Although originally licensed for twice daily dosing (400 mg BD), the ONCEMRK study demonstrated non-inferiority of once daily dosing of 1200 mg in achieving viral suppression. Raltegravir-containing nucleos(t)ide-sparing regimens in therapy-naïve patients may also be effective, such as the combination of twice daily raltegravir and once daily ritonavir-boosted darunavir in patients with a baseline CD4 cell count 4200 cells/uL and a HIV-1 RNA level o100,000 c/ml (NEAT001/ANRS-143 Study). Clinical trials in cART-experienced patients The phase 3 BENCHMRK studies demonstrated that the addition of raltegravir to optimized background therapy (OBT) in INSTI-naïve patients with multi-resistant HIV-1 infection improved virological suppression rates. However, in the SWITCHMRK studies, patients with suppressed plasma HIV-1 RNA switching from ritonavir-boosted lopinavir to raltegravir, both in combination with two nucleos(t) ide inhibitors, were more likely to develop virological failure. This was probably driven by archived NRTI resistance, compromising the NRTI backbone, in conjunction with the switch from a third agent with a high to a low genetic barrier to resistance. In the EARNEST study, patients with HIV-1 viraemia on an NNRTI-containing regimen were switched to ritonavir-boosted lopinavir, either as monotherapy, or with raltegravir, or with two to three NRTIs. Lower levels of viral RNA suppression were seen in those receiving a raltegravir- versus a NRTI-containing regimen, possibly reflecting the short half life of raltegravir and therefore greater susceptibility to development of resistance during episodes of non-adherence.

2012

2013

1st generation INSTI

Dolutegravir 2nd generation INSTI

Elvitegravir

Integrase strand transfer inhibitors Raltegravir 1st generation 2007 INSTI

Twice daily if INSTI resistance Single tablet regimens: DTG/ABC/3TC, DTG/3TC, DTG/RPV

Once daily or

Oral

Nausea, abdominal pain, headache Rarely raised CK, myopathy or neuropsychiatric effects

Toxicity

Mainly metabolised by Nausea, diarrhoea, headache UGT1A1 Neuropsychiatric effects Some metabolism through UGT1A3/A9, CYP3A4, Pgp, BCRP Inducers/inhibitors of these enzymes may ↓/↑DTG levels DTG inhibits OCT2 and MATE1 This may ↑ serum creatinine or metform exposure Caution with polyvalent cations

STARTMRK-1, -2

Examples of phase 3 studies in therapynaïve patients

Low genetic barrier

GS-US-236-0102

ONCEMRK Major RAM include Y143C/H/R, Q148H/R/ K, N155H NEAT001/ANRS-143

Low genetic barrier

Resistance

Viking-3

Dawning

Tango

Spring-2

Flamingo

Gemini-1, -2

Multiple mutations usually required to reduce susceptibility RAM include G148H/R plus G140S

SWORD-1, -2

Striving

Sailing

Single

GS-US-292-0109

EARNEST

SWITCHMRK

BENCHMRK

Examples of phase 3 studies in therapyexperienced patients

High genetic barrier

GS-US-292-0111

Major RAM include GS-US-236-0103 T66I, E92Q, Q148H/R/ K, N155H ↓tubular secretion of creatinine GS-US-292-0104 but normal GFR

Elvitegravir is metabolised by Nausea, diarrhoea, headache CYP450 3A4 Cobicistat inhibits CYP450 3A Rarely neuropsychiatric effects & 2D6

Cobicistat inhibits transporters e.g., P-gp, MATE1, BRCP, OATP1B1, OATP1B3 E/c/TDF/FTC or Avoid with potent CYP450 3A E/c/TAF/FTC inducers Caution with drugs metabolised by CYP450 3A Caution with polyvalent cations

Single tablet regimens:

Once daily

Oral

Avoid with strong UGT1A1 inducers if possible

Once or twice daily Caution with polyvalent cations

Metabolised by UGT1A1

Pharmacokinetics

Oral

Administration

Drug class

Drug name

Year of FDA approval

HIV integrase strand transfer inhibitors and entry inhibitors

Table 1

(Continued )

More insomnia & headache than other INSTI Further data required on association with (i) weight gain (ii) neural tube defects in babies born to women conceiving on DTG

Multiple drug-drug interactions

Taken with food

Longest period of safety data

No single tablet regimen

Other notes

HIV Integrase Inhibitors and Entry Inhibitors 147

Drug class

2nd generation INSTI

Drug name

Bictegravir

Pharmacokinetics

Metabolised by CYP3A and UGT1A1 Once daily Inducers/inhibitors of these enzymes may ↓/↑ BIC levels Metabolised by P-gp and Single tablet BCRP regimen BIC/ TAF/FTC Caution with inhibitors of these transporters

Oral

Administration

2003

2007

2018

2020

CCR5 receptor antagonist

CD4-directed postattachment inhibitor

Maraviroc

Ibalizumabuiyk

Fostemsavir HIV-1 gp120directed attachment inhibitor Twice daily

High genetic barrier

Resistance

GS-US-380-1489

Examples of phase 3 studies in therapynaïve patients

Minimal data

Likely to involve RAM in gp120

Minimal data

May involve RAM in the V5 loop of gp120 None

None

Minimal data available

No clinically significant interactions

Diarrhoea, dizziness, nausea and rash

X4 or X4/R5 dual or MERIT mixed tropism Mutations in the V3 loop of gp120

None

Minimal resistance data None available RAM include G140R, Q148R

RAM in amino acids Injection site reactions, 36-45 within HR1 of diarrhoea, nausea, peripheral gp41 neuropathy, pancreatitis

Injection site reactions (mostly mild)

Multiple mutations usually required to reduce susceptibility ↓ tubular secretion of creatinine RAM include G118R, but GFR normal G148H/R plus G140S, R263K

Rarely neuropsychiatric effects

Headache, nausea, diarrhoea

Toxicity

Metabolised by CYP450 3A4 Nausea, diarrhoea, fatigue, and 3A5 headache Less commonly postural Inducers/inhibitors of hypotension CYP450 may ↓/↑ MVC levels MVC dose adjusted according to presence of these agents

No clinically significant interactions

Oral prodrug of Metabolised by esterasetemsavir mediated hydrolysis, CYP450 3A4, P-gp, BCRP

Once per fortnight

Intravenous injection

Twice daily

Oral

Twice daily

Subcutaneous injection

Investigational Oral tablet, Metabolised by UGT1A1 once daily or Intramuscular injection 4- or 8-weekly

2018

Year of FDA approval

Entry inhibitors Enfuvirtide Fusion inhibitor (T20)

Cabotegravir 2nd generation INSTI

Continued

Table 1

BRIGHTE

TMB-301

MOTIVATE-1, -2

TORO-1, -2

ATLAS-2M

FLAIR

ATLAS

GS-US-380-1878

Examples of phase 3 studies in therapyexperienced patients

Binds extracellular domain of HIV-1 gp120

Monoclonal antibody (IgG isotype 4) against extracellular domain of CD4

Contraindicated in X4, dual or mixed (R5/X4) strains

Rarely used if therapy-naïve

Toxicity, subcutaneous route & twice daily dosing limit use

Ongoing phase 3 treatment trials with CAB/RPV Ongoing phase 3 PrEP trials (HPTN 084)

No long term safety data in real world cohorts

Other notes

148 HIV Integrase Inhibitors and Entry Inhibitors

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149

Elvitegravir Elvitegravir was be approved by the FDA in 2012 and is only available in the single tablet regimens of elvitegravir, cobicistat, emtricitabine and either tenofovir disoproxil fumarate (E/c/TDF/F) or tenofovir alafenamide (E/c/TAF/F). It is used in treatment naïve patients and as a switch option in those with a suppressed HIV-1 RNA without NRTI or INSTI resistance. Elvitegravir is generally well tolerated. The commonest side effects are nausea, diarrhea and headache, and other side effects include rash and abnormal dreams. The main factors limiting its more widespread use are a relatively low genetic barrier to resistance and multiple potential interactions with co-medications. Elvitegravir is coadministered with the boosting agent cobicistat, which has no antiretroviral activity but is a potent inhibitor of cytochrome P450 CYP3A4/5. As elvitegravir is metabolized through this pathway, inhibition of CYP3A4/5 boosts elvitegravir plasma exposure, ensuring therapeutic levels are achieved. Inducers of cytochrome P450, such as rifampicin, greatly reduce elvitegravir plasma concentrations and concomitant administration is contraindicated. Caution is also required with cytochrome P450 3A inhibitors such as itraconazole, which may increase exposure to elvitegravir. Cobicistat also has the potential for multiple interactions. Inhibition of CYP3A by cobicistat results in an increase in plasma levels of drugs metabolized through this pathway. For example, boosting of exogenous corticosteroids may produce iatrogenic Cushing’s disease or suppress the endogenous hypothalamic–pituitary–adrenal axis with consequent adrenal insufficiency. Cobicistat also inhibits certain transporters, such as P-glycoprotein, multidrug and toxin extrusion transporter 1 (MATE 1), breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP). As renal tubular secretion of creatinine is dependent on MATE 1, inhibition of this transporter by cobicistat results in increased serum creatinine and hence a reduced estimated glomerular filtration rate (eGFR), although without a true decline in GFR. Where cobicistat is prescribed with TDF, it is not recommended to be initiated in patients with a creatinine clearance of o70 ml/min, or if coformulated with TAF, o30 ml/min. Clinical trials Phase 3 trials demonstrated high rates of HIV-1 RNA suppression in therapy-naïve patients receiving either E/c/F/TAF or E/c/F/TDF, as well as non-inferiority of E/c/F/TDF by comparison with efavirenz or ritonavir-boosted atazanavir, both combined with TDF/ FTC (Studies 104, 111, 102, 103). High rates of HIV-1 RNA suppression were also observed in therapy-experienced patients with a fully suppressed plasma HIV-1 RNA and no history of resistance to any of the components of the fixed drug combination who switched to E/c/F/TAF (Study 109). Resistance Seven elvitegravir codon mutations have been described in INSTI treatment-naïve and -experienced patients in whom therapy is failing. Elvitegravir shares the Q148 and N155H resistance pathways with raltegravir, whilst the T66 and E92 pathways are predominantly selected by elvitegravir. Y97A is associated with only a 2-fold reduction in elvitegravir susceptibility and may be a polymorphism.

Second Generation INSTI Dolutegravir The second generation INSTI dolutegravir was approved in 2013 and is dosed as a 50 mg oral tablet once daily, or twice daily in individuals with confirmed or suspected INSTI resistance. Dolutegravir is also available as a component of the single tablet regimens of dolutegravir-abacavir-lamivudine, dolutegravir-lamivudine, or dolutegravir-rilpivirine. Its once daily dosing, virological efficacy, tolerability and high genetic barrier to resistance have ensured its incorporation as a preferred option in many treatment guidelines. Although well tolerated, the commonest side effects reported with dolutegravir are nausea, diarrhea and headache. Uncommon but notable associated toxicities include psychiatric disorders such as insomnia and anxiety; suicidal ideation has also rarely been reported. An association between dolutegravir use and weight gain has also recently been described although other factors, such as gender, racial background or co-administered antiretrovirals, may play a role. It is also unclear whether or not the association is pathological or represents a ‘return to health’ phenomenon. A slightly increased risk of neural tube defects in babies born to mothers who conceived whilst receiving dolutegravir has also been reported. In the largest cohort reporting outcomes for women receiving a dolutegravir containing regimen in Botswana, neural tube defects were identified in 0.3% of 1683 deliveries, compared to 0.1% of deliveries born to women receiving a nondolutegravir containing regimen Neural tube closure occurs in the first four weeks after conception and therefore initiation of dolutegravir is relatively contraindicated during conception and in the first six weeks of pregnancy, unless alternative agents are unavailable. However, the mechanism by which dolutegravir might interfere with pregnancy is not established. Dolutegravir is mainly eliminated through the UGT1A1 pathway but is also a substrate of UGT1A3, UGT1A9, CYP3A4, P-glycoprotein and BRCP. Therefore, co-administration of inducers of these enzymes reduces plasma exposure to dolutegravir and twice daily dolutegravir dosing may be required, or, in individuals with INSTI resistant virus, dolutegravir should be avoided. Dolutegravir also inhibits the renal organic cation transporter 2 (OCT 2) and MATE 1. As the secretory fraction of creatinine clearance is dependent on these transporters, a slight decrease in creatinine clearance is observed with dolutegravir. Plasma levels of drugs which are excreted through these transporters, such as metformin, may also increase.

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Clinical trials: Therapy-naïve with 2 NRTI Dolutegravir is highly efficacious in suppressing plasma HIV-1 RNA in randomized controlled trials in therapy-naïve individuals. Non-inferiority has been reported when comparing the performance of dolutegravir against that of raltegravir, and superiority when compared to efavirenz or ritonavir-boosted darunavir (the Spring-2, Single and Flamingo studies, respectively). Clinical trials: Treatment-experienced Trials reporting outcomes of individuals with suppressed HIV-1 RNA who either continued their current ART or switched to a dolutegravir-based regimen, such as the Striving study, also demonstrated non-inferiority in HIV-1 RNA responses. In addition, dolutegravir is highly effective in the treatment of patients failing ART. In the Sailing study, INSTI-naive individuals with unsuppressed plasma HIV-1 RNA on antiretroviral therapy were randomised to receiving OBT with either twice daily raltegravir or once daily dolutegravir for 48 weeks. Greater virological efficacy was reported in the dolutegravir group. The performance of dolutegravir has also been evaluated against a PI in patients with viraemia on first line treatment with NNRTI plus 2 NRTI. In the Dawning study, individuals with virological failure were randomised to receiving dolutegravir once daily or ritonavir-boosted lopinavir, both with two NRTIs, at least one of which was predicted to be fully active according to the resistance profile. At week 48, the proportion with a suppressed (o50 c/ml) HIV-1 RNA was greater in the dolutegravir versus the lopinavir group (84% versus 70%) respectively. However, it remains unclear whether a regimen such as dolutegravir-tenofovir-lamivudine will be effective in individuals with virologic failure who harbor multiple NRTI resistance mutations, such as K65R plus M184V. Efficacy of dolutegravir has also been demonstrated in the difficult to treat population of INSTI-experienced individuals with viraemia and INSTI-resistant virus. In a single arm study performed in this population (Viking-3), addition of twice daily dolutegravir to an optimized background regimen resulted in HIV-1 RNA suppression o50/cml in 69% individuals by week 24. Clinical trials: Dual therapy A novel approach involving therapy with dolutegravir within a NRTI-sparing or single NRTI containing regimen may be advantageous, given the toxicities associated with NRTIs such as TDF or abacavir. The SWORD-1 and -2 studies demonstrated that HIV-1 RNA suppression rates following a switch of triple ART to the single tablet regimen of dolutegravir-rilpivirine was non-inferior to continuing triple therapy in patients with a suppressed HIV-1 RNA and no history of failure or NNRTI/INSTI resistance. In the GEMINI-1 and -2 studies, use of dolutegravir plus lamivudine was shown to be non-inferior in therapy-naïve individuals with a HIV-1 RNA of o500,000 c/ml and no resistance, in comparison to dolutegravir-emtricitabine-TDF. No viral resistance was selected and fewer drug-related adverse events were observed in the dolutegravir-lamivudine group. Dolutegravir-lamivudine may also be indicated as a switch option in patients with suppressed HIV-1 RNA (the TANGO study). Resistance Individuals with virologic failure whilst receiving a first line, dolutegravir-containing regimen, which contains at least one other agent, rarely develop resistance mutations in either integrase or other viral genes. Similarly, INSTI-naïve individuals with a history of virological failure, who initiate dolutegravir containing cART and subsequently fail to achieve virological control, rarely (B1%) develop integrase resistance. By contrast, additional integrase resistance mutations may accumulate in individuals with INSTI resistant virus who switch from a failing first generation INSTI-containing regimen to dolutegravir. For example, in the Viking-3 study, although plasma HIV1 RNA suppressed following the addition of dolutegravir in the majority, 32/183 (17%) patients developed additional INSTI resistance mutations associated with virological failure at week 24, most often in those with a history of Q148 mutations. INSTI resistance also frequently develops in patients receiving dolutegravir monotherapy, a strategy which should therefore be avoided. Several integrase mutations are usually needed to confer high-level resistance to dolutegravir. The mutation G148H/R plus G140S, in combination with L74I/M, E92Q, T97A, E138A/K, G140A or N155H, are associated with 5–20 fold reduced dolutegravir susceptibility. The G118R pathway, usually involving at least one other mutation, is also associated with reduced susceptibility The R263K mutation alone reduces dolutegravir susceptibility by only 2-fold but resistance increases in the context of additional mutations. N155H alone usually yields o2 fold reduction in dolutegravir susceptibility.

Bictegravir Bictegravir is the most recent INSTI to be approved and is administered within a single tablet regimen with TAF and emtricitabine. It is a second generation INSTI and, like dolutegravir, has a high genetic barrier to resistance. Bictegravir is a substrate of CYP3A and UGT1A1 and therefore caution may be required with co-administration of inducers or inhibitors of these enzymes. Bictegravir inhibits OCT2 and MATE1 although it does not appear to increase metformin exposure significantly, in contrast to dolutegravir. Bictegravir also inhibits tubular secretion of creatinine leading to raised serum creatinine levels, although these are not considered to be clinically relevant. The drug is generally well tolerated. The most frequently reported adverse reactions of bictegravir are headache, nausea and diarrhea and less commonly the class-specific neuropsychiatric symptoms. Phase 3 randomized controlled trials In trials involving therapy-naïve HIV-1 infected adults who were randomised to receiving either BIC/TAF/FTC or a dolutegravircontaining regimen, non-inferiority was observed in rates of virological suppression. No treatment emergent resistance was seen in

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151

the bictegravir group. Bictegravir is also effective at maintaining viral suppression in patients switching from other therapies, including in those infected with virus harboring M184V/I mutations. Resistance Bictegravir, like dolutegravir, retains activity against most viral isolates from patients harboring multi-drug resistant virus who have failed an INSTI containing regimen. In vitro, the G118R and R263K mutations confer a 14-fold, and 6-fold decrease in susceptibility, respectively. The G140S þ Q148H combination reduces susceptibility to bictegravir by 5-fold, with further loss of susceptibility conferred by one or more additional mutations (L74M, T97A, S119P/T, E138A/K or Y143C/R/H and G163R). Further resistance data are likely to emerge with bictegravir’s increasing use.

Cabotegravir Cabotegravir is an investigational INSTI with structural similarities to dolutegravir. It may be administered as an oral tablet once daily or by intramuscular injection every four or eight weeks. The injectable route is enabled due to packaging of the drug within nanoparticles, resulting in a long half life (B40 days). In the phase 3 ATLAS and FLAIR studies, individuals with suppressed plasma HIV-1 RNA receiving oral antiretroviral therapy were either switched to injections of cabotegravir and rilpivirine every four weeks or maintained on oral therapy. A comparison of the two groups at 48 weeks demonstrated non-inferiority in HIV RNA suppression rates. Although 21% of participants found injections painful, most of these considered the pain acceptable and overall o1% discontinued injectables. A preference for injectable over oral treatment was reported by 98% of participants. In a subsequent study, ATLAS-2M, therapy-experienced individuals with suppressed HIV-1 RNA were randomised to receiving either 4-weekly or 8-weekly injectable cabotegravir-rilpivirine: after 48 weeks, there was no loss of virological control using the 8-week regimen. Overall, in the ATLAS and FLAIR studies, there were only six (0.5%) virologic failures with resistance in the group receiving injectable therapy, including two individuals infected with virus with pre-existing NNRTI resistance. However, at virological failure, all had NNRTI resistance and 4/6 patients developed INSTI resistance mutations. The development of Q148R or G140R was observed particularly in HIV-1 A1 subtype harboring the L74I integrase polymorphism. Injectable cabotegravir is also a candidate drug for pre-exposure prophylaxis against HIV but acute HIV infection must be carefully excluded before initiating cabotegravir owing to the risk of resistance. In macaques who had been recently infected with a chimeric simian/human immunodeficiency virus, subsequent cabotegravir injections led to selection of INSTI resistance in 3/6 animals, including the mutations G118R, E92G/Q or G140R.

Entry Inhibitors A growing number of antiretroviral agents target steps in virus entry into host cells. Currently approved drugs include the CCR5 coreceptor antagonist, maraviroc, a gp41-directed fusion inhibitor, enfuvirtide, a CD4-directed post-attachment inhibitor, ibalizumab, and an investigational drug, gp120-directed attachment inhibitor, fostemsavir. See the Table 1 for a summary of the key properties of these HIV entry inhibitors.

Maraviroc The first stage in the HIV-1 replication cycle is viral attachment to the cell membrane. The CD4 þ glycoprotein expressed on T lymphocytes and macrophages/monocytes is the major receptor for HIV-1 and binds to the viral envelope protein, gp120. A conformational change in gp120 follows, exposing the V3 loop which then binds a cellular co-receptor, either the C-C chemokine receptor type 5 (CCR5) or the C-X-C chemokine receptor type 4 (CXCR4). Further conformational changes within the gp41 subunit enable fusion of the viral envelope and cell membrane and the release of the viral particle into the cytoplasm. Around 80% of HIV-1 transmission involves CCR5-using viruses. Individuals who are homozygous for a gene encoding a nonfunctional CCR5 protein are therefore relatively resistant to HIV-1 infection. Over time, CXCR4-using viruses emerge in around 50% of untreated individuals, particularly in those with low CD4 þ T lymphocyte counts. X4 virus also frequently predominates in those failing antiretroviral regimens. In some patients, viral strains can use both receptors (dual tropism) or there may be a mixed infection of R5 and X4 viruses. Maraviroc binds to a hydrophobic pocket in the transmembrane helices of CCR5, leading to conformational change in the CCR5 extracellular loops. This prevents binding of CCR5 to the V3 loop of gp120 and thereby blocks virus entry. Maraviroc is ineffective against treating X4 strains and therefore the presence of X4, dual or mixed tropic strains must be excluded prior to its use by sequencing of the V3 loop in HIV-1 RNA. If the RNA level is below the cut off required for reliable amplification, genotypic testing may be performed on proviral HIV-1 DNA. Tropism testing is not routinely undertaken in newly-diagnosed patients, as maraviroc is not a preferred first line agent in therapy-naïve individuals. In addition, tropism may change with time, and therefore the finding of R5 virus at diagnosis may be misleading for future treatment decision-making.

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Maraviroc is approved for oral administration at a usual dose of 300 mg twice daily. However, it is metabolized by cytochrome P450CYP3A4 or 3A5 and therefore the dose is halved in the presence of co-administered inhibitors of these enzymes, such as ritonavir or cobicistat, or doubled in the presence of inducers, such as efavirenz. Although usually well tolerated, the commonest side effects reported in trials are nausea, diarrhea, fatigue and headache. An additional important but uncommon toxicity is postural hypotension. In the phase 3 MERIT trial, maraviroc was compared to efavirenz in therapy-naïve individuals, both co-administered with an NRTI backbone. The primary endpoint of non-inferiority in virological suppression rates was not met, with less viral suppression in the maraviroc arm. However, exclusion of 15% of patients who would have been ineligible by a more sensitive tropism screening assay resulted in similar response rates in the two arms. The benefit of including maraviroc, as an antiretroviral with a novel mechanism of action, in therapy-experienced patients was demonstrated in the MOTIVATE-1 and -2 trials. Here, individuals with R5 HIV-1 infection, who had been treated with and/or had resistance to three antiretroviral drug classes, were randomised to continuing OBT or OBT plus maraviroc. HIV-1 RNA was o50 copies/ml at week 48 in 47% of those in the group receiving twice daily maraviroc, compared to 16% in the placebo group. Thus maraviroc may be included within a salvage regimen in ART-experienced individuals with R5 tropic virus, although it should not be the only fully active agent. Antagonism of CCR5 may also have immunomodulatory effects, and trials are ongoing into the use of maraviroc for the treatment of co-morbidities with an inflammatory component, such as non-alcoholic fatty liver disease, in people with HIV infection. Resistance to maraviroc may develop through one of two mechanisms. X4 using virus or dual or mixed tropic strains may emerge, often representing outgrowth of a pre-existing minority population. Alternatively, mutations within the V3 loop may enable gp120 to bind to CCR5 in the presence of maraviroc, although there is no consensus on the specific signature mutations. Some CCR5 antagonist-resistant viruses selected in vitro have mutations in gp41 only, but the clinical significance of these remains unknown.

Enfuvirtide Enfuvirtide or T20 is the only approved drug in the class of antiretrovirals known as fusion inhibitors. Enfuvirtide binds to an extracellular region, the first heptad repeat (HR1), of the fusion peptide gp41, preventing its structural rearrangement and thereby blocking fusion of the viral envelope and cell membrane. Enfuvirtide is administered by subcutaneous injection, twice daily. The most frequently reported adverse reactions are injection site reactions, diarrhea and nausea. However, numerous other adverse reactions may occur including diabetes mellitus, pancreatitis and peripheral neuropathy. No clinically significant pharmacokinetic interactions have been noted with the CYP450 pathway. However, the subcutaneous route of administration, twice daily dosing requirement and poor tolerability limit the utility of enfuvirtide in most scenarios except as a component of a salvage regimen in multi-resistant HIV-1. The phase 3 TORO-1 and -2 studies demonstrated that the addition of enfuvirtide to optimized OBT resulted in a significantly greater reduction in HIV-1 RNA levels in individuals failing an antiretroviral regimen than in those receiving OBT plus placebo. Resistance may develop in the context of incomplete viral suppression and is predominantly associated with mutations in gp41 HR1. However, mutations in other regions of the envelope gene, as well as co-receptor usage and density, may also affect susceptibility to enfuvirtide.

Ibalizumab-Uiyk Ibalizumab was approved by the FDA in 2018 and is a first in class CD4-directed post-attachment HIV-1 inhibitor. It is a monoclonal antibody of IgG isotype 4 directed against domain 2 of the extracellular portion of the CD4 glycoprotein and is produced by recombinant DNA technology in murine myeloma non-secreting 0 cells. By non-competitive binding to CD4, ibalizumab interferes with post-attachment steps and thereby prevents fusion of the viral envelope and cell membranes. Ibalizumab, unlike maraviroc, is active against both R5 and X4 tropic HIV-1 strains and is indicated for the treatment of HIV-1 infected people with multi-drug resistant infection who are failing their current antiretroviral regimen. Ibalizumab is administered intravenously as a single loading dose followed by a fortnightly maintenance dose. The most common adverse reactions are diarrhea, dizziness, nausea and rash. Drug-drug interactions have not been observed. Of note, ibalizumab binding does not impact CD4 function, given that the target is a domain on CD4 distinct from the binding site of MHC Class II molecules. In the phase 3 TMB-301 study, heavily treatment-experienced HIV-1-infected participants with unsuppressed HIV RNA levels and resistance to at least one NRTI, NNRTI and PI, received ibalizumab with OBT. By week 25, 43% of individuals achieved an HIV-1 RNA level below o50 c/ml. Decreased susceptibility to ibalizumab has been observed in some individuals experiencing virological failure and may be associated with genotypic changes in the HIV-1 envelope gene which results in loss of potential N-linked glycosylation sites (PNGS) in the V5 loop of gp120.

Fostemsavir Fostemsavir is an oral prodrug of temsavir, a first in class attachment inhibitor which binds to the HIV-1 envelope protein gp120. It is an investigational agent, currently undergoing phase 3 clinical trials.

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Temsavir binds to gp120 close to the CD4 binding site and prevents initial attachment to the CD4 receptor. By targeting the first step of viral attachment, before co-receptor binding and fusion, temsavir is unaffected by co-receptor tropism. In one arm of a phase 3 trial in which fostemsavir was added to OBT in heavily treatment experienced patients failing their current cART regimen (the BRIGHTE study), 59% of individuals achieved HIV-1 RNA levels o40 c/ml by week 96. In phase 2 studies, several patients developed emergent substitutions in gp120 associated with reduced temsavir susceptibility although some of these individuals subsequently achieved viral resuppression. Further data on the resistance profile are therefore required.

Pregnancy Raltegravir is the preferred INSTI in pregnant women in view of the availability of long term safety data indicating no increased risk to the fetus. Elvitegravir/cobicistat should not be used owing to limited safety data and the risk of subtherapeutic elvitegravir plasma levels in the second and third trimesters. The slightly increased risk of neural tube defects in babies born to women who conceived on dolutegravir is outlined above. There are currently insufficient data concerning bictegravir for this to be recommended in pregnancy. There are also only limited data on the use of maraviroc and enfuvirtide in pregnancy and no adequate data concerning the use of ibalizumab in pregnant women.

Children and Adolescents An INSTI in conjunction with two NRTI is frequently the preferred regimen for HIV-1 infected children, with longer term safety and efficacy data in children available for raltegravir than other agents. Raltegravir may be administered as oral suspension, granules or chewable tablets and approved for use in infants and children weighing at least 2 kg under the FDA label. In children older than 6 years of age weighing at least 25 kg E/c/F/TAF may be used or, above 15 kg, a dolutegravir-containing regimen. Safety and efficacy of BIC/TAF/FTC in children under 18 years of age is not established. Regarding entry inhibitors, maraviroc is approved for use in children weighing at least 10 kg who are over the age of 2 years. Enfuvirtide may be used in children 6 years of age or older.

Post-Exposure and Pre-Exposure Prophylaxis (PEP and PrEP) INSTI are good candidates for use in PEP with a favorable safety and tolerability profile and the theoretical advantage of acting early in the virus replication cycle to prevent integration of viral DNA. The most frequently recommended regimen for PEP following a significant exposure to HIV-1 is a 28 day course of raltegravir plus tenofovir disoproxil fumarate and emtricitabine although alternative regimens may be prescribed. For example, DTG/TDF/FTC was used successfully in one open-label, single arm study. In cases of exposure to a resistant strain of HIV, the resistance profile may need to be reviewed before determining the optimal PEP regimen. Although the only approved regimen for PrEP requires FTC plus TDF or TAF, phase 3 studies are ongoing into the use of alternative agents including cabotegravir. The injectable formulation is an attractive candidate owing to its long plasma half life and trials are investigating the approach of administration every 8 weeks. However, concerns have been raised that INSTI resistance may develop, for example, following initiation of cabotegravir during unrecognized primary HIV infection or after exposure to HIV during the long pharmacokinetic tail in people discontinuing therapy.

HIV-2 Few trials evaluating the use of antiretroviral agents have been completed in people with HIV-2, including no randomised controlled trials. However, INSTI are active against HIV-2 integrase in vitro and single-arm, open label clinical trials involving raltegravir plus TDF/FTC or EVG/c/TDF/FTC have shown favorable treatment responses in ART naïve individuals with HIV-2 over 48 weeks. A randomized controlled trial comparing the use of raltegravir plus TDF/FTC to lopinavir/ritonavir plus TDF/FTC is currently underway and a single arm open-label trial evaluating the use of dolutegravir with two NRTI in ART-naïve individuals is also ongoing. Maraviroc appears to be active against some HIV-2 isolates; however, there are no FDA-approved assays that can determine HIV-2 co-receptor tropism, and HIV-2 is known to use other minor co-receptors in addition to CCR5 and CXCR4. HIV-2 is intrinsically resistant to enfuvirtide and there are as yet no data on the anti-HIV-2 activity of ibalizumab.

Further Reading Aboud, M., Kaplan, R., Lombaard, J., et al., 2019. Dolutegravir versus ritonavir-boosted lopinavir both with dual nucleoside reverse transcriptase inhibitor therapy in adults with HIV-1 infection in whom first-line therapy has failed (DAWNING): An open-label, non-inferiority, phase 3b trial. Lancet Infectious Diseases 19 (3), 253–264. Cahn, P., Madero, J.S., Arribas, J.R., et al., 2019. Dolutegravir plus lamivudine versus dolutegravir plus tenofovir disoproxil fumarate and emtricitabine in antiretroviral-naive adults with HIV-1 infection (GEMINI-1 and GEMINI-2): Week 48 results from two multicentre, double-blind, randomised, non-inferiority, phase 3 trials. Lancet 393 (10167), 143–155.

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Emu, B., Fessel, J., Schrader, S., et al., 2018. Phase 3 study of ibalizumab for multidrug-resistant HIV-1. The New England Journal of Medicine 379 (7), 645–654. Gallant, J., Lazzarin, A., Mills, A., et al., 2017. Bictegravir, emtricitabine, and tenofovir alafenamide versus dolutegravir, abacavir, and lamivudine for initial treatment of HIV-1 infection (GS-US-380–1489): A double-blind, multicentre, phase 3, randomised controlled non-inferiority trial. Lancet 390 (10107), 2063–2072. Gulick, R.M., Lalezari, J., Goodrich, J., et al., 2008. Maraviroc for previously treated patients with R5 HIV-1 infection. The New England Journal of Medicine 359 (14), 1429–1441. Kozal, M., Aberg, J., Pialoux, G., et al., 2020. Fostemsavir in adults with multidrug-resistant HIV-1 infection. The New England Journal of Medicine 382 (13), 1232–1243. Lennox, J.L., DeJesus, E., Lazzarin, A., et al., 2009. Safety and efficacy of raltegravir-based versus efavirenz-based combination therapy in treatment-naive patients with HIV-1 infection: A multicentre, double-blind randomised controlled trial. Lancet 374 (9692), 796–806. Matheron, S., Descamps, D., Gallien, S., et al., 2018. First-line raltegravir/emtricitabine/tenofovir combination in human immunodeficiency virus type 2 (HIV-2) infection: A phase 2, noncomparative trial (ANRS 159 HIV-2). Clinical Infectious Diseases 67 (8), 1161–1167. Radzio-Basu, J., Council, O., Cong, M.E., et al., 2019. Drug resistance emergence in macaques administered cabotegravir long-acting for pre-exposure prophylaxis during acute SHIV infection. Nature Communications 10 (1), 2005. Rhee, S.Y., Grant, P.M., Tzou, P.L., et al., 2019. A systematic review of the genetic mechanisms of dolutegravir resistance. Journal of Antimicrobial Chemotherapy 74 (11), 3135–3149. Sax, P.E., Erlandson, K.M., Lake, J.E., et al., 2019. Weight gain following initiation of antiretroviral therapy: Risk factors in randomized comparative clinical trials. Clinical Infectious Diseases. Swindells, S., Andrade-Villanueva, J.F., Richmond, G.J., et al., 2020. Long-acting cabotegravir and rilpivirine for maintenance of HIV-1 suppression. The New England Journal of Medicine 382 (12), 1112–1123. Zash, R., Holmes, L., Diseko, M., et al., 2019. Neural-tube defects and antiretroviral treatment regimens in Botswana. The New England Journal of Medicine 381 (9), 827–840.

Management of Respiratory Syncytial Virus Infections (Pneumoviridae) Rachael S Barr, Bristol Royal Hospital for Children, Bristol, United Kingdom Simon B Drysdale, St George’s University Hospitals NHS Foundation Trust, London, United Kingdom and St George’s, University of London, London, United Kingdom r 2021 Elsevier Ltd. All rights reserved.

Introduction and Epidemiology Respiratory syncytial virus (RSV) is a single-stranded RNA virus belonging to the pneumoviridae family (Genus: orthopneumovirus). It has two subtypes, RSV-A and RSV-B, both of which cause acute upper and lower respiratory tract infection. There is no clear association between RSV subtype and disease severity (Green Book, 2015). RSV is one of the leading causes of respiratory infection worldwide with almost all children having been infected by 2 years of age (Glezen et al., 1986). In children under 5 years of age RSV is responsible for 22% of all severe acute lower respiratory tract infections worldwide (Shi et al., 2017). In 2015 it was estimated that globally there were 33.1 million episodes of RSV associated acute lower respiratory tract infection in children under 5 years, resulting in 3.2 million hospital admissions and 59,600 deaths (Shi et al., 2017). RSV also affects adult populations with a systematic review of articles from across the United States showing RSV to be the causative organism in 12% of acute respiratory illnesses reviewed by a medical professional (Colosia et al., 2017). Among adults over the age of 65 years admitted to hospital with RSV, mortality is as high as 6%–8% (Colosia et al., 2017; Falsey et al., 2005) with a similar mortality rate of 8% found in hospitalized younger adults with chronic cardiac or respiratory disease (Falsey et al., 2005). In the United States alone it is estimated that RSV is responsible for 170,000 hospitalizations and 10–17,000 deaths annually in the adult population (Nam and Ison, 2019; Falsey and Walsh, 2000). RSV shows a seasonal pattern which in temperate climates such as the United Kingdom (UK) lies between October and March. The peak number of infections tends to occur within a six week period within this season each year (Green Book, 2015). In more tropical areas, RSV occurs year-round. In each season, one subtype (A or B) tends to predominate.

Pathogenesis and Immunity RSV is transmitted between hosts via droplets or fomites. It initially infects the apical surface of ciliated epithelial cells in both the upper and lower airways via the fusion (F) and attachment (G) proteins. Although there is almost ubiquitous infection by 2 years of age, infection with RSV offers incomplete immunity (Glezen et al., 1986). This results in episodes of repeated infection in both children and adults throughout life (Lambert et al., 2014; Openshaw et al., 2017; Gonzalez et al., 2000). RSV often results in a selflimiting illness, however, there are both environmental and host factors that put individuals at higher risk of more severe clinical manifestations. Host-related risk factors in children include younger age at the start of the RSV season with those under 3 months old at greatest risk, prematurity, low birth weight, bronchopulmonary dysplasia or other underlying respiratory disease, congenital heart disease and immunosuppression (Aujard and Fauroux, 2002; Rossi et al., 2007). Environmental risk factors for infants include having older siblings or living in a household with multiple other children and day care attendance (Rossi et al., 2007; Simoes, 2003; Bulkow et al., 2002). In older adults, underlying cardiopulmonary disease and poor functional status are associated with an increased risk of severe clinical disease (Walsh et al., 2004; Duncan et al., 2009). In the initial weeks of life infants have relative protection against RSV due to transplacental transfer and transfer via breastmilk of protective maternal antibodies. However, these antibodies fall below levels that are protective by approximately three to five months of age (Openshaw et al., 2017). In later childhood, antibody responses to recurrent infection are more brisk and effective than in infancy (Openshaw et al., 2017). Elderly adults are also at higher risk of severe disease due to a combination of physical changes to the lungs over time alongside waning innate and adaptive immunity. Those with low serum neutralizing antibody titers against RSV appear to be at greatest risk of severe disease (Openshaw et al., 2017).

Clinical Features RSV has a range of clinical manifestations, some of which are present exclusively in certain age groups. In all age groups RSV can present as a mild self-limiting upper respiratory tract infection. In children under the age of 1 year, RSV is the most common causative organism of bronchiolitis. Bronchiolitis is defined as inflammation of the small airways and presents with signs and symptoms including increased work of breathing, supplemental oxygen requirement, cough, fever and reduced feeding. In children of pre-school age RSV is a causative organism of viral-induced wheeze. In this illness there is hyper-reactivity of the airways resulting in bronchospasm causing polyphonic wheeze. There may also be increased work of breathing, cough, fever and supplemental oxygen requirements.

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In both children and adult populations RSV can also cause viral pneumonia/pneumonitis. The clinical features associated with this may include cough, increased work of breathing/shortness of breath, supplemental oxygen requirement and fever. Among the adult population underlying cardiovascular disease is a significant risk factor for hospitalization with RSV. RSV in this population may present as an exacerbation of underlying cardiac disease, with 5.4% of all admissions for congestive heart failure during the RSV season thought to be attributable to RSV infection (Nam and Ison, 2019). Studies have also demonstrated an association in older adults between presence of RSV serum IgG and an increased risk of acute myocardial infarction (Ivey et al., 2018; Chuaychoo et al., 2019; Guan et al., 2010).

Treatment Strategies Treatment of RSV is mainly supportive and varies slightly depending on the clinical syndrome. Many of the management strategies laid out below are not specific to RSV but to the clinical manifestations which may equally be caused by a number of other respiratory viruses. With regards to acute viral bronchiolitis in infants, the current NICE guideline states that treatment should be supportive with supplemental oxygen if saturations are persistently less than 92% and by giving fluids via naso/orogastric tube or intravenously if required (National Institute for Health and Care Excellence). Many other treatments have been trialed without success. NICE and the American Academy of Pediatrics (AAP) guidance currently recommend against the routine use of nebulized hypertonic saline, bronchodilators, antibiotics, corticosteroids (systemic or inhaled), nebulized adrenaline and montelukast for the management of acute bronchiolitis (National Institute for Health and Care Excellence; Ralston et al., 2014). Acute viral wheeze in pre-school age children may be caused by RSV and treatment remains mainly supportive. There is evidence to support the use of bronchodilators for symptomatic relief and their use is recommended in this age group by NICE guidance (NICE, 2017; Brand et al., 2008). There is limited evidence for the use of oral corticosteroids in preschool acute viral wheeze. The current consensus from the European respiratory task force is that a trial should only be considered in children with wheeze of such severity that they require admission to hospital, and even in this group, evidence to support benefit is not robust (NICE, 2017; Brand et al., 2008; Grigg, 2010; Brand et al., 2014). Other clinical syndromes including viral pneumonia, pneumonitis and upper respiratory tract infection in all age groups are also managed supportively with anti-pyretics, supplemental oxygen and feeding support as required. One antiviral with activity against RSV has been licensed and is occasionally used in specific clinical scenarios. Ribavirin is a nucleoside inhibitor and is used as an anti-viral medication for a variety of infections, including RSV. A Cochrane review of 12 trials looking at use of ribavirin in infants and children with lower respiratory tract infection found that it may be effective in reducing days of mechanical ventilation in children requiring intensive care, however, the evidence base lacks sufficient power to draw firm conclusions on its efficacy (Ventre and Randolph, 2007). Ribavirin also has significant toxicity associated with it including hemolytic anemia and teratogenicity and so should be prescribed with careful attention to the risks and benefits. Its use is currently not recommended by NICE or the AAP for infants with RSV bronchiolitis (National Institute for Health and Care Excellence; Ralston et al., 2014). Patients who have received haematopoietic stem cell transplant (both adults and children) are at particular risk of severe RSV infection and as such ribavirin has been used as a treatment option in these cohorts. Studies into the use of ribavirin in this cohort have mostly been small but are suggestive that it may be beneficial (Sparrelid et al., 1997; McColl et al., 1998; Boeckh et al., 2007). Further randomised controlled trials are required in this field to establish a true effect. The second treatment that has been investigated is intravenous immunoglobulin (IVIG). A Cochrane review looking at immunoglobulin in treatment of RSV in young children found that IVIG was not effective at reducing mortality or time spent in hospital (Sanders et al., 2019). As with ribavirin, there is minimal supporting evidence for the efficacy of IVIG in haematopoietic stem cell transplant recipients. In the 1990s IVIG was obtained from a pool of patients with high titers of RSV-neutralizing antibodies with the aim to create RSV specific IVIG. One product of this type known as RespiGam was licensed in 1996. This was shown to provide some passive immunity and reduce the rate of hospitalization with RSV by 41%–65% and length of stay in hospital by 53%–59% (Sandritter and Kraus, 1997). However, its manufacture was stopped in 2004 (Halsey et al., 1998; Turner et al., 2014). Small trials of patients (adults an children) undergoing haemopoetic stem cell transplant have also investigated combining ribavirin with IVIG. There seems to be a trend towards improved outcomes but more studies are needed. Some guidelines for this group of patients recommend ribavirin plus IVIG as treatment (Dignan et al., 2016). As discussed below, palivizumab (a monoclonal antibody against RSV) is an effective preventative agent for RSV in infants. It has also been trialed as a treatment in patients with severe LRTI or high-risk patients with URTI. However, a systematic review of palivizumab as treatment in both children and adults did not show any difference in clinical outcomes (Hu and Robinson, 2010). The review included one case report, four case series and two randomised controlled trials (RCTs). Both RCTs investigated pediatric patients only and neither were not adequately powered to assess clinical outcomes with palivizumab (Hu and Robinson, 2010). Palivizumab cannot currently be recommended as a treatment option for patients with RSV infection. There are also a number of novel anti-viral therapies in development. They can be broadly divided into four classes; immunoglobulins, nucleoside analogs, small interfering RNA’s and fusion inhibitors (Xing and Proesmans, 2019). None of these has progressed past a Phase 2 clinical study and thus a new licensed RSV antiviral is remains several years away.

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Prophylaxis A key strategy in the control of RSV infection lies in prophylaxis as currently there is no effective vaccine and treatment is mainly supportive in nature. The only licensed preventative treatment for RSV is palivizumab. Palivizumab is a humanized monoclonal antibody which provides passive immunity by targeting the RSV F protein and preventing fusion with the host cell. A randomised controlled trial of infants born at less than 35 weeks of gestation or infants with bronchopulmonary dysplasia showed that palivizumab reduced rates of hospitalization from RSV by 55% compared to placebo. Those who received palivizumab also had proportionally fewer total days in hospital, fewer days spent on supplemental oxygen and were less likely to be admitted to the intensive care unit as a result of RSV infection (The IMpact-RSV Study Group, 1998). Palivizumab has also been shown to have some benefits in otherwise healthy infants born at 33–35 weeks of gestation. In the MAKI trial, palivizumab prophylaxis resulted in a 67% relative reduction in RSV infections compared with placebo (Blanken et al., 2013). It also resulted in a relative reduction of 61% in number of days of wheezing in the first year of life suggesting an improvement in chronic respiratory morbidity associated with RSV infection in infancy (Blanken et al., 2013). Although it has been shown to be clinically effective, the use of palivizumab remains controversial due to its high cost. The broadly accepted willingness to pay threshold set by the National Institute for Health and Care Excellence (NICE) in the UK is d30,000 per quality adjusted life year (QALY). An initial analysis of available evidence showed that palivizumab was not cost effective in a UK setting if delivered unselectively to all infants with an incremental cost-effectiveness ratio (ICER) of around d60,000 per QALY (Wang et al., 2008). A second systematic review assessed cost effectiveness in specific subgroups of infants and found that palivizumab may be cost effective in infants with chronic lung disease and haemodynamically significant congenital heart disease at certain gestational and corrected gestational ages (Wang et al., 2011). The current guideline for use of palivizumab in the UK has been set out by Public Health England upon guidance from the Joint Committee on Vaccination and Immunization (JCVI) in the Green Book (Green Book, 2015). In the infants for whom it is recommended it is delivered as monthly intramuscular injections at a dose of 15 mg/kg over the RSV season (October to March in the UK). There are other monoclonal antibodies in development (PATH.org). MEDI8897 is a monoclonal antibody in development that has now entered phase 3 trials. Initial results suggest that it is safe to use in otherwise healthy preterm infants and it reduced medically attended RSV LRTI by 70.1% over the course of a year (Pamela Griffin et al., 2019). It may have benefits over palivizumab as it has a longer half-life of 83–94 days and so may be able to be administered as a single dose to cover a 6 month period (Domachowske et al., 2018).

RSV Vaccination Since the 1960s there have been multiple trials looking for an effective RSV vaccine, without any achieving licensure. Proposed populations in whom an RSV vaccine would be beneficial include newborn infants, preschool children, pregnant women and older adults. A cost effectiveness review of different vaccination strategies to protect infants found that the most cost-effective strategy would be a seasonal approach tailored to each specific country, protecting neonates born from just before the RSV season until a few months of age (Cromer et al., 2017). A vaccine could potentially be given to the mother prior to delivery or to a neonate in the first weeks of life (Cromer et al., 2017). A snapshot of the landscape of RSV vaccine development as of August 2019 shows there is one vaccine currently in Phase 3 trials (PATH.org). This is an RSV-F nanoparticle vaccine targeting the maternal population (PATH.org). This trial recently reported headline results suggesting the vaccine is safe and reduced medically significant RSV LRTI by 39.4% (97.5% CI, 1% to 64%) over a 90 day follow up period (Novavax, 2019). Full results are yet to be published at the time of writing. There are multiple vaccines currently undergoing Phase 2 trials which between them target all of the potential population groups mentioned above (PATH.org). The global impact of an effective vaccine for RSV cannot be underestimated. Until that time, RSV will continue to have a significant burden of mortality and morbidity across all age groups but particularly in young children, the elderly and the immunocompromised.

Conclusion RSV causes a significant global burden of disease in both pediatric and adult populations. It particularly affects infants, especially those born prematurely or with other health needs, and the elderly population. It can cause a wide range of clinical signs and symptoms and can result in a minor URTI through to respiratory failure and death. Although there are prophylactic and antiviral options, management is mainly supportive. A safe and effective vaccine is yet to be found, and until that time the ongoing search will remain a public health priority.

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Evaluation of the live attenuated cpts 248/404 RSV vaccine in combination with a subunit RSV vaccine (PFP-2) in healthy young and older adults. Vaccine 18, 1763–1772. Green Book, 2015. Chapter 27a – Respiratory syncytial virus. Grigg, J., 2010. Role of systemic steroids in acute preschool wheeze. Archives of Disease in Childhood 95.doi:10.1136/adc.2009.160994. Guan, X.R., Jiang, L.X., Ma, X.H., et al., 2010. Respiratory syncytial virus infection and risk of acute myocardial infarction. American Journal of the Medical Sciences 340, 356–359. Halsey, N.A., Abramson, J.S., Chesney, P.J., et al., 1998. Prevention of respiratory syncytial virus infections: Indications for the use of palivizumab and update on the use of RSV-IGIV. Pediatrics 102, 1211–1216. Hu, J., Robinson, J.L., 2010. Treatment of respiratory syncytial virus with palivizumab: A systematic review. World Journal of Pediatrics 6, 296–300. Ivey, K.S., Edwards, K.M., Talbot, H.K., 2018. Respiratory syncytial virus and associations with cardiovascular disease in adults. Journal of the American College of Cardiology 71, 1574–1583. Lambert, L., Sagfors, A.M., Openshaw, P.J.M., Culley, F.J., 2014. Immunity to RSV in early-life. Frontiers in Immunology 5.doi:10.3389/fimmu.2014.00466. McColl, M.D., Corser, R.B., Bremner, J., Chopra, R., 1998. Respiratory syncytial virus infection in adult BMT recipients: Effective therapy with short duration nebulised ribavirin. Bone Marrow Transplantation 21, 423–425. Nam, H.H., Ison, M.G., 2019. Respiratory syncytial virus infection in adults. BMJ 10 (366), l5021. National Institute for Health and Care Excellence. Bronchiolitis in children: Diagnosis and management. NICE Guideline [NG9]. NICE, 2017. Cough – Acute with chest signs in children. NICE CKS. Available at: https://cks.nice.org.uk/cough-acute-with-chest-signs-in-children#!scenario. (accessed 09.12.18). Novavax, 2019. New Data from Novavax Phase 3 Prepare™ Trial of ResVax™ Presented at 2019 IDSOG Annual Meeting. Novavax Inc. Available at: https://ir.novavax.com/ news-releases/news-release-details/new-data-novavax-phase-3-preparetm-trial-resvaxtm-presented-0. (accessed 17.02.20). Openshaw, P.J.M., Chiu, C., Culley, F.J., Johansson, C., 2017. Protective and harmful immunity to RSV infection. Annual Review of Immunology 35, 501–532. Pamela Griffin, M., Yuan, Y., Takas, T., et al., 2019. MEDI8897 prevents serious RSV disease in healthy preterm infants. Open Forum Infectious Diseases 6, S27. PATH.org. RSV Vaccine and mAB Snapshot. Available at: https://path.azureedge.net/media/documents/RSV-snapshot-2019_08_28_High_Resolution_PDF.pdf (accessed 16.01.20). Ralston, S.L., Lieberthal, A.S., Meissner, H.C., et al., 2014. Clinical practice guideline: The diagnosis, management, and prevention of bronchiolitis. Pediatrics 134, e1474–e1502. Rossi, G.A., Medici, M.C., Arcangeletti, M.C., et al., 2007. Risk factors for severe RSV-induced lower respiratory tract infection over four consecutive epidemics. European Journal of Pediatrics 166, 1267–1272. Sanders, S.L., Agwan, S., Hassan, M., van Driel, M.L., Del Mar, C.B., 2019. Immunoglobulin treatment for hospitalised infants and young children with respiratory syncytial virus infection. Cochrane Database of Systematic Reviews 8.doi:10.1002/14651858.cd009417.pub2. Sandritter, T.L., Kraus, D.M., 1997. Respiratory syncytial virus-immunoglobulin intravenous (RSV-IGIV) for respiratory syncytial viral infections: Part I. Journal of Pediatric Health Care 11, 284–291. 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, 946–958. Simoes, E.A.F., 2003. Environmental and demographic risk factors for respiratory syncytial virus lower respiratory tract disease. Journal of Pediatrics 143.doi:10.1067/ s0022–3476(03)00511–0. Sparrelid, E., Ljungman, P., Ekelöf-Andström, E., et al., 1997. Ribavirin therapy in bone marrow transplant recipients with viral respiratory tract infections. Bone Marrow Transplantation 19, 905–908. The IMpact-RSV Study Group, 1998. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics 102.doi:10.1542/peds.99.4.645. Turner, T.L., Kopp, B.T., Paul, G., et al., 2014. Respiratory syncytial virus: Current and emerging treatment options. ClinicoEconomics and Outcomes Research 6, 217–225. Ventre, K., Randolph, A., 2007. Ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children. In: Ventre, K. (Ed.), Cochrane Database of Systematic Reviews. Chichester, UK: John Wiley & Sons, Ltd. doi:10.1002/14651858.CD000181.pub3. Walsh, E.E., Peterson, D.R., Falsey, A.R., 2004. Risk factors for severe respiratory syncytial virus infection in elderly persons. Journal of Infectious Diseases 189, 233–238. Wang, D., Bayliss, S., Meads, C., 2011. Palivizumab for immunoprophylaxis of respiratory syncytial virus (RSV) bronchiolitis in high-risk infants and young children: A systematic review and additional economic modelling of subgroup analyses. Health Technology Assessment 15, iii. Wang, D., Cummins, C., Bayliss, S., Sandercock, J., Burls, A., 2008. Immunoprophylaxis against respiratory syncytial virus (RSV) with palivizumab in children: A systematic review and economic evaluation. Health Technology Assessment 12.doi:10.3310/hta12360.

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Xing, Y., Proesmans, M., 2019. New therapies for acute RSV infections: Where are we? European Journal of Pediatrics 178, 131–138.

Further Reading Drysdale, S.B., Kelly, D.F., 2019. How to use…respiratory viral studies. Archives of Disease in Childhood: Education and Practice Edition 104 (5), 274–278. Mazur, N.I., Higgins, D., Nunes, M.C., et al., 2018. The respiratory syncytial virus vaccine landscape: Lessons from the graveyard and promising candidates. Lancet Infectious Diseases 18 (10), e295–e311.

Management of Influenza Virus Infections (Orthomyxoviridae) Bruno Lina, HCL Department of Virology, National Reference Center for Respiratory Viruses, Institute of Infectious Agents, CroixRousse 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 r 2021 Elsevier Ltd. All rights reserved.

Nomenclature ECMO Extracorporeal membrane oxygenation HA Hemagglutinin M2 Matrix protein 2

NA Neuraminidase NAI Neuraminidase inhibitors Pol Polymerase

Introduction The burden of disease related to the annual influenza epidemic is significant in all regions of the world (Wright et al., 2007; Iuliano et al., 2018; Federici et al., 2018; Molinari et al., 2007). Recent estimates suggest that 5%–10% of the population in the western world experience clinical influenza every year, resulting in medical care expenses and burden that have a major impact on public hospitals. Influenza activity observed during pandemics may also lead to increased medical care in both community-based and hospital-based health structures. As a consequence, shortage of available hospital beds (especially in the emergency rooms) and hospitals become overwhelmed by severely ill individuals requiring intensive medical care because of influenza-related complications in both seasonal and pandemic influenza (Wright et al., 2007; Molinari et al., 2007). Although less documented, this impact is also massive in resource-poor and countries (De Francisco Shapovalova et al., 2015). To avoid these crisis situations, annual or pandemic vaccination is promoted. However, the relatively low vaccine effectiveness observed with seasonal influenza vaccines, especially for A(H3N2) viruses, as well as the anticipated long delays before the availability of a pandemic vaccine makes the case for better use of currently available antiviral drugs and further antiviral development (McLean et al., 2016). This need is also stressed in pandemic preparedness plans where the only response in the early stages of a pandemic will be the use of antivirals, especially for severe infections (Monto and Fukuda, 2020; Whitley and Monto, 2006).

Seasonal Influenza Annually, seasonal influenza accounts for about 650,000 deaths, together with millions of severe cases and hospital admissions (Wright et al., 2007). This influenza-related morbidity and mortality is mainly observed in patients with risk factors such as old age, medical comorbidities (pulmonary disease, cardiovascular disease, renal disease, liver disease, diabetes, neuromuscular disease, and cognitive dysfunction), immunocompromised status (stem cell transplant, solid organ transplant, chemotherapy, HIV infection), pregnancy, obesity, and genetic susceptibility (i.e., IFITM3 defects) (Rothberg and Haessler, 2010; Wedzhicha and Seemungal, 2007; Smeeth et al., 2004; Allen et al., 2017). In these patients, symptomatic treatments are often not sufficient to avoid complications and increased risk of morbidity and mortality.

Pandemic Influenza Influenza A viruses have been responsible for four pandemics in the last 100 years (Monto and Fukuda, 2020; Taubenberger et al., 2006; Spreeuwenberg et al., 2018). During these pandemics, additional risk-groups have emerged (i.e., young adults aged 20–40 years in 1918; children younger than two years of age in 1957). In addition, pregnancy has clearly been recognized with a high risk of morbidity, especially in the third trimester during the 1918, 1957, and 2009 pandemics.

Zoonotic Influenza Influenza A is a zoonotic virus that can occasionally be responsible for human infections (Peiris, 2009; Beigel et al., 2005; Koopmans et al., 2004; Peiris et al., 1999). These zoonotic viruses are acquired through exposure to infected livestock (swine, chicken, ducks, and turkeys). In the last 40 years, an increased number of zoonotic influenza cases have been reported, with an occasional very high mortality rate. As these infections can be a pandemic threat, the patients should be handled carefully, and their infection controlled rapidly to avoid the implementation of human to human transmission. The disease management of these cases is also a driving force to develop antivirals. To address all these situations, influenza antivirals have been developed since the 1960s (Wright et al., 2007; Palese and Shaw, 2007; Aoki, 1998; Colman, 1994). Despite showing some efficacy, these products had two major limitations (Wright et al., 2007): a marginal impact on the clinical disease duration in mild or non-severe cases, and (Iuliano et al., 2018) a rapid emergence of resistant viruses, especially in children (Palese and Shaw, 2007; Aoki, 1998; Colman, 1994; Muthuri et al., 2013). However, the clinical benefit in severe

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Sialic acid HA (trimer) NA (tetramer)

M2 ion channel vRNP CAP snatching of a mRNA

Polysome Nuclear import system. PA PB1 PB2 M1 NEP NS1

Fig. 1 Schematic representation of the Influenza replication in the cell and the different targets for antiviral action of molecules currently available or under development. The numbering is describing where the different classes of antiviral may interact with the virus replication cycle in the cell. 1 – inhibition of H þ - mediated decapsidation of vRNPs blocked by M2 ion channel blockers 2 – inhibition of virus release from the cell surface after budding by Neuraminidase inhibitors 3 – inhibition of virus replication and RNA synthesis by polymerase inhibitors 4 – inhibition of HA processing and maturation by blocking HA terminal glycosylation required for HA maturation 5 – inhibition of HA attachment to epithelial cells by removal of sialic acids by a bioavailable sialidase.

cases has been assessed, and antiviral usage has been promoted during the two last pandemics (1968 and 2009) (Aoki, 1998; Colman, 1994; Muthuri et al., 2013). In 1968–1969, Amantadine was positively evaluated and subsequently proposed for the disease management of severe cases, and in 2009, Oseltamivir was also proposed, for the management of both severe and clustered cases. These two antiviral drugs target two virus proteins, M2 (amantadine) and NA (oseltamivir), and aim for rapid virus clearance through impaired virus replication. However, influenza disease is not only the consequence of virus replication. The immune response that develops to combat the infection is responsible for many of the clinical symptoms that are still present after virus clearance (Wright et al., 2007; Palese and Shaw, 2007). This partly explains why the current antivirals, although “virologically effective”, have limitations in the reduction of disease duration in mild cases. This has led to the development of new concepts for influenza infection treatment, with molecules targeting the host response. In this article, we will go through the different classes of currently available antivirals and complete this list of recently developed products, some of which are alternative strategies involving host targeted molecules and immunotherapies.

Antiviral Drugs Available Adamantanes or M2 Blockers (Fig. 1) Adamantanes were the first class of antivirals developed and tested against Influenza during the 1960s (Palese and Shaw, 2007; Aoki, 1998). These molecules are active against influenza A viruses only. Their mode of action involves blocking the proton channel activity of the M2 complex, a function required for the acidification of the virus that allows viral uncoating and disruption of the macromolecular vRNP complex (Fig. 1) (Palese and Shaw, 2007). This disruption results in the release of the vRNP in the cytoplasm before entry into the nucleus of the infected cell. By direct interaction with the domains involved in the ion-channel activity of the M2 complex, adamantanes abort viral replication (Palese and Shaw, 2007; Aoki, 1998). Amantadine, the first molecule of this class, showed a sub-optimal safety profile (neurological disorders) and, as a consequence, a derivative (rimantadine) with an improved safety profile was developed (Wright et al., 2007; Aoki, 1998). These adamantanes were first evaluated and used during the 1968 pandemic with some success; clinical trials have confirmed a superiority of amantadine to placebo (Wright et al., 2007; Aoki, 1998) in disease management. However, they remained poorly used because of the frequent and sometimes severe side effects. In addition, it was observed that resistance could emerge rapidly, related to substitutions in the membrane-spanning region of the M2, especially at amino acids 26, 27, 30, 31, and 34 (Fig. 2)

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Fig. 2 substitutions associated with resistance to Adamantanes in the ion channel of the M2 tetramer of Influenza A viruses. The alpha helix of M2 proteins with S31 substitution has a curvature that allows the ion channel to be efficient despite binding the M2 blocker in its active site (A). As a consequence of this substitution, the size of the channel is increased (B) as depicted in the cartoon. The Adamatane is in red in the cartoon.

(Wright et al., 2007; Palese and Shaw, 2007; Hay et al., 1986; Weinstock and Zuccotti, 2006). This resistance induced no fitness cost and was sustained in the virus progeny. Hence, the resistant viruses were positively selected in a context of antiviral pressure and were readily transmitted to contacts (Hayden et al., 1991; Shiraishi et al., 2003; Schilling et al., 2004). These products remained in use in several countries until the end of the 20th century. However, because of the frequency of the side effects observed and the increasing percentage of influenza A viruses resistant to adamantanes (almost 100% by 2010), these molecules are not recommended anymore (Bright et al., 2005; Deyde et al., 2007; Barr et al., 2007).

Neuraminidase Inhibitors (Fig. 1) The second class of antivirals developed were the neuraminidase inhibitors (NAI). The neuraminidase (NA), a glycoprotein organized as homotetramers accounting for approximately 20% of the glycoproteins of the virus surface, has an enzymatic activity (sialidase) required to cleave the linkage between the terminal sialic acid and the adjacent D-galactose (or D-galactosamine) of the cellular glycoproteins (Palese and Shaw, 2007; Aoki, 1998). This cleavage allows the release of the virus progeny after budding and facilitates the transport of the virus to the epithelial cells through the respiratory mucus containing sialylated proteins (Palese and Shaw, 2007; Aoki, 1998). Despite subtle differences, the structure of the catalytic pockets of influenza A and B NAs are conserved for all viruses (Fig. 3). In addition, preliminary studies have shown that viruses with impaired NA function because of amino acid substitutions altering the sialidase activity (i.e., temperature-sensitive mutants) remained stacked at the surface of the cell (Palese and Shaw, 2007). As a consequence, these viruses cannot be released, and have a very low level of transmission. The identification of the key role of the NA in virus dissemination has led to the development of high-affinity sialic acid analogs with sialidase-inhibiting activity. The first available and tested NA inhibitor (NAI) was Zanamivir (Relanzas, Fig. 4). This molecule

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Fig. 3 Neuraminidase pocket site. The neuraminidase is a tetramer with 4 active sites highly conserved within all NAs. Substitutions associated with resistance are observed either in the catalytic site or in the framework structure. The latter is changing the shape of the catalytic pocket, resulting in a reduced binding of the neuraminidase inhibitors.

Fig. 4 Sialic acids and analogs acting as neuraminidase inhibitors. Chemical structures of the neuraminidase inhibitors (A) DANA, (B) zanamivir, (C) oseltamivir carboxylate, (D) peramivir, and (E) laninamivir. Structures show the differences relative to DANA – the C4–guanidinium group on zanamivir, peramivir, and laninamivir, the C4–amino group on oseltamivir, and the pentyl side chains on oseltamivir and peramivir. From McKimm-Breschkin, 2013. Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance. Influenza Other Respiratory Viruses (Suppl 1), 25–36.

has a very low oral bioavailability and has to be inhaled to be active (Wright et al., 2007). As a consequence, specific inhaling devices have been developed to allow the administration of therapeutic doses of the product. In the clinical studies carried out in patients presenting with mild influenza, Zanamivir demonstrated a clinical benefit with a 24 h reduction of symptom duration compared to placebo, if the drug was administered within 48 h after symptom onset (Gubareva et al., 2000). In addition, it was demonstrated that, as expected, virus shedding was significantly reduced. The complexity of the delivering process (inhalation) and the modest clinical benefit observed resulted in very limited use. A second NAI was introduced, Oseltamivir carboxylate (Tamiflus), a prodrug that could be administered orally (Fig. 4) (Wright et al., 2007). Virological and clinical studies showed similar effects to Zanamivir (similar reduction of virus shedding and similar reduction in clinical symptoms duration) (Gubareva et al., 2000). Later, two additional NAIs were developed, evaluated, and made available: Peramivir (Rapivabs, IV or IM administration, one single dose) and Laninamivir octanoate (Inavirs, inhaled, one single dose) (Fig. 4) (Mancuso et al., 2010; Ikematsu, 2011). The clinical benefits of all these NAI are very similar: A reduction of disease duration by one day. Interestingly, at the early stage of the development of these molecules and based on the preliminary studies conducted on temperature-sensitive mutants (Palese and Shaw, 2007), the risk of emergence of resistance was considered to be very low. In vitro and in vivo studies confirmed a reduction in virus fitness when NA substitutions associated with resistance occurred (Yen et al., 2005; Herlocher et al., 2002; Burnham et al., 2014; Ives et al., 2002). Meanwhile, rare sporadic cases of resistant type A or B influenza viruses with NA harboring substitutions associated with reduced susceptibility were reported (Ferraris et al., 2005; Monto et al., 2006). However, in 2007, a naturally-occurring resistant clone of seasonal influenza emerged with excellent fitness. This resistant clone completely displaced the susceptible parental strain, as a result of combined evolution in NA and HA (epistasis) (Wright et al., 2007; Hurt et al., 2010; Meijer et al., 2009; Hurt et al., 2009; Dharan et al., 2009). Subsequently, surveillance for resistant viruses was reinforced, a better definition of the level of resistance (or reduced susceptibility) was proposed, and phenotypic assays were progressively implemented (chemiluminescent and fluorescent

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assays) (Palese and Shaw, 2007; Ferraris et al., 2005; Nguyen et al., 2012; Okomo-Adhiambo et al., 2014). As a consequence, a normalization was proposed in terms of testing and reporting. Briefly, the NAI susceptibility testing works through the determination of the concentration of drug required to inhibit NA enzymatic activity by 50% (measurement of an IC50 value); the level of NA inhibition being interpreted as a surrogate for virus inhibition. To facilitate the interpretation of the results, IC50 value thresholds (or cut-off) were used to separate drug-resistant and drug-sensitive viruses. To facilitate the interpretation and reporting of NAI assay results, a WHO Expert Working Group introduced a very strict set of criteria to define the antiviral susceptibility of viruses based on the fold change of IC50 values, as compared to a reference IC50 (e.g., a median IC50 of viruses of the same type/subtype) (Table 1). Interpretation of NAI assay data is type-specific (OkomoAdhiambo et al., 2014). This normalized assay has been associated with molecular investigations of viruses showing RI and HRI phenotypes. The substitutions/deletions associated with these phenotypes showed two kinds of changes: within or near the NA catalytic site affecting the binding of one or more NAIs (catalytic substitutions), or responsible for a change in the global folding of the NA, with no changes in the catalytic pocket (structural substitutions) (Ferraris and Lina, 2008; McKimm-Breschkin, 2013). A comprehensive list of substitutions has been prepared, showing differences between the different NAs. Recent surveillance data showed that there is only a very limited number of resistant viruses reported worldwide, mainly in H1N1pdm09 viruses and in children (Lackenby et al., 2018). Individual cases are described, including in immunocompromised patients where chronic infections have been reported. There is no information about the sustained transmission of resistant viruses. A five-year-long study reporting on the resistance to NAI in treated patients showed that Oseltamivir resistance was only detected during antiviral treatment, with the highest incidence occurring among one to five-year-olds. Resistance delayed viral clearance but had no impact on symptom resolution (Lina et al., 2018). Post-licensure studies have been performed to consolidate the potential clinical benefit of the NAI usage in mild and severe influenza. Observational studies have reported the beneficial effects of NAIs, showing – in adult patients with confirmed influenza – a reduction in the likelihood of (Wright et al., 2007) requiring antibiotics because of lower respiratory tract infection (44%) and (Iuliano et al., 2018) being hospitalized (63%) (Katzen et al., 2019; Venkatesan et al., 2017). Similarly, studies conducted during the 2009 pandemic suggested that, when initiated within 48 h of symptom onset in hospitalized patients, NAIs significantly reduced mortality in adults by 62% – this remains significant even when the treatment was started after 48 h (25% overall reduction) (Muthuri et al., 2014). Post-licensure surveillance has also pointed out some very rare adverse effects of NAI, particularly of Oseltamivir. Most minor side effects associated with NAI consumption were gastrointestinal disorders such as nausea and vomiting, but not diarrhea (Doll et al., 2017). Both stop rapidly after treatment cessation. In Japan, some cases of neuropsychiatric adverse events (NPAE) have been reported to be linked with Oseltamivir or Lanimamivir use in adolescents (Nakano et al., 2013). An in-depth analysis carried out in 2008 compared the incidence of cases in Japan and the USA, showing a higher incidence of NPAE reporting rates per 1,000,000 prescriptions in children (aged o16 years) and adults (respectively, 99 and 28 events in Japan and 19 and 8 in the US). This study concluded that there was no link between NPAEs and Oseltamivir usage; the NPAE incidence being equivalent in influenza treated and non-treated cases (Toovey et al., 2008). The mechanism for the development of NPAE remains to be elucidated. Inhaled Zanamivir is contraindicated if there is a risk of bronchospasm, even if remains difficult to quantify (Heneghan et al., 2014). In severe cases as well as in immunocompromised patients, different regimens of NAIs have been tested to increase their beneficial effect. Double or triple doses have been evaluated, showing no additional clinical benefit compared to the standard dose (Marty et al., 2017). These regimens induced more severe and more frequent side effects, including gastrointestinal problems. Secondly, combinations of NAIs have also been tested (Oseltamivir plus Zanamivir). These studies did not report any clinical benefit as compared to Oseltamivir alone, or Zanamivir alone. They even reported a possible antagonistic effect with an overall reduced effectiveness (Escuret et al., 2012; Duval et al., 2010). Post-exposure prophylaxis (PEP) has also been proposed for NAIs (Welliver et al., 2001). Oseltamivir has been approved for this indication, with a seven-day course with a half-dose regimen. A meta-analysis calculated that Oseltamivir PEP had an efficacy of 58.5% (15.6% to 79.6) for households and 68% (34.9%–84.2%) to 89% in contacts of index cases; Zanamivir had a similar performance (Oh et al., 2014). This protection has also been demonstrated in nursing homes (Welliver et al., 2001). However, this half-dose regimen may trigger the emergence of a resistant virus (Wright et al., 2007). Actually, a population dynamical model of pandemic influenza demonstrated that limited post-exposure prophylaxis can provide an optimal strategy in reducing the final size of the pandemic while minimizing the total number of deaths. However, post-exposure prophylaxis of close contacts in the presence of transmissible drug resistance can promote the spread of resistance, especially when combined with aggressive treatment. PEP should be used in the short term, mostly to control epidemics in closed settings (i.e., nursing homes). Lanimamivir has also been favorably evaluated as PEP in nursing homes (Kashiwagi et al., 2016).

Polymerase Inhibitors (Fig. 1) The third class of antivirals is the polymerase (POL) inhibitors. The development of these molecules has been facilitated by the determination of the crystal structure of the Pol complex and subunits ((Pflug et al., 2014; Pflug et al., 2018) Fig. 1). Different modes of action are used by the different POL inhibiting drugs (Fig. 5) (Mifsud et al., 2019).

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Table 1 Summary of the substitution association to reduced or highly reduced inhibition of Neuraminidase activity for seasonal human viruses type A (Table 1A) and type B (Table 1B). These tables are providing the a priori interpretation of the susceptibility to NAI when a specific substitution is observed in the neuraminidase of the tested virus. Ni ¼ normal inhibition; RI ¼ reduced inhibition; HRI ¼ highly reduced inhibition. (Ref: http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/avwg2014_nai_substitution_table.pdf) A Subtype

Amino acid substitution N2 numbering Susceptibility assessed by NA inhibition assays (IC50 fold Change vs Wild type [NAI susceptible virus]) Oseltamivir

Zanamivir

Peramivir

Laninamivir

NI(3) RI (60) RI (17) RI (12–39) RI (28–45) NI (6) NI (4–8) HRI (221–1637) HRI (208) HRI (318–744) HRI (> 10,822) HRI (> 9,100) HRI (1733) HRI (5880) RI (10–43)

HRI (832) HRI (571) NI (6) NI (5–6) RI (10–12) NI (2) NI (2–5) NI (2–6) NI (3) NI (2–3) RI (11) RI (22–27) NI (2) NI (5) NI/RI (3–20)

RI (51) RI (25) NI(2) NI(1–4) ? NI (2) NI (1) RI/HRI (50–751) RI (12) HRI (108) HRI (5,343) HRI (>7500) HRI (1331) HRI (334) NI (4)

? ? ? ? ? ? ? NI (1–2) ? ? RI (11) RI (17) ? ? ?

NI (2) HRI (208) RI/HRI (18–2057) NI (1–7) RI (11) NI (8) NI (9) HRI (> 4,000) HRI (157–175) RI (15) HRI (> 10,000) HRI (300–1879) RI (45) HRI (>6,000) HRI (1,571) HRI (293–2,286) NI(4–8) RI/HRI (3,171) HRI (>300) RI (31) HRI (300) NI (1) RI (10) RI (9) HRI (100–252) RI (12–26) NI/RI (4–10) RI/HRI (15–57) RI (5–8) NI/RI (5–8) NI/RI (5–8) NI (3–6) RI (24) NI/RI (2–12) HRI (> 300)

RI (32) RI (17) NI (1–7) RI/HRI (30–132) NI (2) HRI (164) NI (2) RI (>50) NI (3) HRI (160) NI/RI/HRI (3–134) NI (8) RI (15) HRI (>800) RI (5) NI (2) RI (15–42) RI/HRI (12,538) HRI (>560) RI/HRI (33–560) NI (2) RI (15) RI (25) RI (30) NI/RI/HRI (5–1,000) NI (3–7) NI/RI (3–17) RI (14) RI (5–7) NI (2–4) NI (5) NI (1) RI (39) NI (1) RI (29)

NI (2) NI (3) NI (1–3) ? ? ? ? ? NI (1) ? RI/HRI (14–719) NI (1) ? HRI (>110) ? NI (7) HRI (213–436) HRI (13,700) HRI (>1,598) HRI (>1,598) HRI (531) RI (41) HRI (322) HRI (1321) HRI (214–400) RI (16–18) NI (5) HRI (168) ? ? RI (15–43) NI (4–8) RI (5) RI/HRI (15–110) HRI (502)

? ? NI (3–4) ? ? ? ? ? ? ? ? ? ? HRI (>205) ? ? NI/RI (3–12) ? ? ? ? RI (7) RI (6) RI (9) ? ? ? ? ? ? NI (o3) ? NI (3) ? ? (Continued )

A(H1N1)pdm09 E119G E119V D199G I223K I223R I223V S247N H275Y N295S D199N þ H275Y I223K þ H275Y I223R þ H275Y I223V þ H275Y S247N þ H275Y Q313R þ I427T

119 119 199 222 222 222 246 274 294 198 222 222 222 246 313

E119D E119I E119V Q136K D151E D151V/D I222L R224K Del 245–248 E276D R292K N294S R371K E119V þ T148I E119V þ I222L E119V þ I222V E105K E117A E117A or D E117G E117V Q138R P139S G140R R150K D197E D197N D197Y A200A/T I221L I221T I221V/L A245T H273Y R292K

119 119 119 136 151 151 222 224 245–248 276 292 294 371 119 þ 148 119 þ 222 119 þ 222 110 119 119 119 119 140 141 142 152 198 198 198 201 222 222 222 246 274 292

þ þ þ þ þ þ

274 274 274 275 274 427

A(H3N2)

166 Table 1 A Subtype

Management of Influenza Virus Infections (Orthomyxoviridae) Continued Amino acid substitution N2 numbering Susceptibility assessed by NA inhibition assays (IC50 fold Change vs Wild type [NAI susceptible virus])

N294S K360E R374K A395E G407S D432G G140R þ N144K Y142H þ G145R

294 258–359 371 390 402 429 142 þ 146 144 þ 147

Oseltamivir

Zanamivir

Peramivir

Laninamivir

RI (17–61) NI (2) HRI (101–407) RI (5) NI (4) NI (1) NI (6) RI (5)

NI (1–4) NI (2) RI/HRI (29–145) NI (1) NI (7) NI (1) RI (10) NI (4)

RI (31) HRI (165) HRI (352) RI (5) ? RI (41) HRI (257) HRI (487)

? NI ? NI ? NI ? NI

(o3) (o3) (o3) (o3)

Fig. 5 Structure and mode of action of the different Polymerase inhibitors A describe the normal cap snatching and RNAZ synthesis by the influenza polymerase complex (PB1, PB2, and PA). The three polymerase inhibitors available have different modes of action, as described (Baloxavir inhibits cap snatching, Pimodivir inhibits cap binding, and favipiravir inhibits RNA elongation).

Ribavirine (Ribavirin s ) Historically, this molecule showed a rather large spectrum of activity against RNA viruses, including respiratory syncytial virus (RSV) and hepatitis C. The influenza antiviral effect could be measured in vitro and in pre-clinical studies, but Ribavirine always failed to show clinical efficacy against influenza, in human challenge studies, and clinical studies (Wright et al., 2007; Schofield et al., 1975). The more recently described POL-inhibiting compounds were prepared by drug design. Solving the structure of the three components of the influenza polymerase (PA, PB1, and PB2) allowed the identification of potential targets and the design of inhibiting molecules (Pflug et al., 2014; Pflug et al., 2018). The POL inhibitors interfere with different components of the polymerase complex.

Baloxavir Marboxil (Xofluza s ) Baloxavir Marboxil was developed after solving the structure of the Cap-snatching pocket of the PA (Pflug et al., 2014, 2018). Rapidly, it appeared that a drug that would incorporate this pocket would prevent the mandatory cap-snatching function of the PA and subsequently block RNA synthesis by the influenza polymerase. Baloxavir Marboxil was developed for that purpose and the preliminary in vitro and clinical results (4 log reduction in virus production) were very encouraging (Hayden et al., 2018; Omoto

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Fig. 6 Virological effect of Baloxavir marboxyl as compared to placebo and oseltamivir. Panel A shows the change from baseline (dashed line) in influenza infectious viral load overtime in the baloxavir group (427 patients) and placebo group (210 patients). The mean (7SD) viral loads on day 1 (before the initiation of the trial regimen) were 5.79 7 1.87 and 5.56 7 1.89 log10  50% tissue-culture infective dose (TCID50) per milliliter in the baloxavir and placebo groups, respectively. Asterisks indicate a P value of less than 0.05 for the comparison with placebo. Panel B shows the change from baseline in influenza infectious viral load in adults 20–64 years of age in the baloxavir group (352 patients) and oseltamivir group (359 patients). The mean (7SD) viral loads on day 1 were 5.76 7 1.90 and 5.94 7 1.69 log10 TCID50 per milliliter in the baloxavir and oseltamivir groups, respectively. Asterisks indicate a P value of less than 0.05 for the comparison with oseltamivir. In both panels, I bars indicate standard deviations. From Hayden, F.G., Sugaya, N., Hirotsu, N., et al., 2018. Baloxavir marboxil investigators group. Baloxavir marboxil for uncomplicated influenza in adults and adolescents. The New England Journal of Medicine 379 (10), 913–923.

et al., 2018). This reduction was significantly higher than observed with the INAs, or any other anti-influenza drugs, and was noted in both influenza A and influenza B viruses (Fig. 6). The first data obtained from clinical trials were, initially, not as promising as the in vitro results. (1) First, the clinical impact of treatment is more or less similar to that obtained with the NAIs (roughly a 24 h reduction of symptoms duration), as assessed by two randomized, double-blind, controlled trials involving healthy 12–64 years of age outpatients with acute uncomplicated influenza (Hayden et al., 2018; Omoto et al., 2018). (2) Secondly, although it was speculated that substitutions associated with resistance might be difficult to accommodate for the virus and result in a severe fitness loss, it appeared that in vitro and in vivo resistance were detected rapidly, mostly at position I38 (I38T/M/F variants) of the polymerase acidic protein of A(H3N2) viruses; other substitutions are also associated with

168

Management of Influenza Virus Infections (Orthomyxoviridae) reduced susceptibility (Takashita et al., 2019; Imai et al., 2020), and this resistant virus could be readily transmitted through airborne spread (Imai et al., 2020). The monitoring of the emergence of resistance (by PCR) reported resistance in 2.2% and 9.7% of baloxavir recipients in the phase two and phase three trials, respectively (Takashita et al., 2019).

These clinical results and resistance patterns have been confirmed by additional observational studies; Baloxavir-Marboxyl is now licensed in Japan. It seems that the I38T substitution can emerge, be sustainable, and potentially be transmitted with a higher frequency in A(H3N2) viruses. To address this risk, in vivo studies conducted in animals and in vitro competition experiments confirmed the lack of fitness related to the I38T substitution, for A(H1N1) and for A(H3N2) viruses. In addition, a clinical investigation showed that pre-existing resistant viruses were circulating in the community, as seen for NAI before their usage (Ferraris and Lina, 2008; McKimm-Breschkin, 2013; Takashita et al., 2019; Imai et al., 2020). As with NAIs, children have been reported to be more likely to develop resistance during treatment, especially during A(H3N2) infections. As a consequence, surveillance of baloxavir-resistant I38T PA mutants in seasonal subtypes has been implemented.

Pimodivir This third polymerase inhibitor is a non-nucleoside analog, targeting PB2 and inhibiting the docking of the cap structure in the polymerase subunit to allow RNA synthesis (Mifsud et al., 2019). Because of a major difference between influenza A and B PB2 domains, Pimodivir is only active against influenza A subtypes. It allows, as with Baloxavir, a rapid decrease in virus load both in vivo and in vitro. It can be given orally, twice daily. In a phase 2b clinical trial TOPAZ, the viral load – as measured by qRT-PCR in nasal secretions from baseline to day eight – significantly decreased in all treatment groups compared with the placebo group. A Pimodivir plus Oseltamivir combination resulted in a shorter time to symptom resolution than placebo, suggesting a synergistic effect between the two (Fig. 7) (Finberg et al., 2019). Preliminary data have been collected regarding the risk for Pimodivir resistance. Reduced antiviral activity was reported in viruses with a PB2 F363L substitution. Overall, the PB2 inhibitor gave promising results, and phase three clinical trials are testing its use as monotherapy and in combination with Oseltamivir.

Favipiravir (Avigan s ) The fourth molecule of this class is Favipiravir. It is a purine nucleoside analog that inhibits the PB1 RNA-dependent RNA polymerase (Mifsud et al., 2019). It has demonstrated a large spectrum of action in vitro against influenza A and B viruses, but also against other RNA viruses including Ebola viruses (Furuta et al., 2017). Japan has approved its use to treat infection with NAI-resistant viruses. However, its use is restricted because of potential teratogenicity. No resistance has been observed so far (Hayden and Shindo, 2019). Mechanisms of resistance have been investigated. Two substitutions located in PB1 (K229R) and in PA (P653L) have been detected in viruses resistant to Favipiravir in vitro. The K229R substitution in PB1 has a cost in viral fitness for the virus; the polymerase activity is severely impaired by this substitution. However, when compensated with the P653L substitution in PA, the viral fitness of the resistant virus is restored. This K229R substitution may induce resistance to other POL inhibitors of influenza viruses, such as ribavirin (Goldhill et al., 2018). The common feature of these polymerase inhibitors is their capacity to be associated with NAI and have a synergistic effect for the treatment of influenza. Both Baloxavir and Pimodivir have been tested in combination. Favipiravir has also been tested against influenza viruses resistant to NAI, showing good activity and demonstrating, as for the other pol inhibitors, the lack of crossresistance with NAIs.

Drugs Under Development With Other Mechanisms of Action HA processing blockers (Fig. 1) Nitazoxanide is a drug that was first licensed for the treatment of parasitic diseases. It can also inhibit the replication of a relatively broad range of viruses, including influenza A and B (Beigel et al., 2019; Gamiño-Arroyo et al., 2019). The tizoxanide metabolite of nitazoxanide can block the maturation process of the HA in the cell by impairing its traffic and its insertion in the cellular membrane before the virus budding. In vitro studies have shown antiviral activity at a low concentration (around 30–100 ng/mL), but the clinical trials have failed to show a clinical effect, so far.

HA attachment blockers (Fig. 1) Fludase (DAS – 181) is a recombinant sialidase that showed potent antiviral by preventing the entry of influenza viruses in cells through sialic receptors removal from the epithelium (Beigel et al., 2019). A phase two study conducted provided encouraging results. However, the antiviral effect of fludase might be limited because individuals developed antibodies against the compound during treatment (Zenilman et al., 2015). This will be further investigated but, so far, no further clinical trial has been carried out. Other HA binding inhibitors such as monoclonal antibodies targeting the globular head or the stem of the HA, and small molecules binding to the stem of the HA are in the early stages of development.

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Fig. 7 Clinical impact of pimodivir and pimodivir in association with oseltamivir (Result of the TOPAZ clinical trial, see (Finberg et al.) Viral load over time in full analysis set, as determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis (A) and viral culture (B). The limit of quantification for the viral load assay was 4.0 log10 copies/mL, with a limit of detection of 3.48 log10 copies/mL. Results less than the limit of quantification and greater than the limit of detection (“target detected”) are imputed with 3.75 log10 copies/mL; results less than the LOD (“target not detected”) are imputed with 0 log10 copies/mL. Error bars are 95% confidence intervals (CIs). AUC, area under the curve; OST, oseltamivir; TCID50, median tissue culture infectious dose. From Finberg, R.W., Lanno, R., Anderson, D., et al., 2019. Phase 2b study of pimodivir (JNJ-63623872) as monotherapy or in combination with oseltamivir for treatment of acute uncomplicated seasonal influenza A: Topaz trial. The Journal of Infectious Diseases 219 (7), 1026–1034.

Perspectives at Short and Medium Term In the context of the development of new antiviral drugs, new treatment strategies have been tested, especially during the 2009 pandemic and its aftermath. Actually, monotherapy regimens have shown their added value, but also their limits (lack of very active antiviral and clinical impact). To overcome this problem, novel antiviral strategies have been developed and tested, including host targeted molecules and combined regimens (Beigel et al., 2019; McKimm-Breschkin et al., 2018).

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Host Target Molecules (i.e., Diltiazem) Novel therapeutic approaches to control influenza are currently in development. As successfully proven in antiretroviral therapy (Hütter et al., 2015), targeting the host instead of viral determinants may confer a broad-spectrum antiviral efficacy and a reduced risk of emergence of drug resistance (Ludwig, 2011). In that regard, DNA microarray and, more recently, RNAseq techniques – an in-silico assisted strategy based on transcriptional profiling and signature matching – have been developed. Based on the hypothesis that, instead of focusing on specific factors, the global host gene expression profile can be considered as a “signature” of any specific cell state, including during infection or treatment. It has been supposed that the drug-induced inhibition or reversion of the cellular transcriptomic signature of infection (“proviral status”) could establish a globally unfavorable cellular state for viral replication (“antiviral state”). This hypothesis of signature reversion has been validated through proof-of-concept studies based on the exploitation of global virogenomic signatures of in vitro infections with different human and avian influenza subtypes (Josset et al., 2010). Subsequently, the in vivo global transcriptional signatures of infection directly from paired nasal wash samples from a cohort of untreated influenza A(H1N1)pdm09-infected patients has been determined. Samples were collected during acute infection (infected) and at least three months later to ensure a recovery non-infected state (cured). Then, an in silico drug screening using Connectivity Map (CMAP), a Broad Institute’s publicly available database (more than 7000 drug-associated gene expression profiles obtained after treatment of cells with 1309 different FDA-approved small molecules, (Lamb, 2007)), allowed the identification of a shortlist of high-potential candidate bioactive molecules with signatures anti-correlated with those of the patient’s acute infection state. The putative antiviral properties of these “in silico selected” molecules were confirmed and experimentally validated in classic in vitro models. The most effective compounds identified through this process were compared to the gold standard Oseltamivir for the treatment of influenza infections in mice and in an in vitro model of reconstituted human airway epithelium (hAE), leading to the identification of two drugs: The adrenergic receptor agonist Etilefrine (Effortils) and a calcium channel blocker Diltiazem (Tildiems); the latter being able to stimulate the epithelial antiviral defense during influenza A or B infection (Pizzorno et al., 2019a,b). There is currently a multicenter randomized phase 2b clinical trial trying to assess the effect of this combined therapy compared to the standard of care (oseltamivir monotherapy) for the treatment of severe influenza. This original, host-targeted, drug repurposing strategy constitutes an effective and highly reactive process for the rapid identification of novel anti-infectious drugs, with potentially major implications for the management of antimicrobial resistance and the rapid response to future epidemic or pandemic (re)emerging diseases.

Macrolides The added value of macrolides in treating severe influenza has been discussed for some years, as a result of finding significant antiinflammatory effects when used in adults with severe influenza. This strategy is similar to the host-targeted antiviral approach (Beigel et al., 2019). The mechanism of this anti-inflammatory/immunomodulatory effect is not fully understood, but two interesting trials conducted with Azithromycin and Clarithromycin reported a faster decline of the inflammatory markers in the Oseltamivir-Azithromycin group as compared to the Oseltamivir alone groups, and a trend toward faster symptom resolution although no differences were observed in viral RNA decline. In a second prospective study, two groups of patients received either a two-day combination of Clarithromycin 500 mg, Naproxen 200 mg, and Oseltamivir 75 mg twice daily, followed by three days of Oseltamivir, or Oseltamivir 75 mg twice daily for five days were compared (Hung et al., 2017). The combination treatment was statistically associated with reduced mortality, less clinical severity, and shorter hospital stay. In addition, they also reported a reduction in the virus titers and pneumonia severity index in the combination group. This potential beneficial effect of macrolide adjunction will be further investigated.

Other Immunomodulators Potential immunomodulators have also been tested at least in preclinical studies or suggested after limited observational studies. This includes macrolides, sirolimus, N-acetylcysteine, statins, pamidronate, nitazoxanide, chloroquine, antiC5a antibody, interferons, human mesenchymal stromal cells, mycophenolic acid, peroxisome proliferator-activated receptors agonizts, non-steroidal anti-inflammatory agents, mesalazine, herbal medicine, and the role of plasmapheresis and hemoperfusion as rescue therapy (Beigel et al., 2019; McKimm-Breschkin et al., 2018). All these strategies deserve more investigation preferably by RCTs. Two have been tested in clinical trials, with some interesting results: macrolides (as described above) and sirolimus.

Sirolimus In Taiwan, an open-label prospective randomized controlled trial was conducted in patients with severe H1N1 pneumonia with acute respiratory failure. Patients with confirmed H1N1 pneumonia and on mechanical ventilator support were randomized to receive – in addition to the standard of care – Oseltamivir, an adjuvant treatment of corticosteroids with an mTOR inhibitor, either with Sirolimus for 14 days or without (Jia et al., 2018).

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Biological and clinical monitoring showed that the outcomes were significantly better in the sirolimus group over the nonsirolimus group. The authors suggested a combination treatment with Sirolimus along with Oseltamivir as an immunomodulatory strategy for managing severe influenza.

Combined Treatments (Double, Triple) The observed emergence of resistance in all influenza classes and the larger portfolio of anti-influenza classes are pushing the implementation of combined therapies. Most remain at the stage of preclinical, phase two, or phase three trials, but the preliminary results have already shown the limits or added values of some of the tested combinations.

NAI combinations The potential added value of NAI combinations have been tested before, showing antagonistic effects. NAI combinations have been prohibited.

Triple therapy (amantadine, ribavirine oseltamivir) Preliminary in vitro studies have shown a potential synergistic antiviral effect when Amantadine, Ribavirine, and Oseltamivir were combined, even with naturally resistant Oseltamivir viruses (Nguyen et al., 2009). Although this treatment showed a significant decrease in viral shedding at day three as compared to the monotherapy arm in a clinical trial, no improved clinical benefit was observed (Beigel et al., 2017).

Combined oseltamivir plus monoclonal antibodies Monoclonals targeting the stalk of the HA are under development (Beigel et al., 2019; McKimm-Breschkin et al., 2018). Through binding at this conserved region involved in the conformational change, these antibodies inhibit the HA-mediated fusion. For example, MEDI8852 induced a high level of blockage that resulted in a significant reduction of cell-to-cell spread of the virus. A trial conducted in individuals who were 18–65 years old confirmed the decrease in viral shedding in treated subjects. A combined mAB þ Oseltamivir strategy is currently being investigated in hospitalized patients (Ali et al., 2018).

Combined oseltamivir plus favipiravir This combination of recently developed antivirals has different virus targets (NA for Oseltamivir and PB1 for Favipiravir). The preclinical study conducted in a pharmacologically immunosuppressed mouse model infected with the A(H1N1) pandemic influenza virus showed that the treatment of immunosuppressed mice with high doses of Favipiravir (50 mg/kg) in combination with Oseltamivir (20 mg/kg) resulted in delayed mortality and reduced lung viral titers compared to treatment with a single drug regimen with Oseltamivir (Baz et al., 2018). However, it did not prevent the emergence of Oseltamivir-resistant H275Y neuraminidase variants. Nevertheless, this regimen has been tested in a clinical trial showing interesting results and will be further investigated (Wang et al., 2020).

Combined baloxavir þ oseltamivir

After showing the absence of drug-drug interactions (Kawaguchi et al., 2018), a preclinical study was carried out in a mice model and this treatment has been successfully tested (Hayden et al., 2018).

Pimodivir and oseltamivir This combination has been tested in a human trial (TOPAZ), as described above.

Complementary Treatments for Disease Management Different complementary treatments demonstrated some benefit, such as passive immunotherapy with convalescent plasma and hyperimmune globulin, when administered as an adjunctive therapy for severe influenza. On the other hand, the use of corticosteroids in severe influenza has been discussed intensively. A recently published metaanalysis showed the lack of benefit of high doses of corticosteroids (Hui et al., 2018). However, short-lasting corticosteroid treatment with low doses may be beneficial. This needs further evaluation to adapt the correct dose to the clinical situation and the timing of the infection. These products should be avoided as much as possible during the anergic immune response observed during severe influenza that behaves like a severe sepsis.

ECMO (Extracorporeal Membrane Oxygenation) ECMO has been extensively used during the 2009 pandemic when potentially reversible acute respiratory failure associated with severe influenza A (H1N1) pneumonia was observed. However, ECMO remains an expensive, resource-intensive therapy that needs trained staff with intensive care skills. Despite the value of this intervention, it has a high level of mortality. A review and

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meta-analysis on the use of ECMO reported overall mortality of 37.1% with a median duration for ECMO of ten days, mechanical ventilation of 19 days, and ICU length of stay of 33 days. They concluded that ECMO therapy was associated with better survival if used very early, once the patient had been started on mechanical ventilation, as an adjunct or salvage therapy for severe H1N1 pneumonia with respiratory failure (Sukhal et al., 2017).

Conclusion There is absolutely no doubt about the crucial need for antivirals in influenza disease management. A recently published metaanalysis confirmed the added value of the currently available antivirals (neuraminidase inhibitors). These antivirals represented the standard of care for hospitalized patients, but with some limits inherent to the influenza infections (acute disease with shortlived viral shedding, capacity for the rapid development of resistance, cytokine storm responsible for some of the clinical symptoms), new classes are currently being developed against new targets to tackle some of these problems. First, polymerase inhibitors were tested and showed their synergistic effect in combination with NAIs. In addition, host-targeted strategies or immunomodulators have also been developed and tested in combination with virus-targeted antivirals. All these strategies aim to reduce the burden of influenza, but also to become an efficient first line of treatment in case of a pandemic or zoonotic influenza. The (hopefully) successful development of all these classes of molecules will certainly be of help to reduce the burden of seasonal influenza. They will also be useful in controlling influenza infection and symptoms in frail patients or in patients admitted to the intensive care units because of severe disease. We hope that the use of new combined therapeutic strategies will show a sizeable reduction in disease duration, much better than is currently observed with mild influenza cases when treated with the limited antiviral resources currently available.

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JAMA 285 (6), 748–754. Whitley, R.J., Monto, A.S., 2006. Seasonal and pandemic influenza preparedness: A global threat. The Journal of Infectious Diseases 194 (Suppl 2), S65–S69. Wright, P.F., Neumann, G., Kawaoka, Y., 2007. Orthomyxoviruses. In: Knipe, D.M., Howley, P.M., Griffin, D.E., et al. (Eds.), Fields Virology. vol. 2, fifth ed. Philadelphia: Lippincott Williams and Wilkins, pp. 1691–1740. Yen, H.L., Herlocher, L.M., Hoffmann, E., et al., 2005. Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility. Antimicrobial Agents and Chemotherapy 49, 4075–4084. Zenilman, J.M., Fuchs, E.J., Hendrix, C.W., et al., 2015. Phase 1 clinical trials of DAS181, an inhaled sialidase, in healthy adults. Antiviral Research 123, 114–119.

Further Reading Watanabe, A., Ishida, T., Hirotsu, N., et al., 2019. Baloxavir marboxil in Japanese patients with seasonal influenza: dose response and virus type/subtype outcomes from a randomized phase 2 study. Antiviral Research 163, 75–81.

Management of Herpes Simplex Virus Infections (Herpesviridae) Nicole Samies and Richard Whitley, University of Alabama at Birmingham, Birmingham, AL, United States r 2021 Elsevier Ltd. All rights reserved.

Brief Overview of Available Antiviral Agents for HSV Infections Nucleoside analogs (acyclovir, valacyclovir, and famciclovir) are the drugs of choice for treatment of herpes simplex virus (HSV) infections because of their relative safety profiles. Acyclovir is an acyclic guanine analog selectively phosphorylated by viral thymidine kinase first and then by cellular enzymes to form triphosphate-acyclovir. Once triphosphate-acyclovir is formed, it inhibits the viral DNA polymerase, becomes incorporated into the viral DNA chain, and leads to chain termination. The prodrug of acyclovir, valacyclovir, is synthesized by the addition of L-valine to acyclovir. In comparison to oral acyclovir, valacyclovir’s bioavailability is significantly higher at 51%–54% compared to 15%–30%. The improved bioavailability allows for less frequent dosing in comparison to oral acyclovir. The third nucleoside analog, famciclovir, is a guanine analog and prodrug of penciclovir. Famciclovir is rapidly absorbed and converted to penciclovir, and then penciclovir is phosphorylated by viral thymidine kinase into penciclovir-triphosphate. Unlike acyclovir, penciclovir has a higher affinity for thymidine kinase and does not inhibit DNA chain formation. Famciclovir is preferred over penciclovir secondary to its improved bioavailability of 77%. In addition to nucleoside analogs, foscarnet and cidofovir can be used in the treatment of HSV infections. However because of their toxicity profiles neither is recommended as a first-line antiviral agent. Both foscarnet and cidofovir inhibit the viral DNA polymerase similar to the nucleoside analogs, but they do not require phosphorylation by viral thymidine kinase; thus, they can be used in the treatment of resistant HSV strains which have altered their thymidine kinase preventing the phosphorylation of nucleoside analogs. Foscarnet is a pyrophosphate analog that is capable of binding to a particular site on the DNA polymerase without requiring phosphorylation prior. Cidofovir is a monophosphate cytosine analog that requires phosphorylation for activation, but phosphorylation occurs through cellular kinases rather than by the viral thymidine kinase. Once phosphorylated, the diphosphate form of cidofovir inhibits the viral DNA polymerase.

Management of Infections Beyond the Neonatal Period Orolabial Infections Gingivostomatitis is a common presentation of primary herpes simplex virus (HSV) infection in early childhood commonly confused with herpangina or hand-foot-and mouth disease, and the diagnosis should be confirmed prior to initiating treatment. The infection can be self-limiting with oral lesions lasting about 7–10 days and fever about 3–4 days. The most common complication of the infection is dehydration requiring hospital admission for intravenous fluids secondary to poor oral intake because of the painful oral ulcerations. Although treatment is not necessary, a randomized controlled trial demonstrated a reduction in the duration of oral ulcerations, fever, poor oral intake, and viral shedding when oral acyclovir is administered within the first 72 h of symptom onset. Oral acyclovir should be preferred over intravenous acyclovir if the child is able to take medication orally. A recent study also provided recommendations for valacyclovir dosing in children greater than 3 months of age; this may be a more favorable alternative to acyclovir secondary to the convenience of administration. Bleeding of lesions is common, and reassurance should be provided to parents. Children with gingivostomatitis should be excluded from childcare or school until resolutions of symptoms because of the high risk of transmission from one child to another. Recurrent herpes labialis infections, also commonly referred to as ‘cold sores’ or ‘fever blisters’, is a common manifestation of HSV in adolescent and adult patients. Treatment of these lesions is limited; topical treatments, including acyclovir, penciclovir, and over the counter 10% n-docosanol cream, are not recommended because of limited effectiveness; oral options such as acyclovir, valacyclovir, and famciclovir only have marginal benefit when taken at the initial onset of symptoms. Although oral therapies have only marginal benefit, one randomized clinical trial in adults demonstrated clinical significance in the reduction of recurrent herpes labialis lesions when oral acyclovir was prescribed for suppressive therapy. Thus, patients with at least 6 episodes of recurrent herpes labialis lesions a year may be considered for suppressive therapy for 6 months to 12 months in order to reduce the number of recurrences. Unlike gingivostomatitis, children with recurrent herpes labialis infections do not need to be excluded from school. Other mucocutaneous infections of the upper airway caused by HSV include pharyngitis, tonsillitis, epiglottitis, supraglottitis, and larygyngotracheitis, which occur in both immunocompetent and immunosuppressed patients, can be treated with oral acyclovir.

Cutaneous Infections Herpetic whitlow can be a complication of autoinoculation from oral secretions especially in children who are thumb-suckers or nail-biters or in adults secondary to job exposure as seen with personnel in dentistry, respiratory therapists, and pediatricians. The

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diagnosis of HSV should be confirmed by cultures in order to distinguish it from bacterial felon or paronychia. Treatment with oral acyclovir shows some benefit in the treatment of lesions by reduction in the duration of symptoms and viral shedding as demonstrated in a randomized controlled study that evaluated the use of acyclovir in recurrent hand lesions. Herpes gladiatorum and herpes rugbiorum are two types of cutaneous infections seen in athletes, most notably wresting and rugby as the names suggest. Confirmation of the diagnosis should be made prior to treatment in order to distinguish the infection from impetigo. These infections should be treated with oral acyclovir, valacyclovir, or famciclovir, and athletes should be excluded from practice and/or competition until all lesions have crusted over to prevent the spread of infection. Athletes should also be encouraged to drink plenty of fluids to stay well hydrated while on an antiviral medication due to the risk of reversible nephrotoxicity. In order to prevent the spread of HSV between athletes, coaches and family members should be educated on the appearance of herpes mucocutaneous lesions to prevent those infected from playing in close contact sports, and all mats should be cleaned before, between, and after each practice or match to reduce the risk of transmission to players. Suppressive therapy with these medications has been employed. Patients with eczema and burns are at risk for secondary HSV infections and should be treated with antivirals to prevent further progression to disseminated infection.

Ocular Infections Primary herpes simplex virus ocular infections include blepharitis, follicular conjunctivitis, keratoconjunctivitis, retinitis and can be caused by mucocutaneous infections (e.g., herpes labialis, gingivostomatitis) from either direct contact or nerve route transmission. Children with concerns for HSV ocular infections should be promptly evaluated by an ophthalmologist and treated to prevent further complications as HSV is the most common cause of corneal scarring and vision loss in the United States. Herpetic keratitis is the most common of all the ocular diseases caused by HSV-1 and is often self-limiting. However, treatment reduces duration of symptoms, viral shedding, and risk of corneal scarring and vision loss. The classification into one of the three main subtypes of herpetic keratitis (epithelial, stromal, and endothelial) determines management of the infection. Treatment recommendations for the epithelial subtype are to administer antiviral agents solely whereas stromal and endothelial subtypes should be treated with a combination of antiviral agents and topical corticosteroids to decrease the amount of stromal inflammation. Of note, steroids should never be used alone in treatment of ocular infections. Children should be treated with both systemic acyclovir and ophthalmic antiviral drops or ointments (e.g., 1% trifluridine or 0.15% ganciclovir). Depending on the severity of the infection, the patient may require intravenous acyclovir and then switch to oral acyclovir to complete therapy. Topical vidarabine and iododeoxyuridine were additional antiviral options previously available for use in the treatment of ocular disease, but they are no longer available in the United States. Parents and providers should be aware of the barriers in administration of topical antivirals to children (e.g., crying during administration, skipping doses, or stopping therapy prematurely) and measures should be taken to address these. Children are at increased risk for corneal scarring and blindness with each recurrent episode. Therefore, clinicians should consider placing affected children on suppressive therapy to prevent further reactivation. Suppressive therapy poses the risk of developing resistant HSV strains; however, resistant strains are more commonly detected in immunosuppressed patients. Nonetheless, if a patient’s ocular disease progresses further despite being on adequate therapy, resistance should be suspected. Foscarnet is a second-line option in treatment of ocular disease caused by herpes simplex virus and can be used for treatment of herpes simplex virus when a resistant strain is detected. Foscarnet inhibits viral DNA polymerase, which is a different mechanism of action than the nucleoside analogs (acyclovir, valacyclovir, and famciclovir) which require phosphorylation by the viral thymidine kinase – the usual site of resistance – in order to inhibit HSV replication. Foscarnet does have a significant toxicity profile, which is why it is not first line for treatment. Providers should be aware of its nephrotoxicity and provide adequate pre- and post-hydration to patients when administering the antiviral agent.

CNS Infections Herpes encephalitis is typically caused by reactivation of HSV 1 in adults but can be a primary infection in older children and is a common cause for sporadic encephalitis. Herpes encephalitis can cause fatal complications if diagnosis and treatment are delayed. Patients must be evaluated for herpes encephalitis by cerebrospinal fluid (CSF) HSV polymerase chain reaction (PCR) and should be empirically started on intravenous acyclovir until results are confirmed. Adults and children greater than 12 years of age should receive 10 mg/kg/dose every 8 h and children 3 months to 12 years of age should receive 10–15 mg/kg/dose every 8 h. Once diagnosis is confirmed, patients should be administered intravenous acyclovir for 14–21 days. In addition, all patients diagnosed with herpes encephalitis should have head magnetic resonance imaging (MRI) performed to determine areas of the brain affected and evaluate for a possible infarction; close monitoring of renal function as well as for signs of increased intracranial pressure are essential. Other considerations in the management of patients may be the need for anti-seizure medications and possibly mechanical ventilation. The introduction of intravenous acyclovir for treatment of herpes encephalitis has dramatically improved patient outcomes regarding the reduction in the mortality. Unfortunately, morbidity continues to remain high as a result of neurologic impairment. Even with early diagnosis and initiation of intravenous acyclovir, only about 40%–55% of patients are capable of returning fully to their activities of daily living without the need for assistance. Suppressive therapy following the 14 to

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21-day treatment course is not recommended as a randomized controlled trial demonstrated that 3 months of suppressive therapy with valacyclovir did not improve neurological outcomes as clinically seen in neonates following 6 months of suppressive therapy. The study was terminated at an interim analysis due to clear lack of evidence of clinical benefit. Unlike herpetic encephalitis, Mollaret meningitis, a type of aseptic meningitis caused primarily by recurrent HSV-2 orolabial or genital infections, does not require antiviral therapy as it is usually self-limiting. Differentiation between the diagnoses can be made through the presence of degenerated monocytes in cerebrospinal fluid, which is a diagnostic clue for Mollaret meningitis, and recurrence of orolabial or genital lesions.

Genital Herpes Genital herpes can be acquired through genital-genital, oral-genital, or genital-anal contact with a partner who either has active lesions at time of contact or is asymptomatically shedding virus. Most patients are unaware of their diagnosis allowing for transmission of the virus when patients are asymptomatically shedding the virus. All lesions concerning for HSV should be swabbed and sent for polymerase chain reaction (PCR) testing to confirm the diagnosis. The lesions should also be typed for either HSV-1 or HSV-2 because virus type predicts the frequency of recurrences. Infection caused by HSV-1 is significantly less likely to recur. Serologic testing on each patient should be considered in order to determine if the infection is primary (patient has no antibodies to either type of HSV), first-episode non-primary (patient has antibodies to HSV type 1 or type 2 and newly acquires the other type), or recurrent to provide appropriate counseling in regards to natural history of the infection and risk of recurrence. All patients should be screened for other sexually transmitted infections, particularly human immunodeficiency virus (HIV), as herpes simplex virus is a known risk factor for the transmission of HIV. Treatment of genital infections includes treatment for a primary infection, episodic treatment for recurrent infections, and daily suppressive therapy for prevention of recurrent infections and reduction in subclinical viral shedding. First-line antivirals used in the treatment of genital herpes are nucleoside analogs: acyclovir, valacyclovir, and famciclovir. All three nucleoside analogs have the same efficacy, and patients may prefer one over the other depending on convenience of administration and cost. Oral therapy (or IV if severe infections) is preferred over topical therapy as it is more effective in promptly alleviating symptoms, reducing time to healing, and reducing time of viral shedding if started within 24–72 h of symptom onset. A primary (initial) infection should be treated with antiviral drugs for 7–10 days or longer if lesions are not completely healed. The most convenient antiviral prescribed for treatment of genital lesions is valacyclovir as it only requires daily dosing (Table 1). This is because of the increased bioavailability of valacyclovir when compared to oral acyclovir. However, despite convenience in administration, valacyclovir is more costly than acyclovir, and therefore, may be a contributing factor when determining which antiviral to prescribe. In addition to antiviral therapy, patients may try analgesics and warm sitz baths to aid in alleviation of symptoms. Once patients have completed treatment for their primary infection, the decision on whether to treat each symptomatic recurrence (episodic treatment) or to be placed on suppressive therapy is dependent on each patient’s preference. Episodic treatment is typically a 5-day course started at the first sign of symptoms to treat current reactivation, whereas suppressive therapy is a daily medication regimen prescribed to prevent recurrences and reduce the duration of asymptomatic viral shedding (Table 1). Shorter course therapy has been shown efficacious but may be associated with gastrointestinal upset. As noted above, patients should be aware that lesions secondary to HSV-1 have fewer symptomatic recurrences and this may also aid in decision making. Suppressive therapy does appear to be of benefit to patients and their sexual partners by reducing number of symptomatic occurrences by approximately 80% and subclinical shedding, thus, improving their quality of life. One study by Gupta et al. in 2004 demonstrated the decrease in recurrences and subclinical shedding in participants on acyclovir or valacyclovir in comparison to no treatment (placebo). Although the study did show a reduction in subclinical shedding, patients should be aware that despite taking a daily medication, there is still a risk of asymptomatically shedding the virus and, ergo, transmitting infection. On the other hand, episodic therapy may be preferred by some patients as they are not required to remember to take a daily medication. If a patient chooses episodic treatment, they should be provided with a supply of antiviral drugs in order to start the medication at the earliest onset of prodromal symptoms in order to be the most effective. Resistance to nucleoside analogs is uncommon despite the use of suppressive therapy over long term and is mainly a concern in immunocompromised hosts. Counseling patients on their new diagnosis is as important as prescribing antiviral therapy secondary to the stigma associated with infection and the psychological effects patients experience afterwards. Many patients experience guilt, anger, low self-esteem, and fear of transmitting to the virus to their partner or future child. Counseling should include discussion on preventing transmission of the virus to sexual partners during times of prodromal symptoms or active lesions and even when asymptomatic secondary to subclinical viral shedding. In order to reduce transmission of the virus, patients should consider refraining from sexual activity during times of active lesions, using condoms to prevent transmission, and taking daily antiviral therapy for suppression. Although these practices can reduce the risk of transmission, patients should be aware that none of these measures are completely effective at preventing transmission. Little is known about the use of antivirals for pre-exposure and post-exposure prophylaxis for a seronegative partner; therefore, it is not currently recommended. Patients of long-term relationships should be offered the opportunity to have their partner participate in the counseling session in order to also educate the partner on the disease and ways to prevent transmission. The counseling session should also discuss the ubiquitous nature of the virus in the United States and that acquisition of the virus could have occurred years prior and not be a result of infidelity.

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Table 1

Treatment options for genital herpes

Lesion type

Treatment recommendations

Primary lesion

Oral acyclovir: 400 mg 3 times a day for 7–10 days Treatment duration may be extended past 10 days if lesions are OR incompletely healed 200 mg 5 times a day for 7–10 days Valacyclovir: 1000 mg 2 times a day for 7–10 days Famciclovir: 250 mg 3 times a day for 7–10 days

Recurrent lesion Episodic therapy

Notes

Oral acyclovir: 400 mg 3 times a day for 5 days OR 800 mg 2 times a day for 5 days OR 800 mg 3 times a day for 2 days Valacyclovir: 500 mg 2 times a day for 3 days OR 1000 mg 1 time a day for 5 days Famciclovir: 125 mg 2 times a day for 5 days OR 1000 mg every 12 h for 2 doses OR 500 mg for 1 dose then 250 mg for 2 days

Suppressive therapy Oral acyclovir: 400 mg 2 times a day Valacyclovir: 500 mg 1 time a day OR 1000 mg 1 time a day Famciclovir: 250 mg 2 times a day Pregnancy

Oral acyclovir: 400 mg 3 times a day Valacyclovir: 500 mg 2 times a day

Should begin at 36 weeks gestation

Note: Workowski K.A., Bolan G.A., 2015. Sexually transmitted diseases treatment guidelines. Morbidity and Mortality Weekly Report: Recommendations and Reports 64 (RR-03), 1–137.

Due to the increased risk of vertical transmission of HSV from mother to infant, all pregnant females with a history of genital lesions should be advised to take acyclovir or valacyclovir around 36 weeks gestation, if not already on suppressive therapy, to decrease the risk of developing active lesions at time of delivery. Antiviral suppressive therapy can decrease the probability of active lesions at the time of delivery and therefore, prevent the need for cesarean delivery but it does not completely prevent subclinical viral shedding. Women should be advised that infants are still at risk of perinatal transmission. The American College of Obstetricians and Gynecologists has recommended cesarean delivery for any woman presenting with active genital lesions or prodromal symptoms (burning, tingling, and/or itching in genital area) at time of delivery whether these lesions represent a new primary infection, non-primary first episode infection, or a reactivation. A prospective cohort study demonstrated cesarean delivery at time of active genital lesions greatly reduced transmission of herpes simplex virus from mother to infant, but it did not completely prevent the development of neonatal herpes infection in neonates. Other factors such as duration of rupture of membranes, application of fetal-scalp electrodes, and subclinical shedding contribute to the risk of transmission. Currently the risk of performing cesarean sections in all women with a previous history of genital herpes outweighs the benefit, and thus, cesareans are not recommended unless there are active lesions or prodromal symptoms indicating a reactivation at time of delivery. Also, screening all pregnant women for HSV serologic status is not recommended. Thus, women who are presumed seronegative during pregnancy but are in a relationship with a known seropositive spouse should refrain from sexual contact during their third trimester to prevent acquisition of the virus in order to decrease risk of transmission to their newborn.

Neonatal Herpes Simplex Virus Infection Early recognition and prompt initiation of antiviral therapy in infants has prevented further disease progression and significantly improved morbidity and mortality. Therefore, any neonate presenting with concerns for neonatal HSV infection should undergo a complete evaluation. The evaluation includes performing culture and PCR on any concerning mucocutaneous lesion, obtaining mucous membrane culture and PCR of eye, nasopharynx, mouth, and rectum, obtaining blood HSV PCR and serum alanine aminotransferase (ALT), and performing a lumbar puncture to evaluate cerebrospinal fluid (CSF) for HSV with PCR. Both the use of culture and PCR are currently recommended for the detection of HSV DNA in mucocutaneous lesions and the eye, nasopharynx, mouth, and rectum as the superiority of PCR has not yet been established in the detection of HSV DNA from these sites in

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neonates. However, PCR is quickly replacing viral cultures in many laboratories due to the costs related to performing viral cultures, the known increased sensitivity of PCR in the diagnosis of HSV DNA in genital lesions, and the quicker turn around time (24 h for PCR vs 2-5 days for culture). Once the full evaluation has been completed, all infants should be empirically started on intravenous acyclovir pending results of all the viral HSV studies. Diagnostic results will provide the information needed to classify the infant into a subtype, which will then determine the duration of treatment. The three subtypes of neonatal HSV infection are: skin, eye, and mouth (SEM), central nervous system (CNS), and disseminated disease. Once the diagnosis has been confirmed, all infants regardless of their disease classification should have an ophthalmologic exam and neuroimaging. Infants diagnosed with skin, eye, and mouth (SEM) disease should receive 14 days of intravenous therapy, and those with CNS or disseminated disease should receive a minimum of 21 days of intravenous acyclovir. Treatment was extended to 21 days in infants with CNS and disseminated disease secondary to a large proportion (40%) of infants continuing to detect HSV DNA in the CSF by PCR after 14 days of therapy with intravenous acyclovir. Any infant with CNS involvement requires a repeat lumbar puncture prior to completion of 21 days to determine if the evidence of virus, namely viral DNA, has been cleared from the CSF. If the HSV PCR remains positive near completion of 21 days, intravenous acyclovir should be continued for an additional week in the infant and another lumbar puncture will need to be repeated at the end of the week to reassess for clearance of the virus. Intravenous acyclovir should be continued until the HSV cerebrospinal fluid PCR is negative. Infants that require longer than 21 days of intravenous acyclovir have been shown to have worse outcomes. While on intravenous acyclovir, complete blood cell counts should also be obtained twice a week and renal function closely monitored to evaluate for any signs of neutropenia or renal toxicity respectively. If infants develop severe neutropenia (o500 cells/mL) during the treatment course with intravenous acyclovir, the acyclovir dose can be decreased or granulocyte colony-stimulating factor (G-CSF) can be administered if the absolute neutrophil count remains low despite a reduction in the dose. Dosing recommendations for intravenous acyclovir have been derived from studies performed by the National Institute of Allergy and Infectious Disease (NIAID) Collaborative Antiviral Study over the last 30 years. Acyclovir quickly came into favor in the treatment of neonatal HSV infection because of the ease in administration and less toxic side effects. Initially low dose acyclovir when studied in comparison to vidarabine did not show therapeutic superiority. Later studies demonstrated improved outcomes when high a dose of intravenous acyclovir was used during treatment. Currently acyclovir is in the only nucleoside antiviral agent approved in the use of neonates as further studies are pending on the pharmacokinetics and safety of valacyclovir in neonates. Suppressive therapy with oral acyclovir should be administered after the completion of either the 14- or 21-day treatment course of intravenous acyclovir for a total of 6 months. Suppressive therapy, when given over a 6-month period, has demonstrated a reduction in the number of cutaneous recurrences and improved developmental outcomes in neonates. Infants should be closely monitored for neutropenia during the 6 month course with a complete blood cell counts at the 2nd and 4th week of administration and then monthly thereafter. If the patient develops severe neutropenia (o500 cells/mL), prescribers should consider holding the patient’s acyclovir while waiting for recovery of the patient’s absolute neutrophil count. The suppressive dose should be adjusted monthly to account for the patient’s rapid growth. Although suppressive therapy has improved outcomes for neonates, neurologic impairment in infants diagnosed with encephalitis continues to remain high. Measures to prevent the transmission of HSV to infant include: cesarean delivery in those with active genital lesions limiting the use of fetal scalp electrodes, and preventing exposure to oral secretions in persons with an active cold sore or a younger child with gingivostomatitis. A guidance statement by the American Academy of Pediatrics also recognizes the risk of infants developing HSV disease when born to mothers with active genital lesions at time of delivery and provides recommendations on managing these infants in attempt to diagnose infants with HSV disease early before disease progression occurs. Any genital lesion concerning for HSV should be evaluated and, if positive, typed for HSV-1 or HSV-2. Serologic testing should then be performed on the mother because determination of a mother’s serologic status is imperative in determining the risk of transmission to an infant. A mother with a first-episode primary infection (no antibodies to HSV-1 or HSV-2) is a greater risk of transmitting HSV to their infant than a mother with a first-episode non-primary infection (mother has antibodies to HSV type 1 or type 2 and newly acquires the other type) and recurrent infection (reactivation of virus mother already has antibodies to). All infants born to a mother with an active genital lesion should have a mucous membrane culture and PCR obtained from conjunctiva, nasopharynx, mouth, and rectum and blood HSV PCR performed at 24 h of life. The 24 h of lifetime point is chosen to avoid possible transient maternal contamination. If the infant is born to a mother with known history of genital herpes and the infant remains asymptomatic after birth, acyclovir can be held, and the infant can be discharged as early as 48 h of life as long as all HSV testing remains negative, infant has available access to medical care, and parents receive education on the signs and symptoms of neonatal HSV disease to monitor for. If these criteria cannot be met, the infant should remain hospitalized until culture is confirmed negative or is negative at 96 h, whichever is first. However, if any HSV test returns positive, the infants should undergo further evaluation with lumbar puncture to assess for central nervous system involvement and serum ALT to assess for systemic involvement. If the additional testing is negative, the infant should receive 10 days of empiric treatment with intravenous acyclovir. However, if the results return abnormal, the infant should be treated with intravenous acyclovir for either 14 or 21 days depending on classification. If the infant’s mother has no prior history of genital herpes and presents with active lesions at time of delivery, serologic testing on the mother is extremely helpful in determining infant’s risk as mentioned above. In addition to mucous membrane culture and PCR, the infant should also have a lumbar puncture and serum ATL performed. Intravenous acyclovir should also be initiated. If mother’s genital lesion is confirmed to be primary or non-primary infection, and the infant’s testing is all negative, the infant should be empirically treated for 10 days with intravenous acyclovir. If the testing is abnormal, the infant should be classified into SEM, CNS, or

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disseminated disease and treated accordingly. If mother’s genital lesion is confirmed to be a reactivation, and all HSV testing on the neonate is negative, the infant can be discharged home with parents after they receive education on monitoring for signs and symptoms of neonatal HSV to monitor for. However, if the surface cultures or HSV blood PCR return positive, but the CSF HSV PCR and ALT are negative, the infant should be continued on IV acyclovir for a total of 10 days. If the CSF HSV PCR and/or ALT are abnormal, the infant should be treated based on classification of neonatal HSV disease for a total of 14–21 days.

Immunocompromised Hosts Oral or intravenous acyclovir as well as valacyclovir and famciclovir remain treatments of choice for management of HSV infections in immunocompromised hosts, depending upon severity of infection, risk of dissemination, and degree of immunosuppression. However, immunocompromised patients are known to have a high rate of resistant HSV strains to acyclovir and other nucleoside analogs (valacyclovir, famciclovir) because of their similar mechanism of action when compared to immunocompetent hosts. Thus, other antiviral agents (foscarnet or cidofovir) besides nucleoside analogs should be considered for treatment in these patients if disease progresses despite adequate antiviral therapy. Resistance generally emerges after long-term administration of the antiviral. Resistance can be overcome at times with using higher than the typical recommended doses of acyclovir. However, when resistance cannot be overcome, cidofovir and foscarnet are alternative therapeutic options. These antivirals do not require phosphorylation in order to be active as with nucleoside analogs, and therefore, they are effective alternatives in treating HSV resistant strains. Both cidofovir and foscarnet have significant toxic side effects, limiting their recommendation to a second line treatment alternative.

Immunoprophylaxis Several different vaccines have been studied for effectiveness in both therapeutic and prophylactic trials of HSV-1 and 2, but to date, no vaccine has demonstrated significant efficacy. There are currently several other vaccines contenders being evaluated, but these have not yet been studied in clinical trials.

Further Reading Amir, J., Harel, L., Smetana, Z., Varsano, I., 1997. Treatment of herpes simplex gingivostomatitis with aciclovir in children: A randomised double blind placebo controlled study. BMJ 314 (7097), 1800–1803. Amir, J., Harel, L., Smetana, Z., Varsano, I., 1999. The natural history of primary herpes simplex type 1 gingivostomatitis in children. Pediatric Dermatology 16 (4), 259–263. Azher, T.N., Yin, X.T., Tajfirouz, D., Huang, A.J., Stuart, P.M., 2017. Herpes simplex keratitis: Challenges in diagnosis and clinical management. Clinical Ophthalmology 11, 185–191. Brown, Z.A., Wald, A., Morrow, R.A., et al., 2003. Effect of serologic status and Cesarean delivery on transmission rates of herpes simplex virus from mother to infant. Journal of the American Medical Association 289 (2), 203–209. Gill, M.J., Bryant, H.E., 1991. Oral acyclovir therapy of recurrent herpes simplex virus type 2 infection of the hand. Antimicrobial Agents and Chemotherapy 35 (2), 382–383. Gnann Jr., J.W., Skoldenberg, B., Hart, J., et al., 2015. Herpes simplex encephalitis: Lack of clinical benefit of long-term valacyclovir therapy. Clinical Infectious Diseases 61 (5), 683–691. Gnann Jr., J.W., Whitley, R.J., 2016. Genital herpes. New England Journal of Medicine 375 (19), 1906. Gupta, R., Wald, A., Krantz, E., et al., 2004. Valacyclovir and acyclovir for suppression of shedding of herpes simplex virus in the genital tract. Journal of Infectious Diseases 190 (8), 1374–1381. Kimberlin, D.W., 2004. Neonatal herpes simplex infection. Clinical Microbiology Reviews 17 (1), 1–13. Kimberlin, D.W., 2013. Acyclovir dosing in the neonatal period and beyond. Journal of the Pediactric Infectious Diseases Society 2 (2), 179–182. Kimberlin, D.W., Baley, J., Committee on Infectious Diseases, Committee on Fetus and Newborn, 2013. Guidance on management of asymptomatic neonates born to women with active genital herpes lesions. Pediatrics 131 (2), 383–386. Kolokotronis, A., Doumas, S., 2006. Herpes simplex virus infection, with particular reference to the progression and complications of primary herpetic gingivostomatitis. Clinical Microbiology and Infection 12 (3), 202–211. Rooney, J.F., Straus, S.E., Mannix, M.L., et al., 1993. Oral acyclovir to suppress frequently recurrent herpes labialis. A double-blind, placebo-controlled trial. Annals of Internal Medicine 118 (4), 268–272. Whitley, R., Baines, J., 2018. Clinical management of herpes simplex virus infections: Past, present, and future. F1000Reserach 7. Whitley, R.J., Roizman, B., 2001. Herpes simplex virus infections. Lancet 357 (9267), 1513–1518.

Management of Varicella-Zoster Virus Infections (Herpesviridae) Andreas Sauerbrei, Jena University Hospital, Jena, Germany r 2021 Elsevier Ltd. All rights reserved.

Nomenclature aa Amino acid ABS Adenine triphosphate-binding site bp Base pair(s) BVDU (E)-5-(2-bromovinyl)-20 -deoxyuridine CNS Central nervous system DNA pol Deoxyribonucleic acid polymerase EC50 50% effective concentration EDTA Ethylene diamine tetraacetate ELISA Enzyme-linked immunosorbent assay FAMA Fluorescence antibody membrane antigen test gE Glycoprotein E HIV Human immunodeficiency virus HSV-1 Herpes simplex virus type 1 HSV-2 Herpes simplex virus type 2 i.m. Intramuscular(ly) i.v. Intravenous(ly) IFAT Indirect immunofluorescence test IgA Immunoglobulin A

Glossary Congenital varicella This syndrome may occur in the newborn after maternal chickenpox between the 5th and 24th weeks of gestation. As a consequence of intrauterine VZV infection, the characteristic clinical findings consist of dermatomal skin lesions, neurological defects, eye diseases, and/or limb hypoplasia. Post zoster neuralgia Pain lasting longer than 4 weeks and occurring again after pain-free interval, caused by an irreversible necrosis of ganglion cells.

IgG Immunoglobulin G IgM Immunoglobulin M MMR Measles-mumps-rubella MMRV Measles-mumps-rubella-varicella NBS Nucleoside-binding site pOka Parental Oka PZN Post zoster neuralgia RNA Ribonucleic acid s.c. Subcutaneous(ly) STIKO Standing Vaccine Commission of the Robert Koch Institute, Germany TK Thymidine kinase TK- Thymidine kinase negative TKa Thymidine kinase altered TKr Thymidine kinase reduced UL28 Unique long 28 UL36 Unique long 36 VZIG Varicella-zoster immunoglobulin VZV Varicella-zoster virus

UL28 VZV gene 28 localized in the unique long gene region. UL36 VZV gene 36 localized in the unique long gene region. UN 3373 Label for shipping category B biological substances; category B includes most known microorganisms.

Laboratory Diagnosis Submission of Samples Varicella-zoster virus (VZV)-positive samples are considered as category B, risk group 2 dangerous goods and dispatched in accordance with UN 3373. For this purpose, the primary container with the patient's sample must be shipped with an overpack of adsorbent material in a transportable box. Shipment is possible at room temperature. Cooling is recommended only if the samples are intended for virus isolation in cell culture.

Direct Detection of Virus Acute VZV infection is diagnosed by detection of the virus (Table 1). The method of choice is polymerase chain reaction (PCR) to detect viral genomes in vesicle fluid or swabs. Depending on the clinical manifestations, additional samples such as cerebrospinal fluid (CSF), tissue, bronchoalveolar lavage, ethylene diamine tetraacetate (EDTA) blood, amniotic fluid, aqueous humor, pharyngeal secretion, saliva or tear fluid are collected. PCR is used to examine CSF in acute infections of the central nervous system (CNS) and amniotic fluid in prenatal diagnostics after varicella during pregnancy. In immunosuppressed patients with zoster, the detection of VZV DNA in the blood may be helpful in assessing the potential risk of disseminating infection. In addition, virus isolation from vesicle contents or swabs can be used. However, isolation of VZV is only possible in a few cell types such as human embryonic fibroblasts (Fig. 1). Furthermore, this procedure is time-consuming, requires a high degree of experience and has no

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Overview of methods for direct detection of VZV or VZV DNA

Principle

Method

Patient samples

Detection of viral DNA

Polymerase chain reaction (PCR)

Vesicle content/swab, in 1 ml physiological saline or viral transport medium Cerebrospinal fluid, tissue, bronchoalveolar lavage, EDTA blood, amniotic fluid, aqueous humor, pharyngeal secretion, saliva, tear fluid

Viral isolation

Viral growth in cell culture, detection by monoclonal antibody

Vesicle content in viral transport medium with special swab, tissue, bronchoalveolar lavage; transport under cooling (2–81C)

Virus antigen detection

Immunofluorescence test using monoclonal antibody Reduced sensitivity and specificity

Cell-rich vesicle content in viral transport medium with special swab, tissue

Discrimination between wild-type/ PCR, Restriction fragment length Vesicle content in viral transport medium with special swab, tissue, vaccine strains, genotyping polymorphism analysis, sequencing cerebrospinal fluid, viral isolate

Fig. 1 VZV-induced cytopathic effect in human embryonic lung fibroblasts.

clinically relevant sensitivity. In most cases, only vesicle fluid with a high viral load is suitable for virus isolation. For successful virus isolation, early and careful sampling and optimal transport of the sample are essential. Viral isolates are characterized by immunofluorescence with monoclonal fluorescence-labeled antibodies. The direct qualitative detection of VZV antigens in vesicle content or swabs using commercial detection systems can provide results within a few hours but is characterized by reduced sensitivity and specificity. When interpreting the results, it should be noted that methods for the direct detection of VZV including nucleic acids or antigens do not distinguish between primary and recurrent VZV infections. The distinction between wild-type VZV and vaccine strains can be made by restriction fragment length polymorphism analysis or sequencing (genotyping).

Detection of Antibodies VZV antibody detection (Table 2) is especially indicated when susceptible persons have to be identified to offer protection with active or passive immunoprophylaxis. Past VZV infection is diagnosed by detecting VZV IgG in serum or plasma, and positive VZV IgG indicates immunity. Due to high seroconversion rates, the determination of antibody status after varicella vaccination is not necessary for healthy children, adolescents and adults. In contrast, immune status control is recommended in immunodeficient individuals and healthcare workers after vaccination. In daily laboratory practice, ligand assays or partially immunofluorescence tests to detect VZV-specific IgG are common. Regardless of the test used, any result interpreted as VZV IgG-positive by that laboratory can be used as a criterion for immunity against varicella. Persons with indeterminate VZV IgG results are classified as “non-immune”. Commercial test kits differ in sensitivity so that very low antibody titers are not detected. Therefore, highly sensitive tests such as special glycoprotein enzyme-linked immunoassays (ELISA) or the fluorescence antibody membrane antigen test (FAMA) should be used to determine the immune status especially after varicella vaccination and for vaccine studies. Primary VZV infection (varicella) can normally be diagnosed by determining VZV IgG seroconversion in serum or plasma. This requires the availability of sequentially collected samples where the initial result is VZV IgG-negative. The earliest VZV IgM can be detected from the fourth day after the onset of the disease, usually in combination with VZV IgG. Even though VZV IgM can be used in practice to confirm an active VZV infection, it should be noted that IgM antibody detection will be considerably delayed after the onset of varicella exanthema and is only possible in a maximum of 50%–60% of patients with zoster. Thus,

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Table 2

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Overview of methods for determination of VZV-specific antibodies

Method

Remarks

Ligand assays (ELISA, chemiluminescence immune assay etc.)

Determination and differentiation of Ig classes (IgG, IgM, IgA) in serum, plasma and cerebrospinal fluid, based on whole viral antigen of VZV-infected cell cultures or viral glycoproteins Simple performance, commercially available, automated

Indirect fluorescence antibody test (IFAT)

Determination and differentiation of Ig classes (IgG, IgM, IgA) in serum, plasma and cerebrospinal fluid Simple performance, commercially available, requires experience for evaluation

Fluorescence antibody membrane antigen test (FAMA)

Determination of antibodies against VZV glycoproteins in serum, reference test for determination of immunity

VZV IgG avidity (ELISA or IFAT)

Differentiation between primary infection (varicella) and recurrent infection after virus reactivation (generalized zoster)

Neutralization test

Determination of antibodies against VZV glycoproteins in serum, good correlation with FAMA

this marker is unreliable and unable to distinguish between a primary and a recurrent VZV infection. In addition, numerous commercial VZV IgM immunoassays show reduced sensitivity and may show false-positive results due to cross-reactions with other herpes viruses, especially herpes simplex virus (HSV). VZV IgA can often be determined in latently VZV-infected individuals, but high titer values correlate exclusively with zoster disease. In the case of VZV infection with involvement of CNS, the earliest increase in intrathecal antibody index (cerebrospinal fluid and serum from the same day of collection) can be detected around 10–12 days after symptom onset. Therefore, determining intrathecal VZV-specific IgG is only suitable for the retrospective diagnosis of VZV-associated CNS infections. The determination of VZV IgG avidity allows the differentiation between primary (varicella) and recurrent infections (zoster). However, there has been little experience with this test procedure so far. In principle, serological findings in immunodeficient patients are not reliable and should therefore be interpreted with caution. Despite acute or past infection, antibody findings in these individuals may also be negative.

Antiviral Treatment Antiviral Agents in Clinical Use The administration of antiviral agents may successfully block replication of VZV in infected cells. Early administration, especially in zoster within 72 h after onset of exanthema, can reduce tissue damage and thus diminish or even prevent the destruction of affected ganglion cells. The acyclic nucleoside analogs acyclovir including its prodrug valaciclovir, famciclovir (prodrug of penciclovir) and the cyclic nucleoside analog brivudine ((E)-5-(2-bromovinyl)-20 -deoxyuridine, BVDU) are available for the antiviral treatment of VZV infections (Table 3). The specificity of antiviral activity is based on the phosphorylation by the viral thymidine kinase (TK) to their mono- (acyclovir, penciclovir) or diphosphates (brivudine) while cellular enzymes catalyze the further phosphorylation steps to triphosphate. The activity spectrum is defined by the presence of the key enzyme, the viral TK. The triphosphates of the nucleoside analogs inhibit and fix the viral DNA polymerases (pol) and are incorporated into the growing DNA chain as a “false” substrate. In acyclovir/valaciclovir, this leads to chain termination due to the absence of the hydroxy group in the 30 position, which is essential for further linkage. In other nucleoside analogous compounds, their incorporation into DNA is possible.

Acyclovir Acyclovir is used as the first-line antiviral drug to treat VZV infections. However, a disadvantage is that acyclovir has a low oral bioavailability of about 15%–30%. Varicella in high-risk patients and zoster disease in immunocompetent patients can be treated orally. In severe VZV infections, especially in immunocompromised patients, acyclovir must be administered intravenously (i.v.). Acyclovir is not officially licensed as antiviral therapy during pregnancy, and administration should be especially avoided in pregnant women before the end of the 14th gestational week. However, results from the manufacturer’s pregnancy registries and a Danish population-based retrospective cohort study have not shown an increased rate of major birth defects after oral administration of acyclovir and its topical use. Nevertheless, patients should be informed about the limited data, especially during early pregnancy, and give consent before the drug is used. After i.v. administration of acyclovir, CNS side effects have occasionally been observed, while oral medication may be associated with gastrointestinal side effects. Drugs that can cause renal toxicity should not be combined with acyclovir at the same time. Laboratory kidney and liver parameters must be monitored. If kidney function is impaired, the dose of acyclovir must be reduced.

Valaciclovir Valaciclovir is the l-valyl ester prodrug of acyclovir. After oral administration, valaciclovir is converted into acyclovir by a hepatic enzyme, valaciclovir hydrolase. Since the oral bioavailability of valaciclovir is 54%, the considerably higher acyclovir concentrations compared with oral acyclovir result in longer dosing intervals and higher compliance. Valaciclovir is approved for the

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Table 3

Antiviral drugs against VZV in clinical use

Antiviral drug Administration

Indication and dosage

Acyclovir

Severe and disseminated VZV infections, zoster under immunosuppression; adults: 3  5–10 mg/kg body weight per day for 7–10 days, children: 3  500 mg/m2 body surface per day for 7–10 days Zoster, varicella in risk patients; adults: 5  800 mg per day over 7 days, children: 5  15 mg/kg body weight per day (max. 4000 mg/day) for 7 days Zoster; immunocompetent adults and adults with mild or moderate immunosuppression: 3  1000 mg per day for 7 days Zoster; immunocompetent adults: 3  250 mg per day for 7 days, immunodeficient adults or adults with zoster ophthalmicus: 3  500 mg per day for 7–10 days Zoster; immunocompetent adults: 1  125 mg per day for 7 days VZV infections caused by TK-negative (acyclovir-resistant) VZV strains; adults: 3  40 mg/kg body weight per day for 10 (max. 20) days, children (off label use): 3  60 mg per kg body weight per day for 10 (max. 20) days

intravenously orally

Valaciclovira

orally

Famciclovirb

orally

Brivudine Foscarnet

orally intravenously

a

Prodrug of acyclovir. Prodrug of penciclovir.

b

antiviral treatment of zoster in immunocompetent adults and adults with mild or moderate immunosuppression. The drug is not approved for the antiviral treatment of VZV infections in childhood and adolescence. Valaciclovir is also not licensed in pregnant women. Although available data are reassuring as to the safety of valaciclovir in pregnancy, there is substantially less experience than with acyclovir. Possible side effects are comparable to those after taking acyclovir.

Famciclovir Famciclovir is the inactive diacetyl ester prodrug of penciclovir, which results from the separation of two ester groups in the small intestine and liver. Penciclovir is an acyclic nucleoside analog derived from ganciclovir by replacement of the ether oxygen atom in the acyclic side chain by a methyl bridge. The oral absorption of penciclovir is very low. Therefore, this drug is only used for the antiviral treatment of local HSV infections. After oral administration, famciclovir has a bioavailability of 77%. Compared to acyclovir, the higher stability of penciclovir triphosphate might result in prolonged antiviral efficacy. Famciclovir is used to treat zoster in immunocompetent adults and immunocompromised patients from 25 years of age. As with valaciclovir, famciclovir is not approved in childhood, adolescence and pregnancy because of limited information on safety. In rare cases, famciclovir may cause headache, mental confusion and nausea.

Foscarnet (trisodium phosphonoformate) The pyrophosphate analog foscarnet inhibits the viral DNA pol of numerous DNA and RNA viruses by preventing pyrophosphate exchange. Since foscarnet does not need to be metabolized for its antiviral activity, it is also effective against TK-negative VZV strains that are resistant to nucleoside analogs. For this reason, foscarnet is recommended as an alternative antiviral treatment if clinical resistance to acyclovir is suspected.

Brivudine ((E)-5-(2-bromovinyl)-2 0 -deoxyuridine) The cyclic nucleoside analog brivudine is converted by viral TK into its mono- and diphosphate. Brivudine is administered orally and has a bioavailability of about 40%. It may be used to treat zoster in immunocompetent adults. Since the safety profile is not known due to a lack of studies, brivudine is not approved for antiviral therapy in children and adolescents. Therefore, the risk-benefit ratio should be carefully assessed before the substance is used in younger age groups, and parents must be informed (off-label use). In principle, brivudine is well tolerated. Nevertheless, gastrointestinal disturbances, impairment of kidney function, an increase in liver enzyme values and reversible changes in blood count may occur. Simultaneous administration of 5-fluorouracil or other 5-fluoropyrimidines leads to increased and potentially dangerous toxicity leading to deaths that have been reported in the past.

Development of Resistance Varicella-zoster virus resistance to antiviral drugs such as acyclovir or foscarnet is rare and has been described in the literature only in immunocompromised patients with zoster, e.g., acquired immunodeficiency syndrome or immunosuppression by cancer or transplantation. The disturbed immune response and the long-term administration of antiviral drugs given as treatment or chemoprophylaxis are key for the development of resistance. The weakened immune response leads to prolonged virus replication, and resistant virus mutants can occur more frequently due to an increased number of natural spontaneous mutations. Under antiviral treatment, resistant viruses are selected and cannot be eliminated by the impaired immune response. In general, resistance is associated with non-synonymous mutations located in the gene of the target molecule or in the gene of proteins responsible for the metabolism or efficacy of antiviral agents. For acyclovir and its nucleoside analogs, resistance is based almost exclusively on non-synonymous mutations of the TK gene (UL36) and rarely on amino acid (aa) changes in the DNA pol gene (UL28), mostly associated with resistance to foscarnet. In contrast to TK, which is not required for the replication of VZV, the DNA pol is an essential enzyme within the viral replication cycle. According to current knowledge, VZV strains that are resistant to acyclovir due

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Fig. 2 VZV thymidine kinase (above) and DNA polymerase (below) with functional domains (gray boxes; ABS: ATP-binding site; NBS: Nucleosidebinding site) and conserved regions (black boxes). In the gray boxes above or below the corresponding protein, the confirmed amino acid substitutions from the literature related to the natural gene polymorphism (NP) or resistance (RES) to acyclovir (thymidine kinase) as well as acyclovir, foscarnet and cidofovir, respectively, (DNA polymerase) are summarized.

to mutations in the TK gene are always cross-resistant to famciclovir and brivudine. To date, there are only a few studies in the literature in which the significance of TK and DNA pol mutations has been proven by phenotypic findings.

Thymidine kinase gene The VZV TK gene is 1026 bp in size and encodes 342 aa (Fig. 2). This gene contains two functional domains, a nucleotide (adenosine triphosphate)-binding site (aa 12–29) and a nucleoside (substrate)-binding site (aa 129–145), and three further conserved regions. Unlike the TK of HSV-1 and HSV-2, few natural polymorphisms have been identified in the VZV TK, but it is important to distinguish each of them from resistance mutations. Compared to the frequently used reference VZV strain Dumas (GenBank accession No. X04370.1, clade 1), all European VZV wild-type strains include the aa polymorphism Serine288Leucine. Similar to HSV, the resistance mutations of the VZV TK gene are assigned to three phenotypes: (1) TK negative (TK-, no TK activity detectable, occurs most frequently). (2) TK reduced (TKr, reduced TK activity, 1%–15% of normal activity). (3) TK altered (TKa, altered TK substrate specificity, no phosphorylation of acyclovir and other nucleoside analogs). Resistance to acyclovir may be caused by stop codons, frameshifts (deletions or insertions) or aa substitutions within and outside conserved gene regions. Since only a few validated resistance mutations have been reported in the literature, new or unknown mutations must always be expected in the genotypic analysis of clinically or phenotypically resistant VZV strains.

DNA polymerase gene The VZV DNA pol gene is 3585 bp in size and encodes 1195 aa (Fig. 2). There are eight conserved regions with the designations I to VII and A. Similar to the TK gene, only a few natural polymorphisms have been reported for the DNA pol. Resistance-related aa substitutions are mostly localized in conserved gene centers. So far, little research has been carried out on natural polymorphisms and resistance-related mutations of the VZV DNA pol gene.

Resistance Testing Antiviral therapy failure is assumed for VZV infections if no clinical improvement can be observed within 10–21 days after starting antiviral drugs, usually acyclovir. This means that there is reason to believe that they are resistant virus strains. In these cases, alternative antiviral treatment with foscarnet is indicated. In parallel, genotypic resistance tests of viral TK and possibly DNA pol should be carried out and, if a virus isolate can be detected in the cell culture, phenotypic resistance tests should also be performed. Due to it taking at least 3 weeks to grow, phenotypic resistance testing has usually no clinical relevance, but the procedure can help to characterize new mutations or gene variations that are not yet clear as to their significance for resistance.

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Phenotyping Phenotyping is regarded as the gold standard for antiviral resistance testing, but this procedure cannot be performed in most cases of suspected clinical resistance because VZV isolation in cell culture has a low sensitivity. The plaque reduction assay has been established as the method of choice. After the addition of the antiviral compound to be tested in a descending dilution series, the 50% effective concentration (EC50) is estimated, resulting in 50% inhibition of viral replication. To assess the possible resistance, a sensitive VZV reference strain, e.g., pOka strain, should be tested in each experiment in parallel as a control. It is a decisive advantage that phenotyping allows a clear interpretation of the results. However, the methods used are time-consuming, materialintensive and non-standardized. In practice, phenotypic resistance tests can only be performed if swabs are obtained from vesicle fluid from which the virus can be isolated in cell culture. For results interpretation, the most common and reliable method for nucleoside analogs is to classify VZV strains as resistant if the measured mean EC50 value is three to five times higher than the corresponding value of the sensitive control strain. For resistance to foscarnet, EC50 values 4330 mM were found to be solid.

Genotyping As with HSV, the VZV genotyping resistance test is performed by amplification and sequencing of TK and DNA pol gene sequences. To identify non-synonymous mutations, sequence data must be compared with the published sequences of a sensitive reference strain available in the gene bank (e.g., VZV strain Dumas, accession number X04370.1). The advantages of genotyping are a significantly shorter delay of approximately 2 days compared to phenotyping and a direct examination of patient samples. The limited amount of viral DNA can have a limiting effect. A major drawback is the fact that only a little information is available on proven resistance-associated aa substitutions. Therefore, only stop codons or frameshift mutations can be interpreted with high probability with respect to resistance. Furthermore, the analysis of genotypic resistance can be difficult if a mixture of viral strains with different genotypes is present. Therefore, in clinical practice, it is a problem to define a questionable resistance of VZV strains on the basis of genotypic results. Recently, phenotypic testing of recombinant VZV isolates has been shown to be the best method to validate the significance of any resistance mutations.

Prevention Prevention of Varicella by Vaccination All available varicella vaccines are live-attenuated vaccines, and nearly all are based on the VZV Oka strain. There is only one nonOka vaccine (SuduvaxTM, Green Cross, South Korea) available. The Oka strain (parental Oka, pOka) was isolated in the early 1970s from the vesicle fluid of a three-year-old child with chickenpox, whose surname Oka was used to designate this strain. Okaderived varicella vaccines induce both humoral and T-cell immunity. The seroconversion rate is used as a criterion for assessing vaccine immunity, i.e., the presence of VZV IgG 6–8 weeks after vaccination in individuals who were VZV IgG-negative prevaccination. In healthy individuals, the seroconversion rate was calculated to be more than 95% after a single vaccine dose and 98%–99% after two doses, while in at-risk patients, the rate was 80%–90%. However, clinical efficacy was estimated to be partially lower depending on the duration of the post-vaccination period. Taken together, the efficacy of the vaccine in healthy children was between 80% and 97% and 93%–96% for the first and second doses, respectively. After universal varicella vaccination was introduced in the United States in 1995, the frequency of varicella was reduced by 90% by 2008. In all age groups, the frequency of varicella fell to 71%–84% by the year 2000, and hospitalization due to varicella decreased threefold to fourfold. In the monitored regions, the average vaccination rates for children were approximately 80%. The pronounced herd immunity also led to a reduction of varicella incidence in non-vaccinated individuals. Studies suggest that the antibodies induced by the vaccine persist in a high percentage of vaccinees over decades. As a result of decreasing or insufficient immunity after a one-dose vaccination, a breakthrough disease from 43 days after vaccination can occur after massive virus exposure. Thus, varicella outbreaks in schools and day-care centers, as well as breakthrough diseases in vaccinated persons, have been reported despite high vaccination coverage in the United States after one-dose vaccination. Because there is only adequate protection if the varicella vaccine is administered twice, a two-dose vaccination scheme was introduced in the early 2000s. A shift in varicella to a higher age is not to be expected with vaccination coverage of Z70% comparable to those in the United States and Germany. Reservations against varicella vaccination are mostly justified by the scenario that a decrease in the incidence of varicella can lead to a reduced spread of wild-type viruses and that older people can develop zoster more frequently. In the United States, an influence of varicella vaccination on the incidence of zoster in adults could not be observed, and the current data from different countries confirm these findings. However, zoster has been shown to be 3–12 times less common in children vaccinated against varicella than in unvaccinated children. The disease in vaccinated children tends to be clinically milder and the exanthemas often occur anatomically in the dermatomes corresponding to the sites where the varicella vaccine was given. In July 2004, universal varicella vaccination was included in the vaccination schedule for all children and adolescents in Germany. By 2011, vaccination coverage had risen to almost 70%. Initially, a single vaccination with monovalent varicella vaccines was recommended for children aged between 1 and 13 years. Since 2009, the recommendation of the Standing Vaccine Commission of the Robert Koch Institute (STIKO, Berlin, Germany) has provided for a two-dose schedule as the standard for varicella vaccination. In subsequent years, this recommendation was implemented without reducing vaccination rates. The first dose of varicella vaccine is administered at the age of 11–14 months, either at the same time as the first measles-mumps-rubella (MMR) vaccination or at the earliest 4 weeks later. The second dose of varicella vaccine is recommended at the age of 15–23 months, preferably with the

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Table 4 Risk groups

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Recommendations for indication immunizations against varicella in Germany Persons

Individually (not occupational) increased Seronegative women who want children exposure, disease or complication risk, Seronegative patients before planned protection of others

immunosuppressive therapy or organ transplantation

Remarks

Two doses of varicella vaccine Special hints for vaccination in patients under immunosuppressive therapy without VZV-specific antibodies should be considered

Susceptible persons with severe neurodermitis “Susceptible persons” means: No vaccination and no varicella history or anti-VZV IgG-negative in case of Susceptible persons with close contact to the serologic testing two aforementioned Occupational

Total of 2 vaccinations (if MMR vaccination is indicated, Seronegative persons (including trainees, interns, students and volunteers) in the use MMRV combination vaccine if applicable) following fields of activity: • Medical facilities including facilities of other human medical health care professions • Activities involving contact with potentially infectious material • Care facilities • Community facilities • Facilities for the joint accommodation of asylum seekers, persons obliged to leave the country, refugees and late repatriates

combined measles-mumps-rubella-varicella (MMRV) vaccine. Since a slightly increased risk of febrile seizures was observed 5–12 days after the first vaccination with the combined MMRV vaccine, the information currently available suggests that separate administration of trivalent MMR vaccine and monovalent varicella vaccine should be preferred. For unvaccinated 5–17-year-olds without a varicella history, two-dose varicella or MMRV vaccination is recommended as a catchup vaccination. The minimum interval between two doses of the varicella or MMRV vaccine should be 4–6 weeks. The varicella vaccine is recommended as vaccination for all individuals summarized in Table 4. Currently, the monovalent varicella vaccines Varilrix® from GlaxoSmithKline and Varivax® from MSD Sharp & Dohme GmbH as well as the tetravalent MMRV vaccine PriorixTetra® from GlaxoSmithKline are available in Germany. All vaccines are administered subcutaneously (s.c). The recommendations provide varicella vaccination as post-exposure prophylaxis for non-vaccinated individuals without varicella history after contact with individuals at risk. Vaccination should be carried out within 3 days after the onset of the exanthema in the index case or within 5 days after contact with the index case. Sentinel results from the “Varicella” working group at the Robert Koch Institute showed a decrease in varicella incidence of 80%–90% by 2012, affecting all age groups. However, the age groups most affected were those for which the vaccine recommendations applied (1–4 years) or where the vaccination was highly effective (5–9 years). It is significant that infants susceptible to varicella also benefit from herd immunity. Important contraindications of varicella vaccination are intensive immunosuppressive therapy and pregnancy including the period 4–6 weeks before a planned pregnancy. However, according to current knowledge, there is no risk of prenatal malformations if the varicella vaccination is given accidentally during or shortly before pregnancy. In very rare cases, the transmission of the vaccine virus has been reported from immunocompetent vaccinated individuals to susceptible contacts persons. This is basically possible if the vaccine develops an exanthema with vesicles through which the virus can be transmitted. In contrast, immunodeficient patients with varicella caused by the vaccine virus may have a higher risk of virus transmission. Persons at risk for severe varicella, including pregnant women and neonates of mothers with no history of varicella, should therefore avoid contact with vaccinees. However, pregnancy is not considered a contraindication for vaccinating an unprotected child. After varicella vaccination, side effects such as redness and swelling at the injection site and pain at the injection site may occur in about 4% of vaccinations. 3%–5% of vaccinated children develop skin lesions localized at the injection site. In a further 3%–5% of children, less pronounced varicella-like exanthema may occur 2–6 weeks after vaccination, but adolescents and adults are affected twice as often as children. Varicella-like exanthemas within 2 weeks after vaccination are usually caused by wild-type virus infections. Later exanthemas up to 42 days after vaccination are usually associated with the vaccine virus. Accidental vaccination of seropositive individuals is not associated with an increasing number of side effects.

Passive Immunoprophylaxis of Varicella Passive VZV immunoprophylaxis with varicella-zoster immunoglobulin (VZIG) can prevent the onset of varicella or considerably attenuate the course of the disease. Therefore, the administration of VZIG was recommended for susceptible at-risk patients after exposure to varicella or zoster. These include the groups summarized in Table 5. If non-vaccinated pregnant women without a varicella

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Table 5 Susceptible at-risk patients for whom the administration of VZIG (inclusive dosage and point in time) is recommended after exposure to varicella or zoster in Germany Risk patients

Dosage

Point in time

As soon as possible and Unvaccinated pregnant women without varicella history VZIG i.v.: 25 IU/kg (0.1 ml/kg/h for 10 min, with good not later than 96 h after Immunocompromised patients whose immune status tolerability increase to maximum 1 ml/kg/h) against varicella is uncertain or negative VZIG i.m.: 15 IU (normally 0.2 ml) per kg, divided between exposure

different parts of the body if more than 2 ml for children up to 20 kg, and more than 5 ml for adults Newborns whose mothers develop varicella within 5 days before and 2 days after birth

1 ml/kg i.v. (or 0.5 ml/kg i.m.)

Preterm infants from 28 weeks gestation whose mothers 1 ml/kg i.v. (or 0.5 ml/kg i.m.) have no immunity, after exposure during the neonatal period Preterm infants younger than 28 weeks after exposure during the neonatal period, regardless of the immune status of the mother

Immediately after birth or outbreak of exanthema in the mother As soon as possible and not later than 96 h after exposure

history are passively immunized, it should be noted that the main reason for administering immunoglobulin is to protect them from severe varicella infections and complications. So far, there is no evidence that this cost-intensive approach prevents the spread of fetal disease in the form of congenital varicella syndrome. However, one should be aware that the administration of VZIG is the only way to prevent congenital varicella syndrome after viral exposure of an unprotected woman during the first 20 weeks of pregnancy. An important prerequisite for the efficacy of passive immunoprophylaxis is the timely administration of VZIG. While previous instructions recommended administration within 3 days and a maximum of 10 days after the beginning of exposure, the current STIKO recommendation recommends “as soon as possible and not later than 96 h after exposure”. An “exposure“ was defined as (1) one hour or longer together with an infectious person in a room, (2) personal contact or (3) household contact. For the administration and dosage of VZIG (Table 5), the manufacturer's instructions must be considered. If necessary, the post-exposure application of VZIG can be combined with antiviral chemoprophylaxis.

Prevention of Zoster by Vaccination The age-related increase in zoster incidence correlates with the decrease of specific T-cell immunity. Therefore, an attempt was made to stimulate the specific cellular immunity in elderly people and thus enable the prevention of zoster. Inspired by the success of the varicella vaccine, a live-attenuated zoster vaccine was developed at the beginning of 2000 containing at least 14-fold higher concentrations of the vaccine virus than in the varicella vaccine. A large study has shown that vaccination of adults can reduce the incidence and severity of zoster by approximately 50% and the incidence of post zoster neuralgia (PZN) by 67%. According to the available information, protective immunity is maintained for at least 7 years. The vaccine is well tolerated. Site effects are local reactions at the injection site such as redness, swelling, pain and touch sensitivity. Under the brand name Zostavax®, the first vaccine for the prevention of zoster and PZN for people aged 50 and over was approved in Europe in 2006. Sanofi Pasteur MSD (Lyon, France) holds the European marketing authorization. The vaccine is administered s.c. as a single dose. Like all live vaccines, this zoster vaccine is contraindicated for immunosuppressed patients and pregnant women. The vaccine can be administered at the same time as the inactivated influenza vaccine while it should not be administered at the same time as the pneumococcal vaccine. Zostavax® has been available in Germany since September 2013. Although the zoster vaccination was part of the public vaccination recommendations in different federal states of Germany, the STIKO has not issued an official recommendation to use a live-attenuated zoster vaccine for standard vaccination. Accordingly, the vaccination with the live zoster vaccine was not an affordable service within health insurance. The main reason for this was that the effectiveness of the vaccination decreases with increasing age and ranges from 70% in the 50–59-year-olds to 41% in the 70–79-year-olds to less than 20% in the Z 80-year-olds. Thus, the protection period of the vaccination was documented only for a few years. Furthermore, modeling results from clinical studies showed only a small reduction of the total number of zoster cases by vaccination, which can be between 2.6% (vaccination with 50 years) and 0.6% (vaccination with 80 years) depending on the age of the vaccinated person. In addition, immunosuppressed people who have a significantly increased risk of developing zoster and its complications cannot be vaccinated with the attenuated live vaccine. The epidemiological risk-benefit assessment of the vaccination with Zostavax® did not lead to the recommendation of a standard vaccination in Germany. However, the vaccination of an individual patient can make sense after an individual risk-benefit assessment. Several years ago, a second zoster vaccine based on recombinant adjuvant VZV-specific glycoprotein E (gE) was developed, which is of fundamental importance for the development of VZV-specific immunity. A liposome-based adjuvant serves as an enhancer of immunity. Since March 2018, this new zoster subunit inactivated vaccine designated Shingrix® and manufactured by

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GlaxoSmithKline has been approved in Europe for the prevention of zoster. After administration of two doses, this inactivated vaccine reduced the frequency of zoster between 90%–97% in immunocompetent individuals over 50 years of age in phase III studies, and the vaccine efficacy against PHN was 89% in adults over 70 years of age. So far, two studies on immunodeficient patients have also been conducted. After three vaccine doses at one and three-month intervals, respectively, in 121 adult cancer patients who had undergone an autologous stem cell transplantation, one month after the last immunization (6 months after transplantation), humoral and cellular gE-specific immunity was significantly increased compared to placebo and persisted for up to one year. A similar study of 123 adult HIV patients also demonstrated an increased specific cellular and humoral immune response persisting for 18 months. All available data demonstrated that the adjuvant zoster subunit inactivated vaccine could effectively prevent zoster and PZN in immunocompetent people 50 years of age and older. Efficacy across all age groups from 50 years of age is 92% for protection against zoster and 82% for protection against PZN. Vaccination is safe but very reactive. Local and systemic reactions such as pain at the puncture site or fever, fatigue, headache and myalgia of 1–2 days often occur. It is of great advantage that the vaccine also induces a robust humoral and cellular immune response in immunodeficient individuals. Since December 2018, STIKO, in Germany, has recommended vaccination with the adjuvant zoster subunit inactivated vaccine for general use as a standard vaccine for persons aged 60 years and older and as an indication vaccine for immunosuppressed persons and patients with other severe basic diseases (e.g., rheumatoid arthritis, chronic kidney disease, chronic obstructive lung disease, diabetes mellitus) 50 years and older to prevent zoster, its complications and late sequelae. The vaccination series consists of two intramuscular (i.m.) vaccinations at intervals of at least 2 months and up to a maximum of 6 months. This vaccination is no substitute for an indicated chickenpox vaccination.

Further Reading Cunningham, A.L., 2016. Efficacy of the herpes zoster subunit vaccine in adults 70 years of age or older. New England Journal of Medicine 375, 1019–1032. Damm, O., 2015. Systemic review of models assessing the economic value of routine varicella and herpes zoster vaccination in high-income countries. BMC Public Health 15, 533. De Clercq, E., 2013. Selective ant-herpesvirus agents. Antiviral Chemistry & Chemotheapy 23, 93–101. Robert Koch Institute, 2017. STÄNDIGE IMPFKOMMISSION (STIKO): Wissenschaftliche Begründung für die Entscheidung, die HEPES-ZOSTER-Lebendimpfung nicht als Standardimpfung zu empfehlen. Epidemiologisches Bulletin 36, 391–410. Robert Koch Institute, 2018. STÄNDIGE IMPFKOMMISSION (STIKO): Wissenschaftliche Begründung zur Empfehlung einer Impfung mit dem HERPES-ZOSTER-subunitTotimpfstoff. Epidemiologisches Bulletin 50, 541–567. Robert Koch Institute, 2019. EMPFEHLUNGEN der STÄNDIGEN IMPFKOMMISSION (STIKO) beim ROBERT KOCH-INSTITUT – 2019/2020. Epidemiologisches Bulletin 34, 313–357. Leroux-Roels, I., 2012. A phase 1/2 clinical trial evaluating safety and immunogenicity of a varicella zoster glycoprotein e subunit vaccine candidate in young and older adults. Journal of Infectious Diseases 206, 1280–1290. Lopez, A.S., 2014. Two-dose varicella vaccination coverage among children aged 7 years – Six sentinel sites, United States, 2006–2012. Morbidity and Mortality Weekly Report 63, 174–177. Morfin, F., 2003. Herpes simplex virus resistance to antiviral drugs. Journal of Clinical Virology 26, 29–37. Oxman, M.N., 2005. A Vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. New England Journal of Medicine 352, 2271–2284. Pasternak, B., 2010. Use of acyclovir, valacyclovir, and famcciclovir in the first trimester of pregnancy and the risk of birth defects. Journal of the American Medical Association 304, 859–866. Piret, J., 2016. Antiviral resistance in herpes simplex virus and varicella-zoster virus infections: Diagnosis and management. Current Opinion in Infectious Diseases 29, 654–662. Sauerbrei, A., 2014. Windpocken (Varizellen). In: Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten e.V. (DVV e.V.)., Gesellschaft für Virologie e.V. (GfV e.V.). (Eds.), S2k-Leitlinie Labordiagnostik schwangerschaftsrelevanter Virusinfektionen. Berlin, Heidelberg: Springer. pp. 95–110. Sauerbrei, A., 2001. Resistance testing of clinical varicella-zoster virus strains. Antiviral Research 90, 242–247. Sauerbrei, A., Wutzler, P., 2007. Varicella-Zoster Virus-Infektionen: Aktuelle Prophylaxe und Therapie, second ed. Bremen/London/Boston: Uni-Med.

Treatment and Prevention of Herpesvirus Infections in the Immunocompromised Host Sara H Burkhard and Nicolas J Mueller, University Hospital of Zurich, Zurich, Switzerland r 2021 Elsevier Ltd. All rights reserved.

Introduction Immunodeficiencies are caused by the defective function of one or several components of the immune system. They can be divided into genetically inherited primary deficiencies and acquired secondary immune deficiencies. Primary immunodeficiencies can go unnoticed due to the lack of symptoms or result in serious and recurrent infections. Although severe defects are rare, they often lead to high mortality in early childhood and are predominantly inherited in a recessive and X-chromosome linked manner. Secondary immune defects are much more common. They can be caused by environmental factors such as malnutrition or result from diseases like infections and cancer, or the administration of cytotoxic drugs. In such conditions, the immune defect is often unwanted, while in some cases, the immunosuppression is induced with therapeutic intention. Exemplary, organ transplantation would not be possible without immunosuppressive drugs because a fully functional immune system would cause the rejection of the allograft. Immunodeficiencies can also be distinguished by the nature of the underlying defect. Primary immunodeficiencies are currently classified into nine categories defined by the signaling pathway or immune cell type affected. The study of these distinct genetic defects and associated diseases has aided the understanding of the immune response to various pathogens. We now know that the control of viral infections is mainly dependent on T cell function and, to a lesser extent, Natural killer (NK) cells and antibodies. Secondary immune defects roughly cluster into two main groups and a combination thereof. In the first group, patients are treated with cytotoxic drugs and radiation therapy, which results in the impaired generation of neutrophil granulocytes and mucosal barrier dysfunction. The deficiency in this first line of immune defense usually predisposes individuals to bacterial and fungal infections. The second group includes patients in whom T cells are targeted, yielding a defect in cellular immunity. This is usually the case in patients treated with immunosuppressive drugs to contain autoinflammatory disorders or to prevent the rejection of a transplanted organ. A T cell defect is also the underlying cause of the immunosuppressive state of patients suffering from acquired immune deficiency syndrome (AIDS) caused by the infection with the human immunodeficiency virus (HIV). The HIV pandemic, which started in the 1980s, raised awareness of the diverse types of opportunistic pathogens that take advantage of the impaired T cell function. Viral infections were observed frequently, many of them being caused by members of the Herpesviridae family. The high incidence of herpesvirus infections among AIDS patients is, to some degree, explained by the ability of herpesviruses to latently persist in a large percentage of the population. As seen in HIV infected subjects, the loss of T cell surveillance can then lead to herpes virus reactivation, replication, and subsequent disease. Classically, AIDS patients would present with cytomegalovirus (CMV) retinitis, Epstein–Barr virus (EBV)-associated lymphoma or the human herpesvirus (HHV)-8 associated Kaposi sarcoma. Thanks to more efficient antiretroviral therapies, HIV replication can now be controlled in the great majority of patients, which leads to recovery of T cell function. For patients with access to treatment, this results in a similar susceptibility to infections as seen in the general population. There is a rising number of patients treated with immunosuppressive drugs. This is explained by an increase in the number of conditions requiring immunosuppression and the prolonged survival of patients under improved therapeutic regimens. For autoinflammatory disorders, the blockade of specific pathways involved in the pathogenesis of disease has proven successful. This may be achieved by monoclonal antibodies or small molecule inhibitors. Although such patients may still suffer from opportunistic infections, the therapeutic immune defect is usually more limited, often not resulting in an elevated susceptibility to infection. Transplantation medicine is another field that has seen major advances in the last few decades but still requires a more general immunosuppressive approach. This immunosuppression is achieved by the combination of immunosuppressive drugs that are usually administered for a lifetime. Although subsequent development of a certain immune tolerance to the transplanted organ allows reducing drug dosages, patients are particularly vulnerable in the early months after transplantation. In this phase, induction therapy and initiation of high dose maintenance immunosuppressive therapy prevent acute organ rejection. Just like in antiviral immunity, allograft rejection is mainly mediated by T cells. Upon transplantation, T cells are activated by non-self major histocompatibility complexes (MHC). To limit the cellular immune response, donor and recipient MHC classes are matched before transplantation. However, the lack of organs eligible for transplantation and the urgency of finding a recipient for an organ from a deceased donor limit accurate MHC matching. This, in turn, results in the need for more potent immunosuppression. Also, the adaptive response to minor histocompatibility antigens is not accounted for pre-transplantation and is thought to mirror immunity to viral proteins. It is evident that the immunosuppression that prevents allograft rejection will lead to an increased susceptibility to viral diseases, and again herpes virus infections are at the forefront. Therefore, we will focus on the presentation, management, and treatment of herpesvirus infections in transplanted patients.

Cytomegalovirus (Human Herpesvirus 5) Cytomegalovirus (CMV) is one of the most common infections occurring after organ transplantation and is related to significant morbidity and loss of graft function. In the immunocompromised host, CMV infection is often more severe,

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causing meningoencephalitis, retinitis, gut inflammation, hepatitis, pneumonitis and polyradiculopathy. These end-organ diseases are the result of the invasion and local tissue damage by CMV. However, most often, CMV disease presents with non-specific symptoms such as fever, malaise, decreased leukocyte and platelet count, or elevated liver function values. This symptom complex is usually referred to as a viral syndrome.

Pre-Transplant Risk Stratification The main strategy of disease prevention begins with the risk stratification before transplantation. This is performed by serological testing, which is achieved by measuring donor and recipient CMV IgG. Thereby, the clinician can evaluate whether donor and recipient have been exposed to CMV previously. For seronegative organ recipients, serological testing is periodically repeated before transplantation as primary infection and subsequent seroconversion could occur. Upon solid organ transplantation the highest risk of infection is observed when a seropositive donor is combined with a seronegative recipient (D þ /R). In this case CMV can be transmitted via the organ transplanted into a host lacking CMV immunity. In this setting, the risk of CMV disease post-transplantation is as high as 56%. Intermediate risk is expected in seropositive recipients (R þ ) due to CMV reactivation upon immunosuppression. Simultaneous infections with different CMV serotypes are thought to explain the marginally elevated risk in the D þ /R þ compared to the D/R þ setting. D/R serostatus is linked to the lowest risk in adult recipients, as de novo CMV infection is rare. Prior to hematopoietic stem cell transplantation, recipient immune cells specific for CMV are usually eliminated due to induction therapy. Thus, post-transplantation immunity depends on donor immune cells. Studies have identified a survival advantage for recipients receiving an allotransplant from a seropositive donor compared to those receiving stem cells from seronegative donors. This protective effect was eliminated by T cell depletion, suggesting a central role of CMV specific donor-derived T cells for the control of CMV infection. The serological risk stratification for CMV before transplantation has a direct impact on patient management and prevention of disease after transplantation. It may, for instance, guide the indication and duration of prophylactic measures taken. Due to the inaccuracy of disease prediction by the D/R risk stratification, there is a need for alternative methods. Assessment of the CMV-specific T cell quantity and functionality has been investigated and shows promising results. Methods involve enzyme-linked immunosorbent assays (ELISAs) or enzyme-linked immunosorbent spots (ELISpots) measuring interferon (IFN) g release by T cells activated with CMV peptides, some of which are commercially available. Assays using intracellular cytokine staining or MHC-multimer staining of CMV specific T cells rely on analysis by flow cytometry. Performing such T cell assays before transplantation does not take into consideration the influence of the immunosuppression after transplantation, often severely impairing T cell responses. Currently, there is limited literature on the use of pre-transplantation risk stratification by T cell assays.

Prevention Strategies of CMV Disease After Transplantation The two approaches of “universal prophylaxis” and “pre-emptive therapy” have been proven valuable to prevent CMV disease after solid organ transplantation. For universal prophylaxis, patients are given an antiviral drug in reduced dosage. The administration should be started shortly after transplantation, as this is the time where the threat of acute rejection demands the most potent immunosuppressive regimen. Prophylaxis is continued for the first 3–12 months after transplantation depending on the transplanted organ type, immunosuppressive regimen and serological risk stratification. In the D/R group, with the lowest risk, preventive therapy is not recommended. The prophylactic approach harbors the risk of late-onset CMV disease after discontinuation of antiviral prophylaxis. The late-onset disease affects about 20% of patients and is a particular concern to patients with a D þ /R serostatus, severe immunosuppression and transplant rejection. Another drawback of universal prophylaxis is that patients are exposed to antiviral treatment and adherent adverse effects for a substantial time period. This is particularly critical for subjects undergoing stem cell transplantation where the toxic effects of some antiviral drugs on the bone marrow limit the use of universal prophylaxis. In those populations, clinicians can opt for preemptive therapy. This approach involves the frequent screening for CMV replication after transplantation, followed by antiviral treatment should viral DNA be detected. Early antiviral intervention is essential as it can limit the severity of CMV related disease. Surveillance is usually performed by the measurement of CMV DNA by quantitative PCR in plasma or whole blood. The positive result is commonly referred to as DNAemia, as the majority of CMV DNA detected in the blood was demonstrated to be fragmented and non-functional. Since the World Health Organization standardization of qPCR protocols to measure DNAemia, there is a higher degree of assay reproducibility between laboratories. There is, however, no agreement on what DNA threshold should trigger antiviral treatment and whether this threshold should be adapted to the serological CMV risk. It has been suggested that the change in the level of DNAemia over time should be considered to be relevant in predicting disease. In case CMV qPCR is not available, measurement of the CMV antigen pp65 by ELISA can be a valuable alternative. In contrast, measurement of CMV IgG is not useful for post-transplantation surveillance as serological responses are impaired or delayed due to the immunosuppression. The preemptive strategy was shown to have similar overall outcomes regarding CMV disease and graft function as the universal prophylaxis for most types of transplants. While preemptive therapy is associated with a higher rate

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of CMV DNAemia, late-onset CMV disease and side effects of treatment are more frequently observed in patients receiving prophylactic medication. Thus, the choice of approach is determined by the individual risk for CMV disease and antiviral toxicity. The main drug used for CMV prevention is the oral prodrug valganciclovir, while intravenous ganciclovir is administered on rare occasions. These drugs are ideal for prophylaxis as they also protect against other herpes virus infections, e.g., varicella-zoster virus or herpes simplex virus. Unfortunately, myelosuppressive adverse effects limit the use of ganciclovir and valganciclovir after hematopoietic stem cell transplantation and are frequently observed after solid organ transplantation. Letermovir, an inhibitor of the terminase complex of CMV, was recently approved for CMV prophylaxis in hematopoietic stem cell transplant recipients. In clinical trials using letermovir, hardly any myelotoxicity was observed. Importantly, letermovir has no antiviral activity against other herpes viruses; hence additional administration of prophylactic antiviral compounds may be required. Alternative substances are currently being evaluated in clinical trials. The use of intravenous immunoglobulins or CMV-specific IgG was suggested to reduce the incidence and severity of CMV disease. Due to the better control of CMV replication under ganciclovir or valganciclovir, the sole application of immunoglobulins for prophylaxis has become uncommon. However, it may be useful for high-risk patient populations, given in combination with antiviral compounds or when managing subjects with contraindications for common antiviral drugs. Currently, monoclonal CMV antibodies are in development. Various vaccines are also being engineered and some have proceeded to early clinical studies. Vaccination approaches involve the use of live attenuated virus, viral vectors, recombinant subunits or gene-based vaccines, some of them providing promising results. Several vaccinations were shown to induce the production of neutralizing antibodies, lower DNAemia, preventing infection or reducing CMV episodes.

Diagnosis of CMV Disease and Treatment In the case of a viral syndrome, the association of non-specific symptoms and detection of CMV DNA in the blood usually prompts the initiation of therapy. Tissue invasive CMV disease, in contrast, does not always cause elevated DNAemia. This is most frequently observed in gastrointestinal disease or pneumonitis. Therefore, detection of the virus in biopsy material should be performed whenever possible to confirm the suspected diagnosis. Histological criteria such as inclusion bodies or detection of viral antigen by immunohistochemistry are used to diagnose localized CMV disease. Once the diagnosis has been established, oral valganciclovir and intravenous ganciclovir are the preferred agents. Both antiviral drugs have been shown to achieve comparable long-term outcomes. As oral therapy is more convenient, intravenous treatment with ganciclovir is reserved for severe disease and where oral bioavailability may be impaired, for example, in gastrointestinal disease due to rapid intestinal passage or mucosal damage. Treatment should be continued for a minimum of two weeks. To monitor therapy success, weekly blood CMV DNA load testing is performed. Usually, treatment is only stopped when two negative DNA measurements have been obtained or DNAemia drops below a certain threshold. For tissue invasive disease treatment can be stopped when clinical symptoms have ceased, but a two-week course is usually given to all patients. Recent studies suggest that the assessment of CMV specific T cell immunity may assist the decision to stop the administration of antiviral substances in primary infection. Using a CMV specific ELISA for IFN g release by T cells, antiviral treatment could safely be stopped when a CMV specific immune response had been detected. Similarly, intracellular staining-based assays demonstrated the inability to clear CMV DNAemia in the absence of T cell responses. Apart from pharmacological treatment of CMV disease, optimizing host factors can support immune control. Lower blood concentrations for calcineurin inhibitors – an immune-suppressing agent frequently used after solid organ transplantation – have been associated with more rapid clearance of DNAemia. In contrast, immunosuppressive regimens containing a mammalian target of rapamycin (mTOR) inhibitor may be advantageous due to their suggested direct anti-CMV effect. In severe disease, the reduction of all immunosuppressive agents should be considered. Upon successful treatment of the disease, some patients will develop recurrent CMV disease. After solid organ transplantation, CMV relapses affect up to 35% of patients. Certain risk factors are connected with the development of CMV relapses. These include primary infection, deceased-donor transplantation, high-level CMV DNAemia, delay in CMV DNA regression after the initiation of treatment, multimorbidity, transplant rejection, strong immunosuppressive regimens, thoracic organ transplants and invasive gastrointestinal disease. Patients at risk should be closely monitored for CMV DNAemia and symptoms after discontinuation of therapy. To further limit CMV relapses, the administration of prophylactic medication after resolved CMV disease was trialed. As subsequent studies demonstrated that this approach of secondary prophylaxis failed to prevent relapses, it is not generally recommended. Still, many centers have adopted this strategy.

Antiviral Drug Resistance Antiviral drug resistance is rarely an issue in immunocompetent hosts, as CMV infection does not usually require treatment. In contrast, immunocompromised patients may suffer from a clinical disease that cannot be controlled by the suppressed immune response. Thus, antiviral therapy is initiated and exerts a selection pressure required for drug-resistant viral subpopulations to propagate. Drug-resistant CMV first emerged among patients with AIDS, where the loss of drug susceptibility complicated as many as 20% of cases after 9–12 months of antiviral treatment. Optimizing treatment regimens reduced the frequency of antiviral drug resistance, which now occurs in 5%–12% of transplant recipients.

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Certain host and therapy-related factors are connected with an increased risk of developing CMV drug resistance. These include prolonged antiviral drug exposure and persistent CMV replication under therapy due to impaired CMV immunity. The latter is often observed in the D þ /R risk group, in patients on more potent immunosuppressive regimens or with inadequate CMV treatment. The emergence of ganciclovir resistance seems to be connected to antiviral drug doses used for CMV treatment, especially if it is repeatedly started and stopped, as it is only observed in up to 3% of the patients given prophylactic doses. Drug resistance should be suspected and investigated when patients do not improve or relapse under treatment. Although resistance rarely develops during the first 6 weeks of antiviral therapy, testing is recommended in unresponsive patients after two weeks of therapy. However, it is not unusual that DNAemia increases after initiation of therapy during the first week. Single or multiple mutations lead to different levels of drug resistance and further mutations can be acquired by the virus over time. Testing for resistance is performed by sequencing of viral DNA and comparison with the wild-type sequence. As with all sequence-based resistance testing, mutations can reflect mere sequence polymorphism or confer to drug resistance. The amount of evidence for any given mutation to result in resistance reaches from theoretical predictions to in vitro assays or description of clinical cases. The level of resistance is often reported as the change of drug concentration required to suppress viral propagation by 50%. These fold-changes are then categorized so that a 2–5 fold increase refers to a low-grade, 5–15 fold is moderate and 415 means highlevel resistance. The numbers obtained for single mutations can be added should multiple mutations be detected. Genotypic assessment of resistance requires a certain quantity of CMV DNA. Thus, resistant viral subpopulations of low frequency may not be detected, leading to the assumption that the virus is drug susceptible. Also, resistant CMV subpopulations have been isolated from specific organs, while the mutation was not detected in CMV DNA isolated from blood. Applying technologies such as deep sequencing may improve the detection of drug-resistant subpopulations in future. Sequencing of CMV codons 400–670 and 300–1000 are usually performed as these contain the most known mutations involved in resistance in the UL97 kinase and the UL54 DNA polymerase, respectively. In 90% of patients on ganciclovir treatment, the UL97 kinase mutation appears before the UL54 polymerase mutation. UL54-associated resistance may signify crossresistance to other antiviral drugs and often leads to a loss of viral fitness. If a previously unknown mutation is detected by sequencing, resistance can be assessed by recombinant phenotyping. For this assay, the mutated gene is transferred to a laboratory CMV strain that is then tested for phenotypic resistance to antiviral substances. This is a timely and labor-intensive process and is therefore rarely used clinically. In the case of low-level resistance, an increase in ganciclovir dosage and improving host factors, such as reducing immunosuppression, may be sufficient to overcome resistance. If genetic testing reveals a high-level of resistance, switching the antiviral agent is recommended. In case of life-threatening disease, the antiviral drug foscarnet is added prior to genetic testing for resistance. The salvage therapy using foscarnet is often successful, but metabolic side effects and nephrotoxicity limit its use. Newer compounds such as letermovir, maribavir and brincidofovir are still not approved for the treatment of CMV disease but may be available for compassionate use. The use of CMV hyperimmunoglobulin and intravenous immunoglobulin has thus far only been reported in case studies. The idea of improving cellular immunity towards CMV has fueled the study of adoptive T cell transfer. Recently, this approach was reported to be successful in a number of case studies. An adoptive T cell transfer may play a role in complex cases as the generation of autologous CMV T cells takes several weeks and is costly. Therefore, the creation of HLA matched T cell banks is of great interest. Several agents have been used for off label treatment due to their anti-CMV effects in vitro. Such drugs include the mTOR inhibitors sirolimus and everolimus, leflunomide and artesunate, and addition to antiviral therapy can be considered in complicated cases.

HSV (Herpes Simplex Virus, Human Herpesvirus 1 and 2) Immunosuppressed patients experience more severe herpes simplex virus (HSV)-associated disease and shed virus more frequently. Primary infection post-transplantation has been described, but disease mostly results from reactivation of latent HSV after a previous infection. Therefore, serological testing before transplantation is useful to assess the individual post-transplant risk. Most patients present with typical mucosal and skin lesions, even after immunosuppression, allowing diagnosis on clinical grounds. Lesions can, however, be atypical and include unusual locations. This can render clinical discrimination from lesions caused by other diseases difficult. Consequently, a diagnosis should involve the detection of HSV DNA by PCR from samples collected in mucocutaneous lesions. PCR was shown to be the most sensitive test for HSV detection. Although less sensitive, direct fluorescent antibody assays deliver rapid results. More severe HSV diseases, such as disseminated mucocutaneous disease, visceral disease, esophagitis, hepatitis, and pneumonia, are also observed. Just as in immunocompetent hosts, retrograde reactivation can lead to disease of the central nervous system. Obtaining tissue, cerebrospinal fluid or blood samples for the detection of HSV DNA confirms the diagnosis. Many transplant recipients receive prophylactic valganciclovir or ganciclovir to prevent CMV disease, effectively averting HSV reactivation or infection. For patients not receiving CMV antiviral prophylaxis, or when a preemptive strategy is adopted, HSV prophylaxis may be considered in HSV seropositive transplant recipients. This is also true for the use of agents for CMV prophylaxis that do not have anti-HSV-activity, as is the case with letermovir. Preventive measures should cover the first months after transplantation because this is when the majority of HSV infections occur. Prophylaxis and treatment involve antiviral medication with aciclovir and valaciclovir. With localized disease, oral regimens

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are preferred and are continued for a minimum of 5–7 days and until the HSV lesions have resolved. For severe manifestations, intravenous administration of aciclovir and the reduction of immunosuppressive medication should be considered. As immunosuppressed patients are exposed to longer courses of antiviral treatments, aciclovir resistance is an emerging issue. Frequency of drug resistance in such patients can range from 2.1% to 10.9% and it is more often observed in AIDS patients or after hematopoietic stem cell transplantation compared to patients receiving solid organ transplants. Drug resistance should be considered in the case of treatment-refractory disease and a history of previous exposure to antiviral therapy. Mutations causing aciclovir resistance are most frequently detected within the thymidine kinase UL23, implying a cross-resistance to other antiviral substances such as famciclovir, valaciclovir and ganciclovir. Both genetic testing and phenotypic assays are used to identify mutations causing drug resistance. While waiting for antiviral resistance to be confirmed, the antiviral regimen is usually switched to foscarnet or cidofovir. After successful treatment, aciclovir resistance is sometimes lost in virus strains detected upon HSV reactivation. Recurrent disease is defined as two or more HSV reactivations after the first episode. In this case, suppressive pharmacological therapy can be considered. Such prophylactic treatment was shown to be associated with a lower frequency of aciclovir resistance when compared to repetitive treatment of relapses.

Varicella Zoster Virus (VZV, Human Herpesvirus 3) Primary infection (chickenpox) is most often seen in children and rarely in adults. On the contrary, Varicella-zoster virus (VZV) reactivation (shingles, herpes zoster) is observed frequently particularly in the immunosuppressed host. Most patients with immune defects present with a classical manifestation of VZV infection. The frequency of severe symptoms, such as multi-dermatomal herpes zoster, a prolonged disease course and complications is, however, elevated in immunocompromised patients. Rarely, VZV reactivation can affect internal organs, most commonly causing pneumonia, encephalitis and hepatitis, referred to as visceral herpes zoster. In some cases of visceral disease, patients may lack skin lesions. If skin lesions are present, they are more likely to be atypical in appearance. For more uncommon presentations, PCR is the most sensitive test to detect viral replication in the tissue or body fluids. As a distinction from HSV infection may be difficult clinically, multiplex assays detecting both HSV and VZV have been developed. Alternatively, fluorescent labeling of viral antigen can be used in tissue biopsies or scrapings from vesicles and ulcers. Assessing previous VZV exposure by serology VZV is suggested for patients on transplant waiting lists. The result is used to estimate the post-transplantation risk of infection but also determines if patients are eligible for vaccination and what vaccine should be used. Live attenuated Oka vaccine may be offered to susceptible individuals before transplantation. For seropositive patients above 50 years of age, vaccination with either the live-attenuated Oka herpes zoster vaccine or the adjuvanted subunit herpes zoster vaccine is recommended. These vaccines are partially effective as they can reduce the incidence and severity of disease but fail to prevent herpes zoster completely. Recent recommendations favor the recombinant subunit over the live attenuated vaccine as it shows superior efficacy against herpes zoster and elicits a more long-lasting effect in immunocompetent subjects. Post-transplantation live attenuated vaccines are contraindicated but the subunit vaccine for herpes zoster can still be safely applied. Initiation of herpes zoster treatment is suggested up to 72 h after the appearance of first cutaneous lesions, but later administration can be considered in immunosuppressed patients on an individual basis. Antiviral substances include aciclovir and valaciclovir. Famciclovir and brivudine have not been tested in immunocompromised patients, and with brivudine, interaction with 5-Fluoropyrimidine must be avoided. Disease severity determines whether antiviral drugs should be administered orally or intravenously. Therapy should be continued for a minimum of 7 days and until skin lesions have fully crusted. As for HSV, drugs used to prevent CMV disease, namely ganciclovir, valganciclovir and foscarnet, show anti-VZV activity.

Human Herpes Virus – 6 and 7 After solid organ transplantation, HHV-6 reactivation has been reported in 20% to 82% of transplant recipients. In the majority of reactivations, HHV-6B is identified. Mostly, reactivation occurs early after transplantation and does not cause any symptoms. In the case of symptomatic disease, fever and bone marrow suppression the most common presentations but rashes, hepatitis, gastroduodenitis, colitis and pneumonitis have also been described. Limbic encephalitis caused by HHV-6 infection is mainly associated with hematopoietic stem cell transplantation. For HHV-7, the frequency of reactivation is estimated to be seen in up to 46% of transplant recipients and also occurs in the early phase after transplantation. Symptomatic disease is rare and was primarily reported to cause fever and bone marrow suppression resulting in thrombocytopenia. The pathogenic potential of HHV-7 remains unclear as HHV-7 replication is detected in many asymptomatic immunosuppressed patients. HHV-6 (and probably HHV-7) infections have been associated with fungal infections, accelerated liver fibrosis in hepatitis C virus infections after liver transplantation bronchiolitis obliterans post lung transplantation and allograft dysfunction or rejection. Initially, HHV-6 and -7 were also linked to a higher frequency of CMV-associated disease. Subsequent studies, however, failed to show a correlation between co-infection and the clinical course of CMV disease. The preferred method of diagnosing infection is qPCR from peripheral blood or PBMCs. Due to DNA fragments from lysed cells, HHV-6 DNA detection by PCR from plasma only reaches a specificity of 84% for active viral replication. Furthermore,

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diagnostics are limited by the lack of standardized assays, thresholds and by the fact that viral replication does not imply clinical disease. Consequently, the relationship between HHV-6 DNAemia and clinical symptoms is unclear. Further diagnostic testing such as HHV 6 DNA detection tissue samples or radiological appearance may help establish the diagnosis. Persistent high-level plasma HHV-6 DNA by qPCR may also suggest that integration of the HHV-6 genome into the germline has taken place and, thus, does not reflect viral replication. This rare event can be inherited, clusters within families, and approximately 1% of the European and US American populations are affected. There are no randomized, controlled trials demonstrating antiviral treatment efficacy and recommendations based on in vitro data. Furthermore, no antiviral drugs have been licensed to treat HHV-6 and -7 related diseases. In vitro ganciclovir, foscarnet, cidofovir and brincidofovir have been shown to suppress HHV-6 replication. Several studies have described the successful treatment of HHV-6 using those antiviral drugs after solid organ transplantation. Therapy is recommended in the case of HHV-6 encephalitis and should be considered with a clinical disease that could be linked to HHV-6. In the case of HHV-7, cidofovir and foscarnet have shown anti-viral activity in vitro while ganciclovir and aciclovir did not achieve this effect. Screening for HHV-6 and -7 and prophylactic measures are not routinely recommended.

Epstein–Barr Virus (Human Herpesvirus 4) Epstein–Barr virus (EBV) disease in the immunosuppressed individual ranges from systemic presentations such as infectious mononucleosis to more localized disease including hepatitis, pneumonitis, gastrointestinal, hematological manifestations, and neoplasia. The hematological disorders can be serious, causing hemophagocytic lymphohistiocytosis or macrophage activation syndrome, which results in the uncontrolled activation of immune cells. An important property of EBV is its oncogenic capacity caused by the integration of viral DNA into the host cells' genome. During immunosuppression, the lack of cytotoxic T cell responses allows EBV to transform lymphocytes, potentially resulting in their uncontrolled proliferation. In particular, post-transplantation lymphoproliferative disorders (PTLDs) pose a great challenge. EBV-related PTLD usually arises from B cells but T and NK cell-derived PTLD is also seen. The latter may be caused by increased viral replication in the immunosuppressed, allowing for infection of cells normally not targeted by EBV. About 50% of PTLDs are EBV related and mostly occur within the first year after transplantation. This disease entity is decreasing in incidence, while late presenting EBV-negative PTLD is seen more frequently. A change in induction regimens partly explains this. The application of polyclonal anti-lymphocyte antibodies, which were administered in higher doses in the past, is a known risk factor for PTLD development. An elevated risk for PTLD is seen in EBV seronegative recipients who may contract a primary infection, making the pediatric population particularly vulnerable. This is why the serostatus before transplantation should be determined and most centers measure viral capsid antigen (VCA) IgG and Epstein–Barr nuclear antigen-1 (EBNA-1) IgG. The type of organ transplant is another determinant of PTLD risk and the highest incidence is observed after hematopoietic stem cell transplantation, particularly transplantation of haploidentical cells. Good prediction models are, however, lacking, and the individual risk is difficult to ascertain. Clinical presentation of PTLD can range from asymptomatic to fulminant organ failure and tumor lysis. Early signs include fever, lymph node or tonsil enlargement and changes in the hematogram. Due to organ damage such as hepatitis, colitis, pneumonia and cerebritis, symptoms can be very diverse and mimic an infectious disease process or organ rejection. PTLD frequently involves extranodal sites such as the gut, solid allograft and the central nervous system. Diagnosis is established by histology from lesion biopsy and disease can be categorized into six subclasses defined by the World Health Organization. The detection of EBV nucleic acid or proteins in the tissue establishes a link to the EBV-associated pathogenesis. Monitoring for EBV-related disease is usually performed by measuring plasma EBV DNA by PCR. Generally, highlevel EBV DNAemia or a rapid increase in blood EBV DNA load is correlated with an elevated risk of developing PTLD. However, there is a lack of standardization of thresholds, frequency and time points of viral load measurement. Due to the lack of antiviral drugs with activity against EBV, preemptive strategies and therapy involve reducing the immunosuppressive regimen as an initial step. This conversely risks provoking an acute transplant rejection as seen in 37% of patients followed in a prospective trial. If this approach fails, administration of rituximab – a B cell depleting antibody directed towards CD20 – can be considered in CD20 positive PTLD. Rituximab was the first treatment to improve prognosis drastically. Combined with the reduction of immunosuppression, rituximab achieves a response rate of 44%–79% and complete remissions in 20% to 55%. If the PTLD is still unresponsive to treatment, the next step involves the use of chemotherapy. In rare cases of PTLD such as peripheral T cell lymphoma, Hodgkin’s lymphoma, Burkitt’s lymphoma and central nervous system lymphoma, chemotherapy is considered as first-line therapy. Further treatment modalities include the adoptive transfer of EBV-specific T cells, surgical resection and radiotherapy.

Human Herpes Virus-8 Due to discrepancies in the geographic distribution of HHV-8, seropositivity rates range from approximately 5% to 50%. This explains the variable frequency of HHV-8 associated pathologies. HHV-8-related disease can affect immunocompetent subjects but is more often related to immunosuppression. Sexual transmission is the predominant mode in HIV positive men who have sex

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with men. Besides advanced HIV disease, post-transplant immunosuppression is a risk for progression to HHV-8-associated diseases. After organ transplantation in HHV-8 endemic regions, seroconversion in mismatched donors and recipients is seen frequently. HHV-8 DNAemia, however, usually arises from HHV-8 reactivation after immunosuppression and is rarer. Symptoms include fever, rash, lymphadenopathy and cytopenia but can also be more severe in the case of lymphoproliferative B-cell disorders, gammopathies, bone marrow failure and hemophagocytic syndrome. Similar to EBV, integration of HHV-8 DNA explains the oncogenicity of this virus. Kaposi sarcoma, caused by the uncontrolled proliferation of mesenchymal cells, is the leading clinical presentation linked to the reactivation of HHV-8. Transformation of endothelial cells causes the formation of blood-filled vascular channels, giving the lesions the typical purple color. Kaposi sarcomas can present as blotches or nodules and are usually found on skin or mucosa but can affect visceral organs. HHV-8 also causes lymphoproliferative disease by infecting B cells at different stages of development. This includes multicentric Castleman disease, presenting with enlarged lymph nodes, flu-like symptoms and organ dysfunction, and primary effusion lymphoma, affecting body cavities often without forming a solid tumor mass. Altogether, these lymphoproliferative conditions are rare since the advent of effective antiretroviral treatment for HIV infected individuals. The mortality rate can reach 60% in the case of Kaposi sarcoma and is even higher for body cavity lymphomas. Serologic testing pre-transplantation can be considered to stratify the risk in endemic areas but is not generally recommended. Higher levels of immunosuppression, particularly anti-lymphocyte agents, increase the risk of HHV-8 disease. When disease is suspected, the diagnostic standard involves tissue biopsy and detection of virus-associated antigens or HHV-8 DNA. Management of HHV-8 related disease usually starts by reducing the immunosuppression as this was shown to result in complete remission in 30% of cases. Favoring mTOR inhibitors over calcineurin inhibitors may be beneficial as regression of Kaposi sarcoma was reported with the use of drugs such as sirolimus. As for other herpes viruses, mTOR inhibitors may have a direct anti-HHV-8 activity but may also suppress tumorigenesis by inhibiting vascular growth. Due to the lack of prospective controlled trials, antiviral treatment is not routinely supported, although HHV-8 is susceptible to aciclovir, ganciclovir and cidofovir in vitro. For neoplastic disease, radiotherapy and chemotherapy may be part of treatment. For multicentric Castleman disease, treatment with rituximab appears promising even though the transformed B cells lack CD20 expression.

Further Reading Allen, U.D., Preiksaitis, J.K., 2019. Post-transplant lymphoproliferative disorders, Epstein-Barr virus infection, and disease in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clinical Transplantation 33 (9), e13652. Dierickx, D., Habermann, T.M., 2018. Post-transplantation lymphoproliferative disorders in adults. The New England Journal of Medicine 378 (6), 549–562. Kotton, C.N., Kumar, D., Caliendo, A.M., et al., 2018. The third international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 102 (6), 900–931. Lee, D.H., Zuckerman, R.A., 2019. Herpes simplex virus infections in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clinical Transplantation 33 (9), e13526. Ljungman, P., Boeckh, M., Hirsch, H.H., et al., 2017. Definitions of cytomegalovirus infection and disease in transplant patients for use in clinical trials. Clinical Infectious Diseases 64 (1), 87–91. Lurain, N.S., Chou, S., 2010. Antiviral drug resistance of human cytomegalovirus. Clinical Microbiology Reviews 23 (4), 689–712. Murphy, K., Waever, C., 2017. Chapter 13 – Failures of host defense mechanisms. In: Murphy, K., Weaver, C. (Eds.), Janeway's Immunobiology, ninth ed. Garland Science. Murphy, K., Waever, C., 2017. Chapter 15 – Autoimmunity and transplantation. In: Murphy, K., Weaver, C. (Eds.), Janeway's Immunobiology. Garland Science. Pellett Madan, R., Hand, J., 2019. Human herpesvirus 6, 7, and 8 in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clinical Transplantation 33 (9), e13518. Pergam, S.A., Limaye, A.P., 2019. Varicella zoster virus in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clinical Transplantation 33 (9), e13622. Razonable, R.R., Humar, A., 2019. Cytomegalovirus in solid organ transplant recipients: Guidelines of the American Society of Transplantation Infectious Diseases Community of Practice. Clinical Transplantation 33 (9), e13512. Wang, L., Verschuuren, E.A.M., van Leer-Buter, C.C., et al., 2018. Herpes zoster and immunogenicity and safety of zoster vaccines in transplant patients: A narrative review of the literature. Frontiers in Immunology 9, 1632.

Management of Adenovirus Infections (Adenoviridae) Albert Heim, Hannover Medical School, Hanover, Germany r 2021 Elsevier Ltd. All rights reserved.

Nomenclature c/ml

Copies per milliliter; viral genome equivalents (copies) measured in human body fluids (most frequently blood, cerebrospinal fluid or urine), not harmonized to WHO standard preparations

Glossary DNAemia Presence of viral DNA in peripheral blood, either cell-associated or in plasma. The term is used for viremias and are usually detected by nucleic acid amplification techniques e.g., Polymerase chain reaction (PCR). Latency/latent infection Long term infection of a host with a virus without the production of infectious virus progeny, thus the host is neither affected by a disease nor infectious. On a cellular level, latently infected cells contain complete viral genomes but viral gene expression is usually

HAdV Human (Mast-)Adenovirus LRTI Lower respiratory tract infection URTI Upper respiratory tract infection

restricted to a few genes. Latent infection of a host can be lifelong (as in herpesvirus infections) or limited to a period of several months to years, as seen in adenovirus infections. Latency may lead to reactivation of virus replication with shedding of infectious virus and disease manifestations. Virus load Concentration of virus DNA measured in peripheral blood samples (whole blood, plasma, or serum) for diagnostic purposes by quantitative nucleic acid amplification techniques (e.g., real-time PCR). Values are given as c/ml or if harmonized according to WHO standard preparations as IU/ml.

Taxonomy Family and Genus The seven species of human adenoviruses (HAdVs) (A to G) are members of the Adenoviridae family, genus Mastadenovirus, thus their correct name is human mastadenovirus according to the International Committee on Taxonomy of Viruses. However, their previous (and now vernacular) name HAdV is widely accepted and used in this article. HAdV species were initially defined by phenotypic properties (e.g., hemagglutination) but are nowadays clearly backed by molecular phylogenetic criteria based on complete genomic sequences. Therefore, HAdV types that belong to the same species (A to G, see Table 1) are more closely related to each other and frequently have similar tropism, disease association, and virulence. Tropism of HAdV types is influenced by their use of different cellular receptors they attach to with their highly variable fiber knob. Other factors that can contribute to their different tropism and virulence are the interaction of the penton base with the secondary cellular receptor, and early gene products interacting with regulators of the cell cycle, replicating the viral genome and interfering with the immune system. Moreover, recombination events between HAdV types of the same species are feasible, resulting in novel (geno-)types that may contain sequences coding for immunogenic epitopes of low prevalence (sero-)types within a genomic backbone of a more virulent and prevalent type (Fig. 1). Thus, these recombinant types can be positively selected for by an immune escape mechanism.

Types and Subtypes Typing was based on serology (neutralization testing and as a surrogate technique hemagglutination inhibition testing) up to type 51 (serotypes), but later on, almost all novel types (genotypes) were defined by genomic criteria that include either recombinant phylogeny of genes coding for major capsid proteins or novel genes. Type numbers were assigned chronologically according to the first isolation of a novel HAdV. Therefore, low numbers are somewhat associated with more prevalent types and/or easy propagation on cell cultures but types with closely related numbers do not have to be closely related to each other (for adenovirus species and types, see Table 1). According to more precise but somewhat outdated differentiation techniques, genome types (not to be confused with genotypes) and intermediate types were defined on a subtype level to describe adenoviruses associated with high virulence and unusual disease associations or to study infection chains. Genome types were defined by restriction fragment length polymorphisms and intermediate types by different typing results in neutralization testing (based on the hexon neutralization epitope e) and hemagglutination inhibition testing (based on the fiber knob epitope g). Many of these genome types and intermediate types were reanalyzed by complete genomic sequencing, found to be recombinant adenoviruses fulfilling the genotype criteria for a novel HAdV type and reassigned with a new type number. For example, the genome type 19a – which is associated with epidemic keratoconjunctivitis – was reassigned as type number 64.

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Table 1

HAdV species, types, and major disease associations

Species Types A B C D E F G

Disease

12, 18, 31, 61

Gastroenteritis, disseminated disease in immunosuppressed patients 3, 7, 11, 14, 16, 21, 34, 35, 50, 55, 66, 68, 76–79 URTI, LRTI, conjunctivitis, pharyngoconjunctival fever, cystitis 1, 2, 5, 6, 57, 89 URTI, latency in lymphoid tissue, disseminated disease in immunosuppressed patients 8, 37, 53, 54, 64, 9, 10, 13, 15, 17, 19, 20, 22–30, 32, 33, 36–39, 42–49, 51, 53, 54, Epidemic keratoconjunctivitis, Gastroenteritis, 56, 58–60, 62, 63, 65, 67, 69–75, 80–88, 90–103 asymptomatic infections 4 URTI, LRTI, conjunctivitis, pharyngoconjunctival fever 40, 41 Gastroenteritis 52 Gastroenteritis

Fig. 1 Genome map of the recombinant species HAdV-D type 53, which causes epidemic keratoconjunctivitis. Parts of the genome originating of type 8 in depicted red, of type 22 in blue and of type 37 in orange, and of unknown origin in white. Please note that the main neutralization epitope is coded by type 22 sequences whereas other parts of the genome that code for the usage of cellular receptors and thus determine tropism (e.g., fiber) or are considered to be virulence associated (as e.g., E1, E3, and E4) are derived of virus types associated with epidemic keratoconjunctivitis. Figure from Walsh, M.P., Chintakuntlawar, A, Robinson, C.M., et al., 2009. Evidence of Molecular Evolution Driven by Recombination Events Influencing Tropism in a Novel Human Adenovirus that Causes Epidemic Keratoconjunctivitis. PLOS One 4, e5635. doi:10.1371/journal.pone.0005635.g001.

In this article, the term type will be used throughout for sero- and genotypes. The term subtype will be used for adenovirus isolates with genomes significantly different from the prototype (the virus isolate that was used for labeling of a novel type) but does not fulfill the genotype criteria.

Structure Adenovirus virions consist of a non-enveloped icosahedron with protruding fibers at the vertices. Virions are about 70–75 nm in size, but fibers protruding from the vertices of the virion have a type-specific length (9–33 nm) resulting in different overall sizes. The adenovirus virion consists of 252 capsomers. The 12 apical capsomers are pentons because each is surrounded by five other capsomers. All other 240 capsomers are hexons because they are surrounded by six other capsomers. Pentons consist of a penton base and a fiber, which has a shaft and a terminal knob. The hexon is a trimer of hexon proteins; the penton base is a pentamer consisting of five penton proteins and a trimeric glycoprotein that builds the fiber. Minor capsid proteins stabilize the capsomers or link to the genome and provide additional functions. The genome consists of a single piece of double-stranded DNA, about 35 kbp in size, which codes for structural and multiple non-structural proteins in the early gene regions (E1-E4). Adenovirus mRNAs are spliced; this was discovered while studying adenovirus gene expression. Early gene regions provide gene products with multiple functions – for example, a polymerase for genome replication (E2 region), interference with the cell cycle regulation, and “hiding” infected cells from the immune system (E3 region). Therefore, the E3 region can be deleted from adenovirus genomes

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without compromising their replication in cell cultures. However, this deletion was never observed in clinical isolates because E3 gene products protect infected cells from the action of the immune system.

Epidemiology HAdVs are prevalent worldwide and it can be assumed that every adult has survived infections with multiple types, but of course, not all types. HAdV infections of neonates are observed rarely because of maternal passive immunity against highly prevalent types. However, sporadic infections with types not covered by passive immunity may present as severe lower respiratory tract infections (LRTI) and even disseminated infections (see below) in neonates. As maternal passive immunity wanes at an age of 6–12 months, toddlers get frequently infected with highly prevalent HAdV types of species C (e.g., types 1, 2, and 5), species B (e.g., 3 and 7), and species F (types 40 and 41). Infections with highly endemic species C HAdV types present as upper respiratory tract infections (URTI), sometimes in combination with gastroenteritis. Adenovirus latency in lymphoid cells of the adenoids and gastrointestinal tract is a sequel of these infections and can last for several years after primary infections but is not lifelong in contrast to herpesvirus latency. HAdV latency usually results in intermittent low-level shedding of HAdV DNA and infectious virus with respiratory secretions and feces. The epidemiological significance of this shedding remains obscure, but adenovirus latency may result in clinically significant reactivations in immunosuppressed individuals. Infections with species B types – also associated with respiratory tract infections and occasionally affecting the lower respiratory tract – can be more frequently observed as epidemics. Transmission of respiratory HAdV types is by droplets and smear infections but, in contrast to many other respiratory viruses, not limited to the typical winter season. Adenoviruses are considered to be the third most frequent cause of viral gastroenteritis (after rotavirus and norovirus infection). Infections with species F adenovirus types frequently cause gastroenteritis outbreaks in toddlers and young children. At a slightly higher age (kindergarten and primary school), HAdV types of species A (12, 18, and 31) can cause small gastroenteritis outbreaks at these institutions. Transmission is by the fecal-oral route, frequently by smear infections, and difficult to control due to very high virus loads in feces (up to 1010/ml). Therefore, nosocomial transmission chains can be observed. Older children, adolescents, and young adults are prone to respiratory infections with the less prevalent HAdV species B types with respiratory tropism (e.g., 14, 21, and 55, in addition to 3 and 7) and infections with type 4 (species HAdV-E). These can cause epidemics in summer-camps and military barracks. The same types also cause pharyngoconjunctival fever and conjunctivitis outbreaks and some of these can be traced back to insufficiently chlorinated swimming pools. As many adults are immune to the above-mentioned highly prevalent types, adenovirus infections mostly present as sporadic cases of individuals exposed to the types for which they are susceptible. For example, adults can also be affected in outbreaks of adenovirus types with respiratory tropism (e.g., 3, 4, 7, 14, 21, and 55). Genitourinary infections of adults – urethritis, for example – can be transmitted sexually and are caused by types usually associated with respiratory and eye infections (types 2 and 37, respectively) but these infections appear to be rather sporadic cases or small infection chains. Hemorrhagic cystitis can be observed both in adults and in children and is clearly associated with a few types of species HAdV-B (11, 34, and 35) but cases are rather sporadic. However, it is thought that these infections are underdiagnosed in mild, non-hemorrhagic cystitis cases. Seniors are frequently affected by large outbreaks of epidemic keratoconjunctivitis, which can also affect younger patients. Epidemic keratoconjunctivitis is caused by a few types of species HAdV-D (8, 37, 53, 54, and 64 – previously labeled as 19a). Transmission is by smear infection. Previously, insufficiently disinfected medical devices at ophthalmologists’ offices were a major route of transmission. Nowadays, transmission at ophthalmologists’ offices can still be observed because the crowding of patients in the waiting room facilitates smear infections from non-disinfected surfaces. Control of transmission is not an easy task because of the very high HAdV concentrations in eye secretions. The majority of other HAdV species D types are far less virulent, not associated with eye infections but rather with mild to asymptomatic gastrointestinal and respiratory infections. Adenoviruses can cause severe, sepsis-like, disseminated infections in severely immunosuppressed patients of all age groups. These are most frequently observed in pediatric patients after allogeneic stem cell transplantation. As the majority of these infections are reactivations of latent adenovirus, which has a higher prevalence in pediatric patients, these cannot be completely avoided by hygiene measures.

Clinical Description of Infection Incubation Period The incubation period of HAdV Infections is usually short, within a range of one to two weeks. It may be even shorter in gastroenteritis and URTI. By contrast, many disseminated infections of immunosuppressed patients originate from latent adenovirus infections, which may have been established several years ago. Due to the multitude of HAdV types with different tropism and virulence, a multitude of different diseases are associated with HAdV infections. Clinical manifestations of HAdV infections are also influenced by the age and immune status of the infected person.

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Upper Respiratory Tract Infections Many of these occur early in life and are self-limiting and mild. Usual symptoms are fever, nasal congestion, rhinitis, pharyngitis with sore throat, and cervical lymphadenopathy. Sometimes, otitis media may be present. An exudative tonsillitis may also be observed and can be clinically indistinguishable from a group A streptococcal infection. As C-reactive protein levels are elevated and infections may also occur without the typical seasonality of other respiratory virus infections, these may be falsely diagnosed as bacterial infections and treated unnecessarily with antibiotics. Most of these infections are caused by the few types of HAdV species C and a few types of species B (e.g., 3 and 7).

Lower Respiratory Tract Infections URTI with types of species HAdV-C and -B may progress to LRTI with symptoms of cough, (pseudo-)croup, and bronchitis. Bronchiolitis and pneumonia can be severe and sometimes fatal, particularly in neonates and young children less than two years of age. A pertussis-like syndrome has also been reported, but the etiological significance of HAdV is questionable. In older children and adults, another small group of adenovirus types is clearly associated with severe LRTI (bronchiolitis, pneumonia, ARDS): type 4 of species HAdV-E and several types of species HAdV-B: 14, 21 (subtype 21a), 55, and 66 (previously labeled as genome type 7 h or intermediate strain 7H3) as well as, occasionally, types 3 and 7. Infections with these types account for the outbreaks of severe lower respiratory tract diseases in young military recruits reported most frequently in the US military and occasionally in other militaries. Stress and fatigue in military recruits as well as the cold season promote severe LRTI, including acute respiratory distress syndrome (ARDS). The role of adenoviruses in asthma remains controversial, but long-term sequelae (bronchitis obliterans, bronchiectasis, and chronic atelectasis) of these LRTI are more frequently reported.

Ocular Infections Corneal infection is typical in epidemic keratoconjunctivitis. This may begin with conjunctivitis with a “foreign body in the eye” sensation and progresses with corneal erosions and infiltrates that impair vision and painful edema of the eyelids. Acute inflammatory symptoms resolve in about two weeks. Unfortunately, blurred vision due to chronic infiltrates of the cornea (numuli), photophobia, and a foreign-body sensation may persist for months to years. Most cases of epidemic keratoconjunctivitis are caused only by a few types of species HAdV-D (8, 37, 53, 54, and 64). Several other HAdV types are associated with less severe ocular infections. Acute follicular conjunctivitis resolves without consequence in a few weeks but acute hemorrhagic conjunctivitis may also be observed. Pharyngoconjunctival fever is a typical manifestation of HAdV infection presenting with follicular conjunctivitis in combination with upper respiratory tract symptoms as pharyngitis, fever, and – occasionally – lymphadenopathy and malaise. All these eye infections usually do not affect the cornea and are associated with types 3, 4, and 7 of species HAdV-B and HAdV-E but may be caused by other types occasionally.

Gastroenteritis The enteric HAdV of species F (types 40 and 41) and species A (types 12, 18, 31) are the third most common cause of viral gastroenteritis. In some cases, HAdV upper respiratory tract infections (see above) may also present with gastroenteritis symptoms. Species HAdV-F infections predominate in children below two years old. Symptoms include mild fever, vomiting, abdominal pain, and diarrhea – which is usually watery and non-bloody, lacks fecal leukocytosis, and lasts for about ten days. If dehydration is adequately treated, the vast majority of immunocompetent patients recover uneventfully. Mild enteritis is also observed in many cases of upper respiratory infections with non-enteric types of HAdV (see above). Other gastrointestinal syndromes infrequently associated with non-enteric adenovirus types include intussusception, acute mesenteric lymphadenitis, and appendicitis. However, more studies on these disease associations are required because HAdV DNA detection in these diseases may have been due to coincidental HAdV latency in gut-associated lymphoid tissue.

Genitourinary Diseases Genitourinary diseases – including genital lesions, urethritis, cystitis, and hemorrhagic cystitis – are occasionally caused by HAdV. Cystitis and hemorrhagic cystitis of children and adults is clearly associated with a few types (11, 34, 35) of species HAdV-B. Although usually self-limiting, these infections may progress to graft nephritis in renal transplant recipients. Moreover, there are hints that these infections may also become latent for several years. Genital lesions and urethritis by HAdV can be sexually transmitted but are infrequent manifestations of HAdV infections with types usually associated with other diseases (e.g., type 2 and 37).

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Fig. 2 Pathogenesis of disseminated adenovirus disease in immunocompromised patients. The source of human adenoviruses (HAdV) infection can be either de novo infection or reactivation from latent/persistent infection of the lymphatic tissue, for example, of the gut or the upper respiratory tract. After local replication, for example, in the gastrointestinal tract, HAdV spreads via the peripheral blood to other organs, such as the liver, bone marrow, central nervous system, urogenital, or respiratory tract. Massive HAdV replication in the affected organs leads to tissue destruction and causes the respective organ manifestation, such as enteritis, hepatitis, or pneumonia (here designated in italics). Virus progeny is shed to the blood or stool. Multiple organ involvement as well as disseminated intravascular coagulation might lead to a “septic” picture of disease and result in a very high lethality. From Ganzenmueller, T., Heim, A., 2011. Adenoviral load diagnostics by quantitative polymerase chain reaction: Techniques and application. Reviews in Medical Virology 22 (3), 194–208. doi:10.1002/rmv.724.

Diseases Infrequently Associated With Human Adenoviruses Infections Exanthems are occasionally associated with HAdV infections. Severe neurologic manifestations, such as meningoencephalitis and encephalitis, have been described occasionally and diagnosed by HAdV DNA detection in cerebrospinal fluid. HAdV DNA has also been detected in acute myocarditis of otherwise healthy people but more studies are needed on this topic. Adenovirus type 36 (species HAdV-D) infections have been linked epidemiologically to obesity – although this is controversial – as well as the association of HAdV infections with fetal demise and Kawasaki disease.

Adenovirus Infections of Immunocompromised Patients All of the above-mentioned HAdV diseases may be prolonged and more severe in immunocompromised individuals including, for example. the progression of a respiratory infection from the upper to the lower respiratory tract or from cystitis to graft nephritis. Nevertheless, HAdV infections are only infrequent complications of solid organ transplant recipients and associated with periods of increased immunosuppression. HAdV infections of solid organ transplant recipients may be associated with the graft and are hard to discern from rejection. For example, in liver transplant patients, infections are most often associated with diarrhea, graft hepatitis, and sometimes pneumonia; in renal transplant recipients, acute hemorrhagic cystitis, graft nephritis, and pneumonia. Without proper adenovirus diagnostics, HAdV graft infections can be mixed up with rejection, and increased immunosuppression to treat supposed rejection may result in graft loss. HAdV infections are most frequently observed in pediatric allogeneic hematopoietic stem cell recipients, about 20% may present with an HAdV infection and about 5% succumb before screening and treatment options develop. Disease most frequently starts with endogenous reactivation of a latent HAdV infection in the gastrointestinal tract (Fig. 2) resulting in gastroenteritis or asymptomatic HAdV shedding with the feces. Less frequently, de novo infections of the gastrointestinal tract or respiratory tract are observed. Anyway, these primary localized infections lead to viremia and subsequent organ infections (e.g., hepatitis, pneumonia, pancreatitis, encephalitis), which may be life-threatening. Lymphopenia (o300 ml) and extensive use of immunosuppressive drugs to treat graft versus host disease are risk factors for the progression to disseminated disease, which is indicated by an increase of

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Fig. 3 Typical kinetics of the human adenoviruses (HAdV) blood load, leukocyte counts, and transaminase levels in a stem cell transplant (SCT) recipient with fatal disseminated HAdV disease. The graph depicts the HAdV DNAemia kinetics in peripheral blood of an SCT recipient, who died from multiple organ failure due to disseminated HAdV disease on day 126 after SCT. As a surrogate marker of severe hepatitis, the transaminase levels (aspartate transaminase) are indicated in U/l. Continuously declining leukocyte counts (leukocytes/ml), within parallel increasing HAdV DNAemia, highlight the role of the cellular immune response for the clearance of HAdV infections. From Ganzenmueller, T., Heim, A., 2011. Adenoviral load diagnostics by quantitative polymerase chain reaction: Techniques and application. Reviews in Medical Virology 22 (3), 194–208. doi:10.1002/rmv.724.

virus loads in peripheral blood. High-level viremia (106–1010 copies HAdV DNA/ml) is typical for this disease and associated with sepsis-like symptoms and failure of multiple organs (e.g., hepatitis and pneumonia) resulting in high mortality rates (Fig. 3). Both lysis of cells of visceral organs by HAdV replication and induction of a “cytokine storm” like sepsis by high loads of HAdV in the peripheral blood contribute to its pathophysiology. Disseminated disease of pediatric patients is most frequently caused by types 1 and 2 of species HAdV-C, less frequently by the other types of species C and type 31 of species HAdV-A. In adult allogeneic hematopoietic stem cell recipients, HAdV infections are less prevalent, probably because adults may less frequently have HAdV latency, but disease manifestation is as severe as in the pediatric group. Types of species HAdV-C can be detected, as in pediatric patients, but several types usually associated with cystitis (11, 34, and 35 of species HAdV-B) can almost exclusively be found in this adult age group as an etiology of disseminated disease. Since highly active antiretroviral therapy became available, HAdV complications in HIV infected patients were hardly ever observed. Previously, AIDS patients presented with chronic gastroenteritis, severe hemorrhagic cystitis, adenovirus-associated pneumonia, meningoencephalitis, and hepatitis as well as disseminated disease. In this group of patients, multiple types of species HAdV-D have been observed as etiological agents.

Animal Adenoviruses Adenovirus infections can be found in almost all vertebrae (fish, reptile, bird, and mammal). Although typical zoonotic infections have not been described in humans, several adenovirus types infecting humans or simians share parts of their genomic sequences because of previous recombination events. These results of complete genomic sequencing indicate inter-(host-)species transmission events in the phylogeny of adenovirus types, infecting humans and adenovirus types infecting simians.

Diagnosis Polymerase Chain Reaction (PCR) for Human Adenoviruses DNA Detection In general, detection of HAdV DNA by nucleic acid amplification technologies (usually PCR) is the preferred diagnostic procedure for all types of adenovirus infections. Diagnostic specimens taken from the affected body site are appropriate (e.g., urine in cystitis cases, nasopharyngeal swabs in upper respiratory tract infections), although a few pitfalls have to be considered. The huge genetic diversity of HAdV impedes the detection of all types in a single reaction, but several “generic” PCRs have been published and are also commercially available. However, some other commercially available diagnostic PCRs do not detect all types — sometimes only a very limited number of HAdV types. Many of these PCRs are parts of highly multiplexed PCR panels that detect multiple viral and bacterial pathogens associated with respiratory infections or gastroenteritis. Diagnostic claims and

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intended use of these assays should be carefully checked. For example, a highly multiplexed PCR assay for gastroenteritis diagnostics may merely detect types 40 and 41 of species HAdV-F. Thus, this will underdiagnose gastroenteritis caused by other HAdVtypes (e.g., gastroenteritis outbreaks in older children caused by types of species HAdV-A and sporadic gastroenteritis cases caused by types of species C, D, and G). Moreover, this assay will probably not detect adenovirus gastroenteritis in immunosuppressed patients, where species HAdV-C and -A types predominate. Of course, it is also not appropriate for screening purposes in this group of patients (see below). Another pitfall of HAdV diagnostics by PCR (and to a lesser extent by virus isolation on cell cultures) is the detection of a latent HAdV infections coincident with another acute respiratory or gastrointestinal infection. About 5% of upper respiratory specimens or feces specimens of healthy or otherwise infected children can be HAdV DNA positive if a sensitive PCR protocol is used. Moreover, even EDTA blood specimens of healthy children with HAdV latency can be occasionally positive. Although analytically correct, these results are diagnostically misleading. Adenovirus shedding and DNAemia during latent infections is intermittent and always at low levels, whereas in all acute HAdV infections, an abundance of HAdV DNA is typical. Therefore, the simple solution to this diagnostic problem is either to limit the sensitivity of PCR protocols or to use real-time PCRs (using a cut off for high Ct values).

Quantitative Human Adenoviruses PCRs and Screening of High-Risk Patients Diagnosis of disseminated disease previously required detection of HAdV (by virus isolation or histopathology) at multiple body sites, which was of course a tedious procedure. More recently, high virus loads in peripheral blood were found to be closely associated with disseminated infections. Therefore, testing EDTA blood (or plasma) samples by quantitative PCR became the standard diagnostic procedure for disseminated HAdV disease of immunosuppressed patients. Several generic real-time PCR protocols for HAdV DNA have been published and diagnostic assays are also available commercially. Multiple studies on virus loads in hematopoietic stem cell recipients demonstrated that disseminated disease and significantly higher mortality are associated with high peak virus loads in peripheral blood usually in a range between 106 and 1010 c/ml, whereas peak virus loads o104 c/ml were not. However, all these studies were not harmonized to a WHO standard, and different conclusions – if any – have been made on threshold virus loads for a therapy decision. Anyway, the occasional detection of low HAdV DNA concentrations in feces, respiratory materials, or blood should not be mistaken as the diagnosis of a severe HAdV infection but it rather indicates HAdV latency and a potential risk for severe HAdV infections. In allogeneic hematopoietic stem cell recipients who are at a high risk for a disseminated HAdV infection (reactivation or de novo), weekly screening of blood virus loads is recommended by American and European guidelines to achieve an early diagnosis and facilitate preemptive therapy. Several studies in pediatric HSCT recipients have shown that additional weekly stool screening for HAdV DNA can detect reactivation even earlier and that increasing virus loads in stool precede HAdV DNA detection in peripheral blood by about one week. However, stool screening may fail in case of a de novo HAdV infection. Therefore, it should be performed only in addition to blood screening for HAdV. Real-time PCR protocols can also be used for all other diagnostic applications that usually do not require quantification. Nevertheless, discerning highly positive specimens from weakly positive specimens can be helpful to diagnose acute infections in contrast to low-level HAdV shedding in latency.

Other Diagnostic Methods Detection of HAdV antigens by ELISA and other (e.g., lateral flow) methods is feasible in stool samples and eye swabs with high HAdV loads, using commercial assays. However, their sensitivity and specificity are lower than PCR-based assays. Virus isolation on cell cultures was the previous gold standard of adenovirus diagnostics, but many laboratories stopped performing it because the time of detection is much slower than PCR. A549 cells are appropriate for most adenovirus types with the exception of types 40 and 41, which require Graham 293 cells for replication. Virus isolation is feasible from respiratory tract materials, eye swabs, urine, and stool but several days (up to three weeks) are required for the development of a CPE. An early CPE, observed during the first 24 h after inoculation of the cell culture, is not caused by HAdV replication but by toxic effects of HAdV penton base proteins, which can be present in high concentrations in clinical specimens. Diagnosis of adenovirus infections by serological methods cannot be recommended due to the multiplicity of HAdV types. Type-specific neutralization tests can be regarded as reliable but are more appropriate for seroepidemiological and immunity studies. Adenovirus ELISAs use a group-specific epitope on the internal side of the capsid on the hexon protein but cut-offs are rather arbitrarily set; thus, any result is questionable.

Adenovirus Differentiation and Typing For many diagnostic purposes, precise HAdV typing is not required but, basic differentiation on the HAdV species level can be quite helpful. For example, high virus loads of species HAdV-C in the feces of an HSCT recipient usually predict disseminated disease, whereas species HAdV-F does not. This differentiation can be easily performed with multiplex PCRs (conventional or real-time) for

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the different HAdV species. Sequencing of amplicons of the usual generic diagnostic PCRs also provides rapid information on the HAdV species. Typing is most frequently performed nowadays by sequencing the highly variable loops 1 and/or 2 of the main neutralization determinant e on the hexon protein. This procedure is equivalent to the classical neutralization assays but can be more rapidly performed directly with diagnostic specimens (without prior virus isolation). However, results are equivocal because several of the new (geno-)types (type numbers 453) share neutralization epitope sequences with one of the older (sero-)types. Precise typing requires additional sequencing of the highly variable loops on the fiber knob and penton base. These additional sequence data help identify all HAdV types unequivocally, including the recombinant (geno-)types. By contrast, even a combination of classical serological typing methods (neutralization testing and hemagglutination inhibition testing) may fail with a few of the new (geno-) types. Complete genomic sequencing of HAdV is also feasible to type cell culture isolates or even HAdV DNA directly from diagnostic samples when the viral load is high. By complete genomic sequencing, all types, subtypes, and strains can be identified precisely. Previously, restriction fragment length polymorphisms were used to differentiate genomic variants of HAdV types as genome-types (not to be confused with genotypes) but this approach became obsolete when next-generation sequencing became available.

Treatment and Prevention Antivirals As adenovirus infections of immunocompetent patients are usually mild and self-limiting, these do not really require antiviral treatment – just symptomatic and supportive therapy. Some of these infections may be treated unnecessarily with antibiotics if a diagnosis of HAdV infection has not been made because its symptoms (e.g., fever, elevated c-reactive protein, and tonsillitis) may lead to a suspicion of bacterial infection. A few severe diseases of immunocompetent patients (e.g., pneumonia and epidemic keratoconjunctivitis) would be sensible targets for antiviral treatment; however, this is not established. In a few cases of pneumonia, experimental therapy with cidofovir was reported but its efficacy remains unclear. In immunocompromised patients, a reduction of immunosuppressive therapy, if feasible, is of outstanding importance because disseminated disease and lethal outcomes are clearly associated with high levels of immunosuppressive treatment or lymphopenia. In some cases, HAdV infection may mimic graft rejection (e.g., HAdV nephritis) or graft versus host disease (e.g., HAdV gastroenteritis). Without a diagnostic workup for HAdV, these cases had been treated with high dose immunosuppression resulting in graft loss, disseminated disease, and many fatalities. On the other hand, many immunosuppressed patients with a HAdV infection and viremia lack any specific symptoms until they reach high virus loads (4 106 c/ml). However, antiviral therapy tends to fail in patients with high virus loads. Therefore, a weekly blood (and stool) screening of high-risk patients is essential. As preemptive therapy, a reduction of immunosuppressive therapy and the application of antiviral agents (off label use) should be considered. Several antivirals – including ganciclovir, ribavirin, cidofovir, and its prodrug brincidofovir – have in vitro activities against adenovirus. As many HAdV reactivations and infections were observed during ganciclovir prophylaxis for CMV infections and, in some cases, even during high dose therapy of CMV infections, the clinical use of ganciclovir for HAdV infections should not be considered. Ribavirin may not have activity against all HAdV types but has gained an orphan drug status for HAdV infections. However, results of case reports and case series on ribavirin therapy were rather inconclusive and included therapy failures. Therefore, ribavirin is no longer frequently used for HAdV infections. All HAdV types are susceptible to cidofovir in vitro. Although cidofovir resistance can develop with serial passages, little resistance has so far been detected in isolates from cidofovir-treated patients. Prospective controlled clinical trials have never been published, but several case reports and retrospective studies suggested some efficacy and a reduction of mortality if cidofovir is used as preemptive therapy. However, this is not a proof of the efficacy of cidofovir as other measures (reduction of immunosuppression) may have contributed to the more favorable outcomes. Moreover, HSCT patients can clear their HAdV infection spontaneously with hematopoietic regeneration and rising lymphocyte counts. In contrast to high HAdV loads, low peak HAdV loads in peripheral blood are not associated with mortality. Hence, the use of a threshold virus load (e.g., 104 copies/ml blood) to trigger preemptive cidofovir treatment has been suggested to avoid overtreatment and unnecessary side effects. Other factors supporting the treatment decision are T cell depletion, severe lymphopenia, and rapidly increasing virus loads. A lipid conjugate of cidofovir – brincidofovir (CMX001), which can be administered orally – reaches higher intracellular concentrations and better antiviral activity with lower nephrotoxicity than cidofovir. However, diarrhea is the main side effect of brincidofovir. Although a randomized placebo-controlled clinical trial for the preemptive treatment of asymptomatic HAdVDNAemia in HSCT patients did not show a significant effect on the primary endpoint, a positive trend was observed. In addition, many case studies support the view of a higher efficacy of brincidofovir. Although preemptive antiviral therapy can limit HAdV infection for some time, clearance of HAdV DNAemia is clearly associated with increased lymphocyte counts and the detection of adenovirus specific T cells. Therefore, immunotherapeutic interventions – either using donor lymphocyte infusions or the adoptive transfer of HAdV-specific cytotoxic T lymphocytes expanded from naive T cells – were developed. The latter approach holds promise to avoid graft-versus-host disease associated

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with donor lymphocyte infusion. As the in vitro expansion process requires several days, diagnosis of HAdV infection has to be made early, bridging with antiviral agents as cidofovir is usually required.

Vaccines An oral vaccine against HAdV types 4 and 7 is available only for the US military. This vaccine is effective to prevent respiratory infections, which can be severe in military recruits. Surprisingly, it consists of replication-competent, non-attenuated viruses contained in enteric-coated tablets. After ingestion, the HAdV infection seems to be limited to the gastrointestinal tract, and protective immunity against subsequent respiratory infections with these types is induced.

Germicides Infectious HAdV can survive for long times in liquids or dried on surfaces; moreover, as a non-enveloped virus, it is resistant to common detergents. Therefore, there is a high risk for smear infections. However, HAdV is sensitive to heating and many chemical germicides. These should be certified for their activity against HAdV, but due to the very high virus loads in feces or eye swabs, even these may fail. Therefore, a combination of thorough cleaning and subsequent use of germicides can be considered.

Further Reading Dehghan, S., Seto, J., Liu, E.B., et al., 2019. A zoonotic adenoviral human pathogen emerged through genomic recombination among human and nonhuman simian hosts. Journal of Virology 93. Ganzenmueller, T., Heim, A., 2011. Adenoviral load diagnostics by quantitative polymerase chain reaction: Techniques and application. Reviews in Medical Virology 22 (3), 194–208. doi:10.1002/rmv.724. Heim, A., Hayden, R.T., 2019. Adenoviruses. In: Carroll, K.C., Pfaller, M.A., Landry, M.L., et al. (Eds.), Manual of Clinical Microbiology. Washington, D.C.: ASM Press. Lion, T., 2019. Adenovirus persistence, reactivation, and clinical management. FEBS Letters 593, 3571–3582. Nemerow, G., Flint, J., 2019. Lessons learned from adenovirus (1970–2019). FEBS Letters 593, 3395–3418. Pied, N., Wodrich, H., 2019. Imaging the adenovirus infection cycle. FEBS Letters 593, 3419–3448. San Martin, C., 2012. Latest insights on adenovirus structure and assembly. Viruses 4, 847–877. Walsh, M.P., Chintakuntlawar, A., Robinson, C.M., et al., 2009. Evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis. PLOS One 4, e5635. doi:10.1371/journal.pone.0005635.g001.

Relevant Websites http://hadvwg.gmu.edu/ HAdV Working Group.

Management of Hepatitis A and E Virus Infection Sébastien Lhomme, Florence Abravanel, Jean-Marie Peron, Nassim Kamar, and Jacques Izopet, Toulouse University Hospital, Toulouse, France and Toulouse University Paul Sabatier, Toulouse, France r 2021 Elsevier Ltd. All rights reserved.

Introduction The hepatitis A virus (HAV) and hepatitis E virus (HEV) are small RNA viruses mainly enterically transmitted. There are two forms of infectious particles; one corresponds to non-enveloped particles that are shed in the feces and the other is cloaked in host cell membranes and circulates in the blood. This membrane envelopment protects the HAV and HEV against neutralizing antibodies. Although both are ancient viruses, they are still a frequent cause of disease affecting millions of individuals annually. Many individuals with HAV and HEV infections are asymptomatic or have mild symptoms. However, severe liver disease is also observed. In contrast to HAV and HEV genotypes 1 and 2, HEV genotypes 3 and 4 infections can lead to chronic hepatitis and cirrhosis in immunocompromised patients. For these patients, reduction of immunosuppression or ribavirin therapy can eradicate HEV. Extrahepatic manifestations such as neurological and renal diseases have been described in association with acute and chronic HEV infections. Validated HAV immunoassays are largely available but HEV serological and nucleic acid tests have only recently made available. The improvement of virological tools for HEV diagnosis allowed a deep change in the understanding of the epidemiology and pathology of HEV infection.

Taxonomy Hepatitis A Virus HAV belongs to the family Picornaviridae, genus Hepatovirus. HAV has a primitive capsid related to that of picorna-like viruses (dicistroviruses) that infect insects. The nucleotide sequences of HAV strains vary little over time or place. There are six closely related genotypes (I to VI) but only one serotype. Genotypes I to III, further divided into subgenotypes A and B, are associated with infections in humans, while genotypes IV to VI are simian in origin. Evolutionary ancestral forms have been found in small mammals such as bats, rodents, hedgehogs, and shrews.

Hepatitis E Virus HEV belongs to the Hepeviridae family, which includes two genera (Orthohepevirus and Piscihepevirus) and five species. The species Orthohepevirus A includes HEV that infects humans and several other mammals. Orthohepevirus B infects chickens, while Orthohepevirus C infects rats and ferrets, Orthohepevirus D infects bats, and Piscihepevirus A infects cutthroat trout. Two cases of infection with Orthohepevirus C HEV were reported recently despite its genetic differences from the other human pathogenic strains. The species Orthohepevirus A consists of at least eight distinct HEV genotypes but only one serotype. Genotypes 1 and 2 are strictly human and circulate in developing countries where sanitary conditions are poor. They are responsible for outbreaks. HEV genotype 3 is widely distributed around the world while HEV genotype 4 is found mainly in Asia. Genotypes 3 and 4 have animal reservoirs, mainly pigs, and infect humans in developed countries. HEV genotypes 3 and 4 have been detected in a wide variety of animals, including domestic pigs, wild boar, deer, rabbits, and mongoose. HEV genotypes 5 and 6 have been detected in wild boar in Japan but not yet in humans. HEV genotype 7 was recently detected in dromedary camels; it also caused chronic hepatitis in an immunosuppressed individual who regularly consumed camel milk and meat. Lastly, HEV genotype 8 has been found in Bactrian camels.

Epidemiology and Transmission HAV and HEV are both transmitted mainly by the fecal-oral route. But despite their modes of transmission being quite similar, their epidemiology differs.

Epidemiology Hepatitis A virus HAV is distributed worldwide but its epidemiological pattern varies between populations, depending on sanitation and socioeconomic status. One way of assessing the endemic penetration of HAV is based on the age at which seroprevalence reaches 50% of the population: very high (o5 years), high (5–14 years), intermediate (15–34 years), and low (435 years) (Fig. 1).

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Fig. 1 Geographic distribution of HAV infections. HAV: colors represent different endemic patterns based on the age at which 50% of the population is HAV IgG positive [red: very highly endemic (o 5 years); orange: highly endemic (5–14 years); light green: intermediately endemic (15–34 years); dark green: low endemic (4 35 years)].

Very high and high endemicity pattern The rates of HAV transmission in low-resource countries with poor sanitation, such as most area in Africa and parts of Asia, are very high and HAV is acquired in early childhood. Infections are asymptomatic and lead to universal protection against HAV in later life. Most people are exposed to HAV and become seropositive before they are 5 years old. Low endemicity pattern The circulation of HAV is limited in high-resource countries with good sanitation and hygienic conditions (North America, Europe, Australia, Japan, etc.), and most people in all age groups are susceptible. However, HAV seroprevalence is higher in older people due to a cohort effect. Introduction of HAV into the population of these countries can trigger waves of transmission with symptomatic disease (jaundice), in men who have sex with men (MSM) for example, or through importation of contaminated food. Intermediate endemicity pattern HAV infections are fairly common in intermediate-resource countries like North Africa, South America and Western Asia, where symptomatic disease occurs in children over 5 years old and young adults. Socio-economic development and improved hygiene have led to a change in HAV epidemiology in these countries, with more cases of disease, more hospitalizations, and more deaths, despite reduced HAV transmission. This public health problem has led to increased prevention, including universal childhood HAV vaccination.

Hepatitis E virus HEV genotypes 1 and 2 HEV genotype 1 is highly endemic and prevalent in South, Central, and South-East Asia, as well as in Africa and the Middle East (Fig. 2). HEV genotype 2 is prevalent in western Africa and Mexico. Although HEV genotype 1 was responsible for frequent outbreaks in China over the past few decades, it is now less prevalent. HEV genotype 4 infections have become more prevalent, perhaps due to improved sanitation and increased income. It is estimated that 20.1 million new infections occur annually in Asia and Africa. Determining the real seroprevalence in these areas is difficult as a variety of serology assays with quite different sensitivities have been used. However, published data indicate that 20% to more than 50% of people living in most parts of Asia and Africa have HEV antibodies. Most of the asymptomatic cases are adolescents and young adults. Infection in children is more often asymptomatic than in adults. HEV genotypes 3 and 4 HEV genotype 3 is the main HEV genotype circulating in Europe, North America, and South America, while genotype 4 is predominant in Asia (Fig. 2). Cases of genotype 4 HEV infection in Europe are sporadic and locally acquired while genotype 1 infections are travel-associated. The total number of reported symptomatic cases in Europe increased ten-fold between 2005 (514 cases) and 2015 (5617 cases), but the percentage of cases hospitalized decreased from 80% in 2005 to around 50% in 2015. A meta-analysis showed that the prevalence of IgG in the general population in Europe is influenced by the assay used for the

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Fig. 2 Geographic distribution of HEV infections. HEV: colors represent the predominant HEV genotypes in each area (pink: HEV genotypes 1 and 2; green: HEV genotype 3; blue: HEV genotype 4).

detection. Thus, 52% of the heterogeneity was explained by 3 parameters: the assay used, the geographical location and the cohort studied. Hence, the seroprevalence of IgG can be o10% in countries like Italy, Ireland or Scotland and 420% in others like France, the Netherland or Germany. The estimated seroprevalence of IgG in France was 22.4%, based on more than 10 000 French blood donors. There were geographical differences: rates were higher in the South and North East. But seroprevalence can vary from over 80% to below 20% even in high-rate areas like South-West France. Historical data indicated a seroprevalence of 21% in the United States, while more recent data suggest that the prevalence of HEV IgG is 6%. However, few data are available for the United States, probably due to the lack of FDA-licensed anti-HEV serology assays.

Transmission Hepatitis A virus Although a highly effective, safe hepatitis A vaccine was developed in the early 1990s, HAV is still an important cause of acute viral hepatitis worldwide. The virus is readily transmitted and outbreaks are frequent, especially in developed countries where herd immunity in the population is low. There is no animal reservoir for human HAV strains. Water- and food-borne transmission Most cases of HAV are due to ingesting contaminated water or food, but swimming in water contaminated by adjacent septic systems or sewage is also a risk. A variety of foods have been implicated in hepatitis A outbreaks. Food can be contaminated at any point during processing, harvesting, preparation, or distribution. Seafood, vegetables, and fruit are classic vectors. The largest reported hepatitis A outbreak was associated with consumption of raw clams in Shanghai in 1988: around 300,000 individuals were infected. Hepatitis A outbreaks due to contaminated food remain a public health problem in developed countries with low or intermediate endemicity. Person-to-person transmission Close contact between infected and susceptible people is the most common mode of HAV transmission in developing and developed countries. Transmission is facilitated by the prolonged shedding of HAV in the feces before and after the onset of symptoms, and asymptomatic infections, especially in young children. This type of HAV transmission occurs frequently in close institutions like schools, nurseries or day care centers. Other modes of transmission Outbreaks of HAV infection frequently occur among MSM. HAV is not transmitted by the semen but by oral contact with fecally contaminated sites. The initial introduction of HAV into an MSM community is followed by its rapid spread and subsequent persistence. Outbreaks of HAV infection have been also reported among people who inject drugs. Lastly, HAV can, very rarely, be transmitted by the transfusion of blood or blood products. In some cases, the recipients are HAV IgG positive. Blood donors are not screened for HAV because acute HAV infections are sporadic among blood donors and there are no chronic carriers. However, the plasma industry tests all sources of plasma by testing mini-pools for HAV nucleic acid.

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Hepatitis E virus HEV genotypes 1/2 and genotypes 3/4 are transmitted differently. HEV genotype 1 and 2 infections occur mainly in developing countries, where up to 25% of infected pregnant women can die of severe hepatitis. It can be responsible for outbreaks in areas where it is highly endemic. Drinking contaminated water is the main route of transmission and outbreaks are often linked to fecal contamination of drinking water. Person-to-person transmission of HEV is uncommon. The transmission from infected women to their newborns is well documented, and transmission of HEV genotype 1 through blood transfusions has been reported. HEV genotypes 3 and 4 are found in high-income countries. Since these genotypes have animal reservoirs, the main route of their transmission is from eating HEV-infected animal products, especially undercooked meat. Eating infected pork liver, pork products containing liver, and other raw or undercooked pork meat are the main sources of HEV infections. Infected game meat is another source of human infection. Strains of rabbit HEV have been detected in humans. HEV shed by infected animals can contaminate water sources, leading to the accumulation of HEV in fruit, vegetables, and shellfish. Hence, HEV RNA (HEV genotype 3) has been detected on red fruit, strawberries, salads and in spices, as well as in oysters and mussels. Recent studies in many European countries, China and Japan have also shown that HEV can be transmitted by transfusion of blood products. Most cases of transfusiontransmitted HEV infection are asymptomatic, and only a small proportion of recipients of infected blood products develop symptomatic hepatitis. The frequency of blood donors testing positive for HEV RNA across many countries ranges from 1:600 to 1:14,799. Blood donors are now systematically screened in several European countries.

Clinical Presentation and Course Hepatitis A virus The clinical manifestations of HAV infection usually range from asymptomatic infection to fulminant hepatitis, depending on the age of the patient. Thus, 70% of infected children under 6 years old are asymptomatic while over 70% of infections in adults are symptomatic. Symptoms, including fever, malaise, nausea, vomiting, abdominal pain, dark urine and jaundice, develop after an incubation period of approximately 4 weeks (range 2–7 weeks). Other symptoms like myalgia, pruritus, diarrhea, arthralgia or skin rash are less common. While there is no evidence of chronic liver disease or persistent infection after acute hepatitis, some patients (4%–20%) suffer from prolonged disease or relapsing disease lasting up to 6 months, with prolonged HAV excretion in the feces. Atypical manifestations reported following HAV infection are: relapsing hepatitis, prolonged cholestasis and complicated cases with acute kidney injury or autoimmune hepatitis. Relapsing hepatitis A is defined by a biphasic peak of serum liver enzyme (aminotransferase) with 4–7-week intervals between the first and second peaks. Prolonged cholestatic hepatitis A is associated with pruritus, fatigue, loose stools and weight loss. Prolonged cholestatic cases can be predicted by the detection of HAV RNA in the plasma after 20 days of illness, while relapse is not predictably linked to the presence of plasma HAV RNA. Acute kidney injury occurs in 1.5%–4.7% of patients with non-fulminant hepatitis A. Renal damage may be due to pre-renal factors associated with anorexia, nausea, vomiting, diarrhea and fever as well as the nephrotoxic effect of hyperbilirubinemia, immune complex-mediated nephritis, interstitial nephritis and rare massive intravascular hemolysis. Other rare extra-hepatic manifestations have been reported: autoimmune hemolytic anemia, aplastic anemia, pure red cell aplasia, pleural or pericardial effusion, acute pancreatitis and neurological complications (mononeuritis, mononeuritis multiplex and Guillain-Barré syndrome). Fulminant hepatitis is a rare complication of HAV infection. The incidence varies between 0.015% and 0.5% of acute hepatitis A cases. Older adults with underlying chronic liver disease are at greatest risk of acute liver failure. HAV infection during pregnancy is associated with a great risk of maternal complications and preterm labor, despite the relatively mild features of the disease. Fetal outcome is usually benign despite prematurity.

Hepatitis E Virus Most acute HEV infections are asymptomatic or minor systemic illnesses, whatever the genotype. The typical early phase symptoms of acute hepatitis E that develop after an incubation period of 2–6 weeks are unspecific and include: flu-like myalgia, arthralgia, weakness, and vomiting. Symptoms of acute hepatitis include jaundice, fatigue, itching, nausea, pale stools, and dark urine. The clinical symptoms and signs are indistinguishable from those of other forms of viral hepatitis. Acute hepatitis E in immunocompetent patients usually resolves spontaneously. Although reinfections are possible, they are less likely to develop symptomatic hepatitis than non-immune individuals. A few HEV-infected individuals (0.5%–4%) develop acute liver failure. Patients with pre-existing chronic liver diseases are at increased risk of liver failure and mortality can be as high as 67%. There have been cases that have needed liver transplantation following HEV infection. Pregnant women are at high risk of developing symptomatic disease if they become infected with HEVgenotype 1 during the second and third trimesters. Many progress to acute liver failure, haemorragia and eclampsia, and the mortality rate can reach 15%–25%. Miscarriages, preterm deliveries, stillbirths and perinatal mortality are all more frequent in HEV genotype 1-infected pregnant women. Their new-born babies are also at risk of HEV infection through maternal–fetal transmission and of developing complications, such as anicteric or icteric hepatitis, hypoglycemic and neonatal death. Conversely, pregnant women infected with

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HEVgenotype 3 or HEV genotype 4 do not suffer from such maternal mortality, perhaps because HEV genotype 1 replicates more rapidly in decidual and placental tissues than do other genotypes. Immunocompromised patients, such as those on immunosuppressive therapy following solid organ transplantation, patients with hematological disease on chemotherapy, patients with rheumatic disorders on heavy immunosuppression immunotherapy, and HIV-infected patients with low CD4 T cell counts, can all suffer from chronic HEV infections. Most immunosuppressed patients are asymptomatic. Chronic HEV infection is defined as HEV replication that persists for more than 3 months. Patients infected with HEV genotype 3, HEV genotype 4 or HEV genotype 7 can suffer from chronic HEV infections. A single case of chronic HEV genotype 1 infection has been reported; this needs confirmation. One third of solid organ transplant (SOT) recipients or patients with hematological disease spontaneously clear the virus, while two-thirds develop a chronic infection. The use of tacrolimus (a more potent immunosuppressant than cyclosporine A), a low lymphocyte subset count and less-active anti-HEV-specific T cell responses are more frequently associated with the development of a chronic infection in SOT patients. About 10% of chronically infected patients develop cirrhosis a few years after their infection. Lastly, liver fibrosis can regress after HEV clearance. While HEV reactivation after clearance is very rare, reinfections can occur and can lead to chronic hepatitis. The awareness of extrahepatic effects of HEV infections has increased. Most involve the neurological and renal systems. HEV infections are associated with a range of neurological injuries and cases of neurological injury in HEV genotype 1-infected Asians and HEV genotype 3-infected Europeans have been reported. These neurological injuries seem to be more frequent in immunocompetent patients (22.6%) than in immunocompromised ones (3.2%). The neurological disorders associated with an HEV infection include neuropathic pain, painless sensory disorders, neuralgic amyotrophy (Parsonage-Turner syndrome), GuillainBarre syndrome, encephalitis/myelitis, mononeuritis multiplex, Bell’s palsy, vestibular neuritis, myositis and peripheral neuropathy. All the HEV-infected patients with neurological manifestations generally have normal or modestly abnormal liver function. Thus, the neurological symptoms and signs dominate the clinical picture in these patients. Both acute and chronic HEV infections can lead to impaired renal function. Renal biopsies from patients infected with HEV genotype 1 and HEV genotype 3 show signs of glomerular disease, including membranoproliferative glomerulonephritis with or without cryoglobulinemia and membranous glomerulonephritis. Flare-ups of pre-existing IgA nephropathy may also occur in patients infected with HEV. HEV RNA was detected in the cryoprecipitate from the serum of an immunocompetent patient with an acute HEV infection who presented with cryoglobulinemic membranoproliferative glomerulonephritis. HEV infection has been identified as an independent predictive factor for cryoglobulinemia in SOT recipients. Clearance of HEV is associated with improved renal function and a decline in proteinuria in most patients, which suggests that HEV is probably the cause of the renal injury. Hence, patients with hepatitis who have impaired renal function or proteinuria should perhaps be screened for HEV. Lastly, acutely infected patients may display hematological disorders like thrombocytopenia, autoimmune hemolytic anemia, aplastic anemia, and acute liver failure associated with pure red-cell aplasia. Up to 25% of HEVgenotype 3-infected patients can suffer from asymptomatic monoclonal gammaglobulin but the clinical significance of this observation is uncertain. Acute episodes of pancreatitis have been reported in patients infected with HEV genotype 1 from South-East Asia but not those with HEV genotype 3 or HEV genotype 4 infections.

Diagnosis Clinical manifestations and biochemical tests alone cannot distinguish between an HAV and an HEV infection. Serum alanine aminotransferase activity is elevated during the prodromal phase and during the initial part of the icteric phase (Fig. 3). An HAV/HEV infection can be diagnosed either directly by detecting the genome or antigen in the blood or other body fluids or indirectly by detecting antibodies in the serum. The IgM response is detected around the time that the serum alanine aminotransferase activity increases. Thus, the presence of HAV/HEV IgM in the serum is a key marker of an acute infection. The IgG response appears shortly after the IgM response and can persist for several years.

Antigen Detection Hepatitis A virus HAV antigen can be detected in both blood and fecal samples by immunoassays, but this is less sensitive than nucleic acid assays. No commercial assay is presently available.

Hepatitis E virus Assaying for HEV capsid antigen can be used to diagnose an HEV infection. The sensitivity of the available commercial assay (Wantai Biological Pharmacy, China) is 91% (88% in immunocompetent and 94% in immunocompromised patients) and the specificity is 100%. This assay could be used for the direct diagnosis of an HEV infection by laboratories with no molecular diagnosis facilities.

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Fig. 3 Virus detection (HAV RNA or HEV RNA) at different sites and serologic response (IgM and IgG antibodies). ALT, alanine aminotransferase.

Table 1

Commercial molecular assays for the detecting/quantifying HAV or HEV RNA

Virus Manufacturer

Product name

Type of test

Analytical sensitivity

HAV Altona Diagnostics

Real Star HAV RT-PCR Procleix Parvo/HAV COBAS Taq Screen DPX

In vitro diagnostics real-time PCR qualitative test

46 IU/ml

Transfusion, TMA allowing simultaneous detection of HAV and Parvovirus B19 1 IU/ml Transfusion, Real-time PCR allowing simultaneous detection of HAV and 1 IU/ml Parvovirus B19

Real Star HEV RT-PCR Eurobioplex HEV RNA FTD Hepatitis E RNA Procleix HEV AmpliCube HEV COBAS HEV

In vitro diagnostics quantitative test real-time PCR In vitro diagnostics Quantitative test Real Time PCR In vitro diagnostics quantitative test real-time PCR Transfusion, TMA qualitative test In vitro diagnostics quantitative test real Time PCR Transfusion, real-time PCR qualitative test

Hologic/Grifols Roche

HEV Altona Diagnostics Eurobio Fast-track Diagnostics Hologic/Grifols Mikrogen Diagnostik Roche

20 IU/ml 40 IU/ml 188 IU/ml 7 IU/ml 20 IU/ml 7 IU/ml

Nucleic Acid Detection Hepatitis A virus HAV RNA can be detected in the blood and feces by RT-PCR or TMA before the increase in alanine aminotransferase activity and the appearance of clinical symptoms. Several commercial tests are available, with limits of detections ranging from 1 to 46 IU/ml (Table 1).

Hepatitis E virus HEV RNA can be detected in the blood, other body fluids and feces using nucleic acid amplification. Most RT-PCR assays, including commercial assays, target ORF3. A TMA assay performed on a fully automated platform is well adapted for high throughput testing. The limit of detection of current assays is 7–188 IU/ml (Table 1). Reverse transcription droplet digital PCR gives absolute quantities of HEV RNA without a standard curve. Lastly, the loop-mediated isothermal amplification assay provides a one-step single tube amplification of HEV RNA.

Serological Tests Hepatitis A virus Enzyme or chemiluminescent immunoassays for HAV IgM and IgG are commercially available in either microplate or multiparametric automated formats (Table 2). The diagnostic performance of these immunoassays in terms of sensitivity and specificity is well established (4 95%). No commercial rapid immunochromatographic assay is presently available.

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Table 2

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Commercial immunoassays for detecting anti-HAV and anti-HEV antibodies

Virus

Marker

Manufacturer

Product name

Type of test

HAV

HAV IgM

Abbott

Automate

HAV IgG

Roche Biomerieux Siemens Beckman-Coulter Diasorin Ortho Abbott

Rapid test Rapid test Microplate Microplate Microplate Microplate Microplate Automate Automate Automate Microplate Microplate Microplate Microplate Microplate Automate Automate Automate

HEV

HAV total antibodies

Roche Biomerieux Siemens Beckman-Coulter Diasorin Ortho

ARCHITECT HAVAb-IgM ALINITY HAV Ab IgM COBAS Anti-HAV IgM VIDAS HAV IgM ADVIA Centaur Anti-HAV IgM ACCESS HAV IgM LIAISON HAV IgM VITROS Anti-HAV IgM ARCHITECT HAVAb-IgG ALINITY HAV Ab IgG COBAS Anti-HAV VIDAS Anti HAV Total ADVIA Centaur Anti-HAV Total ACCESS HAV Ab LIAISON Anti-HAV VITROS Anti-HAV Total

HEV IgM

Wantaï MP Diagnostics Wantaï MP Diagnostics Adaltis Mikrogen Euroimmun Biomerieux Orgentec Vircell Wantaï MP Diagnostics Adaltis Mikrogen Euroimmun Biomerieux Orgentec Vircell

HEV IgM Rapid test Assure HEV IgM rapid test HEV Elisa IgM HEV IgM ELISA EIAgen HEV M recomWELL HEV IgM ELISA Anti-Virus HEV IgM VIDAS anti-HEV IgM Alegria Anti-Hepatitis E Virus IgM Virclia Hepatitis E IgM Monotest HEV Elisa IgG HEV IgG ELISA EIAgen HEV G recomWELL HEV IgG ELISA Anti-Virus HEV IgG VIDAS anti-HEV IgG Alegria Anti-Hepatitis E Virus IgG Virclia Hepatitis E IgG Monotest

HEV IgG

Automate Automate Automate Automate Automate Automate Automate Automate Automate Automate Automate Automate Automate

HAV IgM, a key marker of acute infection, is detectable from the time the alanine aminotransferase activity increases and persists for 3–6 months thereafter. False positive results are possible, especially when the clinical picture does not reflect the HAV infection and there is no HAV IgG. The presence of HAV IgG alone is a marker of past infection or vaccination. Testing for HAV IgG can be used to identify the unvaccinated individuals who require immunization, but the cost-effectiveness of screening depends on both age and epidemiologic parameters. Post-vaccination tests are not required due to the high efficacy of HAV vaccines.

Hepatitis E virus Enzyme immunoassays for HEV IgM and IgG are commercially available in Europe and Asia in formats for microplates and multiparametric automated instruments (Table 2). Several immunochromatographic assays are also available in different countries. The antigens used are usually recombinant ORF2 capsid protein and/or ORF3 protein from HEV genotype 1 strain. The diagnostic performance of these immunoassays varies considerably and should be carefully evaluated. The presence of HEV IgM in the serum is a key marker of an acute infection. Using a validated PCR assay as a reference, studies have shown that the sensitivity of IgM immunoassays is 497% for immunocompetent patients and 80%–85% for immunocompromised patients; their specificity is 499.5%. Longitudinal follow-up of patients with acute hepatitis E proven by nucleic acid testing showed HEV IgM persisted for 6–12 months. The presence of HEV IgG alone is a marker of a past infection. It is difficult to compare seroprevalence rates for different populations obtained by different laboratory methods because the analytical sensitivity of IgG immunoassays varies enormously. The limits of detection of commercial HEV IgG assays vary from 0.25 WHO unit/ml to 2.5 WHO unit/ml when measured with an international standard (WHO reference reagent established in 2002; National Institute for Biological Standards and Control, Code 95/584). As highlighted in a meta-analysis of studies of HEV seroprevalence in Europe, the most sensitive immunoassay produced

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the highest estimates of HEV IgG seroprevalence. Determining the HEV IgG concentration could be useful for estimating the risk of reinfection after a natural infection or vaccination.

Vaccines and Antiviral Agents Hepatitis A Virus Prevention against an HAV infection can be afforded by adequate sanitation and housing facilities, together with good personal hygiene. Passive or active immunization, before or after exposure, also provides effective protection against HAV infection.

Immunoglobulin Passive immunization with human immunoglobulins can be used for pre-exposure prophylaxis. An intra-muscular injection of immunoglobulins (0.02–0.06 ml/kg) is effective within hours of injection and for periods of 12–20 weeks. Human immunoglobulins can also be used for post-exposure prophylaxis, especially in immunosuppressed people, individuals at risk of severe disease, and infants less than 1 year old. As the average post-exposure incubation period of hepatitis A is about 28 days (but could be as short as 15 days), the time for intervention is only about 2 weeks. While immunoglobulins may protect against the symptoms of hepatitis A, they may not prevent infection following exposure. In recent years, the decline in the concentrations of HAV antibodies in the plasma pools used to prepare immunoglobulins has raised the question of the efficacy of these immunoglobulins in post-exposure prophylaxis. In addition, the short duration of protection and the increasing cost of immunoglobulins has led to a search for alternatives.

Vaccine Inactivated hepatitis A vaccines contain virus particles produced in cell culture, purified, inactivated with formalin, and adsorbed onto aluminum hydroxide. Vaccines that combine hepatitis A and typhoid protection and hepatitis A and hepatitis B protection have been licensed, as have hepatitis A monovalent vaccines. The immunogenicity of the combination vaccines is equivalent to that of the monovalent hepatitis A vaccine. Live-attenuated HAV vaccines are available in China. Traditionally, two doses of inactivated vaccine at an interval of 6–12 months are recommended, beginning after 12 months of age. The protective efficacy is nearly 95% and protection lasts for at least 10 years. Modeling studies predict that 88% of individuals who were seronegative prior to vaccination will remain protected for at least 30 years. Consequently, the Centers for Disease Control and Prevention now recommend the pre-exposure prophylaxis of 2–40 year-old exposed patients with vaccine instead of immunoglobulins. The threshold for protection from HAV infection in humans is an antibody concentration of 10–33 IU/ml, depending on the assay used. However, clinical experience suggests that vaccination may provide protection even when no HAV antibodies can be detected using standard immunoassays. A positive test for total HAV antibodies signifies immunity to hepatitis A. Optimal strategies for determining the need for HAV vaccination are based on seroprevalence. Vaccination is not routinely recommended for people living in areas where HAV is very prevalent. Almost all such people are asymptomatically infected with HAV in childhood, which effectively prevents clinical hepatitis A in adolescents and adults. Vaccination of all children may protect the health of adolescents and young adults living in countries where HAV prevalence is intermediate. The vaccination of all babies in Argentina with a single dose of inactivated HAV vaccine at 12 months led to marked reduction in the incidence of symptomatic hepatitis A, fulminant hepatitis, and liver transplantation. Targeted vaccination of high-risk groups (travelers to regions of high HAV endemicity, MSM, injecting drug users, immunosuppressed patients, patients infected with HIV, and patients with chronic liver disease) is generally recommended for people living in countries where HAV prevalence is low, but extension to the general population is sometimes an option. The United States began targeted hepatitis A vaccination for high-risk groups and children living in high-incidence communities in 1999. The Centers for Disease Control and Prevention recommended, in 2006, that every child between 12 and 23 months old should be vaccinated. Clinical trials suggest that an inactivated HAV vaccine effectively prevents infection when given within 14 days post-exposure. This has been confirmed by a randomized controlled trial that included 1080 susceptible children and adults. They were given either immunoglobulins or one dose of inactivated HAV vaccine. Symptomatic infection occurred in 3.3% of Ig recipients and in 4.4% of those vaccinated.

Treatment There is no specific treatment for hepatitis A. Recovery from symptoms may be slow, taking several weeks or months. Supportive care includes adequate hydration and nutritional support so as to maintain patient comfort and an adequate nutritional balance, including replacing fluids lost by vomiting and diarrhea. Acetaminophen should not be used to control fever because of its liver toxicity. Anti-vomiting medication should not be given. Considering that acute renal failure or hemolytic anemia may complicate hepatitis A, renal function should be regularly tested, along with complete blood analysis. Individuals with hepatitis A and acute liver failure can be treated with N-acetylcysteine, while liver transplantation is considered for the sickest patients. However, HAV-related fulminant hepatitis resolves spontaneously more frequently than does fulminant hepatitis of other etiologies. This makes the decision to transplant or not rather difficult.

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Hepatitis E Virus Prophylaxis Adequate sanitation and housing, together with personal hygiene, provide the best protection against HEV infection in developing countries. To these can be added avoiding eating uncooked or undercooked meat. “At risk” food should be cooked thoroughly, with an internal temperature of 711C for 20 min to completely inactivate HEV. The high prevalence of viremic blood donations in developed countries and the great proportion of immunocompromised blood recipients has led practitioners to define measures to improve blood safety. The inactivation of pathogens in blood components using amotosalen is not effective for HEV, while treatment with riboflavin (vitamin B2) plus ultraviolet light has a limited effect. Consequently, several developed countries have adopted measures to improve blood safety based on the epidemiology of HEV. All blood donations are screened in Ireland (since 2016), the United Kingdom, the Netherlands (both since 2017) and Switzerland (since 2018). Plasma donations intended for transfusion to patients at risk of severe disease have been selectively screened in France since 2013.

Vaccine Two vaccines against HEV vaccines have been developed; one is a 56-kDa subunit antigen (amino acids 112–607 of ORF2 expressed by cultured insect cells) and the other is a subunit antigen, HEV 239 (amino acids 368–606 of ORF2 expressed by Escherichia coli). Only HEV 239 has been licensed for use in humans, and it is only available in China. The Hecolin® vaccine was developed to elicit protective antibodies against all HEV genotypes and provides cross-protection for both genotypes 1 and 4. The efficacy of this threedose vaccine is 97%, as judged by its ability to prevent episodes of symptomatic acute hepatitis and its long-term efficacy during follow-up. Modeling studies suggest that it can provide protection for up to 30 years. The vaccine seems to be safe for pregnant women. However, its safety and efficacy has not been tested on patients with chronic hepatitis or on immunosuppressed patients. The concentration of HEV IgG needed to prevent infection after a natural infection or vaccination in clinical trials is still unknown, although one study reported that an antibody concentration of 2.5 WHO units/ml is protective. However, both immunocompromised and immunocompetent patients with antibody concentrations below 10 WHO units/ml have suffered reinfection.

Management of acute hepatitis E Most immunocompetent individuals who have acute hepatitis E do not require antiviral therapy with ribavirin because HEV is spontaneously cleared. Whether early treatment with ribavirin helps clear the virus or decreases the risk of liver failure remains to be determined. A few acutely HEV-infected patients have been treated with ribavirin. Their liver enzyme activities were rapidly normalized and they all cleared HEV RNA quickly. However, there was no control group and the ribavirin doses and treatment durations varied greatly. Thus, spontaneous improvement cannot be ruled out. Corticosteroids might decrease the risk of progression to liver failure in patients with fulminant hepatitis E. Some case reports have found that corticosteroid therapy improved liver function. Lastly, some case of HEV infection required liver transplantation.

Management of chronic infections The first-line treatment of organ transplant patients with chronic hepatitis is to reduce their immunosuppressive drugs, especially those targeting T cells (such as calcineurin inhibitors), when possible (Fig. 4). This allows one-third of patients to clear the HEV. The HEV infection in the remaining two-thirds of patients can be efficiently treated by ribavirin monotherapy. Most (78%) patients achieved a sustained virological response after 3-months of ribavirin therapy. Longer treatment (6 months) can allow HEV clearance in relapsers. Persistent HEV shedding in the feces at the end of therapy is associated with HEV relapse. The main side effects of ribavirin treatment are dose-dependent anemia, dry cough and skin reactions. The ribavirin dose should be adapted to maintain hemoglobin and eGFR concentrations because chronically infected patients can suffer from anemia or impaired renal function. It has been suggested that ribavirin inhibits HEV replication by inhibiting inosine monophosphate dehydrogenase (IMPDH), leading to the depletion of guanosine triphosphate pools. Deep sequencing analysis has identified several HEV RNA mutations, such as G1634R. Recent studies have shown that ribavirin increases HEV heterogeneity that seems to be reversible. However, the role of HEV RNA variants and their impact on HEV treatment outcome are uncertain. Pegylated-interferon has also been successfully used to treat HEV infections in liver transplant patients. A 3-month course of pegylated IFNa produced a sustained virological response in a few liver transplant recipients and in a patient with a chronic HEV infection who was on hemodialysis. However, IFNa cannot be used in other organ transplant patients because it increases the risk of acute rejection. Consequently, ribavirin therapy is the main drug used to treat chronic HEV infections. Pegylated-IFNa therapy is restricted to liver transplant patients who do not respond to ribavirin. No other therapy is available for other transplant patients (Fig. 4). Lastly, a few cases of chronic HEV infections in immunocompromised patients other than SOT has been reported. PegylatedIFNa, ribavirin or a combination of the two was used to effectively treat HEV infections in patients with hematological disease receiving chemotherapy and patients who were HIV-positive.

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Fig. 4 Treatment algorithm for immunocompromised patients who are solid organ transplant recipients.

Conclusion While HAV is an ancient virus, it remains a frequent cause of disease that affects millions of individuals annually despite the availability of efficacious, safe hepatitis A vaccines. The worldwide improvements in sanitary and socio-economic conditions has shifted the epidemiological pattern of hepatitis A. The vaccination of infants could be an efficient way of controlling the infection in an entire population. HEV is a major health problem in developing countries, where it is responsible for sporadic outbreaks. Improved sanitary conditions and the availability of a vaccine will help decrease the incidence of infections in these countries. HEV infections also cause several problems in developed countries. These include zoonotic transmission, transfusion transmission, persistence in immunocompromised patients, and severe forms in patient with pre-existing liver diseases. Any meat at risk should be thoroughly cooked to prevent the foodborne transmission of HEV. Strategies to prevent transfusion transmitted infections must be implemented according to the prevalence of HEV in each specific country. The use of ribavirin has been a major advance in the management of chronic infections in immunocompromised patients. However, new anti-HEV compounds are needed to treat patients who relapse or do not tolerate ribavirin.

Further Reading Dalton, H.R., Kamar, N., van Eijk, J.J., et al., 2016. Hepatitis E virus and neurological injury. Nature Reviews Neurology 12, 77–85. European Association for the Study of the Liver, 2018. EASL clinical practice guidelines on hepatitis E virus infection Journal of Hepatology 68, 1256–1271. Hofmeister, M.G., Foster, M.A., Teshale, E.H., 2019. Epidemiology and transmission of hepatitis A vrus and hepatitis E virus infections in the United States. Cold Spring Harbor Perspectives in Medicine 9. Kamar, N., Izopet, J., Pavio, N., et al., 2017. Hepatitis E virus infection. Nature Reviews Disease Primers 3, 17086. 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. Nimgaonkar, I., Ding, Q., Schwartz, R.E., Ploss, A., 2018. Hepatitis E virus: Advances and challenges. Nature Reviews Gastroenterology & Hepatology 15, 96–110. Victor, J.C., Monto, A.S., Surdina, T.Y., et al., 2007. Hepatitis A vaccine versus immune globulin for postexposure prophylaxis. New England Journal of Medicine 357, 1685–1694.

Relevant Websites https://ecdc.europa.eu/en/hepatitis-a Hepatitis A. ECDC. Europa EU.

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https://www.who.int/immunization/diseases/hepatitisA/en/ Hepatitis A. World Health Organization. https://www.who.int/news-room/fact-sheets/detail/hepatitis-a Hepatitis A. World Health Organization. https://www.who.int/immunization/policy/position_papers/hepatitis_a/en/ Hepatitis A position paper. WHO.

Management of Patients With Chronic Hepatitis B (Hepadnaviridae) and Chronic Hepatitis D Infection (Deltavirus) Milan J Sonneveld, Erasmus University Medical Center, Rotterdam, The Netherlands Suzanne van Meer, University Medical Center Utrecht, Utrecht, The Netherlands r 2021 Elsevier Ltd. All rights reserved.

Nomenclature

HBsAg Hepatitis B surface antigen LAM Lamivudine TAF Tenofovir alafenamide TBV Telbivudine TDF Tenofovir disoproxil fumarate

ADV Adefovir CHB Chronic hepatitis B ETV Entecavir HBeAg Hepatitis B e antigen

Glossary HBsAg loss Clearance of HBsAg from serum, functional cure of hepatitis B. NA Nucleo(s)tide analog. Direct acting antivirals that suppress HBV DNA. Examples include entecavir and

tenofovir disoproxil fumarate, and tenofovir alafenamide. PEG-IFN Pegylated interferon alfa, an immunomodulatory used for finite therapy.

Management of Chronic Hepatitis B and Hepatitis D Chronic Hepatitis B Introduction Management of chronic hepatitis B (CHB) requires careful consideration of treatment indications given the requirement for longterm therapy in most patients. For patients who are in need of therapy, options include nucleo(s)tide analogs (NA) and pegylated interferon alfa (PEG-IFN), both with their own advantages and limitations.

Treatment Indications The indications for treatment in patients with CHB are based on a risk profile conferred by a combination of (1) the extent of hepatic fibrosis, (2) the level of viremia and (3) the severity of liver inflammation. They are based on observations from long-term follow-up studies which have shown that these are the main determinants of progression of liver disease to cirrhosis, hepatocellular carcinoma (HCC) and liver-related mortality (Terrault et al., 2016; European Association for the Study of the Liver, 2017; Chen et al., 2006, 2011). Current treatment guidelines from both the European Association for the Study of the Liver (EASL) and the American Association of Study of Liver Disease (AASLD) recognize that all patients with cirrhosis and viremia require urgent treatment. In patients without cirrhosis, a combination of HBV DNA levels, ALT levels and extent of liver inflammation and fibrosis is used to determine indication for treatment (Table 1) (European Association for the Study of the Liver, 2017; Terrault et al., 2018a). Liver biopsy is still the gold standard for assessment of hepatic fibrosis, but it is associated with significant risks to the patient. As a result, it has been mostly supplanted by either non-invasive indices that predict the extent of fibrosis (such as the aspartate aminotransferase – to platelet ratio index [APRI] and fibrosis score based on 4 factors [FIB-4]) and/or liver stiffness-based assessment (e.g., Fibroscan). It is important to note that high rates of misclassification have been reported with both APRI and FIB-4, and that liver stiffness based methods may overestimate the extent of fibrosis in the presence of significant liver inflammation (European Association for the Study of the Liver and Asociacion Latinoamericana para el Estudio del H, 2015; Sonneveld et al., 2019a). In patients not meeting standard treatment criteria, antiviral therapy may be considered in patients at higher risk of HCC, such as those with a family history of HCC, or HBeAg positive patients aged over 30 with high levels of HBV DNA but persistently normal ALT levels (European Association for the Study of the Liver, 2017). Patients with extrahepatic manifestations may also be treated even if they do not meet conventional treatment criteria (European Association for the Study of the Liver, 2017).

Treatment Goals in CHB Eradication of HBV from infected host hepatocytes cannot be achieved with currently available drugs due to persistence of covalently closed circular (ccc)DNA despite antiviral therapy. The aim of treatment is therefore to reduce or stop progression of

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Table 1 Guidelines

AASLD 2018 EASL 2017

Treatment indications HBeAg positive

HBeAg negative

HBV DNA

ALT

Liver Histology

HBV DNA

ALT

Liver histology

N/A >20,000 >2000

N/A >2
ULN >ULN

N/A >2000 >2000

N/A >2
ULN >ULN

>20,000 detectable

>2
ULN N/A

Cirrhosis N/A At least moderate inflammation and/or fibrosis N/A Cirrhosis

>20,000 detectable

>2
ULN N/A

Cirrhosis N/A At least moderate inflammation and/or fibrosis N/A Cirrhosis

Abbreviations: ULN, upper limit of the normal range; AASLD, American Association for the study of Liver Diseases; EASL, European Association for the Study of the Liver; N/A, not applicable.

liver inflammation to liver fibrosis and cirrhosis, and to prevent occurrence of hepatocellular carcinoma (HCC). Various surrogate endpoints are used asses treatment efficacy in clinical practice (Terrault et al., 2016; European Association for the Study of the Liver, 2017). Frequently used endpoints include normalization of ALT (biochemical response), loss of HBeAg from serum with or without seroconversion to anti-HBe (serological response), suppression of HBV DNA to low (o2000 IU/mL) or undetectable levels (virological response), and improvement of liver histology. Complete remission of disease (sometimes called functional cure) is defined by a loss of HBsAg from serum, since the HBsAg negative state is associated with an excellent prognosis with very low relapse rates in immunocompetent patients (Perrillo, 2006; van Zonneveld et al., 2004). For patients treated with NA, HBV DNA kinetics may be further categorized as complete virological response (HBV DNA undetectability) or partial virological response (at least 1 log HBV DNA decrease after commencing therapy, but persistently detectable HBV DNA at 1 year). Virological breakthrough is defined as a 1 log increase of HBV DNA above the nadir and may be the result of emergence of noncompliance or, more rarely, emergence of viral resistance.

Treatment Options Current treatment options for patients with CHB consist of the nucleo(s)tide analogs (NA) and pegylated interferon alfa (PEGIFN). Both classes have different modes of action that translate into different treatment durations and different treatment goals.

Nucleo(s)tide Analogs NA are oral antiviral agents that inhibit the HBV polymerase/reverse transcriptase and cause chain termination. Until recently there were 5 available NA, including entecavir (ETV), tenofovir disoproxil fumarate (TDF), adefovir dipivoxil (ADV), telbivudine (TBV) and lamivudine (LAM). Recently, the novel tenofovir prodrug tenofovir alafenamide has been approved for the management of CHB, based on the results of an ongoing phase 3 trial showing non-inferiority with regard to antiviral efficacy when compared to TDF (Agarwal et al., 2018). Because continuous treatment with LAM, TBV and ADV is associated with high rates of resistance and subsequent therapy failure these drugs are no longer recommended for the treatment of non-pregnant patients with CHB. Among treatment naïve patients long-term therapy with ETV or TDF results in near universal HBV DNA suppression to undetectable levels in compliant patients (Zoutendijk et al., 2011; Buti et al., 2015). Despite long-term HBV DNA suppression the rates of HBsAg loss remain low, often o1% per year (Zoutendijk et al., 2011; Buti et al., 2015). As a result, long-term therapy is required for most patients. Antiviral resistance has not yet been demonstrated definitively for TDF, and resistance rates with ETV appear to be very low among patients not previously exposed to LAM. As a result, failure to achieve undetectable HBV DNA is frequently caused by either non-compliance or may be the result of a delayed response due to high baseline viremia. Continuation of antiviral therapy in the absence of viral breakthrough has been shown to be successful in most patients (Zoutendijk et al., 2011). However, failure to achieve complete HBV DNA undetectability is observed in a small minority of patients. Persistent low level viraemia has been associated with a higher risk of adverse outcomes in patients with cirrhosis, but this has so far not been demonstrated for patients without advanced liver disease (Kim et al., 2017). Treatment adaptation is therefore advised in cirrhotics with low level viraemia (European Association for the Study of the Liver, 2017). For non-cirrhotic patients low levels of detectable HBV DNA (o69 IU/mL) do not have to prompt therapy adaptation and may be sometimes accepted (European Association for the Study of the Liver, 2017).

Safety NA are generally well-tolerated. The most important safety issues are associated with long-term therapy. In particular, long-term ADV and TDF therapy has been associated with reductions in bone mineral density and creatinine clearance, and in rare cases

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Table 2

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Management of NA failure

Failed NA

Options

Lamivudine (LAM) Adefovir (ADV)

Switch to TDF or TAF LAM naïve: switch to entecavir (or TDF or TAF) LAM experienced: switch to TDF or TAF Switch to TDF or TAF Switch to TDF or TAF Switch to ETV Switch to TDF or TAF Consider TDF þ ETV

Telbivudine (TBV) Entecavir (ETV) Tenofovir DF (TDF) or Tenofovir AF (TAF) Multidrug resistance

Abbreviations: TDF, tenofovir disoproxil fumarate; TAF, tenofovir alafenamide; ETV, entecavir; LAM, lamivudine.

development of Fanconi syndrome (Udompap et al., 2018). The risk of renal and bone events may be lower with TAF when compared to TDF, although Fanconi syndrome has also been observed in a patient treated with TAF (Agarwal et al., 2018; Bahr and Yarlagadda, 2019; Grossi et al., 2017) Monitoring of renal function and phosphate levels is therefore advised in patients treated with any of these agents. Among patients with severe liver disease, several case reports have suggested a risk of severe lactic acidosis with ETV therapy, although ETV has also been used safely in patients with decompensated cirrhosis (European Association for the Study of the Liver, 2017; Lange et al., 2009; Hung et al., 2019). In any case, careful monitoring of liver and renal functions is always indicated in patients with advanced liver disease.

Choosing the Appropriate First-Line Agent Among treatment naïve patients without significant comorbidities ETV, TDF and TAF are all excellent options. Based on the possible risk of renal and bone events in patients treated with TDF, patients at increased risk should preferably be treated with ETV or TAF. Among patients with a reduced renal function, ETV (adjusted dose) or TAF (no dose reduction if clearance >15 mL/min) are the best options.

Management of Patients With NA Failure In patients who failed on older generations of NA (i.e., LAM, TBV or ADV), resistance testing may be performed to confirm presence of mutations that confer reduced susceptibility to NA. Mutations associated with LAM resistance may confer crossresistance to ETV, whereas ADV resistance has been associated with somewhat lower response rates to TDF when compared to treatment naïve patients. However, long-term TDF has been successful in the majority of patients with ADV treatment failure. TDF monotherapy has also been effective in patients with ETV failure, with combination therapy only required for a select few patients with previous LAM failure who experience partial responses after long-term TDF therapy (Patterson et al., 2011; Lim et al., 2019; Zoulim et al., 2016). Based on these findings, recommendations have been made for treatment adaptation in patients who failed on NA therapy (Table 2).

Finite NA Therapy All current guidelines recognize that therapy may be discontinued in patients who have confirmed HBsAg negativity (European Association for the Study of the Liver, 2017). Relapse rates after drug withdrawal in HBsAg negative patients are very low in the absence of immune suppression and irrespective of anti-HBs seroconversion. Given the rarity of HBsAg loss with NA therapy, several studies have examined the possibility of discontinuation of NA in HBsAg positive patients. Recent studies show near universal increases in HBV DNA after drug discontinuation, with a subset of patients experiencing significant flares. However, a subgroup of patients achieves sustained HBV DNA levels o2000 IU/mL or even HBsAg loss without a need for further treatment (Berg et al., 2017; Chang et al., 2015; Hadziyannis et al., 2012). Since these favorable outcomes occur in only a minority of patients, current focus is on identifying patients most likely to achieve off-treatment sustained responses. While various factors have been shown to be associated with sustained disease remission, including serum levels of hepatitis B core related antigen (HBcrAg) and HBsAg, no definitive recommendations can yet be made on patient selection (Sonneveld et al., 2019b; Hsu et al., 2019). Nevertheless, guidelines suggest that therapy discontinuation may be considered in select patients without cirrhosis, in particular HBeAg positive patients who experienced HBeAg seroconversion and completed at least 12 months of consolidation therapy, and HBeAg negative patients who have been successfully treated for multiple (at least 3) years (European Association for the Study of the Liver, 2017). Careful monitoring is always indicated after drug withdrawal as major flares may occur. At this time, drug withdrawal is only advised in the context of studies.

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Pegylated Interferon Alfa Interferon alfa (IFN) has both immunomodulatory and direct antiviral effects in patients with CHB. The availability of the pegylated IFN alfa (PEG-IFN) has allowed once weekly subcutaneous dosing. PEG-IFN is used as a finite treatment course, typically for one year. Response is then assessed at 6–12 months post-treatment. Its main advantage is that it offers a chance at finite treatment for a subset of patients with a response, although this is offset by reduced tolerability when compared to the NA. Responses are defined differently for HBeAg positive and HBeAg negative patients and response rates vary widely across subgroups. Careful patient selection is therefore key to effective use of this agent in clinical practice.

Efficacy in HBeAg-Positive Patients HBeAg seroconversion was observed in 29%–30% of patients at 6 months post-treatment in two pivotal trials (Janssen et al., 2005; Lau et al., 2005). A combined endpoint of HBeAg loss with HBV DNA o2000 IU/mL at 6 months post-treatment was achieved in 23% in a recent meta-analysis of 3 global randomized trials (Sonneveld et al., 2013b), and HBsAg seroconversion was achieved in 4%–6% (Janssen et al., 2005; Lau et al., 2005). A long-term follow-up study of a predominantly European cohort showed that serological and virological response rates were sustained in the majority of initial responders through a mean of 3 years of followup, with increasing rates of HBsAg loss up to 30% in patients who became HBeAg negative after treatment (Buster et al., 2008).

Efficacy in HBeAg-Negative Patients In the registration trial for PEG-IFN in HBeAg-negative CHB response was defined as an HBV DNA level o20,000 copies/mL with normalization of ALT at 24 weeks post-treatment. This was observed in 36% (Marcellin et al., 2004). In another study comparing PEG-IFN alone with PEG-IFN plus ribavirin in a predominantly European cohort, response rates (HBV DNA o10,000 with normal ALT) at 24 weeks post-treatment were similar in both groups (16%–20%) (Rijckborst et al., 2010b). Long-term virological remission of HBeAg-negative disease, defined as sustained HBV DNA levels o2000 IU/mL, is achieved in around 23% of treated patients (Marcellin et al., 2009). Patients with an initial response may experience relapse during long-term follow-up, although a subset may also achieve HBsAg loss (Marcellin et al., 2009).

Safety of PEG-IFN in CHB PEG-IFN treatment is associated with considerable side-effects. The most frequently reported side-effects were a flu-like syndrome, myalgia, headache, fatigue and local reactions at the site of subcutaneous injection (Janssen et al., 2005; Lau et al., 2005; Marcellin et al., 2004). PEG-IFN therapy may result in hepatitis flares, which may lead to decompensation and death in patients with advanced liver disease. PEG-IFN is therefore contra-indicated in patients with (a history of) decompensated cirrhosis (Perrillo, 1989; Perrillo et al., 1990; Perrillo, 2001; Flink et al., 2005). PEG-IFN treatment is also associated with mild myelosuppressive effects, but PEG-IFN associated cytopenias are easily managed with dose-reductions and only rarely result in clinically significant infection or bleeding (van Zonneveld et al., 2005). The combination of PEG-IFN with TBV is contra-indicated due to a high risk of myopathy.

Selection of Patients for PEG-IFN Therapy Since response rates to PEG-IFN are limited, baseline selection of patients with the highest probability of response is essential for optimal use of PEG-IFN. Several baseline predictors have been identified that can be used to guide patient selection (Table 3).

HBeAg-Positive Patients Multiple factors have been identified that are associated with response to PEG-IFN in HBeAg-positive patients. Response rates are highest in those with HBV genotype A and those with higher baseline levels of ALT (Janssen et al., 2005; Lau et al., 2005). Various Table 3

Baseline factors associated with higher probability of response to pegylated interferon

HBeAg positive patients

HBeAg negative patients

Genotype A Higher ALT Lower HBV DNA Lower HBsAg

Genotype C Higher ALT Lower HBV DNA Lower HBsAg Younger age

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Table 4 Rapid assessment for eligibility for treatment with pegylated interferon based on a predicted probability of response of >30% HBV genotype

Consider pegylated interferon for HBeAg positive patients with

A B or C D

Either high ALT (Z2
ULN) or low HBV DNA (r9 log copies/mL) Both high ALT (Z2
ULN) and low HBV DNA (r9 log copies/mL) PEG-IFN generally not recommended due to low response rates

Note: Buster, E.H., Hansen, B.E., Lau, G.K., et al., 2009. Factors that predict response of patients with hepatitis B e antigen-positive chronic hepatitis B to peginterferon-alfa. Gastroenterology 137 (6), 2002–2009.

Table 5

Stopping-rule for HBeAg positive patients treated with pegylated interferon based on HBsAg levels during therapy HBV Genotype

Week

A

B

C

D

12 HBsAg 24 HBsAg

No decline >20,000

>20,000 >20,000

>20,000 >20,000

No decline >20,000

Note: At week 12, HBV genotype specific stopping rules are applied. No genotyping is necessary at week 24.

other factors, including lower baseline HBsAg levels, have been identified in subsequent meta-analyses (Buster et al., 2009; Chan et al., 2018). Based on these findings we proposed a simple scoring system to rapidly screen patients for potential PEG-IFN eligibility (Table 4) (Buster et al., 2009). A more extensive scoring system may subsequently be used for individual patient counseling (Buster et al., 2009; Chan et al., 2018). Future studies are focussing on genetic predictors of response, novel cytokines and viral mutants that may together be used to optimized individualized decisions (Sonneveld et al., 2012a,b, 2013a; Brouwer et al., 2019). These are however not yet ready for clinical application.

HBeAg-Negative Patients Less data are available on predictors of response to PEG-IFN in HBeAg-negative patients, which is unfortunate given the even lower response rates in this subgroup. A recent meta-analysis showed higher rates of response in patients with HBV genotype C and younger patients, with inconsistent results regarding HBV DNA levels and HBsAg levels across HBV genotypes (Table 4). A scoring system based on this meta-analysis could be used for assessment of the probability of treatment success (Lampertico et al., 2018).

On-Treatment Prediction of Response HBeAg-Positive Patients On-treatment monitoring of viral replication using HBV DNA, HBeAg and HBsAg levels may help to predict response during therapy. On-treatment HBV DNA and HBeAg decline is associated with higher rates of response, but diagnostic accuracy is suboptimal (Fried et al., 2008; Sonneveld et al., 2013c). Recent studies have shown a strong association between on-treatment HBsAg levels and the probability of response. Failure to achieve on-treatment declines and/or persistently high HBsAg levels were shown to predict failure with high accuracy (Sonneveld et al., 2010, 2013b,d). Based on these findings, HBV genotype specific stopping-rules have been defined for HBeAg-positive patients treated with PEG-IFN (Table 5) (Sonneveld et al., 2013b).

HBeAg-Negative Patients Similar to observations in HBeAg-positive disease, HBV DNA decline on-treatment is associated with treatment response in HBeAg negative CHB patients treated with PEG-IFN (Takkenberg et al., 2009) Subsequent studies have also shown that failure to achieve an on-treatment HBsAg decline is associated with non-response to therapy (Moucari et al., 2009; Marcellin et al., 2010). In the landmark European PARC study, which enrolled predominantly genotype D patients, a combination of HBsAg and HBV DNA levels allowed for reliable prediction of non-response to PEG-IFN. Patients who did not have an HBsAg decline by week 12, and who also failed to achieve a decline of >2 log IU/mL of HBV DNA, had no chance of achieving a sustained response (Rijckborst et al., 2010a). These findings were subsequently validated in two large independent trials, with superior performance among patients with HBV genotype D (Sonneveld et al., 2012a). Current guidelines therefore recognize that patients complying with these criteria should discontinue therapy for futility.

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Optimizing Response Rates: Treatment Prolongation and Combination Therapy The current licensed treatment duration for PEG-IFN is 48 weeks. Reducing the treatment duration has been shown to result in lower response rates (Cheng et al., 2014; Liaw et al., 2011). One study of genotype D HBeAg negative patients showed higher response rates with 96 weeks of therapy (Lampertico et al., 2013). A non-controlled study of HBeAg positive patients treated with PEG-IFN with LAM or ADV for 96 weeks also showed promising results (Cao et al., 2013; Wang et al., 2016). Confirmation of these findings is however required before this can be implemented in clinical practice. Theoretically, combination treatment with PEGIFN and NA could increase response rates through complementary modes of action. Studies have however consistently shown higher on-treatment response rates, including HBsAg loss, which have been off-set by off-treatment relapse (Janssen et al., 2005; Lau et al., 2005; Marcellin et al., 2004; Sonneveld et al., 2016). Combination therapy is therefore not advised.

Optimizing Response to NA With PEG-IFN Various studies have evaluated the efficacy of PEG-IFN add-on therapy to NA. These studies have shown increased HBV DNA, HBeAg and HBsAg declines, and also higher rates of HBeAg seroconversion and HBsAg loss with PEG-IFN add-on when compared to continued NA monotherapy (Brouwer et al., 2015; Bourliere et al., 2017; Lampertico et al., 2019; Chi et al., 2017). However, absolute response rates are low and predictors of sustained response are not yet established. Serum HBsAg levels may be useful to identify a subset more likely to respond, but clinically relevant cut-offs remain elusive (Liem et al., 2019). Current guidelines therefore do not recommend PEG-IFN add-on strategies at this time, although if effective selection tools become available this might be an option for a subset of patients.

Management of CHB in Special Populations Pregnancy All pregnant women should be tested for HBV infection. The risk of perinatal transmission in babies born to HBsAg positive mothers may be profoundly reduced by a combination of passive and active vaccination of the newborn, and combination therapy is considered cost-effective in settings with adequate healthcare infrastructure (European Association for the Study of the Liver, 2017; Chen et al., 2013). In the context of severe cost constraint, HBIG administration is sometimes limited to babies born to highly viremic (HBeAg positive) mothers, although this strategy is generally not recommended in guidelines (European Association for the Study of the Liver, 2017; Chen et al., 2013). The risk of transmission despite vaccination is predominantly determined by the level of viremia. Patients with high HBV DNA levels (>200,000 IU/mL and/or high HBsAg levels (above 4–4.5 log IU/mL)) should be treated with a NA to reduce the probability of perinatal transmission (Song et al., 2019). TDF is the preferred agent based on the extensive clinical experience in CHB and HIV infected patients. In pregnant women with low viremia and without signs of significant liver fibrosis treatment may be deferred until after delivery. Post-partum flares may occur both after therapy discontinuation and in untreated patients.

Immunosuppression Immunosuppression can cause HBV reactivation which may result in fulminant hepatic failure and death. CHB guidelines recommend screening for past or current HBV infection in all patients undergoing significant immunosuppression. The risk of reactivation is determined by HBsAg status (higher in HBsAg positive patients) and with increasing severity immunosuppression. It is recommended that all HBsAg positive patients at risk for reactivation receive prophylactic antiviral therapy. Among HBsAg negative, anti-HBc positive patients, the risk of reactivation should be used to guide decisions on the need for prophylactic therapy. Patients at moderate to high risk should receive prophylaxis.

Chronic Hepatitis Delta Introduction Hepatitis delta virus (HDV) is a defective RNA virus that requires HBsAg to complete virion assembly and secretion after autonomous replication. HDV infection can occur as an acute co-infection with HBV and HDV, which evolves to chronicity in only 2% of patients, or as an HDV superinfection in patients with a CHB infection resulting in a chronic HBV-HDV infection in 70%–90% of patients. In most cases of HDV infection, HBV DNA levels are suppressed HDV. HDV is a highly pathogenic virus and can cause severe liver injury that may result in fulminant hepatic failure and rapid progression to cirrhosis and hepatic decompensation, as well as an increased risk of liver cancer (Hughes et al., 2011).

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Screening The EASL and Asian Pacific Association for the Study of the Liver (APASL) guidelines on management of CHB recommend to rule out HDV coinfection in all patients with a CHB. In the AASLD guideline on hepatitis B screening is only advised in patients with specific risk factors including migrants from regions with high HDV endemicity, a history of intravenous drug use or high-risk sexual behavior, individuals infected with HCV or HIV and patients with elevated aminotransferases with low or undetectable HBV DNA (European Association for the Study of the Liver, 2017; Terrault et al., 2018a; Sarin et al., 2016).

Treatment Indication and Goals Patients with positive HDV RNA levels have an indication for antiviral therapy as liver disease can progress rapidly. However, risks and benefits of initiating therapy should be considered, including severity of liver disease which may be a contraindication to IFN based therapy (Terrault et al., 2018b). A complete virological response of both HBV and HDV infection, thus comprising both HBsAg loss and undectectable HDV RNA levels, is the most desirable endpoint of therapy. However, this occurs only in a small number of patients. The primary aim of anti-HDV therapy is therefore sustained off-treatment HDV RNA undetectability. Due to the high rate of late relapses after initially successful treatment, the definition of “sustained viral response” should be used with caution and long-term monitoring is essential (Yurdaydin et al., 2019).

Treatment Options Interferon Alpha Interferon alpha (IFN) is the only available drug that has been proven to have antiviral efficacy in chronic HDV infection. A Cochrane meta-analysis including five randomized controlled trials comparing IFN treatment for 3–12 months with a notreatment control group showed that a sustained suppression of HDV RNA at six months of follow-up post-treatment was seen in 17% of patients treated with IFN (compared to 5% in the control group, p ¼ 0.02) (Abbas et al., 2011). Response to IFN based therapy is associated with improved clinical outcomes (Wranke et al., 2017; Palom et al., 2019). Currently, standard IFN has been replaced by pegylated interferon alpha (PEG-IFN) because of a prolonged plasma half-life time allowing a once-a-week administration. Studies evaluating the efficacy of PEG-IFN for the treatment of chronic HDV infection showed slightly better results, with sustained suppression of HDV RNA at six months post-treatment in 25%–30% of patients (Wedemeyer et al., 2011; Triantos et al., 2012). Unfortunately, subsequent relapse rates are substantial. Long-term follow-up data from the HIDIT-I trail, the largest randomized trial investigating the safety and efficacy of PEG-IFN with or without adefovir in patients with a chronic HDV infection, showed a late relapse of HDV RNA in more than half of the patients (58%) through approximately four years of followup (Heidrich et al., 2014). Therefore, long-term follow-up with HDV RNA monitoring remains needed in patients with a negative HDV viral load after treatment as long as HBsAg is present in serum (Heidrich et al., 2014). The optimal duration of PEG-IFN therapy is not well defined. Current guidelines recommend treatment with PEG-IFN for at least 48 weeks (European Association for the Study of the Liver, 2017; Terrault et al., 2018a; Sarin et al., 2016). Several studies tried to increase efficacy by prolonging treatment duration. As of yet, treatment prolongation has not been consistently shown to be superior to conventional treatment durations (Heller et al., 2014; Wedemeyer et al., 2019), although some patients may benefit from a prolonged course of treatment (so-called slow responders) (Niro et al., 2016). On the other hand, preliminary studies that investigated shorter treatment durations up to six months showed relapses in almost all patients after discontinuation of therapy (Di Bisceglie et al., 1990; Porres et al., 1989). Based on existing data from previous studies, an accurate prediction of virological response in patients treated for a chronic HDV infection remains difficult. No clear stopping rules have been defined (European Association for the Study of the Liver, 2017; Terrault et al., 2018a; Sarin et al., 2016). However, studies have demonstrated that HDV RNA levels at week 24 of treatment with PEG-IFN are associated with treatment outcome (Keskin et al., 2015; Karaca et al., 2013; Yurdaydin et al., 2008). For example, HDV RNA negativity within 24 weeks of therapy was a strong predictor for sustained HDV suppression at 24 weeks post-treatment with a positive predicted value of 71%. Conversely, a decrease in HDV RNA level of less than 1 log combined with no decrease in HBsAg level effectively identified non-responders (Keskin et al., 2015). Confirmation of these findings is required before they can be implemented in clinical practice.

Nucleo(s)tide Analogs The use of nucleo(s)tide analogs (NA) has been extensively explored in chronic HDV infected patients. Several studies investigated the effect of NA treatment as monotherapy, but no effect on HDV viremia was observed (Wedemeyer et al., 2011; Yurdaydin et al., 2008, 2002; Niro et al., 2005). Furthermore, addition of NA to PEG-IFN therapy did not improve treatment outcome in several randomized trials (Wedemeyer et al., 2011, 2019; Abbas et al., 2016).

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Although NA are not effective against HDV, use of NA is recommended in patients with active HBV replication with persistent elevated HBV DNA levels (>2000 IU/mL), and/or in presence of cirrhosis (European Association for the Study of the Liver, 2017; Sarin et al., 2016; Terrault et al., 2018b), as predominance of both viruses can fluctuate during the natural course of disease and since HBV DNA levels may be associated with more rapid disease progression (Schaper et al., 2010).

Future New therapies are urgently needed for the treatment of chronic HDV, because of the overall poor treatment response and high rate of relapse after treatment with PEG-IFN. Several new drugs targeting various steps of the HBV and HDV life cycle, such as lonafarnib, Myrcludex-B, PEG-IFN lambda and nucleic Acid Polymers (NAPs), are being evaluated in phase 1 and 2 studies and have shown promise. Registration studies are expected to be commencing soon (Yurdaydin et al., 2019).

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Sonneveld, M.J., Hansen, B.E., Piratvisuth, T., et al., 2013b. Response-guided peginterferon therapy in HBeAg-positive chronic hepatitis B using serum hepatitis B surface antigen levels. Hepatology 58, 872–880. Sonneveld, M.J., Rijckborst, V., Zwang, L., et al., 2013c. Hepatitis B e antigen levels and response to peginterferon: Influence of precore and basal core promoter mutants. Antiviral Research 97 (3), 312–317. Sonneveld, M.J., Zoutendijk, R., Flink, H.J., et al., 2013d. Close monitoring of hepatitis B surface antigen levels helps classify flares during peginterferon therapy and predicts treatment response. Clinical Infectious Diseases 56 (1), 100–105. Sonneveld, M.J., Brouwer, W.P., Chan, H.L., et al., 2019a. Optimisation of the use of APRI and FIB-4 to rule out cirrhosis in patients with chronic hepatitis B: Results from the SONIC-B study. The Lancet Gastroenterology and Hepatology 4 (7), 538–544. Sonneveld, M.J., van Oord, G.W., van Campenhout, M.J., et al., 2019b. Relationship between hepatitis B core-related antigen levels and sustained HBeAg seroconversion in patients treated with nucleo(s)tide analogues. Journal of Viral Hepatitis 26 (7), 828–834. Takkenberg, B., Zaaijer, H.L., De Niet, A., et al., 2009. Baseline HBsAg level and on-treatment HBsAg and HBV DNA decline predict sustained virological response in HBeAg negative chronic hepatitis B patients treated with peginterferon alfa-2a and adefovir: An interim analysis. Hepatology 50 (4), Terrault, N.A., Bzowej, N.H., Chang, K.M., et al., 2016. AASLD guidelines for treatment of chronic hepatitis B. Hepatology 63 (1), 261–283. Terrault, N.A., Lok, A.S.F., McMahon, B.J., et al., 2018a. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Clinical Liver Disease 12 (1), 33–34. Terrault, N.A., Lok, A.S.F., McMahon, B.J., et al., 2018b. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 67 (4), 1560–1599. Triantos, C., Kalafateli, M., Nikolopoulou, V., Burroughs, A., 2012. Meta-analysis: Antiviral treatment for hepatitis D. Alimentary Pharmacology & Therapeutics 35 (6), 663–673. Udompap, P., Kim, D., Ahmed, A., Kim, W.R., 2018. Longitudinal trends in renal function in chronic hepatitis B patients receiving oral antiviral treatment. Alimentary Pharmacology & Therapeutics 48 (11–12), 1282–1289. Wang, Z., Sun, L., Wu, Y., Xia, Q., 2016. Extended duration versus standard duration of peginterferon alfa-2a in treatment of chronic hepatitis B: A systematic review and metaanalysis. Clinics and Research in Hepatology and Gastroenterology 40 (2), 195–202. Wedemeyer, H., Yurdaydin, C., Dalekos, G.N., et al., 2011. Peginterferon plus adefovir versus either drug alone for hepatitis delta. New England Journal of Medicine 364 (4), 322–331. Wedemeyer, H., Yurdaydin, C., Hardtke, S., et al., 2019. Peginterferon alfa-2a plus tenofovir disoproxil fumarate for hepatitis D (HIDIT-II): A randomised, placebo controlled, phase 2 trial. Lancet Infectious Diseases 19 (3), 275–286. Wranke, A., Serrano, B.C., Heidrich, B., et al., 2017. Antiviral treatment and liver-related complications in hepatitis delta. Hepatology 65 (2), 414–425. Yurdaydin, C., Abbas, Z., Buti, M., et al., 2019. Treating chronic hepatitis delta: The need for surrogate markers of treatment efficacy. Journal of Hepatology 70 (5), 1008–1015. Yurdaydin, C., Bozkaya, H., Gurel, S., et al., 2002. Famciclovir treatment of chronic delta hepatitis. Journal of Hepatology 37 (2), 266–271. Yurdaydin, C., Bozkaya, H., Onder, F.O., et al., 2008. Treatment of chronic delta hepatitis with lamivudine vs lamivudine þ interferon vs interferon. Journal of Viral Hepatitis 15 (4), 314–321. van Zonneveld, M., Flink, H.J., Verhey, E., et al., 2005. The safety of pegylated interferon alpha-2b in the treatment of chronic hepatitis B: Predictive factors for dose reduction and treatment discontinuation. Alimentary Pharmacology & Therapeutics 21 (9), 1163–1171. van Zonneveld, M., Honkoop, P., Hansen, B.E., et al., 2004. Long-term follow-up of alpha-interferon treatment of patients with chronic hepatitis B. Hepatology 39 (3), 804–810. Zoulim, F., Bialkowska-Warzecha, J., Diculescu, M.M., et al., 2016. Entecavir plus tenofovir combination therapy for chronic hepatitis B in patients with previous nucleos(t)ide treatment failure. Hepatology International 10 (5), 779–788. Zoutendijk, R., Reijnders, J.G., Brown, A., et al., 2011. Entecavir treatment for chronic hepatitis B: Adaptation is not needed for the majority of naive patients with a partial virological response. Hepatology 54 (2), 443–451.

Further Reading Lopatin, U., 2019. Drugs in the pipeline for HBV. Clinical Liver Disease 23 (3), 535–555. doi:10.1016/j.cld.2019.04.006. Seto, W.K., Lo, Y.R., Pawlotsky, J.M., Yuen, M.F., 2018. Chronic hepatitis B virus infection. Lancet 392 (10161), 2313–2324. doi:10.1016/S0140-6736(18)31865-8.

Relevant Websites www.aasld.org AASLD: Home. www.easl.eu EASL. The Home of Hepatology.

Studying Population Genetic Processes in Viruses: From Drug-Resistance Evolution to Patient Infection Dynamics Jeffrey D Jensen, Arizona State University, Tempe, AZ, United States r 2021 Published by Elsevier Ltd.

Glossary Background Selection Reduction in genetic diversity due to purifying selection against deleterious mutations at linked sites. Distribution of Fitness Effects (DFE) The statistical distribution of selection coefficients of newly arising mutations, as compared to a reference genotype.

Mutational Meltdown The accumulation of stochastic effects which may result in population decline and ultimately extinction. Selective Sweep The result of a beneficial mutation being driven to high frequency by positive selection, together with linked variation. Site Frequency Spectrum (SFS) Summary of the frequencies of observed segregating mutations in a population.

In this article, I briefly summarize the population genetic environment in which within-patient viral populations are evolving – discussing the roles of genetic drift as modulated by the infection history of the patient, selection acting on newly arising deleterious and beneficial variants and the related linked selection effects on the genome, and the underlying mutation and recombination/reassortment rates as well as replication behavior. I conclude with a consideration of how an improved understanding of these processes specifically, and evolutionary genomics in general, can inform therapeutic strategies in the future.

The Population Dynamics of Infection The natural starting point in any evolutionary analysis is a consideration of the effects of genetic drift. This stochastic evolutionary force is generally described in terms of the effective population size (Ne) – that is, the idealized size of the population which would experience the observed amount of genetic drift. This is opposed to the census population size (N), that is, the current number of individuals in the population. There are multiple reasons why Ne may be strongly reduced relative to N, including changes in census population sizes over time as well as natural selection itself, as will be described throughout this article. While viral population census sizes are known to often be exceptionally large, it is important to appreciate that viral effective population sizes are much more constrained, owing both to underlying modes of transmission and infection dynamics as described below – with Ne having been estimated to only be on the order of hundreds to thousands (Hughes, 2009; Miyashita and Kishino, 2010; Renzette et al., 2013). This is significant with regards to viral population genetics as it suggests an upper limit on the efficiency of natural selection, given that this efficacy is dictated by Ne. It relatedly serves as a reminder of the pervasively important role of genetic drift in governing evolutionary outcomes, and the generally unjustified adaptation-centric view of virus genome evolution still forwarded in certain sections of the virology community. Thus, before treating the topic of selection, one must firstly consider the neutral population dynamics of the organism in question. In the case of a viral population sampled from a patient, these dynamics likely include a strong population bottleneck (i.e., a temporary reduction in population size) associated with the initial infection – these bottlenecks may be quite severe, with some primary infections being established by a very small number of infecting units. After this initial infection, the viral population will often next enter a phase of rapid population growth. This combination of bottlenecking and growth will leave signatures in the genomic patterns of variation, which in turn may be used to estimate the timing of infection, the severity of the bottleneck, the rate of within-patient population growth, and indeed the relative difference between Ne and N. In certain viruses that compartmentalize, these dynamics might also include population sub-structuring, in which isolated or semi-isolated viral populations are established in different compartments (e.g., in different organs) within a single host. These compartments may or may not be connected by gene flow, via the plasma for example. Multiple approaches have been developed within the field of population genetics to infer these demographic models and their underlying parameters. It is perhaps most helpful to introduce such methodology within the context of a specific application. For this purpose, I will examine recent work in human cytomegalovirus (HCMV), a member of the Herpesviridae family of dsDNA viruses. HCMV is characterized by a particularly large genome (B235,000 base pairs), and is nearly ubiquitous in human populations, with a seroprevalence of 30%–90% in the US and 490% in adults outside of the developed world (Kenneson and Cannon, 2007; Boeckh and Geballe, 2011). Though asymptomatic in most individuals, HCMV can lead to severe symptoms in immunocompromised patients and neonates, and is the most common cause of birth defects resulting from an infectious agent (Hassan and Connell, 2007; Manicklal et al., 2013).

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A large literature has accumulated demonstrating that HCMV has high levels of genetic variation within a host (Spector et al., 1984; Drew et al., 1984; Haberland et al., 1999; Meyer-Kö nig et al., 1998; Faure-Della Corte et al., 2010), despite encoding a DNA polymerase with proofreading activity (Nishiyama et al., 1983), with several recent high-throughput whole-genome studies revealing the full extent of this variation (Renzette et al., 2011, 2013, 2014, 2015, 2016, 2017; Hage et al., 2017; Pokalyuk et al., 2017). Utilizing such wholegenome data sampled from the urine and plasma compartments of five congenitally infected infants, Renzette et al. (2013) found that the sub-structuring effect of compartmentalization was particularly strong – as assessed by FST, a measure of differentiation between populations – with these two compartments from a single patient being as diverged as samples from two unrelated patients. Utilizing the observed frequencies of genome-wide segregating sites in these congenitally infected patient samples – the site frequency spectrum (SFS) – they additionally inferred the demographic model characterizing each infection. Specifically, Renzette et al. inferred a population bottleneck associated with the initial infection (i.e., the movement of the virus from the maternal compartment to the fetal plasma compartment), and a second bottleneck associated with the subsequent infection of the kidney compartment (i.e., as assessed by the urine sample). The severity of the bottleneck characterizing the initial infection was not as strong as earlier results had suggested, with dozens or even hundreds of virions characterizing fetal infection. By estimating the age of the bottleneck, this work also provided the first inference of the timing of fetal infection. In follow-up work with longitudinal sampling of the plasma, urine, and saliva compartments, Pokalyuk et al. (2017) also found evidence for subsequent admixture with maternal HCMV populations after the initial infection and compartmentalization, suggesting re-infection post-birth via, for example, breast milk (Numazaki, 1997; Enders et al., 2011). While these mixed infections can certainly be a significant player in governing levels and patterns of genomic variation as described by Pokalyuk et al., there has been an unfortunate tendency of some authors to only focus on this admixture process while neglecting the variety of other selective and demographic processes which also determine levels of variation (e.g., Cudini et al., 2019; though see Jensen and Kowalik, 2020). Given that no current method exists to prevent maternal-fetal transmission, or to reduce the severity of fetal infection (Britt, 2017), such a characterization of population dynamics may be crucial to future therapeutic strategies. For example, clinicallyimposing a more severe population bottleneck during pregnancy may reduce HCMV variability within the fetus, limiting the pool of variation on which natural selection may subsequently act, thereby potentially improving treatment outcomes. Immunotherapy advances have produced therapeutics capable of reducing the rate of maternal transmission in other viruses, including hepatitis B and HIV, and may thus represent a promising route for HCMV as well (Tseng and Kao, 2017; Voronin et al., 2017).

Direct Selection and Linked Selection Effects With regards to the direct action of natural selection in viral populations, positive selection related to antiviral drug resistance or immune evasion has received particular attention for obvious reasons (see review of Irwin et al., 2016a). This search for adaptive loci is generally based around identifying the patterns of selective sweeps (Maynard Smith and Haigh, 1974) in viral genomes, owing to the genetic hitchhiking effects induced from the rapid rise in frequency of the beneficial mutation towards fixation. However, though it receives comparatively less attention in the virology literature, it is well understood that the vast majority of new fitness-impacting mutations in any organism are deleterious rather than beneficial (e.g., Crow, 1993; Lynch et al., 1999; Bank et al., 2014b; and see reviews of Eyre-Walker and Keightley, 2007; Bank et al., 2014a). The selective removal of these harmful variants by purifying selection will serve to further reduce the effective population size to an extent largely dictated by the rate of recombination and the strength of selection (Charlesworth et al., 1993; Charlesworth, 2013). In addition, genomic linkage to this frequent input of deleterious mutations will reduce the probability of fixation of other linked sites – including reducing the likelihood of adaptation (Hill and Robertson, 1966; and see Pénisson et al., 2017). Importantly, just as the increase in frequency of a beneficial mutation can lead to the genetic hitchhiking of linked variation towards fixation in the genome (leading to a selective sweep effect), so too can the removal of the much larger input of deleterious mutations lead to the genetic hitchhiking of linked neutral variants towards loss. This effect – the loss and reduction in frequency owing to linkage with deleterious variants – is known as background selection (Charlesworth et al., 1993). This effect can be of major consequence in viral populations given their coding-dense genomes (in which most new mutations are expected to be deleterious), particularly in the absence of recombination. Though this is yet to be thoroughly studied in viral populations, initial work has found a much more dominant role for background selection relative to selective sweeps in shaping within-patient genomic variation (e.g., Renzette et al., 2016). Quantifying the relative input of deleterious, neutral, and beneficial mutations, means understanding the shape of the distribution of fitness effects (DFE). There are three general types of methods for performing such inference. The first involves the artificial creation of a mutation, one at a time or in combination, in order to measure the resulting fitness effect on an otherwise wildtype background under lab-controlled environmental conditions (e.g., Fowler et al., 2010; Hietpas et al., 2011, 2012; Bank et al., 2014b). The second approach, mutation-accumulation, maintains a population in a laboratory and allows mutations to naturally occur and accumulate, and fitness is generally quantified and related to this accumulation at multiple time points (e.g., Foll et al., 2014; Ferrer-Admetlla et al., 2016; Lynch et al., 2016; Long et al., 2018). Aside from these two experimental approaches, a third approach is based on the direct sampling of natural population data (e.g., from a patient infection, in the case of viral populations) – using either single time point (e.g., Keightley and Eyre-Walker, 2007; Schneider et al., 2011; Tataru et al., 2017; Johri et al., 2020) or multiple time point data (e.g., Malaspinas et al., 2012; Mathieson and McVean, 2013; Foll et al., 2015;

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Sackman et al., 2019). These approaches generally rely on fitting a population history using patterns of variation at neutral sites, and then using that history to quantify the DFE at putatively selected sites. One particularly important aside here of great relevance to the virology community, is the danger of basing such selection analyses on consensus sequences (i.e., the representation of each patient's viral sample by a single sequence, which represents the most common allele at each site in a given within-patient viral population). While common practice, this tremendous loss of information (e.g., all rare alleles – which represent the vast majority of variation – are neglected), combined with the associated ascertainment issue of only viewing high frequency mutations, can wreak havoc on evolutionary inference. For example, Renzette et al. (2017) demonstrated that earlier work based on consensus sequences had incorrectly arrived at the conclusion that most sampled segregating variation in HCMV was strongly selected owing to this consensus ascertainment – whereas when full withinpatient population-level data was considered, it was clear that most segregating variation was in fact neutral or nearly neutral, consistent with general expectations (Kimura, 1968; Ohta, 1973; and see Jensen et al., 2019; Morales-Arce et al., 2020b). Finally, it is important in this regard to appreciate that the correct evolutionary null model must necessarily account for the population infection dynamics discussed in the first section, together with the purifying and background selection effects discussed above. As viral populations are characterized by changing population sizes owing to infection bottlenecks and subsequent growth, and as the input of deleterious mutations is a constant process, an accounting for these processes is necessary to establish a baseline expectation for levels and patterns of variation, on top of which positively selected outliers may be identified. A neglect of these neutral dynamics can lead to serious mis-inference. For example, Feder et al. (2016) examined consensus HIV-1 population sequence data from 6717 drug-treated patients sequenced over a 24-year span. They found, as expected, that the change in variation observed in the viral population was correlated with the degree of treatment effectiveness. They used this pattern to argue that less effective treatments may be associated with so-called soft selective sweeps (i.e., positive selection on a previously neutrally segregating genomic variant, or on multiple simultaneously occurring and identical genomic variants), whereas more effective treatments may be associated with hard selective sweeps (i.e., positive selection on a de novo beneficial mutation). Perplexingly, they assumed identical strengths of positive selection, and identical within-patient demographic histories, between patients receiving effective and ineffective treatments. Revisiting this data, Harris et al. (2018) demonstrated that if one accounts for these variable fitness effects, and the inherent reality that more effective treatments reduced viral population sizes more than less effective treatments, these data observations can be explained entirely without invoking soft selective sweeps. Hence, it is always crucial to first account for the underlying neutral dynamics.

Other Key Aspects of the Population Genetic Environment Apart from the effects of genetic drift and direct and linked selection described above, multiple other evolutionary processes are necessary to consider in order to develop a more holistic view of within-host viral population dynamics. For example, while the DFE describes the fitness effects of mutations, the rate of input of mutation is itself an important evolutionary parameter, as is the rate of recombination/reassortment, as that dictates the extent to which these newly arising mutations will be linked (and thus experience Hill-Robertson interference). Inference about mutation and recombination is similarly possible from with-patient polymorphism data. Returning to the HCMV example, Renzette et al. (2015) calculated genome-wide rates of mutation and recombination in 500 base pair windows across the genome for 48 longitudinal samples from 18 patients, inferring an average mutation rate of 2.0
10–7 new mutations per site per replication, similar to rates observed from murine cytomegalovirus (Drake and Hwang, 2005; Sanjuan et al., 2010; and see review of Sackman et al., 2018). Importantly, viral mutation rates have generally been thought to be inversely correlated with genome size (Gago et al., 2009), with the general interpretation being, for example, that RNA-based viral genomes are smaller than DNA genomes owing to intrinsically error-prone polymerases (Drake and Holland, 1999; Elena and Sanjuán, 2005). However, it appears more likely that, given that selection operates on the total genome-wide deleterious mutation rate (Kimura, 1967; Lynch, 2008), the selection pressure for replication fidelity (per site) is simply reduced in smaller genomes. This interpretation, as described in the 'drift-barrier hypothesis', has been widely supported across the tree of life (Lynch, 2011, 2012). In other words, genome-wide viral mutation rates are pushed to the lowest levels possible by natural selection, thus predicting the widely-observed correlation between effective population size and mutation rate, as well as between genome size and mutation rate. While some in the virology community retain the notion that viruses may be selected to maintain high mutation rates to better deal with fitness challenges, there is simply neither empirical nor theoretical support for the validity of such a view. Finally, as much of population genetic inference is based on the Wright-Fisher model and Kingman coalescent - in which there is an assumption of a Poisson-shaped progeny distribution in which the variance equals the mean and is small relative to the population size – there is a growing appreciation that violations of this assumption must be accounted for when studying virus populations, which are highly variable in this regard. As such, virus genealogies are better characterized by more generalized Moran and multiple-merger coalescent models (see Donnelly and Kurtz, 1999; Pitman, 1999; Sagitov, 1999; Schweinsberg, 2000; Eldon and Wakeley, 2008; Matuszewski et al., 2018; and see reviews of Tellier and Lemaire, 2014; Irwin et al., 2016b). Specifically, violations of this progeny distribution assumption may elevate linkage disequilibrium even in the presence of frequent recombination (Eldon and Wakeley, 2008; Birkner et al., 2013), and skew estimates of FST (Eldon and Wakeley, 2009). As such, these model violations may be mistaken for either population size change or positive selection if not accounted for (Matuszewski et al., 2018; Sackman et al., 2019).

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Recent theoretical and statistical developments have provided some inroads into estimating, and thus accounting for, progeny skew from within-host pathogen populations. This work has demonstrated an ability to co-estimate the degree of progeny skew together with population size change, mutation rate, and positive selection from within-patient polymorphism data (Eldon et al., 2015; Matuszewski et al., 2018; Sackman et al., 2019; Morales-Arce et al., 2020a), providing a more appropriate null model for the study of pathogen evolution.

An Outlook on Evolutionarily Informed Treatment Strategies The ability of the above described inference approaches to study drug-resistance mechanisms, and thus quantify the likelihood of adaptively evading a given therapeutic strategy within an experimental evolution setting, has been well-justified in the literature. Namely, identifying positively selected mutations in experimental viral populations challenged with possible drug-treatments individually or in combination, may elucidate potential routes to resistance, and highlight drugs requiring more complex (and lower probability) mutational evasion routes. For example, the commonly used influenza drug oseltamivir acts as a competitive inhibitor by binding to a hydrophobic pocked in the viral surface protein – an action which can be effectively blocked by mutations near the binding site (Collins et al., 2008). Initially, the high fitness cost of the most common oseltamivir-resistant mutation, NA H275Y, was hypothesized to make the development of resistance unlikely (Ives et al., 2002) – though resistance spread rapidly in the 2007–8 flu season owing to the limited number of required mutational steps (Moscona, 2009; Bloom et al., 2010), an important drawback that became clearer in subsequent experimental evolution studies (e.g., Foll et al., 2014). Other strategies with wider genomic effects may thus prove more fruitful – ideally ones requiring at least multiple mutation steps in order to confer resistance. Over the past decades, the notion that an excessive input of mutations can drive population extinction has been explored both theoretically and empirically, beginning with the foundational work of Lynch and Gabriel (1990) and Lynch et al. (1993). This possibility owes to the fact that the vast majority of fitness-impacting mutations are deleterious, or said another way, there are many more ways to disrupt rather than improve genomic function. This so-called mutational meltdown is achieved once the mean viability of the population drops to the point that the average individual cannot replace itself, at which time the population begins to decline resulting in a snowball effect leading to ultimate extinction. The cause of this final meltdown-phase is in fact the accelerated reduction in the efficacy of natural selection that results from the decline in Ne – that is, the declining population size allows for the further fixation of deleterious mutations via genetic drift, which in turn further reduces the efficacy of selection, allowing for further deleterious fixations, and so on. Given that many viruses are naturally characterized by high mutation rates, and thus may commonly reside near a threshold of mutational load, a therapeutic increase in mutation rates may indeed induce this phase in which the input of new mutation overwhelms the ability of natural selection to remove this deleterious load. While such a scenario is often referred to as lethal mutagenesis in the virology literature (Bull et al., 2007), the mutational meltdown framework is more general and critically incorporates the additional effects of genetic drift inherent to all populations (see Matuszewski et al., 2017). The ability of an increased mutation rate to induce such extinction in a patient viral population has been relatively well explored and justified in RNA viruses (Loeb et al., 1999; Crotty et al., 2000; Lanford et al., 2001; Crotty et al., 2001; Severson et al., 2003; Airaksinen et al., 2003), using a variety of drugs including ribavirin and, more recently, favipiravir (Furuta et al., 2013; Baranovich et al., 2013; Bank et al., 2016; Ormond et al., 2017). Pertinent to the 2020 pandemic, Sheahan et al. (2020) recently demonstrated that the ribonucleoside analog EEID-1931 has a mutagenic effect in SARS-CoV-2 passaged in cell culture and, working in a mouse model, found a positive correlation between increased viral mutation rates and the degree of therapeutic efficacy. This work lends support to other recent calls to better investigate the potential therapeutic strategy of mutational meltdown in the context of CoV-2 (Jensen and Lynch, 2020; Santiago and Caballero, 2020; Jensen et al., 2020). While this represents a potentially promising and generalized treatment strategy for existing (and future unknown) pathogens, a number of important open questions remain in this regard. These will necessitate further theory work related to the necessary mutation rate turn-up required to induce this effect with realistic DFEs, as well as a more complete exploration of the interplay with underlying recombination rates (when applicable); as well as a more cohesive experimental effort to accurately quantify natural viral mutation rates via mutation-accumulation studies, in order to establish baseline values. What is clear however is that new viral pathogens will continue to emerge, a comprehensive understanding of their evolutionary trajectories will be critical for designing effective clinical treatments and interventions, and hence that a better union of the fields of population genetics and medicine will prove to be of great value.

See also: Antiretroviral Therapy – Nucleoside/Nucleotide and Non-Nucleoside Reverse Transcriptase Inhibitors. Antiviral Classification. HIV Integrase Inhibitors and Entry Inhibitors. Management of Adenovirus Infections (Adenoviridae). Management of Hepatitis A and E Virus Infection. Management of Herpes Simplex Virus Infections (Herpesviridae). Management of Influenza Virus Infections (Orthomyxoviridae). Management of Patients With Chronic Hepatitis B (Hepadnaviridae) and Chronic Hepatitis D Infection (Deltavirus). Management of Respiratory Syncytial Virus Infections (Pneumoviridae). Management of Varicella-Zoster Virus Infections (Herpesviridae). Protease Inhibitors. Treatment and Prevention of Herpesvirus Infections in the Immunocompromised Host

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Molecular Ecology 26, 1980–1990. Renzette, N., Bhattacharjee, B., Jensen, J.D., Gibson, L., Kowalik, T.F., 2011. Extensive genome-wide variability of human cytomegalovirus in congenitally infected infants. PLOS Pathogens 7, e1001344. Renzette, N., Gibson, L., Bhattacharjee, B., et al., 2013. Rapid intrahost evolution of human cytomegalovirus is shaped by demography and positive selection. PLOS Genetics 9, e1003735. Renzette, N., Gibson, L., Jensen, J.D., Kowalik, T.F., 2014. Human cytomegalovirus intrahost evolution – A new avenue for understanding and controlling herpesvirus infections. Current Opinion in Virology 8, 109–115. Renzette, N., Kowalik, T.F., Jensen, J.D., 2016. On the relative roles of background selection and genetic hitchhiking in shaping human cytomegalovirus genetic diversity. Molecular Ecology 25, 403–413. Renzette, N., Pfeifer, S.P., Matuszewski, S., Kowalik, T.F., Jensen, J.D., 2017. On the analysis of intrahost and interhost viral populations: Human cytomegalovirus as a case study of pitfalls and expectations. Journal of Virology 91, e01976-16. Renzette, N., Pokalyuk, C., Gibson, L., et al., 2015. Limits and patterns of cytomegalovirus genomic diversity in humans. Proceedings of the National Academy of Sciences of the United States of America 112, E4120–E4128. Sackman, A., Harris, R.B., Jensen, J.D., 2019. Inferring demography and selection in organisms characterized by skewed offspring distributions. Genetics 211, 1019–1028. Sackman, A.M., Pfeifer, S.P., Kowalik, T.F., Jensen, J.D., 2018. On the demographic and selective forces shaping patterns of human cytomegalovirus variation within hosts. Pathogens 7, 16. Sagitov, S., 1999. The general coalescent with asynchronous mergers of ancestral lines. Journal of Applied Probability 36, 1116–1125. Sanjuan, R., Nebot, M.R., Chirico, N., Mansky, L.M., Belshaw, R., 2010. Viral mutation rates. Journal of Virology 84, 9733–9748. Santiago, E., Caballero, A., 2020. The value of targeting recombination as a strategy against coronavirus diseases. Heredity 125, 169–172. doi:10.1038/s41437-020-0337-5. Schneider, A., Charlesworth, B., Eyre-Walker, A., Keightley, P.D., 2011. A method for inferring the rate of occurrence and fitness effects of advantageous mutations. Genetics 189, 1427–1437. Schweinsberg, J., 2000. Coalescents with simultaneous multiple collisions. Electronic Journal of Probability 5, 1–50. Severson, W.E., Schmalijohn, C., Javadian, A., Jonsson, C., 2003. Ribavirin causes error catastrophe during Hantaan virus replication. Journal of Virology 77, 481–488. Sheahan, T., Sims, A., Zhou, S., et al., 2020. An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Science Translational Medicine 12, eabb5883. Spector, S.A., Hirata, K.K., Neuman, T.R., 1984. Identification of multiple cytomegalovirus strains in homosexual men with acquired immunodeficiency syndrome. Journal of Infectious Diseases 150, 953–956. Tataru, P., Mollion, M., Glémin, S., Bataillon, T., 2017. Inference of distribution of fitness effects and proportion of adaptive substitutions from polymorphism data. Genetics 207, 1103–1119. Tellier, A., Lemaire, C., 2014. Coalescence 2.0: A multiple branching of recent theoretical developments and their applications. Molecular Ecology 23, 2637–2652. Tseng, T.C., Kao, J.H., 2017. Elimination of hepatitis B: Is it a mission possible? BMC Medicine 15, 1–5. Voronin, Y., Jani, I., Graham, B.S., et al., 2017. Recent progress in immune-based interventions to prevent HIV-1 transmission to children. Journal of the International AIDS Society 20, e25038.

Further Reading Charlesworth, B., Charlesworth, D., 2010. Elements of Evolutionary Genetics. USA: Roberts and Company Publishers. Kimura, M., 1983. The Neutral Theory of Molecular Evolution. UK: Cambridge University Press. Provine, W.B., 2001. The Origins of Theoretical Population Genetics. USA: University of Chicago Press. Wakeley, J., 2009. Coalescent Theory: An Introduction. USA: Roberts and Company Publishers. Walsh, B., Lynch, M., 2018. Evolution and Selection of Quantitative Traits. UK: Oxford University Press.

Virus-Based Cancer Therapeutics Roberto Cattaneo, Mayo Clinic, Rochester, MN, United States Christine E Engeland, University Hospital Heidelberg and German Cancer Research Center, Heidelberg, Germany and Witten/Herdecke University, Witten, Germany r 2021 Elsevier Ltd. All rights reserved.

Nomenclature GM-CSF

Granulocyte-macrophage colony-stimulating factor; IFN Interferon; MeV Measles virus;

Glossary Arming Insertion of additional genes into the oncolytic virus genome to increase therapeutic efficacy. Bystander effects Cytotoxic effects on non-infected cells in the vicinity of infected cells.

NDV Newcastle disease virus; RBP Receptor-binding protein; SPECT Single photon emission computed tomography VSV Vesicular stomatitis virus;

Immune checkpoint blockade Treatment with antibodies that block inhibitory signaling in immune cells, especially T cells. Shielding Genetic or chemical modifications to avoid antibody neutralization of viruses. Targeting Modifications of viral tropism to increase tumor specificity.

Definition Oncolytic virotherapy is the treatment of cancer with viruses that replicate preferentially in tumor tissue, damaging it. Selectivity for tumor tissue can occur naturally, and be enhanced by genetic engineering.

Introduction and Historic Notes Virus infection may be responsible for a significant fraction of human cancer deaths. Nevertheless, shortly after the discovery of animal viruses, observing physicians reported cancer regressions coincident with natural infections, most notably in patients with lymphoma or leukemia. These patients were suffering from viral hepatitis, glandular fever, chickenpox, or measles. For example, before a virus was discovered as the causal agent, influenza was associated with leukemia remission. In a 1910 report, the Italian gynecologist De Pace observed tumor lysis and subsequent tumor remission in a cervical cancer patient after rabies vaccination. These and other anecdotal observations inspired early clinical trials in the second half of the 20th century. These trials did not meet current ethical standards for clinical investigations. Infectious body fluids or tissue samples were administered to cancer patients. For instance, in a 1949 trial, 22 patients suffering from Hodgkin’s lymphoma were treated with sera or tissue extracts from patients infected with hepatitis B virus. The first patient died after treatment, and 13 developed hepatitis. Seven patients were reported to show amelioration and four experienced reduction of lymphoma burden. In 1953, a case series was published reporting transient remission of acute leukemia after intramuscular injection of glandular fever sera in three out of five treated patients. Trials with several human pathogenic viruses were conducted, including flaviviruses such as dengue and West Nile virus. In the latter, viremia and intratumoral virus were observed in many patients, with several cases of encephalitis and few cases of transient delay in tumor progression. These approaches to therapy can only be understood in the light of the general hopelessness of what was then cancer treatment. The advent of tissue culture in the 1950s and 1960s allowed viruses to be propagated in a more defined environment. In addition, it became possible to assess the extent of virus spread and virus-induced cell death in cancer cell lines. Based on in vitro experiments, an adenovirus, at that time called adenoidal-pharyngeal-conjunctival virus,emerged as a candidate oncolytic virus. In 1956, this virus was administered to patients with cervical carcinoma by either intratumoral, intra-arterial or intravenous administration. This treatment showed a favorable safety profile. Extensive tumor necrosis occurred in 26 of 40 treated patients, but all patients ultimately succumbed to their tumor within a few months. Another important development in the assessment of the safety and oncolytic efficacy of viruses was the use of models based on transplantable or spontaneous murine tumors. These models were used to characterize the efficacy of animal viruses including two herpes viruses, equine rhinopneumonitis and infectious bovine rhinotracheitis virus, as well as a murine negative sense RNA virus termed “M-P virus” at the time, which was later identified as a lymphocytic choriomeningitis virus, and the poultry pathogen Newcastle disease virus (NDV), a member of the Paramyxoviridae family. Simultaneously with the testing of several animal viruses as oncolytics, cancer regressions coincident with natural human infections continued to be reported. In the 1970s several case reports described coincidence of infections with measles virus (MeV),

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Fig. 1 Lymphoma remission after measles infection. Left panel: patient on admission with Burkitt’s lymphoma; note the swelling around his right eye. Center panel: patient three weeks later after contracting measles by natural infection; note the rash on his skin. Right panel: Patient five weeks later. Tumor and measles exanthem have resolved completely. Reprinted with permission from Bluming, A.Z., Ziegler, J.L., 1971. Regression of Burkitt's lymphoma in association with measles infection. Lancet 2 (7715), 105–106.

another member of the Paramyxoviridae family, with regressions or remissions of leukemia and lymphoma. One of these reports included a striking visual documentation of Burkitt’s lymphoma remission following natural measles (Fig. 1). In 1974, a large clinical trial with a third member of the Paramyxoviridae family naturally infecting humans, mumps virus, was reported. Ninety patients with various terminal cancers were treated by different routes of administration including intravenous or intratumoral injection, local application after scarification of the tumor, oral or rectal administration and inhalation. Side effects were minimal, and 79 of 90 patients experienced tumor remission, including several apparently complete responses. In some patients, signs of increased anti-tumor immunity were observed. However, there was no follow up. In summary, by the end of the 1970s, several clinical trials based on human or non-human viruses reported acceptable toxicities, but limited efficacy. This situation contrasted with the availability of effective chemotherapy and radiation therapy clinical interventions, and resulted in the decline of research activities in the oncolytic virus field. At this crossroads, it was evident that viruses with increased cancer specificity were needed. However, to achieve this goal, three areas of research needed development: methods for directly documenting viral spread in vivo, a deep understanding of the virus tropism determinants, and technology to generate recombinant viruses with specified characteristics.

Re-engineered Viruses: The Beginnings The “natural” oncolytic properties of some viruses were initially inferred by the coincidence of cancer regressions with natural infections. Subsequently, when tissue culture systems for animal cells were established, it became evident that almost every virus grows much better in transformed cancer cell lines than in primary cells. We now know that the multistep process of cancer progression can affect multiple components of the cellular response to viral infection, favoring the replication of many different viruses. Thus, many viruses have “natural” oncolytic properties. On the other hand, oncolytic properties are not universal, but depend on proper matching of virus and host cell. Virus families have evolved specificities for different cell types, and this natural diversity impacts on the oncolytic properties of each virus. To be a cancer therapeutic, viruses need to efficiently enter cells. If this happens, the host cell is per definition susceptible (Fig. 2, center). Following cell entry, depending on the outcome of several interactions between cellular intrinsic immunity proteins and viral proteins, the virus will replicate. In this case, the host cell is per definition permissive to infection and will ultimately be lysed (Fig. 2, right). In the last three decades, research on the tropism determinants for viruses of several different families greatly progressed. This was facilitated by the establishment of methods to easily visualize and quantify virus spread in the host using reporter genes, and to document how virus replication in different locations relates to therapeutic efficacy. In addition, our understanding of tumor biology greatly improved; more sophisticated animal models were included in pre-clinical testing of new therapeutics; and large arrays of diagnostic markers are being used for in-depth evaluation of clinical trial results. Based on this deeper knowledge of the biology of both viruses and cancer, new viruses with improved oncolytic activities have been generated, and many of these recombinant viruses are now in pre-clinical development. In particular, four DNA viruses and five RNA viruses are either approved therapeutics or in different clinical trial phases. Table 1 lists one or two selected examples of approved or experimental cancer therapeutics derived from each of these nine families. It indicates the virus family and name, the genetic changes introduced, the routes of administration, whether the virus was given alone or in combination with another cancer therapeutic, and the clinical trial identifier and time frame.

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Fig. 2 Susceptibility and permissiveness of cancer cells determine the outcome of oncolysis. Top panels: cancer cell that does not express a viral receptor and is therefore not susceptible to the virus. Center panels: cancer cell that expresses a viral receptor, allowing viral entry. However, the cell is not permissive for the virus, which does not replicate and cannot cause oncolysis. Bottom panels: cancer cell both susceptible to, and permissive for the virus. The virus replicates, ultimately leading to oncolysis. (Created with BioRender.com.)

While this table is only a snapshot of current state-of-the-art research, some general conclusions can be drawn. First, virus-based cancer therapeutics are well tolerated, even at the highest doses achievable by current manufacturing processes, after both localized and systemic administration. In addition, although virus shedding was documented in the urinary and respiratory tract, especially after systemic administration, no transmission from a cancer patient to health care providers, or other contacts, has been noted. Second, three of the most successful oncolytic viruses express as immunomodulatory transgene the granulocyte-macrophage colony-stimulating factor (GM-CSF) (Table 1, DNA viruses). This may be due to the combination of replicative oncolysis with GM-CSF-mediated inflammation to induce adaptive immunity against tumor antigens. The third conclusion is cautionary: several viruses have fallen short of the efficacy expectations that were set by pre-clinical models. This fact underscores the importance of improving efficacy of current generation virus-based cancer therapeutics.

Three Classes of Modifications Current clinical trials of oncolysis face several challenges. Insufficient efficacy can be addressed by more aggressive dosing, which is being enabled by continuing technological advances in virus production. However, more aggressive dosing requires improved tumor specificity of future vectors to maintain the current safety profiles. Cancer specificity can be improved by targeting. Targeting, which is based on the introduction of different layers of cancer specificity, either at the cell entry or post-entry levels, can enhance both efficacy and safety of oncolytic vectors. Another key challenge for oncolytic therapy is to avoid immediate neutralization of the vectors through circulating antibodies, which can arise in patient populations via natural contagion, scheduled immunization, or prior administration of an oncolytic virus. Temporary shielding from the host immune response can provide a window of opportunity for the effective initiation of the oncolytic process. Shielding can be achieved by changing or physically masking the epitopes that are recognized by neutralizing antibodies. The third key challenge for oncolytic therapy is the need to reach and eliminate not only all the cells in a primary tumor, but also tumor metastases. This challenge is addressed by arming viruses with proteins that sensitize not only infected tumor cells but also uninfected metastases, to subsequent combination therapies or immune destruction. Arming occurs through the expression of a foreign gene: for example, a prodrug convertase activating an anticancer therapeutic in situ, or an ion channel facilitating radiosensitization, or a cytokine promoting immunostimulation.

VSV-IFNb

Phase I; HCCe and others

Rhabdoviridae

Phase I; NSCLC, HPV-associated MG1 Maraba a.o.

b

Information as of January 2020; for updates see ClinicalTrials.gov. HNSCC, head and neck squamous cell carcinoma. c PDAC, pancreatic ductal adenocarcinoma. d HRV2 IRES, human rhinovirus type 2 internal ribosomal entry site. e HCC, hepatocellular carcinoma.

a

Vocimagene amiretrorepvec Cytosine deaminase (Toca511)

Phase II/III; High-grade glioma

Retroviridae

Reolysin

Phase III completed; HNSCC

Tumor antigen

human IFNb

None

Intravenous

Intratumoral

Into resection cavity

Intravenous

Intratumoral

Reoviridae

b

HRV2 IRESd

PVS-RIPO

Phase II; glioblastoma

Intravenous

Intravenous and intratumoral Intratumoral

human NIS

Picornaviridae

GM-CSF, thymidine kinase deleted

None

MV-NIS

Phase II completed; glioblastoma ParvOryx and PDACc Phase III; hepatocellular Pexastimogene carcinoma devacirepvec (Pexa-Vec)

RNA viruses Paramyxoviridae Phase II completed; multiple myeloma

Poxviridae

Parvoviridae

T-VEC

Phase III; melanoma

Intratumoral

Intravesicular

Route

GM-CSF ICP34.5 Intratumoral deleted, US11 altered, ICP47 deleted As above Intratumoral

E1B-55k deleted

Oncorine (H101)

Talimogene Laherparepvec (T-VEC)

GM-CSF

Genetic changes

CG0070

Virus name

Approved (United States, Europe, 2015); Melanoma

Phase III terminated; bladder cancer Approved (China, 2005); HNSCCb

Phase and type of clinical trial

Selected examples of current clinical trials with viruses from nine familiesa

Herpesviridae

DNA viruses Adenoviridae

Virus family

Table 1

NCT01166542

NCT02986178

NCT02192775

NCT01301430 NCT02653313 NCT02562755

NCT02263508

NCT00769704 NCT00289016 PMID: 17121894

PMID 15601557 29189159

NCT01438112

NCT number/PMID

Adenovirus vaccine NCT02879760: Pembrolizumab NCT03618953: Atezolizumab

None

10/2014 3/2017 6/2018

NCT03618953

8/2012

11/2015-12/2019 1/2020

6/2010-5/2014

6/2017

3/2015-8/2019

9/2011-5/2015 12/2015-5/2018 10/2015

8/2014

Phase III: 4/2009-9/2014 Phase II 12/2005-5/2009 Phase I

3/2014-6/2016

Start-end date

NCT02285816 NCT02879760

NCT01628640

5-flurocytosine NCT02414165 NCT04105374: Temozolomide NCT04105374 and radiation

Carboplatin Paclitaxel

None

Cyclo-phosphamide

Sorafenib

None

Pembrolizumab

None

Chemotherapy (Cisplatin plus 5-FU or adriamycin)

None

Combination therapy

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Fig. 3 Entry and post-entry targeting of oncolytic viruses. Left column: normal cells. Right column: cancer cells. Top panels: receptor targeting. The viral RBP is engineered to no longer recognize its natural receptor, while attaching to a tumor-specific surface protein. This approach has been pioneered with enveloped viruses. Center panels: transcriptional regulation of replication. Virus replication is placed under control of a promoter active in tumor cells, but not in normal cells. This approach has been pioneered with viruses whose replication depends on cellular polymerase II. Bottom panels: posttranscriptional regulation of replication. This process takes advantage of the downregulation of certain microRNAs in tumor cells. Viruses engineered with microRNA target sequences in their genome replicate only in absence of the cognate microRNA. (Created with BioRender.com.)

While not all genetic modifications of viruses fall precisely within one of these classes, the targeting, shielding and arming principles guide the continuous improvement of oncolytic viruses.

Retargeting Virus Tropism The term targeting describes virus modifications that confer greater specificity for tumor cells by improving infection of diseased tissues or decreasing infection of healthy tissues, or both. Tumor specificity can be enhanced either at the stage of virus entry into the cells by modifying receptor choice (Fig. 3, top panels), or post entry during replication. Post-entry targeting can be achieved either at the transcription initiation level through the use of cancer-specific promoters (Fig. 3, center panels), or post-transcriptionally by inserting microRNA (miRNA) target sequences into viral genomes that restrict virus replication in healthy tissues (Fig. 3, bottom panels). For all these targeting strategies, a deeper understanding of tumor biology has facilitated the development of viruses that exploit tumor-specific alterations such as altered expression of cell surface proteins, transcription factors, and microRNAs.

Cell Entry Targeting of Enveloped Viruses All viruses require interactions with surface molecules on host cells to start infection. Surface proteins preferentially expressed on tumor cells can be exploited for entry targeting. This targeting strategy requires modification of the receptor-binding proteins of viruses. These modifications are either genetic and generate chimeric proteins, or they use chemical adapters to link specificity domains to virus particles. Genetic modifications of the receptor-binding proteins (RBP) of viruses are desirable because they are retained after replication. To redirect viral tropism by receptor targeting, their RBP are modified in two ways: first, ablation of binding to the natural receptor, or receptors. Second, engineering of binding to receptors preferentially or exclusively expressed on tumor cells.

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Fig. 3 (top panels) illustrates receptor targeting by RBP genetic engineering. Measles virus (MeV), an enveloped negative strand RNA virus of the Paramyxoviridae family, is used as an example. Receptor targeting is most advanced for enveloped viruses owing to the plasticity of their surface glycoproteins, whose evolution is not restricted by the multiple constraints imposed by icosahedral symmetry on the capsids of other viruses. The Paramyxovirus membrane fusion apparatus is easy to manipulate because receptor binding and membrane fusion functions are mediated by two different proteins. For MeV, the residues required for entry via the natural receptors have been identified and then mutated to disable binding to the natural receptors. Retargeting was completed by adding to the entire ectodomain of the RBP a moiety recognizing a cancer-specific receptor of interest. A flexible linker was introduced between the RBP ectodomain and the targeting moiety to allow its movement. Initially small peptide ligands for growth factor receptors were used to prove that the MeV RBP can bind targeted receptors without losing its membrane fusion support function. It was then shown that single-chain antibodies can also be used as specificity domains. Single-chain antibodies comprise only the variable domains of both light and heavy chains, which confer antigen specificity, and are far smaller than complete antibodies. Single-chain antibody fragments are well suited for retargeting of enveloped viruses, since they share with viral glycoproteins the same intracellular synthesis and transport pathways. Receptor targeting using single-chain antibody fragments has also been developed for the Herpesviridae, although the membrane fusion apparatus of these large DNA viruses is more complex and involves five glycoproteins. On the other hand, the single protein membrane fusion system of the Retroviridae family has proven difficult to retarget without extensive loss of cell entry efficiency.

Cell Entry Targeting of Non-Enveloped Viruses Receptor targeting of non-enveloped viruses with icosahedral capsids is challenging due to the strict structural constraints imposed by icosahedral symmetry. Nevertheless, receptor targeting by genetic modification has been achieved for Adenoviridae. For instance, short peptides or ligands with specificity for tumor surface proteins have been inserted at one end, or into a flexible region, of the adenovirus RBP. Targeting is completed by mutations that ablate binding to the natural receptors. More invasive capsid modifications have had different degrees of success. In addition to the icosahedral symmetry constraints, targeting strategies must take in account that adenovirus capsids are assembled in the cytosol, rather than transiting through the endoplasmic reticulum. Therefore, ligands that require folding, oxidation or other post-translational modifications in the endoplasmic reticulum are unsuitable. As alternative entry targeting strategy, adapter molecules have been developed. These adapters bind with one end to the adenovirus capsid, and with the other end to a cancer cell protein. This approach poses less structural constraints than the addition of specificity domains. However, cell entry occurs through the targeted receptor only during the first round of replication.

Cell Entry Targeting by Protease Activation Another cell entry targeting strategy is based on the selective activation of virus particles in the tumor microenvironment. This strategy, which takes advantage of the requirement of proteolytic cleavage for the activation of fusion proteins, has been used to retarget cell entry of Paramyxoviridae and Retroviridae. Protease targeting requires two modifications: first, inactivation of the cleavage site used by the cognate protease expressed in the natural host cells. Second, introduction of a cleavage site recognized by proteases secreted in the tumor microenvironment, such as matrix metalloproteinases. Since protease cleavage requires not only a specific recognition sequence, but also its accessibility by the cognate protease, engineering of efficient proteolytical activation may not be straightforward and require iterative cycles of optimization through mutagenesis. For example, the protease cleavage sites of the MeV and Sendai virus fusion proteins have been re-targeted following this principle.

Transcriptional Regulation of Replication The most potent oncolytic viruses are arguably wild-type viruses. However, these viruses can kill normal cells, hence causing doselimiting toxicities. Thus, oncolytic therapy has been developed almost exclusively based on attenuated viruses. On the other hand, attenuation is always relative to the target cell. Ideally, oncolytic viruses should be completely attenuated in normal cells, but maintain normal replication in cancer cells. Towards this, two replicational targeting strategies have been developed. The first aims at modulating the transcription of a gene controlling the early phases of replication. The second focuses on the selective transcription of virulence factors expressed late during the viral cycle. These strategies have been developed for viruses whose replication depends on cellular polymerases, in particular the Adenoviridae and the Herpesviridae. Early transcriptional control of replication is best developed for the Adenoviridae (Fig. 3, center panels). It can be achieved through the substitution of the promoter modulating transcription of Adenovirus early proteins by commonly used cancerassociated promoters, such as telomerase, or a prostate-specific antigen promoter in case of prostate cancer. Alternatively, there is now a burgeoning number of tumor-associated promoters that were identified using gene expression profiling in different cancer

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types. Certain elements of these promoters are now being used to control viral replication at the level of early transcription in new vectors proceeding through clinical trials. Another strategy aims at generating recombinant viruses that retain full virulence only in cancer cells by exploiting tumor-associated promoters to selectively activate the expression of virulence factors. This strategy is most advanced for the Herpesviridae, large DNA viruses that code for several virulence factors whose expression can be altered in combination. For example, a glioma-specific promoter is used to control the expression of the neurovirulence gene ICP34.5 of a herpes virus 1 recombinant used for glioma therapy.

Post-Transcriptional Regulation of Replication An alternative strategy for targeting viral replication exploits differential expression of microRNAs between tumors and normal tissues. This approach requires identification of microRNAs that are expressed in non-target tissues, but downregulated in tumor cells. Target sites for microRNAs are inserted into the untranslated regions of viral transcripts. These viral transcripts are degraded by the cellular microRNA machinery in cells that express the cognate microRNAs, abrogating viral replication (Fig. 3, bottom panels). The post-transcriptional regulation of replication principle was initially developed with a picornavirus that was an effective oncolytic but also caused significant toxicities due to myositis. Target sites for muscle-specific microRNAs introduced into the genome of this virus prevented myositis, while therapeutic efficacy was maintained. With extensive knowledge about microRNA expression in tumors, and the relative ease with which these relatively short target sequences can be incorporated into virus genomes without negatively affecting replication, negative targeting via microRNAs has been extended to several oncolytic virus platforms. These include members of the Adenoviridae, Herpesviridae, Paramyxoviridae, Poxviridae, and Rhabdoviridae. Towards this, multiple perfect match microRNA sequences have been expressed in the untranslated regions of essential viral mRNAs.

Preferential Tumor Spread Cancer targeting can also be achieved by favoring preferential spread of viruses in tumor tissue through the exploitation of specific defects in innate immunity. For example, many cancer cells cannot produce interferon (IFN), or cannot respond to IFN stimulation. In these cells, even attenuated viruses with defective IFN control proteins, and thus unable to replicate in normal cells, replicate and spread. This principle has been exploited for the precise targeting of certain viruses for which the mechanisms of interferon inactivation are understood in depth, like the Paramyxoviridae. For example, analyses of the interactions between cellular innate immunity proteins and the MeV proteins controlling their function, have guided the generation of recombinant viruses that retain oncolytic efficacy in tumor tissue, while not spreading in normal cells.

Combination of Targeting Modalities Multiple targeting modifications have been combined in second-generation recombinant oncolytic viruses. Therefore, most viruses that are currently in pre-clinical trials, or in early clinical trials, are targeted according to more than one modality. Targeting combinations allow to sequentially increase tumor specificity, augmenting safety. This is important, especially since armed viruses are being developed to augment the efficacy of virotherapy approaches.

Shielding From the Host Immune Response Like any pathogen, oncolytic viruses are recognized as “non-self” by the host immune system, resulting in an anti-viral immune response. Innate, cellular and humoral anti-viral immunity can hamper efficacy of virotherapy. While innate and cell-mediated responses can limit viral spread, neutralizing antibodies can inactivate the virus before it reaches the tumor. This is a major impediment to intravenous application of oncolytic viruses which, from a clinical perspective, is the preferred route of administration due to its feasibility and the potential to reach disseminated tumor sites. Therefore, avoiding premature viral clearance by neutralizing antibodies has been one focus in the development of advanced oncolytic therapies. Plasmapheresis has been applied to deplete neutralizing antibodies, but this process is complex and costly.

Virus Engineering Virus engineering strategies have been developed to avoid antibody neutralization. These strategies include generating chimeric viruses that are not recognized by neutralizing antibodies. These chimeric viruses, which can be used for sequential rounds of therapy, can be generated by serotype exchange. Serotype exchange is an option for most viruses, such as vesicular stomatitis virus (VSV) and adenovirus. In this approach, the viral proteins that are recognized by neutralizing antibodies, or parts of these proteins, are exchanged. For example, since all neutralizing epitopes of VSV reside within its glycoprotein, it is sufficient to replace it with that of another serotype (Fig. 4, top left panel).

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Fig. 4 Shielding strategies. Left column: virus engineering. Right column: chemical shielding and cell carriers. Left column, top panel. Serotype exchange. For vesicular stomatitis virus (VSV) exchange of the envelope glycoprotein is sufficient to generate a virus that is temporarily not neutralized. Left column, center panel. Multiple epitope replacement. For adenoviruses, epitopes on two capsid proteins must be precisely swapped to generate a virus that is temporarily not neutralized. Left column, bottom panel. Chimeric viruses. For MeV, exchange of both envelope glycoproteins with those of a related virus is required to generate a virus that is temporarily not neutralized. Right column, top panel. Chemical shielding, shown for adenovirus. Small polymers are added to purified virus particles. Right column, bottom panel. Cell carriers, shown for MeV. Cell carriers are infected ex vivo prior to administration. Infection is transmitted to cancer cells either by heterofusion, or by released viral particles. Modified from Miest, T.S., Cattaneo, R., 2014. New viruses for cancer therapy: Meeting clinical needs. Nature Review Microbiology 12, 23–34. (Created with BioRender.com).

In contrast, both hexon and fiber proteins harbor neutralizing epitopes of adenoviruses (Fig. 4, center left panel). To circumvent neutralizing antibodies, hexon-chimeric and fiber-chimeric variants have been generated. This was a complex endeavor because the hexon capsid protein encodes seven distinct loops that all contribute to neutralization; these loops were individually replaced while leaving the backbone of the hexon protein intact. Serotype exchange is not an option for viruses, such as MeV, which have a single serotype. In a variation on this strategy (Fig. 4, bottom left panel), the two MeV envelope glycoproteins were replaced with those of canine distemper virus, a closely related animal Morbillivirus whose proteins are structurally very similar. All these variations on the theme of serotype exchange require a balance between sufficient protein diversity to avoid cross- neutralization, and enough structural similarities to support particle formation.

Chemical Shielding and Cell Carriers An alternative to genetic modification of the viral surface proteins is chemical shielding of viruses (Fig. 4, top right panel). Different hydrophilic polymers have been used for this purpose. A prerequisite for successful application of chemical shielding is

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that viral entry is not impeded. This has been demonstrated for representatives of the Adenoviridae, Poxviridae, and Rhabdoviridae. Chemical shielding can have several positive effects: in addition to reducing sensitivity to pre-existing neutralizing antibodies, it can limit the de novo induction of humoral and cellular immune responses, and uptake into non-target tissues. Processes based on carrier cells have been developed for systemic administration of oncolytic viruses. In addition to protecting viruses from neutralization, cell carriers may possess inherent tumor tropism, thus targeting virus delivery. Cell carriers are infected ex vivo prior to administration. Immune cells, mesenchymal stem cells and also malignant myeloma cells have been used as carriers for oncolytic viruses. Meanwhile mesenchymal stem cells carrying adenovirus or MeV have entered clinical testing. Notably, both chemical shielding and cell carriers do not shield the viral progeny, which may limit the efficacy of these approaches.

Arming Viruses Another key challenge for oncolytic therapy is the need to reach and eliminate not only all the cells in a primary tumor, but also tumor metastases. Oncolytic viruses as standalone therapies have not achieved durable tumor clearance, but their arming with additional therapeutic genes may address this challenge. Most arming strategies rely on “bystander effects”, i.e., the killing of noninfected tumor cells. Bystander cell killing can be elicited by prodrug convertases, radiosensitizers and immunomodulators (Fig. 5).

Prodrug Convertases Prodrug convertases are enzymes that activate non-toxic substrates, converting them into cytotoxic substances (Fig. 5, top panel). Prodrug-encoding viruses derived from Herpesviridae, Adenoviridae, Poxviridae, Paramyxoviridae, and Rhabdoviridae have been developed. The clinically most advanced approach is a replication-competent retrovirus encoding the prodrug convertase cytosine deaminase (Table 1). This enzyme converts a precursor into the active chemotherapeutic 5-fluorouracil (5-FU). 5-FU can diffuse out of the cell and into surrounding cells, exerting toxic effects also on non-infected cells. Compared to systemic chemotherapy, oncolytic virus-encoded prodrug convertases can reduce systemic toxicity by local concentration of the active drug at the site of viral replication, that is within the tumor.

Radiosensitizers for Therapy and Imaging Arming with radiosensitizers aims at local concentration of radioisotopes for ablation of malignant tissue (Fig. 5, center panel). The sodium iodide symporter (NIS), whose physiological role is to concentrate iodide in the thyroid, has been incorporated into multiple viruses to facilitate tumor ablation. Administration of NIS-encoding viruses and certain b-emitting isotopes enables localized radio-virotherapy: radioactive ions are concentrated within infected tumor cells, and these radioisotopes also affect surrounding, non-infected cells. An additional key application of NIS is imaging, which enables real-time tracking of viral gene expression and spread. This is done in pre-clinical and clinical studies using g-emitting isotopes that accumulate at the sites of viral replication. These isotopes, which have much more effective tissue penetration than fluorescence or bioluminescence, have allowed to characterize at high resolution the replication of oncolytic viruses in vivo. In particular, the combination of imaging viral replication by single photon emission computed tomography (SPECT) with tissue imaging by computed tomography (CT) allows to correlate virus replication and distribution with measures of clinical efficacy. In worst case scenarios, SPECT-CT can identify off-target viral replication.

Immunomodulators The successes of cancer immunotherapy in recent years rekindled interest in the immunotherapeutic effects of oncolytic virotherapy. Virus-mediated tumor cell lysis leads to the release of tumor antigens in a highly immunostimulatory context. This can induce anti-tumor immune responses via in situ vaccination effects. Arming oncolytic viruses with immunomodulatory transgenes aims at enhancing and shaping these anti-tumor immune responses (Fig. 5, bottom panel). The immunomodulator most commonly harnessed for this purpose is GM-CSF. GM-CSF recruits and activates antigenpresenting cells, which in turn stimulate T cells. GM-CSF is expressed by three of the clinically most advanced viruses (Table 1). Further immunomodulatory transgenes include cytokines and immune checkpoint blocking antibodies. However, non-specific immunostimulation may result not only in activation of anti-tumor, but also anti-viral immunity. To specifically direct immune responses against tumor cells, bispecific antibodies for recruitment of immune effector cells to tumor cells have been encoded in oncolytic viruses. Furthermore, oncolytic viruses encoding tumor antigens have been generated to prime and expand tumor-specific T cells. This approach has entered clinical development (Table 1). Additional arming strategies include encoding pro-apoptotic factors, or factors which counteract inhibitors of apoptosis to increase tumor cell death. Oncolytic viruses encoding short hairpin RNAs, microRNA mimics, and microRNA decoys to inactivate pro-tumorigenic factors have also been reported. Metabolic reprogramming of the tumor microenvironment is also attempted.

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Fig. 5 Arming strategies. Top panels: prodrug activation. Cancer cells are infected by a virus that expresses a prodrug convertase that activates non-toxic precursors (prodrugs) into therapeutically active toxic metabolites. These metabolites diffuse within the surrounding tissue, killing bystander cells. Center panels: radiosensitization. Cancer cells are infected by a virus that expresses a symporter like NIS, which concentrates radioisotopes in infected cells, leading to radiation-induced death of the infected cell, and of nearby bystander cells. Shown here is radiation-induced DNA damage in a bystander cell. Bottom panels: immuno-stimulation. Cancer cells are infected by a virus that expresses an immunostimulatory gene like GM-CSF. Infected cells activate anti-tumor immune responses. GM-CSF activates dendritic cells to present tumor antigens to T cells via MHC molecules and to express costimulatory molecules such as CD80. Recognition of the presented antigens by a T cell with a cognate T cell receptor (TCR) primes an anti-tumor immune response, leading to T cell-mediated anti-tumor cytotoxicity. Modified from Miest, T.S., Cattaneo, R., 2014. New viruses for cancer therapy: Meeting clinical needs. Nature Review Microbiology 12, 23–34 (Created with BioRender.com).

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Fig. 6 Clinical trials of oncolytic virotherapy in combination with other treatments. Clinical trials initiated in the last 5 years (2015–2019) are categorized by type of combination therapy.

Combination Therapies A paradigm of cancer treatment is that no single drug or therapy will cure cancer. Surgical removal of tumors, chemotherapy, radiation therapy, and now also immunotherapy and oncolytic virotherapy are used in combination to treat cancer patients. Indeed, several current clinical trials combine an oncolytic virus with other therapeutics (Table 1). In particular, more than half of all trials initiated in the years 2015–2019 combine virotherapy with at least one additional treatment modality (Fig. 6) and it is to be expected that oncolytic viruses will increasingly be integrated into combination therapy regimens. For instance, adenovirus H101, the first clinically approved oncolytic virus, is applied in combination with standard chemotherapy for head and neck cancer (Table 1). Several other combinations of chemotherapy and virotherapy are currently in clinical trials. Some of these protocols take advantage of virus-encoded enzymes to locally convert prodrugs into active chemotherapeutics, as outlined above. In pre-clinical studies oncolytic viruses have also been combined successfully with targeted therapies. For example, Talimogene laherparepvec (T-vec) showed synergistic effects with kinase inhibitors (MEK inhibitors) in mouse models of melanoma. So far, radiosensitizers have only been used for therapeutic purposes in pre-clinical models, but several trials have included imaging to assess the biodistribution of the virus. The prospect of inducing anti-tumor immunity through oncolytic vaccination prompts the use of viruses in immunotherapeutic regimens. The combination of oncolytic virotherapy with immune checkpoint blockade appears especially promising and is increasingly pursued in clinical trials (Fig. 6). The proposed mechanism of action for this combination is that oncolytic virotherapy induces tumor-specific T cell responses that are subsequently reinforced by checkpoint blockade. The sheer endless possibilities to combine oncolytic viruses with different cancer treatments require prioritization. Future studies will be guided by better understanding of the mechanisms of action of cancer therapeutics as well as their interactions with other treatments and stringent testing in more refined pre-clinical models. Complex combination therapies raise questions regarding the optimal dosing and scheduling of the individual treatment components. Several groups have attempted to mathematically model combination treatments with oncolytic viruses to inform dosing and scheduling with the aim to predict – in silico – the most effective combination regimens. Surgery is a mainstay in treatment of many cancer types. Incorporating oncolytic viruses into the treatment of these cancers allows for so-called “window of opportunity” clinical trials. Oncolytic viruses are administered in a neoadjuvant setting, that is before surgical removal of the tumor. Histological assessment of the tumor tissue for signs of viral replication and immune infiltration can provide valuable insights into therapeutic effects, or lack thereof, of oncolytic virotherapy. To summarize, the dual mechanism of action of oncolytic viruses, namely direct intratumoral spread followed by inflammatory boosting of the anti-tumor immune response, makes them ideal partners for combination with immune checkpoint inhibitors and other immune-oncology agents. Another current priority is research on synergies with conventional anticancer drugs.

Perspectives Viruses are emerging as a promising new modality in the fight of cancer. While current oncolytic virotherapy approaches are very safe in humans, therapeutic efficacy has been less than expected from pre-clinical studies. This may reflect the fact that many current approaches are based on highly attenuated viruses, and that generalized attenuation interferes with clinical efficacy. Next-generation viruses will seek to retain wild-type replicative competence in cancer tissue, while entry targeting and postentry replication control mechanisms will restrict their spread in off-target tissues. In addition, arming strategies based on chemo-, radio-, or immuno-therapies will be potentiated by enhance virus replication in tumors. Next-generation oncolytic viruses will be deployed strategically, based on deeper understanding the susceptibility of different tumor types, and on progress in stratifying patients. In synergy with approved cancer therapeutics, oncolytic viruses are finding their niches in the treatment of cancer.

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Further Reading Asada, T., 1974. Treatment of human cancer with mumps virus. Cancer 34, 1907–1928. Barry, M.A., Rubin, J.D., Lu, S.C., 2020. Retargeting adenoviruses for therapeutic applications and vaccines. FEBS Letters 594, 1918–1946. doi:10.1002/1873-3468.13731. Bluming, A.Z., Ziegler, J.L., 1971. Regression of Burkitt's lymphoma in association with measles infection. Lancet 2 (7715), 105–106. Cattaneo, R., Miest, T., Shashkova, E.V., Barry, M.A., 2008. Reprogrammed viruses as cancer therapeutics: Targeted, armed and shielded. Nature Reviews Microbiology 6, 529–540. Dorer, D.E., Nettelbeck, D.M., 2009. Targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis. Advanced Drug Delivery Reviews 61, 554–571. Engeland, C.E., Grossardt, C., Veinalde, R., et al., 2014. CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy. Molecular Therapy 22, 1949–1959. Hill, C.A.P., Bau, L., Carlisle, R., 2020. Methods for modification of therapeutic viruses. Methods in Molecular Biology 2058, 7–29. Kelly, E., Russell, S.J., 2007. History of oncolytic viruses: Genesis to genetic engineering. Molecular Therapy 15, 651–659. Miest, T., Cattaneo, R., 2014. New viruses for cancer therapy: Meeting clinical needs. Nature Review Microbiology 12, 23–34. Peters, C., Rabkin, S.D., 2015. Designing Herpes viruses as oncolytics. Molecular Therapy Oncolytics 2, 15010. doi:10.1038/mto.2015.10. Ribas, A., Dummer, R., Puzanov, I., et al., 2017. Oncolytic virotherapy promotes intratumoral T cell infiltration and improves anti-PD-1 immunotherapy. Cell 170, 1109–1119. Ruiz, A.J., Russell, S.J., 2015. MicroRNAs and oncolytic viruses. Current Opinion in Virology 13, 40–48. Schneider, U., Bullough, F., Vongpunsawad, S., Russell, S.J., Cattaneo, R., 2000. Recombinant measles viruses efficiently entering cells through targeted receptors. Journal of Virology 74, 9928–9936. Springfeld, C., von Messling, V., Frenzke, M., et al., 2006. Oncolytic efficacy and enhanced safety of measles virus activated by tumor-secreted matrix metalloproteinases. Cancer Research 66, 7694–7700. Twumasi-Boateng, K., Pettigrew, J.L., Kwok, Y.Y.E., Bell, J.C., Nelson, B.H., 2018. Oncolytic viruses as engineering platforms for combination immunotherapy. Nature Review Cancer 18, 419–432.

Relevant Websites https://clinicaltrials.gov/ ClinicalTrials.gov.

PREVENTION

Surveillance of Infectious Diseases Norman Noah, London School of Hygiene and Tropical Medicine, London, United Kingdom r 2021 Published by Elsevier Ltd.

Glossary Case–fatality rate Number of persons dying of an infection divided by total number of persons with the disease. Thus, CFR of 5% means that 5 of 100 persons with the infection died. It is distinguished from “Mortality Rate” which measures the death rate in the whole population. Endemic An infection continues in a country, though may vary in incidence at different times. Many enteroviruses, adenoviruses and respiratory viruses are endemic. GUM clinics The term GenitoUrinary Medicine clinics is often used for special clinics for sexually transmitted infections (STIs). Incidence The rate of new infections (number by population) in a given time – for example, 5 cases of influenza per 1000 per year. Good for short-term infections. Outbreak and epidemic Most infectious disease epidemiologists do not distinguish between these terms and

use them interchangeably. Generally, an outbreak is defined as a localized increase in cases, whereas an epidemic is more widespread, perhaps affecting a whole country. Outbreak When an infection occurs at a frequency higher than expected for that time or place. It is basically an increased incidence which is usually unexpected. Two linked cases is also theoretically an outbreak. Pandemic This term should be restricted to infections affecting many countries, and may extend virtually worldwide. SARS spared many countries and indeed some continents, but it was a pandemic. SARS-CoV-2 and AIDS undoubtedly caused pandemics. Prevalence The number of infections at any one time in a given population, expressed as a rate. Good for chronic infections such as chronic hepatitis B or C and serological studies (e.g., prevalence of varicella antibody at age 15 in a given population is 95%).

Introduction Surveillance is undoubtedly an essential – indeed critical – ingredient of any disease control program. It is used to paint a picture of the progress and overall burden of infection or disease, so that any preventive or therapeutic action can be measured as it advances. In this way, the impact of an infection, the effect of an intervention or health promotion strategy, health policy, planning, and delivery can be monitored. Surveillance is the ongoing and systematic collection of routine data which are then analyzed, interpreted, and acted upon. It is essentially a practical process, which nevertheless can be useful in other ways. Its main purpose is to analyze time trends – but these can include not simply fluctuations in overall numbers, but also changes in age and sex distributions, geographical locations, and even possibly, in some of the more sophisticated established surveillance systems, at-risk groups (such as particular social, ethnic and occupational groups). Surveillance is essential for evaluating the impact of an intervention, such as mass vaccination, on a population. It can also act as a fairly sensitive system for the early detection of outbreaks.

Essential Characteristics of Surveillance The word ongoing in the description of surveillance helps to distinguish it from a survey, which is usually finite, tends to focus more on one or more groups of persons, and involves a questionnaire. Nevertheless, surveillance does not have to continue forever – when it is no longer useful, it should be stopped. To be systematic and consistent are other important ingredients of surveillance. If reporting centers are not consistent in what they report, nor regular, the data they send in will be uninterpretable, and probably useless. Defining what needs to be reported, its frequency, and agreeing on the criteria for making a reportable diagnosis are necessary to make sense of the data. Timeliness is another important ingredient of a surveillance system. This is especially the case with infection. It is particularly essential for the early recognition of outbreaks, but it is important also in evaluating the success or otherwise of programs to control the spread of an infection e.g., the effect of lockdown on coronavirus disease (COVID-19) on a population. Representativeness is another important ingredient of a surveillance program. The program has to be representative of the effect of that infection on a population. This may not always be possible, and any biases and inconsistency in reporting has to be taken into account in interpreting the data. Completeness is generally less important than representativeness, especially for relatively common infections. Completeness becomes important if or when the number of infections decreases, whether on its own or through a public health intervention. For some serious infections, completeness may also become important. Completeness is discussed in more detail later in this article. The collection of routine data is another important characteristic of surveillance, especially with surveillance of laboratory infections. Generally, testing of samples is done for diagnosis, not primarily for surveillance. As laboratory testing is expensive, making further use of the results by contributing to surveillance makes for more efficient use of information, and the contribution to surveillance itself can

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often justify further testing. Typing echoviruses or coxsackie viruses can seldom be justified on clinical grounds alone, but the surveillance of serotypes can provide valuable information on the epidemiology of these viruses, their clinical characteristics, seasonality, and age and sex distributions – and of course whether there has been an outbreak. Echovirus 4 for example tends to be rare, fairly localized even within a country, but, when it occurs, it has a high aseptic meningitis rate, and causes a short and sharp autumn outbreak. Echovirus 9 on the other hand is far more common, more widespread geographically when it causes an epidemic, more benign, with a macular rash, and with meningitis not an especially common manifestation. The weekly patterns of some of these viruses can provide clues on future resurgences. Echovirus 4 as stated, comes and goes in one season. In the autumn of 1974, a small outbreak of echovirus 19 did not disappear entirely throughout the winter. A further and much larger outbreak in the autumn of 1975 was predictable, and occurred. Although surveillance is essentially a practical exercise, this article attempts to show that surveillance can also be useful in giving us clues about an infection, whether it be its natural history, etiology, severity, or outcome.

Collection of Data: Sources of Data in Surveillance of Viruses Death Certification In most developed and middle-income countries, deaths are certified primarily for legal reasons, but have proved to be an important data source to use for surveillance. Clearly, they tend to be useful mainly for serious infections, and may suffer from inaccuracies, but remain a useful basic data source. Diseases with a high mortality rate and a short duration of illness (the viral hemorrhagic fevers for example) will obviously be better represented by death certification than those with a low mortality. HIV/AIDS, before the era of HAART, had a high mortality but long periods of symptomless and symptomatic infection, so that death certification data needed careful interpretation. It is well known that death certification standards of accuracy vary not only from country to country, but also within countries. In England, in the early years of the HIV epidemic, it was shown that deaths in single men had risen in number although AIDS was not mentioned on the death certificates. With influenza outbreaks there is usually an increase in total mortality in the elderly otherwise unaccounted for. The reporting of deaths from SARS-CoV-2 both within and between countries is also known to be inconsistent. Excess deaths have become a useful measure in estimating mortality from influenza and SARS-CoV-2. Laboratory reporting systems can sometimes be another useful source of information on mortality from infection.

Notifications Surveillance systems such as statutory notification tend to be based mainly on clinical features. These can be very useful for common diseases with distinctive clinical syndromes, such as measles and mumps. It is important however not to make a disease notifiable unless there is a good reason for it: “good” reasons include a mass vaccination program (when surveillance is virtually mandatory), any other mass control program, or serious diseases for which contact tracing, mass or close contact prophylaxis or investigation into source is necessary. Serious less common viral infections such as poliomyelitis are notifiable in most countries. This is because contact tracing and preventive measures can be taken. Broader clinical diagnoses, such as aseptic (viral) meningitis, may on the face of it be less useful to notify, as it is usually impossible at the bedside to distinguish such causes of it as, for example, the coxsackie B viruses, echoviruses, and mumps. Nevertheless, notification of aseptic meningitis can be useful because a rapid rise in notified cases may need to be investigated further. Moreover, the timing of any epidemic may give a clue to etiology – mumps meningitis tends to increase in spring and is usually accompanied by a concurrent outbreak of clinical mumps, while enterovirus epidemics are more likely to occur in autumn.

Other Sources of Data Specific general practitioner (GP) surveillance systems are useful in providing data of epidemiological value for common infections that are not notifiable, such as the common cold or chickenpox (in UK). They are of course clinically based, but are nevertheless useful, and often surprisingly accurate, possibly because those GPs who subscribe to a surveillance system are motivated to do so. GP surveillance systems are often sentinel-based, that is, based on a sample of GPs in a country, region, or area. In the English system, GPs provide data on the base populations of their practices, so that rates of infection can be provided as a routine, a feature that is almost unique amongst surveillance systems. Thus, they are good for common infections which to make notifiable would possibly be wasteful, such as chickenpox. Moreover each sentinel would normally provide complete reporting. GP surveillance systems also tend to be good for timeliness and completeness. Outbreaks of influenza are often first recognized by GPs.

Laboratory Data It is useful to think of laboratory data as being of qualitative rather than quantitative value as they add quality and detail to disease surveillance. Thus, in the example already used for aseptic meningitis, a precise diagnosis of mumps, echovirus, or coxsackie type is

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particularly useful in clustering and outbreaks. Indeed, as the enteroviruses exhibit a strong late summer/autumn seasonal pattern with each enterovirus returning to its baseline in winter, when numbers of a particular type continue to be reported at a level higher than the winter baseline, the return of this virus to cause another epidemic in the following summer can usually be safely predicted. Laboratory data are also essential in qualifying food poisoning and gastroenteritis. Separating viral from bacterial causes is a useful first step, as their management tends to be very different. Norovirus gastroenteritis can be food-borne, but also spreads very easily from person-to-person because an extremely small dose is necessary for infection to occur, and the virus being fairly resistant to the environment will survive for some time. Management therefore must concentrate on hygiene. Rotavirus on the other hand is not commonly known to be foodborne. It can be distinguished from norovirus by its strong winter pattern in temperate climes, and its preponderance in infants. With most bacterial causes of food poisoning, especially the salmonellas, management often depends on the removal of the offending food. This is less common with viruses – only occasionally do hepatitis A or norovirus cause outbreaks of food poisoning. Laboratory surveillance is particularly essential for unraveling the mass of respiratory viral infections that inflict humans, such as respiratory syncytial virus, adenoviruses, parainfluenza viruses, and rhinoviruses. Indeed, it is particularly useful for influenza – not only separating it from influenza-like illness, but also in identifying influenza A and B, and if A, the subtype and variant. The constituents of every influenza vaccine depend on laboratory data.

Surveillance of Outbreaks Surveillance of outbreaks (as opposed to individual infections) can be revealing, and important to allow public health measures. In one incident, routine surveillance revealed two cases of hepatitis B associated with one tattooist. Inspection of the notification data on hepatitis revealed 33 cases in all associated with the tattooist. The existence of the noroviruses was suspected in the UK many years before the organisms were identified. This was because some outbreaks which did not fit the characteristics of known infections but had characteristics of their own had occurred. There are some surveillance systems specifically for outbreaks. One example is EpiWATCH. This is run by the National Health and Medical Research Council (NHMRC) Center for Research Excellence Integrated Systems for Epidemic Response in Australia. Epidemic intelligence systems such as EpiWATCH can be useful for the global surveillance of outbreaks.

Hospital Admissions These can be useful for certain more serious infections, such as hepatitis and encephalitis. They can be unwieldy, generally lack detail, and often are published a year or more after the events have occurred. Special more timely surveillance systems can be created, as for example with SARS-CoV-2 in 2020.

Serological Surveillance Serological surveillance has become increasingly important, and is a useful tool in assessing the immunity of a population, though it can also be used to identify vulnerable individuals. The immunity of a population can be vaccine-induced, and serological surveillance is a valuable adjunct to the methods available to monitor a mass immunization program. Vulnerable age groups can be identified, and booster doses of the vaccine introduced. An example of the importance of serological surveillance in determining public health policy is included below (analysis by person).

Surveillance of Viruses in Nonhuman and Environmental Sources To build a picture of an infection, animals, birds, and the environment have been placed under surveillance. Rabies in foxes and other wildlife, influenza in birds, pigs, and other animals, are examples of important and fairly successful surveillance systems. With the more recent examples of the emerging viruses such as Nipah and the group B coronaviruses, this type of surveillance is becoming important. At the time of writing, there are some surveillance systems for influenza A virus in avians and humans. Environmental surveillance of sewage and other wastewaters for wild and vaccine polio virus, as well as other viruses, exist.

Other Sources of Data Records of sickness absence, absence from school, calls to an emergency room, if available, can provide speedy information that something has happened, but tend to be nonspecific. Surveillance of antiviral resistance will become important. AIDS was first discovered in the US when the use of a drug for treatment of the then rare Pneumocystis carinii infection in gay men began to increase. Surveillance of prescriptions or drug usage as a means of monitoring infection will probably increase in importance.

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Newer Types of Surveillance System Syndromic Surveillance There is no strict definition of this type of surveillance. One description is: “Syndromic surveillance complements traditional public health surveillance by collecting and analyzing health indicators in real time”. It is usually based on an increase in an unusual group of clinical features which may lead to investigation. One of the earliest examples of this, though not a virological one, was the recognition of an unusual cluster of symptoms (subsequently labeled the Eosinophilia–myalgia syndrome) linked to L-tryptophan consumption originating from a single source in Japan. This was a dietary supplement manufactured using genetically-engineered bacteria. The beginning of the outbreak of SARS-CoV-2 was an increase in an association of unusual clinical features in Wuhan – in effect a new syndrome. Although there was some delay in reporting this particular unexpected increase, the primary objective of this type of surveillance is to recognize an outbreak as early as possible. Sudden changes in numbers of doctors’ prescriptions for specific drugs, school or workplace absenteeism may also be used to uncover new outbreaks or new infections. Electronic surveillance systems are now being developed to detect new outbreaks – whether virological, bacteriological, or chemical. The fear of terrorism has stimulated this greater interest in syndromic surveillance. Syndromic surveillance will undoubtedly become more important with time.

Sentinel Surveillance Sometimes, for research purposes or otherwise, more detailed or higher quality data on an infection or infections are required than normally available through a passive system. It may be too much of a burden – and probably wasteful – to expect all laboratories or other sources of data to include or collect this extra information. In this instance a form of active surveillance can be introduced. A sentinel surveillance system can be set up using a selected – and willing – number of motivated laboratories. Chosen laboratories would be expected to diagnose a reasonable number of cases of the infection, be within a fairly large area in which they would be expected to diagnose all cases of the infection, and of course to be technically excellent. Sentinel surveillance systems tend to be time limited, and are unsuitable for rare infections.

Enhanced Surveillance Enhanced Surveillance is a term used for infections of public health importance to combine epidemiological and microbiological data, as well as other types of information necessary. It may include data from a range of sources. For COVID 19, clinical and microbiological as well as social data such as ethnic background have been collected by several countries, including from asymptomatic persons. Serological testing gives information on immunity both in a person as well as in a community. This information can be obtained from primary care and various other settings, including places of education, medical institutions and care homes. Clearly, enhanced surveillance is expensive on costs and manpower, and should only be put into practice for special reasons, such as a pandemic. Further examples covering HIV are given in the next section; many of these are also relevant to Covid19.

Active Surveillance This is a form of enhanced surveillance to ensure completeness and consistency. Reporting sources have to report make negative returns whether or not they have had cases.

Surveillance of HIV/AIDS The association of HIV/AIDS with stigma makes surveillance of this infection especially difficult. It is an example of the importance of tailoring surveillance to a specific serious infection if it becomes necessary to do so. In the UK and some other countries with data protection acts, HIV infection, as with other STIs, is not notifiable. Special confidential surveillance systems through clinicians and GUM clinics, as well as laboratories, are in place. These are especially important for assessing risk factors. Inclusion of risk factors is essential for targeted intervention – for example, the proportions and rates of new diagnoses attributed to men who have sex with men, heterosexual sex, mother-to-infant, blood transfusion, IVDUs, and other needlestick injury. Laboratory reporting is essential. Death certification is useful, though it has been shown that men who have sex with men, and probably those with other risk factors, are under-represented. In the UK, matching reported cases with death certificates is very important, as it allows for detection of deaths due to AIDS (such as pneumonia) as well as deaths associated with AIDS, and which are seen now in HIV-infected individuals – these include liver and cardiovascular disease, overdoses, and malignancies. A surveillance system, based on unlinked anonymous testing of samples of blood taken routinely from certain at-risk population or occupational groups has been shown to provide valuable information on HIV infection in these populations. Specific screening systems for blood donors, military recruits, commercial sex workers, and family planning/termination of pregnancy

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clinics are also useful if these populations are to be targeted. Behavioral surveillance should also be seriously considered, to assist in identifying future trends, healthcare planning, as well as for specific health promotion efforts.

Attributes of Surveillance Systems Completeness Incompleteness is an almost universal drawback of most notification systems. They should never be dismissed for this reason alone. Statutory notification systems can be essential for surveillance and control. For common infections completeness may not be worth striving for, because notifications (assuming consistency in reporting) will generally provide information on trends, as well as fairly accurate information on age, sex and seasonal distributions, and possibly on place. Notifications were able to show a small increase in measles cases in the early 2000s which led to the need for a preschool booster. The effect of mass vaccination programs can also be monitored fairly closely with statutory notification systems, as with measles (Fig. 1) and acute paralytic poliomyelitis (Fig. 2) in the UK. When a mass vaccination or other universal control program reduces the incidence of an infection to low levels, completeness becomes much more essential. For serious infections also, such as SARS or Lassa fever, for which contact tracing or other control measure is necessary, completeness is essential.

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In active surveillance, reporters make negative returns if they have had no cases during the reporting period, to ensure completeness. In some countries, enhanced surveillance has been used to assess more accurately the true incidence of an infection – regions or districts are chosen to report all cases of a particular infection or infections. It is a hybrid of active and sentinel surveillance.

Timeliness Timeliness is important for infections for which urgent public health measures have to be undertaken, such as SARS-CoV-2, poliomyelitis and viral hemorrhagic fever, as well as any outbreak. In some instances, infections, not normally urgent, can become so as an elimination program progresses. In a country with elimination of measles as its goal, a case of indigenous or imported measles needs to be dealt with urgently, as it may lead to an outbreak if not controlled immediately. Laboratory data are often not timely, and hospital data generally even less so, but can make up in accuracy what they lose in timeliness.

Accuracy Accuracy is clearly important, though some minor degrees of inaccuracy can be tolerated in some common infections. Clinical data are most liable to have some inaccuracies, though even laboratory data may be inaccurate. Case definitions and quality control systems can be useful to improve accuracy.

Representativeness For surveillance to provide an accurate picture of the impact of a particular infection, representativeness is essential. It is perhaps the most important quality for any surveillance system. Having a wide coverage of reporting clinicians and laboratories, or a wellchosen sample of sentinel sites, is necessary for the data collected to be representative of an infection in a country. Sometimes it may be necessary to assess data from various sources, such as notifications/GPs (clinical), hospital, laboratory, and death certificates.

Consistency Consistency is another crucial basic attribute of any surveillance system. Reporters must know what to report (case definition) and how often. Otherwise it will not be possible to interpret trends. Active surveillance is one way of ensuring consistency.

Analysis of Data Time The three basic analyzes by time, place, and person should be routine. Computer programs have made analyzes of data quicker but somewhat less flexible. There is no standard period for analysis by time. Depending on what the surveillance intends to show, yearly, quarterly, monthly, four-weekly, or weekly time intervals can be used. In surveillance, time intervals shorter than this are rarely used, although daily data were provided in many countries to monitor SARS-CoV-2. Monthly intervals have the disadvantage of having unequal numbers of days in each month, and are difficult to use when reporting is weekly; for seasonal trends four-weekly periods are better but cannot be divided into quarterly periods. In viral surveillance, four-weekly rather than weekly intervals tend to be most useful in showing seasonal changes. There is more likely to be more variation (“noise”) in weekly intervals, making for less smooth changes. For secular trends, quarterly or annual intervals are generally used. Analyzing by time can reveal regular changes in the periodicity of viruses, enabling some of them to be predicted. A basic knowledge of seasonal and secular patterns makes it easier to detect changes that signify a possible epidemic, and to differentiate these from a random variation. It is important to remember when analyzing laboratory data that there is often an interval, which can be 2 weeks or more, between date of onset and date of reporting.

Person Analysis by age and sex is another basic analysis in surveillance. It can identify those most affected, and vulnerable groups. Changes in age distributions may provide important clues about a changing viral infection, and the effect of mass interventions on the age distribution of an infection can be monitored. Changes in the age distribution of measles in 1994 in the UK signified that an epidemic in older children was imminent, and the vaccine schedule was changed to include an extra booster injection (MR) to children aged 5–16 years. This averted the outbreak and the booster dose became a permanent feature of the routine immunization schedule in the UK. Indeed the changes in age distribution following mass vaccination could be considered an epidemiological side effect of mass vaccination. Requests for occupational groups and travel histories should be selective. For poliomyelitis, SARS, dengue, and the viral hemorrhagic fevers, travel histories are required. Occupational group may be useful for norovirus, and hepatitis types A, B, or C. Specific risk factors may be worthwhile for HIV, hepatitis B and C.

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Table 1

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Stages of a viral infection

Clinical A. Uninfected B. Infected 1. Asymptomatic 2. Symptomatic unreported 3. Symptomatic, sees a doctor 4. Symptomatic, admitted to hospital 5. Symptomatic, dies/survives Note: Chronic carriers can occur at any of the B stages – for example, hepatitis B or C.

Table 2

Stages in laboratory diagnosis

A. Uninfected B. Infected 1. Asymptomatic 2. Symptomatic, unreported 3. Symptomatic, sees a doctor 4. Symptomatic, specimens submitted 5. Symptomatic, specimens positive 6. Symptomatic, specimens reported to surveillance system

Place Analysis by place can pinpoint local outbreaks. Some echoviruses (e.g., echovirus type 4) can cause rare short local outbreaks, other types (e.g., types 9 and 11) are more common and more widespread. Food-borne outbreaks of hepatitis A and norovirus are generally picked up locally through routine surveillance, but sometimes more extensive outbreaks caused by a more widely distributed foodstuff, including shellfish or frozen soft fruit, may be identified.

Interpretation Collection and analysis are generally routine functions; skill is required in interpretation of the data. No statistic is perfect, and surveillance data, like all data, must be interpreted with caution. One must take into account the origins of the data – clinical, laboratory, or hospital. Not only must the reliability, or otherwise, of the data be evaluated, but what the data signify in the natural history of the infection must also be recognized. Every viral infection has its stages and these must be recognized before surveillance data can be sensibly interpreted. At what stage are the data being collected important to understanding and interpretation? Using influenza or hepatitis A as examples, and a defined population (Table 1), only a proportion of persons in the defined population will be infected with the virus. They can only be comprehensively detected by screening, and serological surveillance will identify these persons, or assess population immunity (B1 in Table 1). In HIV/AIDS surveillance, unlinked anonymous testing of samples of blood taken routinely at say, an antenatal clinic, can give vital information on the prevalence of HIV infection, since in this infection presence of antibody denotes infection, not immunity. A smaller proportion will be ill (B2), but only some of these will visit a doctor (B3). Surveillance systems based on GP consultation rates have now been recognized as an important addition to the spectrum of a disease, and many countries have excellent systems. Of those patients that do visit their family doctor, only some will be admitted to hospital (B4). Finally, only some will die (B5). For laboratory data, the stages are slightly different (Table 2), but still important in understanding what the reported data mean. As before, only a proportion of persons will be infected (B), some will be asymptomatic (B1), a smaller proportion will be ill (B2), and an even smaller proportion still visit their doctors (B3). Not all doctors will send specimens to a laboratory (B4), and only a proportion of these specimens (B5), depending on accuracy of the identification process, the method of transport, the fragility of the organism, and the swabbing or other sampling technique, will be positive. Finally, depending on the level of consistency of reporting, only some of these will be reported (B6). It is essential to recognize these stages in interpreting surveillance data. Biases will inevitably occur between these stages. Collection of data in routine surveillance is not normally a scientific process as one has to rely on readily available data – data obtained mostly for other reasons, such as to make a definitive diagnosis. Only the most severe cases die, and death certification thus provides, at best, a limited view of any disease. Similarly, only certain types and severity of cases will be admitted to hospital (some admissions are for social reasons for example) or even visit their family doctor.

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Certain age, sex, and perhaps social or occupational groups are more likely to seek medical help, be investigated and be reported. In laboratory data, more severe cases, or children, are perhaps much more likely to be investigated in detail. These shortcomings of surveillance data do not make them useless – but their strengths and limitations must be recognized.

Feedback If interpretation is turning statistics into information, feedback is getting the information across to those that matter, and those that need to know, so that action – the objective of surveillance – can be taken. Without feedback, surveillance is pointless. Feedback is most likely to be informative if undertaken by those most closely involved in the surveillance cycle, and who understand the significance of the data they are receiving. Feedback should be aimed at contributors and those in public health. Contributors will then be aware of which viruses are circulating and this will help them to know what to look for in their own tests (e.g., what echovirus or adenovirus types are in circulation). Moreover, routine surveillance will undoubtedly uncover outbreaks of infection, which will need further investigation and control at local, national, and even international level. An interesting and welcome side effect of a flourishing microbiological/ feedback surveillance system is that it often stimulates better quality control within reporting laboratories. Regular feedback is essential, not only to contributors, but also to those who can act for the public health. Regular and useful feedback encourages good and regular reporting. Generally, the periodicity of feedback should reflect the frequency of reporting – weekly feedback for weekly reports for example. Regular topic-based reviews are also important.

Evaluation of Surveillance A surveillance system is like a country's train system. Once the rail lines have been built, the goods that will be carried along those lines can be changed according to need. Similarly, in a surveillance network, once the lines of communication have been laid down, the data being reported can be changed according to what is most important at the time (though probably not too frequently). Nevertheless, surveillance systems should ideally be frequently evaluated for usefulness, as well as for accuracy, efficiency, and effectiveness. They should also be sufficiently flexible, so that “new” or emerging infections can be included in an emergency or when the need arises. The successful implementation of international surveillance for SARS was instrumental in controlling it. Emergency surveillance was also essential following the tsunami of 2004, and is also necessary for the successful management of other disasters following earthquakes, hurricanes, and floods. Surveillance systems should be evaluated before they are set up, and again at regular intervals thereafter. Before implementing a surveillance system, is there an adequate public health and administrative infrastructure in place to take action? Are the data to be collected representative and sufficiently timely for the specific infection? Are they useful, and is action being taken on the information? If not, is the feedback inadequate?

Global and International Surveillance The ease and speed of modern travel, the distribution of goods (especially foodstuffs) across increasingly wide parts of the world, and the uncontrollable spread of birds and other wildlife across boundaries has made global and international surveillance essential for outbreak and infection control. Surveillance of viral infections such as influenza and SARS-CoV-2 now requires the expertize of many professionals – epidemiologists, virologists, and vaccinologists, clinicians, statistical modelers, veterinarians, managers, and planners. These occur in many different countries so that information can be exchanged, and attempts made on a global basis, to prevent the next pandemic. Epidemics of Zika virus, dengue and West Nile virus have also spread widely recently. AIDS/HIV was destined to become a global problem almost from the time of its first discovery. On a more positive note, SARS was contained through the use of international surveillance; and surveillance was the backbone of the smallpox eradication program. International surveillance can also be used for the detection of international outbreaks of food poisoning caused by the distribution of foodstuffs across a wide number of countries. An outbreak of hepatitis A in England was caused by frozen raspberries grown and frozen in another country; and another outbreak of hepatitis A, this time in Czechoslovakia (before it became separate republics) was caused by strawberries used to make ice cream; the strawberries had been imported from another Eastern European country. There are now well-established trans-European surveillance systems for salmonella infections and legionnaires’ disease, as well as for viral infections. The need for surveillance will never diminish or disappear. Surveillance systems will only improve, become increasingly sophisticated, and become increasingly relied upon and used. Control of infection will not be possible without it.

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Further Reading Charrel, R.M., de Lamballerie, X., Raoult, D., 2007. Chikungunya outbreaks – The globalization of vectorborne diseases. New England Journal of Medicine 356, 769–771. Chin, J. (Ed.), 2002. Control of Communicable Diseases Manual, seventeenth ed. Washington, DC: APHA. Chorba, T.L., 2001. Chapter 7 – Disease surveillance. In: Thomas, J.C., Weber, D.J. (Eds.), Epidemiologic Methods for the Study of Infectious Diseases. Oxford: Oxford University Press. Colon-Gonzale, F.J., Lake, I.R., Morbey, R.A., et al., 2018. A methodological framework for the evaluation of syndromic surveillance systems: A case study of England. BMC Public Health 18, 544. doi:10.1186/s12889-018-5422-9. Communicable Disease Report, 1994. National measles and rubella immunisation campaign. Communicable Disease Report Weekly 4 (31), 146–150. Heymann, D., Rodier, G.R., 1998. Global surveillance of communicable diseases. Emerging Infectious Diseases 4, 362–365. McCormick, A., 1989. Estimating the size of the HIV epidemic by using mortality data. accessed date: 05.08.89. doi:10.1098/rstb.1989.0081. Noah, N., 1989. Cyclical patterns and predictability in infection. Epidemiology & Infection 102 (2). Noah, N., 2006. Controlling Communicable Disease. Maidenhead, UK: Open University Press, pp. 14–19. (chs. 1–4). UNAIDS, 2003. Introduction to second generation HIV surveillance. Available at: http://www.data.unaids.org/Publications/IRC-pub03/2nd_generation_en.ppt (accessed September 2007). Thomas, M.E.M., Noah, N.D., Tillett, H.E., 1974. Recurrent gastroenteritis in a preparatory school caused by Shigella sonnei and another agent. Lancet 1, 978–981.

Relevant Websites http://www.cdc.gov Centers for Disease Control and Prevention (CDC). http://www.eiss.org EIS Scotland's Largest Teaching Union. http://www.eurosurveillance.org. Eurosurveillance. https://iser.med.unsw.edu.au/epi-watch-outbreak-alerts NHMRC Centre for Research ExcellenceIntegrated Systems for Epidemic Response. http://www.hpa.org.uk Public Health England - GOV.UK. http://www.EWGLI.org Service unavailable. https://www.thedailybeast.com/were-fighting-polio-with-sewage-surveillance We're Fighting Polio With Sewage Surveillance. http://www.who.int World Health Organization: WHO.

Preparing for Emerging Zoonotic Viruses Reina S Sikkema and Marion PG Koopmans, Erasmus Medical Center, Rotterdam, The Netherlands r 2021 Elsevier Ltd. All rights reserved.

Emerging infectious diseases (EID) have been defined as diseases whose incidence increased over the past decades or are predicted to increase in the foreseeable future (“see Relevant Websites section”). This definition includes known infections with new properties, or known infections in new geographic regions, infections that were not previously recognized, and new infections resulting from spillover of pathogens from an animal reservoir to humans. Although difficult to state with certainty, current consensus is that EID outbreaks have increased significantly in the past decades, both in terms of size and number of causative pathogens (Smith et al., 2014; Allen et al., 2017). The majority of EIDs are thought to originate from animals, of which wildlife is the most important source of human outbreaks (Allen et al., 2017; Jones et al., 2008). Such spillovers can occur directly, or through vectors such as mosquitos, ticks and sandflies. Examples of past outbreaks are SARS (Peiris et al., 2004), MERS (Zaki et al., 2012), Avian Influenza (Lai et al., 2016), Ebola (Dudas et al., 2017) and Zika virus (Gubler et al., 2017). Compared to common endemic diseases, the burden of disease may be relatively limited, but the unexpected nature, high case fatality rate, the uncertainty of sources and modes of transmission, and the paucity of medical and non-medical countermeasures make EIDs a threat to global human and animal health. Fear of spread, travel restrictions, and unpredictable self-imposed avoidance of a region can cause serious socio-economic disruptions on local and global level (McCloskey et al., 2014).

Drivers of (Emerging) Zoonotic Diseases Infectious disease emergence has proven extremely challenging to predict, and human cases of a wide range of emerging or reemerging pathogens have been reported all over the world and throughout the year. However, when outbreak data of emerging disease outbreaks in the last decades are combined, some specific conditions or risk factors seem to drive disease emergence (Woolhouse, 2011). The main general drivers that have been described can be summarized as changes in (1) human demographics with the consequential growing demand for food production (Jones et al., 2008), (2) land use, including agricultural changes (Jones et al., 2013; Gortazar et al., 2014) (3) international travel and trade (Gortazar et al., 2014; Randolph and Rogers, 2010), (4) climate change and weather (Allen et al., 2017; Gortazar et al., 2014) (Fig. 1).

Fig. 1 Global changes acting as drivers of infectious disease emergence and spread in the One Health domains.

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In the case of zoonotic EIDs, the first step towards an outbreak of human disease or mortality is the occurrence of spill-over of a pathogen from an animal reservoir host to humans. (Plowright et al., 2017; Karesh et al., 2012; Petersen et al., 2018). The majority of human EIDs originate from wildlife (Jones et al., 2008; Allen et al., 2017). However, humans are usually not in close contact with wildlife. Therefore, occasionally there is an additional intermediate host species involved where a pathogen is amplified and that is in closer contact with humans (Karesh et al., 2012; Plowright et al., 2015). Well known examples are Nipah virus, Hendra virus or SARS-CoV, that originate from bats and amplify in pigs, horses and civet cats used as food source, respectively (Plowright et al., 2015). The risk of animal to human spill-over depends on a plethora of factors. The aforementioned drivers of disease emergence create circumstances that facilitate such spill-over. Additionally, the risk of spillover is influenced by the pathogen itself. In general, RNA viruses are zoonotic more often than DNA viruses (Olival et al., 2017). A possible explanation for this is their genetic plasticity, caused by the replication mechanism: most RNA viruses replicate with the use of virus-encoded RNA polymerases which – in contrast to the host cell DNA polymerase- do not have so-called proof-reading capacity. Proofreading is a mechanism to reduce the amount of errors in the nascent strands of newly produced copies of (pathogen) genomes, and the lack of proofreading leads to copies that contain a higher number of mutations when comparing the progeny genomes with those of the parent sequences. As a consequence, hosts infected with such RNA viruses shed a mutant swarm of viruses. When they then encounter a bottleneck, for instance an exposure of a new host, chances are that within the swarm variants exist that are better adapted (by chance) to infect a new host (Domingo and Perales, 2019). One possible consequence of this flexibility is described as “phylogenetic host breadth” and “host plasticity”, which reflect the diversity of known hosts, and therefore are significant predicting factors for zoonotic potential of viruses (Olival et al., 2017; Kreuder Johnson et al., 2015). Additionally, the dynamics of a pathogen in a reservoir host population is a very important factor for human health risk, and is governed by population structure, host density, host behavior and contact patterns, shedding patterns, stability of pathogens outside a host, levels of immunity, events that stress the population, and more. As a consequence, the resulting risk of infection for humans may vary depending on season, location, weather and host distribution for example (Plowright et al., 2017; Karesh et al., 2012). The second step towards outbreaks of emerging diseases is the ability of a pathogen to transmit between humans. In general, it is thought to be less likely that enveloped viruses and segmented viruses are associated with human-to-human transmission, similar to viruses with acute durations of infection but the evidence for that is largely observational and certainly not black and white (Geoghegan et al., 2016; Walker et al., 2018). For instance, the majority of top ranking epidemic and pandemic threats listed by the world health organization (WHO) are enveloped viruses (“see Relevant Websites section”). Viral host plasticity is not only associated with spill-over but also increases the risk of human-to-human transmission and international spread (Kreuder Johnson et al., 2015). Moreover, the increased interconnectivity and the overall increase of the human population facilitate rapid spread of novel pathogens.

Disease X A recent report by the Global Preparedness Monitoring Board warned that a global Influenza pandemic, similar to the 1918 Spanish flu, could spread around the world within 36–50 h, and would take the lives of 50–80 million people. Such an epidemic could destroy nearly 5% of the global economy (“see Relevant Websites section”). The examples of Ebola, SARS, and avian influenza have shown that zoonotic disease outbreaks can be very disruptive because of fear for spread and uncertainty of how to contain them even when there is limited onward transmission (Bartsch et al., 2015; Qiu et al., 2018). (“see Relevant Websites section”). The devastating Ebola outbreak in West Africa in 2014–1015 led to a specific call for action by the members of the World Health Assembly, charging the WHO to come up with a new vision to be better prepared for the future, given the increasing likelihood of such outbreaks. A list of priority diseases was developed, for which an urgent need for accelerated research and development of countermeasures was identified, due to their potential to cause a public health emergency. (“see Relevant Websites section”). In an update in 2018, the term disease X was used to describe a serious international epidemic caused by a pathogen currently unknown to cause human disease. The rationale behind the inclusion of disease X is that a completely unknown disease etiology limits the ability for development of fast track diagnostics, vaccines and therapeutics. Therefore, preparedness and response in case of disease X requires a significantly different approach compared to the other diseases on the list. According to the International Health Regulations, WHO Member States are obliged to maintain effective disease surveillance and laboratory systems and to report newly emerging diseases that could spread internationally (“see Relevant Websites section”). However, setting up surveillance for diseases that are still unknown is complicated, and extensive surveillance can be costly, especially in the case of diseases that emerge at the human-animal interface. The disease X track therefore is thought to stimulate thinking in terms of generic solutions that increase EID preparedness.

Emerging Disease Detection and the One Health Concept A key component of early control of emerging diseases is their early detection (Bonacic Marinovic et al., 2014; Chan et al., 2010). As part of the WHO Blueprint initiative, the need for diagnostics for the priority diseases has been stressed. The West African Ebola virus outbreak in 2014 had already disseminated widely into three countries by the time it was confirmed by an international

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laboratory, almost 4 months after the index case succumbed to the disease (Baize et al., 2014; Coltart et al., 2017). Similarly, phylogenetic reconstruction of Zika in the Americas predated the time of introduction to almost two years before it was recognized as a health emergency (Thézé et al., 2018). Although easily criticized, the delays in disease detection are not surprising as symptoms of most EID are not specific, and overlap with major “common” diseases that clinicians are familiar with (Sigfrid et al., 2018). Similarly, clinical diagnostic laboratories focus on these common conditions, and may not have the expertize to detect rare and novel causes of disease (Sigfrid et al., 2018). Efforts to increase preparedness for EID are increasingly moving towards riskbased early detection. As most EID come from animal reservoirs, the need for this has reinvigorated the One Health approach. The concept of One Health is described by the US Center of Disease Control (CDC) as follows: “One Health recognizes that the health of people is connected to the health of animals and the environment. It is a collaborative, multisectoral, and transdisciplinary approach— working at the local, regional, national, and global levels—with the goal of achieving optimal health outcomes recognizing the interconnection between people, animals, plants, and their shared environment.” Although the concept has already been known since Hippocrates launched his theories on human health around 400 BCE (“On Airs, Waters, and Places”; Aristotle “Historia Animalium”) The specific term “One Health” however only came up in the 21st century (Evans and Leighton, 2014; Sikkema and Koopmans, 2016). Since then, the One Health concept is increasingly being endorsed and implemented in national and international policy and research. An important milestone was the publication of the FAO-OIE-WHO tripartite concept note, on “Collaboration, sharing responsibilities and coordinating global activities to address health risks at the animal-human-ecosystems interfaces” (“see Relevant Websites section”) Also, the number of scientific publications that mention the term “One Health” showed a major increase in the recent decade (Sikkema and Koopmans, 2016). Although in practice true integration of information and collaboration between different sectors still proves to be challenging (Sikkema and Koopmans, 2016; Farag et al., 2019; Dos et al., 2019; Manlove et al., 2016), the implementation of the One Health concept has great potential for EID preparedness and response. Moreover, there has been a vast increase of (digital) data in the past decade that has great potential to be used for zoonotic disease prediction, early detection and control (Becker et al., 2019; Masri et al., 2019). This is not only caused by the shift of traditional diagnostics to multi-analyte technologies such as next generation sequencing, but also by the large increase of for example environmental, financial and social media information collection (Simonsen et al., 2016; Shi and Wang, 2019). However, the use of this “Big Data” with different data types of multiple origins also makes the analysis much more challenging (Khoury and Ioannidis, 2014). The use of big data has been advocated and explored in some aspects of public health and infectious disease prediction, but this is also mostly restricted to only one of the required domains (clinical, public health, academia) or sectors (human health, animal health, environmental health, informatics, artificial intelligence) instead of a true crosscutting One Health analysis. Moreover, this, again, calls for multidisciplinary collaborations, since the traditional expertize involved in One Health or Public Health, such as veterinary or medical specialists or epidemiologists, generally do not have sufficient experience in working with such large and diverse data sets.

Developing Targeted, Risk Based Sampling To identify risk areas, or so-called hotspots of disease emergence or spread, both knowledge of possible drivers or risk factors for emergence can be used, as well as detailed spatial and quantitative information on these drivers, for example locations where ecosystem disruptions took place or where farming systems or livestock population sizes have gone through significant changes. Other information, that could be mapped to identify risk areas for known and unknown diseases are vectors, animal reservoirs, human population, habitat and climate. (“see Relevant Websites section”). There are currently many open-access databases available online, that contain information that has great potential to be used for zoonotic disease prediction, early detection and control. Examples of currently available information are: animal tracking data (Tian et al., 2015) (“see Relevant Websites section”; “see Relevant Websites section” United States Geological Survey), animal and human outbreak information (Cauchemez et al., 2014) (WAHID; ADNS; WHO), animal populations and trade (Simons et al., 2016) (FAOSTAT), weather and climate (Ryan et al., 2015) (NASA; ECA&D; USGS FEWS NET), country economic data (Farag et al., 2018) (World Bank) and many others. After defining geographical hotspot areas, risk of EID outbreaks also requires a detailed understanding of human and animal populations at risk. For example, past research indicates that humans in contact with animals, especially in the case of wildlife or animal populations that experienced changes in husbandry, population size or other characteristics are at a higher risk of infection with an (emerging) zoonotic pathogen (Jones et al., 2008; Jones et al., 2013; Allen et al., 2017). However, the intensity and nature of the human-animal interface differs greatly, depending on animal species, animal husbandry systems, eating habits, traditions, etc. Additionally, with human demography, migration and travel, current populations and communities are no longer homogeneous or isolated. Selecting a human population at-risk therefore is complex and people-at-risk move in an increasingly large geographical area. (“see Relevant Websites section”). Therefore, there is an increasing need and necessity to tailor surveillance and risk predictions to local circumstances. This calls for the inclusion of social sciences in disease surveillance and infectious disease preparedness (Bedford et al., 2019). (“see Relevant Websites section”). Animal trading patterns and common practices surrounding animal trading have been shown to be particularly important factors in the spread of zoonotic infectious diseases. Livestock farming has undergone massive changes and expansion in the past decades, with increasing international trade (“see Relevant Websites section”). Increasing size and connectivity of the trading networks, including human contacts such as farmers, traders and consumers, result in increased risks of disease transmission and

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spread (“see Relevant Websites section”). Within the poultry production sector, for example, there are large differences between large scale, intensive industrialized production systems and traditional small-scale rural poultry production systems, and associated animal and public health risks. The main type of production system, as well as the organization of the trade chain, and the degree and frequency of human and wildlife contact differs greatly between regions. In South East Asia, the majority of poultry is reared in backyard farms, with complicated subsequent trade chains, involving many traders and live poultry markets (Moyen et al., 2018; Sealy et al., 2019). This influences infectious disease risk and possible spread. Farms that keep their poultry outdoors generally have a higher chance of AI outbreaks, because of their contact with wild birds (Bouwstra et al., 2017; Wibawa et al., 2018). Moreover, trading involving middlemen is associated with higher likelihood of AI spread, as well as density of live-poultry markets (Sealy et al., 2019; Gilbert et al., 2014) Also, it has been shown that the prevalence and risk of infectious diseases, such as AI, differs per stage of the poultry supply chain (Wu et al., 2019). In other regions of the world, such as Europe and the US, backyard farms and live animal markets are very uncommon, and poultry is often transported directly from farm to slaughterhouse. Therefore, risk profiles and set-up of surveillance differ greatly between farming and trading systems in different geographical areas, and detailed information on the local situation is crucial (Martin et al., 2011; Delabouglise et al., 2017). This is not only important for livestock and poultry, but wildlife and wildlife product trading can also pose a significant risk of infectious disease introduction and spread (“see Relevant Websites section”) (Smith et al., 2012). Also, habits and behaviors of humans around animals matter. Live bird markets are not only a risk for AI transmission because of mixing of birds of different origins and species but also because of particular preferences in some regions for fresh poultry meat and on-site slaughtering (Zhou et al., 2015). In-depth knowledge of (cultural) habits and their history, has proven to be extremely valuable in outbreak investigations of emerging diseases. Case investigations of a Nipah outbreak in humans in Bangladesh in 2004–2005 involved anthropological expertize that was used for in-depth interviews and questionnaires in local language to gain knowledge on possible exposures and risk behaviors. It became clear that drinking raw date palm sap was significantly associated with disease. Several interviews pointed towards fruit bats (Pteropus giganteus) as probable source of infection as they drink from the sap collection pots during the night, contaminating the sap with their infectious saliva. (Luby et al., 2006; Halpin et al., 2011). Infrared cameras captured fruit bats feeding on palm sap collection devices, and urinating when flying away, thus potentially contaminating the sap with viruses (Halpin et al., 2011). Protecting the collection devices by a simple bamboo barrier rather than a ban on drinking palm sap was accepted by the local population as intervention (Nahar et al., 2017). MERS-CoV is another example of a disease where an inventory of the local situation revealed very specific traditions and behaviors around dromedary camels, the reservoir host of the virus. Camel racing and associated movement and mixing of camels is thought to be an important risk factor for spread (Farag et al., 2018). Moreover, local customs involve kissing camels, and drinking raw camel milk and urine, all of which should be considered in the investigations of MERS-CoV (Farag et al., 2018; Gossner et al., 2016) Only with extensive knowledge about the history and background of specific risk behaviors, a strategy to discourage or reduce such behavior can be developed. Another clear example of high risk behavior that has proven to be very difficult to change, are burial rituals in Western Africa, that greatly increase the number of people infected with Ebola in times of an outbreak (Masumbuko Claude et al., 2019). The family and community members of the deceased person touch and wash the bodies before the funeral, which has caused a large number of secondary infections because of their contact with Ebola infected bodily fluids (Nielsen et al., 2015).

Sample Collection Appropriate sample collection is essential for reliable surveillance and outbreak investigations. If human, animal or environmental samples are not collected and stored in a technically appropriate manner, subsequent laboratory analyses will not be reliable or will not generate the anticipated results. A deep understanding of kinetics of shedding of a pathogen and of the antibody response is important to decide on the optimal sampling protocol, the use of detection methods and the interpretation of results. Moreover, subject selection (especially in an outbreak situation), appropriate sample type and sufficient and timely metadata collections are all crucial for subsequent analyses. In addition, to be able to compare data between studies or between countries, laboratory analyses, information collection and study designs should be standardized. For example, comparing human serological data to assess zoonotic influenza exposure has proven to be very difficult because there are significant differences between studies when assessing the collection of epidemiological data, laboratory methods used and the study population, amongst other things (Sikkema et al., 2016). Therefore, international organizations and laboratory networks are increasing their efforts to publish guidelines and training materials to stimulate a uniform approach to investigate infectious diseases and increase the comparability of surveillance and research. Not only diagnostic testing methods and quality are carefully assessed and compared (Reusken et al., 2015; Charrel et al., 2017; Pas et al., 2015) (“see Relevant Websites section”) but also the standardized collection of metadata is increasingly important in the era of big data and NGS, for correct interpretation and analysis of the large amount of data that is generated. Therefore, standardized epidemiological questionnaires are being developed and published alongside of the guidelines for infectious disease diagnosis. In practice, complete and timely collection of metadata proves to be challenging. When routine diagnostic data in the Netherlands was assessed for its use for surveillance of arboviral infection in travelers, researchers found that essential information such as vaccination history was only reported in 0.4%, and travel destination was completed in only 42% of patient dossiers

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(Cleton et al., 2014). Especially during outbreaks, medical records and associated epidemiological information is often incomplete or absent. This has been illustrated for example in Sierra Leone during the Ebola outbreak (Owada et al., 2016). Examples of efforts to standardize infectious disease surveillance and clinical research are the global Consortium for the Standardization of Influenza Seroepidemiology (CONSISE) that is aiming to standardize influenza sero-epidemiology and develop investigation protocols (“see Relevant Websites section”) and the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC), a federation of clinical research networks, providing tools and protocols for research response to outbreak-prone infectious disease (“see Relevant Websites section”). Moreover, the WHO, FAO and OIE publish extensive laboratory diagnosis guidelines and are involved in training and capacity building activities.

The Value of Non-Invasive and Bulk Samples Although smart, risk-based strategies can be developed to optimize sampling efficiency, reliable disease monitoring, especially in the case of emerging diseases, requires a huge sample size to reach sufficient population and/or geographical representation. Traditional surveillance of humans and animals make use of invasive sampling, to screen blood samples or swabs for pathogens. However, the use of such samples needs medical-ethical approval that is increasingly difficult to obtain, as well as approval for animal population studies and experiments. Additionally, invasive sampling can cause significant discomfort, can pose an additional risk for infection of healthcare workers or animal care-takers and can only be executed by specialists in the human or veterinary field. Therefore, setting up and validating surveillance using non-invasive sampling can potentially be easier to execute and to scale up as well as better for human and animal welfare. Feces or urine collection and testing can be a practical and animal friendly surveillance method. Especially in the case of wildlife, where catching and sampling animals can be particularly stressful for an animal, fecal or urine surveillance is very suitable. Feces, or even urine, can be collected without handling the animal or even be in the vicinity at the time of sample collection. This has been described for multiple pathogens, such as Lyssaviruses and Paramyxovirus in bats (Begeman et al., 2020; Peel et al., 2019) and tapeworms in foxes (Umhang et al., 2016). In humans, there may also be a large number of pathogens that can be detected via saliva, urine or feces, although it is generally not included in standard diagnostic algorithms (Niedrig et al., 2018). Although alternative, non-invasive sample types increase the ease of sample collection and welfare of the sampled subject, the number of samples will not decrease. Therefore, validation of pooled samples or novel sample types that can be used to monitor groups or populations of humans, animals or a certain geographical area. In Asia, environmental surveillance of live animal markets is routinely implemented as main means of monitoring of Avian Influenza viruses (Bai et al., 2019; Henning et al., 2019; Khan et al., 2018). Various surfaces, such as poultry cages, chopping boards and defeathering machines, are sampled and tested on a regular basis as an indirect measure of poultry infections and public health risk. Similarly, environmental sampling are also being used for the detection of pathogens in healthcare settings (Kim et al., 2016; Kapetshi et al., 2018). Additionally, air sampling may be promising, both in hospital settings, farms and even markets and airports (Scoizec et al., 2018; Zhou et al., 2016; Bailey et al., 2018). Sewage or wastewater monitoring is another example of such a sample type, that can be used to monitor infectious diseases of large numbers of humans. Sewage monitoring has already been used for many years to monitor polio (Asghar et al., 2014) as well as the monitoring and detection of emerging Norovirus and Enterovirus strains (Mabasa et al., 2018; Majumdar and Martin, 2018). However, sewage could have great potential to be used for virus discovery and emerging disease detection (Fernandez-Cassi et al., 2018; Nordahl Petersen et al., 2015). Other sample types, representing large numbers of animals, that can be can be used to monitor circulating of pathogens are: manure composting plants, bulk milk and ponds (Himsworth et al., 2019; Garcia et al., 2014; Balmer et al., 2014). However, the use of non-invasive sampling or pooled sample types may impact the sensitivity of pathogen detection. In the case of non-invasive sampling, each pathogen may have different shedding characteristics, making it impossible to know the optimal sample type to detect emerging diseases. This also impacts the use of sewage for emerging disease surveillance, for example, because not all pathogens are excreted via feces or urine, making it difficult to detect via sewage sampling.

Catch-All Detection Methods A challenge for risk-based surveillance is the huge potential diversity of pathogens that could cause human disease when spilling over from an animal reservoir. Therefore, even if targeting detection efforts through the approaches described above would succeed, the choice of diagnostic method is a critical one. Depending on how the risk-based surveillance was organized, the choice of potential pathogens to target may be limited or more extensive. An interesting development is the use of unbiased thirdgeneration metagenomic sequencing to characterize all genomic material (DNA and RNA) in a sample, without prior knowledge of possible etiologies. Clinical and environmental samples can be processed with procedures that either enrich for bacteria, parasites or viruses, and subsequently be subjected to sequencing (Wilson et al., 2019; Takhampunya et al., 2019; Zolfo et al., 2018; Nieuwenhuijse and Koopmans, 2017; Hendriksen et al., 2019). Analysis generally takes place by assembly of sequence reads and annotating the resulting reads using reference databases of (publicly) known sequences. The metagenomics research field, studying and profiling genetic material abundance in diverse matrices and environments is rapidly growing, as well as the number of novel viruses that are being discovered and described (Simmonds and Aiewsakun, 2018). Often the aim of metagenomics studies of the environment is to map all micro-organisms in

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potential reservoirs in order to identify potential human health threats (Carroll et al., 2018). However, all viruses that have been characterized to date likely only make up a minor fraction (estimated to be around 0.005%) of the estimated total number of viruses on earth (Geoghegan and Holmes, 2017). This means that the number of new viruses and virus families will increase immensely in the coming years. Moreover, the lack of closely related reference virus genomes makes public health risk assessment of novel viruses very complicated (Geoghegan and Holmes, 2017). Moreover, even if viruses belong to a known virus family with known human viruses, this does not necessarily mean that there is a risk of spill-over.

From Genotype to Phenotype: Deriving Meaningful Information From Genomic Data The pathogen genomic sequence alone will often give insufficient information on possible human health risks. Therefore, ideally, metagenomics or NGS approaches are supplemented with phenotypic data such as antigenic characteristics, pathogenicity, virulence, host-specificity and transmission characteristics. For novel variants of known viruses, some indications of phenotype or zoonotic risks can be derived from the sequence, based on experimental study outcomes. The impact of inferring phenotype traits from genotype data can be exemplified by the “H5N1 inventory” compiled by the United States Centers for Disease Control (“see Relevant Websites section”). This inventory summarizes all mutations that have been shown to affect virus replication, virulence, tissue or cellular tropism, host-range, transmission, antigenic properties, immune escape, and antiviral drug resistance. However, in the case of an emerging virus that was previously unknown, novel phenotypic data needs to be generated. This data is important for risk assessment, but information on potentially relevant sequences or epitopes can also be used for development of diagnostics and preventive antiviral strategies. For example, one of the key components influencing ability to spread for an emerging pathogen from a spill-over event is the level of measured or predicted immunity in the population, as this impacts on the likelihood of infection of individuals (humans, livestock, wildlife) and their ability to shed and spread the infection. In silico epitope prediction can predict B-cell epitopes based on sequence data (Potocnakova et al., 2016). The predicted immunogenic virus proteins can then be used to set up serological assays to determine the prevalence and host range of the virus. For this, multiplex antigen arrays can be used to not only measure antibodies to the newly identified antigen, but also to (distantly) related variants of the same virus family, thus providing information on potential cross-protection or disease enhancing antibodies (Mina et al., 2019; Cleton et al., 2017; Freidl et al., 2015). Possible resistance markers can be predicted when analyzing the sequences, for instance for drugs targeting the polymerase or protease genes. Host binding motifs can be predicted from their location on predicted protein structures. However, all such inferences need to be validated as the choice of in vitro systems and animal models can have a significant impact on their validity (Rothenburg and Brennan, 2020; Setoh et al., 2019; Reyes-Ruiz et al., 2019).

Conclusion The risk of emergence of novel human pathogens has increased in the past decades. Early detection is becoming even more important due to the potential rapid spread and impact considering the ever increasing size of human and animal populations and their movements all over the globe. The unpredictable nature of emergence of diseases makes surveillance and preparedness a huge challenge. However, recent history has shown that outbreaks of emerging diseases will happen again, and all sectors involved in infectious disease surveillance and response need to be ready to detect and handle the next disease X. It has been argued that the design of infectious disease surveillance and response should therefore move from crisis response to true integration and evaluation of a strategy to detect and control emerging diseases (Bedford et al., 2019). This involves a One Health approach, integrating not only the humans, animal and environmental health sectors, but also social sciences, bioinformatics and more. Technical developments in recent years, such as the use of Big Data and metagenomic sequencing will aid the rapid detection of novel pathogens on the human-animal interface.

Note Added in Proof: December 2020 SARS-CoV-2 In December 2019 several cases of pneumonia of unexplained etiology in patients in Wuhan city (China) linked to a seafood wholesale market were reported to the WHO (“see Relevant Websites section”). In the beginning of 2020 it became clear that this Disease X that had emerged was also a betacoronavirus, most closely related to SARS, that was already on the WHO Blueprint priority list in public health emergency contexts (“see Relevant Websites section”). In the following months the outbreak developed into a pandemic, with over 68 million human cases and 1,55 million deaths, as of 8 December 2020 (Dong et al., 2020).

One Health and Animal Reservoirs Bats are known to harbor a range of different coronaviruses (Cui et al., 2019). Indeed, closely related SARS-like viruses have previously been detected in horse shoe bats, in China (Zhou et al., 2020; Hu et al., 2018). However, although the SARS-CoV-2 genome is 96% identical to the closest bat sequence, the genetic distance between the closest horseshoe bat SARS-like coronavirus

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and SARS-COV-2 likely reflects several years and even decades of virus evolution (Boni et al., 2020). SARS-related coronaviruses are also found in pangolins, where specifically the RBD domain is closely related to SARS-CoV-2 (Boni et al., 2020). Therefore, the role of bats as SARS-CoV-2 reservoir remains to be proven and other mammalian species are considered as possible intermediate hosts, as was previously the case for SARS, MERS and Nipah (Epstein et al., 2006; Wang et al., 2005; Reusken et al., 2013). To date, an animal intermediate host of SARS-CoV-2 has not been found yet (“see Relevant Websites section”). However, multiple animal species are found to be susceptible for SARS-CoV-2. Ferrets, raccoon dogs, cats and hamsters show infection and virus replication after experimental infection as well as the ability to transmit the virus (Shi et al., 2020; Richard et al., 2020; Sia et al., 2020; Freuling et al., 2020). Other animals that showed infection after experimental inoculation include rabbits, dogs and tree shrews (Shi et al., 2020; Mykytyn et al., 2020; Bosco-Lauth et al., 2020). Additionally, animal infections have been detected in the field, mainly in dogs, cats and mink (Sit et al., 2020; Barrs et al., 2020). Although there is evidence for efficient transmission between cats, infection in these animals is not considered a major concern given the small numbers of animals in households. This is different for the recent outbreaks in mink farms in Europe and the US (Cahan, 2020), with the first described SARS-CoV-2 animal-to-human transmissions (Oude Munnink et al., 2020b). These outbreaks were the first large scale outbreaks in animal populations, and large scale mink passages have specifically sparked concern on the risk of specific mutations, affecting virus properties and possibly resulting in immune escape (Koopmans, 2020). The multiple susceptible animal species and the search for the original animal reservoir, that was at the origin of the SARS-CoV-2 pandemic, clearly shows the need for a One Health approach. Introductions of SARS-CoV-2 into susceptible animal populations can continue to occur, possible resulting in (additional) animal reservoirs and virus evolution and the risk of continuing evolution and spill backs into the human population (Koopmans, 2020; Olival et al., 2020). Therefore, collaboration between different disciplines remains crucial in monitoring, understanding and containing the SARS-CoV-2 pandemic as well as many other (emerging) pathogens.

Emerging Disease Detection, Full Genome Sequencing and Rapid Data Sharing The Chinese authorities officially announced the isolation of a novel coronavirus ( 2019-nCoV) as the causative agent on 7 January 2020 (“see Relevant Websites section”) and the full sequence was released in a publicly accessible discussion forum (“see Relevant Websites section”) 5 days later (“see Relevant Websites section”). Four other sequences were shared on 12 January in the Global Initiative on Sharing All Influenza Data (GISAID) database. A validated real-time RT-PCR assay was developed, validated and published online only 5 days later, on 17 January 2020, and published for PCR validation in a peer-reviewed journal on the 23rd of January (“see Relevant Websites section”) (Corman et al., 2020). Validation panels were available on the European Virus Archive (“see Relevant Websites section”) shortly after. The first validated serological assay followed early April (Okba et al., 2020). This clearly illustrates the technical progress that has been made in the last 17 years, as well as the value of virus sequencing and rapid data and sample sharing. Samples of the first patients in Wuhan city were sequenced using an unbiased deep meta-transcriptomic sequencing method (Wu et al., 2020). When the sequence was known, amplicon-based methods were developed to sequence the new virus in a timely and sensitive manner ((Oude Munnink et al., 2020a) “see Relevant Websites section”) Whole genome sequencing was performed at unprecedented scale, with almost 250.000 SARS-CoV-2 whole genome sequences that had been shared on the GISAID platform as of 8 December 2020. The timely release of unpublished genomic information through GISAID was essential for fast track development of diagnostics, vaccines and therapeutics as well as the application of WGS in outbreak investigation and public health control measures (Oude Munnink et al., 2020a; Voeten et al., 2020). With the ever-increasing numbers of sequences that are being generated, the need for rapid characterization of viruses with specific mutations remains high. A high number of papers are published describing the evolution and mutations in the SARS-CoV2 genome, but assessing the relevance and effect on virus properties, and, consequently, public health, is still challenging (Grubaugh et al., 2020).

Population Surveys and Sero-epidemiological Studies Serologic studies can provide important information for understanding the extent of past transmission, the current state of the epidemic, and future transmission in an affected population. Moreover, serology is a very useful tool on the human-animal interface (Wernike et al., 2020). It can be used to screen potential animal reservoirs, as has been used to determine the likely reservoir species for MERSCoV, (Reusken et al., 2013; Deng et al., 2020). Serological surveys in several animal species did not point towards the SARS-CoV-2 animal reservoir species, but did show significant exposure of dogs and cats in Wuhan and Italy, which were severely affected by SARS-CoV-2 (Zhang et al., 2020; Patterson et al., 2020). Also, serological assays can be an additional tool to gain insight in the potential and scale of animal-to-human transmission, when combined with epidemiological information on exposure, as was done for mink farm employees on SARS-CoV-2 on infected mink farms (Oude Munnink et al., 2020b; Sikkema et al., 2016). However, it is important to realize that coronaviruses including betacoronaviruses are common in many animal species, and can result in cross reactivity and false positives (Franzo et al., 2020; Nemoto et al., 2019; Erles and Brownlie, 2008). In addition to patient diagnostics and surveillance efforts in symptomatic and asymptomatic individuals, wastewater sampling can be used to monitor the circulation of SARS-CoV-2 in the population (Medema et al., 2020; Hart and Halden, 2020). This approach is currently integrated in the national COVID-19 surveillance in multiple counties (“see Relevant Websites section”).

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Concluding Remarks With the emergence and worldwide spread of a novel coronavirus, the disease X scenario that many scientists had warned about came true. The SARS-CoV-2 pandemic lead to drastic government measures worldwide, with massive economic and social consequences. The spread of the virus has proven to be difficult to contain, despite extensive lockdowns, information campaigns, travel bans and compulsory mouth masks. However, previous investments in surveillance, diagnostics, novel laboratory techniques, open data sharing and vaccine platforms did pay off. A cluster of pneumonia of unknown origin was detected in December 2019 in Wuhan, and at the end of January 2020, a set of whole genomes are publicly shared and a validated PCR assay has been set up which was implemented in 24 of 30 EU/EEA countries and many more worldwide (Reusken et al., 2020). Moreover, in December 2020, the first COVID-19 vaccine was licensed (“see Relevant Websites section”). The speed of these key developments in the surveillance and control of the new virus is unprecedented. How the SARS-CoV-2 pandemic will evolve remains to be seen, but recent developments reinforce the call for preparedness research and a One Health approach, as highlighted in the current article.

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Relevant Websites https://reliefweb.int/report/world/world-risk-annual-report-global-preparedness-health-emergencies-global-preparedness A World at Risk: Annual report on global preparedness for health emergencies. Global Preparedness Monitoring Board [EN/AR/RU/ZH]. https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2014.3884 An update on the risk of transmission of Ebola virus (EBOV) via the food chain. https://apps.who.int/iris/bitstream/handle/10665/252646/WHO-OHE-PED-2016.2-eng.pdf Anticipating Emerging Infectious Disease Epidemic. World Health Organization. https://wwwnc.cdc.gov/eid/page/background-goals Background and Goals. CDC. https://isaric.tghn.org/protocols CCP UK & Annual Activation. ISARIC. https://www.who.int/blueprint/priority-diseases/en/ Coronavirus disease (COVID-19) pandemic. http://www.fao.org/3/a-i2198e.pdf Developing sustainable value chains for small-scale livestock producers. https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2 Diagnostic detection of 2019-nCoV by real-time RT-PCR. https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape/ COVID-19 vaccine tracker. http://www.fao.org/avianflu/documents/Economic-and-social-impacts-of-avian-influenza-Geneva.pdf Economic and social impacts of avian influenza. https://www.ecdc.europa.eu/en/about-us/partnerships-and-networks/disease-and-laboratory-networks/evd-labnet Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet). https://life.eurobirdportal.org EuroBirdPortal. LIFE EuroBirdPortal overview. https://EVAg:eur-opean-virus-archive.com European Virus Archive - GLOBAL. https://reliefweb.int/report/world/exploring-machine-learning-map-yellow-fever-risk Exploring Machine Learning to Map Yellow Fever Risk. https://consise.tghn.org Home. CONSISE. https://www.who.int/news-room/feature-stories/detail/how-who-is-working-to-track-down-the-animal-reservoir-of-the-sars-cov-2-virus How WHO is working to track down the animal reservoir of the SARS-CoV-2 virus. http://www.cdc.gov/flu/pdf/avianflu/h5n1-inventory.pdf H5N1 Genetic Changes Inventory. FluTrackers. https://www.who.int/ihr International health regulations. World Health Organization. www.movebank.org Movebank.org. https://doi.org/10.17504/protocols.io.bdp7i5rn nCoV-2019 sequencing protocol v2 (GunIt) V.2. https://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/ Novel Coronavirus China. https://virological.org/t/novel-2019-coronavirus-genome/319 Novel 2019 coronavirus genome. https://www.who.int/activities/prioritizing-diseases-for-research-and-development-in-emergency-context Prioritizing diseases for research and development in emergency contexts. https://www.who.int/csr/don/05-january-2020-pneumonia-of-unkown-cause-china/en/ Pneumonia of unknown cause China. https://www.glopid-r.org/our-work/social-science-research/ Social Science Research. GloPID-R. https://www.who.int/news-room/commentaries/detail/status-of-environmental-surveillance-for-sars-cov-2-virus Status of environmental surveillance for SARS-CoV-2 virus. https://www.who.int/foodsafety/zoonoses/final_concept_note_Hanoi.pdf?ua=1 The FAO-OIE-WHO. http://www.fao.org/3/i0680e/i0680e02.pdf 2. Change in the livestock sector.

Use of Immunoglobulins in the Prevention of Viral Infections Leyla Asadi and Giovanni Ferrara, University of Alberta, Edmonton, AB, Canada r 2021 Elsevier Ltd. All rights reserved.

Introduction In the late 18th century, Emil von Behring and Shibasaburo Kitasato published their ground-breaking work showing that non-immune rabbits could be protected from developing tetanus if they were injected with sera obtained from rabbits infected with Clostridium tetani. This was followed up one week later by another foundational work where Behring used inactivated Corneybacterium diptheriae toxin to prevent diseases in guinea pigs (Kaufmann, 2017). At the same time, the studies of Paul Erlich contributed to understanding the foundations of humoral immunity and allowed the safe production of large volumes of high-quality anti-diphtheria serum from large animals (Graham and Ambrosino, 2015). These combined efforts led to dramatic success in reducing tetanus and diphtheria mortality and were followed by the development of similar therapies for a variety of other bacterial infections, including Neisseria meningitidis, Haemophilus influenzae, and Streptococcus sp. (Casadevall et al., 2004). This pre-antibiotic and preantiviral era was a golden age for “serum therapy” (Casadevall et al., 2004). The first reported use of serum therapy for viruses appears to have been in 1907 when it was used to prevent roseola (Good and Lorenz, 1991). It was also used during the 1918 Spanish influenza pandemic. Indeed, a modern meta-analysis of studies published during the pandemic estimates that there may have been B40% reduction in mortality in those who received Spanish influenzaconvalescent human blood products early in the course of their disease (Luke et al., 2006). Because early immunoglobulin products were often contaminated with animal proteins, serious adverse effects such as serum sickness and anaphylaxis remained a real and constant risk. By the 1930s, it was clear that immunoglobulin therapy could be safer and more effective if antibodies were isolated from pools of normal human sera. Furthermore, new methods of isolating and precipitating human immunoglobulin fractions from placental tissue extracts became available (Good and Lorenz, 1991). Despite these advancements, and also due to the cost and complexity of production, serum therapy for bacteria was supplanted by antibiotic therapy with sulfonamides, then beta-lactams. After World War II, Dr. Edwin Cohn developed a technique that allowed for the purification of antibodies via ethanol fractionation of plasma (Graham and Ambrosino, 2015). His technique – which was further refined by J.L. Oncley (Barahona Afonso and Joao, 2016) – allowed the yield of immunoglobulin for intravenous and subcutaneous use. Since most viral infections remained without treatment, the focus shifted to the use of immunoglobulins for viral pathogens. From there, polyclonal immunoglobulins began to be used for measles, rabies, hepatitis A and B, varicella-zoster virus, and respiratory syncytial virus (RSV). Cohn and Oncley’s techniques (with some additional steps) and the use of polyclonal immunoglobulins for these viruses remain in practice today (Barahona Afonso and Joao, 2016). Since then, several variants of immunoglobulin products have been produced and investigated, in pace with the development of new molecular biology techniques allowing a higher grade of purity and specificity for different uses of the antibodies prepared for the immunoglobulin cocktails. Polyclonal antibodies in the form of intravenous immunoglobulin or hyperimmune immunoglobulin remain the most commonly used form of immunoglobulins in viral infections. Hyperimmune globulins contain a higher proportion of immunoglobulins targeting a specific pathogen. They are produced either by identifying individuals with high titers of the antibody of interest or by vaccination of donors. Given the increasing ease in identifying and manufacturing monoclonal antibodies, they have been the focus of intense research, particularly for the treatment of emerging infectious diseases such as viral hemorrhagic fevers. The hybridoma technique developed in the 1970s was a major advancement that allowed the in-vitro production of monoclonal antibodies. In this method, mice were exposed and immunized with antigens of interest, and the antigen-specific B-cells were harvested from the mice spleens: These cells were then immortalized by fusing them with immortal B-cell cancer cells, allowing expansion and production of the same monoclonal antibody (Marston et al., 2018; Salazar et al., 2017). However, newer and more direct methods of monoclonal antibody production are now available. For instance, if the antigen is known, it can be used to screen a phage display antibody library (Salazar et al., 2017) – with antibodies derived from naturally infected or immunized persons. Alternatively, memory B cells – again from infected or immunized persons – can be sorted by flow cytometry, based on the affinity of their B-cell receptor for defined antigens (Salazar et al., 2017). The identified antibodies can then be cloned. Alternatively, deep sequencing of the B-cell IgG repertoire enables the production of heavy and light chain paired mAbs (McDaniel et al., 2016). Once the antibody has been identified, further techniques are available to stabilize the final product and reduce the occurrence of side effects (e.g., the Fc receptor can be modified to prevent undesirable interactions while maintaining the neutralizing functions of the heavy and light variable chains) (Sapparapu et al., 2016). The possibility to develop monoclonal antibodies targeted to neutralizing epitopes of viral and bacterial molecules enables the production of larger amounts of immunoglobulin without the need to obtain blood products from thousands of donors. It also enables precision medicine in the field, increasing the efficacy and safety of these products that would become “off the shelf” drugs with precise indications. Unfortunately, apart from the few indications already approved (that are reported at the end of the article), this reality is still far for most infectious diseases. Major limitations of this approach include the limited knowledge of the

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humoral response against specific infectious agents (i.e., how to identify the right epitopes and their combination to achieve humoral protection) and the existing high cost of molecular techniques. In this review, we will discuss disease-specific uses of polyclonal and monoclonal antibodies in the prevention of infection.

Polyclonal Immunoglobulins Used in the Prevention of Infectious Diseases Hepatitis A The hepatitis A virus (HAV) typically results in asymptomatic disease or a self-limited illness. It rarely causes fulminant hepatitis, chronic complications, or death; however, those with pre-existing chronic liver disease and those who are immunocompromised are at an increased risk for such complications. Transmission of hepatitis A occurs through the fecal-oral route, either via person-toperson contact or consumption of contaminated food or water (Bennett et al., 2014). Passive immunization against hepatitis began in the 1940s. After seeing the effectiveness of measles passive immunization, Dr. Joseph Stokes administered gamma globulin to children at a camp with an “icteric hepatitis” outbreak. He found that there was a 39% absolute reduction (90% relative reduction) in the development of hepatitis among gamma globulin recipients (Cuthbert, 2001). More recently, a 2009 Cochrane systematic review explored the efficacy of immunoglobulins for pre-exposure and postexposure prophylaxis. For pre-exposure prophylaxis, they found that when compared with placebo, immunoglobulins reduced the risk of hepatitis A infection by 47%–72% (Liu et al., 2009). No trials compared the clinical efficacy of immunoglobulin versus inactivated HAV vaccine; however, both resulted in similar HAV antibody development at four weeks. By 24 weeks, no one in the immunoglobulin group was still seropositive. Only one study looked at giving both agents; there was no benefit to giving both (albeit the study was of poor quality). Concerning post-exposure prophylaxis, they identified two trials. The more recent trial, completed in 2007, compared immunoglobulin to inactivated hepatitis A vaccine by randomizing 1,090 susceptible contacts of HAV cases to inactivated HAV vaccine or immune globulin within two weeks of exposure. There was no difference in disease incidence between the two groups (4.4% versus 3.3%, respectively) (Victor et al., 2007). Separately, studies have shown that the inactivated HAV vaccine is safe and, in healthy adults o40 years of age, confers 95% seropositivity for at least 25 years (Bovier et al., 2010; Hens et al., 2014). However, based on limited data, there may be reduced immunogenicity in older adults, particularly with early seroprotection (within 15 days of vaccination) (Link-Gelles et al., 2018). The vaccine has also not been licensed for use in children o1 year of age and could theoretically interfere with passively acquired maternal antibodies. Nevertheless, due to the high immunogenicity of the HAV vaccine, its ability to induce active immunity, longer duration of protection, ease of administration, and greater acceptability and availability, the vaccine became the preferred method of HAV prevention. Immunoglobulin was primarily reserved for those o12 months of age, >40 years old, or those who are immunocompromised. For the first time, in 2018, the Advisory Committee on Immunization Practice (ACIP) revised their recommendations (Nelson et al., 2018). In contrast to previous recommendations, the vaccine is now preferred for those >40 years of age, and immunoglobulin is only added on a case-to-case basis. They justify this recommendation by pointing at the decreased potency of immunoglobulin, due to reduced HAV antibody titers in the general population from which the immunoglobulin is obtained. This has resulted in a higher volume of immunoglobulin being required for the prevention of HAV infection, making it even less easily available or acceptable. They also now recommend vaccine for pre-exposure prophylaxis in infants traveling internationally between six and 11 months of age (off-label use). This is because the measles, mumps, and rubella (MMR) vaccine and HAV immunoglobulin cannot be given simultaneously. Thus, clinicians should prioritize the prevention of measles, mumps, and rubella by vaccinating with the MMR vaccine and use an inactivated HAV vaccine, as opposed to the immunoglobulin, to try to achieve hepatitis A protection. The remainder of the recommendations are unchanged and described in Table 1, modified from the 2018 MMWR report on HAV (Nelson et al., 2018).

Measles The measles virus is a highly contagious virus that produces a characteristic exanthem after a prodromal phase of conjunctivitis, cough, coryza, and fever. Serious complications include pneumonia, post-infectious encephalitis (one in 1000–2000 cases), subacute sclerosing panencephalitis (SSPE) (one in 100,000 cases), and death (B1–3 in 1000 cases) (Bennett et al., 2014). In developing countries, pediatric mortality rates approach 2%–15%. Pregnant women and those with immunodeficiency are at higher risk of complications (Matysiak-Klose et al., 2018). Before the arrival of the measles vaccine in 1963, major epidemics causing millions of deaths were not uncommon (World Health Organization, 2019a). With the introduction of a highly effective vaccine, global measles rates have plummeted and measles was considered eliminated in most developed countries (World Health Organization, 2019a). One dose of the measles, mumps, rubella (MMR) vaccine provides B93% protection (CDC, 2020). With two doses, effectiveness reaches B97% (CDC, 2020). Measles vaccination is the only approach for pre-exposure prophylaxis and the mainstay of post-exposure prophylaxis (PEP) in immunocompetent individuals >6 months (Plotkin, 2012). The vaccine is approved for those >6 months of age and, because it is a live attenuated virus, its administration is contraindicated for those pregnant, with severe allergic reactions to

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Table 1

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Post-exposure and pre-exposure prophylaxis for hepatitis A virus for healthy individuals

Indication/age group

Hepatitis A vaccine

Immune globulin

o12 months, healthy, post-exposure o 12 months, healthy, pre-exposure
o6 months
6–11 months 12 months – 40 years, healthy, post-exposure and pre-exposure 440 years, healthy, post-exposure and pre-exposure Z12 mos, immunocompromised or chronic liver disease, post-exposure Z12 mos, vaccine contraindicated, post-exposure All ages, immunocompromised or chronic liver disease, pre-exposure 46 months, vaccine contraindicated, pre-exposure

No

0.1 ml/kg

No 1 doseb 1 dosec 1 dosec 1 dose No 1 dose No

0.1–0.2 ml/kga None None 0.1 ml/kgd 0.1 ml/kg 0.1 ml/kg 0.1–02 ml/kg,d,a 0.1–02 ml/kga

a

dose depends on the duration of travel. dose does not count toward routine 2-dose series, which should be started after one year. c only one dose necessary for post-exposure prophylaxis, but two doses for long-term immunity. d risk assessment (severity of exposure and potential for complications) undertaken to determine the need for immune globulin. Note: Modified from Nelson, N.P., et al., 2018. Update: Recommendations of the advisory committee on immunization practices for use of hepatitis A vaccine for postexposure prophylaxis and for preexposure prophylaxis for international travel. Morbidity and Mortality Weekly Report 67 (43), 1216–1220. b

any component of the vaccine, and who are immunocompromised. Thus, immunoglobulins continue to play a role in PEP, particularly for those most vulnerable to complications of the disease. The MMR vaccine, a live vaccine, is not administered in conjunction with immunoglobulins as it results in the negation of each component. In 1926, Zingher showed that convalescent whole blood, serum, or plasma could prevent measles, and in 1940, Janeway confirmed that early immunoglobulin administration could prevent measles in three out of every four people and attenuate disease in the fourth (Plotkin, 2012). Recently, a 2014 Cochrane systematic review examined the effectiveness of post-exposure immunoglobulins (Young et al., 2014). Looking back to the 1920s, they identified 13 studies (3,925 patients), with only one being a randomized controlled trial. 11 studies predated widespread vaccine implementation (so high measles-specific titers in the immunoglobulin preparations would be expected). The studies were heterogeneous and of moderate quality. Nevertheless, the authors concluded that immunoglobulins within seven days of exposure reduces the risk of measles by up to 83% compared to no intervention. Due to high rates of immunity resulting from vaccination, modern PEP studies are challenging; however, since the publication of the Cochrane review, there are at least two new observational studies. A study of 121 contacts who received PEP showed that MMR vaccine effectiveness was at 83.4% (95% CI, 34.4%–95.9%) and immunoglobulin (dosed at 0.5 ml/kg) effectiveness was at 100% (approximately 95% CI, 56.2%–99.8%) (Arciuolo et al., 2017). However, most immunoglobulin recipients were o6 months of age and so they may have had maternal antibodies that provided protection. A 2017 Canadian study of 55 self-reported susceptible individuals found that immunoglobulin effectiveness was B69%; however, the dose administered in this study was only 0.25 ml/kg (Bigham et al., 2017). Both studies excluded pregnant women or immunocompromised patients. Based on these studies, there is consensus that immunoglobulin is beneficial and should be given as quickly as possible (ideally, within six days) if there is a contraindication to the MMR vaccine (Matysiak-Klose et al., 2018). If intramuscular immunoglobulin is available, the dosage should be 0.5 ml/kg; otherwise, IVIg (400 mg/kg) is the agent of choice (Matysiak-Klose et al., 2018; Tunis et al., 2018). Given that individuals weighing 30 kg or more would not have complete protection with IM immunoglobulin at a dosage of 0.5 ml/kg and would therefore require multiple injections, IVIg is the preferred product for most immunocompromised individuals (Tunis et al., 2018; Public Health England, 2020). Given the reduced efficacy of the MMR vaccine when administered as PEP 72 h post-exposure, some jurisdictions (i.e., United States) recommended immunoglobulin for all susceptible individuals (including non-pregnant and immunocompetent hosts) if they present between 72 h and six days (McLean et al., 2013). However, others (i.e., Canada) no longer recommended the use of immunoglobulin for immunocompetent individuals >12 months of age because the measles virus antibodies in immunoglobulin products may no longer be optimally protective (due to waning anti-measles titers in the general population) and because there is a low risk of disease complications in this group (Tunis et al., 2018).

Hyperimmune Immunoglobulins Used in the Prevention of Infectious Diseases Hepatitis B Hyperimmune Globulin Depending on the age of acquisition, hepatitis B virus (HBV) infection may result in acute or chronic hepatitis. Infection in newborns can become chronic in 90% of cases. Chronic disease, in turn, may develop into cirrhosis or hepatocellular carcinoma, resulting in death. Perinatal transmission remains the main mode of HBV transmission but in adults, acquisition primarily occurs via intravenous drug use, sexual activity, or occupational exposure (Bennett et al., 2014). The HBV vaccine – a recombinant vaccine containing yeast-derived hepatitis B surface antigen (HBsAg) – is the cornerstone of HBV prevention (Schillie et al., 2018). The only contraindication is anaphylaxis with having previously received HBV vaccine. The

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seroconversion rate is B95%–100% in children and B90% in adults, with lower response rates in higher ages (Henry and Baclic, 2017). The vaccine’s effect is blunted in the immunocompromised or those with underlying liver disease (Walayat et al., 2015) and B5% of individuals are vaccine non-responders. Hepatitis B hyperimmune IG (HBIG) is administered at a standard dose of 0.06 ml/kg and is used in conjunction with the vaccine in post-exposure settings, in liver transplants and as prevention for maternal-to-child-transmission (MTCT). For known non-responders, HBIG is the only method of protection after an HBV exposure (Schillie et al., 2018). Among susceptible individuals with a high-risk HBV exposure in occupational or non-occupational settings, one dose of HBIG is recommended as soon as possible and up to seven days post-exposure. HBIG provides protection until active immunity from vaccination develops. The evidence for this is from lower quality randomized trials from the 1970s or early 1980s where it appeared that among those given HBIG within seven days of percutaneous exposure to HBsAg positive blood, B75% developed protective antibodies (Schillie et al., 2018). No studies have compared the efficacy of HBIG and the HBV vaccine series or the combination of these two agents in postexposure settings. In addition to immunization of infants and treatment of highly viremic mothers with antiviral therapy, HBIG is also given to prevent vertical transmission. HBV vaccine is 75% effective and HBIG is 71% effective in preventing HBV transmission; when combined, their efficacy reaches 94% (Lee et al., 2006; Schillie et al., 2015; Beasley et al., 1983). Therefore, in cases where the mother is HBsAg positive (and anti-HBe negative) or her hepatitis B status is unknown, vaccine and HBIG are to be administered within 12 h of birth. The addition of HBIG to the vaccine has an incremental cost-effectiveness ratio of $6957 per quality-adjusted life-year (Fan et al., 2016). Despite decreased efficacy after 48 h, HBIG may be given up to seven days post-birth (Henry and Baclic, 2017). A Cochrane review of B30 randomized control trials from China looking at the use of HBIG during pregnancy (antenatal) to prevent MTCT was inconclusive due to very low to low-quality evidence (Eke et al., 2017). A subset of HBsAg-positive liver transplant recipients also require HBIG. As per the 2018 American Association for the Study of Liver Diseases (AASLD) Hepatitis B guidance, in HBsAg-positive liver transplant recipients at low risk of recurrence, “either no HBIG or HBIG for only 5–7 days combined with antivirals long term has been highly effective” (Terrault et al., 2018). However, HBsAg-positive liver transplant recipients who are HIV- or hepatitis delta virus (HDV)-positive, have antiviral drug resistance, or high levels of HBV DNA at the time of transplantation may benefit from indefinite HBIG as they are at higher risk of virological breakthrough and have more limited rescue therapy options (Terrault et al., 2018).

Cytomegalovirus Hyperimmune Immunoglobulin In immunocompetent individuals, cytomegalovirus (CMV) infection is typically asymptomatic. However, in transplant recipients, CMV can cause both direct disease (a viral syndrome or tissue invasive disease) or may indirectly increase the risk of rejection (Bennett et al., 2014). CMV also results in congenital infection and is a leading cause of sensorineural hearing loss and neurological disability (RCOG Scientific Impact Paper, 2018). In the late 1980s, CMV hyperimmune immunoglobulin (CMVIG) was licensed for use in renal transplant recipients to prevent primary CMV disease (Snydman et al., 1987, 1993). In a small randomized trial of renal transplant recipients (Snydman et al., 1987) and then, subsequently, in a small, double-blind, randomized controlled trial (Snydman et al., 1993) involving liver transplant recipients – both against a placebo arm – CMVIG was beneficial in reducing primary CMV disease and severity. However, with the advent of effective antivirals, CMV immunoglobulin use has decreased and there is limited evidence about its efficacy in combination with antivirals or with more modern immunosuppression. While there may be a role for combination therapy in high-risk lung or small bowel recipients (where the donor is CMV-positive and the recipient is CMV-negative) (Florescu et al., 2014; Valantine et al., 2001), the randomized trials supporting combination therapy have not been adequately powered. The latest consensus guidelines on the management of CMV disease in solid-organ transplantation no longer recommends routine use of CMVIG (Kotton et al., 2018). That said, CMVIG has been used in patients who are intolerant of antivirals (i.e., due to prolonged neutropenia) (Kotton et al., 2018) and there are case reports of its use as an adjunct in the treatment of tissue-invasive CMV disease (Schulz et al., 2016). Like its use in transplant recipients, the use of CMVIG in pregnancy has also waned. In the early 2000s, a series of nonrandomized studies studying CMV-specific hyperimmune globulin in pregnant women with primary CMV infection found a decreased rate of mother-to-fetus transmission as well as a lower risk of congenital disease (Nigro et al., 2005, 2012; Visentin et al., 2012). However, in 2014, a placebo-controlled randomized trial of 124 pregnant women did not demonstrate a significant benefit (Revello et al., 2014). In the Congenital Human CMV Infection Prevention (CHIP) trial, not only was there no difference in congenital infections, immunological, or virological parameters, but there was even a trend toward more adverse obstetric events in the immunoglobulin group. While it could be argued that this RCT was underpowered, CMV immunoglobulin is no longer routinely recommended in pregnancy (RCOG Scientific Impact Paper, 2018; Yinon et al., 2018). A large, placebo-controlled, double-blinded, randomized trial of CMV immunoglobulin in pregnancy is currently underway (The George Washington University Biostatistics Center, 2020). Finally, despite the waning use of CMVIG, studies are underway examining CMV monoclonal antibodies. A phase two doubleblinded, placebo-controlled, randomized trial of RG7667, a combination Monoclonal Antibody in high-risk renal transplant recipients showed a reduction of CMV DNAemia and disease (Ishida et al., 2017). A phase II clinical trial of CSJ148, another combination monoclonal antibody, was recently completed among hematopoietic stem cell recipients, but it did not achieve the primary efficacy endpoint (Maertens et al., 2020).

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Rabies Hyperimmune Immunoglobulin After the appearance of clinical signs, rabies, a zoonotic encephalitis caused by a variety of different species of viruses in the Rhabdoviridae family, is almost universally fatal. Because 99% of human cases are caused by dog bites (WHO/Department of Control of Neglected Tropical Diseases, 2018), the primary method of rabies control has been through mass vaccination of dogs. Pre-exposure prophylaxis, with the highly effective rabies vaccine, is recommended only for those at occupational risk or those living in remote endemic settings (WHO/Department of Control of Neglected Tropical Diseases, 2018). Once rabies exposure occurs, post-exposure prophylaxis (PEP) is essential to prevent fatality. PEP consists of thorough wound cleaning, rabies vaccine, and rabies hyperimmune IG (RIG) (WHO/Department of Control of Neglected Tropical Diseases, 2018). RIG is only given to those who have never been immunized. The WHO categorizes rabies exposures into three categories of severity and recommends RIG for severe category three cases. Some national guidelines use RIG in less severe exposure incidents (Human rabies prevention-United States, 2008). Because of the prolonged incubation (at least 45 days) and the latency period of the virus, PEP administration is only too late after the occurrence of clinical signs of rabies. When RIG is warranted, either human RIG (hHRIG), which is from pooled plasma of hyperimmunized donors, equine RIG (eRIG), or the rabies' monoclonal antibody (currently only approved in India) may be given. These products provide immediate virus-neutralizing antibodies and may partially suppress antibody production; therefore, only one dose is recommended and it should be given no more than seven days after the vaccine series is started (Khawplod et al., 1996). Ideally, the full dose of RIG should be infiltrated into the bite wound and surrounding area. The evidence for the addition of RIG to the vaccine series dates to a small study from the 1950s. In 1954, at the Pasteur Institute in Tehran, Iran, 29 rabid wolf bite victims were randomized to receive vaccine-only PEP or vaccine and anti-rabies serum. Among those with severe wounds, 3/5 in the vaccine group and 1/13 in the vaccine and serum group died. From there, RIG with vaccine became the standard of care (Sparrow et al., 2019; Baltazard and Bahmanyar, 1955; Habel and Koprowski, 1955). A more recent study has corroborated these findings by showing significantly higher rabies antibodies at the end of the vaccine series in those who received rabies immunoglobulin (Navarrete-Navarro et al., 1999). Most rabies deaths occur in poor rural populations in Africa and Asia where rabies PEP is not affordable for the population; only 1%–10% of patients requiring RIG receive it (Sparrow et al., 2019). Furthermore, blood-derived products have a short shelflife and there are ethical issues around the use of animals in producing immunoglobulins. Given these issues, there have been global shortages of both hRIG and eRIG, and this has led to the interest and development of rabies monoclonal antibodies. The World Health Organization now recommends the use of monoclonal rabies immunoglobulin (where available) and is partnering with collaborating centers for rabies to facilitate rabies mAb cocktails (World Health Organization (WHO), 2017, 2016). There are currently multiple rabies mAbs in various stages of development (Sparrow et al., 2019). In 2016, Rabishield (SII RMAb), an IgG1 mAb was approved for use in India. In 2/3 phases of randomized controlled trial carried out in India, 200 patients with category three bites from presumed rabid animals were randomized to either vaccine and HRIG or vaccine and RMAb. SII RMab was safe and demonstrated non-inferiority in the level of rabies virus neutralizing activity (Gogtay et al., 2018).

Vaccinia Hyperimmune Globulin Vaccinia immune globulin (VIG) is approved for the treatment of complications associated with the smallpox vaccine. It may also be of some benefit for post-exposure prophylaxis of smallpox. Smallpox, caused by the variola virus, was a highly infectious condition characterized by rash and fever and complicated by a myriad of systemic manifestations, including keratitis, encephalitis, and death. In the late 1960s, using the smallpox vaccine, a global mass vaccination campaign was initiated (Meyer et al., 2020). By 1980, the disease was eradicated globally (World Health Organization, 1979). Despite the eradication of wild type smallpox, two variola virus repositories were maintained at official centers in the United States and Russia for research purposes. Some experts also contend that other countries or groups may also possess or aim to obtain virus samples for bioterrorist aims (Wittek, 2006). The smallpox vaccine is composed of a live attenuated vaccinia virus. The vaccine itself may rarely result in serious complications (Cono et al., 2003). Though historical rates of vaccine adverse effects were higher, after a round of smallpox vaccination of US service members following the September 11, 2001 terrorist attacks, only two serious complications occurred out of B770,000 individuals (Poland et al., 2005). At this time, the only available immunoglobulin formulation for smallpox is an FDA-approved intravenous formulation, produced in Canada and available through the CDC. VIG is given at a dose of 2 ml/kg (100 mg/kg). Because vaccine-related complications are rare, the efficacy of VIG is difficult to study. The largest review on VIG efficacy is a 2004 review of 16 non-controlled studies (Hopkins and Lane, 2004). The studies included in the review were published between 1948 and 2003. These studies suggest that there is potential morbidity and mortality benefit with the use of VIG for progressive vaccinia (vaccinia necrosum) and eczema vaccinatum and severe generalized vaccinia. There is no benefit with post-vaccination encephalitis and VIG is contraindicated in vaccina keratitis. The concern about keratitis is based in part on historical experimental data in rabbits showing that daily administration of VIG in vaccinial keratitis resulted in larger eye lesions with more persistent scarring, possibly due to immune complex disease (Fulginiti et al., 1965). If the smallpox vaccine is given to people with contraindications to the vaccine (i.e., people with eczema, infants, pregnant women, or the immunosuppressed), prophylactic VIG may be of some benefit. However, its use is not approved for prophylaxis (Wittek, 2006).

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Some have explored the use of VIG for post-exposure prophylaxis against smallpox (Hopkins and Lane, 2004). These studies did not meet modern criteria for randomized trials but did point to some benefits. Therefore, given its safety profile, VIG could be considered for individuals at high risk of smallpox complications.

Varicella Hyperimmune Immunoglobulin Varicella-zoster virus causes varicella (chickenpox) and herpes zoster (shingles). Varicella is a self-limited illness characterized by fever and vesicular rash. Rarely, encephalitis, pneumonia, and death may occur. Pregnant women, neonates, and immunocompromised hosts are far more likely to suffer severe manifestations and multi-organ involvement. In neonates whose mothers developed disease five days before or 48 h post-partum, mortality can be as high as 30% (Bennett et al., 2014). Congenital varicella, though rare, is associated with eye and skin abnormalities, hypoplastic extremities, and CNS impairment (Enders et al., 1994). The primary method of pre-exposure and post-exposure varicella prevention is vaccination. In the post-exposure setting, among immunocompetent children, the varicella vaccine is 86% effective against all forms and 100% effective against moderate-to-severe disease (Izurieta et al., 1997). However, because it is a live-attenuated vaccine, it is contraindicated in precisely those at the highest risk of severe disease – neonates, pregnant women, and the severely immunocompromised. It is in this vulnerable group where passive immunization may be warranted. Passive immunization against varicella was attempted as early as the 1940s (Funkhouser, 1948; Schaeffer and Toomey, 1948). In 1962, it was shown that non-specific human serum immunoglobulin limited varicella severity but did not prevent disease (Ross et al., 1962). Then, in a small study with 12 participants, Zoster IG (ZIG), an immunoglobulin formulation produced from convalescent plasma of herpes zoster – appeared to prevent disease transmission (Brunell et al., 1969). However, ZIG was difficult to obtain and was in short supply, and in the early 1980s, varicella-zoster IG (VZIG) – an immunoglobulin prepared from normal donor plasma selected for high titer of antibody to varicella-zoster virus (VZV) – came to market to meet supply shortages. In a double-blind randomized trial, outcomes with ZIG were comparable with outcomes with VZIG (Zaia et al., 1983). The latest preparation of immunoglobulin, VariZig, is equivalent to VZIG in efficacy and safety (Koren et al., 2002). No randomized trials have compared immunoglobulin to placebo. However, observational studies support a change in disease presentation and occurrence. For instance, an observational study from Italy showed that the risk of developing varicella was lower in VZIG recipients than those who did not receive it [42% (21 of 50) versus 72% (13 of 18); p ¼ 0.0263] (Trotta et al., 2018). Though methodologically suboptimal, comparison with historical controls (where IG was not administered) also suggests that IG administration alters the incidence of disease, particularly severe disease (Levin et al., 1986). There are no published data regarding the efficacy of VariZig in immunocompromised hosts nor are there trials comparing immunoglobulin prophylaxis with antiviral prophylaxis (either alone or in combination). However, according to the most recent iteration of the UK varicella PEP guidelines, the administration of VZIG to children with hematologic or oncology diagnoses is variable. This has allowed them to conduct an observational study comparing VZIG and acyclovir. Interim analysis of 73 recruited patients has shown acyclovir is at least equivalent to VZIG in preventing varicella (Public Health England, VZIG Expert Working Group, 2019). National guidelines on PEP varicella immunoglobulin vary. In the UK, where VZIG is in short supply, immunoglobulin is recommended for neonates and women at o20 weeks gestation. Antivirals are preferred after 20 weeks gestational age and for the immunocompromised (Public Health England, VZIG Expert Working Group, 2019). On the other hand, Canadian and US guidelines recommend the use of VariZig after exposure to varicella-zoster virus (varicella or herpes zoster) for neonates, all pregnant women, at-risk premature infants, and immunocompromised children and adults who lack evidence of immunity (National Advisory Committee on Immunization, NACI, 2016; Bialek et al., 2013). VariZig administration is approved for up to ten days after exposure (Bialek et al., 2013). There is limited but increasing evidence of its efficacy beyond 96 h (Levin et al., 2019; Swamy and Dotters-Katz, 2019). The duration of protection is unknown but expected to be at least three weeks. VariZig might extend the incubation period of the virus from 10–21 days to Z28 days.

Monoclonal Antibodies Approved by the Food and Drug Administration Respiratory Syncytial Virus Respiratory syncytial virus (RSV) is the most common cause of pediatric acute lower respiratory tract infections (Nair et al., 2010). Only a small percentage of healthy, term infants develop severe complications; however, in high-risk infants, including those who are preterm or who have chronic lung diseases, congenital heart diseases, Down syndrome, neuromuscular conditions, or immunodeficiencies, the rate of hospitalizations increases to B10% (Feltes et al., 2003; Robinson et al., 2016b; The IMpact-RSV Study Group, 1998). There are no known treatments for RSV and antiviral therapy with ribavirin – which is of questionable efficacy – is reserved for severe cases in the immunosuppressed (American Academy of Pediatrics Committee on Infectious Diseases; American Academy of Pediatrics Bronchiolitis Guidelines Committee, 2014). However, hyperimmune immunoglobulins and monoclonal antibodies have shown promise in the prevention of RSV in vulnerable groups. In the early 1990s, two randomized controlled trials carried out in high-risk infants showed that an RSV immune globulin (RSVIGIV) infused monthly during the RSV season could reduce hospitalizations by 40%–65% (Groothuis and Simoes, 1993; PREVENT

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Study Group, 1997). However, there were reports of an increased risk of morbidity and mortality in infants with congenital heart disease (Simoes et al., 1998), and RSV-IGIV was then replaced by Palivizumab. Palivizumab (Synagis) is a humanized murine monoclonal antibody to the RSV F glycoprotein, a highly conserved glycoprotein among the two serotypes of RSV (serotype A and B) (Resch, 2017). By binding to the F-protein, Palivizumab impairs the fusion of the viral envelope with the host cell membrane, preventing entry into the host cell. Palivizumab also prevents the fusion of the plasma membrane of infected cells, a process that results in syncytium formation. It was the first anti-viral monoclonal antibody approved by the FDA. In the original randomized controlled trial of 1,502 children with prematurity or bronchopulmonary dysplasia (BPD) who were randomized to placebo or monthly IM injections of Palivizumab, there was a 55% reduction in hospitalizations (The IMpact-RSV Study Group, 1998). Hospitalizations were decreased by 78% for children with prematurity without BPD and by 39% for children with BPD. Subsequent studies have also shown reduced hospitalization in infants with hemodynamically significant congenital heart disease (Feltes et al., 2003). A 2014 systematic review of 20 observational studies (Nusrat Homaira et al., 2014) was consistent with these benefits and a 2019 self-controlled case series of 24,329 infants, 1.1% of whom received at least one dose of Palivizumab and 6.2% of whom developed RSV, found that Palivizumab was associated with a 74% reduction in incidence of RSV infection (Moore et al., 2019). While results on the cost-effectiveness of Palivizumab have been inconsistent (Andabaka et al., 2013), the most recent systematic review on the topic showed that, from a payer perspective, Palivizumab was cost-effective in preterm infants, infants with BPD or chronic lung disease, congenital heart disease, and infants in certain remote communities (Mac et al., 2019). There is general agreement among national guidelines that Palivizumab is beneficial in these specific high-risk situations (American Academy of Pediatrics Committee on Infectious Diseases; American Academy of Pediatrics Bronchiolitis Guidelines Committee, 2014; Robinson and Le Saux, 2015; Public Health England, 2015). However, there is some disagreement about adopting its routine use in Down’s syndrome, neuromuscular disorders, cystic fibrosis, or other lung diseases (Luna et al., 2018). In addition to Palivizumab, Motavizumab was a second-generation monoclonal antibody (mAb) derived from Palivizumab (Feltes et al., 2011; Carbonell-Estrany et al., 2010). It was withdrawn from the market after the FDA requested more information on the risks and severity of hypersensitivity reactions (see “Relevant Websites section”). Other monoclonal antibodies with extended half-lives and strong neutralizing activity are currently under investigation, with two entering phase two and three investigations (Aranda and Polack, 2019).

Human Immunodeficiency Virus The emergence of antiretroviral therapies (ART) has helped halt the progression of the Human Immunodeficiency Virus (HIV) epidemic. Treatment with ART controls the progression of the HIV-1 virus among infected individuals and also helps prevent HIV acquisition when taken prophylactically by uninfected individuals. However, for those with multi-drug resistant HIV with limited treatment options, monoclonal antibodies may serve as a salvage therapy. Moreover, until an effective vaccine is found, other treatment modalities, such as broadly neutralizing monoclonal antibodies, may aid in controlling the epidemic. Ibalizumab, a mouse-derived IgG4 monoclonal antibody, was approved by the FDA in 2018 for use in multi-drug resistant HIV (FDA, 2018). Ibalizumab does not act on circulating HIV-1 or cells already infected with HIV-1; instead, it blocks HIV entry into CD4 cells by binding to the CD4 extracellular domain 2 (Emu et al., 2018). In phase three of an open-label study, 40 individuals with multi-drug resistance HIV (defined as triple-class resistance) were treated with at least one active antiviral agent as well as Ibalizumab. 83% of patients had an initial decrease in their viral load and at 25 weeks, 43% of patients had an undetectable viral load (Emu et al., 2018). For patients with MDR-HIV, Ibalizumab may allow the construction of a life-saving treatment regimen. The other antibodies being studied for the prevention and treatment of HIV are classified as broadly-neutralizing monoclonal antibodies (bnMAbs) that act directly on HIV (Caskey et al., 2019). The site of action for these antibodies is the HIV-1 envelope spike (Env) – the only target on the surface of the virus (Caskey et al., 2019). The observation that a small fraction of HIV-1infected persons are able to produce antibodies that neutralize multiple circulating HIV-1 strains indicated the possibility to develop potent bnMAbs (Freund et al., 2017). bnMAbs can both lead to the destruction of HIV-infected cells by engaging with Fc receptors and can boost the endogenous immune system through immune complex formation that increases B-cell and T-cell activation. These features have the potential to both control viral replication and lead to the reduction of viral reservoirs. To date, the clinical information on bNAbs is primarily limited to phase one and phase two studies but holds promise for prophylaxis, maintenance therapy, and potentially even in eliciting prolonged remission or cure (Gruell and Klein, 2018). A recent phase two, randomized controlled trial of VRC01 enrolled 19 individuals with acute HIV infection who started and were well controlled on ART (Crowell et al., 2019). These individuals then underwent an analytic interruption of ART and 14 received VRC01 infusions every three weeks for 24 weeks. However, only one VRC01 recipient achieved the primary efficacy endpoint of viral suppression after ART interruption. The authors postulated that, like antiviral therapy, a combination of bnMAbs may be required to achieved viral suppression.

Experimental Immunoglobulin Therapies Seasonal Influenza Both polyclonal immunoglobulin and monoclonal antibodies have been used in the treatment of seasonal influenza and remain active areas of research interest. A modern systematic review of the impact of early administration of convalescent plasma during

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the 1918 Spanish influenza epidemic hypothesized that the use of the convalescent plasma resulted in a 21% absolute reduction in mortality (Luke et al., 2006). Subsequently, a small cohort study of patients with severe H1N1 influenza suggested a B30% reduction in absolute mortality with administration of H1N1 convalescent plasma (Hung et al., 2011). The same research group followed up with a randomized trial of convalescent plasma-derived immunoglobulin involving 35 patients (Hung et al., 2013). They found a reduced viral load among plasma recipients and on subgroup analysis, immunoglobulin within five days of symptom onset was the only factor independently associated with reduced mortality. A phase two randomized trial of convalescent plasma versus standard of care also demonstrated improvements in clinical parameters (Beigel et al., 2017). These studies, however, had methodological issues including small sample sizes, higher-than-expected mortality levels among controls, or differing baseline characteristics between the study groups. Thus, the findings of two phase III randomized trials were eagerly awaited. Unfortunately, both trials were disappointing and did not support the use of hyperimmune globulin in seasonal influenza. FLU-IVIG was an international double-blind, randomized trial of anti-influenza hyperimmune intravenous immunoglobulin vs standard of care (including neuraminidase-inhibitors) (Davey et al., 2019). Among the 308 participants, there was no benefit with the use of the hyperimmune IVIG. That said, there was clinical benefit among the 27% of patients with influenza B. A randomized trial of high titer plasma (HAI antibody titers of Z1:80) vs low titer plasma (HAI o1:10) in patients with severe influenza A also did not reveal any benefit with the use of high titer plasma (Beigel et al., 2019). However, mAbs may still hold some promise and there are currently multiple influenza-specific molecules being studied (Salazar et al., 2017). MHAA4549A is a broadly neutralizing human IgG1 mAb that targets the highly conserved hemagglutinin stalk region of the influenza A virus. In one phase two study, it resulted in significant improvements in symptoms and viral (McBride et al., 2017). However, though it was safe and well-tolerated, no such benefits were seen in two subsequent phase two trials (Genentech, 2018, 2019). The phase 2A study of another anti-influenza monoclonal antibody, VIS410, suggested that it was well tolerated and that there may be some clinical benefit; therefore, a phase 2B trial is now planned (Visterra, 2019).

Avian Influenza Avian influenza viruses, such as H5N1 and H7N9, are associated with high mortality, with a case fatality rate of B40%–70% in hospitalized patients (Cowling et al., 2013). Neuraminidase inhibitors decrease mortality by B50% (Adisasmito et al., 2010; Kandun et al., 2008) but there are reports of emerging neuraminidase inhibitor resistance (Someya et al., 2005; de Jong et al., 2005) Therefore, adjunctive and alternative therapies are still required. In 2006, convalescent plasma from an H5N1 survivor was successfully used in combination with Oseltamivir to treat a new H5N1 case (Zhou et al., 2007) and another case report described similar success (Kong and Zhou, 2006). Since then, a purified polyclonal immunoglobulin F(FBF001) raised against influenza A/Vietnam/1194/2004 virus (H5N1 subtype) showed activity in animal models and safety in a phase one trial (Bal et al., 2015). H5-specific monoclonal antibodies have also been studied (Chen et al., 2009; Zheng et al., 2011).

Mers-Cov Middle Eastern respiratory syndrome coronavirus (MERS-CoV) is a zoonotic disease (transmitted from camel and bat reservoirs) with occasional human-to-human transmission that emerged in Saudi Arabia in 2012 (World Health Organization, 2019b). Since then, there have been 2458 cases with a fatality rate of 34% (World Health Organization, 2019b). Though cocktails of antiviral therapies are under investigation, no treatment currently exists, and both polyclonal and monoclonal immunotherapies have been explored. MERS-CoV seropositive camel serum was beneficial in mice for both pre- and post-exposure prophylaxis (Zhao et al., 2015), and a polyclonal human anti-MERS coronavirus antibody produced from transchromosomic cattle (SAB-310) was safe and well-tolerated in a phase one double-blind, placebo-controlled trial (Beigel et al., 2018). Several human monoclonal antibodies against MERS-CoV have also been developed (Widjaja et al., 2019). Most of these antibodies target the MERS-CoV S1B receptorbinding domain (RBD). Both RBD and non-RBD antibodies are protective against MERS-CoV in animal models (Widjaja et al., 2019); however, no testing in humans has been initiated.

Ebola and Other Hemorrhagic Fevers The first reported use of immunotherapy for the treatment of the Ebola virus (EBOV) was in 1995 in Kikwit, Democratic Republic of Congo (Mupapa et al., 1999). The idea was explored further, with mixed results, in animal models (Jahrling et al., 2007, 1996; Dye et al., 2012; Sullivan et al., 2011; Qiu et al., 2014; Pascal et al., 2018) Then, owing partly to the scale of devastation and the absence of any therapies, multiple investigational therapies were explored during the 2014 Ebola outbreak in West Africa. The use of convalescent plasma in a small non-randomized study did not improve survival (van Griensven et al., 2016). However, ZMapp, a cocktail of monoclonal antibodies targeting the surface glycoprotein of Ebola, showed promise (Davey et al., 2016). 72 patients were randomized in the PREVAIL randomized controlled study. While the pre-specified statistical threshold for efficacy was not met, there was a 15% absolute reduction in death among those receiving ZMapp (Davey et al., 2016). This survival was sufficiently

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beneficial that ZMapp was considered the standard of care and used in the control arm of the 2018 PAmoja TuLinde Maisha (PALM [together save lives]) Trial (Mulangu et al., 2019). The PALM [together save lives] study is a randomized controlled trial that was carried out as a component of the emergency response to the EBOV outbreak in the Democratic Republic of Congo in 2018. Spearheaded by WHO, this three-arm trial compared one antiviral, Remdesivir, and two monoclonal antibody-based regimens, mAb114 and REGN-EB3, to a ZMapp control. mAb114 was identified from a panel of memory B cells from a survivor of the 1995 Democratic Republic of Congo EBOV outbreak (Corti et al., 2016; Gaudinski et al., 2019). mAb114 binds to a highly conserved region of the EBOV envelope glycoprotein, preventing interaction with the NPC1 receptor, thereby preventing viral entry into the host cell cytoplasm (Corti et al., 2016; Gaudinski et al., 2019). REGN3470-3471-3479 is a cocktail of three fully human IgG1 monoclonal antibodies (REGN3470, REGN3471, and REGN3479 or REGN-B3; each antibody in the cocktail in a 1:1:1 ratio) (Pascal et al., 2018). The three antibodies all act on separate epitopes on the EBOV glycoprotein but they can do so simultaneously (Sivapalasingam et al., 2018). Overall, 53% receiving Remdesivir, B50% of patients receiving ZMap, 35% receiving mAb114, and 33.5% receiving REGN-EB3 died. This study showed that monoclonal antibodies held great promise in reducing EBOV mortality. Other filoviruses are also potential targets for the use of immunotherapies. For Junin Virus, the causative agent of Argentinian hemorrhagic fever, immune or convalescent plasma has been the standard of care since a seminal trial in 1979 showed significant mortality benefit. However, given the costs and challenges of administering immune plasma, recent studies have explored the use of mAbs (Maiztegui et al., 1979). For instance, J199, a mouse-human chimeric mAb produced in Nicotiana benthamiana, administered to guinea pigs provided 100% protection against JUNV six days after infection (Zeitlin et al., 2016). Lassa virus, the causative agent of Lassa fever (LF), results in 100,000 to 300,000 infections annually, with approximately 5000 deaths (Center for Disease Control and Prevention, 2019). In a famous case of passive immunization, Dr. Jordi Casals, the virologist who first isolated and identified the Lassa virus, also inadvertently infected himself with it, but survived after receiving a blood transfusion from the first known survivor of LF, nurse Lilly Lyman Pinneo (Watts, 2012). However, the use of convalescent plasma was not subsequently shown to be beneficial (McCormick et al., 1986). Since 2008, the Viral Hemorrhagic Fever Consortium (Viral Hemorrhagic Fever Consortium, 2020) has studied the protective role of B cells in LF (Robinson et al., 2016a) and created the largest library of huMAbs from convalescent LF patients. Animal models of LF guinea pigs (Cross et al., 2016) and nonhuman primates (Mire et al., 2017) have confirmed the potential efficacy of mAbs in this disease. Another cocktail of three different monoclonal antibodies, FVM04, CA45, and anti-MARV mAb MR191, provided 100% postexposure protection in guinea pigs and non-human primates against Ebola, Sudan, and Marburg viruses (Brannan et al., 2019).

Other Experimental Infections West Nile virus (Hirota et al., 2012), Zika virus (Marston et al., 2018), Chikungunya (Jin and Simmons, 2019), and numerous other viruses are currently being studied and monoclonal antibodies are in the early phases of development. With the recent emergence of SARS-CoV-2 immunoglobulin therapy, polyclonal immunoglobulin therapy has specifically been considered. Not only have immunoglobulins, presumably IVIG, been administered to critically ill patients (Yang et al., 2020), but there are also reports of immunoglobulin therapy with SARS-CoV-2 convalescent plasma (Bloomberg, 2020).

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Further Reading Dixit, R., Herz, J., Dalton, R., Booy, R., 2016. Benefits of using heterologous polyclonal antibodies and potential applications to new and undertreated infectious pathogens. Vaccine 34 (9), 1152–1161. Ferrara, G., Zumla, A., Maeurer, M., 2012. Intravenous immunoglobulin (IVIg) for refractory and difficult-to-treat infections. The American Journal of Medicine 125 (10), (1036. e1,1036.e8). Hey, A., 2015. History and practice: Antibodies in infectious diseases. Microbiology Spectrum 3 (2). Ian Gust, A.O., 2012. Role of passive immunotherapies in managing infectious outbreaks. Biologicals 40 (3), 196–199. Pelfrene, E., Mura, M., Cavaleiro Sanches, A., Cavaleri, M., 2019. Monoclonal antibodies as anti-infective products: A promising future? Clinical Microbiolology and Infection 25 (1), 60–64. Perez, E.E., Orange, J.S., Bonilla, F., et al., 2017. Update on the use of immunoglobulin in human disease: A review of evidence. Journal of Allergy and Clinical Immunology 139 (3), S1–S46. Sparrow, E., Friede, M., Sheikh, M., Torvaldsen, S., 2017. Therapeutic antibodies for infectious diseases. Bulletin of the World Health Organization 95 (3), 235–237.

Relevant Websites https://www.astrazeneca.com/media-centre/press-releases/2010/AstraZeneca-discontinues-motavizumab-RSV-21122010.html# AstraZeneca discontinues development of motavizumab for RSV prophylaxis indication. M2 Pharma.

Vaccine Production, Safety, and Efficacy Thomas J Brouwers, Athena Institute, VU Amsterdam, Amsterdam, The Netherlands Bernard AM Van der Zeijst, Leiden University Medical Center, Leiden, The Netherlands r 2021 Elsevier Ltd. All rights reserved.

Glossary Adjuvant A substance that enhances the body’s immune response to an antigen. Clinical trial A research study in which one or more human subjects are prospectively assigned to one or more interventions (which may include placebo or other control) to evaluate the effects of those interventions on healthrelated biomedical or behavioral outcomes. Correlate of protection Correlates of immunity/ protection to a virus or other infectious pathogen are measurable signs that a person is immune, in the sense of being protected against becoming infected and/or developing disease. For many viruses, antibodies serve as a correlate of immunity.

Herpes zoster Shingles, also known as zoster or herpes zoster, is a viral disease characterized by a painful skin rash with blisters in a localized area. Shingles is due to a reactivation of varicella-zoster virus (VZV) in a person’s body. The disease chickenpox is caused by the initial infection with VZV. Immunization The action of making a person or animal immune to infection, typically by inoculation with a nonpathogenic version of the infectious agent. Synonymous with vaccination. Regulatory authority National regulatory agencies responsible for ensuring that products released for public distribution (normally pharmaceuticals and biological products, such as vaccines) are evaluated properly and meet international standards of quality and safety.

Introduction The development of vaccines is closely linked to the need to protect increasingly larger populations. This started with the transition from populations consisting of hunter-gatherers to agricultural settlements. It went on when urbanization made man even more vulnerable to contagious diseases. Presently about 55% of the world population lives in cities, a situation that is only sustainable with vaccination. Stopping vaccination would result in epidemics. The medical need to prevent (viral) infections is still the major incentive for vaccine development. But whether a vaccine will be developed is also heavily influenced by commercial considerations. The first vaccine against smallpox ended a disease that in the century before its eradication killed 500 million people (Fig. 1). Thanks to vaccination, the average life expectancy has gone up in many countries from about 40 years in 1900 to more than 80 years today. Table 1 gives an overview of available viral vaccines for human use. Additionally, many vaccines for animals have been licensed. Thanks to vaccination, smallpox was eradicated by 1977. Rinderpest, a disease that killed up to 100% of cattle during outbreaks was eradicated by 2010 and polio is very close to eradication. Despite of these successes additional vaccines are needed, some urgently. Table 1 lists these vaccines. Although there have been several “golden ages of vaccines” with important scientific and technical advances bringing vaccine development from a trial-and-error approach to “reverse vaccinology” (Delany et al., 2014), development times of vaccines have only increased. This is especially worrying when we have to develop vaccines against new viral diseases, where pre-existing immunity is completely absent. This article aims to provide an overview of the history, current status, and future of viral vaccine development and use. A full description of all aspects of viral vaccines would require many hundreds of pages (Plotkin et al., 2018; Plotkin, 2011). We have selected some recently developed vaccines to demonstrate the general principles of vaccine development and provide an overview of the long road from vaccine candidates towards market entry. Lastly, several new technologies and opportunities are discussed that are expected to change the vaccine development process for the better, especially by shortening development times.

The History of Vaccination The realization that victims of a severe infectious disease remain immune to the same disease for the rest of their lives was the basis of the first vaccine against smallpox. Actually, a similar approach was already used from about 1570 in China (Plotkin, 2011). The procedure is called variolation and entailed introducing minute amounts of virus-containing pox material into the nostril of a child. This resulted in a light form of the disease. Already, in those times, the balance between efficacy and adverse effects was evaluated. Up to 1% of variolated children died. This was considered acceptable considering the larger risk of contracting smallpox. Vaccination differs fundamentally from variolation in that vaccines are composed of viral antigens that produce protective immunity and immune memory without causing disease. There are three classes of licensed viral vaccines: (1) Live-attenuated

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Fig. 1 Conquering smallpox. The smallpox virus (a) has been responsible for hundreds of millions of deaths. The virus was already present in ancient Egypt (b). It spread, by trade routes, wars and colonization, to all continents. Typically, 3 out of 10 people who got the disease died. Survivors were left with scars; some became blind. An eradication program organized by the WHO was successful by 1977 with the last case of naturally acquired smallpox (c). The virus is now confined to two laboratories. Eradication was possible with a vaccine discovered by Edward Jenner in 1796 (d). Sources: (a) CDC PHIL #1849, (b) and (c) WHO, (d) Wellcome Library no. 546000i. Table 1

Overview of licensed viral vaccines and vaccines under development

Licensed vaccines (Year of market introduction)

Vaccines that are needed (Estimated global death toll /year)

Smallpox (1796) Rabies (1885) Yellow fever (1930) Japanese encephalitis (1930) Influenza (1938) (efficacy 40%–60%) Polio (1954) Adenovirus (1956) Measles (1963) Mumps (1967) Rubella (1969) Varicella (1970) Hepatitis B (1981) Hepatitis A (1991) Rotavirus (2006) HPV (2006) Herpes Zoster (2006) Dengue (2015) (efficacy 44%, adverse events) Ebola (2019)

HIV (770,000) Broadly protecting influenza vaccine (720,000) Hepatitis C (399,000) Norovirus (200,000) Epstein-Barr (143,000) Human respiratory syncytial virus (120,000) Hepatitis E (44,000) Cytomegalovirus (N/A) Improved dengue vaccine (N/A) Herpes simplex Human metapneumovirus (N/A)

vaccines, (2) Inactivated vaccines and (3) Non-replicating protein vaccines produced by recombinant DNA technology. An advantage of live-attenuated vaccines is that production is inexpensive and no adjuvants to boost immune responses are needed. Disadvantages are more adverse effects. These are usually mild, in rare cases severe. For example, smallpox and yellow fever vaccines result in respectively 1–8 and 1–2 deaths per million vaccinations. Another risk is that mutations may lead to regained virulence. This has been a problem for the oral polio vaccine (Amanna and Slifka, 2009). Inactivated and non-replicating vaccines

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are very safe, but a downside is that alone they are often unable to generate robust immunity. These vaccines require adjuvants, substances added to vaccines to enhance the immunogenicity (Di Pasquale et al., 2015). Traditionally aluminum salts, with a strong safety record, were used as adjuvant. Scientific advances have led to additional, more potent, adjuvants with a smaller safety database.

What has Happened When Vaccine Reaches the Market? The Development of Shingrix Shingrix is the herpes zoster (shingles) vaccine. It was licensed in October 2017 in the USA and in 2018 in Europe and Japan. Licensing was based on two large clinical trials with almost 28,000 participants (Maltz and Fidler, 2019; Levin and Weinberg, 2020). Shingrix was the second herpes zoster vaccine. It addressed several limitations of Zostavax, the first vaccine. These limitations included limited protection, especially in individuals over 70 years old, a limited duration of protection, and safety risks for immunocompromised individuals (Dooling et al., 2018). The history of this successful vaccine dates back to basic research in the early 1990s. Researchers looked for promising viral antigens for a new vaccine. They opted for antigens that could induce cell-mediated immunity in addition to virus neutralizing antibodies elicited by Zostavax. Viral glycoprotein E (gE) was selected as the most suitable candidate (Vafai, 1993; Haumont et al., 1996). Thus, at least 24 years passed between the beginning of research into this vaccine and its licensing.

Phases and Timelines in Vaccine Development: From Discovery to Product A development time of several decades is typical for vaccine development which consists of separate phases that can only be carried out after each other. Fig. 2 summarizes this step-wise process.

Fig. 2 Timelines and success rates in vaccine development. The transition successes between the clinical phases are from (Wong, C.H., Siah, K.W., LO, A.W., 2019. Estimation of clinical trial success rates and related parameters. Biostatistics 20, 273–286). The data on duration from (Dimasi, J.A., Florez, M.I., Stergiopoulos, S., et al., 2020. Development times and approval success rates for drugs to treat infectious diseases. Clinical Pharmacology & Therapeutics 107, 324–332) and the cost estimate up to phase 2 from (Gouglas, D., Le, T.T., Henderson, K., et al., 2018. Estimating the cost of vaccine development against epidemic infectious diseases: A cost minimisation study. The Lancet Global Health 6, (e1386–e1396)).

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Preclinical Studies The starting point for vaccine development is the medical need to prevent disease. The development begins with basic research to identify the virus responsible for the diseases and, if possible, the antigen(s) that elicit a protective immune response. In addition, attempts are made to determine which immune responses are protective (antibodies, T cells, for example). This is all carried out in the laboratory. Blood samples are often used to study immune responses ex vivo. In those cases where an animal model is available, candidate vaccines can be tested for protection after challenge with virulent virus. Finally, toxicity data are collected. The preclinical stage ends with the establishment of a production process.

In Human Studies After the preclinical studies have come to a satisfactory end, the vaccine is tested in humans (or animals for veterinary vaccines). ‘First-inhuman’ studies and all other clinical studies are subject to strict rules to protect study participants and ensure quality. Clinical trials in humans are classified into three phases: phase I, phase II and phase III. Phase I studies are carried out on limited numbers, for example, 20 þ healthy adults and are primarily concerned with safety. Phase II studies involve larger numbers of persons belonging to the target population with the aim of getting preliminary information about efficacy, usually immunogenicity and additional safety data. Together, phase I and II trials should give sufficient confidence that a vaccine is efficacious and safe. The real test comes from phase III trials in which a vaccinated population is compared with a control group for protection against the target diseases, resulting in an estimate of vaccine effectiveness. Depending on the prevalence of the disease these studies may require 10,000 or more participants. After the completion of the phase III studies, all results are summarized in a dossier. This dossier also includes data on a consistent manufacturing process and assays to monitor the production and its end products. This dossier is submitted to the regulatory authorities (The Federal Drug Administration in the USA and the European Medicine Agency). After the regulatory authorities have decided that the vaccine is safe and effective, it is licensed and can be released to the market. Exceptionally, a vaccine fails in the phase III stage. This happened in the 1960s with a vaccine against disease caused by respiratory syncytial virus. This vaccine was not efficacious and even unsafe, killing two participants of the trial (Hurwitz, 2011). Other problems that popped up in the past were due to the limited number of participants in the phase III trials. As a consequence, rare adverse effects e.g., occurring 1 in 100,000 times will not be detected. Therefore, post-licensure safety surveillance (phase IV studies) is needed to detect adverse effects following immunization (AEFI). Phase IV studies revealed that a rotavirus vaccine caused intussusception in one or two cases per 10,000 infants vaccinated. This vaccine was withdrawn from the market. Newer rotavirus vaccines are 10-fold safer, but not completely without AEFI (Di Pasquale et al., 2016). Narcolepsy, a sleeping disorder, was identified as a possible AEFI associated with the AS03-adjuvanted influenza vaccine Pandemrix. Although there is a clear association between the receipt of Pandemrix and the development of narcolepsy the causality remains unproven. This incident has shown that there is a need for an internationally coordinated vaccine safety structure that can monitor the safety of the many doses of pandemic vaccines administered in a short period (Edwards et al., 2019).

Costs and Duration of Vaccine Development The exact data on the development of Shingrix are confidential to the developer and producer GlaxoSmithKline. Clinical studies are responsible for the major costs of vaccine development. Thus, costs will vary depending on the complexity of these studies. Estimates of vaccine development expenditure vary from 135 to more than 1000 million USD (Plotkin et al., 2017; DiMasi et al., 2016). An analysis reported a breakdown of the costs of the separate steps in vaccine development (Gouglas et al., 2018). Analysis of databases with clinical trials on vaccines has yielded reliable data on the success rate of vaccine development. Two studies reported an identical success rate of vaccine development from phase I studies to licensing of 33% (DiMasi et al., 2020; Wong et al., 2019). A success rate of 48% in the transition from the preclinical phase to phase 1 studies (Davis et al., 2011) results in a 16% overall success rate. Estimates for the median duration of phase I-III trials during vaccine development are 6.4–10.7 years (Davis et al., 2011; Pronker et al., 2013). A summary of the data is given in Fig. 2.

Vaccine Production Coming back to the Shingrix example: this vaccine is produced by expression, via DNA-recombinant technology, of the antigen in Chinese hamster ovary (CHO) cells. The CHO cells are grown in bioreactors in dedicated production facilities. Typically, these production facilities contain stainless steel bioreactors controlled by sophisticated computer programs to ensure a reproducible production process. After this upstream processing the antigen has to be purified, usually, in various steps, a process called downstream processing. Assays are carried out to check (intermediate) products against the specifications (quality control [QC]) during all steps. At the same time, a quality assurance (QA) team follows the stream of accompanying documentation to ensure that all intended controls were carried out. Production, together with QC and QA, can be a long process of more than one year.

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The decision to build production facilities is usually taken before the regulatory authorities approve the vaccine. This implies a considerable financial risk. Shingrix is a successful vaccine and has reached blockbuster status with annual sales in 2019 of more than 1.5 billion USD. The demand was much higher than foreseen, resulting in the need to build additional production facilities. Since the production process is an integral part of the license, careful calibration of a new production facility and the demonstration of equivalence of the product are required. Alternatives for the traditional bioreactor-based production methods that can speed up production, are scalable and reduce costs are being intensively investigated. See Section “Emerging Viral Diseases”.

More About Vaccine Development Clinical studies are time-consuming and expensive. It would be much easier to measure an immune response that is predictive for protection. Such correlates of protection (CoP) are available for many viral vaccines (Plotkin, 2010). CoPs can be used to license newer vaccines, but also to evaluate existing and new vaccination schedules for protection. Systems vaccinology is a more advanced method to predict the immunogenicity of vaccines. In essence, this links host gene expression (molecular signatures) to protection by vaccines. This approach was very successful for the live attenuated yellow fever vaccine and is, in principle, also useful to predict adverse effects. But more work is needed to move it from ‘promising’ to a standard approach (Raeven et al., 2019).

The Licensing of Influenza Vaccines Follows a Different Procedure The current influenza vaccines are mostly produced from an egg-grown virus. The vaccines have to be matched every year to the predominantly circulating virus strains. This requires a special procedure. The production process is licensed with the possibility to insert the circulating viruses, selected in the Northern hemisphere in February-March by the WHO. The current production process is capable to make the vaccine available in October (Soema et al., 2015).

The Vaccine Industry Over the past decades, the number of vaccine suppliers has decreased considerably due to mergers and acquisitions of pharmaceutical companies. Presently about 90% of global vaccine sales come from four large multi-national corporations: GlaxoSmithKline, Merck, Pfizer and Sanofi Pasteur (Shen and Cooke, 2019). In the 1980s emerging market manufacturers started to enter the vaccine market and assumed a significant role since then. Emerging manufacturers, represented by the Developing Countries Vaccine Manufacturers Network, play a critical role in the supply of vaccines of developing countries. They now supply about half of UNICEF’s vaccine procurement in a volume of doses, representing about 30% of the value of UNICEF’s total vaccine procurement. Even including producers from developing countries, there are relatively few vaccine manufacturers that meet international standards of quality. Many vaccine markets are monopolies or oligopolies. The limited number of vaccine suppliers and production capacities leads to a tenuous balance between demand and supply in many individual vaccine markets and regularly to vaccine shortages.

Taking Stock Due to vaccination, viral diseases remain under control. Two vaccines, hepatitis B and human papillomavirus protect against cancer. Thanks to a functional vaccine industry, vaccines remain available for a largely stable market. In addition, the researchbased vaccine industry is innovative and develops new vaccines to answer unmet medical needs. This also applies to influenza vaccines with a new composition every year. However, problems exist with vaccines against new viral diseases to combat epidemics. The duration of these epidemics is unknown and usually, there is no commercially attractive market. Market forces fail, but vaccines are urgently needed. This will be the topic of the second part of this article.

The Challenge of Emerging Viral Diseases In March 2003, the WHO issued a global alert about a new viral disease, severe acute respiratory syndrome (SARS). In retrospect, the SARS epidemic started in 2002 in China. In several months, SARS spread to 37 countries where it caused 8098 cases with 774 deaths before it came under control. SARS coronavirus (SARS-CoV), the causative agent of the syndrome, was traced back to an intermediate host, the civet cat, which originated as a virus of the horseshoe bat. Small mutations in the viral genome expanded the host range to humans. A new viral disease in an immune naïve population could cause a catastrophe with many fatalities. It is worrying that there are many animal viruses with unknown pandemic potential. The SARS epidemic was the first wake-up call.

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The 2007 World Health Report of the WHO was dedicated to Emerging viral diseases (EVD) and stated that “It would be extremely naive and complacent to assume that there will not be another disease like AIDS, another Ebola, or another SARS, sooner or later” (WHO, 2007). The real wake-up call came only in 2013 after an outbreak of Ebola in West Africa (Guinea, Liberia and Sierra Leone). Ebolavirus had been discovered in 1976. Between 1976 and 2013 it regularly spilled over from its animal reservoir to humans. There were several outbreaks, but these remained small and were contained. In the 2013 outbreak, about 28,000 persons were infected and 11,000 died. In addition, there was transmission to other countries and loss in GDP in the three affected countries. Many discussions in the aftermath of the Ebola outbreak led to the conclusion that more investments in R&D to counter EVD were needed, together with a more active role involving the WHO. As a result, the WHO Blueprint for Action to Prevent Epidemics was developed. The 2018 Blueprint list of priority diseases contains the following viral diseases:

• • • • • • • •

Crimean-Congo hemorrhagic fever Ebola virus disease and Marburg virus disease Lassa fever Middle East respiratory syndrome (MERS-CoV) and SARS Nipah and henipaviral diseases Rift Valley fever Zika Disease X (caused by a pathogen currently unknown)

The coordination and execution of R&D are in the hands of the Coalition for Epidemic Preparedness Innovations (CEPI). CEPI is a public-private partnership that was established during the World Economic Forum Annual Meeting of 2017. CEPI acquired access to 755 million USD and used the money to support work on priority diseases (Lassa, Nipah, MERS, Rift Valley Fever and Chikungunya), selected with the aid of the WHO Blueprint. Two requests for proposals involved priority viruses. The aim is to bring vaccines through phase II studies and have vaccine stockpiles available for at least two pathogens by the end of 2022. Another request for proposals involved platform technologies. Please see the CEPI Business Plan 2019–2022 for more details. Platform technologies have the potential to speed up development and production drastically. The common property of platforms is that they can be used for multiple vaccine antigens. The safety of the platform is known, the only variable is the inserted antigen. This will save time and may lead to regulatory streamlining, comparable to the procedure for influenza virus vaccines. Based on reduced development and production times, the most promising platforms are DNA and RNA vaccines and neutralizing antibodies, as discussed below. DNA vaccines date back to the early 1990s when it was discovered that plasmid DNA, delivered in muscle or skin induces an antibody response to the encoded protein. The DNA has to cross the membranes of the cell and the nucleus. Subsequently, the antigen is synthesized. The initial excitement diminished somewhat after it appeared that immune responses in man were weaker than in mice. However, this problem has been solved by more efficient systems for the delivery and formulations that protect DNA against degradation. In addition to antibodies, DNA vaccines elicit CD4 þ and CD8 þ T cells. Logistically DNA vaccines have many advantages, such as fast, inexpensive and scalable production and short development times. Also, no handling of infectious virus is required during vaccine development. This makes these vaccines ideal as protection against emerging viral diseases (Rauch et al., 2018). Presently vaccine development against various diseases, including Ebola, MERS and Zika, is ongoing (Rauch et al., 2018). A (theoretical) disadvantage of DNA vaccines is that they may integrate into the human genome and lead to undesired gene activation. Presently (2019) no DNA vaccines have been licensed for human use. RNA vaccines consist of mRNA. They have the same advantages as DNA vaccines, but since mRNA has to cross only one membrane, immune responses are stronger. In addition to conventional mRNA, self-amplifying mRNA can be used. mRNA is extremely sensitive to degradation by endonucleases. Therefore, a formulation, e.g., liposomes, which protect the RNA is used. Many vaccines are presently under development and also in clinical trials. But although RNA vaccines are very promising, their development is still at an early stage (Maruggi et al., 2019). Neutralizing antibodies offer an alternative treatment for viral infections. This is what is referred to as passive immunization and was successful in the treatment of Ebola (Saphire et al., 2018). Another new development is the use of broadly neutralizing antibodies directed against conserved viral structures. These antibodies are promising both for prophylaxis and therapy for a range of viruses (Walker and Burton, 2018).

Looking Forward Existing vaccines will continue to play a key role in controlling (viral) diseases. Many vaccines come as a combination vaccine and it will be complicated and very expensive to modify these vaccines. Newly developed vaccines will probably be made using novel approaches (Mascola and Fauci, 2020). We expect that the establishment of CEPI will contribute to these innovations. Paving a regulatory path to the first registration of DNA and RNA vaccines for infectious diseases will be a

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challenge. But nucleic acid vaccines are also a promising approach for the immunotherapy of cancer. This may help in paving this path. Another challenge is the design of clinical trials to test EVD. During the 2013 Ebola outbreak, there was disagreement about the design of clinical studies, particularly on ethical aspects of a placebo group. However, without a placebo group, it is impossible to measure the safety and efficacy of a vaccine. During these discussions the incidence of the disease declined, making it more difficult to carry out the phase III study. Choices about the design of clinical studies have to be made before outbreaks.

The Development of COVID-19 Vaccines Shortly after the completion of the manuscript for this chapter, SARS-CoV-2 was discovered. Its global spread led to an overburdening of health systems and a death toll of about 1.6 million by December 13, 2020. Measures aimed at controlling the disease disrupted daily activities. A widespread hope is that immunity provided by vaccination will be the key to a return to ‘normal life’. The first vaccine, an mRNA vaccine, developed and produced by Pfizer/BioNTech has been approved by now in several countries and vaccination has begun or will so shortly. The approval of a second mRNA vaccine developed by Moderna is expected in January 2021. Vaccine development started on January 11, 2020 when the genomic sequence of the virus, determined by Chinese scientists, became available. According to the WHO, 214 vaccines were under development on December 10, 2020, of which 13 in phase III clinical trials. The approval of more vaccines is expected in the first months of 2021. Estimated efficacies of vaccines are high, more than 90%. Adverse effects are present. But they are of short duration and not categorized as serious. Three vaccine platforms contributed to the most promising COVID-19 vaccines: (1) mRNA vaccines, (2) Adenovirus-based vaccines and (3) Vaccines consisting of inactivated virus. In addition, there are promising results from protein subunit vaccines. The use of these platforms significantly cut the development timelines. Further factors were an abundance of funding, tight collaborations between vaccine developers, governments and regulatory agencies, and the parallelization of activities. Especially the construction of manufacturing facilities parallel to the clinical development saved a lot of time. Several new platforms and procedures were adopted in the development of the COVID-19 vaccines. Licensing of these vaccines will pave the way for more vaccines based on these technologies. This could lead to considerable changes in the development and production of future vaccines.

References Amanna, I.J., Slifka, M.K., 2009. Wanted, dead or alive: New viral vaccines. Antiviral Research 84, 119–130. Davis, M.M., Butchart, A.T., Wheeler, J.R., et al., 2011. Failure-to-success ratios, transition probabilities and phase lengths for prophylactic vaccines versus other pharmaceuticals in the development pipeline. Vaccine 29, 9414–9416. Delany, I., Rappuoli, R., De Gregorio, E., 2014. Vaccines for the 21st century. EMBO Molecular Medicine 6, 708–720. Di Pasquale, A., Bonanni, P., Garcon, N., et al., 2016. Vaccine safety evaluation: Practical aspects in assessing benefits and risks. Vaccine 34, 6672–6680. Di Pasquale, A., Preiss, S., Tavares Da Silva, F., Garcon, N., 2015. Vaccine adjuvants: From 1920 to 2015 and beyond. Vaccines 3, 320–343. Dimasi, J.A., Florez, M.I., Stergiopoulos, S., et al., 2020. Development times and approval success rates for drugs to treat infectious diseases. Clinical Pharmacology & Therapeutics 107, 324–332. Dimasi, J.A., Grabowski, H.G., Hansen, R.W., 2016. Innovation in the pharmaceutical industry: New estimates of R&D costs. Journal of Health Economics 47, 20–33. 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. Edwards, K., Lambert, P.H., Black, S., 2019. Narcolepsy and pandemic influenza vaccination: What we need to know to be ready for the next pandemic. The Pediatric Infectious Disease Journal 38, 873–876. Gouglas, D., Le, T.T., Henderson, K., et al., 2018. Estimating the cost of vaccine development against epidemic infectious diseases: A cost minimisation study. The Lancet Global Health 6.(e1386–e1396). Haumont, M., Jacquet, A., Massaer, M., et al., 1996. Purification, characterization and immunogenicity of recombinant varicella-zoster virus glycoprotein gE secreted by Chinese hamster ovary cells. Virus Research 40, 199–204. Hurwitz, J.L., 2011. Respiratory syncytial virus vaccine development. Expert Review of Vaccines 10, 1415–1433. Levin, M.J., Weinberg, A., 2020. Adjuvanted recombinant glycoprotein E herpes zoster vaccine. Clinical Infectious Diseases 70, 1509–1515. Maltz, F., Fidler, B., 2019. Shingrix: A new herpes zoster vaccine. Pharmacy and Therapeutics 44, 406–433. Maruggi, G., Zhang, C., Li, J., Ulmer, J.B., Yu, D., 2019. mRNA as a transformative technology for vaccine development to control infectious diseases. Molecular Therapy 27, 757–772. Mascola, J.R., Fauci, A.S., 2020. Novel vaccine technologies for the 21st century. Nature Reviews Immunology 20, 87–88. Plotkin, S.A., 2010. Correlates of protection induced by vaccination. Clinical and Vaccine Immunology 17, 1055–1065. Plotkin, S.A., 2011. History of Vaccine Development. New York: Springer. Plotkin, S.A., Orenstein, W.A., Offit, P.A., Edwards, K.M. (Eds.), 2018. Plotkin's vaccines. seventh ed. Elsevier. Plotkin, S., Robinson, J.M., Cunningham, G., Iqbal, R., Larsen, S., 2017. The complexity and cost of vaccine manufacturing – An overview. Vaccine 35, 4064–4071. Pronker, E.S., Weenen, T.C., Commandeur, H., Claassen, E.H., Osterhaus, A.D., 2013. Risk in vaccine research and development quantified. PLOS One 8, e57755. Raeven, R.H.M., Van Riet, E., Meiring, H.D., Metz, B., Kersten, G.F.A., 2019. Systems vaccinology and big data in the vaccine development chain. Immunology 156, 33–46. Rauch, S., Jasny, E., Schmidt, K.E., Petsch, B., 2018. New vaccine technologies to combat outbreak situations. Frontiers in Immunology 9, 1963. Saphire, E.O., Schendel, S.L., Gunn, B.M., Milligan, J.C., Alter, G., 2018. Antibody-mediated protection against Ebola virus. Nature Immunology 19, 1169–1178. Shen, A.K., Cooke, M.T., 2019. Infectious disease vaccines. Nature Reviews Drug Discovery 18, 169–170. Soema, P.C., Kompier, R., Amorij, J.P., Kersten, G.F., 2015. Current and next generation influenza vaccines: Formulation and production strategies. European Journal of Pharmaceutics and Biopharmaceutics 94, 251–263. Vafai, A., 1993. Antigenicity of a candidate varicella-zoster virus glycoprotein subunit vaccine. Vaccine 11, 937–940. Walker, L.M., Burton, D.R., 2018. Passive immunotherapy of viral infections: ‘Super-antibodies' enter the fray. Nature Reviews Immunology 18, 297–308.

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WHO, 2007. The World Health Report 2007: A Safer Future: Global Public Health Security in the 21st Century. Wong, C.H., Siah, K.W., Lo, A.W., 2019. Estimation of clinical trial success rates and related parameters. Biostatistics 20, 273–286.

Relevant Websites http://epidemics.events.nejm.org/#/media/3222 Epidemics going viral: Innovation vs. nature. https://cepi.net/about/governance/ Governance CEPI. www.who.int/blueprint/en/ R&D Blueprint. https://www.reuters.com/article/us-gsk-results-idUSKBN1X91EK Reuters. https://www.weforum.org/events/world-economic-forum-annual-meeting-2017/sessions/cepi-a-global-initiative-to-fight-epidemics World Economic Forum Annual Meeting.

Vaccines Against Viral Gastroenteritis Scott Grytdal and Tyler P Chavers, Centers for Disease Control and Prevention, Atlanta, GA, United States Claire P Mattison, Centers for Disease Control and Prevention, Atlanta, GA, United States and Cherokee Nation Assurance, Arlington, VA, United States Jacqueline E Tate and Aron J Hall, Centers for Disease Control and Prevention, Atlanta, GA, United States Published by Elsevier Ltd.

Glossary Acute gastroenteritis An inflammation of the stomach and/or intestines, often characterized by symptoms of vomiting and diarrhea lasting less than 14 days. Correlate of protection A measurable sign that a person is protected against becoming infected or developing illness from a disease. Genogroup A group of related viruses within a genus, which can then be further broken down into genotypes. Genotype Subclassification of related viruses within a genogroup based on shared genetic sequences. Intussusception Intestinal obstruction that results when a part of the intestine folds into the section next to it.

Mono-/polyvalent Having specific immunologic activity against one or many antigens, viral strains, or diseases (e.g., bivalent, trivalent). Reassortant Having a genome consisting of parts derived from the genomes of two (or more) different viruses. Seroconversion The development of detectable antibodies in the blood that are directed against an infectious agent. Vaccine effectiveness The percentage reduction of disease or outcome in a vaccinated group of people in comparison to an unvaccinated group under typical field or uncontrolled conditions. Vaccine efficacy The percentage reduction of disease or outcome in a vaccinated group of people in comparison to an unvaccinated group under ideal conditions (e.g., a randomized control trial).

Introduction Each year, an estimated 2 billion cases of acute gastroenteritis (AGE) occur worldwide, resulting in more than 1 million deaths. Nearly 40% of AGE cases occur among children o5 years of age. Numerous bacterial, parasitological, and viral pathogens cause AGE, but viruses cause the majority of these infections. Among viruses that cause AGE, rotaviruses and noroviruses contribute the largest burden. Rotavirus disease typically occurs in young children; in contrast, noroviruses causes illness across all age groups. Currently, rotavirus vaccines are the only available preventive vaccines against viral gastroenteritis. Prior to rotavirus vaccine development and availability, rotavirus caused approximately 2 million hospitalizations and a median 440,000 deaths in children o5 years of age annually. By 5 years of age, nearly every child experienced an episode of rotavirus gastroenteritis. To reduce rotavirus burden, in 2006, two rotavirus vaccines were licensed: Rotarix and RotaTeq. In 2009, the World Health Organization (WHO) recommended rotavirus vaccines for priority inclusion in national immunization programs worldwide. Today, 4 WHO pre-qualified rotavirus vaccines have been introduced into the national immunization programs of over 100 countries, with several more in development. These vaccines have been associated with a median 59% reduction in rotavirus hospitalizations and 36% in AGE hospitalizations globally. However, rotavirus is still estimated to cause 258 million episodes of diarrhea and 128,500 deaths among children o5 years of age annually. Approximately half of all rotavirus deaths occur in just four countries: India, Nigeria, Pakistan, and the Democratic Republic of the Congo, while in high-income countries, deaths from AGE are uncommon. With the dramatic decline of rotavirus gastroenteritis, norovirus has become recognized as a leading cause of severe pediatric AGE in some countries that have introduced the rotavirus vaccine. Noroviruses are a diverse group of viruses within the family Caliciviridae and cause an estimated 684 million illnesses and 212,000 deaths worldwide annually. Similar to rotavirus, low- and middle-income countries (LMICs) carry the largest burden, accounting for 82% of norovirus illnesses and 97% of norovirus deaths worldwide. In the United States, it is estimated that norovirus causes an average of 19–21 million illnesses, 1.7–2.9 million outpatient visits, and 650–1100 deaths every year. In June 2016, WHO’s Product Development for Vaccines Advisory Committee identified norovirus as a priority disease for vaccine development. While several candidate vaccines are in development, there is currently no licensed norovirus vaccine.

Rotavirus Vaccines Rotavirus genotypes follow a binary nomenclature describing G and P genotypes, which are based on two viral proteins (VP) expressed on the exterior of the viral capsid. Glycoprotein VP7 forms the outer capsid shell and determines the G genotype, while protease-sensitive VP4 forms spiked structures and determines the P genotype. These two proteins play critical roles in attachment to host cells, pathogenesis, and immune response. As such, these sites, or epitopes, are usually targets against which vaccines

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generate neutralizing antibodies. Rotavirus vaccine development has used both human and reassortant genotypes in rotavirus vaccine formulations. Reassortant rotavirus genotypes arise when human and non-human strains rearrange and combine naturally or artificially (i.e., in a laboratory) due to their segmented genome structure.

Licensed Rotavirus Vaccines In 1998, the first commercially available vaccine for viral gastroenteritis, RotaShield, a tetravalent, human-rhesus reassortant rotavirus vaccine, was licensed and introduced into the routine immunization program in the United States. Use of this vaccine was suspended in 1999 and it was subsequently withdrawn from the United States market due to an observed increased risk of intussusception, a rare condition characterized by an invagination of the intestine, among children who received the vaccine. In 2006, two live, attenuated, oral rotavirus vaccines, Rotarix and RotaTeq were licensed for use. Rotarix is a human, monovalent vaccine comprised of the G1P[8] strain. It is given as a 2-dose series typically at 2 and 4 months of age. In contrast, RotaTeq is a pentavalent human-bovine reassortant vaccine. Each of its five components are made up of a bovine (WC3) backbone combined with either a G1, G2, G3, G4, or P1A[8] human surface protein. This vaccine is given as a 3-dose series at 2, 4, and 6 months of age. After vaccine introduction, promising efficacy against rotavirus with subsequent reductions in rotavirus AGE were observed in randomized controlled trials (RCTs) in early adopting countries such as the United States, Australia, several Latin American countries, and several European countries (range: 85%–98% efficacy against severe rotavirus AGE). Modest efficacy of these vaccines against severe diarrhea was observed in subsequent clinical trials in Africa and Asia (range: 40%–48% efficacy against severe rotavirus AGE). These clinical trials did not find an increased incidence of severe adverse events (SAEs) nor an increase in incidence of intussusception in vaccine recipients compared to placebo recipients. In 2009, WHO officially recommended rotavirus vaccines for inclusion into national immunization programs worldwide. In recent years, two new rotavirus vaccines manufactured in India have completed clinical trials and are joining the global vaccine market. In 2018, Rotavac (Bharat Biotech, India) and Rotasiil (Serum Institute, India) vaccines obtained prequalification from WHO and will soon be incorporated into national immunization programs in Africa and Asia. Rotavac is a live, oral, monovalent vaccine derived from a naturally occurring G9P[11] reassortant human-bovine strain and is given in a 3-dose regimen at 6, 10, and 14 weeks of age. The strain was chosen as a vaccine candidate as it appears naturally attenuated, causing asymptomatic infection among neonates. Rotavac has shown 56% (95% confidence interval [CI]: 37%–70%) efficacy against hospitalization or supervised rehydration for children o1 year of age and 49% (95% CI: 17%–68%) efficacy among children between 1 and 2 years of age. Phase III trials for Rotavac did not have sufficient sample sizes to detect an increased risk of intussusception, but a surveillance network has been set up in India to monitor post-introduction risk of intussusception associated with this vaccine. Rotasiil is a live, oral, pentavalent humanbovine reassortant vaccine comprised of G1, G2, G3, G4, and G9 strains and is also given in a 3-dose regimen at 6, 10, and 14 weeks of age. It is delivered in either a liquid or freeze-dried (lyophilized) form. The lyophilized product is thermostable at 401C for up to 18 months and requires a liquid buffer to be reconstituted at point of use. In RCTs, Rotasiil has shown efficacy against severe rotavirus gastroenteritis of 67% (95% CI: 50%–78%) in Niger, and 36% (95% CI: 12%–54%) in India; neither trial was powered to detect increased risk of intussusception. Both Rotavac and Rotasiil have not shown evidence of interference with other childhood vaccines given concomitantly, such as oral polio vaccine (OPV). Two other rotavirus vaccines have been used within individual countries but are not available globally. The Lanzhou lamb rotavirus vaccine (Lanzhou Institute of Biological Products, China) is a live, attenuated oral vaccine using a monovalent G10P[12] rotavirus strain isolated from lamb. It has been sold exclusively through the private market in China since 2000. In Vietnam, the internally developed Rotavin M1 (POLYVAC, Center for Research and Production of Vaccines and Biologicals, Vietnam) was first used in 2012. Rotavin M1 is a live, attenuated, frozen oral vaccine that includes a G1P[8] strain, sold exclusively to the Vietnamese private healthcare market. Neither of these vaccines are licensed outside of their respective countries or have been evaluated for further use by WHO.

Vaccine Effectiveness and Impact Post-licensure studies which measure rotavirus vaccine effectiveness (VE) have consistently shown strong protection in upper and upper-middle income countries, and robust albeit lower protection in LMICs. A recent review of global rotavirus impact studies in children o5 years of age found that after rotavirus vaccines were introduced, rotavirus AGE hospitalizations in countries using vaccines were reduced by a median of 59%. Countries with rotavirus vaccine coverage above 85% demonstrated the greatest impact. Reductions also differed by child mortality levels, demonstrating a 66% reduction in low, 59% reduction in medium, and 50% reduction in high child mortality countries. Globally, Rotarix has demonstrated a median VE against any healthcare utilization of 84% in low child mortality countries, 75% in medium child mortality countries, and 57% in high child mortality countries; RotaTeq demonstrated a median VE of 90% in low child mortality countries and 45% in high child mortality countries. Potential explanations for disparity in VE and impact between high and low child mortality countries include differences in nutritional status, gut microbiota, and interference by other childhood vaccinations such as OPV that reduce seroconversion. Despite a lower VE, due to high rotavirus burdens in LMICs, rotavirus vaccines do still prevent and mitigate the impact of rotavirus illness, preventing severe diarrhea, healthcare visits, and deaths in these countries.

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Both Rotarix and RotaTeq are used in the United States where overall rotavirus vaccine coverage was estimated at 73.2% (95% CI: 71.8%–74.6%) in 2015, notably lower than other routine childhood immunizations. In the United States, rotavirus vaccines are associated with a median 80% decline in rotavirus-associated hospitalizations and 57% in rotavirus-related emergency department visits along with indirect protection of unvaccinated age groups and a decrease in health-care costs. Biennial peaks of rotavirus detection have emerged since rotavirus vaccines were introduced into the U.S. immunization program. Protection from rotavirus infection does not require an exact match between wild-type and vaccine genotypes. Studies have demonstrated that both the monovalent vaccine Rotarix and pentavalent vaccine RotaTeq show cross-protection against dissimilar genotypes. Prior to vaccine introduction, the G1P[8] strain was the most common rotavirus genotype in humans. A recent systematic review showed that the most common strains after vaccine introduction were G2P[4] (50%) and G1P[8] (22%) in countries using Rotarix, and G1P[8] (33%) and G2P[4] (30%) in countries using RotaTeq, although dominant subtypes vary by time and location. Vaccine performance may also be modulated by the presence of maternal antibodies against rotavirus transferred through the placenta or present in breast milk. These antibodies typically lessen severity and reduce incidence of disease in newborns up to approximately 2 months of age. It has been hypothesized that these maternal antibodies may also neutralize rotavirus vaccine particles before the child can initiate an immune response and thus lower their effectiveness. Further studies are required at this time, as different studies have shown either reduced vaccine seroconversion when high levels of rotavirus-specific IgG were present or no impact on vaccine response when breastfeeding was withheld.

Rotavirus Vaccines in Development Several rotavirus vaccine candidates are currently in development. RV3-BB is a vaccine that aims to confer immunity in the neonatal period even in the presence of maternal antibodies; it recently completed Phase IIb trials in Indonesia. Other vaccines in development are parenterally administered, non-replicating vaccines, which potentially bypass issues of maternal antibody interference, intestinal replication (potential risk of intussusception), and interference with other childhood vaccines such as OPV. The P2-VP8-P[8] and VAC 041, P2-VP8-P[4]P[6]P[8] subunit vaccines have completed dose escalation trials and Phase I/II trials are currently underway in South Africa. Other non-replicating candidates in early development stages include a VP6 subunit that uses norovirus virus-like particles (VLP) in a combination vaccine; two inactivated rotavirus vaccines (IRV) using the G1P[8] strain; a truncated VP4 vaccine derived from the Lanzhou Lamb vaccine; and an inactivated polio vaccine(IPV)-IRV combination using the G1P[8] strain.

Rotavirus Vaccine Safety Monitoring After a decade of recommendation by WHO, Rotarix and RotaTeq have demonstrated substantial impact on reducing the global burden of rotavirus disease; the largest effect has been observed on severe disease and hospitalization. Post-licensure safety evaluations have detected a low-level risk of intussusception in some high- and middle-income countries, although the risk-benefit ratio remains favorable towards rotavirus vaccinations due to their substantial impact on disease burden. Additionally, no increased risk of intussusception has been observed in low-income countries to date; however, current safety data are limited and additional studies are on-going. WHO continues to recommend rotavirus vaccine use in all countries as the small increased risk in intussusception remains greatly outweighed by the benefits of rotavirus vaccination.

Norovirus In 2016, WHO identified norovirus as a priority disease for vaccine development. While there is currently no vaccine available to prevent norovirus disease, many candidate norovirus vaccines exist, including those in Phase I and Phase II trials, as well as many others in preclinical stages. The wide diversity of noroviruses and the limited understanding of naturally acquired norovirus immunity have complicated vaccine development. In addition, there is no single, established correlate of protection, although many candidates have been explored, including serum histo-blood group antigen (HBGA)-blocking antibodies, serum hemagglutination inhibition antibodies, salivary, serum, and fecal IgA, and virus-specific IgG memory B-cells. Noroviruses are an extremely diverse group of viruses. Based on the phylogenetic clustering of the major capsid protein VP1, there are 10 genogroups, of which GI, GII, and GIV infect humans. Genogroup GII is the predominant norovirus genogroup circulating worldwide; within that genogroup, genotype GII.4 is the most common genotype in many parts of the world, with a new GII.4 strain typically emerging every 2–4 years, sometimes associated with an increase in norovirus activity. Recently, norovirus genotyping expanded to include simultaneous typing of both the capsid and polymerase genes, allowing surveillance to monitor for recombinant strains. Individual susceptibility to norovirus infection varies based on the fucosyltransferase-2 (FUT2) gene, which controls the expression of HBGAs to which norovirus binds. Individuals with polymorphisms in the FUT2 gene are known as “non-secretors” and make up 20%–30% of African and European populations. Non-secretors are less susceptible (but not completely immune) to norovirus GII.4 infection than secretors; however, they can still become infected with non-GII.4 norovirus strains.

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Naturally acquired immunity to norovirus post-infection is not well understood. Challenge studies in adults have shown short-term, strain-specific immunity, lasting from 6 months to 2 years, while modeling studies have estimated immunity lasts 4–8 years. Immunity is thought to be homotypic, with greater protection from strains in the same genogroups compared to other genogroups. Repeat infections by the same genotype are rare, but repeated infections by the same genogroup have been observed, suggesting that immunity is genotype specific. Since norovirus immunity is thought to be genogroup- or genotype-specific, determining which genotypes to include in vaccine formulations is an important consideration. Current vaccine candidates have used both GII.4 as well as GI.1 genotypes; however, norovirus vaccine candidates may need to consider the addition of other genogroups, or update formulations periodically as new strains emerge.

Norovirus Vaccine Development Norovirus candidate vaccines utilize a variety of different technologies, including non-replicating VLPs, P particles, and recombinant adenoviruses. VLPs are multi-protein structures that have no genetic material and are therefore noninfectious. However, they can interact with cellular receptors and elicit an immune response. P particles resemble the P domain of norovirus, which is the domain that binds to HBGAs. Recombinant adenoviruses expressing the major norovirus capsid protein VP1, through which they are able to elicit an immune response. Due to the lack of a cell culture system prior to 2016, whole killed or live-attenuated viruses have not been previously pursued for norovirus vaccine development. The recently developed human intestinal enteroid (“mini-gut”) system for in vitro norovirus replication is expected to provide many new avenues of norovirus research and enhance norovirus vaccine development.

Norovirus Vaccines in Human Clinical Trials There are currently four single- and multi-dose norovirus vaccine candidates in human clinical trials. A bivalent intramuscular GI.1/GII.4 VLP vaccine being developed by Takeda Pharmaceutical Company Limited is in Phase IIb trials; an oral vaccine being developed by Vaxart, Inc. is in Phase I trials; a tetravalent vaccine is being developed by the Chinese Academy of Sciences; and a bivalent GI.1/GII.4 vaccine is being developed by the National Vaccine & Serum Institute in China. Takeda’s GI.1/GII.4 vaccine was initially developed as a monovalent GI.1 VLP vaccine. In Phase I trials, two GI.1 vaccine doses 21 days apart induced a 4.8-fold increase in norovirus-specific IgG and a 9.1-fold increase in norovirus-specific IgA, while also increasing norovirus-specific IgG and IgA memory B-cells in 92%–100% of participants. When challenged with a homologous GI.1 strain, vaccinated participants had a 47% reduction in norovirus illness and a 26% reduction in norovirus infection. In pre-clinical trials in rabbits, the reformulated bivalent vaccine containing GII.4 and GI.1 VLPs induced broadly reactive antibodies to heterologous GI.1, GII.1, GII.3, and GIV noroviruses. Phase II studies have focused on the bivalent GI.1/GII.4 formulation and have trialed both single-dose and multi-dose vaccines. The bivalent vaccine has demonstrated a rapid immune response, including higher pan-Ig, IgA, and HBGA-blocking antibodies. While the duration of elevated immune responses is unknown, antigen-specific IgG memory B-cells persist until at least 180 days post-vaccination, while other responses have lasted over a year. In a GII.4 norovirus challenge study, two intramuscular injections of the bivalent vaccine did not significantly decrease the incidence of pre-defined norovirus disease compared to placebo. However, reductions in mean viral shedding and illness severity were observed among vaccinated participants. No SAEs related to vaccination were reported in any of the aforementioned studies. Data from a Phase IIb field efficacy study in military recruits (NCT02669121) were recently reported. Recruits were randomized to receive a placebo or single intramuscular injection of bivalent GI.1/GII.4 vaccine and followed for 45 days afterwards. Vaccine efficacy against moderate or severe AGE caused by any norovirus strain was 61.8% (95% CI: 20.8–81.6); however, due to the low number of norovirus infections in recruits, vaccine efficacy against homotypic infection could not be calculated. Multiple other Phase II trials have recently been completed, including a trial in infants 6 weeks to children 8 years old (NCT02153112), and an evaluation of safety and immune responses in those older than 60 years (NCT02661490), as well as a 5-year Phase II trial in adults and the elderly (NCT03039790). A second norovirus vaccine currently in human clinical trials is an oral vaccine being developed by the biotechnology company Vaxart, Inc. This single dose vaccine formulation would be delivered as an oral tablet for adults or as a liquid for children and is stable at ambient temperature, an advantage over other vaccines. The vaccine uses a recombinant adenovirus expressing the norovirus GI.1 major capsid protein (VP1). Two Phase I trials have been completed. A Phase I trial testing the safety and immunogenicity of both low- and high-dose formulations found they were well tolerated and no SAEs were reported. After one administration of the high dose vaccine, 78% of participants showed a significant immune response with a 2-fold increase in HBGA-blocking assays (BT50s), increased IgA and IgG memory B-cells, and increased fecal IgA. Vaxart has reported that the vaccine has been well tolerated with no SAEs reported. Vaxart has also trialed a bivalent GI.1/GII.4 VP1 vaccine in a Phase Ib trial, alongside monovalent GII.4 and GI.1 formulations. The trial reported no SAEs and demonstrated robust immunogenicity. It was also reported that the study met all primary endpoints with no SAEs reported. A Phase II dose confirmation study is planned to begin in 2020. The two other vaccines are beginning clinical trials. The tetravalent GI.1/GII.3/GII.4/GII.17 vaccine being developed by the Institut Pasteur of Shanghai under the Chinese Academy of Sciences received a permit in May 2019 to begin clinical trials. In

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addition, at its National Vaccine & Serum Institute, China is recruiting participants aged 6 months to 59 years in a Phase I clinical trial for a bivalent intramuscular GI.1/GII.4 VLP vaccine.

Norovirus Vaccines in Pre-Clinical Development An intramuscular, trivalent norovirus GI.3, GII.4, and rotavirus VP6 vaccine is being developed by the University of Tampere, Finland, the Daiichi Sankyo Company Limited, & UMN Pharma Inc., Japan. When vaccinated with the trivalent vaccine, mice showed an immune response that persisted for 24 weeks post-vaccination. The addition of rotavirus VP6 protein showed an adjuvant effect, increasing cross-reactive IgG and norovirus-specific blocking antibodies. Recently, research has evaluated the impact of including CVB1 VLPs to the vaccine to protect against coxsackievirus B (CVB), an enterovirus that can cause severe disease in infants. Addition of CVB1 VLPs did not interfere with immune responses to the norovirus or rotavirus VLPs. A trivalent vaccine containing a fusion of hepatitis E, norovirus GII.4, and astrovirus P particles has been designed by the Cincinnati Children’s Hospital Medical Center, University of Cincinnati, and Virginia Polytechnic Institute and State University. This candidate vaccine has been shown to produce a 1.9-fold higher norovirus IgG titer than immunization with norovirus P particle alone in mice and demonstrates the potential for a P particle vaccine to vaccinate against multiple diseases. The Ohio State University recently published the results of a pre-clinical trial of an oral, lactic acid bacteria based GII.4 norovirus vaccine candidate on gnotobiotic piglets. Vaccination was found to induce a norovirus-specific immune response and prevent norovirus infection in pig intestines when challenged. The Chinese Academy of Sciences in Shanghai has tested a VLP-based bivalent vaccine against norovirus GII.4 and enterovirus 71 (one of the viruses that causes hand, foot, and mouth disease) in mice. Vaccination produced significant increases in both enterovirus 71- and norovirus GII.4-specific antibody responses for up to 14 weeks. Using a recombinant adenovirus expression system, a candidate vaccine expressing the norovirus GII.4 major capsid protein VP1 has been developed by the Chinese Center for Disease Control and Prevention. One study in mice found that this intranasal vaccine increased norovirus IgG and IgA immune responses. The researchers found that administering the recombinant adenovirus expressing norovirus GII.4 capsid protein vaccine before norovirus VLP vaccination produced higher norovirus-specific antibody levels in mice when compared to previous administration of the VLP or multiple VLP vaccinations. These studies show that a recombinant adenovirus expressing norovirus proteins may be able to produce a comparable immune response to norovirus VLP formulations.

Future Considerations for Norovirus Vaccines While norovirus causes a tremendous morbidity burden worldwide, factors such as the vaccine’s cost, effectiveness, and duration of protection will all end up impacting a vaccine’s viability. A modeling study of the potential impact of a norovirus vaccine in the United States found that a vaccine with 50% efficacy and a 12-month duration would avert 1.0–2.2 million illnesses per year but cost $400 million to $1 billion annually; in contrast, one that conferred protection for 48 months could save up to $2.1 billion annually. It is also currently unclear who the target population will be for norovirus vaccination. Modeling studies have shown vaccination would have the greatest impact if it targeted the young (under 5 years old) or the elderly (over 65 years old). Additional potential target populations include those working in health care, childcare, and food service.

CDC Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Names of specific vendors, manufacturers, or products are for identification only and do not imply endorsement by the Centers for Disease Control and Prevention or the US Department of Health and Human Services.

Further Reading Bartsch, S.M., Lopman, B.A., Hall, A.J., Parashar, U.D., Lee, B.Y., 2012. The potential economic value of a human norovirus vaccine for the United States. Vaccine 30 (49), 7097–7104. Bartsch, S.M., Lopman, B.A., Ozawa, S., Hall, A.J., Lee, B.Y., 2016. Global economic burden of norovirus gastroenteritis. PLoS One 11 (4), e0151219. Burke, R., Mattison, C., Pindyck, T., et al., 2020. The burden of norovirus in the United States, as estimated based on administrative data: Updates for medically attended illness and mortality, 2001 – 2015. Clinical Infectious Diseases. ciaa438. Burke, R., Tate, J., Kirkwood, C., Steele, A., Parashar, U., 2019. Current and new rotavirus vaccines. Current Opinion in Infectious Diseases 32 (5), 435–444. Burnett, E., Jonesteller, C., Tate, J., Yen, C., Parashar, U., 2017. Global impact of rotavirus vaccination on childhood hospitalizations and mortality from diarrhea. Journal of Infectious Diseases 215 (11), 1666–1672. Burnett, E., Parashar, U.D., Tate, J.E., 2020. Global impact of rotavirus vaccination on diarrhea hospitalizations and deaths among children o5 years old: 2006–2019. Journal of Infectious Dieases. jiaa081. Cohen, D., Muhsen, K., 2019. Vaccines for enteric diseases. Human Vaccines & Immunotherapeutics 15 (6), 1205–1214. Cortes-Penfield, N., Ramani, S., Estes, M., Atmar, R., 2017. Prospects and challenges in the development of a norovirus vaccine. Clinical Therapeutics 39 (8), 1537–1549.

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GBD Diarrhoeal Diseases Collaborators, 2017. Estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: A systematic analysis for the Global Burden of Disease Study 2015. Lancet Infectious Diseases 17 (9), 909–948. Hallowell, B., Parashar, U., Hall, A., 2019. Epidemiologic challenges in norovirus vaccine development. Human Vaccines & Immunotherapeutics 15 (6), 1279–1283. Jonesteller, C., Burnett, E., Yen, C., Tate, J., Parashar, U., 2017. Effectiveness of rotavirus vaccination: A systematic review of the first decade of global postlicensure data, 2006–2016. Clinical Infectious Diseases 65 (5), 840–850. Lucero, Y., Vidal, R., O’Ryan, G., 2018. Norovirus vaccines under development. Vaccine 36 (36), 5435–5441. Mattison, C., Cardemil, C., Hall, A., 2018. Progress on norovirus vaccine research: Public health considerations and future directions. Expert Review of Vaccines 17 (9), 773–784. Pindyck, T., Tate, J., Parashar, U., 2018. A decade of experience with rotavirus vaccination in the United States – Vaccine uptake, effectiveness, and impact. Expert Review of Vaccines 17 (7), 593–606. Troeger, C., Khalil, I., Rao, P., et al., 2018. Rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5 years. JAMA Pediatrics 172 (10), 958–965.

Human Papillomavirus (HPV) Vaccines and Their Impact Jade Pattyn, Pierre Van Damme, and Alex Vorsters, University of Antwerp, Antwerp, Belgium r 2021 Elsevier Ltd. All rights reserved.

Nomenclature AAHS Amorphous aluminum hydroxyphosphate sulfate AS04 Aluminum hydroxide and 3-O-desacyl-40 monophosphoryl lipid A CFS Chronic Fatigue Syndrome CIN Cervical intraepithelial neoplasia CRPS Complex regional pain syndrome DNA Deoxyribonucleic acid GACVS Global Advisory Committee for Vaccine Safety GMTs Geometric mean titers

Glossary Herd immunity Herd immunity is a form of immunity that occurs when a sufficient proportion of a population (or herd) is immune, through disease or vaccination, conferring indirect protection for individuals who have not developed immunity. Vaccine effectiveness Vaccine effectiveness is the ability of the vaccine to prevent outcomes of interest in the ‘real world’.

HIV Human immunodeficiency virus HPV Human papillomavirus hr High-risk IARC International Agency for Research on Cancer IgG Immunoglobulin G POTS Postural orthostatic tachycardia syndrome RRP Recurrent respiratory papillomatosis VLPs Virus-like particles WHO World Health Organization

Vaccine efficacy Vaccine efficacy is the percentage reduction of disease in a vaccinated group of people compared to an unvaccinated group, using the most favorable conditions (¼ clinical trial conditions). Vaccine immunogenicity Immunogenicity refers to the ability of a vaccine to induce an immune response (antibody- and/or cell-mediated immunity) in a vaccinated individual.

HPV and HPV-Related Diseases Human papillomaviruses (HPV) are the most common viral infections of the reproductive tract. These viruses are easily transmitted. Most sexually active women and men will be infected by HPV at some point in their lives. The peak time for acquiring infection for both women and men is shortly after becoming sexually active (Moscicki, 2007). HPV infections are primarily transmitted by mucosal contact, usually during sexual intercourse. However penetrative sex is not required for transmission, also manual-genital and oral-genital contact is a recognized mode of transmission (Liu et al., 2016). Although most infections with HPV do not cause symptoms or disease, persistent HPV infection with certain high-risk genotypes can lead to precancerous lesions that may progress to become cancerous (zur Hausen, 1977). Cancers caused by HPV include virtually all cancers of the cervix, most anal cancers (88%), and for a substantial fraction vaginal (78%), vulvar (15%–48% depending on age), penile (51%), and oropharyngeal (13%–60% depending on region) cancers (Bray et al., 2018; World Health Organization, 2017). Worldwide, HPV causes up to 4.5% (643,000 cases) of all new cancer cases worldwide (8.6% females and 0.9% males). The global burden of cervical cancer (530,000 cases) is substantially higher than other HPV-related cancers (113,000 cases) (de Sanjose et al., 2018). HPV types 16 and 18 account together for about 70% of cervical cancers in every world region, and HPV31/33/45/52/58 for another 20% (de Martel et al., 2017). In HPV-positive cancers of other anogenital sites, HPV16 is the most common HPV type. HPV6 and 11 have been classified as Group 3 agents (i.e., “not classifiable as to its carcinogenicity to humans”) by the IARC Monographs Program (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012), but are responsible for almost 100% of anogenital warts and recurrent respiratory papillomatosis (RRP) (Gillison et al., 2012). RRP is a rare disease in children and young adults that causes wart-like growths in the upper aero-digestive tract with the risk of airway obstruction (Fortes et al., 2017).

HPV Vaccines Three prophylactic HPV vaccines are currently in use to prevent HPV infection and HPV-related diseases, a bivalent, a quadrivalent, and a nonavalent vaccine (summarized in Table 1). The bivalent (HPV16/18) and quadrivalent (HPV6/11/16/18) vaccines were licensed in 2006–2007 and the nonavalent vaccine (HPV6/11/16/18/31/33/45/52/58) in 2014. Currently available HPV vaccines are made from the purified HPV L1 structural protein that self-assembles to form HPV type-specific empty shells, called virus-like

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particles (VLPs) (Lowy and Schiller, 2006). The VLPs are structured geometrically and antigenically almost identical to the actual virus but contain no viral DNA, and therefore cannot reproduce or cause disease. HPV vaccines are not therapeutic and are not indicated for the treatment of HPV infection or HPV-related disease (Schiller and Lowy, 2018). Hence, all vaccines against HPV are most effective when administered to individuals not yet exposed to HPV, i.e., before sexual debut (Arbyn et al., 2018; Xu et al., 2019). Most countries offer HPV vaccines to girls aged 9–14 years. HPV vaccines are mainly targeted towards girls because cancer of the cervix is the most common HPV-associated cancer. In addition, some countries also offer routine or catch-up vaccination in boys, older adolescent females and young women. The vaccination schedule depends on the age of the vaccine recipient (o15 years ¼ 2-dose schedule, Z15 years ¼ 3-dose schedule). By October 2019, 98 countries (global 51%) had introduced the HPV vaccine in their national immunization program for girls and 16 countries for boys (global 8%) (WHO, 2017).

Vaccine Immunogenicity HPV vaccines prevent HPV-related disease by preventing infection. The mechanism of protection by HPV vaccines is assumed to be mediated by neutralizing antibodies against the major HPV coat protein, L1 (Schiller and Lowy, 2018). Possibly due to better immune activation by parental vaccination than by mucosal infection, and by the use of adjuvants, serological response after vaccination is much stronger (10- to 100-fold higher) than the response after natural HPV infection (Stanley, 2010). Furthermore, long-lived plasma cells continuously generate antibodies and are responsible for long-term HPV-specific antibody persistence shown in several clinical trials (Einstein et al., 2014; Joura et al., 2015; Schwarz et al., 2017). Systemic antibodies generated by HPV immunization are thought to reach the site of infection by active antibody transudation at least in the female genital tract, and by passive exudation at sites of trauma that are supposed to be required for initiation of HPV infection (Pattyn et al., 2019). With HPV vaccines, practically all adolescent and young vaccinees who were initially naïve to vaccine-related HPV types develop an antibody response after vaccination (Einstein et al., 2014; Joura et al., 2015; Schwarz et al., 2017). Data have shown that these vaccine-induced antibody titers peak after the final dose, decline within the first year, then stabilize at a plateau titer (Schiller and Lowy, 2018). Antibody titers after HPV immunization remain high for at least 10 years for the bivalent vaccine with 100% seropositivity (Schwarz et al., 2017; Naud et al., 2014), for at least 9.9 years (Nygard et al., 2015) for the quadrivalent vaccine and at least 5 years for the nonavalent vaccine (World Health Organization, 2017; Joura et al., 2015). The immunogenicity of the bivalent and quadrivalent vaccines was compared in a head-to-head trial. After 60 months, geometric mean titers (GMTs) were consistently higher in those receiving the bivalent vaccine across all age strata (18–45 years) (Einstein et al., 2014). However, the clinical relevance of these findings is unclear as vaccine failures have not yet been unequivocally identified in clinical studies, and so no antibody threshold or other immune measurement that correlates with protection has been defined. In general, the different choices of placebo recipients or control subjects, immunological assays and populations analyzed precluded the most direct comparison of results among the trials with the different HPV vaccines.

HPV Vaccine Impact As the development of cervical cancer may occur decades after HPV infection, regulatory authorities have accepted the use of vaccine-related persistent HPV infections and cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN2–3) as virologic and clinical endpoints in vaccine efficacy trials instead of invasive cervical cancer (Group, 2014). Moreover, using cervical cancer as the outcome in those trials was precluded for ethical reasons. HPV vaccines were licensed based on the demonstration of their clinical efficacy in young adult women. Collecting cervical specimens from girls or young adolescents is generally considered unethical. Results of immunobridging studies comparing vaccine immunogenicity in females aged 9–14 years with that in females aged 15–26 years were therefore used to infer clinical efficacy in the younger age group (World Health Organization, 2017).

Impact on HPV Infection and Precancerous Lesions A meta-analysis by (Drolet et al., 2019) provided updated data on more than 100 studies investigating HPV vaccination impact (Drolet et al., 2019), an impact that was far beyond initial expectations. Clinical trials and post-marketing surveillance of the licensed prophylactic HPV vaccines have demonstrated high efficacy against new persistent high-risk HPV types contained in the vaccine and precancerous cervical abnormalities (Arbyn et al., 2018). Notably, the bivalent vaccine demonstrated a high degree of cross-protection against types 31, 35, and 45 (Lehtinen and Dillner, 2013; Palmer et al., 2019). While the quadrivalent vaccine established more restricted cross-protection, most notably against type 31 (Brown et al., 2009). From a public health perspective, current evidence suggests that the 3 licensed HPV vaccines have relatively similar effectiveness in preventing cervical cancer, mainly caused by HPV16 and 18 (Arbyn et al., 2018). At non-cervical sites, HPV vaccines have been shown to prevent the majority of anal, vulvar and vaginal HPV infections and lesions associated with HPV16 and 18 (Xu et al., 2019). Importantly, reported data on the impact of HPV vaccines is thoroughly consistent across different regions and settings. The protective efficacy of the three vaccines has been maintained throughout their respective observation periods and protection in the long term is likely (Stanley, 2017).

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Impact on HPV-Related Cancer Due to the long latency period between infection and HPV-related cancers, it is still too early to observe a clear vaccination effect on the existing HPV-related cancer incidences. Nevertheless, some indicative observations of a reduced incidence of cervical cancer in vaccinated populations exist (Guo et al., 2018; Luostarinen et al., 2018; Scotland, 2019). Generally, it is expected that the existing HPV vaccines could prevent up to 90% of precursor cancer lesions and HPV-associated cancers (Hartwig et al., 2017).

Impact on Anogenital Warts HPV vaccination programs have also shown to effectively reduce the incidence of anogenital warts (Drolet et al., 2019). The quadrivalent and nonavalent vaccine, which include HPV6 and HPV11, provides a high-level protection against anogenital warts in men and women. In several countries, the introduction of the HPV vaccine was followed by a rapid drop in the prevalence of genital warts. Furthermore, in countries with even moderate vaccination coverage (Z50%), there was evidence of vaccine herd effects, with significant reductions in anogenital wart diagnoses among unvaccinated boys and older women (World Health Organization, 2017; Drolet et al., 2019). Australian active surveillance of pediatric reports suggests a continuous decline in juvenile-onset recurrent respiratory papillomatosis, a disease caused by the perinatal transmission of HPV6/11 infection from an infected mother to her infant, likely due to a very low post-vaccination prevalence of maternal HPV 6/11 infection in Australia (Novakovic et al., 2018; Brotherton, 2019).

Current Status and Future Directions At least 15 countries now have data demonstrating vaccine effectiveness and/or showing falls in vaccine-targeted types, and crossprotective types especially for the bivalent vaccine, following HPV vaccination (Brotherton, 2019). Falls are the largest with higher coverage and multiple cohorts vaccinated (World Health Organization, 2017). These results reinforce the need for vaccinating a high proportion of the targeted population to maximize and accelerate the direct and herd effects of HPV vaccination (Drolet et al., 2019), such as successfully demonstrated in the Australian mass catch up program (Drolet et al., 2019). Notably, HPV vaccines can be used in persons who are immunocompromised and/or HIV-infected. Nevertheless, although the evidence base to support the immunogenicity of HPV vaccines in immunocompromised and/or HIV-infected persons is reasonable, the evidence base to support the efficacy of HPV vaccines in these populations remains inconsistent (Lacey, 2019). Recently, studies found high vaccine effectiveness following a single dose of bivalent or quadrivalent vaccine (Kreimer et al., 2018; Safaeian et al., 2018; Sankaranarayanan et al., 2018; Stanley and Dull, 2018). However, the interpretation of trials, which included women with incomplete vaccination schedules, is limited by several factors including that women were not randomized by the number of doses, the sample size, and the low number of incident or persistent infections. Several randomized controlled trials are ongoing to evaluate the efficacy of a one-dose regimen. If effective, a reduction in program costs achieved by implementing a onedose schedule would make it a very attractive option to public health authorities and governments. Some other prospects to improve immune-based prevention of HPV-associated cancers include: Delivery of HPV vaccination as part of childhood immunization, development of lower-cost locally manufactured vaccines (WHO, 2019), and L2- vaccines, which might have potential for broad HPV coverage since the minor capsid protein L2 contains type-common epitopes (Brotherton, 2019). Of importance, HPV vaccination does not eliminate the need for effective cervical cancer screening in the HPV-vaccinated cohorts, mainly because the vaccines do not protect against all oncogenic HPV types. However, vaccination has the potential to greatly decrease the intensity, cost, and morbidity associated with screening. It is clear that, together, HPV vaccination (the ultimate long-term preventive strategy) and screening using more sensitive HPV tests could dramatically alter the landscape of HPV-related cancers. However, the effective implementation of HPV vaccination and screening globally remains a challenge, as well as vaccineimpact evaluations (Wong et al., 2011).

Vaccine Safety and Confidence Data from clinical trials and post-licensure surveillance conducted in several continents are reviewed on a regular basis. Data from all sources continue to be reassuring regarding the safety of HPV vaccines. Over 12 years of research and monitoring have shown that HPV vaccines are very safe (Arbyn et al., 2018; Suragh et al., 2018; Bonaldo et al., 2019). The side-effects of the licensed HPV vaccines are comparable with those of other vaccines. These include predominantly mild and transient local reactions at the site of injection (erythema, pain or swelling). Reported systemic adverse events of minor nature included headache, dizziness, myalgia, arthralgia, and gastrointestinal symptoms (nausea, vomiting abdominal pain). No patterns of serious adverse events were reported in trials or postlicensure surveillance suggesting a causal relation to the vaccine (World Health Organization, 2017; Arbyn et al., 2018). Although case reports have identified a range of new-onset chronic conditions occurring after vaccination, including autoimmune diseases, a wellconducted population-based study on post-licensure safety surveillance showed no association between HPV vaccine and such conditions. Concerns have been raised about complex regional pain syndrome (CRPS), postural orthostatic tachycardia syndrome (POTS) and Chronic Fatigue Syndrome (CFS) following HPV vaccination (World Health Organization, 2017; Arbyn et al., 2018). Despite the

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difficulties in diagnosing these disorders, reviews of pre-and post-licensure data provide no evidence that there is a causal association with HPV immunization. The safety of the HPV vaccine has been also regularly reviewed by the Global Advisory Committee for Vaccine Safety (GACVS) of the World Health Organization (WHO) who have not identified any safety concerns and classified HPV vaccines as “extremely safe”, which is encouraged by the statements of many other national agencies. Unfortunately, misinformation, insufficient preparedness and inadequate crisis response have led to controversy about the safety of HPV vaccines (Vorsters and Damme, 2018). Although the benefits of HPV vaccines are already very apparent in countries where the HPV vaccination programs have been implemented effectively, and despite robust evidence of HPV vaccine safety, uptake has remained low, also in many high-income countries (Bloem and Ogbuanu, 2017). To increase uptake, the lack of HPV vaccination confidence among families and healthcare providers needs to be overcome and decision-makers need to be up to date with the impact and importance of HPV vaccination.

Models on Impact and Cost-Effectiveness of HPV Vaccination HPV vaccination programs have been shown to be cost-effective in a wide range of settings worldwide (World Health Organization, 2017). Nevertheless, assessment of the cost-effectiveness of HPV vaccines is heavily influenced by parameters including vaccine price, operational costs, HPV prevalence, number of vaccine doses, and uptake of cancer screening and treatment, especially in resource-constrained settings. Global cost-effectiveness analysis informed by country-based evidence suggests that vaccinating preadolescent girls is usually cost-effective, particularly in resource-constrained settings where alternative cervical cancer prevention and control measures often have limited coverage (World Health Organization, 2017). Although extending vaccination to older ages (known as HPV-FASTER (Bosch et al., 2016)) or vaccinating boys as well as girls, could increase the speed and extent of the impact on HPV-related cancer outcomes, the cost-effectiveness of such strategies need to be established by public health authorities in the context of their own disease burden, economic and social priorities.

References Arbyn, M., Xu, L., Simoens, C., Martin-Hirsch, P.P., 2018. Prophylactic vaccination against human papillomaviruses to prevent cervical cancer and its precursors. Cochrane Database of Systematic Reviews 5, Cd009069. doi:10.1002/14651858.CD009069.pub3. Bloem, P., Ogbuanu, I., 2017. Vaccination to prevent human papillomavirus infections: From promise to practice. PLOS Medicine 14 (6), e1002325. doi:10.1371/journal. pmed.1002325. Bonaldo, G., Vaccheri, A., D'Annibali, O., Motola, D., 2019. Safety profile of human papilloma virus vaccines: An analysis of the US Vaccine Adverse Event Reporting System from 2007 to 2017. British Journal of Clinical Pharmacology 85 (3), 634–643. Bosch, F.X., Robles, C., Diaz, M., et al., 2016. HPV-FASTER: Broadening the scope for prevention of HPV-related cancer. Nature Reviews Clinical Oncology 13 (2), 119–132. Bray, F., et al., 2018. Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians 68 (6), 394–424. Brotherton, J.M.L., 2019. Impact of HPV vaccination: Achievements and future challenges. Papillomavirus Research 7, 138–140. doi:10.1016/j.pvr.2019.04.004. Brown, D.R., et al., 2009. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16–26 years. The Journal of Infectious Diseases 199 (7), 926–935. de Martel, C., et al., 2017. Worldwide burden of cancer attributable to HPV by site, country and HPV type. International Journal of Cancer 141 (4), 664–670. de Sanjose, S., et al., 2018. Burden of human papillomavirus (HPV)-related cancers attributable to HPVs 6/11/16/18/31/33/45/52 and 58. JNCI Cancer Spectrum 2 (4), pky045. Drolet, M., Benard, E., Perez, N., Brisson, M., 2019. Population-level impact and herd effects following the introduction of human papillomavirus vaccination programmes: Updated systematic review and meta-analysis. Lancet 394 (10197), 497–509. doi:10.1016/s0140-6736(19)30298-3. Einstein, M.H., et al., 2014. Comparison of long-term immunogenicity and safety of human papillomavirus (HPV)  16/18 AS04-adjuvanted vaccine and HPV-6/11/16/18 vaccine in healthy women aged 18–45 years: End-of-study analysis of a Phase III randomized trial. Human Vaccines & Immunotherapeutics 10 (12), 3435–3445. Fortes, H.R., et al., 2017. Recurrent respiratory papillomatosis: A state-of-the-art review. Respiratory Medicine 126, 116–121. Gillison, M.L., et al., 2012. Human papillomavirus and diseases of the upper airway: Head and neck cancer and respiratory papillomatosis. Vaccine 30 (Suppl 5), F34–F54. Group, I.H.W., 2014. Primary End-Points for Prophylactic HPV Vaccine Trials. International Agency for Research on Cancer. Guo, F., Cofie, L.E., Berenson, A.B., 2018. Cervical cancer incidence in young U.S. females after human papillomavirus vaccine introduction. American Journal of Preventive Medicine 55 (2), 197–204. Hartwig, S., et al., 2017. Estimation of the overall burden of cancers, precancerous lesions, and genital warts attributable to 9-valent HPV vaccine types in women and men in Europe. Infectious Agents and Cancer 12, 19. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012. Biological agents. Volume 100 B. A review of human carcinogens. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 100 (Pt B), 1–441. Joura, E.A., et al., 2015. A 9-valent HPV vaccine against infection and intraepithelial neoplasia in women. The New England Journal of Medicine 372 (8), 711–723. Kreimer, A.R., et al., 2018. Evidence for single-dose protection by the bivalent HPV vaccine – Review of the Costa Rica HPV vaccine trial and future research studies. Vaccine 36 (32 Pt A), 4774–4782. Lacey, C.J., 2019. HPV vaccination in HIV infection. Papillomavirus Research 8, 100174. Lehtinen, M., Dillner, J., 2013. Clinical trials of human papillomavirus vaccines and beyond. Nature Reviews Clinical Oncology 10 (7), 400–410. doi:10.1038/nrclinonc.2013.84. Liu, Z., Rashid, T., Nyitray, A.G., 2016. Penises not required: A systematic review of the potential for human papillomavirus horizontal transmission that is non-sexual or does not include penile penetration. Sex Health 13 (1), 10–21. Lowy, D.R., Schiller, J.T., 2006. Prophylactic human papillomavirus vaccines. Journal of Clinical Investigation 116 (5), 1167–1173. Luostarinen, T., et al., 2018. Vaccination protects against invasive HPV-associated cancers. International Journal of Cancer 142 (10), 2186–2187. Moscicki, A.B., 2007. HPV infections in adolescents. Disease Markers 23 (4), 229–234. Naud, P.S., et al., 2014. Sustained efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine: Final analysis of a long-term follow-up study up to 9.4 years post-vaccination. Human Vaccines & Immunotherapeutics 10 (8), 2147–2162.

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Novakovic, D., et al., 2018. A prospective study of the incidence of juvenile-onset recurrent respiratory papillomatosis after implementation of a national HPV vaccination program. The Journal of Infectious Diseases 217 (2), 208–212. Nygard, M., et al., 2015. Evaluation of the long-term anti-human papillomavirus 6 (HPV6), 11, 16, and 18 immune responses generated by the quadrivalent HPV vaccine. Clinical and Vaccine Immunology 22 (8), 943–948. Palmer, T., et al., 2019. Prevalence of cervical disease at age 20 after immunisation with bivalent HPV vaccine at age 12–13 in Scotland: Retrospective population study. The BMJ 365, l1161. Pattyn, J., et al., 2019. Infection and vaccine-induced HPV-specific antibodies in cervicovaginal secretions. A review of the literature. Papillomavirus Research 8, 100185. Safaeian, M., et al., 2018. Durability of protection afforded by fewer doses of the HPV16/18 vaccine: The CVT Trial. Journal of the National Cancer Institute 110 (2). Sankaranarayanan, R., et al., 2018. Can a single dose of human papillomavirus (HPV) vaccine prevent cervical cancer? Early findings from an Indian study. Vaccine 36 (32 Pt A), 4783–4791. Schiller, J., Lowy, D., 2018. Explanations for the high potency of HPV prophylactic vaccines. Vaccine 36 (32 Pt A), 4768–4773. doi:10.1016/j.vaccine.2017.12.079. Schwarz, T.F., et al., 2017. Ten-year immune persistence and safety of the HPV-16/18 AS04-adjuvanted vaccine in females vaccinated at 15–55 years of age. Cancer Medicine 6 (11), 2723–2731. Scotland, I., 2019. Cancer Statistics – Female Genital Organ Cancer. Available at: https://www.isdscotland.org/Health-Topics/Cancer/Cancer-Statistics/Female-Genital-Organ. Stanley, M., 2010. HPV – Immune response to infection and vaccination. Infectious Agents and Cancer 5, 19. Stanley, M., Dull, P., 2018. HPV single-dose vaccination: Impact potential, evidence base and further evaluation. Vaccine 36 (32 Pt A), 4759–4760. doi:10.1016/j.vaccine.2018.02.076. Stanley, M., 2017. Tumour virus vaccines: Hepatitis B virus and human papillomavirus. Philosophical Transactions of the Royal Society B: Biological Sciences 372 (1732), doi:10.1098/rstb.2016.0268. Suragh, T.A., et al., 2018. Safety of bivalent human papillomavirus vaccine in the US vaccine adverse event reporting system (VAERS), 2009–2017. British Journal of Clinical Pharmacology 84 (12), 2928–2932. Vorsters, A., Van Damme, P., 2018. HPV immunization programs: Ensuring their sustainability and resilience. Vaccine 36 (35), 5219–5221. doi:10.1016/j.vaccine.2018.06.066. WHO, 2017. Immunization,Vaccines and Biologicals database as of 31 March 2017. Available at: https://www.who.int/entity/immunization/monitoring_surveillance/VaccineIntroStatus.pptx. (Accessed October 2019). WHO, 2019. Global Market Study: HPV Vaccines. World Health Organization. Wong, C.A., et al., 2011. Approaches to monitoring biological outcomes for HPV vaccination: Challenges of early adopter countries. Vaccine 29 (5), 878–885. World Health Organization, 2017. Human papillomavirus vaccines: WHO position paper. Vaccine 35 (43), 5753–5755. doi:10.1016/j.vaccine.2017.05.069. Xu, L., Selk, A., Garland, S.M., et al., 2019. Prophylactic vaccination against human papillomaviruses to prevent vulval and vaginal cancer and their precursors. Expert Review of Vaccines 18 (11), 1157–1166. zur Hausen, H., 1977. Human papillomaviruses and their possible role in squamous cell carcinomas. Current Topics in Microbiology and Immunology 78, 1–30.

Relevant Websites https://www.hpvcentre.net HPV INFORMATION CENTRE. https://www.cdc.gov/hpv/ HPV, the Vaccine for HPV, and Cancers Caused by HPV|CDC. https://www.hpvworld.com HPVWorld. - HPV Research Articles. www.hpvboard.org HPV Prevention and Control Board. https://www.who.int/immunization/policy/position_papers/hpv/en/ Human Papillomavirus (HPV) position paper - WHO. https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-agents/hpv-vaccine-fact-sheet Human Papillomavirus (HPV) Vaccines.

Influenza Vaccination Topi Turunen, Infectious Disease Unit, Espoo, Finland and Finnish Institute for Health and Welfare, Helsinki, Finland r 2021 Elsevier Ltd. All rights reserved.

Introduction Influenza is of great public health significance. The yearly epidemics and occasional pandemics of this respiratory illness give rise to a considerable burden of disease worldwide. Seasonal influenza vaccines are the principal method of preventing influenza infection and complications in humans. Full coverage from the influenza virus and its epidemiology is covered elsewhere in the encyclopedia. Here, the characteristics and epidemiology of influenza viruses are discussed mainly as they relate to vaccination. The bulk of this chapter focuses on seasonal influenza vaccines; vaccination against pandemic influenza is described in the end.

Influenza as a Disease The symptoms of influenza illness include fever, myalgia, headache, chills, sore throat, general malaise, and cough, although children and the elderly may present with less specific symptoms. People at both extremes of age are also more prone to developing complications of influenza such as secondary bacterial pneumonia or exacerbation of an existing pulmonary or cardiovascular condition. Understanding the transmission of influenza is crucial to preventive efforts. The illness is spread by aerosols and droplets when infected people cough or sneeze, as well as by fomites. Notably, viral shedding in infected individuals begins before symptoms appear, and viruses can be transmitted to others during this period as well as during asymptomatic infections that may comprise a large part of all influenza infections. In clinical settings, the diagnosis of influenza illness is typically based on symptoms, possibly aided by laboratory tests. Real-time reverse-transcription polymerase chain reaction (RT-PCR) is often considered the gold standard but antigen tests, serology, and viral culture diagnostic methods are also available. The sensitivity and specificity of tests used for diagnosis are relevant not only for the correct diagnosis and treatment of the patient but also for estimating influenza vaccine effectiveness (discussed in more depth below).

Epidemiology of Influenza Influenza viruses are divided into types A, B, and C – as well as subtypes and lineages – based on their genetics and surface antigens. Typically, there are several types and subtypes of influenza A and B viruses cocirculating during any given season, necessitating the inclusion of several viral strains in each seasonal vaccine. Circulating viruses since the 2009 pandemic have included the A(H1N1)pdm09, A(H3N2), B/Victoria, and B/Yamagata viruses. Influenza viruses cause annual global epidemics. In the temperate regions of the northern and southern hemispheres, influenza activity is typically pronounced in the fall and winter seasons while in the tropics, influenza activity can occur around the year. Influenza viruses possess the capacity for antigenic drift: A constant change in their surface antigens. This gives circulating influenza viruses an evolutionary edge because the changed viruses may not be recognized by immune systems based on previous exposure (whether natural infection or vaccination). Antigenic drift, combined with waning immunity post-vaccination, necessitates annual reformulation and administration of seasonal influenza vaccines. Occasionally, antigenic shift between different viruses (such as human and animal influenza viruses) occurs and results in a completely novel type of virus. Such viruses, if transmitted easily between humans, can give rise to global pandemics of varying severity. While seasonal influenza vaccines may sometimes provide cross-protection against pandemic influenza strains, the prevention of pandemic influenza will typically require a specifically developed pandemic vaccine.

Brief History of Influenza Vaccines Influenza viruses were first isolated in 1933 and the development of the first influenza vaccines began shortly thereafter. The first commercial vaccines were developed and tested in the United States in the shadow of the Second World War and approved for use in 1945. The first vaccines were composed of whole viruses. Other forms of vaccines (variations of split virion, subunit, and live attenuated vaccines) have been introduced over the years. For most of the time, eggs have been used as a growth medium for vaccine viruses; cell culture became available later. Production methods have been refined over the years; for example, purification steps have been added to reduce the number of unwanted proteins and other substances in the finished product.

Production of Influenza Vaccines Timeline of the Production Process Due to the epidemiology of influenza – in particular, its seasonal nature – the strain composition of the vaccines is updated annually for both northern and southern hemispheres. The strain selection and the ensuing production process take place according to a certain schema each year.

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The choice of virus strains is based on an international surveillance effort. National influenza centers in various countries around the world monitor the circulating viruses and send representative samples to the World Health Organization’s (WHO) collaborating centers. The surveillance system aims to detect patterns in genetic and antigenic changes of the viruses that would be the most relevant for protection in the next influenza season. Viral isolates thought to represent the best candidates for the vaccine virus strains are collected, grown in culture media, and distributed to vaccine manufacturers for scaling up production. The production (notably, the growth of viruses in eggs) takes several months and needs to be finished in time for the produced vaccines to be distributed and administered before the next seasonal epidemic. As a result, the consultation to select vaccine strains usually takes place in February for the northern hemisphere and in September for the southern hemisphere. The production delay sometimes gives rise to antigenic mismatch if the circulating strains’ evolution unfolds in an unexpected way through antigenic drift and shift. This may have a negative impact on vaccine effectiveness.

Production Steps Production capacity for influenza vaccines exists in several countries but is mostly centered in the global North and in a handful of private companies. However, nationally manufactured influenza vaccines are also used in many parts of the world. Each manufacturer of influenza vaccines uses its own proprietary process. While production processes differ in some details, all share certain key steps and result in vaccines of comparable safety; immunogenicity can be verified using standardized methods. The key production steps are as follows (please note that recombinant, cell-based, and live-attenuated vaccines do not necessarily share all of these):

• • • • •

Propagation in hen eggs (or other media); Disruption of viral lipid envelope using one of several possible detergents; Purification steps; Establishing the immunogenicity of monovalent vaccine components and combining them in a multivalent (typically, tri- or quadrivalent) formulation; and Insertion in vials, labeling, packaging, allocation of lot numbers.

Dependence on hen eggs for growing the viruses leads to several potential problems. Most obviously, the large number of eggs required presents a limit to how many vaccine doses may be produced. A surprising unavailability of eggs, due to, say, an outbreak of avian influenza, may halt production. On the other hand, a problem of “egg adaptation” – where egg-grown viruses change in a way that does not resemble the circulating ones – has been suspected to partially account for lower vaccine effectiveness against some strains – A(H3N2), in particular. The production of live-attenuated influenza vaccines (LAIV) is somewhat different. LAIV is based on attenuated master donor strains that are cold-adapted (i.e., can replicate in the relatively cooler temperature of human upper airways but not the lower airways) and thus, should not cause significant illness in humans. The production process makes use of the segmented genome of the virus that permits genetic reassortment. Surface antigen-encoding genes of newly emergent wild-type viruses are inserted into master donor viruses; the resulting virus passes the attenuation characteristic to its progeny but expresses the relevant antigens for the season.

Influenza Vaccine Composition All influenza vaccines contain viral proteins, of which hemagglutinin (HA) is thought to be the main immunogen. Standardization processes aim to keep HA levels in vaccines constant. Other influenza surface and structural viral antigens present in vaccines include neuraminidase (NA), matrix protein, and nucleoprotein. They may contribute to the vaccine-elicited immune response but their levels are typically not quantified. Adjuvants are used in certain vaccine formulations to increase the immunogenicity and effectiveness of the vaccine. Commerciallyused adjuvants include emulsions of oil and water (AS03 and MF59), aluminum compounds, and influenza virosomes (where the presence of influenza viral lipids is thought to contribute to a greater immune response). Modern vaccine production techniques use sequential purification steps to reduce the number of unnecessary or unwanted substances in the vaccine. However, depending on the specific production method of the vaccine in question, trace amounts of the following may be detectable:

• • • •

Egg proteins: In vaccine products grown in hen eggs, trace amounts of proteins, such as ovalbumin, may be present. The amount is typically low enough to be insignificant even in most cases of egg allergy, but may be relevant if past anaphylactic reaction (see Section “Safety and Contraindications” below). Antibiotics are sometimes used to prevent bacteria from growing in the virus cultures. In the case of influenza vaccines, these are typically aminoglycosides such as gentamicin or neomycin. Penicillins, cephalosporins, and sulfonamides are not used in vaccine production. Detergents used to disrupt viruses (largely removed in purification stage). Formaldehyde, from formalin used to inactivate viruses in inactivated vaccines. While toxic in larger quantities, the trace amounts per vaccine dose are insignificant compared to formaldehyde production in human metabolism and environmental exposure.

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Gelatin may be used as a stabilizer (i.e., to keep the vaccine effective during storage until use). While gelatin is derived from pigs, the form used in vaccines has been purified and hydrolyzed, therefore different from gelatin found in foodstuffs. Muslim and Jewish religious communities have generally accepted the administration of gelatin-containing vaccines. Thiomersal is a mercury-based compound used earlier to prevent the growth of microbes during the production process or in multi-dose vaccine vials. There is no evidence that thiomersal in vaccines cause harm, but it has largely been removed from vaccines in the US and Europe as a precaution. Consequently, most current vaccine products contain no thiomersal even in trace amounts.

Valency Influenza vaccines can be characterized by their valency, or how many viral components they contain. In the history of seasonal influenza vaccines, as little as one (monovalent) and many as five (pentavalent) components have been used, but most seasonal vaccines are nowadays tri- or quadrivalent – that is, they contain both circulating influenza A components (currently, H1N1 and H3N2) and either one or both of the two influenza B components (B/Victoria and B/Yamagata). Pandemic vaccines are usually monovalent. The valency of the vaccine has practical implications if the circulating strains turn out to be different from the ones included in the vaccine. Choosing the “correct” influenza B lineage to be included in trivalent influenza vaccines prove difficult at times. In some influenza B-mismatched seasons, this has resulted in suboptimal vaccine effectiveness but in some seasons, even the trivalent vaccine has demonstrated cross-protection to both influenza B lineages. On the other hand, in seasons with low influenza B activity or a good match to vaccine lineage, the valency of the vaccine has mattered little.

Dosage The HA content of current vaccines is standardized and quantified through a process called single radial immunodiffusion (SRID). The HA level measured by SRID correlates with the immunogenicity of the vaccine. Standard-dose vaccines contain 15 mg of HA per viral strain included in the vaccine (generally three or four for the seasonal trivalent and quadrivalent vaccines, respectively). More recently, high-dose vaccines that contain 60 mg of each strain’s HA have been licensed. The high-dose vaccines are discussed in more depth below.

Formulations of Seasonal Influenza Vaccines Nowadays, many different forms of influenza vaccines exist; others are in development. Many products seek to improve the efficacy and effectiveness of the vaccines, especially in key target groups such as the elderly where the effectiveness of traditional vaccines may be suboptimal. It would be of great interest to public health decision-makers to know if some vaccine products are better than others and which represent the best effectiveness-safety profile for the various target groups of vaccination. However, as of now, few head-to-head comparisons have been made, and drawing too many conclusions from individual studies is problematic for reasons explored in Section “Evaluating the Impact of Influenza Vaccines”.

Traditional Inactivated Vaccines These are the most commonly used influenza vaccines worldwide: egg-based, nonadjuvanted, standard-dose inactivated vaccines. All the other vaccines discussed below differ from these “traditional” vaccines regarding one or more of the above attributes. Most available evidence of influenza vaccine efficacy, effectiveness, and safety derives from studies with these “typical” inactivated vaccines. Even inside this group, inactivated vaccines differ by their valency (see Section “Production of Influenza Vaccines”). Trivalent and quadrivalent seasonal influenza vaccines are most commonly used today; many countries are shifting towards using quadrivalent vaccines.

Recombinant and Cell-Based Influenza Vaccines These vaccine types circumvent the egg-growing phase; all other influenza vaccines are currently grown in hen eggs. This approach is presumed to avoid some of the pitfalls with using eggs (see Section “Production of Influenza Vaccines”). A trivalent recombinant vaccine was licensed in the US in 2013 and has since been replaced by a quadrivalent version. In preparing recombinant vaccines, the virus nucleic acid encoding the surface protein HA is combined with a baculovirus vector that transports the gene into a host cell line to produce the antigen, then collected and purified. Recombinant vaccines have been licensed for people aged 18 and above. There is limited evidence on the comparative efficacy or effectiveness of recombinant vaccines, but one randomized controlled trial (RCT) conducted in the A(H3N2)-predominant 2014-2015 season found a 30% lower probability of influenza infection (in relative terms) in recombinant versus egg-based vaccines. Cell-based vaccines are grown in mammalian cells. A cell-based influenza vaccine production method was first approved by the US Food and Drug Administration (FDA) in 2012. At the time, it still involved egg-grown candidate vaccine viruses but since 2016,

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the process has been changed to be completely egg-free. As with recombinant vaccines, evidence of comparative effectiveness is limited. One observational study looking at the A(H3N2)-dominated 2017–2018 influenza season found a quadrivalent cell-based vaccine to have a relative vaccine effectiveness of approximately 10% points higher than a quadrivalent egg-based vaccine.

Live Attenuated Influenza Vaccines LAIV use cold-adapted weakened live influenza viruses and are administered through an intranasal spray. Because of the cold-adaptation, the viruses can replicate in the upper respiratory tract and cause an immune response similar to that evoked by a natural infection. They do not replicate in the lower airways (thus, limiting the risk of vaccine-induced lower respiratory infection). Live attenuated influenza vaccines have been used in countries of the former Soviet Union since the 1980s. In 2003, another type of LAIV was licensed in the US in a trivalent formulation that has since been replaced with a quadrivalent version. The effectiveness of LAIV seems generally the best in younger age groups, and in the EU, the vaccines are only indicated for people aged 2–17 years (in the US, 2–49 years). Being live virus vaccines, they have somewhat different contraindications than inactivated vaccines. The effectiveness has generally been comparable to that of inactivated vaccines. After the 2009 pandemic, some studies showed reduced effectiveness against A(H1N1), leading US authorities to temporarily recommend against its use in 2016. This recommendation was eventually dropped after the A(H1N1) component of the vaccine was changed.

Adjuvanted Vaccines Adjuvants have been used to increase the immunogenicity of vaccines and result in increased post-vaccination antibody titers. Besides increased immunogenicity, a potential benefit of adjuvanted vaccines is dose-sparing in the face of a large global demand for vaccines such as in the event of a pandemic. However, injection site reactions may be more pronounced with adjuvanted vaccines. The clinical efficacy and effectiveness of adjuvanted vaccines have compared favorably to nonadjuvanted inactivated vaccines, but evidence from direct head-to-head comparisons is scarce. The use of adjuvanted seasonal influenza vaccines focuses on the elderly population. Studies have also been performed in pediatric populations. AS03 and MF59 -adjuvants were used in the 2009 H1N1 pandemic vaccines which were widely used across age groups.

High-Dose Influenza Vaccines A high-dose vaccine resembles the standard-dose inactivated vaccine in composition but contains four times the antigen per viral strain. The higher dose antigen dose is intended to evoke a stronger immune response in the elderly, for whom the vaccine is indicated. To date, available evidence of comparative efficacy/effectiveness is limited, but high-dose vaccines have been associated with better immunogenicity (higher antibody titers post-vaccination) and somewhat better efficacy against laboratory-confirmed influenza (LCI) and hospitalizations. A trivalent high-dose vaccine was licensed for use in the United States in 2009. A quadrivalent formulation has since been developed and FDA approved in 2019. The vaccines are indicated for use in adults aged 65 years and older.

Intradermal Vaccines When a vaccine is injected intradermally (into the dermal layer of skin instead of the muscle), similar immunogenicity can be achieved using less antigen. The vaccination process uses a smaller needle, which some vaccinees will perceive as an upside but may generate more localized redness and swelling. In the United States, intradermal vaccines are available for use in adults aged 18–64 years. A trivalent form has been in use since 2012 and a quadrivalent formulation was approved in 2014.

The Future of Influenza Vaccines The need for annual revaccination and the occasionally suboptimal vaccine performance due to mismatch and other reasons have led to hopes of “universal” influenza vaccines. These would be broadly cross-protective vaccines against conserved antigenic targets on influenza viruses such as the HA stalk domain and other surface components. The idea is that one vaccine could protect against several types of influenza across several seasons, and possibly even pandemic strains. In a universal vaccine, a conserved antigenic target could be presented to the immune system conjugated to another molecule or in a carrier system such as a virus-like particle. Virus-like particles contain viral lipid membrane-anchored proteins in their native state but lack the virus nucleic acid for safety reasons. Some universal vaccine candidates have shown some promise in preclinical and clinical studies but currently, public health professionals must accept that universal vaccines will not be available for some time. Meanwhile, further development of existing vaccines can take place as well as research to identify the vaccine products that have the best safety-effectiveness profile for each of the various target groups.

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New administration methods such as a microneedle patch have been investigated. Patches with small dissolvable needles have demonstrated good immunogenicity and safety in studies. Such a vaccine, much like the current LAIV, could be self-administered, potentially reducing the need for health care personnel in vaccination campaigns. How to address the rare but possible severe allergic reactions should be addressed before launching such campaigns.

Vaccination in Practice Administration Routes Inactivated influenza vaccines are most commonly given by intramuscular, subcutaneous, or intradermal route. Also, intranasal and oral routes have been studied. Live attenuated vaccines are given intranasally.

Timing of Programs Influenza vaccination programs typically begin between October and December in the northern hemisphere and between April and July in the southern hemisphere. In some years, programs may be delayed due to holdup in vaccine production.

Vaccine Uptake Vaccine uptake varies widely between countries and vaccination target groups but is seldom as high as recommended. Efforts beyond the mere biomedical domain are needed to understand why some people at high risk for severe influenza choose to remain unvaccinated even when offered vaccination.

Duration of Protection Most vaccinees develop protective serum antibody levels around two weeks post-vaccination. In principle, the duration of protection is dependent on at least two aspects: changes in the circulating viruses and whether existing antibodies are still protective against the drifted viruses, and waning immunity or reduced antibody levels over time. Protection against influenza for longer than one year has been documented both post-vaccination and after natural infection with influenza virus. Based on evidence from past epidemics and pandemics, natural infection in childhood or young adulthood may provide protection to antigenically similar viruses even several decades later. However, annual immunization is recommended because of reductions in serum antibody levels and because one or more vaccine antigens are usually updated each year. Concerns have been raised that protection conferred by the vaccine might wane even during a given season. This has sometimes been observed in studies that track vaccine effectiveness (VE) at several points during a season. Potential explanations include intraseasonal evolving mismatches between circulating viruses, waning immunity (especially in the elderly), and methodological bias. Regarding the latter, the unvaccinated may develop symptomatic or asymptomatic influenza infections during the season and gain protection from these natural encounters. Cumulative natural encounters in the population may therefore bias VE towards null.

Safety and Contraindications Overview Seasonal influenza vaccines have been prepared in similar ways for decades and their safety is well established. Adverse effects tend to be mild and benign, and serious adverse events are rare. This chapter includes both safety and contraindications of influenza vaccines.

Local and Systemic Inflammatory Reactions The most frequent adverse events of influenza vaccines are local reactions. Characteristic signs of inflammation (pain, redness, swelling) can be observed in up to 65% of vaccinees. The local reactions rarely affect daily activities and generally do not persist for more than one or two days. Systemic inflammatory reactions (fever, myalgia, headache) occur less frequently, in o15% of vaccinees. They are more common in children than adults. Febrile seizures have occasionally been reported in small children, mostly in children under five years of age. The occurrence of local and systemic inflammatory reactions is dependent on vaccine dosage and the presence of adjuvant.

Allergic Reactions Allergic reactions to influenza vaccines may be immunoglobulin E (IgE)-mediated or non-IgE-mediated. Potential antigens that can lead to allergic reactions include egg proteins (such as ovalbumin), trace amounts of the preservative thiomersal (increasingly rare), and possibly – though unlikely – latex or other rubber compounds found in the vaccine vial and introduced in tissue through the needle, puncturing the vial seal.

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Anaphylaxis, a potentially life-threatening but rare serious adverse event of influenza vaccines, is an example of an IgE-mediated reaction and may take place in approximately one per 1,000,000 vaccinations. Persons with a history of anaphylaxis following the administration of an influenza vaccine should not be revaccinated with a similar vaccine. It is, however, important to note that most allergic reactions are not anaphylaxis and rarely prevent revaccination when the vaccine is otherwise indicated. If needed, the vaccine can be given under the physician’s observation, or with sufficient follow-up time.

Rare Adverse Events: Neurological Syndromes Guillain-Barré syndrome (GBS) is a rare condition that has been associated with many infections of the respiratory and gastrointestinal tracts, including naturally-occurring influenza infection. Its symptoms include ascending muscle paralysis and paresthesia – which, when not treated with assisted ventilation, can be fatal. However, most GBS patients do recover. During the 1970s, an increased incidence of GBS (of roughly 1:100,000) was reported in vaccines. This finding was not replicated for several years until, in the 1990s, a small increase (incidence of 1:1,000,000) was observed in vaccinees. In some studies, a protective effect of seasonal influenza vaccines on GBS has been seen. The exact relationship between seasonal influenza vaccination and GBS has been difficult to untangle because of the very low incidence of GBS. It should be noted that even if the one-in-a-million risk of GBS was consistently observed, it would still be outweighed by the protective effect of the vaccine on influenza illness and its severe complications.

Evaluating the Impact of Influenza Vaccines Studies of influenza vaccines’ impact apply various outcome measures. These include immunogenicity, real-life prevention of LCI, and various less specific outcomes such as acute respiratory illness (specifically defined influenza-like illness or any medically-attended respiratory infection), hospitalization, overall mortality, or seroconversion to circulating influenza strains.

Immunogenicity Vaccination induces the production of protective antibodies targeted at influenza virus antigens, most notably HA. The level of HA antibodies can be detected by various methods. In addition to the humoral immune response, the cellular T cell response plays a role in influenza immunity. The strength of the immune response depends on age and previous exposure to influenza viruses and influenza vaccines, as well as underlying health conditions and the formulation of the vaccine (such as dosage and presence of an adjuvant). Usually, antibody levels peak at around two weeks after vaccination but the response may take longer to form and be less pronounced in the elderly. The same is true of persons without any previous exposure; this is why two doses are recommended for vaccine-naïve children. While HA antibodies generally correlate with protection from influenza illness, the correlation between antibody levels and clinically significant protection from influenza illness are not always clear. Therefore, vaccine efficacy and effectiveness are more relevant for vaccination policy-makers, something that has been recently reflected in the regulatory field.

Vaccine Efficacy Vaccine efficacy refers to the percentage reduction of clinical illness between the vaccinated and unvaccinated groups under favorable conditions. This type of information is typically collected in RCTs where the investigators set up an unvaccinated (or vaccinated with a non-influenza vaccine) control group and have control over many variables of the study design. In the presence of vaccination policies recommending annual influenza vaccines to various target groups, running placebocontrolled RCTs on influenza vaccine efficacy has become increasingly rare and the role of real-life vaccine effectiveness studies is becoming more and more important.

Vaccine Effectiveness VE refers to the percentage reduction of clinical illness between the vaccinated and unvaccinated groups under conditions considered typical – or “real-world” situations. This type of information is obtained from observational studies where the vaccinated and unvaccinated groups are not assigned by investigators. Observational study designs used to measure influenza vaccine effectiveness include:

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Cohort studies, where groups of vaccinated and unvaccinated people are followed (either prospectively or retrospectively) and those ill with influenza are identified. Case-control studies, where the vaccination status of people ill with influenza (cases) is compared to those who were not ill (controls). A popular variant is the test-negative design, where people with influenza-like symptoms are screened for the virus and assigned as cases or controls depending on the test result. Screening method, which combines the influenza-positive cases’ individual vaccination status and ecologic data on vaccine coverage in the population where the individual came from.

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Observational study designs have strengths and weaknesses compared to RCTs. RCTs are, by design, better at managing bias and confounding than observational study designs. However, proper choices at study planning, execution, and data analysis stages can considerably mitigate the risk of bias in observational studies. Considering also the cost and feasibility as well as ethical issues, data from observational studies are widely used as a basis for public health policy and are increasingly recognized by regulators. To be informative, observational studies need to obtain the necessary sample size and statistical power. Failure to recruit enough patients is compounded when studying VE in various individuals (e.g., children, adults, elderly) or the VE of several vaccine products to assess brand-specific effectiveness. Multicenter studies with compatible protocols are one way to circumvent this problem. Another approach is to use the comprehensive registers for vaccinations, laboratory findings of influenza, and other covariates needed for analysis that many countries and regions have nowadays.

Understanding Vaccine Efficacy and Effectiveness Influenza vaccines, unlike any other vaccines, need to be administered annually and have variable efficacy and effectiveness. The effectiveness varies across several dimensions – from year to year and between groups of vaccine recipients. Before delving deeper into available evidence on vaccine effectiveness, it is important to recognize some determinants that may influence the results of the studies. Both naturally-occurring variations in vaccine efficacy and effectiveness and topics related to study design and analytical methods must be considered. First, there are biological factors that influence the actual vaccine efficacy or effectiveness:

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Level of match/mismatch between circulating and vaccine viruses. Population studied: Immune responses are typically better in children and healthy adults than with the elderly and the chronically ill. Vaccine type used: Valency, dose, presence of adjuvant, and other topics (see Section “Formulations of Seasonal Influenza Vaccines”) may affect the immune response. Waning protection (see Section “Duration of Protection”). Repeated vaccinations: It has been postulated that vaccinations repeated in subsequent years can cause positive or negative interference with an increased or decreased immune response. Some studies have observed signals of negative interference with certain seasons and viral strains; however, a meta-analysis found no overall evidence that prior season vaccination would impact current-season VE negatively. An increasing number of studies now include vaccination in one or more previous seasons as a covariate in order to better understand this phenomenon. Second, there are factors related to study design and execution that may explain why studies sometimes yield different results:

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Outcome studied: Vaccine effectiveness against LCI is different from vaccine effectiveness against influenza-like illness or overall mortality (only a fraction of which is attributable to influenza). Study power: The optimal sample size depends on the chosen study design, influenza attack rate, vaccine coverage, and expected vaccine effectiveness. Lack of statistical power leads to uncertainty represented by wide confidence intervals which mean the results should be interpreted with caution. Bias that may arise from many different sources and cause the perceived VE to be different from true VE. Are the data on influenza and vaccination status accurate and complete, or can there be misclassification? Are the subjects selected in a representable way? Are vaccinated people on average healthier or more frail than unvaccinated? Are they more or perhaps less likely to seek healthcare services?. Confounding and its adjustment in study design and statistical analysis process. In influenza vaccine studies, typically “crude” and “adjusted” figures are given, with the latter representing correction for confounding and thus likely more representative of a true causal relationship between vaccination and influenza (rather than mere statistical correlation). Multi-center studies and pooling. Often, data from several smaller studies are pooled together in a meta-analysis. This increases the probability to detect an effect of vaccination but requires the individual studies to be sufficiently heterogeneous.

Vaccination of Specific Population Groups As an annually recurring epidemic that routinely attains attack rates of 10% or more and results in many hospitalizations and deaths, influenza is of great public health importance. Effective preventive measures to lessen its impact on the population and health systems are therefore valuable. Among preventive measures, annual seasonal influenza vaccination is likely the single most important one. Who, however, should be vaccinated against influenza, and how effective are the vaccines in the different population groups? Vaccination policy varies between different countries. The United States has recommended annual influenza vaccination of all persons aged six months and above since 2010, but such universal vaccination recommendations are globally rare. Many countries have implemented vaccination programs that address specific risk groups. In the global south, influenza vaccines are seldom prioritized among public health measures and generally utilized less.

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Vaccination policy is a varied and evolving topic. It is not practical to list here all vaccination recommendations around the world; instead, some target groups for vaccination and the rationale for vaccinating them will be discussed. There are some unique challenges related to estimating and communicating the impact of influenza vaccines, but it is crucial to understand that even a suboptimally effective vaccine can provide a large public health impact because of the high incidence of influenza and the potential decrease in disease severity and complications, even when the vaccine does not completely prevent the clinical illness.

Elderly The elderly (often defined as people aged 65 years and above) have long been recognized as a high-risk group for severe influenza and influenza complications. They are among the most universally recommended target groups to receive the annual influenza vaccination. However, antibody responses to vaccination are decreased with age and decreased protective VE has been shown in studies. This puts the elderly in a seemingly paradoxical situation: They are among the ones that would benefit the most from vaccination and also the ones who seem to get the least protection. Because annual influenza vaccination has been recommended to the elderly for a long time, there are few recent placebo-controlled trials. Annual data of vaccine effectiveness are gathered from observational studies. The elderly generally benefit from annual influenza vaccination. Vaccine efficacy or effectiveness against symptomatic LCI has been investigated in many studies and is commonly in the 20%–60% range with considerable season-to-season variation. However, in some seasons, especially when a significant mismatch occurs, statistically significant effects of influenza vaccines have not been found for this age group. Influenza vaccination might also reduce the risk for influenza-related hospitalizations. Some studies have looked into less specific outcomes such as all-cause mortality or any hospitalizations, but these studies run a high risk of bias and should generally be interpreted with caution. The desire to improve vaccine effectiveness among the elderly has led to the development of vaccines intended to promote a better immune response in this population. Besides traditional inactivated vaccines, some vaccines of interest in the elderly group are adjuvanted, recombinant, and high-dose vaccines described more in Section “Formulations of Seasonal Influenza Vaccines”. Still, most effective data in the elderly population are derived from traditional inactivated vaccines.

Underlying Conditions Underlying conditions such as chronic pulmonary, cardiovascular, metabolic, renal, hepatic, neurologic, malignant, and immunosuppressive conditions (due to illness or iatrogenic) are risk factors for complications with influenza and often considered indications for influenza vaccination. However, data on which conditions pose the greatest risk and where might the influenza vaccination be especially important are sometimes sparse and often heterogeneous. Influenza can trigger exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Evidence of the protective effect of influenza vaccines against chronic pulmonary conditions focuses on asthma, where some studies have shown protection against hospitalization and decreased use of bronchodilators and systemic steroid medicines commonly used to alleviate exacerbations of asthma and, therefore, a proxy of clinical benefit. Respiratory infections may also trigger acute vascular events in persons with coronary artery disease, and several studies have suggested a protective effect of influenza vaccination against “heart attacks”. The effect is more pronounced the more severe the underlying atherosclerosis. In some studies, the statin medicines used to improve lipid profiles and decrease coronary event risk have been associated with poorer VE, but data are ambiguous and more research on the topic is needed. Influenza vaccine immunogenicity, efficacy, and effectiveness in patients with renal and hepatic conditions as well as autoimmune conditions such as rheumatic arthritis and inflammatory bowel disease have generally been comparable to that of healthy controls. However, immune responses to vaccination in patients with the latter conditions can be reduced if they are receiving concomitant immunomodulatory therapies. Malignancies are a very heterogeneous group of diseases where the response to the influenza vaccine is greatly dependent on the level of immunocompromise. In conditions such as HIV infection, vaccine immunogenicity correlates with CD4 þ T-lymphocyte cell counts.

Pregnancy In their 2012 position paper, the WHO recommended that countries considering the initiation or expansion of seasonal influenza vaccination programs would give the highest priority to pregnant women. The basis of the recommendation is two-fold: First, pregnancy is an immunosuppressive condition and severe influenza infections can occur during this time, and second, newborns are a risk group for severe influenza but are not eligible to receive influenza vaccine; passive transfer of influenza antibodies from vaccinated women as well as the “cocooning” (i.e., when the child is protected by not having their mother fall ill with influenza) provide the newborns with protection. Both RCTs and observational studies have demonstrated significant protection of mothers and their newborn children against LCI, with vaccine efficacy or effectiveness of 40%–60% VE and above in the infants when the mother is vaccinated.

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Healthy Children During influenza epidemics, higher attack rates have often been observed in children than in adults. It has been postulated that children might be “super spreaders” who drive influenza epidemics in a population. On the other hand, while many children’s influenza infections are mild and self-limiting, influenza can be severe and even life-threatening in small children. For these reasons, some countries such as the United Kingdom and Finland have adopted universal influenza vaccination programs for healthy children (e.g., all children from six months to six or ten years). In the position paper referenced above, the WHO included children aged 6–59 months in additional risk groups to be considered for vaccination. Besides traditional inactivated vaccines, LAIV is relevant in this target group. Where multiple vaccine types are available, vaccination recommendations are sometimes preferential to one particular type of vaccine and sometimes non-preferential (i.e., parents may choose). When a child is vaccinated for the first time with a seasonal influenza vaccine, two doses are recommended to ensure sufficient protection. This is particularly true of inactivated vaccines, where vaccine effectiveness has been shown to be lower among children who received only one dose in their first year of vaccination compared to children who received two doses. With the Ann Arbor-backbone LAIV, one dose has been shown to provide roughly 90% of the protection observed after two doses.

Healthy Adults Few countries prioritize non-pregnant healthy adults as a target group for influenza vaccination. However, this is not to say the vaccine does not work (indeed the vaccine effectiveness is typically higher in adults aged o65 years than in the elderly). Healthy adults can be vaccinated especially if they want to protect themselves from influenza (e.g., because they have a close contact whose age or health condition requires protection from influenza, because of a holiday trip, or if falling ill with influenza would be particularly bad for their work or social situation). Among adults, healthcare workers are an important target group for vaccination, again prioritized by the WHO. The rationale for vaccinating medical personnel is two-fold: Through clinical work, they become exposed to influenza more than the general population, and may also transmit influenza to their patients, some of whom may be frail and at risk of serious infection.

Pandemic Influenza Vaccines Influenza viruses have a considerable pandemic potential because of their capacity for antigenic shift, adaptation to infect multiple species, and their mode of transmission. The occurrence of pandemics is inherently unpredictable but in recent history, influenza pandemics have been observed every couple of decades: 1918, 1957, 1968, and 2009. Much of what is said above about seasonal influenza vaccines applies to pandemic influenza vaccines. A key point to understand regarding vaccine production is that especially in the age of air travel, pandemics may spread very rapidly, and without proper forward planning and production capacity, vaccines may not be available until it is too late (though with several waves of pandemic influenza activity spread over some time, the vaccine may be available for the second wave if not the first one). Ways to enhance preparedness between pandemics include the preparation of collections of candidate vaccine viruses such as H5, H7, and H9 viruses. Regulators such as the FDA and the European medicines agency (EMA) have developed procedures for fast-track licensure of pandemic vaccines. Among other hurdles, pandemic vaccine producers face increased safety concerns. A high-pathogenicity donor virus would have to be grown under high-containment conditions, so manufacturers have turned to other methods, including recombinant techniques and baculovirus vector-expressed vaccine antigens in cell cultures. The use of aluminum or lipid emulsion adjuvants has often been deemed necessary to achieve sufficient immunogenicity. Another appealing aspect of adjuvants is dose sparing (i.e., extending the supply and availability of vaccine doses through less requirement of viral protein per dose). At the time of writing, the most recent pandemic vaccine preparation effort was in the 2009 A(H1N1) pandemic. Here, a vaccine was developed, produced, and administered over only 8 months – though large-scale production was not attained as quickly as initially hoped. The pandemic itself turned out to be relatively mild, though in some cases (especially in clinical risk groups) resulted in severe viral pneumonitis, bacterial sequelae, and death. Compared to seasonal epidemics, there were relatively more severe infections in children and young adults than in the elderly. The adjuvanted vaccines during the 2009 pandemic attained very good effectiveness in many studies. In general, the pandemic vaccines of 2009 had a safety profile comparable to seasonal vaccines and unusual safety issues were not observed in most countries. However, others experienced an increase in narcolepsy, a rare condition characterized by impaired ability to regulate sleep-wake cycles leading to daytime sleepiness and involuntary sleep episodes. Cases of narcolepsy appeared to be associated with the AS03-adjuvanted pandemic H1N1 vaccine in Finland, Sweden, and Iceland. For reasons currently unknown, this was not observed in other countries using that vaccine. Notably, narcolepsy has never been linked to seasonal influenza vaccines. Overall, the experience with the 2009 pandemic demonstrated that adjuvanted pandemic influenza vaccines are immunogenic, effective, and have an acceptably low rate of adverse events. The pandemic response may have contributed to improved preparedness for influenza pandemics in general and reduced barriers to large-scale vaccine manufacture and vaccination program implementation.

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Further Reading Fiore, A.E., Bridges, C.B., Katz, J.M., Cox, N.J., 2018. Inactivated Influenza Vaccines, in Plotkin’s Vaccines, 7th ed. Philadelphia, PA: Elsevier. World Health Organization, 2012. Position Paper on Influenza. Weekly epidemiological record 23 November 2012, 87th year. No. 47, vol. 87, pp. 461–476. Geneva: WHO. World Health Organization, 2017. Evaluation of Influenza Vaccine Effectiveness: A Guide to the Design and Interpretation of Observational Studies. Geneva: WHO.

Relevant Websites https://www.cdc.gov/flu/professionals/acip/immunogenicity.htm Immunogenicity, Efficacy, and Effectiveness of Influenza Vaccines. https://vk.ovg.ox.ac.uk/vk/vaccine-ingredients Vaccine ingredients at the Vaccine Knowledge Project.

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Background An ancient disease first documented in the 2nd millennium BCE and feared worldwide through much of the modern 20th century, polio is nearing eradication. Derived from the Greek words polio (gray) and myelon (marrow), poliomyelitis describes the predilection of poliovirus (genus Enterovirus, family Picornaviridae) to target motor neurons within the gray matter in the anterior horn of the spinal cord, leading to the classic manifestation of poliovirus-induced acute paralysis. Poliovirus is highly infectious, with replication primarily occurring in the intestinal tract of infected individuals. Humans are the only reservoir for poliovirus, with transmission occurring from person-to-person mainly through the fecal-oral route. Over 70% of poliovirus infections in children are asymptomatic, though even infected individuals without symptoms shed virus in stool and can transmit the virus to others. Approximately 24% of infected children develop minor, nonspecific illness without evidence of central nervous system invasion. Aseptic meningitis occurs in 1%–5% of poliovirus-infected children, typically followed by complete recovery. Less than 1% of all poliovirus infections result in acute flaccid paralysis (AFP) and these are classified into three types. Spinal poliomyelitis is most common, characterized by asymmetric paralysis most often involving the legs. Bulbar polio describes the weakness of muscles innervated by cranial nerves. Bulbospinal polio is a combination of bulbar and spinal paralysis. In 1988, wild poliovirus (WPV) was present on all continents except Australia, and estimates of case burden exceeded 350,000 annually. That same year, the Global Polio Eradication Initiative (GPEI) was launched following the early successes of polio eradication efforts in the Americas (initiated by the Pan American Health Organization in 1985) where the last case occurred in 1991. GPEI is a public-private partnership driven by the objective to eradicate polio worldwide, led by national governments and other key partners including the World Health Organization (WHO), US Centers for Disease Control and Prevention, UNICEF, and Rotary International. These initial spearheading partners were later joined in 2007 by the Bill and Melinda Gates Foundation and by GAVI in 2019. By 2011, the number of countries with WPV circulation was only 16, and by 2014 the regions of the Americas, Europe, Southeast Asia and the Western Pacific were certified to be free of WPV. The only countries with endemic WPV cases in 2019 were in South Asia (Pakistan and Afghanistan). The African Region reported its last case of WPV in 2016, and Africa was declared free of WPV in 2020. It is estimated that the efforts of the GPEI in combination with the use of two highly effective poliovirus vaccines have prevented over 16 million cases of paralytic poliomyelitis over the past 30 years. Polioviruses comprise three antigenically distinct serotypes, types 1, 2, and 3. Infection with one serotype confers protection only against viruses of that same serotype. Type 2 wild poliovirus (WPV2) was last detected in 1999 and was declared eradicated in 2015. Type 3 wild poliovirus (WPV3) was last detected in 2012 and it was declared eradicated in 2019. Since 2012, the only WPVs detected have been of type 1 (WPV1). Initial eradication efforts focused on WPVs (i.e., those that circulated naturally in human populations). While GPEI focused on the eradication of WPV, it later became clear that the live, attenuated Sabin oral vaccine strains (see Section “Vaccines”) could very rarely revert and reacquire neurovirulence and transmissibility. For all three serotypes, key sites of reversion include attenuating mutations in the 50 untranslated region as well as additional sites of reversion within the capsid. Reversion can happen quickly during replication within the human gut. These reverted vaccine viruses, termed vaccine-derived poliovirus (VDPV) can emerge in areas with very low vaccine coverage, causing paralytic disease indistinguishable from WPV. A VDPV was originally defined operationally as a poliovirus that was derived from the oral vaccine strains and that diverged 41% from the parental strain in the nucleotide sequence encoding the VP1 capsid protein (i.e., Z10 changes in the B900 nucleotide VP1 region). The threshold of 1% is somewhat arbitrary but is consistent with prolonged replication of the virus. Based on genetic information from several large VDPV2 outbreaks, the threshold for the definition of VDPV2 was modified to a nucleotide divergence 40.6% from the parental OPV strain (Z6 changes in VP1). VDPV are classified as circulating VDPV (cVDPV) when there is evidence for person-to-person transmission; immunodeficiency-associated VDPV (iVDPV) if isolated from a person with primary immune deficiency (primarily deficiencies in humoral immunity, such as hypogammaglobulinemia, combined variable immunodeficiency, or severe combined immunodeficiency); or ambiguous VDPV when the virus cannot be conclusively classified as either cVDPV or iVDPV. To fully eradicate polio, all sources of the virus (wild and vaccine-derived) must be eliminated. Surveillance and vaccination continue to be the most important pillars in efforts toward ensuring poliovirus becomes the second human virus to be eradicated.

Vaccines Two key tools, Sabin live oral poliovirus vaccine (OPV) and Salk inactivated poliovirus vaccine (IPV), have been central to the global eradication effort. Both IPV and OPV contain a representative strain for each of the three poliovirus serotypes (though the type 2 component was removed from OPV in 2016; see below), with vaccination resulting in the production of neutralizing

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antibodies that protect recipients from the disease. Each vaccine has advantages and disadvantages, and appropriate circumstances exist for the use of each. IPV consists of WPV strains that have been chemically inactivated. It provides excellent systemic immunity and is very safe since no infectious virus is present. First licensed in 1955, IPV reduced the number of polio cases by over 99% in the US. IPV has been shown to be capable of eradicating poliomyelitis in settings where the overall burden of viral exposure is low, and the rate of immunization coverage is high (490%). However, IPV does have disadvantages. It is more expensive to produce than the live vaccine and the use of large amounts of wild seed virus in production comes with potential hazards. In addition, while IPV protects vaccinees from disease, it does not induce the intestinal immunity required to prevent person-to-person transmission of poliovirus. In developing countries where viral exposure is more intense, live-attenuated vaccines imitating natural infection often provide better protection. Accordingly, Sabin OPV was introduced in the early 1960s, containing live strains of poliovirus that have been attenuated via passage in animals and cell cultures. OPV provides strong systemic immunity, limiting poliovirus infection and disease in vaccinees. Unlike IPV, OPV also induces the mucosal immunity necessary to interrupt person-to-person transmission. Since recipients excrete vaccine virus into the environment, OPV expands community protection by secondary infection of contacts of vaccinees. OPV is inexpensive, easy to administer, and has been the cornerstone of the global eradication program. It has been demonstrated as an effective vaccine tool to interrupt polio epidemics and break transmission, resulting in the eradication of the virus from entire regions across the world. While vaccination has played a central role in the dramatic decline of poliomyelitis cases, unexpected challenges have become apparent during the final articles of eradication, particularly those associated with the use of Sabin OPV. Much is now known regarding the key mutations underlying the attenuation of Sabin OPV strains and their inherent genetic instability. During replication in the human gut, the attenuated strains are under pressure to adapt and evolve. In rare instances, reversion of key attenuating “gatekeeper” mutations restores virulence to OPV strains, resulting in one case of vaccine-associated paralytic poliomyelitis (VAPP) for approximately every 2.4 million doses of OPV administered, with the highest rate after the first dose. A fundamental piece of the poliovirus eradication strategy is achieving and maintaining high levels of routine immunization. Within that context, VAPP cases are not associated with outbreaks of poliomyelitis. However, in areas of low-vaccine coverage and poor sanitation, where OPV strains have the opportunity to replicate unchecked and transmit for longer durations, the genetic instability of OPV strains can lead to transmission of circulating vaccine-derived polioviruses and spur outbreaks, the most prevalent being type 2 VDPVs. Accordingly, WHO coordinated a synchronized global withdrawal of the type 2 component of OPV in April 2016, replacing trivalent OPV with a bivalent formulation containing only the Sabin type 1 and 3 strains. Prior to the switch, WHO recommended that all countries introduce at least one dose of IPV into their routine immunization program to provide immunity to type 2, which has, in turn, strained the global supply of IPV. Recent clinical trials highlighting the efficacy of using fractional doses (one-fifth the typical amount) of IPV (fIPV) have offered a way forward that may help alleviate both the supply and cost issues for IPV used in this manner. Several countries have already begun to use fIPV for routine immunization. After global eradication of all three serotypes has been certified, all use of OPV in routine immunization is expected to cease. OPV cessation is considered an essential step to eliminate the risk of VAPP and remove the source of potential future VDPV emergence. At that point, continued immunization with IPV will be critical to maintaining eradication.

Poliovirus Surveillance Essential to the eradication program is surveillance for cases of AFP, defined for the purpose of polio surveillance as sudden onset of paralysis or weakness in any part of the body in a child less than 15 years of age. There are multiple etiologies of AFP, including poliovirus, other infectious agents, toxins, autoimmunity, trauma, and congenital conditions. While all these etiologies are medically important and approaches to patient care must consider the cause (or suspected cause) of the illness, GPEI is focused specifically on paralytic cases associated with poliovirus infection. Virologic surveillance (laboratory confirmation that poliovirus is associated with an AFP case) is essential to identifying WPV reservoirs, directing vaccination campaigns, assessing overall program progress, and global certification of eradication. Surveillance quality indicators include timely case detection, notification, and investigation, as well as adequate stool collection and timely transport to the laboratory. The Global Polio Laboratory Network (GPLN) was launched in 1989 to ensure laboratory support for every country’s surveillance system. The GPLN currently consists of 146 laboratories in 92 countries; some large countries have multiple polio laboratories and some laboratories serve neighboring countries that do not have a polio laboratory of their own. Stool specimens collected from children with AFP are tested for the presence of poliovirus by virus isolation in cell cultures and any viruses detected are characterized by poliovirus-specific polymerase chain reaction assays and genomic sequencing. In a typical year, GPLN laboratories test approximately 200,000 stool specimens from AFP cases. Quality of laboratory results is assured through standardized protocols and reagents, annual proficiency testing, ongoing training, and a WHO-led accreditation program. Methods to detect and characterize poliovirus are constantly evolving. Environmental surveillance (detection of poliovirus in sewage-affected waters) is an important adjunct to AFP surveillance, permitting virologic surveillance of a relatively large population (usually tens of thousands to a few million). While environmental surveillance has played a key role in the detection of poliovirus transmission, especially in the latter stages of eradication, it cannot identify the individuals who are shedding the virus. Its main advantage is its ability to detect virus from asymptomatic virus excreters because those individuals would not be identified through AFP surveillance. Environmental surveillance expanded dramatically from

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Fig. 1 Global wild and circulating vaccine-derived poliovirus cases with onset of paralysis from 1 January to 31 December 2019. Red, WPV1 (n ¼ 143, in 2 countries); orange, cVDPV1 (n ¼ 8, in 3 countries); green, cVDPV2 (n ¼ 241, in 15 countries). Not shown: Viruses detected from environmental surveillance. Data from the World Health Organization, as of 31 December 2019.

2013 to 2019. Environmental surveillance is currently conducted in hundreds of sites in more than 50 countries, and expansion to additional countries is planned for 2020–2022. Continued environmental surveillance, especially in high-risk areas prone to VDPV emergence, will be needed to assure final eradication.

Status of Eradication The number of WPV1 cases globally had declined to less than 50 per year in 2016–2018 but the 2019 global total was more than 150 cases, in Pakistan and Afghanistan (Fig. 1). As noted, no WPV has been detected anywhere in Africa since 2016. WPV2 and WPV3 were declared eradicated in 2015 and 2019, respectively. Polio eradication has been difficult to accomplish in countries with environments conducive to poliovirus replication and spread (a high biological infectivity index) and weak routine immunization programs. In recent years, however, the primary barriers have not been biological or environmental. Rather, the difficulties encountered by GPEI have largely been man-made. Some of these include the inability to immunize children in areas under the control of rebels or other anti-government elements, the spread of misinformation regarding vaccine safety, violence against vaccinators and their accompanying security teams, corruption and dishonest practices, as well as simple poor-quality of immunization campaigns. In 2018–2019, immunization was again prohibited by the Taliban in parts of Afghanistan after several years of cooperation. The targeting of polio immunization staff by violence, starting in 2011 in Pakistan, impeded eradication progress, forcing the GPEI to change strategies for advertising immunization campaigns and delivering vaccines. Despite these challenges in the immunization side of the program, AFP surveillance quality in Pakistan and Afghanistan has generally been high since 2015, although there are pockets of lower quality surveillance in areas of insecurity and civil unrest. In 2018 and 2019, there were significantly more paralytic polio cases associated with VDPV than with WPV (Fig. 1). There were 104 cVDPV cases in 2018 and B250 cVDPV cases in 2019, compared to 33 and B150 WPV cases in the two years, respectively. The number of cases associated with VDPV still pales in comparison to the 416 million cases of paralytic polio that have been prevented by GPEI over the past 3 decades. After the switch from Trivalent oral polio vaccine (tOPV) to Bivalent oral polio vaccine (bOPV), cVDPV2 from an ongoing outbreak was detected in Nigeria. Other cVDPV2 outbreaks originating prior to the switch were detected in 2017 (Horn of Africa) and in 2019 (Philippines). Since late 2016, Monovalent type 2 oral polio vaccine (mOPV2) from a global vaccine stockpile has been used on an emergency basis in response to cVDPV2 outbreaks. The WHO Director-General must approve use of mOPV2 in order to limit the exposure to the population of a live OPV strain that would have the potential risk of creating new circulating VDPV2. New emergences were apparently seeded by the use of mOPV2 in outbreak responses, and the cycle continued to increase the number of cVDPV2 outbreaks in African countries, including Nigeria and the Democratic Republic of Congo, which each experienced

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several concurrent outbreaks. After the switch, the number of emergences of cVDPV2 increased each year. Although some cVDPV2 outbreaks were stopped in countries such as the Democratic Republic of Congo, other outbreaks continued. In 2019, cVDPV2 was detected in 12 countries in Africa. Nigeria, the Democratic Republic of the Congo, Somalia, and the Philippines exported cVDPV2 to surrounding countries. Three years after the switch, over 250 million doses of mOPV2 have been used in cVDPV2 outbreak responses. In addition to type 2 cVDPV outbreaks, separate cVDPV1 outbreaks were detected in Papua New Guinea and Indonesia in 2018, and in Myanmar and the Philippines in 2019. Late in 2019, cVDPV1 genetically linked to the cVDPV1 outbreak in the Philippines was detected in Malaysia.

New Vaccines When the poliovirus eradication program began, it was assumed that OPV and IPV would be sufficient for the successful achievement of the goal. Indeed, the use of OPV has reduced poliomyelitis cases by more than 99% worldwide. However, with the growing knowledge of the current risks associated with OPV, and the inability of IPV use alone to fully achieve global eradication, the availability of additional novel vaccine options has become highly desirable. In recent years, efforts to develop novel vaccine platforms have included noninfectious DNA-based vaccines, virus-like particles, and immunogenic peptides, among others. Additionally, to more safely produce IPV, some manufacturers have replaced the traditional wild seed strains used in the production of IPV with attenuated Sabin strains. More recently, the knowledge gained about poliovirus biology over the last half-century has led to the development of novel OPV (nOPV) strains engineered to be safer and more genetically stable than traditional Sabin strains. These rationally redesigned nOPV strains stabilize known reversion hotspots within the Sabin virus genome, while also offering new attenuating strategies targeting replicative fitness, polymerase fidelity, and recombination. The development of these nOPV strains has been deemed necessary by global eradication partners, with a particular interest in using monovalent nOPV2 in outbreak settings, to reduce the risk of spurring additional VDPV emergences. Clinical testing of these nOPV strains is currently underway, with initial results demonstrating nOPV2 is viable, immunogenic, and safe in human recipients. In November 2020, WHO granted provisional emergency use listing to nOPV2, permitting its initial use in outbreak responses.

Antivirals In countries where prophylactic immunoglobulin therapy is available, patients with a primary immune deficiency are generally protected from paralytic disease even if they become infected with a poliovirus vaccine strain. However, an iVDPV can replicate for years in some immunodeficient persons and the virus can revert to a virulent phenotype (iVDPV) and continue to evolve, accumulating additional nucleotide substitutions throughout the genome. One such patient has excreted virulent poliovirus for over 30 years. Continued replication of virulent iVDPV puts the patient at risk for paralysis, even in the face of immunoglobulin therapy, and excretion of iVDPV into the environment poses a risk of transmission in the community, especially in areas of low vaccine coverage or in the post-eradication era when OPV will no longer be used. To minimize the risks to both the patient and the community, antiviral drugs are being developed to treat chronically infected primary immune deficiency patients. One drug, pocapavir, has completed a Phase II trial with positive results, and a second drug is being developed to provide a safeguard against the development of drug resistance, in the form of a combination product.

Containment Once complete eradication of all three serotypes is achieved and vaccination campaigns are no longer implemented, population immunity to polioviruses will decrease. At that point, live poliovirus will exist only in settings such as laboratories and vaccine manufacturing facilities. Therefore, the consequences of any poliovirus introduction into communities from a facility containment breach, even if unlikely, would be severe. This is the case for PV2 now, in 2020, apart from ongoing cVDPV2 circulation. As a result, WHO has developed a “Global Action Plan to minimize poliovirus facility-associated risk after type-specific eradication of WPVs and sequential cessation of oral polio vaccine use” (GAPIII) to provide guidance for facilities that need to retain live polioviruses for essential functions such as vaccine production and critical research. Currently, all type 2 polioviruses must be contained in facilities that comply with GAPIII, with slightly different requirements for vaccine virus compared to WPV2 and VDPV2. Now that WPV3 has been declared eradicated, it also falls under GAPIII containment requirements. At present (in 2020), Sabin 3 is still included in the oral polio vaccine, so the vaccine strain is not yet subject to containment. In recent years, there have been several known breaches of containment in vaccine manufacturing facilities, underlining the risks associated with retaining live poliovirus materials post-eradication. Fortunately, none of the incidents resulted in community transmission. To limit the risk involved in the use of WPV seed strains for IPV production, multiple manufacturers are developing IPV products based on liveattenuated vaccine strains. As of the end of 2019, Japan was the only country with a licensed Sabin IPV product.

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Transition of Global Polio Eradication Initiative Assets Post-Eradication Once global eradication has been achieved, certain GPEI activities and functions will still be needed to ensure the world remains polio-free for the long term. Four key activities that must continue are containment, disease surveillance, outbreak response capacity, and vaccination (with IPV only once OPV use has ceased). Vaccination with IPV is expected to continue for the foreseeable future, as a barrier to disease caused by VDPVs (e.g., from an immunodeficient chronic excreter) or transmission of WPV that could result from exposure in a facility that retains live virus, such as a vaccine manufacturing facility. Even with heightened awareness and effort, it is expected that maintaining high vaccine coverage will be challenging, especially in resourcelimited settings where GPEI has already faced difficulties in achieving levels of population immunity sufficient to interrupt transmission. Poliovirus containment has been described in some detail above; it will be increasingly important as the years pass after eradication given that reintroduction of WPV into a population with reduced immunity would be catastrophic. The capacity to detect polio cases or outbreaks should they occur will need to be integrated into the existing community- and event-based surveillance systems to trigger appropriate investigations and outbreak responses. Vaccine stockpiles will need to be generated and maintained to supply outbreak response vaccination campaigns. While significant capacity in all these areas has been built over more than three decades and the program’s assets have been used to support many other high-priority public health programs, GPEI as a stand-alone program is not sustainable posteradication. This polio infrastructure – notably the trained global public health workforce that includes field surveillance officers, laboratory scientists, communications and logistics experts, and others, at local to global levels – will be invaluable to other public health programs. Integrating polio assets into other health programs will help maintain the capacity to detect and respond to a polio outbreak while also enhancing the capacity to investigate and respond to other priority health threats.

Further Reading Bandyopadhyay, A.S., Garon, J., Seib, K., Orenstein, W.A., 2015. Polio vaccination: Past, present and future. Future Microbiology 10, 791–808. Greene, S.A., Ahmed, J., Datta, S.D., et al., 2019. Progress toward polio eradication – Worldwide, January 2017-March 2019. MMWR Morbidity and Mortality Weekly Report 68, 458–462. Heymann, D.L., Sutter, R.W., Aylward, R.B., 2005. A global call for new polio vaccines. Nature 434, 699–700. Hampton, L.M., Farrell, M., Ramirez-Gonzalez, A., et al., 2016. Cessation of trivalent oral poliovirus vaccine and introduction of inactivated poliovirus vaccine – worldwide, 2016. MMWR Morbidity and Mortality Weekly Report 65, 934–938. Jorba, J., Diop, O.M., Iber, J., et al., 2019. Update on Vaccine-Derived Poliovirus Outbreaks – Worldwide, January 2018-June 2019. MMWR Morbidity and Mortality Weekly Report 68, 1024–1028. Kew, O., Pallansch, M., 2018. Breaking the last chains of poliovirus transmission: Progress and challenges in global polio eradication. Annual Review of Virology 5, 427–451. McKinlay, M.A., Collett, M.S., Hincks, J.R., et al., 2014. Progress in the development of poliovirus antiviral agents and their essential role in reducing risks that threaten eradication. Journal of Infectious Diseases 210 (Suppl. 1), S447–S453. Van Damme, P., De Coster, I., Bandyopadhyay, A.S., et al., 2019. The safety and immunogenicity of two novel live attenuated monovalent (serotype 2) oral poliovirus vaccines in healthy adults: A double-blind, single-centre phase 1 study. The Lancet 394, 148–158. World Health Organization, 2019. Polio Endgame Strategy 2019–2023: Eradication, Integration, Certification and Containment (WHO/Polio/19.04). Geneva: WHO.

Relevant Websites http://polioeradication.org/wp-content/uploads/2016/12/GAPIII_2014.pdf GAPIII. http://polioeradication.org/wp-content/uploads/2019/06/english-polio-endgame-strategy.pdf Polio Endgame Strategy. http://polioeradication.org/wp-content/uploads/2018/04/polio-post-certification-strategy-20180424-2.pdf Polio Post-Certification Strategy. http://www.polioeradication.org The Global Polio Eradication Initiative.

Subject Index Note This index is in letter-by-letter order, whereby hyphens and spaces within index headings are ignored in the alphabetization, and it is arranged in set-out style, with a maximum of three levels of heading. Cross-reference terms in italics are general cross-references, or refer to subentry terms within the main entry (the main entry is not repeated to save space). Location references refer to the volume number, in bold, followed by the page number. Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A AAEV see Anacridium aegyptium EV (AAEV) AAP see antiaptotic proteins (AAP) AAV see adeno-associated virus (AAV) AAV-2 see adeno-associated parvoviruses (AAV-2) AAvV-1 see Avian Avulavirus 1 (AAvV-1) ABLV see Australian bat lyssavirus (ABLV) ABPV see acute bee paralysis virus (ABPV) ABV see acidianus bottle-shaped virus (ABV) ABVV see American bat vesiculovirus (ABVV) Acanthamoeba polyphaga 1:372–373 acanthoma, defined 2:629 accessory gene, defined 4:98 accessory proteins 3:575–576 accessory RNAs associated with mitovirus infections 4:602 accuracy 5:252 ACD see Amasya cherry disease (ACD) ACE2 see angiotensin-converting enzyme 2 (ACE2) ACEV see Anomala cuprea EPV (ACEV) acidianus bottle-shaped virus (ABV) 4:361 Acidianus filamentous virus 1 (AFV1) 1:368, 4:363, 1:435–436 Acidianus tailed spindle virus (ATSV) 1:370f Acidianus two-tailed virus (ATV) 4:361, 4:422 acidic hot springs, viruses of 4:422 acidophilic, defined 4:359 AcMNPV, see Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) AConSLA, see Ageratum conyzoides symptomless alphasatellite (AConSLA) acquired immune deficiency syndrome (AIDS) 5:190, 2:56, 1:567–568 actin filaments (microfilaments), defined 3:32 active surveillance 5:250 acute bee paralysis virus (ABPV) 4:771, 4:772, 4:773

acute flaccid paralysis (AFP) 5:310 defined 2:256 acute gastroenteritis, defined 5:289 acute lower respiratory tract infections (ALRI) 2:747–748 acute otitis media (AOM) 2:761 defined 2:757 acute respiratory distress syndrome (ARDS) 2:559 defined 2:551, 2:825 ACV, see Aeropyrum coil-shaped virus (ACV) acyclovir 5:183, 5:179 adamantanes/M2 blockers 5:161–162 adaptation vs. evolution, defined 1:71 ADCC see antibody-dependent cellular cytotoxicity (ADCC) ADCP see antibody-dependent cellular phagocytosis (ADCP) additional strand, conserved E (ASCE) 4:160 additional strand conserved glutamate (ASCE) 4:148–149, 4:149 defined 4:148 adefovir dipivoxil (ADV) 5:218 Adelaide River virus (ARV) 2:875 adeno-associated parvoviruses (AAV-2) 1:268–269 adeno-associated virus (AAV) 1:411–412, 1:211, 1:533, 1:536 AAV2 4:845 adenosine triphosphate (ATP) 1:495, 1:488 Adenoviridae adenoviruses as vectors 2:15 classification 2:3–6 clinical features 2:10–11 HAdV–A 2:11 HAdV–B1 2:11 HAdV–B2 2:11 HAdV–C 2:11 HAdV–D 2:11 HAdV–E 2:11 HAdV–F 2:11–12 HAdV–G 2:12 diagnosis 2:14–15

epidemiology 2:9–10 epizootiology 2:12 avian adenoviruses 2:13 bovine, ovine, caprine, and other ruminant adenoviruses 2:13 canine and other carnivoran adenoviruses 2:12 equine adenoviruses 2:12–13 frog and fish adenoviruses 2:14 porcine adenoviruses 2:12 reptilian adenoviruses 2:13–14 simian adenoviruses 2:12 genome 2:8 life cycle 2:8–9 pathogenesis 2:14 prevention 2:15 taxonomy of 2:4t–6 treatment 2:15 virion structure 2:6–8 adenovirus 26 (Ad26) 2:243 adenovirus death protein (ADP) 4:761 adenoviruses (ADVs) 1:401, 2:3, 5:109, 5:105, 5:239, 1:660 external minor coat protein in 1:340f general features of 1:329 human adenovirus type 5, structure of 1:329–331 core proteins 1:335 fiber 1:331–333 hexon 1:331 penton base protein 1:331 protein IIIa 1:333 protein VI 1:333 protein VIII 1:333 protein IX 1:333–335 structural relatives 1:337 asymmetric features 1:340 capsid size determination in DJR lineage 1:340–342 double jelly roll fold, variants of 1:337–339 double jelly roll lineage 1:337 double jelly roll lineage, genomes in 1:340

315

316

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adenoviruses (ADVs) (continued) double jelly roll lineage, vertex structures in 1:339 membranes and membrane proteins 1:339–340 polintons and evolutionary pathway of DJR viruses 1:342–343 variants 1:335–337 external minor coat proteins 1:337 fiber shafts and heads 1:335–337 as vectors 2:15 adenovirus fiber 1:331–333 adenovirus infections, management of clinical description of infection 5:199 animal adenoviruses 5:202 diseases and human adenoviruses infections 5:200–201 gastroenteritis 5:200 genitourinary diseases 5:200 immunocompromised patients, adenovirus infections of 5:201–202 incubation period 5:199 lower respiratory tract infections 5:200 ocular infections 5:200 upper respiratory tract infections 5:199–200 diagnosis 5:202–203 adenovirus differentiation and typing 5:203–204 diagnostic methods 5:203 polymerase chain reaction (PCR) for human adenoviruses DNA detection 5:202–203 quantitative human adenoviruses PCRs and screening of high-risk patients 5:203 epidemiology 5:199 structure 5:198–199 taxonomy 5:197 family and genus 5:197 types and subtypes 5:197–198 treatment and prevention 5:204–205 antivirals 5:204–205 germicides 5:205 vaccines 5:205 adenovirus vaccine 2:216 adjuvant, defined 5:281 ADP see adenovirus death protein (ADP) adsorption, defined 1:402, 4:53 adsorption rate, defined 4:387 adult T-cell leukemia (ATL) 2:532–533 ADV see adefovir dipivoxil (ADV); adenoviruses (ADVs) advanced light microscopy approaches 1:210–211 multiphoton imaging 1:211–213 quantitative spectral imaging 1:211 real-time single virus tracking (SVT) in live cells 1:211 adverse effects following immunization (AEFI) 5:283 Aedes aegypti 1:575, 2:894 Aedes pseudoscutellaris dinovernavirus 1 (ApDNV-1) 4:870

AEFI see adverse effects following immunization (AEFI) AEP see archaeo-eukaryotic primases (AEP) Aeropyrum coil-shaped virus (ACV) 1:436, 4:362 Aeropyrum pernix Bacilliform Virus 1 (APBV1) 1:369, 4:361 aeropyrum pernix ovoid virus 1 (APOV1) 4:362 AF4 see asymmetrical flow field-flow fractionation (AF4) affimers, defined 3:692, 3:364 affine extension, defined 1:248 affinity, defined 4:136 AFM see atomic force microscopy (AFM) A-form, defined 4:387, 4:359 AFP see acute flaccid paralysis (AFP) Africa, CLCuD in 3:356, 3:357 African cassava mosaic disease 3:24–26 classification of cassava mosaic begomoviruses 3:27 control 3:28–29 disease symptoms and yield losses 3:26–27 epidemiology 3:27 geographical distribution 3:24–26 recent outbreak of CMD in South East Asia 3:27–28 African horse sickness virus (AHSV) 1:545 classification 2:17 clinical features 2:18 diagnosis 2:19–20 epidemiology 2:17–18 life cycle 2:17 pathology 2:18–19 treatment, control, and prevention 2:20–21 virion structure and genome 2:17 African swine fever virus (ASFV) 1:159–160, 2:22–23 ASFV genes and their functions 2:26t–27 classification 2:22–23 clinical features 2:31 diagnosis and control 2:32–33 epidemiology 2:30–31 genome of 2:24f genome organisation 2:24–25 DNA repair 2:27–28 DNA replication 2:25 helicases 2:25 inhibitors of apoptosis 2:28 inhibitors of type I interferon and inflammatory responses 2:28 multigene families 2:25 nucleotide metabolism 2:25–27 other enzymes 2:28 polymerases 2:25 proteins that inhibit host defences 2:28 virion morphogenesis and structural proteins 2:28 immune responses 2:32 life cycle 2:28–30 assembly and morphogenesis 2:30 DNA replication 2:30 entry 2:29–30 transcription 2:30

pathogenesis 2:31–32 replication cycle of 2:29f structure 2:23f virion structure 2:23–24 AFV1, see Acidianus filamentous virus 1 (AFV1) AfV-F, see Aspergillus foetidus dsRNA mycovirus (AfV-F) AfV-S2, see Aspergillus foetidus slow virus S2 (AfV-S2) Agallia constricta 4:772 agar gel immunodiffusion (AGID) 2:134 Agaricus bisporus 4:431 Agaricus bisporus virus 1 (AbV1) 4:528–529, 4:437 biological properties (virus host relationships) 4:529–530 control 4:530–532 diagnostics and identification 4:530 epidemiology 4:530 genome organization 4:529 taxonomy and classification 4:529 virion structure and composition 4:529 Agaricus bisporus virus 16 (AbV16) 4:532 classification 4:532 diagnosis 4:533 epidemiology 4:533 genome and virion structure 4:532 viral expression and disease development 4:532–533 agarose slides, preparation of 5:11 Ageratum conyzoides symptomless alphasatellite (AConSLA) 3:360–361 Ageratum enation virus 3:750 ageratum yellow vein betasatellite (AYVB) 3:361 Ageratum yellow vein India alphasatellite (AYVINA) 3:360–361 Ageratum yellow vein virus (AYVV) 3:357 aggregates 3:777 aggresome, defined 1:495 AGID see agar gel immunodiffusion (AGID) AGO1 protein see Argonaute 1 (AGO1) protein AGO proteins see Argonaute (AGO) proteins AGP see antigenome promoter (AGP) agricultural research centers 1:646 agrobacterium-mediated genetic transformation, defined 3:123 agroinfection, defined 3:768, 3:461 agroinfiltration, defined 3:743, 3:123 agro-inoculation 3:749 21238:p0015 3:667, 3:768, 3:313 agro-terrorism, defined 1:644 agro-warfare 1:647–648 AHF see argentinian hemorrhagic fever (AHF) AHS see arthrogryposis-hydranencephaly syndrome (AHS) AHSV see African horse sickness virus (AHSV) AHV see avian herpesviruses (AHV) AIDS see acquired immune deficiency syndrome (AIDS) airborne transmission 1:561

Subject Index defined 1:559 AIV see avian influenza viruses (AIV) akabane virus 1:545, 2:34 classification 2:34 clinical features 2:36 diagnosis 2:38 epidemiology 2:35–36 genome 2:34–35 pathogenesis 2:36–38 prevention 2:38–39 viral replication 2:35 virion structure 2:34 akt, defined 2:875 alanine aminotransferase (ALT) 2:896 Albetovirus 3:581, 3:581–582 Alfalfa mosaic virus (AMV) 3:261, 3:265 history 3:132 taxonomy and classification 3:132–133 genome structure 3:133–134 host range and economic significance 3:137–139 interaction between viral RNA and coat protein 3:134 particle structure and composition 3:132–133 replication of viral RNA 3:135–136 role of coat protein in the AMV replication cycle 3:136–137 translation of viral RNA 3:134–135 virus encapsidation and movement 3:136 virus-host relationships 3:137 transmission 3:139 epidemiology and control 3:139 Alfamovirus 3:260, 3:262t characteristics of RNA genome in 3:261t alfavirus, defined 3:229 algal double-stranded DNA viruses 4:685–686 genus Dinodnavirus 4:686 algal double-stranded RNA viruses 4:684–685 genus Mimoreovirus 4:684–685 algal single-stranded DNA viruses 4:685 genus Bacilladnavirus 4:685 algal single-stranded RNA viruses 4:684 genus Dinornavirus 4:684 algal viruses belonging to a subgroup within Mimiviridae family general properties 4:678–681 history 4:677–678 proposed subfamily Mesomimivirinae 4:681 unclassified algae-infecting members in the family Mimiviridae 4:681–682 defined 4:677 alkaline midgut, defined 4:858 alkaliphilic, defined 4:368 AlkB protein, defined 3:642 allele, defined 3:69, 3:554 Allium virus X (AlVX) 3:628 allogeneic, defined 2:778, 2:441 allograft, defined 2:441 Alloherpesviridae 2:308f alloherpesvirus, defined 2:306

allosteric inhibitors, defined 5:121 Almendravirus 4:715–716 almendraviruses genome 4:885 alphachryso-P3 4:564 alphaentomopoxvirus 4:859 Alphaflexiviridae 3:539, 3:140 alphaflexiviruses (Alphaflexiviridae) 3:140 diagnosis 3:148 epidemiology and control 3:146 replication and propagation 3:146 satellite rnas associated with infections of 3:148 taxonomy, phylogeny of family members, and evolution 3:140–141 transmission, host range 3:146 virion structure 3:141–145 genome organization 3:142–145 properties and functions of gene products 3:145–146 virus–host relationships 3:146–148 alpha helix (a-helix), defined 4:457, 1:345 Alphaherpesvirinae 1:321 alphaherpesvirus 2:442, 2:445 defined 2:441 alpha-HPV E6 proteins 2:498 alpha-like viruses, defined 3:839 Alphamesonivirus 4:804, 4:805t, 4:807 Alphamesonivirus 1 4:804 alphamesonivirus 4 4:804 Alphanemrhavirus 4:715–716 alphanodaviruses 4:709–710 Alphanudivirus 4:827, 4:827–828 alphapartitivirus 4:635 alphapleolipovirus, defined 4:380 Alphaproteobacteria 4:332–334 marine Pelagibacterales, phages of 4:332–334 marine Rhodobacteraceae, phages of 4:334–335 SAR116 phages 4:334 alphasatellite components, CLCuD-associated 3:359–361 alphasatellites (Alphasatellitidae) 3:149, 3:753 classification 3:149–150 defined 3:470 life cycle/epidemiology 3:150–152 likely origins of 3:150 pathogenesis 3:152–153 structure of 3:150 Alphasatellitidae 3:150 Alphatetraviridae 4:897, 4:717 alphatetravirus 4:900, 4:900f alphavirus assembly and budding 1:470–472, 1:471f alphaviruses 1:266 alphaviruses causing encephalitis clinical features of infection 2:46 epidemiology 2:44–45 evolution 2:43–44 future 2:47 genetics 2:43 genome, properties of 2:40–41 geographic and seasonal distribution 2:42–43

317

history 2:40 host range and virus propagation 2:43 pathology and histopathology 2:46–47 prevention, treatment and control 2:47 replication 2:42 taxonomy and classification 2:40 transmission and tissue tropism 2:45–46 pathogenicity 2:45–46 viral proteins, properties of 2:41 nonstructural proteins 2:41 structural proteins 2:41–42 virion, properties of 2:40 alphavirus life cycle 1:469 alphavirus RNA synthesis 2:176 alphavirus unique domain (AUD) 2:177 alphavirus virion structure 1:469–470 Alphavodaviruses 4:822 ALPV see aphid lethal paralysis virus (ALPV) ALRI see acute lower respiratory tract infections (ALRI) ALT see alanine aminotransferase (ALT) Alternaria alternata 4:545–546 alternaria alternata vius 1 (AaV1) 4:544 alternaviruses 4:544, 4:545t biological effects of 4:545–546 chrysoviruses, evolutionary relationships among 4:547–548 genome organization 4:544 3’ poly (A) structure of 4:547 proteins encoded by 4:546–547 virion properties 4:544–545 ALV see avian leukosis virus (ALV) AlVX see Allium virus X (AlVX) Amalgaviridae 3:154 amalgaviruses (Amalgaviridae) classification 3:154 diagnosis 3:155–157 epidemiology 3:155 genome 3:154 life cycle 3:154–155 pathogenesis 3:155 prevention 3:157 treatment 3:157 virion structure 3:154 amantadine 5:161, 5:171 Amasya cherry disease (ACD) 4:646–647 ambisense, defined 4:764, 2:765, 4:835 ambisense genome, defined 3:507 ambisense polarity, defined 3:719 Amblyomma hebraeum 1:547 Amblyomma variegatum 2:893–894 American bat vesiculovirus (ABVV) 2:875 AMEV see Amsacta moorei EPV (AMEV) AMGs see auxiliary metabolic genes (AMGs) 2-aminopurine (2-AM) 4:876 aMLVs see amphotropic MLV (aMLVs) ampelovirus, defined 3:336 amphibian alloherpesviruses classification 2:306–307 clinical features 2:311–313 diagnosis 2:313 epidemiology 2:310 genome 2:307–309 life cycle 2:309

318

Subject Index

amphibian alloherpesviruses (continued) management and treatment 2:313–314 pathogenesis 2:311 prevention 2:314–315 virion structure 2:307 amphotropic MLV (aMLVs) 2:646 amplicon sequencing 1:178–179 Ampullaviridae 4:361 AMR see analytical measurement range (AMR) Amsacta moorei EPV (AMEV) 4:858 AMV see Alfalfa mosaic virus (AMV) Anacridium aegyptium EV (AAEV) 4:865 analogy, defined 1:87 analytical measurement range (AMR) 5:72–73 anamorph, defined 4:450 anaphylaxis 5:305 anastomosis 4:520, 4:589, 4:557, 4:450, 4:601, 4:607, 4:648, 4:552 Anatid herpesvirus 1 (AnHV1) 2:113 Anelloviridae 2:48 classification 2:48–49 clinical features 2:52 diagnosis 2:54 epidemiology 2:51–52 life cycle 2:50–51 pathogenesis 2:52–54 treatment and prevention 2:54–55 virion structure and genome 2:49–50 anelloviruses (AV) 2:48 angiotensin-converting enzyme 2 (ACE2) 2:199–200 A˚ngstro¨m, defined 4:10, 1:257 anguillid perhabdovirus 2:325 AnHV1 see Anatid herpesvirus 1 (AnHV1) ANI see Asymptomatic Neurocognitive Impairment (ANI) animal adenoviruses 5:202 animal morbilliviruses see morbilliviruses animal papillomaviruses see papillomaviruses Animal Reservoir, defined 2:397 animal rhabdoviruses see rhabdoviruses animal viruses 1:6–7 vector transmission of 1:542–543 arthropod vectors of animal arboviruses 1:543–545 co-evolution of viruses and vectors 1:545–546 co-infection of arthropods with symbiotic bacteria 1:549–550 environmental/anthropogenic factors contributing to vector-borne virus spread 1:546–547 replication and transmission of arboviruses in their arthropod vector 1:547–548 role of vector in genetic diversity of animal arboviruses 1:550 virus–vector interactions and role of arthropod innate immune response 1:548–549 ankyloses, defined 2:34 ankyrin repeat (ANK)/F-box genes 2:672 Anomala cuprea EPV (ACEV) 4:859

Anopheles A serogroup 2:662 antagonism, defined 4:419 antagonist, defined 4:776 Antennaviruses 2:507 anteriograde, defined 2:738 anthrax toxin receptor I (ANTXRI) 1:659 anthropogenic factors, defined 1:542 antiaptotic proteins (AAP) 4:813 antibiotic resistance, defined 4:252 antibiotic resistance genes (ARG) 1:642 Antibiotic Resistant (AR) bacteria 4:252 antibiotics 4:252, 5:301 Antibiotic Susceptibility Test (AST) 4:252–253 defined 4:252 antibodies in antiviral humoral immunity 1:591 antibody-dependent cellular cytotoxicity (ADCC) 1:591–592 neutralization 1:591 opsonization 1:591 antibody, defined 2:441 antibody assay, neutralizing 5:19 antibody avidity, defined 2:778 antibody-dependent cellular cytotoxicity (ADCC) 1:584, 1:591–592 antibody-dependent cellular phagocytosis (ADCP) 1:591 antibody-dependent enhancement, defined 2:899 antibody prevalence, defined 2:362 antibody repertoire, sequencing analysis of 5:20 anti-CRISPR (Acr) genes, defined 4:400 anti-CRISPR proteins 4:248–249 of archaeal viruses 4:405 guilt-by association bioinformatic approach 4:249–250 lytic phage dependent functional approach 4:250 prophage-dependent functional approach 4:249 self-targeting bioinformatic approach 4:250 antigen, defined 2:441 antigen detection 5:210 hepatitis A virus 5:210 hepatitis E virus 5:210 antigenemia, defined 2:789 antigenic complex, defined 2:765 antigenic drift, defined 2:551 antigenicity and antibody interactions 1:287–288 antigenicity and antigenic variation dengue virus (DENV) envelope 1:598–599 enterovirus A71 capsid 1:599–600 human immunodeficiency virus type 1 envelope glycoprotein 1:599 influenza A virus hemagglutinin 1:597–598 antigenic shift, defined 2:551 antigenome, defined 2:747 antigenome promoter (AGP) 2:69–70 antigen presentation 1:602–603 MHC-I antigen presentation 1:603 MHC-II antigen presentation 1:603–604

viral subversion of 1:604–605 antigen-presenting cells (APCs) 1:584, 1:601–602, 1:662, 2:512–513, 1:601 anti-hypovirus defense mechanisms 4:592–593 antiretroviral therapy (ART) 2:468 non-nucleoside reverse-transcriptase inhibitors (NNRTIs) 5:135 chemical structures of 5:135f clinical uses of 5:135–136 drug-drug interactions with 5:136 mechanisms of NNRTI resistance 5:136–137 and their mechanism of action 5:135 toxicities 5:136 nucleoside/nucleotide reversetranscriptase inhibitors (NRTIs) 5:132–133 clinical uses of 5:133 drug-drug interactions with 5:134 mechanisms of NRTI resistance 5:134–135 new drugs 5:135 and their mechanism of action 5:132–133 toxicities 5:133–134 reverse transcriptase structure and functions 5:131 reverse transcription, process of 5:131–132 reverse transcriptase (RT) 5:132 RNase H 5:132 tRNA primer 5:131–132 antisense RNA technology 3:362 antiviral agents 1:478–479 antiviral classification 5:121 hepatitis B virus (HBV) 5:128 HBV DNA polymerase 5:128 hepatitis C virus (HCV) 5:123 HCV NS3/4A protease 5:123 HCV NS5A phosphoprotein 5:123 HCV NS5B polymerase 5:123 herpes simplex virus (HSV) 5:124 HSV DNA polymerase UL30 5:124 HSV envelope protein 5:124–125 human cytomegalovirus (HCMV) 5:125 HCMV DNA polymerase UL54 5:125 HCMV terminase UL56 5:125 human immunodeficiency virus (HIV) 5:121 HIV GP120 5:122–123 HIV GP41 5:122 HIV integrase 5:122 HIV protease 5:121–122 HIV reverse transcriptase 5:121 human influenza virus 5:125 matrix protein 2 5:127 neuraminidase 5:125–127 viral RNA polymerase 5:125 respiratory syncytial virus (RSV) 5:123–124 RSV Fusion glycoprotein 5:124 RSV RNA polymerase 5:123–124 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 5:127 SARS-CoV-2 polymerase 5:127 varicella zoster virus (VZV) 5:127–128

Subject Index VZV DNA polymerase 5:128 variola virus (human smallpox) 5:127 VP37 envelope wrapping protein 5:127 antiviral defense mechanisms 4:394–397 CRISPR-Cas systems 4:397 toxin-antitoxin (TA) system 4:397–398 antiviral drug resistance, defined 5:121 antiviral drugs 1:286–287 antiviral gene silencing, defined 3:116 antiviral immunity, defined 3:116 antiviral innate immunity 1:577 antiviral cytokines 1:578 cellular innate immunity 1:578 complement system 1:577–578 defensins 1:577 direct antiviral effects of Type I IFNs 1:580–581 good cop–bad cop 1:582 indirect antiviral effects of Type I IFNs 1:581 innate immunity memory 1:581 interferon induction 1:579–580 new kids in the gut: the Type III interferons 1:578–579 tonic IFN Levels 1:581 Type I IFN signaling 1:580 viral counterstrategies 1:581–582 antivirals 5:204–205 antiviral therapies, viral factories as targets for 1:499 ANTXRI see anthrax toxin receptor I (ANTXRI) Anulavirus 3:260, 3:262t characteristics of RNA genome in 3:261t AOM see acute otitis media (AOM) AP1, defined 2:875 aparavirus 4:768, 4:769t, 4:770t, 4:774 APBV1, see Aeropyrum pernix Bacilliform Virus 1 (APBV1) APCI MS see atmospheric pressure chemical ionization (APCI) MS APCs see antigen-presenting cells (APCs) ApDNV-1, see Aedes pseudoscutellaris dinovernavirus 1 (ApDNV-1) aphid-borne, defined 3:594 aphid lethal paralysis virus (ALPV) 4:771 aphid transmission factor 3:318 aphid-transmitted caulimoviruses, case of 3:112 aphid-transmitted cucumoviruses, case of 3:112 aphid-transmitted luteovirids, case of 3:107–108 aphid-transmitted potyviruses, case of 3:112 apical membrane, defined 1:529 aplastic anemia, defined 2:362 APMV-1 see avian paramyxovirus 1 (APMV-1) APOBEC, defined 4:835 APOBEC3 2:65 apolipoprotein E (ApoE) 2:386–387 apoptosis 2:895–896, 4:858, 2:182, 2:22, 2:738, 1:577, 2:428, 4:724, 4:732 inhibitor of 2:672 apoptosome, defined 3:60 apoptotic bodies, defined 1:577, 4:724 aposymbiotic, defined 4:780

APOV1 see aeropyrum pernix ovoid virus 1 (APOV1) APVs see avipoxviruses (APVs) aquareovirus 1:306, 4:871 Arabis mosaic virus (ArMV) 3:628–629 ARAV see Aravan lyssavirus (ARAV) Aravan lyssavirus (ARAV) 2:738 AR bacteria see Antibiotic Resistant (AR) bacteria “arbitrium” system 1:638 arboviruses 2:891, 1:542–543, 2:899, 1:542, 2:218, 2:173, 2:40, 4:764, 2:654 arthropod vectors of animal arboviruses 1:543–545 replication and transmission in their arthropod vector 1:547–548 role of vector in genetic diversity of animal arboviruses 1:550 in their invertebrate vectors 4:722, 4:720t Archaea 1:367–369 phage egress in 1:517–518 Archaea, antiviral defense mechanisms in 4:400 archaeal innate antiviral systems 4:405–406 CRISPR-Cas 4:400–401 anti-CRISPR proteins of archaeal viruses 4:405 archaeal Type I CRISPR-Cas systems 4:401–402 archaeal Type III CRISPR-Cas systems 4:402–405 CRISPR loci and crRNA biogenesis, expression of 4:401 novel archaeal CRISPR-Cas systems 4:405 spacer acquisition and its regulation in archaea 4:400–401 future perspectives 4:406 Archaea, virus-host interactions in 4:387–388 antiviral defense mechanisms 4:394–397 CRISPR-Cas systems 4:397 toxin-antitoxin (TA) system 4:397–398 counterdefense mechanisms 4:398 genome integration 4:391–392 genome replication 4:390–391 transcription 4:392–393 control 4:393 regulators 4:392–393 virion egress 4:393–394 cell membrane disruption 4:393–394 membrane disruption, viral release without 4:394 virus entry 4:388–389 cell-surface, interaction with 4:389–390 cellular appendages, interaction with 4:388–389 kinetics of virus entry 4:390 archaeal innate antiviral systems 4:405–406 archaeal Type I CRISPR-Cas systems 4:401–402 archaeal Type III CRISPR-Cas systems 4:402–405 archaeal virus, see also bacterial and archaeal viruses

319

defined 1:402, 4:380 ecology and evolution shaping 4:424–425 in hot spring environments using viral metagenomics 4:407 learning 4:410–412 next generation sequencing of samples 4:409 post sequence analysis 4:409–410 sample collection and processing 4:407–409 and viral metagenomes 4:414–415 archaeo-eukaryotic primases (AEP) 1:433 Archimedean lattices 1:248, 1:252f architecture of viruses 1:475 ARDS see acute respiratory distress syndrome (ARDS) arenaviruses, human pathogenic 2:508f clinical features 2:513 Lassa fever (LF) and Lujo hemorrhagic fever (LHF) 2:513 lymphocytic choriomeningitis (LCM) 2:513–514 New World (NW) Mammarenaviral HFs 2:514 diagnosis 2:514 Lassa fever (LF) 2:514 lymphocytic choriomeningitis (LCM) 2:514–515 New World (NW) mammarenaviral HFs 2:515 history and classification 2:507 human pathogenic mammarenaviruses, epidemiology of 2:511 argentinian hemorrhagic fever (AHF) 2:512 Bolivian hemorrhagic fever (BHF) 2:512 Lassa fever (LF) 2:511 Lujo hemorrhagic fever (LHF) 2:511 lymphocytic choriomeningitis (LCM) 2:511–512 Venezuelan hemorrhagic fever (VeHF) 2:512 mammarenavirus genome organization and proteins 2:507–509 mammarenavirus life cycle 2:509–510, 2:509f assembly and budding 2:511 cell attachment and entry 2:509–510 expression and replication of the viral genome 2:510–511 mammarenavirus virion structure 2:507 pathogenesis and pathology 2:512–513 prevention and control 2:515 antibody therapy 2:515–516 antiviral drugs 2:515 medical management 2:515 vaccines 2:516 ARG see antibiotic resistance genes (ARG) argentinian hemorrhagic fever (AHF) 2:512, 2:507, 2:514 arginine finger 4:166 defined 4:148 arginine toggle 4:164 Argonaute (AGO) proteins 3:43 argonaute, defined 3:52, 3:594, 3:123 Argonaute 1 (AGO1) protein 3:758, 3:692

320

Subject Index

arming, defined 5:233 arming viruses 5:241 immunomodulators 5:241 prodrug convertases 5:241 radiosensitizers for therapy and imaging 5:241 ArMV see Arabis mosaic virus (ArMV) ART see antiretroviral therapy (ART) art and literature, viruses in 1:679 Arteriviridae 1:499 arteriviruses 1:499 arthralgia, defined 2:173 arthrogryposis, defined 2:34 arthrogryposis-hydranencephaly syndrome (AHS) 2:36 arthropod-borne viruses 2:891 of vertebrates 2:417 arthropods, bunyaviruses of see bunyavirus: of arthropods arthropod-specific virus, defined 4:764 arthropod transmission cycles, influence of 1:563–564 arthropod vectors of animal arboviruses 1:543–545 artificially established viral cross infections between plants and fungi 4:445–447 artificial microRNAs, defined 3:293 Artoviridae 4:703 ARV see Adelaide River virus (ARV) AS1 see Asymmetric Leaves gene 1 (AS1) ASCE see additional strand, conserved E (ASCE); additional strand conserved glutamate (ASCE) ascocarp, defined 4:450 ascomycete/basidiomycete, defined 4:568 ascomycetous fungi, defined 4:594 ascospores, defined 4:607, 4:450 Ascoviridae 4:703, 4:724 ascoviruses 4:724 distribution and taxonomy 4:724–725 future perspectives 4:730 history 4:724 origin and evolution 4:730 pathology and pathogenesis 4:728 cytopathology and cell biology 4:728–729 signs of disease 4:728 tissue tropism 4:729 replication and virion assembly 4:729–730 transmission and ecology 4:727–728 host range 4:728 virion structure and composition 4:725–727 aseptic meningitis 2:812 ASFV see African swine fever virus (ASFV) ASGPR see asialoglycoprotein receptor (ASGPR) ASHBV see ashy-headed sheldgoose (ASHBV) ashy-headed sheldgoose (ASHBV) 2:100 asialoglycoprotein receptor (ASGPR) 1:537 Asia Minor, defined 3:586 ASLV see Avian Sarcoma and Leukosis Virus (ASLV) aspartate aminotransferase (AST) 2:896

Aspergilli, diversity of mycoviruses in 4:450 aspergillus foetidus mycovirus complex 4:453–454 chrysoviruses 4:454 genomes 4:451–453 narnaviruses and mitoviruses 4:454 partitiviruses 4:454 phenotypes 4:455 polymycoviruses 4:454–455 prevalence 4:450–451 transmission 4:451 Aspergillus flavus 4:520 Aspergillus foetidus dsRNA mycovirus (AfV-F) 4:544 Aspergillus foetidus slow virus S2 (AfV-S2) 4:660 assay, defined 4:175 assay verification and validation 5:70–72 accuracy 5:72 linearity/analytical measurement range (AMR) 5:72–73 precision 5:72 reportable range and limit of detection (LoD) 5:72 assembly, defined 1:382 assembly code embedded within viral genetic message 1:254–255 assembly of virion 1:480 icosahedral capsid assembly, structural principles in 1:480–481 modes of assembly 1:481–483 capsid self-assembly 1:483 scaffolding protein-assisted capsid assembly 1:483–486 viral genome-assisted capsid assembly 1:486 synthesis of nascent capsid assembly and assembly intermediates 1:481 assembly of viruses 1:468 antigenic sites 1:478 antiviral agents 1:478–479 architecture of viruses 1:475 assembly 1:477–478 evolution 1:477 helical viruses, atomic structure of 1:475–476 host receptor recognition site 1:478 icosahedral enveloped viruses 1:468–469 alphavirus assembly and budding 1:470–472 alphavirus life cycle 1:469 alphavirus virion structure 1:469–470 flavivirus assembly and budding 1:473 flavivirus life cycle 1:472 flavivirus virion structure 1:472–473 nucleic acid–protein interaction 1:476–477 spherical viruses, atomic structure of 1:476 structure determination, methods of 1:475 viral envelope 1:468 assembly pathways 4:49 AST see Antibiotic Susceptibility Test (AST); aspartate aminotransferase (AST) asthma 2:761–762 astrocytes, defined 2:778 Astroviridae classification 2:92–93

clinical features 2:95 animal disease 2:95–96 human disease 2:95 diagnosis 2:98 epidemiology 2:95 classical HAstV 2:95 novel HAstV 2:95 genome 2:93–94 life cycle 2:94 assembly and release 2:94–95 binding and entry 2:94 uncoating and replication 2:94 pathogenesis 2:96 immune response 2:97–98 mechanism of disease 2:96–97 obstacles 2:96 prevention 2:98 treatment 2:98 virion structure 2:93 asunaprevir 5:123 asymmetrical flow field-flow fractionation (AF4) 1:171, 1:162, 1:172f Asymmetric Leaves gene 1 (AS1) 3:245 asymmetric unit, defined 1:248 Asymptomatic Neurocognitive Impairment (ANI) 2:471 Atadenovirus 2:13 atazanavir 5:141 atherosclerosis, defined 2:778 atherosclerotic vascular disease 2:454 ATL see adult T-cell leukemia (ATL) atmosphere 4:354–355 atmospheric pressure chemical ionization (APCI) MS 5:92 atomic force microscopy (AFM) of biological specimens 1:219–220 complex viruses, dissection of 1:227–230 defined 4:53 icosahedra, triangulation numbers of 1:222–223 infection and budding from host cells 1:226 initial observations from 1:220–222 principles and technology 1:218–219 recombinant and mutant virus particles 1:223 specific and special structural features 1:226–227 viral assembly 1:232 viral nucleic acid, visualization of 1:230–232 virus fibers and membranes 1:223–226 atomic level structures, classification of viral world based on 1:153–154 case study 1:158–160 future perspectives 1:160–161 incorporating different levels of data 1:157 inferring phylogeny based on structural data 1:158 methods to compare (viral) proteins 1:154–157 structure-based classification, databases for 1:157–158 ATP see adenosine triphosphate (ATP) ATPase, defined 4:160, 4:105 ATPase gp16, defined 4:302

Subject Index ATP-driven genome packaging, defined 1:488 ATSV, see Acidianus tailed spindle virus (ATSV) attachment, defined 1:382 attachment factor, defined 1:388 ATV, see Acidianus two-tailed virus (ATV) AUD see alphavirus unique domain (AUD) audits, internal and external 5:70 Aumaivirus 3:581, 3:582 Australian bat lyssavirus (ABLV) 2:738, 2:740 autochthonous transmission, defined 2:173 Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) 1:654–655 autoimmune disease, defined 2:884 autoimmune vasculitis 2:454 autophagy 4:824 defined 1:529, 2:738, 3:420, 2:428, 1:495 autophagy-related vesicle-mediated release of virus 1:539 auxiliary metabolic genes (AMGs) 1:621 auxotrophic complementation 4:522 AV see anelloviruses (AV) avian adenoviruses 2:13 Avian Avulavirus 1 (AAvV-1) 2:648 life cycle 2:649f avian hepadnaviruses classification 2:100 diagnosis 2:107–108 endogenous avian hepadnavirus elements 2:109–111 epidemiology 2:106 genome 2:101–103 life cycle 2:103–106 pathogenesis and clinical features 2:106–107 treatment 2:108–109 virus structure 2:100–101 avian herpesviruses (AHV) classification 2:112–113 clinical features 2:115 diagnosis 2:116 epidemiology 2:114–115 genome 2:113–114 host range 2:113 pathogenesis 2:115–116 prevention 2:116 replication cycle 2:114 virion structure 2:113 avian hosts 2:805, 2:813 avian influenza 5:274 avian influenza viruses (AIV) 2:117 clinical features and pathogenesis 2:118–119 epidemiology 2:118 life cycle 2:117–118 prevention 2:119–121 taxonomy 2:117 virion structure and genome 2:117 avian leukosis virus (ALV) 2:122 avian paramyxovirus 1 (APMV-1) 2:648 Avian Sarcoma and Leukosis Virus (ASLV) 1:211, 2:122 avidity, defined 4:136

avipoxviruses (APVs) 2:343 classification 2:343 control and treatment 2:347 use of APV as recombinant vaccine vectors 2:347 epidemiology 2:346 genome 2:344–345 life cycle 2:345–346 pathogenesis 2:346–347 host range 2:346–347 immunomodulation 2:347 replication cycle of 2:345f virion structure 2:343–344 avirulence, defined 3:69 avirulence determinant, defined 3:761 avirulence gene, defined 3:60 Avsunviroidae 3:852 AYVB see ageratum yellow vein betasatellite (AYVB) AYVINA, see Ageratum yellow vein India alphasatellite (AYVINA) AYVV, see Ageratum yellow vein virus (AYVV)

B Bacilladnavirus 4:685 bacilli double-stranded DNA bacteriophages, replication of 4:61–63 of j29-like viruses 4:66–67 of SPO1-like viruses 4:63–65 of SPP1-like viruses 4:65–66 Bacillus subtilis phage SPP1 4:206 Background Selection, defined 5:227 backyard production systems (BPS) 2:118 bacteria 1:664 bacterial and archaeal viruses in different sizes 1:162–164 genome replication of DNA replication mechanisms 1:435–436 prokaryotic dsDNA viruses, genome replication of 1:429–430 prokaryotic RNA viruses, genome replication of 1:437 prokaryotic ssDNA viruses, genome replication of 1:436 prokaryotic viruses, genomes of 1:429 protein-primed DNA replication 1:433–434 RNA-dependent RNA polymerase, replication using 1:437 RNA-primed DNA replication 1:430–433 rolling circle DNA replication (RCR) 1:434–435 rolling circle replication 1:436–437 isolation and culturing of 1:164–166 plaque morphologies 1:165f polyphyletic group of viruses 1:162–164 precipitation methods 1:167–168 preparative ultracentrifugation methods 1:168–170 chromatographic methods 1:170–171

321

flow field-flow fractionation methods 1:171–172 purification performance, assessing 1:172 purification of viruses 1:166–167 ultrafiltration methods 1:167, 1:167f bacterial and archeal virus entry 1:402, 1:404f genome delivery mechanisms of phages 1:405–406 filamentous DNA viruses 1:407 icosahedral DNA viruses with an internal membrane 1:406–407 icosahedral enveloped dsRNA bacterial viruses 1:407–408 icosahedral ssDNA bacterial viruses 1:407 icosahedral ssRNA bacterial viruses 1:408 icosahedral tailed dsDNA bacterial viruses 1:405–406 pleomorphic DNA viruses 1:407 host cell barriers 1:402 host recognition and adsorption 1:403 viral enzymes in capsule and cell wall penetration 1:403–405 virion as a genome delivery devise 1:402–403 bacterial artificial chromosome, defined 2:306 bacterial evolution, role of bacteriophages in coevolution defense mechanisms 1:641–642 integration sites 1:641 in the patient 1:641 polylysogeny 1:638–640 prophage remnants 1:640–641 war and peace 1:642 lysis-lysogeny decision 1:638 synergistic phage-bacterium relationships 1:633–634 synergistic phage-bacterium relationships 1:634–638 bacteria-like versus virion-emphasizing modes of existence 4:318 bacterial virus 1:6 defined 1:402 bacteriophage 4:253, 4:252–253, 4:291–292, 1:552–553, 4:277, 4:77, 4:80–81 defined 5:5, 4:291, 4:302 discovery of phages 4:3–4 of human microbiome 4:283–284 clinical utility of 4:290 dynamics and implications of human phage community 4:287–290 human phageome distribution and composition 4:284–287 phage and biotechnology 4:7–8 phage biochemistry 4:6–7 phage development as diagnostics for bacterial infections 4:254–255 as therapeutics for bacterial infections 4:253–254

322

Subject Index

bacteriophage (continued) phage diagnostics, additional challenges for 4:257 intracellular bacteria 4:257 market acceptance 4:257 multiplex testing 4:257 phage manipulation 4:257 sample interference 4:257 phage ecology and evolution 4:8 phage genetics 4:5–6 phage technology, major advantages of 4:255 self-replication 4:255 specificity 4:255 phage technology, major limitations of 4:255 narrow host range 4:255 phage resistance 4:255 phage therapy 4:4 phage therapy, additional challenges for 4:255–256 chemistry, manufacturing, and controls (CMC) 4:256 dosing strategies 4:256 environmental impact 4:256–257 preclinical data translation 4:255–256 regulatory pathway 4:256 safety and efficacy 4:256 phage typing 4:4–5 structure and function 4:46 capsids 4:46 tails and tail fibers 4:46–48 T4 4:70–72 T7 4:72 early transcription 4:72 elongation 4:74 phage RNAP and late gene transcription 4:72 termination 4:74 transcription cycle 4:72–74 temperate versus virulent bacteriophages 4:48 bacteriophage 186 4:81–83 bacteriophage adherence to mucus (BAM) 1:556–557 bacteriophage diversity 4:265–266 examples of 4:268–269 bottom up studies 4:269 contribution of prophages to phage diversity 4:270 Enterobacteriales tailed phage example 4:269–270 human phageome 4:268–269 marine phageome 4:269 top down studies 4:268–269 two large diversity studies 4:269 genome mosaicism 4:272 genetic exchange among superclusters 4:272 and horizontal exchange of genetic information 4:270–272 nature of horizontally exchangeable mosaic section alleles 4:270–272 phage-host relationships and phage diversity 4:272–273 narrow and wide host range phages 4:273–274

phage cluster-host species relationships with Enterobacteriales 4:272–273 strategies for studying 4:267–268 tailed phage diversity and phage classification 4:266–267 bacteriophage DNA packaging nanomotors 4:302–305 application of 4:308 biological nanopore channel, applications of 4:309–312 mechanism of single pore sensing 4:308–309 Phi29 connector 4:308 SPP1 connector 4:308 T7 connector 4:308 revolving mechanism of biomotors in bacteriophages 4:305 revolution mechanism without rotation 4:305 revolving mechanisms determined by motor channel sizes 4:305–306 revolving motors distinguished by chirality 4:306–307 special aspects of revolving motor actions 4:307–308 Bacteriophage Exclusion (BREX) 1:608 bacteriophage l red recombination system 4:291 biological roles 4:293 DNA repair 4:293–294 genetic exchange among similar phages 4:293 role in DNA replication 4:293 classical models of l recombination 4:294 homologous recombination systems in E. coli and l phage 4:292–293 bacteriophage-phage, defined 4:283 bacteriophage plaques 4:315f bacteriophage receptor proteins of gram-negative bacteria history and overview 4:175 phage-host interactions, ecology and evolution of 4:181–182 phage-receptor interactions’ relevance to phage therapy 4:182–183 properties of receptor proteins 4:176–181 techniques and methods for studying phage receptors 4:175–176 techniques for studying 4:177t–178 bacteriophages, general ecology of 4:314 phage, microscopic determination of 4:315–316 phage community ecology 4:320 phage ecosystem ecology 4:320–321 phage isolation and host range 4:315 phage organismal ecology 4:317–318 bacteria-like versus virion-emphasizing modes of existence 4:318 phage population ecology 4:318–320 bacteriophage tail fibers, structures of 4:195–199 bacteriophage tailspikes, structures of 4:199–201 bacteriophage vaccines 4:259 in vitro VLP assembly 4:261–262

phage T4 VLP vaccines 4:261–262 phage T4, architecture of 4:259 phage T4 as a vaccine platform 4:259–261 in vivo VLP assembly 4:259–261 phage vaccine platforms 4:262–263 Bacteroidetes 4:336–337 Baculoviridae 4:703–705 baculovirus 4:747 acquisition of genes 4:733–735 baculoviral insecticides 4:745 baculovirus expression technology 4:745 baculovirus transmission and host behavioral manipulation 4:744–745 defined 4:827 evolution 4:743–744 gene expression 4:752–754 early gene expression 4:752–754 late and very late gene expression 4:754 temporal regulation of transcription 4:752–754 genome organization and content 4:749–752 genomes, gene content, organization 4:741–743 historical perspective 4:739 host range genes 4:735–736 infection cycle 4:744, 4:747–748 dissemination of OBs 4:749 oral and systemic infection 4:748–749 two virion phenotypes 4:748 morphology 4:740–741 nomenclature, taxonomy, and classification 4:739–740 replication 4:733 viral DNA replication 4:754–755 virion morphogenesis and protein composition 4:755 BV morphogenesis 4:755–756 F protein properties 4:756 GP64 glycoprotein characteristics 4:756 nucleocapsids 4:755 ODV assembly and ODV envelopes 4:756–757 tegument proteins 4:757 virus defense and resistance to 4:736 baculovirus expression vector system (BEVS) 4:705, 4:704 baculovirus-host interactions 4:732 hosts acquiring genes from infecting viruses 4:732–733 viruses acquiring genes from their hosts 4:732 baculovirus of cricket 4:827–828 baculovirus X 4:827–828 badnavirid, defined 3:274 badnaviruses (Caulimoviridae) 3:158, 3:159t–161, 3:162f, 3:165, 3:167 classification 3:158 clinical features 3:166 defined 3:274 diagnosis 3:167–168 disease symptoms caused by 3:166f epidemiology 3:165–166 genome 3:163 life cycle 3:163–165 pathogenesis 3:166–167

Subject Index prevention 3:168 virion structure 3:158–163 Baloxavir + Oseltamivir 5:171 Baloxavir marboxil 2:559 Baloxavir – Marboxyl (Xofluzas) 5:166–168 BALT see bronchus-associated lymphoid tissue (BALT) baltimore classes, evolutionary status of 1:44 baltimore classification (1971) 1:29 BAM see bacteriophage adherence to mucus (BAM) Bamboo Mosaic Virus (BaMV) 1:364–365 BaMV see Bamboo Mosaic Virus (BaMV) banana bunchy top virus (BBTV) 3:169 diagnosis 3:173–174 epidemiology and control of 3:174–175 genome organization and functions of gene products 3:170–171 geographical distribution 3:174 historical records and possible origins 3:174 host range and transmission 3:171–172 taxonomy, phylogeny, and evolution 3:169–170 virion structure 3:170 virus–host relationships 3:172–173 banana streak disease complex (BSD) 3:166 banana streak GF virus (BSGFV) 3:167 banana streak IM virus (BSIMV) 3:167 banana streak MY virus (BSMYV) 3:167 banana streak OL virus (BSOLV) 3:167 banana viral diseases 3:90 bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) 1:76 Banna virus 4:881 barley yellow dwarf viruses (Luteoviridae) 3:176 control 3:182–183 diagnosis 3:182 epidemiology 3:181–182 genome organization and expression 3:177–180 host range and transmission 3:180–181 replication 3:181 taxonomy and classification 3:176 virion properties and composition 3:176–177 virus–host relationships 3:181 barnaviruses (Barnaviridae) 4:549 MBV evolutionary relationships 4:550 MBV genome organization and expression 4:549–550 MBV transmission and host range 4:550 MBV virion properties 4:549 MBV virion structure and composition 4:549 base-calling, defined 5:27 baseplate-hub protein (BHP) pb3 4:191 Basic local alignment search tool (BLAST) 5:33, 1:101 basic reproductive number, defined 1:53 basolateral membrane, defined 1:529 bat coronavirus (BtCoV) 512 2:245 bathypelagic layer, defined 4:322 BBB see blood-brain barrier (BBB)

BBLV see Bokeloh bat lyssavirus (BBLV) BBMV see Broad bean mottle virus (BBMV) BBSV see beet black scorch virus (BBSV) BBTV see banana bunchy top virus (BBTV) B cell receptors (BCRs) 1:585–587, 1:590–591, 1:663 B cells 1:590–591 BCMD see Brown Cap Mushroom Disease (BCMD) BCMV and BCMNV see Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) BCoV see bovine coronavirus (BCoV) BCRs see B cell receptors (BCRs) BCTV see beet curly top virus (BCTV) BD see border disease (BD) Beak and feather disease virus (BFDV) 2:184 Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) diagnosis 3:190 host range and symptomatology 3:190 reverse transcription-polymerase chain reaction 3:190 serological techniques 3:190 geographic distribution 3:185 history 3:184–185 host range and transmission 3:185–187 pathogenicity, pathology, and resistance genes 3:188–189 prevention and control 3:190–191 properties of the virion and genome 3:187 properties of particles 3:187 properties of the genome and replication 3:188 serological relationships 3:187–188 recombination and variability 3:189–190 taxonomy and classification 3:184–185 bean dwarf mosaic virus (BDMV) 3:561 bean golden mosaic virus and bean golden yellow mosaic virus (Geminiviridae) 3:192–194, 3:561 diagnosis 3:197 management 3:197–198 taxonomy, phylogeny, and evolution 3:194–195 transmission, host range, and epidemiology 3:196 virion structure, genome organization, properties and functions of gene products 3:195–196 virus-host relationships 3:196–197 beans, geminivirus resistance in 3:561–563 becurtovirus 3:411–412 beet black scorch virus (BBSV) 3:795 beet curly top virus (BCTV) 3:200, 3:363 changes in BCTV strain prevalence 3:205 classification and nomenclature 3:201–203 experimental systems for infection of plants by 3:209–210 genome organization and gene expression 3:205–206 geographic and seasonal distribution 3:205 history 3:200–201

323

host range and pathogenesis 3:207–208 management of curly top disease caused by 3:210 cultural practices 3:210–211 IPM for curly top disease 3:211 management of the leafhopper vector with insecticides 3:211 resistant varieties 3:210 sanitation 3:211 origin of BCTV and the beet leafhopper vector 3:203–205 replication 3:206–207 resistance to 3:560–561 transmission of BCTV by the beet leafhopper vector 3:208–209 virus movement 3:207 beetles, plant viruses transmission by 3:112–113 beet necrotic yellow vein virus (Benyviridae) classification 3:213 diagnosis 3:217 disease symptoms 3:216–217 epidemiology 3:215–216 genome organization and functions of gene products 3:213–215 prevention 3:217–218 transmission 3:215 virion structure 3:213 virus–host relationships 3:217 beet poleroviruses 3:599–600 BEFV see bovine ephemeral fever virus (BEFV) begomoviruses 3:13–14, 3:412, 3:357 CLCuD in Africa 3:357 CLCuD in Indian subcontinent 3:357–359 behavioral fever, defined 2:306 Belpaoviridae 3:654 benyviruses (Benyviridae) control 3:226–227 defined 3:213, 3:219 diagnosis 3:226 disease, host range, and epidemiology 3:224–226 history 3:219 organization of genome and properties of encoded proteins 3:220–224 similarities and dissimilarities with other taxa 3:227 transmission 3:226 virion structure 3:219–220 Berrimah virus (BRMV) 2:875 b-barrel, defined 3:692, 3:364, 3:348 b-barrel fold, defined 1:329 bC1 3:239, 3:244, 3:245 beta chemokines, defined 2:827 betaentomopoxvirus 4:859 betaflexiviruses (Betaflexiviridae) 3:229, 3:805 genome organization 3:234–235 geographical distribution 3:237–238 members of the family 3:232 properties and functions of gene products 3:235 replication and propagation 3:235–237 taxonomy and phylogeny 3:229–232 transmission and host range 3:237

324

Subject Index

betaflexiviruses (Betaflexiviridae) (continued) virion structure 3:232–234 beta-helical tailspikes, structures of 4:199f b-helix, defined 1:329 Betaherpesvirinae 1:321 betaherpesvirus 2:442, 2:451f defined 2:441 beta-hexamer 3:262–263 Betairidovirinae 4:707–708 Malacoherpesviridae 4:708 Mesoniviridae 4:708–709 Nimaviridae 4:709 Nodaviridae 4:709–710 Nudiviridae 4:710 Nyamiviridae 4:710–711 Betanodaviruses 4:821 Betanudivirus 4:827, 4:827–828 betapartitivirus 4:635 betapleolipovirus, defined 4:380 betasatellites (Tolecusatellitidae) 3:239 classification and nomenclature 3:239–241 CLCuD-associated 3:361 defined 3:554 genetic diversity and center of origin for 3:241–244 functional role of bC1 in disease development 3:245 life cycle, epidemiology 3:244 replication 3:244–245 resistance against betasatellites 3:245 genome organization 3:241, 3:243f phylogenetic trees for 3:242f species of the genera Betasatellite 3:240t–241 beta sheet, defined 1:345 b-spiral, defined 1:329 beta-structure, defined 4:194 Betatetravirus 4:897 BEVS see baculovirus expression vector system (BEVS) BFDV, see Beak and feather disease virus (BFDV) bhanja virus 2:776 Bhendi yellow vein mosaic virus (BYVMV) 3:756 BHF see Bolivian hemorrhagic fever (BHF) BIBD see boid inclusion body disease (BIBD) Bicaudaviridae 1:369–370, 4:361 bicaudavirus ATV 4:393 bicistronic, defined 3:461, 3:439, 3:154 bictegravir 5:150–151 Phase 3 randomized controlled trials 5:150–151 resistance 5:151 bidensoviruses 4:705 classification 4:759 genome organization and expression strategy 4:759 pathology of silkworm associated with BmBDV 4:759–761 viral non-structural proteins 4:761 viral replication 4:761–762 viral structural protein 4:761 virus evolution 4:762 bidirectional transcription, defined 2:144

Bidnaviridae 4:759, 4:705–706 big-vein disease of lettuce, defined 3:833 biochemical phase, of virology 1:6 bio-crime, defined 1:644 biological control, defined 4:615, 4:493, 4:468, 4:892 biological warfare, defined 1:644 biomotor/nanomotor, defined 4:302 biopesticide, defined 4:892 biosafety and biosecurity in diagnostic laboratories 5:82 biorisk management system and legislation 5:89 diagnostic laboratories 5:82 laboratory biosafety 5:82–83 biological agents 5:82–83 biosafety cabinets 5:84–85 decontamination and waste management 5:85–86 laboratory accidents 5:86–87 laboratory biosafety levels 5:83 occupational health and special groups 5:87 personal protective equipment (PPE) 5:84 risk assessment 5:83 routes of transmission and infective dose 5:83–84 shipment of clinical specimens 5:87–88 laboratory biosecurity 5:88–89 Biosafety Level, defined 2:891 Biosafety level 4 (BSL-4), defined 2:355 bioterrorism, defined 1:644 bioterrorism, viruses and their potential for 1:644 agro-warfare 1:647–648 biological agents for good or evil 1:648–649 bioweapons, viruses as 1:646–647 effects of viral introductions 1:649 entomological warfare 1:647 20th century state sponsored biological weapons programs 1:645–646 unconventional bioweapons 1:644 use of diseases in early history 1:644–645 bioweapons 1:676–677 viruses as 1:646–647 bipartite begomoviruses 3:753, 3:758, 3:244 birds, retroviruses of classification 2:122–123 clinical features 2:125 diagnosis 2:126 endogenous retroviruses 2:126 epidemiology 2:124–125 genome 2:123–124 history 2:122 life cycle 2:124 pathogenesis 2:125–126 treatment 2:126 virion structure 2:123 Birnaviridae 2:544, 4:776–777 birnaviruses of invertebrates (entomobirnavirus) 4:706 Dicistroviridae 4:706 Hytrosaviridae 4:706–707 Iflaviridae 4:707

birth defects, defined 2:797 BIV see bovine immunodeficiency virus (BIV) bivalent oral polio vaccine (bOPV) 5:312–313 BK see bradykinin (BK) BK polyomavirus (BKPyV) 2:522, 5:105, 5:108 clinical features 2:522 infectious cycle 2:522–523 BKPyV see BK polyomavirus (BKPyV) Black queen cell virus (BQCV) 4:768–770, 4:771, 4:772, 4:773 blood-brain barrier (BBB) 1:536, 2:847 defined 2:884 blood virome 1:556 bluetongue virus (BTV) 2:127, 1:545, 1:303 classification 2:127 diagnosis 2:134 epidemiology 2:132–133 future perspectives 2:135–136 life cycle 2:129–130 assembly of genomic RNA and packaging 2:132 core assembly 2:131–132 transcription and replication 2:130–131 virion maturation and egress 2:132 virus attachment and entry into cells 2:129–130 pathogenesis and clinical features 2:133–134 replication cycle 2:130f structure of 2:129f vaccination and control 2:134–135 virion structure and genome 2:127–129 blunervirus 3:247 diagnosis 3:251 diseases, epidemiology, and control 3:250–251 genome organization and gene product function 3:248–249 members of the family 3:247 replication and propagation 3:249–250 taxonomy, phylogeny, and evolution 3:247 transmission and host range 3:250 virion structure 3:247–248 BLV see bovine leukemia virus (BLV) B lymphocytes 1:590–591 BM2 2:566–567 BmBDV see Bombyx mori bidensovirus (BmBDV) BmDNV see Bombyx mori densovirus (BmDNV) BMV see brome mosaic virus (BMV) bnMAbs see broadly-neutralizing monoclonal antibodies (bnMAbs) bocaviruses clinical features 2:424 epidemiology 2:422–424 boceprevir 5:123 boid inclusion body disease (BIBD) 2:507 Bokeloh bat lyssavirus (BBLV) 2:738 Bolivian hemorrhagic fever (BHF) 2:507, 2:512 Bombyx mori bidensovirus (BmBDV) 4:759, 4:839

Subject Index Bombyx mori densovirus (BmDNV) 4:759–760 bombyx mori latent virus 3:825 properties and distinguishing characteristics 3:825 bona fide viruses 1:14 bOPV see bivalent oral polio vaccine (bOPV) border disease (BD) 2:153, 2:161 control of BD of sheep 2:163 Borell bodies, defined 2:343 borna disease 2:139 borna disease virus and related bornaviruses 2:137 classification 2:137 control and prevention 2:142–143 diagnosis 2:142 endogenous bornavirus-like elements 2:141 epidemiology and clinical features 2:139 bornavirus associated encephalitis in humans 2:139–140 proventricular dilatation disease (PDD) of birds 2:140–141 staggering disease in cats 2:139 future prospects 2:143 genome 2:137 life cycle 2:137–139 pathogenesis 2:141–142 virus structure 2:137 botryoid, defined 2:182 Botrytis cinerea, hypovirulence in 4:476 Botrytis virus F (BVF) 4:478–479, 4:437–438, 4:462 biological properties 4:479 family –Gammaflexiviridae 4:479 genome structure 4:479 genus –Mycoflexivirus 4:479 phylogenetic relationships 4:479–480 virion morphology 4:479 Botrytis Virus X (BVX) 4:480, 4:438 biological properties 4:480–481 family –Alfaflexiviridae 4:480 genome structure 4:480 genus –Botrexvirus 4:480 phylogenetic relationships 4:481 virion morphology 4:481 bottleneck, defined 4:457 bottle-shaped ampullaviruses 1:433–434 botybirnaviruses 4:552 biology 4:554–556 genome organization and replication 4:552–553 taxonomy and similarity with other viruses 4:553–554 transmission and distribution 4:554 virion properties 4:552 bovine, ovine, caprine, and other ruminant adenoviruses 2:13 bovine coronavirus (BCoV) 2:198 bovine ephemeral fever virus (BEFV) 2:875 classification 2:875 control and treatment 2:882–883 epidemiology 2:880–881 life cycle 2:878–879 assembly and budding 2:879–880

attachment, entry and uncoating 2:878–879 gene expression and replication 2:879 inhibition and modification of host cell functions 2:880 pathogenesis and clinical features 2:881–882 virus structure 2:875–878 genomes and proteins 2:877–878 bovine immunodeficiency virus (BIV) 2:56–57, 2:66 bovine leukemia virus (BLV) 2:317 classification 2:144 diagnosis 2:144–145 epidemiology and clinical features 2:144 genome organization and expression 2:148–150 life cycle 2:145 pathogenesis 2:150 prevention and treatment 2:145 virion structure 2:145–148 bovine papular stomatitis (BPS) 2:668, 2:668f bovine papular stomatitis virus (BPSV) 2:666, 2:170–171 bovine RSV (bRSV) 2:754 infections 2:748 bovine viral diarrhea 2:153 and border disease 2:158–159 control of 2:163 and mucosal disease 2:160 acute BVDV infection 2:160 fetal infection with BVDV 2:160 mucosal disease 2:160–161 persistent BVDV infection 2:160 vaccines against 2:162 bovine viral diarrhea virus (BVDV) 1:425 BPCV1 see bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) BPD see bronchopulmonary dysplasia (BPD) BPS see backyard production systems (BPS); bovine papular stomatitis (BPS) BPSV see bovine papular stomatitis virus (BPSV) BPXV see buffalopox virus (BPXV) BQCV see Black queen cell virus (BQCV) brachygnathia, defined 2:34 bracovirus 4:712, 4:849 Bradford Hill criteria for causation 1:567, 1:568t bradykinin (BK) 2:353 breaching epithelial barriers 1:535–536 Brevihamaparvovirus 4:836 BREX see Bacteriophage Exclusion (BREX) bridge amplification 1:177 bridge vector, defined 2:805 brivudine ((E)-5-(2-bromovinyl)-2’deoxyuridine) 5:184 BRMV see Berrimah virus (BRMV) Broad bean mottle virus (BBMV) 3:266 broadly neutralizing antibodies (bNAbs) 1:213 broadly-neutralizing monoclonal antibodies (bnMAbs) 5:273

325

brome mosaic virus (BMV) 1:463, 3:261–262, 3:262–263, 3:263, 3:264f, 3:266, 3:252 application in biotechnology 3:259 coat proteins (CP) 3:266 future perspectives 3:259 genome organization 3:253–254 genome structure, gene expression and sequences 3:253–254 noncoding regions, cis signals, and subgenomic promoter 3:254–255 host genes 3:257 inducing/stabilizing spherules 3:257–258 regulating lipid metabolism and membrane composition 3:257 regulating translation of viral proteins and 1a targeting 3:258 required for viral replication activity 3:258 transmission and host range 3:258–259 properties and functions of viral gene products 3:256–257 BMV 1a and viral replication compartments 3:256–257 replication and propagation 3:255–256 in vitro and in vivo replication studies 3:255–256 RNA recombination 3:256 sub-genomic mRNA synthesis 3:256 virion structure and RNA encapsidation 3:252–253 Bromoviridae 3:371, 3:132, 3:260 evolution of 3:263f genera and species in 3:262t phylogeny and biodiversity of 3:261 bromovirids 3:261 bromoviruses (Bromoviridae) biology 3:266–267 characteristics of RNA genome in 3:261t genome organization and expression 3:264–266 genomic RNA replication and recombination 3:266 phylogeny and biodiversity of the family Bromoviridae 3:261 virion properties and structure 3:261–264 bronchiolitis and recurrent wheezing 2:761 bronchopulmonary dysplasia (BPD) 5:273 bronchus-associated lymphoid tissue (BALT) 2:76 Brown Cap Mushroom Disease (BCMD) 4:532 classification 4:532 diagnosis 4:533 epidemiology 4:533 genome and virion structure 4:532 viral expression and disease development 4:532–533 Brownian motor, defined 4:105 bRSV see bovine RSV (bRSV) BSD see banana streak disease complex (BSD) BSGFV see banana streak GF virus (BSGFV) BSIMV see banana streak IM virus (BSIMV)

326

Subject Index

BSMYV see banana streak MY virus (BSMYV) BSOLV see banana streak OL virus (BSOLV) BTV see bluetongue virus (BTV) budded virus (BV) 4:704 defined 4:739, 4:747 budding, defined 1:345 budding, virus 1:519–520 assembly 1:519–520 cell-to-cell transmission 1:525 envelopment 1:521 ESCRT-dependent budding 1:522–523 intracellular budding 1:523–524 maturation 1:524–525 quasi-enveloped viruses 1:524 buffalopox virus (BPXV) 2:858 Bunyamwera serogroup 2:662 Bunyavirales 3:495 bunyavirales glycoproteins 1:348 bunyavirus 1:499 of arthropods 4:764 genome and coding strategies of viral genomes 4:764–766 host associations, virus maintenance cycles and pathogenicity 4:767 viral replication cycle 4:766–767 virion structure 4:764 defined 4:764 buoyant densities in CsCl, defined 4:632 Bursa of Fabricius, defined 2:182, 2:540 burst, defined 4:314 burst assay, defined 4:98 BV see budded virus (BV) BVDV see bovine viral diarrhea virus (BVDV) BVF see Botrytis virus F (BVF) BVX see Botrytis Virus X (BVX) Bwamba serogroup 2:662 bymoviruses (Potyviridae) classification 3:268 control 3:272 defined 3:268 diagnosis 3:271–272 member species 3:268 nucleic acid properties and differentiation of bymoviruses 3:268–269 organization of the genome and properties of the encoded proteins 3:269–271 symptoms, epidemiology and host range 3:271 virion structure, serological- and cytological properties 3:268 virus transmission and movement 3:271 bystander effects, defined 5:233 BYVMV see Bhendi yellow vein mosaic virus (BYVMV)

C cabotegravir 5:151 cacao swollen shoot disease (CSSD) 3:90 cacao swollen shoot Togo B virus (CSSTBV) 3:167 cacao swollen shoot virus 3:274

control of the disease 3:284 diagnosis 3:280–283 PCR diagnosis 3:283–284 serological diagnostics 3:281–283 epidemiology: geographical distribution of viral molecular diversity 3:278–280 genome organization 3:277 host range and symptomatology 3:274–276 phylogeny 3:277–278 taxonomy 3:274 transmission 3:276–277 virion structure 3:277 cactus-infecting viruses 3:623–624 Cactus virus X (CVX) 3:623–624, 3:628 cadherin-related family member 3 (CDHR3) 2:758–759, 1:410 CAEV see caprine arthritis encephalitis virus (CAEV) Cafeteria roenbergensis 1:85 CAGE-Seq see cap analysis gene expression sequencing (CAGE-Seq) calcium 4:523 Caliciviridae 1:278–279 Californian myxoma virus 2:731–732 California serogroup 2:662–663 Caligrhavirus 4:715–716 Calliptamus italicus EPV (CIEV) 4:865 Calmodulin-like proteins 3:245 calnexin 1:603 Calvusvirinae 3:788 CAM see cell adhesion molecules (CAM) CaMV see cauliflower mosaic virus (CaMV) cancer 2:454 cancer therapeutics, virus-based arming viruses 5:241 immunomodulators 5:241 prodrug convertases 5:241 radiosensitizers for therapy and imaging 5:241 combination therapies 5:241–243 definition 5:233 historic notes 5:233–234 re-engineered viruses: the beginnings 5:234–235 shielding from host immune response 5:239–240 chemical shielding and cell carriers 5:240–241 virus engineering 5:239–240 three classes of modifications 5:235–237 virus tropism, retargeting 5:237–238 cell entry targeting by protease activation 5:238 cell entry targeting of enveloped viruses 5:237–238 cell entry targeting of non-enveloped viruses 5:238 combination of targeting modalities 5:239 post-transcriptional regulation of replication 5:239 preferential tumor spread 5:239 transcriptional regulation of replication 5:238–239 cancer vaccine, defined 1:658

cancer vaccines and therapeutics, VLPs as 1:669–670 canine and other carnivoran adenoviruses 2:12 Canine circovirus (CanineCV) 2:185 canine coronavirus (CCoV) 2:245, 2:198 CanineCV, see Canine circovirus (CanineCV) canine distemper virus (CDV) 2:74 canine parvovirus (CPV) 2:684 CPV-2 2:684 emergence of classification (compact) 2:683 diagnosis 2:687 epidemiology 2:684–686 life cycle 2:683–684 pathogenesis and clinical features 2:686–687 prevention 2:687 treatment 2:687 virion structure and genome 2:683 host range variation, its control, and the emergence of 2:685–686 Canis familiaris papillomavirus type 1 (CPV-1) 2:85 Canna yellow mottle virus (CaYMV) 3:166 canonical/cap-dependent MRNA translation 1:444–445 elongation and termination phases 1:446 initiation phase 1:444–445 initiation factor eIF4E 1:445 protein kinase R (PKR) 1:444–445 scanning and assembly of the 80S ribosome 1:445–446 canonical initiation of virus mRNA translation 1:446–447 “imprisonment” of cellular mRNAs within the nucleus 1:447 microRNAs (miRNAs) 1:455 non-canonical initiation of virus mRNA translation 1:447–449 cap-independent translation enhancers (CITEs) 1:449 initiation at non-AUGs 1:450 internal ribosome entry sites (IRESes) 1:447–449 leaky scanning 1:450 ribosome reinitation 1:450–452 ribosome ‘shunting’ (discontinous scanning) 1:450 virus alternatives to components of initiation 1:449–450 translational ‘recoding’: non-canonical elongation and termination 1:452 ribosomal bypassing (‘hopping’) 1:452 ribosomal ‘frame-shifting’ 1:452–454 ribosome stop codon ‘read-through’ 1:454 virus-encoded proteinases 1:454–455 ‘stopgo’/‘stop carry-on’/ribosomal ‘skipping’ 1:454 CAPA see corrective actions and preventative actions (CAPA) cap analysis gene expression sequencing (CAGE-Seq) 1:178 cap-independent translation enhancers (CITEs) 1:449

Subject Index capping, defined 2:475 caprine arthritis encephalitis virus (CAEV) 2:56–57, 2:65, 2:66–67 capripoxviruses (CPPV) 2:165 capsid 1:257, 1:495, 2:441, 1:402, 1:362, 4:387, 4:359, 1:382, 4:26, 4:276, 4:98, 2:92, 4:115, 3:545, 4:568, 4:105, 4:167 capsid architecture, general principles of 1:257–261 helical symmetry 1:261 icosahedral symmetry 1:258–261 capsid assembly 1:263–266, 4:49–50 enveloped icosahedral RNA viruses 1:266–268 large icosahedral dsDNA viruses 1:269–271 non-enveloped icosahedral RNA viruses 1:265–266 small icosahedral ssDNA viruses 1:268–269 tailed bacteriophages 1:271–272 capsid-associated tegument complex (CATC) 1:321, 1:322 capsid-encoding organisms 1:14 capsid/head, defined 4:45 capsid layers, structural organization of 1:306–307 double-stranded RNA (dsRNA) viruses, cell entry of 1:307–309 outer capsid layer 1:306–307 capsidless particles 2:442–443 capsid or head, defined 1:318 capsid or phage head, defined 4:136 capsid protein (CP) 3:163, 4:594, 3:318, 4:821, 1:353, 1:355–356, 2:57 defined 4:658 structural folds of 1:262–263 four-helix bundle 1:263 HK97 fold 1:263 immunoglobulin-like fold 1:263 jelly-roll b-barrel 1:262–263 serine protease fold 1:263 capsid protein 2:184, 2:884–885 capsid self-assembly 1:483 capsid strategy 3:112 aphid-transmitted cucumoviruses, case of 3:112 capsid vertex specific complex (CVSC) 2:715 cap snatching, defined 4:764, 3:719, 2:765 capsomer, defined 4:115, 3:461, 2:441 capsomere, defined 1:257 capsomers, defined 1:248, 2:22 capsule, defined 1:402 capulavirus 3:412–413 CAR see coxsackievirus B3-receptor (CAR) Carajas virus (CARV) 2:875 Carcinus mediterraneus reovirus W2 (CMRV) 4:878 cardoreovirus 4:878–879, 4:878 carmo-like viruses (Tombusviridae) 3:285 distribution, host range, transmission, and economic significance 3:288–289 genome structure and protein functions 3:289–290 replication and gene expression 3:290

satellites, defective-interfering RNAs 3:290–291 taxonomy, classification, and evolutionary relationships 3:285–288 virion structure and assembly 3:289 virus–host interaction 3:291 Carmotetraviridae 4:898, 4:717 carnivora, defined 2:683 carnivores, parvoviruses of classification (compact) 2:683 diagnosis 2:687 epidemiology 2:684–686 life cycle 2:683–684 pathogenesis and clinical features 2:686–687 prevention 2:687 treatment 2:687 virion structure and genome 2:683 carrier state, defined 4:26 cART see combinatorial antiretroviral therapy (cART) CARV see Carajas virus (CARV) case-control study and cohort studies 1:564–565 defined 1:559 case fatality rate (CRF) 2:212, 2:608 defined 5:247 cash crops viral diseases 3:94–95 ornamentals and orchids viral diseases 3:95–96 sugarcane viral diseases 3:94–95 casing, defined 4:549, 4:528 Caspar Klug theory (CKT) 1:250 defined 1:248 caspase, defined 4:724 Casphalia extranea 4:839 cassava brown streak disease (CBSD) current status in Africa 3:295 engineering CBSD resistance 3:299–300 evolution, diversity and distribution of viruses Involved in 3:297–298 genome organization and gene functions 3:295–297 management and control of 3:298 sources of CBSD resistance and their introgression 3:298–299 cassava brown streak viruses (Potyviridae) 3:293 aetiology, host range and transmission 3:293–295 Cassava common mosaic virus (CsCMV) 3:628, 3:628–629 cassava mosaic geminiviruses (CMGs), resistance to 3:556–558 cassava mosaic viruses (Geminiviridae) 1:649 control 3:309 cultural methods 3:310–311 biosafety considerations 3:311 virus resistance by engineering 3:310–311 diagnosis 3:308–309 economic importance 3:309 epidemiology 3:306 distribution of CMGs in Asia 3:306–307 diversity and distribution 3:306 distributionoftheAfricanCMGs,21523: s0065

327

recombination and the CMD pandemic 3:307–308 field-level epidemiology 3:308 genome 3:303 history 3:301–302 host plant resistance 3:309–310 intraspecific diversity 3:301 pathogenesis and symptoms 3:305–306 regional epidemiology 3:308 replication 3:303–304 taxonomy 3:301 transmission 3:304–305 virion structure 3:302–303 cassava viral diseases 3:83–85 Cassava virus X (CsVX) 3:623, 3:623–624, 3:628 CAT1 see cationic amino acid transporter 1 (CAT1) catalytic RNA, defined 3:852 CATC see capsid-associated tegument complex (CATC) catch-all detection methods 5:260–261 category A-C Priority pathogens, defined 2:765 cationic amino acid transporter 1 (CAT1) 2:145 Caudovirales 1:319, 4:186, 4:186–187 caudovirales, defined 4:276, 4:342 cauliflower mosaic virus (CaMV) 3:164, 1:462 properties and functions of CaMV gene products 3:316–318 aphid transmission factor (P2) 3:318 capsid protein (P4) 3:318 movement protein (MP) 3:317–318 protease/reverse transcriptase (P5) 3:318 transactivator/viroplasmin (P6) 3:318 virion-associated protein (P3) 3:318 caulimoviruses (Caulimoviridae) 3:313–314 genome organization 3:314–316 replication and propagation 3:318–320 taxonomy, phylogeny, and evolution 3:314 transmission, host range 3:320 virion structure 3:314 virus-host relationships 3:320–321 causation, Bradford Hill criteria for 1:567, 1:568t CAV see chicken anemia virus (CAV); coxsackie A virus (CAV) caveolae, defined 1:529 caveolin-1 (CAV-1) 1:532–533 caveolin-dependent endocytosis 1:532–533 CaYMV see Canna yellow mottle virus (CaYMV) CBP see chitin-binding protein (CBP) CBSD see cassava brown streak disease (CBSD) CCAAT box 2:58–59 C-C chemokine receptor type 5 (CCR5) 5:151, 2:60, 2:827 CCD see colony collapse disorder (CCD) CCHBV see crane hepatitis B virus (CCHBV) CCHFV see Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV)

328

Subject Index

CcIRV-5 see Ceratitis capitata idnoreovirus-5 (CcIRV-5) CCMV see cowpea chlorotic mottle virus (CCMV) CCoV see canine coronavirus (CCoV) CCR5 see C-C chemokine receptor type 5 (CCR5) CCRS see Cherry chlorotic rusty spot disease (CCRS) CCS see closed circular consensus (CCS) CD3 1:584 CD4 2:60, 2:778, 2:463, 2:464, 1:396, 5:139, 1:213, 1:213–215, 1:383 CD4 T cells 2:229, 1:585, 2:456, 2:825 CD4+ T cells 2:469, 2:470, 2:471, 2:229, 2:392, 1:589, 1:590, 1:595–596, 1:587, 2:65, 2:66, 2:827, 2:56, 1:399–400, 2:14, 1:604 CD4+, defined 2:827 CD8 2:778 CD8 T cells 2:229, 2:455, 2:457, 2:825 CD8+ cytotoxic T cells (CTL) 1:589 CD8+ T cells 1:587, 2:229, 1:589, 1:590, 2:392, 1:603 CD14+ lineage-committed granulocytemacrophage progenitors 2:456 CD40 2:613–614 CD28 1:589 CD34+ stem cells 2:456–457, 2:457 CD46 1:396, 2:778, 1:412 CD55 1:284 CD56 1:584 CD80 2:613–614 CD81 2:390 CD86 2:613–614 CD134 2:60, 2:66, 2:778 CD155 1:659, 1:410, 2:688 CD163 2:703 CD206 2:60 CDHR3 see cadherin-related family member 3 (CDHR3) CDR see complementarity-determining regions (CDR) CDV see canine distemper virus (CDV) cELISA see competitive enzyme-linked immunosorbent assay (cELISA) cell adhesion molecules (CAM) 1:394 cell-associated enveloped virion (CEV) 2:165 cell-based vaccines 5:302–303 cell biology phase, of virology 1:6–7 cell-cycle link protein, defined 3:169 cell entry targeting of enveloped viruses 5:237–238 of non-enveloped viruses 5:238 by protease activation 5:238 cell envelope 1:503–504 cell envelope, defined 1:402 cell-free assembly (CFA) system 2:131–132 cell-free protein synthesis (CFPS) 1:665 cell line, defined 4:888 cell-mediated immunity (CMI) 2:516, 2:639 defined 2:79 cell polarity, defined 1:529 cell-surface, interaction with 4:389–390 cell-to-cell movement, defined 3:140

cell-to-cell movement protein, defined 3:545 cell-to-cell transmission 1:525 cell tropism, defined 2:198 cellular adenosine deaminases 1:55–56 cellular appendages, interaction with 4:388–389 cellular cytidine deaminases 1:55 cellular electron cryotomography 1:238–240 cellular immune response contraction of 1:590 VLPs and the induction of 1:662 cellular “rotting” 1:503 cell wall, defined 1:402 central nervous system (CNS) 1:498–499 infections 5:102 assays 5:102 background 5:102 clinical impact 5:102–103 CEPI see Coalition for Epidemic Preparedness Innovations (CEPI) Ceratitis capitata idnoreovirus-5 (CcIRV-5) 4:875 cereal viral diseases 3:83 Certification Program, defined 3:430 certified reference materials (CRM) 5:76 cervidpoxvirus 2:171 cesium chloride gradients 5:11 CEV see cell-associated enveloped virion (CEV) CFA system see cell-free assembly (CFA) system CfCPV-16 see Choristoneura fumiferana cypovirus 16 (CfCPV-16) CFPS see cell-free protein synthesis (CFPS) CFS see Chronic Fatigue Syndrome (CFS) CFTR see cystic fibrosis transmembrane conductance regulator (CFTR) Chagas disease 4:771 chagres virus 2:771 chain-termination sequencing 5:28 Chandipura virus (CHPV) 2:875 chaperones, defined 3:32 Chaphamaparvovirus 4:836 chapparvovirus 4:836 CHB see chronic hepatitis B (CHB) chemical composition of viruses 2:415 chemical fixation 1:243 chemiosmotic theory 4:215 chemistry, manufacturing, and control (CMC) 4:252, 4:256 chemokine, defined 2:56 chemokine binding protein (vCBP) 2:671–672 chemokine receptor, defined 2:827 cheraviruses classification 3:322 clinical features and pathogenesis 3:325 diagnosis 3:325 genome organization 3:322 geographic distribution 3:324 life cycle and epidemiology 3:322–324 host range 3:324 prevention 3:326 transmission 3:324–325 treatment 3:325–326

virion structure 3:322 cherax quadricarinatus densovirus (CqDV) 4:840 Cherry chlorotic rusty spot disease (CCRS) 4:646–647 chestnut mosaic virus (ChMV) 3:165 CHF see Crimean hemorrhagic fever (CHF) chicken anemia virus (CAV) 2:48 Chickpea chlorotic dwarf virus (CpCDV) 3:358–359 Chikungunya virus (CHIKV) 1:263, 1:546–547, 1:239, 5:275 classification 2:173 clinical features 2:178–180 diagnosis, treatment, and prevention 2:180 epidemiology 2:178 genome organization and features 2:173–174 CHIKV 3’ UTR 2:175 non-coding elements 2:174–175 protein-coding regions 2:174 life cycle 2:175–176, 2:175f attachment and entry 2:175–176 non-structural protein expression and viral replication 2:176–177 structural protein expression and virion assembly 2:177–178 pathogenesis 2:180 virion structure 2:173 CHIKV see Chikungunya virus (CHIKV) ChiLCD see chilli leaf curl disease (ChiLCD) ChiLCV see Chilli leaf curl virus (ChiLCV) chili leaf curl betasatellite (ChLCB) 3:357 chilli leaf curl disease (ChiLCD) 3:756 Chilli leaf curl India virus 3:750 Chilli leaf curl virus (ChiLCV) 3:756, 3:750 chilli viral diseases 3:86–87 Chilo iridescent virus (CIV) 1:269–270 Chinese hamster ovary (CHO) cells 2:857–858, 1:668, 5:284 chitin-binding protein (CBP) 4:813 ChLCB see chili leaf curl betasatellite (ChLCB) ChMV see chestnut mosaic virus (ChMV) CHO cells see Chinese hamster ovary (CHO) cells Chordopoxvirinae 4:858 chordopoxviruses (ChPVs) 4:860–861 chordovirus, defined 3:805 Choristoneura fumiferana cypovirus 16 (CfCPV-16) 4:874 CHPV see Chandipura virus (CHPV) ChPVs see chordopoxviruses (ChPVs) chromatographic methods 1:170–171 chromosomal integration of HHV–6 (CIHHV–6) 2:778, 2:781, 2:787, 2:784–785 and potential for disease in immunocompetent 2:784–785 and potential for disease in immunocompromised 2:785 chromoviruses, defined 3:653 Chronic Fatigue Syndrome (CFS) 5:297–298

Subject Index chronic hepatitis B (CHB) 5:217 management in special populations 5:222 immunosuppression 5:222 pregnancy 5:222 treatment goals in 5:217–218 chronic hepatitis B and chronic hepatitis D infection chronic hepatitis delta 5:222 future 5:224 management of 5:217 nucleo(s)tide analogs 5:218–219 choosing the appropriate first-line agent 5:219 finite NA therapy 5:219 management of patients with NA failure 5:219 safety 5:218–219 nucleo(s)tide analogs 5:223–224 on-treatment prediction of response 5:221 HBeAg-negative patients 5:221 HBeAg-positive patients 5:221 optimizing response rates: treatment prolongation and combination therapy 5:221–222 optimizing response to NA with PEG-IFN 5:222 pegylated interferon alfa 5:219–220 efficacy in HBeAg-negative patients 5:220 efficacy in HBeAg-positive patients 5:220 safety of PEG-IFN in CHB 5:220 screening 5:222–223 selection of patients for PEG-IFN therapy 5:220–221 HBeAg-negative patients 5:221 HBeAg-positive patients 5:220–221 treatment indications 5:217 and goals 5:223 treatment options 5:218, 5:223 interferon alpha (IFN) 5:223 chronic hepatitis C, defined 2:386 chronic obstructive pulmonary disease (COPD) 2:762 chronic persistent infection, defined 2:697 chronic release, defined 4:314 chryso-P4 4:564 chrysoviruses (Chrysoviridae) 4:557, 4:505–506, 4:454 biology and effects of chrysoviruses on fungal hosts 4:565–566 evolutionary relationships among 4:547–548 genome expression and replication 4:562–563 alphachryso-P3 4:564 chryso-P4 4:564 chrysovirus CPs 4:563–564 chrysovirus RdRps 4:563 replication of chrysoviruses 4:564 genome organization 4:559–562 taxonomy and phylogenetic analysis 4:564–565 virion properties 4:557 virion structure and composition 4:557–559

CHV1 see cryphonectria hypovirus 1 (CHV1) CHV4 see cryphonectria hypovirus 4 (CHV4) chytrid fungi, defined 3:567 Cicadulina spp. 3:461 cidofovir 2:458–459 CIEV see Calliptamus italicus EPV (CIEV) CIHHV–6 see chromosomal integration of HHV–6 (CIHHV–6) cilevirus 3:247 diagnosis 3:251 diseases, epidemiology, and control 3:250–251 genome organization and gene product function 3:248–249 members of the family 3:247 replication and propagation 3:249–250 taxonomy, phylogeny, and evolution 3:247 transmission and host range 3:250 virion structure 3:247–248 Circoviridae 4:718 classification 2:182 clinical features 2:186–187 diagnosis 2:190–191 disease control 2:191 epidemiology 2:185–186 genome 2:184 life cycle 2:184–185 pathogenesis 2:187–190 virion structure 2:182–184 circoviruses 2:186 circularly permuted, defined 4:276 circular permutation, defined 4:368 circulating recombinant forms (CRFs) 1:108 circulating vaccine-derived poliovirus (cVDPV) 5:310, 2:692–695 circulative, propagative transmission, defined 3:761 circulative non-propagative transmission 3:107–108 aphid-transmitted luteovirids, case of 3:107–108 leafhopper-transmitted mastreviruses, case of 3:108 whitefly-transmitted begomoviruses, case of 3:108–110 circulative propagative transmission 3:110 circulative transmission, defined 3:169 circumsporozoite protein (CSP) 1:665 cis-acting replication element (Cre) 2:757 cis-acting RNA elements (CREs) 2:387 CITEs see cap-independent translation enhancers (CITEs) 3’CITE, defined 3:456 citopathology, defined 3:788 citrivirus, defined 3:805 citrus tristeza virus (CTV) 3:90–91, 3:327–328 defective RNAs 3:332 diagnosis 3:334–335 economic costs of 3:329 gene functions 3:331–332 CTV sub-genomic RNAs 3:332 genome 3:330–331

329

untranslated regions (UTRs) 3:331 geographic distribution 3:328–329 history 3:328 host range and cytopathology 3:329–330 CTV virions 3:330 taxonomy and classification 3:328–329 transgenic resistance to CTV infection 3:334–335 transmission 3:333–334 CTV control measures 3:334 virus-virus interactions 3:332–333 CIV see Chilo iridescent virus (CIV) cKS see classical Kaposi’s sarcoma (cKS) CKT see Caspar Klug theory (CKT) cladogram 1:117–118 classical Kaposi’s sarcoma (cKS) 2:606 classical swine fever (CSF) 2:32, 2:158, 2:159 acute CSF 2:159 chronic CSF 2:159 control of in domestic pigs 2:162 in wild boar 2:162–163 late onset CSF 2:159–160 vaccines against 2:161–162 classical swine fever viruses 2:153 classification, defined 1:28 classification, virus 1:33 taxa and viruses, differentiating 1:33 Class I fusion glycoproteins 1:420, 1:419f Class I common characteristics 1:420–421 influenza HA 1:420 Class II fusion glycoproteins 1:421, 1:422f Class II common characteristics 1:421–423 several cellular fusogens have the class II fold 1:423–424 TBEV E 1:421 Class III fusion glycoproteins 1:424, 1:423f Class III common characteristics 1:424 VSV G 1:424 class switch recombination (CSR) 1:591 clathrin-coated pits, defined 1:529 clathrin-independent mechanisms 2:176 clathrin-mediated endocytosis (CME) 2:176 defined 2:22 Clavaviridae 4:361–362 Claviviridae 1:369 CLCAlaV, see Cotton leaf curl Alabad virus (CLCAlaV) CLCBurA, see Cotton leaf curl Burewala alphasatellite (CLCBurA) CLCGezA, see Cotton leaf curl Gezira alphasatellite (CLCGezA) CLCGezB, see Cotton leaf curl Gezira betasatellite (CLCGezB) CLCGezV, see Cotton leaf curl Gezira virus (CLCGezV) CLCKokV, see Cotton leaf curl Kokhran virus (CLCKokV) CLCKokV-Burewala strain (CLCKokV-Bur) 3:357 CLCMulA, see Cotton leaf curl Multan alphasatellite (CLCMulA) CLCMulB, see Cotton leaf curl Multan betasatellite (CLCMulB)

330

Subject Index

CLCuD see cotton leaf curl disease (CLCuD) cleavage, defined 3:797 cleavage/packaging, defined 2:441 CLEM see correlative light and electron microscopy (CLEM) Clinical and Laboratory Standards Institute (CLSI) 5:55 clinical diagnostic virology 5:98–99 central nervous system infections 5:102 assays 5:102 background 5:102 clinical impact 5:102–103 gastrointestinal infections (GI) 5:101 assays 5:101–102 background 5:101 clinical impact 5:102 hepatitis E virus (HEV) infection 5:103 respiratory tract infections (RTI) 5:99–100 assays 5:100–101 background 5:99–100 clinical impact 5:101 clinical signs, defined 2:697 clinical trial, defined 5:281 clinical utility 5:73–74 clink, defined 3:470 clonal expansion, defined 2:144 clonal interference, defined 1:53 clonality, defined 2:528 Clonal virus strains 2:453 cloned recessive resistance genes 3:73–74 cloning 1:8–9 closed circular consensus (CCS) 1:181 closely related viruses independently infecting plants and fungi 4:445 Closteroviridae 3:336 closteroviruses (Closteroviridae) 3:336 classification 3:336–338 defined 3:336 diagnosis 3:346 disease symptoms 3:345 epidemiology 3:343–345 genome organization 3:338–341 infectious cycle 3:341–343 pathogenesis 3:345–346 prevention 3:346 treatment 3:346 Clover yellow mosaic virus (ClYMV) 3:628 CLSI see Clinical and Laboratory Standards Institute (CLSI) clustered regularly interspaced short palindromic repeat (CRISPR) 1:623, 1:624f, 5:93, 5:113, 1:60, 1:633, 4:423, 4:343, 4:242–243 CRISPR-associated (Cas) genes 1:633 CRISPR-associated nuclease 9, defined 3:293 CRISPR-Cas 1:610–613, 4:252, 3:411, 3:52, 3:123 CRISPR-Cas3 see Type I CRISPR-Cas systems CRISPR/Cas9 technology, 1:498, 3:420, see also Type II CRISPR-Cas systems CRISPR-Cas immunity, defined 4:419 CRISPR-Cas system 4:229, 4:397, 4:400

clustered regularly interspaced short palindromic repeats-CRISPR associated 9 (CRISPER-Cas9) 3:759 clustered regularly interspaced short palindromic repeats with the enzyme Cas (CRISPR-Cas) 5:113–114 ClYMV see Clover yellow mosaic virus (ClYMV) CM see cytoplasmic membrane (CM) CMC see chemistry, manufacturing, and control (CMC) CMD see covert mortality disease (CMD) CME see clathrin-mediated endocytosis (CME) CMI see cell-mediated immunity (CMI) CMRV see Carcinus mediterraneus reovirus W2 (CMRV) CMs see convoluted membranes (CMs) CMV see cucumber mosaic virus (CMV) CMV see cytomegalovirus (CMV) CMVIG see cytomegalovirus hyperimmune immunoglobulin (CMVIG) CNS see central nervous system (CNS) Coalition for Epidemic Preparedness Innovations (CEPI) 2:516, 2:361, 5:286 coat protein (CP) 4:262–263, 4:105, 3:140 cocal virus (COCV) 2:875 COCV see cocal virus (COCV) Codon 129 polymorphism, defined 2:707 coevolution defense mechanisms 1:641–642 defined 2:3, 2:349 integration sites 1:641 in the patient 1:641 polylysogeny 1:638–640 prophage remnants 1:640–641 war and peace 1:642 cohort study, defined 1:559 cold/hot tumor, defined 1:658 cold tumor 1:659 coliphage N4 virions 4:213–214 collection of routine data 5:247–248 colletotrichum camelliae filamentous virus 1 4:483–484 biological properties 4:484 family –Unclassified 4:483–484 genome structure 4:483–484 genus –Unclassified 4:483–484 phylogenetic relationships 4:484 virion morphology 4:484 colony collapse disorder (CCD) 4:772 Colorado tick fever virus (CTFV) 1:546 Columbiformes 2:811 combination therapy, defined 1:658 combinatorial antiretroviral therapy (cART) 2:460 commerce and tulip mania 1:675 common cold 2:761 common source transmission 1:561 defined 1:559 comoviruses 3:348 economic importance 3:353 epidemiology 3:353 expression of the viral genome 3:351–352

functions of the viral proteins 3:352 genome structure 3:351 geographic distribution 3:352 host range 3:352–353 physical properties of viral particles 3:349–350 relationships with other viruses 3:352 symptomatology and cytopathology 3:353 taxonomy and classification 3:348–349 transmission 3:353 use in biotechnology 3:352 viral structure 3:350–351 competitive enzyme-linked immunosorbent assay (cELISA) 2:134 complementarity-determining regions (CDR) 1:587, 1:288 complementary RNA (cRNA) 2:289, 2:553–554 complement control proteins 1:396–397 complement fixation 5:20 complement system, defined 1:577 completeness 5:247, 5:251–252 complex regional pain syndrome (CRPS) 5:297–298 complex viruses, dissection of 1:227–230 concatamer, defined 2:599, 1:318, 4:368, 4:136, 4:148, 4:61, 2:441 concatemeric DNA, defined 4:105 concatemeric replication products 4:137–138 conformational recognition, defined 4:136 conformational selection model, defined 2:707 congenital infection, defined 2:797 congenital varicella, defined 5:181 congenital Zika syndrome (CZS) 2:899, 2:906 defined 2:899 conidia, defined 4:601, 4:607, 4:648, 4:552 conidiospore, defined 4:450 conidium, defined 4:557 conjugation, defined 4:242, 4:53 connector/nanopore, defined 4:302 consensus genome, defined 1:71 conserved sequence elements (CSEs) 2:174 defined 2:173 CONSISE see Consortium for the Standardization of Influenza Seroepidemiology (CONSISE) consistency 5:252 Consortium for the Standardization of Influenza Seroepidemiology (CONSISE) 5:260 contagious pustular dermatitis (CPD) 2:667–668 contagium vivum fluidum 1:3–4 contig, defined 5:27 continuous professional development (CPD) 5:67–68 continuum mechanics theory 4:206 Contractile injection system 4:186 contractile long-tailed phages 4:211 contractile tail 4:206 defined 4:45 contrast transfer function (CTF) information 1:233

Subject Index convalescent fatigue syndrome 2:812 convergent evolution, defined 4:359, 4:457 convoluted membranes (CMs) 1:498–499, 1:499 cooperativity 1:427 co-option, defined 4:732 CoP see correlate of protection (CoP) COPD see chronic obstructive pulmonary disease (COPD) COPI/COPII, defined 3:32 core gene, defined 2:441, 4:359 core proteins 1:335 Coronavirus (CoV) 1:266, 2:193, 1:499, 2:198–199, 2:245–246, 2:253–254, see also COVID-19 pandemic accessory proteins 2:203–204 avian coronavirus IBV 2:195f background 2:428–429 classification 2:429 CoV-ectoenzyme interactions 1:397 diagnosis 2:438 diseases and host range 2:196–197 epidemiology and clinical features 2:437 HCoV-229E 2:437 HCoV-HKU1 2:437–438 HCoV-NL63 2:437 HCoV-OC43 2:437 future perspectives 2:206 genome 2:430–431 accessory proteins 2:431–432 non-structural proteins 2:431 genome organization and expression 2:194–195 genome replication and recombination 2:196 HCoV-host interactions 2:434–435 apoptosis 2:435–436 autophagy 2:435 ER stress response 2:435 MAP kinase pathway 2:435 translational control 2:434–435 life cycle 2:432–433 assembly and release of virion 2:434 attachment 2:432–433 formation of the replicationtranscription complex 2:433 viral entry and uncoating 2:433 viral RNA synthesis 2:433–434 manipulating CoV genomes using RNA recombination and reverse genetics 2:204–205 molecular features of 2:199–201 pathogenesis 2:436–437 prevention 2:439 replication and transcription of CoV RNA 2:201–203 replication cycle 2:195–196, 2:202f taxonomy and classification 2:193 treatment 2:438–439 vaccines and antiviral drug development 2:205–206 virion properties 2:193 virion structure 2:429–430 virion structure and composition 2:193–194 Coronavirus RNAs 1:464

corpora allata/cardiac, defined 4:780 corrective actions and preventative actions (CAPA) 5:70 correlate of protection (CoP) 5:285 defined 5:289, 5:281 correlative light and cryo tomography in cells 1:215–216 correlative light and electron microscopy (CLEM) 1:215, 1:497 defined 1:208, 1:495 workflows 5:12 Corynebacteriales 1:514 cos, defined 4:61 cos-clearance 4:130–131 nucleotide switch for 4:131 cosmopolitan viruses 1:20 co-suppression, defined 3:123 co-translational disassembly, defined 3:727 Cotton leaf curl Alabad virus (CLCAlaV) 3:357 Cotton leaf curl Burewala alphasatellite (CLCBurA) 3:360–361 cotton leaf curl disease (CLCuD) 3:355 components of CLCuD complex 3:357 begomoviruses associated with CLCuD 3:357 CLCuD-associated alphasatellite components 3:359–361 CLCuD-associated betasatellite 3:361 engineered resistance to CLCuD complex 3:362 genome editing strategy 3:363 history of 3:355–356 in Africa 3:356 elucidation of etiology 3:356–357 etiology 3:356 in Southern Asia 3:356 in the Indian sub-continent 3:29 causal agent and disease classification 3:29–30 control 3:30–31 disease symptoms and yield losses 3:29 epidemiology 3:30 geographical distribution 3:29 long term strategies 3:362 genetics of host plant resistance to CLCuD 3:362 non-pathogen derived resistance 3:363 pathogen derived resistance (PDR) 3:362 antisense RNA technology 3:362 RNAi technology 3:362–363 short term strategies 3:361–362 strategies to control CLCuD complex 3:361 Cotton leaf curl Gezira alphasatellite (CLCGezA) 3:357, 3:360–361 Cotton leaf curl Gezira betasatellite (CLCGezB) 3:357 Cotton leaf curl Gezira virus (CLCGezV) 3:357 Cotton leaf curl Kokhran virus (CLCKokV) 3:357, 3:756 Cotton leaf curl Multan alphasatellite (CLCMulA) 3:360–361 Cotton leaf curl Multan betasatellite (CLCMulB) 3:361, 3:244–245

331

cotton leaf curl virus in cotton (CLCuVs), resistance to 3:563–565, 3:565f Cotton yellow mosaic virus (CYMV) 3:357 Councilman bodies 2:895–896, 2:896 counterdefense mechanisms 4:398 counter-evolution by phages to avoid parasitism by satellites 4:102–103 CoV see Coronavirus (CoV) covert mortality disease (CMD) 4:822 COVID-19 pandemic, 1:671, 5:262, 1:205, 5:33, 5:115, 5:3, 1:566, see also Coronavirus (CoV) COVID-19 vaccines 5:286–287 cowpea chlorotic mottle virus (CCMV) 1:664, 1:667, 1:488–490, 3:262–263, 3:263–264, 3:264f capsids 3:263–264 cowpea mosaic virus (CPMV) 1:279, 3:364 biology 3:364 expression of the viral genome 3:366–367 functions of the viral proteins 3:367–368 genome structure 3:366 physical properties of viral particles 3:364–365 replication 3:368–369 taxonomy and classification 3:364 use in biotechnology 3:369 viral structure 3:365–366 cowpox virus (CPXV) 2:854 coxsackie A virus (CAV) 1:395 Coxsackievirus A10 (CV-A10) 1:284 coxsackievirus B (CVB) 5:293 Coxsackievirus B3 (CVB3) 1:287 coxsackievirus B3-receptor (CAR) 1:284 coxsackieviruses A24 (CVA24) 1:392 CP see capsid protein (CP); coat protein (CP) CpCDV, see Chickpea chlorotic dwarf virus (CpCDV) CPD see contagious pustular dermatitis (CPD); continuous professional development (CPD) CpDV 4:845 CPE see cytopathic effects (CPE) CPMV see cowpea mosaic virus (CPMV) CPPV see capripoxviruses (CPPV) CPV see canine parvovirus (CPV); cytopathic vacuoles (CPVs) CPV-1, see Canis familiaris papillomavirus type 1 (CPV-1) CPXV see cowpox virus (CPXV) CqDV see cherax quadricarinatus densovirus (CqDV) crane hepatitis B virus (CCHBV) 2:100 crawlers 3:165 Cre, see cis-acting replication element (Cre) Crenarchaeota, viruses of 4:360t CREs see cis-acting RNA elements (CREs) CRF see case fatality rate (CRF); circulating recombinant forms (CRFs) cricket baculovirus 4:827–828 Cricket paralysis virus (CrPV) 4:768–770, 4:771, 4:772, 4:773 Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) 2:208, 2:209f

332

Subject Index

clinical features 2:212–213 diagnosis 2:214–215 epidemiology 2:211–212 human vaccines against 2:216t pathogenesis 2:213–214 prevention 2:215 adenovirus vaccine 2:216 DNA vaccines 2:215 inactivated vaccines 2:215–216 modified vaccinia virus ankara (MVA) 2:216 replication cycle 2:210f treatment 2:216 virion structure, genome and life cycle 2:208–209 L segment 2:209–210 M segment 2:209 S segment 2:208–209 virus life cycle 2:210–211 Crimean hemorrhagic fever (CHF) 2:208 crinivirus, defined 3:336 cripavirus 4:768, 4:769t, 4:770t, 4:774 CRISPR see clustered regularly interspaced short palindromic repeat (CRISPR) CRISPR-Cas systems and anti-CRISPR proteins 4:244f, 3:554 anti-CRISPR proteins 4:248–249 guilt-by association bioinformatic approach 4:249–250 lytic phage dependent functional approach 4:250 prophage-dependent functional approach 4:249 self-targeting bioinformatic approach 4:250 diversity and classification of CRISPR-Cas 4:245–246 history of CRISPR-Cas 4:244–245 mechanisms of CRISPR-Cas immunity 4:246 adaptation 4:246 defense 4:246–248 phage-host arms race 4:242–244 CRM see certified reference materials (CRM) cRNA see complementary RNA (cRNA) cro gene 4:81 Crohn’s disease 2:454 cross-kingdom virus infection 4:443 artificially established viral cross infections between plants and fungi 4:445–447 closely related viruses independently infecting plants and fungi 4:445 contribution to virus evolution 4:448 fungal DNA virus that replicates in an insect 4:447–448 natural transmission of a plant virus to a fungus 4:447 viruses that replicate in both plants and insects 4:443–445 cross-protection 3:260, 3:586, 3:862, 3:327, 3:371, 3:439, 3:839 Croton yellow vein mosaic virus (CYVMV) 3:756 CRPS see complex regional pain syndrome (CRPS)

CrPV see Cricket paralysis virus (CrPV) cryoconite hole, defined 4:342 cryo-electron microscopy (cryo-EM) 1:242, 5:12–13, 1:490–491, 1:318, 3:420, 1:329, 1:362, 3:456, 4:900, 4:902, 4:206, 4:211, 4:218 cryo-electron microscopy (cryo-EM) structures of viruses global symmetry, beyond 1:235–236 envelope glycoproteins 1:236–237 genome structures 1:237–238 symmetry mismatch in DNA portals 1:235–236 historical background 1:233 in-situ structural virology 1:238–240 cellular electron cryotomography 1:238–240 near-atomic resolution, cryo-EM at 1:233–235 data collection and image reconstruction 1:233–235 near-atomic resolution maps, interpretation of 1:235 side chain interactions and virus assembly 1:235 cryoelectron microscopy reconstructions 4:901f cryo-electron tomography (cryo-ET) 1:244–245, 1:213–215, 4:206, 4:211, 4:218 of cell lamellae or vitreous sections 1:245–246 of cell periphery 1:244–245 cryo-EM see cryo-electron microscopy (cryo-EM); cryogenic electron microscopy (cryo-EM) cryo-ET see cryo-electron tomography (cryo-ET) cryogenic electron microscopy (cryo-EM) 2:182, 4:342 cryo-immobilization 1:243–244 cryopeg, defined 4:342 cryo-preparation techniques 1:496–497 cryosphere, defined 4:342 cryphonectria hypovirus 1 (CHV1) 4:431–432, 4:433, 4:461, 4:658 cryphonectria hypovirus 4 (CHV4) 4:433 Cryphonectria parasitica, hypovirulence in 4:472–474 artificial application of hypovirulence 4:474–475 exclusive transmissible hypovirulence 4:473–474 cryptotope, defined 3:727 cryspovirus 4:635–636 crystallography, virus 1:199–200, 4:184 future outlook 1:206 Nitty Gritty 1:200–201 crystal handling and cryoprotection 1:201 crystallization and automation 1:201 data collection and automation 1:201–202 structure solution and model building 1:202–204 targets, virus families and predisposition to crystallization 1:200–201

synergies 1:204 understanding evolution through structural anatomy 1:204 vaccines, anti-virals and therapeutics 1:204–206 CsCMV see Cassava common mosaic virus (CsCMV) CSEs see conserved sequence elements (CSEs) CSF see classical swine fever (CSF) CSP see circumsporozoite protein (CSP) CSR see class switch recombination (CSR) CSSD see cacao swollen shoot disease (CSSD) CSSD see cacao swollen shoot virus (CSSD) CSSTBV see cacao swollen shoot Togo B virus (CSSTBV) CsVX see Cassava virus X (CsVX) CTCL see cutaneous T-cell lymphoma (CTCL) C terminus, defined 1:345 CTF information see contrast transfer function (CTF) information CTFV see Colorado tick fever virus (CTFV) CThTV see curvularia thermal tolerance virus (CThTV) CTL see cytotoxic T lymphocytes (CTL) CTV see citrus tristeza virus (CTV) C-type lectin, defined 2:22 cucumber green mottle mosaic virus 3:9 causal agent and classification 3:9 control 3:10 disease symptoms and yield losses 3:9–10 epidemiology 3:10 geographical distribution 3:9 cucumber mosaic virus (CMV) 3:371, 4:440, 4:525, 3:266, 3:266–267, 3:267f cytopathology 3:373 epidemiology and control 3:380–382 genome organization 3:374–375 geographic distribution 3:372 host range 3:372 infection cycle 3:375–377 cell-to-cell and long-distance movement 3:377 replication 3:375–377 transmission 3:377–379 properties and functions of gene products 3:375 symptoms and diagnosis 3:372–373 taxonomy and phylogeny 3:371–372 variation and evolution 3:379–380 virion structure 3:373–374 cucumber vein yellowing virus (CVYV) 3:296–297 Cucumovirus 3:260, 3:260–261, 3:261, 3:265, 3:262t characteristics of RNA genome in 3:261t cucurbit viral diseases 3:87–88 Culex mosquito species 2:805 Culex nigripalpus 2:810–811 Culex p. pipiens 2:810–811 Culex p. quinquefasciatus 2:810–811 Culex pipiens 2:810, 2:812 Culex restuans 2:810 Culex salinarius 2:810

Subject Index Culex tarsalis 2:810, 2:812 Culex vectors 2:810 Culicoides 1:545 Culicoides midges 1:545 cultivation of animal viruses 2:415 culture-independent studies 4:378 curettage, defined 2:629 curly top disease, defined 3:200 current virus taxonomy (1971–present) 1:29–32 curtovirus 3:413 curvularia thermal tolerance virus (CThTV) 4:441 cutaneous T-cell lymphoma (CTCL) 2:419 CV-A10 see Coxsackievirus A10 (CV-A10) CVA24 see coxsackieviruses A24 (CVA24) CVB see coxsackievirus B (CVB) CVB3 see Coxsackievirus B3 (CVB3) cVDPV see circulating vaccine-derived poliovirus (cVDPV) CVSC see capsid vertex specific complex (CVSC) CVX see Cactus virus X (CVX) CVYV see cucumber vein yellowing virus (CVYV) C-X-C chemokine receptor type 4 (CXCR4) 5:151 CXCR4 see C-X-C chemokine receptor type 4 (CXCR4) Cyanobacteria 4:331–332 filamentous cyanobacteria, phages of 4:332 unicellular cyanobacteria, phages of 4:331–332 cyanophages 4:347 cyclic oligoadenylates (cOA) signal transduction pathway 4:400 Cymbidiummosaic virus (CymMV) 3:628, 3:628–629 CymMV see Cymbidiummosaic virus (CymMV) CYMV, see Cotton yellow mosaic virus (CYMV) cypoviruses 1:306, 4:868, 4:714–715, 4:870, 4:870–871, 4:871–875, 4:877 cysteine-rich protein, defined 3:642 cystic fibrosis transmembrane conductance regulator (CFTR) 2:789 cytokines, defined 1:577 cytomegalovirus (CMV) 5:105, 5:190–191 antiviral drug resistance 5:192–193 characteristics and functions of CMV gene products 2:446t–450 classification 2:442 clinical features 2:454–455 congenital disease 2:454–455 immunocompromised host 2:455 CMV inclusion disease (CID) 2:454, 2:454–455, 2:455 diagnosis 2:458 diagnosis of CMV disease and treatment 5:192 epidemiology 2:452–453 patterns of infection 2:453 virus propagation 2:453–454 future perspectives 2:459 genome 2:443–445

evolution 2:445 structure 2:443–445 life cycle 2:445–451 host range 2:445–451 replication steps 2:451–452 pathogenesis 2:455–456 immunity 2:457 modulation of adaptive immune response to infection 2:458 modulation of cell autonomous response to infection 2:457–458 modulation of innate immune response to infection 2:458 persistence and latency 2:456–457 transmission 2:456 pre-transplant risk stratification 5:191 prevention 2:459 prevention strategies of CMV disease after transplantation 5:191–192 prophylaxis and treatment 2:458–459 virion structure 2:442–443 cytomegalovirus hyperimmune immunoglobulin (CMVIG) 5:270 cytopathic effects (CPE) 2:337, 2:757, 2:316, 4:776, 4:839, 2:17 cytopathic vacuoles (CPVs) 1:499, 2:173 cytopathology, defined 3:247, 2:441 cytoplasmic male sterility 3:388 cytoplasmic membrane (CM) 4:42 cytoplasmic polyhedrosis virus (CPV), 4:868, see also cypoviruses cytoskeleton 1:495 cytotoxic CD4+ T cells 1:588–589 cytotoxicity 1:590 cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) 1:660 cytotoxic T lymphocytes (CTL) 4:824, 2:304, 2:283, 1:662 CYVMV see Croton yellow vein mosaic virus (CYVMV) CZS see congenital Zika syndrome (CZS)

D DAAs see direct-acting antivirals (DAAs) DAdV–1 see duck adenovirus 1 (DAdV–1) DAF see decay-accelerating factor (DAF) DALY see disability-adjusted life year (DALY) DAMPs see danger-associated molecular patterns (DAMPs) danger-associated molecular patterns (DAMPs) 1:659 danoprevir 5:123 darunavir 5:140–141 database, viral analysis and visualization capabilities 1:146 analysis and visualization of 3D protein structures 1:148 genome annotation using VIGOR 1:148–149 HA subtype numbering conversion 1:149

333

metadata-driven comparative genomics 1:148 phylogenetic tree reconstruction 1:147–148 sequence annotation 1:146 sequence search and alignments 1:146–147 virus genotype/clade classification 1:149 workbench 1:149–150 and analytical tools 1:141–142 types of bioinformatics webtools 1:142–143 types of databases 1:141–142 data curation 1:146 data retrieval 1:146 application programming interfaces (API) 1:146 search interface 1:146 data summary 1:144–145 influenza research database (IRD) 1:143 significance of 1:141 sources of data 1:143 data aggregated from public data archives 1:143 derived and predictive data 1:143–144 direct submission of novel data 1:143 usage statistics 1:150 user support 1:150 virus pathogen database and analysis resource (ViPR) 1:143 DBDs see DNA binding domains (DBDs) DC see dendritic cells (DC) DCLs see dicer-like proteins (DCLs) DC-SIGN see dendritic cell-specific ICAM–grabbing non-integrin (DC-SIGN) DCV see Drosophila C virus (DCV) DDBJ see DNA Data Bank of Japan (DDBJ) DdDp see DNA-dependent DNA-polymerase (DdDp) DdRp see DNA dependent RNA polymerase (DdRp) death certification 5:248 decapping, defined 4:664 decay-accelerating factor (DAF) 1:397, 1:659 decontamination and waste management 5:85–86 deep-sea hydrothermal vents 4:347–348 defined 4:342 deep sequencing, defined 5:27 defective-interfering (DI) viruses assay for DI particles 1:618–619 biological effects 1:619 cyclic variations of defective interference 1:618 defective interfering versus defective viruses 1:618 defectiveness 1:617 experimental animals, DI particles in 1:619 future perspectives 1:620 generation of DI genomes 1:617 history 1:617 interference 1:617–618 natural infections, DI particles in 1:619–620

334

Subject Index

defective-interfering (DI) viruses (continued) structure 1:617 defective interfering particles (DIPs) 4:830 defective-interfering RNAs 1:465–467 defined 3:285, 3:839, 1:53 defective RNA, defined 3:327 defense island system associated with restriction modification (DISARM) system 1:608–610 defense systems 1:606 classification and diversity of 1:606–607 CRISPR-Cas 1:610–613 self versus nonself recognition innate immunity systems 1:607–610 toxins-antitoxins 1:607 unclassified defense systems 1:613 genomic organization and evolution of 1:613–615 association of defense systems with programmed cell death components 1:615 defense islands and a tight link between defense and mobilome genes 1:613–615 gene and domain shuffling and sharing 1:615 degenerate primers, defined 5:27 Delphi process 1:573 deltaentomopoxvirus 4:859 deltapapillomavirus DNA 2:88 deltapartitivirus 4:636–637 deltasatellites (Tolecusatellitidae) 3:239, 3:245 classification and nomenclature 3:239–241 genome organization 3:245–246, 3:243f phylogenetic trees for 3:242f species of the genera Deltasatellite 3:240t–241 trans-replication of 3:246 DEmARC (DivErsity pArtitioning by hieRarchical Clustering) 1:101–102 dendritic cells (DC) 1:584, 2:229, 2:671 dendritic cell-specific ICAM–grabbing nonintegrin (DC-SIGN) 1:399, 2:765 dengue hemorrhagic fever (DHF) 2:218 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) 2:218 dengue virus (DENV) 1:498–499, 1:598–599 clinical features of infection 2:227–228 DENV-1 2:218, 2:219 DENV-2 2:218, 2:219 DENV-3 2:218, 2:219 DENV-4 2:218, 2:219 epidemiology 2:223–224 evolution 2:222–223 future 2:231 genetics 2:222 geographic and seasonal distribution 2:221 history 2:218–219 host range and virus propagation 2:221–222 immune response 2:228–229 pathogenesis 2:224–227

pathology and histopathology 2:228 prevention and control of dengue 2:229–231 serologic relationships and variability 2:223 structures 2:220f taxonomy and classification 2:219–221 transmission and tissue tropism 2:224 vaccines 2:229–231 de novo sequencing, defined 5:27 dense body, see also capsidless particles defined 2:441 density ultracentrifugation 1:169–170 densonucleosis viruses (DNVs) 4:835 densoviral capsid structures 4:844f Densovirinae 4:711–713 densoviruses (DVs) 4:839, 4:838t, 4:835, 4:836 biochemical properties and purification of 4:840–841 biophysical features and functions associated with DV capsid 4:843–844 discovery, taxonomy and evolution of 4:835–837 genome structure and replication 4:844–845 shrimp 4:839–840 fenneropenaeus chinensis hepandensovirus (FcHDV) 4:840 new invertebrate DVs 4:840 penaeus stylirostris penstyldensovirus 1 (PstDV1) 4:839–840 DENV see dengue virus (DENV) deoxynucleotide triphosphate (dNTP) 4:15 deoxyuridine triphosphatase (dUTPase) 2:25–27 dermatome, defined 2:860 design qualification (DQ) 5:69 detective quantum efficiency (DQE) 1:233–234 detergents 5:301 detritus, defined 2:629 DEV see duck enteritis virus (DEV) DFE see distribution of fitness effects (DFE) DHBV see duck hepatitis B virus (DHBV) DHF see dengue hemorrhagic fever (DHF) DHF/DSS see dengue hemorrhagic fever/ dengue shock syndrome (DHF/DSS) DHIS see drug-induced hypersensitivity syndrome (DHIS) DHS see drug hypersensitivity syndrome (DHS) Diadromus pulchellus idnoreovirus 1 (DpIRV-1) 4:870, 4:875 diagnosis of viral infection clustered regularly interspaced short palindromic repeats with the enzyme Cas (CRISPR-Cas) 5:113–114 future perspectives 5:115 matrix-assisted laser desorption/ ionization-time of flight (MALDITOF) mass spectrometry 5:114 microfluidics 5:115 multiplex and point of care tests (POCT) 5:114–115

next-generation sequencing (NGS) 5:112–113 polymerase chain reaction (PCR) and its evolution 5:112 diagnostic assays, standardization of 5:52 batch testing 5:58 commercial and laboratory-developed tests 5:60–61 external quality assessment/proficiency testing 5:58–59 international standards in clinical practice 5:61–62 laboratory accreditation 5:59–60 physical standards 5:52 preparation and evaluation of standards 5:52–55 quality assurance (QA) 5:55–57 use of controls 5:57–58 virus isolation 5:61 written standards and guidelines 5:55 diagnostic molecular based assays, development of isothermal amplification 5:22 non-isothermal PCR based technologies 5:22–25 diagnostics, viral 5:3 diagnostic transmission electron microscopy 5:6–9 dianthovirus (Tombusviridae) 3:383 cell-to-cell and systemic movement 3:386 host factors in dianthovirus RNA replication 3:385–386 RNA replication 3:385 RNA silencing suppression 3:386–387 taxonomy and classification 3:383 genome organization 3:383 virions, transmission, and epidemiology 3:383–385 gene expression 3:384–385 diapause, defined 2:805 Diaporthe RNA virus (DRV) 4:438 DIC see disseminated intravascular coagulation (DIC) dicer, defined 3:788, 3:383, 3:123 dicer-like protein-1 (DCL1) 3:758 dicer-like proteins (DCLs) 3:43 Dicistroviridae 4:706, 1:279, 4:768 dicistroviruses 4:768 biophysical properties 4:768–770 geographic and strain variation 4:773–774 host range 4:771–772 organization of dicistrovirus genome 4:770f organization of the dicistrovirus genome 4:770–771 pathology and transmission 4:772–773 relationships within the family 4:774 similarity with other taxa 4:774–775 taxonomy and classification 4:768 virus replication and genome expression 4:771 dicotyledon, defined 3:545 differentiating infected from vaccinated animals (DIVA) 2:117 differentiation of naturally infected versus vaccinated animals (DIVA) 2:134

Subject Index differentiation state-dependent process, defined 2:441 diffracted X-ray tracking (DXT) 1:195–197, 1:191 diffraction Limit, defined 5:5 digital epidemiology 1:566 defined 1:559 digital PCR (dPCR) 5:75–76, 5:76, 5:112 Digoxigenin-labeled RNA probes 3:629 Diltiazem 5:169–170 Dinodnavirus 4:686 Dinornavirus 4:684 Dinovernavirus 4:869, 4:871, 4:876, 4:877 diphtheritic infections 2:346 DIPs see defective interfering particles (DIPs) diptera 4:867 dipteran, defined 4:768 direct-acting antivirals (DAAs) 1:575, 2:515, 2:394, 2:395f defined 2:386 direct contact transmission 1:561 defined 1:559 direct “rabies immunoperoxidase test” (dRIT) 2:743 direct repeat, defined 4:276 DISA see disabled infectious single-animal (DISA) disability-adjusted life year (DALY) 1:678 disabled infectious single-animal (DISA) 2:135 vaccine 2:20–21 disabled infectious single-cycle (DISC) vaccines 2:135, 2:20–21 DISARM system see defense island system associated with restriction modification (DISARM) system discontinuous transcription, defined 2:198 DISC vaccines see disabled infectious singlecycle (DISC) vaccines disease prevention, implications for 1:568 disease X 5:257 disseminated intravascular coagulation (DIC) 2:513, 2:239, 2:613–614 distribution of fitness effects (DFE) 5:228–229, 5:227 DIVA see differentiating infected from vaccinated animals (DIVA); differentiation of naturally infected versus vaccinated animals (DIVA) divavirus, defined 3:805 diversity, defined 4:283 diversity-generating retroelements 1:55 DI viruses see defective-interfering (DI) viruses DJR see double jelly roll (DJR) DLP see double-layered particle (DLP) DmIRV-5, see Drosophila melanogaster idnoreovirus-5 (DmIRV-5) DMVs see double membrane vesicles (DMVs) DNA, defined 2:441 DNA, persistence of 4:77–78 extrachromosomal 4:78–79 integration into the chromosome 4:77–78 DNA binding domains (DBDs) 4:140–141

DNA-condensing agents 4:206 DNA Data Bank of Japan (DDBJ) 5:33 DNA degradation (DND) 1:610 DNA-dependent DNA-polymerase (DdDp) 2:62, 2:60 DNA dependent RNA polymerase (DdRp) 4:813–814 defined 4:808 DNA-dependent RNA-polymerase (RdDp) 2:62 DNAemia, defined 5:197 DNA-filled head, energetics of 4:167 intra-capsid DNA equilibrium energy and structure of 4:167–169 metastability of 4:169–170 mobility of packaged genome controls ejection dynamics 4:170–171 pressure-driven release of viral genome into a host cell 4:171–172 DNA injection 4:48 DNA microarrays 5:92 DNA mycovirus 4:493 DNA packaging 4:136–137, 4:160, 4:148 characteristics 4:161–162 force generation 4:162–163 packaging speed 4:161–162 pausing and slipping 4:163 conformational variation of DNA 4:139f defined 4:36, 4:45 and DNA binding protein 4:18–19 DNA “crunching” or “compression” translocation model 4:155 DNA wrapping/bending 4:146 future studies 4:157–159 genome replication and selection mechanisms 4:137 concatemeric replication products 4:137–138 monomeric replication products 4:137 “inchworm” translocation model 4:153–154 “lever” translocation model 4:154–155 mechanisms 4:163–164 force generation 4:164–166 mechanochemical coupling 4:163–164 motor coordination 4:166 motor-substrate interactions 4:164 molecular structure 4:160–161 Phi29 motors 4:151–152 protein-DNA binding 4:139–140 DNA binding domains (DBDs) 4:140–141 multiple binding events regulate complex assembly 4:141 sequence specific and non-specific binding 4:140 shape of DNA 4:138–139 terminase and Phi29 motors 4:148–151 terminase motors 4:152–153 termination of packaging 4:155–157 viral genomic DNA, recognition of 4:141 concatemeric DNA and terminase proteins 4:141–142 monomeric genomes and terminal proteins 4:141

335

DNA phosphorotioation (DPT) system 1:610 DNA polymerase gene 5:185 DNA portals, symmetry mismatch in 1:235–236 DNA repair enzymes 4:238 DNA replication 4:15 of j29-like viruses 4:66–67 of SPO1-like viruses 4:63–65 of SPP1-like viruses 4:65–66 DNA sequencing 1:178–179 DNA synthesis, defined 4:61 DNA vaccines 2:215, 5:286 double-stranded 1:43–44 single-stranded 1:43 DNA viruses origin of 1:19–20 recombination in 1:460–462 DNA virus evolution, mechanisms of 1:71–72 co-divergence with hosts as a driver of DNA diversification 1:74 contributions of DNA virus persistence and chronic infections 1:73–74 DNA virus genome types, diversity of 1:72 DNA virus hosts vary from single cells to complex multi-cellular organisms 1:73 duplication and deletions of genes and genome segments 1:76–77 fluctuations in tandem repeat copy number as a mechanism of evolution 1:75 host cell biology and availability of host enzymes constrains virus evolution 1:72–73 host-virus exchange via horizontal gene transfer and transposable elements 1:77 in vivo observations of within-host diversity and adaptation of DNA viruses 1:75 large DNA viruses undergo frequent recombination 1:75–76 recombination at different frequencies for small DNA virus genomes 1:76 single nucleotide differences as a measure of evolutionary change 1:74–75 time frames 1:73 DND see DNA degradation (DND) dNTP see deoxynucleotide triphosphate (dNTP) DNVs see densonucleosis viruses (DNVs) docosanol 5:124–125 document control 5:65–66 dolutegravir 5:149–150 clinical trials dual therapy 5:150 therapy-naı¨ve with 2 NRTI 5:149–150 treatment-experienced 5:150 resistance 5:150 domestic pigs, control of classical swine fever in 2:162 dominant allele, defined 3:69, 3:554 Donnan’s effect, defined 4:105 doravirine 5:136

336

Subject Index

double antibody sandwich, defined 3:539 double jelly roll (DJR) 1:329 capsid size determination in DJR lineage 1:340–342 DJR lineage 1:337 genomes in 1:340 vertex structures in 1:339 polintons and the evolutionary pathway of DJR viruses 1:342–343 variants of DJR fold 1:337–339 double-layered particle (DLP) 1:309 double-membraned bodies, defined 3:396 double membrane vesicles (DMVs) 1:498–499, 1:499, 2:388–389, 2:390, 2:251, 2:201–202, 2:198 double-stranded DNA (dsDNA) 2:855, 1:385, 1:235, 1:501–502, 1:318, 1:319, 3:158, 1:257, 4:160, 1:19, 1:43–44, 1:386 dsDNA recombineering 4:298, 4:295–296 double-stranded RNA (dsRNA) 1:444–445, 1:385, 4:26, 1:499, 1:492–493, 1:303, 1:310f, 1:312f, 3:555 cell entry of 1:307–309 classification and host range 4:26–27 Cystoviridae family 4:26 dsRNA mycoviruses 4:433–437 recombination and reassortment 4:33–34 applications and intellectual property 4:34 carrier state 4:33–34 reverse genetics and self assembly 4:34 replication cycle 4:31–32 membrane acquisition and host cell lysis 4:33 NC maturation 4:33 PC assembly and packaging 4:33 plasma membrane penetration and NC entrance into the cytoplasm 4:31–32 transcription 4:32–33 structures of 1:304f virion structure and properties 4:27 architecture of the polymerase complex and the nucleocapsid 4:27–28 genome organization and sequence similarity 4:31 host cell attachment complex 4:30–31 membrane envelope 4:30 P1 protein 4:28 P2 protein 4:28–30 P4 protein 4:30 P7 protein 4:30 P8 protein 4:30 physico-chemical properties 4:27 protein components of the cystovirus portal complex and nucleocapsid 4:28 double-stranded RNA (dsRNA) mycoviruses, structure of 4:504 chrysoviruses 4:505–506 evolutionary relationships based on structural comparisons 4:509 future perspectives 4:511–512 megabirnaviruses 4:508–509 partitiviruses 4:508 quadriviruses 4:506–508 RdRp and dsRNA organization within mycovirus capsids 4:509–511

totiviruses 4:504–505 double-stranded RNA (dsRNA) virus core and endogenous transcription 1:309 viral innermost capsid layer that encloses the genome 1:309 viral RNA capping 1:311–313 viral RNA-dependent RNA polymerase (RdRp) 1:309–311 double-stranded RNA (dsRNA) virus families 1:314–315 dPCR see digital PCR (dPCR) DpIRV-1 see Diadromus pulchellus idnoreovirus 1 (DpIRV-1) DplDV see dysaphis plantaginea densovirus (DplDV) DPT system see DNA phosphorotioation (DPT) system DQ see design qualification (DQ) DQE see detective quantum efficiency (DQE) dRIT see direct “rabies immunoperoxidase test” (dRIT) droplet transmission, defined 1:559 Drosophila C virus (DCV) 4:768, 4:772, 4:773, 1:549 Drosophila melanogaster 4:772 Drosophila melanogaster idnoreovirus-5 (DmIRV-5) 4:875 drug hypersensitivity syndrome (DHS) 2:786 drug-induced hypersensitivity syndrome (DHIS) 2:786 drug repurposing 1:499 drug resistance 1:60 drug susceptibility, defined 5:121 DRV see Diaporthe RNA virus (DRV) dsDNA see double-stranded DNA (dsDNA) dsRNA see double-stranded RNA (dsRNA) duck adenovirus 1 (DAdV–1) 2:13 duck enteritis virus (DEV) 2:113 duck hepatitis B virus (DHBV) 2:100, 2:100–101, 2:102–103 DNA synthesis 2:105f genome organization 2:103f duck plague see duck viral enteritis (DVE) duck viral enteritis (DVE) 2:113 Duplodnaviria 1:20 dUTPase see deoxyuridine triphosphatase (dUTPase) Duvenhage lyssavirus (DUVV) 2:738, 2:740–741 DUVV see Duvenhage lyssavirus (DUVV) DVE see duck viral enteritis (DVE) DVs see densoviruses (DVs) DXT see diffracted X-ray tracking (DXT) dye-terminator sequencing 5:28 dynamin, defined 1:529 dysaphis plantaginea densovirus (DplDV) 4:835, 4:845 dysbiosis, defined 1:552, 4:283 dysplasia, defined 2:316 dystocia, defined 2:34

E E1 glycoprotein 1:266–267

E1 protein 2:497–498 E1ˆE4 protein 2:498 E2 protein 2:498, 1:266–267 E5 protein 2:498 E6 Associated Protein (E6AP) 2:498 E6 protein 2:498 E7 protein 2:498–499 E8ˆE2 protein 2:498 early gene, defined 2:441 early taxonomic developments (1886–1971) 1:28–29 Eastern equine encephalomyelitis virus (EEEV) 1:66, 1:263, 2:40, 2:43–44, 2:44, 2:45, 2:46 EAV see equine arteritis virus (EAV) EBHSV see European brown hare syndrome virus (EBHSV) ebola and other hemorrhagic fevers 5:274–275 Ebola hemorrhagic fever (EHF) 2:232 Ebola virus (EBOV) 1:399, 1:401, 2:610, 1:121, 2:232, 2:233t, 1:367f classification 2:232 clinical features 2:239 early disease 2:239 peak disease 2:239 recovery 2:239 virus persistence 2:239 diagnosis 2:241 epidemiology 2:232–234 genome 2:234–235 genome structure and morphology 2:234f pathogenesis 2:240f pathogenesis 2:239–241 prevention 2:232–234 adenovirus 26 (Ad26) and modified vaccinia ankara (MVA) 2:243 vesicular stomatitis virus 2:243 replication cycle 2:236–237, 2:237f assembly and budding 2:238 immune evasion 2:238–239 primary transcription and translation 2:237–238 replication 2:238 virus attachment and fusion 2:236–237 treatment 2:241–242 monoclonal antibody therapy 2:241–242 small interfering RNAs (siRNAs) 2:242 small-molecule compounds 2:242 virion structure 2:235–236 Ebola virus disease (EVD) 1:591, 2:232 EBOV see Ebola virus (EBOV) EBV see Epstein–Barr virus (EBV) EC50, defined 2:561 ecdysteroids, defined 4:858 Echovirus 30 (E30) 1:284 ECMO see extracorporeal membrane oxygenation (ECMO) ecology and global impacts of viruses 1:621–622 viral community composition, drivers of 1:625 viral community ecology, methods for studying 1:622–623 viral impacts on carbon, nutrient, and biogeochemical cycling 1:623–625

Subject Index virus-host linkages and ecology 1:623 ECRA vaccines see Entry Competent Replication Abortive (ECRA) vaccines ecthyma contagiosum 2:667–668 ectoenzymes 1:397 CoV-ectoenzyme interactions 1:397 ectotherm, defined 2:306 EDS see egg drop syndrome (EDS) EEEV see Eastern equine encephalomyelitis virus (EEEV) eel virus American (EVA) 2:325 eel virus European X (EVEX) 2:325 EEV see extracellular enveloped virion (EEV); external enveloped virions (EEVs) EFC see entry fusion complex (EFC) EFF-1 see epithelial fusion failure 1 (EFF-1) efficiency of plating (EOP) 1:162 EFM see epifluorescence microscopy (EFM) EGCG see epigallactechin-3-gallate (EGCG) EGFR see epidermal growth factor receptor (EGFR); epithelial growth factor receptor (EGFR) egg drop syndrome (EDS) 2:13 egg proteins 5:301 E glycoprotein 2:891–892 egress, defined 2:441 EGTA see ethylene glycol tetraacetic acid (EGTA) Egyptian rousette bats (ERBs) 2:609 EHF see Ebola hemorrhagic fever (EHF) EHM see equine herpesvirus myeloencephathy (EHM) EIA see enzyme immunoassay (EIA) EIAV see equine infectious anemia virus (EIAV) EID see emerging infectious diseases (EID) eIF4E binding proteins (eIF4E-BPs). 1:445 eIF4E-BPs. see eIF4E binding proteins (eIF4E-BPs). eIF4E-mediated recessive resistance characteristics of 3:74–76 molecular mechanisms of 3:76–77 eIF4E phosphorylation 1:444 eIFs see eukaryotic translation initiation factors (eIFs) EIOS see Epidemic Intelligence from open sources (EIOS) ejection/E protein, defined 4:219 electron microscopy (EM) 3:629, 5:3, 1:497, 1:498–499 for viral diagnosis 5:6 biological EM challenges and sample preparation 5:9–10 cryo electron microscopy 5:12–13 diagnostic transmission electron microscopy 5:6–9 negative staining, general protocol for the study of viruses by 5:10–11 negative staining for diagnosis of fecal samples 5:11 scanning electron microscopy, advances in 5:13–14 traditional processing techniques, limitations of 5:11–12

Electron Multiplied Charged Coupled Device (EM-CCD) sensor 1:208 electron tomography (ET) 1:496–497, 1:499 electron tomography (ET), analysis of viruses in the cellular context by 1:242–243 cryo-electron tomography (cryo-ET) 1:244–245 of cell lamellae or vitreous sections 1:245–246 of cell periphery 1:244–245 room temperature-electron tomography (RT-ET) 1:244 sample preparation 1:243 chemical fixation 1:243 cryo-immobilization 1:243–244 electron transport chain (ETC) 1:212 electrophoretic mobility shift assay (EMSA) 4:392 elegant-crested tinamou hepatitis B virus (ETHBV) 2:100 elimination program, defined 2:797 ELISA see enzyme-linked immunosorbent assay (ELISA) ELISpots see enzyme-linked immunosorbent spots (ELISpots) elvitegravir 5:146–149 clinical trials 5:149 resistance 5:149 E lysis 1:515 EM see electron microscopy (EM) EMBL Nucleotide Sequence Data Library 5:33 EMCV see Encephalomyocarditis Virus (EMCV) emergence, defined 3:8 emergent viruses 1:569 Emerging disease, defined 3:411 emerging infectious diseases (EID) 5:256, 5:257 emerging viral diseases (EVD) 5:285–286 challenge of 5:285–286 emerging viruses, prevention and control of 1:572–574 diagnosis 1:573–574 mosquito vectors, control of 1:575 treatment 1:574–575 vaccination 1:575 EMSA see electrophoretic mobility shift assay (EMSA) ENA see enzootic nasal adenocarcinoma (ENA) enation, defined 3:200 encapsidation, defined 2:441 encapsulation defined 4:849 loading of virus-like particles by 1:667–668 encephalitis 2:812 alphaviruses causing see alphaviruses causing encephalitis defined 2:40, 2:778, 2:404, 2:884, 2:675 encephalomyelitis, defined 2:34 Encephalomyocarditis Virus (EMCV) 1:445 endemic, defined 2:218, 1:569, 5:247 endemicity, defined 2:654

337

endocytosis, defined 1:529, 1:417 endogenisation 1:79, 1:79–80, 1:80f endogenous avian hepadnavirus elements 2:109–111 endogenous bornavirus-like elements 2:141 endogenous lentivirus 2:64 defined 2:56 endogenous retroviruses (ERVs) 1:628, 1:630–631, 1:627, 2:122, 2:64 evolutionary impact of 1:81–83 exaptation of 1:630–631 ERV-derived non-coding elements 1:631–632 ERV-derived proteins 1:632 ERV-encoded envelope glycoproteins 1:630–631 origin of 1:631f endogenous viral element (EVE) 4:792, 3:158, 4:835 endogenous virus, defined 2:643 endolysin, defined 4:276 endoparasitic wasps, defined 4:724 endophyte, defined 3:388 endoplasmic reticulum (ER) 2:386–387, 1:268, 2:463, 2:554–555, 1:411–412, 1:444, 1:603, 1:498–499, 1:499, 1:664, 2:399, 3:140 endoplasmic reticulum-Golgi intermediate compartment (ERGIC) 2:201–202, 2:196, 2:452, 2:441 endoreduplication, defined 3:200 Endornaviridae 3:388 endornaviruses (Endornaviridae) 3:388 fungal 3:389 gene products 3:391–392 genome organization 3:390–391 horizontal transmission 3:393 from other eukaryotic kingdoms 3:389–390 pathogenesis 3:394 plant 3:388–389 regulation of copy number 3:393–394 replication (RdRp Activity) 3:392–393 vertical transmission 3:393 virus-host relationship 3:394–395 endosomal sorting complexes required for transport (ESCRT) 2:511, 1:282, 3:32, 2:238, 2:879–880, 2:132, 1:529, 1:539 -dependent budding 1:522–523 endosomes, defined 3:32 endothelial cell, defined 1:529 energy-driven genome packaging in doublestranded DNA and giant viruses 1:493 energy-independent genome packaging 1:488 in larger (ss)RNA viruses 1:490–492 in small single-stranded viruses 1:488–490 enfuvirtide 5:152 enhanced RSV disease (ERD) 2:754 Enhanced Surveillance 5:250 enhancer, defined 2:56 EnMBV1 see Entoleuca megabirnavirus 1 (EnMBV1) enrichment strategies 1:179 negative selection-based enrichment 1:179–180

338

Subject Index

enrichment strategies (continued) positive selection-based enrichment 1:179 entecavir (ETV) 5:218 Enterobacteriales, phage cluster-host species relationships with 4:272–273 Enterobacteriales tailed phage example 4:269–270 enterovirus 71 (EV-A71) 1:284, 1:599–600 enteroviruses (EVs) 1:530, 1:284, 1:392 classification (compact) 2:256–257 clinical features 2:261–263 diagnosis 2:263–264 epidemiology 2:261 genome 2:258–259 life cycle 2:259–261 pathogenesis 2:263 prevention 2:265 treatment 2:264–265 virion structure 2:257–258 enthalpy, defined 4:167 Entoleuca megabirnavirus 1 (EnMBV1) 4:600 entomobirnavirus 4:706 classification (compact) 4:776–777 clinical features 4:778–779 Dicistroviridae 4:706 epidemiology 4:778 genome 4:777–778 Hytrosaviridae 4:706–707 Iflaviridae 4:707 life cycle 4:778 pathogenesis 4:779 virion structure 4:777 entomological warfare 1:647 entomopathogen, defined 4:892 Entomopoxvirinae 4:858, 4:713 entomopoxviruses (EPVs) 4:858, 4:861, 4:862, 4:865 entropy, defined 4:167 entry, virus 1:561 Entry Competent Replication Abortive (ECRA) vaccines 2:135 entry fusion complex (EFC) 2:855, 2:22 entry inhibitors 2:472 entry receptor, defined 1:388 ENTV see enzootic nasal tumor virus (ENTV) Env, defined 3:96 envelope 1:495, 2:441, 1:257, 1:362, 1:382, 3:507 envelope (E) protein 2:885 enveloped icosahedral phages – double-stranded RNA (dsRNA) 4:26 classification and host range 4:26–27 Cystoviridae family 4:26 recombination and reassortment 4:33–34 applications and intellectual property 4:34 carrier state 4:33–34 reverse genetics and self assembly 4:34 replication cycle 4:31–32 membrane acquisition and host cell lysis 4:33 NC maturation 4:33 PC assembly and packaging 4:33

plasma membrane penetration and NC entrance into the cytoplasm 4:31–32 transcription 4:32–33 virion structure and properties 4:27 architecture of the polymerase complex and the nucleocapsid 4:27–28 genome organization and sequence similarity 4:31 host cell attachment complex 4:30–31 membrane envelope 4:30 P1 protein 4:28 P2 protein 4:28–30 P4 protein 4:30 P7 protein 4:30 P8 protein 4:30 physico-chemical properties 4:27 protein components of the cystovirus portal complex and nucleocapsid 4:28 enveloped icosahedral RNA viruses 1:266–268 enveloped particles 1:468 icosahedral enveloped viruses 1:468–469 alphavirus assembly and budding 1:470–472 alphavirus life cycle 1:469 alphavirus virion structure 1:469–470 flavivirus assembly and budding 1:473 flavivirus life cycle 1:472 flavivirus virion structure 1:472–473 viral envelope 1:468 enveloped virion, defined 2:797 enveloped viruses cell entry targeting of 5:237–238 defined 1:417 enveloped virus membrane fusion 1:417–418 fusion machinery, working of 1:425 common mechanism of fusion 1:425 cooperativity 1:427 structural intermediates during conformational changes 1:426–427 target membrane, interaction with 1:425–426 specific lipid requirements 1:420 viral fusion glycoproteins, structure of 1:420 Class I fusion glycoproteins 1:420, 1:419f Class II fusion glycoproteins 1:421, 1:422f Class III fusion glycoproteins 1:424, 1:423f premature triggering, regulating the conformational change to avoid 1:425 viral fusion site in the cell 1:418–420 envelope glycoproteins 1:236–237 envelopment, defined 2:441 env gene 1:358–360, 2:57–58, 2:61t Env-like functions in plants 3:102 enzootic, defined 2:899 enzootic nasal adenocarcinoma (ENA) 2:581 enzootic nasal tumor virus (ENTV) 2:575, 2:581–582

enzyme immunoassay (EIA) 5:5 enzyme-linked immunosorbent assay (ELISA) 2:144–145, 1:9, 2:341, 5:17–18, 5:5, 2:896, 3:6, 2:743, 5:105–106, 3:322, 3:470, 5:94, 2:651–652, 5:191 enzyme-linked immunosorbent spots (ELISpots) 5:191 enzymes of lipid metabolism 1:499 EOP see efficiency of plating (EOP); equipment operating procedures (EOPs) Ephemeroviruses 2:878 epicardium, defined 2:875 epidemic, defined 3:355, 1:569, 5:247, 1:559 Epidemic Intelligence from open sources (EIOS) 1:566 epidemiological studies 1:564–565 case-control studies and cohort studies 1:564–565 qualitative studies 1:565 time series analysis 1:565 epidemiology, defined 1:559, 2:884 epidermal growth factor receptor (EGFR) 2:498, 2:390 epifluorescence microscopy (EFM) 1:622, 1:622f epifluorescent microscopy, defined 4:314 epigallactechin-3-gallate (EGCG) 4:817 epipelagic layer, defined 4:322 episomal element, defined 4:387 episome, defined 4:827, 2:599, 2:778 epithelial cells, defined 4:858, 1:529 epithelial fusion failure 1 (EFF-1) 1:95 epithelial growth factor receptor (EGFR) 2:450–451 epizootic, defined 1:542 epizootic haemorrhagic disease virus 1:545 E protein 1:421, 2:196 capsids with large numbers of E proteins distributed throughout the DNA 4:222–223 enigmas of 4:224–225 encapsidation of E proteins 4:225–226 impacts of E proteins on capsid DNA packaging and ejection 4:226 impacts of E proteins on DNA ejection 4:226–227 phages with yet to be identified E proteins 4:224–225 potential of E protein delivery from capsid derived nanocontainers 4:227 low copy number E proteins with specific locales in the capsid 4:221 podoviral E proteins 4:221–222 Epstein Barr nuclear antigen - 1 (EBNA-1) IgG 5:195 Epstein–Barr virus (EBV) 1:532–533, 1:533, 5:195, 1:178, 2:785, 5:105, 5:107–108, 1:321, 1:620, 1:461 -associated diseases 2:272 Burkitt’s lymphoma 2:273 diffuse large B-cell lymphomas 2:273 EBV-associated T Cell and NK cell lymphomas 2:274

Subject Index gastric carcinoma and other epithelial tumors 2:275–276 Hodgkin’s lymphoma 2:273–274 infectious mononucleosis 2:272 lymphomas in immunosuppressed individuals 2:272–273 multiple sclerosis 2:272 nasopharyngeal carcinoma 2:274–275 attachment 1:396 classification 2:267 control and prevention of EBV infection 2:276 epidemiology and natural history of infection 2:267–270 future perspectives 2:276 history 2:267 immune response 2:271–272 strain variation 2:270–271 virion and genome structure 2:267 EPVs see entomopoxviruses (EPVs) EQA see external quality assessment (EQA) equid herpesvirus type 1 (EHV-1) 2:278, 2:278–279, 2:284 equid herpesvirus type 2 (EHV-2) 2:278, 2:278–279, 2:284 equid herpesvirus type 3 (EHV-3) 2:278, 2:278–279, 2:284 equid herpesvirus type 4 (EHV-4) 2:278, 2:278–279 equid herpesvirus type 5 (EHV-5) 2:278, 2:278–279, 2:284 equid herpesvirus type 9 (EHV-9) 2:278, 2:278–279 equine, canine and swine influenza classification 2:287 clinical features 2:292 diagnosis 2:292 epidemiology 2:290–291 genome 2:287–288 life cycle 2:288–290 pathogenesis 2:291–292 prevention 2:293 treatment 2:292–293 virion structure 2:287 equine adenoviruses 2:12–13 equine arteritis virus (EAV) 2:245, 2:697, 1:499 classification 2:697 clinical features 2:702 diagnosis 2:705 epidemiology 2:701–702 genome organization 2:698–700 immunity 2:704 life cycle 2:700–701 nonstructural and structural proteins of 2:699t pathogenesis 2:703–704 prevention and control 2:705–706 taxonomy of 2:697t virion structure 2:697–698 equine encephalitis viruses 2:808 equine herpesviruses 2:278 classification 2:278–279 clinical features and pathology 2:283–285 control 2:285 epidemiology 2:282–283

future perspectives 2:285 genomes 2:279–281 latency 2:282 life cycle 2:281–282 pathogenesis and immune response 2:283 virion structure 2:279 equine herpesvirus myeloencephathy (EHM) 2:283 equine infectious anemia virus (EIAV) 2:56–57, 2:64, 2:67 equipment calibration 5:69 equipment maintenance and monitoring 5:69–70 equipment operating procedures (EOPs) 5:65 equipment qualification 5:69 ER see endoplasmic reticulum (ER) eragrovirus 3:413 ER-associated degradation (ERAD), defined 4:534 ERBs see Egyptian rousette bats (ERBs) ERCC4-like nuclease, defined 2:22 ERD see enhanced RSV disease (ERD) ER exit sites, defined 3:32 ERF4 see ethylene response factor-4 (ERF4) ERGIC see endoplasmic reticulum-Golgi intermediate compartment (ERGIC) Eriocheir sinensis reovirus 4:878–879 errantivirus, defined 3:653 error catastrophe, defined 1:53 error catastrophe and lethal mutagenesis of viruses 1:58 error-prone repair polymerases 1:55 error-prone replication 1:53–54 lack of proofreading 1:54–55 polymerase selectivity, factors determining 1:54 viral polymerases, intrinsic selectivity of 1:54 ER stress, defined 2:428 ERVs see endogenous retroviruses (ERVs) Escherichia coli 1:639–640, 1:637f–638, 4:291, 4:128 ESCRT see endosomal sorting complexes required for transport (ESCRT) ESRF see European Synchrotron Radiation Facility (ESRF) ET see electron tomography (ET) ETC see electron transport chain (ETC) ETHBV see elegant-crested tinamou hepatitis B virus (ETHBV) ethylene glycol tetraacetic acid (EGTA) 4:40 ethylene response factor-4 (ERF4) 3:758 ethylene-signaling genes 3:758 etiology, defined 3:200 ETV see entecavir (ETV) etymology and evolution of the meaning of virus 1:671–672 eubiosis, defined 4:283 eukaryotic translation initiation factors (eIFs) 3:794 eIF4E 1:311, 3:293 eukaryotic viruses 1:552 Euprosterna elaeasa virus (EeV) 4:897–898 European bat 1 lyssavirus (EBLV-1) 2:738, 2:740

339

European bat 2 lyssavirus (EBLV-2) 2:738, 2:740 European brown hare syndrome virus (EBHSV) classification 2:724 clinical features 2:726 diagnosis 2:728 epidemiology 2:726 genome 2:725–726 pathogenesis and host immunity 2:726–728 prevention and control 2:728–729 treatment 2:729 virion structure 2:724–725 European hedgehog (Erinaceus europaeus) 2:896 European rabbits, defined 2:730 European Synchrotron Radiation Facility (ESRF) 1:202 euryarchaeal viruses 4:368–370 culture-independent studies 4:378 icosahedral internal membrane-containing viruses 4:374–376 icosahedral tailed viruses 4:370–373 icosahedral tailed viruses of halophilic euryarchaea 4:371–373 tailed viruses of methanogenic euryarchaea 4:373–374 pleomorphic, spindle-shaped, and spherical viruses 4:376 pleomorphic viruses 4:376 spherical virus MetSV 4:377–378 spindle-shaped viruses 4:376–377 EV see extracellular virions (EV) EVA see eel virus American (EVA) EVD see Ebola virus disease (EVD); emerging viral diseases (EVD) EVE see endogenous viral element (EVE) EVE-derived immunity (EDI), defined 4:835 EVEX see eel virus European X (EVEX) evidenced based medicine, defined 5:98 evolutionarily informed treatment strategies 5:230 evolutionary arms race 2:65 evolutionary lineage, defined 4:457 evolution steered by structure architectural and functional similarities with cellular structures 1:96–98 evolution of viruses 1:87–88 structure-based virus classification 1:90–92 inferring viral evolutionary relationship through 1:92–94 viral envelope glycoproteins 1:94–96 viral evolutionary relationships from sequence analysis 1:88–90 evolvability, defined 1:62 EVs see enteroviruses (EVs); extracellular vesicles (EVs) exanthem, defined 2:868 exaptation, defined 1:627 exclusion, defined 4:276 exocyst, defined 1:529 exocytosis, defined 1:529 exogenous lentivirus, defined 2:56 exogenous retrovirus, defined 1:627 exogenous virus, defined 2:643

340

Subject Index

exonuclease, defined 4:291 exophthalmia, defined 2:324 exosome, defined 1:529, 2:362 exosome-like release of quasi-enveloped viruses 1:538–539 experimental immunoglobulin therapies 5:273–274 avian influenza 5:274 seasonal influenza 5:273–274 extant viral families, calibrating the longterm evolutionary history of 1:81 extensible tail 4:206 external enveloped virions (EEVs) 4:858 external quality assessment (EQA) 5:76f, 5:72, 5:76, 5:64–65, 5:55 design and objectives 5:76–78 and performance criteria 5:76 extracellular enveloped virion (EEV) 2:165 extracellular vesicle-mediated export of viruses 1:538 autophagy-related vesicle-mediated release of virus 1:539 exosome-like release of quasi-enveloped viruses 1:538–539 nonlytic release of virus in extracellular vesicles 1:538 extracellular vesicles (EVs) 1:538, 1:16–17 extracellular virions (EV) 2:857 extracorporeal membrane oxygenation (ECMO) 5:171–172 extrafollicular B cells 1:591 extra small virus (XSV) 4:716 extreme environments acidic hot springs, viruses of 4:422 ecology and evolution shaping archaeal viruses 4:424–425 host-virus interaction, environment impact on 4:420–422 host virus-interactions, complexity of 4:422–424 viral fitness, aspects of 4:419–420 virus interactions impacting ecology and evolution 4:425–426 extreme environments, ecology of phages in 4:342–343 atmosphere 4:354–355 hypersaline environments, phages in 4:343–345 polar and other cold environments, phages in 4:349–352, 4:354 glaciers 4:352–353 permafrost 4:353–354 polar lakes 4:353 polar oceans 4:352 sea ice 4:353 thermal environments, phages in 4:345–347 deep-sea hydrothermal vents 4:347–348 hot deserts 4:348–349 terrestrial hot springs 4:345–347 extremophile, defined 4:342 extremophilic bacteria, defined 4:53 extrinsic incubation period 2:893, 2:218, 2:805 Eyach virus (EYAV) 1:546

EYAV see Eyach virus (EYAV)

F fabaviruses 3:348 economic importance 3:353 epidemiology 3:353 expression of the viral genome 3:351–352 functions of the viral proteins 3:352 genome structure 3:351 geographic distribution 3:352 host range 3:352–353 physical properties of viral particles 3:349–350 relationships with other viruses 3:352 symptomatology and cytopathology 3:353 taxonomy and classification 3:348–349 transmission 3:353 use in biotechnology 3:352 viral structure 3:350–351 FACS see fluorescence-activated cell sorting (FACS) FAIDS see feline acquired immune deficiency syndrome (FAIDS) FAMA see fluorescence antibody membrane antigen test (FAMA) famciclovir 5:184, 5:175 farnesyl pyrophosphate synthetase (FPPS) 2:150 fas, defined 2:875 FAT see fluorescent antibody test (FAT) fatty acid synthase 2:391 Faustovirus 1:270–271 Favipiravir (Avigans) 5:168 FAVN test see fluorescent antibody virus neutralisation (FAVN) test F-box, defined 3:594 FCCS see Fluorescence Cross-Correlation Spectroscopy (FCCS) FcHDV see fenneropenaeus chinensis hepandensovirus (FcHDV) FCS see Fluorescence Correlation Spectroscopy (FCS) FCV see feline calicivirus (FCV) FD see fusion domain (FD) febrile fits or convulsions, defined 2:778 febrile headache 2:812 febrile status epilepticus, defined 2:778 fecal microbial transplantation or transplant (FMT) 4:283 fecundity, defined 4:314 FEGSEM see field emission scanning electron microscope (FEGSEM) feline acquired immune deficiency syndrome (FAIDS) 2:66 feline calicivirus (FCV) 1:450–452, 2:245 classification 2:294 control and prevention 2:298 disease control 2:298 vaccination 2:298 epidemiology 2:297 life cycle 2:296–297 pathogenesis 2:297–298 virus characteristics 2:294–296

genome organisation and viral proteins 2:294–296 non-structural proteins 2:296 feline immunodeficiency virus (FIV) 2:301, 2:56–57, 2:66 feline infectious peritonitis virus (FIPV) 2:198 feline leukemia virus (FeLV) 1:631 clinical features and pathology 2:302 envelope gene variation and pathogenicity 2:302–303 epidemiology 2:301 FeLV oncogenesis 2:303–304 FeLV sequences (FeLIX) 2:301–302 FeLV subgroups and host range 2:301–302 future perspectives 2:305 geographic distribution 2:301 history 2:300 immune response 2:304 prevention and control 2:304–305 replication 2:300–301 taxonomy and classification 2:300 transmission 2:304 virion and genome structure 2:300 feline morbillivirus (FmoPV) 2:68 feline panleukopenia virus (FPV) 2:684 FeLV see feline leukemia virus (FeLV) fenneropenaeus chinensis hepandensovirus (FcHDV) 4:839, 4:840 Feral, defined 2:730 F glycoprotein 2:749–750 FHB, see Fusarium head blight disease (FHB) FHV see Flock House Virus (FHV) fiber, defined 00057:p0050 FIB milling see focused ion beam (FIB) milling fibroblast cultures 2:453 fibroma viruses 2:730 Californian myxoma virus 2:731–732 classification 2:730 clinical disease in European rabbits 2:732–733 diagnosis 2:734–735 epidemiology 2:731 genome 2:730 hare fibroma virus 2:736 life cycle 2:730–731 myxomatosis in European rabbits 2:732 myxoma virus 2:731 pathogenesis of myxomatosis in European rabbits 2:733–734 prevention of myxomatosis 2:735 rabbit fibroma virus 2:735–736 squirrel fibroma virus 2:736 treatment 2:735 virion structure 2:730 field emission scanning electron microscope (FEGSEM) 5:13–14, 5:7f fijivirus 4:870, 4:871, 3:546–547, 3:551 filamentous, defined 4:53 filamentous bacteriophage 1:365–366 filamentous DNA viruses 1:407 filamentous phage 4:77 biotechnological applications of 4:59–60 structure of 4:55–56

Subject Index filamentous viruses 4:363–364 Tokiviricetes 4:363–364 filamentous viruses 4:484–485 filiformism, defined 3:371 filiform symptom, defined 3:862 filopodia, defined 2:22 Filoviridae family classification 2:233t filoviruses 2:235 Fimoviridae 3:396 fimoviruses (Fimoviridae) 3:396 classification 3:396 diagnosis 3:403 epidemiology 3:400–402 genome 3:398–400 life cycle (replication 3:400 symptomatology and economic impact 3:402–403 treatment and prevention 3:403 virion structure 3:396–398 finfish, defined 2:324 fingerlings, defined 2:544 FIPV see feline infectious peritonitis virus (FIPV) fIPV see fractional doses of inactivated poliovirus vaccine (fIPV) first generation sequencing technology 5:28–29 first sequencing methods 1:175–176 FI-RSV vaccine-enhanced disease see formalin-inactivated RSV (FI-RSV) vaccine-enhanced disease FISH see fluorescent in situ hybridization (FISH) fish alloherpesviruses classification 2:306–307 clinical features 2:311 diagnosis 2:313 epidemiology 2:309–310 genome 2:307–309 life cycle 2:309 management and treatment 2:313–314 pathogenesis 2:310–311 prevention 2:314–315 virion structure 2:307 fish retroviruses isolation and sequencing of retroviruses from WDS and WEH lesions 2:318 isolation of a retrovirus from salmon swimbladder sarcoma 2:320–321 phylogeny of 2:322–323 retroviruses and their life cycle 2:316–317 salmon swimbladder sarcoma-associated retrovirus 2:320 salmon swimbladder sarcoma virus (SSSV) pathogenesis of 2:322 prevalence, seasonality, and transmission of 2:321–322 sequence comparisons of SSSV with other retroviruses 2:321 seasonal cycle of disease, control of 2:319 skin tumors in walleye and their associated retroviruses 2:317–318 snakehead retrovirus 2:319–320 walleye dermal sarcoma virus (WDSV) accessory proteins 2:318–319 zebrafish endogenous retrovirus 2:322

fish rhabdoviruses classification 2:324–325 clinical features 2:328–329 diagnosis 2:329–330 epidemiology 2:328 genetic diversity 2:326–327 genome 2:326 life cycle 2:327–328 pathogenesis and immune response 2:329 prevention 2:330 virus structure 2:325–326 fitness, defined 1:62, 1:53 fitness landscape, defined 1:53 FIV see feline immunodeficiency virus (FIV) fivivirus, defined 3:805 flaviviruses 1:498–499, 2:810–811, 2:891, 2:891–892 assembly and budding 1:473, 1:473f life cycle 1:472 flavivirus particle organization 1:290–292, 1:291f, 1:293f acid sensitivity 1:293–295 flavivirus precursor polyprotein and the derived structural proteins 1:290–292 icosahedral surface lattices, thermodynamic transition between 1:297–299 immature flavivirus particle 1:292–293 mature particle organization 1:295–296 physical principles of 1:296–297 flavivirus virion structure 1:472–473 Flavobacterium-infecting, lipid-containing phage (FLiP) 1:159–160 Flavobacterium psychrophilum 4:354 flexible, filamentous plant viruses 1:364–365 FLIM see Fluorescence Lifetime Imaging Microscopy (FLIM) FLiP, see Flavobacterium-infecting, lipid-containing phage (FLiP) Flock House Virus (FHV) 1:409–410, 1:499 flock house virus 1:549 Floodwater Aedes spp. 2:765 florid, defined 2:629 florida clade 1 (FC1) viruses 2:291 florida clade 2 (FC2) viruses 2:291 flow field-flow fractionation methods 1:171–172 Flt3L see Fms-like tyrosine kinase 3 ligand (Flt3L) fludase 5:168 fluorescence-activated cell sorting (FACS) 1:179 fluorescence-activated virus sorting 1:187 DNA decontamination and fluorescenceactivated virus sorting preparation 1:187 preparation of 384-well plates for sorting 1:187 sorting 1:187 fluorescence antibody membrane antigen test (FAMA) 5:182 Fluorescence Correlation Spectroscopy (FCS) 1:208, 1:211 Fluorescence Cross-Correlation Spectroscopy (FCCS) 1:208

341

Fluorescence Lifetime Imaging Microscopy (FLIM) 1:211, 1:208 fluorescent antibody test (FAT) 2:743 fluorescent antibody virus neutralisation (FAVN) test 2:743 fluorescent in situ hybridization (FISH) 2:781, 2:782f, 1:498 fluorescent protein (FP) 1:209, 1:208 fluoro- (or fluorescence) immunoassay (FIA) 5:17–18 FMD see foot-and-mouth disease (FMD) FMDV see foot-and-mouth disease virus (FMDV) FmoPV see feline morbillivirus (FmoPV) Fms-like tyrosine kinase 3 ligand (Flt3L) 1:604 FMT see fecal microbial transplantation or transplant (FMT) FNV see French neurotropic vaccine (FNV) focused ion beam (FIB) milling 1:239–240 focus reduction neutralisation test (FRNT) 2:641 fomite, defined 1:559, 2:92 foodborne transmission 2:400 foot-and-mouth disease (FMD) 1:5 foot-and-mouth disease virus (FMDV) 1:647, 1:279–280, 1:284–285, 1:285, 2:414, 1:199, 1:202, 1:205, 4:659 classification 2:332 diagnosis 2:341 epidemiology 2:338 future perspectives 2:341–342 genetics and evolution 2:339 genome 2:334–336 leader protein 2:336 P1 region 2:336 P2 region 2:336 P3 region 2:336 protein products 2:335–336 history 2:332 host range 2:338–339 immune response 2:340–341 life cycle 2:336–337 cell attachment and entry 2:337 post-translation processing 2:337–338 RNA replication 2:337 translation 2:337 virus assembly and release 2:338 pathogenesis and clinical features 2:340 prevention and control 2:341 serological relationships and variability 2:339–340 virion structure 2:332–334 formaldehyde 5:301 formalin-inactivated RSV (FI-RSV) vaccineenhanced disease 2:754 forma specialis, defined 3:528 Forster Resonance Energy Transfer (FRET) 1:211, 1:208 forward genetics, defined 3:123 foscarnet 5:184, 2:458–459 fosmid, defined 4:322, 4:414 fossilized or endogenized viral genome, defined 1:71 fostemsavir 5:152–153

342

Subject Index

Foundation Block, defined 3:430 founder effect, defined 2:778 four-helix bundle 1:263, 1:329 454 (pyrosequencing) 1:176–177 fowl adeno virus 4 (FAdV–4) 2:13 fowlpox virus (FWPV) 2:343 classification 2:343 control and treatment 2:347 use of APV as recombinant vaccine vectors 2:347 epidemiology 2:346 genome 2:344–345 life cycle 2:345–346 pathogenesis 2:346–347 host range 2:346–347 immunomodulation 2:347 virion structure 2:343–344 FP see fluorescent protein (FP) FpMyV2 see fusarium poae mycovirus 2 (FpMyV2) FPPS see farnesyl pyrophosphate synthetase (FPPS) F protein, defined 2:747 FPV see feline panleukopenia virus (FPV) fractional doses of inactivated poliovirus vaccine (fIPV) frameshift, defined 3:788, 3:383 frameshifting, defined 3:778 Freeze Etching 5:5 Freeze Fracture 5:5 French neurotropic vaccine (FNV) 2:897 French viscerotropic virus (FVV) 2:897 Frenkel method 2:341 FRET see Forster Resonance Energy Transfer (FRET) FRNT see focus reduction neutralisation test (FRNT) frog and fish adenoviruses 2:14 fruit bats (flying foxes) 2:358 fruitbody, defined 4:528 fruit virus diseases 3:89–90 banana viral diseases 3:90 cacao swollen shoot disease (CSSD) 3:90 citrus tristeza virus (CTV) 3:90–91 papaya ring spot virus 3:91–92 plum pox virus (Sharka) disease of stone fruits 3:92 fry, defined 2:544 fucosyltransferase-2 (FUT2) gene 5:291 fulminant hepatitis, defined 2:397 functional genomics, defined 3:123 fungal DNA virus, see also DNA mycovirus replicating in insect 4:447–448 fungal endornaviruses 3:389 fungal gene expression, effects of mycoreoviruses on 4:612–613 fungal partitiviruses (Partitiviridae) 4:568 classification 4:568–569 epidemiology 4:572–573 genome 4:569–570 life cycle 4:570–572 pathogenesis 4:573–574 taxonomic and phylogenetic considerations 4:574–576 virion structure 4:569 fungal resistance in plants 4:515–516

fungal viruses 4:431, 4:434t–436 biological properties 4:431–432 host range 4:431–432 mixed infections 4:433 symptom expression 4:432 transmission 4:432–433 future perspectives 4:439–440 cross-kingdom infection by viruses and viroids 4:439–440 host defense against mycoviruses and their counter-defense 4:440–441 mycovirus 4:441–442 plant-fungal mutualistic associations, role of mycoviruses in 4:441 virus neo-lifestyles 4:440 yeast as a (model) host to study viral replication 4:440 recent technical advances in fungal virology 4:439 replication and gene expression strategy 4:438–439 taxonomy 4:433–437 dsRNA mycoviruses 4:433–437 single-stranded (ss) RNA mycoviruses 4:437–438 unassigned ssRNA viruses 4:438 fungi 4:544 fungi and protists, plant viruses transmission by 3:113 furovirus, defined 3:405 classification 3:405 control 3:409 diagnosis 3:408–409 nucleic acid properties and differentiation of 3:405–408 organization of the genome and properties of the encoded proteins 3:406–408 similarities and dissimilarities with other taxa 3:409 symptoms, epidemiology and host range 3:408 virion structure 3:405 virus transmission and movement 3:408 Fusarium graminearum, hypovirulence in 4:477 fusarium graminearum negative-stranded RNA virus 1 4:483 biological properties 4:483 family –Mymonaviridae 4:483 genome structure 4:483 genus –Unclassified 4:483 particle morphology 4:483 phylogenetic relationships 4:483 Fusarium head blight disease (FHB) 4:441 fusarium poae mycovirus 2 (FpMyV2) 4:660 fusariviruses 4:577 future prospective 4:580 gene expression 4:579 genome organization 4:578–579 taxonomy and classification 4:577 virion property 4:577–578 virus–host interactions 4:579–580 virus transmissions 4:579 Fuselloviridae 1:369–370, 4:362, 4:365

fusion domain (FD) 1:424 fusion machinery, working of 1:425 common mechanism of fusion 1:425 cooperativity 1:427 structural intermediates during conformational changes 1:426–427 elongated trimeric intermediate for class I and class II fusion glycoproteins 1:426–427 monomeric intermediates for vesiculovirus G 1:427 target membrane, interaction with 1:425–426 fusion peptide or loop, defined 1:417 fusion pore, defined 1:417 fusion protein 2:70–71 defined 1:382 fusogen, defined 1:87 fusolin, defined 4:858 FUT2 gene see fucosyltransferase-2 (FUT2) gene FVV see French viscerotropic virus (FVV) FWPV see fowlpox virus (FWPV)

G GACVS see Global Advisory Committee for Vaccine Safety (GACVS Gag (group-specific antigen), defined 3:96 gag gene 2:57–58, 2:59, 2:61t gag-pol gene 2:61t Gag-Pol polyprotein 1:455 Gag proteins 1:354 gag proteins and retroviral virion structure 1:352–354 GAGs see glycosaminoglycans (GAGs) GaHV1 see gallid herpesvirus type 1 (GaHV1) GaHV2 see Gallid herpesvirus 2 (GaHV2) Gallid herpesvirus 2 (GaHV2) 2:113 gallid herpesvirus type 1 (GaHV1) 2:112 gamma activation site (GAS) 2:65 gammaentomopoxvirus 4:859 Gammaflexiviridae 4:437–438 Gammaherpesvirinae 1:321 gammaherpesviruses 2:441, 2:442, 2:445 defined 2:441 gammapleolipovirus, defined 4:380 Gammaproteobacteria 4:335–336 pseudoalteromonas phages 4:336 vibriophages 4:335–336 ganciclovir 2:454, 2:458–459 Gannoruwa bat lyssavirus (GBLV) 2:738 GAS see gamma activation site (GAS) gastroenteritis 5:200 gastrointestinal infections (GI) 5:101 assays 5:101–102 background 5:101 clinical impact 5:102 gastrointestinal tract virome 1:553–555 GAV see gill-associated virus (GAV) gazelle herpesvirus 1 see equid herpesvirus type 9 (EHV-9) GBLV see Gannoruwa bat lyssavirus (GBLV)

Subject Index GBS see Guillain-Barre´ syndrome (GBS) GB virus C (GBV-C) 1:556 GCRV see grass carp reovirus (GCRV) GCV see Giardia canis virus (GCV) GDarSLA, see Gossypium darwinii symptomless alphasatellite (GDarSLA) GDGT see glycerol dibiphytanyl glycerol tetraether (GDGT) GDGT lipids see glycerol dialkyl glycerol tetraether (GDGT) lipids gelatin 5:302 geminate virion, defined 3:411 Geminiviridae 3:461, 3:768, 3:21, 3:301, 3:244–245 geminiviruses (Geminiviridae) 3:411–412, 3:21 African cassava mosaic disease 3:24–26 classification of cassava mosaic begomoviruses 3:27 control 3:28–29 disease symptoms and yield losses 3:26–27 epidemiology 3:27 geographical distribution 3:24–26 recent outbreak of CMD in South East Asia 3:27–28 control 3:418–419 cotton leaf curl disease in the indian subcontinent 3:29 causal agent and disease classification 3:29–30 control 3:30–31 disease symptoms and yield losses 3:29 epidemiology 3:30 geographical distribution 3:29 defined 3:21 diagnosis 3:415–417 DNA satellites associated with 3:415 genome and viral proteins 3:415 life cycle 3:417–418 pathogenicity 3:418 taxonomy, phylogeny, and evolution 3:411–412 becurtovirus 3:411–412 begomovirus 3:412 capulavirus 3:412–413 curtovirus 3:413 eragrovirus 3:413 grablovirus 3:413 mastrevirus 3:413 topocuvirus 3:413 turncurtovirus 3:413 tomato leaf curl new delhi disease 3:23–24 component capturing and exchange of helper components 3:24 control 3:24 disease symptoms and yield losses 3:24 epidemiology 3:24 geographical distribution 3:23–24 tomato yellow leaf curl disease 3:21–22 causal agent and taxonomy 3:22–23 control 3:23 disease symptoms, host range and yield losses 3:22 epidemiology 3:23 geographical distribution 3:21–22

virion structure 3:413–415 geminiviruses, plant resistance to 3:555, 3:557t in beans 3:561–563 beet curly top virus (BCTV), resistance to 3:560–561 cassava mosaic geminiviruses (CMGs), resistance to 3:556–558 to cotton leaf curl virus (CLCuVs) in cotton 3:563–565, 3:565f to maize streak virus (MSV) 3:565–566, 3:565f to mungbean yellow mosaic viruses (MYMIV) 3:563, 3:564f natural resistance to viruses in plants 3:555–556 in tomato 3:558–559 characterization of Ty genes 3:559–560 tomato yellow leaf curl virus (TYLCV) resistance 3:559, 3:560 geminivirus infection 3:750, 3:753, 3:758 gemonoviridae, defined 4:493 GenBank 3:245 defined 4:45 gene end (GE) sequence 2:752 gene expression defined 1:382 host interactions and regulation of 4:48 classes of genes 4:48 early genes 4:48–49 late genes 4:49 microarrays 1:498 gene-for-gene resistance, defined 3:60 general practitioner (GP) surveillance system 5:248 gene silencing, defined 3:293, 3:520, 3:116, 3:594 gene silencing, viral suppressors of 3:116 antiviral gene silencing pathway 3:116–117 assays to identify and characterize gene silencing suppressors 3:120–121 effect of gene silencing suppressors on plant development 3:121–122 evolution of viral suppressors of gene silencing 3:121 mechanisms of 3:119–120 cellular transcription, activation of 3:120 DNA methylation, interference with 3:120 homeostasis of gene silencing components 3:120 inhibition of siRNA biogenesis 3:120 RNA-induced silencing complexes, inhibition of assembly/activity of 3:120 silencing amplification, inhibition of 3:120 small RNA binding 3:119–120 silencing suppressors mediate viral synergism 3:122 small interfering RNAs direct antiviral immunity 3:117–118 viral suppressors in support of biotechnology 3:122

343

viral suppressors of gene silencing need cellular factors 3:121 viral suppressors of gene silencing promote infection 3:118–119 gene-silencing pathways 3:43 antiviral RNA silencing (av-RNAi), view of applications of 3:49–51 antiviral RNA silencing 3:45–47 antiviral silencing in crops 3:47–49 diverse gene-silencing pathways in plants 3:43–45 viral and plant RNA-silencing suppressors 3:47 viral siRNA-mediated antiviral RNAi in mammalian cells 3:49 gene therapy and cancer therapy 1:676 genetic barrier to resistance, defined 5:121 genetic clade, defined 2:117 genetic complementation, defined 1:53 genetic engineering, defined 4:291 Genetic engineering technique 3:759 genetic fusion 1:665–666 genetic hitch-hiking, defined 1:53 genetic organization and virion proteins 2:575–576 genetic parasite 1:606 Geneva Protocol (1925) 1:645 genital and urinary tract viromes 1:556 genital herpes, treatment options for 5:178t genitourinary diseases 5:200 GenitoUrinary Medicine (GUM) clinics, defined 5:247 genogroup, defined 5:289 genome, defined 4:175, 4:276, 3:327, 2:441, 2:747 genome activation 3:260 defined 3:439 genome editing, defined 3:293, 3:69 genome editing strategy 3:363 genome integration 4:391–392 genome maturation, defined 4:136 genome maturation complex 4:128–129, 4:130 cos-cleavage reaction 4:129–130 lambda cohesive end site and assembly of 4:128–129 model for the assembly of 4:129 genome mosaicism, defined 4:265 genome packaging 1:488 defined 1:318 double-stranded RNA viruses 1:492–493 energy-driven genome packaging in double-stranded DNA and giant viruses 1:493 energy-independent genome packaging in larger (ss)RNA viruses 1:490–492 in small single-stranded viruses 1:488–490 genome packaging complex 4:130 assembly of the packaging motor 4:130 cos-clearance 4:130–131 nucleotide switch for 4:131 genome promoter (GP) 2:69–70 Genome Relationships Applied to Virus Taxonomy (GRAViTy) 1:102 genome replication 4:390–391

344

Subject Index

defined 1:382 genomes and genomics 4:50 chromosome diversity and replication 4:50 common ancestry 4:51 common themes in genome structure 4:50–51 diversity in genome size and organization 4:50 horizontal exchange of genes is widespread 4:51 genome structure 4:601–602, 1:237–238 genomic architecture, defined 1:62 genomic precursor, defined 4:26 genomics-based taxonomy of viruses, emergence of 1:50–51 genomic segment, defined 4:26 Genomoviridae 4:718 genotype, defined 1:62, 5:289, 2:789, 2:22 genotyping 5:186 geodesic polyhedra 1:250 geometric mean titers (GMTs) 5:296 germicides 5:205 germline insertions 1:630–631 endogenous retroviruses (ERVs), exaptation of 1:630–631 ERV-derived non-coding elements 1:631–632 ERV-derived proteins 1:632 ERV-encoded envelope glycoproteins 1:630–631 GE sequence see gene end (GE) sequence GFP see green fluorescent protein (GFP) gfp gene, defined 4:858 G glycoprotein 2:750–752 GI see gastrointestinal infections (GI) giant viruses 1:493, 1:372 diversity and distribution of 1:373–375 genomics and proteomics of 1:376–377 life style of giants 1:375–376 origin and evolution 1:377–379 origin of 1:20–22 parasites of viruses 1:379–381 serendipitous discovery of 1:372–373 Giardia canis virus (GCV) 4:582, 4:583, 4:584 Giardia intestinalis/duodenalis 4:582 Giardia lamblia 4:582 Giardia lamblia virus (GLV) 4:582, 4:582–583, 4:584, 4:585, 4:586 giardiaviruses history 4:582 host range and geographic distribution 4:582–583 infection and replication 4:586–587 organization and molecular biology of GLV and GCV dsRNA genomes 4:585–586 physical and biochemical characteristics 4:583–585 taxonomy, classification and evolution 4:582 gill-associated virus (GAV) 2:245 Gillespie-type algorithms, defined 1:248 gingivostomatitis 5:175, 2:404

GISAID see Global Initiative on Sharing All Influenza Data (GISAID) GISRS see Global Influenza Surveillance and Response System (GISRS) glaciers 4:352–353 glecaprevir 5:123 glioblastoma multiforme 2:454 Global Advisory Committee for Vaccine Safety (GACVS 5:297–298 Global Influenza Surveillance and Response System (GISRS) 2:559, 2:561 Global Initiative on Sharing All Influenza Data (GISAID) 1:141–142, 5:262 Global Polio Eradication Initiative (GPEI) 5:310 global polio eradication program 2:690–692 Global Polio Laboratory Network (GPLN) 5:311 Globuloviridae 4:362 Glossinavirus 4:781 glutamic protease, defined 3:692 GLV, see Giardia lamblia virus (GLV) glycan receptors 1:390–391 enteroviruses (EVs) 1:392 glycan-based antiviral strategies 1:393–394 glycosaminoglycan receptors 1:393 histo–blood group antigen receptors (HBGAs) 1:392–393 noroviruses 1:392–393 rotaviruses 1:393 influenza viruses 1:391–392 polyomaviruses (PyVs) 1:392 sialic acid receptors 1:390–391 glycerol dialkyl glycerol tetraether (GDGT) lipids 1:368–369 glycerol dibiphytanyl glycerol tetraether (GDGT) 4:364 glycoprotein (GP) 2:612, 3:575, 1:417 glycoprotein precursor (GPC) 2:507, 2:508 glycosaminoglycan receptors 1:393 glycosaminoglycans (GAGs) 1:390, 2:475 glycosylation, defined 4:380 Glyphodes pyloalis 4:839 GMTs see geometric mean titers (GMTs) gnotobiotic mice, defined 4:283 Goatpox virus (GTPV) 2:165, 2:167 Gokushovirus genomes 4:18 Goldberg polyhedra 1:250 Goldberg polyhedral, defined 1:248 “gold-standard” procedure 1:234–235 Golgi apparatus 2:185 Gomphocerus sibiricus EPV (GSEV) 4:865 gonad-specific virus (GSV) 4:829 gonotrophic cycle, defined 2:805 gooseberry vein-banding associated virus (GVBaV) 3:165 Gossypium darwinii symptomless alphasatellite (GDarSLA) 3:360–361 Gossypium hirsutum 3:355, 3:356f, 3:563–564 Goule´ako virus 4:764 GP see genome promoter (GP); glycoprotein (GP) GPC see glycoprotein precursor (GPC)

GPCRs see G-protein-coupled receptors (GPCRs) GPEI see Global Polio Eradication Initiative (GPEI) GPI anchor, defined 3:32 GPLN see Global Polio Laboratory Network (GPLN) G protein, defined 2:747 G-protein-coupled receptors (GPCRs) 4:527, 2:167 GpSGHV transmission dynamics in the tsetse fly 4:788–789 GP surveillance system see general practitioner (GP) surveillance system grablovirus 3:413 graft-versus-host disease, defined 2:778 gram-negative bacteria 1:513 bacteriophage receptors in 4:179t gram-positive hosts, MGL in 1:513–514 granular cells (GCs) 4:816 granule, defined 4:739 granulin, defined 4:739 granulocyte-macrophage colony stimulating factor (GM-CSF) 1:659, 5:235 granulovirus (GV) 4:739, 4:747, 4:699 grapevine pinot gris virus 3:16 causal agent and classification 3:16 control 3:16–17 disease symptoms and yield losses 3:16 epidemiology 3:16 geographical distribution 3:16 grapevine roditis leaf discolorationassociated virus 3:15 causal agent and classification 3:15 control 3:16 disease symptoms and yield losses 3:15 epidemiology 3:15–16 geographical distribution 3:15 grapevines viruses 3:14–15 grapevine pinot gris virus 3:16 causal agent and classification 3:16 control 3:16–17 disease symptoms and yield losses 3:16 epidemiology 3:16 geographical distribution 3:16 grapevine roditis leaf discolorationassociated virus 3:15 causal agent and classification 3:15 control 3:16 disease symptoms and yield losses 3:15 epidemiology 3:15–16 geographical distribution 3:15 ornamental viruses: rose rosette virus 3:19 causal agent and classification 3:19 control 3:20 disease symptoms and yield losses 3:19 epidemiology 3:19–20 geographical distribution 3:19 staple crops: cassava brown steak disease 3:17 causal agent and classification 3:17 control 3:18–19 disease symptoms and yield losses 3:17–18 epidemiology 3:18

Subject Index geographical distribution 3:17 grapevine vein clearing virus (GVCV) 3:165 graphical user interface (GUI) 1:111 grass carp reovirus (GCRV) 1:306 GRAViTy see Genome Relationships Applied to Virus Taxonomy (GRAViTy) grazoprevir 5:123 greater virus world and its evolution 1:38–40 baltimore classes, evolutionary status of 1:44 double-stranded DNA viruses 1:43–44 evolutionary relationships within and between baltimore classes 1:40–42 reverse-transcribing viruses 1:42–43 RNA viruses 1:40–42 single-stranded DNA (ssDNA) viruses 1:43 virus hallmark genes 1:39–40 green fluorescent protein (GFP) 1:209–210, 1:208 groundnut rosette virus (GRV) 3:795 GRV see groundnut rosette virus (GRV) Gryllus bimaculatus 4:827–828 GSEV see Gomphocerus sibiricus EPV (GSEV) GSV see gonad-specific virus (GSV) GTPV see Goatpox virus (GTPV) guanyl transferase, defined 2:22 GUI see graphical user interface (GUI) Guillain-Barre´ syndrome (GBS) 5:305, 2:899 guilt-by association bioinformatic approach 4:249–250 Guttaviridae 4:362 GV see granulovirus (GV) GVBaV see gooseberry vein-banding associated virus (GVBaV) GVCV see grapevine vein clearing virus (GVCV) gyrated lattice, defined 1:248

H HA see hemagglutinin (HA) HAART see highly active antiretroviral therapy (HAART) HA assay see hemagglutination (HA) assay HAdV–A 2:11 HAdV–B1 2:11 HAdV–B2 2:11 HAdV–C 2:11 HAdV-C5 see human adenovirus type 5 (HAdV-C5) HAdV–D 2:11 HAdV–E 2:11 HAdV–F 2:11–12 HAdV–G 2:12 HAdVs see human adenoviruses (HAdVs) hAE see human airway epithelium (hAE) HAE-ALI, defined 2:419 haematophagous arthropods, defined 1:542 HAI assay see hemagglutination inhibition (HAI) assay half-life, defined 2:697

haloarchaea, defined 4:380 haloarchaeal pleomorphic viruses (HRPVs) 4:380–381 Haloarcula californiae icosahedral virus 1 (HHIV-1) 1:337–339 Haloarcula hispanica icosahedral virus 2 (HHIV-2) 1:337–339 Haloarcula hispanica pleomorphic virus 1 (HHPV-1) 1:434–435 Haloarcula sinaiiensis tailed virus 1 (HSTV-1) 1:323 halophile 4:342 halophilic, defined 4:368 haloquadratum walsbyi, viruses of 4:416–417 Halorubrum pleomorphic virus 1 (HRPV-1) 1:434–435 halorubrum pleomorphic virus 6 (HRPV-6) 4:390 Halorubrum sodomense tailed virus 2 (HSTV-2) 1:252–253 HAM1-like sequence, defined 3:293 Hamaparvovirinae 4:711–713 Hamiltonian Paths Analysis (HPA) 1:249, 1:248 Hamiltonian Paths Approach (HPA) 1:254 applications of 1:254–255 assembly code embedded within viral genetic message 1:254–255 icosahedral symmetry, beyond 1:255 virus assembly mechanisms 1:255 hammerhead structure, defined 3:852 hand, foot-and-mouth disease (HFMD) 1:284 hantavirus, defined 2:349 hantavirus cardiopulmonary syndrome (HCPS) 2:349 hantaviruses clinical picture and pathogenesis 2:351–353 diagnostics and prevention 2:353 ecology and epidemiology 2:350–351 historical introduction 2:349 life cycle of 2:351f virology 2:349–350 haplotype, defined 4:601, 4:607 harnessing of viruses by humans 1:675 bioweapons 1:676–677 commerce and tulip mania 1:675 gene therapy and cancer therapy 1:676 phage therapy 1:676 vaccines 1:675–676 HaSV see helicoverpa armigera stunt virus (HaSV) HAV see hepatitis A virus (HAV) HBcAg see hepatitis B virus core antigen (HBcAg) HBeAg see hepatitis B e antigen (HBeAg) HBE cultures see human bronchial epithelial (HBE) cultures HBGAs see histo-blood group antigens (HBGAs) HBIG see Hepatitis B hyperimmune IG (HBIG) HBsAg see hepatitis B surface antigen (HBsAg)

345

HBsAg loss, defined 5:217 HBV see hepatitis B virus (HBV) HBVDb see Hepatitis B Virus Database (HBVDb) Hbz gene 2:535–536 HCC see hepatocellular carcinoma (HCC) HCMVCMV see human cytomegalovirus (HCMV), see also cytomegalovirus (CMV) HCoV see human coronavirus (HCoV) HCPS see hantavirus cardiopulmonary syndrome (HCPS) HCRSV see hibiscus chlorotic ringspot virus (HCRSV) HCs see hyaline cells (HCs) HCV see hepatitis C virus (HCV) HDFs see host dependency factors (HDFs) HDL see high-density lipoproteins (HDL) HDV see hepatitis delta virus (HDV) HE see hemagglutinin-esterase (HE) head, dealing with 4:219–221 capsids with large numbers of co-localized E proteins 4:223–224 capsids with large numbers of E proteins distributed throughout the DNA 4:222–223 E proteins, enigmas of 4:224–225 encapsidation of E proteins 4:225–226 impacts of E proteins on capsid DNA packaging and ejection 4:226 impacts of E proteins on DNA ejection 4:226–227 phages with yet to be identified E proteins 4:224–225 potential of E protein delivery from capsid derived nanocontainers 4:227 tailed phage capsids with low numbers of E proteins in specific locales 4:220–221 low copy number E proteins with specific locales in the capsid 4:221 podoviral E proteins 4:221–222 headful packaging, defined 4:61, 4:368 headful termination, defined 4:136 head-tail connector, defined 4:105 heartland banyangvirus (HRTV) 2:775–776 heating, ventilation, and air conditioning (HVAC) systems 5:69 heat therapy, defined 3:430 HEF protein see hemagglutinin-esterasefusion (HEF) protein helical and filamentous phages 4:53 biotechnological applications of filamentous phage 4:59–60 biotopes 4:53–55 infection process 4:56 M13 (fd, f1), biosynthesis of 4:56 assembly and secretion 4:58–59 coat proteins 4:56 major coat protein gp8 4:56–57 minor coat proteins gp3 and gp6 4:57–58 minor coat proteins gp7 and gp9 4:57 replication by the rolling circle mechanism 4:56 structure of filamentous phages 4:55–56

346

Subject Index

helical and filamentous phages (continued) helical symmetry 1:261, 1:362, 3:229 helical viruses, structure of 1:362, 1:364t Archaea, Rudiviridae and Lipothrixviridae, viruses of 1:367–369 atomic structure 1:475–476 Claviviridae 1:369 filamentous bacteriophage 1:365–366 flexible, filamentous plant viruses 1:364–365 hemorrhagic filoviridae 1:366–367 rod-shaped helical plant viruses 1:362–364 spindle-shaped viruses 1:369–370 helicase-primase, defined 2:441 helicoverpa armigera stunt virus (HaSV) 4:897, 4:901–902 Heliothis zea nudivirus 4:827–828 heliothsi zea nudivirus 1 (HzNV-1) 4:710 4-Helix bundle 1:362 6-helix bundle (6HB) 2:750 Helminthosporium victoriae 4:432 hypovirulence in 4:476–477 helper component, defined 3:703, 3:106 Helper component proteinase, defined 3:293 helper phage, defined 4:98 helper strategy 3:112 aphid-transmitted caulimoviruses, case of 3:112 aphid-transmitted potyviruses, case of 3:112 hemagglutination (HA) assay 5:93–94 hemagglutination inhibition 5:20 hemagglutination inhibition (HAI) assay 5:93–94 hemagglutination inhibition (HI) test 2:292 hemagglutinin (HA) 1:570–571, 1:420, 5:301, 2:565–566, 1:668 hemagglutinin-esterase (HE) 2:251–252 hemagglutinin-esterase-fusion (HEF) protein 2:561 hematopoietic stem cell transplantation (HSCT) 2:453, 2:455, 5:105, 2:779, 2:778 hemifusion 2:289, 1:417 hemipteran, defined 4:768 hemocoel, defined 3:176, 3:447 hemocyte, defined 4:849 hemogram, defined 2:899, 3:176, 3:200, 3:447, 3:106 hemorrhagic fever with renal syndrome (HFRS) 2:349, 2:350, 2:350–351 hemorrhagic filoviridae 1:366–367 Hendra virus (HeV) 2:355 henipaviruses 2:355–356 classification 2:355 clinical features 2:358–359 diagnosis 2:360 epidemiology 2:357–358 genome 2:356 life cycle 2:356–357 pathogenesis 2:359–360 prevention 2:361 treatment 2:360–361 virion structure 2:355–356

Hepadnaviridae 2:373 classification 2:373–374 clinical features 2:381 acute HBV infection 2:381 chronic HBV infection 2:381 course of chronic infection 2:381–382 mutant viruses and chronic infection 2:382 diagnosis 2:383 epidemiology 2:379 genome 2:375–376 core and polymerase 2:376 surface proteins 2:375–376 transcription and translation 2:375–376 X protein 2:376 genotypes 2:379 life cycle 2:376 attachment 2:376 egress 2:378–379 encapsidation 2:377–378 maturation 2:378 penetration and uncoating 2:376 transcription/translation 2:376–377 pathogenesis 2:382–383 prevention 2:384 subtypes 2:379 treatment 2:383–384 resistance 2:384 variants 2:379–381 virion structure 2:374–375 Hepanhamaparvovirus 4:836 heparan sulfate 1:285, 2:751 heparan sulfate proteoglycans (HSPG) 2:390, 2:399 hepatitis A 5:268 hepatitis A virus (HAV) 5:206–207, 5:213, 2:397, 1:530, 5:268, 1:282 cell culture and growth characteristics 2:366 clinical features and pathology 2:366–367 common course of infection 2:367 extrahepatic manifestations 2:368 fulminant hepatitis A 2:368 pathology 2:368–369 prolonged hepatitis A 2:367 relapsing hepatitis A 2:367–368 clinical presentation and course 5:209 diagnosis 2:370–371 antigen detection 5:210 nucleic acid detection 5:210–211 serological tests 5:211–212 epidemiology 2:371 intermediate endemicity pattern 5:207 low endemicity pattern 5:207 very high and high endemicity pattern 5:206–207 genome organization and expression, replication, morphogenesis 2:363–366 history 2:362 host range, transmission and tissue tropism 2:366 immunoglobulin 5:213 innate and adaptive immune response 2:369–370 adaptive immune response 2:370

innate immune response 2:369–370 morphology and physicochemical properties 2:363 prevention and control 2:371 taxonomy 5:206 taxonomy and evolutionary origin 2:362–363 transmission 5:208 other modes of transmission 5:208 person-to-person transmission 5:208 water- and food-borne transmission 5:208 treatment 5:213 vaccine 5:213 hepatitis B, see Hepadnaviridae hepatitis B e antigen (HBeAg) 2:374 hepatitis B hyperimmune globulin 5:269–270 Hepatitis B hyperimmune IG (HBIG) 5:270 hepatitis B surface antigen (HBsAg) 5:269–270, 2:374 hepatitis B virus (HBV) 1:521, 1:266, 1:537, 1:668, 1:490, 5:128, 2:100 HBV DNA polymerase 5:128 hepatitis B virus core antigen (HBcAg) 1:664 Hepatitis B Virus Database (HBVDb) 1:141–142 hepatitis B virus genotypes, recombination among 1:112–113 hepatitis C virus (HCV) 1:11, 1:537, 1:178, 1:180, 5:61–62, 5:123, 4:28–30, 1:498–499 activation and inhibition of innate and adaptive immunity by 2:391–392 classification 2:386 clinical features 2:392–393 diagnosis 2:393–394 epidemiology 2:392 formation of the HCV replication organelle 2:390 genome organization and viral proteins 2:387–389 HCV NS3/4A protease 5:123 HCV NS5A phosphoprotein 5:123 HCV NS5B polymerase 5:123 HCV viremia, defined 2:386 infection 1:68 life cycle 2:389–390 pathogenesis 2:393 prevention 2:394 replication cycle host cell factors contributing to various steps of 2:390–391 host cell lipids of relevance to 2:391 treatment 2:394 virion structure and properties 2:386–387 hepatitis delta virus (HDV) 4:623 hepatitis E virus (HEV) 5:110, 5:207, 5:213–214, 2:397, 2:400, 2:400–401, 2:402, 2:398t clinical presentation and course 5:209–210 diagnosis antigen detection 5:210 nucleic acid detection 5:211 serological tests 5:212–213

Subject Index epidemiology HEV genotypes 1 and 2 5:207 HEV genotypes 3 and 4 5:207–208 infection 5:103 management of acute hepatitis E 5:214 management of chronic infections 5:214 prophylaxis 5:214 taxonomy 5:206 transmission 5:208–209 vaccine 5:214 hepatocellular carcinoma (HCC) 5:62, 2:106 hepatocytes 2:390, 2:104 hepatopancreatic parvovirus (HPV) see fenneropenaeus chinensis hepandensovirus (FcHDV) Hepatoviruses 1:279–280 Hepeviridae classification 2:397 clinical features 2:401–402 diagnosis 2:402 epidemiology 2:399–401 genome 2:397–399 life cycle 2:399 pathogenesis 2:402 prevention 2:402 treatment 2:402 virion structure 2:397 Herbert virus 4:764 herd immunity, defined 5:295, 1:559 herpes encephalitis 5:176–177 herpes gladiatorum 5:176 herpes rugbiorum 5:176 herpes simplex virus (HSV) 1:266, 1:178, 5:182–183, 5:109, 5:124, 2:714–715 -associated disease 5:193–194 HSV DNA polymerase UL30 5:124 HSV envelope protein 5:124–125 herpes simplex virus (HSV) infections available antiviral agents for 5:175 immunocompromised hosts 5:180 immunoprophylaxis 5:180 management beyond neonatal period 5:175 CNS infections 5:176–177 cutaneous infections 5:175–176 genital herpes 5:177–178 ocular infections 5:176 orolabial infections 5:175 neonatal herpes simplex virus infection 5:178–180 herpes simplex virus 1 (HSV-1) 1:424, 1:658–659, 1:604, 1:446, 1:252–253 herpes simplex virus 1 and 2 (HSV-1, HSV-2) 1:321 classification 2:404 clinical features 2:409–410 genital herpes 2:410 gingivostomatitis 2:409–410 herpes simplex encephalitis 2:411 herpes simplex virus infections in the immunocompromised host 2:411 herpetic keratitis 2:410 mucocutaneous infections 2:409–410 neonatal herpes simplex virus infection 2:410–411

skin manifestations 2:410 diagnosis 2:411–412 epidemiology 2:408–409 life cycle 2:404–407 epigenetic regulation 2:407 host factors 2:407 latent infection 2:407–408 lytic infection 2:406–407 pathogenesis 2:411 prevention 2:412 treatment 2:412 virion structure 2:404 genome 2:404 Herpesviridae 2:404, 2:267 herpesvirus 2:442, 1:321–322 defined 2:441 herpesviruses virions, structure of 1:322f herpesvirus infections in immunocompromised host 5:190 cytomegalovirus (human herpesvirus 5) 5:190–191 antiviral drug resistance 5:192–193 diagnosis of CMV disease and treatment 5:192 pre-transplant risk stratification 5:191 prevention strategies of CMV disease after transplantation 5:191–192 Epstein Barr virus 5:195 herpes simplex virus (HSV)-associated disease 5:193–194 human herpes virus 5:194–195 human herpes virus-8 5:195–196 Varicella Zoster Virus (VZV) 5:194 herpes virus of turkeys (HVT) 2:652–653, 2:542 herpes zoster, defined 5:281 Hershey-Chase experiment 4:206 heteroduplex DNA, defined 4:291 hetero-encapsidation, defined 3:528 heterokaryon, defined 4:557, 4:534, 4:450 heterokaryosis 4:520 heterokaryotic/homokaryotic, defined 4:568 heterospecific antibodies, defined 3:727 heterothallic fungus, defined 4:450 heterotrimeric G proteins 4:523 HeV see Hendra virus (HeV) HEV see hepatitis E virus (HEV) hexagonal peroxisome protein (Hex1) 4:432 hexamers 1:250–251 hexon 1:331 defined 2:441 HFMD see hand, foot-and-mouth disease (HFMD) H form 1:414 HFRS see hemorrhagic fever with renal syndrome (HFRS) HGT see horizontal gene transfer (HGT) hhi1 transcript 4:832–833 HHIV-1, see Haloarcula californiae icosahedral virus 1 (HHIV-1) HHIV-2, see Haloarcula hispanica icosahedral virus 2 (HHIV-2) HHPV-1, see Haloarcula hispanica pleomorphic virus 1 (HHPV-1) HHV–6 see human herpesvirus 6 (HHV–6) HHV-8 see human herpesvirus-8 (HHV-8)

347

hibiscus chlorotic ringspot virus (HCRSV) 1:667 Hidden Markov Model (HMM) 1:127 HIF pathway see hypoxia-inducible factor (HIF) pathway high-density lipoproteins (HDL) 2:386–387 higher-order RNA structures, defined 3:778 highly active antiretroviral therapy (HAART) 5:133, 2:455, 5:121 highly pathogenic avian influenza (HPAI) 2:119, 2:117 high mobility group box protein 1 (HMGB1) 2:139, 1:660 high-pressure freezing (HPF) 5:13 high throughput sequencing (HTS) 1:50, 5:92–93, 1:47, 3:192, 5:27, 1:175–176, 1:177, see also next generation sequencing (NGS) applications of high-throughput sequencing in virology 1:177 DNA sequencing – amplicon sequencing 1:178–179 enrichment strategies 1:179 negative selection-based enrichment 1:179–180 positive selection-based enrichment 1:179 first sequencing methods 1:175–176 454 (pyrosequencing) 1:176–177 illumina sequencing 1:177 metagenomic approaches 1:177–178 next generation sequencing and applications in virology 1:176 RNA-sequencing 1:178 single cell sequencing 1:180 using single cell sequencing to investigate cellular heterogeneity and its impact on viral infection 1:180 using single cell sequencing to investigate viral heterogeneity 1:180 single molecule sequencing (Pacific Biosciences and nanopore) 1:180–181 merging SRS and LRS 1:182 Oxford Nanopore technologies 1:181–182 Pacific Biosciences 1:181 higrevirus 3:247 diagnosis 3:251 diseases, epidemiology, and control 3:250–251 genome organization and gene product function 3:248–249 members of the family 3:247 replication and propagation 3:249–250 taxonomy, phylogeny, and evolution 3:247 transmission and host range 3:250 virion structure 3:247–248 Himetobi P virus (HiPV) 4:771 HiPV see Himetobi P virus (HiPV) histo-blood group antigens (HBGAs) 1:307–308, 1:392–393, 1:390 noroviruses 1:392–393 rotaviruses 1:393

348

Subject Index

histo-blood group antigens (HBGAs) (continued) histology, defined 2:629 histone deubiquitinase (DUB) activity 4:592 history of virology 2:414, 3:3 arthropod-borne viruses of vertebrates 2:417 biochemical/biophysical age 3:4–5 biochemistry 2:415–416 biological age 3:4 chemical composition of viruses 2:415 cultivation of animal viruses 2:415 discovery of phages 4:3–4 early investigations 2:414 foot-and-mouth disease virus 2:414 yellow fever virus 2:414 future 2:417 HIV-AIDS, recognition of 2:417 immunology, impact on 2:416–417 molecular biology age 3:5–7 phage and biotechnology 4:7–8 phage biochemistry 4:6–7 phage ecology and evolution 4:8 phage genetics 4:5–6 phage therapy 4:4 phage typing 4:4–5 physical studies of viruses 2:414–415 prehistory 3:3 recognition of viral entity 3:3–4 structure of the virion 2:416 taxonomy and nomenclature 2:417 tumor virology 2:416 vaccines and disease control 2:417 word virus 2:414 HI test see hemagglutination inhibition (HI) test HIV see human immunodeficiency virus (HIV) HJ see Holliday junction (HJ) HK97 fold 1:263 H/KDEL receptors, defined 4:534 HLA see human leukocyte antigen (HLA) HMGB1 see high mobility group box protein 1 (HMGB1) HMG-CoA see 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) HMM see Hidden Markov Model (HMM) HMP see Human Microbiome Project (HMP) hMPV see human metapneumovirus (hMPV) HoCV-1 see homalodisca coagulata virus 1 (HoCV-1) Holliday junction (HJ) 4:64, 2:857 Holliday junction resolvase, defined 4:387 homalodisca coagulata virus 1 (HoCV-1) 4:773 homeologous DNA, defined 4:291 homogenous wash-free immunoassays 5:19 homologous, defined 1:62 homologous DNA, defined 4:291 homologous gene products, defined 2:441 homologous recombination, defined 4:387, 4:359

homologous repeat (hr), defined 4:808, 4:747 Homologous Structure Finder (HSF) software 1:157 homologs, defined 4:242 homology, defined 1:87 homothallic fungus, defined 4:450 hordeiviruses (Virgaviridae) 3:420 function and relatedness of 3:422–423 coat protein (CP) 3:423 cytopathology and replication 3:424–426 pathogenesis 3:426–427 pathogenesis protein 3:424 replicase proteins 3:422–423 TGB1 3:423 TGB2 protein 3:423–424 TGB3 protein 3:424 future perspectives 3:428 taxonomy and characteristic 3:420–421 genome structure and expression 3:421–422 virus particle structure 3:421 hordeivirus-host interactions 3:427 gb interference with host defenses 3:427 autophagy responses 3:427 RNA silencing pathways 3:427 ROS bursts 3:427 hordeivirus applications to cereal genomic analyses 3:428 post-translational modifications of BSMV proteins 3:427–428 horizontal gene exchange, defined 4:45 horizontal gene transfer (HGT) 1:613, 1:77, 1:71, 4:242, 1:87, 4:732 horizontal mycovirus transmission, defined 4:468 horizontal transmission, defined 4:520, 2:778, 4:419, 1:559, 3:388, 3:430, 3:106, 4:780, 4:768 hospital admissions 5:249 host, clinical status of 1:562–563 host, defined 4:276 Hosta virus X (HVX) 3:628 host cell recognition 4:201–203 host dependency factors (HDFs) 1:628–629, 1:627, 1:629 host enzymes, viral hyper-mutation mediated by 1:55 cellular adenosine deaminases 1:55–56 cellular cytidine deaminases 1:55 host factor, defined 3:252 host genes with indirect effects on retrovirus infection, spread or disease 1:630 host immune response, shielding from 5:239–240 chemical shielding and cell carriers 5:240–241 virus engineering 5:239–240 host immunity 1:630 host-pathogen coevolution, defined 2:730 host plant resistance, defined 3:355 host plasticity 5:257 host population immunity, influence of 1:563

host range, defined 4:265, 4:276, 4:732 host-virus exchange via horizontal gene transfer and transposable elements 1:77 host-virus interactions complexity of 4:422–424 environment impact on 4:420–422 hot deserts 4:348–349 hot tumor 1:659 house finches (Haemorhous mexicanus) 2:805 housefly, MdSGHV transmission dynamics in 4:789 house sparrows (Passer domesticus) 2:805 HPA see Hamiltonian Paths Analysis (HPA); Hamiltonian Paths Approach (HPA) HPAI see highly pathogenic avian influenza (HPAI) HPeV1 see human parechovirus 1 (HPeV1) HPF see high-pressure freezing (HPF) HRPV-1, see Halorubrum pleomorphic virus 1 (HRPV-1) HRPV-6 see halorubrum pleomorphic virus 6 (HRPV-6) HRPVs see haloarchaeal pleomorphic viruses (HRPVs) HRTV see heartland banyangvirus (HRTV) HRV see human rhinoviruses (HRV) HRV14 see human common cold virus strain 14 (HRV14) HRV1A see human rhinovirus 1A (HRV1A) HSCT see hematopoietic stem cell transplantation (HSCT) HSF software see Homologous Structure Finder (HSF) software HSPG see heparan sulfate proteoglycans (HSPG) HSTV-1 see Haloarcula sinaiiensis tailed virus 1 (HSTV-1) HSTV-2 see Halorubrum sodomense tailed virus 2 (HSTV-2) HSV see herpes simplex virus (HSV) HTLV-1 see human T-cell leukemia virus-1 (HTLV-1) HTSNGS see high throughput sequencing (HTS), see also next generation sequencing (NGS) HTUs see hypothetical taxonomy units (HTUs) huaiyangshan banyangvirus 2:771–775 Hubei sclerotinia RNA virus 1 (HuSRV1) 4:466 human adenoviruses (HAdVs) 5:197, 5:199, 5:198t, 5:202f human adenovirus type 5 (HAdV-C5) 1:329–331 core proteins 1:335 fiber 1:331–333 hexon 1:331 minor coat proteins 1:333 protein IIIa 1:333 protein VI 1:333 protein VIII 1:333 protein IX 1:333–335 penton base protein 1:331 human airway epithelium (hAE) 5:170

Subject Index human and animal viral diseases, epidemiology of digital epidemiology 1:566 disease occurrence and outcome, assessment of 1:564–565 epidemiological studies 1:564–565 molecular epidemiology 1:565–566 disease prevention, implications for 1:568 factors influencing the spread of viral diseases 1:561 arthropod transmission cycles, influence of 1:563–564 clinical status of the host, influence of 1:562–563 host population immunity, influence of 1:563 modes of virus transmission 1:561–562 perpetuation of viruses in nature 1:561 population size, influence of 1:563 virulence of the virus, influence of 1:563 virus entry 1:561 virus shedding 1:561 zoonotic transmission cycles, influence of 1:563 mathematical modeling 1:566–567 data sharing, data privacy and ethics 1:567 proof of causation 1:567–568 sentinel studies 1:566 seroepidemiologic studies 1:566 vaccine trials 1:566 human bronchial epithelial (HBE) cultures 2:749 human common cold virus strain 14 (HRV14) 1:259–260, 1:265–266, 1:266 human coronavirus (HCoV) background 2:428–429 classification 2:429 diagnosis 2:438 epidemiology and clinical features 2:437 HCoV-229E 2:437 HCoV-HKU1 2:437–438 HCoV-NL63 2:437 HCoV-OC43 2:437 genome 2:430–431 accessory proteins 2:431–432 non-structural proteins 2:431 HCoV 229E and NL63 2:245 HCoV-host interactions 2:434–435 apoptosis 2:435–436 autophagy 2:435 ER stress response 2:435 MAP kinase pathway 2:435 translational control 2:434–435 HCoV-OC43 1:122 life cycle 2:432–433 assembly and release of virion 2:434 attachment 2:432–433 formation of the replicationtranscription complex 2:433 viral entry and uncoating 2:433 viral RNA synthesis 2:433–434 pathogenesis 2:436–437 prevention 2:439 treatment 2:438–439

virion structure 2:429–430 human cytomegalovirus (HCMV) 5:62, 5:227, 1:604, 5:106–107, 5:125, 1:321, 1:321–322, see also cytomegalovirus (CMV) HCMV DNA polymerase UL54 5:125 HCMV terminase UL56 5:125 human herpes virus 5:194–195 human herpesvirus 5 5:190–191 antiviral drug resistance 5:192–193 diagnosis of CMV disease and treatment 5:192 pre-transplant risk stratification 5:191 prevention strategies of CMV disease after transplantation 5:191–192 human herpesvirus 6 (HHV–6) 2:778–779, 2:779f, 2:780t, 2:780, 2:781, 2:783, 2:784, 2:787–788 and disease after organ transplant 2:786 HHV–6A 2:781, 2:783 and HHV–6B 2:779–780 chromosomal integration of 2:781–782 vertical transmission of 2:783 HHV–6B 2:781, 2:783 and disease after organ transplant 2:786 chromosomal integration of 2:781–782 vertical transmission of 2:783 HHV–6B and HHV–7 primary infection and disease 2:785 encephalitis 2:785 exanthem subitum 2:785 FSE and temporal lobe epilepsy 2:785 and HHV–7 disease in older children and adults 2:785 Alzheimer’s disease 2:786 and the possibility of encephalitis 2:785 delayed primary infection 2:785 HHV–6 and multiple sclerosis 2:786 HHV–6B and DRESS (drug rash with eosinophilia and systemic symptoms) 2:785–786 and HHV–7 laboratory tests 2:786–787 antibody detection 2:787 CIHHV–6, diagnosis of 2:787 CIHHV–6 and misdiagnosis of active infection 2:787 virus detection – HHV–6A, HHV–6B and HHV–7 2:787 and HIV 2:786 horizontal transmission of 2:783 transmission of HHV–6 by organ donation 2:783–784 human herpesvirus-8 (HHV-8) 2:599, 5:195–196, 1:179 human immune deficiency virus (HIV) integrase inhibitors 5:147t–148 children and adolescents 5:153 entry inhibitors 5:151–152 enfuvirtide 5:152 fostemsavir 5:152–153 ibalizumab-uiyk 5:152 maraviroc 5:151–152 HIV-2 5:153 integrase strand transfer inhibitors (INSTI) 5:145–146

349

first generation INSTI 5:146 second generation INSTI 5:149–150 virology 5:145–146 post-exposure and pre-exposure prophylaxis (PEP and PrEP) 5:153 pregnancy 5:153 human immunodeficiency virus (HIV) 1:266, 2:56, 2:56–57, 1:399, 1:229, 1:121, 5:273, 1:178, 1:605, 5:121, 5:190 classification 2:460 clinical features 2:469–470 diagnosis 2:468–469 early phase 2:464 attachment and entry 2:464 uncoating, reverse transcription, nuclear localization, and integration 2:464 epidemiology 2:466–468 modes of transmission and risk factors 2:467–468 prevalence 2:468 genome, organization of 2:461 HIV-AIDS recognition of 2:417 surveillance of 5:250–251 HIV-associated dementia (HAD) 2:470–471, 2:471 HIV associated Kaposi’s sarcoma 2:606 HIV GP120 5:122–123 HIV GP41 5:122 HIV integrase 5:122 HIV protease 5:121–122 HIV reverse transcriptase 5:121 late phase 2:464–465 budding and maturation 2:465–466 gene expression and assembly 2:465 life cycle 2:464 origin of HIV-1 2:460–461 pathogenesis 2:470 coreceptor switch 2:470 drug resistance, evolution of 2:471 latency 2:471 macrophage-tropic HIV-1 and infection of CNS 2:470–471 prevention 2:473 biomedical prevention 2:473 Pre-Exposure Prophylaxis (PrEP) 2:473–474 social and educational prevention 2:474 treatment 2:471–472 entry inhibitors 2:472 integrase inhibitors 2:473 Protease Inhibitors (PIs) 2:473 reverse transcriptase inhibitors (RTIs) 2:472–473 viral genes, expression of 2:461–463 viral proteins 2:463–464 virion proteins 2:463 human immunodeficiency virus type 1 (HIV-1) 1:620, 1:209, 1:567–568, 1:445, 1:383, 2:460, 1:599, 2:56–57, 2:57f, 2:58f, 2:57–58, 2:62, 2:64 HIV-1 centralized vaccines, recombination in the design of 1:112 HIV-1 fitness recovery, influence of recombination on 1:112

350

Subject Index

human immunodeficiency virus type 1 (HIV1) (continued) HIV-1 gp120, analyzing adaptation of 1:113 human immunodeficiency virus type 2 (HIV-2) 2:827–828, 2:56–57, 2:57f, 2:64, 5:153 clinical features 2:832 defined 2:827 epidemiology of 2:830–831 diagnosis of HIV-2 2:831–832 risk and transmission of HIV-2 2:831 genome 2:828–829 lifecycle 2:828–829 immunity to 2:832–833 HIV-2 cellular immunity 2:833 HIV-2 humoral immunity 2:833 interactions of HIV-2 and HIV-1 in vivo 2:834–836 HIV-2 protection from HIV-1 2:834–836 in vitro evidence for HIV-2 protection from HIV-1 2:835–836 pathogenesis of 2:832 prevention of 2:834 taxonomy and classification 2:828 treatment of 2:833–834 virion structure 2:828 human influenza virus 5:125 matrix protein 2 5:127 neuraminidase 5:125–127 viral RNA polymerase 5:125 human leukocyte antigen (HLA) 2:639–640 human metapneumovirus (hMPV) classification 2:475 diagnosis 2:481 prevention 2:481–482 treatment 2:481 epidemiology and clinical features 2:477–479 genome 2:475–476 life cycle 2:476–477 entry of hMPV 2:476–477 viral particle assembly and spread 2:477 viral RNA synthesis 2:477 pathogenesis 2:479–480 cellular responses 2:479–480 pattern recognition receptors (PRRs) and antagonism of immune responses 2:480–481 virion structure and virus proteins 2:475 human microbiome bacteriophages of see bacteriophages: of human microbiome defined 1:552, 4:283 Human Microbiome Project (HMP) 1:553 human noroviruses (HuNoVs), 1:535, see also norovirus human papillomavirus (HPV) 1:588, 1:668, 1:179 functions of the HPV proteins 2:497t genome 2:493–494 host range and tissue tropism 2:494–496 individual viral genes, functions of 2:497–498

clinical features, pathogenesis and histopathology of HPV infection 2:499 diagnosis 2:500 E1 protein 2:497–498 E1ˆE4 protein 2:498 E2 protein 2:498 E5 protein 2:498 E6 protein 2:498 E7 protein 2:498–499 E8ˆE2 protein 2:498 HPV-associated cancers 2:500 immune response 2:500 L1 protein 2:499 L2 protein 2:499 natural history, transmission and epidemiology 2:499 oncogenic HPVs 2:499–500 prevention 2:501 treatment 2:500–501 life cycle 2:496–497 early stages of infection 2:497 entry 2:496–497 late stages of infection 2:497 maintenance phase of infection 2:497 phylogenetic tree of 2:494f taxonomy, classification, and evolutionary relationships 2:493 virion structure 2:493 human papillomaviruses (HPV) vaccines 5:295–296 current status and future directions 5:297 HPV and HPV-related diseases 5:295 immunogenicity 5:296 impact 5:296 on anogenital warts 5:297 on HPV infection and precancerous lesions 5:296 on HPV-related cancer 5:296–297 models on impact and cost-effectiveness of 5:298 vaccine safety and confidence 5:297–298 human parechovirus 1 (HPeV1) 1:282 human pathogenic arenaviruses see arenaviruses, human pathogenic human polyomaviruses see polyomaviruses (PyVs) human rhinovirus 1A (HRV1A) 1:286 human rhinoviruses (HRV) 1:395, 1:202 human sapovirus see sapovirus human T-cell leukemia virus-1 (HTLV-1) 2:316, 2:317, 1:352 human T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2) classification 2:528–529 clinical features 2:532–533 adult T-cell leukemia (ATL) 2:532–533 HAM/TSP 2:533 HTLV-2-associated symptoms 2:533 diagnosis and prevention 2:538 epidemiology 2:532 transmission 2:532 genome 2:529–531 immune response 2:537–538 life cycle 2:531 assembly, budding, and maturation 2:531–532

viral gene transcription 2:531 viral translation 2:531 pathogenesis 2:533–535 Hbz gene 2:535–536 in vivo animal models 2:537 Tax proteins 2:533–535 viral clonality 2:536–537 treatment 2:538 virion structure 2:529 viral entry 2:529 human virome, defined, 1:552, 2:48, see also virome, human humoral and T cell-mediated immunity to viruses adaptive immune response to viral infections 1:585–587 T cell recognition of viral antigens 1:587 antibodies in antiviral humoral immunity 1:591 antibody-dependent cellular cytotoxicity (ADCC) 1:591–592 neutralization 1:591 opsonization 1:591 dysregulation of immune response to viruses 1:595–596 humoral immune response to viral infections 1:590–591 B cells 1:590–591 innate immune system, role of 1:584 barrier immunity 1:584 crosstalk between innate and adaptive immune cells 1:585 innate immune effectors 1:584 innate immune recognition of viruses 1:584–585 mounting an effective T cell-mediated immune response to viral infections 1:589 cellular immune responses, contraction of 1:590 cytotoxicity 1:590 immune memory, formation of 1:590 T cell help 1:589–590 T cell priming 1:589 T cell effectors in the response to viral infections 1:587 CD4+ T Cells 1:587 CD8+ cytotoxic T cells (CTL) 1:589 cytotoxic CD4+ T cells 1:588–589 regulatory T cells (Tregs) 1:588 T follicular helper (Tfh) cells 1:587–588 T helper 1 (Th1) cells 1:587 vaccination, induction of immunity to viruses by 1:592–595 humoral immune response to viral infections 1:590–591 B cells 1:590–591 VLPs and the induction of 1:662–663 humoral immunity, defined 2:79 HuSRV1 see Hubei sclerotinia RNA virus 1 (HuSRV1) HVAC systems see heating, ventilation, and air conditioning (HVAC) systems HVD see hypervariable domain (HVD) HVT see herpes virus of turkeys (HVT) HVX see Hosta virus X (HVX)

Subject Index hyaline cells (HCs) 4:816 Hyalomma marginatum tick-adapted TBEV 1:550 hyaluronidase 2 (HYAL2) 2:576 hybridization probe, defined 3:192 hydranencephaly, defined 2:34 hydrodynamic model for phage genome ejection in vivo 4:216, 4:217f, 4:217 hydrophobic, defined 1:345 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) 2:391 hymenoptera, defined 4:867 hymenopteran viruses 4:771, 4:772 defined 4:768 hyperarid 4:342 hyperendemic, defined 2:899 hyperendemicity, defined 2:218 hyperhalophiles, defined 4:387 hyperimmune immunoglobulins 5:269–270 cytomegalovirus hyperimmune immunoglobulin 5:270 hepatitis B hyperimmune globulin 5:269–270 rabies hyperimmune immunoglobulin 5:270–271 vaccinia immune globulin (VIG) 5:271–272 varicella hyperimmune immunoglobulin 5:272 hyperparasite, defined 3:581 hyperplasia, defined 2:316, 3:200, 4:780 hyperplastic lesion, defined 2:306 hypersaline environments, phages in 4:343–345 Hypersensitive Reaction, defined 3:612 hypersensitive response, defined 3:60 hyperthermophiles, defined 4:387 hyperthermophilic, defined 4:359 hyperthermophilic archaeal viruses, diversity of 4:359–360 family Fuselloviridae 4:365 genomes 4:365 morphology and structure 4:360 order Ligamenvirales 4:365–366 evolutionary relationships 4:366 particular morphologies, viruses with 4:360–361 family Ampullaviridae 4:361 family Bicaudaviridae 4:361 family Clavaviridae 4:361–362 family Fuselloviridae 4:362 family Guttaviridae 4:362 family Spiraviridae 4:362 filamentous viruses 4:363–364 spherical viruses 4:362 hypertrophy, defined 3:200, 4:780 hypervariable domain (HVD) 2:177 hyphal anastomosis, defined 4:431, 4:568, 4:528, 4:468 hypocalcemia, defined 2:875 hypothetical taxonomy units (HTUs) 1:117 hypovirulence 4:431, 4:461, 4:589, 4:594, 4:577, 4:568, 4:615, 4:493, 4:552, 4:468

in Botrytis cinerea 4:476 in Cryphonectria parasitica 4:472–474 artificial application of hypovirulence 4:474–475 exclusive transmissible hypovirulence 4:473–474 in Fusarium graminearum 4:477 in Helminthosporium victoriae 4:476–477 in Ophiostoma novo-ulmi 4:475 in Rosellinia necatrix 4:476 in Sclerotinia sclerotiorum 4:475–476 hypoviruses (Hypoviridae) 4:589 anti-hypovirus defense mechanisms 4:592–593 hypovirus functional domains 4:591–592 hypovirus gene expression strategy 4:589–591 taxonomy and genetic organization 4:589 hypoxia-inducible factor (HIF) pathway 2:672 hytrosa, defined 4:780 Hytrosaviridae 4:706–707, 4:780 hytrosaviruses 4:707, 4:780–781 diagnosis of SGHV infections 4:789–790 genome organization 4:783 host range 4:786 major structural proteins 4:784–786 management of SGHV infections 4:790 modes of infection and gene expression 4:783–784 pathogenesis in other host tissues 4:787–788 pathogenesis in the salivary glands 4:787 similarities with other virus taxa 4:781–783 taxonomy and classification 4:781 transmission and epidemiology 4:788–789 GpSGHV transmission dynamics in the tsetse fly 4:788–789 MdSGHV transmission dynamics in the housefly 4:789 viral latency 4:788 virion structure 4:783 Hz-1 baculovirus 4:827–828 Hz-1 virus (Hz-1V) 4:827–828 HzNV-1 see heliothsi zea nudivirus 1 (HzNV-1)

I IAF see International Accreditation Forum (IAF) IAP see Intracisternal A Particle (IAP) IAPV see Israeli acute paralysis virus (IAPV) IAS see internal activation sequence (IAS) iatrogenic Kaposi’s sarcoma 2:606 iatrogenic transmission 1:562, 1:559 IAV see intracellular annular viruses (IAV) Ibalizumab 5:273 ibalizumab-uiyk 5:152 IBDV see infectious bursal disease virus (IBDV) IBs see inclusion bodies (IBs)

351

IBV see infectious bronchitis virus (IBV); intranuclear bacilliform viruses (IBVs) IC50 see inhibitory concentration 50 (IC50) ICAM-1 see intercellular adhesion molecule1 (ICAM-1) ICAM-5 see intercellular adhesion molecule 5 (ICAM-5) ICD see immunogenic cell death (ICD) ICER see incremental cost-effectiveness ratio (ICER) ICEs see integrative and conjugative elements (ICEs) ichnoviruses 4:712–713, 4:849 Ichtadenovirus 2:14 Ichthamaparvovirus 4:836 ICI see immune checkpoint inhibitors (ICI) iCIHHV–6 see inherited CIHHV–6 (iCIHHV–6) ICOS see inducible T cell co-stimulator (ICOS) icosahedra, triangulation numbers of 1:222–223 icosahedral asymmetric unit, defined 1:329, 1:278 icosahedral capsid, defined 2:797 icosahedral capsid architecture, mathematical models of 1:249–250 implications for viral evolution 1:253–254 non-quasi-equivalent trihexagonal lattice architectures 1:253 overarching framework for icosahedral architectures 1:251–253 practical applications 1:251 predictions and limitations of quasiequivalence theory 1:250 quasi-equivalence theory 1:249–250 viral tiling theory 1:250–251 virus structure in 3D – The Hamiltonian paths approach 1:254 icosahedral capsid assembly, structural principles in 1:480–481 icosahedral DNA viruses with an internal membrane 1:406–407 icosahedral enveloped dsRNA bacterial viruses 1:407–408 icosahedral enveloped viruses 1:468–469 alphavirus assembly 1:469, 1:471f alphavirus assembly and budding 1:470–472 alphavirus life cycle 1:469 alphavirus virion structure 1:469–470 flavivirus assembly 1:472, 1:473f flavivirus assembly and budding 1:473 flavivirus life cycle 1:472 flavivirus virion structure 1:472–473 icosahedral head/capsid 1:319 icosahedral internal membrane-containing viruses 4:374–376 icosahedral particle, defined 3:712 icosahedral phages DNA packaging and DNA binding protein 4:18–19 DNA replication 4:15 evolution and evolutionary studies 4:19 evolution of a two scaffolding protein system 4:19–20

352

Subject Index

icosahedral phages (continued) gene expression 4:15–17 history 4:10 host cell recognition, attachment, eclipse and penetration 4:12–15 lysis 4:19 morphogenesis 4:17–18 jX174-like viruses as a model system for experimental evolution 4:20 virion morphology and genome content 4:10–12 icosahedral ssDNA bacterial viruses 1:407 icosahedral ssRNA bacterial viruses 1:408, 4:21–24 background 4:21–24 phage lifecycle 4:21–24 icosahedral surface lattices, thermodynamic transition between 1:297–299 icosahedral symmetry 1:258–261, 1:255 defined 1:362, 1:248, 4:568 viral geometry and 1:249 icosahedral tailed dsDNA bacterial viruses 1:405–406 icosahedral tailed viruses 4:370–373 of halophilic euryarchaea 4:371–373 icosahedral tailed viruses of halophilic euryarchaea 4:371–373 tailed viruses of methanogenic euryarchaea 4:373–374 icosahedron 1:258–259, 2:540, 4:115, 3:818, 3:545 ICTV see International Committee On Taxonomy Of Viruses (ICTV) idaeoviruses (Idaeovirus) 3:430–431 diagnosis and detection 3:436 reverse transcriptase 3:436 serological testing 3:436 disease symptoms and effects 3:431–433 naturally infected plants 3:432–433 experimentally infected plants 3:433 genome 3:433–435 geographical distribution 3:431 natural and experimental transmission 3:431 prevention and control 3:436–437 relationships with other viruses 3:437–438 therapy 3:436 viral proteins 3:435 coat protein 3:435 movement protein 3:435 replicase protein 3:435–436 virion structure 3:433 identity, defined 4:276 Idnoreovirus 4:869 idnoreoviruses 4:868, 4:871, 4:875–876, 4:877 IDV see intracellular dense viruses (IDV) IEDB see Immune Epitope Database (IEDB) IEM see immuno-electron microscopy (IEM) IEV see intracellular enveloped virion (IEV) IF see immunofluorescence (IF) IFA see immunofluorescence assay (IFA) Iflaviridae 4:707, 4:792 iflaviruses (Picornavirales, Iflaviridae, Iflavirus)

classification 4:792 clinical signs 4:794–795 diagnosis/detection methods 4:795 epidemiology 4:794 genome 4:792 life cycle 4:792–794 pathogenesis 4:795 prevention 4:795 virion structure 4:792 IFN see interferon alpha (IFN) IFT see indirect Fourier transform (IFT) IG domains see immunoglobulin (IG) domains IgG avidity, defined 2:419 IGLP see immunoglobulin-like proteins (IGLP) IgSF receptors, virus interactions with 1:395–396 IHC see immunohistochemistry (IHC) IHNV see infectious hematopoietic necrosis viruses (IHNV) IHR see International Health Regulations (IHR) IHR see iterative helical reconstruction (IHR) Ikoma lyssavirus (IKOV) 2:738 IKOV see Ikoma lyssavirus (IKOV) ILAC see International Laboratory Accreditation Cooperation (ILAC) Ilarvirus 3:260, 3:261, 3:265, 3:262t characteristics of RNA genome in 3:261t ilarviruses (Bromoviridae) 3:439 classification 3:439–440 control 3:443–444 diagnosis 3:445–446 genome 3:440–442 infection cycle 3:442 replication 3:442–443 symptoms and pathogenesis 3:444–445 transmission 3:443 virion structure 3:440 illumina sequencing 1:177 ILTV see infectious laryngotracheitis virus (ILTV) ILVs see intralumenal vesicles (ILVs) IMC-HZ-I-NOV 4:827–828 immature intracellular virions 2:891–892 immediate early gene, defined 2:441 immortalization, defined 2:528 immune checkpoint blockade, defined 5:233 immune checkpoint inhibitors (ICI) 1:660, 1:658 Immune Epitope Database (IEDB) 1:141 immune escape 1:59–60 immune memory, formation of 1:590 immunity, defined 4:276, 2:441 immunity repressor 4:81, 4:276 immunization, defined 5:281 immunoassays 5:17–18 homogenous wash-free immunoassays 5:19 immunoblotting 5:18 immunofluorescence microscopy 5:18–19 lateral-flow immunoassays 5:19

solid-phase microwell immunoassays 5:17–18 immunoblot assays 5:94 immunoblotting 5:18 immunocapture-RT-PCR 3:629 immunocompetent, defined 2:441 immunocompromised, defined 2:441 immunocompromised hosts 5:180 immunocompromised patients, adenovirus infections of 5:201–202 immunodeficiencies 5:190, 2:441, 2:56 immunodeficiency-associated VDPV (iVDPV) 5:310, 5:313 immunodeficient vaccine-derived polioviruses (IVDPVs) 2:695 immuno-electron microscopy (IEM) 5:11 immunofluorescence (IF) 2:640–641 immunofluorescence assay (IFA) 5:18–19, 2:215, 5:94 immunofluorescence microscopy 5:18–19 immunogenic cell death (ICD) 1:659, 1:658 immunoglobulin (IG) domains 1:606 immunoglobulin 2:441, 1:533–534 immunoglobulin-like fold 1:263 immunoglobulin-like proteins (IGLP) 1:556–557 immunoglobulins in the prevention of viral infections 5:267–268 experimental immunoglobulin therapies 5:273–274 avian influenza 5:274 seasonal influenza 5:273–274 hyperimmune immunoglobulins 5:269–270 cytomegalovirus hyperimmune immunoglobulin 5:270 hepatitis B hyperimmune globulin 5:269–270 rabies hyperimmune immunoglobulin 5:270–271 vaccinia immune globulin (VIG) 5:271–272 varicella hyperimmune immunoglobulin 5:272 Middle Eastern respiratory syndrome coronavirus (MERS-CoV) 5:274–275 ebola and other hemorrhagic fevers 5:274–275 experimental infections 5:275 monoclonal antibodies 5:272–273 human immunodeficiency virus 5:273 respiratory syncytial virus (RSV) 5:272–273 polyclonal immunoglobulins 5:268 hepatitis A 5:268 measles 5:268–269 immunoglobulin superfamily proteins 1:394–396 IgSF receptors, virus interactions with 1:395–396 immunogold electron microscopy, defined 2:22 immunohistochemistry (IHC) 2:190, 2:518, 2:79 immunology, impact on 2:416–417 immunomodulation 2:784, 2:373

Subject Index immunomodulators 5:241 immunoprophylaxis 5:180 immunosenescence 2:454 immunosorbent electron microscopy, defined 3:539 immunosuppressed individuals 5:105 adenoviruses (AdVs) 5:109 Epstein-Barr virus (EBV) 5:107–108 hepatitis E virus 5:110 herpes simplex virus (HSV) 5:109 human cytomegalovirus (HCMV) 5:106–107 polyomaviruses (PyV) 5:108–109 Varicella-Zoster Virus (VZV) 5:109–110 viral diagnostic tests 5:105–106 immunosuppression, defined 2:48, 2:441 immunotherapy, defined 1:658 implications for viral evolution 1:253–254 IMV see intracellular mature virion (IMV) inactivated polio vaccine (IPV) 2:690, 2:695, 1:668, 5:310–311 inapparent, defined 2:441 inbred mice, defined 2:643 “inchworm” translocation model 4:153–154 incidence, defined 5:247, 2:362 incidence risk (or attack rate), defined 1:559 inclusion bodies (IBs) 2:738–739, 2:343 inclusion principle, defined 1:28 incremental cost-effectiveness ratio (ICER) 5:157 indel, defined 1:627 Indian subcontinent, CLCuD in 3:357–359 indirect Fourier transform (IFT) 1:192–193 inducible T cell co-stimulator (ICOS) 1:591 ICOS ligand (ICOSL) 1:591 induction, defined 4:88, 4:253, 2:441 infectious bronchitis virus (IBV) 2:193–194, 2:194f, 2:198 infectious bursal disease virus (IBDV) 1:492–493 classification 2:540 diagnosis and control 2:542 electron micrograph of 2:541f pathogenesis and clinical features 2:541–542 virion structure and genome 2:540–541 infectious clone, defined 2:193 infectious diseases, surveillance of analysis of data 5:252 person 5:252 place 5:252–253 time 5:252 collection of data 5:248 death certification 5:248 hospital admissions 5:249 laboratory data 5:248–249 notifications 5:248 other sources of data 5:248 other sources of data 5:249 serological surveillance 5:249 surveillance of outbreaks 5:249 surveillance of viruses in nonhuman and environmental sources 5:249

essential characteristics of surveillance 5:247–248 evaluation of surveillance 5:254 feedback 5:254 global and international surveillance 5:254 interpretation 5:253–254 surveillance system, newer types of 5:249–250 active surveillance 5:250 Enhanced Surveillance 5:250 sentinel surveillance 5:250 surveillance of HIV/AIDS 5:250–251 syndromic surveillance 5:250 surveillance systems, attributes of 5:251–252 accuracy 5:252 completeness 5:251–252 consistency 5:252 representativeness 5:252 timeliness 5:252 infectious hematopoietic necrosis viruses (IHNV) 2:324, 2:328 infectious hypodermal and hematopoietic necrosis virus (IHHNV) see penaeus stylirostris penstyldensovirus 1 (PstDV1) infectious laryngotracheitis virus (ILTV) 2:113 infectious pancreatic necrosis (IPN) virus 2:544, 2:546f classification 2:544 control 2:549–550 epidemiology 2:548 life cycle 2:547–548 molecular determinants of virulence 2:548–549 pathogenesis and clinical features 2:548 virion structure and genomic organization 2:544–547 infectious subviral particle (ISVP) 1:308–309 inflammasome, defined 3:60 influenza 1:668, 5:300 as a disease 5:300 epidemiology of 5:300 Influenza A 5:160 influenza A virus (IAV) 2:291, 2:288f, see also human influenza virus classification of 2:551 clinical features 2:558 diagnosis 2:559 evolution of 2:556 genome 2:552 genomic organization 2:552 reassortment and mutagenesis 2:552 IAV epidemics and pandemics 2:556–558 immunological responses and immune evasion 2:555–556 adaptive immune responses 2:556 immune evasion strategies of IAV 2:556 innate immune responses 2:555–556 life cycle 2:552–554, 2:289f attachment and entry 2:553–554 genome replication 2:555 new virions, budding of 2:555

353

transcription and translation 2:554–555 viral assembly 2:555 pathogenesis 2:558–559 structure of 2:552f treatment and prevention 2:559 antivirals 2:559 vaccines 2:559–560 virion structure 2:551–552 influenza A virus hemagglutinin 1:597–598 influenza B, C and D viruses classification (compact) 2:561 clinical features 2:569, 2:573 diagnosis 2:569, 2:573 epidemiology 2:567–569, 2:572–573 functions of HEF glycoprotein during life cycle 2:571–572 life cycle and functions of viral proteins 2:564–566 BM2 2:566–567 hemagglutinin (HA) 2:565–566 matrix protein (M1) 2:566 NB 2:566 NEP 2:567 neuraminidase (NA) 2:566 non-structural protein 1 (NS1) 2:567 pathogenesis 2:569, 2:573 prevention 2:570, 2:573 treatment 2:569–570, 2:573 influenza B virus 2:561–562 genome 2:562–564 virion structure 2:562 influenza C and D viruses 2:570–571 virion structure and genome 2:570–571 influenza HA 1:420 influenza polymerase 5:125 influenza research database (IRD) 1:143 influenza vaccines evaluating the impact of 5:305 immunogenicity 5:305 understanding vaccine efficacy and effectiveness 5:306 vaccine effectiveness 5:305–306 vaccine efficacy 5:305 history of 5:300 pandemic influenza vaccines 5:308 production of 5:300–301 dosage 5:302 influenza vaccine composition 5:301–302 production steps 5:301 timeline of the production process 5:300–301 valency 5:302 safety and contraindications 5:304 allergic reactions 5:304–305 local and systemic inflammatory reactions 5:304 rare adverse events: neurological syndromes 5:305 seasonal 5:302 adjuvanted vaccines 5:303 high-dose influenza vaccines 5:303 intradermal vaccines 5:303 live attenuated influenza vaccines 5:303 recombinant and cell-based influenza vaccines 5:302–303

354

Subject Index

influenza vaccines (continued) the future of influenza vaccines 5:303–304 traditional inactivated vaccines 5:302 specific population groups, vaccination of 5:306–307 elderly 5:307 healthy adults 5:308 healthy children 5:307–308 pregnancy 5:307 underlying conditions 5:307 vaccination in practice 5:304 administration routes 5:304 duration of protection 5:304 timing of programs 5:304 vaccine uptake 5:304 influenza viruses 1:266, 1:391–392 influenza virus infections, antiviral management of 5:160 adamantanes/M2 blockers 5:161–162 complementary treatments for disease management 5:171–172 ECMO (extracorporeal membrane oxygenation) 5:171–172 drugs under development with other mechanisms of action 5:168 HA attachment blockers 5:168 HA processing blockers 5:168 neuraminidase inhibitors 5:162–164, 5:163f pandemic influenza 5:160 perspectives at short and medium 5:168–170 combined Baloxavir + Oseltamivir 5:171 combined Oseltamivir plus Favipiravir 5:171 combined Oseltamivir plus monoclonal antibodies 5:171 host target molecules 5:169–170 macrolides 5:170 neuraminidase inhibitors (NAI) combinations 5:171 Pimodivir and Oseltamivir 5:171 Siromilus 5:170–171 triple therapy (Amantadine, Ribavirine, and Oseltamivir) 5:171 polymerase (POL) inhibitors 5:164–166 Baloxavir – Marboxyl (Xofluzas) 5:166–168 Favipiravir (Avigans) 5:168 Pimodivir 5:168 Ribavirine (Ribavirins) 5:164–166 seasonal influenza 5:160 zoonotic influenza 5:160–161 inherited CIHHV–6 (iCIHHV–6) 2:781 inhibitory concentration 50 (IC50) 2:561 INIBs see intranuclear inclusion bodies (INIBs) injection 4:48 DNA Injection 4:48 protein injection 4:48 innate immune system, role of 1:584 barrier immunity 1:584 crosstalk between innate and adaptive immune cells 1:585 innate immune effectors 1:584

innate immune recognition of viruses 1:584–585 innate immunity 4:524, 4:520, 2:404, 1:577, 2:428 INSDC see International Nucleotide Sequence Database Collaboration (INSDC) insect cells 1:664 insects, plant viruses transmission by 3:106–107 beetles, transmission by 3:112–113 circulative transmission 3:107–108 circulative non-propagative transmission 3:107–108 circulative propagative transmission 3:110 history of typology of transmission modes 3:107 non-circulative transmission 3:110–112 capsid strategy 3:112 helper strategy 3:112 insects, rhabdoviruses of classification 4:883 clinical features 4:886 diagnosis 4:887 epidemiology 4:886 distribution, spatial and temporal 4:886 population dynamics 4:886 prevalence 4:886 transmission 4:886 genome 4:884–885 almendraviruses genome 4:885 sigmavirus genome 4:885 life cycle 4:885–886 integration into the host genome 4:885–886 pathogenesis 4:886–887 virion structure 4:883–884 insects, tetraviruses of see tetraviruses of insects insect-specific virus, defined 4:764 insertional mutagenesis, defined 2:122 in silico, defined 3:388 in situ hybridization (ISH) 4:808, 2:190, 3:629, 2:79 in-situ structural virology 1:238–240 cellular electron cryotomography 1:238–240 installation qualification (IQ) 5:69 INSTI see integrase strand transfer inhibitors (INSTI) integrase, defined 4:387, 4:276, 4:380, 4:368 Integrase function (Int), defined 3:96 integrase inhibitors 2:473 integrase strand transfer inhibitors (INSTI) 5:145–146 first generation INSTI 5:146 elvitegravir 5:146–149 raltegravir 5:146 second generation INSTI 5:149–150 bictegravir 5:150–151 cabotegravir 5:151 dolutegravir 5:149–150 virology 5:145–146 integration, defined 2:643

integrative and conjugative elements (ICEs) 1:613 intein 4:677 interactomics, defined 3:586 intercellular adhesion molecule-1 (ICAM-1) 1:659, 1:266 intercellular adhesion molecule 5 (ICAM-5) 1:410 interferon alpha (IFN) 5:223, 5:220 interferons 2:555–556 defined 1:577 interferon-stimulated genes (ISGs) 2:70 interferon-stimulated response element (ISRE) 1:577 intergenic region, defined 3:507, 4:768 internal activation sequence (IAS) 4:78 internal quality assessment (IQA) 5:74 internal quality control procedures and monitoring assay performance over time 5:74–75 internal ribosome entry site (IRES) 2:688, 3:692, 2:757–758, 2:757, 2:387, 2:335, 1:440, 2:386, 4:808, 4:699, 4:792, 1:447–449, 4:770–771, 4:771, 2:675 IRES-like RNA structure 2:399 IRES trans-activating factors (ITAFs) 2:337 International Accreditation Forum (IAF) 5:70 International Committee On Taxonomy Of Viruses (ICTV) 4:433, 1:47, 1:122, 1:32, 4:699 organization 1:32 taxonomic process 1:32–33 International Health Regulations (IHR) 2:895 International Laboratory Accreditation Cooperation (ILAC) 5:55, 5:70 International Nucleotide Sequence Database Collaboration (INSDC) 5:33 International Organization for Standardization (ISO) 5:55 International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) 5:260 International Standard, defined 5:52 international surveillance 5:254 International Unit, defined 5:52 interspecies transmission, factors in 1:569–570 epidemiological/ecological barriers 1:570 host–pathogen interactions 1:570 viral factors 1:570–571 ability to adapt 1:571–572 receptor specificity 1:570–571 virulence 1:572 intra-capsid DNA equilibrium energy and structure of 4:167–169 metastability of 4:169–170 intracellular annular viruses (IAV) 4:767 intracellular budding 1:523–524 intracellular dense viruses (IDV) 4:767 intracellular enveloped virion (IEV) 2:165 intracellular mature virion (IMV) 2:165 Intracisternal A Particle (IAP) 1:82

Subject Index intralumenal vesicles (ILVs) 1:538, 1:529 intranuclear bacilliform viruses (IBVs) 4:827 intranuclear inclusion bodies (INIBs) 4:840 intravenous immunoglobulin (IVIG) 5:156 intravenous pathogenicity index (IVPI) 2:118–119, 2:117 intrinsic resistance, defined 2:404 introgression, defined 3:768 intussusception, defined 5:289, 2:789 invertebrate rhabdoviruses 4:716 invertebrates, iridoviruses of 4:797 classification of iridovirids 4:797–798 genome organization and codon usage 4:800 host range and pathology 4:799–800 IIV-6 persistence and sensitivity to external factors 4:799 induction/inhibition of apoptosis in infections 4:802 morphology and composition 4:798–799 promoter elements and transcriptional regulation 4:802 transcriptional regulation 4:801–802 viral entry, replication, and release strategy 4:800–801 virion proteins 4:800 invertebrates, parvoviruses of see parvoviruses: of invertebrates invertebrates, viruses of 4:700t–702 arboviruses in their invertebrate vectors 4:722, 4:720t Artoviridae 4:703 Ascoviridae 4:703 Baculoviridae 4:703–705 Bidnaviridae 4:705–706 birnaviruses of invertebrates 4:706 Dicistroviridae 4:706 Hytrosaviridae 4:706–707 Iflaviridae 4:707 iridoviruses of invertebrates 4:707–708 Malacoherpesviridae 4:708 Mesoniviridae 4:708–709 Nimaviridae 4:709 Nodaviridae 4:709–710 Nudiviridae 4:710 Nyamiviridae 4:710–711 parvoviruses of insects 4:711–713 Polydnaviridae 4:712–713 plant viruses in their invertebrate vectors 4:722, 4:721t poxviruses of invertebrates 4:713 reoviruses of invertebrates 4:713–715 retrotransposons associated with invertebrates 4:722 rhabdoviruses of invertebrates 4:715–716 Roniviridae 4:716 Sarthroviridae 4:716 Solinviviridae 4:716–717 taxa of other viruses of invertebrates 4:717–722 tetraviruses 4:717 inverted terminal repeats (ITRs) 4:365, 4:43, 2:669, 4:387, 4:359 in vivo vs. in vitro, defined 1:71 IPN virus see infectious pancreatic necrosis (IPN) virus IPV see inactivated polio vaccine (IPV)

IQ see installation qualification (IQ) IQA see internal quality assessment (IQA) IRES see internal ribosome entry site (IRES) iridovirid, defined 4:797 iridoviruses of invertebrates (Betairidovirinae) 4:707–708, 4:797 classification of iridovirids 4:797–798 genome organization and codon usage 4:800 host range and pathology 4:799–800 IIV-6 persistence and sensitivity to external factors 4:799 induction/inhibition of apoptosis in infections 4:802 Malacoherpesviridae 4:708 Mesoniviridae 4:708–709 morphology and composition 4:798–799 Nimaviridae 4:709 Nodaviridae 4:709–710 Nudiviridae 4:710 Nyamiviridae 4:710–711 promoter elements and transcriptional regulation 4:802 transcriptional regulation 4:801–802 viral entry, replication, and release strategy 4:800–801 virion proteins 4:800 Irkut lyssavirus (IRKV) 2:738 IRKV see Irkut lyssavirus (IRKV) Irodoviridae 4:708 ISARIC see International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) Isfahan virus (ISFV) 2:875 ISFV see Isfahan virus (ISFV) ISGs see interferon-stimulated genes (ISGs) ISH, see in situ hybridization (ISH) ISO see International Organization for Standardization (ISO) isogenic line, defined 3:388 isolate, defined 3:8 isothermal amplification 5:22 i-spanin, defined 1:501 Israeli acute paralysis virus (IAPV) 4:768–770, 4:771, 4:772, 4:773 ISRE see interferon-stimulated response element (ISRE) ISVP see infectious subviral particle (ISVP) iterative helical reconstruction (IHR) 1:364–365 Iterons 3:239 ITRs see inverted terminal repeats (ITRs) iVDPV see immunodeficiency-associated VDPV (iVDPV) IVDPVs see immunodeficient vaccinederived polioviruses (IVDPVs) IVIG see intravenous immunoglobulin (IVIG) IVPI see intravenous pathogenicity index (IVPI)

J Jaagsiekte sheep retrovirus (JSRV) 2:322 classification 2:575 enJSRVs 2:578–579

355

enzootic nasal tumor virus (ENTVs) 2:581–582 future perspectives 2:582 genetic organization and virion proteins 2:575–576 history 2:575 ovine pulmonary adenocarcinoma (OPA) 2:579–580 replication cycle 2:576–578 virus-induced cell transformation, mechanisms of 2:580–581 Jacalin-type lectin gene (JAX1) 3:623, 3:626–628, 3:629 JAK inhibitors, defined 2:825 JAM-1 see junctional adhesion molecule 1 (JAM-1) JAM-A see junctional adhesion molecule-A (JAM-A) Japanese encephalitis virus (JEV) 2:810–811 classification 2:583 clinical features 2:594 diagnosis 2:595 clinical diagnosis 2:595 laboratory diagnosis 2:595 epidemiology 2:591–593 genetic diversity 2:593 geographic distribution and epidemiologic patterns 2:592–593 gene expression 2:586–587 nonstructural proteins 2:587–589 structural proteins 2:586–587 genome structure 2:585–586 life cycle 2:589–590 viral entry 2:589–590 viral morphogenesis 2:591 viral translation and RNA replication 2:590–591 pathogenesis 2:594–595 prevention 2:595–596 transmission 2:593 non-vector-borne transmission 2:593–594 vector-borne transmission 2:593 treatment 2:595 virion structure 2:583 extracellular mature virion 2:583 intracellular immature virion 2:583–584 JAX1 see Jacalin-type lectin gene (JAX1) JCI see Joint Commission International (JCI) JC polyomavirus (JCPyV) 2:523–524, 5:109 clinical features 2:523–524 infectious cycle 2:524 JCPyV see JC polyomavirus (JCPyV) JD see Jembrana disease (JD) Jdvgp5 gene 2:61t jelly-roll, defined 1:87, 4:457 jelly-roll fold, defined 4:387, 4:359 jelly-roll b-barrel 1:262–263 Jembrana disease (JD) 2:66 JEV see Japanese encephalitis virus (JEV) JH see juvenile hormone (JH) JNK, defined 2:875 Joint Commission International (JCI) 5:3 JSRV see Jaagsiekte sheep retrovirus (JSRV)

356

Subject Index

jumbo phages 4:229 advanced capabilities 4:237–238 DNA repair enzymes 4:238 LPS biosynthesis 4:239 NAD+ salvage pathway 4:238–239 phage nucleus 4:239–240 RNA polymerases 4:238 evolution 4:240 genome features 4:236 genome nucleotide composition 4:236–237 nucleotide modifications and substitutions 4:237 ORFan genes 4:237 terminal redundancies 4:236 transfer RNA genes 4:237 history 4:229–230 isolation and characterization 4:230–234 virion structure 4:234 head and tail fibers 4:235–236 icosahedral geometries 4:234 morphotypes 4:234 virion DNA density 4:234–235 junctional adhesion molecule 1 (JAM-1) 1:410 junctional adhesion molecule-A (JAM-A) 1:308–309 jungle yellow fever 2:894 JUNV see Junı´n virus (JUNV) Junı´n virus (JUNV) 2:507, 2:512 Jurona virus (JURV) 2:875 JURV see Jurona virus (JURV) juvenile hormone (JH) 4:862, 4:858

K Kalanchoe top spotting virus (KTSV) 3:165 Kallithea virus (KV) 4:829 Kaposi’s sarcoma (KS) 2:604, 2:605, 2:606, 2:599 classical Kaposi’s sarcoma (cKS) 2:606 HIV associated 2:606 post-transplantation/iatrogenic 2:606 Kaposi’s sarcoma-associated herpesvirus (KSHV) classification 2:599 clinical features 2:604 Kaposi’s sarcoma (KS) 2:604 multicentric Castleman’s disease (MCD) 2:604–605 primary effusion lymphoma 2:604 diagnosis 2:606 Kaposi’s sarcoma 2:606 multicentric Castleman’s disease (MCD) 2:606 primary effusion lymphoma 2:606 epidemiology 2:604 genome 2:599 life cycle 2:599–601 binding and entry of the virus to the host cell 2:600–601 genome segregation 2:603 host cytoplasm, transport of virus in 2:601–602

interaction of KSHV with the host immune system 2:603–604 latent KSHV episome, replication of 2:602–603 lytic activation 2:603 nucleus, entry of viral DNA into 2:601–602 pathogenesis 2:605 Kaposi’s sarcoma 2:605 KSHV Inflammatory syndrome (KICS) 2:605–606 multicentric Castleman’s disease (MCD) 2:605 primary effusion lymphoma 2:605 prevention 2:607 treatment 2:606 Kaposi’s sarcoma 2:606 multicentric Castleman’s disease (MCD) 2:606–607 primary effusion lymphoma 2:606 vaccination 2:607 virion structure 2:599 Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8), defined 2:599 Kaposi’s sarcoma-associated virus (KSV) 1:321 Karelian fever 2:840 Kashmir bee virus (KBV) 4:771, 4:772, 4:773 KBLV see Kotalahti bat lyssavirus (KBLV) KBV see Kashmir bee virus (KBV) keratitis, defined 2:404 Khujand lyssavirus (KHUV) 2:738 KHUV see Khujand lyssavirus (KHUV) KICS see KSHV Inflammatory syndrome (KICS) killer immunoglobulin receptors (KIRs) 1:584 killer phenomena 4:513 killer proteins, comparison of 4:517–518 killer virus-infected yeast, self-protection in 4:542 killer virus system, defined 4:534 “Killing-the-Winner” model 1:638 Kimberley virus (KIMV) 2:875 KIMV see Kimberley virus (KIMV) kinetics of virus entry 4:390 KIRs see killer immunoglobulin receptors (KIRs) Kitaviridae 3:247 knockdown, defined 3:123 knockout, defined 3:123 kobuviruses 1:279–280 Koch’s postulates, defined 3:355 Koinobiont parasitoid, defined 4:849 Koolpinyah virus (KOOLV) 2:875 KOOLV see Koolpinyah virus (KOOLV) Kotalahti bat lyssavirus (KBLV) 2:738 Kotonkan virus (KOTV) 2:875 KOTV see Kotonkan virus (KOTV) KP1 4:516–517 KP4 blocks L-type voltage gated Ca2+ channels 4:515 KP4 protein 4:513–514 atomic structure of 4:514f effect of KP4 on plants 4:515

evolutionary origin of 4:516 possible application of 4:515–516 on U. maydis cells 4:514–515 KP6 4:516 atomic structure of 4:516, 4:517f KREMEN-1 see Kringle-containing transmembrane protein-1 (KREMEN-1) Kringle-containing transmembrane protein1 (KREMEN-1) 1:284 KS see Kaposi’s sarcoma (KS) KSHV see Kaposi’s sarcoma-associated herpesvirus (KSHV) KSHV Inflammatory syndrome (KICS) 2:605–606 KSV see Kaposi’s sarcoma-associated virus (KSV) KTSV see Kalanchoe top spotting virus (KTSV) Kunjin virus 1:292–293 Kupffer cells 2:895–896 KV see Kallithea virus (KV) kyphosis, defined 2:34

L L1 protein 2:499 L2 protein 2:499 labeling, virus 1:209–210 laboratory biosafety 5:82–83 biological agents 5:82–83 biosafety cabinets 5:84–85 decontamination and waste management 5:85–86 laboratory accidents 5:86–87 laboratory biosafety levels 5:83 occupational health and special groups 5:87 personal protective equipment (PPE) 5:84 risk assessment 5:83 routes of transmission and infective dose 5:83–84 shipment of clinical specimens 5:87–88 laboratory biosecurity 5:82, 5:88–89 laboratory data 5:248–249 laboratory developed tests (LDTs) 5:72, 5:60–61 Laboratory Information Management System (LIMS) 5:65, 5:68–69 laboratory specimen retention times 5:67 LACA see last archaeal common ancestor (LACA) lactate dehydrogenase-elevating virus (LDV) 2:245 Lactobacillus casei 4:84 La France Disease 4:528–529 biological properties (virus host relationships) 4:529–530 control 4:530–532 diagnostics and identification 4:530 epidemiology 4:530 genome organization 4:529 taxonomy and classification 4:529 virion structure and composition 4:529

Subject Index Lagenaria mild mosaic virus (LaMMoV) 3:623, 3:623–624 Lagos bat lyssavirus (LBV) 2:738 LAIV see live attenuated influenza vaccines (LAIV) lake trout rhabdovirus (LTRV) 2:325 LAM see lamivudine (LAM) lambda CI protein 4:81 l genome ejection 4:215 lambda lysis, looking under the hood of 1:505–507 lambda lysis cassette 1:505 lambda lysogeny, maintenance of 4:95f lambda MGL, operational outline of 1:505 lambda prophage as a lysis platform 1:504–505 l Red system, defined 4:291 lambda system 4:126 Escherichia Coli integration host factor 4:128 TerL DNA packaging domain 4:127 terminase enzyme of phage lambda 4:126 l TerL maturation domain 4:127–128 l TerS subunit 4:126–127 lambda “switch” 4:94 l TerL maturation domain 4:127–128 l TerS subunit 4:126–127 laminin-binding protein (LBP) 2:843 lamivudine (LAM) 5:218 LaMMoV see Lagenaria mild mosaic virus (LaMMoV) LAMP see loop-mediated isothermal amplification (LAMP) LANA see latency associated nuclear protein (LANA) Laodelphax striatellus 4:771 large icosahedral dsDNA viruses 1:269–271 Lassa fever (LF) 2:507, 2:511, 2:513, 2:514 Lassa virus (LASV) 2:507, 2:516 last archaeal common ancestor (LACA) 1:17 last bacterial common ancestor (LBCA) 1:17 last eukaryotic common ancestor (LECA) 1:19 LASV see Lassa virus (LASV) late gene, defined 2:441 latency, defined 2:599, 2:306, 1:71, 2:528, 2:441 latency associated nuclear protein (LANA) 2:602 latency/latent infection, defined 5:197 latent infection, defined 4:827, 2:404 latent period, defined 4:314 latent reservoir, defined 2:460 lateral bodies, defined 4:858 lateral flow assays (LFA) 5:94 lateral flow detection, defined 3:839 lateral flow immunoassay (LFIA) 3:629, 5:19 lateral gene transfer 4:6 latex agglutination 5:19–20 LAV see live-attenuated vaccines (LAV) laves lattice 1:248, 1:253 LBA see long branch attraction (LBA)

LBCA see last bacterial common ancestor (LBCA) LBM see live bird markets (LBM) LBP see laminin-binding protein (LBP) LBV see Lagos bat lyssavirus (LBV) LCDV see lymphocystis disease virus (LCDV) LCGs see lysogenic conversion genes (LCGs) LCM see lymphocytic choriomeningitis (LCM) LCMV see lymphocytic choriomeningitis virus (LCMV) LDL see low-density lipoproteins (LDL) LDLR see low-density lipoprotein receptor (LDLR) LDTs see laboratory developed tests (LDTs) LDV see lactate dehydrogenase-elevating virus (LDV) leader and trailer, defined 3:567 leafhopper-transmitted mastreviruses, case of 3:108 leafhopper vectors 3:467–468 leaky late (LL) viral gene 2:452 leaky scanning, defined 3:528, 3:285, 3:839, 3:594 leaky scanning of ribosomes, defined 3:712 LECA see last eukaryotic common ancestor (LECA) lectin, defined 3:486 legume viral diseases 3:92–94 Leidenfrost Effect 5:5 leiomyosarcoma, defined 2:316 Lentinula edodes virus (LeV) dsRNA 4:627 lentiviruses 2:56–57, 2:63–64, 2:64, 2:65, 2:56 gene products of 2:61t lepidoptera, defined 4:867 lepidopteran, defined 4:768 Leporid herpesvirus 4 2:734 letermovir 2:458–459 lethal mutagenesis, defined 1:53 leucine-rich repeats (LRR) 1:606, 4:522 leucopenia, defined 2:875 leukemia, defined 2:528 leukocytopenia, defined 2:362 leukodepletion, defined 2:441 LeV dsRNA see Lentinula edodes virus (LeV) dsRNA “lever” translocation model 4:154–155 Levinthal’s Paradox in protein folding, defined 1:248 Leviviridae, single gene lysis (SGL) of 1:515–516 LF see Lassa fever (LF)) LFA see lateral flow assays (LFA) LFA-1 see lymphocyte function-associated antigen-1 (LFA-1) LFIA see lateral flow immunoassay (LFIA) LHF see Lujo hemorrhagic fever (LHF) life history, defined 1:62 Ligamenvirales 4:365–366 evolutionary relationships 4:366 light microscopy (LM) 1:495–496 limbic encephalitis, defined 2:778 limit of detection (LoD) 5:72

357

LIMS see Laboratory Information Management System (LIMS) LIN see lysis inhibition (LIN) linker peptides 1:356 lipidome (virus), defined 1:388 lipid raft, defined 2:475, 2:56 lipid transfer proteins (LTPs) 1:499 lipobox, defined 1:501 lipopolysaccharide (LPS) 1:556–557, 4:194, 4:175 lipopolysaccharide, defined 1:402 Lipothrixviridae 1:368, 1:367–369 Lispiviridae 4:718 live, attenuated vaccine, defined 2:797 live attenuated influenza vaccines (LAIV) 5:301 live-attenuated vaccines (LAV) 2:516, 2:20 live bird markets (LBM) 2:118 liver dysfunction 2:896 liver necrosis 2:895–896 living status of viruses 1:26–27 Ljungan virus (LV) 1:279–280, 1:282 LLEBV see Lleida bat lyssavirus (LLEBV) Lleida bat lyssavirus (LLEBV) 2:738 LLoQ see Lower Limit of Quantitation (LLoQ) LL viral gene see leaky late (LL) viral gene LM see light microscopy (LM) lobule, defined 2:629 LoD see limit of detection (LoD) long branch attraction (LBA) 1:121 long contractile tails, baseplates of 4:190–191 long-distance movement, defined 3:140 long non-contractile tails, tail tip complex of 4:191 long read sequencing (LRS) 1:182 “long-read sequencing” parallel technologies 5:31 long terminal repeats (LTR) 2:58–59, 3:96, 2:122 different genera of LTR retrotransposons 3:100–101 life cycle of LTRs retrotransposons 3:99–100 long-term non progressors, defined 2:827 loop-mediated isothermal amplification (LAMP) 5:98, 3:629, 3:759 lopinavir 5:141 lordosis, defined 2:34 low-density lipoprotein receptor (LDLR) 1:395, 2:758–759, 2:390 low-density lipoproteins (LDL) 2:386–387 Lower Limit of Quantitation (LLoQ) 5:73–74 lower respiratory tract infections (LRTI) 5:199, 5:200 low pathogenic avian influenza virus (LPAI) 2:117 LPAI see low pathogenic avian influenza virus (LPAI) L polymerase protein 2:752–753 L protein defined 2:747, 3:833, 3:567 encoded on RNA1 3:834–835 of herbeviruses 4:766

358

Subject Index

L protein (continued) LPS see lipopolysaccharide (LPS) LRR see leucine-rich repeats (LRR) LRS see long read sequencing (LRS) LRTI see lower respiratory tract infections (LRTI) LSDV see Lumpy skin disease virus (LSDV) L segment 2:209–210 LTPs see lipid transfer proteins (LTPs) LTR see long terminal repeats (LTR) LTR-retroelement, defined 3:653 LTRV see lake trout rhabdovirus (LTRV) Lujo hemorrhagic fever (LHF) 2:511, 2:513 Luminex GPP assay 5:102 Lumpy skin disease virus (LSDV) 1:547, 2:165 lumpy skin disease virus 2:168 clinical features 2:168 diagnosis 2:169 epidemiology 2:168 pathogenesis 2:168–169 pathology 2:168 prevention 2:169 treatment 2:169 Luteoviridae 3:594 luteoviruses (Luteoviridae) 3:447 control 3:454–455 diagnosis 3:454 epidemiology 3:454 evolutionary relationships 3:452 genome organization and expression 3:449–452 host range and transmission 3:452–453 replication 3:453 taxonomy and classification 3:447–448 virion properties 3:448–449 virion structure and composition 3:449 virus–host relationships 3:453–454 LV see Ljungan virus (LV) LVX 3:628 lymphocystis disease virus (LCDV) 1:104 lymphocyte function-associated antigen-1 (LFA-1) 1:601 lymphocytic choriomeningitis (LCM) 2:511–512, 2:513–514, 2:514–515 lymphocytic choriomeningitis virus (LCMV) 2:507, 2:512–513, 2:514, 1:445, 1:604 lymphoid progenitor cells 1:587 lymphoma, defined 2:528 lymphopenia, defined 2:814, 2:825 lyophilization 5:5 Lyphogel method 5:11 lysis 4:19, 4:50 defined 1:621 lysis inhibition (LIN) 1:510 lysis-lysogeny decision 1:638 lysogen, defined 4:242, 4:77, 4:368 lysogenic, defined 4:276 lysogenic conversion, defined 4:77 lysogenic conversion genes (LCGs) 1:636 lysogenic cycle, defined 4:252 lysogenic life cycle, defined 4:387 lysogenic or temperate phage, defined 4:283 lysogenic pathway, defined 4:88 lysogenic state, maintenance of 4:79–81

bacteriophage 186 4:81–83 bacteriophage l 4:80–81 host protein as immunity repressor 4:83–84 integration-dependent bacteriophage immunity 4:83 prophage induction 4:84–85 RNA to maintain lysogeny 4:84 lysogeny 4:77 defined 1:621, 1:162, 4:276, 1:71 DNA, persistence of 4:77–78 extrachromosomal 4:78–79 integration into the chromosome 4:77–78 evolutionary and phenotypical effects of 4:85 gene disruption 4:85 genomic rearrangement 4:85–87 lysogenic conversion 4:85 lyssaviruses 5:260 classification (compact) 2:738 clinical features 2:740–741 diagnosis 2:742–743 epidemiology 2:739–740 genome 2:738 life cycle 2:738–739 pathogenesis 2:741–742 prevention 2:744–745 treatment 2:743–744 virion structure 2:738 Lytic (virulent) phage, defined 4:98 lytic, defined 4:276 lytic cycle, defined 4:252 lytic life cycle, defined 1:162, 4:387 lytic pathway, defined 4:88 lytic phage, defined 4:69, 4:283 lytic transcription 4:69 developmental pathways and transcription control 4:70–72 by N4 4:74–75 by T4 4:70–72 by T7 4:72, 4:73f host RNA polymerase, transcription initiation and elongation by 4:69–70 inhibition of host RNAP by T4, T7, and N4 4:75

M M2–1 protein, defined 2:747 M2–2 protein, defined 2:747 MA see matrix protein (MA) machlomovirus classification 3:456 diagnosis 3:459 epidemiology 3:458–459 genomes 3:456–457 hosts and distribution 3:457–458 life cycle 3:458 pathogenesis 3:459 prevention 3:459 virion structure 3:456 machrobrachium rosenbergii nodavirus (MrNV) 4:820, 4:716

Machupo virus (MACV) 2:507, 2:512 Macrobrachium nipponense reovirus 4:879 macrodomain 2:177 macrolides 5:170 Macronovirus 4:889 macrophage, defined 2:56 macropinocytosis 1:533 defined 1:529 defined 2:22 macroptilium yellow spot virus (MaYSV) 3:194, 3:561 maculaviruses 3:824 properties and distinguishing characteristics 3:824 MACV see Machupo virus (MACV) Madariaga virus (MADV) 2:40 Madin-Darby canine kidney (MDCK) cells 1:531 MADV see Madariaga virus (MADV) maize chlorotic mottle virus (MCMV) 3:792–793 maize streak disease (MSD) 3:565–566 control of 3:467 epidemiology 3:467 maize streak virus (MSV) 3:461, 3:468, 1:649 complementary sense genes (Rep and RepA) 3:465 diversity and evolution 3:462–463 future threat 3:468 genome organization 3:463 history and taxonomy 3:461 host range and symptoms 3:461–462 leafhopper vectors 3:467–468 long intergenic region 3:463–465 molecular biology of 3:465–466 replication 3:465–466 particle assembly and movement 3:466–467 particle structure 3:463 plant hosts 3:468 resistance to 3:565–566, 3:565f short intergenic region 3:465 transmission 3:463 virion sense genes (MP and CP) 3:465 major capsid protein (MCP) 1:374, 4:266–267, 2:442–443, 4:115, 1:153, 1:252–253, 1:319, 1:322–323, 1:325 major coat protein (MCP) 1:337 major histocompatibility complex (MHC) 2:455, 1:584, 5:190 defined 2:441, 2:778 MHC-I antigen presentation 1:603 MHC-II antigen presentation 1:603–604 major immediate early promoter (MIEP) 2:443–445, 2:452 major Interspersed Genomic Element (MIGE) 4:677 major tail protein (MTP) 1:320 Malacoherpesviridae 4:708 MALDI MS see matrix-assisted laser desorption–ionization (MALDI) MS MALDI-TOF mass spectrometry see matrixassisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry

Subject Index Malpais Spring virus (MSP) 2:875 malpighian cells, defined 2:316 malpighian tubules, defined 4:858 mammalian cells 1:664 mammarenavirus genome organization and proteins 2:507–509 mammarenavirus life cycle 2:509–510, 2:509f assembly and budding 2:511 cell attachment and entry 2:509–510 expression and replication of the viral genome 2:510–511 Mammarenavirus L proteins 2:508 mammarenavirus RNA replication and gene transcription 2:510f mammarenavirus virion structure 2:507 Mammarenavirus Z proteins 2:508–509 MAPK see mitogen activated protein kinases (MAPK); mitogen-activated phosphate kinases (MAPKs) mapping of epitopes, defined 3:727 marafiviruses 3:822–823 infection and transmission 3:824 properties and distinguishing characteristics 3:823 virion structure, genome organization, replication cycle, and phylogenetic relationships 3:823–824 maraviroc 5:151–152 Marburg and Ravn viruses (MARV) 2:611f, 2:611t, 2:875 classification 2:609 clinical features 2:613 diagnosis 2:615 epidemiology 2:608–609 genome 2:610–611 glycoprotein (GP) 2:612 life cycle 2:613 MARV L 2:613 nucleoprotein (NP) 2:611–612 pathogenesis 2:613–615 prevention 2:616–618 treatment 2:615–616 viral protein 24 (VP24) 2:612–613 viral protein 30 (VP30) 2:612 viral protein 35 (VP35) 2:612 viral protein 40 (VP40) 2:612 virion structure 2:609–610 virulence factors for 2:614f Marburg hemorrhagic fever (MHF) 2:232 Marburg virus disease (MVD) 2:232 Marek’s disease (MD) 2:113 Marek’s disease virus (MDV) 2:652–653, 2:113 maribavir 2:458–459 marine microbial ecology, RNA viruses in 4:674–675 marine phage ecology 4:337–338 auxiliary metabolic genes in marine phages 4:340 diel rhythms of phage infections in the marine environment 4:339–340 marine phages as factors driving bacterial mortality and diversity 4:338–339 phage micro- and macro-diversity in the marine environment 4:337–338

transfer RNAs (tRNAs) 4:340–341 marine phages 4:322 genomic diversity of 4:324–325 linking phage genomes with host identity 4:329–330 long read viromics 4:326–328 metagenome assembled viral genomes (MAVGs) 4:328–329 phage marker genes 4:324–325 single virus genomics 4:325–326 ssDNA phages in the marine environment 4:330 viral contigs from fosmid libraries 4:325 viral metagenomics (viromics) 4:325 morphological diversity of 4:322–324 marine protists, RNA viruses infecting 4:671 marine Pseudoalteromonas spp. phage PM2 (Corticoviridae) 4:208 marine RNA virus quasispecies 4:674 marker-assisted selection (MAS) 3:557 Marnaviridae 1:279 marine RNA virus quasispecies 4:674 RNA viruses infecting marine protists 4:671 RNA viruses in marine microbial ecology 4:674–675 taxonomic framework, development of 4:671–674 MARV see Marburg and Ravn viruses (MARV) massively parallel/deep sequencing see next generation sequencing (NGS) mass spectrometry, defined 4:252 Mastadenovirus 2:8 Mastrevirus 3:358–359, 3:413 mathematical modeling 1:566–567 data sharing, data privacy and ethics 1:567 defined 1:559 proof of causation 1:567–568 mathematical modeling of virus architecture 1:249 genetic economy, principle of 1:249 Hamiltonian paths approach, applications of 1:254–255 assembly code embedded within viral genetic message 1:254–255 icosahedral symmetry, beyond 1:255 virus assembly mechanisms 1:255 icosahedral capsid architecture, mathematical models of 1:249–250 3D, virus structure in 1:254 implications for viral evolution 1:253–254 non-quasi-equivalent trihexagonal lattice architectures 1:253 overarching framework 1:251–253 practical applications 1:251 predictions and limitations of quasiequivalence theory 1:250 quasi-equivalence theory 1:249–250 viral tiling theory 1:250–251 icosahedral symmetry, viral geometry and 1:249 matrix-assisted laser desorption–ionization (MALDI) MS 5:92 matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry 5:114

359

matrix protein (MA) 2:325–326, 3:574–575, 2:566, 1:353, 1:354–355, 1:382, 1:349, 2:57, 5:127 maturation, defined 1:382, 4:115 maturation protein 1:238 mature extracellular virions 2:891–892 MAVGs see metagenome assembled viral genomes (MAVGs) MAVS see mitochondrial antiviral signaling protein (MAVS) maximum likelihood (ML) 1:119–120, 2:183f maximum parsimony (MP) 1:119–120 MaYSV see macroptilium yellow spot virus (MaYSV) MBV see monodon baculovirus (MBV); see mushroom bacilliform virus (MBV) MBV1 see mulberry badnavirus 1 (MBV1) MC29, defined 2:122 MCD see multicentric Castleman’s disease (MCD) MCF viruses see mink cell focus-inducing (MCF) viruses MCM see minichromosome maintenance (MCM) MCMV see maize chlorotic mottle virus (MCMV) MCP see major capsid protein (MCP); major coat protein (MCP) MCPyV see Merkel cell polyomavirus (MCPyV) MCS proteins 1:325 MCV see molluscum contagiosum virus (MCV); mud crab virus (MCV) MD see Marek’s disease (MD) MDA see multiple displacement amplification (MDA) MDA5 see melanoma differentiationassociated gene 5 (MDA5) MDCK cells see Madin-Darby canine kidney (MDCK) cells MdSGHV transmission dynamics in the housefly 4:789 MDV see Marek’s disease virus (MDV) MEAM1 see Middle East Asia minor 1 (MEAM1) measles 5:268–269 measles, mumps, and rubella (MMR) vaccine 5:268 measles virus classification 2:619 clinical features 2:623–625 complications 2:624–625 immune responses 2:625 immunosuppression 2:625 persistent infections 2:625 diagnosis 2:626 epidemiology 2:623 genome 2:619–622 envelope complex 2:622–623 replicative complex 2:621–622 transcription and replication 2:622 life cycle 2:623 MeV-based vaccines and cancer therapeutics 2:627

360

Subject Index

measles virus (continued) pathogenesis 2:625–626 prevention 2:627 treatment 2:626–627 virion structure 2:619 mechanical transmission, defined 1:542 MED1 see Mediterranean 1 (MED1) Mediterranean 1 (MED1) 3:757 megabirnaviruses (Megabirnaviridae) 4:594, 4:630–631, 4:464, 4:508–509 biological properties 4:596–597 future perspectives 4:600 functions of megabirna-P3 and -P4 4:600 genome expression and replication 4:594–595 genome organization 4:594 taxonomic and phylogenetic considerations 4:597–600 virion properties 4:594 virion structure 4:595–596 megaphages, origin of 1:20–22 megavirales, defined 1:372 melanization, defined 4:849 Melanoconion 2:810 melanoma differentiation-associated gene 5 (MDA5) 1:455, 2:555 Melanoplus sanguinipes EPV (MSEV) 4:861, 4:865 meleira 4:662 membrane-containing icosahedral DNA bacteriophages 4:36–37 genomes and genomics 4:43 life cycle 4:40–42 cell lysis 4:42–43 genome replication 4:42 particle assembly 4:42 receptor recognition and DNA delivery 4:42 virion structure and properties 4:37–40 membrane and DNA 4:40 overall structure 4:37–40 membrane insertion, defined 4:53 membranes and membrane proteins 1:339–340 membranous web 2:388–389 memory inflation 2:458 Mendelian gene 2:62 meningitis, defined 2:884, 2:675 meningoencephalitis, defined 2:675 meristem tip-culture, defined 3:430 Merkel cell polyomavirus (MCPyV) 2:524–525 clinical features 2:524–525 infectious cycle 2:525 MERS see Middle East respiratory syndrome (MERS) MERS-CoV see Middle Eastern respiratory syndrome coronavirus (MERSCoV) Mesoniviridae 4:804, 4:708–709 mesoniviruses classification 4:804 genome 4:804–806 genome organization 4:806f life cycle 4:807

ORF1a/ORF1b 4:806 particle morphology and spike protein architecture 4:806f structural proteins 4:806–807 virion structure 4:804 mesopelagic layer, defined 4:322 messenger RNA (mRNA) 1:437, 1:382, 2:35 metagemone, defined 4:265, 4:776 metagenome assembled viral genomes (MAVGs) 4:328–329, 4:409 cultivated phages infecting main groups of 4:330–332 marine Alphaproteobacteria, phages of 4:332–334 marine Bacteroidetes, phages of 4:336–337 marine Cyanobacteria, phages of 4:331–332 marine Gammaproteobacteria, phages of 4:335–336 metagenomically characterized viruses, classification of 1:50 metagenomics 1:127, 4:342, 1:177–178, 1:621, 4:314, 1:133, 4:283, 1:184 applied to viruses 1:133 pioneering viral metagenomics, one gene at a time 1:133 scaling up from fragmented genes to complete genomes 1:133–134 future of 1:139–140 global viral diversity, characterizing 1:137 identifying globally dominant bacteriophages 1:137 leveraging time series to track virus populations dynamics 1:138–139 ssDNA and RNA viruses, revealing the extraordinary diversity of 1:138 unveiling new uncultivated giant viruses 1:137–138 viral metagenomics in the clinic 1:134–136 epidemiological surveillance and environmental monitoring 1:136–137 metagenomic discovery of new viral pathogens 1:136 metal-tagging TEM (METTEM) 1:497 metaphor, virus as 1:672 metapneumovirus see human metapneumovirus (hMPV) metapopulation, defined 4:419 metastability, defined 4:167 metastable, defined 1:388 metaviridae (Ty3/gypsy retrotransposons) 3:101 Metaviridae 3:654 metavirus, defined 3:653 methanogenic, defined 4:387, 4:368 Methanosarcina Spherical Virus (MetSV) 1:433–434 methyl-directed mismatch repair (MMR) 4:291 MetSV see Methanosarcina Spherical Virus (MetSV) METTEM see metal-tagging TEM (METTEM) MEV see mink enteritis virus (MEV)

MGEs see mobile genetic elements (MGEs) MGL see multigene lysis (MGL) MHC see major histocompatibility complex (MHC) MHF see Marburg hemorrhagic fever (MHF) MHV see murine hepatitis virus (MHV) microarray, defined 4:808 microbial loop 4:320–321, 4:321f microcephaly, defined 2:899 microfluidics 5:115 micromyelia, defined 2:34 microRNAs (miRNA) 2:743–744, 2:144, 2:48, 3:293, 2:602, 1:455, 3:52, 3:123, 3:563 microRNA-122 (miR122) 2:391 microtiter serum neutralization (MTSN) 2:134 microtubule organizing center (MTOC) 1:498 microtubules, defined 3:32 microvesicle, defined 1:529 Microviridae 1:268–269 capsid assembly pathway in 1:270f microviruses 4:207 Middle East Asia minor 1 (MEAM1) 3:359, 3:757 Middle Eastern respiratory syndrome coronavirus (MERS-CoV) 5:274–275, 2:193, 1:121, 5:259 ebola and other hemorrhagic fevers 5:274–275 experimental infections 5:275 Middle East respiratory syndrome (MERS) 2:815f, 2:816f, 1:12 animal infection models 2:821 clinical features 2:819–820 epidemiology ecology, animal reservoir and zoonotic transmission 2:818–819 human-to-human transmission 2:819 laboratory diagnosis 2:820–821 pathogenesis 2:822 treatment 2:822–823 vaccines and immunity 2:823 virology 2:814–815, 2:815, 2:815–816 phylogeny 2:816–818 virus receptors 2:818 MIEP see major immediate early promoter (MIEP) MIGE see major Interspersed Genomic Element (MIGE) Mild Neurocognitive Disorder (MND) 2:471 mild-strain cross-protection, defined 3:520 Milker’s nodule 2:668 milt, defined 2:544 MIMIVIRE, defined 1:372 Mimiviridae family, algal viruses belonging to a subgroup within general properties 4:678–681 history 4:677–678 proposed subfamily Mesomimivirinae 4:681 unclassified algae-infecting members in the family Mimiviridae 4:681–682 mimivirus 1:493, 1:224 mimivirus virophage resistant element 1:380–381

Subject Index Mimoreovirus 4:684–685 minichromosome, defined 3:158 minichromosome maintenance (MCM) 4:390–391, 2:602 minigenome, defined 2:747 Minimum Information about an Uncultivated Virus Genome (MIUViG) 1:127 mink cell focus-inducing (MCF) viruses 2:646 mink enteritis virus (MEV) 2:684 minor coat proteins 1:333 protein IIIa 1:333 protein IX 1:333–335 protein VI 1:333 protein VIII 1:333 minor variant, defined 1:71 (–)-sense RNA (minus-sense RNA) 1:382 miRNA see microRNAs (miRNA) mites, plant viruses transmission by 3:113 mitochondria 1:495, 1:499 mitochondrial antiviral signaling protein (MAVS) 1:455, 2:556, 2:391–392 mitogen-activated phosphate kinases (MAPKs) 2:254 mitogen activated protein kinases (MAPK) 3:245, 2:428 mitoviruses 4:465–466, 4:454 accessory RNAs associated with infections of 4:602 codon usage and implications for biology and evolution of 4:605 engineering mitoviruses for infectivity 4:604 genome structure 4:601–602 host defense 4:604–605 origin and evolution 4:605 phenotypic effects of mitovirus infection 4:602 phylogenetic relationships 4:602–603 plant mitoviruses 4:605–606 taxonomy and nomenclature 4:603–604 transmission 4:604 mitoxantrone dihydrochloride 5:124 MIUViG see Minimum Information about an Uncultivated Virus Genome (MIUViG) ML see maximum likelihood (ML) MLA see multilateral recognition arrangements (MLA) MMR see methyl-directed mismatch repair (MMR) MMR vaccine see measles, mumps, and rubella (MMR) vaccine MMV see mouse mammary tumor virus (MMV); multi-membraned vesicles (MMVs) MND see Mild Neurocognitive Disorder (MND) mobile genetic elements (MGEs) 1:14, 4:98, 1:38, 4:400 modified vaccinia ankara (MVA) 2:243, 2:216 modular evolution, defined 4:457 MOI see multiplicity of infection (MOI) 20969:p0030

Mokola lyssavirus (MOKV) 2:738 MOKV see Mokola lyssavirus (MOKV) molecular clock, defined 3:520 molecular diagnostics, defined 4:252 molecular epidemiology 1:565–566, 1:382 molecular strain typing, defined 2:707 molluscum contagiosum virus (MCV) clinical features 2:631 diagnosis 2:632 epidemiology 2:630–631 genome 2:629–630 history 2:629 life cycle 2:630 pathogenesis 2:631–632 prevention 2:633 treatment 2:632–633 virion structure 2:629 Moloney leukemia murine virus (MoMlv) 2:576 monkeypox virus (MPXV) classification and structural morphology 2:868–869 clinical features 2:870–871 diagnosis 2:872 epidemiology 2:871–872 history 2:868 pathogenesis 2:869–870 prevention 2:872–873 treatment 2:873–874 viral life cycle 2:869 mono-, bi- and tripartite, defined 4:632 monocistronic, defined 3:516, 4:632 monoclonal antibodies 2:230, 5:272–273, 2:778, 2:441 human immunodeficiency virus 5:273 respiratory syncytial virus (RSV) 5:272–273 therapy 2:241–242 monocot, defined 3:778 monocotyledon, defined 3:545 monodon baculovirus (MBV) 4:828–829 monomeric replication products 4:137 Mononegavirales 2:232, 3:495 mononucleosis, defined 2:441 monopartite, defined 3:703 monophyletic, defined 1:28, 3:653 monophyly, defined 4:835 mono-/polyvalent, defined 5:289 monosense, defined 4:835 monovalent type 2 oral polio vaccine (mOPV2) 5:312–313 Mopeia virus (MOPV) 2:516 MOPV see Mopeia virus (MOPV) mOPV2 see monovalent type 2 oral polio vaccine (mOPV2) morbilliviruses classification 2:68 clinical features 2:74–75 diagnosis and prevention 2:76–77 epidemiology and host range 2:72–73 aquatic mammal morbilliviruses 2:74 canine distemper virus (CDV) 2:74 emerging morbilliviruses 2:74 Peste-des-petitsruminants virus (PPRV) 2:73–74 rinderpest virus (RPV) 2:73

361

genome 2:69–70 life cycle 2:73f, 2:71–72 pathogenesis 2:75–76 proteins 2:70–71 rinderpest eradication 2:77–78 treatment 2:77 virion structure 2:68–69, 2:69f morphogenetic signal, defined 4:53 morphologies, viruses with 4:360–361, 1:258f family Ampullaviridae 4:361 family Bicaudaviridae 4:361 family Clavaviridae 4:361–362 family Fuselloviridae 4:362 family Guttaviridae 4:362 family Spiraviridae 4:362 filamentous viruses 4:363–364 proposed class Tokiviricetes 4:363–364 spherical viruses 4:362 Family Globuloviridae 4:362 Family Ovaliviridae 4:362 Family Portogloboviridae 4:362–363 Family Turriviridae 4:363 mosaicism 4:277 in Myoviridae 4:280 in Podoviridae 4:280 in Siphoviridae 4:278–280 mosquitoes 2:893 mosquito vectors, control of 1:575 most recent common ancestor (MRCA) 1:117–118 motif, defined 3:797 mourning doves (Zenaida macroura) 2:805 mouse mammary tumor virus (MMV) 1:352, 2:643 movement of viruses in plants 3:32–33 intercellular transport 3:39–40 intracellular movement 3:33–39 systemic transport 3:40–41 virus movement and plant defense responses 3:41 movement protein (MP) 3:317–318, 4:437–438, 3:727, 3:229, 3:719, 3:140, 3:456 MP see maximum parsimony (MP); movement protein (MP) MpDV see myzus persicae densovirus (MpDV) M protein 2:891–892 MPXV see monkeypox virus (MPXV) MRCA see most recent common ancestor (MRCA) mRNA see messenger RNA (mRNA) MrNV see machrobrachium rosenbergii nodavirus (MrNV) MSD see maize streak disease (MSD) M segment 2:209 MSEV, see Melanoplus sanguinipes EPV (MSEV) MSPV see Malpais Spring virus (MSP)V MSV see maize streak virus (MSV) MTOC see microtubule organizing center (MTOC) MTP see major tail protein (MTP) MTSN see microtiter serum neutralization (MTSN)

362

Subject Index

mud crab virus (MCV) 4:772 mulberry badnavirus 1 (MBV1) 3:163 Muller’s ratchet, defined 1:53 multicentric Castleman’s disease (MCD) 2:604–605, 2:605, 2:606, 2:606–607, 2:599 multigene families, defined 2:22 multigene lysis (MGL) 1:501–502, 1:501, 1:504–505 diversity 1:512 in gram-positive hosts 1:513–514 lambda lysis, looking under the hood of 1:505–507 lambda lysis cassette 1:505 lambda MGL, operational outline of 1:505 lambda prophage as a lysis platform 1:504–505 regulation of 1:509–510 dual start holin genes 1:509–510 real-time regulation of holin function 1:510–512 multilateral recognition arrangements (MLA) 5:70 multi-membraned vesicles (MMVs) 1:498–499 multiphoton imaging 1:211–213 multiple displacement amplification (MDA) 4:330, 1:184 multiplex and point of care tests (POCT) 5:114–115 multiplex PCR 5:92, 5:98 multiplex RT-PCR 3:629 multiplicity of infection (MOI) 4:83, 4:90–91, 4:88 multivesicular bodies (MVB) 1:522, 2:210–211, 3:32, 2:399, 3:132, 2:879–880 multivesicular endosomes (MVEs) 1:538, 1:529 MuLVs/MLVs see murine leukemia virus (MuLVs/MLVs) mumps virus antibody response 2:641 clinical features of primary infection 2:637 detection of mumps virus 2:641 differential diagnosis 2:640 epidemiology 2:637 geographic and seasonal distribution 2:637 transmission and tissue tropism 2:637 evolution and genetics 2:637 genome, properties of 2:635 history 2:634 immune response 2:639–640 laboratory diagnosis 2:640–641 MuV proteins, properties of 2:635 neutralisation tests 2:641 pathogenesis 2:639 physical properties 2:635–636 prevention and control 2:642 future perspectives 2:642 properties of the proteins of 2:636t replication 2:636 serologic relationships and variability 2:636–637 structure of 2:634f

systemic manifestations 2:637 cardiac symptoms 2:639 genitourinary tract 2:638–639 musculoskeletal system 2:639 neurological disease 2:637–638 pancreas 2:638 parotid glands 2:637 taxonomy and classification 2:634 utilization of assays 2:641–642 primary infection and primary vaccine failure 2:641–642 secondary infection and secondary vaccine failure 2:642 virion, properties of 2:634–635 MuNoVs see murine noroviruses (MuNoVs) mungbean yellow mosaic viruses (MYMIV), resistance to 3:563, 3:564f murine astroviruses (MuAstV) 2:96 murine hepatitis virus (MHV) 4:804, 1:492 murine leukemia virus (MuLVs/MLVs) 1:631, 1:627, 1:178, 1:223, 1:454 classification 2:643 endogenous 2:647 genome and structure 2:643–644 history 2:643 host-virus interactions 2:645–646 pathology 2:646 replication 2:644–645 vectors 2:646–647 murine noroviruses (MuNoVs) 1:535 Murine Sarcoma Viruses (MuSVs/MSVs) pathology 2:646 Murray Valley encephalitis virus 2:812 Muscavirus 4:781 mushroom bacilliform virus (MBV) 4:437 MBV evolutionary relationships 4:550 MBV genome organization and expression 4:549–550 MBV transmission and host range 4:550 MBV virion properties 4:549 MBV virion structure and composition 4:549 mushroom compost, defined 4:528 MuSVs/MSVs see Murine Sarcoma Viruses (MuSVs/MSVs) Mutational Meltdown, defined 5:227 mutation rate, defined 1:53 mutation-selection balance 1:57–58, 1:53 mutualism, defined 4:419, 4:658 MVA see modified vaccinia ankara (MVA) MVB see multivesicular bodies (MVB) MVD see Marburg virus disease (MVD) MVEs see multivesicular endosomes (MVEs) Mxra8 2:176 myalgia, defined 2:675 Myc, defined 2:122 Mycoflexivirus 4:437–438 mycoreovirus 1 (MyRV1) 4:658 mycoreovirus 2 (MyRV2) 4:433 mycoreoviruses 4:433 coinfections of mycoreovirus and other viruses 4:613 on fungal gene expression 4:612–613 genome rearrangements 4:613 genome structures, organizations, and relationships 4:609–610

mycoreovirus 1 4:610–611 expression of MyRV1 gene products 4:610–611 mycoreovirus 2 4:611 mycoreovirus 3 4:611–612 sclerotinia sclerotiorum reoviruses 4:612 mycoreovirus 4 4:612 sclerotinia sclerotiorum reovirus 1 4:612 structure-function relationships 4:607–609 taxonomy and nomenclature 4:609 mycorrhiza, defined 3:388 mycoviruses 4:441–442, 4:461, 3:388, 4:432, 4:439, 1:621, 4:544, 4:431, 4:594, 4:648 coinfection and evolution 4:459–460 diversity in Aspergilli 4:450 aspergillus foetidus mycovirus complex 4:453–454 chrysoviruses 4:454 genomes 4:451–453 narnaviruses and mitoviruses 4:454 partitiviruses 4:454 phenotypes 4:455 polymycoviruses 4:454–455 prevalence 4:450–451 transmission 4:451 evolutionary history and drivers of evolution 4:458–459 horizontal transmission of 4:525–526 host divergence in the evolution 4:459 infections in Sclerotinia sclerotiorum 4:461, 4:462t co-infecting hypovirulent strain SCH941 4:463–464 hypovirulent strain SZ-150 4:463 megabirnavirus 4:464 mitoviruses 4:465–466 possible reasons for co-infections 4:466 strain AH98 4:463 strain Ep-1PN 4:461–462 strain sunf-M 4:464–465 strain SX247 4:462–463 three strains of S. sclerotiorum harbor multiple unassigned mycoviruses/ dsRNA elements 4:466 structural evolution 4:459–460 mycoviruses with filamentous particles 4:478 Botrytis virus F (BotV-F) 4:478–479 biological properties 4:479 family –Gammaflexiviridae 4:479 genome structure 4:479 genus –Mycoflexivirus 4:479 phylogenetic relationships 4:479–480 virion morphology 4:479 Botrytis Virus X (BotV-X) 4:480 biological properties 4:480–481 family –Alfaflexiviridae 4:480 genome structure 4:480 genus –Botrexvirus 4:480 phylogenetic relationships 4:481 virion morphology 4:481 colletotrichum camelliae filamentous virus 1 4:483–484 biological properties 4:484

Subject Index family –Unclassified 4:483–484 genome structure 4:483–484 genus –Unclassified 4:483–484 phylogenetic relationships 4:484 virion morphology 4:484 fusarium graminearum negative-stranded RNA virus 1 4:483 biological properties 4:483 family –Mymonaviridae 4:483 genome structure 4:483 genus –Unclassified 4:483 particle morphology 4:483 phylogenetic relationships 4:483 relationships between filamentous viruses from fungi and plants 4:484–485 sclerotinia sclerotimonavirus 4:481 biological properties 4:481 family –Mymonaviridae 4:481 genome structure 4:481 genus –Sclerotimonavirus 4:481 phylogenetic relationships 4:483 virion morphology 4:481–483 mycovirus-mediated biological control 4:468 basic concepts for 4:468–469 future perspectives 4:477 general procedure for developing 4:469 biocontrol testing in the field 4:472 biological control agents, mycoviruses as 4:472 biosafety issues 4:472 testing for phenotypic effects of the mycovirus 4:469 transmission properties 4:469–472 virus detection and characterization 4:469 hypovirulence in Botrytis cinerea 4:476 hypovirulence in Cryphonectria parasitica 4:472–474 artificial application of hypovirulence 4:474–475 exclusive transmissible hypovirulence 4:473–474 hypovirulence in Fusarium graminearum 4:477 hypovirulence in Helminthosporium victoriae 4:476–477 hypovirulence in Ophiostoma novo-ulmi 4:475 hypovirulence in Rosellinia necatrix 4:476 hypovirulence in Sclerotinia sclerotiorum 4:475–476 plant pathogenic fungi, disease cycle of 4:468 mycovirus transmission 4:525 between VCGs 4:526–527 myelomonocytic lineage cells 2:456 Mymonaviridae 4:615 genomic structure 4:617–618 host and distribution 4:618–620 impact on the host 4:618 phylogenetic status of 4:615 species and tentative species in 4:615 virion 4:615–617 mymonaviruses, defined 4:615 myositis, defined 2:675 Myoviridae 1:320–321, 4:186, 4:276, 4:280

myovirus 4:342 myristoylation, defined 2:757, 2:22 myxomatosis in European rabbits 2:732 myxoma virus 2:731 myzus persicae densovirus (MpDV) 4:845

N N4 (lytic podoviridae phage) 4:74–75 NA see neuraminidase (NA) nAChR see nicotinic acetylcholine receptor (nAChR) NAC transcription factors 3:758 NAI see neuraminidase inhibitors (NAI) nanobiotechnology, defined 3:364 nano-carriers, virus-like particles as 1:665–666 conjugation of antigens and VLPs 1:665–666 chemical and affinity conjugation 1:666–667 genetic fusion 1:665–666 SpyTag/SpyCatcher system 1:667 Encapsulation, loading of virus-like particles by 1:667–668 Nanohaloarchaea, viruses of 4:417 nanoparticle, defined 3:778 nanopore sequencing, defined 1:175 Nanopore Sequencing Technology, defined 4:322 nanotechnology, defined 3:364 Nanoviridae 3:471 nanoviruses (Nanoviridae) 3:470–471 classification and taxonomy 3:471–472 diagnosis 3:479–480 epidemiology and control 3:478–479 genome organization 3:472–474 nanovirid-associated alphasatellites 3:476–477 nanovirid evolution 3:476 nanovirid replication 3:474–475 particle properties 3:472 properties and functions of nanovirid proteins 3:474 taxonomy and phylogeny 3:475–476 transmission and host range 3:477–478 host range 3:478 tissue tropism and means of transmission 3:477–478 transmission by aphids 3:478 Narcissus mosaic virus (NWV) helix 3:625 narnaviruses (Narnaviridae) 4:621, 4:454 cis-acting signals for replication 4:623–624 cis-signals for formation of ribonucleoprotein complexes 4:624–625 generation of narnaviruses in vivo 4:623 historical background 4:621 narnavirus persistence in the host 4:625–626 replication intermediates 4:623 ribonucleoprotein complexes as a viral entity 4:622–623 viral genomes 4:621–622

363

NASBA see nucleic acid sequence-based amplification (NASBA) NASH assay see nucleic acids spot hybridization (NASH) assay NAT see nucleic acid testing (NAT) National Immunization Days (NIDs) 2:690–692 natural killer (NK) cells 1:584 naturally occurring recessive resistance 3:70–73 natural transformation defined 4:242 of a plant virus to a fungus 4:447 Natural Vector method 1:102 Naı¨ve, defined 1:542 NB 2:566 NB-LRR protein, defined 3:60, 3:554 NC see nucleocapsid (NC); nucleocapsid protein (NC) NCAM see neuronal cell adhesion molecule (NCAM) NCLDV see nucleocytoplasmic large DNA viruses (NCLDV) NCVOG see Nucleo-Cytoplasmic Virus Orthologous Groups (NCVOG) NDV see Newcastle disease virus (NDV) near-atomic resolution, cryo-EM at 1:233–235 data collection and image reconstruction 1:233–235 near-atomic resolution maps, interpretation of 1:235 side chain interactions and virus assembly 1:235 NEAR technology see nicking enzyme amplification reaction (NEAR) technology necro-like viruses (Tombusviridae) cytopathology 3:484 genome organization and expression 3:481–483 intraspecific relationships 3:483 satellites 3:483–484 taxonomy and classification 3:481 transmission and host range 3:484 virion properties, structure and composition 3:481 necrotic syndrome of lettuce, defined 3:833 nef gene 2:61t negative-contrast electron microscopy, defined 4:632 negative predictive value (NPV) 5:74 negative selection-based enrichment 1:179–180 Negative sense (–) strand, defined 1:429 negative sense genome, defined 3:507 negative-sense RNA viruses 1:499, 3:567 negative single stranded RNA viruses (NSRVs) 1:345–346 classification of NSRVs and genome organization 1:346 general features of NSRV virion structures 1:346 inside the virion 1:348–349 matrix proteins 1:349 nucleoproteins 1:349–350

364

Subject Index

negative single stranded RNA viruses (NSRVs) (continued) phosphoproteins 1:350–351 polymerase 1:350 viral ribonucleoproteins/nucleocapsids assemblies 1:349 virion surface, structure of 1:346–348 accessory proteins in the virion surface 1:348 structure and function of virion glycoproteins 1:347–348 Negative Stain 5:5 negative staining (NS) 5:9, 5:9–10 for diagnosis of fecal samples 5:11 agarose slides, preparation of 5:11 general protocol for the study of viruses by 5:10–11 neighbor-joining (NJ) method 1:118–119 nematodes, plant viruses transmission by 3:113 NendoU function 2:253 Neodiprion sertifer 4:704 neonatal herpes simplex virus infection 5:178–180 neoplasia, defined 2:316 neotope, defined 3:727 NEP see nuclear export protein (NEP) nepoviruses (Secoviridae) cell-to-cell and systemic movement in the plant 3:491 diseases, economic considerations and control 3:493 genome organization and function of viral proteins 3:489–490 historical perspective 3:486 host range, symptom determinants and interaction with plant defense responses 3:491 population structures 3:493 production of viral proteins 3:490 taxonomy, phylogeny, and evolution 3:486 transmission 3:491–493 viral rna replication 3:490–491 virion structure 3:486–489 neuraminidase (NA) 2:566, 5:125–127 neuraminidase inhibitors (NAI) 5:162–164, 5:163f, 5:171 neurodevelopmental, defined 2:441 neuroinvasion 1:536 neuroinvasive disease, defined 2:884 neuronal cell adhesion molecule (NCAM) 2:738–739 neuronal progenitor cells (NPCs) 2:906 neuropilin-2 2:450–451 neuropsychiatric adverse events (NPAE) 5:164 neurotropic disease 2:891, 2:896 neutralization 1:591 neutralizing antibodies 5:286 Newcastle disease virus (NDV) 1:191 classification 2:648 clinical features 2:651 diagnosis 2:651–652 epidemiology 2:650 genome 2:648–649

life cycle 2:649–650 pathogenesis 2:650–651 prevention 2:652–653 virion structure 2:648 new invertebrate DVs 4:840 New-World (NW) begomoviruses 3:751 New World (NW) mammarenaviral HFs 2:515, 2:514 next generation sequencing (NGS) 2:673, 5:76, 5:112–113, 2:652, 4:414, 1:553 analyzing recombination in sequences derived from 1:111–112 and applications in virology 1:176 defined 1:552, 3:293, 1:175 N-fold symmetric assembly 4:186 NF-kB inhibitors 2:672 defined 2:875 NF-kB signaling pathway see nuclear factor kappa B (NF-kB) signaling pathway N-Glycosylation, defined 4:380 NGS see next generation sequencing (NGS) NHPs see nonhuman primates (NHPs) nicking enzyme amplification reaction (NEAR) technology 5:46 Nicotiana benthamiana 3:753, 3:244f, 3:244–245, 3:556f nicotinic acetylcholine receptor (nAChR) 2:738–739, 2:741–742 Nidovirales 4:804 nidovirales 2:193 diseases associated with nidoviruses 2:245–246 effect of nidovirus infection on the host cell 2:253–254 genome organization 2:247–251 replicase 2:249–251 host cell, effect of nidovirus infection on 2:253–254 origin of nidoviruses 2:253 origin of nidoviruses 2:253 replicase 2:249–251 replication 2:252 structural and accessory protein genes 2:251–252 taxonomy and phylogeny 2:245 transcription 2:252–253 virus structure 2:246–247 nidovirus classification and prototype members 2:246f genome structure 2:249f phylogeny 2:247f replicase genes 2:250f structure 2:248f Nidovirus endoribonuclease (NendoU) 2:250 NIDs see National Immunization Days (NIDs) Niemann-Pick C1 (NPC1) 2:237, 2:613 NIENV see Nilaparvata lugens endogenous nudivirus (NIENV) Nilaparvata lugens 4:771 Nilaparvata lugens endogenous nudivirus (NIENV) 4:829 Nimaviridae 4:808, 4:709 nimaviruses 4:808

apoptosis 4:815–816 clinical features and pathology 4:816–817 epidemiology 4:816 genome and phylogeny 4:809–813 hemocyte responses to WSSV infection 4:816 life cycle 4:813–815 protection of shrimp against WSSV infection 4:817 taxonomy 4:808 virion structure and composition 4:808–809 90–90–90 Initiative, defined 2:460 Nipah virus (NiV) 2:355, 1:121 NIS see sodium iodide symporter (NIS) nitazoxanide 5:168 NiV see Nipah virus (NiV) NJ method see neighbor-joining (NJ) method NK cells see natural killer (NK) cells NLRs, defined 4:520 NLS see nuclear localization sequences (NLS); nuclear localization signal (NLS) NM infection, defined 3:743 NNRTIs see non-nucleoside reversetranscriptase inhibitors (NNRTIs) Noble Prizes awarded for discoveries relating to viruses 1:674t NOBs see non-occluded baculoviruses (NOBs) Nodaviridae 4:709–710 nodaviruses of invertebrates and fish classification (compact) 4:819 proposed six clade taxonomic structure 4:819–820 provisional nodaviruses 4:819 clinical features 4:822–823 viral covert mortality disease (VCMD) 4:823 viral nervous necrosis (VNN) 4:822–823 white tail disease (WTD) 4:823 diagnosis 4:824–825 viral covert mortality disease (VCMD) 4:825 viral nervous necrosis (VNN) 4:824–825 white tail disease (WTD) 4:825 epidemiology 4:822 genome 4:820 immune responses to nodavirus infection 4:823–824 adaptive immune responses in nodavirus infection 4:824 innate immune responses 4:823–824 life cycle 4:821–822 pathogenesis 4:823 physical properties 4:821 prevention 4:825 viral covert mortality disease (VCMD) 4:825 viral nervous necrosis (VNN) 4:825 white tail disease (WTD) 4:825 proteins 4:820–821 capsid proteins 4:821

Subject Index proteins B1 and B2 4:821 RNA-dependent RNA polymerase (RdRp) 4:820–821 treatment 4:825 virion structure 4:820 NOD-like receptor family pyrin domain containing 3 (NLRP3) 2:555 nomenclature, defined 1:28 nonanucleotide, defined 3:301 non-canonical initiation of virus mRNA translation 1:447–449 cap-independent translation enhancers (CITEs) 1:449 initiation at non-AUGs 1:450 internal ribosome entry sites (IRESes) 1:447–449 leaky scanning 1:450 ribosome reinitation 1:450–452 ribosome ‘shunting’ (discontinous scanning) 1:450 virus alternatives to components of initiation 1:449–450 alternative to eIF4F 1:450 alternative to the m7g cap structure 1:449–450 nonchromosomal gene, defined 4:487 non-circulative, defined 3:371 non-contractile long-tailed phages (Siphoviridae) 4:209–211 non-enveloped eukaryotic virus 1:409–410 complex 1:412–414 exploitation of virus entry pathway 1:414–415 simple 1:410–412 non-enveloped icosahedral RNA viruses 1:265–266 nonenveloped particles antigenic sites 1:478 antiviral agents 1:478–479 architecture of viruses 1:475 assembly 1:477–478 evolution 1:477 helical viruses, atomic structure of 1:475–476 host receptor recognition site 1:478 nucleic acid–protein interaction 1:476–477 spherical viruses, atomic structure of 1:476 structure determination, methods of 1:475 non-enveloped viruses, cell entry targeting of 5:238 nonhuman and environmental sources, surveillance of viruses in 5:249 nonhuman primates (NHPs) 2:232, 2:232–234 non-insect vectors, plant viruses transmission by 3:113 fungi and protists, transmission by 3:113 mites, transmission by 3:113 nematodes, transmission by 3:113 non-isothermal PCR based technologies 5:22–25 nonlytic release of virus in extracellular vesicles 1:538 non-NB-lRR resistance 3:66–67

non-nucleoside reverse-transcriptase inhibitors (NNRTIs) 2:472–473, 5:135, 5:121 clinical uses of 5:135–136 drug-drug interactions with 5:136 mechanisms of NNRTI resistance 5:136–137 and their mechanism of action 5:135 toxicities 5:136 non-occluded baculoviruses (NOBs) 4:827 non-pathogen derived resistance 3:363 non-persistent, defined 3:371 non-persistent manner, defined 3:81 non-pharmaceutical interventions, defined 2:825 non-propagative manner, defined 3:81 non-propagative transmission, defined 3:200 non-quasi-equivalent trihexagonal lattice architectures 1:253 non-retroviral endogenous RNA (NERVE) 4:601 non-retroviral EVE, evolutionary impact of 1:83–84 nonsense suppressor tRNA, defined 4:487 non-structural (NS) proteins 1:498, 2:296, 2:40–41, 2:806–808 NS1 2:567 NS5A 5:123 NSP3 1:446 nonsynonymous, defined 1:62 non-template strand, defined 4:69 non-translated regions (NTRs) 2:387 nOPV strains see novel oral poliovirus vaccine (nOPV) strains norovirus 2:483, 1:392–393 clinical features 2:487 diagnosis 2:488–489 epidemiology 2:486–487 genome 2:483–484 immunity 2:488 life cycle 2:485–486 pathogenesis 2:487–488 prevention 2:489–490 treatment 2:489 viral classification 2:483 virion structure 2:484–485 vaccine 5:291–292 development 5:292 future considerations for 5:293 in human clinical trials 5:292–293 in pre-clinical development 5:293 nosocomial transmission 1:562 defined 1:559 notifications 5:248 novel oral poliovirus vaccine (nOPV) strains 5:313 NP see nucleoprotein (NP) NPAE see neuropsychiatric adverse events (NPAE) NPC1 see Niemann-Pick C1 (NPC1) NPCs see neuronal progenitor cells (NPCs) NPV see negative predictive value (NPV); nucleopolyhedrovirus (NPV) NRTIs see nucleoside/nucleotide reversetranscriptase inhibitors (NRTIs)

365

NS see negative staining (NS) NS1 protein, defined 2:747 NS2A 2:844 NS2B 2:844 NS2 protein, defined 2:747 ns3 gene 4:761 NS proteins see non-structural (NS) proteins N-Succinimidyl S-Acetylthioacetate (SATA) 1:667 N terminus, defined 1:345 NTRs see non-translated regions (NTRs) nuclear export protein (NEP) 2:552, 2:290, 2:567 nuclear factor kappa B (NF-kB) signaling pathway 2:672 nuclear inclusion protein a, defined 3:293 nuclear inclusion protein b, defined 3:293 nuclear localization sequences (NLS) 2:289 nuclear localization signal (NLS) 2:602, 3:164, 2:561 nuclear shuttle protein, defined 3:169 nuclease, defined 4:242 nucleic acid 1:229f, 5:5, 4:242 nucleic acid detection 5:210–211 hepatitis A virus 5:211 hepatitis E virus 5:211 nucleic acid–protein interaction 1:476–477 nucleic acid satellites, defined 3:411 nucleic acid sequence-based amplification (NASBA) 5:98, 5:22, 5:23f nucleic acid sequence dependent strategies for known viruses 5:31–32 nucleic acid sequence independent strategies for sequencing 5:32 nucleic acids spot hybridization (NASH) assay 3:759 nucleic acid testing (NAT) 5:17, 5:98, 5:91–92 clustered regularly interspaced short palindromic repeats (CRISPER) platforms 5:93 DNA microarrays 5:92 high throughput sequencing 5:92–93 multiplex PCR 5:92 polymerase chain reaction assays 5:92 VirCapSeq-VERT 5:93 Nucleo(s)tide analog (NA), defined 5:217 nucleocapsid (NC) 2:610, 1:491, 4:747, 4:827, 1:257, 1:362, 2:747, 1:382, 4:26, 3:507, 2:441 nucleocapsid core, defined 3:567 nucleocapsid protein (NC) 1:353, 3:572–573, 1:356, 2:57 nucleocytoplasmic large DNA viruses (NCLDV) 1:621, 1:493, 1:372, 1:379f, 4:677, 1:159, 1:72, 4:797, 1:269 Nucleo-Cytoplasmic Virus Orthologous Groups (NCVOG) 4:677 nucleopolyhedrovirus (NPV) 4:739, 4:747, 4:699 nucleoprotein (NP) 2:611–612, 2:648, 3:154, 1:349–350, 2:552 nucleos(t)ide analogue, defined 2:373

366

Subject Index

nucleoside/nucleotide reverse-transcriptase inhibitors (NRTIs) 5:132–133, 5:133, 2:472–473, 5:140, 5:121 clinical uses of 5:133 drug-drug interactions with 5:134 mechanisms of NRTI resistance 5:134–135 new drugs 5:135 and their mechanism of action 5:132–133 toxicities 5:133–134 nucleus, virocells with 1:25 nudaurelia capensis omega virus (NoV) 4:900, 4:901–902 nudaurelia capensis b virus (NbV) 4:897 Nudiviridae 4:827, 4:710 members of 4:827–829 nudiviruses 4:828t, 4:710 classification 4:827 criteria for classification 4:827 history 4:827 members of Nudiviridae 4:827–829 core genes 4:832 defined 4:827 gene regulation for switching productive and latent infections 4:832–833 genome 4:830–832 gene content 4:832 general features 4:831–832 negative impacts and potential applications 4:833 phylogeny 4:829 transmission and pathogenesis 4:829–830 virion structure 4:829 virus life cycle 4:830 Number and Brightness (N&B) 00108: p0130 1:208 NWV helix see Narcissus mosaic virus (NWV) helix Nyamiviridae 4:710–711 nyamiviruses 4:711 nystagmus, defined 2:34

O OAEV see Oedaleus asiaticus EPV (OAEV) OaPV-3 DNA see ovis aires papillomavirus type 3 (OaPV-3) DNA OAS see original antigenic sin (OAS) oat chlorotic stunt virus (OCSV) 3:790–791 OB see occlusion body (OB) obligate endoparasite, defined 3:528 Obodhiang virus (OBOV) 2:875 OBOV see Obodhiang virus (OBOV) occlusion body (OB) 4:739, 4:747 occlusion body, defined 4:827 occlusion-derived virus (ODV) 4:704, 4:739, 4:747 Ockelbo disease 2:840 OCSV see oat chlorotic stunt virus (OCSV) ocular infections 5:200 odonstyle, defined 3:486 odontophore, defined 3:486 ODV see occlusion-derived virus (ODV) Oedaleus asiaticus EPV (OAEV) 4:865

OELCA, see Okra enation leaf curl alphasatellite (OELCA) OELCV, see Okra enation leaf curl virus (OELCV) Okra enation leaf curl alphasatellite (OELCA) 3:360–361 Okra enation leaf curl virus (OELCV) 3:358–359 okra viral diseases 3:88 Old-World (OW) 3:751 Old World primates, defined 2:827 Oleavirus 3:260, 3:262t characteristics of RNA genome in 3:261t oligo-mediated recombineering, mechanism of 4:297–298 oligonucleotide, defined 4:291 oligonucleotide primer, defined 5:27 Olpidium virulentus 3:833 Omegatetravirus 4:897 omp, defined 4:175 oncogene, defined 2:316, 2:122 oncogenesis, defined 2:528 oncogenic HPVs 2:499–500 oncolytic virotherapy, defined 2:619 oncolytic viruses (OVs) 1:658–659, 1:658 combination therapies 1:660 modes of action 1:659 tumor targeting by OVs 1:659–660 oncoproteins, defined 2:79 126-kDa protein, defined 3:839 onychomadesis, defined 2:256 oomycete, defined 3:388 OPA see ovine pulmonary adenocarcinoma (OPA) open reading frame (ORF) 2:184, 4:677, 2:757, 2:79, 3:388, 3:778, 3:140, 4:776, 2:779, 4:845, 3:163 defined 2:182 ORF1 2:48–49 operational taxonomy units (OTUs) 1:117, 3:653 Ophiostoma novo-ulmi, hypovirulence in 4:475 ophioviruses (Aspiviridae) 3:495 cytopathology 3:505 diagnosis 3:503–504 genome organization and replication 3:497–498 pathogenicity and geographic distribution 3:500–503 properties and functions of gene products 3:498–500 taxonomy, phylogeny and evolution 3:495–497 transmission, experimental host-range and control 3:504–505 virion structure 3:497 opportunist, defined 2:441 opportunistic pathogen, defined 2:441 opsonization 1:591 Opuntia virus X (OpVX) 3:623–624, 3:628 OPV see oral poliovirus vaccine (OPV) OpVX see Opuntia virus X (OpVX) OR14l1 2:450–451 oral cavity and respiratory tract viromes 1:555–556

oral poliovirus vaccine (OPV) 5:310–311 orbivirus 1:303 orchiodynia, defined 2:675 ORF see open reading frame (ORF) ORF1a/ORF1b 4:806 orfA gene 2:61t ORFan genes 4:237, 4:229, 4:528 ORFV, see orf virus (ORFV) orf virus (ORFV) 2:169, 2:666 clinical features and gross pathology 2:169–170 diagnosis 2:170 epidemiology 2:169 histopathology 2:170 pathogenesis 2:170 prevention 2:170 treatment 2:170 organelle remodeling proteins 1:499 original antigenic sin (OAS) 2:419 origin of assembly, defined 3:734 origin of viruses 1:14 DNA viruses 1:19–20 evolutionary connection between viruses and mobile genetic elements 1:14 giant viruses and megaphages 1:20–22 reverse-transcribing RNA and DNA viruses 1:18–19 RNA viruses 1:14–15 infecting eukaryotes 1:17–18 scenario for 1:16–17 selfish RNA replicons, origin of 1:15–16 Orinoco sheldgoose (OSHBV) 2:100 ornamentals and orchids viral diseases 3:95–96 ornamental viruses 3:19 causal agent and classification 3:19 control 3:20 disease symptoms and yield losses 3:19 epidemiology 3:19–20 geographical distribution 3:19 ortervirales, defined 3:653 orthobunyaviruses 2:654 classification 2:654 phylogenetic analysis by sequencing 2:654 serological classification 2:654 clinical features and pathogenesis 2:662 Anopheles A 2:662 Bunyamwera 2:662 Bwamba 2:662 California 2:662–663 Simbu 2:663 diagnosis 2:663 polymerase chain reaction (PCR) detection of viral RNA 2:663–664 serological assays 2:663 epidemiology 2:660–661 host and viral determinants of diseasespecies barriers 2:661–662 orthobunyavirus outbreaks and tracking 2:661 surveying viral endemic ranges and tracking viral spread 2:661 viral discovery 2:660–661 genome 2:654–659 genome and encoded proteins 2:655–659

Subject Index life cycle 2:659–660 reassortment 2:660 effects of 2:660 evidence of 2:660 transmission 2:660 vector 2:660 vertebrate hosts 2:660 treatment and prevention 2:664 orthohantavirus structure of 2:350f types of 2:352t orthologous genes, defined 4:387 orthopteran, defined 4:768 orthoreovirus 1:305–306, 4:871 orthotospoviruses (Tospoviridae) 3:12 detection and diagnosis 3:514 genetics and evolution 3:513–514 genome properties 3:511–512 protein properties 3:512 geographic distribution 3:510 history 3:507 host-range and virus propagation 3:510–511 virion properties 3:510–511 pathogenicity and cytopathology 3:512–513 prevention and control 3:514–515 future perspectives 3:515 replication 3:512 taxonomy and classification 3:507–510 transmission and epidemiology 3:513 oryzaviruses 3:551, 3:547–548 Oseltamivir 5:170, 5:164, 5:171 plus Favipiravir 5:171 plus monoclonal antibodies 5:171 OSHBV see Orinoco sheldgoose (OSHBV) osmolyte, defined 4:167 osmotic pressure, defined 4:167 o-spanin, defined 1:501 OTUs see operational taxonomy units (OTUs) ourmiaviruses 3:516 diagnosis 3:518 epidemiology and control 3:518 genome organization 3:516 properties and functions of gene products 3:516–517 replication and propagation 3:517–518 taxonomy, phylogeny and evolution 3:516 transmission, host range 3:518 virion structure 3:516 virus–host relationships 3:518 outbreak, defined 1:569, 5:247 Ovaliviridae 4:362 overarching framework for icosahedral architectures 1:251–253 overwintering 2:893 ovine pulmonary adenocarcinoma (OPA) 2:575, 2:579–580 oviposition, defined 3:461, 4:849 ovis aires papillomavirus type 3 (OaPV-3) DNA 2:87 OVs see oncolytic viruses (OVs) OW see Old-World (OW) Oxford Nanopore technologies 1:181–182

P P1 protein 4:28 P2 protein 4:28–30 P3 movement protein 3:574 P4 protein 4:30 P7 protein 4:30 P8 protein 4:30 p24 protein 2:145–148 p75 neurotrophin receptor (p75NTR) 2:738–739 PA see protective antigen (PA); protein antibiotics (PA) pac, defined 4:61 Pacheco’s parrot disease (PPD) 2:113 Pacheco’s parrot disease virus (PPDV) 2:113 Pacific Biosciences 1:181 packaging, defined 4:26, 4:136 packaging, termination of 4:132–133 terminase ejection and virion completion 4:133–134 unit length packaging motors 4:133 packaging signal, defined 1:248, 1:488 packaging signal mediated assembly, defined 1:248, 1:488 packaging signals (PSs) 1:249, 1:254–255 PACR protein see poxvirus APC/cyclosome regulator (PACR) protein PaFlV1 see penicillium aurantiogriseum foetidus-like virus 1 (PaFlV1) pag1 miRNAs 4:832–833 PAGE see polyacrylamide gel electrophoresis (PAGE) pAGO see prokaryotic Argonaute (pAGO) pairwise evolutionary distances (PED) 4:804 pairwise sequence comparison (PASC) 1:103f, 2:507 advantages of 1:104–105 description and application 1:102–104 limitations of 1:105–107 sequence-based virus classification methods 1:101–102, 1:105t DEmARC (DivErsity pArtitioning by hieRarchical Clustering) 1:101–102 Genome Relationships Applied to Virus Taxonomy (GRAViTy) 1:102 Natural Vector method 1:102 Sequence Demarcation Tool (SDT) 1:102 ViCTree 1:102 ViPTree 1:102 sequence comparison methods 1:100–101 virus classification methods 1:100 PaLCV, see Papaya leaf curl virus (PaLCV) Palenque lineage 2:809 paleovirology 1:79 endogenous retroviruses, evolutionary impact of 1:81–83 extant viral families, calibrating the longterm evolutionary history of 1:81 host-virus interactions 1:85–86 ecology of 1:84–85 non-retroviral EVE, evolutionary impact of 1:83–84 patterns and mechanisms underlying viral integration 1:79–81

367

palivizumab 5:157, 5:124, 2:754, 5:273 PAM see protospacer adjacent motifs (PAM) PAM-dependent DNA interference, defined 4:400 PAMPs see pathogen-associated molecular patterns (PAMPs) PAMV see Potato aucuba mosaic virus (PAMV) pandemic, defined 1:569, 5:247, 3:21, 3:301 pandemic influenza 5:160 pandoravirus 1:257 panicoviruses classification 3:456 diagnosis 3:459 epidemiology 3:458–459 genomes 3:456–457 hosts and distribution 3:457–458 life cycle 3:458 pathogenesis 3:459 prevention 3:459 virion structure 3:456 Papanivirus 3:581, 3:582–584 Papaya leaf curl virus (PaLCV) 3:357 Papaya mosaic virus (PapMV) 3:628 papaya ringspot virus (PRSV) 3:91–92 diagnostics 3:523–524 general properties and genome of 3:521 history and taxonomy 3:520 host range, symptomatology, and geographic distribution 3:521 molecular determinants for biological properties 3:523 “PRSV cluster” 3:520–521 sequence diversity and evolution; molecular clock analyzes 3:521–523 transmission and epidemiology 3:521 virus control 3:524 breeding for resistance 3:524 cross-protection 3:524 cultural practices: prophylactic measures 3:524 pathogen-derived resistance for controlling PRSV 3:524–525 transgenic resistance 3:525 papaya sticky disease 4:662 papillomas 2:83–84, 2:79 cats 2:85 diagnosis 2:89–90 dogs 2:85 horses 2:84–85 ruminants 2:83–84 treatment 2:90 papillomaviruses 2:493, 2:86, see also human papillomavirus (HPV) classification 2:79–81 clinical features 2:83–84 diagnosis 2:88–90 papillomas 2:89–90 sarcoids 2:90 squamous cell carcinomas and other neoplasms 2:90 viral plaques 2:90 epidemiology 2:83 genome 2:81–82 life cycle 2:82–83 neoplasms 2:88 papillomas 2:83–84

368

Subject Index

papillomaviruses (continued) cats 2:85 dogs 2:85 horses 2:84–85 ruminants 2:83–84 pathogenesis 2:88 prevention 2:91 sarcoids 2:87–88 cats 2:88 horses 2:87–88 squamous cell carcinomas 2:86–87 cats 2:87 dogs 2:87 horses 2:87 rabbits 2:87 ruminants 2:86–87 treatment 2:90 papillomas 2:90 squamous cell carcinomas and sarcoids 2:90–91 viral plaques 2:90 viral plaques 2:85 cats 2:85–86 dogs 2:85 horses 2:85 virion structure 2:81 PapMV see Papaya mosaic virus (PapMV) papule, defined 2:868 paraesthesia, defined 2:738 parainfluenza virus (PIV) 1:191 classification 2:502 clinical features 2:504–505 diagnosis 2:505 epidemiology 2:503–504 genome 2:502 life cycle 2:502–503 pathogenesis 2:505 treatment and prevention 2:505–506 virion structure 2:502 parallel sequencing technologies 5:29–31 application of NGS in virology 5:30–31 paralog, defined 3:69 Paramecium bursaria chlorella virus 1 (PBCV-1) 1:269–270, 1:270 Paramyxovirus 5:260 paraphyletic clade, defined 3:653 paraphyly, defined 4:835 parapoxviruses (PPVs) 2:666, 2:165 clinical features 2:667–668 bovine papular stomatitis (BPS) 2:668, 2:668f Milker’s nodule, paravaccinia, pseudocowpox 2:668 orf, scabby mouth, contagious pustular dermatitis (CPD), ecthyma contagiosum 2:667–668 red deer, parapox of 2:668 diagnosis 2:673 genomes 2:669 host range, epidemiology, and virus propagation 2:666–667 immune response to infection 2:673 known or putative genes involved in pathogenesis and virulence 2:670–671

ankyrin repeat (ANK)/F-box genes 2:672 apoptosis, inhibitor of 2:672 chemokine binding protein (vCBP) 2:671–672 NF-kB Inhibitors 2:672 poxvirus APC/C regulator (PACR) 2:672 VEGF-E 2:670–671 viral dUTPase 2:671 viral GM-CSF inhibitory factor (GIF) 2:671 viral IL-10 2:671 viral interferon resistance protein 2:671 oncolytic potency 2:674 ORFV vector vaccines 2:673–674 pathogenesis and the host immune response to infection 2:672–673 physical properties 2:668 prevention and control 2:674 of red deer in NZ 2:171 serologic relationships 2:667 taxonomy and classification 2:666 viral proteins 2:670 viral replication 2:666–667 virion properties 2:668–669 pararetrovirus, defined 3:667, 3:274 parasitism 1:606 parasitoid wasp, defined 4:849 paravaccinia 2:668 parechoviruses 1:279–280 classification 2:675 clinical features 2:679–680 virus genetics and association to disease 2:680 diagnosis 2:680–681 epidemiology 2:677–679 Australia 2:679 Japan 2:679 parechovirus species B-D 2:679 seroprevalence 2:678–679 genome 2:676–677 life cycle 2:677 pathogenesis 2:680 treatment and prevention 2:681 virion structure 2:675–676 pariacoto virus 1:250, 1:477, 1:478f paritaprevir 5:123 parr, defined 2:544 Parrot hepatitis B virus (PHBV) 2:100 Partitiviridae 4:568 partitiviruses 4:544, 4:508, 4:454 partitiviruses (Partitiviridae) – fungal 4:568 classification 4:568–569 epidemiology 4:572–573 genome 4:569–570 life cycle 4:570–572 pathogenesis 4:573–574 taxonomic and phylogenetic considerations 4:574–576 virion structure 4:569 Parton genome 2:808–809 Parvoviridae 4:836, 4:837f, 2:419 classification 2:419–420 clinical features 2:424–425 human bocaviruses 2:424–425 human protoparvoviruses 2:425

diagnosis 2:425–426 epidemiology 2:422–424 human bocaviruses 2:422–424 human protoparvoviruses 2:424 genome 2:420–422 life cycle 2:422 pathogenesis 2:425 prevention 2:426 virion structure 2:420 parvoviruses (PVs) 4:835 of carnivores classification (compact) 2:683 diagnosis 2:687 epidemiology 2:684–686 life cycle 2:683–684 pathogenesis and clinical features 2:686–687 prevention 2:687 treatment 2:687 virion structure and genome 2:683 of insects 4:711–713 Polydnaviridae 4:712–713 of invertebrates 4:835–837 biochemical properties and purification of densoviruses 4:840–841 biophysical features and functions associated with densovirus capsid 4:843–844 densovirus genome structure and replication 4:844–845 discovery, taxonomy and evolution of densoviruses 4:835–837 expression strategies 4:845–848 general features 4:839 shrimp densoviruses 4:839–840 structural features of virions 4:841–843 PASC see pairwise sequence comparison (PASC) Passeriform birds 2:810–811 Passeriformes 2:811 passerine, defined 2:343 PAT1 see persistency-associated transcript 1 (PAT1) patatin-like phospholipase-1 (PLP-1) 4:524–525 pathogen, defined 2:441 pathogen-associated molecular patterns (PAMPs) 2:603, 2:475, 1:9, 1:585, 1:659, 1:601–602, 1:577 pathogen derived resistance (PDR) 3:362 antisense RNA technology 3:362 RNAi technology 3:362–363 pathogen-derived resistance, defined 3:355, 3:520 pathogenesis, defined 2:884, 2:442 pathogenic bacteria, recombineering in 4:298–299 pathogenicity, defined 2:697, 4:53, 4:888, 4:768 pathogenicity determinant protein 3:239 pathogenicity determinants, defined 3:116 pathogen recognition receptors (PRRs) 2:603 pathognomonic, defined 2:814 pathogroup, defined 3:184 pathosystem, defined 3:8

Subject Index pathotype, defined 3:184 pattern recognition receptors (PRRs) 2:611–612, 2:480–481, 1:577, 1:658 pattern-triggered immunity (PTI) 3:32 PBCV-1 see Paramecium bursaria chlorella virus 1 (PBCV-1) PBL see peripheral blood leukocytes (PBL) PBMCs see peripheral blood mononuclear cells (PBMCs) PBS see primer binding site (PBS) PCD see programmed cell death (PCD) PCNA see proliferating cell nuclear antigen (PCNA) PCPV see pseudocowpox virus (PCPV) PCR see polymerase chain reaction (PCR) PCV-2 systemic disease (PCV-2-SD) 2:187 PCVD see porcine circovirus diseases (PCVD) PDA see potato dextrose ager (PDA) PDB see Protein Data Bank (PDB) PDD see proventricular dilatation disease (PDD) PDGFR see platelet-derived growth factor receptor (PDGFR) PDNS see porcine dermatitis and nephropathy syndrome (PDNS) PDR see pathogen derived resistance (PDR) PDVs see polydnaviruses (PDVs) PeaLDV 3:751 pecluviruses (Virgaviridae) assembly of virus particles 3:534 cultural practices 3:537 host plant genetic resistance 3:537 detection and diagnosis 3:534–535 immunoassays 3:535 molecular assays 3:535 disease control 3:536–537 epidemiology and disease cycle 3:535–536 disease development cycle 3:535–536 future perspectives 3:537 geographic and seasonal distribution 3:529 history 3:528 host range and symptoms 3:529–531 molecular properties 3:532 5’ and 3’ noncoding sequences 3:532–533 coding sequences 3:532 genome structure and expression strategies 3:532 sequence comparisons and phylogeny 3:533–534 physical properties 3:531–532 taxonomy and classification 3:528–529 transmission 3:531 seed and sap transmission 3:531 soil-borne vector transmission 3:531 vector 3:535 virion morphology 3:531 PED see pairwise evolutionary distances (PED) PEDV see porcine epidemic diarrhea virus (PEDV) PEG-IFN see pegylated interferon alpha (PEG-IFN)

PEG precipitation see polyethylene glycol (PEG) precipitation PEGylated, defined 2:814 pegylated interferon alpha (PEG-IFN) 5:223, 5:217 PEL see primary effusion lymphoma (PEL) Pelagibacterales 4:332–334 PemoNPV see penaeus monodon nucleopolyhedrovirus (PemoNPV) PEMS see poult enteritis mortality syndrome (PEMS) penaeid, defined 4:768 penaeid shrimp virus 2:245 penaeus monodon baculovirus 4:828–829 penaeus monodon nucleopolyhedrovirus (PemoNPV) 4:828–829 penaeus monodon nudivirus (PmNV) 4:828–829 penaeus monodon singly enveloped nuclear polyhedrosis virus (PmSNPV) 4:828–829 penaeus stylirostris penstyldensovirus 1 (PstDV1) 4:839–840 penetration, defined 1:382 penicillium aurantiogriseum foetidus-like virus 1 (PaFlV1) 4:660 Penicillium chrysogenum 4:547–548 penstyldensoviruses 4:848 Penstylhamaparvovirus 4:836 pentamers 1:250–251 penton, defined 2:442 penton base protein 1:331 PEP see post-exposure prophylaxis (PEP) pepino mosaic virus (PepMV) 3:539, 1:649, 1:364–365, 1:365f, 3:626–628, 3:628–629 diagnostic 3:543 genome organization and expression 3:540 host range and symptomatology 3:540–541 taxonomy and classification 3:539 virion properties 3:539–540 virus damage and control 3:543–544 virus isolates and strains 3:541–542 virus transmission and epidemiology 3:542–543 PepMV see pepino mosaic virus (PepMV) pepper, potato virus Y (PVY) in 3:619–620 Pepper leaf curl Lahore virus 3:750 peptide-loaded MHC complexes (pMHC) 1:585 peptidoglycan, defined 1:402, 4:194 perch rhabdovirus (PRV) 2:325, 2:328 percutaneous, defined 2:373 performance qualification (PQ) 5:69 pericardial fluid, defined 2:875 perinatal, defined 2:373 Perinet virus (PERV) 2:875 periodontal disease 2:454 peripheral, defined 2:778 peripheral blood leukocytes (PBL) 2:283 peripheral blood mononuclear cells (PBMCs) 2:189, 2:778, 2:148 Periplaneta fuliginosa 4:839 peritrophic matrix, defined 4:780

369

peritrophic membrane, defined 4:858 permafrost 4:342, 4:353–354 Permutotetraviridae 4:897–898, 4:717 permutotetravirus REPs 4:897–898 per os infectivity factor (PIF) 4:832 perpetuation of viruses in nature 1:561 persistence, defined 2:442 persistence length, defined 4:167 persistency-associated gene 1 (pag1) 4:830 persistency-associated transcript 1 (PAT1) 4:830 persistent infection, defined 4:827, 1:542, 2:797 persistent lymphocytosis (PL) 2:144 persistent or chronic infection, defined 1:71 persistent viral transmission, defined 3:719 personal protective equipment (PPE) 5:82, 5:84, 5:68 person-to-person transmission 5:208 PERV see Perinet virus (PERV) Peste-des-petitsruminants virus (PPRV) 2:73–74 pestiviruses classification 2:153 clinical features and pathogenesis 2:159 border disease (BD) 2:161 bovine viral diarrhea and mucosal disease 2:160 classical swine fever (CSF) 2:159 epidemiology 2:158 bovine viral diarrhea and border disease 2:158–159 classical swine fever (CSF) 2:158 genome structure and polyprotein processing 2:153–154 life cycle 2:157–158 prevention and control 2:161 border disease of sheep, control of 2:163 bovine viral diarrhea, vaccines against 2:162 bovine virus diarrhea, control of 2:163 classical swine fever, vaccines against 2:161–162 classical swine fever in domestic pigs, control of 2:162 classical swine fever in wild boar, control of 2:162–163 diagnosis 2:161 vaccines 2:161–162 virion structure 2:154–156 Npro 2:156 NS2 2:156 NS2-3 2:156–157 NS3 2:156 NS4A 2:157 NS4B 2:157 NS5A 2:157 NS5B 2:157 p7 2:156 PFGE see pulsed-field gel electrophoresis (PFGE) PFS see protospacer-flanking site (PFS) PFV1 see pyrobaculum filamentous virus 1 (PFV1) pgRNA see pregenomic RNA (pgRNA)

370

Subject Index

phage 4:342, see also bacteriophages adsorption 4:255 biochemistry 4:6–7 and biotechnology 4:7–8 defined 4:242 development as diagnostics for bacterial infections 4:254–255 as therapeutics for bacterial infections 4:253–254 discovery of 4:3–4 ecology 4:316–317 and evolution 4:8 phage community ecology 4:320 phage ecosystem ecology 4:320–321 phage organismal ecology 4:317–318 phage population ecology 4:318–320 genetics 4:5–6 in hypersaline environments 4:343–345 in polar and other cold environments 4:349–352, 4:354 glaciers 4:352–353 permafrost 4:353–354 polar lakes 4:353 polar oceans 4:352 sea ice 4:353 structure and assembly 4:24–25 therapy 4:4 in thermal environments 4:345–347 deep-sea hydrothermal vents 4:347–348 hot deserts 4:348–349 terrestrial hot springs 4:345–347 typing 4:4–5 phage cluster, defined 4:265 phage diagnostics, additional challenges for 4:257 intracellular bacteria 4:257 market acceptance 4:257 multiplex testing 4:257 phage manipulation 4:257 sample interference 4:257 phage display, defined 4:252 phage diversity examples of 4:268–269 bottom up studies 4:269 contribution of prophages to phage diversity 4:270 Enterobacteriales tailed phage example 4:269–270 human phageome 4:268–269 marine phageome 4:269 top down studies 4:268–269 two large diversity studies 4:269 and horizontal exchange of genetic information 4:270–272 nature of horizontally exchangeable mosaic section alleles 4:270–272 strategies for studying 4:267–268 types of bacteriophages 4:266t phage existence within environments 4:314–315 microscopic determination 4:315–316 phage isolation and host range 4:315 phage genome and protein ejection in vivo contractile long-tailed phages 4:211

ejection of proteins into the host cell at the initiation of infection 4:206–207 factors affecting phage genome translocation 4:211–214 non-transcriptional genome internalization in vivo 4:214–215 transcription-mediated genome internalization in vivo 4:213–214 models of 4:215–218 non-contractile long-tailed phages (Siphoviridae) 4:209–211 protein ejection and trans-envelope channel formation 4:206–207 short-tailed phages (Podoviridae) 4:208–209 tail-less phages 4:207–208 phage-host interactions, ecology and evolution of 4:181–182 phage-inducible chromosomal islands (PICIs) 4:98–99, 4:99–100, 4:101 defined 4:98 PICI-like elements (PLEs) 4:99–100, 4:98 phage lambda 1:460, 1:635 post-infection decision of 4:88–90 phage lysis, definition of 1:502–503 phageome, defined 4:265 phage-receptor interactions’ relevance to phage therapy 4:182–183 phage receptors properties of 4:176–181 techniques and methods for studying 4:175–176 phage satellites, mobilization of 4:98–99 counter-evolution by phages to avoid parasitism by satellites 4:102–103 genetic organization 4:99 in human health and disease 4:103–104 integration and excision, gene induction, and replication 4:99–101 packaging and horizontal transmission 4:101 prevalence of 4:102 strategies PICIs use to interfere with their helper phages 4:101–102 phage T4 lysis inhibition 1:510–512 phage technology major advantages of 4:255 self-replication 4:255 specificity 4:255 major limitations of 4:255 narrow host range 4:255 phage resistance 4:255 phage therapy 1:676 additional challenges for 4:255–256 chemistry, manufacturing, and controls (CMC) 4:256 dosing strategies 4:256 environmental impact 4:256–257 preclinical data translation 4:255–256 regulatory pathway 4:256 safety and efficacy 4:256 defined 4:283 phage typing 4:254 defined 4:252 phagocytosis 1:584 defined 1:577

pham (or phamily), defined 4:276 Phase 3 compost, defined 4:528 phase variation system, defined 4:387 Phasmaviridae 4:718 PHBV see Parrot hepatitis B virus (PHBV) phenotype, defined 1:62 phenotyping 5:185–186 Phenuiviridae 4:718, 3:720 Phi29 connector 4:308 Phi29 motors 4:151–152 CryoEM structure of 4:149f terminase and 4:148–151 PhiH repressor, defined 4:380 phleboviruses in the candiru antigenic complex 2:771 phlegiviruses 4:627 biological properties 4:630 evolutionary relationships among phlegiviruses 4:630–631 future perspectives 4:631 genome organization 4:628–630 virion properties 4:627–628 phloem, defined 3:456 phloem-limited, defined 3:594 phloem-limited virus, defined 3:200 phosphatidylethanolamine 4:40 phosphatidylglycerol 4:40 phosphoinositide 3-kinase (PI3K) 1:534 defined 2:875 phospholipids, defined 1:345 phosphoproteins 1:350–351, 3:573–574, 2:442–443 phosphorodiamidate morpholino oligomers (PMOs) 2:615–616 photolyase, defined 2:343 Phycodnaviridae 4:687–688 phycodnaviruses (Phycodnaviridae) ecology 4:692–693 genomes 4:690–691 history 4:687 phycodnavirus genes 4:693–694 resistance to phycodnavirus infections 4:693 taxonomy and classification 4:687–688 virion structure and composition 4:688–689 carbohydrates 4:690 lipids 4:689–690 morphology 4:688–689 nucleic acids 4:689 physicochemical and physical properties 4:689 proteins 4:689 virus replication 4:691–692 virus transcription 4:692 phylodynamics, defined 1:87 phylogenetic analysis 1:101 phylogenetic clade, defined 4:776 phylogenetic diversity 4:677 phylogenetic host breadth 5:257 phylogenetics 4:677, 2:3 phylogenetic tree, defined 2:182 phylogeny, defined 1:62, 4:858, 3:818 phylogeny of viruses 1:117f applications 1:121–122 evolution, phylogeny, and viruses 1:116

Subject Index phylogenetic analysis 1:118–121 tree definitions 1:116–118 physical studies of viruses 2:414–415 physico-chemical phase, of virology 1:5–6 phytopathogen, defined 3:388 phytoreovirus 1:303, 4:878, 4:879–881, 3:548, 3:551 phytosanitation, defined 3:293 phytoviruses, defined 3:106 p-p stacking, defined 4:10 PI3K see phosphoinositide 3-kinase (PI3K) PIC see pre-integration complex (PIC) PICIs see phage-inducible chromosomal islands (PICIs) Picobirnavirus 1:253 Picornavirales 4:792, 4:768 Picornaviridae 1:253–254 classification of 2:688–689 picornaviruses 2:757–758, 1:452–454, 1:278, 1:279–282, 1:280f capsid assembly 1:280–282 genome encapsidation 1:282–283 host interactions 1:283–285 piercing-sucking insects, defined 3:106 Pieris rapae 4:772 PIF, see per os infectivity factor (PIF) PIF proteins, defined 4:747 “Piggyback-the-Winner” model 1:638 Pimodivir 5:168 and Oseltamivir 5:171 pinholins and SAR endolysins 1:507–508 endolysin diversity 1:512–513 holin diversity 1:512 lysis in mycobacteria 1:514 second type of spanin 1:508–509 spanin diversity 1:513 pinwheel, defined 3:631 PIPO see Pretty Interesting Potyviral ORF (PIPO) PIRYV see Piry virus (PIRYV) Piry virus (PIRYV) 2:875 PIs see protease inhibitors (PIs) piscine myocarditis virus (PMCV) 4:582 Pitaya virus X (PiVX) 3:623–624, 3:628 pithovirus 1:257 PIV see parainfluenza virus (PIV) PiVX see Pitaya virus X (PiVX) PKR see protein kinase R (PKR) PL see persistent lymphocytosis (PL) PlAMV 3:628 Plantain virus X (PlVX) 3:623, 3:623–624 plant and protozoal partitiviruses 4:632–633 current taxonomy of partitiviridae 4:633 genome organization and replication strategy 4:637 5’ and 3’UTRs 4:637–639 genome organization 4:637 replication strategy 4:640 RNA1640 transmission 4:640–641 virion properties 4:633–635 alphapartitivirus 4:635 betapartitivirus 4:635 cryspovirus 4:635–636 deltapartitivirus 4:636–637 virus-host-interaction 4:641

plant antiviral defense: gene-silencing pathways 3:43 antiviral RNA silencing (av-RNAi) 3:45–47 applications of 3:49–51 antiviral silencing in crops 3:47–49 diverse gene-silencing pathways in plants 3:43–45 viral and plant RNA-silencing suppressors 3:47 viral siRNA-mediated antiviral RNAi in mammalian cells 3:49 plant endornaviruses 3:388–389 plant-fungal mutualistic associations, role of mycoviruses in 4:441 plant reoviruses (Reoviridae) 3:545 diagnosis and control 3:552–553 distribution 3:551 future 3:553 host range and virus transmission 3:551 fijiviruses 3:551 oryzaviruses 3:551 phytoreoviruses 3:551 pathogenicity 3:551–552 replication and gene expression 3:548–551 taxonomy and classification 3:545–546 virion structure and genome organization 3:546–547 fijivirus 3:546–547 oryzavirus 3:547–548 phytoreovirus 3:548 plant resistance to viruses 3:60, 3:52–53, 3:69–70 application of recessive resistance in agriculture 3:78–79 benefits of virus-resistant transgenic plants to agriculture 3:54–55 cloned recessive resistance genes 3:73–74 eIF4E-mediated recessive resistance characteristics of 3:74–76 molecular mechanisms of 3:76–77 environmental and human health safety issues 3:55–59 field resistance and commercial adoption 3:54 historical perspective on engineered resistance against plant viruses 3:53 mechanisms of recognition 3:62–63 molecular underpinnings of engineered resistant to plant viruses 3:53–54 naturally occurring recessive resistance 3:70–73 new recessive resistance genes found in natural diversity 3:77–78 non-NB-lRR resistance 3:66–67 resistosome 3:64–66 R gene products 3:60–61 R gene responses to viruses 3:61–62 signaling mechanisms 3:63–64 plant rhabdoviruses see rhabdoviruses plants and plant cell culture 1:664–665 plant satellite viruses 3:581 Albetovirus 3:581–582 Aumaivirus 3:582 effect of mixed infections on host and helper viruses 3:584

371

Papanivirus 3:582–584 plant satellite viruses 3:584–585 Virtovirus 3:584 plant viral diseases 3:81–83 cash crops viral diseases 3:94–95 ornamentals and orchids viral diseases 3:95–96 sugarcane viral diseases 3:94–95 cereal viral diseases 3:83 fruit virus diseases 3:89–90 banana viral diseases 3:90 cacao swollen shoot disease (CSSD) 3:90 citrus tristeza virus (CTV) 3:90–91 papaya ring spot virus 3:91–92 plum pox virus (Sharka) disease of stone fruits 3:92 legume viral diseases 3:92–94 root and tuber crops viral diseases 3:83–85 cassava viral diseases 3:83–85 potato viral diseases 3:85–86 sweet potato viral diseases 3:86 vegetable viral diseases 3:86–87 chilli viral diseases 3:86–87 cucurbit viral diseases 3:87–88 okra viral diseases 3:88 tomato viral diseases 3:88–89 plant viruses 1:5–6, 3:3 biochemical/biophysical age 3:4–5 biological age 3:4 host and vector ‘manipulation’ by 3:113–115 molecular biology age 3:5–7 prehistory 3:3 recognition of viral entity 3:3–4 in their invertebrate vectors 4:722, 4:721t plant viruses, emerging and re-emerging 3:8–9 grapevines viruses 3:14–15 grapevine pinot gris virus 3:16 grapevine roditis leaf discolorationassociated virus 3:15 ornamental viruses: rose rosette virus 3:19 staple crops: cassava brown steak disease 3:17 vegetable viruses 3:9 begomoviruses 3:13–14 cucumber green mottle mosaic virus 3:9 orthotospoviruses 3:12 squash leaf curl virus 3:14 tobamoviruses 3:9 tomato brown rugose fruit virus (ToBRFV) 3:10–11 tomato spotted wilt virus 3:12 plant viruses transmission by insects 3:106–107 beetles, transmission by 3:112–113 circulative transmission 3:107–108 circulative non-propagative transmission 3:107–108 circulative propagative transmission 3:110 history of typology of transmission modes 3:107

372

Subject Index

plant viruses transmission by insects (continued) non-circulative transmission 3:110–112 capsid strategy 3:112 helper strategy 3:112 plant viruses transmission by non-insect vectors 3:113 fungi and protists, transmission by 3:113 mites, transmission by 3:113 nematodes, transmission by 3:113 plant virus vectors 3:106 plaque, defined 1:162, 4:314, 4:276 plaque assay, defined 4:265 plaque-forming unit (Pfu), defined 4:21, 2:442 plaque reduction neutralization test (PRNT) 2:907, 2:641, 5:94 plasmid, defined 4:276 plasmodesmata 1:495, 3:788, 3:383, 3:313, 4:478, 3:456 plasmodiophorida, defined 3:528 plastochron, defined 3:461 platelet-derived growth factor receptor (PDGFR) 2:450–451 Plautia stali 4:771 Plautia stali intestine virus (PSIV) 4:771 pleiotropic mutation, defined 3:69 Pleolipoviridae 4:382–384 pleolipovirus, defined 4:380 pleolipovirus His2 1:433–434 pleolipovirus infectivity, stability of 4:384 salinity 4:384 temperature and other significant factors 4:384–385 pleolipovirus virion structure 4:382f pleomorphic, defined 2:738 pleomorphic, spindle-shaped, and spherical viruses 4:376 pleomorphic viruses 4:376 spherical virus MetSV 4:377–378 spindle-shaped viruses 4:376–377 pleomorphic DNA viruses 1:407 pleomorphic viruses 4:376, 4:359 pleomorphism 2:68–69 Pleosporales megabirnavirus 1 (PMbV1) 4:600 PLP-1 see patatin-like phospholipase-1 (PLP-1) plum pox virus (Potyviridae) 3:586 detection 3:591 ecology and control 3:592 economical importance 3:586 history and geographical distribution 3:586–587 host range and symptomatology 3:587 virion structure and genome properties 3:587 strains/molecular groups 3:587–591 virus spread 3:591–592 plum pox virus (Sharka) disease of stone fruits 3:92 (+)-sense RNA (plus-sense RNA) 1:382 plus-strand RNA virus, defined 2:797 Plutella xylostella 4:772 PLV see puma lentivirus (PLV) PlVX see Plantain virus X (PlVX)

PMbV1 see Pleosporales megabirnavirus 1 (PMbV1) PMCV see piscine myocarditis virus (PMCV) PMF see proton-motive force (PMF) pMHC see peptide-loaded MHC complexes (pMHC) PML see progressive multifocal leukoencephalopathy (PML) PmNV see penaeus monodon nudivirus (PmNV) PMOs see phosphorodiamidate morpholino oligomers (PMOs) p/mpMLVs see polytropic and modified polytropic MLVs (p/mpMLVs) PmSNPV see penaeus monodon singly enveloped nuclear polyhedrosis virus (PmSNPV) pneumonia 2:762 pneumonia virus of mice (PVM) 2:754 Pneumoviridae genome organization 2:748f poaceae, defined 3:456 pock, defined 2:868 Podoviridae 1:320, 4:186, 4:276 mosaicism in 4:280 podoviruses 1:406, 4:342 Pogosta disease 2:840 poinsettia mosaic virus 3:824–825 properties and distinguishing characteristics 3:825 Point-of-Care (PoC) clinical impact 5:48–50 Point-of-Care (PoC) menus 5:48 Point-of-Care (PoC) strategy and implementation 5:46 Laboratory Grade 1A 5:46 Laboratory Grade 1B 5:46 Laboratory Grade 2A 5:46 Laboratory Grade 2B 5:46–47 Laboratory Grade 3 5:47–48 Point-of-Care (PoC) technologies 5:45–46, 5:47f point-of-care serodiagnostics 5:20 Point-of-Care-tests, defined 5:98 polar and other cold environments, phages in 4:349–352, 4:354 glaciers 4:352–353 permafrost 4:353–354 polar lakes 4:353 polar oceans 4:352 sea ice 4:353 polar lakes 4:353 polar oceans 4:352 poleroviruses (Luteoviridae) 3:594–595 beet poleroviruses 3:599–600 defined 3:594 diagnosis 3:600–601 disease management 3:601 genome organization and gene expression 3:595–596 phylogenetic diversity 3:596–597 physical properties 3:595 potato leafroll virus 3:599 silencing suppression by 3:599 sugarcane yellow leaf virus 3:599 transmission 3:597–598 virus-virus interactions 3:598–599 pol gene 2:57–58, 2:61t

POL inhibitors see polymerase (POL) inhibitors polintons, defined 1:329 polintons and evolutionary pathway of DJR viruses 1:342–343 polintovirus, defined 1:372 polio 1:668–669 eradication antivirals 5:313 background 5:310 containment 5:313 new vaccines 5:313 poliovirus surveillance 5:311–312 status of eradication 5:312–313 transition of global polio eradication initiative assets post-eradication 5:313–314 vaccines 5:310–311 poliomyelitis 2:808 polioviruses 2:688, 2:689 cessation of vaccination 2:695 global polio eradication program 2:690–692 pathogenesis and disease 2:689 Picornaviridae, classification of 2:688–689 poliovirus receptor 2:688 Sabin vaccine strains, attenuation of 2:690 vaccine-derived poliovirus 2:692–695 vaccines 2:689–690 virus genome 2:688 virus particle 2:688 poliovirus receptor (PVR) 1:284 pollen-borne, defined 3:430 poly(A) binding protein (PABP) 1:444, 3:132 polyacrylamide gel electrophoresis (PAGE) 3:586 polyadeniration, defined 4:544 Polycipiviridae 4:718 polycistronic RNA, defined 4:21 polyclonal immunoglobulins 5:268 hepatitis A 5:268 measles 5:268–269 Polydnaviridae 4:712–713, 4:849 polydnaviruses (PDVs) 4:712 classification (compact) 4:849 fate of PDV packaged genome in parasitized insect 4:853–854 function of the genes encoded by PDV packaged genome 4:853 life cycle of 4:849–850 morphogenesis of PDV particles 4:851 organization of PDV proviral segments in the wasp genome 4:854–856 packaged genome 4:851–853 proviral genome maintained in the wasp genome 4:854 viral machineries 4:856–857 virion structure 4:850–851 polyethylene glycol (PEG) precipitation 1:167 polyextreme environment 4:342 polyhedrin, defined 4:739, 4:747 polyhedron, defined 1:248, 4:739 polyhedrosis 4:704 polylysogeny 1:639–640, 1:638–640

Subject Index polymerase (POL) inhibitors 5:164–166 Baloxavir – Marboxyl (Xofluzas) 5:166–168 Favipiravir (Avigans) 5:168 Pimodivir 5:168 Ribavirine (Ribavirins) 5:164–166 polymerase 1:350 polymerase chain reaction (PCR) 1:8, 2:3, 1:9, 1:622–623, 2:301, 5:91, 2:640–641, 2:820, 2:722–723, 2:735, 4:324–325, 5:98, 3:192 assays 5:92 -based amplification 3:759 -based methods 3:629 defined 5:27, 3:430 detection of viral RNA 2:663–664 for human adenoviruses DNA detection 5:202–203 and its evolution 5:112 technique 1:673 testing 5:177 polymerase complex, defined 4:26 polymerase protein 3:575 polymerase selectivity, factors determining 1:54 polymeric immunoglobulin receptor (pIgR) 1:529, 1:531, 1:533 polymycoviruses 4:454–455 polyomavirus associated nephritis (PVAN) 5:108 polyomaviruses (PyVs) 2:518–519, 5:108–109, 1:498, 1:392, 2:526–527 BK polyomavirus (BKPyV) 2:522 clinical features 2:522 infectious cycle 2:522–523 genome 2:519–521 infectious cycle 2:521–522 JC polyomavirus (JCPyV) 2:523–524 clinical features 2:523–524 infectious cycle 2:524 Merkel cell polyomavirus (MCPyV) 2:524–525 clinical features 2:524–525 infectious cycle 2:525 of skin 2:526 virion structure 2:519 polypeptide III see penton base protein polypeptide IV see adenovirus fiber Polypeptide V 2:8 polyphyletic, defined 1:28, 4:457, 3:653 polyphyletic group of viruses 1:162–164 polyphyly, defined 4:835 polyprotein, defined 2:386, 1:382, 3:388, 3:631, 3:703, 3:797, 4:768 polyprotein processing, defined 2:362 polypurine tract (PPT) 2:59, 2:62–63, 3:96 polypyrimidine tract-binding protein (PTB) 2:252 polyribonucleotidyltransferase (PRNTase) 1:350 polythetic, defined 1:28 polythetic class, defined 1:47 polytropic and modified polytropic MLVs (p/mpMLVs) 2:646 pomoviruses (Virgaviridae) 3:603 genome properties and functions of the encoded proteins 3:604–605

host range, geographical distribution, transmission by vector and variability 3:606–609 physical properties of particles 3:603–604 serological relationships, diagnosis, and control 3:609–610 taxonomy and classification 3:603 virus–host interactions and movement 3:605–606 population genetic processes, studying direct selection and linked selection effects 5:228–229 evolutionarily informed treatment strategies 5:230 key aspects of population genetic environment 5:229–230 population dynamics of infection 5:227–228 populations, viral 1:53 basic population genetic processes shaping viral diversity 1:57–58 adaptation via directional selection of beneficial mutations 1:58 error catastrophe and lethal mutagenesis of viruses 1:58 frequency-dependent selection 1:58–59 mutation-selection balance 1:57–58 random genetic drift 1:59 viral quasispecies 1:58 diversity-generating retroelements 1:55 error-prone replication 1:53–54 lack of proofreading 1:54–55 polymerase selectivity, factors determining 1:54 viral polymerases, intrinsic selectivity of 1:54 post-replicative repair, role of 1:55 error-prone repair polymerases 1:55 repair avoidance 1:55 recombination 1:56 in DNA viruses 1:56–57 reassortment 1:56 template switching 1:56 viral disease, implications for 1:59 drug resistance 1:60 immune escape 1:59–60 short-term pathogenesis 1:59 viral hyper-mutation mediated by host enzymes 1:55 cellular adenosine deaminases 1:55–56 cellular cytidine deaminases 1:55 population size, influence of 1:563 porcine adenoviruses 2:12 Porcine circovirus 1 (PCV-1) 2:184 Porcine circovirus 2 (PCV-2) 2:184, 2:189f Porcine circovirus 3 (PCV-3) 2:185 porcine circovirus diseases (PCVD) 2:187, 2:188–189 porcine dermatitis and nephropathy syndrome (PDNS) 2:187 porcine epidemic diarrhea virus (PEDV) 2:198, 2:245 classification 2:850 clinical features 2:850–852 diagnosis 2:852 epidemiology 2:850

373

genome 2:850 genome organization of 2:851f life cycle 2:850 pathogenesis 2:852 prevention 2:852 replication cycle 2:851f treatment 2:852 vaccines for 2:852t virion structure 2:850 porcine reproductive and respiratory syndrome (PRRS) 2:697 porcine reproductive and respiratory syndrome virus (PRRSV) 1:447, 2:245 classification 2:697 clinical features 2:702 diagnosis 2:704–705 epidemiology 2:701–702 genome organization 2:698–700 immunity 2:703 life cycle 2:700–701 nonstructural and structural proteins of 2:699t pathogenesis 2:703 prevention and control 2:705–706 taxonomy of 2:697t virion structure 2:697–698 porcine respiratory disease complex (PRDC) 2:187 porencephaly, defined 2:34 portal, defined 1:318, 4:45, 2:442 portal proteins 1:323–324, 4:105, 4:107–108 actively assisting DNA packaging coupling with ATP hydrolysis 4:111 bound with adapter proteins facilitates tail attachment 4:109 as a check-valve preventing DNA from slipping out 4:110 help to spool condensed genomic DNA in the capsid 4:109–110 main techniques used to study protal protein structure and function 4:112 biochemical methods 4:112 molecular dynamics simulation 4:113 optical tweezers: a technique to observe DNA packaging in real time 4:113 planar bilayer membrane (BLM) technology 4:113 techniques for structural analysis 4:112–113 as a nucleator in the assembly of the prohead with proper morphology 4:108 portal directly-mediated packaging mode 4:111 lengthwise channel expansion and contraction in the portal protein may apply forces to DNA 4:111 rotation of the portal protein may apply forces to DNA 4:111 sequential movement of tunnel loops may apply force to DNA 4:111–112 portal indirectly-mediated packaging mode 4:112

374

Subject Index

portal proteins (continued) DNA translocation driven by a brownian motor inherent in the portal protein 4:112 DNA translocation driven by the energy from the DNA deformation in the portal protein 4:112 portal proteins actively assists DNA packaging at the late stage of DNA packaging 4:111 providing binding sites for terminases 4:108–109 as a sensor to control the DNA packaging 4:110–111 structural features of 4:107 terminase-driven mechanism plays a key role at the initial stage of DNA packaging 4:111 portal vertex 4:105–106 DNA packaging into the prohead 4:106 formation of a prohead 4:105–106 virion maturation 4:106–107 Portogloboviridae 4:362–363 positional cloning, defined 3:69 positive predictive value (PPV) 5:74 positive selection-based enrichment 1:179 positive sense (+) strand, defined 1:429 positive-sense, defined 4:776 positive-sense, single-stranded RNA virus 3:388 positive-sense RNA viruses 1:499 positive-strand RNA virus, defined 2:386 Pospiviroidae 3:852 post-exposure and pre-exposure prophylaxis (PEP and PrEP) 5:153 post-exposure prophylaxis (PEP) 5:164, 2:744 Post-F, defined 2:747 postfusion conformation, defined 1:417 post-larvae, defined 4:888 post-replicative repair, role of 1:55 error-prone repair polymerases 1:55 repair avoidance 1:55 post-transcriptional gene silencing (PTGS) 3:361, 4:632, 3:362–363, 3:788, 3:116, 3:149, 3:528, 3:123, 3:555 post-translational modifications (PTMs) 3:420, 1:664 post-transplantation/iatrogenic Kaposi’s sarcoma 2:606 post-transplantation lymphoproliferative disorders (PTLDs) 5:195 postural orthostatic tachycardia syndrome (POTS) 5:297–298 post zoster neuralgia, defined 5:181 potato, potato virus Y (PVY) in 3:616–618 Potato aucuba mosaic virus (PAMV) 3:623, 3:628 potato dextrose ager (PDA) 4:522 potato leafroll virus 3:599 potato viral diseases 3:85–86 potato virus X (PVX) 3:623, 3:626f, 3:627f, 3:626–628, 3:628, 3:628–629, 3:4, 1:68 potato virus Y (PVY) 3:612, 3:4, 1:68 control methods 3:618–619

host range 3:614–615 in pepper 3:619–620 phylogeny and evolution 3:616 in potato 3:616–618 serology 3:616 in tobacco 3:621–622 in tomato 3:620–621 transmission 3:615–616 viral particle, genome and cytopathology 3:612–614 Potexviruses (Alphaflexiviridae) diagnosis 3:629 genome organization 3:625–628 properties and functions of gene products 3:626–628 replication and propagation 3:628 species demarcation criteria 3:625 symptoms of potexvirus-infected plants 3:627f taxonomy, phylogeny, and evolution 3:623–625, 3:626f transmission, host range 3:628–629 epidemiology and control 3:628–629 virion structure 3:625 virus species in the genus Potexvirus 3:624t POTS see postural orthostatic tachycardia syndrome (POTS) potyvirid, defined 3:631, 3:456 Potyviridae 3:295, 3:296–297, 3:520, 3:520–521, 3:526, 3:586, 3:862, 3:631, 3:268, 3:184, 3:797, 3:612 potyviruses (Potyviridae) 3:631 diagnosis 3:640 epidemiology and control 3:638–640 pathogenicity 3:637–638 taxonomy, phylogeny and evolution 3:631–632 virion structure 3:632–634 expression of overlapping gene products 3:636–637 properties and functions of gene products derived from polyprotein 3:634–636 properties of genome 3:633–634 replication and propagation 3:637 poult enteritis mortality syndrome (PEMS) 2:96 power stroke, defined 4:148 poxvirus APC/cyclosome regulator (PACR) protein 2:672 poxviruses 2:165, 1:498 cervidpoxvirus 2:171 classification 2:165 genome 2:165–166 of invertebrates 4:713 life cycle 2:166–167 bovine papular stomatitis virus (BPSV) 2:170–171 lumpy skin disease virus 2:168 orf virus 2:169 parapoxvirus of red deer in NZ 2:171 pseudocowpoxvirus (PCPV) 2:171 sheeppox and goatpox virus 2:166–167 of ruminants 2:165t vaccinia virus (VACV) 2:171 virion structure 2:165

poxviruses of insects 4:858 classification 4:858–859 entomopoxvirus phylogeny 4:865 genomics 4:862–865 conserved and shared genes 4:863–865 gene families 4:865 replication in larvae 4:860–862 virus/midgut interactions 4:859–860 PPD see Pacheco’s parrot disease (PPD) PPDV see Pacheco’s parrot disease virus (PPDV) PPE see personal protective equipment (PPE) PpNSRV-1 see pteromalus puparum negative-strand RNA virus 1 (PpNSRV-1) P protein, defined 2:747 PPRV see Peste-des-petitsruminants virus (PPRV) PPT see polypurine tract (PPT) PPV see positive predictive value (PPV) PPVs see parapoxviruses (PPVs) PPXY late domain 1:522–523 PQ see performance qualification (PQ) practical applications 1:251 PRANC (Pox protein Repeats of Ankyrin Cterminal) domain 2:672 PRC see primary replicase complex (PRC) PRD1 genes 4:36, 4:37, 4:37–39, 4:38t PRD1 infection pathway 4:208f PRD1-like viruses 1:337–339 PRDC see porcine respiratory disease complex (PRDC) precipitation methods 1:167–168 pre-emptive therapy 5:191 Pre-Exposure Prophylaxis (PrEP) 2:473–474 Pre-F, defined 2:747 prefusion conformation, defined 1:417 pregenomic RNA (pgRNA) 3:164, 2:104 pre-integration complex (PIC) 2:644, 2:464 PrEP see Pre-Exposure Prophylaxis (PrEP) preparative ultracentrifugation methods 1:168–170 chromatographic methods 1:170–171 flow field-flow fractionation methods 1:171–172 purification performance, assessing 1:172 preprimosome, defined 4:61 preprotoxin, defined 4:534 Pretty Interesting Potyviral ORF (PIPO) 3:631 prevalence, defined 5:247, 1:559 previrology 1:3 primary effusion lymphoma (PEL) 2:604, 2:605, 2:606, 2:599 primary replicase complex (PRC) 2:130 primer binding site (PBS) 3:96 priming, defined 1:417 primosome, defined 4:61 principle of genetic economy, defined 1:248 prion, defined 2:707 prion incubation period, defined 2:707 prion protein (PrP), defined 2:707 prions of vertebrates 2:707 aberant prion protein metabolism 2:707–708

Subject Index future perspectives 2:712 human prion diseases 2:708 neuronal cell death in prion disease 2:712 prion disease pathogenesis 2:710–711 prion strains 2:711–712 protein-only hypothesis of prion propagation 2:708–709 structural properties of PrPSc 2:709–710 structure and putative function of PrPC 2:709 prion strain, defined 2:707 prM protein 2:885 PRNP, defined 2:707 PRNT see plaque reduction neutralization test (PRNT) PRNTase see polyribonucleotidyltransferase (PRNTase) procapsid, defined 4:10, 4:26, 4:160, 4:302, 1:318, 4:45 Procedovirinae 3:788, 3:481 processing bodies, defined 3:761 prodrome, defined 2:868 pro-drug, defined 2:404 prodrug convertases 5:241 productive/lytic infection, defined 4:827 proficiency testing (PT) 5:72, 5:76 progeny positive-strand RNAs 4:771 progeny virions 2:452 programmed cell death (PCD) 1:606, 1:607, 4:724 programmed death receptor 1 (PD-1) 1:660 programmed death receptor ligand-1 (PD-L1) 1:660 progressive multifocal leukoencephalopathy (PML) 5:109, 1:74 prohead 4:115–117 assembly 4:117–118 assembly parasites 4:123 defined 4:219, 4:105 major capsid protein before capsid assembly 4:117 portal 4:120–121 scaffolding protein 4:118–119 accelerating assembly 4:119 delay MCP transformation 4:119–120 exclude host factors 4:120 promote correct geometry 4:119 scaffold involvement in portal incorporation 4:120 stability of 4:123 structure of 4:121–123 prokaryotes, defined 4:242 prokaryotic Argonaute (pAGO) 1:608 prokaryotic cells, release of phages from 1:501–502 cell envelope 1:503–504 multigene lysis (MGL) systems 1:504–505 lambda lysis, looking under the hood of 1:505–507 lambda lysis cassette 1:505 lambda MGL, operational outline of 1:505 lambda prophage as a lysis platform 1:504–505 phage lysis, definition of 1:502–503 pinholins and SAR endolysins 1:507–508

endolysin diversity 1:512–513 holin diversity 1:512 lysis in mycobacteria 1:514 MGL diversity 1:512 MGL in gram-positive hosts 1:513–514 regulation of MGL 1:509–510 second type of spanin 1:508–509 spanin diversity 1:513 single gene lysis (SGL) systems 1:514–515 Archaea, phage egress in 1:517–518 extrusion 1:517 of Leviviridae 1:515–516 mysterious L 1:516–517 jX174 E 1:514–515 prokaryotic dsDNA viruses, genome replication of 1:429–430 prokaryotic RNA viruses, genome replication of 1:437 prokaryotic ssDNA viruses, genome replication of 1:436 prokaryotic viruses 1:165–166 classification of 1:430t genomes of 1:429 proliferating cell nuclear antigen (PCNA) 4:390–391, 4:366 proliferation, defined 2:528 promoter, defined 2:442, 2:56, 4:69 promoters, viral 1:652 use 1:655–657 viral examples 1:653–655 prophage 4:291, 4:265, 1:552, 4:276, 4:242, 4:77, 4:88, 4:368, 4:283, 4:342 prophage induction 4:84–85, 4:77 prophage remnants 1:640–641 prophage reservoir, defined 4:283 prophages 1:636 pro-retroviruses, defined 3:653 protease, defined 4:776 protease activation, cell entry targeting by 5:238 protease inhibitors (PIs) 5:139, 2:473 -based regimens 5:140 atazanavir 5:141 darunavir 5:140–141 dual therapy 5:140 lopinavir 5:141 new PIs 5:141 triple therapy 5:140 classes of 5:140t in combination with other ART classes 5:140 drug–drug interactions 5:141 mechanism of action 5:139 pharmacodynamics and pharmacokinetics in 5:139–140 protease inhibitors in clinical practice 5:143 protease inhibitors in low resource settings 5:142–143 sub-Saharan Africa 5:142–143 resistance to PIs 5:141–142 side effects 5:141 cardiovascular 5:141 dyslipidemia, lipodystrophy and insulin resistance 5:141 gastrointestinal 5:141

375

hepatotoxicity 5:141 kidney stones 5:141 tolerability of 5:141 protease protein, defined 3:96 protease/reverse transcriptase (P5) 3:318 protective antigen (PA) 4:262 protein antibiotics (PA) 1:515, 1:501–502 protein cluster, defined 4:265 protein-coding genes 2:457 protein-coding regions 2:444f Protein Data Bank (PDB) 1:141, 1:153 protein-DNA binding 4:139–140 DNA binding domains (DBDs) 4:140–141 multiple binding events regulate complex assembly 4:141 sequence specific and non-specific binding 4:140 protein domain, defined 1:345 protein ejection and trans-envelope channel formation 4:206–207 protein glycosylation 4:387, 4:359 protein IIIa 1:333 protein injection 4:48 Protein IX 1:337, 1:333–335 protein kinase R (PKR) 2:857–858, 1:444–445 protein loops, defined 1:345 protein-only hypothesis, defined 2:707 protein-primed DNA replication 1:433–434, 4:36, 1:429, 4:368 protein receptors 1:394–396 complement control proteins 1:396–397 ectoenzymes 1:397 CoV-ectoenzyme interactions 1:397 immunoglobulin superfamily proteins 1:394–396 virus interactions with IgSF receptors 1:395–396 Protein V 1:335 protein VI 1:333 protein VIII 1:333 proteomics 1:498 protist, defined 3:106 protohelix, defined 3:734 proton-motive force (PMF) 1:504, 4:206 proto-oncogene, defined 2:316 protoparvoviruses 2:684f clinical features 2:425 epidemiology 2:424 protoplast, defined 4:601, 4:607, 3:456, 3:507, 4:648 protospacer adjacent motifs (PAM) 4:398 protospacer-flanking site (PFS) 4:248 prototype virus, defined 4:776 proventricular dilatation disease (PDD) 2:137, 2:140–141 providence virus 4:898, 1:250 proviral DNA 2:62 proviral load, defined 2:528 provirophage 1:380 provirophage, defined 1:372 provirus, defined 1:162, 2:643, 2:144, 4:387, 1:627, 2:528, 2:56, 4:359 PrP, defined 2:707 PrPC, defined 2:707 PrPSc, defined 2:707

376

Subject Index

PRRs see pathogen recognition receptors (PRRs); pattern recognition receptors (PRRs) PRRS see porcine reproductive and respiratory syndrome (PRRS) PRRSV see porcine reproductive and respiratory syndrome virus (PRRSV) PRSV see papaya ringspot virus (PRSV) prunevirus, defined 3:805 pruritis, defined 2:738 PRV see perch rhabdovirus (PRV) P-selectin glycoprotein ligand-1 (PSGL-1) 1:410, 1:284 Pseudaletia separata EPV (PSEV) 4:859 pseudocowpox 2:668 pseudocowpox virus (PCPV) 2:666, 2:171 pseudoknot, defined 3:447, 3:818, 4:648, 3:734 pseudolysogeny, defined 4:314 Pseudomonas aeruginosa 1:640–641 pseudomurein endoisopeptidase, defined 4:387 pseudorabies virus (PrV) 2:715f classification 2:714 clinical features and pathogenesis 2:720–721 epidemiology and prevention 2:722–723 future perspectives 2:723 genome 2:716f history 2:714 immunity 2:721–722 latency 2:722 ORFs 2:717t–718 pseudorabies virus as a tool in neurobiology 2:723 replication cycle 2:719f replication cycle 2:715–720 virion structure 2:714–715 capsid 2:715 envelope 2:715 genome 2:714–715 tegument 2:715 pseudo-recombinant, defined 3:301 pseudo-recombination, defined 3:371, 3:81, 3:411 Pseudoviridae 3:654, 3:100–101 pseudovirus, defined 1:208, 3:653 PSEV see Pseudaletia separata EPV (PSEV) PSGL-1 see P-selectin glycoprotein ligand-1 (PSGL-1) PsHV1 see psittacid herpesvirus type 1 (PsHV1) psittacid herpesvirus type 1 (PsHV1) 2:112, 2:113 psittacine, defined 2:343 psittacine BFDV 2:187 PSIV see Plautia stali intestine virus (PSIV) PSs see packaging signals (PSs) P(S/T)AP late domain 1:522 PstDV1 see penaeus stylirostris penstyldensovirus 1 (PstDV1) PT see proficiency testing (PT) PTB see polypyrimidine tract-binding protein (PTB) pteromalus puparum negative-strand RNA virus 1 (PpNSRV-1) 1:104

pteropid bats, defined 2:355 PTGS see post-transcriptional gene silencing (PTGS) PTI see pattern-triggered immunity (PTI) PTLDs see post-transplantation lymphoproliferative disorders (PTLDs) PTMs see post-translational modifications (PTMs) pulsed-field gel electrophoresis (PFGE) 1:622 puma lentivirus (PLV) 2:56–57 infections 2:66 PLVA 2:66 PLVB 2:66 punta toro and cocle viruses 2:771 purification of viruses 1:166–167 purification performance, assessing 1:172 purifying selection, defined 4:457 pustule, defined 2:868 Puumala virus (PUUV) 2:353 PUUV see Puumala virus (PUUV) PVAN see polyomavirus associated nephritis (PVAN) PVM see pneumonia virus of mice (PVM) PVR see poliovirus receptor (PVR) PVs see parvoviruses (PVs) PVSRIPO 1:659 PVX see Potato virus X (PVX) PVY see potato virus Y (PVY) PVYNTN strain of Potato virus Y 1:649 pyrobaculum filamentous virus 1 (PFV1) 4:363 Pyrobaculum oguniense 4:394 PyVs see polyomaviruses (PyVs)

Q QA see quality assurance (QA) QMS see quality management system (QMS) qPCR see quantitative polymerase chain reaction (qPCR) Q protein 4:89–90 quadriparticulate virus, defined 4:642 quadripartite genome, defined 4:642 quadriviruses (Quadriviridae) 4:642, 4:506–508 genome organization and expression 4:644–645 molecular and biological properties 4:645–646 phylogenetic and evolutionary relationships of RnQV1 to other dsRNA mycoviruses 4:646–647 virion structure and composition 4:642–644 qualitative studies 1:565 quality assurance (QA) 5:55–57 quality assurance (QA) and quality management (QM), principles of 5:64 document control 5:65–66 laboratory specimen retention times 5:67

personnel training, competency, and continuous professional development (CPD) 5:67–68 quality management documentation 5:64–65 quality management system (QMS) 5:64, 5:65f specimen management 5:66 transportation and storage of specimens 5:66–67 quality assurance (QA) in the clinical virology laboratory assay verification and validation 5:70–72 accuracy 5:72 linearity/analytical measurement range (AMR) 5:72–73 precision 5:72 reportable range and limit of detection (LoD) 5:72 EQA design and objectives 5:76–78 equipment calibration 5:69 equipment maintenance and monitoring 5:69–70 equipment qualification 5:69 external quality assessment and performance criteria 5:76 future aspects of QA to virology laboratory 5:78–81 internal and external audits 5:70 internal quality assessment (IQA) 5:74 internal quality control procedures and monitoring assay performance over time 5:74–75 laboratory facilities and equipment 5:68 sensitivity, specificity, and clinical utility 5:73–74 suitable standards, reference materials and controls to support quality assurance in virology 5:75–76 quality management documentation 5:64–65 quality management system (QMS) 5:56–57, 5:64, 5:65f quantitative polymerase chain reaction (qPCR) 2:651 quantitative spectral imaging 1:211 quantitative trait loci, defined 3:69, 3:554, 3:555 Quantum Dot, defined 1:208 quasi-enveloped viruses 1:524 quasi-enveloped virus particles, defined 2:397 quasi-equivalence, defined 1:248, 1:278, 4:115 quasi-equivalence theory 1:249–250 predictions and limitations of 1:250 quasi-equivalent conformers, defined 4:21 quasispecies concept 1:68–69 defined 2:373 theory 1:58 viral 1:58 quasispecies-like populations, evolution as 3:100 quiescent infection 2:456–457 quinviruses (Betaflexiviridae) 3:642

Subject Index diagnosis 3:652 epidemiology and control 3:651–652 genome organization 3:643–648 properties and functions of gene products 3:648–649 replication and propagation 3:649 taxonomy and phylogeny 3:642–643 transmission and host range 3:649–651 virion structure 3:643

R rabbit hemorrhagic disease virus (RHDV) 1:665–666, 1:669, 1:648 classification 2:724 clinical features 2:726 diagnosis 2:728 epidemiology 2:726 genome 2:725–726 pathogenesis and host immunity 2:726–728 prevention and control 2:728–729 treatment 2:729 virion structure 2:724–725 rabbit myxoma virus and fibroma viruses 2:730 Californian myxoma virus 2:731–732 classification 2:730 clinical disease in European rabbits 2:732–733 diagnosis 2:734–735 epidemiology 2:731 genome 2:730 life cycle 2:730–731 myxomatosis in European rabbits 2:732 myxoma virus 2:731 pathogenesis of myxomatosis in European rabbits 2:733–734 prevention of myxomatosis 2:735 treatment 2:735 virion structure 2:730 rabies and lyssaviruses classification (compact) 2:738 clinical features 2:740–741 diagnosis 2:742–743 epidemiology 2:739–740 genome 2:738 life cycle 2:738–739 pathogenesis 2:741–742 prevention 2:744–745 treatment 2:743–744 virion structure 2:738 rabies hyperimmune immunoglobulin 5:270–271 rabies immunoglobulin (RIG) 2:744 rabies lyssavirus (RABV) 2:738 RABV see rabies lyssavirus (RABV) raccoon parvovirus (RPV) 2:684 RACE see rapid amplified of cDNA ends (RACE) Rac prophage, defined 4:291 radiological abnormalities, defined 2:814 radiosensitizers for therapy and imaging 5:241 Radish leaf curl betasatellite (RaLCB) 3:245

Radi virus (RADV) 2:875 RADV see Radi virus (RADV) RaLCB see Radish leaf curl betasatellite (RaLCB) raltegravir 5:146 clinical trials in cART-experienced patients 5:146 clinical trials in cART naı¨ve individuals 5:146 resistance 5:146 randomized controlled trial, defined 5:98 RAP see replicase-associated protein (RAP) rapid amplified of cDNA ends (RACE) 4:544 rapid fluorescent focus inhibition test (RFFIT) 2:743 Rathayibacter toxicus 1:649 Ravn virus (RAVV) 2:609 RAVV see Ravn virus (RAVV) RBD see receptor binding domain (RBD) RBPs see receptor-binding proteins (RBPs) RBS see receptor-binding site (RBS) RBSDV see rice black streaked dwarf virus (RBSDV) RCA see regulators of complement activity (RCA); rolling circle amplification (RCA) RCR see rolling circle DNA replication (RCR); rolling circle replication (RCR) RCRE see replication initiation endonucleases (RCRE) RdDp see DNA-dependent RNA-polymerase (RdDp); RNA dependent DNA polymerase activity (RdDp) RDF see recombination directionality factor (RDF) RDG, defined 2:475 RDR see recombination-dependent replication (RDR) RdRp see RNA-dependent RNA polymerase (RdRp) RDS see runt deformity syndrome (RDS) RDV see rice dwarf virus (RDV) reactivation, defined 2:442 reactive oxygen species (ROS) 3:420 read, defined 4:322 read of insert (ROI) 1:181 readthrough, defined 3:788, 3:481, 3:285 readthrough protein, defined 3:106 real-time PCR assays, validating 5:35, 3:629 analytical specificity and sensitivity 5:40 analytical verification stage 5:38 reference materials and sample numbers 5:38 template, primers and probes 5:38–39 clinical validation stage 5:42 precision, accuracy and trueness 5:42 commercial assays 5:36–37 consultation stage 5:37 experimental optimization 5:40 gold standard comparative study 5:42–43 inhibition study 5:40–41 inter-laboratory testing 5:43 linear dynamic range or reportable range 5:41–42

377

maintaining the validated status and re-validation 5:43–44 normalization 5:40 PCR efficiency 5:41 preliminary considerations 5:37 quantification standards, choice of 5:39 reaction controls 5:39 reverse transcription PCR 5:39 study without a gold standard 5:43 validation, need for 5:35–36 in-house assays 5:35–36 validation plan 5:37–38 Westgard rules 5:39–40 real-time polymerase chain reaction (RT-PCR) 1:497 reassortant, defined 5:289, 2:349, 3:371 reassortment, defined 1:62, 1:569, 3:516, 4:607, 2:654 receptor, defined 1:402, 1:382, 4:380, 2:518 receptor binding domain (RBD) 2:70–71 receptor-binding proteins (RBPs) 1:129, 5:237, 1:402 receptor-binding site (RBS) 1:525 receptor-mediated, clathrin-dependent endocytosis 1:533 receptor-mediated endocytosis 1:389 receptor-mediated virus uncoating 1:389 receptors, viral 1:388–389 attachment factors 1:399–400 co-receptors for infection 1:397–399 evolution of virus-recept or interactions and viral tropism 1:400–401 glycan receptors 1:390–391 enteroviruses (EVs) 1:392 glycan-based antiviral strategies 1:393–394 glycosaminoglycan receptors 1:393 histo–blood group antigen receptors (HBGAs) 1:392–393 influenza viruses 1:391–392 polyomaviruses (PyVs) 1:392 sialic acid receptors 1:390–391 identification of 1:389–390 protein receptors, interactions with viruses 1:394–396 complement control proteins 1:396–397 ectoenzymes 1:397 immunoglobulin superfamily proteins 1:394–396 in virus cell entry 1:389 attachment 1:389 receptor-mediated endocytosis 1:389 receptor-mediated virus uncoating 1:389 recessive allele, defined 3:69, 3:554 RecET, defined 4:291 recombinant and mutant virus particles 1:223 recombinant nematode anticoagulant protein c2 (rNAPc2) 2:616 recombinant vesicular stomatitis virus (rVSV)-based Ebola vaccine 2:616 recombinase, defined 4:291 recombination 2:63–64, 1:56, 1:62, 1:569, 4:291, 1:71, 3:411, 2:256

378

Subject Index

computational estimation of 1:108–109 analyzing recombination in sequences derived from next generation sequencing 1:111–112 estimation of recombination rates 1:111 identification of recombination breakpoints 1:109–111 phylogenetic recombination networks, reconstruction of 1:111 rapid tests to detect the presence of recombination 1:108–109, 1:110f defective-interfering RNAs 1:465–467 in DNA viruses 1:56–57 homologous and non-homologous, defined 4:276 reassortment 1:56 recombination in DNA viruses 1:460–462 recombination in RNA viruses 1:462–465 template switching 1:56 recombination-dependent replication (RDR) 4:62–63, 3:753–754 recombination directionality factor (RDF) 4:77–78 recombination in virus genetic data 1:112 hepatitis B virus genotypes, recombination among 1:112–113 HIV-1 centralized vaccines, recombination in the design of 1:112 HIV-1 fitness recovery, influence of recombination on 1:112 HIV-1 gp120, analyzing adaptation of 1:113 recombineering combining with other gene modification technologies 4:299 development of 4:294–295 with dsDNA 4:295–296 dsDNA recombineering, mechanism of 4:298 in vivo cloning 4:297 markerless gene deletions and counterselection schemes 4:296 oligo-mediated recombineering, mechanism of 4:297–298 recombinase-independent recombination 4:299–300 recombineering in pathogenic bacteria 4:298–299 with ssDNA 4:296–297 recombineering, defined 4:291 recycling endosome pathway (REP) 2:72 red deer, parapox of 2:668 regulators of complement activity (RCA) 1:396 regulatory authority, defined 5:281 regulatory T cells (Tregs) 1:588 reinfection, defined 2:442 release, defined 1:382 Reoviridae 3:545 Reoviridae family viruses, overall structure of 1:303 Sedoreovirinae 1:303 orbivirus 1:303 phytoreovirus 1:303 rotavirus 1:303 Spinareovirinae 1:303–306

aquareovirus 1:306 cypovirus 1:306 orthoreovirus 1:305–306 viral genome, general features of 1:306 reoviruses 1:303, 4:867, 4:714 capsid layers, structural organization of 1:306–307 cell entry of dsRNA viruses 1:307–309 outer capsid layer 1:306–307 double-stranded RNA (dsRNA) virus core and endogenous transcription 1:309 viral innermost capsid layer that encloses the genome 1:309 viral RNA capping 1:311–313 viral RNA-dependent RNA polymerase (RdRp) 1:309–311 double-stranded RNA (dsRNA) virus families 1:314–315 genome replication and packaging 1:313 replication and packaging 1:313–314 viroplasm/replication factories 1:313 Reoviridae family viruses, overall structure of 1:303 general features of the viral genome 1:306 Sedoreovirinae 1:303 Spinareovirinae 1:303–306 reoviruses of invertebrates 4:713–715 Sedoreovirinae 4:877–879 cardoreovirus 4:878–879 phytoreovirus 4:879–881 seadornavirus 4:881 Spinareovirinae 4:868–870 antigenic and genetic relationships 4:876–877 historical overview 4:870 host range, diseases, transmission, and distribution 4:870–871 virion properties, genome, and replication 4:871–875 taxonomy 4:867 REP see recycling endosome pathway (REP) RepA gene 3:465 repeat region 2:58–59 Rep gene 3:465 Rep/Helicase, defined 4:849 replicase-associated protein (RAP) 3:792–793 replicase proteins 3:265 replication, defined 4:136, 2:442 Replication and Transcription Activator (RTA) 2:601 replication cycle, defined 1:162 replication initiation endonucleases (RCRE) 4:390–391, 4:391 replication initiator protein, defined 3:470 replication of viruses assembly 1:386 attachment 1:383–384 double-stranded DNA 1:385 double-stranded DNA with RNA intermediate 1:386 double-stranded RNA 1:385 entry 1:384 maturation 1:386 release 1:386–387

single-stranded (–)-sense RNA 1:385 single-stranded (+)-sense RNA 1:385 single-stranded DNA 1:385 single-stranded RNA with DNA intermediate 1:386 transcription and genome replication 1:384–385 uncoating 1:384 replication organelle (RO) 1:495, 1:498 defined 1:495 replication origin (ori), defined 4:61 replicon, defined 2:386, 2:747 replisome, defined 4:61 Rep protein 3:360 representational difference analysis, defined 2:48 representativeness 5:247, 5:252 reproduction number, defined 2:825 reptilian adenoviruses 2:13–14 RER see rough endoplasmic reticulum (RER) reservoir, defined 2:117 resistance, defined 3:192 resistance breaking strain, defined 3:355 resistance gene, defined 3:60, 3:554, 3:555 resistance gene analog (RGA) marker 3:563 resistant, defined 4:276 resistosome 3:64–66, 3:60 respiratory syncytial virus (RSV) 1:205, 5:272–273, 5:123–124 animal models of 2:753–754 antiviral agents 2:755 classification 2:748–749 disease 2:747–748 F glycoprotein 2:749–750 genome transcription and replication 2:752–753 G glycoprotein 2:750–752 history 2:747 live attenuated vaccines 2:754–755 management of RSV infections clinical features 5:155–156 epidemiology 5:155 pathogenesis and immunity 5:155 prophylaxis 5:156–157 treatment strategies 5:156 vaccination 5:157 maternal vaccination 2:754 non-structural viral proteins 2:753 passive immunity and prophylactic therapy 2:754 protection from RSV disease 2:754 vaccination 2:754 RSV Fusion glycoprotein 5:124 RSV RNA polymerase 5:123–124 third viral glycoprotein 2:753 treatment of 2:755 vectored RSV vaccines 2:755 virion organization 2:749f virus entry 2:749 virion 2:749 respiratory tract infections (RTI) 5:99–100 assays 5:100–101 background 5:99–100 clinical impact 5:101 lower 5:200

Subject Index upper 5:199–200 restriction factor (RF) 1:627, 2:442, 1:628–629, 1:629–630 restriction fragment length polymorphisms (RFLPs) 1:75, 3:192, 3:200 restriction-modification system, defined 4:229 reticulons, defined 3:32 retinoblastoma protein, defined 2:79 retinoic-acid-inducible gene 1 (RIG-I) 2:391–392, 2:214, 2:555 retroelements, classification of 3:98t retrograde, defined 2:738 retrotranscription 2:62 retrotranscytosis 1:529 retrotransposons associated with invertebrates 4:722 defined 3:274 retrotransposons of plants Env-like functions in plants 3:102 general classification 3:96–99 long terminal repeat (LTR) retrotransposons different genera of 3:100–101 life cycle of 3:99–100 Metaviridae (Ty3/gypsy retrotransposons) 3:101 nomenclature issue 3:102–103 Pseudoviridae (Ty1/copia retrotransposons) 3:100–101 quasispecies-like populations, evolution as 3:100 retroviral cycle, high similarities with intracellular steps of 3:99–100 unclassified LTR retrotransposons 3:101–102 retroviral cycle, high similarities with intracellular steps of 3:99–100 Retroviridae 2:56 animal lentiviral infections 2:65–66 bovine immunodeficiency (BI) and Jembrana disease (JD) 2:66 equine infectious anemia (EIA) 2:67 feline immunodeficiency and puma lentiviral infections 2:66 visna-maedi (VM) and caprine arthritis and encephalitis (CAE) 2:66–67 biological properties 2:63–64 natural hosts 2:64 transmission 2:64 tropism 2:64 variability 2:63–64 classification 2:56–57 encoded proteins 2:59–60 genome 2:57–59 pathogenic processes 2:64–65 cell defenses and viral mechanisms 2:65 immune deficiency 2:65 inflammation 2:65 replication cycle 2:60–63 reverse transcription 2:62–63 virion structure 2:57 Retroviridae 2:827–828 clinical features 2:829–830 defined 2:827 epidemiology 2:829

genome 2:828–829 lifecycle 2:828–829 immunity to 2:830 taxonomy and classification 2:828 virion structure 2:828 retroviruses 1:525, 1:491–492, see also fish retrovirusesof birds classification 2:122–123 clinical features 2:125 diagnosis 2:126 endogenous retroviruses 2:126 epidemiology 2:124–125 genome 2:123–124 history 2:122 life cycle 2:124 pathogenesis 2:125–126 treatment 2:126 virion structure 2:123 in cellular evolution 1:627 germline insertions 1:630–631 molecular consequences of retrovirus integration 1:627–629 as selective agents 1:629f and their life cycle 2:316–317 retrovirus integration, molecular consequences of 1:627–629 host dependency factors (HDFs) 1:629 host genes with indirect effects on retrovirus infection, spread or disease 1:630 host immunity 1:630 restriction factors (RFs) 1:629–630 retrovirus virions, structure of env gene 1:358–360 gag proteins and retroviral virion structure 1:352–354 immature retroviral virion, assembly of 1:356–358 individual gag domains, structures of 1:354–355 capsid protein (CA) 1:355–356 linker peptides 1:356 matrix protein (MA) 1:354–355 nucleocapsid protein (NC) 1:356 reverse genetics, defined 4:26, 3:123 reverse-transcribing RNA and DNA viruses, origin of 1:18–19 reverse-transcribing viruses (RTV) 1:42–43, 1:15–16 genome organization and functions of gene products 3:654–656 host range and evolutionary dynamics 3:662–664 impact and relationships of LTR retrotransposons and retroviruses with their hosts 3:664–665 life cycle and replication 3:659–662 LTR retrotransposons and reversetranscribing viruses 3:654 taxonomy, phylogeny and members of the families 3:656–659 reverse transcriptase (RT) 2:464, 5:132, 2:62f, 2:63f, 3:313 reverse transcriptase inhibitors (RTIs) 2:472–473 reverse transcriptase protein, defined 3:96

379

reverse transcriptase structure and functions 5:131 reverse transcription (RT)-PCR 3:629 reverse transcription, defined 2:460, 5:27, 3:158 reverse transcription, process of 5:131–132 reverse transcriptase (RT) 5:132 RNase H 5:132 tRNA primer 5:131–132 reverse transcription loop-mediated rapid isothermal amplification (RT-LAMP) detection kit 4:825 reverse transcription polymerase chain reaction (RT-PCR) 2:241, 5:98, 5:300, 2:704, 2:705, 2:743, 2:38, 2:812–813, 3:322, 5:29, 2:214, 2:341, 2:542, 3:539, 2:514 rev gene 2:61t Rev Response Element (RRE) 2:463 RF see restriction factor (RF) RFFIT see rapid fluorescent focus inhibition test (RFFIT) RFLPs see restriction fragment length polymorphisms (RFLPs) RFS see ribosomal frameshift motif (RFS) R-gene 3:758–759 R gene products 3:60–61 R gene responses to viruses 3:61–62 R genes 3:555 RGHV see Ross’ goose hepatitis virus (RGHV) Rhabdoviridae 4:715–716, 3:567 rhabdoviruses 3:567–568 classification 2:875 control and treatment 2:882–883 cytopathology and replication 3:576 cell-to-cell movement 3:577–578 entry 3:576 morphogenesis 3:576–577 transcription and genome replication 3:576 epidemiology 2:880–881 genome structure and organization 3:568–572 of invertebrates 4:715–716 Roniviridae 4:716 Sarthroviridae 4:716 Solinviviridae 4:716–717 life cycle 2:878–879 assembly and budding 2:879–880 attachment, entry and uncoating 2:878–879 gene expression and replication 2:879 inhibition and modification of host cell functions 2:880 particle morphology and composition 3:568 pathogenesis and clinical features 2:881–882 taxonomy and classification 3:568 vector relationships, distribution and evolution 3:578–579 viral proteins, properties of 3:572–573 accessory proteins 3:575–576 glycoprotein 3:575 matrix protein 3:574–575

380

Subject Index

rhabdoviruses (continued) nucleocapsid protein 3:572–573 P3 movement protein 3:574 phosphoprotein 3:573–574 polymerase protein 3:575 virus structure 2:875–878 genomes and proteins 2:877–878 rhabdoviruses of insects classification 4:883 clinical features 4:886 diagnosis 4:887 epidemiology 4:886 distribution, spatial and temporal 4:886 population dynamics 4:886 prevalence 4:886 transmission 4:886 genome 4:884–885 almendraviruses genome 4:885 sigmavirus genome 4:885 life cycle 4:885–886 integration into the host genome 4:885–886 pathogenesis 4:886–887 virion structure 4:883–884 rhabdovirus glycoprotein G 1:348 RHDV see rabbit hemorrhagic disease virus (RHDV) Rhinorrhea, defined 2:814 Rhinovirus C 1:410 rhinoviruses 2:758t classification 2:757 clinical features 2:761 acute otitis media (AOM) 2:761 asthma 2:761–762 asymptomatic infections 2:761 bronchiolitis and recurrent wheezing 2:761 chronic obstructive pulmonary disease (COPD) 2:762 common cold 2:761 pneumonia 2:762 diagnosis 2:762 culture 2:762–763 nucleic acid detection 2:762 sampling 2:762 typing 2:763 epidemiology 2:760 genome 2:757–758 life cycle 2:758–759 assembly 2:760 entry 2:759 receptor 2:758–759 replication 2:759–760 translation 2:759 uncoating 2:759 pathogenesis 2:760–761 prevention 2:764 receptor usage of 2:759t structure 2:758f treatment 2:763–764 virion structure 2:757 Rhipicephalus appendiculatus 1:547 Rhizoctonia solani 4:461 Rhizoctonia solani megabirnavirus 1 (RsMBV1) 4:600

Rhizoctonia solani RNA virus HN008 (RsRV-HN008) 4:600 rhizomania, defined 3:213, 3:219 Rhodobacteraceae 4:334–335 Rhopalosiphum padi virus (RhPV) 4:771 RhPV see Rhopalosiphum padi virus (RhPV) RHR see rolling hairpin replication (RHR) ribavirin, defined 2:475 ribavirine 5:171, 5:164–166 Ribonuclease H (RNase H) 2:62, 5:132 ribonucleoprotein (RNP) complexes 5:6, 2:237–238, 3:507, 4:776, 2:138–139 cis-signals for formation of 4:624–625 as a viral entity 4:622–623 Ribosomal-1 frameshifting, defined 4:594 ribosomal bypassing (‘hopping’) 1:452 ribosomal frameshifting 2:193, 3:594, 3:154, 4:664, 3:712, 4:648, 4:892 ribosomal frameshift motif (RFS) 4:804–806 ribosomal read-through, defined 3:594 ribosomal RNA (rRNA) 1:377 ribosomal ‘frame-shifting’ 1:452–454 ribosome-protected RNA fragments (RPFs) 1:178 ribosome reinitation 1:450–452 ribosome shunt, defined 3:313 ribosome ‘shunting’ 1:450 ribosome stalling, defined 2:362 ribosome stop codon ‘read-through’ 1:454 Riboviria 3:481 defined 3:788 ribovirocells 1:25 ribozyme, defined 4:621, 3:852 rice black streaked dwarf virus (RBSDV) 1:313 rice dwarf virus (RDV) 1:303 rice tungro bacilliform virus (RTBV) 3:669, 3:164, 4:662 biotechnological applications 3:672 control 3:671–672 functions of proteins 3:671 genome 3:669–671 genome variability 3:671 structure 3:669 taxonomy 3:669 transmission and host range 3:669 virus-host relationships 3:671 rice tungro disease 3:667–669, 3:667 rice tungro spherical virus (RTSV) 3:672, 1:450, 4:662 control 3:673 gene functions 3:672 genome 3:673 genome diversity 3:672–673 structure 3:672 taxonomy 3:672 transmission and host range 3:672 virus-host relationships 3:673 rice tungro virus 1:649 rice yellow mottle virus (Solemoviridae) 3:675 biological properties 3:676–677 diagnostic and identification 3:677 diversity and evolution 3:678–679

epidemiological aspects 3:677–678 organization of the genome 3:675–676 relationships of the species with other taxa 3:676 resistance 3:555 virion properties 3:675 rift valley fever virus 2:766, 2:768–770, 1:617 bhanja virus 2:776 chagres virus 2:771 heartland banyangvirus (HRTV) 2:775–776 huaiyangshan banyangvirus 2:771–775 nonstructural proteins, functions of 2:767–768 phleboviruses in the candiru antigenic complex 2:771 punta toro and cocle viruses 2:771 rift valley fever virus 2:768–770 sandfly fever naples and sandfly fever sicilian viruses 2:770–771 toscana virus 2:771 virion structure, genome, and strategy of replication 2:766–767 RIG see rabies immunoglobulin (RIG) RIG-I see retinoic-acid-inducible gene 1 (RIG-I) rinderpest virus (RPV) 2:73 ring vaccination 1:575 RIP see RNAP inhibitory protein (RIP) RISC see RNA-induced silencing complex (RISC) RLPS see rough lipopolysaccharide (RLPS) RMSD see Root Mean Square Deviation (RMSD) 3’ RNA, defined 1:345 RNA dependent DNA polymerase activity (RdDp) 2:60 RNA-dependent RNA polymerase (RdRp) 4:806, 2:177, 2:389, 1:128, 4:820–821, 1:437, 3:623, 4:771, 1:62, 4:544, 4:867, 4:699, 3:388, 3:336, 3:545, 1:441, 3:555, 3:559 containing evolutionarily conserved architecture 1:441 coordinating transcription, capping, and polyadenylation 1:442–443 different RdRp and template conformations distinguish transcription from replication 1:441–442 replication using 1:437 RNA drug, defined 3:43 RNAi see RNA interference (RNAi) RNA-induced silencing complex (RISC) 3:792–793, 4:592, 3:788 RNA-induced silencing complex, defined 3:52, 3:123 RNA interference (RNAi) 4:832, 3:777, 4:808, 3:192, 2:616, 4:821, 4:772, 3:667, 3:60, 3:52, 3:123, 4:776, 3:362–363, 3:554 RNAP see RNA polymerase (RNAP) rNAPc2 see recombinant nematode anticoagulant protein c2 (rNAPc2) RNAP inhibitory protein (RIP) 4:393

Subject Index RNA polymerase (RNAP) 4:393, 2:25, 4:238 RNA-primed DNA replication 1:430–433, 1:429 RNA recombination and reverse genetics, manipulating CoV genomes using 2:204–205 RNase H see Ribonuclease H (RNase H) RNA-Seq see sequencing of total RNA (RNASeq) RNA-sequencing 1:178 RNA silencing 3:260, 3:628–629, 3:192, 3:293, 3:388, 3:285, 3:439, 4:457, 4:601, 4:607, 4:648, 3:719 RNA silencing suppressor 3:260, 3:623, 3:628–629, 3:439, 3:229, 3:719, 3:642 RNA splicing, defined 2:460 RNA vaccines 5:286 RNA viruses 1:40–42 infecting marine protists 4:671 in marine microbial ecology 4:674–675 origin of 1:14–15 infecting eukaryotes 1:17–18 recombination in 1:462–465 RNA virus evolution, mechanisms of additional constraints on RNA virus evolution 1:69 coevolution with hosts 1:67–68 genetic drift 1:68 genetic exchange 1:64–65 detecting recombination 1:65 rates of recombination 1:65 reassortment 1:65 recombination 1:64–65 recombination and reassortment, evolutionary outcomes for 1:65–66 mutation rates of RNA viruses 1:62–64 natural selection in viruses 1:66–68 quasispecies concept 1:68–69 viruses under selection by factors other than host cells 1:68 RnFV1 see Rosellinia necatrix fusarivirus 1 (RnFV1) RnMBV2 see Rosellinia necatrix megabirnavirus 2 (RnMBV2) RnMBV3 see Rosellinia necatrix megabirnavirus 3 (RnMBV3) RNP complexes see ribonucleoprotein (RNP) complexes RnPV1 see Rosellinia necatrix partitivirus 1 (RnPV1) RO see replication organelle (RO) rod-shaped helical plant viruses 1:362–364 ROI see read of insert (ROI) rolling circle, defined 3:768 rolling circle amplification (RCA) 3:168, 3:749, 3:753–754, 3:411 rolling circle DNA replication (RCR) 1:434–435, 1:429 rolling circle replication (RCR) 2:184, 1:436–437, 2:182, 4:387, 4:53, 3:470, 3:149 rolling hairpin replication (RHR) 4:836 Roniviridae 4:716 Roniviruses 2:245

room temperature electron microscopy (RT-EM) 1:242 room temperature-electron tomography (RT-ET) 1:244 root and tuber crops viral diseases 3:83–85 cassava viral diseases 3:83–85 potato viral diseases 3:85–86 sweet potato viral diseases 3:86 Root Mean Square Deviation (RMSD) 1:154 root-nodules, defined 4:632 ROS see reactive oxygen species (ROS) Rosellinia necatrix 4:461 hypovirulence in 4:476 Rosellinia necatrix fusarivirus 1 (RnFV1) 4:526, 4:527 Rosellinia necatrix megabirnavirus 2 (RnMBV2) 4:597, 4:599–600 Rosellinia necatrix megabirnavirus 3 (RnMBV3) 4:600 Rosellinia necatrix partitivirus 1 (RnPV1) 4:433 roseolaviruses 2:442 defined 2:442 roseolovirus cell tropism 2:781 HHV–6A and HHV–6B 2:781 HHV–7 2:781 roseoloviruses 1:321 chromosomal integration of HHV–6A and HHV–6B 2:781–782 chromosomal integration, mechanism of 2:781–782 CIHHV–6 and disease 2:784–785 CIHHV–6 and potential for disease in the immunocompetent 2:784–785 CIHHV–6 and potential for disease in the immunocompromised 2:785 epidemiology 2:783 HHV–6, HHV–7 and immunomodulation 2:784 HHV–6 and HHV–7 and antiviral therapy 2:787–788 HHV–6 and HHV–7 disease in older children and adults 2:785 Alzheimer’s disease 2:786 delayed primary infection 2:785 HHV–6 and HHV–7 and the possibility of encephalitis 2:785 HHV–6 and multiple sclerosis 2:786 HHV–6B and DRESS (drug rash with eosinophilia and systemic symptoms) 2:785–786 HHV–6 and HHV–7 laboratory tests and diagnosis of infection 2:786–787 antibody detection – HHV–6 and HHV–7 2:787 CIHHV–6 and misdiagnosis of active infection 2:787 diagnosis of CIHHV–6 2:787 virus detection – HHV–6A, HHV–6B and HHV–7 2:787 HHV–6 and HIV 2:786 HHV–6B and disease after organ transplant 2:786 HHV–6B encephalitis after HSCT 2:786 HHV–6B and HHV–7 primary infection and disease 2:785

381

encephalitis 2:785 exanthem subitum 2:785 FSE and temporal lobe epilepsy 2:785 HHV–7 and disease after organ transplant 2:786 immune responses 2:784 occurrence 2:783 chromosomal integration 2:783 HHV–6A 2:783 HHV–6B and HHV–7 primary infections 2:783 roseolovirus cell tropism 2:781 HHV–6A and HHV–6B 2:781 HHV–7 2:781 tissue distribution 2:783 transmission 2:783 horizontal transmission of HHV–6 and HHV–7 2:783 transmission of HHV–6 by organ donation 2:783–784 vertical transmission of HHV–6A and HHV–6B 2:783 viral genomics 2:779–780 HHV–6A and HHV–6B 2:779–780 HHV–7 2:780 viral latency 2:782 CIHHV–6 and the possibility of reactivation 2:783 reactivation from latency 2:782–783 state of viral genome during latency 2:782 virus structure and replication 2:780–781 rose rosette virus 3:19 causal agent and classification 3:19 control 3:20 disease symptoms and yield losses 3:19 epidemiology 3:19–20 geographical distribution 3:19 Rossmann fold 1:306–307, 4:148 Ross River virus 1:263 Ross’ goose hepatitis virus (RGHV) 2:100 rotaviruses 1:393, 1:303 classification 2:789 clinical features 2:792 diagnosis 2:793 epidemiology 2:791–792 genome 2:790 immune response and prevention 2:793–795 life cycle 2:790–791 pathogenesis 2:792–793 treatment 2:793 vaccines 5:289–290 in development 5:291 licensed 5:290 safety monitoring 5:291 vaccine effectiveness and impact 5:290–291 virion structure 2:789–790 rough endoplasmic reticulum (RER) 2:185 rough lipopolysaccharide (RLPS) 4:27 Rousettus aegyptiacus 2:608f Rous sarcoma virus (RSV) 1:675, 1:653–654, 1:352, 1:452, 2:122 RPFs see ribosome-protected RNA fragments (RPFs)

382

Subject Index

RpRp 3:790 RPV see raccoon parvovirus (RPV) RRE see Rev Response Element (RRE) rRNA see ribosomal RNA (rRNA) RsMBV1 see Rhizoctonia solani megabirnavirus 1 (RsMBV1) RSV see respiratory syncytial virus (RSV); Rous sarcoma virus (RSV) RT see reverse transcriptase (RT) RTA see Replication and Transcription Activator (RTA) RTase/RNaseH, defined 3:274 RTBV see rice tungro bacilliform virus (RTBV) RT-EM see room temperature electron microscopy (RT-EM) RT-ET see room temperature-electron tomography (RT-ET) RTI see respiratory tract infections (RTI) RTIs see reverse transcriptase inhibitors (RTIs) RT-LAMP detection kit see reverse transcription loop-mediated rapid isothermal amplification (RT-LAMP) detection kit RT-PCR see real-time polymerase chain reaction (RT-PCR) RT-PCR see reverse transcription polymerase chain reaction (RT-PCR) RTSV see rice tungro spherical virus (RTSV) RTV see reverse-transcribing viruses (RTV) rubella virus (RUBV) 1:263, 2:797, 1:499 classification 2:797 clinical features of infection 2:801–802 diagnosis 2:803 epidemiology 2:801 future 2:804 genetics and evolution 2:800–801 genomic organization 2:798–799 host range and virus propagation 2:797–798 immune response and serodiagnosis 2:802–803 intracellular replication cycle 2:799–800 pathogenesis, pathology, and histopathology 2:802 prevention and control of rubella 2:803–804 properties of the virion 2:798 serologic relationships and variability 2:801 transmission and tissue tropism 2:801 Rubus yellow net virus (RYNV) 3:165 RUBV see rubella virus (RUBV) Rudiviridae 1:367–369 rule-of-six, defined 2:355 runt deformity syndrome (RDS) 4:839 rVSV-based Ebola vaccine see recombinant vesicular stomatitis virus (rVSV)based Ebola vaccine Rx gene 3:629, 3:623 rymoviruses 3:797 biotechnological application 3:803 diagnosis 3:802 disease management 3:802 cultural practice 3:802

genetic resistance 3:802–803 functions of viral proteins 3:799 genome organization 3:799 life cycle and epidemiology 3:799–800 disease cycle 3:801–802 host range 3:800 seed transmission 3:800–801 vector transmission 3:800 pathogenicity 3:802 taxonomy and classification 3:797 virion structure 3:797–799 RYNV see Rubus yellow net virus (RYNV)

S S2 gene 2:61t sabin vaccine strains, attenuation of 2:690 Saccharolobus solfataricus 4:388–389 Saccharomyces cerevisiae 4:431, 4:440 dsRNA viruses and killer phenotype expression in 4:535–536 Saccharomyces cerevisiae virus L-A (ScV-L-A) 4:432 S-adenosylmethionine (SAM) 1:309, 2:177 sadwaviruses classification 3:322 clinical features and pathogenesis 3:325 diagnosis 3:325 genome organization 3:322 geographic distribution 3:324 life cycle and epidemiology 3:322–324 host range 3:324 prevention 3:326 transmission 3:324–325 treatment 3:325–326 virion structure 3:322 SAGs see single amplified genomes (SAGs) Saint Louis encephalitis virus (SLEV) 2:805, 2:805–806 classification/genetic characterization 2:808–809 diagnosis 2:812–813 epidemiology 2:810–812 history 2:805–808 host range 2:810–812 life cycle 2:809–810 pathogenesis/clinical features 2:812 prevention 2:813 virion structure/genome 2:806–808 salmonid, defined 2:324 salmon swimbladder sarcoma -associated retrovirus 2:320 isolation of a retrovirus from 2:320–321 salmon swimbladder sarcoma virus (SSSV) pathogenesis of 2:322 prevalence, seasonality, and transmission of 2:321–322 sequence comparisons of SSSV with other retroviruses 2:321 SAM see S-adenosylmethionine (SAM) sandfly fever naples and sandfly fever sicilian viruses 2:770–771 sanger sequencing technique 5:28

SaPIs, see Staphylococcus aureus pathogenicity island (SaPIs) SAPNs see self-assembling protein nanoparticles (SAPNs) sapovirus 2:490 cell culture 2:491 classification 2:490 clinical features 2:491 diagnosis 2:491 epidemiology 2:490–491 genome 2:490 immunity 2:491 life cycle 2:490 pathogenesis 2:491 prevention 2:491–492 virion structure 2:490 sarcoids 2:87–88 cats 2:88 diagnosis 2:90 horses 2:87–88 treatment 2:90–91 SAR endolysins, defined 1:501 SARS see severe acute respiratory syndrome (SARS) SARS-CoV see severe acute respiratory syndrome coronavirus (SARS-CoV) SARS-CoV-2 see severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Sarthroviridae 4:716, 4:889 sarthroviruses 4:888 clinical signs of WTD and histopathology 4:888 control measures 4:890–891 diagnostic tools 4:890 genome organization 4:888–889 geographical distribution 4:888 host range 4:890 morphology of virus 4:888 pathogenicity and transmission of disease 4:889–890 susceptibility of cell line to XSV 4:890 taxonomic position 4:889 satellite conserved region 3:239 satellite DNA 3:239 satellite dsRNA, defined 4:534 satellite like molecule, defined 3:149 satellite-like RNA, defined 3:681 satellite nucleic acids and viruses 3:681 classification 3:682 expression of foreign sequences from satellite vectors 3:691 general properties and effects of satellites 3:682–685 satellite viruses 3:682–685 geographical distribution 3:682 history 3:681–682 replication and structure 3:688–690 satellite RNAs 3:688–690 satellite DNAs 3:690 satellite-mediated control of viruses 3:691 satellite nucleic acids and satellite-related nucleic acids 3:685–688 sequence variation and evolution 3:690–691 satellite phage 4:90

Subject Index defined 4:98 satellite RNAs (satRNAs) 3:795, 3:692, 3:712, 3:581, 3:371, 3:285, 3:456, 3:681 satellites, defined 3:470, 3:21 satellite tobacco mosaic virus (STMV) 3:740 satellite tobacco necrosis virus (STNV) 1:249 satellite virus, defined 3:581, 3:681, 3:456, 4:888 satRNAs see satellite RNAs (satRNAs) SAXS see small angle X-ray scattering (SAXS) SBL technology see sequencing-by-ligation (SBL) technology SBMV see southern bean mosaic virus (SBMV) SBV see schmallenberg virus (SBV) scab, defined 2:343 scabby mouth 2:667–668 scaffolding protein 1:444, 4:118–119 accelerating assembly 4:119 -assisted capsid assembly 1:483–486 defined 4:10, 4:105 delay MCP transformation 4:119–120 exclude host factors 4:120 promote correct geometry 4:119 scaffold involvement in portal incorporation 4:120 scanning electron microscopy, advances in 5:13–14 Scanning Electron Microscpe (SEM) 5:5 SCARB2 see scavenger receptor class B member 2 (SCARB2) scavenger receptor class B member 1 (SRB1) 2:390 scavenger receptor class B member 2 (SCARB2) 1:284, 1:410 SCC see squamous cell carcinoma (SCC) Schlumbergera virus X (SchVX) 3:623–624, 3:628 schmallenberg virus (SBV) 2:34, 1:573–574 classification 2:34 clinical features 2:36 diagnosis 2:38 epidemiology 2:35–36 genome 2:34–35 pathogenesis 2:36–38 prevention 2:38–39 viral replication 2:35 virion structure 2:34 SchVX see Schlumbergera virus X (SchVX) science, viruses and 1:672–674 harnessing of viruses by humans 1:675 bioweapons 1:676–677 commerce and tulip mania 1:675 gene therapy and cancer therapy 1:676 phage therapy 1:676 vaccines 1:675–676 impact of science and technology on virology 1:673–674 impact of viruses on science 1:674–675 Sclerophthora macrospora virus A (SmV-A) 4:438 Sclerophthora macrospora virus B (SmV-B) 4:438

sclerotia, defined 4:552 sclerotinia sclerotimonavirus 4:481, 4:438 biological properties 4:481 family –Mymonaviridae 4:481 genome structure 4:481 genus –Sclerotimonavirus 4:481 phylogenetic relationships 4:483 virion morphology 4:481–483 Sclerotinia sclerotiorum 4:441, 4:615, 4:493 hypovirulence in 4:475–476 mycoviruses’ infections in see mycoviruses’ infections in Sclerotinia sclerotiorum Sclerotinia sclerotiorum botybirnavirus 1 (SsBV1) 4:464 Sclerotinia sclerotiorum debilitationassociated RNA virus (SsDRV) 4:438, 4:462 Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) 4:520 distribution of 4:498 exploring SsHADV-1 to control fungal disease 4:501–503 extracellular entry of 4:495–496 genome and proteins of 4:494–495 host range of 4:495 mutualistic interaction with mushroom sciarid fly 4:496–497 taxonomy of 4:498–501 transmission of 4:497–498 Sclerotinia sclerotiorum hypovirus 1 (SsHV1) 4:463 Sclerotinia sclerotiorum integrin-like gene (SSITL) 4:462 Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1) 4:597, 4:599 Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4) 4:520 Sclerotinia sclerotiorum negative-sense RNA virus 1 (SsNSRV-1) 4:463 Sclerotinia sclerotiorum reoviruses 4:612 mycoreovirus 4 4:612 sclerotinia sclerotiorum reovirus 1 4:612 sCMOS camera, defined 1:208 scoliosis, defined 2:34 SCP see smallest capsid protein (SCP) screening for viral infections 5:91 culture 5:91 high throughput serology, multiplex platforms for 5:94–95 nucleic acid tests 5:91–92 clustered regularly interspaced short palindromic repeats (CRISPER) platforms 5:93 DNA microarrays 5:92 high throughput sequencing 5:92–93 multiplex PCR 5:92 polymerase chain reaction assays 5:92 VirCapSeq-VERT 5:93 pathology 5:91 serology 5:93–94 enzyme-linked immunosorbent assays (ELISA) 5:94 hemagglutination inhibition assays 5:93–94

383

immunoblot assays 5:94 immunofluorescent assays (IFA) 5:94 lateral flow assays (LFA) 5:94 plaque reduction neutralization (PRNT) assays 5:94 “scrunchworm” model 4:165 ScV-L-A, see Saccharomyces cerevisiae virus L-A (ScV-L-A) Scylla serrata reovirus 4:879 SD see sleeping disease (SD) SDA see Strand Displacement Amplification (SDA) SDS-PAGE see sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDT see Sequence Demarcation Tool (SDT) seadornavirus 4:881 sea ice 4:353 seasonal cycle of disease, control of 2:319 seasonal influenza 5:273–274, 5:160 sea star-associated densovirus (SsaDV) 4:840 sea-star wasting disease (SSWD) 4:839, 4:840 secondary structure, defined 1:62 second generation/parallel sequencing technologies 5:29–31, 1:175 application of NGS in virology 5:30–31 Secoviridae 3:322, 3:486, 3:703, 3:364, 3:348 secoviruses (Secoviridae) 3:692 biotechnological applications 3:701 diagnosis 3:701–702 epidemiology and control 3:700–701 genome organization 3:697–698 properties and functions of gene products 3:698–699 replication and propagation 3:699–700 taxonomy, phylogeny, and evolution 3:692–694 members of the family 3:694 transmission, host range 3:700 virion structure 3:694–697 sedimentation rate, defined 1:162 Sedoreovirinae 1:303, 4:877–879, 4:713–715 cardoreovirus 4:878–879 orbivirus 1:303 phytoreovirus 1:303 phytoreovirus 4:879–881 rotavirus 1:303 seadornavirus 4:881 seed and sap transmission 3:531 segmental mobility in proteins, defined 3:727 segmented negative strand (sNS) RNA genomes 2:507–508 select agent, defined 2:765 selection coefficient, defined 1:53 selection/counterselection, defined 4:291 Selective Sweep, defined 5:227 SELEX, defined 1:278 self-assembling protein nanoparticles (SAPNs) 1:251 self-assembly, defined 4:26 selfish RNA replicons, origin of 1:15–16

384

Subject Index

self versus nonself recognition innate immunity systems 1:607–610 SEM see Scanning Electron Microscpe (SEM) semigranular cells (SGCs) 4:816 semi-persistent transmission, defined 3:667, 3:313, 3:229, 3:703 Semliki Forest virus (SFV) 1:546, 1:499 semotivirus, defined 3:653 sensitivity 5:73–74 analytical 2:697 diagnostic 2:697 sentinel studies 1:566 sentinel surveillance 5:250 sepsis, defined 2:675 sequelae, defined 2:868 sequence-based virus classification methods 1:101–102 DEmARC (DivErsity pArtitioning by hieRarchical Clustering) 1:101–102 Genome Relationships Applied to Virus Taxonomy (GRAViTy) 1:102 Natural Vector method 1:102 Sequence Demarcation Tool (SDT) 1:102 ViCTree 1:102 ViPTree 1:102 sequence-characterized amplified region (SCAR) marker 3:562 sequence comparison methods 1:100–101 Sequence Demarcation Tool (SDT) 1:102 sequence identity, defined 2:442 Sequence Independent Single Primer Amplification (SISPA) 1:178 sequence specific binding, defined 4:136 sequencing-by-ligation (SBL) technology 5:30 Sequencing by Synthesis Technology, defined 4:322 sequencing library, defined 1:175 sequencing of total RNA (RNA-Seq) 1:178 sequencing strategies 5:27–28 first generation sequencing technology 5:28–29 sample preparation strategies for sequencing viruses 5:31–32 nucleic acid sequence dependent strategies for known viruses 5:31–32 nucleic acid sequence independent strategies for sequencing 5:32 second generation/parallel sequencing technologies 5:29–31 application of NGS in virology 5:30–31 third generation sequencing technologies 5:31 virus databases 5:32–34 sequiviruses 3:703 classification 3:703–704 diseases and management 3:710 genome organization 3:707–708 properties of viral proteins 3:708–709 properties of virions 3:707 transmission 3:709–710 variation of isolates and strains 3:704–707 serine-like protease, defined 2:362 serine protease fold 1:263 seroconversion, defined 5:289

serodiagnosis, defined 2:797 seroepidemiologic studies 1:566 serogroup, defined 3:184 serological approaches for viral diagnosis 5:15 antibody assay, neutralizing 5:19 antibody repertoire, sequencing analysis of 5:20 complement fixation 5:20 future perspectives 5:20–21 general considerations and sources of error 5:17 restrictions and limitations 5:17 hemagglutination inhibition 5:20 IgG avidity 5:16–17 immunoassays 5:17–18 homogenous wash-free immunoassays 5:19 immunoblotting 5:18 immunofluorescence microscopy 5:18–19 lateral-flow immunoassays 5:19 solid-phase microwell immunoassays 5:17–18 latex agglutination 5:19–20 point-of-care serodiagnostics 5:20 principles of serological assays 5:15–16 serological differentiation index, defined 3:727 serological surveillance 5:249 serological tests 5:211–212 hepatitis A virus 5:211–212 hepatitis E virus 5:212–213 seronegative, defined 2:442 seropositive, defined 2:442 seroprevalence, defined 2:518, 2:442 serotype, defined 2:789, 3:184 Serpentovirales 3:495 serum neutralization tests (SNT) 2:285 serum therapy 5:267 SEV1 see sulfolobus ellipsoid virus 1 (SEV1) 17D Yellow fever virus vaccine 2:897 78-kD protein, defined 2:765 severe acute respiratory syndrome (SARS) 2:815f, 2:816f, 5:285, 1:12, 5:7, 1:499 animal infection models 2:821 clinical features 2:819–820 epidemiology ecology, animal reservoir and zoonotic transmission 2:818 history 2:814 emergence of SARS 2:814 human-to-human transmission 2:819 laboratory diagnosis 2:820–821 pathogenesis 2:822 treatment 2:822 vaccines and immunity 2:823 virology 2:814–815, 2:815, 2:815–816 phylogeny 2:816–818 virus receptors 2:818 severe acute respiratory syndrome coronavirus (SARS-CoV) 4:804, 2:198, 2:200f, 2:825, 2:825–826, 2:826, 1:121, 2:508, 2:193

severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1:13, 5:286–287, 5:3, 5:261, 5:127 severe fever with thrombocytopenia syndrome virus (SFTSV) see huaiyangshan banyangvirus sexually transmitted infections (STIs) 2:467 SFFV see spleen focus-forming virus (SFFV) SFS see Site Frequency Spectrum (SFS) SFV see Semliki Forest virus (SFV) SFV1, see Sulfolobus filamentous virus 1 (SFV1) SGCs see semigranular cells (SGCs) SGHBV see snow goose hepatitis B virus (SGHBV) SGL systems see single gene lysis (SGL) systems sGP see soluble glycoprotein (sGP) sgRNA see sub-genomic RNA (sgRNA) sharka disease, defined 3:586 shedding, virus 1:561 sheeppox and goatpox virus 2:166–167 clinical features and pathology 2:167 diagnosis 20934:s0060 epidemiology 2:166–167 histopathology 2:167 pathogenesis 2:167 prevention 20934:s0070 treatment 20934:s0065 Sheeppox virus (SPPV) 2:165, 2:167 sheldgoose hepatitis B virus 2:100 Shewanella piezophila 4:55 SHFV infecting monkeys see simian hemorrhagic fever virus (SHFV) infecting monkeys SHIBV see Shimoni lyssavirus (SHIBV) shielding, defined 5:233 Shigella flexneri 4:200 Shimoni lyssavirus (SHIBV) 2:738 Shingrix, development of 5:282 SHIV, defined 2:827 SHM see somatic hypermutation (SHM) S-hole 1:505 short non-contractile tails 4:191–192 short read sequencing (SRS) 1:182, 1:180–181 short-tailed phages (Podoviridae) 4:208–209 short-term pathogenesis 1:59 shotgun metagenomics 1:623 shotgun sequencing 1:125, 1:175, 5:27, 1:552 SHP see Structural Homology Program (SHP) SH protein see small hydrophobic (SH) protein shrimp densoviruses 4:839–840 fenneropenaeus chinensis hepandensovirus (FcHDV) 4:840 new invertebrate DVs 4:840 penaeus stylirostris penstyldensovirus 1 (PstDV1) 4:839–840 Siadenovirus 2:14 sialic-acid-binding immunoglobulin-like lectin-1 (Siglec-1) 1:399–400 sialic acid receptors 1:390–391 sialoadhesin 2:703

Subject Index Sibine fusca 4:839 sida micrantha mosaic virus (SiMMV) 3:194 SiDV see solenopsis invicta densovirus (SiDV) s replication, defined 4:61 Sigmavirus 4:715–716 sigmavirus genome 4:885 Sigmodontine-borne hantaviruses 2:351 Signal Peptidase I (SPI) 1:501 Signal Peptidase II (SPII) 1:501 signal transducer and activator of transcription (STAT) 2:555–556 silencing suppression, defined 3:293 silencing suppressor, defined 3:788, 3:667, 3:383, 3:285, 3:456, 4:632, 3:594 Simbu serogroup 2:663 simeprevir 5:123 simian 40 virus (SV40) 1:675, 1:653–654 simian adenoviruses 2:12 simian hemorrhagic fever virus (SHFV) infecting monkeys 2:245 simian immune deficiency virus (SIV) 2:56–57, 2:64, 1:121, 2:827–828, 2:460 clinical features 2:829–830 defined 2:827 epidemiology 2:829 genome 2:828–829 lifecycle 2:828–829 immunity to 2:830 taxonomy and classification 2:828 virion structure 2:828 similarity, defined 4:276 similarity score, defined 1:153 SiMMV see sida micrantha mosaic virus (SiMMV) Sindbis-like viruses 1:66 Sindbis virus (SINV) 1:263 classification 2:837 clinical features 2:840–841 diagnosis 2:841 epidemiology 2:838–840 genome 2:838 geographical distribution of 2:837f life cycle 2:838, 2:839f pathogenesis 2:841 prevention 2:841 SINV proteins and their key functions 2:838t treatment 2:841 virion structure 2:837–838 single amplified genomes (SAGs) 4:329–330 single cell sequencing 1:180 defined 1:175 to investigate cellular heterogeneity and its impact on viral infection 1:180 to investigate viral heterogeneity 1:180 single gene lysis (SGL) systems 1:514–515, 1:501–502 Archaea, phage egress in 1:517–518 extrusion 1:517 of Leviviridae 1:515–516 mysterious L 1:516–517 jX174 E 1:514–515 small interfering RNA (siRNA) 3:555, 3:560

single jelly roll (SJR) 1:16 single molecule Fo¨rster resonance energy transfer (smFRET) 4:57, 1:213–215 Single Molecule Localization Microscopy, defined 1:208 single molecule sequencing 1:180–181 defined 1:175 merging short read sequencing (SRS) and long read sequencing (LRS) 1:182 Oxford Nanopore technologies 1:181–182 Pacific Biosciences 1:181 single nucleotide polymorphism (SNP) 4:865, 1:410, 1:74, 1:75 defined 1:71 single particle analysis (SPA) 1:364–365, 5:5 Single Primer Isothermal Amplification (SPIA) 1:178 single radial immunodiffusion (SRID) 5:302 single-step growth, defined 4:314 single-strand annealing, defined 4:291 single-stranded (+)-sense RNA 1:385 single-stranded (–)-sense RNA 1:385 single-stranded DNA (ssDNA) mycoviruses 4:493 discovery of DNA mycoviruses 4:493–494 host of DNA mycovirus 4:493 Sclerotinia sclerotiorum hypovirulenceassociated DNA virus 1 (SsHADV-1) distribution of 4:498 exploring SsHADV-1 to control fungal disease 4:501–503 extracellular entry of 4:495–496 genome and proteins of 4:494–495 host range of 4:495 mutualistic interaction with mushroom sciarid fly 4:496–497 taxonomy of 4:498–501 transmission of 4:497–498 single-stranded DNA (ssDNA) viruses 1:43, 1:138, 1:385, 4:835, 2:182, 2:186, 1:19, 4:296–297 single-stranded DNA binding protein (SSB) 1:433–434 single-stranded RNA (ssRNA) 3:555 single-stranded RNA (ssRNA) bacterial viruses 5:131, 3:158, 4:621, 2:130 biotechnological applications 4:25 icosahedral ssRNA bacterial viruses 4:21–24 background 4:21–24 phage lifecycle 4:21–24 phage structure and assembly 4:24–25 single-stranded RNA (ssRNA) mycoviruses 4:437–438 unassigned ssRNA viruses 4:438 single-stranded RNA with DNA intermediate 1:386 single-virus genomics best practices and protocol for 1:184–185 sample size and preservation 1:185 sequencing 1:186 staining and single-virus separation (sorting) 1:185

385

viral capsid lysis and whole genome amplification 1:185–186 fluorescence-activated virus sorting 1:187 DNA decontamination and fluorescence-activated virus sorting preparation 1:187 preparation of 384-well plates for sorting 1:187 sorting 1:187 new tool for viral discovery 1:184 sample collection and preservation 1:186 viral staining 1:186–187 whole-genome amplification 1:187–188 DNA decontamination 1:188 reagent preparation 1:188 single virus tracking (SVT) 1:211 SINV see Sindbis virus (SINV) SINV-1 4:771, 4:772, 4:772–773 Siphoviridae 1:320, 4:186, 4:276 mosaicism in 4:278–280 siphovirus 4:342 siRNA see small interfering RNA (siRNA) Siromilus 5:170–171 SIRV, see Sulfolobus islandicus rod-shaped virus (SIRV) SISPA see Sequence Independent Single Primer Amplification (SISPA) Site Frequency Spectrum (SFS) 5:227 SIV see simian immune deficiency virus (SIV) Sjogren’s syndrome 2:454 SJR see single jelly roll (SJR) skin, polyomaviruses of 2:526 skin tumors in walleye and their associated retroviruses 2:317–318 skin virome 1:555 S-layer, defined 4:387 S-Layer protein (archaeal), defined 4:380 SLC7A1 see solute carrier family 7 member 1 (SLC7A1) SLCCNV see Squash leaf curl China virus (SLCCNV) sleeping disease (SD) 4:772 SLEV see Saint Louis encephalitis virus (SLEV) SLIPTA see Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA) SLMTA see Strengthening Laboratory Management Toward Accreditation (SLMTA) SMAC see supramolecular adhesion complex (SMAC) small angle X-ray scattering (SAXS) 1:191 as a structural tool 1:192–194 time-resolved 1:194–195 smallest capsid protein (SCP) 2:442–443 small hydrophobic (SH) protein 2:749 small icosahedral ssDNA viruses 1:268–269 small icosahedral viruses, structures of 1:278 basic structure 1:278 diversity 1:278–279 picornaviruses 1:279–282, 1:280f capsid assembly 1:280–282 genome encapsidation 1:282–283

386

Subject Index

small icosahedral viruses, structures of (continued) host interactions 1:283–285 uncoating 1:285–287 antigenicity and antibody interactions 1:287–288 antiviral drugs 1:286–287 small interfering RNA (siRNA) 2:743–744, 1:498, 3:43, 3:52, 3:123, 2:242, 3:788, 3:116, 3:293, 3:456 small interfering RNAs direct antiviral immunity 3:117–118 small ruminant lentiviruses (SRLV) 2:56–57, 2:65, 2:66–67 small soluble glycoprotein (ssGP) 2:234–235, 2:238 smFRET see single molecule Fo¨rster resonance energy transfer (smFRET) smokybrown cockroach 4:839 smolts, defined 2:544 SMYEV see Strawberry mild yellow edge virus (SMYEV) snakehead retrovirus 2:319–320 SNALPs see stable nucleic acid-lipid particles (SNALPs) SNARE proteins 1:534 snow goose hepatitis B virus (SGHBV) 2:100 SNP see single nucleotide polymorphism (SNP) sNSRV polymerases 1:350 SNT see serum neutralization tests (SNT) society, impact of viruses on 1:677–678 art and literature, viruses in 1:679 human disease 1:677–678 impact of viruses on livestock and crops 1:678–679 impact on human capital in the era of COVID-19 1:678 origins and historical demographics of viral infections in humans 1:677–678 viral epidemics in the past century 1:678 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 1:172, 5:94 sodium iodide symporter (NIS) 5:241 Sogatella furcifera 4:771 soil-borne vector transmission 3:531 Solemoviridae 3:675, 3:676, 3:712 solemoviruses (Solemoviridae) 3:712 genome 3:714–716 host range and transmission 3:717–718 pathological and epidemiological aspects 3:718 phylogenetic relationships 3:716–717 replication and movement 3:717 virion structure 3:712–714 Solenopsis geminate 4:771 Solenopsis invicta 4:771, 4:772–773 solenopsis invicta densovirus (SiDV) 4:840 Solenopsis invicta virus 1 4:771 solid organ transplantation (SOT) 5:105 solid organ transplant recipients 2:455 solid-phase microwell immunoassays 5:17–18 SOLiD sequencing 1:177

Solinviviridae 4:716–717, 4:892 classification 4:892 diagnosis 4:895–896 epidemiology 4:894–895 genome 4:893–894 life cycle 4:894 pathogenesis 4:895 prevention 4:896 virion structure 4:892–893 solinviviruses 4:717 soluble glycoprotein (sGP) 2:234–235 solute carrier family 7 member 1 (SLC7A1) 2:145 solution-state small angle X-Ray scattering 1:191–194 diffracted X-ray tracking (DXT) 1:195–197 SAXS as a structural tool 1:192–194 time-resolved SAXS 1:194–195 X-ray footprinting mass spectrometry 1:197 somatic hypermutation (SHM) 1:591 somatic incompatibility, defined 4:568 SOPs see standard operating procedures (SOPs) SOR see Standard Operation Record (SOR) SOT see solid organ transplantation (SOT) Southern Asia, CLCuD in 3:356 southern bean mosaic virus (SBMV) 1:259–260, 1:262–263, 1:278 SPA see single particle analysis (SPA) spanin 1:505 spatial structure, defined 4:314 species 1:51 assignment of viruses to 1:47–48 criteria used in species definitions 1:48–49 development of species level in virus taxonomy 1:47–48 genomics-based taxonomy of viruses, emergence of 1:50–51 metagenomically characterized viruses, classification of 1:50 species and genotypes 1:49–50 species assignments and genetic relationships 1:48 in biology 1:47 defined 1:28 nomenclature 1:51 species-jump, defined 2:730, 1:559 specificity 5:73–74 analytical 2:697 diagnostic 2:697 specimen management 5:66 spherical lattices 1:297 triangulation and derivation of 1:299f spherical viruses 4:362 atomic structure of 1:476 family Globuloviridae 4:362 family Ovaliviridae 4:362 family Portogloboviridae 4:362–363 family Turriviridae 4:363 MetSV 4:377–378 spheroid, defined 4:858 spheroplasts, defined 4:594, 4:534 spherules, defined 2:173, 3:252 SPI see Signal Peptidase I (SPI)

SPIA see Single Primer Isothermal Amplification (SPIA) SPII see Signal Peptidase II (SPII) spike 1:257 defined 4:380 spike protein 4:385–386 spillover, defined 2:117 Spinareovirinae 1:303–306, 4:868–870 antigenic and genetic relationships 4:876–877 cypoviruses 4:877 dinovernaviruses 4:877 idnoreoviruses 4:877 aquareovirus 1:306 cypovirus 1:306 historical overview 4:870 host range, diseases, transmission, and distribution 4:870–871 aquareovirus 4:871 cypoviruses 4:870–871 dinovernavirus 4:871 fijivirus 4:871 idnoreoviruses 4:871 orthoreovirus 4:871 orthoreovirus 1:305–306 virion properties, genome, and replication 4:871–875 cypoviruses 4:871–875 dinovernavirus 4:876 idnoreoviruses 4:875–876 Spinaviridae 4:713–715 spindles, defined 4:858 spindle-shaped salterprovirus His1 1:433–434 spindle-shaped viruses 1:369–370, 4:376–377 spine helix 1:263 Spiraviridae 4:362 Spirea yellow leaf spot virus (SYLSV) 3:165 spleen focus-forming virus (SFFV) 2:322 split sampling 5:74 SPMMV see sweet potato mild mottle virus (SPMMV) SPO1-like viruses, DNA replication of 4:63–65 Spodoptera ornithogalli 4:772 spore balls, defined 3:603 SPP1 connector 4:308 SPP1-like viruses, DNA replication of 4:65–66 SPPV see Sheeppox virus (SPPV); sweet potato pakakuy virus (SPPV) spraing, defined 3:743, 3:603 spring viraemia of carp virus (SVCV) 2:324–325, 2:328 S protein 2:197 sputnik virus 1:379–380 SPV1 see sulfolobus polyhedral virus 1 (SPV1) SpyTag/SpyCatcher system 1:667 SQPV see squirrel poxvirus (SQPV) squamata, defined 2:3 squamous cell carcinoma (SCC) 2:84–85, 2:86, 2:87, 2:86–87 cats 2:87 diagnosis 2:90

Subject Index dogs 2:87 horses 2:87 rabbits 2:87 ruminants 2:86–87 treatment 2:90–91 Squash leaf curl China virus (SLCCNV) 3:756 squash leaf curl virus 3:14 causal agent and classification 3:14 control 3:14 disease symptoms and yield losses 3:14 epidemiology 3:14 geographical distribution 3:14 squash vein yellowing virus (SqVYV) 3:296–297 squirrel poxvirus (SQPV) 2:666 SqVYV see squash vein yellowing virus (SqVYV) SRB1 see scavenger receptor class B member 1 (SRB1) Src, defined 2:122, 2:875 SRID see single radial immunodiffusion (SRID) SRLV see small ruminant lentiviruses (SRLV) SRS see short read sequencing (SRS) SsaDV see sea star-associated densovirus (SsaDV) SSB see single-stranded DNA binding protein (SSB) SsBV1, see Sclerotinia sclerotiorum botybirnavirus 1 (SsBV1) ssDNA mycoviruses see single-stranded DNA (ssDNA) mycoviruses ssDNA viruses see single-stranded DNA (ssDNA) viruses SsDRV, see Sclerotinia sclerotiorum debilitation-associated RNA virus (SsDRV) S segment 2:208–209 ssGP see small soluble glycoprotein (ssGP) SsHADV-1, see Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) SsHV1, see Sclerotinia sclerotiorum hypovirus 1 (SsHV1) SSITL, see Sclerotinia sclerotiorum integrin-like gene (SSITL) SsMBV1, see Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1) SsMYRV4, see Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4) SsNSRV-1, see Sclerotinia sclerotiorum negative-sense RNA virus 1 (SsNSRV-1) SSP see stable signal peptide (SSP) SSPE see subacute sclerosing panencephalitis (SSPE) ssRNA bacterial viruses see single-stranded RNA (ssRNA) bacterial viruses ssRNA mycoviruses see single-stranded RNA (ssRNA) mycoviruses SSSV see salmon swimbladder sarcoma virus (SSSV) SSTV see Swedish sea trout virus (SSTV) SSV, see Sulfolobus spindle-shaped viruses (SSV)

SSV1 see sulfolobus spindle-shaped virus 1 (SSV1) SSWD see sea-star wasting disease (SSWD) St. Louis encephalitis virus 2:809–810 stable nucleic acid-lipid particles (SNALPs) 2:616 stable signal peptide (SSP) 2:507 standard operating procedures (SOPs) 5:56–57, 5:65 Standard Operation Record (SOR) 5:65 standing variation, defined 1:71 Staphylococcus aureus pathogenicity island (SaPIs) 4:98 Staphylococcus aureus pathogenicity island 1 4:98–99 staple crops: cassava brown steak disease 3:17 causal agent and classification 3:17 control 3:18–19 disease symptoms and yield losses 3:17–18 epidemiology 3:18 geographical distribution 3:17 start-feeding, defined 2:544 steckling, defined 3:200 STED see stimulation emission depletion (STED) Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA) 5:60 sternal recumbency, defined 2:875 sterol regulatory element–binding protein (SREBP) 2:391 STHBV see stork hepatitis B virus (STHBV) stimulation emission depletion (STED) 1:497 STIs see sexually transmitted infections (STIs) STIV see sulfolobus turreted icosahedral virus (STIV) STMV see satellite tobacco mosaic virus (STMV) STNV see satellite tobacco necrosis virus (STNV) stochastic optical reconstruction (STORM) 1:497 stokes shift, defined 1:208 stop-and-start model, defined 3:833 stork hepatitis B virus (STHBV) 2:100 STORM see stochastic optical reconstruction (STORM) strabismus, defined 2:34 strain, defined 3:184, 3:8 strain sunf-M 4:464–465 strand-coupled genome replication, defined 4:387 strand-displacement, defined 4:387 Strand Displacement Amplification (SDA) 5:75–76 stratum spinosum, defined 2:629 Strawberry crinkle virus 3:628–629 Strawberry mild yellow edge virus (SMYEV) 3:623, 3:628, 3:628–629 Strawberry mottle virus 3:628–629 Strawberry pallidosis-associated virus 3:628–629 Strawbery polerovirus 1 3:628–629

387

Strengthening Laboratory Management Toward Accreditation (SLMTA) 5:60 Streptococcus thermophilus 1:633, 1:634f structural comparison of tailed phages and herpesviruses 1:322–323 major capsid proteins (MCPs) fold 1:322–323 portal protein 1:323–324 structural genomics, defined 4:359 Structural Homology Program (SHP) 1:94 structural lineage, defined 1:329 structural proteins 1:257 structure-based virus classification 1:90–92 inferring viral evolutionary relationship through 1:92–94 structure of virus, principles of 1:257 capsid architecture, general principles of 1:257–261 helical symmetry 1:261 icosahedral symmetry 1:258–261 capsid assembly 1:263–266 enveloped icosahedral RNA viruses 1:266–268 large icosahedral dsDNA viruses 1:269–271 non-enveloped icosahedral RNA viruses 1:265–266 small icosahedral ssDNA viruses 1:268–269 tailed bacteriophages 1:271–272 capsid proteins, structural folds of 1:262–263 four-helix bundle 1:263 HK97 fold 1:263 immunoglobulin-like fold 1:263 jelly-roll b-barrel 1:262–263 serine protease fold 1:263 methods for studying virus structures 1:261–262 structure of virus in 3D 1:254 STSV2 see sulfolobus tengchongensis spindle-shaped virus 2 (STSV2) SU see surface proteins (SU) subacute sclerosing panencephalitis (SSPE) 5:268, 1:562–563, 2:619 subclinical infection, defined 2:707 sub-genomic RNA (sgRNA) 1:450–452, 3:260, 2:92, 4:577, 3:252, 3:327, 3:285, 3:439, 3:818, 4:699 sub-Saharan Africa 5:142–143 substitution rate, defined 1:62 subviral RNAs 3:785–786 sucrose method 5:11 sugarcane viral diseases 3:94–95 sugarcane yellow leaf virus 3:599 Sulfolobus acidocaldarius 4:421 sulfolobus ellipsoid virus 1 (SEV1) 4:362, 1:433–434 Sulfolobus filamentous virus 1 (SFV1) 1:368 Sulfolobus islandicus 4:421 Sulfolobus islandicus rod-shaped virus (SIRV) 4:419, 4:422 SIRV1 1:434–435 SIRV2 1:367, 1:367–368, 1:368f, 4:388, 4:363 SIRV3 4:397

388

Subject Index

Sulfolobus islandicus rod-shaped virus (SIRV) (continued) Sulfolobus polyhedral virus 1 (SPV1) 4:363 Sulfolobus spindle-shaped viruses (SSV) 4:420, 4:422, 4:424 SSV1 4:388, 4:362, 4:411 Sulfolobus tengchongensis spindle-shaped virus 2 (STSV2) 4:388 Sulfolobus turreted icosahedral virus (STIV) 4:388, 4:388–389, 4:363, 1:204, 4:419–420 STIV1 4:411 sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SulfoSMCC) 1:667 support quality assurance, controls to 5:75–76 suppressors of gene silencing, defined 3:116 supraglacial (top) biotopes 4:352–353 supramolecular adhesion complex (SMAC) 1:601 surface layer (s-layer), defined 1:402 surface proteins (SU) 2:57 surveillance, essential characteristics of 5:247–248 surveillance of outbreaks 5:249 surveillance system attributes of 5:251–252 accuracy 5:252 completeness 5:251–252 consistency 5:252 representativeness 5:252 timeliness 5:252 newer types of 5:249–250 active surveillance 5:250 Enhanced Surveillance 5:250 sentinel surveillance 5:250 surveillance of HIV/AIDS 5:250–251 syndromic surveillance 5:250 sustained virological response (SVR) 2:386 SV40 see simian 40 virus (SV40) S value/Svedberg, defined 4:10 SVCV see spring viraemia of carp virus (SVCV) Svedberg unit 1:169 defined 1:162 SVR see sustained virological response (SVR) SVT see single virus tracking (SVT) Swedish sea trout virus (SSTV) 2:325 sweet potato mild mottle virus (SPMMV) 3:296–297 sweet potato pakakuy virus (SPPV) 3:163 sweet potato viral diseases 3:86 SYBR Green, defined 2:757 SYLSV see Spirea yellow leaf spot virus (SYLSV) sylvatic, defined 2:899 sylvatic cycle, defined 2:22 sylvatic cycle of CHIKV transmission, defined 2:173 sylvatic rabies, defined 2:738 symbiosis, defined 4:419 symptomatology, defined 3:247 syncytia, defined 2:619 syncytium, defined 2:475

syndromic surveillance 5:250 synergism/synergistic disease, defined 3:456 synergistic phage-bacterium relationships 1:633–634, 1:634–638 synergy, defined 3:703 synonymous, defined 1:62 synovial membranes, defined 2:875 synovitis 2:173 synteny 4:278 defined 4:359, 4:276 Synthetic Fluorescent Dyes, defined 1:208 systemic infection, defined 3:743, 2:797 systemic lupus erythematosus 2:454

T T–3 Icosahedron, defined 2:92 T4 (bacteriophage) 4:260f, 4:261f, 4:70–72, 1:319 architecture of 4:259 structure of 1:272f as a vaccine platform 4:259–261 in vivo VLP assembly 4:259–261 T5 genome 4:214 T7 (bacteriophage) 4:72, 4:73f connector 4:308 early transcription 4:72 elongation 4:74 gene expression 4:213 phage RNAP and late gene transcription 4:72 termination 4:74 transcription cycle 4:72–74 TAAs see tumor-associated antigens (TAAs) TABS, see trans-activator binding site (TABS) TABV see Tamana bat virus (TABV) Tacaribe virus (TCRV) 2:507 TAdV–3 see Turkey adenovirus 3 (TAdV–3) TAF see tenofovir alafenamide (TAF) tail, defined 1:318, 4:175, 4:276, 4:45 tail assembly 4:49 tailed bacteriophages 1:271–272 tailed bacteriophages 4:194, 4:195f tailed dsDNA phages 4:45 bacteriophage structure and function 4:46 capsids 4:46 tails and tail fibers 4:46–48 gene expression, host interactions and regulation of 4:48 classes of genes 4:48 early genes 4:48–49 late genes 4:49 genetic mosaicism in 4:277 clusters and superclusters 4:280–281 complete bacteriophage sequences, comparative genomics of 4:278 different phage clusters acquiring new genes at different rates 4:281 DNA sequencing 4:277–278 evidence for genome mosaicism before advent of DNA sequencing 4:277 mosaicism, hybrids, and single gene surveys 4:281–282

mosaicism in the Myoviridae 4:280 mosaicism in the Podoviridae 4:280 mosaicism in the Siphoviridae 4:278–280 genomes and genomics 4:50 chromosome diversity and replication 4:50 common ancestry 4:51 common themes in genome structure 4:50–51 diversity in genome size and organization 4:50 horizontal exchange of genes is widespread 4:51 injection 4:48 DNA Injection 4:48 protein injection 4:48 lysis 4:50 structure of 4:45–46 temperate versus virulent bacteriophages 4:48 virion assembly 4:49 assembly pathways 4:49 capsid assembly 4:49–50 tail assembly 4:49 tailed phage capsids 4:220–221 low copy number E proteins with specific locales in the capsid 4:221 podoviral E proteins 4:221–222 tailed phages 1:318–319 icosahedral head/capsid 1:319 infective viral cycle, structural insights into 1:324–325 final virion assembly 1:327 genome ejection 1:324–325 genome packaging 1:326–327 genome replication 1:325 procapsid morphogenesis 1:325–326 Myoviridae 1:320–321 Podoviridae 1:320 Siphoviridae 1:320 tailed viruses of methanogenic euryarchaea 4:373–374 tail fiber defined 4:194, 4:276, 4:45 structures of 4:197f tail-less phages 4:207–208 tail protein complex, defined 4:105 tailspike, defined 4:194 tail structure and dynamics 4:186–187 Caudovirales 4:186–187 long contractile tails, baseplates of 4:190–191 long non-contractile tails, tail tip complex of 4:191 organization and assembly of long tails 4:187–189 sheath 4:189–190 short non-contractile tails 4:191–192 tailspikes, tail fibers and phage-host interaction 4:192–193 tube 4:189 tail tip protein (TTP) pb9 4:191 Taiwanese Bat Lyssavirus (TBLV) 2:738 Tamana bat virus (TABV) 1:546 tandem repeats, defined 1:71

Subject Index tape measure, defined 4:276 tape measure protein (TMP) 1:320 targeted therapy, defined 1:658 targeting, defined 5:233 target recognition domain (TRD) 1:608 TAS see TNF activation site (TAS) TAstV2 see Turkey astrovirus type-2 (TAstV2) TA system see toxin-antitoxin (TA) system TATA binding protein 2:445 TATA box 2:58–59 tat gene 2:61t Taura syndrome virus (TSV) 4:772, 4:774 taxon, defined 1:28 taxonomic richness 4:677 taxonomy, defined 1:28 taxonomy, virus classification, virus 1:33 taxa and viruses, differentiating 1:33 future developments 1:35 history of 1:28–29 baltimore classification (1971) 1:29 current virus taxonomy (1971–present) 1:29–32 early taxonomic developments (1886–1971) 1:28–29 International Committee on Taxonomy of Viruses (ICTV) 1:32 organization 1:32 taxonomic process 1:32–33 nomenclature of viruses 1:34–35 virus taxa 1:33–34 tax proteins 2:533–535 TBEV see tick-borne encephalitis virus (TBEV) TBEV E 1:421 TBLV see Taiwanese Bat Lyssavirus (TBLV) TBSV see tobacco bushy stunt virus (TBSV); tomato bushy stunt virus (TBSV) TBTV see tobacco bushy top virus (TBTV) TBV see telbivudine (TBV) T cell-dependent (TD) B cell 1:591 T cell effectors in the response to viral infections 1:587 CD4+ T Cells 1:587 CD8+ cytotoxic T cells (CTL) 1:589 cytotoxic CD4+ T cells 1:588–589 regulatory T cells (Tregs) 1:588 T follicular helper (Tfh) cells 1:587–588 T helper 1 (Th1) cells 1:587 T cell help 1:589–590 T-cell immunoglobulin and mucin domain 1 (TIM-1) 1:410 T cell immunoglobulin and mucin domain 3 (TIM-3) 1:660 T cell priming 1:589 T cell receptors (TCRs) 1:585–587, 1:585, 1:587 T cell recognition of viral antigens 1:587 TCRs see T cell receptors (TCRs) TCRV see Tacaribe virus (TCRV) TCV see turnip crinkle virus (TCV) TDF see tenofovir disoproxil fumarate (TDF) T-DNA vector, defined 3:132 TE see transposable elements (TE)

TEC see transcriptional enzyme complex (TEC) tectiviruses 4:36 TED see translation enhancer domain (TED) tegument 1:321 defined 2:599, 1:318, 2:442 tegument proteins 2:452 teichoic acid, defined 4:194 telaprevir 5:123 telbivudine (TBV) 5:218 teleomorph, defined 4:450 telomeres 2:778 TEM see transmission electron microscopy (TEM) TEM8 see tumor endothelial marker 8 (TEM8) temperate, defined 1:621, 4:36, 4:276 temperate bacteriophage, defined 4:77 temperate phage decision making by 4:88 counting by infecting phages 4:90–93 decision to remain dormant 4:94–97 post-infection decision of phage lambda 4:88–90 single cell, view from 4:93–94 defined 4:88, 4:98 temperate versus virulent bacteriophages 4:48 temperate virus, defined 1:162, 4:368 template strand, defined 4:69 temporal lobe 2:778 temporal lobe epilepsy (TLE) 2:785, 2:778 tenofovir alafenamide (TAF) 5:133, 5:134 tenofovir disoproxil fumarate (TDF) 5:218, 5:133, 5:134 tenuiviruses (Phenuiviridae) 3:719–720 biological properties 3:724–725 host range and geographic distribution 3:725 classification 3:720 components 3:720–723 cytopathology 3:725 disease symptoms and control 3:725–726 genome organization 3:723 insect vector and type of transmission 3:726 physical and physicochemical properties 3:720 relation to other taxa 3:726 replication and transcription 3:723–724 taxonomy 3:720 virion structure 3:720 morphology 3:720 tepovirus, defined 3:805 TER see transepithelial electrical resistance (TER) TerL DNA packaging domain 4:127 TerL subunits 4:125–126 terminally redundant DNA, defined 4:77 terminal protein (TP) 4:761–762, 2:8, 4:62–63, 1:433–434 Terminal Protein Region 1 (TPR1) and 2 (TPR2) 1:433–434 terminal redundancy, defined 4:229 terminase, defined 4:277, 4:160, 4:105

389

terminase holoenzymes 4:126, 4:124 terminase motors 4:152–153 terminase protomer, defined 4:124 termination upstream ribosome-binding site (TURBS) 2:875 terrestrial dust 4:355 terrestrial hot springs 4:345–347 TerS subunits 4:125 tetherin 2:65 tetravirus capsid capsid maturation, auto-proteolysis and dynamics 4:901–902 capsid structure 4:900–901 tetraviruses 4:717 tetraviruses of insects 4:897 genome organization 4:897 Alphatetraviridae 4:897 Carmotetraviridae 4:898 Permutotetraviridae 4:897–898 pathology 4:902 host range 4:902 persistent infections 4:902–903 symptoms and transmission 4:902 taxonomic classification 4:898t tetravirus capsid capsid maturation, auto-proteolysis and dynamics 4:901–902 capsid structure 4:900–901 viral replication 4:898–900 TEV see Tobacco Etch Virus (TEV) T-even capsids 4:215, 4:216 T follicular helper (Tfh) cells 1:587–588 TfR see transferrin receptor (TfR) TGB proteins see Triple gene block (TGB) proteins TGEV see transmissible gastroenteritis virus (TGEV) TGMV see tomato golden mosaic virus (TGMV) TGN, see trans-Golgi network (TGN) TGS see transcriptional gene silencing (TGS) THC see total hemocyte count (THC) Theiler’s murine encephalomyelitis virus (TMEV) 1:476f, 1:447 T helper 1 (Th1) cells 1:587 Theobroma cacao 3:165 thermal environments, phages in 4:345–347 deep-sea hydrothermal vents 4:347–348 hot deserts 4:348–349 terrestrial hot springs 4:345–347 thermophilic, defined 4:359, 4:368 thermoproteus spherical piliferous virus 1 (TSPV1) 4:362 Thermoproteus tenax virus 1 (TTV1) 4:419 Thermus thermophilus 1:320 y replication, defined 4:61 thiomersal 5:302 third generation sequencing technologies 5:31 30K superfamily, defined 3:229 thoracic fluid, defined 2:875 Thosea asigna virus (TaV) 4:897–898 3’ cap-independent translation enhancers (3’ CITEs) 1:449

390

Subject Index

3C-like chymotrypsin-like protease (3CLpro) domain 4:806 3C-like protease, defined 3:703 3Dpol 4:771 threshold detection nucleic acids 2:455 thymidine kinase gene 5:185 tick-borne encephalitis virus (TBEV) 1:290 classification 2:843 clinical features 2:846–847 diagnosis 2:847–848 ecological cycle 2:847f epidemiology 2:844–846 genome strategy of 2:846f life cycle 2:843 pathogenesis 2:847 phylogenetic tree based on the complete coding regions of 2:844f prevention 2:848 structure of TBEV virion 2:845f treatment 2:848 viral proteins 2:843–844 virion structure 2:843 ticks 1:549 tilling, defined 3:69 timeliness 5:247, 5:252 time-resolved SAXS 1:194–195 time series analysis 1:565 Tipula paludosa baculovirus (TpBV) 4:828–829 Tipula paludosa nucleopolyhedrovirus (TpNPV) 4:828–829 TIRF microscopy see total internal reflection fluorescence (TIRF) microscopy tissue Culture, defined 3:430 tissue tropism, defined 2:518 TLE see temporal lobe epilepsy (TLE) TLRs see toll-like receptors (TLRs) TL viral gene see true late (TL) viral gene TM see transmembrane (TM) TMA see transcription-mediated amplification (TMA) TMEV see Theiler’s murine encephalomyelitis virus (TMEV) TMP see tape measure protein (TMP) TMV see tobacco mosaic virus (TMV) tmx gene 2:61t TNF activation site (TAS) 2:65 T number, defined 4:45 tobacco, potato virus Y (PVY) in 3:621–622 tobacco bushy stunt virus (TBSV) 1:259–260 tobacco bushy top virus (TBTV) 3:795 Tobacco curly shoot virus 3:750 Tobacco Etch Virus (TEV) 1:449 tobacco mosaic virus (TMV) 1:5, 1:363f, 5:6, 1:475–476 antigenicity of 3:730–731 beginnings of virology 3:727–728 bionanotechnology applications of 3:732–733 biotechnology applications of 3:732 physical and chemical properties of 3:728–730 replication and cell-to-cell movement of 3:732 self-assembly of TMV Particles 3:730

structure of 1:261f taxonomy and classification 3:731–732 virus disassembly 3:730 tobacco ringspot virus (TRSV) 1:279 tobamoviruses 3:9 cis-acting sequences 3:740 evolution 3:735–736 genome organization 3:737–739 history of tobamovirus research 3:734 host range and symptomology 3:740–741 interactions between viral and host proteins 3:739–740 satellite tobacco mosaic virus (STMV) 3:740 taxonomy and classification 3:734–735 transmission 3:741 viral proteins 3:739 virus replication 3:740 virus structure and composition 3:736–737 tobraviruses (Virgaviridae) diseases caused by tobraviruses 3:747–748 genome structure and expression 3:744–745 expression of virus genes 3:745 recombination and sequence deletions in tobravirus RNAs 3:745–746 RNA sequences 3:744–745 M-type and NM-type infections 3:743–744 taxonomy and characteristics 3:743 tobraviruses as gene expression/silencing vectors 3:747 viral proteins 3:746 RNA1 3:746 RNA2 3:746–747 virus particle production and structure 3:743 ToBRFV see tomato brown rugose fruit virus (ToBRFV) Togaviridae 1:469, 1:499 togavirus spherules 1:499 toggle switch 4:94–96 Tokiviricetes 4:363–364 ToLCA, see Tomato leaf curl alphasatellite (ToLCA) ToLCB see tomato leaf curl betasatellite (ToLCB) ToLCBanV, see Tomato leaf curl Bangalore virus (ToLCBanV) ToLCD see Tomato leaf curl disease (ToLCD) ToLCNDV, see Tomato leaf curl New Delhi virus (ToLCNDV) ToLCRnV, see Tomato leaf curl Ranchi virus (ToLCRnV) ToLCV, see Tomato leaf curl virus (ToLCV) Tolecusatellitidae 3:240t–241 tolerance, defined 3:749 toll-like receptors (TLRs) 1:601–602 TLR3 pathway 2:391–392 VLPs and the stimulation of 1:663–664 tomato, geminivirus resistance in 3:558–559 tomato yellow leaf curl virus (TYLCV) resistance 3:559 breeding for 3:559

genetic engineering for 3:560 Ty genes, characterization of 3:559–560 tomato, potato virus Y (PVY) in 3:620–621 tomato brown rugose fruit virus (ToBRFV) 3:10–11 causal agent and classification 3:11 control 3:12 disease symptoms and yield losses 3:11 epidemiology 3:11–12 geographical distribution 3:11 tomato bushy stunt virus (TBSV) 1:257–258, 1:258–259, 1:262–263, 1:278, 3:790–791 tomato golden mosaic virus (TGMV) 3:194 Tomato leaf curl alphasatellite (ToLCA) 3:360–361 Tomato leaf curl Bangalore virus (ToLCBanV) 3:357, 3:750 tomato leaf curl betasatellite (ToLCB) 3:361 Tomato leaf curl disease (ToLCD) 3:245 and virus biodiversity on tomato in the Indian subcontinent 3:750–751 Tomato leaf curl Gujarat virus 3:750 Tomato Leaf Curl New Delhi Disease 3:23–24 component capturing and exchange of helper components 3:24 control 3:24 disease symptoms and yield losses 3:24 epidemiology 3:24 geographical distribution 3:23–24 Tomato leaf curl New Delhi virus (ToLCNDV) 3:358–359, 3:751 diagnosis and management 3:759 genome organization and function of gene products 3:751–753, 3:755f host range of 3:755–756 maximum-likelihood dendrogram 3:752f phylogeny and molecular diversity 3:751 synergism and genetic changes leading to begomovirus emergence and evolution 3:753–755 ToLCNDV2 (JQ897969) 3:751 ToLCNDV4 (KF551592) 3:751 ToLCNDV5 (EF450316) 3:751 transmission and epidemiology 3:756–757, 3:757f virus-host interactions 3:757–758 basal level of defense 3:758 counter defense RNA silencing 3:758 resistance gene/R-gene mediated defense 3:758–759 small non-coding RNAs 3:758 Tomato leaf curl Pune virus 3:750 Tomato leaf curl Ranchi virus (ToLCRnV) 3:756 Tomato leaf curl virus (ToLCV) 3:358–359, 3:245 Tomato ringspot virus (ToRSV) 3:628–629 tomato spotted wilt virus (Tospoviridae) 1:649, 3:12 causal agent and classification 3:12 classification 3:761 control 3:13 diagnosis 3:764 disease symptoms and yield losses 3:12

Subject Index epidemiology 3:764, 3:12–13 genome 3:762 geographical distribution 3:12 pathogenesis 3:764 plant resistance 3:765–766 prevention 3:764–765 resistance breaking strains 3:13 transmission 3:762–764 virion structure 3:761–762 tomato viral diseases 3:88–89 tomato yellow leaf curl disease 3:21–22 causal agent and taxonomy 3:22–23 control 3:23 disease symptoms, host range and yield losses 3:22 epidemiology 3:23 geographical distribution 3:21–22 tomato yellow leaf curl viruses (TYLCVs) 3:768, 3:359 bipartite 3:771 breeding for resistance 3:775 diagnosis 3:774 immunodetection 3:774 microscopy 3:774 nucleic acid amplification 3:774 nucleic acid hybridization 3:774 sequencing 3:774–775 symptom observation 3:774 epidemiology 3:772–773 genetic engineering 3:775–776 CRISPR/Cas9 3:776 expression of viral genes 3:775–776 RNAi 3:776 genome 3:768–770 genome evolution 3:771 geographical distribution and spread 3:772–773 hosts 3:772 life cycle 3:771–772 monopartite 3:769–770 pathogenesis 3:773–774 prevention 3:775 biological control of whiteflies 3:775 chemical control of whiteflies 3:775 cultures under cover 3:775 planting in whitefly-free period, eradication of inoculum 3:775 satellite DNAs associated with 3:771 treatment 3:775 TYLCV transmission to tomato and host response 3:776 involvement of heat shock and quality control proteins 3:777 response of plants to TYLCV infection 3:776–777 transcriptome and metabolome 3:777 transmission by whiteflies 3:776 TYLCV-whitefly vector relationship 3:773 effects of TYLCV on life expectancy and fertility of whitefly vector 3:773 path of the virus in the insect 3:773 replication of the virus in the insect vector 3:773 retention of the virus in the insect vector 3:773 virion structure 3:768

tombusvirid, defined 3:788, 3:778, 3:285, 3:456 Tombusviridae 3:828–829, 3:778, 3:481, 3:286–287, 3:456 Tombusviruses (Tombusviridae) 3:789t–790, 1:499 classification 3:788 cytopathology 3:794 genome organization and replication strategy 3:792–794 life cycle 3:794 pathogenesis 3:795–796 subviral molecules 3:794–795 taxonomy, phylogeny and evolution 3:788–790 virion structure 3:790–792 Tombusvirus-like viruses (Tombusviridae) 3:778 applied aspects 3:786–787 gene expression 3:783 gene function 3:781–783 genome replication 3:783–785 genome structure 3:781 host range and geographic distribution 3:786 subviral RNAs 3:785–786 symptoms and transmission 3:786 taxonomy, phylogeny, and evolution 3:778–779 virion structure, assembly, and disassembly 3:779–781 Tombusvirus polymerase molecules 1:497 tomography 5:5 tonoplast, defined 3:371 topocuvirus 3:413 tOPV see trivalent oral polio vaccine (tOPV) Toroviruses 2:246, 2:253 torquetenomidivirus (TTMVD) 2:48 torquetenominivirus (TTMV) 2:48 Torque Teno Viruses (TTVs) 1:556, 5:106 torradoviruses classification 3:322 clinical features and pathogenesis 3:325 diagnosis 3:325 genome organization 3:322 geographic distribution 3:324 life cycle and epidemiology 3:322–324 host range 3:324 prevention 3:326 transmission 3:324–325 treatment 3:325–326 virion structure 3:322 ToRSV see Tomato ringspot virus (ToRSV) torticollis, defined 2:34 toscana virus 2:771 Tospoviridae 4:718, 3:761, 3:509 total hemocyte count (THC) 4:816 total internal reflection fluorescence (TIRF) microscopy 1:497, 1:211 Total Quality Management (TQM) 5:64 Totiviridae 4:648 totivirids (Totiviridae) 4:648–649 biological properties 4:654–655 defined 4:648 genome organization and expression 4:652–654

391

virus replication cycle 4:654 taxonomy and evolutionary relationships among 4:649 virion properties 4:649 virion structure and composition 4:649–652 virus–host relationships 4:655–656 totiviruses 4:630–631, 4:513, 4:504–505 defined 4:648 Toursvirus 4:724 toxin-antitoxin (TA) system 4:397–398 toxins-antitoxins 1:607 TP see terminal protein (TP) TQM see Total Quality Management (TQM) tracheoblasts, defined 4:858 tracrRNA see trans-activating crRNA (tracrRNA) traditional processing techniques, limitations of 5:11–12 trans-acting siRNA, defined 3:52, 3:123 trans-activating crRNA (tracrRNA) 4:245–246, 1:611–613 trans-activator binding site (TABS) 3:790 transactivator/viroplasmin (P6) 3:318 transcription, defined 3:778 transcriptional elongation, defined 2:475 transcriptional enzyme complex (TEC) 1:306 transcriptional gene silencing (TGS) 3:361, 3:362–363, 3:561 defined 3:116, 3:149, 3:554 transcriptional interference, defined 4:77 transcriptional regulatory sequence (TRS) 2:202–203, 2:198 transcription-dependent CRISPR interference, defined 4:400 transcription factor binding sites (TBS) 2:58–59 transcription factor II H, defined 2:765 transcription-mediated amplification (TMA) 5:98 transcription-mediated genome internalization 4:214 transcription regulating sequences (TRSs) 4:806, 4:699, 4:709, 2:195, 2:252–253 transcytosis 1:529 defined 2:789 endocytic mechanisms leading to 1:531–533 caveolin-dependent endocytosis 1:532–533 immunoglobulins as mediators of transcytosis 1:533–534 macropinocytosis 1:533 receptor-mediated, clathrin-dependent endocytosis 1:533 and viral pathogenesis 1:534–536 breaching epithelial barriers 1:535–536 fetal infections 1:537–538 neuroinvasion 1:536 viral hepatitis 1:536–537 transduction, defined 4:98 trans-encapsidation/hetero-encapsidation, defined 4:658

392

Subject Index

transepithelial electrical resistance (TER) 1:531, 2:92 transfection, defined 4:468 transferrin receptor (TfR) 1:410, 1:268–269 transfer RNAs (tRNAs) 1:377 transformation, defined 2:528 transfusion, defined 2:442 transfusion transmitted (TT) virus 1:409, 2:48, 2:51, 2:51–52 transgene, defined 3:313 transgenic P12 tobacco, defined 3:132 transgenic pathogen-derived resistance, defined 3:327 transgenic plant, defined 3:52, 3:293 trans-Golgi network (TGN) 1:498, 1:425 translational ‘recoding’ 1:452 ribosomal bypassing (‘hopping’) 1:452 ribosomal ‘frame-shifting’ 1:452–454 ribosome stop codon ‘read-through’ 1:454 ‘stopgo’/‘stop carry-on’/ribosomal ‘skipping’ 1:454 virus-encoded proteinases 1:454–455 translational repression, defined 4:21 translation enhancer domain (TED) 1:449 translesional polymerases, defined 2:683 translocating motor 4:131–132 coordinated ATP hydrolysis by terminase motors 4:132 mechanochemical coupling and DNA translocation 4:132 structure of 4:131–132 transmembrane (TM) 2:57 transmissible gastroenteritis virus (TGEV) 2:194, 2:197, 2:245, 2:198 classification 2:850 clinical features 2:850–852 diagnosis 2:852 epidemiology 2:850 genome 2:850 genome organization of 2:851f life cycle 2:850 pathogenesis 2:852 prevention 2:852 treatment 2:852 vaccines for 2:852t virion structure 2:850 transmission, defined 1:559 transmission barrier, defined 2:707 transmission electron microscopy (TEM) 1:622, 5:5, 3:629, 1:495–496, 1:497 transmission of virus, modes of 1:561–562 transmission propensity, defined 3:862 transovarial, defined 2:654 transovarial transmission, defined 3:200, 3:545, 4:724 transplacental, defined 2:442 transplant, defined 2:442 transportation and storage of specimens 5:66–67 transposable elements (TE) 1:79–80 defined 1:71 transposon, defined 4:175 transpoviron, defined 1:372 Transwell assays 1:531 traveling salesman problem, defined 1:248 TRD see target recognition domain (TRD)

trD see trimerization domain (trD) Treatment as Prevention (TasP) 2:473 Tregs see regulatory T cells (Tregs) triangulation, defined 1:248 triangulation number (T-number) 1:259–260, 1:250, 1:319, 4:342, 4:115, 4:36, 1:278, 1:318, 3:778, 4:21, 4:229 triangulation number series (T-number series), defined 1:248 triangulation numbers of icosahedra 1:222–223 Triatoma infestan 4:771 triatoma virus (TrV) 4:768–770 triatovirus 4:768, 4:769t, 4:770t, 4:774 Trichomonas vaginalis virus 1 (TVV1) 4:585 Trichoplusia ni 4:772 Trichoplusia ni virus (TnV) 4:902 trichovirus, defined 3:805 triggering, defined 1:417 trihexagonal lattice series (tt-number series), defined 1:248 TRIM5a 2:65, 2:827, 1:629–630 trimerization domain (trD) 1:424 tripartite genome 3:260, 3:260–261, 3:439 tripartite RNA genome, defined 3:252 Triple gene block (TGB) proteins 3:623, 3:623–624, 3:625–626 triple gene block, defined 4:478, 3:528, 3:140, 3:642 triradiate lumen, defined 3:486 triskelion 1:292 tritimoviruses 3:797 biotechnological application 3:803 diagnosis 3:802 disease management 3:802 cultural practice 3:802 genetic resistance 3:802–803 functions of viral proteins 3:799 genome organization 3:799 life cycle and epidemiology 3:799–800 disease cycle 3:801–802 host range 3:800 seed transmission 3:800–801 vector transmission 3:800 pathogenicity 3:802 taxonomy and classification 3:797 virion structure 3:797–799 trivalent oral polio vaccine (tOPV) 5:312–313 triviruses (Betaflexiviridae) 3:805 cellular localization of trivirus particles and viral replication 3:811–813 classification 3:805–810 diagnosis 3:815–816 genomic organization and properties of encoded proteins 3:810–811 host range and distribution of triviruses 3:813–814 phylogenetic relationships of the different triviruses 3:811 prevention and treatment 3:816 symptomology, impact and vectors 3:814–815 virion structure 3:810 tRNA-like structure, defined 3:336

tRNA primer 5:131–132 tRNAs see transfer RNAs (tRNAs) trophallaxis, defined 4:892 tropism, defined 1:382, 2:442 TRS see transcriptional regulatory sequence (TRS) TRSs see transcription regulating sequences (TRSs) TRSV see tobacco ringspot virus (TRSV) true flies see diptera true late (TL) viral gene 2:452 Trypanosoma cruzi 4:772–773 tsetse fly, GpSGHV transmission dynamics in 4:788–789 TSPV1 see thermoproteus spherical piliferous virus 1 (TSPV1) TSV see Taura syndrome virus (TSV) TTMV see torquetenominivirus (TTMV) TTMVD see torquetenomidivirus (TTMVD) TTV1, see Thermoproteus tenax virus 1 (TTV1) TT virus see transfusion transmitted (TT) virus TTVs see Torque Teno Viruses (TTVs) Tulip virus X (TVX) 3:628 tumor-associated antigens (TAAs) 1:659, 1:658 tumor endothelial marker 8 (TEM8) 1:659 tumorigenesis, defined 2:528 tumor virology 2:416 TURBS see termination upstream ribosomebinding site (TURBS) turgor 4:216 Turkey adenovirus 3 (TAdV–3) 2:13 Turkey astrovirus type-2 (TAstV2) 2:96 turncurtovirus 3:413 turnip crinkle virus (TCV) 3:790–791 turnip yellow mosaic virus (TYMV) 1:258–259 Turriviridae 4:363 TVV1, see Trichomonas vaginalis virus 1 (TVV1) TVX see Tulip virus X (TVX) 20th century state sponsored biological weapons programs 1:645–646 2A-like motif, defined 4:658 TYLCV-IR 3:244 TYLCVs see tomato yellow leaf curl viruses (TYLCVs) Tymoviridae 3:818 tymoviruses (Tymoviridae) 3:818–821 bombyx mori latent virus 3:825 properties and distinguishing characteristics 3:825 capsid structure 3:821 family and its distinguishing features 3:818 genome organization 3:821–822 infection and transmission 3:822 maculaviruses 3:824 properties and distinguishing characteristics 3:824 marafiviruses 3:822–823 infection and transmission 3:824 properties and distinguishing characteristics 3:823 virion structure, genome organization, replication cycle, and phylogenetic relationships 3:823–824

Subject Index phylogenetic relationships and species demarcation 3:822 poinsettia mosaic virus 3:824–825 properties and distinguishing characteristics 3:825 properties and distinguishing characteristics 3:821 replication cycle 3:822 TYMV see turnip yellow mosaic virus (TYMV) Type I CRISPR-Cas systems 4:247 Type I interferon, defined 2:428 Type 1 wild poliovirus (WPV1) 5:310 Type II CRISPR-Cas systems 4:247–248 Type 2 wild poliovirus (WPV2) 5:310 Type III CRISPR-Cas systems 4:248 Type 3 wild poliovirus (WPV3) 5:310 Type IV CRISPR-Cas systems 4:248 Type 4 pili, defined 4:387 type species, defined 4:632 5’ tyrosyl-DNA phosphodiesterase 2 (TDP2) 2:336

U UK Biological weapons program 1:645–646 UL28, defined 5:181 UL36, defined 5:181 ultracentrifugation 5:11 ultrafiltration methods 1:167, 1:167f ultrastructural imaging 1:496–497 Umbravirus 3:788 umbraviruses (Calvusvirinae, Tombusviridae) 3:827 cell-to-cell movement function 3:830 classification 3:827 genome organization and expression 3:827–829 host range 3:831 interaction with assistor virus 3:831 involvement of nucleolus in umbravirus systemic infection 3:831 phloem-dependent long-distance movement function 3:830–831 prevention and control 3:831–832 replication 3:829 satellite RNA 3:829–830 similarity with other taxa 3:829 transmission 3:831 virion structure 3:827 UN 3373, defined 5:181 uncoating, defined 1:388, 1:382, 2:92 unconventional bioweapons 1:644 unfolded protein response, defined 2:428 unit length versus headful packaging mechanisms 4:124–125 universal prophylaxis 5:191 unrooted tree 1:117–118 untranslated regions (UTRs) 3:331 defined 2:757, 4:21, 4:792 3’ untranslated region (3’UTR) 2:892, 2:892f upper respiratory tract infections (URTI) 5:199, 5:199–200

upstream regulatory region (URR) 2:493 urban cycle of CHIKV transmission, defined 2:173 uridylation, defined 2:757 URR see upstream regulatory region (URR) URTI see upper respiratory tract infections (URTI) US28 signaling 2:457 u-spanin, defined 1:501 Ustilago maydis viruses and their killer toxins atomic structure of KP6 4:516 comparison of the killer proteins 4:517–518 effect of KP4 on plants 4:515 effects of KP4 on U. maydis cells 4:514–515 evolutionary origin of KP4 4:516 killer phenomena 4:513 KP1 4:516–517 KP4 blocks L-type voltage gated Ca2+ channels 4:515 KP4 protein 4:513–514, 4:514f KP6 4:516, 4:517f possible application of KP4: fungal resistance in plants 4:515–516 totiviruses 4:513 Ustilago maydis virus H1 (UmV-H1) 4:647 UTRs see untranslated regions (UTRs)

V VAC see viral assembly compartment (VAC) vaccination, induction of immunity to viruses by 1:592–595 vaccine-associated paralytic poliomyelitis (VAPP) 5:311, 2:692 vaccine-derived poliovirus (VDPV) 5:310 vaccine design, biotechnology approaches to virus-like particle (VLP)-based vaccines for humans 1:668 HPV and HBV 1:668 influenza 1:668 polio 1:668–669 virus-like particles (VLPs) 1:662 virus-like particles (VLPs), expression systems to produce 1:664 bacteria 1:664 cell-free protein synthesis (CFPS) 1:665 insect cells 1:664 mammalian cells 1:664 plants and plant cell culture 1:664–665 yeast 1:664 virus-like particles (VLPs), immunological properties of 1:662 cellular immune response, VLPs and induction of 1:662 humoral immune response, VLPs and induction of 1:662–663 toll-like receptors, VLPs and the stimulation of 1:663–664 virus-like particles (VLPs) as cancer vaccines and therapeutics 1:669–670

393

virus-like particles (VLPs) as nano-carriers 1:665–666 conjugation of antigens and virus-like particles 1:665–666 loading of virus-like particles by encapsulation 1:667–668 vaccine effectiveness, defined 5:289, 5:295 vaccine efficacy, defined 5:289, 5:295 vaccine for coronaviruses (CoVs) 2:205–206 vaccine immunogenicity, defined 5:295 vaccine production, safety, and efficacy 5:281 costs and duration of vaccine development 5:283–284 emerging viral diseases, challenge of 5:285–286 history of vaccination 5:281–282 in human studies 5:283 influenza vaccines, licensing of 5:285 more about vaccine development 5:285 phases and timelines in vaccine development: from discovery to product 5:282 preclinical studies 5:282–283 Shingrix, development of 5:282 taking stock 5:285 vaccine industry 5:285 vaccine production 5:284–285 vaccines 1:675–676, 5:205 vaccines against viral gastroenteritis CDC disclaimer 5:293 norovirus 5:291–292 future considerations for norovirus vaccines 5:293 norovirus vaccine development 5:292 norovirus vaccines in human clinical trials 5:292–293 norovirus vaccines in pre-clinical development 5:293 rotavirus vaccines 5:289–290 licensed rotavirus vaccines 5:290 rotavirus vaccine safety monitoring 5:291 rotavirus vaccines in development 5:291 vaccine effectiveness and impact 5:290–291 vaccines and antiviral agents 5:213 hepatitis A virus 5:213 immunoglobulin 5:213 treatment 5:213 vaccine 5:213 hepatitis E virus 5:213–214 management of acute hepatitis E 5:214 management of chronic infections 5:214 prophylaxis 5:214 vaccine trials 1:566 vaccinia immune globulin (VIG) 5:271–272 vaccinia virus (VACV) 2:666, 2:171, 1:498, 1:461 antiviral drugs 2:859 classification 2:854 genome 2:855 host range 2:857–858 immunomodulators 2:858 life cycle 2:856f, 2:855 assembly 2:857

394

Subject Index

vaccinia virus (VACV) (continued) entry 2:855 genome replication 2:856–857 transcription and translation 2:855–856 origin of 2:854 smallpox vaccine 2:858–859 vaccine vectors 2:859 virion structure 2:854–855 virulence 2:858 VACV see vaccinia virus (VACV) valaciclovir 5:183–184 valganciclovir 2:458–459 vaniprevir 5:123 VAP see virion-associated protein (VAP); virus-associated pyramids (VAP) VAPP see vaccine-associated paralytic poliomyelitis (VAPP) variable regions (VRs) 4:841 varicella, defined 2:860 varicella hyperimmune immunoglobulin 5:272 varicella-zoster immunoglobulin (VZIG) 5:187–188, 5:272 varicella zoster virus (VZV) 5:127–128, 5:109–110, 5:194, 1:321 classification 2:860 clinical features 2:863–864 diagnosis 2:865 DNA polymerase 5:128 epidemiology 2:862–863 genome 2:860–861 life cycle 2:861–862 pathogenesis 2:864–865 prevention 2:866 treatment 2:865–866 virion structure 2:860 varicella-zoster virus (VZV) infections 5:182t antiviral agents in clinical use 5:183 acyclovir 5:183 brivudine ((E)-5-(2-bromovinyl)-2’deoxyuridine) 5:184 famciclovir 5:184 foscarnet (trisodium phosphonoformate) 5:184 valaciclovir 5:183–184 development of resistance 5:184–185 DNA polymerase gene 5:185 thymidine kinase gene 5:185 laboratory diagnosis 5:181 detection of antibodies 5:182–183 direct detection of virus 5:181–182 submission of samples 5:181 prevention 5:186–187 passive immunoprophylaxis of varicella 5:187–188 prevention of varicella by vaccination 5:186–187 prevention of zoster by vaccination 5:188–189 resistance testing 5:185–186 genotyping 5:186 phenotyping 5:185–186 varicosaviruses (Rhabdoviridae) 3:833–834 genetics and evolution 3:834–835 L protein encoded on RNA1 3:834–835 proteins encoded on RNA2 3:835–836

transcription termination/initiation strategy 3:836–837 host range and geographic distribution 3:837 taxonomy and classification 3:834 transmission and vectors 3:837–838 Varidnaviria 1:20 variola virus (VARV) 2:666, 5:127 classification and structural morphology 2:868–869 clinical features 2:870–871 diagnosis 2:872 epidemiology 2:871 history 2:868 pathogenesis 2:869–870 prevention 2:872–873 treatment 2:873–874 viral life cycle 2:869 VP37 envelope wrapping protein 5:127 VariZIG, defined 2:860 VariZig administration 5:272 Varroa destructor 4:771 VARV see variola virus (VARV) vascular endothelial growth factor (VEGF) 2:671 vCBP see chemokine binding protein (vCBP) VCGs see vegetative compatibility groups (VCGs) VCMD see viral covert mortality disease (VCMD) vCyclin 2:602 VDJ rearrangement 1:590–591 VDPV see vaccine-derived poliovirus (VDPV) vector-borne transmission 1:562, 1:559 vector capacity, defined 1:542 vector competence, defined 1:542, 2:805 vectored RSV vaccines 2:755 VEEV see Venezuelan equine encephalitis virus (VEEV) vegetable viral diseases 3:86–87 chilli viral diseases 3:86–87 cucurbit viral diseases 3:87–88 okra viral diseases 3:88 tomato viral diseases 3:88–89 vegetable viruses 3:9 begomoviruses 3:13–14 cucumber green mottle mosaic virus 3:9 causal agent and classification 3:9 control 3:10 disease symptoms and yield losses 3:9–10 epidemiology 3:10 geographical distribution 3:9 orthotospoviruses 3:12 squash leaf curl virus 3:14 causal agent and classification 3:14 control 3:14 disease symptoms and yield losses 3:14 epidemiology 3:14 geographical distribution 3:14 tobamoviruses 3:9 tomato brown rugose fruit virus (ToBRFV) 3:10–11 causal agent and classification 3:11

control 3:12 disease symptoms and yield losses 3:11 epidemiology 3:11–12 geographical distribution 3:11 tomato spotted wilt virus 3:12 causal agent and classification 3:12 control 3:13 disease symptoms and yield losses 3:12 epidemiology 3:12–13 geographical distribution 3:12 resistance breaking strains 3:13 vegetative Compatibility, defined 4:457 vegetative compatibility groups (VCGs) 4:520 vegetative incompatibility 4:601, 4:431, 4:520, 4:589, 4:607, 4:493, 4:648, 4:552, 4:528, 4:468, 4:522–523 vegetative incompatibility, in filamentous fungi 4:520 genetics of 4:522–523 HET protein involved in NLR-mediated innate immunity in fungi 4:523–525 microscopic and macroscopic analyses of hyphal fusion 4:521 mycoviruses, horizontal transmission of 4:525–526 mycovirus transmission 4:525 potential approaches to enhance mycovirus transmission between VCGs 4:526–527 signaling pathways of 4:523 vegetative compatibility groups (VCGs), identification of 4:521–522 auxotrophic complementation 4:522 barrage assay 4:521–522 detecting the PCD of fused cells 4:522 microscopy 4:522 visualization of labeled proteins during co-culture 4:522 VEGF see vascular endothelial growth factor (VEGF) VEGF-E 2:670–671 VeHF see Venezuelan hemorrhagic fever (VeHF) velarivirus, defined 3:336 velogenic viruses 2:650 Venezuelan equine encephalitis virus (VEEV) 2:40, 2:43, 2:44–45, 2:45, 2:45–46, 2:46, 1:645–646, 1:536, 1:491, 1:546 Venezuelan hemorrhagic fever (VeHF) 2:512 vertebrates, prions of see prions of vertebrates vertex, defined 1:402 vertical mycovirus transmission, defined 4:468 vertical transmission 2:778, 2:218, 2:899, 4:419, 3:388, 3:430, 3:106, 4:780, 4:888, 4:768 vertical/transplacental transmission 1:562, 1:559 Verticillium albo-atrum 4:523 Verticillium dahliae 4:523 very low-density lipoprotein receptor (VLDL-R) 1:284 very low-density lipoproteins (VLDL) 2:386–387

Subject Index vesicle, defined 2:860, 2:868 vesicle-like archaeal viruses 4:380–381 genomic characteristics 4:381–382 gene content and conserved genes 4:382 new isolates and haloarchaeal genomic regions 4:384 Pleolipoviridae 4:382–384 pleolipovirus infectivity, stability of 4:384 salinity 4:384 temperature and other significant factors 4:384–385 structural proteins 4:385–386 spike protein 4:385–386 virion structure and viral life cycle 4:381 vesicle-mediated transcytosis and export of viruses 1:529–530 endocytic mechanisms leading to transcytosis 1:531–533 caveolin-dependent endocytosis 1:532–533 immunoglobulins 1:533–534 macropinocytosis 1:533 receptor-mediated, clathrin-dependent endocytosis 1:533 endocytic sorting and vectorial transport of vesicles 1:534 extracellular vesicle-mediated export of viruses 1:538 autophagy-related vesicle-mediated release of virus 1:539 exosome-like release of quasi-enveloped viruses 1:538–539 nonlytic release of virus in extracellular vesicles 1:538 in vitro models of viral transcytosis 1:531 secretion of cargo 1:534 transcytosis 1:530–531 transcytosis and viral pathogenesis 1:534–536 breaching epithelial barriers 1:535–536 fetal infections 1:537–538 neuroinvasion 1:536 viral hepatitis 1:536–537 vesicle packets (VPs) 1:498–499, 1:499 vesicular stomatitis Alagoas virus (VSAV) 2:875 vesicular stomatitis Indiana virus (VSIV) 2:516, 2:875 vesicular stomatitis New Jersey virus (VSNJV) 2:875, 2:881f vesicular stomatitis virus (VSV) 1:64, 2:243, 1:10, 5:94, 1:659, 1:180, 5:239, 1:617, 2:808 classification 2:875 control and treatment 2:882–883 epidemiology 2:880–881 life cycle 2:878–879 assembly and budding 2:879–880 attachment, entry and uncoating 2:878–879 gene expression and replication 2:879 inhibition and modification of host cell functions 2:880 pathogenesis and clinical features 2:881–882

virus structure 2:875–878 genomes and proteins 2:877–878 VSV G 1:424 VFs see virus factories (VFs) VHF see viral hemorrhagic fever (VHF) VHGs see Virus Hallmark Genes (VHGs) VHSV see viral hemorrhagic septicemia viruses (VHSV) ViCTree 1:102 vif gene 2:61t VIG see vaccinia immune globulin (VIG) VIGS see virus-induced gene silencing (VIGS) ViPR see Viral Pathogen Resource (ViPR); virus pathogen database and analysis resource (ViPR) ViPTree 1:102 viral assembly 1:231f, 1:232 viral assembly compartment (VAC) 2:452 defined 2:442 viral capsid, defined 2:79, 1:248 viral capsid antigen (VCA) IgG 5:195 viral core, defined 2:643 viral covert mortality disease (VCMD) 4:823, 4:825, 4:822 viral disease, implications for 1:59 drug resistance 1:60 immune escape 1:59–60 short-term pathogenesis 1:59 viral DNA packaging machines, enzymology of 4:124–125 genome maturation complex 4:128–129, 4:130 lambda cohesive end site and assembly of 4:128–129 model for the assembly of 4:129 The cos-cleavage reaction 4:129–130 genome packaging complex 4:130 assembly of the packaging motor 4:130 cos-clearance: transition to a translocating motor 4:130–131 nucleotide switch for cos-clearance? 4:131 lambda system 4:126 Escherichia Coli integration host factor 4:128 l TerL maturation domain 4:127–128 l TerS subunit 4:126–127 TerL DNA packaging domain 4:127 terminase enzyme of phage lambda 4:126 packaging, termination of 4:132–133 terminase ejection and virion completion 4:133–134 unit length packaging motors 4:133 translocating motor 4:131–132 coordinated ATP hydrolysis by terminase motors 4:132 mechanochemical coupling and DNA translocation 4:132 structure of 4:131–132 unit length versus headful packaging mechanisms 4:124–125 viral terminase enzymes 4:125 TerL subunits 4:125–126 terminase holoenzymes 4:126

395

TerS subunits 4:125 viral dUTPase 2:671 viral envelope 1:468, 1:402, 4:359 viral evolutionary relationships from sequence analysis 1:88–90 viral factory, defined 1:372 viral fibers 1:225f viral fitness 4:419, 4:419–420 viral genome-assisted capsid assembly 1:486 viral genomic DNA, recognition of 4:141 concatemeric DNA and terminase proteins 4:141–142 cohesive ends 4:142–144 direct terminal repeats 4:142 headful bacteriophages, genome recognition in 4:144–146 monomeric genomes and terminal proteins 4:141 viral GM-CSF inhibitory factor (GIF) 2:671 viral hemorrhagic fever (VHF) 5:66 viral hemorrhagic septicemia viruses (VHSV) 2:324, 2:328 viral hepatitis 1:536–537 viral IL-10 2:671 viral infected myeloid cells 2:812 viral interferon resistance protein 2:671 viral killer toxins 4:534–535 dsRNA viruses and killer phenotype expression in Saccharomyces cerevisiae 4:535–536 endocytosis and intracellular transport of K28 virus toxin 4:538–539 ER exit and nuclear entry of the K28 virus toxin 4:539 K28 affecting DNA synthesis, cell-cycle progression, and induces apoptosis 4:539–541 killer virus-infected yeast, self-protection in 4:542 lethality of membrane damaging viral killer toxins 4:541–542 viral preprotoxin processing and toxin maturation 4:536–538 viral replication cycle 4:536 viral late transcription factor 3 (VLTF3) 1:377–378 viral membrane assembly proteins (VMAPs) 2:857 viral membranes 1:225f viral metagenomes archaeal viruses and 4:414–415 from hypersaline environments 4:415–416 viral metagenomics, defined 1:552 viral mutants, defined 3:727 viral nervous necrosis (VNN) 4:822–823, 4:819, 4:825 viral nucleic acid, delivery of 1:230f visualization of 1:230–232 viral operational taxonomic unit (vOTU) 1:621, 1:622 Viral Pathogen Resource (ViPR) 1:141 viral plaques 2:85 cats 2:85–86 diagnosis 2:90 dogs 2:85 horses 2:85

396

Subject Index

viral plaques (continued) treatment 2:90 viral protein 24 (VP24) 2:612–613 viral protein 30 (VP30) 2:612 viral protein 35 (VP35) 2:612 viral protein 40 (VP40) 2:612 viral protein-genome linked 3:293, 3:692, 3:364, 3:348 viral proteins, translation of 1:444 canonical/cap-dependent MRNA translation 1:444–445 elongation and termination phases 1:446 initiation phase 1:444–445 ‘imprisonment’ of cellular mRNAs within the nucleus 1:447 microRNAs (miRNAs) 1:455 non-canonical initiation of virus mRNA translation 1:447–449 cap-independent translation enhancers (CITEs) 1:449 initiation at non-AUGs 1:450 internal ribosome entry sites (IRESes) 1:447–449 leaky scanning 1:450 ribosome reinitation 1:450–452 ribosome ‘shunting’ 1:450 virus alternatives to components of initiation 1:449–450 translational ‘recoding’ 1:452 ribosomal bypassing (‘hopping’) 1:452 ribosomal ‘frame-shifting’ 1:452–454 ribosome stop codon ‘read-through’ 1:454 ‘stopgo’/‘stop carry-on’/ribosomal ‘skipping’ 1:454 virus-encoded proteinases 1:454–455 viral quasispecies, defined 5:27, 1:53 viral reactivation, defined 2:306 viral replicase proteins 1a and 2a 3:265 viral replication 4:898–900 viral replication complex (VRC) 1:495 defined 1:495 viral ribonucleoproteins (vRNPs) 2:118, 2:561 viral ribonucleoproteins/nucleocapsids assemblies 1:349 viral RNAs (vRNAs) 4:897 viral silencing suppressors (VSRs) 3:43 viral species, defined 2:48 viral suppressor of RNA silencing (VSR) 3:420, 3:545, 3:623 Viral Tiling theory (VTT) 1:251, 1:251f, 1:248, 1:250–251 viral titer detection 3:749 viral transcription 1:439 general principles of 1:439 5’ and 3’ ends promote translation, features of viral transcripts at 1:439–440 host transcription machinery, degree of dependence on 1:439 overall genome organization orchestrating viral transcriptional program 1:440–441 transcription strategies determining mechanisms of host shutoff 1:440

RNA-directed RNA polymerase (RdRp) 1:441 containing evolutionarily conserved architecture 1:441 coordinating transcription, capping, and polyadenylation 1:442–443 different RdRp and template conformations distinguish transcription from replication 1:441–442 VirCapSeq-VERT 5:93 viremia 2:896, 2:805, 2:173, 2:48, 2:884 Virgaviridae 3:743, 3:603, 3:528–529, 3:839–843 virgaviruses (Virgaviridae) 3:839–843 diagnosis 3:849 epidemiology and control 3:850 genome organization 3:847 infectious cycle 3:849 pathogenicity 3:849–850 replication 3:847–849 taxonomy, phylogeny and evolution 3:843–844 unassigned virgaviruses 3:850 virion structure 3:844–847 virion assembly 4:49 assembly pathways 4:49 capsid assembly 4:49–50 tail assembly 4:49 defined 2:442 as a genome delivery devise 1:402–403 structure 4:769f, 2:806–808 virion-associated protein (VAP) 3:163, 3:318 virion-containing vesicles, defined 4:724 virion egress 4:393–394 cell membrane disruption 4:393–394 membrane disruption, viral release without 4:394 virion infectivity factor (Vif) 1:68 virion-producing organisms 1:14 virions, structural features of 4:841–843 virion sense genes (MP and CP) 3:465 virion structure and viral life cycle 4:381 virocell 1:625, 1:14, 1:621 virocell concept critics of 1:24–25 diversity of virocells 1:25 virocells and ribovirocells 1:25 virocells with nucleus 1:25 implications of 1:25–26 enumeration of viruses in the environment 1:26 living status of viruses 1:26–27 viruses as cradles of new genes 1:25–26 virion/virus paradigm 1:23 critics of 1:23–24 virocontrol, defined 4:431, 4:461 viroids (Pospiviroidae and Avsunviroidae) classification 3:853–855 host range and transmission 3:855 genome structure 3:852–853 movement 3:859–860 pathogenicity 3:860–861 implications for host range 3:861 origin and evolution 3:861

replication 3:858–859 symptomatology 3:855 epidemiology and control 3:855–858 geographic distribution 3:855 molecular biology 3:858 taxonomy 3:852 virology 1:3 biochemical phase 1:6 cell biology phase 1:6–7 dawn of 1:3–5 drugs 1:10–11 epidemiology 1:9 nobel prizes awarded for work of relevance to 1:4t pathogenicity and host defense mechanisms 1:9 physico-chemical phase 1:5–6 previrology 1:3 selected historical highlights in 1:5t sequencing phase 1:8–9 structural phase 1:7–8 vaccines 1:10 viral control 1:9–10 viral diversity 1:7 virus emergence and human activities 1:11–13 virome 4:342 defined 4:265 virome, human composition and diversity of 1:553–555 blood virome 1:556 gastrointestinal tract virome 1:553–555 genital and urinary tract viromes 1:556 oral cavity and respiratory tract viromes 1:555–556 skin virome 1:555 definition of 1:552–553 in human health and immunity 1:556–557 technological development and limits for the description of 1:553 viromics, defined 1:621, 1:133 virophage 4:342, 4:677, 1:372, 3:681 viroplasm, defined 2:343, 3:545, 3:567, 3:158 viroporin, defined 2:428 virosphere, defined 1:87 Virtovirus 3:581, 3:584 virulence, defined 1:569, 2:697, 3:371 virulence gene, defined 3:60, 4:849 virulence of the virus, influence of 1:563 virulent, defined 4:36 virulent systemic strains of FCV (VS-FCV) 2:298 virulent virus, defined 1:162, 4:368 viruliferous, defined 3:200, 3:461 viruliferous whitefly 3:749, , defined, 3:768 virus, defined 1:28 virus-associated nucleic acid, defined 3:681 virus-associated pyramids (VAP) 4:394, 4:395f virus attachment protein, defined 1:382 virus bioinformatics 1:124 diagnostics, tools for 1:124–125 evolution and phylogenetics 1:128–129 genome sequencing 1:125–127

Subject Index host transcriptomics 1:130–131 machine learning as an opportunity 1:129–130 RNA secondary structures in viruses 1:127 technology and bioinformatics drive discoveries 1:124 viral metagenomics 1:127–128 virus-host interactions 1:129 virus disassembly, defined 3:727 virus-encoded proteinases 1:454–455 virus-encoded suppressors of host RNAi (VSRs) 4:772 virus factories (VFs) 1:495 defined 1:495 main components and functions of 1:496f methods to study 1:495–498 representative examples of 1:498–499 as targets for antiviral therapies 1:499 Virus Hallmark Genes (VHGs) 1:38–39, 1:39–40 virus immunity, defined 3:554 virus-induced gene silencing (VIGS) 3:124, 3:564, 3:555 advantages and limitations 3:130 application 3:128–130 defined 3:420, 3:554 establishment of a VIGS System 3:124–126 post-transcriptional gene silencing (PTGS) and 3:124 post-transcriptional gene silencing (PTGS) and VIGS 3:124 vectors 3:126–130 virus-induced gene silencing, defined 3:116 virus infected cells 1:226f virus interactions impacting ecology and evolution 4:425–426 virus like particle (VLP) 1:257, 3:96, 2:493, 1:662, 4:342, 5:295–296, 1:282–283, 2:844, 2:70, 4:259, 1:26, 4:343, 4:347, 4:348f antigen display on the surface of 1:669f -based vaccines for humans 1:668 human papillomavirus (HPV) and hepatitis B virus (HBV) 1:668 influenza 1:668 polio 1:668–669 biotechnology uses of 1:663f cell-specific cargo delivery using 1:670f defined 2:173 expression systems to produce 1:664 bacteria 1:664 cell-free protein synthesis (CFPS) 1:665 insect cells 1:664 mammalian cells 1:664 plants and plant cell culture 1:664–665 yeast 1:664 immunological properties of 1:662 and induction of cellular immune response 1:662 and induction of humoral immune response 1:662–663 and stimulation of toll-like receptors 1:663–664 virus-like particles as nano-carriers 1:665–666

conjugation of antigens and virus-like particles 1:665–666 chemical and affinity conjugation 1:666–667 genetic fusion 1:665–666 SpyTag/SpyCatcher system 1:667 loading of virus-like particles by encapsulation 1:667–668 virus load, defined 5:197 virus neutralisation test (VNT) 2:651–652, 2:77, 2:360 virus-neutralizing antibodies (VNAs) 2:741–742 virus pathogen database and analysis resource (ViPR) 1:143 virus reactivation 2:778 virus re-assortment, defined 3:692, 3:348 virus replication complexes, defined 3:692, 3:364, 3:348 virus resistance, defined 3:554 virus response 1 (vr1) 4:432 virus self-assembly, defined 3:727 virus symptomatology, defined 3:539 virus tolerance, defined 3:554 viscerotropic disease 2:891 visna-maedi virus (VMV) 2:56–57, 2:65, 2:66–67 vitellogenesis, defined 4:780 vitivirus, defined 3:805 vitrification 5:5, 4:342 VLDL see very low-density lipoproteins (VLDL) VLDL-R see very low-density lipoprotein receptor (VLDL-R) VLP see virus like particle (VLP) VLTF3 see viral late transcription factor 3 (VLTF3) VMAPs see viral membrane assembly proteins (VMAPs) VMV see visna-maedi virus (VMV) VNAs see virus-neutralizing antibodies (VNAs) VNN see viral nervous necrosis (VNN) VNT see virus neutralisation test (VNT) vOTU see viral operational taxonomic unit (vOTU) voxilaprevir 5:123 VP3-Like protein, defined 4:380 VP4-Like protein, defined 4:380 VPg 4:770–771 defined 4:768 vpr gene 2:61t, 2:62 VPs see vesicle packets (VPs) Vps4 1:499 vpu gene 2:61t vpw gene 2:61t vpx gene 2:61t vpy gene 2:61t VRC see viral replication complex (VRC) vRNAs see viral RNAs (vRNAs) vRNPs see viral ribonucleoproteins (vRNPs) VRs see variable regions (VRs) VSAV see vesicular stomatitis Alagoas virus (VSAV) VSIV see vesicular stomatitis Indiana virus (VSIV)

397

VSNJV see vesicular stomatitis New Jersey virus (VSNJV) VSR see viral suppressor of RNA silencing (VSR) VSRs see viral silencing suppressors (VSRs) VSRs see virus-encoded suppressors of host RNAi (VSRs) VSV see vesicular stomatitis virus (VSV) VTT see Viral Tiling theory (VTT) VZIG see varicella-zoster immunoglobulin (VZIG) VZV see varicella zoster virus (VZV) VZV infections see varicella-zoster virus (VZV) infections

W waikaviruses 3:703 classification 3:703–704 diseases and management 3:710 genome organization 3:707–708 properties of viral proteins 3:708–709 properties of virions 3:707 transmission 3:709–710 variation of isolates and strains 3:704–707 walleye dermal sarcoma (WDS) 2:317 walleye dermal sarcoma virus (WDSV) 2:316, 2:318–319 walleye discrete epidermal hyperplasia (WEH) 2:317 walleye epidermal hyperplasia virus type 1 (WEHV-1) 2:316 walleye retroviruses 2:316 wamavirus, defined 3:805 water- and food-borne transmission 5:208 watermelon mosaic virus and zucchini yellow mosaic virus biotechnological application 3:870 classification 3:862–863 control methods 3:867 cross-protection 3:867 prophylactic measures 3:867 resistant cultivars 3:867–868 diagnostic methods 3:865 epidemiology 3:866 efficient vectors 3:866 pattern of spread 3:866–867 virus sources 3:866 genetics and evolution 3:868–870 geographic distribution 3:865 history and taxonomy 3:862 host range 3:865 symptomatology 3:863 synergism and antagonism 3:864–865 variability 3:868 vector relationships 3:865–866 WCBV see West Caucasian bat lyssavirus (WCBV) WClMV see White clovermosaic virus (WClMV) WDS see walleye dermal sarcoma (WDS) WDSV see walleye dermal sarcoma virus (WDSV)

398

Subject Index

weak crystallization theory 1:297–299 weapons of mass destruction, defined 1:644 WEE complex of viruses see Western equine encephalomyelitis (WEE) complex of viruses WEEV see Western equine encephalitis virus (WEEV) WEH see walleye discrete epidermal hyperplasia (WEH) WEHV-1 see walleye epidermal hyperplasia virus type 1 (WEHV-1) Weighted Gene Coexpression Network Analysis (WGCNA) 1:143–144 West Caucasian bat lyssavirus (WCBV) 2:738 Western equine encephalitis virus (WEEV) 2:40, 2:43, 2:44, 2:45, 2:46 Western equine encephalomyelitis (WEE) complex of viruses 1:66 Westgard Rules 5:39–40, 5:57–58 West Nile virus (WNV) 1:572, 1:180, 5:275, 1:649, 2:848, 2:810–811, 2:812 classification 2:884 clinical manifestations 2:888–889 diagnosis 2:889 epidemiology 2:887–888 genome and viral proteins 2:884–885 capsid (C) protein 2:884–885 envelope (E) protein 2:885 prM protein 2:885 life cycle 2:886–887 NS1 2:885 NS2A and NS2B 2:885–886 NS3 2:886 NS4A and NS4B 2:886 NS5 2:886 pathogenesis 2:889 treatment and prevention 2:889 virion structure 2:884 WGCNA see Weighted Gene Coexpression Network Analysis (WGCNA) WGS see whole genome sequencing (WGS) wheat streak mosaic virus 1:649 Whispovirus 4:808 White clovermosaic virus (WClMV) 3:628 whiteflies biological control of 3:775 chemical control of 3:775 defined 3:768 whitefly-transmitted begomoviruses, case of 3:108–110 white spot disease (WSD) 4:709 white spot syndrome virus-CN (WSSV-CN) 4:709 white spot syndrome viruses (WSSVs) 4:808, 4:813, 4:816, 4:809f, 4:810f, 4:811t, 4:709 white tail disease (WTD) 4:823, 4:825 whole-genome amplification 1:187–188 defined 1:184 DNA decontamination 1:188 reagent preparation 1:188 DLB preparation 1:188 DNA decontamination of DLB and stop solution 1:188

master mix preparation 1:189 stop solution 1:188 whole genome reaction procedure 1:189 whole genome sequencing (WGS) 1:565–566, 2:673 wHTH see winged HTH domain (wHTH) wild boar, control of classical swine fever in 2:162–163 wild poliovirus (WPV) 5:310 wild-type (WT) capsids 4:118–119 winged HTH domain (wHTH) 4:144 WNV see West Nile virus (WNV) WPV see wild poliovirus (WPV) WRKY 3:758 WSD see white spot disease (WSD) WSSV-CN see white spot syndrome virus-CN (WSSV-CN) WSSVs see white spot syndrome viruses (WSSVs) WT capsids see wild-type (WT) capsids WTD see white tail disease (WTD)

X xenotropic MLVs (xMLVs) 2:646 xenotropic murine leukemia virus-related virus (XMRV) 2:647 XFMS see X-ray footprinting and mass spectrometry (XFMS) Xinmoviridae 4:718–722 xMLVs see xenotropic MLVs (xMLVs) XMRV see xenotropic murine leukemia virus-related virus (XMRV) X protein 2:376 X-ray crystallography 4:900, 1:8 X-ray diffraction, defined 1:362 X-ray footprinting and mass spectrometry (XFMS) 1:197, 1:191 XSV see extra small virus (XSV) Xylella fastidiosa 4:772–773

Y yado-kari, defined 4:658 yado-kari virus 1 (YkV1) 4:440, 4:658 future directions 4:663 genome characteristics of 4:659–660 -like virus combinations in other fungi 4:663 molecular entities sharing similar YkV1-like interactions 4:661–663 phylogenetic placements of 4:660 predicted past and future of 4:663 proposed replication model for 4:660–661 virion morphology 4:658 yado-nushi, defined 4:658 yado-nushi virus 1 (YnV1) 4:658 future directions 4:663 genome characteristics of 4:658–659 -like virus combinations in other fungi 4:663 molecular entities sharing similar YnV1-like interactions 4:661–663

phylogenetic placements of 4:660 proposed replication model for 4:660–661 virion morphology 4:658 Yam virus X (YVX) 3:628 Yata virus (YATV) 2:875 YATV see Yata virus (YATV) YBV see Yug Bogdanovac virus (YBV) yeast 1:664, 4:440 yeast and fungi, prions of anti-prion systems 4:491 biological roles of prions: a help or a hindrance? 4:490–491 chaperones and prions 4:490 enzyme as prion 4:492 genetic signature of a prion 4:487 history 4:487 infectious prion amyloids have in-register parallel folded b-sheet architecture 4:489–490 inositol polyphosphates and prion propagation 4:491–492 prion generation, and [PIN]: a prion that gives rise to prions 4:490 prion variant information templating mechanism 4:490 prion variants and the species barrier 4:490 self-propagating amyloid as the basis for most yeast prions 4:487–489 shuffleable prion domains suggests parallelin-register b-sheet structure 4:489 shuffling prion domains and amyloid structure 4:489 yeast L-A virus 4:664 genome organization 4:664–665 history 4:664 L-A genetics 4:667–668 L-BC 4:668 replication cycle 4:665–666 RNA packaging 4:666 RNA replication 4:666 transcription 4:665–666 20S RNA 4:668 23S RNA 4:668 viral translation 4:666–667 virion structure 4:664 YEL-AND see yellow fever vaccine associated neurotropic disease (YEL-AND) YEL-AVD see yellow fever vaccine-associated viscerotropic disease (YEL-AVD) yellow fever vaccine associated neurotropic disease (YEL-AND) 2:897–898 yellow fever vaccine-associated viscerotropic disease (YEL-AVD) 2:897–898 yellow fever virus (YFV) 1:542, 2:414, 2:245 classification 2:891 clinical features 2:895–896 diagnosis 2:896–897 epidemiology 2:894–895 genome 2:892–893 life cycle 2:893–894 prevention 2:897–898 treatment 2:897 vaccine 1:575 virion structure 2:891–892

Subject Index yellowing, defined 3:371 yellow mosaic disease (YMD) 3:563 YFV see yellow fever virus (YFV) YPXnL late domains 1:522 Yug Bogdanovac virus (YBV) 2:875 YVX see Yam virus X (YVX)

Z Zanamivir 5:164 ZBD see zinc-binding domain (ZBD) zebrafish endogenous retrovirus 2:322 zero-mode waveguides (ZMW) 1:181, 5:27 Zika virus (ZIKV) 2:899, 1:253, 5:275, 1:121, 5:3, 1:267–268, 1:268f, 1:498–499 classification 2:899–900 clinical features 2:905–906 diagnosis 2:907 epidemiology 2:904–905 brief history 2:904–905 potential factors contributing to ZIKV emergence 2:905 ZIKV lineages and disease association 2:905 life cycle 2:902–903 ecology and transmission cycles 2:902–903 virus replication cycle 2:903–904 pathogenesis 2:906–907 prevention and control 2:907–908 replication cycle 2:904f treatment 2:907

virion structure and genome organization 2:900–902 ZIKV see Zika virus (ZIKV) zinc-binding domain (ZBD) 2:253 ZMW see zero-mode waveguides (ZMW) zone of emergence, defined 2:899 zoonosis 1:569, 1:570f defined 1:569, 2:355, 1:542, 1:559 emerging viruses, prevention and control of 1:572–574 diagnosis 1:573–574 mosquito vectors, control of 1:575 treatment 1:574–575 vaccination 1:575 factors in increasing emergence of viral diseases 1:572 interspecies transmission, factors in 1:569–570 epidemiological/ecological barriers 1:570 host–pathogen interactions 1:570 viral factors 1:570–571 zoonotic infection, defined 2:397 zoonotic influenza 5:160–161 zoonotic transmission cycles, influence of 1:563 zoonotic viruses catch-all detection methods 5:260–261 developing targeted, risk based sampling 5:258–259 disease X 5:257 drivers of 5:256–257 emerging disease detection, full genome sequencing and rapid data sharing 5:262

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emerging disease detection and the one health concept 5:257–258 from genotype to phenotype 5:261 non-invasive and bulk samples, value of 5:260 one health and animal reservoirs 5:261–262 population surveys and seroepidemiological studies 5:262–263 sample collection 5:259–260 SARS-CoV-2 5:261 zoster, defined 2:860 zucchini yellow mosaic virus biotechnological application 3:870 classification 3:862–863 control methods 3:867 cross-protection 3:867 prophylactic measures 3:867 resistant cultivars 3:867–868 diagnostic methods 3:865 epidemiology 3:866 efficient vectors 3:866 pattern of spread 3:866–867 virus sources 3:866 genetics and evolution 3:868–870 geographic distribution 3:865 history and taxonomy 3:862 host range 3:865 symptomatology 3:863–864 synergism and antagonism 3:864–865 variability 3:868 vector relationships 3:865–866 Zygocactus virus X (ZyVX) 3:623–624, 3:628 ZyVX see Zygocactus virus X (ZyVX)