Zika Virus Biology, Transmission, and Pathways Volume 1: The Neuroscience of Zika 9780128202685, 9780323858649

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Zika Virus Biology, Transmission, and Pathways The Neuroscience of Zika Virus

Zika Virus Biology, Transmission, and Pathways The Neuroscience of Zika Virus Volume 1

Edited by

Colin R. Martin Institute for Clinical and Applied Health Research (ICAHR), University of Hull Hull, United Kingdom

Caroline J. Hollins Martin Edinburgh Napier University Edinburgh, Scotland, United Kingdom

Victor R. Preedy King’s College London, London, United Kingdom

Rajkumar Rajendram Department of Medicine, King Abdulaziz Medical City, Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-820268-5 SET ISBN: 978-0-323-85864-9 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Nikki Levy Acquisitions Editor: Natalie Farra Editorial Project Manager: Timothy Bennett Production Project Manager: Paul Prasad Chandramohan Cover Designer: Mark Rogers Typeset by SPi Global, India

Dedication The book is dedicated to my daughter Dr. Caragh Brien, whom I am so proud of and also to my wonderful late father. Colin R. Martin

Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Quinn H. Abram (83), Department of Biochemistry, McGill University, Montreal, QC, Canada Marcelo Addas-Carvalho (467), Division of Transfusion Medicine, Hematology and Transfusion Medicine Center, University of Campinas, Campinas, SP, Brazil Sergio P. Alpuche-Lazcano (397), Department of Medicine, Division of Experimental Medicine at McGill University, Lady Davis Institute for Medical Research, Montreal, QC, Canada Maria Joa˜o Alves (431), Centre for Vectors and Infectious Diseases Research, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge, ´ guas de Moura, Portugal A Soniza Vieira Alves-Leon (199), Department of Neurology, Laboratory of Translational Neuroscience/Biomedical Institute, Federal University of the State of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Fa´tima Amaro (431), Centre for Vectors and Infectious Diseases Research, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge, ´ guas de Moura, Portugal A Jos
elio Maria Galva˜o de Arau´jo (19), Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Mariana Araujo-Pereira (221), Department of Pathophysiology and Toxicology, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Maı´ra Cardoso Aspahan (255), Department of Neurology, Madre Teresa Hospital, Belo Horizonte, Brazil Maria Helena de M. Barbosa (245), D’Or Institute for Research and Education, Rio de Janeiro, RJ, Brazil

Bruno Deltreggia Benites (467), Division of Transfusion Medicine, Hematology and Transfusion Medicine Center, University of Campinas, Campinas, SP, Brazil Walter Orlando Beys-da-Silva (307), Faculty of Pharmacy and Post-Graduation Program in Cellular and Molecular Biology, Federal University of Rio Grande do Sul; Experimental Research Center, Clinical Hospital of Porto Alegre, Porto Alegre, Brazil Marshall E. Bloom (299), Biology of Vector-Borne Viruses Section, Laboratory of Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States Guy Boivin (289), Research Center in Infectious Diseases, Laval University, Quebec City, QC, Canada Aaron C. Brault (443), Division of Vector-borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, United States Otavio Cabral-Marques (221), Department of Immunology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Luiz Carlos de Caires Junior (179), Department of Genetics and Evolutionary Biology, Human Genome and Stem Cells Research Center, Institute of Bioscience, University of Sao Paulo, Sao Paulo, Brazil Cynthia Chester Cardoso (235), Department of Genetics, Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil Marlen Yelitza Carrillo-Herna´ndez (117), Grupo de Investigacio´n en Ciencias Animales-GRICA, Universidad Cooperativa de Colombia, Bucaramanga, Colombia Leila Chimelli (155), Laboratory of Neuropathology, State Institute of Brain, Rio de Janeiro, RJ, Brazil

Trisha R. Barnard (83), Department of Microbiology & Immunology, McGill University, Montreal, QC, Canada

Paulo Pereira Christo (255), Department of Neurology, Clinical Hospital, Federal University of Minas Gerais, Belo Horizonte, Brazil

C
ecile Beck (453), UMR 1161 Virology, ANSES, INRAe, ENVA, ANSES Animal Health Laboratory, EURL for equine diseases, Maisons-Alfort, France

Dinh-Toi Chu (367, 377), Center for Biomedicine and Community Health, International School, Vietnam National University Hanoi, Cau Giay, Hanoi, Vietnam

xix

xx Contributors

Kevin M. Coombs (319), Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada Fabio T.M. Costa (267), Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas (Unicamp), Sa˜o Paulo, SP, Brazil Lucia Maria Costa Monteiro (279), National Institute of Woman, Adolescent and Child Health—Fernandes Figueira/Fiocruz, Rio de Janeiro, Brazil Juan Cristina (409), Laboratory of Molecular Virology, Centro de Investigaciones Nucleares, Universidad de la Repu´blica, Montevideo, Uruguay Marielton Dos Passos Cunha (221), Department of Pathophysiology and Toxicology, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Lia Giraldo da Silva Augusto (43), Oswaldo Cruz Fundation, IAM, Recife, PE, Brazil Philippe Despre`s (129), La Reunion University, INSERM U1187, CNRS UMR 9192, IRD UMR 249, Infectious Processes in Tropical Island Environment (PIMIT) Laboratory, Technology Platform CYROI, Sainte-Clotilde, La Reunion Island, France Adria´n Diaz (3), Laboratory of Arbovirus, Institute for Virology “Dr. J. M. Vanella”, Faculty of Medicine, National University of Co´rdoba, Co´rdoba, Argentina Finn Diderichsen (43), Department of Public Health, University of Copenhagen, Copenhagen, Denmark Alberto Jos
e da Silva Duarte (341), Laboratory of Medical Investigation LIM-3, Hospital das Clinicas da FMUSP, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil Marine Dumarest (453), UMR 1161 Virology, ANSES, INRAe, ENVA, ANSES Animal Health Laboratory, EURL for equine diseases, Maisons-Alfort, France Nadia El Khawanky (221), Department of Hematology, Oncology, and Stem Cell Transplantation, Faculty of Medicine, University of Freiburg, Freiburg, Deutchland Jos
e Verı´ssimo Fernandes (19), Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil

Gilles Gadea (129), La Reunion University, INSERM U1187, CNRS UMR 9192, IRD UMR 249, Infectious Processes in Tropical Island Environment (PIMIT) Laboratory, Technology Platform CYROI, Sainte-Clotilde, La Reunion Island, France Luciana Guerra Gallo (63), Centre for Tropical Medicine, University of Brasilia, Brası´lia, DF, Brazil Anne Gatignol (397), Department of Medicine, Division of Experimental Medicine at McGill University; Department of Microbiology and Immunology at McGill University, Lady Davis Institute for Medical Research, Montreal, QC, Canada David Alejandro Cabrera Gayta´n (387), Coordination of Epidemiological Surveillance, Mexican Social Security Institute (IMSS), Mexico City, M
exico Gaelle Gonzalez (453), UMR 1161 Virology, ANSES, INRAe, ENVA, ANSES Animal Health Laboratory, EURL for equine diseases, Maisons-Alfort, France Ernesto Goulart (179), Department of Genetics and Evolutionary Biology, Human Genome and Stem Cells Research Center, Institute of Bioscience, University of Sao Paulo, Sao Paulo, Brazil Ernesto R. Gregorio Jr (75), Department of Health Promotion and Education, College of Public Health, University of the Philippines Manila, SEAMEOTROPMED Regional Center for Public Health, Hospital Administration, and Environmental and Occupational Health, Manila, Philippines Paulo Marcos Matta Guedes (19), Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Rodolphe Hamel (453), UMR MIVEGEC, IRD—French National Research Institute for Sustainable Development, CNRS, University of Montpellier, Montpellier, France Cristina Barroso Hofer (555), Department of Infectious Diseases, School of Medicine, Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, RJ, Brazil Amni Adilah Ismail (209), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Anne J. J€a€askel€ainen (95, 107), University of Helsinki and Helsinki University Hospital, Helsinki, Finland

Simone G. Fonseca (267), Department of Microbiology, Immunology, Parasitology and Pathology, Federal University of Goias, Goi^ania, GO, Brazil

Soe Hui Jen (209), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Fabrı´cia Lima Fontes-Dantas (199), Department of Neurology, Laboratory of Translational Neuroscience/Biomedical Institute, Federal University of the State of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Carla Judice (267), Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas (Unicamp), Sa˜o Paulo, SP, Brazil

Contributors

Carolini Kaid (327), Human Genome and Stem-Cell Center (HUG-CELL), Biosciences Institute, University of S. Paulo, Sa˜o Paulo, SP, Brazil Jun Kobayashi (75), Department of Global Health, School of Health Sciences, University of the Ryukyus, Nishihara, Okinawa, Japan Sylvie Lecollinet (453), UMR 1161 Virology, ANSES, INRAe, ENVA, ANSES Animal Health Laboratory, EURL for equine diseases, Maisons-Alfort, France Marcio Leyser (169), University of Iowa Stead Family Department of Pediatrics, Division of Developmental and Behavioral Pediatrics, Iowa City, IA, United States Maria Clara de Magalha˜es-Barbosa (245), D’Or Institute for Research and Education, Rio de Janeiro, RJ, Brazil Rishya Manikam (209), Department of Trauma & Emergency, University of Malaya Medical Centre, University of Malaya, Kuala Lumpur, Malaysia Fernanda J.P. Marques (143, 169), Department of Neurology and Neuroscience, Fluminense Federal University; SARAH Network of Rehabilitation Hospitals, Rio de Janeiro, Brazil Marlen Martı´nez-Guti
errez (117), Grupo de Investigacio´n en Ciencias Animales-GRICA, Universidad Cooperativa de Colombia, Bucaramanga, Colombia Felipe Martins (221), Department of Pathophysiology and Toxicology, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Anto´nio Pedro Alves de Matos (431), Centre for Electron Microscopy and Histopathology, Egas Moniz Interdisciplinary Research Centre, Egas Moniz, Cooperativa de Ensino Superior CRL, Campus Universita´rio-Quinta da Granja, Caparica, Portugal Erin M. McDonald (443), Division of Vector-borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, United States Eyal Meltzer (351), The Center for Geographic Medicine, Department of Medicine C, The Chaim Sheba Medical Center, Tel Hashomer; Sackler Faculty of Medicine at the Tel Aviv University, Tel Aviv, Israel Teresita Rojas Mendoza (387), Technical Input Control Coordination, Mexican Social Security Institute (IMSS), Mexico City, M
exico

xxi

Helder I. Nakaya (221, 267), Department of Pathophysiology and Toxicology, School of Pharmaceutical Sciences; Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Andrezza Nascimento (341), Laboratory of Medical Investigation LIM-56, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil Gilmara Lima Nascimento (63), Federal District Health Department, Setor Escolar Lote 04 – Cruzeiro VelhoCruzeiro, Brası´lia, DF, Brazil Manuela Sales Lima Nascimento (19), Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Osvaldo J.M. Nascimento (143, 169), Department of Neurology and Neuroscience, Fluminense Federal University, Rio de Janeiro, Brazil Tiep Tien Nguyen (377), College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk, South Korea Lumumba Arriaga Nieto (387), Coordination of Epidemiological Surveillance, Mexican Social Security Institute (IMSS), Mexico City, M
exico Maria Regina Fernandes de Oliveira (63), Centre for Tropical Medicine, School of Medicine, University of Brasilia, Brası´lia, DF, Brazil Katarzyna Owczarek (419), Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Cracow, Poland Alfonso Vallejos Para´s (387), Coordination of Epidemiological Surveillance, Mexican Social Security Institute (IMSS), Mexico City, M
exico Vinood B. Patel (479), School of Life Sciences, University of Westminster, London, United Kingdom Henry Maia Peixoto (63), Centre for Tropical Medicine, School of Medicine, University of Brasilia, Brası´lia, DF, Brazil Marianoel Pereira-Go´mez (409), Laboratory of Molecular Virology, Centro de Investigaciones Nucleares, Universidad de la Repu´blica; Laboratory of Experimental Evolution of Viruses, Institut Pasteur de Montevideo, Montevideo, Uruguay

Luwanika Mlera (299), BIO5 Institute, University of Arizona, Tucson, AZ, United States

Irmtraut Araci H. Pfrimer (267), Department of Master in Environmental Sciences and Health, School of Medical, Pharmaceutical and Biomedical Sciences, Pontifical Catholic University of Goia´s, Goi^ania, GO, Brazil

Concepcio´n Grajales Mun˜iz (387), Technical Input Control Coordination, Mexican Social Security Institute (IMSS), Mexico City, M
exico

Atchara Phumee (359), Department of Medical Technology, School of Allied Health Sciences, Walailak University, Nakhon Si Thamarat, Thailand

Thiago Mitsugi (327), Human Genome and Stem-Cell Center (HUG-CELL), Biosciences Institute, University of S. Paulo, Sa˜o Paulo, SP, Brazil

xxii Contributors

Jocelyne Piret (289), Research Center in Infectious Diseases, Laval University, Quebec City, QC, Canada Arnaldo Prata-Barbosa (245), D’Or Institute for Research and Education, Rio de Janeiro, RJ, Brazil Victor R. Preedy (479), Diabetes and Nutritional Sciences Research Division, Faculty of Life Science and Medicine, King’s College London, London, United Kingdom Krzysztof Pyr
c (419), Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Cracow, Poland Rajkumar Rajendram (479), College of Medicine, King Saud bin Abdulaziz University for Health Sciences; Department of Medicine, King Abdulaziz Medical City, Ministry of National Guard—Health Affairs, Riyadh, Saudi Arabia; Diabetes and Nutritional Sciences Research Division, Faculty of Life Science and Medicine, King’s College London, London, United Kingdom Chandramathi Samudi Raju (209), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia ´ Atila Duque Rossi (235), Department of Genetics, Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil Fernanda Cristina Rueda-Lopes (199), Department of Radiology, University Hospital Antonio Pedro, Federal Fluminense University, Nitero´i, RJ, Brazil Julian Ruiz-Saenz (117), Grupo de Investigacio´n en Ciencias Animales-GRICA, Universidad Cooperativa de Colombia, Bucaramanga, Colombia Selena M. Sagan (83), Departments of Microbiology & Immunology and Biochemistry, McGill University, Montreal, QC, Canada Amanda Costa Ayres Salmeron (19), Edmond and Lily Safra International Institute of Neuroscience, Santos Dumont Institute, Macaiba, Rio Grande do Norte, Brazil Sabri Saeed Sanabani (341), Laboratory of Medical Investigation LIM-56; Laboratory of Medical Investigation LIM-3, Hospital das Clinicas da FMUSP, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil Luc
elia Santi (307), Faculty of Pharmacy and PostGraduation Program in Cellular and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil

Wilo Victor dos Santos (19), Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Viviane Schuch (221), Department of Pathophysiology and Toxicology, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Shamala Devi Sekaran (209), Deputy Dean, Faculty of Medicine and Helath Sciences, UCSI University, Kuala Lumpur, Malaysia Matt Sherwood (327), School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton, United Kingdom Prateek Saurabh Shrivastava (31), Department of Community Medicine, Shri Sathya Sai Medical College and Research Institute, Sri Balaji Vidyapeeth (SBV)—Deemed to be University, Chengalpet, Tamil Nadu, India Saurabh RamBihariLal Shrivastava (31), Medical Education Unit Coordinator and Member of the Institute Research Council, Department of Community Medicine, Shri Sathya Sai Medical College and Research Institute, Sri Balaji Vidyapeeth (SBV)—Deemed to be University, Chengalpet, Tamil Nadu, India Padet Siriyasatien (359), Vector Biology and Vector Borne Disease Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Nguyen Thai Son (367, 377), Department of Microbiology, Vietnam Military Medical University, Hanoi, Vietnam Juliana Miranda Tatara (307), Post-Graduation Program in Cellular and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil Felipe Ten Caten (221), Department of Pathophysiology and Toxicology, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil Ho Huu Tho (367), Institute of Biomedicine & Pharmacy, Vietnam Military Medical University, Hanoi, Vietnam Wildriss Viranaicken (129), La Reunion University, INSERM U1187, CNRS UMR 9192, IRD UMR 249, Infectious Processes in Tropical Island Environment (PIMIT) Laboratory, Technology Platform CYROI, Sainte-Clotilde, La Reunion Island, France Daniela Pires Ferreira Vivacqua (55), Department of Infectious Diseases, Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, RJ, Brazil

Contributors

Dan Xu (189), Fujian Key Laboratory of Molecular Neurology, Institute of Neuroscience, Fujian Medical University, Fuzhou, China Zhiheng Xu (189), State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

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Mayana Zatz (179, 327), Department of Genetics and Evolutionary Biology, Human Genome and Stem Cells Research Center, Institute of Bioscience, University of Sao Paulo, Sao Paulo, Brazil Libia Z
e-Z
e (431), Centre for Vectors and Infectious Diseases Research, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge, ´ guas de Moura, Portugal A

Foreword Zika virus is a disease of great concern for a great many reasons, although the fundamental issues relating to Zika infection are the current lack of medication or vaccination and the potentially devastating birth defects impacting babies following transmission of the virus during pregnancy. Spread mostly by the Aedes species mosquito, there are also other known transmission routes, for example, through unprotected sexual intercourse with an infected partner and potential transmission routes such as blood donation. Symptomatically, as with other viral infections, Zika may be accompanied by fever, headache, and joint and muscle pain among other symptoms. However, again, similar to several other viral infections, there may be no symptoms whatsoever and thus the infected person will have no awareness of their infection status. The recent Covid-19 pandemic has focused International interest and awareness on the devastating effect of viral infections at the population level, but interestingly, within the context of Zika, the impact of birth defects associated with this particular virus can be devastating and lifelong. Fortunately, Zika can be diagnosed by a blood or urine test and these offer some confidence in both a confirmatory diagnosis, the context for the development of interventions for those affected, and a fertile basis for proactive research enquiry and the nurturing of a robust evidence base. There is so much that we don’t know about Zika and it is thus a great pleasure for me to write this Foreword for this excellent work by Professors Martin, Hollins Martin, Preedy, and Dr. Rajendram in bringing together leading experts in the Zika disease field to provide an evidence-based and state-of-the art account of the latest research insights into the biology and pathology that circumscribe Zika virus. Understanding the underlying biological mechanisms that underpin the transmission of Zika and the consequential physiological impact on the individual is critical in both the development of preventative strategies and innovation in the treatment for those experiencing this potentially devastating infection. I thus commend these editors, and the authors, in producing Zika Virus Biology, Transmission and Pathways, a comprehensive “one-stop shop” book which is accessible to both the clinician and the researcher alike and relevant to all those working in this area. Finally, we should always be minded that as we innovate in treatment, particularly in the case of the more extreme consequences of Zika infection, there is a person and individual impacted by the diagnosis and the potential consequential impact on their quality of life and life course development and aspirations, and it is hoped that this book will help treatment at both the global and the individual level. Alan St Clair Gibson Alan St Clair Gibson is an associate dean (Research) at the University of Hull. Formerly, he was the head of the School of Medicine at the University of the Free State in South Africa, dean of the School of Health and Human Performance Sciences at the University of Waikato in New Zealand, and a research fellow at the National Institute of Neurological Disorders and Stroke, NIH, in Washington DC in the USA. He received MBChB, PhD, and MD degrees from the University of Cape Town in South Africa, and believes in the importance of all of teaching and training, clinical work, and research in best combating diseases and pathology which afflict the world’s population.

xxv

Preface Zika virus is a member of the family Flaviviridae and was first identified in the 1940s. In 2007, the first outbreak of Zika virus disease was reported and this was followed by substantial outbreaks of the viral infection from 2013. In 2015, a large outbreak was reported in Brazil. Concomitant associations with Guillain-Barre syndrome and microcephaly (presently termed congenital ZIKV syndrome) were also reported. The gravity of the situation was recognized by the World Health Organization and declared the Zika virus a Public Health Emergency of International Concern. In 2018, the Zika virus was listed on the World Health Organization’s list of diseases that merits priority attention and research. The Zika virus is transmitted by Aedes mosquitoes and clinical symptoms can be mild. However, in some adults, there may be adverse consequences of Zika virus infection. Inflammatory conditions, nerve damage, and triggering of GuillainBarre syndrome as a consequence of Zika virus can also arise. Zika virus infection during pregnancy can impact the developing fetus, resulting in offspring with microcephaly. These conditions are only one end of the spectrum of disorders and conditions associated with Zika virus. There is also psychological impact on the mother carrying the child with microcephaly to term. The family unit and community at large are also adversely affected. From a public health point of view, there needs to be strategies to prevent and treat Zika virus using emerging and established scientific knowledge and methods. For example, it has recently been shown that the Zika virus can be transmitted sexually and via breast milk. However, there is very little known about the long-term consequences of Zika virus infection. In order to provide a framework for the broader understanding of Zika virus, it is necessary to gather all the relevant information in one single place. However, hitherto, information on the Zika virus and associated conditions has been sporadic and within different scientific domains. Much of the material is targeted toward experts working within specific disciplines without regard for the novice or those newly embarking upon Zika virus-related research. To address these issues, we have compiled two books on Zika virus as follows: Book 1: Zika Virus Biology, Transmission, and Pathways Book 2: Zika Virus Impact, Diagnosis, Control, and Models In the book Zika Virus Biology, Transmission, and Pathways, we have over 40 chapters divided into the following sections: Section Section Section Section Section

A: Zika virus: Introductory chapters B: Effects on neurological and body systems C: International aspects D: Features of the virus and transmission E: Resources

There are of course always difficulties in ascribing chapters to different sections and placing them in order. Some chapters are equally at home in more than one section. However, the excellent indexing system allows material to be rapidly located. To aid understanding across the different disciplines, we have introduced the following features within each chapter An Abstract: published online. Mini-dictionary: to aid the novice and to bridge the transdisciplinary and transintellectual divides. Key Facts: focused areas of interest. Policy and Procedure: recommendations or suggestions for research, strategies, guidelines, prevention, and treatment. Summary Points: encapsulating the whole chapter in a series of single sentences. Zika Virus Biology, Transmission, and Pathways is designed for research and teaching purposes. It is suitable for neurologists, virologists, health scientists, public health workers, doctors, pharmacologists, and research scientists. It is valuable as a personal reference book and also for academic libraries that covers the domains of virology, neurology, and health sciences. Contributions are from leading national and international experts, including those from worldrenowned institutions. It is suitable for undergraduates, postgraduates, lecturers, and academic professors. The Editors xxvii

Chapter 1

Flaviviruses and where the Zika virus fits in: An overview Adria´n Diaz Laboratory of Arbovirus, Institute for Virology “Dr. J. M. Vanella”, Faculty of Medicine, National University of Co´rdoba, Co´rdoba, Argentina

Abbreviations °C BBB C CNS DENV E ER IFN ISF JEV M MBF ml NKVF NS1 NS2A NS2B NS3 NS4A NS4B NS5 PFU RNA SLEV TBEV TBF TGN USA USUV VERO WHO YFV ZIKV

Celsius degrees blood-brain barrier capsid protein central nervous system dengue virus envelope protein endoplasmic reticulum interferon insect-specific flavivirus Japanese encephalitis virus membrane protein mosquito-borne flavivirus milliliters no-known vector flavivirus nonstructural protein 1 nonstructural protein 2A nonstructural protein 2B nonstructural protein 3 nonstructural protein 4A nonstructural protein 4B nonstructural protein 5 plaque forming units ribonucleic acid St. Louis encephalitis virus tick-borne encephalitis virus tick-borne flavivirus trans Golgi network United States of America Usutu virus kidney green monkey cells World Health Organization yellow fever virus Zika virus

Classification, diversity, and evolution There are more than 50 viruses belonging to the Flavivirus genus, including those with high public health concern such as yellow fever, dengue, Japanese encephalitis, West Nile, and Zika (ICTV, 2019). Members of this genus are vectored by

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00001-8 Copyright © 2021 Elsevier Inc. All rights reserved.

3

4 SECTION

A Zika virus: Introductory chapters

mosquitoes and ticks and especially those transmitted by mosquitoes have a wide geographical distribution (i.e., West Nile, dengue, Zika). Whole genome phylogenetic analyses indicate that the Flavivirus genus originated
85,000 or 120,000 years ago in Africa and thereafter dispersed through human, rodents, and/or birds around the globe (Pettersson & Fiz-Palacios, 2014). Four main groups can be recognized: (1) insect-specific flaviviruses (ISF) isolated exclusively from mosquitoes in nature. ISFs can be divided into two distinct phylogenetic groups. The first group includes cell fusing agent virus, Kamiti River virus, and Culex flavivirus. The second group includes Chaoyang virus, Nounane virus, and Lammi virus (Table 1) (Blitvich & Firth, 2015); (2) no-known vector flaviviruses (NKVF) grouped viruses mainly associated with either bats or rodents and infect vertebrates without an apparent arthropod vector transmitting the viruses (Table 1) (Blitvich & Firth, 2017); (3) mosquito-borne flaviviruses (MBFs) commonly, but not exclusively, vectored by either Aedes or Culex mosquitoes (Table 2) (Kuno, 2007), and (4) tick-borne flaviviruses (TBFs), the only monophyletic group of the genus, vectored by ticks, and are further subdivided into a mammal and a seabird group based on their host specificity (Table 3) (Gritsun, Nuttall, & Gould, 2003). Two eco-epidemiological groups can be distinguished in the MBF: (1) Aedes-borne flaviviruses group, frequently amplified by mammals (humans, primates) and associated with polyarthritis and hemorrhagic fever (DENV, YFV). ZIKV is an exception causing neurological disorders in humans. (2) Culex-borne flaviviruses, which are etiological agents of neurological diseases and hosted by birds (JEV, WNV, SLEV, USUV) (Table 2) (Gould, de Lamballerie, Zanotto, & Holmes, 2003). Mosquito-borne flaviviruses are present in every continent except Antarctica. Although their genetic relationship is highly correlated with geographic location some members such as DENV, WNV, and ZIKV are widely distributed around the globe (Coffey, Forrester, Tsetsarkin, Vasilakis, & Weaver, 2013). On the other hand, TBF have been detected mainly in the Northern Hemisphere, with TBEV distributed from Siberia to Eastern Europe. All the viruses within the mammalian tick-borne virus group (Table 3) have evolved as a complex of genetically very closely related viruses despite their wide distribution. These viruses evolve less quickly than the MBF and therefore appear to be more constrained by their vectors (Coffey et al., 2013). While virus undoubtedly replicates in the ticks, it never reaches high titers, thus reducing the likelihood of variation due to RNA-dependent RNA polymerase errors and total virus turnover is relatively low in ticks, compared with the mosquito-borne flaviviruses because of the prolonged life cycle of the tick (Gritsun et al., 2003).

Biology of transmission and persistence of MBF and TBF Vector-borne flaviviruses are transmitted and maintained in a complex network of interactions between vertebrate hosts and vector arthropods (Fig. 1). These interactions are dynamics and are influenced by vector and host abundance and bloodfeeding preference of the vector (Diaz, Flores, Quaglia, & Contigiani, 2013). Even more, arboviruses need cycling between host and vector to achieve a maximum fitness, thereby increasing their chances to survive in nature (Ciota & Kramer, 2010). The arthropod vector acquires the virus from a viremic host during the feeding process. Once the virions are ingested, they cross the digestive tube and infect the midgut epithelium cells. After replication, viral particles come across the basal lamina and disseminates through hemolymph and neurons to other organs. Finally, the virus reaches the salivary glands where it causes infection and replicates (Fig. 2). Along with the saliva, virus can get into the new susceptible vertebrate host and start the transmission cycle again (Kenney & Brault, 2014). Flaviviruses produce an acute self-limited infection in the vertebrate host where the virus persists in the bloodstream for a short time period. After the viremia, the host immune system produces neutralizing antibodies that clear the present infection and protect the host in case of further infections. Most of the flavivirus infections in host are unapparent but sometimes can manifest clinical symptoms like encephalitis (WNV, SLEV, JEV, TBEV) and hemorrhages (DENV, YFV) (Gould & Solomon, 2008). An effective host must develop long-lasting and higher viremias enough to infect the vector, to be abundant and cohabit with the main arthropod vector, and to be attractive to blood-seeking arthropods vectors (Diaz et al., 2013). Humans are often regarded as dead-end hosts for the majority of flaviviruses (WNV, SLEV, JEV) where low-level viremia in the body prevents the virus from being transmitted to another host (Fig. 1). However, for DENV, ZIKV, and YFV humans are true amplifying host infecting mosquitoes during the acute phase of infection. In tropical and subtropical areas arboviruses can persist all year round through vector transmission because of the availability of vector and susceptible host abundance. However, in areas with dry and cold season, arboviruses can develop alternative strategies to persist (overwinter mechanisms). A few hypotheses have been postulated to explain how an arbovirus overwinters: (a) alternative vectors as ticks that are stable and have long lasting life cycles, (b) annual introduction through viremic migratory birds, (c) hibernating infected mosquito females that transmit the virus in the next season, and (d) vertical transmission from an infected female to the progeny (Rosen, 1987).

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TABLE 1 Inspect-specific flaviviruses and no-known vector flavivirus recognized by the International Committee on Taxonomy of Viruses. Virus

Geographic distribution (year of isolation)

Natural host

Insect-specific flaviviruses Aedes flavivirus

Japan (2003), Italy (2008), USA (2011), Thailand (2012)

Ae. albopictus, Ae. flavopictus, Cx. pipiens

Aedes galloisi flavivirus

Japan (2003)

Ae. galloisi

Calbertado virus

Canada (2003), USA (2006)

Cx. tarsalis, Cx. pipiens

Cell fusing agent virus

Laboratory (1975), Puerto Rico (2002), Indonesia (2004), Mexico (2007), Thailand (2008), United States (2012)

Ae. albopictus, Ae. aegypti, Culex spp.

Culex flavivirus

Japan (2003), Indonesia (2004), China (2006), Guatemala (2006), USA (2006), Mexico (2007), Trinidad (2008), Uganda (2008), Argentina (2009)

Cx. interrogator, Cx. maxi, Cx. nigripalpus, Cx. pipiens, Cx. quinquefasciatus, Cx. restuans, Cx. tarsalis, Cx. tritaeniorhynchus, Cx. usquatus

Culex theileri flavivirus

Spain (2006), Portugal (2009–2010), Greece (2010), Thailand (date not specified)

Cx. fuscocephala, Cx. pipiens, Cx. theileri

Hanko virus

Finland (2005), Spain (2006), Italy (2007), Portugal (2007)

Ae. caspius, Ae. detritus, Ae. vexans, Cx. pipiens, Cx. perexiguus, Cx. theileri

Kamiti River virus

Kenya (1999)

Ae. macintoshi

Nakiwogo virus

Uganda (2008)

Mansonia africana nigerrima

Nienokoue virus

Cote d’Ivoire (2004)

Culexspp.

Palm Creek virus

Australia (2010)

Coquillettidia xanthogaster

Quang Binh virus

Vietnam (2002), China (2009)

An. sinensis, Cx. tritaeniorhynchus

Bat-associated no-known vector flaviviruses Batu Cave virus

Malaysia (1971)

Cynopterus brachyotis (lesser short-nose fruit bat), Eonycteris spelaea (dawn bat)

Bukalasa bat virus

Senegal, Uganda (1963)

Chaerephon pumila (little free-tailed bat), Tadarida (Mops) condylurus (Angolan free-tailed bat)

Carey Island virus

Malaysia (1970)

Cynopterus brachyotis (lesser short-nosed fruit bat), Macroglossus lagochilus (lesser long-tongued fruit bat)

Dakar bat virus

Central African Republic, Madagascar, Senegal, Nigeria, Uganda (1962)

Chaerephon pumilus (little free-tailed bat), Scotophilus nigrita (giant house bat), Tadarida (Mops) condylurus (Angolan free-tailed bat), Taphozous perforatus (Egyptian tomb bat), Homo sapiens (human)

Entebbe bat virus

Uganda (1957)

Chaerephon (Tadarida) pumilus (little free-tailed bat)

Montana myotis leukoencephalitis virus

USA (1958)

Myotis lucifugus (little brown bat)

Phnom Penh bat virus

Cambodia, Malaysia (1969)

Cynopterus brachyotis (Lesser short-nosed fruit bat), Eonycteris spelaea (dawn bat)

Rio Bravo virus

USA, Mexico, Trinidad (1954)

Eptesicus fuscus (big brown bat), Molossus rufus (black mastiff bat), Tadarida brasiliensis mexicana (Mexican free-tailed bat)

Sokoluk virus

Kyrgyzstan, Russia (1970)

Pipistrellus spp. bats

Tamana bat virus

Trinidad (1973)

Pteronotus parnellii (Parnell’s mustached bat) Continued

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TABLE 1 Inspect-specific flaviviruses and no-known vector flavivirus recognized by the International Committee on Taxonomy of Viruses—cont’d Virus

Geographic distribution (year of isolation)

Natural host

Yokose virus

Japan (1971)

Miniopterus fuliginosus (eastern bent-wing bat)

Rodent-associated no-known vector flaviviruses Apoi virus

Japan (1954)

Apodemus and/or Clethrionomys spp.

Cowbone Ridge virus

USA (1965)

Sigmodon hispidus (hispid cotton rat)

Jutiapa virus

Guatemala (1969)

Sigmodon hispidus (hispid cotton rat)

Modoc virus

USA, Canada (1958)

Peromyscus maniculatus (deer mouse)

Sal Vieja virus

USA (1978)

Peromyscus leucopus (white-footed mouse)

San Perlita virus

USA (1971)

Sigmodon hispidus (hispid cotton rat)

Modified from Blitvich, B. J., & Firth, A. E. (2015). Insect-specific flaviviruses: A systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization. Viruses, 7(4), 1927–1959; Blitvich, B. J., & Firth, A. E. (2017). A review of flaviviruses that have no known arthropod vector. Viruses, 9(6), 154.

TABLE 2 Mosquito borne flaviviruses recognized by the International Committee on Taxonomy of Viruses.

Virus

Geographic distribution

Year of isolation

Diseases in human or other animals

Unknown

South America

1972

Unknown

Culex spp

Monkeys

South America

1956

Fever

Iguape virus (IGPV)

Unknown

Birds?

South America

1979

Unknown

Naranjal virus (NJLV)

Unknown

Unknown

South America

1976

Unknown

Dengue virus 1 (DENV1)

Aedes aegypti

Nonhuman primates/ humans

Hawaii

1944

Fever, rash, vasculopathy

New Guinea

1944

Dengue virus 3 (DENV3)

Philippines

1957

Dengue virus 4 (DENV4)

Philippines

1957

Subtype

Vector

Host

Aroa virus (AROAV)

Unknown

Buusuquara virus (BSQV)

Aroa virus group Aroa virus

Dengue virus group Dengue virus

Dengue virus 2 (DENV2)

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TABLE 2 Mosquito borne flaviviruses recognized by the International Committee on Taxonomy of Viruses—cont’d

Virus

Subtype

Vector

Host

Geographic distribution

Year of isolation

Diseases in human or other animals

Japanese encephalitis virus group Cacipacor e virus

Cacipacore virus (CPCV)

Unknown

Birds

South America

1977

Fever

Japanese encephalitis virus

Japanese encephalitis virus (JEV)

Culex tritaeniorhynchus

Human beings, birds, and pigs

Japan

1935

Encephalitis

Koutango virus

Koutango virus (KOUV)

Unknown

Rodents?

Senegal Taterakempi Senegal

1969

Fever, rash

Murray Valley encephalitis virus

Alfuy virus (ALFV)

Unknown

Wild birds and fowl?

Australia

1966

Fever?

Murray Valley encephalitis virus (MVEV)

Culex annulirostris

Human beings and birds

Australia

1951

Encephalitis

St. Louis encephalitis virus

St. Louis encephalitis virus (SLEV)

Culex spp.

Birds

American continent

1933

Fever, Encephalitis

Usutu virus

Usutu virus (USUV)

Culex spp.

Birds

Europe, Africa

1959

Fever, rash

West Nile virus

West Nile virus (WNV)

Culex spp.

Birds

Worldwide

1937

Fever, Encephalitis

Australia

1960

Kunjin virus (KUNV) Yaounde virus

Yaounde virus (YAOV)

Culex spp

Birds? Wild rodents?

Cameroon, Central Africa Republic

1968

Unknown

Kokobera virus (KOKV)

Culex spp.

Mammals?

Australia

1960

Fever, polyarthritis

Stratford virus (STRV)

Aedes spp.

Mammals?

Australia

1961

Bagaza virus

Bagaza virus (BAGV)

Culex spp.

Unknown

Africa

1966

Fever

Ilheus virus

Ilheus virus (ILHV)

Aedes, Psorophora, Culex spp. mosquitoes?

Birds

South and Central America

1944

Fever

Rocı´o virus (ROCV)

Culex spp.? Aedes scapularis?

Birds

Brazil

1975

Encephalitis

Israel turkey meningoencephalitis virus

Israel turkey meningoencephalitis virus (ITV)

Culex spp.

Birds?

Israel, South Africa

1959

Unknown

Ntaya virus

Ntaya virus (NTAV)

Mosquitoes

Unknown

Africa

1943

Fever

Tembusu virus

Tembusu virus (TMUV)

Culex spp.

Birds

Malaysia, Thailand, Taiwan

1955

Unknown

Kokobera virus group Kokobera virus

Ntaya virus group

Continued

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TABLE 2 Mosquito borne flaviviruses recognized by the International Committee on Taxonomy of Viruses—cont’d Diseases in human or other animals

Geographic distribution

Year of isolation

Monkeys, human beings

Africa, Asia, Central and South America

1947

Fever, rash, microcephaly,

Mansonia septempunctata?

Unknown

New Guinea

1966

Fever

Wesselsbron virus (WESSV)

Aedes spp

Unknown

Africa, Asia

1955

Unknown

Yellow fever virus (YFV)

Aedes spp., Haemagougus spp., Sabethes spp.

Human beings, monkeys

Africa, South America

1927

Hemorrhagic fever

Unknown

Senegal, Central Africa Republic

1972

Unknown

Virus

Subtype

Vector

Host

Zika virus

Zika virus (ZIKV)

Aedes spp

Sepik virus

Sepik virus (SEPV)

Wesselsbron virus Yellow fever virus

Yellow fever virus group

Probably mosquito-borne flavivirus, Kedougou virus group Kedougou virus

Kedougou virus (KEDV)

Aedes spp.

Probably mosquito-borne flavivirus, Edge Hill virus group Banzi virus

Banzi virus (BANV)

Culex spp

Unknown

Africa

1956

Fever

Bouboui virus

Bouboui virus (BOUV)

Aedes spp.

Unknown

Africa

1967

Unknown

Edge Hill virus

Edge Hill virus (EHV)

Aedes spp.

Wallabies?

Australia

1969

Unknown

Jugra virus

Jugra virus (JUGV)

Aedes spp.

Bats?

Malaysia

1968

Unknown

Saboya virus

Potiskum virus (POTV)

Unknown

Unknown

Nigeria

1969

Unknown

Saboya virus (SABV)

Unknown

Unknown

Senegal

1968

Unknown

Uganda S virus (UGSV)

Aedes spp.

Mammals?

Africa, potentially Southeast Asia

1947

Unknown

Uganda S virus

Virion structure, genome, and viral protein function Mature flaviviruses virions are small enveloped icosahedral particles measuring around 40–60 nm in diameter (Fig. 3). Infectious virions contain three structural proteins: capsid (C) protein, membrane (M) protein, and envelope (E) protein. The C protein packages the viral RNA genome constituting nucleocapsid. Surrounding the nucleocapsid is located the host cell-derived lipid bilayer where proteins M and E are anchored (Mukhopadhyay, Kuhn, & Rossmann, 2005) (Fig. 3). Flavivirus genome consists in one positive sense single-stranded RNA molecule of around 11,000 nucleotides in length. The single long open reading frame (ORF) is flanked by 30 and 50 noncoding region and is translated into a polyprotein comprising three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Figs. 3 and 4). The 30 and 50 untranslated terminal regions contain multiple conserved RNA structures that interact to form a panhandle structure that is required for the initiation of minus strand RNA synthesis with the 50 terminal structure functioning as the promoter (Brinton & Basu, 2015). The positive single-stranded RNA works as a single

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TABLE 3 Tick-borne flaviviruses recognized by the International Committee on Taxonomy of Viruses.

Virus specie

Virus name

Vector

Host

Geographic distribution

Year of isolation

Diseases in human or other animals

Mammals tick-borne flaviviruses group Gudgets Gully virus (GGYV)

Gudgets Gully virus (GGYV)

Ixodes uriae

Unknown

Australia

1976

Unknown

Karshi virus (KSIV)

Karshi virus (KSIV)

Ornithodoros papillipes

Unknown

Uzbekistan, Kazachsta

1972

Unknown

Kyassanur Forest disease virus (KFDV)

Kyassanur Forest disease virus (KFDV)

Haemaphysalis spinigera

Monkeys? Rats?

India

1957

Hemorrhagic fever, encephalitis Hemorrhagic fever in monkeys

Alkhurma hemorrhagic fever virus (AHFV)

Ixodidae (Ornithodoros spp.?)

Camels and sheep?

Arabian Peninsula

1995

Hemorrhagic fever, encephalitis

Langat virus (LGTV)

Langat virus (LGTV)

Ixodidae (Ixodes spp.)

Rattus spp.?

South-east Asia, Siberia

1956

Fever, encephalitis

Louping-ill virus

Louping-ill virus (LIV)

Ixodes ricinus

Red deer? Hares?

Scotland, N. England, N. Ireland

1929

Encephalitis

British subtype

Unknown

British Islands, Norway

Irish subtype

Unknown

Spanish subtype

Unknown

IberianPeninsula

Unknown

Greece

1969

Turkish sheep encephalitis virus subtype

Unknown

Turkey

1969

Negishi virus

Unknown

Japan

1948

Dermacentor spp.

Unknown

Western Siberia

1947

Hemorrhagic fever Encephalitis in muskrats, bank voles, various species of birds

Ixodes cookie, Ixodes persulcatus

Woodchuck? Squirrels?

Canada, United States, Far Eastern Siberia

1958

Deer

United States

Encephalitis Encephalitis in horses, rarely in dogs

Afghanistan

1968

Unknown

Europe

1937

Encephalitis Rarely encephalitis in dogs, fever in calves and lambs

Greek goat encephalitis virus subtype

Omsk hemorrhagic fever virus (OHFV) Powassan virus (POWV)

Powassan virus(POWV)

Ixodes gibbosus

Deer tick virus Royal Farm virus (RFV) Tick-borne encephalitis virus (TBEV)

Argasidae (Argas spp.) European subtype TBEV-Eu

Ixodes ricinus

Unknown

Continued

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TABLE 3 Tick-borne flaviviruses recognized by the International Committee on Taxonomy of Viruses—cont’d

Virus specie

Host

Geographic distribution

Year of isolation

Diseases in human or other animals

Virus name

Vector

Far Eastern subtype TBEV-FE

Ixodes persulcatus

Far Eastern Asia

Encephalitis

Siberian subtype TBEV-Sib

Ixodes persulcatus

Finland, European part of Russia, Siberia

Encephalitis

Seabird tick-borne flavivirus group Kadam virus (KADV)

Ixodidae (Rhipicephalus pravus)

Unknown

East Africa (Uganda), Arabian Peninsula

1967

Unknown

Meaban virus (MEAV)

Argasidae (Ornithodoros spp.)

Seabirds?

France (Britanny)

1981

Unknown

Saumarez Reef virus (SREV)

Ixodidae (Ixodes spp.), Argasidae (Ornithodoros capensis)

Seabirds (Larus spp.)?

Australia

1974

Unknown

Tyuleniy virus (TYUV)

Ixodidae (Ixodes spp.)

Seabirds, seals?

Northern Russia (Kola Peninsula), Norway, Western USA

1969

Unknown

RNA messenger containing one ORF which is traduced into a polyprotein (Fig. 4). Nonstructural proteins are necessary in the viral genome replication, virion packaging, and immune evasion (Barrows et al., 2018). NS1 is a homodimer involved in the replication process of viral RNA and viral particle assembly (Rastogi, Sharma, & Singh, 2016). NS2A is a small hydrophobic protein thought to intervene in the RNA replication and immune evasion. NS2B is a small membrane-associated protein and acts as a cofactor necessary for the NS2B-NS3 complex serine-protease activity. NS3 is a big cytoplasmic protein that intervenes in several enzymatic activities (protease, helicase). NS4A and NS4B are small hydrophobic membrane-associated proteins intervening in the viral RNA replication and immune evasion (Barrows et al., 2018). NS5 is the biggest and most conservative protein throughout the genera. It has a methyltransferase activity on its N-terminal region and an RNA-dependent RNA polymerase activity on the C-terminal motifs (Fig. 4) (Chong, Leow, Abdul Majeed, & Leow, 2019).

Viral infection and replication cycle The entry of flaviviruses into the host cells is mediated by the Envelope protein. Besides viral receptors, cellular attachment factors (glycosaminglycans, mannose receptor, phosphatidylserine receptors, DC-SIGN lectins) promote viral infection by increasing the duration of viral particle and cells surface interaction. Once flavivirus virion is attached to the cell membrane they can move across until the encounter of preexisting clathrin-coated pit (Pierson & Diamond, 2013). Virion particles are then internalized in endosomes where viral fusion occurs thanks to conformational changes in the viral Envelope protein triggered by acidic environment in the endosomes. Then nucleocapsid is released into the cytoplasm and viral replication starts. Viral RNA replication occurs in perinuclear spots and implies the synthesis of a single complementary negative strand that works as a template for the synthesis of positive strand molecule (Chong et al., 2019). The genomic viral RNA is used directly as a messenger and is completely translated from its 50 end to produce one polyprotein that is later

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FIG. 1 Hypothetical transmission networks for Culex-borne and Aedes-borne flaviviruses. Arrows represent the viral flow between vectors and hosts involved in the flavivirus maintenance network. The thickness of the arrow represents the amount of virus existing between the particular connection of host and vector (which is determined by the vector-host preference, vector-host population density, vector and host competence). The spotted line arrows represent viral flow to a dead-end host. The colored arrows represent the season in which the vector-host interaction takes place (green: spring, red: summer, orange: fall).

cleaved to generate each individual viral protein (Brinton & Basu, 2015). The new viral particle assembly occurs in the endoplasmic reticulum (ER), where immature particles are generated (containing prM) and then transported to the cell exterior through the exocytic pathway. Evidence shows that the acidic pH in the trans-Golgi network (TGN) produces conformational changes in the prM-E complex that are necessary for particle maturation. Once the cleavage of the complex is produced by the action of cellular furins, the mature particles are released through exocytosis (Pierson & Diamond, 2013).

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FIG. 2 Infection process of flavivirus in the mosquito vector. Once the flavivirus particles are ingested with the blood meal, virions infect the midgut cells (midgut infection barrier) (1) and overcome the basal lamina (midgut escape bBarrier) (2). Right after crossing the midgut, viral dispersion starts and the virus infects the salivary glands (salivary glands infection barrier) (3). Next the mosquito feed on a host, viral particles will be injected through the salivary secretion (salivary gland escape barrier) into the vertebrate host.

FIG. 3 Schematic representations of flavivirus virion (A) and viral genome (B). Flavivirus are enveloped single-stranded positive RNA viruses. The viral genome has an open reading frame (ORF) that encodes a total of 10 proteins and is flanked by a 50 and 30 untranslated region (UTR) extremes. Structural viral proteins are membrane (M), capside (C), and envelope (E). Nonstructural viral proteins include nonstructural 1 (NS1), nonstructural 2A (NS2A), nonstructural 2B (NS2B), nonstructural 3 (NS3), Nonstructural 4A (NS4A), nonstructural 4B (NS4B), and nonstructural 5 (NS5).

Host infection, immune response, and pathogenesis Flavivirus virions are injected into the host through salivary secretion during arthropod vector feeding. Skin is likely the primary site for local viral replication where intradermal immune cells such as dendritic cells, monocytes, keratinocytes, and macrophage are primary target for viral replication (Wu, Yu, Wang, & Cheng, 2019). Vector saliva contains protein factors (34-kDa protein LTRIN, serine protease) that suppress interferon signaling and influence viral entry into host cells ( Jin et al., 2018). Saliva injection also produces an inflammatory response promoting viral transmission by enhancing the infection in the host’s virus permissive neutrophils and myeloid cells (Pingen et al., 2016). Active flavivirus replication can

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FIG. 4 Schematic representation of a flaviviral polyprotein topology and transmembrane domains. Flavivirus polyprotein is integrated into the endoplasmic reticulum (ER) membrane. Viral proteins prM, envelope (E), and nonstructural 1 (NS1) are mainly on the luminal side and capside (C), nonstructural 3 (NS3), and nonstructural 5 (NS5) on the cytoplasmic side. Proteins nonstructural 2A (NS2A), nonstructural 2B (NS2B), nonstructural 4A (NS4A), and nonstructural 4B (NS4B) have several transmembrane domains spanning across the ER.

be detected within 1 day of the infection at the subcutaneous site and virus spreads to the lymph node. Flavivirus disseminate to local lymph node associated with migratory infected dendritic cells or as free virus (Pierson & Diamond, 2013). Macrophage located in the lymph node capture viral particles efficiently serving as virus amplification infection and initiators of innate and adaptive immune responses. Viral particles producing in the lymph node spread to intravascular venous compartment via lymphatic drainage. Virus in the bloodstream can directly infect blood cells or visceral tissues which results in further dissemination and secondary viremia (Pierson & Diamond, 2013). Flaviviruses can also infect the central nervous system (CNS) by crossing the blood-brain barrier (BBB). These viruses replicated in brain microvascular endothelial cells (BMECs), which induced down regulation of tight junction proteins and increased the permeability of the BBB. Virus replication and activation of astrocytes and microglia also affect the endothelial barrier permeability. All these events are associated with the expression and secretion of inflammatory mediators, which are believed to recruit leukocytes to the CNS. The leukocyte infiltrate could further mediate viral invasion through a “Trojan horse” mechanism and might contribute to BBB breakdown and neurological alterations (Mustafa´, Meuren, Coelho, & de Arruda, 2019). Flaviviruses infection induces both humoral and cellular immune responses in the host. The earliest host cell responses against flavivirus infection involve secretion of type I interferons (IFN), an antiproliferative, antiviral, and immunomodulatory cytokine (Thurmond, Wang, Song, & Hai, 2018). IFN production results in the transcription of IFN-inducible genes such as interferon-stimulated genes, the double-stranded RNA-activated protein kinase, and 20 ,50 -OAS leading to the induction of antiviral pathways (Chong et al., 2019). However, flaviviruses are still capable of proliferating, suggesting that there are virus-specific mechanisms in which the induction of IFN pathways can be altered, inhibited, or bypassed (Chong et al., 2019). Flavivirus nonstructural proteins such as NS2A and NS5 have been observed interfering with this antiviral mechanism (Thurmond et al., 2018; Tu et al., 2012). In humoral immunity, neutralizing antibodies are produced primarily to target epitopes located on the E glycoprotein. Specific antibodies against prM and nonstructural (NS) proteins have also been reported. These antibodies inhibit viral attachment, internalization, or replication within cells (Pierson & Diamond, 2013). Besides the humoral immune response, cellular immunity in the infected host is also induced by flavivirus infection. T-cell responses play a major role in viral clearance of flavivirus infections (Brien, Uhrlaub, & Nikolich-Zugich, 2008).

Flavivirus emergence and global burden Starting in the 1980s several emerging infectious disease events occurred around the globe. Those events originated by a zoonotic infectious agent were located in areas from developing countries where anthropogenic activities (deforestation, mining, cattle, agriculture) cause environmental disturbances ( Jones et al., 2008). Disturbances from anthropogenic activities modify the vector and host community composition, their abundance, and ecological interactions affecting the arbovirus transmission networks (Diaz et al., 2013). Moreover, climatic changes, human movements through airplanes, animal trade and migration, and genetic mutations causing spillovers are some factors promoting the emergence of arboviruses (Huang, Higgs, & Vanlandingham, 2019). The majority of arboviruses remain silent in their enzootic transmission network with no effect on human populations. The permanence of human beings in wild areas increases the likelihood of getting infected and ill by an arbovirus. Moreover, if the humans can produce high enough viremia to infect urban mosquitoes, the virus can be dispersed and introduced into an urbanized area (Weaver & Reisen, 2010).

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Human civilization is under global expansion, building settlements everywhere. In most developing countries this expansion is taking place without urban planning. New settlements around urban periphery have no water service, so people create temporary water reservoirs that represent breeding sites for vector mosquitoes like Ae. aegypti and Ae. albopictus, increasing the risk for outbreaks of Aedes-borne viruses such as DENV, YFV, and ZIKV (Kraemer et al., 2019). Moreover, the huge mass transportation of humans by airplanes experienced in the last decade has caused the introduction and dispersion of several flaviviruses including DENV, ZIKV, and YFV.

Emergence of Aedes-borne flaviviruses Dengue virus is transmitted by female mosquitoes of the species Ae. aegypti and Ae. albopictus. Dengue is widespread throughout the tropical and subtropical areas. It causes a wide spectrum of disease, ranging from subclinical disease to severe flu-like symptoms. Some people develop severe dengue (severe bleeding, organ impairment, and/or plasma leakage) (Simmons, Farrar, Nguyen, & Wills, 2012). The number of dengue cases reported to WHO increased over 15-fold over the last two decades, from 505,430 cases in 2000 to over 2,400,138 in 2010 and 3,312,040 in 2015. Actual estimations indicate 390 million dengue virus infections per year (WHO, 2020). Zika virus was isolated in 1947 in Zika forest of Kampala (Uganda) from a rhesus monkey. In 2013–2014, an outbreak of ZIKV was reported in French Polynesia, followed by an increase in the prevalence of Guillain-Barre Syndrome in infected residents (Leonhard et al., 2020). In 2015, the transmission of ZIKV in South America was associated with congenital microcephaly outbreaks mainly in northern areas of Brazil (de Arau´jo et al., 2018). A total of 18 Brazilian states reported autochthonous circulation of ZIKV and 440,000  1,300,000 suspected infection cases were reported during the outbreak. More than 4300 cases of fetal abnormalities including microcephaly were reported up to February 2016 (de Arau´jo et al., 2018). After a worldwide immunization program against YFV was established, this virus was successfully controlled. Nowadays, small enzootic foci remain in some countries of South America and small urban outbreaks still occur in some countries of Africa (Maguire & Heymann, 2016; Monath & Vasconcelos, 2015). Enzootic activity in the Amazon basin (Brazil) is very dynamic and is influenced by rainfall and likely dispersed by nonhuman primates and rural worker movements (Monath & Vasconcelos, 2015). A big concern about YFV reurbanization emerged since viral activity was detected close to very densely populated areas of Bahia, Minas Gerais, Sao Paulo, and Rio de Janeiro states. Between 2016 and 2019, more than 15,000 nonhuman primate epizootics were reported in Brazil in sylvatic, rural, and urban areas and at least 2251 human cases and 772 deaths were confirmed (Silva et al., 2020). The 2016–2019 YF epidemics have been considered the most significant outbreaks of the last 70 years in the country, and the number of human cases was 2.8 times higher than total cases in the previous 36 years (Silva et al., 2020). Although not completely understood, human and nonhuman primate movements dispersing new lineage of YFV, high mosquito abundance in peri-urban areas, and poor vaccination coverage in resident human populations could be some of the factors promoting this reemergence of YFV in Brazil.

Emergence of Culex-borne flaviviruses Japanese encephalitis virus is the most important neurotropic flavivirus and a major causative agent of encephalitis worldwide. JEV is prevalent in 24 countries of Southeast Asia and the Western Pacific where 68,000 clinical cases are reported annually, and the case fatality rate is 25%–30%. About 30%–50% of JE survivors have permanent neurological sequelae, imposing a heavy burden on public health and society (Banerjee & Tripathi, 2019). It is maintained in enzootic cycles involving water birds (rural areas) and pigs (domestic areas) as amplifying hosts and Culex vishnui complex mosquitoes as vectors. Irrigated rice fields are important amplification hotspots because of the abundance of Cx. tritaeniorhynchus, its primary vector (van den Hurk, Ritchie, & Mackenzie, 2009). Changes in its epidemiology have been observed during the last years. Five genotypes (G1-G5) have been described for JEV. JEV G1 has replaced G3 as the dominant genotype in the traditional epidemic areas in Asia. G3 has spread from Asia to Europe and Africa. G2 and G5, which were originally endemic in Malaysia, exhibited great geographical displacement: G2 migrated southward and was introduced into Australia, while G5 emerged in China and South Korea after decades of silence. JEV started to emerge in nontraditional epidemic regions (Africa, Europe) posing a greater burden for new countries (Gao et al., 2019). West Nile virus was first isolated in Africa and recognized as human pathogen years later. It is transmitted by Culex mosquito species and amplified by birds. Endemic in the Old World (Asia, Africa, Europe) its epidemiology changed dramatically in 1999 when it was introduced and successfully established in the American continent. Its introduction seriously

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affected bird, horses, and human populations. In the United States, 51,747 human cases, approximately 48% being neuroinvasive diseases, and 2454 deaths were reported between 1999 and 2019 (CDC, 2020). Around 25 wild bird species (New World sparrows, finches, vireos, blue jay, bluebirds) were negatively affected by the introduction of WNV representing a serious conservation threat for some of those (George et al., 2015). In 2018, WNV infections in Europe increased dramatically compared to previous transmission seasons. A total of 2083 human cases were reported with the largest number detected in Italy, Serbia, and Greece. This reemergence could be a consequence of an earlier onset of the transmission season due to warm temperatures during spring, resulting in abundance of vector Culex mosquitoes (Pacenti et al., 2020). St. Louis encephalitis virus (SLEV) is a causative agent of encephalomyelitis especially in elderly (Reisen, 2003). Historically, it was the most important neurotrophic flavivirus in the United States until WNV arrived (Diaz, Coffey, BurkettCadena, & Day, 2018). In the rest of the American continent, it was not associated with encephalitis outbreak in human populations. In 2002, it emerged as a human pathogen in southern area of Brazil (Sao Paulo state) and central region of Argentina (Cordoba) (Rocco et al., 2005; Spinsanti et al., 2008). Ecological studies that have been focused to understand factors driving its emergence in Argentina suggested that the introduction of a more virulent and pathogenic strain from Brazil (potentially through migratory birds) and environmental changes (urbanization, expansion of soybean production area) led to an abundance of Culex quinquefasciatus and Eared Doves (Zenaida auriculata) (Diaz et al., 2018; Rivarola, Tauro, Llina´s, & Contigiani, 2014). Interestingly, reemergence of SLEV in the west coast of United States was associated with a viral reintroduction likely from Cordoba, Argentina (White et al., 2016). This finding pointed out the role of migratory bird as dispersers of virus (Diaz et al., 2018).

Policy and procedures Vector-borne flaviviruses diagnoses Massive human transportations in our days have globally dispersed flaviviruses, so the likelihood of coinfection is increasing in human populations. Importantly, the frequency of coinfection in nature and its clinical and epidemiologic implications are poorly understood. Because of cross-reactions among flaviviruses, serological results are hard to determine especially when a person has been infected by more than one flavivirus species. Most of commercially available kits are sensible but not specific. The gold standard technique for flavivirus diagnose is the neutralization test. Although expensive, time consuming, and requiring well-trained personnel, this technique gives us the highest specificity in arbovirus serological diagnose. To confirm an arbovirus infection by a serological technique we need to detect a seroconversion between acute and convalescent sera samples or by detection of a 4 times titer increment between sera samples. Plaque reduction neutralization test: 1. Before performing the neutralization assay you have to titrate your viral stock in order to inoculate around 100 theoretical plaque forming unit (PFU). 2. Transfer 100 μL of your sera sample into a plastic tube and add equal volume of your viral stock dilution containing 100 PFU. 3. Then incubate the sera-virus mix for 60 min at 37 °C for neutralization process. 4. Prepare 48-h-old confluent monolayer of VERO cells in a polycubet plate. 5. After the neutralization process, in each well inoculate 100 μL of your sera-virus mix onto VERO cells monolayer. 6. Incubate inoculated cells for 60 min at 37°C 7. Add around 1 mL of 1% agarose dilution in each well. 8. Check daily for the presence of plaque forming focus on cell monolayers. The incubation period and plaque appearance vary depending on viral strains. 9. After incubation period (5 days for West Nile virus, 7 days for Saint Louis encephalitis virus, 5 days for yellow fever virus, 6 days for Zika virus, and 7 days for dengue virus) fix the cells with 10% formaldehyde for 2 h at room temperature 10. Rinse the plates with running tap water to remove the agar layer. Remove excess water by gently tapping and by using paper towels. 11. Stain cells with crystal violet solution for 15 min. Rinse with tap water. Dry stained cells. 12. Count plates on each well. Positive samples are those that neutralized more than 80% or 90% of the total PFU inoculated.

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Mini-dictionary of terms Emerging infectious disease: An old disease that is increasing its geographic distribution or a new one that appears for the first time. Examples of emerging arboviral diseases are Zika, dengue, Saint Louis encephalitis, Japanese encephalitis, and West Nile fever. Arbovirus: Viruses that are biologically vectored by arthropods. It is an ecologically clustered rather than a phylogenetic group. Alphavirus, Flavivirus, Orthobunyavirus, Asfarvirus, and Reovirus among others are viral genera with arbovirus members. Vector: An hematophagous arthropod (mosquito, ticks, midges, sand flies) that allow viral infection, replication, and dissemination transmitting arboviruses in nature from one host to another. Host: An arbovirus host is a vertebrate that allow viral replication and amplification high enough to infect an arthropod vector during feeding process. A broad diversity of vertebrates (birds, rodents, primates, human) is used as host by flaviviruses. Virion: Refers to viral particle comprising a capsid and a viral genome. Enveloped virions also have a lipid envelope. Virus is a population of virions.

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Members of Flavivirus genus account for more than 50 viral species and based on phylogeographic evidence they originated in Africa approximately 120,000 years ago. Flaviviruses can be grouped into mosquito-borne flaviviruses, tick-borne flaviviruses, insect-specific flaviviruses, and no known-host flaviviruses. Flavivirus-vector interactions are more specific compared with other arboviruses such as Alphavirus. All their members have a single-stranded positive viral RNA molecule and encode for 10 proteins (3 structural proteins and 7 nonstructural proteins). The envelope protein is the viral receptor that interacts with the cellular receptors.

Summary points l

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Flaviviruses originated around 120,000 years ago in Africa and then dispersed globally through human migration and animal trade. Mosquito-borne and tick-borne flaviviruses are the most important flaviviruses from a public health perspective. They cause thousands of human deaths and economic loss annually around the globe. The global burden of flaviviruses is based their nature to cause severe diseases such as hemorrhagic fever, polyarthritis, neuroinvasive diseases (encephalomyelitis), and hepatitis, among others. Flavivirus infection in humans is an acute self-limited infection controlled by both native and adapted immune system. Antibody response is a neutralizing and permanent mechanism that will control further infections. Deforestation, urbanization, massive human transportation, animal trade, and climatic changes are some of the factors that modify our environment and biological communities of arbovirus vectors and hosts promoting the emergence of flavivirus diseases.

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Global trends in emerging infectious diseases. Nature, 451(7181), 990–993. Kenney, J. L., & Brault, A. C. (2014). The role of environmental, virological and vector interactions in dictating biological transmission of arthropod-borne viruses by mosquitoes. Advances in Virus Research, 89, 39–83. Kraemer, M., Reiner, R. C., Jr., Brady, O. J., Messina, J. P., Gilbert, M., Pigott, D. M., … Golding, N. (2019). Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus. Nature Microbiology, 4(5), 854–863. Kuno, G. (2007). Host range specificity of flaviviruses: Correlation with in vitro replication. Journal of Medical Entomology, 44(1), 93–101. Leonhard, S. E., Bresani-Salvi, C. C., Lyra Batista, J. D., Cunha, S., Jacobs, B. C., Brito Ferreira, M. L., … M.F. (2020). Guillain-Barre syndrome related to Zika virus infection: A systematic review and meta-analysis of the clinical and electrophysiological phenotype. PLoS Neglected Tropical Diseases, 14 (4), e0008264. Maguire, H. C., & Heymann, D. L. (2016). Yellow fever in Africa. BMJ, 354, i3764. Monath, T. P., & Vasconcelos, P. F. (2015). Yellow fever. Journal of Clinical Virology, 64, 160–173. Mukhopadhyay, S., Kuhn, R. J., & Rossmann, M. G. (2005). A structural perspective of the flavivirus life cycle. Nature Reviews. Microbiology, 3(1), 13–22. Mustafa´, Y. M., Meuren, L. M., Coelho, S., & de Arruda, L. B. (2019). Pathways exploited by flaviviruses to counteract the blood-brain barrier and invade the central nervous system. Frontiers in Microbiology, 10, 525. Pacenti, M., Sinigaglia, A., Franchin, E., Pagni, S., Lavezzo, E., Montarsi, F., … Barzon, L. (2020). Human West Nile virus lineage 2 infection: Epidemiological, clinical, and virological findings. Viruses, 12(4), E458. Pettersson, J. H., & Fiz-Palacios, O. (2014). Dating the origin of the genus flavivirus in the light of Beringian biogeography. The Journal of General Virology, 95(Pt 9), 1969–1982. Pierson, T. C., & Diamond, M. S. (2013). Flaviviruses. In D. M. Knipe, & P. M. Howley (Eds.), Fields virology (6th ed., pp. 747–794). Philadelphia, US: Lippincott Williams and Willkins. Pingen, M., Bryden, S. R., Pondeville, E., Schnettler, E., Kohl, A., Merits, A., … McKimmie, C. S. (2016). Host inflammatory response to mosquito bites enhances the severity of arbovirus infection. Immunity, 44(6), 1455–1469. Rastogi, M., Sharma, N., & Singh, S. K. (2016). Flavivirus NS1: A multifaceted enigmatic viral protein. Virology Journal, 13, 131. https://doi.org/10.1186/ s12985-016-0590-7. Reisen, W. K. (2003). Epidemiology of St. Louis encephalitis virus. Advances in Virus Research, 61, 139–183. https://doi.org/10.1016/s0065-3527(03) 61004-3. Rivarola, M. E., Tauro, L. B., Llina´s, G. A., & Contigiani, M. S. (2014). Virulence variation among epidemic and non-epidemic strains of Saint Louis encephalitis virus circulating in Argentina. Memo´rias do Instituto Oswaldo Cruz, 109(2), 197–201.

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Rocco, I. M., Santos, C. L., Bisordi, I., Petrella, S. M., Pereira, L. E., Souza, R. P., … Suzuki, A. (2005). St. Louis encephalitis virus: First isolation from a human in Sa˜o Paulo State, Brazil. Revista do Instituto de Medicina Tropical de Sa˜o Paulo, 47(5), 281–285. Rosen, L. (1987). Overwintering mechanisms of mosquito-borne arboviruses in temperate climates. The American Journal of Tropical Medicine and Hygiene, 37(3 Suppl), 69S–76S. Silva, N., Sacchetto, L., de Rezende, I. M., Trindade, G. S., LaBeaud, A. D., de Thoisy, B., & Drumond, B. P. (2020). Recent sylvatic yellow fever virus transmission in Brazil: The news from an old disease. Virology Journal, 17(1), 9. Simmons, C. P., Farrar, J. J., Nguyen, V. V., & Wills, B. (2012). Dengue. The New England Journal of Medicine, 366(15), 1423–1432. Spinsanti, L. I., Dı´az, L. A., Glatstein, N., Arsela´n, S., Morales, M. A., Farı´as, A. A., … Contigiani, M. (2008). Human outbreak of St. Louis encephalitis detected in Argentina, 2005. Journal of Clinical Virology, 42(1), 27–33. Thurmond, S., Wang, B., Song, J., & Hai, R. (2018). Suppression of type I interferon signalling by flavivirus NS5. Viruses, 10(12), 712. Tu, Y. C., Yu, C. Y., Liang, J. J., Lin, E., Liao, C. L., & Lin, Y. L. (2012). Blocking double-stranded RNA-activated protein kinase PKR by Japanese encephalitis virus non-structural protein 2A. Journal of Virology, 86(19), 10347–10358. van den Hurk, A. F., Ritchie, S. A., & Mackenzie, J. S. (2009). Ecology and geographical expansion of Japanese encephalitis virus. Annual Review of Entomology, 54, 17–35. Weaver, S. C., & Reisen, W. K. (2010). Present and future arboviral threats. Antiviral Research, 85(2), 328–345. White, G. S., Symmes, K., Sun, P., Fang, Y., Garcia, S., Steiner, C., … Coffey, L. L. (2016). Reemergence of St. Louis encephalitis virus, California, 2015. Emerging Infectious Diseases, 22(12), 2185–2188. World Health Organization. (2020). https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue. (Accessed 20 April 2020). Wu, P., Yu, X., Wang, P., & Cheng, G. (2019). Arbovirus lifecycle in mosquito: Acquisition, propagation and transmission. Expert Reviews in Molecular Medicine, 21, e1.

Chapter 2

The innate immune response during Zika virus infection Manuela Sales Lima Nascimentoa, Wilo Victor dos Santosa, Amanda Costa Ayres Salmeronb, Jos elio Maria Galva˜o de Arau´joa, Jos e Verı´ssimo Fernandesa, and Paulo Marcos Matta Guedesa a

Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil, b Edmond and Lily Safra

International Institute of Neuroscience, Santos Dumont Institute, Macaiba, Rio Grande do Norte, Brazil

Abbreviations AIM2 Akt (PKB) BCL2 CARD CASP-1 CCL CD209 cGAS CXCL CZS DAMPs DC DC-SIGN DDX58 DNA ds EGF EIF2 EIF2AK2 HemeOX HFF1 cells IFITM IFN IFNAR IL IRF ISGF ISGs JAK LC3 LCs LGP2 MAPK MAVS MDA mTOR MX1 NF-κB

absent in melanoma 2 protein kinase B B-cell lymphoma 2 caspase activation and recruitment domains Caspase-1 chemokine (CdC motif) ligand 1 cluster of differentiation 209 cyclic GMP–AMP synthase chemokine (C-X-C motif) ligand congenital Zika syndrome damage-associated molecular patterns dendritic cells dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin DExD/H-Box helicase 58 deoxyribonucleic acid double- stranded epidermal growth factor eukaryotic initiation factor 2 eukaryotic translation initiation factor 2-alpha kinase 2 humans oxidative stress human foreskin fibroblast cell line IFN-induced transmembrane protein interferon human type I interferon receptor interleukin interferon regulatory factor IFN-stimulated gene factor interferon-stimulated genes janus kinases microtubule-associated protein 1 light chain 3 epidermal langerhans cells laboratory of genetics and physiology 2 mitogen-activated protein kinase mitochondrial antiviral signaling melanoma differentiation-associated gene mammalian target of rapamycin myxovirus resistance 1 factor nuclear kappa B

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00002-X Copyright © 2021 Elsevier Inc. All rights reserved.

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NLRP3 NLRs NMD NOD NS OAS p62 (SQSTM1) PAMP PKR PRRs ER RIG RLRs RNA RSAD2 SAMHD1 SOD2 ss STAT STB STING TBK1 TIM-1 TLRs TNF-α TNFR TRAF TRIF TRIM TYK2 U87-MG UPF1 ZIKV

A Zika virus: Introductory chapters

NOD-like receptor pyrin domain containing 3 NOD-like receptors nonsense-mediated mRNA decay nucleotide oligomerization domain-containing protein nonstructural 20 5’-Oligo A synthetase selective autophagy receptor sequestosome 1 pathogen-associated molecular patterns protein kinase R pattern recognition receptors endoplasmic reticulum retinoic acid-inducible gene RIG-like receptors ribonucleic acid radical S-adenosyl methionine domain-containing protein 2 sterile alpha motif and histidine-aspartic domain containing protein 1 superoxide dismutase 2 single-stranded signal transducer and activator of transcription proteins SyncyTiotrophoBlast. stimulator of interferon genes TANK-Binding kinase 1 transmembrane immunoglobulin and mucin domain 1 toll-like receptors tumor necrosis factor tumor-necrosis factor receptor TNFR-associated factor TIR-Domain-containing adapter-inducing interferon-β tripartite motif-containing. tyrosine-protein kinase 2 human cell line derived from malignant glioma used in neural studies up-frameshift protein 1 Zika virus

Introduction The immune system of mammals provides protection against invaders and guarantees homeostasis in a healthy organism. It can be divided into two arms that act together to provide immediate and long-term immunity: the innate and adaptive responses, respectively. For the purposes of this chapter, we focus only on the innate immune response mechanisms and how they are is coordinated to defend the host against Zika virus (ZIKV) infection. Innate immunity comprises a set of defense mechanisms that exists prior to the contact with a pathogen. It is prepared to act promptly and therefore provides the first line of defense to the body. It is composed of physical and chemical barriers, such as epitheliums and antimicrobial peptides; tissue resident cells such as macrophages, dendritic cells (DCs), mast cells, innate lymphoid cells; recruited cells such as neutrophils, monocytes, natural killers, eosinophils; and a wide variety of cell membrane or cytosolic receptors and soluble mediators. During infection, ZIKV has to cross multiple barriers depending on the transmission route and the infection site. In an immunocompetent host, at those sites, innate immunity components, e.g., keratinocytes and fibroblasts in the skin, glial cells in the brain, or Hofbauer cells in the placenta, are ready to respond and immediately prevent the establishment of the infection. Several molecules from ZIKV can activate the innate immunity through pattern recognition receptors (PRRs), thus acting as pathogen-associated molecular patterns (PAMPs). Then, ZIKV-infected cells produce and release type I interferon (IFN-I), which acts in autocrine and, most important, paracrine ways, inducing the expression of enzymes that blocks viral replication, the so-called antiviral state. In addition to the activation of innate immunity being critical for the early control of viral replication, it is also indispensable for the instruction of adaptive response in secondary lymphoid organs. Here, we explore in detail the current knowledge of those anti-ZIKV innate mechanisms, its features in different and specialized tissues, and the escape strategies developed by the virus.

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Importance of pattern recognition receptors (PRRs) in sensing ZIKV PRRs are responsible for recognizing conserved structures of microorganisms called PAMPs and also damage-associated molecular patterns (DAMPs). Many PRR families have been identified, including transmembrane proteins such as toll-like receptor (TLR) and C-type lectin receptors (CLR), cytoplasmic proteins such as RIG-like receptors (RLRs) and NOD-like receptors (NLRs), and cytosolic DNA sensors (CDS). They are expressed in several cell types and their activation induces a downstream signaling cascade that starts transcription of proinflammatory cytokines (TNF-α, IL-1, and IL-6), IFN-I (IFN-α and IFN-β), chemokines, and antimicrobial proteins (Takeuchi & Akira, 2010). ZIKV is a single-stranded (ss) RNA virus that infects skin cells, such as fibroblasts and keratinocytes; myeloid cells, such as DCs and macrophages; neurons and cells from the placenta, testis, and eye (Miner & Diamond, 2017). The endocytosis-mediated entry and then the escape to the cytosol directly activate two classes of evolutionarily conserved germline-encoded receptors: TLRs and RLRs. The viral RNA can be recognized by endosomal receptors such as TLR3, TLR7, and TLR8 (da Silva et al., 2019; Hamel et al., 2015), and when the virus gains the cytoplasm, RIG-I and melanoma differentiation-associated protein (MDA)5 can also detect its ssRNA (Fig. 1). LGP2 is another RLR present in mammalian cells that is activated by ZIKV infection. As this molecule does not express the CARD signaling domain, it only works as a negative and positive regulator of RIG-I and MDA5, respectively (Sanchez David et al., 2019). Recent studies using gene silencing through short interfering RNAs in the human foreskin fibroblast cell line (HFF1 cells) showed a ZIKV accumulation when TLR3, RIG-I, and MDA5 were blocked, but this effect was not observed when blocking TLR7 (Hamel et al., 2015). However, ZIKV replication in human myeloid cells is blocked using a TLR7/8 agonist by the induction of the antiviral protein viperin (Vanwalscappel, Tada, & Landau, 2018). Studies in patients during acute

FIG. 1 Innate immune receptors involved in the protective response against ZIKV infection. ZIKV infection activates PRRs such as TLR3, TLR7, TLR8, RIG1, MDA5, LGP2, and cGAS through different PAMPs and DAMPS. The activation of these receptors induces downstream intracellular signaling cascades that activate their adaptor molecules such as TRIF, Myd88, TRAF, TRIM, and STING. This leads to the activation of transcription factors such as IRF3, IRF5, IRF7, and NF-kB to induce the production of proinflammatory and antiviral mediators, including IFN-α and IFN-β. Those cytokines bind to the IFNAR autocrinally and paracrinously, inducing the phosphorylation-activation of tyrosine kinases JAK1 and TYK2 that activate STAT1, STAT2, and IRF-9 and lead to ISGs production and antiviral state. Blue and red colors indicate receptors and molecules involved in the ZIKV protective immune response and pathogenesis, respectively.

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ZIKV infection (up to 5 days after the onset of symptoms) showed an increase of mRNA expression of TLR3 correlated with an upregulation of IFN-α and IFN-β in peripheral blood mononuclear cells, although a high viral load was correlated with high expression of TLR8, RIG-1, MDA-5, IFN-α, and IFN-β (da Silva et al., 2019). The NLRs (NLRP1, NLRP3, AIM2) and CDS (cyclic GMP-AMP synthase/cGAS) are also described to be indirectly activated during ZIKV infection (Serman & Gack, 2019). Despite being an RNA virus, ZIKV infection induces citopathic effects that conduce to a host DNA mislocalization. This is detected by cyclic GMP-AMP synthase (cGAS) that catalyzes the synthesis of cyclic GMP-AMP (cGAMP). cGAMP interacts with endoplasmic transmembrane protein called Stimulator of Interferon Genes (STING) that activates the downstream transcription factors IRF3/5/7 and NF-κB, leading to cytokines and chemokines production (Ivashkiv & Donlin, 2008; Liu et al., 2018). On the other hand, the upregulation of TLR3, NLRP1, NLRP3, and AIM2 is related to neurological damage in Congenital ZIKV Syndrome (CZS) or Guillain-Barre pathogenesis (Malkki, 2016).

Type I interferon in ZIKV infection In ZIKV infection, the endosomal receptor TLR3 and the cytosolic receptors, RIG-I and MDA5, are the major PRRs described to act promoting the antiviral state in infected cells from different sites, such as the brain, placenta, skin, testis, kidney, and retina (Hamel et al., 2015). Activation of these receptors starts a complex event characterized by increased expression of IFN-α and IFN-β, which binds to its receptor a heterodimer consisting of two subunits, IFNAR1 and IFNAR2, expressed on the surface of all nucleated mammalian cells (De Weerd & Nguyen, 2012). When interacting with IFN-I, these receptors activate a signaling cascade by the JAK/STAT pathway. The JAK family proteins, janus tyrosine kinase (TyK2) and JAK1, are recruited and activated, inducing phosphorylation of STAT1 and STAT2 proteins, which are translocated to the nucleus to associate with p48 and IRF9, forming a transcriptional complex called the IFN-stimulated gene (ISG) factor (Velazquez et al., 1992). After that, several hundreds of antiviral proteins are induced in an attempt to protect the cell against the virus infection and, additionally, recruit leukocytes (Cedillo-Barro´n et al., 2018). The ISG complex activates the expression of antiviral-associated proteins, such as PKR and EIF2 α, that induces the inhibition of ZIKV protein synthesis; 20 , 50 -oligo A synthetase (OAS) and RNAse L, which promotes the degradation of viral RNA. The ISG complex also stimulates IFN-induced transmembrane protein (IFITM), which prevents ZIKV-induced cell death and blocks virus internalization by the host cell lipid bilayer (Tanaka et al., 2004). There are three different types of IFITM expressed in humans, IFITM 1, IFITM 2, and IFITM 3 (Cedillo-Barro´n et al., 2018). The main ISGs involved in inhibiting ZIKV replication are IFITM 1 and IFITM 3, IFITM 3 being more implicated in interrupting early stages of ZIKV replication. This protein is expressed at high levels during infection to prevent stress on the endoplasmic reticulum (ER), with a subsequent autophagic vesicle (Monel et al., 2017). In mouse models, Ifnar1/ or Ifnar1//Ifngr/ double knockouts are used to study the role of IFN-I in zika fever and neuropathology (Lazear et al., 2016). These models are able to mimic features of human infection, since wild rodents are refractories to ZIKV infection. While wild-type C57BL/6 pregnant mice infected with ZIKV are capable of inhibiting the virus replication in the placenta (Dias et al., 2016), immunocompromised pregnant Ifnar1/ infected with ZIKV results in intrauterine growth restriction, microcephaly, and/or fetal demise (Yockey et al., 2017). This highlights the importance of IFN-I in preventing viral replication and disease sequels.

Innate immunity against ZIKV in the skin The human skin is a physical and immunological barrier. During ZIKV transmission by the Aedes mosquito, the epidermis and dermis are punctured and the virus and arthropod saliva are inoculated. Although ZIKV usually leads to an asymptomatic infection, usually, symptomatic patients develop skin rash (Cordel et al., 2017) where high levels of ZIKV RNA is found (Cordel et al., 2018). Following the virus inoculation, the innate antiviral response is initiated in an attempt to inhibit the virus spread. There are three major cell populations that became infected early in the skin: keratinocytes, fibroblasts, and immature DCs (Hamel et al., 2015). Also, mast cells are activated and degranulate, which increases blood vessel permeability leading to plasma leaking and edema (Pingen et al., 2017). Keratinocytes represent the main population in the epidermis, and have a key innate role in the detection and defense against ZIKV because of the expression of PRRs and also their ability to produce antimicrobial peptides, chemokines, and cytokines (Briant et al., 2014). The ZIKV infection in human keratinocytes and fibroblasts results in the upregulation of RLRs, leading to the production of antiviral mediators (Kim et al., 2019). In primary human dermal fibroblasts, ZIKV upregulates the expression of RIG-1, MDA5, TLR3, IFN-α, IFN-β, IFN-g, and chemoattractants to leukocytes such as CXCL10, CXCL11 (both CXCR3 ligands), and CCL5 (a CCR5 ligand) (Hamel

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et al., 2015). Moreover, once infected and stimulated with IFN-I, fibroblasts increase the expression of ISGs such as OAS2, ISG15, MX1, IFIT1, IFIT3, and IFI16, as well as defense response proteins DDX58, STAT1, OAS3, EIF2AK2, and SAMHD1 (Wichit, Hamel, Yainoy, et al., 2019). All of those factors are involved in the restriction of ZIKV replication in the skin by the innate immunity. DCs have critical roles in sampling the microenvironment, detecting, and capturing pathogens, migrating through the lymphatic vessels to the draining lymph nodes to present antigens and induce effector T cells. Human skin contains two types of DC subsets: epidermal Langerhans cells (LCs) and dermal DCs, which are distinguished by the expression of Langerin and DC-SIGN, respectively. Several receptors are described to promote ZIKV binding or entry into the cell, such as DC-SIGN, also called CD209, Axl, Tyro3, and TIM-1 receptors, expressed in other target cells (Hamel et al., 2015; Meertens et al., 2017; Tabata et al., 2017). The interaction with different entry receptors is likely to provide an evolutionary advantage for the ZIKV, and, as a result, the virus is able to infect a wide range of target cells. Besides resident’s skin and mucosal DCs, peripheral blood circulating myeloid DCs are also susceptible to ZIKV infection (Bowen et al., 2017).

Innate immunity against ZIKV in the placenta From the site of ZIKV inoculation, it disseminates through the blood vessels and targets immune privileged sites such as brain, testicles, and placenta. To succeed, the virus needs to cross blood-tissue barriers where the specialties include restricting the access of pathogens, leukocytes, and some solutes (Khaiboullina et al., 2019). By crossing the maternal-fetal barrier, the virus reaches the fetus and cause a spectrum of abnormalities, the CZS (Walker et al., 2019). ZIKV vertical transmission can occur from basal decidua to chorionic villi and then to fetal circulation, and also from parietal decidual to amniochorionic membranes, amniotic fluid and finally reaching the fetus. Many cell types within the umbilical cord and placenta are susceptible to ZIKV infection, such as umbilical mesenchymal cells (El Costa et al., 2016), decidual macrophages (dMacs, CD14 + CD16 + CD206 +) (Quicke et al., 2016), monocytes (CD14 +), cytotrophoblasts, endothelial cells, fibroblasts and Hofbauer cells (CD163 +) in the chorionic villi, and amniotic epithelial cells and trophoblast progenitors in amniochorionic membranes (Tabata et al., 2017). The expression of the viral entry cofactors Axl, Tyro3, and/or TIM1 receptors is responsible for its permissiveness. Primary human trophoblasts are the barrier cells of the placenta. They constitutively produce type III IFN (IFN-λ1 or IL-29) that protects trophoblast and nontrophoblast cells from ZIKV infection (Bayer et al., 2016). Thus, for ZIKV to infect syncytiotrophoblasts (STB, the outer layer in contact with the maternal blood), it must evade the anti-ZIKV effect imparted by IFN-λ1 and other STB-specific antiviral factors and then gain access to the fetal compartment. On the other hand, ZIKV infection in full-term human placentas explant also induces IFN-λ4, IFN-γ, IL-1β, IL-10, IL-6, and TNF-α (Ribeiro et al., 2018). In addition, NFKB1, IL-1β, IL-1RA, EGF, TNF-α, TLR2, TLR4, CXCL8, CCL5, CXCL10, NLRP3, and NLRP6 are upregulated in the placental tissue after the delivery of baby, from a patient infected in the first gestational trimester (Lum et al., 2019). A massive infiltration of neutrophils into the placenta was also observed in this patient, while monocytes were found to infiltrate the placenta of a patient infected by ZIKV during the third trimester (Lum et al., 2019). The inflammatory milieu generated by ZIKV infection in the placenta of susceptible pregnant women results in apoptosis via caspase-3 after the TNFR signaling pathway is activated (Ribeiro et al., 2018). IL-1β secretion indicates inflammasome activation and, together with other inflammatory mediators, is involved in placental dysregulation and might be associated with fetal brain injury because it allows more virus to translocate through the inflamed placenta (Lei et al., 2019).

Innate immunity against ZIKV in the brain ZIKV can cause infections with mild symptoms, or progress to several neurological disorders, including Guillain-Barre syndrome, meningoencephalitis, myelitis, and fetal central nervous system injuries (White et al., 2016). The neurotropism of ZIKV can lead, among other consequences, to diffuse astrogliosis and microglia activation (Mlakar et al., 2016). The Axl plays an important role in the entrance of ZIKV in neural cells and mediates signaling through its tyrosine kinase domain to decrease IFN-I signaling, facilitating the infection, and mediating innate immunity suppression in glial cells. The Axl is present in radial glia cells, astrocytes, endothelial cells, and microglia, suggesting that these populations are vulnerable to infection (Nowakowski et al., 2016). Glial cells are the main cell types involved in ZIKV-induced neuroinflammation. ZIKV-infected astrocytes produce IL-6, IL-1α, IL-4, IP-10, CCL5, TGF-β1 and also increase the activation of the autophagy pathway. In human microglia, the downregulation of TLR3 decreases the viral titers and the secretion of inflammatory molecules, suggesting that TLR3 regulates ZIKV replication as well as ZIKV-induced secretion of inflammatory molecules in glial cells (Ojha et al., 2019). Also, in vitro experiments using the ZIKV-infected human cell line derived from malignant glioma (U87MG) showed

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increase of IL-1β, NLRP3, CASP1, SOD2, and HemeOX genes expression (Tricarico et al., 2017). Therefore, ZIKV is able to successfully replicate in neural cells causing oxidative stress and inflammasome activation. Different ZIKV strains induce distinctive antiviral responses. In vitro studies demonstrated that the Asian strain leads to the expression of TLRs, NLRs, and RLRs, which was associated with the expression of IFN-β, ISGs, chemokines, and gene products involved in the inflammasome activation in the first hours of astrocytes infection, whereas the African strain induced those factors after 24 h. Also, induction of MAPK, SPP1, TRAF3, and TBK1 genes is upregulated to a greater extent by the Asian strain (Hamel et al., 2017). Additionally, only the Asian lineage has been associated with fetal microcephaly and other neurological abnormalities (Cao-Lormeau et al., 2016).

Involvement of autophagy Autophagy is a physiological process that occurs in every living eukaryotic cell to remove long-lived cytoplasmic components, damaged organelles, and redundant proteins. Basically, this highly conserved catabolic pathway sequesters self-unwelcome cytoplasmic proteins through the formation of double-membraned autophagosomes and then fuses with lysosomes to form the single-membraned autophagolysosomes that degrade or recycle their contents (Klionsky & Emr, 2000). Autophagy is also considered an innate host defense response and, in the presence of any cellular stress signal, the autophagy signaling pathway is potentialized (Levine & Kroemer, 2008). The autophagy process can be confirmed by the formation of microtubule-associated protein 1 light chain 3 (LC3)-II spots that usually interact with its adaptor p62. On the other hand, the mammalian target of rapamycin (mTOR) pathway is responsible for preventing the autophagy process (Klionsky et al., 2008). As a participant of the innate response, the autophagy aims to destroy internalized microorganisms directly, to promote antigen presentation, and also it controls inflammation and inflammasome activation (Chiramel, Brady, & Bartenschlager, 2013). Nevertheless, ZIKV has evolved to take advantage of this process and the autophagy vesicle formation can assist the assembly and release of mature viral particles to the extracellular environment (Gratton et al., 2019). ZIKV induces two different pathways of autophagy, the traditional degradative autophagy (reticulophagy) and secretory autophagy (Chiramel & Best, 2018). The reticulophagy is activated by FAM134 A, B, and C and it restricts ZIKV replication. Nevertheless, the ZIKV NS2B3 protein induces cleavage of the FAM134 B receptor inhibiting the host antiviral mechanism (Lennemann & Coyne, 2017) (Fig. 2). After ZIKV internalization, the endocytic vesicles-containing virus approaches the nucleus. During this process, the lowered internal pH and high TLR3/TRIF/Beclin-1 activation cause conformational changes in viral envelope proteins, which fuse with the endocytic membrane and help the release of viral genetic material to the host cell cytoplasm for its translation (Yu et al., 2008). The replication machinery used by the virus is settled at the endoplasmic reticulum (ER) membrane, which undergoes an NS4A and NS4B-dependent rearrangement, originating the vesicle packets. Meanwhile, the virus immature particles are assembled, causing an ER stress (Ojha et al., 2018). The ER stress is the main mechanism by which autophagy is induced by ZIKV infection. Additionally, NS4A and NS4B proteins also inhibit Akt phosphorylation and the mTORC pathway inactivation, thus inducing the upregulation of autophagosome formation and the autophagy process itself (Liang et al., 2016). Ultimately, ZIKV uses the secretory autophagy machinery to facilitate the release of mature viral particles through the golgi network vesicles and then exocytosis (Fig. 2). The use of the autophagy pathway to facilitate virus replication has been observed in several cell types, e.g., HeLa, human fetal neural stem cells, human umbilical vein endothelial cells, astrocytes, and primary fibroblasts (Liang et al., 2016; Monel et al., 2017; Peng et al., 2018). Because autophagy inhibition reduces ZIKV production, targeting the autophagy pathway for therapy purpose is under investigation (Gratton et al., 2019).

Innate immunity evasion by ZIKV The outcome of ZIKV infection is established by competition between the host immune response and the viral replication. While the innate immune response uses a highly complex machinery to detect the virus and produce antiviral cytokines, ZIKV has evolved to antagonize some of these mechanisms. This is done mainly by its nonstructural proteins, which directly inhibit the reticulophagy (Fig. 2) and the innate immunity receptors, or it inhibits the downstream signaling pathway to evade from the IFN-I response (Fig. 3) (Serman & Gack, 2019). The ZIKV NS4a protein acts directly binding RLRs (Ma et al., 2018), and NS3 interacts with the scaffold proteins (14– 3-3ε and 14–3-3η) to prevent the translocation of RIG-I and MDA5 from the cytosol to mitochondria (Riedl et al., 2019), blocking their interaction with its adaptor protein, the mitochondrial antiviral signaling (MAVS), and then inhibiting the

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FIG. 2 Autophagy process in ZIKV infection. Following TLR3, exacerbated activation of TRIF adapter protein is recruited, leading to the dissociation of Beclin 1 and BCL2 protein complex. Beclin 1 initiates the autophagy pathway by promoting nucleation, enveloping, and elongation of the ER membrane. Synergistically, viral proteins NS2b, NS4a, and NS4b assist the ER curvature to promote the assembly of immature viral particles. Those proteins inactivate the mTORC pathway, preventing Akt phosphorylation. Immature viral particles go to the golgi apparatus where it maturates and enter in the secretory autophagy pathway to be released. On the other hand, reticulophagy is activated by FAM134 A, B, and C receptors in ER, leading ZIKV degradation. This process can be inhibited by NS2b3. Red and blue colors indicate the autophagy pathways involved in the release of mature viral particles and destruction (reticulophagy) of the ZIKV, respectively.

downstream signaling that would lead to IFN-I and ISGs induction. Downstream from the MAVS pathway, NS1, NS2a, NS2b, and NS4b have been found to suppress TBK1 activation, and NS1, NS2a, NS2b, NS4a, NS4b, and NS5 can inhibit IRF3 (Xia et al., 2018). cGAS/STING is an important cytosolic sensor against ZIKV. The NS2b protein cleaves the STING, inhibiting the downstream signaling (Ding et al., 2018). In addition, NS1 and NS5 are implicated in inflammasome activation that induces CASP1 and cleaves cGAS, reducing antiviral response (Zheng et al., 2018). Also, ZIKV increases the cell expression of NLRP1, NLRP3, and AIM2 (de Sousa et al., 2018), which induces IL1β, IL-18, and IL-33, contributing to the inflammatory process involved in ZIKV pathogenesis (Zheng et al., 2018). Furthermore, TLR3 activation by ZIKV infection reduces human organoid growth (de Sousa et al., 2018). ZIKV proteins (NS2b and NS5) also inhibit signaling downstream of the IFNα/β receptor (IFNAR), and NS2b3 degrades the kinase JAK1 in a proteasome-dependent manner (Wu et al., 2017), while NS5 inhibits STAT1 activation and induces the proteasomal degradation of STAT2 (Grant et al., 2017).

Policy and procedures mRNA expression of innate immune receptors determined by real time PCR The RNA is extracted and purified using a RNA Isolation System kit and stored at 80°C. The RNA concentration and quality should be analyzed by a Nanodrop. The complementary DNA (cDNA) is synthesized from 2 μg of the total RNA using a cDNA synthesis kit. PCR reactions are performed in triplicate using specific primers and the SYBR Green reagent in a Real Time PCR machine. A housekeeping gene as β-actin or GAPDH must be used for normalization. The expression levels of the transcripts are calculated by the mean of the cycle threshold (CT) from test sample relative to the control by the

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FIG. 3 Innate immunity evasion by ZIKV. ZIKV viral nonstructural proteins are involved in the antagonism of innate immune receptors (RIG1, MDA5, cGAS) and/or blocking their interaction with adaptor proteins. Those PAMPs also induce inflammasome (NLRP1, NLRP3, and AIM2) activation, IL-1β, IL-18, IL-33 production, inflammation, and pathology. NS2b, NS4a, NS4b inhibit akt/mTORC and, together with high TLR3 activation, induce autophagy through the secretory pathway. The IFNα/β signaling pathway is blocked by NS2b3, which degrades the kinase JAK1, while NS5 inhibits STAT1 and STAT2. Blue and red colors indicate receptors and molecules involved in the ZIKV protective immune response and immune evasion/pathogenesis, respectively.

formula: 2-ΔΔCT, where ΔCT is the CT from the target gene – CT from the housekeeping gene and ΔΔCT is the ΔCT from tested sample – mean of the ΔCT from the control group.

Measuring innate immune receptors protein expression by flow cytometry A single cell suspension obtained from tissues or culture (approximately 1  106 cells/mL) have the unspecific bidding sites blocked with 5% rabbit serum in PBS. Surface molecules are stained with a fluorochrome-labeled specific antibody diluted in the block buffer according to the manufacture instruction or better titration, followed by 30 min incubation in the dark at 4°C. Intracellular receptors and adaptive molecules are stained after cell fixation and permeabilization performed with commercial kits or with 2% paraformaldehyde and 0.5% saponin solutions. After 30 min incubation of the intracellular fluorochrome-conjugated antibody under the same conditions as for surface antibodies, the cells need to be washed twice and then suspended in 100 μL of PBS if acquired in the same day or 1% paraformaldehyde for acquisition in the next day and if the sample was not previously fixed. After collecting 50,000–200,000 events/sample in a Flow Cytometer, the data need to be analyzed using the FlowJo software (Treestar, USA) or equivalent. The gates strategies will unveil the percentage of PRRs positive cells or other targets.

Analyzing innate immune receptors localization and function PRRs localization can be performed in both tissues previously dehydrated through sequential exposure to solutions of 10%, 20%, and 30% sucrose, mounted in a cryomold with OCT (optimal cutting temperature) compound (Tissue-Tek, Sakura) and 7 μm sectioned; and also in isolated cells adhered to a slide. The sample is fixed with 2% paraformaldehyde and, if necessary, permeabilized with 0.5% Triton X-100. Nonspecific sites are blocked with 1% bovine serum albumin plus 10%

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rat serum. Specific fluorochrome-conjugated antibodies are incubated overnight at 4°C. The slides are washed and then mounted with Prolong containing DAPI, to be acquired using a confocal microscopy and analyzed with Leica QWin Quantitative Imaging software (Leica Microsystems, Germany) or similar. To evaluate the role of innate immunity receptors, several techniques of silencing or blocking gene expression can be used, such as knockout animals, blockers antibodies, CRISPCAS9 system, and interference RNA (iRNA).

Mini-dictionary of terms Chemokines: soluble mediators involved in cell migration. Cytokines: soluble mediators responsible for immune cells signaling. Nonstructural proteins: proteins produced in the infected cell, mainly enzymes, which participate in viral replication. Pattern recognition receptors: innate immune receptors that recognize conserved structures among microorganisms. Pathogen-associated molecular patterns: conserved antigens among microorganisms, such as viral RNA.

Key facts of innate immune response in ZIKV infection l l l l l l

Differential innate immune receptor activation by ZIKV infection can lead to protective immune response or pathology. Innate immune receptors activation leads to IFN-I and ISGs production culminating in antiviral state. IFN-III protects the placenta cells against the virus. ZIKV uses the Axl receptor to invade neural cells and decrease IFN-I signaling. Autophagy can conduce to viral destruction or release mature ZIKV particles. ZIKV evades the protective immune response mainly using nonstructural proteins.

Summary points l

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l

l

l

l

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Innate immune receptors (TLR3, TLR7, TLR8, MDA5, RIG1, LGP2, cGAS) are activated directly or indirectly by ZIKV and protective molecules are induced, such as IL-6, IL-12, TNFα, IFNα, IFNβ, chemokines, and ISGs. NLRP1, NLRP3, and AIM2 activation induces CASP-1, cGAS degradation, and cleavage of pro-IL-1β, pro-IL-18, and pro-IL-33 contributing to ZIKV inflammation and pathology. IFN-I is produced by ZIKV-infected cells and it acts through IFNRA by inducing antiviral proteins such as OAS, RNAse L, PKR, EIF2 α, and IFITM. Keratinocytes, fibroblasts, and immature DCs are the major cell population infected in the skin and several ISGs are induced. IFN-III is produced by trophoblasts and protects cells in the placenta from the ZIKV infection. On the other hand, an inflammatory microenvironment is involved in placental dysregulation and might be associated with fetal injury. ZIKV infection can progress to neurological disorders. The Axl receptor plays a dual role regulating innate immunity and the entry of ZIKV into neural cells. Glial cells can serve as a ZIKV reservoir in the CNS, contributing to neuroinflammation. ZIKV induces two different autophagy pathways: the degradative autophagy (reticulophagy) and secretory autophagy. Viral particle release by secretory autophagy is induced by high TLR3 activation and modulated by nonstructural ZIKV proteins (NS2a, NS4a, NS4b). Nonstructural ZIKV proteins (NS1, NS2a, NS2b, NS4a, NS4b, NS5) act directly or indirectly on PRRs or their signaling pathways by deactivating them and inhibiting the immune response.

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Chapter 3

The public health perspective of Zika virus infection Saurabh RamBihariLal Shrivastavaa and Prateek Saurabh Shrivastavab a

Medical Education Unit Coordinator and Member of the Institute Research Council, Department of Community Medicine, Shri Sathya Sai Medical

College and Research Institute, Sri Balaji Vidyapeeth (SBV)—Deemed to be University, Chengalpet, Tamil Nadu, India, b Department of Community Medicine, Shri Sathya Sai Medical College and Research Institute, Sri Balaji Vidyapeeth (SBV)—Deemed to be University, Chengalpet, Tamil Nadu, India

Abbreviations GBS IDSP IHR PHEIC WHO ZVD

Guillaine Barrie syndrome Integrated Disease Surveillance Programme International Health Regulations Public Health Emergency of International Concern World Health Organization Zika virus disease

Introduction Zika virus disease (ZVD) is a mosquito-borne illness and has emerged as one of the major public health concerns, especially due to its attribution with severe birth defects in newborns and adverse neurological complications among the affected adults in the 2015–16 outbreak in South America (Hasan, Saeed, Panigrahi, & Choudhary, 2019; Oliveira Melo et al., 2016). Zika virus is an arbovirus from the genus Flavivirus of the family Flaviviridae and for the first time, it was isolated from a rhesus monkey in the Zika forest of Kampala in Uganda (Dick, Kitchen, & Haddow, 1952). Subsequently, the virus was also isolated from the Aedes africanus mosquitoes in the same forest (Dick et al., 1952). However, the initial seropositive cases in humans were reported from Tanzania and Uganda in 1952, while the virus was first isolated from humans in Nigeria in 1953 (MacNamara, 1954). On the phylogenetic front, Zika virus has been linked with two lineages, namely African and Asian (identified in the 2015 outbreak in Latin American nations) (Pettersson et al., 2016; Wang et al., 2016). In the African region, the life cycle of virus generally occurs by propagating between the simian species and mosquito, which acts as a vector, while humans are the accidental hosts. On the contrary, in the Asian region, humans have been identified as the predominant host (Haddow et al., 2012; Rodriguez-Morales, Bandeira, & FrancoParedes, 2016). The laboratory diagnosis of infection is established by the detection of the virus and demonstration of viral RNA using reverse transcriptase-polymerase chain reaction (Bonaldo et al., 2016). However, in the absence of no definitive treatment or vaccine, the principal method for the prevention of the disease is through either preventing mosquito bites or via using barrier modes of contraception to prevent sexual transmission (Shrivastava, Shrivastava, & Ramasamy, 2016a; Sookaromdee & Wiwanitkit, 2019). It would not be wrong to document that till the entire epidemiology and mechanism of transmission of the virus is understood, it will be ideal to adopt and practice universal precautions (Marano, Pupella, Vaglio, Liumbruno, & Grazzini, 2016). Zika virus infection has been regarded as a global public health concern owing to its magnitude, the presence of the causative vector in all the continents, propensity to get transmitted to all the regions through travelers and attributed life-threatening complications in the affected newborn (Hasan et al., 2019; Oliveira Melo et al., 2016; Shrivastava, Shrivastava, & Ramasamy, 2016b). In order to contain the situation and effectively respond to the outbreaks of the disease, multiple strategies, including strengthening of the public health sector have been envisaged (Sharma & Lal, 2017; Shrivastava et al., 2016b). In fact, the World Health Organization has launched an app to ensure that the awareness about Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00003-1 Copyright © 2021 Elsevier Inc. All rights reserved.

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the infection, its mode of transmission, and the preventive approaches can be enhanced among the general population (Lea˜o, Gueiros, Lodi, Robinson, & Scully, 2017).

Timeline of the outbreaks Since the isolation of Zika virus from rhesus monkey in Zika forest, the infections have remained sporadic among humans for almost six decades. The sporadic cases were reported in nations such as Indonesia, Malaysia, Thailand, etc., as depicted in Table 1 (Darwish et al., 1983; Hammon et al., 1958; Smithburn, 1954). As a matter of fact, the first case of Zika virus infection outside the African region in humans was in Indonesia and the virus was isolated from the vector in Malaysia (Marchette et al., 1969; Olson et al., 1981). In 2007, the first major outbreak of the infection was reported in the Yap Islands in the Western Pacific, in which almost three-fourth of the general population was infected (Duffy et al., 2009). Subsequently, the virus spread to French Polynesia in 2013–14 and outbreaks were reported in different parts of the Pacific islands (Dupont-Rouzeyrol et al., 2015; Roth et al., 2014; Tognarelli et al., 2016). The virus reached the American region between 2013 and 2015, and an explosive outbreak

TABLE 1 Timeline of Zika virus outbreaks. Year

Event with region and number of cases

Reference

1947

Discovery of Zika virus in Rhesus Monkey in Zika forest, Uganda

Dick et al. (1952)

1948

Isolation of Zika virus from Aedes mosquito, Uganda

1953

First isolation of Zika virus from human in Nigeria, Tanzania, and Uganda

MacNamara (1954)

1950s– 1980s

Occasional cases reported in nations from Asian region (viz., Indonesia, India, Malaysia, Thailand, etc.)

Darwish, Hoogstraal, Roberts, Ahmed, and Omar (1983), Hammon, Schrack, and Sather (1958), Marchette, Garcia, and Rudnick (1969), Olson, Ksiazek, Suhandiman, and Triwibowo (1981), and Smithburn (1954)

2007



Duffy et al. (2009)



First major outbreak in Yap Island in the Federated States of Micronesia 49 Confirmed cases and more than 7350 suspected cases

2013–14

    

Outbreak in French Polynesia Evidence of the Guillaine-Barrie syndrome (GBS) Evidence of sexual transmission French Polynesia: Average 29,000 cases New Caledonia: 1400 cases

Dupont-Rouzeyrol et al. (2015), Musso and Gubler, (2016), Roth et al. (2014), and (Tognarelli et al., (2016))

2015–16



Outbreak in Brazil, Columbia, El Salvador, Mexico, Paraguay, etc. Brazil: Average 870,000 cases Columbia: More than 51,000 cases

Hennessey, Fischer, and Staples (2016) and Zanluca et al. (2015)

  2016

Outbreak in Continental USA Florida (4 cases) and Texas

McCarthy (2016)

2017

  

First outbreak in India Tamil Nadu: 3 cases Gujarat: 1 case

Bhardwaj, Gokhale, and Mourya (2017) and WHO (2017)

2018

 

Biggest outbreak of India Rajasthan and Madhya Pradesh: 289 cases

IDSP (2018)

Various Zika virus infections are depicted in the chronological order in the various parts of the world. In addition, number of infected cases in the outbreak has also been mentioned.

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was reported in Brazil in 2015 (Zanluca et al., 2015). In the year 2016, four cases were reported in Florida (McCarthy, 2016). By the end of the 2015 year, 18 states in the nation itself reported autochthonous transmission. The first infection cases were reported from Rio Grande do Norte in the early months of 2015 (Zanluca et al., 2015). On an average, a total of 8,70,000 suspected infected cases were reported in this outbreak alone (Hennessey et al., 2016; Musso & Gubler, 2016). Further, a significant rise in the incidence of microcephaly and fetal abnormalities was also reported from the virus affected parts of the nation. The magnitude of the outbreak was so huge that it was declared as a Public Health Emergency of International Concern (PHEIC) by the Emergency Committee (Shrivastava et al., 2016b). This was followed by the appearance of the virus in India in the year 2017, even though, there was no history of travel among the infected persons. However, considering the low levels of transmission, the number of infected cases was restricted to only 4 (Bhardwaj et al., 2017; WHO, 2017). This was followed by a major outbreak of the disease in Rajasthan and Madhya Pradesh in 2018, wherein more than 150 cases were reported, which also included more than 60 pregnant women (IDSP, 2018).

Epidemiology: Distribution and determinants Geographical distribution According to the recent estimates available, in 87 nations and territories, evidence of autochthonous mosquito-borne transmission of Zika virus has been reported. These nations are distributed in the African, American, South-East Asia, and Western Pacific region across the globe (WHO, 2019a). Ethiopia was the most recent nation added to the list in the year 2018, wherein such evidence of transmission has been reported (Mengesha Tsegaye et al., 2018). Even though the incidence of infection in the American region showed a significant rise in the year 2016, a significant decline was subsequently reported in the next 2 years (WHO Region of the Americas/Pan American Health Organization, 2019). In fact, apart from three nations, in the Americas, the transmission of the virus was observed in all the nations, which was quite alarming (WHO Region of the Americas/Pan American Health Organization, 2019). At present, the information pertaining to the epidemiological distribution of the disease in the remaining three affected WHO regions is limited, and the findings of different epidemiological studies have provided valuable evidence. In a study done among children in Indonesia through serosurvey, it was observed that close to 10% of the children had prior exposure to the viral infection before they attained the age of 5 years (Sasmono et al., 2018). On a similar note, in another study done among asymptomatic blood donors in Lao People’s Republic, it was found that 10% had been exposed to the virus in the past (Pastorino et al., 2019). Furthermore, the outbreak of the disease was reported in Rajasthan (WHO, 2018a; Yadav et al., 2019). The findings of different studies have reported that the strain of the virus isolated in the American region has been detected in Angola in the cluster of children diagnosed with microcephaly in the period of 2017 and 2018 (Hill et al., 2019; Sassetti et al., 2018). Further, cases of congenital malformations, microcephaly, and even fetal deaths have been attributed to the viral infection in different nations in the Asian region (Moi et al., 2017; Wongsurawat et al., 2018). It is worth noting that across the six regions of the globe, despite the presence of the vectors in more than 60 nations, no cases of Zika virus infections have been reported (WHO, 2019a). Even though it is an encouraging thing, there is a potential risk of the spread of the disease to a greater number of nations. At the same time, the possibility of failure to detect the ongoing viral transmission or chance for reemergence or reintroduction of the virus in regions cannot be ruled out (WHO, 2019a). The ground reality is that the precise estimates and epidemiological distribution of the disease is not comprehensive and uniform across the globe. This is predominantly because of the fact that the majority of infections are asymptomatic and often the initial symptoms of the disease are mild and nonspecific, and thus there is a definite chance of not being detected or reported to the health care authorities. Another important dimension has been the weaknesses prevailing in the surveillance system of the various nations, especially in the low-and-middle-income nations. This has accounted for the absence of comprehensive information about the disease and thus lack of research has been encouraged as it cannot be identified as a public health priority (Badolo et al., 2019; Lanciotti et al., 2008).

Epidemiological determinants Agent: Zika virus is a mosquito-borne flavivirus from the genus Flavivirus of the family Flaviviridae (Hasan et al., 2019; Oliveira Melo et al., 2016). It has been isolated first time in the Zika forest of Kampala in Uganda from rhesus monkeys (Dick et al., 1952; Kindhauser, Allen, Frank, Santhana, & Dye, 2016).

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Vectors: The virus has been isolated in the African region among Aedes, Anopheles, and Mansonia genera of mosquitoes, while in the Southeast Asian and the Pacific region, it was isolated only from Aedes. However, it is important to note that Aedes albopictus has been reported from multiple nations in Central Africa and Mediterranean regions and continues to spread even further (Vorou, 2016). Host: The virus has been isolated in monkeys, nevertheless, antibodies to the virus have been detected in domestic sheep, goats, horses, cows, ducks, rodents, bats, orangutans, and carabaos (Vorou, 2016). Humans are also the host acquiring infection from the bite of an infected mosquito (WHO, 2018b). Environment: The incidence and the spread dynamics of the infection is primarily dependent on the parameters accounting for the mosquito vector occurrence and their density, and human population attributes such as international travel and social status, as reported in Table 2 (Zhang et al., 2017). As far as the density of the vector is concerned, it is dependent on climatic characteristics such as rainy seasons, accounts for a higher abundance of them (Hono´rio, Castro, Barros, Magalhaes, & Sabroza, 2009). With regard to the temperatures, a range of temperatures between 16°C and 35°C is ideal for the reproduction and survival of the vector (Carrington, Armijos, Lambrechts, Barker, & Scott, 2013). It is important to note that temperatures in the higher range curtails the time span of the extrinsic incubation period and thus facilitates increased rates of infection transmission (Tjaden, Thomas, Fischer, & Beierkuhnlein, 2013). It is quite obvious that the probability of detection of the virus is more in regions with a greater number of health-care establishments. Poverty, in terms of poor housing standards and the absence of window screens has been linked with higher rates of transmission of Aedes-borne pathogens in human beings (Brunkard et al., 2007; Hotez, Murray, & Buekens, 2014). At the same time, poverty is often linked with poor health-seeking behaviors and thus the rates of detection of the disease are being extremely low. In addition, the factor of global trade and population mobility also affects the rate of transmission in the regions which have not reported about the transmission of the infection (Stoddard et al., 2009). Mode of transmission: The most common mode of transmission of the infection is through the bite of an infected mosquito from the Aedes genus, especially Aedes aegypti (Kindhauser et al., 2016). From the prevention and control perspective, it is important to note that the Aedes mosquitoes generally bite in the daytime, with peaks observed in the early morning and late afternoon or evening. In addition, the virus can also be transmitted from the mother to fetus during pregnancy (congenital Zika infection) or through sexual transmission, or during the process of transfusion of blood and its products and organ transplantation (Brooks et al., 2016). Complications: The infection with the virus during antenatal period has been linked with the development of congenital anomalies and microcephaly in the developing fetus and newborn (Moi et al., 2017; Oliveira Melo et al., 2016; Wongsurawat et al., 2018). In addition, the complications such as fetal loss, stillbirth, and preterm birth have also been linked to the infection during the antenatal period (Moi et al., 2017; Wongsurawat et al., 2018). Further, the infection has also been associated with the development of Guillain-Barre syndrome, neuropathy, and myelitis, especially among the affected adults and older children (WHO, 2018b).

TABLE 2 Epidemiological determinants of Zika virus infection. Epidemiological determinants

Parameters

Agent

Mosquito-borne flavivirus

Host

  

Environment

   

Virus isolation from monkeys Antibodies to the virus detected in domestic sheep, goats, horses, cows, ducks, rodents, bats, orangutans, and carabaos Humans Factors influencing mosquito vector occurrence and density (a) Rainy season (b) Temperature: 16–35°C International travel Poverty Poor housing standards

The epidemiological determinants of the Zika virus infections have been enumerated.

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Public health consequences Public Health Emergency of International Concern: The 2015 outbreak of Zika virus infection was linked with a simultaneous and significant rise in the number of congenital anomalies, fetal complications, and neurological complications. Amidst the threat of the spread of the infection to various Latin American nations, the Emergency Committee declared the outbreak as a Public Health Emergency of International Concern (PHEIC) in 2016 (Shrivastava, Shrivastava, & Ramasamy, 2017b; WHO, 2016a). This called for an immediate need to collaborate with different national and international stakeholders to understand the magnitude of the problem, minimize the threat of the disease in the affected regions, and strengthen implementation of measures to interrupt the risk of international spread of the disease (Shrivastava et al., 2017b; Vorou, 2016; WHO, 2016a). The recommendation to declare the disease as a PHEIC was the need of the hour, as various stakeholders were working in isolation and there was a need to have a collaborative response to contain the situation in the affected nations (Musso & Gubler, 2016; WHO, 2016a). The need to strengthen the primary health care was reemphasized and it was decided to respond to all the shortcomings in the existing health-care delivery system (Shrivastava et al., 2016a; WHO, 2016a). In addition, it has been proposed to intensify the vector control, surveillance, clinical care, risk communication, travel, and research activities (WHO, 2016a). Health-care delivery system: The 2015–16 outbreak of the Zika virus infection in the American region has exposed the weaknesses prevailing in the health-care delivery system and the lack of preparedness by the public health authorities and other concerned stakeholders (Shrivastava et al., 2017b). It is an alarming fact that the disease which has been detected first in the 1950s still does not have any cure or vaccine, which can control the disease (Shrivastava, Shrivastava, & Ramasamy, 2016c; WHO, 2018b). This indirectly reflects the dearth of research in the field, whether it is with regard to the understanding of the various modes of transmission or with regard to measures which can effectively control the transmission of the disease (WHO, 2018b). Acknowledging the fact that the incidence of population mobility and international travel has increased immensely in the last few decades amidst the impending globalization, it would not be wrong to admit that none of the region or nation can be considered as full proof (Shrivastava et al., 2016c; WHO, 2019b). There have been reports of reoccurrence and reemergence of the infections in the areas where the infections have been contained earlier and it again signifies the need that the prevention and control measures have to be consistent and sustainable (WHO, 2019a). Another important dimension has been the lack of preparedness by the stakeholders such as the health sector, environmental, engineering, international health regulations at seaport or airports, etc., as depicted in Table 3 (Shrivastava et al., 2017b). This has led to not only the emergence of local outbreaks but also its spread to various nations (Mengesha Tsegaye et al., 2018; Moi et al., 2017; Sasmono et al., 2018; Wongsurawat et al., 2018). From the diagnostic perspective, most of the health-care facilities lack the setup for offering relevant diagnostic services and it is being offered in only selected centers in most of the developing nations as highlighted in Table 3 (WHO, 2018b). It is important to consider the situation of health-care facilities and the uptake of available health services in

TABLE 3 Identified weakness in the public health response. Factor

Weaknesses/challenges

Lack of preparedness

   

Absence of environmental modification/engineering Lacunae in the risk communication mechanism Poor surveillance system Lack of awareness about the disease among the general population

Health system

    

Lack of research (a) No curative option (b) No vaccine Absence of diagnostic facilities in most of the health care facilities Nonadherence to standard infection control practices Poor uptake of the available services Insufficient logistics/resources

 

Poor implementation Availability of the health care facilities in all the airports and seaports

International Health Regulations

Important weaknesses and challenges have been highlighted which cumulatively are affecting the public health response to the Zika virus infection.

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low-and-middle-income nations. These nations are struggling with their numerous other existing public health challenges, and thus the policymakers have failed to ensure that the services reach those who need them the most. In fact, the prevailing lacunae in the surveillance system is one of the main reasons due to which Zika infection has not become a public health priority in most of the nations (Shrivastava et al., 2016a). Thus, there is an immense need to improve the existing mechanism of disease reporting, but we have to accept that close to four-fifths of the infected cases are asymptomatic (WHO, 2018b). Social consequences: The rise in the incidence of microcephaly and other congenital malformations has affected the general population in the affected regions to a great extent (Moi et al., 2017; Oliveira Melo et al., 2016; Wongsurawat et al., 2018). There has been a sense of fear, feeling of distress till congenital anomaly being ruled out, and disappointment among the women who have delivered congenitally malformed children and the fear has spread among all the antenatal mothers. Even though some of the youth groups and other welfare organizations have come forward to contain the situation, but an extensive amount of damage has already been done (WHO, 2018b). Further, as environmental and social factors play a big role in the causation and the transmission of the infection, the health authorities have to come forward and work with a solitary aim of ameliorating the socioeconomic status of the general population (Brunkard et al., 2007; Carrington et al., 2013; Hotez et al., 2014; WHO, 2018b).

Prevention and control of Zika virus infections Better and sustained preparedness For a disease which was known for more than 6 decades, in the year 2016, all the stakeholders, including the general population were shell-shocked after seeing the disheartening images of newborns with microcephaly (Moi et al., 2017; Oliveira Melo et al., 2016; WHO, 2019a). At the same time, there was a significant amount of fear among the people of the affected nations that a mosquito bite can result in life-threatening complications (Chan, 2017). In order to avert any such outbreaks in the future, in any part of the world, it is quite essential to be well prepared, which essentially includes strengthening of the health system, implementation of measures for better vector control, and increase in the awareness activities to empower the general population about the modes of transmission of the disease and ways to prevent the same as mentioned in Table 4 (Musso & Gubler, 2016; Shrivastava et al., 2017b; WHO, 2016a). In addition, it is extremely essential to improve the sanitation facilities and the living conditions of the people from low socioeconomic status (Chan, 2017). However, the preparedness activities also include more investment in the field of research to bridge the existing gaps in the field of prevention and treatment of the disease (WHO, 2016a).

TABLE 4 Areas to focus for better preparedness and public health response. Areas

Parameters

Health Care Delivery System

       

Availability of diagnostic facilities Provision of safe antenatal care Extending support to child born with microcephaly Counseling Strengthening of the surveillance system Ensuring availability of desired resources Intensifying advocacy, communication and social mobilization activities Training of health workers to identify the potential cases and clear the myths associated with the infection

Vector control

 

Integrated vector control measures Implementation of the environmental modification/engineering measures

Others

  

Stringent implementation of International Health Regulations Establishment of the risk communication mechanism Intensification of research activities—discovery of an effective drug and potential vaccine

It describes the important domains which needs to be focused so that we are better prepared to launch a public health response to the outbreak of the infection.

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Implementation of strategic response framework The World Health Organization (WHO) formulated a Strategic Response Framework with an aim to ensure better implementation of the prevention and control activities of the disease in both affected and unaffected regions (WHO, 2016b). This framework has been designed with an aim to accomplish three objectives, namely strengthening of the surveillance mechanism, institution of a better response through risk communication, community involvement, sensitization of the health team workers, and expedite research work by exploring the association between the resulting complications and simultaneous rise in the incidence of Zika virus infection, and speedup the development of rapid diagnostics, vaccines, and therapeutics (WHO, 2016b). The proposed framework emphasizes the improvement in the surveillance (in terms of case detection and identification of all complications) and response activities and also has provision to encourage research by promoting more coordination between national and international funding agencies for the development of vaccines and rapid diagnostic tools (Hajra, Bandyopadhyay, & Hajra, 2016; WHO, 2016b). Furthermore, measures have been proposed to enhance the national capacity to discharge better risk communication, improvement in the vector control measures and expansion of the laboratory network to ensure early detection of the infection (WHO, 2016b). In addition, specific guidelines have been formulated to promote clinical care and follow-up in both the high-risk groups as well as the general population (WHO, 2016b).

Strengthening of International Health Regulations: Advice to travelers Based on the nature of ongoing transmission (viz., active transmission or sporadic cases or no recent evidence of the transmission), the potential risk to acquire or transmit the infection will vary in any nation or region (WHO, 2019b). At the same time, considering the fact that a major proportion of infections are asymptomatic, it is wise to not conclude that no transmission is occurring in a region just because no case has been reported (WHO, 2016a, 2016b). Amidst the current dynamics of the disease, the WHO has released that there are no general restrictions on travel or trade with those nations which have reported transmission of the disease (Hajra et al., 2016; WHO, 2019b). However, the standard recommendation pertaining to the vector control and surveillance activities should be implemented in accordance with the International Health Regulations (IHR) at airports, including disinfection of aircraft (WHO, 2019b). Further, the travelers have to be sensitized about different aspects (viz., measures to prevent mosquito bites like using insect repellent/mosquito nets/full clothing, practice safe sex or abstinence, blood donation practices) pertaining to the infection before, during, and after travel to areas with positive evidence of transmission of the infection (Hajra et al., 2016; WHO, 2019b). For pregnant women or women who are planning to conceive within a couple of months of travel, the women should be explained about the risk and if possible, should delay the travel (Shrivastava et al., 2016c; WHO, 2019b). Even after returning home, the travelers should continue the practice to use insect repellent for at least 3 weeks to avoid being bitten and thus the risk of potential transmission to other people. However, returning travelers who are symptomatic with the initial symptoms of the disease such as rash, fever, painful joints, etc., should consult the health professionals and get diagnosed and avail clinical care, if need be (WHO, 2019b).

Strengthening of the sexual and reproductive health services Even though mosquito-borne transmission is the most common route for the transmission of the infection, sexual transmission is one which attracted loads of attention, especially in cases of pregnant women due to the fear of life-threatening complications (Brooks et al., 2016). This essentially calls for the strengthening of the routine sexual and reproductive health care which is being offered from the health-care facilities. Apart from the health education to the general population about the risk of transmission of infection through sexual route, it is very essential to motivate people to practice safer sex, which refers to correct and the consistent use of condoms or other modes of barrier contraceptives (Brooks et al., 2016; Shrivastava, Shrivastava, & Ramasamy, 2017a). Even abstinence has been advocated for the couples or pregnant women who have a history of travel to a region with positive evidence of virus transmission (WHO, 2018b). Further, owing to the known predisposition that poverty and poor socioeconomic status increases the risk of acquisition of the infection, it becomes very essential to ensure that the available services reach vulnerable sections of the community (Brooks et al., 2016). This factor gets even more emphasis as these women tend to have a high unmet need for family planning and therefore the chance of unplanned pregnancies becomes quite high. However, for this to translate into

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practice, the health workers have to go beyond the boundaries of health centers into the communities and ensure that all the desired sexual and reproductive services are readily available and affordable (Brooks et al., 2016; Shrivastava et al., 2017a; WHO, 2018b).

Vector control, surveillance, risk communication, and promotion of research In order to control the magnitude of the infection as well as prevent the future occurrence of the outbreaks, there is a significant need to strengthen the essential components of vector control, surveillance mechanism, therapeutic care, and revamp the mechanism of risk communication (Shrivastava, Shrivastava, & Ramasamy, 2016a; Singh et al., 2018; WHO, 2016b). Keeping the breeding and biting practices in mind about the causative vector, appropriate antilarval and antiadult measures should be implemented (Kindhauser et al., 2016; Roth et al., 2014; Singh et al., 2018). Improving the surveillance practices, including the notification of all the cases and vector surveillance has been envisaged by all the stakeholders, as then only evidence-based decisions can be taken by the policymakers (Chan, 2017; WHO, 2016b). There is an immense need to improve the various facets of risk communication, which includes identifying the needs of the society, chances of sexual transmission, strategies to enhance community engagement, improvement in the notification rates, and implementation of the preventive measures in the affected regions (Shrivastava et al., 2016a). Research activities need to be fast-tracked with an aim to enhance the understanding between the viral infection and clusters of complications, genetic sequencing, development of new diagnostics, and development of an effective vaccine as mentioned in Table 5 (Abbink, Stephenson, & Barouch, 2018; Bhardwaj et al., 2017). The World Health Organization has also come forward to support research activities, capacity building of laboratories, vector control monitoring, and for extending care to the children and family members affected by the complications of the infection (WHO, 2016b, 2018b).

Bridging the gap between social and health inequalities The incidence of the infection has been extremely high in areas with favorable breeding conditions for mosquitoes such as poor sanitation facilities, which is quite common in areas with poor socioeconomic status (Ali et al., 2017). It won’t be wrong to document that women from poor socioeconomic status have lower levels of literacy and are often not aware of the various modes of transmission of the disease, especially in term of lower awareness about sexuality, knowledge about family planning, access to contraceptives, utilization of antenatal care, etc. (Ali et al., 2017; Chan, 2017; Stoddard et al., 2009). The problem is further compounded with the minimum number of health-care facilities in these regions (Chan, 2017). The need is to bridge this gap of social and health inequality and this essentially requires a multisectoral and a multipronged approach (Ali et al., 2017). The entire team of health professionals has to understand the local prevailing problems and based on the identified needs it has to be sorted out. However, the main focus has to be toward capacity building and strengthening of the existing health facilities. The environment engineering sector can look for the elimination of the breeding places of the causative vector (Ali et al., 2017). Moreover, it is important to acknowledge that the gains won’t be sustainable unless we are involving the local communities in all the strategies (WHO, 2016b).

TABLE 5 Potential candidate vaccines for Zika virus infection. Potential candidate vaccine 

Zika virus purified inactivated virus vaccine



DNA-based vaccine



Adenovirus-based vaccine



mRNA-based vaccine



Modified vaccinia virus Ankara



Measles virus-based vaccine



Zika virus live-attenuated vaccine

The above list shows the potential candidate vaccines which are in the different stages of the clinical trials.

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Conclusion In conclusion, Zika virus infection is a global public health concern and it requires extensive effort from all the concerned sectors for the prevention and control of the infection. The need of the hour is to strengthen the health-care delivery system and support the same with the standard infection control, International Health Regulations, and research activities.

Policy and procedures Measuring head circumference: Microcephaly is defined as the head circumference less than two standard deviations from the general population. The standard charts for head circumference in children are available on the link: www. who.int/childgrowth/standards/hc_for_age/en. Head circumference has also been looked upon as a surrogate measure of brain development and correlates with cognitive measures in later life. Materials: A flexible but nonstretch tape such as the reusable Lasso-o tape. Methodology: The Lasso-o tape is placed over the head of the child, then above the ears and midway between the eyebrows and the headline to the occipital prominence at the back of the head (Great Ormond Street, 2019).

Mini-dictionary of terms Autochthonous virus transmission. It is the transmission of virus originating from indigenous sources and not from migrants. Extrinsic incubation period. It is the time span required for a microorganism to complete its development in the intermediate host. Microcephaly. It is a condition in which a newborn is born with a small head or the head stops growing after birth. Public Health Emergency of International Concern. It refers to “an extraordinary event which is determined to constitute a public health risk to other States through the international spread of disease and to potentially require a coordinated international response,” formulated when a situation arises that is “serious, sudden, unusual or unexpected,” which “carries implications for public health beyond the affected State’s national border,” and “may require immediate international action.”

Key facts of the Zika virus infections l l l l l

Zika virus is a flavivirus and was isolated for the first time in Uganda. Almost 80% of the Zika virus infections are asymptomatic. No vaccine is available for the prevention of the infection. Zika virus infections can be transmitted through the bite of infected mosquitoes or via sexual transmission. International Health Regulations need to be strengthened to contain the international spread of the infection.

Summary points l l

l

l l

This chapter focuses on the public health perspective of the Zika virus infection. Even though the virus has been isolated for more than 6 decades, there is no therapeutic tool or vaccine available for the containment of the same. Zika virus infections have been simultaneously associated with the rise in the incidence of microcephaly and GuillainBarre syndrome. The infection can be sexually transmitted, and thus safer sexual practices are advocated. Strengthening of the health-care delivery system and vector control measures are crucial components for the control of the infection.

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The spatial distribution of Aedes aegypti and Aedes albopictus in a transition zone, Rio de Janeiro, Brazil. Cadernos de Sau´de Pu´blica, 25, 1203–1214. Hotez, P. J., Murray, K. O., & Buekens, P. (2014). The Gulf Coast: A new American underbelly of tropical diseases and poverty. PLoS Neglected Tropical Diseases, 8, 4–6. Integrated Disease Surveillance Programme. (2018). 44th week—Weekly outbreak report. Retrieved from https://idsp.nic.in/WriteReadData/l892s/ 442018.pdf. Kindhauser, M. K., Allen, T., Frank, V., Santhana, R. S., & Dye, C. (2016). Zika: The origin and spread of a mosquito-borne virus. Bulletin of the World Health Organization, 94, 675–686C. Lanciotti, R. S., Kosoy, O. L., Laven, J. J., Velez, J. O., Lambert, A. J., Johnson, A. J., … Duffy, M. R. (2008). Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerging Infectious Diseases, 14, 1232–1239. Lea˜o, J. C., Gueiros, L. A., Lodi, G., Robinson, N. A., & Scully, C. (2017). Zika virus: Oral healthcare implications. Oral Diseases, 23, 12–17. MacNamara, F. N. (1954). Zika virus: A report on three cases of human infection during an epidemic of jaundice in Nigeria. Transactions of the Royal Society of Tropical Medicine & Hygiene, 48, 139–145. Marano, G., Pupella, S., Vaglio, S., Liumbruno, G. M., & Grazzini, G. (2016). Zika virus and the never-ending story of emerging pathogens and transfusion medicine. Blood Transfusion [Trasfusione del sangue], 14, 95–100. Marchette, N. J., Garcia, R., & Rudnick, A. (1969). Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia. American Journal of Tropical Medicine and Hygiene, 18, 411–415. McCarthy, M. (2016). Four in Florida are infected with Zika from local mosquitoes. British Medical Journal, 354, i4235. Mengesha Tsegaye, M., Beyene, B., Ayele, W., Abebe, A., Tareke, I., Sall, A., … Tayachew, A. (2018). Sero-prevalence of yellow fever and related Flavi viruses in Ethiopia: A public health perspective. BMC Public Health, 18, 1011. Moi, M. L., Nguyen, T. T. T., Nguyen, C. T., Vu, T. B. H., Tun, M. M. N., Pham, T. D., … Hasebe, F. (2017). Zika virus infection and microcephaly in Vietnam. Lancet Infectious Diseases, 17, 805–806. Musso, D., & Gubler, D. J. (2016). Zika virus. Clinical Microbiology Reviews, 29, 487–524.

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Oliveira Melo, A., Malinger, G., Ximenes, R., Szejnfeld, P., Alves Sampaio, S., & Bispo de Filippis, A. (2016). Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: Tip of the iceberg? Ultrasound in Obstetrics & Gynecology, 47, 6–7. Olson, J. G., Ksiazek, T. G., Suhandiman, & Triwibowo. (1981). Zika virus, a cause of fever in Central Java, Indonesia. Transactions of the Royal Society of Tropical Medicine & Hygiene, 75, 389–393. Pastorino, B., Sengvilaipaseuth, O., Chanthongthip, A., Vongsouvath, M., Souksakhone, C., Mayxay, M., … Dubot-Pere`s, A. (2019). Low zika virus seroprevalence in Vientiane, Laos, 2003-2015. American Journal of Tropical Medicine and Hygiene, 100, 639–642. ˚ ., Falconar, A. K., Gaunt, M. W., … de Lamballerie, X. (2016). How did Zika virus emerge in Pettersson, J. H., Eldholm, V., Seligman, S. J., Lundkvist, A the Pacific Islands and Latin America? mBio, 7, e01239-16. Rodriguez-Morales, A. J., Bandeira, A. C., & Franco-Paredes, C. (2016). The expanding spectrum of modes of transmission of Zika virus: A global concern. Annals of Clinical Microbiology and Antimicrobials, 15, 13. Roth, A., Mercier, A., Lepers, C., Hoy, D., Duituturaga, S., Benyon, E., … Souares, Y. (2014). Concurrent outbreaks of dengue, chikungunya and Zika virus infections—An unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014. Euro Surveillance, 19, 20929. Sasmono, R. T., Dhenni, R., Yohan, B., Pronyk, P., Hadinegoro, S. R., Soepardi, E. J., … Soebandrio, A. (2018). Zika virus seropositivity in 1-4-year-old children, Indonesia, 2014. Emerging Infectious Diseases, 24, 1740–1743. Sassetti, M., Ze-Ze, L., Franco, J., Cunha, J. D., Gomes, A., Tome, A., & Alves, M. J. (2018). First case of confirmed congenital Zika syndrome in continental Africa. Transactions of the Royal Society of Tropical Medicine & Hygiene, 112, 458–462. Sharma, A., & Lal, S. K. (2017). Zika virus: Transmission, detection, control, and prevention. 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World Health Organization. (2019b). Information for travellers visiting countries with Zika virus transmission. Retrieved from https://www.who.int/csr/ disease/zika/information-for-travelers/en/. Yadav, P. D., Malhotra, B., Sapkal, G., Nyayanit, D. A., Deshpande, G., Gupta, N., … Mourya, D. T. (2019). Zika virus outbreak in Rajasthan, India in 2018 was caused by a virus endemic to Asia. Infection, Genetics and Evolution, 69, 199–202. Zanluca, C., Melo, V. C., Mosimann, A. L., Santos, G. I., Santos, C. N., & Luz, K. (2015). First report of autochthonous transmission of Zika virus in Brazil. Memo´rias do Instituto Oswaldo Cruz, 110, 569–572. Zhang, Q., Sun, K., Chinazzi, M., Piontti, A. P. Y., Dean, N. E., Rojas, D. P., … Vespignani, A. (2017). Spread of Zika virus in the Americas. Proceedings of the National Academy of Sciences of the United States of America, 114, E4334–E4343.

Chapter 4

Inequality in Zika virus and congenital Zika syndrome Finn Diderichsena and Lia Giraldo da Silva Augustob a

Department of Public Health, University of Copenhagen, Copenhagen, Denmark, b Oswaldo Cruz Fundation, IAM, Recife, PE, Brazil

Abbreviations CHIKV CZS DENV GINI-index HDI NCD UNDP ZIKV

chikungunya virus congenital Zika Syndrome dengue virus measure of the relative inequality in income between individuals human development index noncommunicable disorders United Nations Development Programme Zika virus

Inequalities in health Today socioeconomic inequalities are observed in almost all aspects of somatic and mental health, whereby people living in more socioeconomically disadvantaged conditions experience worse health than their more advantaged peers. Health inequalities are observed across rich and poor countries and across socioeconomic groups within both rich and poor countries (Arcaya, Arcaya, & Subramanian, 2015). Most socioeconomic health inequalities are both unfair and avoidable. It is in particular true for inequalities found among children who have little control over their health and conditions that influence it (Pearce, Dundas, Whitehead, & Taylor-Robinson, 2019). The same can be said about those inequalities generated by unequal exposure to many environmental conditions. For diseases where there is no effective treatment available, social inequalities become more alarming. For diseases strongly clustering in disadvantaged groups, it is impossible to reduce the overall morbidity without addressing the socioeconomic inequalities in their occurrence. The causes that need to be addressed to reduce inequalities of a disease might however be different from those needed to reduce its average occurrence. Causes that generate an unequal risk of a disease might not be the same as causes that create unequal consequences of the disease. It all depends on the inequalities in the distribution of causes and inequalities in the effect of the different causes (Diderichsen, Hallqvist, & Whitehead, 2019). Inequalities exist for a very broad range of different disorders, not least for infectious diseases. Inequality in infectious disease has however not been in focus in the last decades of intensive scholarship on health inequalities that primarily addresses disparities in NCDs (Quinn & Kumar, 2014). When specifically dealing with an emerging infectious disease we might also tend to focus on emerging causes rather than on the social determinants that have been there all the time (Farmer, 1996). Many infectious diseases show, however, steep socioeconomic gradients (Semenza & Giesecke, 2008) including those where the pathogens are arbovirus like dengue virus (DENV), chikungunya (CHIKV), and Zika virus (ZIKV). UNDP has formulated the issue with these words “….there is a profound equity challenge at the core of the Zika epidemic. The impact is disproportionate on the poorest countries, as well as on the poorest and most vulnerable groups, especially poor women in peri-urban communities” (UNDP, 2017). For many diseases, the association between socioeconomic circumstances and health is clear and well described, but the pathways linking them are more complex. The social epidemiology of ZIKV is still in its early phases, and we lack both descriptive and analytical studies. The purpose of this chapter is to consider, based on the limited data available, how

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00004-3 Copyright © 2021 Elsevier Inc. All rights reserved.

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Social Posi on Housing policy

Context Housing condi ons Vector control

Differen al exposure

Vector density Enhanced immunity Differen al vulnerability

Policy Poli cs

Zika infec on Unknown ? Differen al intrauterine suscep bility

Congenital Zika Syndrome Care, habilita on Differen al consequences

Social consequences FIG. 1 Model of pathways and policy entry points linking social context and individual’s social position to Zika related disorders. Depending on housing policies, there will be a varying association between an individual’s social position (education, occupation, and income) and housing conditions, including water supply and sanitary conditions. These conditions will create unequal vector density (differential exposure) that, depending on immunity and other factors (differential vulnerability) will influence the risk of Zika infection. Depending on unknown interacting causes creating differential intrauterine susceptibility, the Zika infection in pregnant mothers may lead to congenital Zika syndrome. Depending on access to medical and social care the disabled child might generate a range of social consequences for the family.

inequalities in morbidity related to Zika virus emerge and provide public health professionals dealing with them, a better understanding of what might be done to alleviate those differences. Since infection with ZIKV in the vast majority of cases is an asymptomatic or mild disorder, the main burden related to ZIKV is the teratogenic and neurological effects i.e., the congenital Zika syndrome (CZS) and Guillain-Barr
e syndrome. The social epidemiology on GBS is extremely limited, and we shall, therefore, focus on CZS. We shall start our review by describing what is known about the social inequalities in CZS. We then move backward in the causal pathway from CZS to ZIKV infection, to transmission by arthropod vectors and other pathways, to vector density and finally to housing and sanitary conditions essential for the vector ecology. For each step, we shall examine the social inequalities in the exposure, and in the vulnerability to the effect of those causes. We apply a model we have presented earlier (Diderichsen, Augusto, & Perez, 2019) (see Fig. 1).

The macro-drivers of the ZIKV The transformation from a “sleeping epidemic” to a global crisis, as seen for DENV in 1990s, CHIKV from 2004, and ZIKV in 2015–16 is driven by complex and fast changes with poorly planned urbanization and globalization together with large economic inequalities and ineffective prevention (Ali et al., 2017; Depoux et al., 2018; Gubler, 2011). The fast growth of cities in low- and middle-income countries (LMIC) has led to a spread of informal settlements or slums driven by the search for jobs and rising housing cost in more central parts of the cities. The political governance and regulation of housing markets and costs to ensure that the poor have access to affordable healthy housing has not worked properly in many countries. These areas typically lack regular access to clean water and have substandard sanitation and garbage collection. There is high population density as well as low durability of housing material and insecure tenure (Ezeh et al., 2017).

Socioeconomic inequality in the prevalence of CZS The epicenter of the Zika epidemic in Brazil 2015–16 was Recife, the metropolitan of Pernambuco state, build on the deltas of the Capibaribe and Beberibe rivers in Northeast Brazil. Recife has a very high income-inequality (GINI-coefficient ¼ 0.65) and has a long history of water-related infectious diseases primarily affecting poor neighborhoods (Castro, 2015).

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TABLE 1 Prevalence rate ratios of newborns with microcephaly by socio-environmental conditions (HDI) and vector density (Breteau index). Socio-environmental variable

Prevalence rate ratio (95% confidence interval)

Mothers education Not completed college

1 (reference)

Completed college

0.26 (0.16–0.41)

Municipal human development index 0.710–0.809

1 (reference)

0.692–0.709

2.69 (1.28–5.66)

0.671–0.691

3.67 (1.79–7.53)

0.494–0.670

3.90 (1.88–8.06)

Vector density (Breteau index) 0.0–1.3

1 (reference)

1.4–2.5

1.70 (0.86–3.37)

2.6–4.5

2.03 (1.04–3.97)

4.6–9.3

2.31 (1.19–4.50)

Prevalence rate ratio (95% confidence intervals) of microcephaly among 17,990 newborns in Recife born August 2015 through May 2016. HDI ¼ Human Development Index. Breteau Index is the number of water tanks tested positive for Ae.Aegypti divided by the number of households examined (Souza et al., 2018).

Souza et al. registered over 90% of the 19,000 live births in the Recife area from August 2015 to May 2016 (Souza et al., 2018). They found that 1.1% of the infants could be classified as having microcephaly. They had mothers who less often had college education (RR ¼ 0.26; 95% CI: 0.16–0.41), and more often lived in municipalities with lowest compared to highest human development index—HDI (RR ¼ 3.9 (1.9–8.1), and in areas with high larvae density of Ae.Aegypti, bad or no sewage system or garbage collection (Table 1). Similar findings have been made in ecological studies in the same city (de Souza et al., 2018). Case–control studies from Pernambuco (de Arau´jo et al., 2018) and the neighboring Paraiba (Krow-Lucal et al., 2018) show only weak association with mother’s education. That might partly be due to selection bias since controls were selected from the same public hospitals and areas as cases. These studies were primarily designed to reduce confounding in the search for a causal relationship between CZS and ZIKV, which they did very convincingly.

Inequality in the exposure to ZIKV Social inequalities in CZS can involve two different—not mutually exclusive—mechanisms. It can be due to unequal exposure to ZIKV and/or unequal susceptibility to the effects of ZIKV. A few studies have shown a negative association between household- or area-level income and ZIKV infections (Krystosik et al., 2017; Scarbrough, Holt, Hill, & Kafle, 2019). Others have not been able to confirm that, maybe because of bias due to low access to care and detection in poor areas (McHale et al., 2019; Rees, Petukhova, Mascarenhas, Pelcat, & Ogden, 2018). Since ZIKV share the main vector with DENV (Ae.Aegypti), the social and geographical patterns found for DENV might also have relevance for the understanding of ZIKV epidemiology. Several ecological studies have found a strong negative association between area income and DENV infections (Kikuti et al., 2015; Farinelli, Baquero, Stephan, & Chiaravalloti-Neto, 2018; MacCormack-Gelles et al., 2018), but there is much heterogeneity in those findings (Mulligan, Dixon, Sinn, & Elliott, 2015). The epidemiology of ZIKV incidence is suffering from severe underreporting and misclassification. Particularly in 2015, it is assumed that a large proportion of ZIKV cases were reported as DENV (Ministerio da Sau´de, 2019).

Inequality in susceptibility to the effect of ZIKV on CZS With ZIKV as a necessary cause of CZS, the differential exposure to the virus plays a major role in the social inequality in CZS infections. There are, however, strong indications that other interacting causes might generate a differential

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susceptibility to the teratogenic effects of ZIKV. Most cases of microcephaly observed globally in 2015–2016 clustered as mentioned in the Northeast region of Brazil. Cases of ZIKV infections were, however, observed all over Latin America and in Asia and Africa. The incidence rate of ZIKV was presumably higher in the Northeast (Brady et al., 2019; de Oliveira et al., 2017; Lowe et al., 2018), but that difference cannot explain the clustering of CZS cases (Barbeito-Andr
es, SchulerFaccini, & Garcez, 2018; Sampaio et al., 2019). The pattern of reported cases does certainly not indicate that (Ministerio da Sau´de, 2019), but the degree of underreporting may vary strongly (Brady et al., 2019) (Fig. 2). With an assumption that 10% of all pregnant women across all states of Brazil were infected with ZIKV, the risk for them to have a baby with microcephaly in 2015/16, was for Sergipe (in Northeast Brazil) estimated to be 3.85%, Paraiba 2.80%, and Pernambuco 2.28% compared to 0.63% in Rio de Janeiro and 0.03% in Parana´ ( Jaenisch et al., 2017) (see Table 2). These huge differences in the susceptibility to the effect of ZIKV indicates that even if ZIKV is a component cause in almost all cases of microcephaly observed in that period, there must have been some other interacting cause that is more prevalent among pregnant women in the Northeast. Since the risk of CZS is very low in most parts of the world, where ZIKV has been epidemic, the interacting factors would account for a large proportion of microcephaly cases too (Amaral et al., 2019). There might not only be a geographical and social dimension in the unequal susceptibility but also a time dimension. The peak in ZIKV cases in the middle of 2015 was followed 5 months later by a peak in microcephaly but a new peak of ZIKV cases reported in early 2016 was not (de Oliveira et al., 2017; Ministerio da Sau´de, 2019). This has contributed to a focus on interacting comorbidities with temporal variations. Northeast and other parts of Brazil experienced a peak of DENV in 2015–2016, and in particular, the Northeast experienced a peak of CHIKV in 2016 (Ministerio da Sau´de, 2019).

Sources of differential susceptibility The hypothesis of the interacting virus has found some support in ecological studies showing a link between CZS and CHIKV in areas with high poverty rates (Campos et al., 2018). DENV-cases in Recife have earlier been found to cluster in the same impoverished areas as CZS (Braga et al., 2010). Coinfection with DENV or other flavivirus could thus play a role in the differential susceptibility to ZIKV (Paixa˜o, Teixeira, & Rodrigues, 2017). Laboratory studies have shown that antibodies generated after DENV infection bind with ZIKV, significantly enhance pathogenicity but fail to counteract the virus (Priyamvada et al., 2016). There is some in vitro evidence that prior DNV infection might increase the risk of clinical complications of ZIKV infection, but a recent review found little evidence for that in human studies (Masel et al., 2019). Other known causes of microcephaly are genetic (e.g., Downs syndrome) as well as other infections such as toxoplasmosis, cytomegalovirus, rubella, varicella, syphilis, herpes, and HIV. Maternal exposure to heavy metals, alcohol, smoking, and severe malnutrition are also contributing causes. None of them shows, however, the relevant geographical patterns to explain the CZS clustering. A study from Rio Grande do Sul (in South Brazil) with no ZIKV outbreaks registered and with a normal low prevalence of microcephaly (3.8 per 10.000 births) found that approximately 50% could be attributed to infections, and the other half was part of genetic syndromes (Herber et al., 2019). Because many pesticides are designed to cause adverse effects in the nervous system of the pest species, these chemicals have been suspected of contributing to neurodevelopmental disorders (Audouze, Taboureau, & Grandjean, 2018). Several insecticides have during decades been used for vector control. Pyriproxifen is a frequently used larvicide in water storages to protect against DENV, ZIKV, etc. So far, however, there is no evidence of an association with CZS. Genetic causes have been investigated by Caires-Ju´nior et al. (2018). They have in a twin study shown that neural progenitor cells derived from nonaffected and CZS-affected twins have different gene expression signature of neural development and that this may contribute to the different susceptibility to ZIKV. This opens to the possibility of epigenetic responses to environmental exposures. There was no single locus associated with CZS. Brady et al. (2019) tested in their study of 5 million births 2015–2017 in Brazil but could not verify any association between CZS and area-level data on water quality, DENV, Yellow Fever of Bovine Viral Diarrhea. Only ZIKV infection in the first two trimesters of pregnancy was found to be associated with CZS.

Inequality in vector density Even if the data are scarce it would not be unexpected if there are social inequalities in the occurrence of ZIKV infections. Not only are the incidence data on DENV and ZIKV pointing in that direction but also the preferred urban poor housing and bad sanitation. The spatial distribution of vector density is determined by the interaction between environmental, socioeconomic, and meteorological conditions. Associations with adverse socioeconomic living standards are fairly consistent

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FIG. 2 Estimated Zika transmission risk in Latin America (A) and cases of microcephaly in Brazil (B). (A) Risk for Zika virus transmission estimated by ecological, meteorological, and socioeconomic conditions (Cunze, Kochmann, Koch, Genther, & Klimpel, 2019). (B) Spatial distribution of confirmed cases of microcephaly in Brazil 2015–16 (Lowe et al., 2018).

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TABLE 2 Unequal susceptibility to the teratogenic effects of Zika infection during pregnancy. No. of live births

Assuming 10% Zika infection rate during pregnancy in all states

Assuming 50% Zika infection rate during pregnancy in all states

Rio de Janeiro

213,745

0.63

0.13

Pernambuco

171,402

2.28

0.46

Paraiba

53,586

3.40

0.68

Santa Catarina

85,452

0.05

0.009

Sergipe

32,225

4.96

0.77

Parana´

147,382

0.03

0.005

State

Estimated absolute risk (%) of confirmed cases of microcephaly in a baby born to a women infected by Zika virus during pregnancy. States in Brazil November 2015 to October 2016 ( Jaenisch et al., 2017).

across studies, while the association with environmental factors is less so, probably due to large variation between studies in design and parameters used. High population and housing density, as well as low income levels, have in several studies shown to be associated with high vector density of Ae.Aegypti and Ae. Albopictus (Sallam, Fizer, Pilant, & Whung, 2017; Whiteman et al., 2019). Bad and irregular water supply leads to more use of uncovered inappropriate water-storage containers that open more standing water surfaces providing breeding places for Aedes mosquitos. Poor areas typically have more irregular water supply. Bad fitting of doors and window screens, and abandoned household debris (e.g., disposed tires, packages, broken bowls, and cups) provide breeding sites that are perfect for Aedes mosquitoes. Studies from Recife have identified ZIKV infected Culex Quinquefascitus mosquitoes (Guedes et al., 2017). Culex Q. is a very abundant mosquito living close to humans. Studies indicate that Culex Q has the potential of being a competent vector for ZIKV, but its vector capacity is still unknown. Studies from Recife on lymphatic filariasis, where the vector is Culex Q., show a very strong clustering to slum areas in the city (Braga et al., 2001). Culex Q. breeds in contrast to Ae.Aegypti in dirty waters. Since many poor areas suffer from nonexistent or dysfunctional sanitation and sewage systems there is plenty of dirty water. The possibility of Culex Q. as an important vector therefore has the potential of being relevant for the socioeconomic inequality of ZIKV and the policies to prevent it. The translation of vector density to incidence has not been examined quantitatively for ZIKV but has been studied carefully for DENV. The association is far from clear and it has not been possible to quantify based on existing studies (Bowman, Runge-Ranzinger, & McCall, 2014). Since DENV transmission is reported even in areas with low vector density, it is possible that socioeconomic factors modify that relationship (see below), but it has not been examined. Sexual transmission also plays a role in ZIKV infections but estimates indicate that it only represents a small proportion (4.8%) of the overall reproductive number (Maxian, Neufeld, Talis, Childs, & Blackwood, 2017).

Inequality in the vulnerability to the effect of vector density on ZIKV incidence For ZIKV, as well as for other infectious diseases, there is a range of host factors that in addition to immunity influence who might be infected from exposure to a pathogen. Malnutrition has historically played an important role, but in today’s urban environment, focus has shifted to prolonged social and psychological stress. It is since long well known that it might compromise several physiological systems, which might increase susceptibility to disease (Glaser & Kiecolt-Glaser, 2005; Segerstrom & Miller, 2004). Evidence from animal models and human studies suggests a considerable modulation of the hypothalamic–pituitary–adrenal axis in response to stress, with altered biological functions such as compromised immunity (e.g., impaired humoral and cell-mediated immunity) and increased inflammatory reactivity. Chronic stress affects susceptibility to infectious disease because it can suppress protective immune responses and/or exacerbate pathological immune responses and make immune cells resistant to cortisol regulation (Dhabhar, 2014). Large population-based epidemiological studies have confirmed this (Song et al., 2019). Stress has also been found to influence immunological reactions related to neurotropic viruses, which has relevance for ZIKV (Ives & Bertke, 2017).

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The life in slums with high population density, violence, and poverty means chronically high stress levels and living in these environments, therefore, does not only mean a high level of exposure to pathogens like ZIKV but also high levels of vulnerability to be infected (Lilford et al., 2017).

The fundamental determination of ZIKV inequalities The fast growth of the sprawling cities of low- and middle-income countries has proven to be preferred environments for many disease vectors. The development is exacerbated by segregation, i.e., the large disparities in income across the spatial distribution of the population. Low-income groups are often settled in peripheral segregated spaces disconnected from urban infrastructure with houses and sanitation build with inappropriate materials and construction. In many cities in Latin America, the slum (in Brazil: favelas) is the only affordable housing available for the poor. These areas are often located on hazardous places, with bad communications, and low tenure security and the population suffer from poor education, stigmatization, and discrimination (Ezeh et al., 2017; Young, 2017). Precarious housing and settlements, fragile socio-environmental conditions, and exposure to natural and anthropogenic hazards are elements that combine and contribute to a continuous process of vulnerability. The residents of informal settlements tend to be marginalized from the more “formal” development processes of cities, limiting their collective capabilities. All the three elements of vulnerability—exposure to hazards, susceptibility to their effects, and lack of capability to change or cope (Diderichsen, Hallqvist, & Whitehead, 2019) dominates the lives of low-income people in the cities.

Socioeconomic implications UNDP (2017) has estimated the economic consequences of the Zika epidemic. The challenge is that the clinical picture of children with microcephaly and other types of CZS is very complicated with multiple comorbidities and disabilities. Looking after a child with severe developmental disorders and complex needs of care places enormous emotional, social, and economical demands on families, with a high risk of paternal abandonment and mothers forced to leave paid employment. Families with CZS children are often poor and the sick child puts further financial pressure on the family through lost income and extra costs for travel and care. The unequal socioeconomic consequences of CZS, therefore, contribute to a vicious circle of increasing inequalities. Studies have documented the steep difficulties mothers with microcephaly children are facing even in Brazil with a universal health care system organized across the country (Albuquerque et al., 2019). The care offered is often insufficient, with lack of collaboration across specialties, and with fragmented follow-up routines. Inequities in access, costs, and quality have been found. The added costs are far higher than the extra Bolsa-Familia support the Brazilian government has provided for these mothers (UNDP, 2017). The austerity recently imposed on health care in many Latin American countries including Brazil also means that the health workers helping families with CZS children are working under difficult conditions. They try to help mothers with care but face limitations due to very low salaries, lack of educated staffing, long working hours, etc. (Nunes, 2019).

Major knowledge gaps Morbidities related to Zika virus is a new recently emerged public health problem and the diagnostic tools and research in the area are still far from developed. This review of processes generating social inequalities in ZIKV and CZS make some unanswered research questions more visible: l

l

l

l

The most obvious gap in our knowledge is the causes of CZS that interacts with ZIKV. If they are associated with social conditions, they will be important causes of the differential susceptibility to the effects of ZIKV (Brady et al., 2019). The clustering comorbidities of arbovirus have the potential of interaction between diseases. This happens in a context of social inequalities, but our understanding of this suffers from knowledge gaps on asymptomatic cases, the effects of coinfections and the interactions with socioeconomic host factors (Eder et al., 2018; MacDonald & Holden, 2018). The area would benefit from a syndemic approach with both anthropological and epidemiological studies (Singer, 2017). Social host factors with impact on the immune system are potentially important for the unequal burden of ZIKV, but much knowledge is needed for a better understanding of the specific exposure and mechanisms involved. There is no lack of knowledge about the fundamental role of housing conditions, water supplies, and sanitary conditions. Causes and cost-effective interventions are well known (WHO, 2017). We need a better understanding of the political and economic processes preventing the implementation of effective policies that can tackle the structural issues of territorial and economic segregation in the urban outskirts and ensure communities better control over the determinants of their health.

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Policies to tackle the inequalities The five steps of the causal processes described above also indicate five different policy entry points to tackle the inequalities in Zika related morbidity: l

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l

l

Regulating housing conditions: Making healthy housing affordable for poor people is a critical element in any sustainable public housing policies to reduce vector-borne disease including ZIKV. Regular safe water supply, proper sanitary installations, and removal of containers with stagnant water as essential elements (WHO, 2017). Careful re-urbanization of these areas, upgrading the access to public services is particularly important for the most vulnerable populations (Corburn & Sverdlik, 2017; Lilford et al., 2017). Controlling the vectors: The history of vector control has since the 1930s shown many successful examples of the first decades but has more recently turned worryingly ineffective when the focus has been narrowed down to the mosquitos. The increasing resistance to the growing list of insecticides, larvicides, and growth regulators is part of the problem (L€ owy, 2017; Martı´n et al., 2010). The policy response to the Zika epidemic was mainly more of the same, framing it as a “war” against the mosquito (Matta, Nogueira, Rabello, & Silva, 2019). Health education programs are important but tend to increase inequalities since they require the mobilization of an individual’s resources and will often primarily benefit those with more resources (Abel & Frohlich, 2012). Other sustainable methods of vector control are tested, but still not available. Reducing susceptibility to ZIKV. Since the causes and mechanisms generating the differential susceptibility, the effect of ZIKV on CZS is still unknown is not possible to suggest specific policies. If comorbidity is part of mechanism the environmental policies mentioned above have the advantage that they might prevent other vector-borne diseases at the same time. Reducing stress on the immunological system. The origins of the stress of living with overcrowding, poverty, and violence together with mental and somatic multimorbidity by reducing poverty, and upgrade the urban infrastructure of slum areas. Strengthening the capacity of primary care resources to enable to meet the syndemics of clustering and interacting comorbidities among poor patients is important. Stress research has shown that increasing individuals’ and communities’ capabilities can reduce some of the adverse effects of social stress (Cooper & Quick, 2017). Providing for the medical and social needs of families with a disabled child. Social policies are needed to reduce the socioeconomic consequences of CZS including the economic impact on families due to reduced work capacity and costs of traveling to receive care. They will need access to a health care system that can ingrate the complex help CZS disabled children need, and ensure an integrated follow-up and habilitation. But these qualities of a health care system are exactly where many systems have their weakest point including the Brazilian health care system (Gurgel Jr., de Sousa, de Oliveira, Santos, & Diderichsen, 2017).

Mini-dictionary of terms Comorbidity: The cooccurrence of two or more disorders in the same individual at the same time. Confounding: A cause of a disease that is associated with the exposure under study. Differential exposure: Unequal exposure to the cause of disease. Ecological studies: Studies where population rates are compared rather than individual cases and controls. Epidemiology: The study of the determinants and distribution of health-related states or events in specific populations. Epigenetic: A process where the function of a gene changes without an alteration in DNA sequence. Gene expression: The process by which information from a gene is used in the synthesis of a functional gene product. Herd immunity: When a large percentage of a population become immune to an infection. Human Development Index: A measure of human development combing average education, income, and longevity. Hypothalamic–pituitary–adrenal axis: A complex set of influences and feedbacks among three organs in the brain (hypothalamus and the pituitary gland) and the adrenal glands. Inequality in health: Systematic differences in occurrence and consequences of ill health between groups. Interaction: Interaction between causes means that one cause modifies the effect on the disease of another cause. When diseases interact, one disease modifies the course and consequences of another disease. Misclassification: When a measurement of diseases or exposure includes false positive and/or false negative measurements. Neural progenitor cells: Cells of the central nervous system that give rise to glial and neuronal cell types. Neurotropic virus: A virus that is capable of infecting nerve cells.

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Pesticides: Chemical compounds used to kill insects, rodents, fungi, and unwanted plants. Susceptible: Sensitive to health effects of exposures. Syndemic: When two or more diseases cluster and interact in a context of social inequality. Vector: Mosquito that transmits virus between humans and/or animals. Vulnerability: When people are more exposed to hazard, more sensitive (susceptible) to their health effects, and/or less capable to change, adapt to, or cope with hazards.

Key facts l

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Congenital Zika syndrome (CZS) including microcephaly occurs 4 times more often in children of mothers without a college education. Cases of CZS clustered in 2015–2016 to Northeast Brazil, partly due to high incidence of Zika infection, but the geographical pattern indicates that other factors might have modified the teratogenic effect of Zika virus. Zika virus infection is strongly associated with high poverty rates, population density, and substandard housing, water supply, and sanitary conditions. Fast urbanization, large income inequalities, segregation and lack of effective housing, and sanitary policies have led to the accumulation of poor people in precarious housing areas where mosquito vectors thrive.

Summary points l

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Congenital Zika syndrome occurs more frequently among infants to low educated mothers—and most of the cases occurring 2015–2016 clustered in poor areas in Northeast Brazil. The clustering was only partially caused by a higher incidence of infection with Zika virus in those areas, and may partially be due to unknown causes in those areas, interacting with Zika virus in its teratogenic effect. The higher incidence of Zika virus infection in poor areas is partially due to a higher density of mosquito vectors on those areas, but comorbidity and stress due to high population density, poverty, and violence might contribute to the inequality. The higher vector density of Ae. Aegypti mosquito is created by more larvae breeding sites in standing water surfaces in water storage containers and debris that accumulate in poor areas due to irregular water supply and garbage collection. The fundamental determination is poverty and unregulated fast urbanization, driven by a search for jobs and a housing policy that makes it impossible for poor people to afford healthy housing. Caring for a child with microcephaly put enormous emotional, social, and economic demands on the often already poor families.

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Scarbrough, A. W., Holt, M. M., Hill, J., & Kafle, J. C. (2019). Is there a relationship between income and infectious disease: Evidence from Cameron County. International Journal of Community Well-Being, 2, 3–13. https://doi.org/10.1007/s42413-019-00020-2. Segerstrom, S. C., & Miller, G. E. (2004). Psychological stress and the human immune system: A meta-analytic study of 30 years of inquiry. Psychological Bulletin, 130(4), 601–630. https://doi.org/10.1037/0033-2909.130.4.601. Semenza, J. C., & Giesecke, J. (2008). Intervening to reduce inequalities in infections in Europe. American Journal of Public Health, 98(5), 787–792. https://doi.org/10.2105/AJPH.2007.120329. Singer, M. (2017). The spread of Zika and the potential for global arbovirus syndemics. Global Public Health, 12(1), 1–18. https://doi.org/10.1080/ 17441692.2016.1225112. Song, H., Fall, K., Fang, F., Erlendsdo´ttir, H., Lu, D., Mataix-Cols, D., et al. (2019). Stress related disorders and subsequent risk of life-threatening infections: Population-based sibling controlled cohort study. BMJ, 367, I5784. https://doi.org/10.1136/bmj.I5784. Souza, A. I., Siqueira, M. T., Ferreira, A. L. C. G., Freitas, C. U., Bezerra, A. C. V., Riberiro, A. G., et al. (2018). Geography of microcephaly in the Zika era: A study of newborn distribution and socio-environmental indicators in Recife, Brazil, 2015-2016. Public Health Reports, 133(4), 461–471. https:// doi.org/10.1177/0033354918777256. UNDP. (2017). A socio-economic impact assessment of the Zika virus in Latin America and the Caribbean: With a focus on Brazil, Colombia and Suriname. New York: UNDP. Available at https://www.undp.org/content/undp/en/home/librarypage/hiv-aids/a-socio-economic-impactassessment-of-the-zika-virus-in-latin-am.html. Whiteman, A., Gomez, C., Rovira, J., Ghen, G., McMillan, O. W., & Loaiza, J. (2019). Aedes Mosquito infestation in socioeconomically contrasting neighborhoods of Panama City. EcoHealth, 16(2), 210–221. https://doi.org/10.1007/s10393-019-01417-3. WHO. (2017). Keeping the vector out: Housing improvements for vector control and sustainable development. Geneva: World Health Organization. https://www.who.int/social_determinants/publications/keeping-the-vector-out/en/. Young, A. F. (2017). Zika outbreak in 2016: Understanding Brazilian social inequalities through urban spatial analysis and their consequences to health. MOJ Ecology & Environmental Sciences, 2(4), 00032. https://doi.org/10.15406/mojes.2017.02.00032.

Chapter 5

Lifestyle factors and congenital Zika syndrome: Drugs, alcohol, and beyond Daniela Pires Ferreira Vivacquaa and Cristina Barroso Hoferb a

Department of Infectious Diseases, Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, RJ, Brazil, b Department of Infectious Diseases, School of Medicine, Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, RJ, Brazil

List of abbreviations CMV CZS SUS TORCH WHO ZIKAV

cytomegalovirus congenital Zika syndrome ´ nico de Sau´de (Brazil’s Public Health system) Sistema U toxoplasmosis, other (syphilis, varicella-zoster, parvovirus B19), rubella, cytomegalovirus, and herpes World Health Organization Zika virus

Introduction During the outbreak of Zika infection in Brazil, microcephaly and later congenital Zika syndrome (CZS) were described as consequences of maternofetal transmission of the Zika virus (ZIKAV) (Brasil et al., 2016) (Fig. 1). Newborns whose mothers had infection during pregnancy (Miranda-Filho et al., 2016), mostly during the first two trimesters of gestational age (Lima et al., 2019), present higher chance of developing this syndrome. Among newborns with CZS, the severity of the syndrome varied: from asymptomatic to severe microcephaly, with several malformations (Franc¸a et al., 2018). The factors (other than the Zika virus infection) that could possibly contribute to the CZS severity variation are not well understood. The mothers are infected with ZIKAV due to the transmission of the virus via the bite of mosquitoes, especially Aedes aegypti, very common in tropical regions (Dick, 1952). As other mosquito-borne diseases, it often affects developing countries more severely, which, in addition to having a more favorable climate for the spread of mosquitoes, tend to have disorderly construction of residential neighborhoods, which generates potential mosquito breeding grounds (Hemme, Thomas, Chadee, & Severson, 2010; Liu-Helmersson, Br€annstr€om, Sewe, Semenza, & Rockl€ov, 2019). In addition, these diseases also tend to affect the most vulnerable strata of the population: less educated and poor. This observation is a consequence of failure of several steps for infection prophylaxis: as prevention involves the use of repellents (which are expensive) and wearing long sleeve clothes, which is not culturally usual in warm climates, besides peridomiciliary cleaning to avoid water accumulation (population with less access to public cleaning services, sanitary education, and installations). As the World Health Organization has recognized, “the burden of the Zika falls on the poor” (WHO, n.d.).

Socioeconomic condition Several studies have reported a higher prevalence of dengue fever, yellow fever, and other mosquito-borne diseases in lowincome populations, especially within slum communities with high levels of absolute poverty (Kikuti et al., 2015; Vieira et al., 2019). In the case of Zika, this observation is more recent as prior to the large outbreak reported between 2014 and 2016 in Brazil, little was published about the disease (Gardner, Bo´ta, Gangavarapu, Kraemer, & Grubaugh, 2018). The largest number of cases of the disease occurred in the northeast region of Brazil, regions with the lowest per capita income in the country (Franc¸a et al., 2018). After stratification of this region, it was possible to conclude that most microcephaly cases occurred in the poorest regions (de Souza et al., 2018). Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00005-5 Copyright © 2021 Elsevier Inc. All rights reserved.

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TIMELINE OF ZIKA VIRUS HISTORY 1947

2013-201

Nov, 2015

May, 2017

Zika virus identified in Uganda

Outbreak at French Polynesia (suspected associated malfromations).

Public Health Service emergency (ZIKV found in fetus tissues)

End of Public health service emergency in Brazil

2007

May, 2015

Sep 2016

Human Outbreak: Yap Islands: attack rate -73% dos residents

ZIKAV confirmed in Brazil, WHO Epidemiologic alert

WHO declares association of maternal ZIKAV infection and congenital malformations

FIG. 1 Timeline of zika virus history. Evolution of important facts about Zika virus outbreak and its association with congenital malformations.

The virus targets people who live in conditions favoring mosquito breeding, such as precarious housing without sewage systems and regular water supply (Rasanathan, MacCarthy, Diniz, Torreele, & Gruskin, 2017). The concentration of mosquito-borne diseases is higher in marginalized neighborhoods, where the water supply is irregular. Because of that, it is very common for each family to have its own water tank, which if in poor condition or not covered can become excellent breeding sites for Aedes aegypti larvae. In addition, garbage collection is not regular in many districts of Brazil and several discarded items can also become reservoirs for mosquitoes (Leseri & Kitron, 2016). Besides that, it is known that the Aedes aegypti mosquito travels, on average, 100 m in its lifetime (Hemme et al., 2010). In the densely populated slums, a single 100 m2 space could contain more than 100 housing structures, with two to three floors, each one with a different family, which could include a population of 1000 people. These residents can be infected multiple times by mosquitoes circulating in such neighborhoods. Wealthy residents of high-rise apartment buildings have mosquito screens, air conditioning, and insecticides. Even if located adjacent to these favelas, they are more protected against mosquitoes (Snyder et al., 2017). The only way to solve this problem is through public health response. Their ethical design and implementation require incorporation of equity, responsibility, solidarity, and transparency. Equity entails effort to ensure that the poor and disadvantaged are not disproportionately burdened by the outbreak. Equity is essential to vector control (Pan American Health Organization, 2016). Additionally, the poor cannot afford repellent and nets to protect themselves. The Zika virus and microcephaly epidemic led to a 49% jump in sales of repellents in 2016, compared with 2015, in Brazil (Alvarenga, 2017). Repellents are generally used as a preventive measure in pregnant women with higher levels of schooling and fewer children. The relativity high cost of repellents is the main reason for nonuse (Dantas Melo, Santos Silva, & La Corte, 2019).

Lifestyle In addition to the prevalence of acquired infection, some factors may increase the mother-fetus transmission rates of this disease or possibly the pathogenesis of the CZS. Previously, several determinants were described in the literature as etiology of microcephaly: other congenital infections (such as TORCH), genetic abnormalities, and exposure to alcohol, tobacco, and illicit drugs (Alvarado-Socarras et al., 2018). Alcohol is the teratogenic agent responsible for the fetal alcohol syndrome, being a major nongenetic cause of intellectual disability and behavioral problems, as well as microcephaly (Abel & Sokol, 1987; Momino et al., 2012). Illicit drug consumption during pregnancy is another public health problem involving a potential embryo-fetal effect, including low birth weight, intrauterine growth restriction, and placental abruption, as well as premature birth or spontaneous abortion (Holbrook & Rayburn, 2014). Besides being independent teratogenic substances, a study in Rio de Janeiro, Southeast of Brazil, related the use of alcohol and illicit drugs with the presence of CZS. Mothers of Zika virus in utero exposed children live in situation of very high social vulnerability in Brazil, many are unemployed, divulged using alcohol and illicit drugs during pregnancy, and reported sexual abuse before or during pregnancy (Hofer, Lima, Vivacqua, Abreu, & Frota, 2019) (Table 1).

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TABLE 1 Maternal demographics factors associated with the development of congenital Zika syndrome. Total population (n 5 42)

Controls (n 5 18)

Cases (n 5 24)

P-value

0.43 (0.25–0.71)

0.33

0.46

0.60

Tobacco

6/40 (15.00%)

2/18 (11.11%)

4/22 (18.18%)

0.67

Alcohol

10/41 (24.39%)

4/18 (22.22%)

623 (26.09%)

1

llicit drugs

2/40 (5.00%)

0/18 (0%)

2/22 (9.09%)

0.49

a

b

Household income per capita , median (IQR ) (missing data 15) Substance use during pregnancy (%)

Lifestyle factors that can affect the mother to child transmission of Zika virus during pregnancy in a study in Rio de Janeiro, Brazil. a Household income was presented in monthly Brazilian minimum wages (approximately $290.00 US dollars). b IQR—interquartil range. Adapted with authorization from Lima, G. P., Rozenbaum, D., Pimentel. C. et al. (2019). Factors associated with the development of Congenital Zika Syndrome: A case–control study. BMC Infectious Disease, 1, 277, doi:https://doi.org/10.1186/s12879-019-3908-4.

This study also showed that women who did not plan the pregnancy had more babies affected by Zika virus. This may be justified by the increased care that women wanting to get pregnant often take of their health, in this case trying to protect themselves more from mosquito bites (Hofer et al., 2019). Although, in the bivariate analysis of a case–control study, maternal alcohol use during pregnancy was associated with microcephaly, this association was not confirmed when adjusting for Zika virus infection (de Arau´jo et al., 2018). Indeed, in a small cohort study of long-term follow-up of children with microcephaly associated with Zika infection, there is no evidence that the use of alcohol, tobacco, and/or illicit drugs during pregnancy have any role in the pathogenesis of worse prognosis of this infection and their consequences (Gouv^ea, 2020). Considering the whole spectrum of CZS, the potential role of other injuries in the pathogenesis of this infection was not completely investigated, probably due to the novelty of the syndrome, as well as small sample size of the studies. Probably, with analysis of pool data, this role can be elucidated (Wilder-Smith et al., 2019).

Access to information/health education Accordingly to the Health Impact Assessment, published at World Health Organization website, there are several factors that can be considered determinants of health. These determinants are based on the socioeconomic and physical environment, and the individual’s behaviors. The combination of these factors will determine the individual’s health. The individual’s motivation is not a determinant in that matter, since it is unlikely that one person can directly control many of these factors associated with health. Determinants such as income and social status (higher income and social status are proxy for better health situation), education (low education levels and poor medical literacy are linked with worse health outcomes), physical environment (safe water, sewage and clean air, healthy home and workplaces, safer communities and roads all contribute to better health), employment and working conditions (people in employment are healthier, although healthier people have more chance to be employed) are much more important to health than motivation per se (WHO, 2020) (Fig. 2). Regarding the guidelines on preventive measures for the infection, a survey questioned the population in some regions of Brazil about their knowledge of ZIKAV and the sources from where they received this information. The second main source of information was repellent advertisements that had the power to reach all strata of the population, being an important source of information (Alvarenga, 2017). This shows how the preventive health education system is flawed in low-income countries where many times, the population receives information from advertisement and media, instead of receiving from their health-care professionals.

Health-care access One factor that may increase this discrepancy in the prevalence of CZS in underprivileged populations is access to contraceptive methods. During the 2014–2016 ZIKAV outbreak, World Health Organization instructed that women who could postpone pregnancy should do so (Wenham et al., 2019), but in developing countries access to effective contraceptive methods

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FIG. 2 Determinants of health. Determinants of health are factors that combined will determine the individual’s health. One factor is related to the other and all of them are interdependent. (Data adapted from WHO (2020, 01 14). Health Impact Assessment (HIA). Retrieved from World Health Organization: https://www.who.int/hia/evidence/doh/en/index1.html.)

is difficult for the general population and culturally there is still some prejudice with some of the options made available by governments. As many as 56% of pregnancies in Latin American countries are unintended (Rights, 2000). On the other hand, in many countries of Latin America, young women who have unwanted pregnancies are from middle and upper class, while the poorest young women’s pregnancies are due to the lack of a different lifestyle option, most of which are reported as desired (Velez & Diniz, 2016). A study of the mothers of whose babies were born with microcephaly, showed that most of them had more than one child already, and were interested in tubal ligation. This is common in Brazil, where access to effective contraceptive methods ´ nico de Sau´de (SUS)-dependent population. In addition to the can be difficult for the Public Health System—Sistema U cost, there is also a sexist behavior that ends up leaving to woman all the responsibility to convince the partner to use condoms or to choose a more definitive method such as tubal ligation, and never a vasectomy (Carneiro & Fleischer, 2018; Vlassoff & John, 2019). Most women who have had babies with microcephaly have been young, single, black, poor, and tend to live in small cities or on the outskirts of big ones (Butler, 2016).

Abortion legislation Many of the countries most severely affected by the Zika outbreak have laws that prevent abortion in most cases (Aiken et al., 2016) (Table 2). While abortion is routinely provided as health-care service in Cuba and Uruguay, for example, it is completely illegal in Dominican Republic, Nicaragua, and El Salvador, countries with documented cases of Zika, and women who seek abortion may face considerable penalties. Other countries, such as Colombia and Brazil, have decriminalized abortion only under exceptional circumstances (e.g., after rape or in case of a life-threatening condition for the mother). However, although ZIKAV may cause anencephaly, abortion due to Zika/CZS alone is not currently legal (Carabali, Austin, King, & Kaufman, 2018). In Latin America, abortion is mostly a clandestine or quasi-clandestine procedure (Velez & Diniz, 2016). During the Zika outbreak, requests for online abortion pills (from a nonprofit organization) from women in Brazil doubled between November 2015 and March 2016 and increased by more than a third in El Salvador and Colombia (Aiken et al., 2016). The use of these clandestine/quasi-clandestine procedures is linked with high maternal mortality, due to hemorrhage and infection.

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TABLE 2 Abortion legislation in Latin America. Brazil

Decriminalized abortion under exceptional circumstances

Colombia

Decriminalized abortion under exceptional circumstances

Cuba

Legal

Dominican Republic

Illegal

EI Salvador

Illegal

Nicaraqua

Illegal

Uruguay

Legal

Example of abortion legislation in some Latin American countries. Data adapted from Aiken, A. R., Scott, J. G., Gomperts, R. et al. (2016). Requests for Abortion in Latin America in the Wake of Zika Virus. New England Journal of Medicine, 4, 396–398, doi:https://doi.org/10.1056/ NEJMc1605389.

This may bias the analysis of rates of CZS when comparing with countries that allow abortion. As the syndrome is often severe with the presence of various malformations, especially microcephaly, many people with higher financial status have traveled to countries where abortion is permitted to perform this procedure, but there is no official information about that number. Besides the law concerning the abortion, there are culture and religious beliefs against abortion in many Latin American countries, especially among the low-income women (Carneiro & Fleischer, 2018). There is prejudice against women who consider abortion in these regions. Religion, education, socioeconomic status, and access to health care may play a role in the reproductive decisions of Zika-infected women, whether this decision concerns the termination or continuation of a current pregnancy, or the postponement of a future pregnancy (Carabali et al., 2018).

Human rights Some human rights principles are very important in a situation like the ZIKAV outbreak: right to health, right to freedom of information, nondiscrimination, right to participation, legal and policy context, and accountability. Every human being should have access to health and the right to receive information about your condition so that they can make informed decisions about their care. In ZIKAV outbreak the right to freedom of information is very important as women could be informed of the risk of infection and the right to health necessary so that they could decide if they did want to get pregnant during that time. The reality was that wealthy women could decide to not get pregnant or to travel to countries without ZIKAV transition during the pregnancy while the poor women were vulnerable to the ZIKAV infection and their baby’s to CZS. It is responsibility of the governments to create an environment that provides equality and that recriminates the discrimination of all kinds (Rasanathan et al., 2017; UN Committee on Economic, Social and Cultural Rights (CESCR), 2000). Sexual and reproductive health inequalities place a disproportional burden on certain groups of women. In view of this, the Zika infection and its consequences will continue to affect the same women for whom access to comprehensive reproductive health-care service is restricted (Velez & Diniz, 2016). Comprehensive information should be based on solid sexual education programs so that women become aware of their sexual and reproductive rights. Only in this context, it is possible to recommend postponing a pregnancy without burdening women with the responsibility of avoiding the consequences of a Zika infection. For those who choose to get pregnant in a context of a great uncertainty of a health outcome, all possible support should be granted for their mental health (Velez & Diniz, 2016).

Policy and procedures Multidisciplinary follow-up Infants affected by ZIKAV in utero may have different kinds of malformations and limitations. It is important that their families rely in a medical service that can provide multidisciplinary care for the infant and their family during the diagnosis, as well as the therapeutic period. For that, the medical service must include medical doctors (with expertise in pediatrics,

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infectious diseases, neurology, ophthalmology, and genetics), psychologists, social workers, and therapists (that will work with speech, physiotherapy, and occupational therapy). The diagnosis and follow-up must be done in a way that the infants and their families’ go to the health center a minimum amount of time so that they can have a more normal everyday life. A protocol needs to be followed where the infant is seen by every professional that he or she needs in only one visit to the health center.

Multicenter follow-up of cohorts During the 2014–2016 Zika virus outbreak, different cohorts were created for a follow-up of the patients infected with the virus and its consequences. Most of these studies are local studies with data of a specific region. For a better understanding of the CZV, it is important that more studies are done with bigger pools of infants. The faster and easier way to do that is to coordinate multicenter follow-ups of the cohorts already existing in the affected countries. This is important for a better understanding of how the virus behave in different environments and regions and what kind of external factors are important for the spectrum of presentation of the CZV.

Mini-dictionary of terms Congenital Zika syndrome: It is a syndrome that affects newborns of mothers who were infected by Zika virus during the gestational period. The syndrome can cause only microcephaly or is accompanied by other malformations like congenital clubfoot. Favelas (slums): An overcrowded urban neighborhood in low-income countries, usually without sewage systems and regular water supply. Microcephaly: It is an alteration in the size of the head. It is noticeable at birth or can occur when the growth of the head does not follow the expected curve of growth. Microcephaly was defined as a head circumference less than two standard deviations at The International Fetal and Newborn Growth Consortium for the 21st Century—Intergrowth and severe microcephaly less than three standard deviations. Mosquito-borne disease: It is an infectious disease that is transmitted to humans by mosquito bite. Sistema U´nico de Sau´de (SUS)—Brazil’s Public Health System: In Brazil, the government is responsible for the health care of all population. It is the right of all people living in Brazil to have access to a system that provides any health care they need.

Key facts Key facts of birth control methods l l

l l

l

l

As many as 56% of pregnancies in Latin American countries are unintended. Brazil has decriminalized abortion under exceptional circumstances; however, abortion due to Zika/congenital Zika syndrome alone is not currently legal. Abortion is routinely provided as health-care service in Cuba and Uruguay. Abortion is completely illegal in Dominican Republic, Nicaragua, and El Salvador and women who seek abortion may face considerable penalties. In Brazil, as the access to effective contraceptive methods can be difficult for part of the population, many women stay in line for a tubal ligation. During the Zika outbreak, requests for online abortion pills (from a nonprofit organization) from women in Latin American countries where abortion is illegal doubled.

Key facts of causes of mal formation in newborns l

l l

Several congenital infections were described in the literature as etiology of microcephaly, for example, toxoplasmosis and cytomegalovirus. Some genetic syndromes can cause microcephaly and other malformations. Exposure to alcohol, tobacco, and illicit drugs were already described as causes of malformations in newborns.

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Exposure to alcohol can cause a specific group of malformations in infants called fetal alcohol syndrome that includes brain damage and growth problems. There is no amount of alcohol that is known to be safe to consume during pregnancy.

Summary points l l l l

l

The mosquito-borne infections affect more intensely the developing countries. The Zika virus outbreak was more intense in low-income populations. The second main source of information about preventive methods for Zika infection was repellent advertisements. Most women who have had babies with microcephaly during de Zika virus outbreak have been young, single, black, poor, and tend to live in small cities or on the outskirts of big ones. There is no biological plausibility on this assertion, but these women were more exposed to the mosquito bites, than women from higher socioeconomic status strata. Women who did not plan the pregnancy had more babies affected by Zika virus.

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Chapter 6

Economic impact of Zika virus infection and associated conditions Henry Maia Peixotoa, Luciana Guerra Gallob, Gilmara Lima Nascimentoc, and Maria Regina Fernandes de Oliveiraa a

Centre for Tropical Medicine, School of Medicine, University of Brasilia, Brası´lia, DF, Brazil, b Centre for Tropical Medicine, University of Brasilia,

Brası´lia, DF, Brazil, c Federal District Health Department, Setor Escolar Lote 04 – Cruzeiro VelhoCruzeiro, Brası´lia, DF, Brazil

Abbreviations CEA COI CUA CZS DALY GBS HE HEE HLY ICER ID-NAT MID MP-NAT QALY Ro RTA RT-PCR ULV UNDP USA WHO ZIKV

cost-effectiveness analyses cost-of-illness cost-utility analysis congenital Zika syndrome disability-adjusted life year Guillain-Barr
e syndrome health economics health economic evaluation healthy life years incremental cost-effectiveness ratio Individual Donation Nucleic Acid Testing intelligent dengue monitoring system nucleic acid testing of mini-pool universal quality-adjusted life year reproduction number relative transmissibility of asymptomatic infection reverse-transcription polymerase chain reaction ultralow volume United Nations Development Program United States of America World Health Organization Zika virus

Introduction The outbreak of Zika virus (ZIKV) infection identified in Brazil in 2015 astonished the global health and scientific communities. The ZIKV was discovered in 1947, and until the Brazilian epidemic approximately 80% of the documented cases of infections in humans were considered mild or asymptomatic (Brasil, 2015; Ioos et al., 2014; Zanluca et al., 2015). In the recent years, ZIKV infections have been increasingly associated with serious consequences of congenital infection, severe neurological complications such as Guillain-Barr
e syndrome (GBS), sexual transmission, and transfusion transmission (O’Neill, 2018). The health sector faced an emerging health crisis of large and unexpected socioeconomic impact. Thus, ZIKV infection was responsible for the fifth statement of a Public Health Emergency of International Concern in the history of the World Health Organization (WHO). ZIKV is an arbovirus, whose main vector is Aedes aegypti, a widely distributed mosquito adapted to many countries around the world. A. aegypti infestation is closely related to the living and health conditions of the exposed populations (Ioos et al., 2014; Zanluca et al., 2015). These circumstances, in addition to other environmental conditions conducive to the proliferation of urban arboviruses, raise a warning to this health crisis and its potential health emergencies. The

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environmental setting as well as the demographic susceptibility of the population of numerous developing countries favor rapid arboviral spread (Kraemer et al., 2015), allowing large epidemics of ZIKV. Since it is an emerging problem, countries around the world are underprepared to respond to a possible ZIKV epidemic and its consequences. Many questions pertaining to the epidemiological, social, and economic implications of the ZIKV infection are still unanswered. The goal of this chapter is to present and discuss the state of the art of the economic impact of ZIKV infection and its associated conditions.

Brief history of ZIKV The ZIKV was first isolated in 1947 in nonhuman primates in the Zika forest in Uganda. The infection was first identified in humans in 1954, and most of its cases were asymptomatic. The rare documented sporadic outbreaks of symptomatic cases presented cutaneous rash, characterized as an acute, benign, self-limited, and short-term feverish disease (Brasil, 2015; Ioos et al., 2014). In 1954, the antibody seroprevalence for ZIKV was estimated at 0.5% in French Equatorial Africa; and at 38% in Nigeria between 1971 and 1975. Decades later, in 2007 an outbreak of an acute infection with manifestations of rash, conjunctivitis, and arthralgia was attributed to ZIKV in the Yap Islands (Duffy et al., 2009). From this outbreak, new cases were identified, with serological evidence in Nigeria, Senegal, Egypt, India, Malaysia, Thailand, Vietnam, and the Philippines (Duffy et al., 2009). Between October 2013 and February 2014, French Polynesia reported 8264 suspected cases, and of the 746 samples referred for laboratory diagnosis, 53.1% tested positive by molecular biology techniques—the largest outbreak of ZIKV infection to date. At the same time, other manifestations were reported as neurological complications and other health conditions (thrombocytopenia; ophthalmologic; and cardiac complication). There were 42 cases of GBS, among which 41 had ZIKV-positive IgM and IgG (Cao-Lormeau et al., 2016). In New Caledonia, the first cases of ZIKV (imported from French Polynesia) were reported at the end of November 2013. The incidence of autochthonous cases was reported in January 2014 and in February of the same year, an outbreak of infection was reported (Dupont-Rouzeyrol et al., 2015). Two years later, in February 2015, a large outbreak of undetermined exanthematic syndrome cases was detected in the Northeast region of Brazil. The initial clinical characteristics of self-limited mild fever and cutaneous rash led to numerous causal hypotheses, including dengue and chikungunya fever. The first ZIKV infection confirmed by the identification of the ZIKV RNA molecule in Brazil was reported in March 2015 (Brasil, 2015; Zanluca et al., 2015). The association of ZIKV with microcephaly was only reported in the outbreak in Brazil, up to that time. After such a finding, a review study in French Polynesia led to the conclusion that the number of babies born with malformations of the central nervous system have increased in the years following the 2013–2014 epidemic (Cauchemez et al., 2016). Considering the 61 countries where A. aegypti is widely distributed, but did not report ZIKV yet, in addition to the sexual transmission of the virus, there are a huge number of susceptible people. In this context, the risk for a new epidemic and an emergency crisis remains a global threat.

Health economics Health economics (HE) is a broad and growing area and is considered one of the most important factors in the health decision-making process. The growing and diverse health needs, as well as the development of new technologies, often face complex epidemiological scenarios, with limited and competing financial resources. Thus, health economics provides tools that facilitate efficiency through the decision-making process in public health management (Drummond et al., 2007). The HE involves the health sector financing, cost management, budgeting, budget impact analysis facing the introduction of new technologies, and health economic evaluation studies. One of the purposes of HE, through full health economic evaluation (HEE), is to compare technology outcomes (interventions, programs, policies, equipment, drugs, etc.) in relation to the cost of its implementation. These studies are classified into four types: cost-effectiveness, cost–benefit, cost-minimization, and cost-utility analysis. The main difference between them is the way the effectiveness (outcome) of technology is measured (see Fig. 1). In cost-effectiveness analysis, the effectiveness of the technology can be measured by various types of outcomes, such as life years saved, deaths prevented, correct diagnosis, and so forth. In cost-minimization analysis, the effectiveness of the technologies compared through the same outcome is similar, however, the costs are different, and such difference is their final product (Drummond, Sculpher, Claxton, Stoddanrt, & Torrance, 2015). In cost-utility analysis, outcomes are measured in terms of utility measures, which imply people’s preferences [disability-adjusted life years (DALY) or quality-adjusted life years

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Full health economic evaluation studies Cost-effectiveness analysis

Cost-utility analysis

Cost-minimization analysis

Cost-benefit analysis

Effectiveness (outcomes used to compare technologies) Natural health outcomes (e.g., life-years saved, prevented deaths and correct diagnosis)

Utility outcomes (e.g., QALY and DALY)

The outcomes are measured in monetary units

Only costs are compared (technologies with equivalent outcomes)

Cost is measured in monetary units (all methods) FIG. 1 Types of full health economic evaluation: effectiveness and costs.

(QALY)]. When the interest is to compare the outcomes evaluated in monetary values, cost–benefit studies are performed (Drummond et al., 2007). The rise of ZIKV infection brought several unknown serious consequences, such as microcephaly and GBS. A considerable amount of public and private resources has been used in health actions whose epidemiological and economic impact is not yet adequately known. In this scenario, knowledge about the effectiveness of health actions, the cost of infection, its fatal outcomes, complications, and sequelae has fundamental importance for public health planning and management. The previous steps for performing complete HEE are characterized by the measurement of costs and outcomes (effectiveness, utility) of the technologies analyzed (Drummond et al., 2007). In this sense, cost-of-illness (COI) studies constitute a type of partial HEE, whose main objective is to measure the economic impact of a disease on society, being particularly important for characterizing the burden of emerging diseases such as ZIKV infection. Through a variety of costing methods, such studies highlight both direct and indirect disease costs. Direct costs are assigned to the health system (prevention, diagnosis, treatment, rehabilitation) and can be classified as medical costs (exams, laboratory tests, drugs, consultations, etc.) and nonmedical direct costs, which are related to other needs for the management of disease and/or its complications but are not directly related to the health sector, such as home adaptations, transportation, food, etc. The costs considered indirect represent the loss of productivity (healthy workforce) in society due to the disease. COIs are essential to establish parameters for resource forecasting and allocation in research, prevention, and care, contributing to the formulation and prioritization of health policies (Rice, 2000). There are few studies that quantify the disease cost of ZIKV infection and its consequences, which are discussed in the following sections. Even with few articles available in the literature, which assess costs in different scenarios (high-, medium-, and low-income settings), what is available points to high costs with significant impact in economic terms, in addition to impacts on population morbidity and mortality.

Economic and social impacts of recent outbreaks ZIKV epidemics affect the physical health of populations and their local economies. The neurotropic behavior of the ZIKV produces health outcomes that harm families and affects communities, producing short- and long-term as well as direct and indirect costs. The epidemic cost depends mostly on the attack rate and the size of the population, but the burden also depends on the type of health system (private or public-funded), clinical protocols, and local economic base. Additionally, most countries that have suffered from ZIKV epidemics also face epidemics of other arboviruses that share the same vector. In these cases, especially in prevention activities, it may be difficult to understand the costs of ZIKV infection. In a very conservative estimate, the potential economic burden of ZIKV in the United States, considering only the six states at greatest risk of Zika epidemics and an attack rate of 0.05% in an epidemic that spans 230 days, maybe up to US$558.8 million (US$15,858 per infected person), if only the direct medical costs plus productivity losses are considered (Lee et al., 2017). Applying the same attack rate for Latin America and the Caribbean, the yearly short cost of Zika epidemic was estimated at US$2.32 billion. In this scenario, the highest absolute cost would rely on Brazil (14% of the total costs of the region), but the highest economic impact would fall on the poorest countries, such as Haiti and Belize (UNDP, 2017). In this regard, attention should be paid to the uneven impact of the ZIKV epidemics. A higher incidence of Zika and a higher burden of its effect are reported in poor areas and populations. Economically vulnerable people often cannot access sexual and family planning services, nor could afford preventive measures against mosquitoes (air-conditioning, window screens, insect repellents, or even piped water and sanitation) (WHO, 2016b).

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The short-term costs of the ZIKV epidemics includes the direct and indirect costs associated with the detection, diagnosis, and treatment of the individuals (either uncomplicated symptomatic ZIKV infection or its severe effects), productivity losses due to absence from work, and the costs of reduction in the tourism revenue, even in nonaffected areas. Regarding the latter, the ZIKV outbreaks happened mostly in tropical areas, where tourism accounts for an important part of the local economies. Considering a 3-year scenario, initial estimates of the impact point to a decrease in the international tourism revenue that could account for more than 80% of the short-term costs—US$2,489,000 in the Caribbean and a total of US$9,011,000 in Latin-America (UNDP, 2017). The long-term consequences of the ZIKV outbreaks should not be underestimated. It includes direct and indirect costs associated with disabilities attributable to ZIKV infection, and macroeconomic implications for countries, especially the loss of productivity and lost earnings of people with congenital Zika syndrome who may not be able to participate in the country’s workforce (UNDP, 2017). Concerning the social impact of congenital Zika syndrome, although the care and research are still primarily focused on children, the high burden borne by women should also be taken into consideration (UNDP, 2017). Most often mothers are the primary caretakers and responsible for the affected child’s daily routine, which is full of appointments and therapeutic activities. To handle the child’s needs, these mothers have to give up their projects and quit their jobs. It has also been observed that often the family structure crumbles under the stress of caring for a child born with microcephaly, with fathers abandoning their homes. Other challenges, such as mobility and transportation to health and social services, and the accumulation of domestic activities with the childcare schedule add to the mother’s burdens, which can lead to mental health issues such as an increase in stress, anxiety, and depression (Bailey & Ventura, 2018; UNDP, 2017). For all the cited burden of ZIKV infection (see Fig. 2) and its impact on the macroeconomic scenario and the family perspective, it should be understood that ZIKV outbreaks may perpetuate or deepen the cycle of social poverty.

Latin America and Caribbean US$ 2.32 billion

Higher burden on poor communities and families

High productivity losses

FIG. 2 Economic and social impacts of Zika virus.

Deepening social and economic inequalities

Economic and social impact of ZIKV

Tourism decline

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The cost of vector control Aedes mosquitoes are the major ZIKV vectors, A. aegypti is considered the main vector. This mosquito thrives in urban environments, and threatens over 3.5 billion people worldwide. Thus, vector surveillance and control are important public health interventions to reduce the incidence and spread of ZIKV. To monitor the mosquito population, evaluate the vector control programs, and detect new areas of vector introduction, the WHO recommends daily inspection of houses and commercial areas (WHO, 2016a). The expenses related to vector control programs include larvicides and insecticides (supplies and equipment), transportation (equipped cars, maintenance, and fuel), human resources (wages, protection equipment, and uniforms), utilities (electricity, water, internet, and telephone), laboratory activities (gloves, microscope, and supplies), and social mobilization activities (office supplies). The human resources usually account for two-thirds of the total costs, excluding the cost of insecticides (Taliberti & Zucchi, 2010). Studies conducted in countries with year-round A. aegypti infestation reported that the cost with insecticide represents between 3.8% and 10.6% of the total budget of the vector control program (Baly et al., 2012; Packierisamy et al., 2015; Salinas-Lo´pez, Soto-Rojas, & Ocampo, 2018; dos Santos et al., 2015). Community participation is an essential tool for mosquito control. In order to enable social mobilization, it is important to encourage community-directed initiatives, such as working groups, education campaigns, community empowerment, and home visits. These interventions account for 26% of the total cost of vector control program (Salinas-Lo´pez et al., 2018). Well-trained personnel, including admin and general duty staff, as well as trained mosquito control professionals are vital to the implementation of these strategies. Workforce cost (including wages and general expenses) accounts for up to 60.7% of the total vector control program budget (Packierisamy et al., 2015). The total cost of the vector control and surveillance interventions may vary depending on the scope of intervention activities as well as the affected infection setting (size of the human population, spread of the mosquito, and health system type) (Baly et al., 2012; Constenla, Garcia, & Lefcourt, 2015). Studies from the 2000 decade estimate a yearly cost per capita of US$0.20 (total cost of US$638,000) in Cambodia (2005) (Suaya et al., 2007), US$1.56 (total cost of US$9,816,456) in Panama (2005) (Armien et al., 2008), US$2.68 in Malaysia (total cost of US$73.45 million) (2010) (Packierisamy et al., 2015), and US$20.04 in Cuba (total cost in the municipality of Guanta´namo of US$4,891,764) (2006) (Baly et al., 2007; Baly et al., 2012). More recent data from Latin America points to a cost of US$0.88 and US$0.99 per capita (total cost of US$97,936 and US$146,651) in 2016 considering two Colombian municipalities (Salinas-Lo´pez et al., 2018) and US$0.22 per capita (total cost of US$47,567,417.13) in Brazil (2017) (Bueno, Almeida, Castro, Retamero, & Clark, 2017). Moreover, it should not be forgotten that the vector population is also impacted by securing intersectoral support on environmental interventions, such as basic sanitation infrastructure and garbage collection, and these efforts should be part of a sustainable strategy of vector control and management. Finally, it is essential to highlight that controlling the A. aegypti population is an effective preventive measure since this intervention reduces not only the risk of ZIKV infection but also the risk of other arboviruses transmitted by the mosquito (Bueno et al., 2017).

The cost of diagnosis ZIKV infection and its consequences need to be better understood. Epidemics in recent years have led to severe outcomes in scenarios of limited diagnostic methods. The possibility of vertical transmission, sexual transmission, and transmission by blood transfusion has led to the expansion of situations in which laboratory confirmation of infection is mandatory (do Souza et al., 2016), but is very costly to public health systems. Laboratory diagnosis is mainly based on molecular biology (RT-PCR) and serology methods, depending on the time between the onset of symptoms and probable exposure to the virus until testing (Zanluca et al., 2015). Recent studies predicting the social and economic impacts of epidemics in the Americas, and regarding willingness-to-pay for a possible ZIKV vaccine, have estimated the average cost of US$150 per person tested (Harapan et al., 2019). Another study that evaluated the cost of Zika-associated GBS in Brazil estimated the cost of ZIKV RT-PCR at US$28.40 and ZIKV serology at US$19.90 per person (Peixoto, Romero, De Arau´jo, & Fernandes de Oliveira, 2019). However, the wide clinical spectrum presented by the ZIKV infection may substantially increase the cost of diagnosis. Let us examine cases of newborns suspected of congenital syndrome, whose mothers were not diagnosed during pregnancy. In Brazil, in order to investigate suspected cases of congenital syndrome, newborns underwent clinical, epidemiological, and neurological evaluations; ultrasound and computerized tomography; and specific and nonspecific laboratory tests. The etiological investigation is very complex, especially for short duration of viremia and the involvement of tests for

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other differential diagnoses. Therefore, the cost of diagnosing ZIKV infection does not depend solely on the techniques utilized by the laboratories. There are sparse studies in the international literature that quantify the costs of diagnosing ZIKV broadly, including all direct costs for the diagnosis of the full known clinical spectrum of the infection. Such knowledge is essential for the development of comprehensive health economic assessments to enable decision-making on resources applied in research, prevention, and other technologies that contribute to minimizing the impact of epidemics.

The cost of ZIKV-associated microcephaly Microcephaly is a rare condition where the baby’s head circumference is smaller than expected when compared to babies of the same sex and age. Microcephaly due to ZIKV exposure during pregnancy was the first congenital abnormality proven to be caused by the virus. It is considered a manifestation of intracranial abnormalities that affects about 6% of the pregnancies exposed to ZIKV and the most severe presentation of congenital Zika syndrome. Still, Zika-associated microcephaly is considered a small part of a larger problem (Rice et al., 2018; UNDP, 2017). The Zika-associated microcephaly is commonly concomitant with other malformations, demanding specialized care and needs. The average lifetime direct medical cost of Zika-associated microcephaly per case was estimated at around US$120,769 in the Caribbean (UNDP, 2017). In Brazil, the estimated yearly cost of Zika-associated microcephaly to the national health-care system was US$10,218 per case (2015) (Morone, Silva, Morone-Pinto, & Araujo, 2019). From a societal perspective, another research in Brazil assessed the costs of the first year of life. It has shown a mean cost of US$9,588.24 per microcephaly case (Gallo, 2020). In Puerto Rico, the lifetime direct medical and nonmedical costs are estimated at US$3,788,843 per case (Li et al., 2017). In Texas (United States), each microcephaly case hospitalization costs an average of US$25,471.46, considering only the health-care system perspective (Shewale et al., 2019). Although these numbers seem to be high, the lifetime direct medical and nonmedical expenses account for only 12% of the total lifetime cost of Zika associated-microcephaly (UNDP, 2017). The loss of productivity due to premature mortality and lowered caregiver productivity amounts to massive lifetime costs to families and entire communities. The estimated lifetime cost per case of microcephaly (2015) in Latin-America was estimated to range from US$580,000 (Nicaragua) to US$1,430,000 (US Virgin Islands) (UNDP, 2017). In the United States, the lifelong costs per microcephaly case are estimated at US$10,000,000 (2008–2015) (see Fig. 3) (WHO, 2016b). It is important to note that Zika-associated microcephaly is an emergent condition and its effects and hardships are not fully understood. Most of the studies conducted so far considered a first-year mortality rate of 20% and a life expectancy of

The Health care System Perspective Brazil US$ 10,218/ case

Cost of ZIKVassociated microcephaly Lifetime cost per case Latin-America US$ 580,000 US$ 1,430,000 FIG. 3 Cost per case of ZIKV-associated microcephaly.

Lifetime cost per case United States US$ 10,000,000/ per case

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35 years after the first year. This assumption implies that the caregiver, who would withdraw from the workforce, would later face challenges when trying to return to work. In this regard, the life expectancy can also be understood as the period in which the microcephaly case will demand more expenses on the health-care system but after that, the family may continue to require government financial assistance. Therefore, the real long-term demands of the family and the impact on social burden are not yet known and are most likely underestimated at this point. Currently, the only certainty is that the human cost of Zika-associated microcephaly is high and long-lasting (Bailey & Ventura, 2018).

The cost of congenital Zika syndrome Currently, the full cost of congenital Zika syndrome (CZS) is undetermined. Still, it is possible to understand the economic impact of this condition by understanding its implications. The full spectrum of the CZS is under study and the magnitude of its impairment remains uncertain. Yet, research findings show that ZIKV exposure during pregnancy results in 14% of newborns with Zika-associated abnormalities— approximately 5% of these children die during the first year of life. Among the children with CZS, approximately 64% exhibit severe irritability, 24%–55% present ophthalmologic anomalies, 20% experience seizures, 5%–20% present arthrogryposis or other congenital contractures, 14%–77% experience difficulties in feeding, and 9% have a hearing impairment. Cardiac, digestive, and genitourinary disorders are also reported. In addition, learning impairments, neurodevelopmental disorders, and other developmental may become issues later in life (Franca et al., 2016; Gordon-Lipkin & Peacock, 2019; Rice et al., 2018). All the CZS-related conditions have to be carefully diagnosed, treated, and followed with a multidisciplinary approach. In some areas, the health-care system is not sufficiently arranged to accommodate affected children and may need soft and hard technologies to offer appropriate care. Organizing the health system to serve children with CZS may cost millions of dollars, but the built infrastructure will benefit a high number of children with complex needs. Although the management of children with CZS would vary depending on the health-care system and the children’s condition (individual needs should define the follow-up), there are some general recommendations that would impact costs. Besides the comprehensive physical exam and the test for ZIKV infection, at least one head ultrasound (US$390) and other image diagnoses such as computed tomography (US$525) and magnetic resonance imaging (US$1261) to investigate brain impairment, an ophthalmologic screening by dilated fundoscopic examination (US$238), and a hearing screening by an auditory brainstem response test (US$250) are recommended. For any abnormal results, the child should be referred to a specialist. In any case, it is important to maintain close monitoring of the Zika-affected children (US$234 for each “complex medical problem” appointment) to guarantee early referral to services when any special needs are detected, aiming to improve the outcomes (Adebanjo et al., 2017; Brasil, 2016; Salvador & de Salud, 2016; Healthcare Bluebook, 2019). Despite their significant cost, these interventions only represent short-term medical needs. The estimated long-term cost of CZS is substantial, especially the indirect costs that rely on the family and society. The lifelong burden includes, but are not limited to economic impact due to the need for a complex multidisciplinary care system, possible hospitalizations, surgeries, and medical treatments, as well as the potential loss of productivity, stigma, stress, and mental health issues faced by parents and caregivers (Bailey & Ventura, 2018). It is important to note that Zika-affected children without microcephaly can also have severe problems, albeit often less severe. Cases of CZS without microcephaly are generally less costly. Nevertheless, the CZS impacts a wider range of families and represents an important burden to the community and its costs should be addressed by future research.

The costs of ZIKV-associated Guillain-Barr
e syndrome GBS is a serious autoimmune disorder often preceded by an infection, such as that caused by ZIKV. The typical clinical presentation is characterized by paresthesia, followed by progressive muscle weakness, which may progress to paralysis of the muscles of the legs, arms, and chest, leading to severe complications and death. Thus, due to the high risk of complications, patients with GBS should be hospitalized and closely monitored by multidisciplinary teams. Studies have shown that the highest GBS costs are due to hospitalization and loss of productivity, especially those associated with treatment and early mortality (Frenzen, 2008; Peixoto et al., 2019; UNDP, 2017). According to a study presented by the United Nations Development Program (UNDP), the lifetime costs per ZIKVassociated case during the epidemic in Latin America and the Caribbean (2015–2017) amounted to US$34,103 including direct medical costs alone, and US$206,020 when including lost productivity costs. The total cost has been estimated at US$242,000,000 and could reach up to 10 billion US dollars, depending on the number of estimated cases. The scarcity of

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Latin America and Caribbean (2015 - 2017) US$ 242 million

Cost of GBS associated to ZIKV

Brazil (2016) US$ 12 million US$ 12,368 per case

FIG. 4 Cost of ZIKV-associated Guillain-Barr
e syndrome.

regional data at the time of the study may have led to an overestimation of costs, mainly due to the use of GBS costs derived from the US reality. Based on Brazilian national data, Peixoto et al. estimated the costs of ZIKV-associated GBS, considering cases that occurred in 2016 in the country. The researchers demonstrated that the cost per patient corresponded to US$12,368.27, of which US$4871.11 were medical costs and US$7497.15 indirect costs. The total estimated cost, considering 977 cases, was US$11,997,225.85, of which US$4,724,992.4 were direct costs, with a predominance of human immunoglobulin therapy costs (US$3,263,210.50) and US$7,272,233.45 were indirect costs, especially the cost of lost productivity associated with premature death (US$4,398,551.72) (Peixoto et al., 2019). The epidemics caused by ZIKV substantially increased the number of GBS cases, causing a significant economic impact on health-care systems, families, and societies (see Fig. 4), especially due to the need for specialized hospital care, long recovery, and early mortality, which can be further aggravated by lack of access to effective treatment and proper care.

Understand the economic evaluation of health technologies We have observed in previous topics that the epidemics caused by ZIKV produce harmful socioeconomic and epidemiological consequences, which continue to reverberate for years, mainly affecting economically disadvantaged communities and families, deepening social and economic inequalities, especially in poor and developing populations. Within this framework, health economics researchers have been challenged to evaluate the efficiency of health technologies for the prevention, diagnosis, and care of patients during ZIKV infection and on the onset of an associated condition. These assessments can contribute to efficient resource allocation by providing critical information for decision-making on whether to incorporate health technologies. The most appropriate studies for this purpose are full HEEs, especially cost-effectiveness analysis (CEA) and costutility analysis (CUA), which assess efficiency by analyzing the differences between costs and health outcomes (QALY, DALY, cases avoided, life years gained, etc.) of the health technologies evaluated. The main measure presented by the CEA and CUA studies is called incremental cost-effectiveness ratio (ICER), calculated by dividing the cost differences (incremental cost) and health outcomes (incremental health effect) of two technologies under evaluation, expressing the cost per additional unit of health effect, for example, cost per additional case of microcephaly avoided. Results expressed by ICERs should be compared to a cost-effectiveness threshold, which represents a willingness-to-pay. The choice of this threshold may vary widely depending on the decision-maker, the importance attributed to the health outcome, and the resources available (Owens, 1998). Because ZIKV has only recently been recognized as a serious public health concern, several technologies are still under development or have not yet been evaluated for efficiency. However, a few cost-effectiveness analyses on the subject have been published in recent years, some of which will be highlighted in the next section.

Economic evaluation of current and emerging health technologies To assess the cost-effectiveness of ZIKV control strategies, Alfaro-Murillo et al. (2016) proposed an interactive tool for informing policies on ZIKV control. The tool is available on the web and allows the insertion of epidemiological parameters and costs associated with microcephaly and GBS caused by ZIKV. The authors fed the tool with data from Latin America and the Caribbean and estimated that interventions that prevent 10,000,000 ZIKV infections across the region (1.6% of the population) would be very cost-effective with expenditures below US$409 million, indicating that substantial financial resources directed to control are justified due to the high burden associated with ZIKV.

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Considering the vaccine prioritization of the World Health Organization (WHO) for women of reproductive age, Shoukat et al. examined the efficiency of a potential ZIKV vaccine candidate from a Colombian government perspective. However, because vaccines are still under development and their efficacy is not yet known, the researchers assumed that protection efficacy is in the range 60%–90% against infection. Different simulations were performed, demonstrating that in a favorable transmission scenario, reproduction number (Ro) corresponding to 2.2 and relative transmissibility of asymptomatic infection (RTA) at 10% and without considering herd immunity, the vaccine would be cost saving (ICE 95 °C), annealing (the primers attach to single-stranded DNA; temperature depends on the targeted sequence and primer melting temperature, usually 50–60°C), and extension (enzyme builds up the missing DNA strand starting from primers; usually occurring in 60–70°C) (Fig. 2). Denaturation, annealing, and extension together comprise one thermal cycle, i.e., amplification cycle. This amplification reaction is carried as long as thermal cycling is ongoing and typically there are 40–45 cycles of these in the PCR program. In order to detect these amplification products, i.e., PCR products, we need hydrolysis probes, which can specifically detect if the correct ZIKV specific PCR product is amplified during the PCR thermal cycling. The ZIKV-specific probe anneals to its targeted single-stranded DNA after the denaturation step. The probe has an attached fluorescence reporter dye that has been silenced using a quencher. After annealing, the extension reaction is carried out by the enzymes for it, and during this reaction, the enzyme builds up repetitively the single-stranded DNA to a double-stranded DNA. The probe annealed to single-stranded DNA is processed away (hydrolysis) by the enzyme during the extension reaction and the fluorescence dye is released during this process (Fig. 2). This dye release reaction happens in every amplification cycle if the probe anneals to its targeted DNA and the extension reaction is successfully carried out. The fluorescence reporter dye detachment is detected by the device used for thermal cycling and fluorescence detection, and the device plots and saves the fluorescence data in real-time along with the thermal cycling process. The sample is positive for ZIKV if there is a correct accumulation of a fluorescent signal above the background level. The cycle threshold (Ct) value of positive reaction is determined from the number of amplification cycles required for the accumulated fluorescent probe signal to cross the general fluorescent background level (threshold level of reaction; background level).

Multiplex real-time RT-PCRs Nowadays, there are both published methods and commercial multiplex real-time PCRs, targeted for simultaneous detection of ZIKV, DENV, and CHIKV (Boga et al., 2019; Colombo et al., 2019; Mansuy et al., 2018; Mishra et al., 2019; Wu et al., 2018; Table 1). Some of these tests are still aimed at research use only, but many of these can be used in vitro diagnostics, too. The multiplex-based methods for simultaneous detection of several different viruses, benefit over the single real-time RT-PCR targeted just for one virus in diagnostic laboratories. Using multiplex-based methods, laboratories can save time, possibly reagents costs, and sample amount. As ZIKV, DENV1–4, and CHIKV can cause similar kinds of tropical disease and are transmitted by mosquitoes and located in the same tropical areas, it is recommended to test all of these agents. Coinfections of these viruses have also been reported (Carrillo-Herna´ndez et al., 2018; Pess^ oa et al., 2016), and these would be missed without multiplex testing or several parallel single target RT-PCRs.

Conventional RT-PCRs In addition to real-time RT-PCRs, there are conventional RT-PCRs, or combination of these two, which can amplify, for example, any flavivirus nucleic acids present in the sample (Lambert & Lanciotti, 2009; Moureau et al., 2007, 2010). Typically, these are used in settings where you do not know which flavivirus is the causative agent (pan-flavivirus approach) or for research purposes. These methods usually need more time and are laborious, as the amplified PCR products have to be visualized using, i.e., agarose gel electrophoresis and send for sequencing to confirm which of the flavivirus was amplified. These broad-range pan-flavivirus RT-PCRs are often nested-RT-PCRs (Fig. 3) in which there are one reverse transcriptase reaction (RT) and two separate PCR reactions. In first PCR reaction, primers targeted for flavivirus amplify a PCR product using the template produced in RT reaction from the RNA extracted from the patient sample. The PCR product in the first PCR reaction is typically longer, about 500–700 base pairs long. In the second PCR reaction, this PCR product from the first PCR reaction is used as a template for the second round PCR reaction. The broad-range RT-PCRs are not as sensitive as, for example, real-time RT-PCRs, and another amplification round is needed for visualizing the PCR product and sequencing. However, the nested-RT-PCR protocols are vulnerable because contamination and therefore false-positive results may occur (Fig. 3). It is not recommended to use these assays for daily diagnostics with large number of patient samples due to contamination risk, but instead, these can be used for research purposes or on a single occasion in

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2nd PCR:

1st PCR: Reverse transcriptase (RT): RNA is transcribed to DNA

DNA from RT reacon is used as template for 1st PCR; typically 40 cycles of amplificaon.

Amplified DNA from 1st PCR is used as a template for the 2nd round PCR; typically 30-40 cycles of amplificaon. High risk of contaminaon!

FIG. 3 Steps in nested-RT-PCR protocol.

diagnostics if other methods fail to detect the causative flavivirus (i.e., new flaviviruses appear). This protocol is slower as compared to real-time RT-PCRs which will have the confirmed results at the end of running the PCR and no extra steps are needed, i.e., agarose gel electrophoresis and sequencing.

Validation criteria To validate the molecular-based diagnostic tests, the laboratories should test the molecular methods using different sample materials (serum/plasma, whole blood, urine, etc.) from patients with diagnosed ZIKV infection in parallel with samples originated from acute DENV infection. In addition to PCR method, the laboratories should validate the nucleic acid extraction methods along with the used molecular test (Fig. 4). The combination of these two key elements, nucleic acid extraction and genome amplification, for example using real-time RT-PCR, should be tested together in order to confirm the compatibility of both elements together for the diagnosis of acute ZIKV infection. The WHO, CDC, and ECDC recommend tests based on NAATs because of confirmed and specific detection of ZIKV, and discrimination of ZIKV from DENV. However, not in all cases, the sampling is possible in the time window optimal for the NAATs and therefore after a week post-onset of symptoms, the diagnostic tests should also include the serological methods, e.g., immunoassays.

Validaon of ZIKV NAAT process Extracon of RNA What sample types needed? Whole blood Serum/plasma Urea

Different extracon methods for different sample types?

NAAT Validate your NAAT for detecon of ZIKV RNA from different sample types.

CSF All combinaons of sample type and extracon method needed should be tested during this process.

FIG. 4 Facts to remember during the validation of NAAT for diagnosis of ZIKV infection. NAAT, nucleic acid amplification test; CSF, cerebrospinal fluid.

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Policy and procedures International authorities, as World Health Organization (WHO), Centers for Disease Control and Prevention (CDC), and European Center for Disease Prevention and Control (ECDC), have provided guidance and recommendations for diagnostic tests to be carried out in case of a possible ZIKV infection. There is a specific concern about ZIKV infection (both asymptomatic and symptomatic infection) and pregnancy, and detailed guidelines for molecular testing are available: l

l

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CDC, guidance: National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Division of Vector-Borne Diseases (DVBD). https://www.cdc.gov/zika/hc-providers/testing-guidance.html Updated in November 2019. ECDC, guidance: Laboratory tests for Zika virus diagnostic and Interim guidance for healthcare providers and Zika virus laboratory diagnosis. Updated in 2016. https://www.ecdc.europa.eu/sites/default/files/media/en/publications/ Publications/zika-virus-guidance-healthcare-providers-and-laboratory-diagnosis.pdf. WHO, guidance: Laboratory testing for Zika virus infection. Interim guidance. March 23, 2016. WHO/ZIKV/LAB/ 16.1.

Mini-dictionary of the terms RT-PCR: Process where RNA is transcribed to DNA using reverse transcriptase enzyme and suitable primer/primers (RT) followed by another reaction where a certain part of the transcribed DNA is amplified multiple times using primers and PCR enzyme (PCR). Real-time RT-PCR: Same process as described above but the amplified PCR product is detected and identified using, for example, hydrolysis probe with fluorescence dye during the PCR run. The amplification can be monitored in real time during the process. Ct-value: The cycle threshold (Ct) value is determined from the number of PCR amplification cycles required for the fluorescent signal (probe-based) to cross the general fluorescent background level of the reaction. One PCR cycle consists of denaturation, annealing, and extension with probe hydrolysis. NAAT: General term for nucleic acid amplification tests, such as conventional RT-PCRs, real-time RT-PCRs, loopmediated isothermal amplification methods (RT-LAMP), nucleic acid sequence-based amplification (NASBA), and recombinase polymerase amplification (RPA) assays. IQR: Interquartile range (i.e., mid-spread, middle 50%, or H-spread), is a measure where the data set includes the middle 50% of all of data (data between 75th and 25th percentiles, or between upper and lower quartiles).

Key facts of molecular tests used for diagnosis of ZIKV infection l l

l l

l

l

There are a variety of commercial molecular methods validated to the diagnosis of ZIKV infection. Most common molecular methods are real-time RT-PCRs with a hydrolysis probe detection system which can be used for monitoring the PCR reaction in real time during the analysis. The conventional RT-PCR methods are generally not as sensitive and fast as probe-based real-time RT-PCRs. Advantage of conventional pan-flavi RT-PCRs is that these PCRs target a wide variety of different flaviviruses while probe-based real-time RT-PCRs target usually only a few flaviviruses. Pan-flavi RT-PCRs are usually nested-RT-PCRs and this increases the risk of contaminations and therefore falsepositive results and it is not recommended for daily use in diagnostic laboratories. Multiplex real-time RT-PCRs are recommended to be used for in parallel detection of multiple viral targets such as Zika virus, dengue viruses, and chikungunya viruses, which all cause clinically similar disease and are endemic in the same geographical areas.

Summary points l

l l l l

Diagnosis of Zika (ZIKV) and dengue virus (DENV) infection should be carried out together using preferably molecular method detecting nucleic acids for both ZIKV and DENV. The most reliable method for diagnosis of ZIKV infection is a molecular-based test. In acute phase, ZIKV and DENV infection can be discriminated from each other using molecular methods. There are commercial tests for molecular detection and in vitro diagnosis of ZIKV infection. Molecular tests used for diagnosis of ZIKV infection should be validated in their real context; specificity and sensitivity may vary depending on the sample types used, for example, serum/plasma, urine, whole blood, cerebrospinal fluid.

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References Balmaseda, A., Zambrana, J. V., Collado, D., Garcı´a, N., Saborı´o, S., Elizondo, D., … Harris, E. (2018). Comparison of four serological methods and two reverse transcription-PCR assays for diagnosis and surveillance of Zika virus infection. Journal of Clinical Microbiology, 56, e01785-17. Barzon, L., Percivalle, E., Pacenti, M., Rovida, F., Zavattoni, M., Del Bravo, P., … Baldanti, F. (2018). Virus and antibody dynamics in travelers with acute Zika virus infection. Clinical Infectious Diseases, 66, 1173–1180. Boga, J. A., Alvarez-Arguelles, M. E., Rojo-Alba, S., Rodrı´guez, M., de On˜a, M., & Melo´n, S. (2019). Simultaneous detection of Dengue virus, Chikungunya virus, Zika virus, yellow fever virus and West Nile virus. Journal of Virological Methods, 268, 53–55. Carrillo-Herna´ndez, M. Y., Ruiz-Saenz, J., Villamizar, L. J., Go´mez-Rangel, S. Y., & Martı´nez-Gutierrez, M. (2018). 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Laboratory tests for Zika virus diagnostic and Interim guidance for healthcare providers and Zika virus laboratory diagnosis. ECDC. https:// www.ecdc.europa.eu/sites/default/files/media/en/publications/Publications/zika-virus-guidance-healthcare-providers-and-laboratory-diagnosis.pdf. Erra, E. O., Korhonen, E. M., Voutilainen, L., Huhtamo, E., Vapalahti, O., & Kantele, A. (2013). Dengue in travelers: Kinetics of viremia and NS1 antigenemia and their associations with clinical parameters. PLoS One, 8, e65900. Estrela, P. F. N., Mendes, G. M., de Oliveira, K. G., Baila˜o, A. M., Soares, C. M. A., Assunc¸a˜o, N. A., & Duarte, G. R. M. (2019). Ten-minute direct detection of Zika virus in serum samples by RT-LAMP. Journal of Virological Methods, 271, 113675. Euliano, E. M., Hardcastle, A. N., Victoriano, C. M., Gabella, W. E., Haselton, F. R., & Adams, N. M. (2019). Multiplexed adaptive RT-PCR based on LDNA hybridization monitoring for the detection of Zika, dengue, and chikungunya RNA. Scientific Reports, 9, 11372. Frankel, M. B., Pandya, K., Gersch, J., Siddiqui, S., & Schneider, G. J. (2017). Development of the Abbott RealTime ZIKA assay for the qualitative detection of Zika virus RNA from serum, plasma, urine, and whole blood specimens using the m2000 system. Journal of Virological Methods, 246, 117–124. Gonza´lez-Gonza´lez, E., Mendoza-Ramos, J. L., Pedroza, S. C., Cuellar-Monterrubio, A. A., Ma´rquez-Ipin˜a, A. R., Lira-Serhan, D., … Alvarez, M. M. (2019). Validation of use of the miniPCR thermocycler for Ebola and Zika virus detection. PLoS One, 14, e0215642. Gouel-Cheron, A., Lumbard, K., Hunsberger, S., Arteaga-Cabello, F. J., Beigel, J., Belaunzara´n-Zamudio, P. F., … Ruiz-Palacios, G. (2019). Serial realtime RT-PCR and serology measurements substantially improve Zika and Dengue virus infection classification in a co-circulation area. Antiviral Research, 172, 104638. Herrada, C. A., Kabir, M. A., Altamirano, R., & Asghar, W. (2018). Advances in diagnostic methods for Zika virus infection. Journal of Medical Devices, 12, 408021–4080211. J€a€askel€ainen, A. J., Korhonen, E. M., Huhtamo, E., Lappalainen, M., Vapalahti, O., & Kallio-Kokko, H. (2019). Validation of serological and molecular methods for diagnosis of zika virus infections. Journal of Virological Methods, 263, 68–74. Lambert, A. J., & Lanciotti, R. S. (2009). Consensus amplification and novel multiplex sequencing method for S segment species identification of 47 viruses of the Orthobunyavirus, Phlebovirus, and Nairovirus genera of the family Bunyaviridae. Journal of Clinical Microbiology, 47, 2398–2404. L’Huillier, A. G., Lombos, E., Tang, E., Perusini, S., Eshaghi, A., Nagra, S., … Gubbay, J. B. (2017). Evaluation of Altona diagnostics RealStar Zika virus reverse transcription-PCR test kit for Zika virus PCR testing. Journal of Clinical Microbiology, 55, 1576–1584. Lin, X., Wu, M., Wang, W., Gao, Y., Zhang, W., Wu, D., … Li, G. (2019). Visual detection of Zika virus by isothermal nucleic acid amplification combined with a lateral-flow device. Analytical Methods, 11, 1795–1801. Lozier, M. J., Rosenberg, E. S., Doyle, K., Adams, L., Klein, L., Mun˜oz-Jordan, J., … Paz-Bailey, G. (2018). Prolonged detection of Zika virus nucleic acid among symptomatic pregnant women: A cohort study. Clinical Infectious Diseases, 67, 624–627. Mansuy, J. M., Lhomme, S., Cazabat, M., Pasquier, C., Martin-Blondel, G., & Izopet, J. (2018). Detection of Zika, dengue and chikungunya viruses using single-reaction multiplex real-time RT-PCR. Diagnostic Microbiology and Infectious Disease, 92, 284–287. Mansuy, J. M., Mengelle, C., Pasquier, C., Chapuy-Regaud, S., Delobel, P., Martin-Blondel, G., & Izopet, J. (2017). Zika virus infection and prolonged Viremia in whole-blood specimens. Emerging Infectious Diseases, 23, 863–865. Meaney-Delman, D., Oduyebo, T., Polen, K. N., White, J. L., Bingham, A. M., Slavinski, S. A., … U.S. Zika Pregnancy Registry Prolonged Viremia Working Group. (2016). Prolonged detection of Zika virus RNA in pregnant women. Obstetrics & Gynecology, 128, 724–730. Mishra, N., Ng, J., Rakeman, J. L., Perry, M. J., Centurioni, D. A., Dean, A. B., … Lipkin, W. I. (2019). One-step pentaplex real-time polymerase chain reaction assay for detection of zika, dengue, chikungunya, west nile viruses and a human housekeeping gene. Journal of Clinical Virology, 120, 44–50. M€ ogling, R., Zeller, H., Revez, J., Koopmans, M., ZIKV Reference Laboratory Group, & Reusken, C. (2017). Status, quality and specific needs of Zika virus (ZIKV) diagnostic capacity and capability in National Reference Laboratories for arboviruses in 30 EU/EEA countries, May 2016. Eurosurveillance, 7, 22. Moureau, G., Ninove, L., Izri, A., Cook, S., De Lamballerie, X., & Charrel, R. N. (2010). Flavivirus RNA in phlebotomine sandflies. Vector Borne and Zoonotic Diseases, 10, 195–197.

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Moureau, G., Temmam, S., Gonzalez, J. P., Charrel, R. N., Grard, G., & de Lamballerie, X. (2007). A real-time RT-PCR method for the universal detection and identification of flaviviruses. Vector Borne and Zoonotic Diseases, 7, 467–477. Pess^ oa, R., Patriota, J. V., de Souza, M. D. L., Felix, A. C., Mamede, M., & Sanabani, S. S. (2016). Investigation into an outbreak of dengue-like illness in Pernambuco, Brazil, revealed a cocirculation of Zika, Chikungunya, and dengue virus type 1. Medicine, 95. Petridou, C., Simpson, A., Charlett, A., Lyall, H., Dhesi, Z., & Aarons, E. (2019). Zika virus infection in travellers returning to the United Kingdom during the period of the outbreak in the Americas (2016-17): A retrospective analysis. Travel Medicine and Infectious Disease, 29, 21–27. Pyke, A. T., Daly, M. T., Cameron, J. N., Moore, P. R., Taylor, C. T., Hewitson, G. R., … Gair, R. (2014). Imported zika virus infection from the Cook Islands into Australia, 2014. PLoS Current Outbreaks, 2, 6. Ren, P., Ortiz, D. A., Terzian, A. C. B., Colombo, T. E., Nogueira, M. L., Vasilakis, N., & Loeffelholz, M. J. (2017). Evaluation of Aptima Zika Virus Assay. Journal of Clinical Microbiology, 55, 2198–2203. Santiago, G. A., Sharp, T. M., Rosenberg, E., Sosa Cardona, I. I., Alvarado, L., Paz-Bailey, G., & Mun˜oz-Jorda´n, J. L. (2019). Prior dengue virus infection is associated with increased viral load in patients infected with dengue but not Zika virus. Open Forum Infectious Diseases, 6, 7. Santiago, G. A., Va´zquez, J., Courtney, S., Matı´as, K. Y., Andersen, L. E., Colo´n, C., … Mun˜oz-Jordan, J. L. (2018). Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses. Nature Communications, 9, 1391. Sharp, T. M., Fischer, M., Mun˜oz-Jorda´n, J. L., Paz-Bailey, G., Staples, J. E., Gregory, C. J., & Waterman, S. H. (2019). Dengue and Zika virus diagnostic testing for patients with a clinically compatible illness and risk for infection with both viruses. Recommendations and Reports, 68, 1–10. Tozetto-Mendoza, T. R., Avelino-Silva, V. I., Fonseca, S., Claro, I. M., Paula, A. V., Levin, A. S., … Mayaud, P. (2019). Zika virus infection among symptomatic patients from two healthcare centers in Sao Paulo state, Brazil: Prevalence, clinical characteristics, viral detection in body fluids and serodynamics. The Revista do Instituto de Medicina Tropical de Sa˜o Paulo, 61, e19. Vasileva Wand, N. I., et al. (2018). Point-of-care diagnostic assay for the detection of Zika virus using the recombinase polymerase amplification method. Journal of General Virology. https://doi.org/10.1099/jgv.0.001083. Voermans, J. J. C., Pas, S. D., van der Linden, A., GeurtsvanKessel, C., Koopmans, M., van der Eijk, A., & Reusken, C. B. E. M. (2019). Whole-blood testing for diagnosis of acute Zika virus infections in routine diagnostic setting. Emerging Infectious Diseases, 25, 1394–1396. World Health Organization (WHO). (2016). Laboratory testing for Zika virus infection. Interim guidance. 23 March 2016. WHO/ZIKV/LAB/16.1. Accessed 26th of December, 2019. Wu, W., Wang, J., Yu, N., Yan, J., Zhuo, Z., Chen, M., … Xia, N. (2018). Development of multiplex real-time reverse-transcriptase polymerase chain reaction assay for simultaneous detection of Zika, dengue, yellow fever, and chikungunya viruses in a single tube. Journal of Medical Virology, 90, 1681–1686.

Chapter 11

Coinfection of Zika with Dengue and Chikungunya virus Marlen Yelitza Carrillo-Herna´ndez, Julian Ruiz-Saenz, and Marlen Martı´nez-Guti
errez Grupo de Investigacio´n en Ciencias Animales-GRICA, Universidad Cooperativa de Colombia, Bucaramanga, Colombia

Abbreviations Ae CHIKV CPE CSF DENV ELISA GBS MAYV NGS OROV PCR PRNT ZIKV

Aedes Chikungunya virus cytopathic effects cerebrospinal fluid Dengue virus enzyme-linked immunosorbent assay Guillain–Barr
e syndrome Mayaro virus new generation sequencing Oropuche virus polymerase chain reaction plaque reduction neutralization test Zika virus

Introduction Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV) are arboviruses that share a common vector in urban transmission—specifically, mosquitoes of the genus Aedes (Ae.), such as Ae. Aegypti and Ae. albopictus. Ae. aegypti has been reported as the primary mosquito species responsible for the transmission of arboviruses due to its wide geographical distribution (Vogels et al., 2019). The emergence of these viruses in new locations is owed to numerous factors, including climate change, human behaviors, patterns, and living conditions of present populations (e.g., humans invading undeveloped areas where Aedes species already reside), poor sanitation, inadequate vector control, the exchange of goods and international trips that facilitate exposure, and the transmission and dissemination of infectious agents, among other factors (Gould & Higgs, 2009). Moreover, the efficient and continuous circulation of CHIKV and ZIKV (which have emerged in numerous new tropical and subtropical countries over the last few years) have caused various cases of coinfection in humans to be reported and characterized. In this study, we describe the distribution of ZIKV coinfections with other arboviruses, the diagnostic methods used for the identification of such viruses, and the potential implications of these coinfections regarding the improvement or severity of the associated diseases.

Timeline of coinfections Coinfection is defined as the simultaneous presence of two or more infections. In the case of viruses, this exists as the simultaneous infection of two or more viruses in the same cell or organism (Kumar, Sharma, Barua, Tripathi, & Rouse, 2018). Coinfections between two arboviruses such as DENV and CHIKV were first described several decades ago. In 1962, a study assessed 150 patients from Thailand who had a diagnosis of chikungunya or dengue fever and found four cases of coinfection with DENV and CHIKV (Nimmannitya, Halstead, Cohen, & Margiotta, 1969). However, coinfections with ZIKV began to occur more recently. The expansion of this virus to other geographic areas initiated the reporting of these coinfections (Fig. 1). Coinfections with ZIKV and DENV were first reported during the ZIKV outbreak of 2014 in New Caledonia in the Pacific Islands. One such case was found in a 14-year-old child who had traveled to French Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00011-0 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Timeline of ZIKV coinfections with other arboviruses from 2014 to 2016. This figure shows the first appearances of coinfections around the world. Maps were obtained from mapchart.net (https://mapchart.net/world.html).

Polynesia (coinfection ZIKV/DENV-3), and the other case was found in a 38-year-old woman who had not reported a history of travel (coinfection ZIKV/DENV-1) (Dupont-Rouzeyrol et al., 2015). A coinfection with ZIKV and CHIKV was described for the first time in 2016 in a study carried out in Guayaquil, Ecuador. In that case, the coinfection was detected in a 57-year-old woman with a medical history of hyperthyroidism who presented with loss of deep tendon reflexes and was diagnosed with Guillain-Barr
e syndrome (Zambrano et al., 2016). The first case of a triple coinfection with ZIKV, DENV, and CHIKV was reported in 2016 in a 49-year-old man from Colombia who did not develop complications. He presented with fever (38 °C), bilateral conjunctivitis, and a maculopapular rash (Villamil-Go´mez et al., 2016). In the same year and region, an additional triple coinfection was detected in a pregnant woman coinfected with ZIKV, DENV-2, and CHIKV; neither she nor her pregnancy experienced complications (Villamil-Go´mez, Gonza´lez-Camargo, Rodriguez-Ayubi, Zapata-Serpa, & Rodriguez-Morales, 2016). Additionally, coinfections have been reported with other arboviruses such as Mayaro virus (MAYV) (de Souza Costa et al., 2019) and Oropuche virus (OROV) (Martins-Luna et al., 2020). In the case of OROV, Peru reported nine coinfections of ZIKV with OROV and one triple coinfection of ZIKV, OROV, and DENV (Table 1).

Distribution of coinfections Of the 87 countries and territories with reports of local ZIKV transmission (Malla, Shanmugaraj, & Ramalingam, 2020), only nine have reported coinfections with other arboviruses (Fig. 2). These coinfections were first registered in 2015 when ZIKV spread outside the African and Asian continents, reaching to the Pacific Islands (specifically to Yap, the Federated States of Micronesia, French Polynesia, and New Caledonia), causing an outbreak in 2013 and 2014 (Campos, Bandeira, & Sardi, 2015). Thereafter, the first case of coinfection with ZIKV and DENV was registered in New Caledonia (Dupont-Rouzeyrol et al., 2015). Subsequently, ZIKV expanded into the American continent; the first autochthonous case was declared in February 2015 on Easter Island (Chile) (Lessler et al., 2016). In May 2015, the Brazilian Ministry of Health recognized the circulation of ZIKV in Northwestern Brazil (Cardoso et al., 2015), where it has since spread to more than 18 states (PAHO/ WHO, 2015). Of the 37 studies regarding ZIKV coinfections described in Table 1, Brazil is the country with the highest occurrence of coinfections, with 20 reported cases (54.1%,); it is followed by Colombia, with five cases (13.5%), Haiti, with three cases (8.1%), Ecuador, Singapore, and Peru, with two cases each (5.4%), and New Caledonia, Nicaragua, and Thailand, with one case each (2.7%) (Fig. 3). Furthermore, regarding the proportions of coinfections found in these studies, the rate of

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TABLE 1 Chronological report of co-infections of ZIKV with other arboviruses (DENV/ZIKV, CHIKV/ZIKV, ZIKV/PROV, ZIKV/MAYV, DENV/CHIKV/ZIKV, and PROV/DENV/ZIKV) in different countries from 2015.

Country

Year

Study type

Number of samples

New Caledonia

2015

Case series

NA

DENV

NA

Dupont-Rouzeyrol et al. (2015)

Colombia

2016

Case report

NA

DENV/ CHIKV

NA

Villamil-Go´mez, Rodrı´guez-Morales, et al. (2016)

Colombia

2016

Case report

NA

DENV/ CHIKV

NA

Villamil-Go´mez, Gonza´lez-Camargo, et al. (2016)

Brazil

2016

Cross sectional

77

DENV

2/2.6%

Pess^ oa et al. (2016)

Brazil

2016

Cohort

345

CHIKV

3/0.87.0

Brasil et al. (2016)

Nicaragua

2016

Cross sectional

263

CHIKV

16/4.6

Waggoner et al. (2016)

DENV

6/1.7

DENV/ CHIKV

6/1.7

Coinfection ZIKV +

Cases/ percentage

Reference

Colombia

2016

Case report

NA

CHIKV

NA

Cherabuddi et al. (2016)

Ecuador

2016

Case series

NA

CHIKV

NA

Zambrano et al. (2016)

Brazil

2016

Cross sectional

30

CHIKV

1/3.3

Cabral-Castro, Cavalcanti, Peralta, and Peralta (2016)

Brazil

2016

Cross sectional

15

CHIKV

2/13.3

Sardi et al. (2016)

Peru´

2016

Cross sectional

139

DENV

1/0.7

Alva-Urcia et al. (2017)

Singapore

2017

Cross sectional

163

DENV

5/3.0

Chia et al. (2017)

Haiti

2017

Case report

NA

DENV

NA

Iovine et al. (2016)

Brazil

2017

Surveillance

433

DENV

4/0.9

Terzian et al. (2017)

Brazil

2017

Case report

NA

CHIKV

NA

Brito, Azevedo, Cordeiro, Marques Jr, and Franca (2017)

Singapore

2017

Case series

NA

DENV

NA

Li et al. (2017)

Brazil

2017

Cross sectional

263

CHIKV

2/0.76

Magalhaes et al. (2017)

Brazil

2017

Cross sectional

273

CHIKV

36/13.2

da Costa et al. (2017)

Brazil

2017

Cross sectional

433

DENV

4/0.92

Colombo et al. (2017)

Ecuador

2017

Cross sectional

16

CHIKV

4/25.0

Acevedo et al. (2017)

DENV/ CHIKV

4/25.0

Case report

NA

CHIKV

NA

Brazil

2018

Prata-Barbosa et al. (2018) Continued

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TABLE 1 Chronological report of co-infections of ZIKV with other arboviruses (DENV/ZIKV, CHIKV/ZIKV, ZIKV/PROV, ZIKV/MAYV, DENV/CHIKV/ZIKV, and PROV/DENV/ZIKV) in different countries from 2015—cont’d

Country

Year

Study type

Colombia

2018

Cross sectional

Brazil

2018

Surveillance

Number of samples

Coinfection ZIKV +

Cases/ percentage

157

CHIKV

8/5.1

DENV

10/6.3

DENV/ CHIKV

3/1.9

CHIKV

16/17.5

DENV

1/1.1

91

Reference Carrillo-Herna´ndez, Ruiz-Saenz, Villamizar, Go´mez-Rangel, and Martı´nez-Gutierrez (2018)

De Souza et al. (2018)

Brazil

2018

Case report

NA

CHIKV

NA

Silva et al. (2018)

Brazil

2018

Cross sectional

134

DENV

18/13.4

Azeredo et al. (2018)

Haiti

2018

Cross sectional

82

CHIKV

6/7.3

White et al. (2018)

Brazil

2018

Case series

NA

CHIKV

NA

Mehta et al. (2018)

Thailand

2019

Cross sectional

182

DENV

1/0.6

Suwanmanee et al. (2018)

Brazil

2019

Cross sectional

948

DENV

1/0.1

Silva et al. (2019)

Brazil

2019

Cross sectional

193

CHIKV

6/3.1

Bagno et al. (2019)

DENV

1/0.5

DENV/ CHIKV

1/0.5

Brazil

2019

Cross sectional

9

DENV

1

de Arau´jo, Ju´nior, Teno´rio, and dos Santos (2019)

Colombia

2019

Surveillance

23,871

CHIKV

28/0.1

Mercado-Reyes et al. (2019)

DENV

3/0.1

Brazil

2019

Surveillance

1254

DENV

12/0.9

Estofolete et al. (2019)

Brazil

2019

Case report

NA

DENV

NA

Slavov et al. (2019)

Brazil

2019

Cross sectional

453

MAYV

1/0.2

de Souza Costa et al. (2019)

Peru

2020

Cross sectional

943

OROV

9/0.9

Martins-Luna et al. (2020)

DENV/ OROV

1/0.1

NA, not applicable.

coinfection with ZIKV and DENV was documented to be between 0.013% (Colombia, 23,871 samples analyzed) and 13.4% (Brazil, 134 samples analyzed); the rate of coinfection with ZIKV and CHIKV was between 0.12% (Colombia, 23,871 samples analyzed) and 25% (Brazil, 16 samples analyzed); and the rate of triple coinfection with ZIKV, DENV, and CHIKV was between 0.52% (Brazil, 193 samples analyzed) and 1.91% (Colombia, 157 samples analyzed).

Detection of arboviruses in coinfections The infections caused by ZIKV, DENV, and CHIKV are indistinguishable at the acute disease stage. They produce similar clinical signs and symptoms, including fever, myalgia, rash, arthralgia, headache, and lymphatic node hypertrophy;

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FIG. 2 The global distribution of reports of ZIKV coinfection, including coinfection types. This map shows the countries with coinfections of ZIKV with other arboviruses in red. The colored diamonds show the different types of coinfections reported in each country. Figure generated using mapchart.net (https://mapchart.net/world.html).

FIG. 3 Distribution of studies reporting ZIKV coinfections with other arboviruses, by country. This map shows the number of reports of coinfection in each country. The countries with the most reports are Brazil (20), Colombia (5), and Haiti (3). Following are Ecuador, Peru, and Singapore (2 each) and New Caledonia, Nicaragua, and Thailand (1 each). Map was generated using mapchart.net (https://mapchart.net/world.html).

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therefore, making a specific diagnosis through clinical manifestations is difficult, and the cases are frequently diagnosed as an acute undifferentiated febrile syndrome or without focus. DENV is the most commonly contracted arbovirus, and its diagnosis is primarily based on clinical signs and symptoms. Coinfections of ZIKV with other pathogens (particularly DENV) may be relevant for the recovery and treatment of patients infected with DENV but are not usually detected by laboratory techniques. In addition, when a region experiences an epidemic or endemic of one of these arboviruses, clinical diagnoses are made by the relevant epidemiological association. This implies that the true burdens of these diseases are underestimated. When it is necessary to detect arboviruses in instances of acute febrile syndrome, close consideration must be given to the stage of the infection. Different types of laboratory tests may be effective depending on the stage of the disease. In the acute stage (prior to 7 days after the onset of symptoms), arboviruses can be isolated from serum samples by inoculating samples into cell lines from mosquitoes or mammals. However, this technique is not highly recommended for the identification of coinfections. Isolation presents a “bottleneck” effect; several passes are required before the appearance of cytopathic effects (CPEs), and the different replications of the virus or a viral interference may eliminate one of them (Kumar et al., 2016). Similarly, if culture conditions are more favorable for one virus than for the other, then one of the two viruses may be overlooked. In addition, it should be considered that not all viruses can generate CPE; therefore, one virus in a coinfection may induce CPE, while the other may not (Kumar et al., 2016). In acute disease stages, molecular methods such as single RT-PCR, multiplex RT-PCR, and RT-qPCR should be used. These techniques yield results more quickly and are more sensitive than viral isolation methods. They have higher specificity and are most commonly used in coinfection studies. However, molecular techniques are slightly more expensive and require specific training. Furthermore, though qRT-PCR is highly sensitive, it has a limited number of fluorophores used in the probes. This restricts the maximum reliable detection limit to 4–5 viruses (Acevedo et al., 2017). Because antibodies take time to appear after initial infection, detection in the later stages of infection is based on serological methods (Furuya-Kanamori et al., 2016). Included in those methods is the ELISA technique, which is used to distinguish between acute and convalescent infections by detecting IgM and/or IgG antibodies. Although this serological test is widely used for laboratory diagnostics in health institutions due to its inexpensive nature and its availability in regions with limited resources, the major difficulty it proposes is the cross-reactivity of serum antibodies between DENV and ZIKV (Arrieta, Mattar, Villero-Wolf, Gomezcaceres, & Doria, 2019). Another technique more recently used to identify causal agents of the febrile syndrome is new generation sequencing (NGS), which allows the detection of all pathogens in clinical samples as well as the discovery of new viruses (Kumar et al., 2018). This method is able to simultaneously characterize various viruses that are not considered in conventional tests as “known” or “divergent” by the detection of sequences from almost all organisms impartially. The study of coinfections with this technique reveals genetic information and allows the study of the evolution of a virus, the discovery of new clades/viral lineages, and/or the analysis or monitoring of the geographical spread of a virus (Sardi et al., 2016). Among the 37 studies regarding coinfections with ZIKV described in Table 1, the most commonly used technique was RT-PCR (n ¼ 35; 94.6%), followed by the detection of IgM/IgG by ELISA (n ¼ 15; 40.5%), the detection of Ag by ELISA (specifically for DENV, NS1) (n ¼ 5; 13.5%), viral isolation (n ¼ 6; 16.2%), and NGS (n ¼ 2; 5.4%).

Clinical outcomes of coinfections The clinical outcomes of coinfections have not been sufficiently studied. Moreover, because studies on coinfections have not addressed serious clinical symptoms, no coinfection-specific clinical symptoms have been defined, which may allow us to differentiate coinfections from monoinfections. Despite these difficulties, it is essential to recognize the different clinical signs and symptoms displayed in patients with coinfections that include ZIKV.

Coinfection with ZIKV and DENV In general, this coinfection has not been observed to cause severe synergistic effects, because patients coinfected with ZIKV and DENV have generally had mild symptoms and have recovered without requiring hospitalization (Pess^oa et al., 2016). However, patients in some studies have presented with more severe clinical manifestations than those described in a monoinfection, including severe arthralgia and joint inflammation (Iovine et al., 2016). Notably, a study of coinfected patients in Singapore reported that 60% of the patients experienced diarrhea, and one teenaged patient presented with thrombocytopenia (platelet count of 74  109/L) (Chia et al., 2017). In addition, a separate study reported that seven coinfected patients presented with abrupt decreases in platelet counts, mucosal bleeding, gingival bleeding, hepatomegaly, and liver enlargement (Lobkowicz et al., 2020). Other clinical manifestations were also reported in the upper respiratory tract in

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13%–25% of the cases studied (Lobkowicz et al., 2020). Finally, two cases have been reported of pregnant women coinfected with ZIKV and DENV. In one case, the fetus displayed functional plagiocephaly, while in the other case, the newborn died of respiratory failure (Azeredo et al., 2018).

Coinfection with ZIKV and CHIKV In a study carried out in Bahia, Brazil, the clinical manifestations associated with coinfected patients were related to the virus with the higher viral titer. For example, a patient coinfected with ZIKV and CHIKV had a higher serum viral titer of CHIKV than of ZIKV, and his symptoms were in line with those of chikungunya fever. Another patient had a higher serum viral titer of ZIKV than CHIKV, and his clinical symptoms were in line with those of Zika fever (fever, rash, myalgia, and conjunctivitis) (Sardi et al., 2016). Other patients coinfected with ZIKV and CHIKV displayed gastrointestinal (GI) symptoms (Ball et al., 2019; Cabral-Castro et al., 2016). Finally, in Colombia, two cases were reported in which the patients experienced sepsis and multiorgan failure and died. These coinfections were detected by PCR (Lobkowicz et al., 2020). Nonetheless, it should be noted that diverse clinical presentations have been reported in patients coinfected with ZIKV and CHIKV. For example, in some cases in Ecuador in which RNA of ZIKV was detected in the patients’ CSF, the patients suffered prolonged ZIKV viremia, and one case of Guillain-Barr
e syndrome was presented (Zambrano et al., 2016). In other studies, patients coinfected with ZIKV and CHIKV presented with prolonged symptoms of arthritis or arthralgia (Cherabuddi et al., 2016). In patients with a history of comorbidities and/or rheumatologic disorders, it was observed that this type of coinfection may be associated with the appearance or exacerbation of arthralgia/arthritis; with the progression of the disease, this may lead to death (K. R. Silva et al., 2018). For example, three fatal cases were described in Colombia in which adults were coinfected with ZIKV and CHIKV and presented with the neurological syndrome and neurological failure (Mercado-Reyes et al., 2019). Two of these three patients suffered comorbidities such as hypertension, diabetes mellitus, and obesity. Upon autopsy, the histopathological findings included tubular interstitial nephritis, acute demyelinating polyneuropathy, pneumonia, and changes related to systemic inflammatory response syndrome (SIRS) (Mercado-Reyes et al., 2019). Likewise, some coinfections with ZIKV and CHIKV have evolved with severe neurological manifestations. In 2016 in Recife, Brazil, RT-qPCR detected both ZIKV and CHIKV in the CSF of a 74-year-old man. In this case, the coinfection caused meningoencephalitis associated with peripheral polyneuropathy, where the cellular and protein content of the CSF indicated the presence of inflammatory cells. It is likely that after replication of both viruses in the Central Nervous System, this inflammatory profile contributed to the neurological symptoms since these viruses are highly neurotropic (Brito et al., 2017). Accordingly, the patient required hospitalization and treatment with intravenous human immunoglobulin (IVIG). He later displayed neurological improvement and was discharged. Another patient coinfected with ZIKV and CHIKV in the same country (Campo Grande, Brazil) presented with disorientation and blurred vision, was hospitalized, and was subsequently discharged because did not have any other complications (Azeredo et al., 2018). During the ZIKV outbreak in Colombia, in a study carried out by Mercado et al., two cases of fetal mortality associated with coinfection of ZIKV and CHIKV were described in two 22-year-old mothers (Mercado-Reyes et al., 2019). In the first case, the ultrasound performed at week 15 of the pregnancy showed that the fetus had acrania and anencephaly. The second case was diagnosed 34 weeks into the pregnancy owing to the lack of heart rate when the echocardiography was performed. The subsequent ultrasound showed the annulment of the skull bones (Mercado-Reyes et al., 2019). Similarly, there was another case of fetal death associated with the coinfection of ZIKV and CHIKV in a 20-year-old pregnant Brazilian woman who displayed a diffuse maculopapular rash and a high fever (39°C). Four days after the appearance of her symptoms, a routine ultrasound revealed the absence of a fetal heartbeat. The autopsy of the fetus did not show abnormalities, but the weight of the fetus was between the 3rd and 10th percentiles for its gestational age. Moreover, upon examination of the tissues, placental and renal calcifications were observed. Finally, RT-PCR indicated the presence of CHIKV in the placenta, and the kidney tested positive for ZIKV (K. R. Silva et al., 2018).

Triple coinfection with ZIKV, DENV, and CHIKV There is little established information describing the symptoms in cases of triple coinfection. In the first case reported, the patient presented with general weakness, nonpurulent conjunctivitis in both eyes, mild cervical lymphadenopathy, and mild edema of the lower extremities. His heart rate was 110 beats/min, his blood pressure was 140/90 mmHg, and no hemorrhagic or neurological findings were found; nor were signs of Guillain-Barr
e syndrome (GBS). Therefore, this patient did not require hospitalization, and he recovered after a mild clinical course (Villamil-Go´mez, Gonza´lez-Camargo, et al., 2016).

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The second case was a pregnant woman who presented with bilateral nonpurulent conjunctivitis, intense pain in the upper extremities, an itchy maculopapular rash on the thorax and abdomen, headache, arthralgia, mild-to-severe bilateral pain in the wrist, and limb edema. No neurological complications were reported, and the results of obstetric ultrasounds from 14.6 to 29 weeks were normal for the gestational age (Villamil-Go´mez, Rodrı´guez-Morales, et al., 2016). Finally, one case series in Ecuador included 16 patients with neurological symptoms. Four of them were coinfected with ZIKV, CHIKV, and DENV. Two of these patients had GBS, one had meningitis, and the last one had encephalitis, which resulted in death (Lobkowicz et al., 2020). As observed, the impacts that coinfections have on a patient’s prognosis are still unknown. The interactions between viruses are complex and variable, and the outcome of the related disease may be due to either the synergistic effect between them (Vogels et al., 2019) or to the viral interference that can result from the elimination of one of the viruses in the coinfection (Kumar et al., 2018). This potential competition between viruses regularly replicating in the same cell types can result in the establishment of one viral infection that interferes with the second (Badolato-Corr^ea et al., 2018). Moreover, it is likely that if one virus replicates more quickly than the other, that virus can infect cells first and use all of the cellular resources available for its replication (Sardi et al., 2016). Finally, it is possible that the viruses may not compete, since viruses can replicate within a host as if no other virus were present, causing no significant impact on the replication of the other virus (Vogels et al., 2019).

Conclusion Current literature reports some coinfections of ZIKV with other microorganisms, but coinfections with the arboviruses CHIKV and DENV have been more frequently studied. Some of these studies have shown that these coinfections generally do not cause a complicated clinical presentation; however, these studies are mostly case reports and small-scale crosssectional studies. Results from such studies do not allow us to determine whether the simultaneous presence of two or more infections can aggravate clinical symptoms. Therefore, the examination of coinfections opens a wide range of research opportunities. Further research is necessary to better understand the complications associated with transmission, cocirculation, and infections of arboviruses in endemic areas.

Policy and procedures Laboratory diagnoses of ZIKV, DENV, and CHIKV infections are accomplished by serologic methods, viral isolation, and molecular techniques. However, to identify coinfections, the majority of studies use RT-PCR due to its sensitivity and specificity. In the case of DENV, single and multiplex RT-PCR were described by Lanciotti in 1992 (Lanciotti, Calisher, Gubler, Chang, & Vorndam, 1992) and modified by Chien in 2006 to make RT-qPCR (Chien et al., 2006); for CHIKV, RT-qPCR has been described by several authors (Lanciotti et al., 2007; Pfeffer, Linssen, Parker, & Kinney, 2002); the same is true for ZIKV (Lanciotti et al., 2008). Moreover, multiplex RT-PCR has recently been reported to identify three viruses in the same sample (Mansuy et al., 2018; Pabbaraju et al., 2016; Santiago et al., 2018; Waggoner et al., 2016). Finally, tests are commercially available for detecting one, two, or three viruses in the same sample (Mardekian & Roberts, 2015).

Mini-dictionary of terms l l l l l

Coinfection: Simultaneous presence of two or more infection-causing agents in the same host. Monoinfection: Presence of only one infection-causing agent in a host. Cocirculation: Presence of two or more viruses in the same geographical region. Cytopathic effect: The visible morphological alterations in a cell line susceptible to infection by a virus. Case series: Report of more than two cases detailing their symptoms, complications, diagnoses, and treatment.

Key facts l

The first coinfections of ZIKV and DENV; ZIKV and CHIKV; and ZIKV, DENV, and CHIKV were reported in New Caledonia (2014), Ecuador (2016), and Colombia (2016), respectively.

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Coinfections of ZIKV with another virus (MAYV and OROV) have been reported more recently (2019–2020). The country with the highest incidence of coinfections is Brazil (ZIKV/DENV, 13%; ZIKV/CHIKV, 25%). The impacts that coinfections have on the prognosis of a patient with the associated disease are still unknown.

Summary points l l l l l l

This chapter is focused on coinfection of ZIKV with other arboviruses (DENV and CHIKV, principally). The coinfection has been reported in nine countries around the world. Brazil and Colombia are the countries with the major number of ZIKV coinfection. The coinfection of ZIKV and DENV is the most frequent. Most of the studies are case reports and small-scale cross-sectional studies. The impact that coinfections have either on the severity or recovery of the disease is still unknown.

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Santiago, G. A., Va´zquez, J., Courtney, S., Matı´as, K. Y., Andersen, L. E., Colo´n, C., … Villanueva, J. M. (2018). Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses. Nature Communications, 9(1), 1–10. Sardi, S. I., Somasekar, S., Naccache, S. N., Bandeira, A. C., Tauro, L. B., Campos, G. S., & Chiu, C. Y. (2016). Coinfections of Zika and chikungunya viruses in Bahia, Brazil, identified by metagenomic next-generation sequencing. Journal of Clinical Microbiology, 54(9), 2348–2353. Silva, K. R., Bica, B. E., Pimenta, E. S., Serafim, R. B., Abreu, M. M., Gonc¸alves, J. L., … Cavalcanti, M. G. (2018). Fatal human case of Zika and chikungunya virus co-infection with prolonged viremia and viruria. Diseases, 6(3), 53. Silva, M. M., Tauro, L. B., Kikuti, M., Anjos, R. O., Santos, V. C., Gonc¸alves, T. S., … Campos, G. S. (2019). Concomitant transmission of dengue, chikungunya, and Zika viruses in Brazil: Clinical and epidemiological findings from surveillance for acute febrile illness. Clinical Infectious Diseases, 69(8), 1353–1359. Slavov, S. N., Ferreira, F. U., Rodrigues, E. S., Gomes, R., Covas, D. T., & Kashima, S. (2019). Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit. Journal of Vector Borne Diseases, 56(2), 166. Suwanmanee, S., Surasombatpattana, P., Soonthornworasiri, N., Hamel, R., Maneekan, P., Miss
e, D., & Luplertlop, N. (2018). Monitoring arbovirus in Thailand: Surveillance of dengue, chikungunya and zika virus, with a focus on coinfections. Acta Tropica, 188, 244–250. Terzian, A. C. B., Schanoski, A. S., Mota, M. T. D. O., Da Silva, R. A., Estofolete, C. F., Colombo, T. E., … Kalil, J. (2017). Viral load and cytokine response profile does not support antibody-dependent enhancement in dengue-primed Zika virus–infected patients. Clinical Infectious Diseases, 65(8), 1260–1265. Villamil-Go´mez, W. E., Gonza´lez-Camargo, O., Rodriguez-Ayubi, J., Zapata-Serpa, D., & Rodriguez-Morales, A. J. (2016). Dengue, chikungunya and Zika co-infection in a patient from Colombia. Journal of Infection and Public Health, 9(5), 684–686. Villamil-Go´mez, W. E., Rodrı´guez-Morales, A. J., Uribe-Garcı´a, A. M., Gonza´lez-Arismendy, E., Castellanos, J. E., Calvo, E. P., … Musso, D. (2016). Zika, dengue, and chikungunya co-infection in a pregnant woman from Colombia. International Journal of Infectious Diseases, 51, 135–138. Vogels, C. B., R€ uckert, C., Cavany, S. M., Perkins, T. A., Ebel, G. D., & Grubaugh, N. D. (2019). Arbovirus coinfection and co-transmission: A neglected public health concern? PLoS Biology, 17(1), e3000130. Waggoner, J. J., Gresh, L., Mohamed-Hadley, A., Ballesteros, G., Davila, M. J. V., Tellez, Y., … Pinsky, B. A. (2016). Single-reaction multiplex reverse transcription PCR for detection of Zika, chikungunya, and dengue viruses. Emerging Infectious Diseases, 22(7), 1295. Waggoner, J. J., Gresh, L., Vargas, M. J., Ballesteros, G., Tellez, Y., Soda, K. J., … Harris, E. (2016). Viremia and clinical presentation in Nicaraguan patients infected with Zika virus, chikungunya virus, and dengue virus. Clinical Infectious Diseases, 63(12), 1584–1590. ciw589. White, S. K., Mavian, C., Elbadry, M. A., De Rochars, V. M. B., Paisie, T., Telisma, T., … Morris, J. G., Jr. (2018). Detection and phylogenetic characterization of arbovirus dual-infections among persons during a chikungunya fever outbreak, Haiti 2014. PLoS Neglected Tropical Diseases, 12(5), e0006505. Zambrano, H., Waggoner, J. J., Almeida, C., Rivera, L., Benjamin, J. Q., & Pinsky, B. A. (2016). Zika virus and chikungunya virus coinfections: A series of three cases from a single center in Ecuador. The American Journal of Tropical Medicine and Hygiene, 95(4), 894–896.

Chapter 12

Zika virus, pathology, and control: Zika vaccine strategies in development Gilles Gadea, Wildriss Viranaicken, and Philippe Despre`s La Reunion University, INSERM U1187, CNRS UMR 9192, IRD UMR 249, Infectious Processes in Tropical Island Environment (PIMIT) Laboratory, Technology Platform CYROI, Sainte-Clotilde, La Reunion Island, France

Abbreviations A.D.E CZS DENV E EDI EDII EDIII GBS GMP JEV LAV LNP NS TBEV UTR VLP VSV WHO WNV YF-17D YFV ZIKV

antibody-dependent enhancement congenital Zika Syndrome dengue virus envelope protein antigenic domain I of the envelope E protein antigenic domain II of the envelope E protein antigenic domain III of the envelope E protein Guillain-Barre syndrome good manufacturing practice Japanese encephalitis virus live-attenuated viral vaccine lipid-nanoparticle nonstructural protein Tick-Borne encephalitis virus untranslated region virus-like particle vesicular stomatitis virus World Health Organization West Nile virus live-attenuated 17D strain of yellow fever virus yellow fever virus Zika virus

Introduction Mosquito-borne Zika virus (ZIKV) belongs to the flavivirus genus (Flaviviridae family), which includes other medically important viruses such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Tick-borne encephalitis (TBE) virus (Choumet & Despre`s, 2015). For more than a century, deserving efforts have been devoted to the development of antiflavivirus vaccines and licensed vaccines for preventing infection with YFV, JEV, DENV, and TBE are available (Bos, Gadea, & Despre`s, 2018; Ishikawa, Yamanaka, & Konishi, 2014). Most of them are live-attenuated or inactivated vaccine strains. The emergence of Asiatic strains of ZIKV in the South Pacific (2007–2014) and then in the Americas in 2015 has been associated with unprecedented complications in humans as congenital Zika syndrome (CZS) and Guillain-Barr
e syndrome (GBS) (Cao-Lormeau et al., 2016; Baud, Gubler, Schaub, Lanteri, & Musso, 2017; Krauer et al., 2017; Pierson & Diamond, 2018). ZIKV transmission in humans classically involves a blood meal by infected female mosquitoes from the Aedes genus but sexual contact, blood transfusion, and intrauterine transmission have also been documented as nonusual transmission routes (Runge-Ranzinger, Morrison, Marique-Saide, & Horst, 2019). ZIKV can be detected in human biological fluids for prolonged periods after the infection (Paz-Bailey et al., 2018). Such findings highlight the need for effective disease prevention measures, especially with regard to the risk of CZS and the presence of viral reservoirs in human fluids such as semen ( Joquet et al., 2017; Mead, Hills, & Brooks, 2018). Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00012-2 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Zika virus routes of transmission. Zika vaccines are intended to prevent the conventional (mosquito bite) and nonconventional (contacts with infected body fluids) routes of ZIKV transmission in humans.

Vaccines are a part of the prevention strategies against Zika disease but the correlates of protection that relate to vaccine development still remain elusive. Numerous vaccine candidates are currently in the pipeline in response to the declaration of Public Health Emergency of International Concern by the World Health Organization (WHO, 2016). The development of vaccine candidates for preventing ZIKV infection in women of reproductive age has been claimed as a public health priority by the WHO. It is presumed that such a vaccine could prevent the onset of viremia in individuals infected by ZIKV after the bite of a mosquito or following close contact with contaminated body fluids, including semen, regardless of whether the viral strains belong to the African or Asian/American genetic lineage (Fig. 1). So far, it is worth noting that all contemporary epidemic ZIKV strains belong to the Asian lineage (Beaver, Lelutiu, Habiv, & Skountzou, 2018). The development of vaccine candidates makes essential the knowledge of correlates and surrogates required for a rapid and long-term protection against ZIKV infection in vaccinated individuals. An emerging consensus claims that efficient vaccination involves elicitation of a robust humoral response based on the production of neutralizing antibodies against the envelope E protein, which is present on the ZIKV surface (Heinz & Stiasny, 2017). However, the possibility of a crossantigenic reactivity between ZIKV and other flaviviruses raises the open question of immune enhancement leading to more severe ZIKV infection in individuals with preexisting immunity against related flaviviruses. A vaccine candidate should ideally avoid such a risk, especially for DENV infections for which the antibody-dependent enhancement process contributes to more severe forms of the disease during secondary infections with heterologous serotypes (Bos et al., 2018). In the present chapter, we summarize the more important questions related to the development of safe and effective ZIKV vaccines for humans. The current ZIKV vaccine platforms and challenges for the preclinical and clinical vaccine studies are discussed.

Adaptive immunity to ZIKV infection The humoral immune response to ZIKV Both envelope E and nonstructural NS1 proteins are considered as two major targets for eliciting protective humoral immunity against ZIKV infection (Bailey et al., 2018; Heinz & Stiasny, 2017) (Fig. 2). The production of neutralizing anti-ZIKV antibodies relates to the accessibility of neutralizing antibody epitopes on virus’s surface (Yang, Dent, Lai, Sun, & Chen, 2017). The E protein that mediates the early steps of ZIKV replication in the host cell is the main target for neutralizing antibodies. The E ectodomain is divided into three distinct antigenic domains, EDI, EDII, and EDIII, each of which has been targeted by neutralizing anti-ZIKV antibodies (Heinz & Stiasny, 2017). The EDI comprises a glycanloop, which could play a role in the accessibility of neutralizing antibody epitopes (Frumence et al., 2019). The EDII includes a pH-dependent fusion loop that mediates fusion between the viral envelope protein and intracellular cell

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FIG. 2 Zika genomic RNA organisation. Genetic organization of Zika genomic RNA and expression of viral proteins encoded by the structural (C, prM/ M, and E) and nonstructural (NS1 to NS5) protein regions. The open arrowheads indicate the cleavage sites by the viral and cellular proteases. Both E and NS1 proteins are major antigen targets for eliciting protective immunity against ZIKV.

membranes. The EDIII contains flavivirus type-specific epitopes that are recognized by neutralizing anti-ZIKV antibodies (Gallichotte et al., 2019). It has been demonstrated that immunization with a dimeric E protein stimulates the production of neutralizing anti-ZIKV antibodies that target complex epitopes on virus particles (Collins et al., 2019; Metz et al., 2019). ZIKV NS1 is expressed as a dimer on the cell surface and secreted as a hexameric lipoprotein particle (Hilgenfeld, 2016). It has been reported that human NS1-specific antibodies have protective efficacy against ZIKV in the mouse model (Bailey et al., 2018) (Fig. 2). Immunization with recombinant NS1 can confer partial protection against ZIKV in the absence of neutralizing antibodies (Brault et al., 2017; Li,Dong, et al., 2018). An advantage of protective NS1-specific antibodies could be to reduce the potential risk of the antibody-dependent enhancement (A.D.E.) phenomenon observed with the E-specific antibodies directed against ZIKV and DENV.

The T-cell immune response to ZIKV A robust T cell-mediated immunity can take place during ZIKV infection (Lai et al., 2017; Pardy & Richer, 2019). Using mouse models, it has been shown that both CD4 + and CD8 + T-cells may play an important role in the T-cell response to ZIKV ( Jurado et al., 2018). Immunogenic T-cell epitopes have been detected in almost all viral proteins, structural proteins being targeted by CD4 + T-cells and nonstructural proteins by CD8 + T-cells. To date, it is still unclear whether the CD8 + Tcell response contributes to ZIKV pathogenesis in humans (Pardy & Richer, 2019). Consequently, it is urgent to better understand the role of T-cell-mediated immunity in ZIKV pathogenesis in order to determine whether effectiveness of a Zika vaccine candidate necessitates the induction of a robust CD4 +/CD8 + T-cell immunity toward ZIKV immunodominant epitopes.

Key challenges facing Zika vaccine development Target populations for ZIKV vaccination We learned with the emergence of ZIKV how it is recognized as a singular arbovirus, which does not make so easy the targeting of populations for Zika vaccination. Several of the ZIKV specificities were highlighted during the recent epidemics in the South Pacific and the Americas. We have now evidence that ZIKV infection can result in neurological disorders such as GBS and, more importantly, in congenital abnormalities (termed CZS), emblematically represented by microcephaly (Krauer et al., 2017). In 2013, during the French Polynesia outbreak that lasted from October 2013 to April 2014, cases of GBS were related to ZIKV infection (Cao-Lormeau et al., 2016). Dissemination of ZIKV in South and Central America was associated with an increase in cases of GBS among individuals diagnosed for ZIKV infection (Nascimento & da Silva, 2017). Intrauterine fetal infection by ZIKV was first highlighted in Brazil in 2016 by the sudden increase of microcephaly rate concomitantly with virus spread in South America in 2015. We now have evidence that ZIKV infection is associated with

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severe birth defects and pediatric neurodevelopmental abnormalities (Moore et al., 2017). The threat of fetal infection requires the development of ZIKV vaccines that could protect the female reproductive tract and reduce the risk of the fetus acquiring CZS (Schwartz, 2018). The first reporting of a potential sexual ZIKV transmission in humans has been documented in the USA in 2008 (Sherley & Ong, 2017). During the outbreaks in French Polynesia and the Americas, many other cases came to support the evidence that ZIKV is a newly identified sexually transmitted infection (STI) (Magalhaes, Foy, Marques, Ebel, & Weger-Lucarelli, 2018). Several months after the acute infection, the detection of ZIKV RNA in both the male urogenital and female reproductive tracts highlighted the risk of genital persistence. A long-term shedding of ZIKV from the male reproductive tract increases the risk of sexual transmission. Understanding the contribution of asymptomatic cases of ZIKV infection in STI incidence represents a new priority in terms of public health (Moghadas et al., 2017). It is now well admitted that ZIKV can persist in different human tissues including both the male urogenital and female reproductive tracts. Such ZIKV persistence has also been observed in other immune-privileged organs such as the brain and retina. Indeed, the virus has been detected in the liver as well as in the kidneys, leading to a prolonged viral shedding in the urine (Alcendor, 2018). ZIKV persistency also raises the transplantation risk in immunocompromised hosts receiving grafts from infected donors (Nogueira et al., 2017). It has been recently proposed that ZIKV infection could lead to fatal nonneurological complications in older patients with underlying comorbidities (Rajahram et al., 2019).

The protection afforded by ZIKV vaccination The development of ZIKV vaccine candidates is simplified by the existence of a unique ZIKV serotype, which is not the case for DENV. The development of animal models for ZIKV infection such as mice and nonhuman primates led to preclinical vaccine studies and thus demonstrated the effectiveness of ZIKV vaccine candidates from various platforms (Morrison & Diamond, 2017). The respective role of the humoral and T-cell responses in the protective immunity against ZIKV is still debated (Lai et al., 2017). Elicitation of neutralizing antibodies is considered as a major immune correlate for protection against ZIKV infection but it is not well understood whether the establishment of a sterilizing humoral immunity is needed. The exact role of the T-cell responses in the physiopathology of ZIKV infection is an important issue that also remains to be investigated ( Jurado et al., 2018; Pardy & Richer, 2019). It has been reported that cross-reactive dengue antibodies could have a detrimental effect on ZIKV infection by promoting the ADE phenomenon (Bardina et al., 2017; Langerak et al., 2019). The existence of cross-reactive T-cells between DENV and ZIKV has been documented from tests on patient blood specimens (Wen et al., 2017). The possibility that preexisting DENV-specific immune responses might have an influence on the effectiveness of a Zika vaccine in regions where both DENV and ZIKV are cocirculating is still an open debate. Preclinical ZIKV vaccine studies have shown that protection was correlated with serum neutralizing activity, passive transfer studies demonstrating protection against ZIKV challenge in mice and nonhuman primates (Abbink, Stephenson, & Barouch, 2018; Barrett, 2018; Poland et al., 2018). Indeed, most efforts are directed on the production of vaccines leading to strong neutralizing antibody response. Such antibodies mainly target E protein quaternary epitopes on virus particles (Collins et al., 2019). The scientists’ challenge now is to maintain the E quaternary structure displayed on the virus particle, which is thought to be determinant to raise an effective protection. It is of note that expression of antigenic domain EDIII, which is one major target of neutralizing antibodies, has the ability to elicit protective humoral immune response against ZIKV (Gallichotte et al., 2019). Lastly, ZIKV vaccination could be achieved by an immunization against a recombinant NS1 protein in order to produce anti-NS1 antibodies showing protective effect against ZIKV infection (Bailey et al., 2018). The development of ZIKV vaccine candidates has been initiated when the epidemics started in the Americas in late 2015 (Beaver et al., 2018; Makhluf & Shresta, 2018; Maslow, 2019). In this chapter, our objective is not to provide an exhaustive list of the numerous ZIKV vaccine candidates (there are many publications on this subject) but rather to give an overview of the vaccine strategies which could be considered as the most promising with respect to target populations to be vaccinated against ZIKV. Two major platforms of ZIKV vaccine candidates have been privileged: the whole virus-based vaccines and subunit virus-based vaccines (Fig. 3).

Zika vaccine platforms Whole virus-based vaccines Whole virus-based vaccines consist of viruses that are treated in such way a that they have lost or reduced virulence within the host. The first strategy involves inactivation of virus particles by exposure to chemical treatment such as formaldehyde

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FIG. 3 Zika vaccine design strategies. Anti-Zika vaccine platforms and their strategy.

or heat. Classically, killed viral vaccines require adjuvants such as aluminum salts or oil to provide a better immune response in vaccinated individuals. The second strategy consists of live-attenuated vaccines (LAV) that are generated by reducing the pathogenic properties of the virus. Still, they retain their ability to induce an effective immune response as in natural infection and have intrinsic adjuvant properties. Several LAV have been successfully raised for prophylaxis of medically important flaviviruses such as LAV of YFV, JEV, or TBEV or chimeric dengue-yellow fever live attenuated vaccine (Heinz & Stiasny, 2012). Nevertheless, virus virulence evaluation can be subjected to interpretation with a nonnegligible impact on the safety and effectiveness of LAV (Ishikawa et al., 2014). New generation of LAV for ZIKV can be achieved by the mutational approach based on a reverse genetic method (Table 1). Most of them have been tested in a mouse model. LAV candidates containing a short deletion in viral capsid or into the 30 end of the untranslated region (30 UTR) have been shown to induce a long-term protective immunity against ZIKV (Shan et al., 2017; Xie et al., 2018). A nonglycosylated chimeric ZIKV with historical African strain MR766 as the backbone and structural proteins from the epidemic Brazilian strain BeH819015 enables the production of anti-ZIKV antibodies that neutralize different viral strains (Frumence et al., 2019). Such a chimeric ZIKV has been developed to allow a prime-boost strategy where a live virus is used for the first dose and purified exosomes presenting viral antigens for the second dose with the aim of stimulating a long-term protective immunity against ZIKV infection (Wilder-Smith et al., 2018; Frumence et al., 2019). Immunization with LAV 17D of YFV (YF-17D) and SA14–14-2 of JEV elicits a long-lasting immune protection against YFV or JEV and the two vaccines are currently used for human prophylaxis (Heinz & Stiasny, 2012). It has been reported that chimeric YFV-ZIKV and JEV-ZIKV, which contain the ZIKV envelope protein region inserted into genomic RNA of LAV YF-17D or SA14–14-2, can confer protection against ZIKV infection (Kum et al., 2018; Li, Ke, et al., 2018). Recently, a codon pair deoptimization strategy has been successfully applied to generate a live attenuated ZIKV for inducing an immune response against ZIKV and protection in pregnancy (Li, Ke, et al., 2018).

Subunit virus-based vaccines Subunit virus-based vaccines differ from whole virus-based vaccines as they only contain the antigenic parts of the virus of interest, which are thought to be necessary to elicit a protective immunity. As they contain no live component of the pathogen of interest, they are considered as very safe. Depending on their vectorization system, subunit-based vaccines can also require incorporation of adjuvants to elicit a strong protective immune response, because the antigens themselves

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TABLE 1 Whole virus-based Zika vaccine candidates in development. Zika virus vaccine candidate

Viral strain (origin)

Mouse strain

Immunization age

Boost

Correlate of protection

Laboratory-adapted virus variant

H/PF/2013 (French Polynesia)

A129

Adult

No

Mouse protection including fetus to virus challenge

1

Laboratory-adapted virus variant

PA259249 (Panama)

A129

Adult

No

n.d.

2

Inactivated virus particles

PRVABC59 (Puerto Rico)

BALB/c

Young

Yes

Mouse protection to virus challenge

3

Live virus expressing nonglycosylated NS1protein

KU955593.1 (Cambodia)

A129

Adult

No

Mouse protection including fetus to virus challenge

4

Live virus expressing both nonglycosylated E and NS1 proteins

MR766 (Uganda)

A129

Young

No

Mouse protection to virus challenge

5

Chimeric virus expressing nonglycosylated E protein

MR766 (Uganda) and BeH819015 (Brazil)

BALB/c

Adult

Yes

In vitro neutralization test

6

Referencea

a

1. Kwek et al. (2018); 2. Li et al. (2019); 3. Baldwin et al. (2018); 4. Richner et al., 2017 ; 5. Annamalai et al. (2019); 6. Frumence et al. (2019).

are not sufficient to induce adequate long-term immunity. Several technologies are currently in use to generate subunitbased vaccines against ZIKV and were shown to be successful in mice to elicit immune response against ZIKV and confer protection in pregnancy (Table 2). For ZIKV, the expression of viral protein subunits mainly concerns the E and NS1 proteins as recombinant antigens driving a protective humoral immune response against ZIKV. There is a large variety of expression systems, prokaryotes, or eukaryotes such as bacteria, yeast, mammalian, or insect cells, with different advantages, allowing the production of large scales of viral proteins with respect to their antigenic properties (To et al., 2018). Virus-like particles (VLPs) are multimeric nanostructures assembled from native viral structural proteins but are devoid of any genetic material. ZIKV VLPs are composed of high-density displays of viral surface proteins. Such ZIKV VLPs are stable and show excellent adjuvant properties to stimulate a protective immune response against ZIKV (Boigard et al., 2017). The nucleic acid-based technology involves directly delivering the naked viral genetic material encoding the major antigens into the vaccinated recipient. The body’s own cells are required to produce viral antigens and then elicit a specific immune response. It has been demonstrated that lipid-encapsulated mRNAs encoding ZIKV envelope proteins are effective for inducing a sterilizing immunity against ZIKV in a mouse model (Richner et al., 2017). Advances in the fields of molecular biology and genetic engineering had allowed to create live recombinant vectors capable of delivering heterologous antigens by the introduction of viral antigen-encoding genes. This approach takes advantage of the infection capacity and the immunological properties of a live vector such as measles virus vaccine, vesicular stomatitis virus, adenovirus, or vaccinia to produce recombinant ZIKV antigens and induce a robust anti-ZIKV immune response (Hassan et al., 2019; Li, Dong, et al., 2018; Perez et al., 2018).

Clinical evaluation of Zika vaccine candidates With the large number of ZIKV vaccines that have been and are still in development, ten have already progressed into clinical trials (Table 3). The ZIKV vaccine clinical development involves a three-phase process (Gruber et al., 2017). Phase I study is required to provide information on safety and immunogenicity of the ZIKV vaccine candidate in a small number of adult volunteers without any risk of ZIKV infection. Phase II study pursues Phase I with a much greater number of volunteers. Phase III study evaluates the ability of the ZIKV vaccine candidates to provide large protection against Zika disease for people living in regions where ZIKV is endemic or epidemic.

TABLE 2 Subunit virus-based Zika vaccine candidates in development. Experimental model

Immunization age

Boost

Correlate of protection

Referencea

H/PF/2013 (French Polynesia)

Mouse

Adult

Yes

Protection

1

prM-E

2007 (YAP)

Mouse

Young

No

Protection mice and fetuses

2

Chimpanzee adenovirus

M-E

FSS13025 (Cambodia)

Mouse

Young

No

Protection (including testis)

3

Measles virus

prM-E (soluble E—no stem anchor)

KU365777.1 (Brazil)

Mouse

Adult

Yes

Protection mice and fetuses

4

YF chimera

prM-E

H/PF/2013 (French Polynesia)

Mouse

Young

No

Protection

5

Adenovirus

prM-E + NS1

KX056898.1 (China)

Mouse

Young

Yes

Protection of 6-week-old pups

6

Adenovirus ChAdOX1

prM-E

Asian consensus sequence

Mouse

Young

Yes

Protection

7

VSV

prM-E

KU681081.3 (Thailand)

Mouse

Adult

No

Protection

8

VSV or DNA

prM-E + NS1

FSS13025 (Cambodia)

Mouse

Young

Yes

Protection

9

Vaccinia virus Ankara strain

prM-E

KU312312 (Suriname)

Mouse

Young

Yes

Protection

10

DENV chimera

EDIII

H/PF/2013 (French Polynesia)

Human

Adult

–

Reactivity against chimeric virus

11

–

CprME + NS2B/NS3

H/PF/2013 (French Polynesia)

Mouse

Adult

Yes

N.d.

12

–

EDIII (mutant 375)

KU321639.1 (Brazil)

Mouse

Adult

Yes

Protection mice and fetuses

13

–

E

PRVABC59 (Puerto Rico)

NHP

Adult

Yes

Passive transfer in mice

14

–

EDIII

KU312312 (Suriname)

Mouse

Young

Yes

Passive transfer in mice

15

–

E

H/PF/2013 (French Polynesia)

Mouse

Young

Yes

Passive transfer in A129 mice

16

Naked DNA

NS1

PRVABC59 (Puerto Rico)

Mouse

Adult

Yes

Passive transfer in STAT2 /  mice

17

Naked DNA

prM-E (prM signal from JEV)

H/PF/2013 (French Polynesia)

Mouse

Adult

Yes

Protection

18

mRNA in LNP

prM-E

EU545988 (Micronesia)

Mouse

Adult

Yes

Protection of fetuses

19

self-replicating RNA

prM-E

Rio-S1 (KU926310.1)

Mouse

Adult

Yes

Protection

20

Strategy

Vector

Zika protein

ZIKV strain (origin)

Recombinant vectors

Gorilla adenovirus

prM-E

YF chimera

Viral antigens

Nucleic acids

a

1. Hassan et al. (2019); 2. Kum et al. (2018); 3. Xu et al. (2018); 4. Nurnberger et al. (2019); 5. Touret et al. (2018); 6. Liu et al. (2018); 7. Lopez-Camacho et al. (2018); 8. Emanuel et al. (2018); 9. Li, Yu, et al. (2018); 10. Perez et al. (2018); 11. Gallichotte et al. (2019); 12. Boigard et al. (2017); 13. Tai et al. (2019); 14. Medina et al. (2018); 15. Zhang et al. (2019); 16. Metz et al. (2019); 17. Bailey et al. (2019); 18. Jagger et al. ; 19. Richner et al. (2017); 20. Zhong et al. (2019).

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TABLE 3 Zika vaccine candidates in clinical trials. Vaccine type

Vaccine name

Clinical trial phase

Last update

Sponsor

Inactivated

ZPIV

Phase I / recruiting

July 26, 2019

National Institute of Allergy and Infectious Diseases

NCT03008122 NCT02963909 NCT02952833 NCT02937233

PIZV

Phase I / active, not recruiting

March 26, 2019

Takeda

NCT03343626

VLA-1601

Phase I / completed

July 26, 2019

Valneva Austria GmbH

NCT03008122

Attenuated live virus

rZIKV/ D4Δ30–713

Phase I / completed

October 29, 2019

National Institute of Allergy and Infectious Diseases

NCT03611946

DNA

GLS-5700

Phase I / completed

December 5, 2018

GeneOne Life Science, Inc.

NCT02809443 NCT02887482

ZKADNA085– 00-VP

Phase II / active, not recruiting

July 25, 2019

National Institute of Allergy and Infectious Diseases

NCT03110770

ZIKA001

Phase I / recruiting

November 6, 2019

University of Oxford

NCT04015648

mRNA-1325

Phase I / completed

December 9, 2019

ModernaTX, Inc.

NCT03014089

mRNA-1893

Phase I / recruiting

November 13, 2019

ModernaTX, Inc.

NCT04064905

MV-Zika

Phase I / recruiting

August 13, 2019

Themis Bioscience GmbH

NCT04033068 NCT02996890

mRNA

Measles vaccine

Clinical Trials. gov Identifier

At this stage of our knowledge on Zika disease, ZIKV vaccination concerns different populations living in endemic areas as priority targets, such as the pregnant and women of childbearing age, individuals exposed to STI, recipients for solid organ transplantation, or older patients with comorbidities. It has been proposed inclusion of pregnant women in the clinical trials of ZIKV vaccine candidates (Schwartz, 2018).

Concluding remarks The development of Zika vaccines has been initiated in response to the first epidemics of Zika disease in the Americas with the aim of preventing the risks of CZS as well as nonconventional transmission of the pathogen. Using different vaccine platforms, numerous ZIKV vaccine candidates were designed mainly based on the production of neutralizing antibodies as a surrogate of protective adaptive immunity. Mutational approaches based on the reverse genetic method, nucleic acidbased technologies, vector-based vaccines, and viral antigen subunits have been used to develop ZIKV vaccine candidates and most of them have been successfully validated in preclinical studies. To date, about 10 ZIKV vaccine candidates have been in Phase I clinical trials and three are planned to enter Phase II (Barrett, 2018; Garg, Mehmetogly-Gurbuz, & Joshi, 2018; Maslow, 2019). It is noteworthy that some of them are inactivated ZIKV and their inoculation necessitates an adjuvant. However, all these vaccine candidates will have to face major issues for their validation. Although it is widely admitted that neutralizing anti-ZIKV antibodies are the major correlates of protection against ZIKV infection, it is still unclear whether they will be sufficient to avoid the nonvector-borne-transmission of ZIKV from human-to-human in link with the unexpected tropism of the pathogen toward sexual organs (Eckels, De La Barrera, & Putnak, 2019; Poland et al., 2018). Due to the significant decline in the Zika disease incidence in endemic areas, evaluation of vaccine efficacy has become increasingly difficult. The ZIKV vaccine candidates are now facing up to a double challenge, where they must not only be able to elicit a sterilizing and long-term immunity to ZIKV but also to dispose of a robust clinical validation model with respect to the target populations for ZIKV vaccination (Gruber et al., 2017; Wilder-Smith et al., 2018).

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Policy and procedures Evaluation of safety for each vaccine candidate firstly demands implementation of Good Manufacturing Practices (GMP) requirements in vaccine production according to the WHO GMP guidelines for pharmaceutical products (WHO, 2016). Classically, a vaccine clinical development requires a three-phase process. Phase I study is expected to provide information on safety and immunogenicity of a vaccine candidate in a small number of adult volunteers similar to individuals for whom the candidate vaccine is intended. Phase II study extends Phase I with a much greater number of volunteers. Lastly, Phase III study assesses the ability of vaccine candidates to provide large protection against disease in a great number of people.

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Vaccine: A vaccine is an antigenic agent administered to stimulate the immune system of a living organism to elicit long-term protection against a pathogen. Whole virus-based vaccines: A whole virus-based vaccine consists of virus particles that are treated in such a way that they have lost or strongly reduced their pathogenic characteristics toward the target hosts. Subunit virus-based vaccine: A subunit vaccine only contains the antigenic parts of the virus of interest, which are thought to be necessary to elicit protective immunity. Congenital syndrome: A congenital syndrome is a disease, with or without malformation, that appears in utero and is often detected at birth. Guillain-Barr
e syndrome: Guillain-Barr
e syndrome (GBS) is an autoimmune disease responsible for peripheral nerve damage and paralysis. ADE: The antibody-dependent enhancement (ADE) phenomenon can occur when preexisting humoral immunity from a primary flavivirus infection potentiates a secondary infection by a related flavivirus.

Key facts on Zika vaccine l

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Effective vaccination against the Zika virus necessitates a robust and long-term protective immune response mostly based on the production of anti-Zika virus neutralizing antibodies. A vaccine against Zika virus should avoid the risk of an antibody-dependent enhancement phenomenon in individuals with preexisting immunity against other flaviviruses, especially dengue virus. An effective prevention strategy against Zika virus should avoid congenital Zika syndrome and virus persistence in human body fluids. An effective vaccine should prevent the onset of viremia in individuals infected by Zika virus after the bite of a mosquito or following a close contact with contaminated human body fluids, including semen. A priority is the development of vaccine candidates for preventing Zika virus infection in women in reproductive age. The development of ZIKV vaccine candidates is orientated toward the generation of live-attenuated viruses and expression of recombinant viral antigens or viral nucleic acids.

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This chapter focuses on vaccine strategies against the emerging mosquito-borne Zika virus. The women in procreative age are considered as one of the main target population for Zika virus vaccination. The protective immunity against Zika virus infection is mainly based on the production of antibodies against the envelope E and nonstructural NS1 proteins. The production of neutralizing anti-Zika virus antibodies is a correlate of protective immunity against Zika virus infection. A Zika vaccine must not promote antibody-dependent enhancement facilitation toward related flaviviruses, including dengue viruses. The current Zika vaccine platforms consist of whole-based and subunit-based vaccines. Several Zika vaccine candidates are currently challenged in clinical trial Phases I and II.

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Kum, D. B., Mishra, N., Boudewijns, R., Glawyn-Ng, I., Alfano, C., Ma, J., … Dallmeier, K. (2018). A yellow fever-Zika chimeric virus vaccine candidate protects against Zika infection and congenital malformations in mice. npj Vaccines, 3, 56. Kwek, S. S., Wanabe, S., Chan, K. R., Ong, E. Z., Tan, H. C., Ng, W. C., … Ooi, E. E. (2018). A systematic approach to the development of a safe live attenuated Zika vaccine. Nature Communications, 9(1031). https://doi.org/10.1038/s41467-018-03337-2. Lai, L., Rouphael, N., Xu, Y., Natrajan, M. S., Beck, A., Hart, M., … Emory Zika Patient Study Team. (2017). Innate, T-, and B-cell responses in acute human Zika patients. Clinical Infectious Diseases, 66, 1–10. Langerak, T., Mumtaz, N., Tolk, V. I., Van Gorp, E. C. M., Martina, B. E., Rockx, B., & Koopmans, M. P. G. (2019). The possible role of cross-reactive dengue virus antibodies in Zika virus pathogenesis. PLoS Pathogens, 15, e1007640. 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Incorporation of NS1 and prM/M are important to confer effective protection of adenovirus-vectored Zika virus vaccine carrying E protein. NPJ Vaccines, 3, 29. Lo´pez-Camacho, C., Abbink, P., Larocca, R. A., Dejnirattisai, W., Boyd, M., Badamchi-Zadeh, A., … Reyes-Sandoval, A. (2018). Rational Zika vaccine design via the modulation of antigen membrane anchors in chimpanzee adenoviral vectors. Nature Communications, 9, 2441. Magalhaes, T., Foy, B. D., Marques, E. T. A., Ebel, G. D., & Weger-Lucarelli, J. (2018). Mosquito-borne and sexual transmission of Zika virus: Recent development and future directions. Virus Research, 254, 1–9. Makhluf, H., & Shresta, S. (2018). Development of Zika virus vaccines. Vaccine, 6, 7. Maslow, J. N. (2019). Zika vaccine development-current progress and challenges for the future. Tropical Medicine and Infectious Disease, 4, E104. Mead, P. S., Hills, S. L., & Brooks, J. T. (2018). Zika virus as a sexually transmitted pathogen. 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Shan, C., Muruato, A. E., Nunes, B. T. D., Luo, H., Xie, X., Mediros, D. B. A., … Si, P. Y. (2017). Nature Medicine, 23, 763–767. Sherley, M., & Ong, C. W. (2017). Sexual transmission of Zika virus: A literature review. Sexual Health, 15, 183–199. Tai, W., Chen, J., Zhao, G., Geng, Q., He, L., Chen, Y., … Dua, L. (2019). Rational design of Zika virus subunit vaccine with enhanced efficacy. Journal of Virology, 93(17), e02187–18. To, A., Medina, L., Mfuh, K. O., Leberman, M., Wong, T. A. S., Namekar, M., … Lehrer, A. T. (2018). Recombinant Zika virus subunits are immunogenic and efficacious in mice. mSphere, 3, e00576-17. Touret, F., Gilles, M., Klitting, R., Aubryde Lamballerie, F. X., & Nougaire‘de, A. (2018). Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone. Emerging Microbes & Infections, 7, 161. Wen, J., Elong Ngono, A., Regal-Nava, J. A., Kim, K., Gorman, M. J., Diamond, M. S., & Shresta, S. (2017). Dengue virus-reactive CD8+ T cells mediate cross-protection against subsequent Zika virus challenge. Nature Communications, 8, 1459. WHO. (2016). GMP for biological products. In WHO technical report series, n° 999. Wilder-Smith, A., Vannice, K., Durbin, A., Hombach, J., Thomas, S. J., Thevarjan, I., & Simmons, C. P. (2018). Zika vaccine and therapeutiocs: Landscape analysis and challenge ahead. BMC Medicine, 16, 84. Xie, X., Kum, D. B., Xia, H., Luo, H., Shan, C., Zou, J., … Shi, P. Y. (2018). A single dose live attenuated Zika virus vaccine with controlled infection rounds that protects against vertical transmission. Cell Host & Microbe, 24, 487–499. Xu, K., Song, Y., Dai, L., Zhang, Y., Lu, X., Xie, Y., … Gaoa, G. F. (2018). Recombinant chimpanzee adenovirus vaccine AdC7-M/E protects against Zika virus infection and testis damage. Journal of Virology, 92(6), e01722–17. Yang, M., Dent, M., Lai, H., Sun, H., & Chen, Q. (2017). Immunization of Zika virus envelope protein domain III induces specific and neutralizing immune responses against Zika virus. Vaccine, 35, 4287–4294. Zhang, W., Qu, P., Li, D., Zhang, C., Liu, Q., Zou, G., … Huang, Z. (2019). Yeast-produced subunit protein vaccine elicits broadly neutralizing T antibodies that protect mice against Zika virus lethal infection. Antiviral Research, 170, 104578. Zhong, Z., Paulo Portela Catani, J., Mc Cafferty, S., Couck, L., Van Den Broeck, W., Gorle´, N., … Sanders, N. N. (2019). Immunogenicity and protection efficacy of a naked self-replicating mRNA-based Zika virus vaccine. Vaccines, 7, 96.

Chapter 13

The adult brain and neurologic manifestations of the Zika virus Fernanda J.P. Marques and Osvaldo J.M. Nascimento Department of Neurology and Neuroscience, Fluminense Federal University, Rio de Janeiro, Brazil

Abbreviations ADEM CHKV CSF DENV GA1 GBS MR imaging PRNT RT-PCR ZIKV

acute disseminated encephalomyelitis chikungunya cerebral spinal fluid dengue virus antiglycolipid antibody Guillain-Barre syndrome magnetic ressonance imaging plaque reduction neutralization test reverse transcription polymerase chain reaction Zika virus

Introduction During the recent outbreaks of Zika virus infection, neurological complications, which include congenital microcephaly and Guillain-Barre syndrome (GBS), were reported (Araujo, Ferreira, & Nascimento, 2016; Krauer et al., 2017); scientists all over the world mobilized their efforts to explain the pathophysiology of such involvement. The dramatic scenario of congenital Zika virus infection has received worldwide attention (Garcez et al., 2016). However, serious neurological involvement in adults are also reported and cannot be neglected, as it may be responsible for permanent sequelae, long term care as well as important socioeconomic impact (Nascimento & da Silva, 2017). The most common and documented neurologic disorder associated with ZIKV infection is GBS. GBS in an acute, immune-mediated polyradiculoneuropathy typically occurring after viral and bacterial infections. In Guillain-Barre syndrome, the body’s immune system attacks part of the peripheral nervous system. The syndrome can affect the nerves that control muscle movement as well as those that transmit pain, temperature, and touch sensations. This can result in muscle weakness and loss of sensation in the legs and/or arms. It is a rare condition, and while it is more common in adults and in males, people of all ages can be affected. It is a potentially treatable condition, requiring prompt suspicion on clinical grounds to allow early treatment (Anaya et al., 2017; Krauer et al., 2017). In addition, the recent ZIKV outbreaks have triggered the occurrence of a spectrum of neurological disorders, such as transverse myelitis, meningoencephalitis, and ophthalmologic manifestations (Broutet et al., 2016; Nascimento, Frontera, Amitrano, de Filippis, & Da Silva, 2017). It is of high urgency that neurologists and physicians in general familiarize themselves with this new agent causing neuronal injury. With that purpose, the objective of this chapter is to describe the most common neurological disorders recently documented to be associated with ZIKV in adults.

Zika virus and the nervous system: NeuroZika The NeuroZika spectrum represents a heterogeneous group of clinical neurologic manifestations associated with Zika virus infection, including the Guillain-Barre syndrome, radiculomyelitis, and meningoencephalitis. (Krauer et al., 2017; Yasri & Wiwanitkit, 2019). Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00013-4 Copyright © 2021 Elsevier Inc. All rights reserved.

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B Effects on neurological and body systems

Initially, what appeared to be an agent responsible for a mild virus disease in humans turned out to be responsible for serious neurological consequences. The identification of ZIKV in an anatomopathological study of an embryo with important malformations (Mlakar, Korva, Tul, Popovic, et al., 2016), and the ZIKV neurotropism to progenitor cells demonstrated in neurospheres (Garcez et al., 2016, 2017), were some of the primordial studies that established the cause-effect relationship between ZIKV and neurological disorders.

Guillain-Barr e syndrome The Guillain-Barre syndrome is an acute, immune-mediated polyradiculoneuropathy that occurs after viral and bacterial infections. The clinical spectrum affects motor function, with an ascending and progressive paralysis that can affect cranial nerves and respiratory function (da Silva, Frontera, & do Nascimento, 2016). GBS is generally considered an immune-mediated, post exposure polyradiculoneuropathy that occurs after viral and bacterial infections or any other stimuli, leading to an increased immune response and molecular mimicry between an inciting agent and nerve antigens, targeting peripheral nerves and their respective spinal roots. The clinical spectrum affects motor function, with an ascending and progressive paralysis that can affect cranial nerves and respiratory function (Barbi, Coelho, Alencar, & Crovella, 2018; da Silva et al., 2016). The diagnosis is based on the Brighton Collaboration GBS working group criteria which help to indicate the accuracy of the diagnosis (Sejvar et al., 2011). It is divided into three levels: - Level 1: diagnosis supported by nerve-conduction studies and the presence of albuminocytologic dissociation in cerebral spinal fluid (CSF); - Level 2: CSF white-cell count of less than 50 cells per cubic millimeter or nerve conduction studies consistent with GBS; - Level 3: clinical features without support from nerve-conduction or CSF studies. Increased numbers of cases classified as GBS were reported in many countries in South America after ZIKV epidemics. ZIKV-GBS has redefined some of the current knowledge since, in most studies, the weakness had its onset 6–10 days after the viral symptoms, which is relatively fast for an autoimmune reaction to a first exposure to a virus. A possible explanation is that ZIKV-GBS may be a parainfectious disorder; the virus may trigger a process of immune molecular mimicry against nervous system antigens before the viral symptoms appear. In addition, ZIKV can promote an immune dysregulation that can lead to a polyradiculoneuropathy through mechanisms not associated with molecular mimicry. Besides, the virus may produce a direct neuropathogenic injury that is not yet fully understood (Parra et al., 2016). Among the molecular mechanisms contributing to the pathogenesis of the Guillain-Barre syndrome, a broad range of antiglycolipid IgG antibodies, notably directed to gangliosides, has been previously described, particularly in axonal variants of the disease. Cao-Lormeau et al. in a study using both ELISA and combinatorial microarray techniques, found less than 50% of sera at admission with a significant autoimmune response against glycolipids, including gangliosides or glycolipid complexes. These findings suggest that there might be autoantibodies in this post-Zika virus Guillain-Barre syndrome cohort that cannot be fully identified by current methods. Moreover, complementary analysis of sera with reactivity against GA1 did not show any competition between GA1 and Zika virus proteins, thus suggesting the absence of antigenic mimicry between Zika virus antigens and GA1 in these patients with Guillain-Barre syndrome as well as casting doubt on the relevance of the anti-GA1 antibodies to neuropathy pathogenesis. The disease might not be anti-glycolipid antibody-mediated, but rather be mediated by other autoantibody specificities or unknown neurotoxic factors. Alternatively, viral neurotoxicity might contribute a more direct but as yet unexplained role (Cao-Lormeau et al., 2016). The first reported case of ZIKV-GBS was of a female patient during the French Polynesian outbreak in 2014. Later, in 2015–2016, some studies in South America reported significant increases in hospital admissions for GBS, especially in Brazil and Colombia (Cao-Lormeau et al., 2016; da Silva et al., 2016; Parra et al., 2016; Roze et al., 2017; Simon et al., 2018; Uncini et al., 2018). A prospective cohort study in Brazil analyzed the neurological complications following ZIKV infection; of 40 patients admitted to a tertiary neurological referral center in Rio de Janeiro, Brazil, 35 (88%) had molecular and/or serological evidence of recent ZIKV infection, based on serum and/or cerebrospinal fluid (CSF) testing. Compared to historical records from the same institution, the GBS admissions had increased from an average of 1.0/month to 5.6/month. Table 1 describes the ZIKV-GBS findings reported in the literature (Nascimento & da Silva, 2017).

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TABLE 1 Studies associating Zika virus and Guillain–Barr e syndrome.

Country

Year

No. of patients

Symptom

Sample tasted

ZIKV detection method

IMG findings

References

French Polynesia

2013

1

GBS

Serum

Anti-ZIKV IgG ELISA and PRNT

AIDP

Oehler et al. (2014)

Brazil

2015

4

GBS

Serum or CSF

RT-PCR and/or anti-ZKV IgM/IgG ELISA

Not reported

Araujo, Silva, and Araujo (2016)

French Polynesia

2013– 2014

42

GBS

Serum

Seroneutralization assay and antiZIKV IgM/IgG ELISA

AMAN

CaoLormeau et al. (2016)

Colombia

2016

68

GBS

Blood, urine, and CSF

RT-PCR and antiflavivirus antibody ELISA

36 with ACP, 4 equivocal, 2 normal, 3 unexcitable, and 1 AMAN

Parra et al. (2016)

Brazil

2017

35

GBS, Encephalitis, Myelitis

Serum and CSF

RT-PCR/ MAC-ELISA

18 with AIDP, 2 AMAN, 6 AMSAN, and 1 MFS

da Silva, Frontera, de Filippis, and do Nascimento (2017)

Colombia

2015– 2016

19

GBS

Seam

RT-PCR

AMAN

Arias et al. (2017)

Canada

2015– 2016

2

GBS

Not reported

RT-PCR, anti-ZIKV IgM ELISA, and seroneutralization assay

Not reported

Boggild et al. (2017)

Brazil

2014

1

GBS

Serum, saliva, CSF, and urine

RT-PCR and antiZIKV IgM/G ELISA

Without findings

Brasil et al. (2016)

Puerto Rico

2015– 2016

5

GBS

Seam

RT-PCR and/or anti-ZIKV IgM ELISA

AIDP

Dirlikov et al. (2016)

Brazil

2015

2

GBS

Not reported

RT-PCR, ant-ZIKV IgM ELISA, and PRNT

AIDP

do Rosario et al. (2016)

United States

2016

1

GBS

Urine

RT-PCR

AIDP

Fabrizius et al. (2016)

Brail

2016

1

GBS

Not reported

Not reported

AIDP

Fontes, Dos Santos, and Marchiori (2016)

Suriname

2016

3

GBS

Urine

RT-PCR, seroneutralization assay, and antiZIKV IgG ELISA

AIDP

Langerak et al. (2016)

New Zealand

2016

1

GBS

Seam

RT-PCR and AntiZIKV IgM/G ELISA

AIDP

Siu et al. (2016) Continued

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TABLE 1 Studies associating Zika virus and Guillain–Barr e syndrome—cont’d

Country

Year

No. of patients

Symptom

Sample tasted

ZIKV detection method

Colombia

2016

16

IMG findings

References

GBS

Not reported

RT-PCR

Not reported

VillamilGomez et al. (2017)

Martinique

2015

2

GBS

Urine

RT-PCR

AIDP

Roze et al. [2017]

AIDP, acute inflammatory demyelinating polyradiculoneuropathy; AMAN, acute motor axonal neuropathy; AMSAN, acute motor and sensory axonal neuropathy; CSF, cerebrospinal fluid; EMG, electromyography; GBS, Guillain–Barre ´ syndrome; IgG, immunoglobulin G; IgM, immunoglobulin M; MAC, IgM antibody capture; MFS, Miller Fisher syndrome; PRNT, plaque reduction neutralization test; RT-PCR, reverse transcriptase PCR; ZIKV, Zika virus.

The subtype of GBS may vary from country to country; in the first study documenting a series of patients with GBS associated to ZIKV from French Polynesia, the most common electromyography pattern was from the AMAN type, characterized by distal motor nerve involvement, absence of patterns and levels of antiglycolipids antibodies, and faster recovery. In contrast, in the report of cases of ZIKV-GBS from Colombia, electrophysiological studies were consistent with the AIDP form of GBS in the majority of patients. Evolutionary changes of the virus and/or host-dependent factors in different countries, differences in the distribution of HLA alleles, and associated immunological response might be explanations to such differences (Parra et al., 2016). Extensive investigation based on ancillary tests (nerve conduction studies and cerebrospinal fluid examination) to further document this syndrome might be unavailable in some regions. In 2014, the GBS Classification Group proposed a diagnostic classification for GBS and its variants, such as Miller Fischer syndrome (MFS), based exclusively on clinical grounds. This classification is very simple to apply and useful for the prompt recognition of GBS and MFS, especially in a possible context of a widespread epidemic (Nascimento and da Silva, 2017). Fig. 1 shows the clinical presentations of GBS in general.

ZIKV-GBS viral diagnosis ZIKV infection can mimic the clinical signs and symptoms of dengue virus (DENV) and chikungunya (CHKV), and there is a lack of a simple laboratory test for a prompt diagnosis. These three viral infections might present with an acute exanthematous fever, normally associated with a maculopapular skin rash, conjunctivitis, and edema, along with headache, Classic Guillan-Barré syndrome (GBS)

Paraparetic GBS

Pharyngeal-cervical-brachial Bifacial weakness with weakness paraesthesias

Miller Fisher syndrome

Bickerstaff’s brainstem encephalitis

ZZZZZ

FIG. 1 Clinical presentations of GBS (Nascimento & da Silva, 2017).

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myalgia, and arthralgia, with subtle differences in the presentation (www.who.int). The rash might be more prominent with ZIKV, whereas DENV leads to more pronounced headaches and generalized pain, with CHKV frequently causing incapacitating arthralgia and/or arthritis (Mancera-Pa´ez, Roma´n, Pardo-Turriago, Rodrı´guez, & Anaya, 2018). RT-PCR for ZIKV is the gold standard test to prove acute ZIKV infection; however, its positivity is very transient, usually persisting no longer than 5–7 days in the blood and CSF. Its positivity might be observed in urine samples for a few weeks (Araujo, Silva, & Araujo, 2016). Regarding the postinfectious syndromes, RT-PCR might be negative, but ZIKV IgM antibody capture ELISA might remain positive in the blood and CSF samples for a longer period. Diagnosing acute ZIKV infection with the aid of serological testing is complicated in regions with endemic dengue virus as cross-reactivity is known to occur. PRNT can be performed to help differentiate anti-ZIKV antibodies from cross-reacting antibodies (Ferreira et al., 2017; Savino et al., 2017).

ZIKV-GBS neuroimaging Neuroimaging of the CNS and peripheral nervous system might be useful in the setting of ZIKV-GBS (Fig. 2). In a Brazilian prospective cohort, two patients developed GBS with associated CNS lesions, notably located in bilateral cerebellar peduncles, resembling West Nile Virus encephalitis. Moreover, in the same study, brain and spine MR imaging were performed in 21 of the ZIKV-GBS cases, with cranial nerve enhancement and cauda equina or nerve root contrast enhancement present in 19%. These findings of nerve enhancement did not differ from what is occasionally observed in patients with GBS (Nascimento & da Silva, 2017).

GBS-Zika virus treatment and prognosis Immunoglobulin was the most commonly used treatment in most series, usually administered within 7 days after onset of symptoms. Admission to an ICU was needed for 59%, 38%, and 15% of the patients in the Colombian, French Polynesian, and Brazilian series, with fatal outcomes in 4%, 0%, and 4%, respectively. Treatment-related fluctuations were observed in FIG. 2 Radiological findings in ZIKV-GBS. Upper left: MRI of the thoracic spine (T2WI) disclosing hypersignal of the bilateral cortical-spinal tracts. Upper right: MRI of the lumbar spine (T1WI with gadolinium) showing enhancement of the nerve roots. Lower left: MRI of the brain (T2WI) showing hypersignal of the bilateral cerebellar peduncles. Lower right: Ultrasonography of the median nerve showing enlarged sectional area of the nerve (white arrow) of an acute infectious polyneuropathy ZIKV PCR-positive case (Nascimento & da Silva, 2017).

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one case in the Brazilian series. Twenty-nine % of the French Polynesian patients, 31% in the Colombian series, and 7% in the Brazilian cohort required mechanical ventilation. The prognosis was generally favorable in all the series. Finally, the ZIKV-associated GBS combined outcomes of the three major studies did not significantly differ from what had been observed in the largest prospective cohort of patients with GBS secondary to other causes.

Others neurological complications ZIKV has been associated with various neurologic complications in adults, including not only GBS but also transverse myelitis, meningoencephalitis, and ophthalmologic manifestations (Mun˜oz et al., 2017; Munoz, Barreras, & Pardo, 2016). Though some of these syndromes may be due to a postinfectious (molecular mimicry) mechanism, a direct viral pathogenic mechanism may be responsible (da Cruz, Nascimento, Lopes, & Da Silva, 2018). Nascimento et al. (2017) reported a ZIKV-associated acute transient polyneuritis, where three patients showed signs and symptoms of distal pain, stocking-glove hypoesthesia, mild distal weakness, and hyporeflexia within 1–2 days of ZIKV symptoms onset, findings consistent with a mild, self-limited, distal, sensorimotor neuropathy (da Cruz et al., 2018). Other neurologic complications in adults are illustrated in Figs. 3–7: Although there is no definitive evidence of an association between myelitis and ZIKV infection, few reports have suggested that this association is likely to occur. Flaviviruses are neurotropic viruses, and DENV, Japanese encephalitis virus, and West Nile virus infections are usually responsible for the occurrence of extensive transverse and longitudinal myelitis (da Cruz et al., 2018). Meningoencephalitis is another condition that can have ZIKV as a causative agent. Clinical presentation includes progressive somnolence, seizures, and focal deficits, and in rare cases, may evolve into deep coma or brain death. No specific neuroimaging pattern has been found suggesting the occurrence of this condition (da Cruz et al., 2018). Acute disseminated encephalomyelitis is an immune-mediated inflammatory demyelinating disorder targeting mostly the white matter of the brain and, less frequently, the gray matter and the spinal cord. Patients with these conditions can present symptoms related to an acute or subacute encephalopathy with multiple neurologic deficits; it is typically monophasic and self-limiting. Clinical symptom onset generally develops within 2–3 weeks after a viral infection. Flaviviruses are also responsible for this neurological condition, and ZIKV is now one more agent that must be investigated, especially during epidemics. The diagnosis is based on clinical and imaging findings, as well as on excluding other conditions that can mimic ADEM. Although it can occur at any age, it is more common in children and young adults (da Cruz et al., 2018).

FIG. 3 Acute myelitis in a patient with Zika virus infection. Spine MR imaging of a 38year-old woman with unsteadiness and weakness in the lower limbs. Hyperintense, ill-defined lesions are seen in sagittal (A, arrows) and axial (C and D, arrows) T2-weighted images of the cervicothoracic and middle thoracic spinal cord, causing mild expansion of the cord (A). The lesions demonstrate contrast enhancement in the postgadolinium sagittal T1-weighted image (B, arrows) (da Cruz et al., 2018).

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FIG. 4 Zika virus-related Guillain-Barre syndrome associated with brain stem encephalitis and myelitis (encephaloradiculomyelitis). Brain MR imaging of a 35-year-old man positive for Zika infection with Guillain-Barre syndrome who presented with progressive ascending paralysis evolving to respiratory distress and decreased level of consciousness. The patient had skin rashes preceded by flulike symptoms 1 week before the development of neurologic symptoms. Axial and coronal T2-weighted brain images show bilateral hyperintensity (arrows) in the middle cerebellar peduncles (A) and corticospinal tracts bilaterally (B and C). Brain and spine MR imaging performed 2 months after treatment demonstrate improvement of the middle cerebellar peduncle and cortico-spinal tract hyperintensity (D and E). An absence of contrast enhancement is seen in the conus medullaris and cauda equina nerve roots in the postcontrast sagittal fat-suppressed T1-weighted image (F). Axial T2-weighted spine image reveals improvement of the hyperintensity in the anterior horns of the thoracic spinal cord (G) (da Cruz et al., 2018).

As we have described, ZIKV is a novel agent responsible for severe neurological complications in children and adults. The intense neurotropism of the virus, associated to a direct and indirect effect through immune-mediated mechanisms brings special attention to ZIKV, especially during an epidemics period (Beckham, Pastula, Massey, & Tyler, 2016; Garcez et al., 2016). It is important to try to establish differential diagnosis with other causative agents, especially flaviviruses such as West Nile virus, Japanese B encephalitis virus, yellow fever and dengue virus, with can mimic ZIKV symptoms and neurological consequences. As we now live in a connected world, physicians must be aware about ZIKV’s possible consequences when facing patients with neurological conditions associated to virus infection (Carod-Artal, 2018).

General implications ZIKV is a recognized and well-documented agent responsible for severe neurological complications in children and adults. The most common neurological consequences are congenital abnormalities and GBS. Research efforts might have focused on congenital abnormalities because clusters of affected infants were so unusual, especially in Brazil, and also because GBS

150

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FIG. 5 Zika virus-related brain stem encephalitis in a 40-year-old man. Axial fluid-attenuated inversion recovery image shows diffuse hyperintensity in the brain stem, especially in the pons and cerebral peduncle (A and B). Axial T2weighted images show bilateral hyperintensity in the middle cerebellar peduncles (C, arrows). Hyperintensity is also seen in the corticospinal tracts bilaterally in axial (D) and coronal (E) T2-weighted images. These lesions showed no enhancement on postcontrast T1-weighted images (not shown) (da Cruz et al., 2018).

FIG. 6 Axial T2-weighted images of a 52-year-old man with Zika virus-related brain stem encephalitis and myelitis. Hyperintense lesions are seen in the upper portion of the lateral columns of the cervical spine (A, arrow) and anterior portion of the medulla bilaterally (B, arrow). The brain stem is diffusively involved (C–G), as are the middle cerebellar peduncles (C) and the bilateral corticospinal tracts (G, arrows) (da Cruz et al., 2018).

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FIG. 7 Brain and spine MR imaging of a 48-year-old woman with Zika virus infection and encephalitis and myelitis. Axial T2-weighted image of the brain shows hyperintensity of the middle cerebellar peduncles bilaterally (A, arrows). Short tau inversion recovery sagittal image (B) demonstrates a hyperintense lesion in the upper portion of the anterior spine without enhancement in the sagittal postcontrast T1-weighted image (C). Follow-up MR imaging was performed 2 weeks later (da Cruz et al., 2018).

is a well-known neurological disorder. Nevertheless, neurological affections must not be neglected; studying the outbreaks, producing scientific knowledge that could aid in further understanding of the pathophysiology, the clinical course, and therapy response of GBS is fundamental for better conduction of such cases. It is imperative that medical societies and public health agencies urgently develop training programs for health personnel on clinical suspicion and treatment of GBS and other neurological complications. In addition, the creation of collaborative networks among neurological centers and referral hospitals would be advisable.

Policy and procedures Using the Brighton collaboration GBS working group criteria for GBS diagnosis l

l

l

Level 1: diagnosis supported by nerve-conduction studies and the presence of albuminocytologic dissociation in cerebral spinal fluid (CSF); it is the most high level of accuracy for GBS diagnosis; Level 2: CSF white-cell count of less than 50 cells per cubic millimeter or nerve conduction studies consistent with GBS; Level 3: clinical features without support from nerve-conduction or CSF studies.

Assessments are used to understand the types and severity of GBS. It is important to help make predictions about the future, to assist with planning interventions, and to measure the outcomes or effectiveness of interventions and therapies (outcome measures).

Mini-dictionary of terms l

l

l

NeuroZika: heterogeneous group of clinical neurologic manifestations associated with Zika virus infection, including Guillain-Barre syndrome, radiculomyelitis, and meningoencephalitis. Guillain-Barre syndrome: acute, immune-mediated polyradiculoneuropathy that occurs after viral and bacterial infections. Zika virus: a virus transmitted by mosquitoes which typically causes asymptomatic or mild infection (fever and rash) in humans, identified originally in Africa and later in other tropical regions, including South America, where it may be

152

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B Effects on neurological and body systems

associated with an increased incidence of microcephaly in babies born to mothers infected during pregnancy and neurological complications in adults. Meningoencephalitis: inflammation of the brain and meninges that can be responsible for clinical symptoms, such as progressive somnolence, seizures, and focal deficits, and in rare cases, coma or brain death. Acute disseminated encephalomyelitis (ADEM): immune-mediated inflammatory demyelinating disorder targeting mostly the white matter of the brain and, less frequently, the gray matter and the spinal cord.

Key facts of NeuroZika l l

l

l l

ZIKV is a novel agent responsible for severe neurological complications in children and adults. ZIKV should be deemed a global threat that can cause devastating neurologic complications among individuals in all age ranges. ZIKV is a recognized and well-documented agent responsible for severe neurological complications in children and adults. The most common neurological consequences are congenital abnormalities and GBS. ZIKV has been associated with various neurologic complications in adults, including GBS transverse myelitis, meningoencephalitis, and ophthalmologic manifestations.

Summary points l

l l

l

l

ZIKV is a recognized and well-documented agent responsible for severe neurological complications in children and adults. The most common neurological consequences are congenital abnormalities and GBS. Neurological affections associated with ZIKV infection must not be neglected and ZIKV etiology should be considered especially during epidemic periods. NeuroZika spectrum represents a heterogeneous group of clinical neurologic manifestations, including Guillain-Barre syndrome, radiculomyelitis, and meningoencephalitis. Research efforts might focus on congenital abnormalities because clusters of affected infants were so unusual, especially in Brazil, and as GBS is a known neurological disorder. However, neurological affections must not be neglected; studying the outbreaks, producing scientific knowledge that could aid in further understanding of the pathophysiology, clinical course, and therapy response of GBS, is fundamental for a better conduction.

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da Cruz, L. H., Nascimento, O. J. M., Lopes, F. P. P. L., & Da Silva, I. R. F. (2018). Neuroimaging findings of Zika virus-associated neurologic complications in adults. American Journal of Neuroradiology, 39(11), 1967–1974. da Silva, I. R. F., Frontera, J. A., & do Nascimento, O. J. M. (2016). News from the battlefront: Zika virus-associated Guillain-Barre syndrome in Brazil. Neurology, 87(15), e180–e181. da Silva, I. R. F., Frontera, J. A., de Filippis, A. M. B., & do Nascimento, O. J. M. (2017). Neurologic complications associated with the Zika virus in Brazilian adults. JAMA Neurology, 74(10), 1190–1198. Dirlikov, E., Major, C. G., Mayshack, M., et al. (2016). Guillain-Barre syndrome during ongoing Zika virus transmission: Puerto Rico, January 1–July 31, 2016. Morbidity and Mortality Weekly Report, 65, 910–914. do Rosario, M. S., de Jesus, P. A., Vasilakis, N., et al. (2016). Guillain-Barre syndrome after Zika virus infection in Brazil. American Journal of Tropical Medicine and Hygiene, 95, 1157–1160. Fabrizius, R. G., Anderson, K., Hendel-Paterson, B., et al. (2016). Guillain-Barre syndrome associated with Zika virus infection in a traveler returning from Guyana. American Journal of Tropical Medicine and Hygiene, 95, 1161–1165. ´ . J. P., de Morais Machado, M.I´., Henriques-Souza, A., Cordeiro, M. T., … Pena, L. J. (2017). Guillain– Ferreira, M. L. B., de Brito, C. A. A., Moreira, A Barre syndrome, acute disseminated encephalomyelitis and encephalitis associated with Zika virus infection in Brazil: Detection of viral RNA and isolation of virus during late infection. The American Journal of Tropical Medicine and Hygiene, 97(5), 1405–1409. Fontes, C. A., Dos Santos, A. A., & Marchiori, E. (2016). Magnetic resonance imaging findings in Guillain-Barre syndrome caused by Zika virus infection. Neuroradiology, 58, 837–838. Garcez, P. P., Loiola, E. C., da Costa, R. M., Higa, L. M., Trindade, P., Delvecchio, R., … Rehen, S. K. (2016). Zika virus impairs growth in human neurospheres and brain organoids. Science, 352(6287), 816–818. Garcez, P. P., Nascimento, J. M., De Vasconcelos, J. M., Da Costa, R. M., Delvecchio, R., Trindade, P., … Sequeira, P. C. (2017). Zika virus disrupts molecular fingerprinting of human neurospheres. Scientific Reports, 7, 40780. Krauer, F., Riesen, M., Reveiz, L., Oladapo, O. T., Martinez-Vega, R., Porgo, T. V., … WHO Zika Causality Working Group. (2017). Zika virus infection as a cause of congenital brain abnormalities and Guillain–Barre syndrome: Systematic review. PLoS Medicine, 14(1), e1002203. Langerak, T., Yang, H., Baptista, M., et al. (2016). Zika virus infection and Guillain-Barre syndrome in three patients from Suriname. Frontiers in Neurology, 7, 233. Mancera-Pa´ez, O., Roma´n, G. C., Pardo-Turriago, R., Rodrı´guez, Y., & Anaya, J. M. (2018). Concurrent Guillain-Barre syndrome, transverse myelitis and encephalitis post-Zika: A case report and review of the pathogenic role of multiple arboviral immunity. Journal of the Neurological Sciences, 395, 47–53. Mlakar, J., Korva, M., Tul, N., Popovic, M., et al. (2016). Zika virus associated with microcephaly. The New England Journal of Medicine, 374(10), 951–958. Munoz, L. S., Barreras, P., & Pardo, C. A. (2016, September). Zika virus-associated neurological disease in the adult: Guillain–Barre syndrome, encephalitis, and myelitis. Seminars in Reproductive Medicine, 34(5), 273–279. Mun˜oz, L. S., Parra, B., Pardo, C. A., & Neuroviruses Emerging in the Americas Study. (2017). Neurological implications of Zika virus infection in adults. The Journal of Infectious Diseases, 216(Suppl 10), S897–S905. Nascimento, O. J., & da Silva, I. R. (2017). Guillain–Barre syndrome and Zika virus outbreaks. Current Opinion in Neurology, 30(5), 500–507. Nascimento, O. J., Frontera, J. A., Amitrano, D. A., de Filippis, A. M. B., & Da Silva, I. R. (2017). Zika virus infection-associated acute transient polyneuritis. Neurology, 88(24), 2330–2332. Oehler, E., Watrin, L., Larre, P., Leparc-Goffart, I., Lastere, S., Valour, F., … Ghawche, F. (2014). Zika virus infection complicated by Guillain-Barre syndrome – Case report, French Polynesia, December 2013. Eurosurveillance, 19(9), 20720. Parra, B., Lizarazo, J., Jimenez-Arango, J. A., Zea-Vera, A. F., Gonza´lez-Manrique, G., Vargas, J., … Rizcala, K. H. (2016). Guillain–Barre syndrome associated with Zika virus infection in Colombia. New England Journal of Medicine, 375(16), 1513–1523. Roze, B., Najioullah, F., Ferge, J. L., Dorleans, F., Apetse, K., Barnay, J. L., … Valentino, R. (2017). Guillain-Barre syndrome associated with Zika virus infection in Martinique in 2016: A prospective study. Clinical Infectious Diseases, 65(9), 1462–1468. Savino, W., Messias, C. V., Mendes-da-Cruz, D. A., Passos, P., Ferreira, A. C. A., & Nascimento, O. J. (2017). Zika virus infection in the elderly: Possible relationship with Guillain-Barre syndrome. Gerontology, 63(3), 210–215. Sejvar, J. J., Kohl, K. S., Gidudu, J., Amato, A., Bakshi, N., Baxter, R., … Heininger, U. (2011). Guillain-Barre syndrome and fisher syndrome: Case definitions and guidelines for collection, analysis, and presentation of immunization safety data. Vaccine, 29(3), 599. Simon, O., Acket, B., Forfait, C., Girault, D., Gourinat, A. C., Millon, P., … Hoinard, D. (2018). Zika virus outbreak in New Caledonia and Guillain-Barre syndrome: A case-control study. Journal of Neurovirology, 24(3), 362–368. Siu, R., Bukhari, W., Todd, A., et al. (2016). Acute Zika infection with concurrent onset of Guillain-Barre syndrome. Neurology, 87, 1623–1624. Uncini, A., Gonza´lez-Bravo, D. C., Acosta-Ampudia, Y. Y., Ojeda, E. C., Rodrı´guez, Y., Monsalve, D. M., … Molano-Gonza´lez, N. (2018). Clinical and nerve conduction features in Guillain Barre syndrome associated with Zika virus infection in Cu´cuta, Colombia. European Journal of Neurology, 25(4), 644–650. Villamil-Gomez, W. E., Sanchez-Herrera, A. R., Hernandez, H., et al. (2017). Guillain-Barre syndrome during the Zika virus outbreak in Sucre, Colombia, 2016. Travel Medicine and Infectious Disease, 16, 62–63. Yasri, S., & Wiwanitkit, V. (2019). Concurrent Guillain-Barre syndrome, transverse myelitis and encephalitis post-Zika and multiple arboviral immunity. Journal of the Neurological Sciences, 397, 183.

Chapter 14

Postmortem studies: Contribution to understand the pathogenesis of congenital Zika syndrome Leila Chimelli Laboratory of Neuropathology, State Institute of Brain, Rio de Janeiro, RJ, Brazil

Abbreviations CNS CZS GL PC RG ZIKV

central nervous system congenital Zika syndrome glia limitans progenitor cells radial glia Zika virus

Introduction From the second semester of 2015, several babies were born with microcephaly in Brazil, coinciding with maternal infection with the Zika virus (ZIKV) during pregnancy. Cerebral malformations and calcification were detected in utero with ultrasonography (Ho et al., 2018; Melo, Malinger, et al., 2016; Oliveira-Szejnfeld et al., 2016). Additional neuroimaging methods provided more information to this new syndrome, identified as congenital Zika syndrome (CZS), consisting of severe microcephaly with a partially collapsed skull, arthrogryposis, marked early hypertonia and symptoms of pyramidal and extrapyramidal involvement, thin cerebral cortex with subcortical calcifications, and ocular manifestations with important consequences on vision. Hearing abnormalities and other neurologic symptoms such as seizures have also been reported (Araga˜o et al., 2016; Marques et al., 2019; Melo, Aguiar, et al., 2016; Moore et al., 2017). The most common structural neuroimaging abnormalities include brain calcifications, especially at the corticosubcortical junction, cortex malformations, ventriculomegaly, and reduced brain volumes, followed by brainstem hypoplasia, cerebellar hypoplasia, and corpus callosum abnormalities (Araga˜o et al., 2016; Hazin et al., 2016; OliveiraSzejnfeld et al., 2016; Pool et al., 2019). These lesions vary according to the trimester of maternal ZIKV infection; most of the affected infants were born to mothers infected in the first trimester (Pool et al., 2019). Few neonates died just after birth and postmortem case reports from various centers confirmed the severity of the cerebral lesions (Martines et al., 2016; Schwartz, 2016; Solomon, Milner, & Folkerth, 2016), including a detailed neuropathological description of a fetus whose mother was infected in Brazil, and pregnancy was interrupted when she went back to her country in Europe (Mlakar et al., 2016; Sˇtrafela et al., 2016). From February 2016, postmortem examinations with thorough neuropathological analyses were conducted in a single center in Brazil, reaching a series of 10 cases in November of the same year (Chimelli et al., 2017). It was possible to define a spectrum of cerebral lesions, but the most important was that the pathogenesis of the changes observed in CZS was understood. Various degrees of destructive lesions, calcification, hypoplasia, and migration disturbances, which were more severe when the infection occurred in the first trimester of gestation, led to different patterns of central nervous system (CNS) lesions. In the following years, further series with neuropathological analyses of fetuses, stillborns, and neonates were published, with similar results (Araga˜o et al., 2019; Azevedo et al., 2018; Sousa et al., 2017), also from Puerto Rico (Da´vila-Castrodad et al., 2018) and Colombia (Sa´nchez, Martı´nez, Dı´az-Martı´nez, & Mojica, 2019). In addition, Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00014-6 Copyright © 2021 Elsevier Inc. All rights reserved.

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postmortem examination of an infant who survived longer (5 months) not only confirmed the severity of the lesions but also verified persistence of ZIKV and progression of the cerebral damage (Chimelli et al., 2018).

Pathogenesis of the lesions in CZS The following clinical and neuroimaging observations in CZS could be explained with postmortem studies. Microcephaly is due to massive destruction of progenitor cells (PC) of neural and glial lineage, resulting in extremely small brains (Fig. 1) and consequent small heads (Ferraris et al., 2019; Tang et al., 2016). With immunohistochemistry, immunofluorescence, electron microscopy and in situ hybridization, ZIKV was detected in degenerating glial cells and neurons in the periventricular germinal matrix (Chimelli et al., 2017; Martines et al., 2016; Mlakar et al., 2016) where neural PC are concentrated. Experimental infections in organoids showed that ZIKV causes productive replication, infects neural/glial PC, decreases both populations, causes premature differentiation, induces apoptosis, and reduces their size (Garcez et al., 2016; Sutarjono, 2019). The fact that there are fewer axons crossing the hemispheres also explains the hypoplasia or hypogenesis of the corpus callosum (Fig. 2) described in neuroimaging studies (Araga˜o et al., 2016; Lage et al., 2019; Oliveira-Szejnfeld et al., 2016), and confirmed in autopsies (Chimelli et al., 2017; Mlakar et al., 2016). Radiological descriptions also state that the skull is collapsed with overlapping cranial bones and the posterior fossa and cisterna magna are large (Araga˜o et al., 2016; Oliveira-Szejnfeld et al., 2016). The changes described in these bone structures are in fact due to excedent space in the cranial cavity. The size of posterior fossa and cisterna magna is actually normal, but appears large because they are partly empty due to cerebellar and brainstem hypoplasia, in particular the basis pons, which is flat due to lack of descending fibers (Fig. 3). Ventriculomegaly—Despite the fact that the brains are very small, and babies are born with microcephaly, the ventricles are usually large (ex-vacuo ventriculomegaly) because the hemispheric cerebral tissue is thinner than usual (Fig. 2). However, not all babies with severely damaged/small brains are born with a small head/microcephaly. Obstructive ventriculomegaly/hydrocephalus also occurs and is due to brainstem damage and distortion of the midbrain, which is deformed, calcified and the aqueduct is closed. This change occurred in 50% of the autopsies in neonates (Chimelli et al., 2017). Therefore, even with little brain tissue, represented by a thin cortex and white matter, sometimes represented by a membrane (Fig. 4), the head circumference may be normal or enlarged, despite the fact that the brain weight is much lower than expected. In fact, there are ultrasound reports that even during gestation, head circumference may increase after a previous diagnosis of microcephaly (van der Linden et al., 2019). This may be due to aqueduct obstruction and worsening of the ventriculomegaly. Brainstem damage and intracranial hypertension, interfering with breathing, may be the cause of death of neonates with obstructive hydrocephalus just after birth. In addition, a sudden increase in

FIG. 1 Brain from a neonate with microcephaly. Macroscopic appearance of the convexity of an unfixed small brain with few and wide gyri.

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FIG. 2 Cerebral hemisphere from a neonate with microcephaly. Slice of a smooth and thin cerebral hemisphere with consequent large ex-vacuo ventriculomegaly. Limits between cortex and white matter are not defined and the corpus callosum is very thin (arrow).

FIG. 3 Hypoplasia of cerebellum and brainstem. Macroscopic appearance of a small cerebellum with a smooth surface (left) and a brainstem (right) with a flat pons (black arrow) and flat pyramids (white arrows).

head circumference occurred in an infant at 4 months necessitating ventricle-peritoneal shunt placement, complicating with bacterial meningitis, sepsis, and death at 5 months. Progressive gliosis and microgliosis in the midbrain may have contributed to aqueduct compression and subsequent hydrocephalus (Chimelli et al., 2018). Calcification—Dystrophic calcification, initially assessed in utero with the ultrasound (Melo, Malinger, et al., 2016) and then postnatally (Araga˜o et al., 2016; Lage et al., 2019), is prominent in postmortem tissue. It is present throughout the neuroaxis, particularly in the hemispheres, basal ganglia and brainstem (Fig. 5) and occurs following the massive PC death. However, despite extensive destruction, lymphocytic infiltration is mild, but microglial activation and reactive gliosis are evident (Fig. 6). Curiously, unequivocal recent necrosis, as seen in congenital toxoplasmosis, is not seen although apoptotic bodies are present (Chimelli et al., 2017).

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FIG. 4 Obstructive ventriculomegaly—hydrocephalus. Slice of a brain with very large and fused lateral ventricles and very thin (and folded) cerebral hemispheres. Some foci of calcification are shown with the arrows.

Migration disturbances and epilepsy—Epilepsy is a frequent manifestation in children with CZS (Lage et al., 2019; Marques et al., 2019; Pool et al., 2019; Silva et al., 2016), which could be explained by the abnormal migration and organization of neurons in the cerebral cortex as demonstrated in postmortem studies (Chimelli et al., 2017; Sˇtrafela et al., 2016). As mentioned previously, PC are concentrated in the germinal matrix, located close to the ventricular surface. During brain development, radial glia (RG) extends a long basal process that contacts the pial surface and a short apical process to the ventricular surface. PC loss leads to proliferation and migration disorders through disruption of cell polarity, imbalanced proliferation and/or differentiation rates, which, in turn, lead to disruption of the cytoarchitecture of the ventricular zone, protrusion of cells into the ventricles, cortical thinning, and ventriculomegaly/hydrocephalus (Bustamante et al., 2019; Ferraris et al., 2019). Actually, astrocytes, the most abundant cells in the CNS, which control axonal guidance, synaptic signaling, neurotransmitter trafficking, and maintenance of neurons are targeted by ZIKV (Ledur et al., 2020). Several astrocyte proteins involved in diverse functions including cell migration and differentiation are significantly dysregulated by ZIKV infection (Sher, Glover, & Coombs, 2019). Morphologically, damage of RG leads to cortical dyslamination/dysplasia, polymicrogyria, and agyria/lissencephaly (Chimelli et al., 2017), also observed radiologically (Araga˜o et al., 2016; Oliveira-Szejnfeld et al., 2016). Furthermore, damage of glia limitans (GL) with interruption of the pia mater, is the cause of overmigration of cells to the subarachnoid space, also called glioneuronal leptomeningeal heterotopia (Chimelli et al., 2017), represented macroscopically by the cobblestone appearance of the cortical surface (Fig. 7), a change that could not be detected with computed tomography (Araga˜o et al., 2019). Clinically, in addition to seizures, migration disturbances also explain the irritability, hyperexcitability, clonus following external stimulation, sleep disorders, and focal or multifocal epileptiform discharges (on electroencephalography), all observed in CZS (Pereira et al., 2020; Pessoa et al., 2018), indicate highly disorganized brain activity. It is worth mentioning that the malformed/simplified hippocampus, seen in most cases as an ill-defined structure with few and disorganized nerve cells (Fig. 8), may also contribute to the epileptic seizures (Chimelli et al., 2017, 2018). In fact, experimental infection with ZIKV results in a pronounced reduction in NeuN immunoreactivity in all hippocampal subfields indicating an overall demise/damage of different neuronal populations, both in early and late generated neurons and independent on the maturation state of nerve cells (B€uttner, Heer, Traichel, Schwemmle, & Heimrich, 2019). Arthrogryposis/fetal akinesia syndrome—Most of the babies with CZS are born with fixed joints and deformed limbs/arthrogryposis (Brasil et al., 2016; Chimelli et al., 2017; Lage et al., 2019; Melo, Malinger, et al., 2016). Postmortem studies indicated that the spinal motor nerve cell loss, which may be accompanied by calcification and the consequent neurogenic muscular atrophy (Fig. 9), are responsible for intrauterine akinesia. Lack of movements in utero fixes the joints. This is reinforced by the fact that a neuroimaging study has described that in infants with arthrogryposis the thickness of the spinal cord and of the anterior roots was reduced (Araga˜o et al., 2017). Once again, severe lesions occur when the infection happens early in gestation; the only neonate who was not born with arthrogryposis in the series of 10 autopsies was infected late—the mother had Zika at the 28th gestational week (Chimelli et al., 2017). Dysphagia is observed in most clinical reports of CZS (Leal et al., 2017; Marques et al., 2019; Nielsen-Saines et al., 2019; Silva et al., 2016; Soares et al., 2019). Considering the fact that brainstem damage with nerve cell loss of motor nuclei

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(C) FIG. 5 Cerebral calcification. (A) Macroscopic appearance of the cerebral hemispheres with almost smooth cortical surface and several foci of calcification (arrows), (B) microscopic appearances of calcification in the cerebral cortex (B), and in the brainstem (C) are shown with the arrows.

and distortion of axons affect the formation of cranial nerves (Fig. 10), it is expected that, together with high cervical spinal motor nerve cell loss, innervation of pharyngeal muscles is impaired and cause dysphagia. Moreover, denervation of the tongue with the replacement of muscle fibers by fat cells (personal unpublished observation) in one infant who survived 3 years, may also explain the difficulty in swallowing. This possibility has been postulated clinically, suggesting that children with neurologic damage from congenital ZIKV infection may be at heightened risk of dysphagia, arising from dysfunction in tongue movement and the pharyngeal phase of swallowing (Leal et al., 2017). Lung disease (pneumonia with massive pulmonary aspiration, probably secondary to dysphagia, choking, and reflux), and sepsis have been reported as the cause of death (Araga˜o et al., 2019). In addition, from what has been observed in autopsies by Chimelli et al. (2017), brainstem malformations and spinal motor nerve cell loss, it is expected that neurogenic atrophy of pharyngeal and tongue

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FIG. 6 Microglial proliferation and reactive gliosis. (A) Histological section of the midbrain showing numerous activated microglial cells immunopositive with CD68. (B) histological section of the white matter showing excessive amount of reactive astrocytes exhibiting abundant GFAP positive cytoplasmic processes.

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FIG. 7 Cortical migration disturbances. (A) Cortical dysplasia (disorganization of neuronal lamination) and overmigration of the neurons to the leptomeninges due to interruption of the pia mater (arrows), resulting in a cobblestone macroscopic appearance of the cortical surface (B).

muscles may also interfere with suckling, which together with dysphagia, contribute to the undernutrition observed in babies affected with CZS (Santos et al., 2019). Concerning motor development, both pyramidal and extrapyramidal functions are affected in children with CZS (Franc¸a et al., 2018; Moore et al., 2017; Nielsen-Saines et al., 2019; Pereira et al., 2020; Pessoa et al., 2018; Silva et al., 2016) and include hypertonia, clonus, hyperreflexia, increased archaic reflexes, tonus fluctuation, and asymmetric dyskinesias in the extremities that are absent during sleep. Pereira et al. (2020) have grouped the neurological motor involvement in corticospinal, neuromuscular and dyskinetic signs and symptoms, presented either isolated or concomitant. From what has been observed in autopsies, all motor symptoms can be explained: the pyramidal dysfunction may be due to the absence of descending fibers in the cerebral hemispheres, resulting in hypoplastic basis pontis, pyramids, and lateral corticospinal tracts (Fig. 11). Motor neuronal loss in the spinal cord, resulting in thin ventral roots, also interfere with the motor development, particularly the neuromuscular ones, confirmed by the evidence of neurogenic atrophy in skeletal muscles (Chimelli et al., 2017, 2018). Added to cerebellar hypoplasia with cortical dysplasia and deficient or delayed cerebral

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FIG. 8 Malformed/simplified hippocampus. The various hippocampal subfields cannot be identified due to the disorganization of neurons. The fascia dentata is straight and has just one layer of neurons (arrows).

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FIG. 9 Spinal motor nerve cell loss and neurogenic muscular atrophy. (A) Histological section at the level of the spinal ventral horn with hardly any motor neuron between the black lines. A few are seen between the blue lines. The arrow shows a focus of calcification. (B) Atrophic muscle fibers due to the lack of motor neurons (and axons).

myelination, documented with myelin staining and Olig2 immunohistochemistry (Chimelli et al., 2017, 2018), it is not surprising that many infants with CZS do not walk or do it late and with difficulty. In addition, it is possible that neurogenic atrophy of neck muscles explain the fact that most babies do not sustain the head or do it much later than expected in normal development. As for the extrapyramidal manifestations described in CZS, they are also expected, because in most cases there are calcifications in the basal ganglia and other extrapyramidal nuclei, which are not well-formed or are very small, sometimes hardly recognizable, both in neuroimaging and in autopsies (Araga˜o et al., 2019; Chimelli et al., 2017, 2018; Oliveira-Szejnfeld et al., 2016). Visual disturbances, including macular lesions, focal pigment mottling of the retina, chorioretinal atrophy, optic nerve abnormalities, microphthalmia, nystagmus, strabismus, iris coloboma, lens subluxation, cataract, and congenital glaucoma, have all been reported in children with CZS (Dias et al., 2018; Marques et al., 2019; Ventura, Ventura Filho, & Ventura, 2019; Zin et al., 2018). Clinical follow-up verified abnormal visual function in 30% of the infants and this was significantly associated with eye and CNS abnormalities (Zin et al., 2018). The fact that the optic nerve and chiasm are ill-defined and atrophic in most of the autopsies (Chimelli et al., 2017; Dias et al., 2018), favors a central origin for the visual problems although the only eye sampled in the series of autopsies described by Chimelli et al. (2017) showed retinal degeneration and

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(C) FIG. 10 Malformed brainstem, neuronal degeneration, and calcification along the traject of cranial nerve axons. (A) Disorganized brainstem structures at the level of the medulla (Luxol Fast Blue staining for myelin). (B) Nerve cell degeneration and calcification in a brainstem nucleus. (C) Bundles of axons (black arrows) close to clusters of calcium (white arrows), on their way to form cranial nerves.

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FIG. 11 Flat pyramids and lack of lateral corticospinal tracts. (A) Ventral aspect of a brainstem section at the level of the medulla showing that both pyramids are flat (arrows) due to lack of descending axons. (B) The lateral corticospinal tracts are missing (arrows) in this transverse section of the spinal cord stained with Luxol Fast Blue for myelin.

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FIG. 12 Retinal degeneration and calcification. Histological section at the level of the retina which is degenerated and calcified.

calcification both in the retina (Fig. 12) and in the lens. These observations can explain part of the visual impairment, including the cataract. It is possible that if more eyes were sampled and analyzed in autopsies, the other various visual manifestations could be better understood. Hearing loss associated with CZS has also been described. Sensorineural, retrocochlear or central origin cannot be ruled out (Barbosa et al., 2019; Lage et al., 2019; Peloggia, Ali, Nanda, & Bahamondes, 2018). The damage and nerve cell loss of cranial nerve nuclei or disruption/distortion of axons by calcification and malformation of the brainstem, observed in autopsies (Chimelli et al., 2017), could in part explain this clinical manifestation. However, since postmortem analyses did not include auditory organs, the pathogenesis of auditory changes is not yet completely understood. The lesions caused by ZIKV during pregnancy could be sufficient to explain the clinical outcomes, but it has been documented that the virus persists and the lesions can even progress. Some children were born with normal head circumference, but later developed microcephaly (Pereira et al., 2020). In addition, virus persistence and late astrocyte and microglia activation/reaction have been observed by Chimelli et al. (2018) in an infant who survived 5 months. Viruses manipulate cell biology to utilize monocytes/macrophages as vessels for dissemination, contributing to the establishment of viral genome long-term persistence, latency, and replication within tissues (Nikitina, Larionova, Choinzonov, & Kzhyshkowska, 2018). The fact that ZIKV can replicate in various neuronal populations, born both prenatally and postnatally, lead to the assumption that other factors than differentiation state determine susceptibility/vulnerability (B€ uttner et al., 2019). Therefore, considering all the changes associated with CZS, it is expected, as stated by Pardy and Richer (2019), that the impacts on the infants can be devastating, with potential for lifelong implications for both infants and their families.

Conclusions Postmortem studies confirmed neuroimaging and clinical observations that the severity of the lesions is related to the time of infection in pregnancy. Most mothers of neonates with severely affected brains, either with microcephaly or normal/ enlarged heads due to obstructive ventriculomegaly, are infected in the first trimester of gestation. On the other hand, mild cerebral lesions coincide with late gestational infection. Concerning neurodevelopmental outcomes in infants with CZS reported in the last 4 years, neurosensory deficits, below-average motor neurodevelopment, abnormal visual, hearing or language functions, have all been described. The low performance of children with CZS, both in motor and cognitive developmental domains is consistent with the severity and topography of CNS damage and the lack of axonal tracts, all observed in autopsies. Various morphological evidences of abnormal neuronal migration and hippocampal malformation, responsible for the epileptic manifestations, were also demonstrated in postmortem analyses.

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Although the lesions caused by ZIKV during pregnancy could be sufficient to explain the clinical outcomes, the fact that ZIKV can replicate in neuronal populations born both prenatally and postnatally, and the documentation of astrocyte and microglia activation and viral persistence within postmortem tissue, at least 5 months postnatally, explain the fact that some children have developed microcephaly or hydrocephalus after birth.

Policy and procedures Performing postmortem examinations. Postmortem examinations or autopsies are considered the gold standard to verify the precise diagnosis of several diseases and the cause of death. The first requirement is to obtain the consent of the family. Autopsies should be performed by pathologists: medical doctors specialized in the macroscopic and microscopic examinations of the various organs, both normal and altered by disease. Specimens should be collected as soon as possible after death, to avoid autolysis, and immediately fixed in formalin before paraffin embedding. If required, specimens should be also frozen or fixed in special solutions for electron microscopy. As in congenital Zika syndrome, the main target is the developing central nervous system, it is important that the pathologist has experience in neuropathology and basic knowledge of clinical neurology and neuroradiology, to make clinicopathological correlations that allow understanding the pathogenesis (mechanism) of the lesions and of the disease. Postmortem examinations require various methods to appropriately prepare material for microscopic analysis and this includes the work of experienced technicians who prepare the slides, the immunohistochemical reactions, and other special methods, including molecular/genetic techniques.

Mini-dictionary of terms Arthrogryposis: Congenital fixation of the limb joints as a consequence of limited fetal movement (fetal akinesia syndrome). Cortical dysplasia: Abnormal organization of neurons of the cerebral due to abnormal migration, resulting in various morphological presentations. Dystrophic calcification: Pathological deposition of calcium in body tissues in areas that have been injured, usually following degeneration or necrosis. Dysphagia: Difficulty to swallow. Gliosis: Nonspecific reaction of the astrocytes in response to CNS insult, resulting in changes in morphological appearance, either by hypertrophy (increase in cell size) or hyperplasia (increased cell division). Hypoplasia: Underdevelopment or incomplete development of a tissue or organ which remain below the usual size or in an immature state. Microgliosis: Reaction of microglial cells to pathogenic insults, usually infectious agents, implicating in proliferation and altered cell morphology. Neurogenic atrophy: Muscular atrophy due to lack of innervation by motor axons. Organoids: Miniorgans produced experimentally (in vitro) from stem cells. Artificial grown tridimensional clusters of cells in culture media that resembles an organ. Pathogenesis: Biological mechanisms and chain of events leading to disease. Origin (genesis) of a disease (pathos). Progenitor cells: Undifferentiated cells that arise from stem cells and are capable to differentiate into specific types of cells. In the developing nervous system, they are located predominantly at the proximity of the lateral ventricles forming the periventricular germinal matrix.

Key facts Key facts of congenital Zika syndrome (CZS) l

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Calcification and microcephaly were the first observations during pregnancy that suggested a possible relationship with ZIKV infection. Other cerebral malformations, such as signs of abnormal neuronal migration and ventriculomegaly, were detected both with neuroimaging and postmortem studies after birth. Arthrogryposis and symptoms of pyramidal and extrapyramidal involvement are motor presentations of children with CZS. Visual manifestations, hearing impairment and other neurological symptoms, such as seizures, have also been reported.

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Postmortem studies added important information to explain the malformations and the various clinical presentations of CZS. The earlier the intrauterine infection, the more severe are the lesions. Not all babies are born with microcephaly, despite the fact that their brain is severely affected. Obstruction of the cerebral aqueduct due to severe brainstem damage may cause hydrocephalus and an enlarged head. Viral persistence may explain progression of the lesions after birth.

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This chapter focuses on the mechanisms by which ZIKV causes lesions in fetuses and neonates. Microcephaly and calcification follow massive destruction of progenitor cells although some babies are born with normal or enlarged head circumference. Abnormal neuronal migration due to radial glia and glia limitans loss causes epilepsy. Arthrogryposis is due to intrauterine lack of movements because of the loss of spinal motor neurons. Dysphagia may be due to nerve cell loss of brainstem nuclei impairing innervation of pharyngeal muscles. Reduction of hemispheric descending axons explains pyramidal symptoms, while the extrapyramidal ones may be related to malformed basal ganglia. Optic nerve and chiasm malformation, and/or degeneration/calcification of the retina and lens, explain part of the visual disturbances.

Acknowledgments Acknowledgments to the mothers, for their consent to perform the postmortem examinations, and to the Institutions where the specimens were collected for neuropathological studies: Research Institute Prof. Amorim Neto, Campina Grande, PB; Fernandes Figueira Institute—FioCruz, Rio de Janeiro, RJ; Antonio Pedro University Hospital, Fluminense Federal University, Nitero´i, RJ; and Municipal Hospital Jesus, Rio de Janeiro, RJ, Brazil.

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Dias, J. R. O., Ventura, C. V., Freitas, B. P., Prazeres, J., Ventura, L. O., Bravo-Filho, V., et al. (2018). Zika and the eye: Pieces of a puzzle. Progress in Retinal and Eye Research, 66, 85–106. Ferraris, P., Cochet, M., Hamel, R., Gladwyn-Ng, I., Alfano, C., Diop, F., et al. (2019). Zika virus differentially infects human neural progenitor cells according to their state of differentiation and dysregulates neurogenesis through the Notch pathway. Emerging Microbes & Infections, 8, 1003–1016. Franc¸a, T. L. B., Medeiros, W. R., Souza, N. L., Longo, E., Pereira, S. A., Franc¸a, T. B. O., et al. (2018). Growth and development of children with microcephaly associated with congenital Zika virus syndrome in Brazil. International Journal of Environmental Research & Public Health, 15, 1990. Garcez, P. P., Loiola, E. C., Costa, R. M., Higa, L. M., Trindade, P., Delvecchio, R., et al. (2016). Zika virus impairs growth in human neurospheres and brain organoids. Science, 352, 816–818. Hazin, A. N., Poretti, A., Cruz, D. D. C. S., Tenorio, M., van der Linden, A., Pena, L. J., et al. (2016). Computed tomographic findings in microcephaly associated with Zika virus. New England Journal of Medicine, 374, 2193–2195. Ho, C.-Y., Castillo, N., Encinales, L., Porras, A., Mendoza, A. R., Lynch, R., et al. (2018). Second-trimester ultrasound and neuropathologic findings in congenital Zika virus infection. Pediatric Infectious Disease Journal, 37, 1290–1293. Lage, M.-L. C., Carvalho, A. L., Ventura, P. A., Taguchi, T. B., Fernandes, A. S., Pinho, S. F., et al. (2019). Clinical, neuroimaging, and neurophysiological findings in children with microcephaly related to congenital Zika virus infection. International Journal of Environmental Research & Public Health, 16, E309. Leal, M. C., van der Linden, V., Bezerra, T. P., Valois, L., Borges, A. C. G., Antunes, M. M. C., et al. (2017). Characteristics of dysphagia in infants with microcephaly caused by congenital Zika virus Infection, Brazil, 2015. Emerging Infectious Disease, 23, 1253–1259. Ledur, P. F., Karmirian, K., Pedrosa, C. D. S. G., Souza, L. R. Q., Assis-de-Lemos, G., Martins, T. M., et al. (2020). Zika virus infection leads to mitochondrial failure, oxidative stress and DNA damage in human iPSC-derived astrocytes. Scientific Reports, 10, 1218. Marques, V. M., Santos, C. S., Santiago, I. G., Marques, S. M., Brasil, M. D. G. N., & Lima, T. T. (2019). Neurological complications of congenital Zika virus infection. Pediatric Neurology, 91, 3–10. Martines, R. B., Bhatnagar, J., Ramos, A. M. O., Davi, H. P., Iglezias, S. D., Kanamura, C. T., et al. (2016). Pathology of congenital Zika syndrome in Brazil: A case series. Lancet, 388, 898–904. Melo, A. S., Aguiar, R. S., Amorim, M. M., Arruda, M. B., Melo, F. O., Ribeiro, S. T., et al. (2016). Congenital Zika virus infection: Beyond neonatal microcephaly. Journal of the American Medical Association Neurology, 73, 1407–1416. Melo, A. S. O., Malinger, G., Ximenes, R., Szejnfeld, P. O., Sampaio, S. A., & Filippis, A. M. B. (2016). Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: Tip of the iceberg? Ultrasound in Obstetrics & Gynecology, 47, 6–7. Mlakar, J., Korva, M., Tul, N., Popovic, M., Poljsˇak-Prijatelj, M., Mraz, J., et al. (2016). Zika virus associated with microcephaly. New England Journal of Medicine, 374, 951–958. Moore, C. A., Staples, J. E., Dobyns, W. B., Pessoa, A., Ventura, C. V., Fonseca, E. B., et al. (2017). Characterizing the pattern of anomalies in congenital Zika syndrome for pediatric clinicians. Journal of American Medical Association Pediatrics, 171, 288–295. Nielsen-Saines, K., Brasil, P., Kerin, T., Vasconcelos, Z., Gabaglia, C. R., Damasceno, L., et al. (2019). Delayed childhood neurodevelopment and neurosensory alterations in the second year of life in a prospective cohort of ZIKV-exposed children. Nature Medicine, 25, 1213–1217. Nikitina, E., Larionova, I., Choinzonov, E., & Kzhyshkowska, J. (2018). Monocytes and macrophages as viral targets and reservoirs. International Journal of Molecular Medicine, 19, 2821. Oliveira-Szejnfeld, P. S., Levine, D., Melo, A. S., Amorim, M. M., Batista, A. G., Chimelli, L., et al. (2016). Congenital brain abnormalities and Zika virus: What the radiologist can expect to see prenatally and postnatally. Radiology, 281, 203–218. Pardy, R. D., & Richer, M. J. (2019). Zika virus pathogenesis: From early case reports to epidemics. Viruses, 11, 886. Peloggia, A., Ali, M., Nanda, K., & Bahamondes, L. (2018). Zika virus exposure in pregnancy and its association with newborn visual anomalies and hearing loss. International Journal of Gynaecology and Obstetrics, 143, 277–281. Pereira, H. V. F. S., Santos, S. P., Am^ancio, A. P. R. L., Oliveira-Szejnfeld, P. S., Flor, E. O., Tavares, J. S., et al. (2020). Neurological outcomes of congenital Zika syndrome in toddlers and preschoolers: A case series. The Lancet Child & Adolescent Health. https://doi.org/10.1016/S23524642(20)30041-9. Pessoa, A., van der Linden, V., Yeargin-Allsopp, M., Carvalho, M. D. C. G., Ribeiro, E. M., Van Naarden Braun, K., et al. (2018). Motor abnormalities and epilepsy in infants and children with evidence of congenital Zika virus infection. Pediatrics, 141(Suppl. 2), S167–S179. Pool, K. L., Adachi, K., Karnezis, S., Salamon, N., Romero, T., Nielsen-Saines, K., et al. (2019). Association between neonatal neuroimaging and clinical outcomes in Zika-exposed infants from Rio de Janeiro, Brazil. Journal of the American Medical Association Network Open, 2, e198124. Sa´nchez, L. A. G., Martı´nez, D. K. S., Dı´az-Martı´nez, L. A., & Mojica, C. H. B. (2019). Zika virus infection: A correlation between prenatal ultrasonographic and post-mortem neuropathologic changes. Neuropathology, 39, 434–440. Santos, S. F. M., Soares, F. V. M., Abranches, A. D., Costa, A. C. C., Gomes-Ju´nior, S. C. D. S., Fonseca, V. M., et al. (2019). Nutritional profile of newborns with microcephaly and factors associated with worse outcomes. Clinics (Sa˜o Paulo, Brazil), 74, e798. Schwartz, D. A. (2016). Autopsy and postmortem studies are concordant: Pathology of Zika virus infection is neurotropic in fetuses and infants with microcephaly following transplacental transmission. Archives of Pathology and Laboratory Medicine, 141, 68–72. Sher, A. A., Glover, K. K. M., & Coombs, K. M. (2019). Zika virus infection disrupts astrocytic proteins involved in synapse control and axon guidance. Frontiers in Microbiology, 10, 569. Silva, A. A. M., Ganz, J. S., Sousa, P. D., Doriqui, M. J., Ribeiro, M. R., Branco, M. D., et al. (2016). Early growth and neurologic outcomes of infants with probable congenital Zika virus syndrome. Emerging Infectious Disease, 22, 1953–1956. Soares, F., Abranches, A. D., Villela, L., Lara, S., Arau´jo, D., Nehab, S., et al. (2019). Zika virus infection in pregnancy and infant growth, body composition in the first three months of life: A cohort study. Scientific Reports, 9, 19198.

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Solomon, I. H., Milner, D. A., & Folkerth, R. D. (2016). Neuropathology of Zika virus infection. Journal of Neuroinfectious Disease, 7, 220. Sousa, A. Q., Cavalcante, D. I. M., Franco, L. M., Arau´jo, F. M. C., Sousa, E. T., Valenc¸a-Junior, J. T., et al. (2017). Postmortem findings for 7 neonates with congenital Zika virus infection. Emerging Infectious Diseases, 23, 1164–1167. Sˇtrafela, P., Vizjak, A., Mraz, J., Mlakar, J., Pizˇem, J., Tul, N., et al. (2016). Zika virus-associated micrencephaly: A thorough description of neuropathologic findings in the fetal central nervous system. Archives of Patholology and Laboratory Medicine, 141, 73–81. Sutarjono, B. (2019). Can we better understand how Zika leads to microcephaly? A systematic review of the effects of the Zika virus on human brain organoids. Journal of Infectious Diseases, 219, 734–745. Tang, H., Hammack, C., Ogden, S. C., Wen, Z., Qian, X., Li, Y., et al. (2016). Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell, 18, 587–590. Van Der Linden, V., Petribu, N. C. L., Pessoa, A., Faquini, I., Paciorkowski, A. R., van der Linden, H., Jr., et al. (2019). Association of severe hydrocephalus with congenital Zika syndrome. Journal of the American Medical Association Neurology, 76, 203–210. Ventura, C. V., Ventura Filho, M. C., & Ventura, L. O. (2019). Ocular manifestations and visual outcome in children with congenital Zika syndrome. Topics in Magnetic Resonance Imaging, 28, 23–27. Zin, A. A., Tsui, I., Rossetto, J. D., Gaw, S. L., Neves, L. M., Zin, O. A., et al. (2018). Visual function in infants with antenatal Zika virus exposure. Journal of American Association for Pediatric Ophthalmology and Strabismus, 22, 452–456. e1.

Chapter 15

Developmental trajectories in infants and toddlers born with congenital Zika syndrome Fernanda J.P. Marquesa,b, Osvaldo J.M. Nascimentob, and Marcio Leyserc a

SARAH Network of Rehabilitation Hospitals, Rio de Janeiro, Brazil, b Department of Neurology and Neurosciences, Fluminense Federal University, Rio

de Janeiro, Brazil, c University of Iowa Stead Family Department of Pediatrics, Division of Developmental and Behavioral Pediatrics, Iowa City, IA, United States

Abbreviations AIMS CZS GMFCS WHO ZIKV

Alberta Infant Motor Scale Congenital Zika Syndrome Gross Motor Function Classification System World Health Organization Zika virus

Introduction Congenital infectious diseases can lead to malformations during the brain’s cortical development and are often accompanied by poor neurodevelopmental outcomes (Levine, Jani, Castro-Aragon, & Cannie, 2017; Marques et al., 2018). In 2016, a new congenital infectious disease emerged amidst an epidemic. Congenital Zika syndrome (CZS) causes malformations of both central and peripheral nervous systems (De Oliveira Melo et al., 2016). The congenital Zika spectrum of neurological manifestations goes beyond microcephaly. A high association between severe brain abnormalities, congenital contractures, visual and hearing impairments, epilepsy, dysphagia, neurodevelopmental disorders, and Zika virus have been already demonstrated (Moore et al., 2017). Additionally, CZS is unequivocally associated with syndromic presentations that underpin the diagnostic criteria for cerebral palsy (Marques et al., 2018). Families of infants who were born with congenital Zika syndrome (CZS) have been facing uncertainties when it comes to their children’s developmental outcomes (Bailey & Ventura, 2018). Today, we have a generation of children and families in need of multiple types of support and treatments. According to the World Health Organization (WHO) (www.who.int), early child development is the foundation of economic productivity, social equality, and welfare of the nations. In that regard, CZS significantly affects the proper acquisition of children’s developmental milestones and the affect-related developmental trajectories (Alves, Paredes, Silva, Mello, & Alves, 2018; Carvalho et al., 2019; De Oliveira Melo et al., 2016; Einspieler et al., 2019; Marques et al., 2018; Pessoa et al., 2018; Satterfield-Nash et al., 2017; Wilder-Smith et al., 2019). While assessing children’s development is difficult and time-intensive, such evaluations at early stages of life are imperative in children with CZS so that adequate multidisciplinary care can be provided aiming at changes in developmental outcomes. Given the continuous need to further advance knowledge about this novel congenital infectious disease, including how CZS may affect children’s overall growth, global development, and well-being in the long term, the aim of this chapter is to describe the neurodevelopmental trajectories in infants born with CZS. The early identification of infants at risk is fundamental for planning and coordination of early intervention strategies. The term “infant” is typically applied to young children under 1 year of age (https://www.cdc.gov/ncbddd/ childdevelopment/positiveparenting/infants.html); however, definitions may vary and may include children up to 2 years of age (https://www.who.int/social_determinants/themes/earlychilddevelopment/en/). In this chapter, we will consider the term “infant” for children under 12 months of age and the term “toddler” for children between 12 and 24 months of age. Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00015-8 Copyright © 2021 Elsevier Inc. All rights reserved.

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Zika virus and the developing brain The first studies that established a cause-effect relationship between Zike virus (ZIKV) and microcephaly were conducted by Mlakar et al., who identified significant malformations in an embryo (Mlakar, Korva, Tul, Popovic, et al., 2016), and Garcez et al., who showed the neurotropism of ZIKV to progenitor cells through the visualization of neurospheres inside of the progenitor cells (Garcez et al., 2016). The pathophysiology of the central nervous system involvement is heterogeneous and complex (Aragao et al., 2016; Mulkey et al., 2019). Some fundamental mechanisms responsible for brain malformations are described below: l

l

Vascular insufficiency and neuroprogenitor cell dysfunction during fetal development and multifocal dystrophic calcifications. Necrosis and gliosis of highly vascularized deep gray matter tissue and abnormal migration of neuroprogenitor cells (Fig. 1).

Congenital Zika syndrome Congenital ZIKV syndrome emerges as a novel diagnosis because of the range of its peculiar clinical, neurological, and imaging aspects (Moore et al., 2017). The most frequent clinical presentations include: l

Variations in cranial morphology: severe microcephaly, overlapping cranial sutures, prominent occipital bone, redundant scalp skin, and neurological impairment.

FIG. 1 Typical neuroimaging findings in CZS. Patients 2 (A, E), 3 (F), 4 (G, H), 5 (I, J), 10 (K), and 11 (L) underwent fetal ultrasound (USG), magnetic resonance imaging (MRI), and computed tomography (CT). (A) Multiple and aligned subcortical calcifications (arrowheads) at 29 weeks of gestation. (B) Reduced brain volume, corpus callosum hypoplasia, vermin hypoplasia (arrowhead), and enlarged large cistern. (C) Abnormal cortical development (pachygyria). (D) Calcifications in basal and subcortical ganglia (arrowheads) in postpartum images. (E) Microcephaly in three-dimensional postnatal reconstruction (3D). (F) Abnormal cortical development, ex-vacuum ventriculomegaly, and ventricular septa between the atrium and occipital horn (arrowhead) with points suggestive of thalamus calcification at 29 weeks of gestation. (G) Subcortical calcifications (arrowheads) at 24-week gestation. (H) Significant reduction in cerebral parenchyma volume associated with lisencephaly (blue arrowhead) and marked underdevelopment of the diencephalon, brainstem, and cerebellum (yellow arrows) at 36 weeks of gestation. (I) Remarkable underdevelopment of the cerebral parenchyma and aligned subcortical calcifications (arrowheads) at 37 weeks of gestation. (J) Ventriculomegaly and abnormal cortical development with polymicrogyria (arrowheads) in postpartum images. (K) Subcortical calcifications (white arrowheads) and cranial deformity (red arrows) on postnatal imaging. (L) reduced brain volume, ventriculomegaly, and aligned subcortical calcifications (arrowheads) on postnatal imaging. (From Melo et al. (2016), with permission.)

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l

l l

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Brain abnormalities: thinning of the cerebral cortex, neuronal migration abnormalities, ventriculomegaly, subcortical calcifications, corpus callosum abnormalities, reduced white matter, and cerebellar vermis hypoplasia. Eye anomalies: structural abnormalities (microphthalmia, coloboma), cataracts, chorioretinal atrophy, focal pigment spots, and optic nerve hypoplasia/atrophy. Congenital contractures: unilateral or bilateral clubfoot and congenital multiple arthrogryposis. Neurological and neurodevelopmental disorders: motor deficits, cognitive disabilities, hypertonia/spasticity, hypotonia, irritability/excessive crying, tremors and extrapyramidal symptoms, swallowing dysfunction, visual impairment, hearing loss, and epilepsy.

Motor development Many factors can contribute to a poor motor prognosis in infants with CZS. The combination of congenital contractures, early signs of extrapyramidal involvement, and cognitive impairment are some mechanisms liable for extremely delayed infants and their significant limitations in properly attaining motor development milestones. Several authors have previously documented the gross motor skills development in this population (Alves et al., 2018; Carvalho et al., 2019; Da Silva Pone et al., 2018; De Oliveira Melo et al., 2016; Einspieler et al., 2019; Marques et al., 2018; Pessoa et al., 2018; Satterfield-Nash et al., 2017; Wilder-Smith et al., 2019). In all case-series illustrated in the literature, cerebral palsy (CP) was a frequent coexisting secondary outcome (Fig. 2).

Cognitive development In addition to sensory-motor involvement, severe-to-profound cognitive disability is expected in this population (Franc¸a et al., 2016), as showed in 1991 when a direct link between small head size and intellectual disability (ID) was shown (Dolk, 1991). Besides the underlying brain lesions, one justification for poor cognitive outcomes in CZS is that children with gross and fine motor delays are less likely able to explore the environment, a key determinant for cognitive development. Moreover, their hearing and visual deficits may interfere with the overall cognitive analysis Table 1 represents the neurological and neurodevelopmental evaluations undertaken in 35 infants born with CZS followed in a rehabilitation center in Rio de Janeiro, Brazil. The Bayley III scales were utilized to assess these children’s developmental milestones at 12 and 24 months of age. Regarding the cognitive domain, the discrepancy between the expected development in typically developing children and children born with CZS worsened over time. The milestones Average AIMS 20

16

15.77 14.13

Average

12 9.74

8

4

0 AIMS 6 months

AIMS 12 months

AIMS 18 months

FIG. 2 AIMS average over time in 39 infants with congenital Zika syndrome. The mean difference is significant at the level 0.05 (Bonferroni test) (Marques et al., 2018).

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TABLE 1 Developmental milestones based on the results of the Bayley III scales (Albers & Grieve, 2007). 12 months

24 months

Head balance

8 (22.9%)

10 (28.6%)

Rolls from back to sides

4 (11.4%)

5 (14.3%)

Crawls

2 (5.7%)

5 (14.3%)

Sits without support

3 (8.6%)

5 (14.3%)

Reciprocal creeping

2 (5.7%)

5 (14.3%)

Stands with support

2 (5.7%)

5 (14.3%)

Walks

0

2 (5.7%)

Regards object for 3 s

19 (54.3%)

21 (60.0%)

Eyes-follow

15 (42.9%)

17 (48.6%)

Reacts to sounds in the environment

31 (88.6%)

34 (97.1%)

Finds hidden object

1 (2.9%)

5 (14.3%)

Two vowel sounds

25 (42.9%)

22 (62.9%)

Responds to name

11 (31.4%)

15 (42.9%)

Directs attention of other

4 (11.4%)

6 (8.6%)

Uses one-word approximations

3 (8.6%)

5 (14.3%)

Social smile

24 (68.6%)

34 (97.1%)

Demonstration of relational play

–

5 (14.3%)

Uses a two-word utterance

–

1 (2.9%)

Only two (5.7%) infants could walked independently at 24 months. Five (14.3%) infants demonstrated understanding of relational playing, and one (2.9%) infant produced at least two-word utterance with different concepts.

achieved essentially did not change. Carvalho et al. also reported very low cognitive scores in the vast majority of infants assessed with the Bayley III scales (Table 2). Although severe cognitive delays are expected in this population, the results from this assessment’s subset should be interpreted carefully because of the impaired sensory issues (Bayley, 1969). Hearing and visual deficits are also features that can be part of the clinical manifestations of CZS and, therefore, might interfere with the overall cognitive analysis (Nielsen-Saines et al., 2019; Russell, Weaver, Bull, Weinbaum, & Opitz, 1984). Besides, the cognitive assessment depends upon fine motor manipulation, which is limited in most children with CZS because of significant spasticity and/or arthrogryposis (Lopes Moreira et al., 2018; Morgan, Honan, Allsop, Novak, & Badawi, 2018).

Communication skills When compared to other domains, communication showed the lowest progression over time. The widespread malformation of the brain’s cortical development might be responsible for language impairment (Aragao et al., 2016). Table 3 shows deficits in communication skills in a population of infants born with CZS (Alves et al., 2018).

General implications Because of multiple variabilities and severe developmental delays, infants born with CZS are likely to become extremely limited in terms of their overall functioning. This contributes to lifelong complex and challenging care (Wheeler, 2018; Wilder-Smith et al., 2019). Over the last decades, perspectives on children with disabilities have been revisited to take what we know as “rehabilitation treatment” to the next level. While refusing to accept the continuity of old rehabilitation models, abandoning

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TABLE 2 Neurodevelopmental evaluation with the Bayley III scales in 82 infants with probable congenital Zika syndrome. Characteristic

No. (%) a

Cognitive score Extremely low

78/82 (95.1)

Borderline

2/82 (2.4)

Low average

2/82 (2.4) a

Language score Extremely low

80/82 (97.6)

Low average

2/82 (2.4)

a

Motor score

Extremely low

80/82 (97.6)

Borderline

2/82 (2.4)

Muscular tone abnormalities

b

Generalized hypertonia

41/80 (51.2)

Upper extremities predominant hypertonia

33/80 (41.2)

Lower extremities predominant hypertonia

1/80 (1.3)

Unilateral hypertonia

4/80 (5.0)

Hypotonia

1/80 (1.3)

Abnormal persistence of primitive reflexes

70/74 (94.6)

Presence of expected postural reactions

5/73 (6.8)

c

HC categories 5 days; hyperbilirubinemia, perinatal asphyxia; aminoglycoside administration for more than 5 days, and other perinatal risk fators. In utero infections, such as CMV, herpes, rubella, syphilis, and toxoplasmosis. Craniofacial anomalies, including those that involve the external ear structures and the temporal bone.

To see the complete list of the risk indicators, refer to: https://digitalcommons.usu.edu/jehdi/vol4/iss2/1, where access to the Joint Committee on Infant Hearing’s Position Statement is also available in full. Children who do not pass the hearing screening should be referred for a complete audiological evaluation by 2 months. This assessment includes family history collection, ENT examination, observation of the child’s response to sound, and diagnostic audiological examinations, namely FS-ABR, click-evoked ABR with cochlear microphonics testing as needed, and tympanometry. Once the hearing loss is diagnosed, appropriate intervention should begin as soon as possible, no later than 6 months of age. The Algorithm for Hearing Screening is available at: https://www.asha.org/practice-portal/professional-issues/ newborn-hearing-screening/#collapse_1. Fig. 2 shows a simplified flow chart for the Universal Hearing Screening.

Policy and procedures: Major hearing assessment tests and their uses There are a number of tests to evaluate the hearing function and the indications to perform each of them will depend on some factors such as the patient’s age, level of cooperation, and what is intended to be evaluated. The combination of tests increases the accuracy of the diagnosis of hearing alterations. These include: Electrophysiological: l l

Otoacoustic emissions (OAE) Auditory Brainstem Responses (ABR) l Automated ABR (aABR) l Click-evoked ABR l Frequency-specific ABR (FS-ABR)

Physiological: l

Imitanciometry l Tympanometry l Acoustic reflex thresholds

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FIG. 2 Flow chart for newborn hearing screening. Newborn hearing screening is mandatory for all children and should be carried out preferably before discharge. Special attention must be given to those in the risk group, who should be screened by automated ABR. This flow chart illustrates the pathway to adequate management in the hearing assessment. OAE, otoacoustic emissions; aABR, automated Auditory Brainstem Response; ABR, Auditory Brainstem Response.

Psychoacoustical: l

Audiometry l Conventional audiometry l Behavioral audiometry l Visual reinforcement audiometry l Conditioned Play Audiometry

To learn more about the types of tests used to evaluate hearing in children and adults, visit: https://www.asha.org/public/ hearing/Types-of-Tests-Used-to-Evaluate-Hearing/.

Mini-dictionary of terms Neurotropism. Affinity for neural tissue. A neurotropic infectious agent may cause damage to central or peripheral nervous system organs or may lie dormant in these regions without any clinical repercussions. Hearing threshold. Minimum level of sound intensity at which the individual is able to perceive the stimulus in a quiet and ideal environment. The normal hearing threshold for the frequencies audible to the human ear ranges between 0 and 25 dB.

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Conductive hearing loss. Characterized by the impedance of the sound wave being transmitted properly through the vibration of the tympanic-ossicular system to the cochlea—where the mechanical energy will be transformed into electrical energy. Can be caused by tympanic perforations, ossicular chain stiffness, presence of fluid in the middle ear, and external ear canal cerumen plugs, among other causes. Sensorineural hearing loss. Is characterized by dysfunction of cochlear sensory cells or neural auditory pathways. This condition has numerous causes, including certain congenital infections, aging, the use of ototoxic medications, and loud noise injuries, among others. Cochlear hair cells. Sensory cells located in the Organ of Corti, inside the cochlea. Responsible for the reception, amplification, and mecanoelectrical transduction of the sound stimulus, through the deflection of their stereocilia. Outer and inner hair cells exist, each group with a specific function. Auditory neuropathy. Dissynchrony of the electrical conduction of auditory stimuli by the auditory nerve. Its expression occurs through a spectrum of clinical manifestations. This condition may have genetic, infectious, perinatal, and morphological causes, among others. Cochlear microphonics. Consists in preneural electrical activity originating in the outer hair cells of the cochlea, and its presence indicates cochlear activity. It is detected by ABR, may be more evident when ABR has no replicable waves (altered neural conduction), and tends to last longer in individuals presenting with auditory neuropathy spectrum disorder.

Key facts of hearing loss l

l

l

l

l

Hearing loss is currently the fourth leading cause of years lived with disability worldwide, which represents a substantial health and economic burden for individuals and society (GBD 2016 Disease and Injury Incidence and Prevalence Collaborators, 2017). Hearing deprivation negatively impacts language acquisition in children, as well as the ability to communicate using spoken language, the maintenance of an adequate auditory processing, increasing educational level, cognition, and social and emotional development ( Joint Committee on Infant Hearing, 2019). Universal newborn hearing screening has raised the opportunity for early diagnosis and intervention in congenital hearing losses, improving the prognosis in these cases. The regular surveillance of the hearing development is also important, and parents and health-care agents should be aware of developmental milestones (Harlor & Bower, 2009). Prevention strategies are the most cost-effective way to reduce the burden of hearing loss, and the World Health Organization estimates that around 50% of the hearing losses could be prevented (Wilson et al., 2017). Hearing loss in older people has been linked with increased chances of developing dementia (Thomson, Auduong, Miller, & Gurgel, 2017).

Summary points l

l

l

l

l

l

l

The quality of scientific evidence for the causal association between ZIKV infection and hearing loss is still insufficient regarding both congenital and acquired infections. Some studies have reported a hearing impairment frequency in microcephalic children exposed to prenatal ZIKV, similar to other risk groups for hearing impairment. There appears to be a relationship between the degree of clinical impairment of patients with prenatal ZIKV exposure and hearing loss. In ZIKV-acquired infection, reported hearing loss is transient and sensorineural, but the evidence is limited to a few case reports. The pathogenic mechanisms of possible auditory impairment in ZIKV infection have not yet been clarified. It is assumed that a mechanism of neural damage may be involved, considering ZIKV neurotropism, but further studies are required. Most authors recommend that congenital ZIKV infection be included in the congenital hearing impairment risk group and screening should include ABR. There is still controversy regarding the need for mandatory follow-up hearing tests. Evidence is still lacking to establish a hearing monitoring recommendation for acquired infection. The need to clarify many gaps regarding hearing loss in ZIKV infection opens the space for future research in the area.

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References Adebanjo, T., Godfred-Cato, S., Viens, L., Fischer, M., Staples, J. E., Kuhnert-Tallman, W., … Moore, C. A. (2017). Update: Interim guidance for the diagnosis, evaluation, and management of infants with possible congenital Zika virus infection—United States, October 2017. MMWR. Morbidity and Mortality Weekly Report, 66, 1089–1099. Aragao, M. F. V., Van der Linden, V., Brainer-Lima, A. M., Coeli, R. R., Rocha, M. A., Da Silva, P. S., … Valenca, M. M. (2016). Clinical features and neuroimaging (CT and MRI) findings in presumed Zika virus related congenital infection and microcephaly: Retrospective case series study. BMJ, 353, 1–10. Barbosa, M. H.d. M., Magalha˜es-Barbosa, M. C.d., Robaina, J. R., Prata-Barbosa, A., Lima, M. A.d. M. T.d., & Cunha, A. J. L. A.d. (2019). Auditory findings associated with Zika virus infection: An integrative review. Brazilian Journal of Otorhinolaryngology, 85, 642–663. Barbosa, M. H.d. M., Magalha˜es-Barbosa, M. D.d., Robaina, J. 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Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. The Lancet, 390, 1211–1259. Gely-Rojas, L., Garcı´a-Fragoso, L., Negro´n, J., Deynes, D., Garcı´a-Garcı´a, I., & Zorrilla, C. D. (2018). Congenital Zika syndrome in Puerto Rico, beyond microcephaly, a multiorgan approach. Puerto Rico Health Sciences Journal, 37, S73–S76. Harlor, A. D. B., & Bower, C. (2009). Hearing assessment in infants and children: Recommendations beyond neonatal screening. Pediatrics, 124, 1252–1263. Joint Committee on Infant Hearing. (2019). Year 2019 Position Statement: Principles and Guidelines for Early Hearing Detection and Intervention Programs. The Journal of Early Hearing Detection and Intervention, 4(2), 1–44. https://doi.org/10.15142/fptk-b748. In this issue. Julander, J. G., Siddharthan, V., Park, A. H., Preston, E., Mathur, P., Bertolio, M., … Morrey, J. D. (2018). Consequences of in utero exposure to Zika virus in offspring of AG129 mice. Scientific Reports, 8, 1–11. Kim, C.-H., Choi, H., & Shin, J. E. (2016). Characteristics of hearing loss in patients with herpes zoster oticus. Medicine, 95, 1–5. Kim, J., Jung, J., Moon, I. S., Lee, H.-K., & Lee, W.-S. (2008). Statistical analysis of pure tone audiometry and caloric test in herpes zoster oticus. Clinical and Experimental Otorhinolaryngology, 1, 15–19. Lage, M. L. C., Nascimento-Carvalho, C., Fernandes, A., Carvalho, A., Ventura, P., Taguchi, T., … Santos-Junior, O. (2019). Clinical, neuroimaging, and neurophysiological findings in children with microcephaly related to congenital Zika virus infection. International Journal of Environmental Research and Public Health, 16, E309–E318. Leal, M. D. C., Muniz, L. F., Caldas Neto, S. D. S., van der Linden, V., & Ramos, R. C. F. (2016). Sensorineural hearing loss in a case of congenital Zika virus. Brazilian Journal of Otorhinolaryngology. pii:S1808-8694(16)30127-6. Leal, M. C., Muniz, L. F., Ferreira, T. S. A., Santos, C. M., Almeida, L. C., Van der Linden, V., … Caldas Neto, S. S. (2016). Hearing loss in infants with microcephaly and evidence of congenital Zika virus infection—Brazil, November 2015–May 2016. MMWR. Morbidity and Mortality Weekly Report, 65, 917–919. Leal, M. D. C., Ramos, D. S., & Caldas Neto, S. S. (2019). Hearing loss from congenital Zika virus infection. Topics in Magnetic Resonance Imaging, 28, 19–22. Leite, R. F. P., Santos, M. S. A., Pessoa, A. L. S., Ribeiro, E. M., Cavalcanti, L. P. D. G., Giacheti, C. M., & Lewis, D. R. (2018). Hearing screening in children with congenital Zika virus syndrome in Fortaleza, Ceara´, Brazil, 2016. Epidemiologia e Servic¸os de Sau´de, 27, 1–10.

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Lin, F., Resnick, S., Kraut, M. A., Ferrucci, L., & An, Y. (2014). Hearing loss linked to accelerated brain tissue loss. Retrieved from https://www. hopkinsmedicine.org/news/media/releases/hearing_loss_linked_to_accelerated_brain_tissue_loss_. Martins, O. R., Rodrigues, P. D. A. L., Santos, A. C. M. D., Ribeiro, E. Z., Nery, A. F., & Lima, J. B. (2017). Otological findings in patients following infection with Zika virus: Case report. Audiology: Communication Research, 22, 1–9. Miranda-Filho, D. B., Martelli, C. M. T., De Alencar Ximenes, R. A., Arau´jo, T. V. B., Rocha, M. A. W., Ramos, R. C. F., … Rodrigues, L. C. (2016). Initial description of the presumed congenital Zika syndrome. American Journal of Public Health, 106, 598–600. Mittal, R., Fifer, R. C., & Liu, X. Z. (2017). A possible association between hearing loss and Zika virus infections. JAMA Otolaryngology. Head & Neck Surgery, 144, 3–4. Nielsen-Saines, K., Brasil, P., Kerin, T., Vasconcelos, Z., Gabaglia, C. R., Damasceno, L., … Moreira, M. E. (2019). Delayed childhood neurodevelopment and neurosensory alterations in the second year of life in a prospective cohort of ZIKV-exposed children. Nature Medicine, 25, 1213–1217. Nogueira, M. L., Nery Ju´nior, N. R. R., Estofolete, C. F., Bernardes Terzian, A. C., Guimara˜es, G. F., Zini, N., … Ko, A. I. (2017). Adverse birth outcomes associated with Zika virus exposure during pregnancy in Sa˜o Jose do Rio Preto, Brazil. Clinical Microbiology and Infection, 24, 646–652. Peloggia, A., Ali, M., Nanda, K., & Bahamondes, L. (2018). Zika virus exposure in pregnancy and its association with newborn visual anomalies and hearing loss. International Journal of Gynecology & Obstetrics, 143, 277–281. Pichora-Fuller, M. K., & Singh, G. (2006). Effects of age on auditory and cognitive processing: Implications for hearing aid fitting and audiologic rehabilitation. Trends in Amplification, 10, 29–59. Pool, K.-L., Adachi, K., Karnezis, S., Salamon, N., Romero, T., Nielsen-Saines, K., … Pone, M. (2019). Association between neonatal neuroimaging and clinical outcomes in Zika-exposed infants from Rio de Janeiro, Brazil. JAMA Network Open, 2, e198124. Racicot, K., VanOeveren, S., & Alberts, A. (2017). Viral hijacking of formins in neurodevelopmental pathologies. Trends in Molecular Medicine, 23, 778–785. Rao, V. B., Maneesha, K., Sravya, P., Franchito, S. H., Dasari, H., & Gan, M. A. (2019). Future increase in extreme El Nin˜o events under greenhouse warming increases Zika virus incidence in South America. npj Climate and Atmospheric Science, 2, 2–8. Rodriguez-Barraquer, I., Nascimento, E. J. M., Marques, E. T. A., Costa, F., Azar, S. R., Adhikarla, H., … Cruz, J. (2019). Impact of preexisting dengue immunity on Zika virus emergence in a dengue endemic region. Science, 363, 607–610. Sanz Cortes, M., Rivera, A. M., Yepez, M., Guimaraes, C. V., Diaz Yunes, I., Zarutskie, A., … Parra Saavedra, M. (2018). Clinical assessment and brain findings in a cohort of mothers, fetuses and infants infected with ZIKA virus. American Journal of Obstetrics and Gynecology, 218, 440.e1–440.e36. Satterfield-Nash, A., Kotzky, K., Allen, J., Bertolli, J., Moore, C. A., Pereira, I. O., … Peacock, G. (2017). Health and development at age 19-24 months of 19 children who were born with microcephaly and laboratory evidence of congenital Zika virus infection during the 2015 Zika virus outbreak—Brazil, 2017. MMWR. Morbidity and Mortality Weekly Report, 66, 1347–1351. Silva, M. F. A. D. A., & Mendonc¸a de Arau´jo, F. C. (2018). Hearing screening in children exposed to Zika virus. In II Congresso Brasileiro de Ci^ encias Da Sau´de, (1), Campina Grande—Paraı´ba. Available at www.conbracis.com.br. Soares de Oliveira-Szejnfeld, P., Levine, D., Melo, A. S. D. O., Amorim, M. M. R., Batista, A. G. M., Chimelli, L., … Tovar-Moll, F. (2016). Congenital brain abnormalities and Zika virus: What the radiologist can expect to see prenatally and postnatally. Radiology, 281, 203–218. Tappe, D., Nachtigall, S., Kapaun, A., Schnitzler, P., G€unther, S., & Schmidt-Chanasit, J. (2015). Acute Zika virus infection after travel to Malaysian Borneo, September 2014. Emerging Infectious Diseases, 21, 911–913. Thomson, R. S., Auduong, P., Miller, A. T., & Gurgel, R. K. (2017). Hearing loss as a risk factor for dementia: A systematic review. Laryngoscope Investigative Otolaryngology, 2, 69–79. Van Der Linden, V., Filho, E. L. R., Lins, O. G., Van Der Linden, A., Araga˜o, M. D. F. V. V., Brainer-Lima, A. M., … Ramos, R. C. (2016). Congenital Zika syndrome with arthrogryposis: Retrospective case series study. BMJ, 354, i3899. Ventura, C. V., Maia, M., Dias, N., Ventura, L. O., & Belfort, R. (2016). Zika: Neurological and ocular findings in infant without microcephaly. The Lancet, 387, 2502. Ventura, C. V., Maia, M., Ventura, B. 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Chapter 23

Neuromyelitis optica spectrum disorder: What it is and how it relates to Zika virus Maı´ra Cardoso Aspahana and Paulo Pereira Christob a

Department of Neurology, Madre Teresa Hospital, Belo Horizonte, Brazil, b Department of Neurology, Clinical Hospital, Federal University of Minas

Gerais, Belo Horizonte, Brazil

Abbreviations Anti-MOG APS AQP4 AQP4-Abs AQP4-IgG AZA BBB CBA CHIKV CNS DENV FLAIR IL-6 LETM MMF MS NMO NMOSD ON RTX STIR ZIKV

anti-myelin oligodendrocyte glycoprotein area postrema syndrome anti-aquaporin 4 anti-aquaporin 4 antibody anti-aquaporin 4 antibody azathioprine blood-brain barrier cell-based assays chikungunya virus central nervous system dengue virus fluid attenuated inversion recovery interleukin-6 longitudinal extensive transverse myelitis mycophenolate mofetil multiple sclerosis neuromyelitis optica neuromyelitis optica spectrum disorder optic neuritis rituximab sagittal short T1 inversion recovery Zika virus

Introduction Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease of the central nervous system (CNS). It was described by Eugene Devic (1858–1930) and Fernand Gault (1873–1936) in 1894 as a syndrome characterized by myelitis and optic neuritis of severe courses ( Jarius & Wildemann, 2013). Originally known as Devic disease, it was for many years considered part of the multiple sclerosis (MS) spectrum until the discovery of a disease-specific marker: the anti-aquaporin 4 antibody (AQP4-Abs) (Lennon et al., 2004). This antibody is a protein against aquaporin 4 (AQP4) channels that are present in the spinal cord, optic nerves, as well as in the periaqueductal and periventricular regions of the brain (Marignier, Giraudon, Vukusic, Confavreux, & Honnorat, 2010). These channels are related to water transport and, for this reason, are essential to understand processes such as CNS edema and blood-brain barrier (BBB) disruption. Also, their expression is upregulated in astrocytes during neuroinflammation (Fukuda & Badaut, 2012). The discovery of AQP4-Abs allowed NMOSD to be considered an immune-mediated disease that differs from MS in its pathogenesis, clinical symptoms, imaging, progression, and treatment. More recently, another antibody, the anti-myelin

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oligodendrocyte glycoprotein (anti-MOG), was related to NMOSD. Anti-MOG works against the glycoprotein located on the myelin sheath and on the oligodendrocytes (Vourc’h & Andres, 2004) and some authors referred to this syndrome as anti-MOG encephalomyelitis (Weber, Derfuss, Metz, & Br€uck, 2018). Currently, other antibodies are linked to NMOSD: anti-neurofascin antibody ( Jia et al., 2019), connexine 43 (Masaki et al., 2013), and anti-aquaporin 1 (Tzartos et al., 2013). Many discoveries about NMOSD have led to definition updates in recent years. AQP4-Ab was first reported in 2004 and was further included in the diagnostic criteria in 2006 (Wingerchuk, Lennon, Pittock, Lucchinetti, & Weinshenker, 2006). In 2007, the term NMO spectrum disorder was created to include patients with initial or limited disease, and in 2015 the International Panel for NMO Diagnosis proposed the current revised criteria (Wingerchuk et al., 2015). These new criteria divide groups of patients with clinical symptoms and radiological characteristics into two different groups: AQP4-Abs positive and AQP4-Abs negative or unknown (Table 1 and Fig. 1). Under these new criteria, brain symptoms that were previously not part of the spectrum of the disease were included, such as the area postrema syndrome (APS), which is characterized by nausea, vomiting, and incoercible hiccups. Now, it is possible to determine the diagnosis of NMOSD with a single clinical event of myelitis, encephalitis, or optic neuritis in the presence of AQP4-Abs, thus allowing early diagnosis and treatment. The immunopathogenesis of positive AQP4-Abs NMOSD involves astrocyte injury and, consequently, loss of oligodendrocytes, demyelination, and axonal involvement. Several infections associated with the development of NMOSD have been reported in the medical literature, including Zika virus (ZIKV) (Aspahan et al., 2019) and dengue virus (DENV) (Puccioni-Sohler et al., 2017). ZIKV displays tropism for astrocytes (Sher, Glover, & Coombs, 2019) that are present in great quantities in the spinal cord, optic nerves, and brain. Also, ZIKV has been associated with myelitis, optic neuritis, and encephalitis, among several parainfectious and/or postinfectious neurological complications, which raises the discussion about the possibility of direct action of this infectious agent in the CNS as well as its role as an autoimmune disease trigger.

TABLE 1 NMOSD diagnostic criteria for adult patients. Diagnostic criteria for NMOSD with AQP4-IgG 1. At least 1 core clinical characteristic 2. Positive test for AQP4-IgG using best available detection method (cell-based assay strongly recommended) 3. Exclusion of alternative diagnoses Diagnostic criteria for NMOSD without AQP4-IgG or NMOSD with unknown AQP4-IgG status 1. At least 2 core clinical characteristics occurring as a result of one or more clinical attacks and meeting all of the following requirements: a. At least 1 core clinical characteristic must be optic neuritis, acute myelitis with LETM, or area postrema syndrome b. Dissemination in space (2 or more different core clinical characteristics) c. Fulfillment of additional MRI requirements, as applicable 2. Negative test(s) for AQP4-IgG using best available detection method, or testing unavailable 3. Exclusion of alternative diagnoses Core clinical characteristics 1. Optic neuritis 2. Acute myelitis 3. Area postrema syndrome: episode of otherwise unexplained hiccups or nausea and vomiting 4. Acute brain stem syndrome 5. Symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSD-typical diencephalic MRI lesions 6. Symptomatic cerebral syndrome with NMOSD-typical brain lesions Additional MRI requirements for NMOSD without AQP4-IgG and NMOSD with unknown AQP4-IgG status 1. Acute optic neuritis: requires brain MRI showing (a) normal findings or only nonspecific white matter lesions OR (b) optic nerve MRI with T2-hyperintense lesion or T1-weighted gadolinium-enhancing lesion extending over >1/2 optic nerve length or involving optic chiasm 2. Acute myelitis: requires associated intramedullary MRI lesion extending over 3 contiguous segments (LETM) or 3 contiguous segments of focal spinal cord atrophy in patients with prior history compatible with acute myelitis 3. Area postrema syndrome: requires associated dorsal medulla/area postremalesions 4. Acute brain stem syndrome: requires associated periependymal brain stem lesions Abbreviations: AQP4-IgG, aquaporin 4 IgG; LETM, longitudinally extensive transverse myelitis; MRI, magnetic resonance imaging; NMOSD, neuromyelitis optica spectrum disorder. From Wingerchuk, D. M., Banwell, B., Bennett, J. L., Cabre, P., Carroll, W., Chitnis, T., … International Panel for NMO Diagnosis (2015). International consensus diagnostic criteria for neuromyelitis optica spectrum disorders. Neurology, 85(2), 177–189. https://doi.org/10.1212/WNL.0000000000001729, with permission.

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FIG. 1 NMOSD diagnostic criteria for adult patients (Wingerchuk et al., 2015). AQP4-Abs, aquaporin-4 antibodies; LETM, longitudinally extensive transverse myelitis.

Epidemiology NMOSD is a worldwide disease that affects all ethnic groups and, differently from MS, has no relation to the country’s latitude. Recent Japanese studies (Mori, Kuwabara, & Paul, 2018) have shown that the estimated prevalence of NMOSD is 1.64–3.42 per 100,000 in Japan and the disease prevalence does not differ significantly by region, hardly ever exceeding 5 per 100,000. However, differences in ethnic groups may occur regarding disease characteristics, age of onset, and aggressiveness of attacks (Kim et al., 2018). The prevalence of sex strongly depends on the serological status. Positive AQP4-Abs patients are mainly women, affecting up to 10 times more females than males, thus representing 80% of the total NMOSD cases (Waters et al., 2012). However, in cases of seronegative NMOSD, this proportion is lower, despite its continuous tendency to affect women, with a prevalence of 2 women affected per man. Although the disease can affect people from 3 to 80 years of age, the mean age onset of this condition is 40 years (Weinshenker & Wingerchuk, 2017). A small portion of NMOSD patients may be positive for anti-MOG. However, they tend to be younger, with preferential and more severe optic nerve involvement as well as uncommon recurrence of attacks (Wu, Zhong, & Geng, 2019).

Pathogenesis The pathophysiology of NMOSD is based on the humoral characteristics of the disease and its prototype is based on the positive AQP4-Abs NMOSD. AQP4-Abs is an IgG1 isotype that acts against aquaporin channels, which are highly concentrated in the optic nerves, spinal cord, and brain (mainly in the periventricular and periaqueductal regions, where the cerebrospinal fluid [CSF] is in contact with the brain parenchyma) (Kim, Kim, Huh, & Kim, 2012). In NMOSD patients, high levels of peripheral B lymphocytes have already been identified via CD27highCD38highCD180 CD19 markers (Weinshenker & Wingerchuk, 2017). These cells display plasmablast characteristics, being involved in AQP4-Abs synthesis and stimulated by interleukin-6 (Chihara et al., 2011). The concentration of these antibodies is higher in serum than

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it is in the CSF, suggesting that they are synthesized peripherally and later cross the BBB (Wu et al., 2019). They interact with AQP4 channels at the astrocyte feet, thus initiating an immune cascade that involves complement activation and recruitment of natural killer cells, neutrophils, macrophages, and eosinophils (Wu et al., 2019). Serum AQP4-Abs levels can also be parameters for disease follow-up, as high titers are indicative of an active disease state. NMOSD lesions are associated with disruption of water intake in the CNS. This process leads to damage of astrocytes, myelin sheath, and axon. It may also activate B lymphocytes, leading to immunoglobulin production, interleukin-6 (IL-6) release, and subsequent vascular proliferation (Huda et al., 2019). This inflammatory reaction increases endothelial permeability, which results in necrosis accompanied by an infiltrate of lymphocytes. Some infectious agents have been associated with NMOSD such as ZKV, dengue virus (DENV), cytomegalovirus, HIV, and varicella zoster virus among others. Regarding ZKV, its association with NMOSD may have a direct action of the virus at the onset of the disease. ZIKV is highly neurotropic, and frequently PCR for this virus is positive in biological samples during acute optic neuritis, acute myelitis, and acute encephalitis that are secondary to this infection. However, patients with ZIKV-associated NMOSD may continue to display relapses even after PCR turns negative for the virus in biological samples. It is suggested that the clinical manifestation of ZIKV-associated NMOSD is due to an association of direct virus injury and postinfectious immune response. Case reports of Zika-associated NMOSD are still limited. Further understanding of the mechanism of action between these associations is needed. Positive anti-MOG cases with clinical criteria for NMOSD may display an overlap of clinical presentations (Weinshenker & Wingerchuk, 2017) and, for some authors, anti-MOG disease is a totally different disease (Weber et al., 2018). Generally, NMOSD anti-MOG patients display distinct clinical characteristics as well as pathophysiology and prognosis in comparison to those individuals who are only AQP4-Abs positive (Weinshenker & Wingerchuk, 2017). Anti-MOG antibody levels are higher in the periphery as compared to intrathecal concentrations (Wu et al., 2019). This suggests that this antibody is initially synthesized by peripheral B-lymphocytes (Weber et al., 2018) and further gains access to the central nervous system via MOG-specific T-cell activation (Wu et al., 2019). Even though histological studies are limited, patients with anti-MOG neurological disease do not show eosinophilic inflammatory infiltrate that is normally seen in acute attacks in AQP4-Abs patients (Weber et al., 2018). In addition, there is evidence of myelin sheath loss accompanied by relative axonal and astrocyte preservation and significant impairment of oligodendrocytes (Weber et al., 2018). Anti-MOG optic neuritis and myelitis have also been reported on their own, even without meeting the criteria for NMOSD diagnosis (Dos Passos et al., 2018).

Clinical features of neuromyelitis optica spectrum disorder The most prominent clinical manifestations associated with NMOSD are due to their preferred anatomical involvement: spinal cord, optic nerves, and brain (especially area postrema). According to the 2015 diagnostic criteria (Wingerchuk et al., 2015), the central clinical features of NMOSD are: optic neuritis, acute myelitis, APS, acute brainstem syndrome, symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSD-typical diencephalic MRI lesions, and symptomatic cerebral syndrome with NMOSD-typical brain lesions. According to the 2015 diagnostic criteria, one single episode of one of the core clinical (Fig. 2) manifestations accompanied by a positive AQP4-Abs would be enough for diagnosis. However, in cases of negative AQP4-Abs or unknown immune status, two clinical manifestations would be necessary for diagnosis, as long as the radiological characteristics (Fig. 3) are also present and alternative diagnoses are excluded.

Clinical core symptoms of NMOSD and its relationship to ZIKV Longitudinally extensive transverse myelitis associated with Zika virus Myelitis related to NMOSD usually presents as a longitudinally extensive transverse myelitis (LETM), which is a definition attributed to myelitis that affects most of the spinal cord diameter and extends into three or more consecutive vertebral segments (Fig. 4A and D). LETM is associated with white and gray matter inflammatory damage, demyelination, and axonal injury with a centrilobular distribution on the spine. LETM manifestations lead to spinal cord syndrome with clinical characteristics related to the affected segment of the spinal cord (cervical, thoracic, and/or lumbar). Due to this reason, patients with LETM may present a broad spectrum of clinical manifestations with sensory changes, paraplegia, quadriplegia, urinary/fecal retention, dysautonomia, and/or respiratory failure. Patients with AQP4-Abs and acute transverse myelitis do not need to achieve LETM radiological characteristics to meet NMOSD criteria.

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FIG. 2 Core clinical characteristics of NMOSD. Neuromyelitis optica spectrum disorder (NMOSD).

FIG. 3 Additional MRI requirements for NMOSD without AQP4-IgG and NMOSD with unknown AQP4-IgG status (Wingerchuk et al., 2015). LETM, longitudinally extensive transverse myelitis.

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FIG. 4 Spine, brain, and orbit MRI of a patient with neuromyelitis optica spectrum disorder associated with ZIKV. (A) Sagittal short T1 inversion recovery (STIR) spine MRI shows hyperintense, nonenhancing lesions in the T1–T4 and T6–T9 segments. (B) Fluid-attenuated inversion recovery axial brain MRI demonstrates hyperintensity in the pons, superior and middle left cerebellar peduncle, as well as periependymal lesion. (C) Axial orbit MRI T1 postcontrast shows extensive gadolinium enhancing lesion compromising at least 50% of the right nerve length compatible with optic neuritis. (D) Hyperintensities at level C3–C6 are depicted on the sagittal STIR spine MRI. (From Aspahan, M. C., Leonhard, S. E., Gomez, R. S., Rocha, E. da S., Vilela, M. R. da S., Alvarenga, P. P. M., … Meira, F. (2019). Neuromyelitis optica spectrum disorder associated with Zika virus infection. Neurology Clinical Practice, 9(1), e1–e3. https://doi.org/10.1212/CPJ.0000000000000546, with permission.)

In endemic countries for ZIKV, there have been several reports of LETM associated with this virus. In many of these cases, PCR for ZIKV was positive in biological samples. Also, many of these patients were also seropositive antibodies to previous arbovirus infections such as DENV and chikungunya (CHIKV) (Roma´n, Anaya, Mancera-Pa´ez, Pardo-Turriago, & Rodrı´guez, 2019). The mechanism that leads to these neurological complications is not fully elucidated. The possibility of a direct infection due to this virus’s neurotropism is discussed. However, the infection could possibly trigger the synthesis of polyclonal autoantibodies due to molecular mimicry and superantigen-mediated disease (Krishnan, Kaplin, Deshpande, Pardo, & Kerr, 2004), process that combines cellular and humoral immunities. Neurological complications secondary to Zika infection appear to be present or even exacerbated when the patient has a history of previous flavivirus infection (Oliveira et al., 2019). This preceding infection could increase ZKV’s viremia by antibody-dependent enhancement (ADE) immune response (Oliveira et al., 2019), in which poorly neutralizing antibodies facilitate the entry

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of the virus into monocytes. In this way, viremia and the severity of infection increase. For this reason, ADE immune response due to previous arbovirus infection facilitates cellular infection due to preexisting antibodies (Khandia et al., 2018). In the literature, Zika-related LETM was also reported to be associated with anti-MOG (Neri et al., 2018), even without meeting diagnostic criteria for NMOSD. Other demyelinating disorders, such as multiple sclerosis (MS) and acute disseminated encephalomyelitis (ADEM), have also been associated with LETM, outlining the importance of paying attention to these pathological entities in the differential diagnosis of myelitis (Acosta-Ampudia et al., 2018). Further studies are necessary to understand the relationship between ZIKV, LETM, and NMOSD.

Optic neuritis associated with ZIKV Optic neuritis (ON) is an inflammation of the optic nerve, which causes visual acuity reduction, eye movement pain, dyschromatopsia, and afferent pupillary defect. The ophthalmoscopy is normal when ON is retrobulbar and the optic disc can be blurred in cases of papillitis. The clinical diagnosis may be complemented by orbit MRI, visual evoked potential, and optical coherence tomography. In NMOSD, although MRI may also be normal, NO is radiologically T2-weighted or T1-induced gadolinium uptake in at least half of the optic nerve or with optic chiasm involvement. Fig. 4C shows an ON in the brain MRI of a patient with NMOSD associated with ZIKV. Cases of unilateral and bilateral optic neuritis were associated with ZKV infection. This neurological manifestation may occur at the end of the fever period or from 2 weeks to 1 month after the onset of the infection (Zaidi et al., 2018).

Encephalitis associated with ZIKV There are several reports of ZKV-associated encephalitis, rhomboencephalitis, and encephalomyelitis (Macedo et al., 2018). However, the presence of only one of the core manifestations is enough for NMOSD diagnosis, as long as the individual is also AQP4-Abs positive. Despite the characteristic clinical symptoms of Zika such as fever, skin rash, myalgia, arthralgia, and conjunctivitis, patients from endemic areas with encephalitis should be investigated for AQP4-Abs if they exhibit the following characteristics: Postrema area syndrome (PAS), which is the most characteristic brain manifestation of NMOSD. The patients display intractable hiccups, nausea, and vomiting. In PAS, the radiological involvement occurs in the dorsal surface of the medulla and extends to the most caudal portion of the fourth ventricle. The posterior area is considered a sensory circumventricular organ that mediates the CSF flow between ventricles and the cerebral parenchyma. This area has no specific BBB (Miller & Leslie, 1994). Brainstem syndrome, which occurs in NMOSD most typically with lesions adjacent to the fourth ventricle on brain MRI (Fig. 4B), affecting especially the pons and cerebral peduncle. However, the syndrome may happen even without radiological abnormalities (Kim et al., 2012). Patients can exhibit different symptoms regarding the topography of the lesion, such as ocular movement disorders, nystagmus, dysarthria, dysphagia, hemiparesis, tetraparesis, sensory loss, and ataxia, among others. There have been cases of isolated rhomboencephalitis and even rhomboencephalitis secondary to myelitis and Guillain-Barre syndrome, both related to ZKV (Cruz, Nascimento, Lopes, & Silva, 2018). Symptomatic narcolepsy or acute diencephalic clinical syndrome, which is related to dysfunction of the hypothalamus and/or thalamus. Although the lesions may be seen in brain MRI in asymptomatic NMOSD patients, such dysfunction can lead to hypersomnia, narcolepsy, anorexia, hypothermia, hyponatremia, hyperprolactinemia, irregular menstruation, and behavioral changes (Kim et al., 2015). Studies conducted on mice suggest that the hypothalamus is affected by ZIKV infection (Wu et al., 2018). Cerebral syndromes, which may occur along with focal deficits, hemiparesis, hemisensory loss, encephalopathy, visual field impairment, and cortical vision loss, is often associated with extensive, irregular, confluent lesions in subcortical areas or in deep white matter. Typical NMOSD radiological lesions may be asymptomatic (Kim et al., 2015). Figs. 5 and 6 shows a silent white matter lesion in the brain MRI of a patient with NMOSD associated with ZIKV.

Treatment NMOSD treatment is based on its humoral pathophysiology and is divided between the acute phase and long-term treatments.

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FIG. 5 Brain MRI of a patient with ZIKV-associated neuromyelitis optica spectrum disorder. Axial FLAIR demonstrates periependymal hyperintensity involving the right lateral ventricle.

FIG. 6 Brain MRI of a patient with ZIKV-associated neuromyelitis optica spectrum disorder. Sagittal FLAIR shows a deep parietal white matter lesion.

Acute treatment In the acute phase, methylprednisolone 1 g/day for 5 days is indicated with 5–7 plasmapheresis cycles if response to corticosteroid treatment was not satisfactory (Weinshenker, 2016). In severe cases, especially in LETM, treatment with corticosteroids with plasmapheresis may be initiated earlier (Bonnan et al., 2018). Adequate clinical support with strict observation of the breathing pattern is important, mainly in patients with cervical myelitis due to the risk of respiratory failure.

Long-term treatment Maintenance treatments to prevent new attacks of NMOSD are based on the pathophysiology of the disease and have undergone many updates in recent years considering the emergence of new drugs. Untreated NMOSD patients have a higher recurrence of outbreaks and higher mortality, mainly secondary to respiratory failure. Therefore, it is important

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to provide treatment for AQP4-Abs positive patients since diagnosis, whereas patients with unknown or seronegative AQP4-Abs should be evaluated individually, considering that many of these cases may have a monophasic course. The pharmacological treatment of NMOSD may be influenced by patient profile, gender, comorbidities, and social context, among others. The most common oral drugs used for long-term treatment of NMOSD are azathioprine (AZA) and mycophenolate mofetil (MMF), whereas the most commonly used intravenous drug is rituximab. Azathioprine is administered at a dose of 2.5–3 mg/kg/day, once daily, and MMF is administered for adults at a dose of 750–1500 mg twice a day. Both drugs have a latency period of 4–6 months when prednisone is used as an adjuvant. Rituximab (RTX) is a monoclonal human antibody anti-CD20. It is an intravenous option for long-term treatment of NMOSD. The administration for adults is 1 g on Day 1 and 1 g on Day 14, repeating the cycle at 6 months if the CD19 + B lymphocyte index remains greater than 1% of the total B lymphocytes. Considering the increasing amount of knowledge around the pathophysiology of NMOSD AQP4-Abs, other humanized monoclonal antibodies have also been developed and studied. Examples of these antibodies are: Eculizumab (anticomplement C5), Inebilizumab (anti-CD19 + [B cells]), Tocilizumab (anti-IL6), Satralizumab (anti-IL6), and Bortezomib (26S proteasome inhibitor). Prospects for AQP4-Abs specific therapies include Aquaporumab that is a human monoclonal IgG antibody that binds to AQP4 channels. The drug inhibits the inflammatory cascade activated by AQP4-Abs. This drug is still under study in vitro and may be a potential therapeutic strategy for NMOSD AQP4-Abs patients (Duan, Tradtrantip, Phuan, Bennett, & Verkman, 2020).

Policy and procedures Serum screening of AQP4-Abs by cell-based assays (CBA) (sensitivity 75% and specificity 95%–100%) (Weinshenker & Wingerchuk, 2017) and anti-MOG in patients with suspected NMOSD should be considered. Patients from an arbovirus endemic area or history of travel to these areas should undergo PCR investigation and/or IgM/IgG antibodies for ZIKV, DENV, CHIKV, and yellow fever virus (check for recent yellow fever immunization). Investigation for other infectious agents, such as cytomegalovirus, varicella zoster, Epstein-Barr virus, viral hepatitis, HIV, and tuberculosis, is also important since all these agents have also been related to NMOSD. Laboratory evaluation should be performed including blood count, liver function, renal function, and screening for autoimmune conditions that may be differential diagnoses or coexist with NMOSD, such as autoimmune thyroiditis, lupus erythematosus, Sjogren’s syndrome, sarcoidosis, Behcet syndrome, and others. Patients should undergo brain, orbit, and spine MRI. Other exams may be performed according to the clinical presentation, such as visual evoked potential and optical coherence tomography in some cases of NO. All NMOSD patients should undergo lumbar puncture for CSF evaluation, with cytometry, biochemical parameters, protein electrophoresis, oligoclonal bands research, and infectious agents screening. CSF may present normal cellularity in half of the patients or may display pleocytosis with a predominance of lymphocytes and/or monocytes. CSF may have normal or increased protein levels and normal glucose rates. The presence of oligoclonal bands may occur in a minor group of NMOSD patients but in general, oligoclonal bands are negative. The presence of oligoclonal bands is a stronger marker for MS. Investigating MS is important to differentially diagnose NMOSD, which requires magnetic resonance imaging, CSF examination, and individualized evaluation. This differentiation is important because of the treatment of these two neurological conditions that tend to be different. Additionally, some medications used to treat MS may worsen NMOSD. Acute Disseminated Encephalomyelitis is another important differential diagnosis that has also been related to ZIKV. NMOSD has also been described to relate to paraneoplastic involvement. This hypothesis should be investigated according to the clinical suspicion.

Mini-dictionary of terms Neuromyelitis optica spectrum disorder. Autoimmune inflammatory disease affecting the spine, optic nerves, and brain. Optic neuritis. Inflammation of the optic nerve. Myelitis. Inflammation of the spine. Encephalitis. Inflammation of the brain, cerebellum, and/or brainstem. Area postrema syndrome. Intractable nausea, vomiting, and hiccups due to a lesion on the dorsal surface of the medulla oblongata.

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Key facts of NMOSD and ZIKV l

l l l l l l

NMOSD is an autoimmune disease that causes acute myelitis, optic neuritis, and encephalitis, with the prominence of the area postrema syndrome. Postrema area syndrome is characterized by incoercible nausea, vomiting, and hiccups. ZKV infection may lead to neurological complications such as myelitis, optic neuritis, and encephalitis. Zika virus may act directly on the central nervous system and/or trigger autoimmune neurological diseases. In NMOSD, AQP4-Abs acts against the aquaporin channels in astrocyte feet. ZKV is an infectious disease that displays tropism to astrocytes. Astrocytes are the most abundant glial cells of the central nervous system and their feet are responsible for their starlike shape.

Summary points l

l l

l

l

Neurological impairment may occur in patients with ZIKV infection in the absence of fever, skin rash, arthralgia, headache, myalgia, and conjunctivitis. NMOSD can be a parainfectious and/or postinfectious complication associated with ZIKV. Patients with core symptoms of NMOSD (myelitis, optic neuritis, and encephalitis) should consider investigating their AQP4-Abs status, since its positivity is enough to confirm NMOSD diagnosis. Patients with NMOSD symptoms from endemic areas or with a travel history to those areas should consider investigating ZIKV and other arboviruses. The acute treatment of ZIKV-associated NMOSD does not differ from the treatment of this syndrome on its own.

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Chapter 24

Zika virus infection and cytokines Simone G. Fonsecab, Irmtraut Araci H. Pfrimerc, Carla Judiced, Fabio T.M. Costad, and Helder I. Nakayaa a

Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil, b Department of ^ Microbiology, Immunology, Parasitology and Pathology, Federal University of Goias, Goiania, GO, Brazil, c Department of Master in Environmental ^ Sciences and Health, School of Medical, Pharmaceutical and Biomedical Sciences, Pontifical Catholic University of Goia´s, Goiania, GO, Brazil d

Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas (Unicamp), Sa˜o Paulo, SP, Brazil

Abbreviations CZS IFN IL IP-10 RANTES TNF-α

congenital Zika syndrome interferon interleukin interferon gamma-induced protein 10 regulated upon activation, normal T cell expressed and secreted tumor necrosis factor

Introduction Zika virus (ZIKV) is an emerging virus from Flaviviridae family and genus Flavivirus that caused significant outbreaks around the world since 2007 (Rajah, Pardy, Condotta, Richer, & Sagan, 2016). Although the virus is mostly spread by Aedes mosquitoes, transmission can also occur by sexual and congenital routes (D’Ortenzio et al., 2016; Petersen, Jamieson, Powers, & Honein, 2016). In general, ZIKV infection causes mild symptoms with the majority of the cases being asymptomatic. The most frequent symptoms are cutaneous rash (exanthema), fever, headache, conjunctivitis, hyperemia, swelling, itching, and nausea (Rajah et al., 2016). In recent outbreaks, ZIKV infection was associated with severe neurological symptoms, such as Guillain-Barre syndrome (GBS) in adults and congenital Zika syndrome (CZS) in newborns (Cao-Lormeau et al., 2016; de Arau´jo et al., 2016). The initial immune response to ZIKV infection is critical to determine the subsequent clinical manifestations and outcomes. Different cytokines, chemokines, and growth factors are produced following the infection and may each have a role in controlling both virus replication and dissemination, as well as being involved in the immunopathogenesis of the virus. This chapter describes which cytokines are induced by ZIKV infection during acute and convalescent phases and during pregnancy and discuss their putative associations with symptoms, virus entry in host cells, and adaptive immune responses. Cytokines involved in nonneurological conditions such as ocular problems, hearing loss, placenta and testicular infections are highlighted. The better understanding of the cytokines involved in ZIKV infection may improve disease treatment and prevention of complications.

Cytokine profiling in acute ZIKV infection Early immune response plays an essential role in controlling ZIKV replication and spread. The first work to describe the cytokines produced during acute phase of ZIKV infection was performed with only six individuals who traveled to endemic area and got infected (Tappe et al., 2016). When compared to noninfected healthy controls, these six infected individuals showed higher sera levels of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-9, IL-10, IL-13, IL-17, and interferon-γ-induced protein 10 (IP-10 or CXCL-10), regulated upon activation normal T cell express sequence (RANTES or CCL5), macrophage inflammatory protein 1 alpha (MIP-1α), and the vascular endothelial growth factor (VEGF). In the recovery phase (>10 days after the onset of symptoms), increased levels of IL-1β, IL-6, IL-8, IL-10, IL-13, IP-10/CXCL10, RANTES, MIP-1α, MIP-1β, VEGF, fibroblast growth factor (FGF), and granulocyte-monocyte colony-stimulating factor

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00024-9 Copyright © 2021 Elsevier Inc. All rights reserved.

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(GM-CSF) were observed in ZIKV-infected individuals compared to healthy controls. However, no significant difference was observed in the levels of plasma cytokines between acute and recovery phases (Tappe et al., 2016). More robust studies with larger cohorts of infected individuals and living in endemic areas were reported. Kam et al. (2017) profiled the cytokines induced in a cohort of 95 individuals, including pregnant women and newborns from ZIKVinfected mothers. They showed an increase of plasma proinflammatory cytokines such as IL-18, IL-8, IL-6, IL-7, TNF-α, IFN-γ, and growth regulated oncogene-alpha (GRO-α); antiinflammatory cytokines such as IL-10, interleukin-1 receptor antagonist (IL-1Ra), and IL-4; chemokines as IP-10/CXCL-10, monocyte chemoattractant protein-1 (MCP-1), MIP-1β, eotaxin, and stromal cell-derived factor 1 (SDF-1α or CXCL12); growth factors and others immune mediators as brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), leukemia inhibitory factor, stem-cell factor, and FGF-2 and platelet-derived growth factor-BB (PDGF-BB) (Kam et al., 2017). In contrast, Barros et al. (2018) showed that only IP-10/CXCL-10, RANTES/CCL5, IFN-γ, IL-9, IL-7, IL-5, and IL-1Ra presented significantly higher plasma levels in acute ZIKV-infected individuals from an endemic area compared to healthy donors, suggesting the involvement of different subsets of T cells in the immune response. This study showed that IP-10/ CXCL10 was detected in almost all ZIKV-infected individuals in acute phase and it was the only one that decreased in convalescent phase (around 3 weeks after onset of symptoms), when the viremia was undetectable (Barros et al., 2018). Naveca et al. (2018) followed subjects in different days of acute ZIKV infection and showed higher plasma levels of IL1β, IL-6, TNF-α, IFN- γ, IL-17, IL-4, IL-1Ra, CXCL8, CCL11, CCL3, CCL4, CCL2, RANTES/CCL5, and IP-10/ CXCL10, and growth factors as G-CSF, GM-CSF and FGF-basic, PDGF, VEGF. Importantly, the levels of IL-4 and IL-1Ra, two antiinflammatory cytokines, were higher in ZIKV-infected patients when compared to controls. Decreased levels of CCL3, IP-10/CXCL10, IL-6, and FGF-basic at day 3 of symptom onset were observed, coinciding with the drop of viremia. A decision-tree analysis revealed that IP-10/CXCL10 was the best biomarker of acute ZIKV infection (Naveca et al., 2018). Another study (Lum et al., 2018) showed increase plasma inflammatory cytokines (IL-12p40, IFN-γ, IL-1β, IL-18, IL-6, TNF-α, IL-17, IL-9, IL-22, IL-2), antiinflammatory cytokines (IL-1Ra, IL-10, IL-4, IL-5, IL-21), chemokines (IP-10/ CXCL10, MCP-1, SDF-2, MIP-1β, GRO-α/CXCL-1), growth factors as BDNF, epidermal growth factor (EGF), PDGF-BB, placental growth factor (PIGF), HGF, and GM-CSF in 55 individuals infected with ZIKV when compared to 31 healthy controls, who were nonfebrile and had no signs of acute disease during recruitment, negative for ZIKV viral RNA and ZIKV-specific antibodies (Lum et al., 2018). The summary of cytokines, chemokines, and growth factors detected in the plasma of acutely infected individuals from these four studies is shown in Table 1. The only mediators consistently increased in all studies were the inflammatory mediators, IP-10/CXCL10 and IFN-γ, and an antiinflammatory cytokine, IL-1Ra (Fig. 1). This suggests that acute ZIKV infection induces systemic immune activation, involving both proinflammatory and immunoregulatory mediators that may participate in virus control. IP-10/CXCL-10 is involved not only in viral clearance but also in virus dissemination and brain damage in infection models such as murine cerebral malaria (Campanella et al., 2008) and Japanese encephalitis (Wang et al., 2018). As ZIKV infects primarily monocytes in human blood, these infected activated monocytes may not only produce IP-10/CXCL10 but also migrate to different tissues disseminating the virus (Foo, Chen, Chan, Bowman, et al., 2018; Michlmayr, Andrade, Gonzalez, & Balmaseda, 2018). The increase of IL-1Ra could be related to a compensatory mechanism for the excessive inflammation observed in these patients.

Cytokines associated with symptoms of ZIKV infection Cytokines and chemokines may be strongly associated with the symptoms observed during acute phase of ZIKV infection. Higher levels of IP-10/CXCL10 were associated with exanthema, whereas lower levels of G-CSF were detected in subjects with fever, suggesting that cytokines can be either inducing or controlling some of the clinical symptoms (Barros et al., 2018). ZIKV-infected subjects who had headache showed higher plasma levels of TNF-α, IL-17A, IL-2, IL-9, IL-12p70, MIP-1α, G-CSF, GM-CSF, VEGF, and more significantly with IL-5 levels compared to those without headache. In addition, individuals with nausea showed higher plasma levels of IL-5, IL-1p70, IL-8, and PDGF-BB compared to those without nausea. ZIKV-infected subjects with arthralgia showed higher levels of IL-1Ra, while those with hyperemia showed lower plasma levels of IL-6, IL-7, and VEGF. Finally, increase in IL-1Ra in ZIKV-infected individuals suggests that IL-1Ra could be controlling the excess of inflammation observed in these individuals (Barros et al., 2018). A more robust analysis showed that the high levels of IP-10/CXCL10 could strongly predict the presence of exanthema. Headache was predicted mainly by plasma levels of IL-5 and lesser by MIP-1β, and arthralgia by plasma levels of IL-1Ra and IL-7. G-CSF was associated with fever and IL-13 with myalgia (Barros et al., 2018).

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TABLE 1 Cytokines, chemokines, growth factors detected in plasma of ZIKV-infected individuals during acute phase in different regions. Number of individuals ZIKV +

Region

Endemic area

Tappe et al. (2016)

6

Europe

Kam et al. (2017)

95

Barros et al. (2018)

Studies

Growth factors

Cytokines

Chemokines

No

IL-1beta, IL-2, IL-4, IL-6, IL-9, IL-10, IL-13, IL-1

IP-10/CXCL-10, RANTES (MIP-1a)

VEGF

Brazil (Campinas—SP)

Yes

IL-18, IL-8, IL-6, IL-7, TNF-α, IFN-γ, GRO-α; IL-10, IL-1RA, IL-4

IP-10/CXCL-10 MCP-1, MIP-1β, eotaxin, SDF-1α

HGF, FGF-2, PDGF-BB

36

Brazil (Goi^ania—GO)

Yes

IFN-γ, IL-7, IL-9, IL-5, IL-1RA

IP-10/CXCL-10, RANTES

Naveca et al. (2018)

54

Brazil (Manaus—AM)

Yes

IL-1b, IL-6, TNF α, IFN-γ, IL-17, and IL-4, IL-1RA

CXCL8, CCL11, CCL3, CCL4, CCL2, CCL5, IP10/CXCL-10

G-CSF and GM-CSF, FGF-basic, PDGF, VEGF

Lum et al. (2018)

55

Singapore

Yes

IL-12p70, IFN-γ, IL-1β, IL-18, IL-6, TNFα, IL-17A, IL-9, IL-22, IL-2, IL-1RA, IL-10, IL-4, IL-5, IL-21

IP-10/CXCL-10, RANTES, MCP-1, SDF-1α, MIP-1β, GRO-α

BDNF, EGF, PDGF-ββ, PIGF-1, HGF, GM-CSF

Cytokines, chemokines, growth factors were measured in plasma of infected individuals, in general between 1st to 7th days after symptoms onset. ZIKV were confirmed by real time RT-PCR, and in some cases by ELISA. The plasma cytokines, growth factors were quantified by multiplex and/or ELISA.

FIG. 1 Venn diagram representation of common immune signature of acute ZIKV infection of four studies from endemic areas. Plasma cytokines, chemokines, and growth factors were measured in plasma of acute infected individuals using multiplex and ELISA. All acute ZIKVinfected individuals were symptomatic. In general, the blood was collected between 1st to 7th days of symptoms onset.

In general, viral load did not correlate with (the number of) clinical manifestations in ZIKV infection, maybe due to the low and transient viremia (Barros et al., 2018; Lum et al., 2018). Conversely, it has been also shown that viremic patients with few clinical manifestations showed high levels of IP-10/CXCL10, MCP-1, IL-1Ra, IL-8, and PIGF-1 and reduced numbers of peripheral CD8+, CD4+, and CD4-CD8- T cells (Lum et al., 2018). They also showed that high levels of IP-10/CXCL-10, IFN-γ, and IL-10 were detected in ZIKV patients at recovery, suggesting the involvement of T cells in this process (Lum et al., 2018).

ZIKV infection and interferon production ZIKV enters host cells through clathrin-mediated endocytosis which is triggered by an interaction of ZIKV E glycoprotein with cell surface receptors (Agrelli, de Moura, Crovella, & Branda˜o, 2019). Several receptors such as the innate immune receptor DC-SIGN, transmembrane protein TIM-1 and TAM receptors (TYRO3, AXL, MER), have been shown to

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facilitate ZIKV entrance and enhance of infection (Agrelli et al., 2019). Once the virus invades the cell, a complex defense system quickly initiates an antiviral response. Interferons are among the cytokines that are early produced and have antiviral activity. The IFN superfamily is divided into type I IFN, IFN-α, and IFN-β that signaling through IFNα/β receptor (IFNAR); type II or IFN-γ, produced by T cells and NK cells; and type III IFNs, IFN-λ1, IFN-λ2, IFN-λ3 (IL-29, IL-28A, and IL-28, respectively), and IFN-λ4 (McNab, Mayer-Barber, Sher, Wack, & O’Garra, 2015). Almost all nucleated cells can produce type I interferons in response to viral infections, following the recognition of viruses by cellular pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and melanoma-associated differentiation antigen 5 (MDA5) (Kawai & Akira, 2006). The interaction of these PRRs with viral nucleic acids leads to the activation of several transcription factors such as interferon regulatory factors (IRFs) and NF-κB that participate in inducing more interferons and various other inflammatory cytokines and chemokines required for an effective immune response. It has been suggested that ZIKV can activate TLR3, TLR7, RIG-I, and MDA5, leading to type I IFN production (da Silva et al., 2019). Also, ZIKV has been shown to trigger the cGAS-STING pathway (Aguirre et al., 2017; Hamel et al., 2015). Although some other sensors can be involved in the ZIKV recognition, RIG-I and MDA5 may bind and recognize RNA viruses in the cytosol. After binding, RIG-I/MDA5 recruits the adaptor mitochondrial antiviral-signaling protein (MAVS) to trigger activation of the noncanonical IKK-related kinases, IKKε, and TBK1, which in turn activates IRF3, IRF7, and NF-κB to induce type I IFN production. Endosomal TLR7/TLR8 and TLR9 can recognize, respectively, single-stranded (ss) RNA and unmethylated CpG sequences in DNA and trigger the myeloid differentiation factor 88 (MyD88) pathway. MyD88 activates IL-1 receptor-associated kinase (IRAK) family kinases, leading to phosphorylation of IFN regulatory factor (IRF)7, which is necessary for IFN production (Levy, Marie, & Durbin, 2012). Double-stranded (ds) RNA activates TLR3, which is expressed in myeloid Dendritic cells (DCs) and epithelial cells. Following activation, TLR3 recruits the TIR-domain-containing adapter inducing IFN-β (TRIF) pathway for IFN production (Kawai & Akira, 2006) (Fig. 2A). Mitochondrial DNA may be sensed by the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase-stimulator of IFN genes (cGAS-STING) resulting in IFN production (Keating, Baran, & Bowie, 2011). Recognition of cytoplasmic DNA by cGAS triggers the synthesis of cyclic GMP-AMP (cGAMP) (Ma et al., 2015). This cGAMP binds to STING and enables it to recruit TBK1 that activates IRF3, leading to IFN production (Ma et al., 2015) (Fig. 2A). Once interferons are produced by the infected cell, they can bind to the cognate receptors on cell surface and promote an antiviral state. Type I interferons bind to IFNAR, a heterodimer consisting of IFNAR1 and IFNAR2 chains. Type III IFNs can signal through a heterodimeric receptor comprising IFNLR1 and IL10Rb (Fig. 2B). Type II interferon family consists only of IFN-γ that signals as a dimer through a tetrameric receptor comprising IFNGR1 and IFNGR2. The binding of IFNs to their receptors starts a signaling cascade through the Janus kinase-signal transducer and activation of transcription (JAK-STAT) pathway. The JAK kinases, JAK1 and TYK2, phosphorylate and activate STAT1 and STAT2, followed by heterodimerization of the activated STAT1 and STAT2 and association with another DNA-binding protein, interferon regulatory factor 9 (IRF-9), to form the interferon-stimulated gene factor 3 (ISGF-3). The ISGF-3 complex promotes gene expression by binding to interferon-stimulated response elements (ISREs) of type I interferon-dependent genes. The interferon-stimulated genes (ISGs) interfere in several cellular processes, including RNA processing, protein stability, and cell viability, modifying specific stages of the viral cycle and virus replication. ISGs are also important for the activation of other cells such as DCs, macrophages/monocytes, T and B cells, which impact the magnitude and quality of the adaptive immune response and virus clearance. The IFNs can bind to ISG promoters and induce expression of many genes which are involved in different functions (Schneider, Chevillotte, & Rice, 2014). The modifications in gene expression allow the cell to generate an environment where viral replication is inhibited and the possibilities of virus dissemination are decreased. The importance of type I interferon in the control of viremia in ZIKV infection has been further demonstrated using mice deficient to type I interferon receptor (IFNAR knockout mice). While wild-type C56BL/6 mice are resistant to ZIKV infection, IFNAR knockout mice are highly susceptible to the virus (Yockey et al., 2016, 2018). In addition, human placental trophoblasts can control ZIKV replication by producing Type III IFNs (IFN-λ1) (Bayer et al., 2016; Yockey et al., 2018). It has been shown that the administration of IFN-λ2 to ZIKV-infected IFNAR KO pregnant mice decreased the viral load in the fetus (Chen et al., 2017). In a model of ZIKV transmission in utero using type I IFN-deficient mice, placenta and fetus were more susceptible to ZIKV infection at earlier phases of pregnancy ( Jagger et al., 2017). ZIKV infection at day 6 resulted in placental insufficiency and fetal demise. When the ZIKV infection occurred at middle pregnancy stage, it led to decreased in cranial size. Later infection in pregnancy did not cause fetal demise. Also, mice deficient of type III IFN-λ signaling showed increase of ZIKV replication in the placenta and fetus and treatment of pregnant mice with IFN-λ2 decreased ZIKV infection ( Jagger et al., 2017). In addition, ZIKV-infected wild-type pregnant mice treated with

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FIG. 2 ZIKV infection and interferon responses. (A) ZIKV can enter the host cell by clathrin-mediated endocytosis which is triggered by an interaction of ZIKV with different receptors. Following ZIKV entry, viral RNA can be sensed by cellular pattern recognition receptors such as TLR, RIG-I, or mitochondrial (mt) DNA activating downstream pathways that induce several transcription factors such as IRFs and NF-Kb, leading to production of IFNs and other cytokines. (B) Type I and type III IFNs bind to their receptors on cell surface and phosphorylate and activate JAK-STAT pathways. IRF9 joins to the STAT1/STAT2 heterodimer to form ISGF3 complex that enters the nucleus inducing ISRE which leads to ISG expression. Abbreviations: ssRNA, singlestranded RNA; RIG-I-like receptor, TLR, Toll-like receptor; MAVS, mitochondrial antiviral-signaling protein; cGAS, cyclic GMP-AMP synthase; cGAMP, cyclic GMP-AMP; STING, stimulator of interferon genes; IRF, interferon regulatory factor; ISGF, interferon-stimulated gene factor; ISRE, interferonstimulated response element.

anti-IFNAR blocking antibody and IFN-λ2, showed decrease of viral load in the fetus ( Jagger et al., 2017). These results showed that the type III IFN has a role in controlling ZIKV replication. ZIKV proteins may inhibit the type I interferon pathway in different ways, as described for other viruses. Table 2 summarizes the virus proteins and their actions in inhibiting the host interferon system. The type I IFN response seems to have a dual role in ZIKV infection. In one hand, it is important for protection since it helps controlling viral replication (Yockey et al., 2016). However, the type I IFN response may also be pathogenic. Miner and Diamond (2017) have shown that female mice lacking type I IFN signaling (IFNAR / ) crossed to wild-type males generated heterozygous fetuses (IFNAR +/ ) with similar immune status of human fetuses. The ZIKV infection in these pregnant mice caused fetal demise that was associated with placenta and fetal brain infection. The antibody blockade of Ifnar1 signaling in wild-type pregnant mice increased ZIKV placenta infection but did not cause fetal death, suggesting a pathogenic role of type I IFN (Miner & Diamond, 2017).

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TABLE 2 ZIKV factors that inhibit the interferon system. ZIKV factors

Target pathway

NS1

Actions

References

Inhibit cGAS-STING

Promote stability of caspase 1, that cleaves cGAS

(Zheng et al., 2018)

NS2A

IFN signaling

Inhibition MDA5/RIG-I signaling pathway

(Ngueyen, Kim, Lee, and Myoung 2019)

NS4A

IFN signaling

Inhibition MDA5/RIG-I signaling pathway

(Ngueyen, Kim, Lee, and Myoung 2019)

NS4B

IFN signaling

Negatively regulate RIG-I-like receptor by inhibiting phosphorylation of TBK1

(Wu et al., 2017)

NS5

IFN signaling

STAT2 degradation, increase CXCL10

(Chaudhary et al., 2017)

RIG-I, retinoic-acid inducible gene I; MDA5, retinoic-acid inducible gene I; cGAS, cyclic GMP-AMP synthase, STING, stimulator of interferon genes.

Role of T cells in ZIKV infection The importance of T cell response in ZIKV infection has been evaluated in different mouse models. A study using IFNAR deficient mice showed that specific CD8+ T cell responses to ZIKV were polyfunctional and cytotoxic (Elong Ngono et al., 2017). The depletion of CD8+ T cells in this model increased viral load in serum and in several tissues, and adoptive transfer of CD8+ T cells from ZIKV-infected mice to naı¨ve mice promoted decrease of viral load (Elong Ngono et al., 2017). Depletion of CD4+ T cells in IFNAR deficient mice also led to increase of viral loads, severe paralysis, and survival decrease (Hassert et al., 2018; Lucas et al., 2018). T cell immune responses may also play a critical role in human ZIKV infection (Cimini et al., 2017; El Sahly et al., 2019; Lai et al., 2018). El Sahly et al. (2019) collected CD4+ and CD8+ T cells from 45 infected individuals and stimulated them in vitro with peptides from all ZIKV proteins. They showed that both CD4+ and CD8+ T cells produced IFN-γ, IL-2, and TNF-α (El Sahly et al., 2019). This polyfunctional virus-specific T cell immune response was detected even after 10– 12 months after infection, suggesting that natural infection with ZIKV generates functional memory T cells (El Sahly et al., 2019).

Cytokines involved in neurological complications The prominent neurotropism of ZIKV may lead to neuroinflammation and neuronal cell death in humans. In addition to GBS in adults (Oehler et al., 2014), ZIKV is also associated with other neurological complications, such as meningoencephalitis and acute myelitis (Cao-Lormeau et al., 2016; Carteaux et al., 2016; Mecharles et al., 2016). Although the immunomodulation underlying ZIKV pathology is not fully understood, there is evidence that IP-10/CXCL10 may be involved with some of the neurological complications caused by ZIKV infection. Studies focusing on chemokines in GBS have most consistently reported increased levels of IP-10/CXCL10 in plasma, cerebrospinal fluid, or nerve biopsies from GBS patients (Chiang & Ubogu, 2013). A similar finding was found in the plasma of ZIKV patients with neurological complications (Kam et al., 2017), suggesting that IP-10/CXCL10 may also act as an important modulator of ZIKV neurological damage. Moreover, recent study on Zika virus-associated encephalitis in children showed that although discrete, plasma IL10 levels were significantly increased in these patients compared to healthy controls (Salgado et al., 2020).

Cytokines involved in nonneurological complications Congenital ZIKV syndrome is characterized by severe complications, including vision and hearing loss, seizures, and microcephaly. Infection in pregnancy is also associated with placental damage, leading to intrauterine growth restriction (IUGR) and fetal demise (Yockey et al., 2018). Yockey et al. (2018) using a mice model of ZIKV infection during pregnancy with heterozygous fetuses (Ifnar +/ ), showed that after ZIKV infection, the activation of IFNAR pathway in the fetus inhibits the development of the placental labyrinth leading in abnormal architecture of the maternal-fetal barrier.

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In addition, explants of mid-gestation human chorionic villous treated with type I IFN, but not type III, showed modifications in the morphology and structure of the placenta. These data suggest that type I IFNs may be a possible mediator of pregnancy complications (Yockey et al., 2018). Infants with congenital ZIKV syndrome show a broad spectrum of ocular complications, but the most distinctive are the pigment mottling and chorioretinal atrophy that are commonly seen in the macular region. It has been shown that retinal endothelial cells, retinal pericytes of the inner blood-retinal barrier, and pigmented epithelial cells of the outer blood-retinal barrier in humans are fully permissive for ZIKV lytic replication and are the primary target cells in the retinal barriers for ZIKV infection in the eyes (Roach & Alcendor, 2017). The exposure of retinal endothelial cells and retinal pericytes to ZIKV caused a discrete increase in levels of β2-microglobulin, GM-CSF, and MCP1; a moderate increase of ICAM-1, IL-6, and VCAM-1 expression but a strong increase of RANTES/CCL5 expression when compared to controls that may contribute to ocular inflammation in mice (Roach & Alcendor, 2017). Unlike other arboviruses, ZIKV can be sexually transmitted and may persist in the male reproductive tract. Siemann, Strange, Maharaj, Shi, and Verma (2017) demonstrated that primary human Sertoli cells were highly susceptible to ZIKV infection compared to the dengue virus and that induced the expression of IFN-α and cell adhesion molecules VCAM-1 and intracellular adhesion molecule ICAM-1 (Siemann et al., 2017). Ma et al. (2016) challenged different types of testicular cells from mice with ZIKV. Sertoli cells and Leydig cells secreted high levels of TNF-α, IL-6, IFN-β, and CXCL10 and low levels of IFN-α upon infection (Ma et al., 2016). Since these cytokines were not produced by ZIKV-infected germ cells or peritubular cells, it has been suggested that Sertoli cells are one of the major sources of inflammatory cytokines in human and mouse testis.

Immunoprofiles associated with fetal abnormalities in ZIKV-positive pregnancies Recent reports have attempted to explore the correlation between altered maternal immunity and fetal abnormality of ZIKV patients. Kam and colleagues demonstrated that pregnant women previously infected with ZIKV and carrying a fetus with development anomalies had high levels of IL-8, IL-18, IL-4, IL-22, IL-23 and IL-27, MCP-1, TNF-α, IP-10/ CXCL-10, epidermal growth factor, eotaxin, and FGF-2 when compared with those carrying fetuses without development anomalies (Kam et al., 2017). In this context, levels of IL-22, MCP-1, IP-10/CXCL-10, and TNF-α were significantly higher during the acute phase of the disease and could be useful prognostic biomarkers to predict the possible outcome in infants born to ZIKV-infected mothers. Increased levels of CCL2, IP-10/CXCL10, IL-6, IL-8, VEGF, and G-CSF were also detected in the amniotic fluid of ZIKV-positive pregnant women whose infants had microcephaly (Ornelas et al., 2017). Recently, Foo and colleagues showed that IP-10/CXCL10, CCL2, and CCL8 chemokines were specifically associated with symptomatic ZIKV-positive infection during pregnancy and that increased CCL2/CD163, CCL2/TNFRSF1A, and CCL2/CCL22 ratios in ZIKV-positive pregnancy is correlated with abnormal birth outcomes (Foo, Chen, Chan, Lee, et al., 2018). On the other hand, profile in infants showed that higher levels of IL-18 and IP-10 and lower levels of HGF were associated with congenital neurological abnormalities compared to healthy infants born to ZIKV-infected mothers (Kam et al., 2017). ZIKV also infects the human fetal microglia, which are cells involved in inflammatory and immune responses at the central nervous system. Indeed, microglia when infected with ZIKV increase the levels of proinflammatory immune mediators typically found during neuroinflammation (IL-6, MIP-1α, MIP-1β, MCP-1, TNF-α, IL-1Ra, IL-1α, IL-1β, IL-8, and IL-12p70) (Lum et al., 2017). Furthermore, a case report of a ZIKV-positive fetus associated with GBS and spontaneous retained abortion demonstrated high levels of the proinflammatory cytokines IFN-γ and TNF-α. RANTES/CCL5 and VEGFR2 were also described as involved in placental inflammation and dysfunction (Rabelo et al., 2018).

Concluding remarks The involvement of cytokines in ZIKV infection discussed in this chapter is summarized in Fig. 3. ZIKV may induce different cytokines, chemokines, and growth factors depending on the cell types and the tissues. These mediators can be involved with both control of virus replication and its own pathogenesis. Further studies are needed to clarify the mechanisms underlying cytokine functions which will open possibilities for therapeutical interventions and vaccine development.

FIG. 3 Cytokine production in ZIKV infection. Following ZIKV infection, several cytokines, chemokines, and growth factors are produced by different cell types and can be detected in the peripheral blood or tissues (Skin, placenta). Cytokines may be associated with symptoms such as exanthema, fever, headache, arthralgia, myalgia, and conjunctivitis. Infected pregnant women may transmit the ZIKV to their fetuses and it can lead to several abnormalities. Some cytokines produced during acute phase have been detected in the plasma of pregnant women and were associated with fetal abnormalities. Specific immune responses to ZIKV induce cytokine production that may be involved in the protection. However, some cytokines can also lead to immunopathogenesis.

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Policy and procedures Assays for cytokine measurements Several assays have been developed to measure cytokines in biological fluids including serum, plasma, and amniotic fluid. In plasma or serum, the techniques more frequently used are enzyme-linked immunosorbent assay (ELISA) and multiplex platform. ELISA is a plate-based assay technique developed for quantifying soluble substances such as cytokines. In this case, the primary antibody against a given cytokine is immobilized on a solid surface (plate) and the secondary antibody is complexed with an enzyme. The cytokine detection is accomplished by measuring the enzyme activity by incubating it with the adequate substrate that produces a color product that can be measured. ELISA can measure one cytokine at a time. Multiplex (Luminex) methodology uses the capture antibody attached to a bead. Usually, this technology uses a micron magnetic microsphere, which are internally dyed with red and infrared fluorophores of different intensities. Each bead is given a unique number allowing to differentiate one bead to another and is covalently bound to an anticytokine antibody. Beads can be mixed in the same assay, using a 96-well microplate format, allowing the measurement of many cytokines simultaneously. Multiplex (Luminex) methodology has shown some advantages since, as several studies have already compared it with ELISA (which is a method limited by the ability to measure only a single biomarker) and demonstrated that the results obtained by the two methods were similarly comparable (Breen, Perez, Olmstead, Eisenberger, & Irwin, 2014; duPont, Wang, Wadhwa, Culhane, & Nelson, 2005; Richens et al., 2010).

Mini-dictionary of terms IFN (interferons). A class of cytokines produced on the challenge to the host defense and are crucial for immune responses to pathogens. IFNs are divided into three different types: Type I (IFN-α, IFN-β, and less representative for IFN-ω, and IFN-τ); Type II or IFN-γ; and type III that includes IFN-λ1 (IL-28A), IFN-λ2 (IL-28B), and IFN-λ3 (IL-29). Pathogen-associated molecular pattern (PAMPs). Are derived from microorganisms and recognized by pattern recognition receptor (PRR)-bearing cells of the innate immune system. Retinoic acid-inducible gene I (RIG-I). Is a cytosolic pattern receptor (PRR) responsible for type I interferon response, which is implicated in virus recognition. Toll-like receptors (TLR). Are germline-encoded pattern recognition receptors (PRRs) that can play a central role in host cell recognition and responses to microbial pathogens. Viremia. Is the presence of viruses in the blood circulating in a living being.

Key facts l

l

l

l

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Cytokines are molecules produced by one cell and acts on the same cell as autocrine manner or on another cell as paracrine manner or both. Cytokines are often called interleukins (IL). Chemokines are a group of small molecules, ranging from 8 to 12 kDa, able to induce chemotaxis in several types of cells including neutrophils, monocytes, lymphocytes, eosinophils, fibroblasts, and many others. Cytokine and chemokine are redundant secreted proteins with growth, differentiation, and activation functions that regulate and determine the nature of immune responses and control immune cell trafficking and the distribution of cells in immune organs. Cytokine and chemokine act on specific receptors on the cells that they affect. Interferons (IFNs) can function as the first line of immune defense against viral infection. However, recently, they have been shown to cause immunopathology in some acute viral infections. The major acute innate cytokines are IL-1, TNF-α, IL-6, CXCL8 (IL-18), C-CSF, and GM-CSF. Th1 cytokine profile includes predominantly IFN-gamma, TNF-α, and IL-2. Th2 cytokine profile includes predominantly IL-4, IL-5, and IL-13; Th17 cytokine profile includes IL-17A, IL-17F, IL-22, IL-21.

Summary points l

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During acute phase of ZIKV infection proinflammatory and antiinflammatory cytokines, chemokines, and growth factors are produced. Symptoms are associated with cytokine production. IP10/CXCL10 can predict exanthema; headache was predicted by IL-5, arthralgia predicted by IL-1Ra and IL-7.

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ZIKV can activate TLR3, TLR7, RIG-I, and MDA5 leading to type I IFN production, which inhibit virus replication and dissemination. Also, type I IFN can be pathogenic causing placental damage. ZIKV infection induces a robust polyfunctional specific CD4+ and CD8+ T cells responses. Neuronal damage can be mediated by CD8+ T cell response. ZIKV infection is associated with nonneurological complications such as ocular damage (probably involving increase of RANTES), hearing loss, and persists in male reproductive tract. CCL2, CD163, TNFRSF1A, and CCL2 seem to be associated with fetal and newborn anomalies caused by ZIKV infection during pregnancy.

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Hamel, R., Dejarnac, O., Wichit, S., Ekchariyawat, P., Neyret, A., Luplertlop, N., … Misse, D. (2015). Biology of Zika virus infection in human skin cells. Journal of Virology, 89(17), 8880–8896. https://doi.org/10.1128/jvi.00354-15. Hassert, M., Wolf, K. J., Schwetye, K. E., DiPaolo, R. J., Brien, J. D., & Pinto, A. K. (2018). CD4+ T cells mediate protection against Zika associated severe disease in a mouse model of infection. PLoS Pathogens, 14(9), 1–24. https://doi.org/10.1371/journal.ppat.1007237. Jagger, B. W., Miner, J. J., Cao, B., Arora, N., Smith, A. M., Kovacs, A., … Diamond, M. S. (2017). Gestational stage and IFN-λ signaling regulate ZIKV infection in utero. Cell Host & Microbe, 22(3), 366–376. https://doi.org/10.1016/j.physbeh.2017.03.040. Kam, Y. W., Leite, J. A., Lum, F. M., Tan, J. J. L., Lee, B., Judice, C. C., … Costa, F. T. M. (2017). 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Chapter 25

Urological sequels in the scope of the Congenital Zika Syndrome Lucia Maria Costa Monteiro National Institute of Woman, Adolescent and Child Health—Fernandes Figueira/Fiocruz, Rio de Janeiro, Brazil

Abbreviations CMG CNC CZS DMSA DSD DTPA EMG FIOCRUZ ICCS LPP LUT MBC NB SUS UDS UI US UTI VCUG VUR WHO

cystometry or cystometrogram central nervous system congenital Zika syndrome dimercaptosuccinic acid detrusor-sphincter dyssynergia diethylenetriamine pentaacetic acid electromyography Fundac¸a˜o Oswaldo Cruz International Children’s Continence Society leak point pressure lower urinary tract maximum bladder capacity neurogenic bladder Brazilian Unified Health System urodynamic studies urinary incontinence ultrasound urinary tract infection voiding cystourethrography vesicoureteral reflux World Health Organization

Introduction Zika virus (ZIKV) is a mosquito-borne flavivirus that affects pregnant women and their infants by causing severe fetal abnormalities (CDC Report, 2018; Cugola et al., 2016; Delaney et al., 2018; Einspieler et al., 2019; Gulland, 2016b; Melo et al., 2016). Although the virus is known for a long time, it was discovered in Africa in 1947 and first reported in Asia in 1966, its potential effect on public health was not recognized until the first outbreaks in the Pacific, starting in 2007 (Musso, Ko, & Baud, 2019). The virus increased in the Americas around 2015, when the association between Zika virus and fetal malformation was exposed during its first epidemic infection in Brazil. It was detected when medical and epidemiological records confirmed a rise in the number of babies born with microcephaly in the northeast region of the country. The association was then investigated, what was possible due to a combination of factors related to the design of the Brazilian Unified Health System (SUS) that includes a solid epidemiological surveillance, an open-to-all public health system and a permanent concern with research-based evidence to guide the national health policies. Fig. 1 shows the distribution of cases of Congenital Zika Virus in Brasil, according to the last available Epidemiological bulletin from the Brazilian Ministry of Health (Health Surveillance Secretariat j Ministry of Health, 2019). Further studies confirmed the association of microcephaly, and other congenital abnormalities, to mothers exposed to Zika virus during pregnancy (Brasil et al., 2016; Campos, Bandeira, & Sardi, 2015; Dyer, 2015; Lebov et al., 2019; Rasmussen, Jamieson, Honein, & Petersen, 2016; Reynolds et al., 2017; Schuler-Faccini et al., 2016) and the term Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00025-0 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Distribution of congenital Zika syndrome 2015–19 in Brazil. Distribution of suspected, under investigation and confirmed cases of congenital Zika syndrome between the epidemiological weeks 45/2015 and 40/2019. Brazil, 2015–19. There are 3474 confirmed cases of children affected by the virus. Other 2659 children are still under investigation to confirm diagnosis. (Source: Health Surveillance Secretariat j Ministry of Health Special Number (November 2019). Sı´ndrome cong^enita associada a` infecc¸a˜o pelo vı´rus Zika situac¸a˜o epidemiolo´gica, ac¸o˜es desenvolvidas e desafios de 2015 a 2019 ^ (Boletim epidemiologico special issue Nov 2019; congenital Zika syndrome). Secretaria de Vigilancia em Sau´de, Minist erio da Sau´de. https://docu mentcloud.adobe.com/link/track?uri¼urn:aaid:scds:US:3a5d2319-d281-4f81-af6c-aef3f0a3f084.)

congenital Zika syndrome (CZS) was adopted. CZS was then considered a worldwide health emergency by WHO (Gulland, 2016a) triggering a Brazilian National Response Effort (Ministerio da Saude, 2017a, 2017b) in collaboration with international research groups. The investigation focused on understanding the link between maternal Zika virus infection and the development of fetal congenital central nervous system malformations and the possibility of subsequent development of postnatal organ dysfunction. Several congenital brain abnormalities were identified in the affected babies (Horovitz, da Silva Pone, Moura Pone, Dias Saad Salles, & Bastos Boechat, 2016; Oliveira-Szejnfeld et al., 2016; Vasco Aragao et al., 2016). The CNC anatomic areas responsible for controlling the lower urinary tract were also affected, therefore urological sequels were investigated, and the first positive cases of neurogenic bladder were established (Costa Monteiro et al., 2018). This was the first report of a possible association of congenital neurogenic bladder and a mosquito-borne disease in the world.

Neurogenic bladder Neurogenic bladder is a urinary dysfunction associated with neurological malfunction. It happens when an injury to the nervous system reaches the micturition centers, as had happened with the babies born with congenital Zika syndrome. This will compromise the function of the bladder and the urethral sphincter and it will interfere with the physiological mechanism of micturition. Consequently, bladder filling and/or emptying will be affected, unbalancing the hydrodynamic flow of the urinary system. Patients with neurogenic bladder will suffer from a degree of urological dysfunctions, including urinary incontinence and urinary tract infections (UTI). The pathogenesis of UTI includes urinary retention and bladder wall ischemia caused by high bladder pressures (Vigil & Hickling, 2016). Frequent UTIs also increase the risk of nephropathy. However, common UTI symptoms, such as fever and dysuria, tend to be silent or misinterpreted in neonates and toddlers and failure to recognize and treat these urinary infections can quickly lead to life-threatening situations including sepsis. In the other hand, overtreatment is also common and contributes to antibiotic resistance (McKibben, Seed, Ross, & Borawski, 2015). UTI remains one of the most difficult conditions to diagnose, treat, and prevent in the CNB setting. Urinary incontinence (UI) is another common symptom in the neurogenic bladder that frequently remains unnoticed during the first years of life because of the regular use of diapers by babies in general. UI is caused mainly by low bladder capacity and by the increase in the number and intensity of bladder contractions during the filling phase (overactive bladder). Both were diagnosed in the urodynamic tests of children affected by ZIKV. Failure to recognize and treat it will lead to chronic urinary incontinence, a recognized life-long setback to patients and families due to its well-known social associated problems. Both symptoms (UTI and UI) impose a great burden to patient’s health and quality of life if left untreated.

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The preventive approach, with early start to treatment, is paramount to preserve kidney function on neurogenic bladder patients (Costa Monteiro, Cruz, Fontes, Vieira, et al., 2017; Dik, Klijn, van Gool, de Jong-de Vos van Steenwijk, & de Jong, 2006). Delays in diagnose and treatment may cause progressive urinary system damage, advancing to chronic stages and renal failure, depending on the severity of the bladder malfunctioning (Kari, 2006; Olandoski, Koch, & Trigo-Rocha, 2011; Rodrı´guez-Ruiz, Somoza, & Curros-Mata, 2016). Furthermore, chronicity increases morbidity and raises the costs of treatment, including kidney transplantation.

Diagnose of neurogenic bladder The possible association with congenital neurogenic bladder must be considered in patients with congenital Zika syndrome. The criteria for the urological investigation are published (Costa Monteiro, Cruz, Fontes, & Boechat, 2017) and must include the urodynamic study to correctly access bladder and urethral sphincter behavior. Urodynamic studies (UDS) are the gold diagnose standard and have become a major tool in evaluating neurogenic bladder in children because it allows the identification of risk factors to the urinary tract and help to guide treatment. Urodynamic findings are considered normal when patients present a bladder with appropriate capacity and good compliance, with no detrusor overactivity that increases the bladder pressure during the filling phase, followed by a complete voiding, with no postvoid residual left during emptying. A normal urodynamic pattern is presented at Fig. 2. Abnormal findings that increase the risk for the urinary tract include lower bladder capacity and compliance and higher detrusor pressure during the filling phase. The test also identifies urethral sphincter malfunction such as the detrusorsphincter dyssynergia (DSD) that compromises voiding and increases the risk of UTIs and incontinence related to higher postvoid residual.

Urodynamic studies in children with special needs Patients with congenital neurogenic bladder, and especially children with congenital Zika syndrome, will demand special care to allow the test to be accomplished without increasing stress to families and patients. The pediatric urodynamic test must be performed in the absence of UTI, by professionals specially trained to assist children and adolescents and appropriately designed equipment and material. It is important to schedule a clinic visit before the day of the test to familiarize parents and patients with the urodynamic room and procedure. The examination room needs to be adapted to promote a child-friendly environment, which includes toys and video entertainment/movies. Parents must be encouraged to stay in the room during the entire procedure so as to comfort and support the child. The basic urodynamic tests for neurogenic bladder include a cystometrogram (CMG) and an electromyography (EMG). CMG requires urethral catheterization, and it is important to choose a size-appropriate double-lumen pediatric catheter and use topical anesthesia with lidocaine gel (1%). It is important to measure any urinary residual volume before starting the exam, taking into account markers of recent voiding such as diaper wetness and/or voiding before or during catheterization. Filling rate is calculated as 5% of the expected bladder capacity for age (mL/min). Bladder overactivity is defined by the presence of involuntary detrusor contraction during bladder filling. In general, children with neurogenic bladder do not express a strong urge to void to estimate the bladder capacity (Drzewiecki & Bauer, 2011). The maximum bladder capacity is measured just before the child leaked or voided, or in the absence of leak, when the child displayed discomfort, when bladder baseline pressures steadily stayed above 40 cm H2O or when the volume infused was 1.5 times the expected capacity for the patient’s age. The maximum bladder pressure is measured at the bladder capacity. Patch EMG electrodes are more comfortable and work very well for EMG in children. When the sphincter function is normal (detrusor sphincter synergy), the sphincter activity remains present during the filling phase and reduces following an increase in bladder pressure during contraction, or during a leak or a void. Detrusor sphincter dyssynergia (DSD) is considered when the sphincter failed to relax, or increase its activity following a high bladder pressure during contraction, or during a leak or a void. It is important to highlight that a dyscoordination of the detrusor-sphincteric unit may occur during micturition of healthy infants (Guerra, Leonard, & Castagnetti, 2014). During the voiding phase, it is important to check if any residual urine was left. The leak point pressure is measured during the first leak and the voiding pressure is measured when a more sustained emptying is observed. The urodynamic diagnosis is established based on bladder and urethral sphincter behavior during filling and voiding phases, considering bladder and sphincter activity, maximum bladder pressure and micturition/leak point pressure, bladder capacity, and bladder compliance. Fig. 3 shows a pattern of overactive bladder, the most common type of neurogenic bladder.

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FIG. 2 Normal urodynamic study showing a relaxed bladder characterized by a steady, lower measurement of the bladder pressure during the entire filling phase (observed in the pink line). The voiding phase started at the end of the study, as expected in a normal UDS, characterized by an sustained bladder contraction increasing the bladder pressure, followed by a completed voiding that was not entirely measured by the graph at this time. Pves, vesical pressure, pink line; PAbd, abdominal pressure, purple line; Pdet, detrusor pressure, green line; EMG, eletromiography, red line.

FIG. 3 Overactive bladder. Urodynamic study confirmed the presence of high-pressure bladder contractions during the filling phase (Pves: vesical pressure, pink line), causing early emptying of the bladder, and increasing the risk of malfunction to the upper urinary tract.

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Neurogenic bladder as a urological sequel of congenital Zika syndrome The Fernandes Figueira Institute (www.iff.fiocruz.br), from FIOCRUZ (www.fiocruz.br) is the Brazilian National Institute of Women, Children, and Adolescent Health and supported the Ministry of Health during the Zika epidemic period in Brazil. During the period of 2015–19, the institution monitored a cohort of 108 patients with confirmed congenital Zika syndrome and 97 patients, 54 female, and 43 male, started urological evaluation as part of the ethically approved research protocol to investigate associate sequels (Costa Monteiro et al., 2019). By the time this chapter was written, 79 patients had completed the urological assessment and the results are presented in Table 1. Since this was a new disease and urodynamic evaluation is the golden standard for diagnosing congenital neurogenic bladder, it was included in the protocol and it was performed in all 79 patients. The majority (70 patients, 87% of UDS done) presented overactive bladder, with bladder contractions frequently recorded during the filling phase increasing the bladder pressure. The average maximum bladder pressure was 64.8 cm H2O. Patients also presented reduced bladder capacity and compliance. The maximum bladder capacity was, in average, 72.8% of the expected capacity for the age group. Not all patients were able to void to completion and postvoid residual was also observed. Only six patients (8%) presented a normal urodynamic pattern, with a relaxed bladder during the filling phase and a sustained bladder contraction triggering the voiding phase. However, their bladder capacity was smaller or larger than the expected for the age, and/or they presented a larger postvoid residual. There is still some debate related to the definition of normal bladder function in children. For a long time, the bladder function in nontoilet-trained children was considered to be triggered by a spinal cord reflex arc without CNS inhibition. However, recent urodynamic studies in infants showed that their bladders are frequently stable during the filling phase. It is now believed that under ideal conditions, most infant bladders should be stable, and significant uninhibited contractions should be considered abnormal (Guerra et al., 2014; Neveus & Sillen, 2013). In relation to other urological complications, 19 patients (22%) presented at least one episode of UTI confirmed by urine culture. However, it is possible that the actual number of patients infected is higher since ITU symptoms may be

TABLE 1 Characteristics of patients with congenital Zika syndrome and microcephaly with completed urological evaluation (n 5 79). General features

Number

Percentage (%)

Female

46

58.23

Male

33

41.77

Average

Gender

Age

15.53 months

Under 12 months

28

35.44

13–24 months

46

58.23

Over 24 months

5

6.33

Overactive bladder

70

88.61

Underactive bladder

1

0.89

Normal bladder

6

7.59

19

24.05

Abnormal bladder wall

15

18.99

Hydronephrosis

3

3.80

Vesical ureteral reflux

1

1.27

Urodynamic evaluation

Lab tests At least one UTI episode confirmed US and VCGU

Profile of the patients with congenital Zika syndrome and microcephaly with completed urological evaluation. The majority of patients were female, age between 13 and 24 months old and presented an abnormal urodynamic study with overactive bladder.

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misinterpreted and febrile episodes were not frequently investigated. All children were treated with antibiotics according to susceptibility testing. Renal US confirmed hydronephrosis in 3 (4%) and a thickened bladder wall (above 0.4 cm) in 15 patients (19%). VUR was confirmed in one patient.

Treatment of neurogenic bladder in congenital Zika syndrome Treatment of neurogenic bladder is based on urodynamic diagnosis. In pediatric patients, oxybutynin continues to be the drug of choice to control overactive bladder and to increase bladder compliance. Interaction with other medicaments and side effects must be observed, especially in patients with CZS. Clean intermittent catheterization (CIC) is indicated whenever bladder empty is ineffective, and this can be estimated based on the postvoid residual urine. There is a lot of stigma related to the use of CIC and to increase adherence it is important to train, support, and reassure parents to confidently perform the techniques. If the patient presents UTI, antibiotics are prescribed based on the susceptibility test. Patients with congenital neurogenic bladder who are diagnosed and treated within the first year of life tend to respond better by reducing the urodynamic risk factors and triplicates the chance of enhancing prognosis (Costa Monteiro, Cruz, Fontes, Vieira, et al., 2017). Early proactive management, including clean intermittent catheterization (CIC) and anticholinergics, stabilized as early as possible, will prevent UTI, incontinence, and deterioration of renal function (Filler et al., 2012; Lehnert, Weisser, Till, & Rolle, 2012; Verpoorten & Buyse, 2008). The follow-up of the cohort patients confirmed an average reduction in bladder pressure from 64.8 cm H2O in the first UDS to 48.5 H2O in the second one. However, the average maximum bladder capacity did not change between the two evaluations. A long-term follow-up will be necessary to better assess the entire results of treatment in these specific populations and also to assess its sustainability.

Further studies related to Zika virus in the urological system With the new investigations, the literature starts to report possible urological sequels of ZIKV infection. That is what was founded by now. The virus persists longer and has a higher load in urine than in serum and probably urine will be more frequently used on diagnostic tests in the future (Lamb et al., 2018). New published experimental investigations, developed in animal models, confirmed that ZIKV can infect the kidney including glomerular podocytes, renal glomerular endothelial, and mesangial cells (Chen et al., 2017; Liu et al., 2019). It was also experimentally proved that mice inoculated with ZIKV presented viral replication in both renal glomeruli and tubules, and the author concluded that ZIKV-induced renal pathogenesis may occur in humans (Alcendor, 2018). In terms of human outcomes, a complete absence of the urinary bladder with severe bilateral renal hypoplasia was confirmed by the autopsy in a fetus of a 22-year-old pregnant woman, and this may be related to the virus (Villamil-Go´mez et al., 2019). However, further research is necessary to better understand the outcomes of this new disease in the urological system.

Policy and procedures This is a summary of the criteria to evaluate neurogenic bladder in children with congenital Zika syndrome. The initial steps are presented in Fig. 4. A detailed version is published (Costa Monteiro, Cruz, Fontes, & Boechat, 2017). 1. Urological evaluation: a. Extended clinical history including prenatal and maternal history to identify the period of exposure to Zika and perinatal complications, bladder and bowel habits (number of diapers per day, wetness of diapers and dry intervals between diapers), and UTI symptoms (changes in the color and aspect of urine, fever, and dysuria). b. Comprehensive physical examination including blood pressure and evaluation of CNS, spinal cord, and bladder and bowel. c. Lab tests: urinalysis and culture during the initial assessment and in all febrile episodes to exclude UTI, blood count, blood urea nitrogen, and serum creatinine. d. Urinary ultrasonography including kidney, ureter, and bladder evaluation (bladder wall, contents, volume, and postvoid residual). e. Urodynamic studies.

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FIG. 4 Initial urological assessment for children with confirmed congenital Zika syndrome. Stages for the initial urological screening and follow-up recommendations for patients with CZS.

2. Treatment: CZS patients with confirmed urological sequels must be followed by specialized professionals and treated by the same criteria applied to all patients with similar diagnoses. The treatment with chloride of oxybutynin or anticholinergic drugs is beneficial to control overactive bladder and low bladder compliance. The use of intermittent clean catheterization is important whenever bladder emptying is ineffective (Lehnert et al., 2012), in children with recurrent urinary tract infections, renal and ureteral dilatation, or vesicoureteral grade IV or V reflux. Antibiotics are restricted to treat confirmed urinary tract infections, according to the susceptibility test. Prophylaxis can be used at clinical discretion, according to the clinical and laboratory history of recurrent urinary tract infections. 3. Follow-up: The follow-up will depend on the severity of each case. In general, patients are clinically reevaluated every three to 6 months. Parents are oriented to observe changes in the color and/or smell of the patient’s urine and also to seek advice during fever episodes without a known cause. During follow-up, new urodynamic studies and renal bladder ultrasound may be ordered to evaluate urological improvemts related to treatment or any possible evolutive changes related to worseing of urological symptoms. When the patient presents a ureteral or pelvic dilatation at the renal US and high bladder pressure, a voiding cystourethrography (VCUG) is useful to determine the presence or absence of bladder and urethral abnormalities, including vesicoureteral reflux (VUR).

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When the patient presents abnormal renal US, repeated UTI, pyelonephritis, and higher detrusor pressure with urethral sphincter dyssynergia, a renal scan is useful to evaluate renal morphology, particularly for the detection of scarring and pyelonephritis (DMSA) and kidney excretion (DTPA).

Mini-dictionary of terms Congenital Zika syndrome. A distinct pattern of birth defects/disabilities associated with a Zika virus infection during pregnancy. It includes severe microcephaly with partial skull collapse, decreased brain tissue and subcortical calcifications, congenital contractures, and hypertonia, among other anomalies. Urodynamic studies. Aim to replicate the physiological hydrodynamics of the lower urinary tract (LUT) in a laboratory situation (D’Ancona, Gomes, & Rosier, 2017). Age specific technical details must be considered as well as correct interpretation is clinical relevance. Cystometry or cystometrogram (CMG). Measures the storage function of the bladder during the filling phase. CMG needs to be complemented with the study of the voiding phase, to evaluate the bladder capacity to completely evacuate all urine that was produced during the filling. These are very important parts of a urodynamic study. We recommended the use of a double-lumen pediatric urodynamic catheter, to reduce invasiveness. Electromyography. Evaluates the striated component of the external urethral sphincter and pelvic floor musculature. We recommended patch EMG electrodes to reduce invasiveness Overactive bladder. Is defined by the presence of any involuntary detrusor contraction during bladder filling. Maximum bladder capacity. Is measured in milliliter, during CMG, at the end of the filling phase, and before voiding starts. If the child is unable to express a strong desire to void, in the absence of voiding, the MBC is considered before the baseline pressure raised and stayed above 40 cm H2O during filing or if the child was in pain or uncomfortable. Maximum bladder pressure. Is measured at the maximum bladder capacity. Note that this is not necessarily the maximum pressure measured during the UDS study. Expected capacity for the age group. Bladder capacity in children varies with weight and age. Therefore, it is important to calculate the expected capacity for age (EBC). There are several formulas to calculate it but we recommended the one used by the ICCS: age (years)  30 + 30 (expressed in mL) (Bauer et al., 2015).

Key facts The five steps to diagnose neurogenic bladder in children: l l l l l

Clinical history including bladder and bowel habits. Physical examination. Urinalysis and culture, to be repeated in all febrile episodes to exclude UTI. Urinary ultrasonography based on kidney, ureter and bladder evaluation, including measurement of postvoid residual. Urodynamic studies.

Summary points l

l

l

l

l

l

l

The extent of urological sequels related to the Zika virus (ZIKV) needs to be further investigated but neurogenic bladder was a common condition in our cohort. Neurogenic bladder is one of the genitourinary tract abnormalities that cause chronic renal disease (CKD) in children, a worldwide public health problem that imposes a high resource burden on the health-care system. The prevalence of CKG in the world’s population exceeds 10% and may reach 50% in high-risk subpopulations. Children born with CZS are included in the high-risk subpopulations. Proactive management of congenital neurogenic bladder, with treatment onset within the first year of life improves urodynamic prognosis and triplicates the chance of preventing renal disease. The treatment delay can cause progressive urinary system damage, advancing to chronic stages which may involve a lifelong burden of recurrent urinary tract infections, urinary incontinence, and ultimately CKD. Neurogenic bladder is one of the few morbidities that can be treated/prevent in the settings of CZV and its proactive management may help mitigate the disease burden for these patients and families. Therefore, we recommend urological investigation for patients with congenital Zika syndrome, including the urodynamic studies to explore neurogenic bladder.

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Acknowledgments The author expresses sincerest gratitude to CAPES, CNPq, and DECIT (funding agencies); the researchers; and coauthors of previous publications related to the investigation of urological sequels in congenital Zika syndrome, the urodynamic pediatric team and the Zika team of Fernandes Figueira Institute.

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Emerging Infectious Diseases, 21(10), 1885–1886. https://doi.org/ 10.3201/eid2110.150847. CDC Report. (2018). CDC reports increase in birth defects potentially related to Zika in US areas with local transmission. https://www.cdc.gov/ pregnancy/zika/key-findings/birth-defects-increase-and-zika.html. Chen, J., Yang, Y., Chen, J., Zhou, X., Dong, Z., Chen, T., … Zhu, T. (2017). Zika virus infects renal proximal tubular epithelial cells with prolonged persistency and cytopathic effects: ZIKV infects hRPTEpiCs. Emerging Microbes & Infections, 6(1), 1–7. https://doi.org/10.1038/emi.2017.67. Costa Monteiro, L. M., Cruz, G. N. D. O., Fontes, J. M., & Boechat, M. C. B. (2017). Criteria to evaluate neurogenic bladder in children with congenital Zika syndrome. Protocols.io. https://www.protocols.io/view/criteria-to-evaluate-neurogenic-bladder-in-patient-k5vcy66. Costa Monteiro, L. M., Cruz, G. N. D. O., Fontes, J. M., de Araujo, G. F., Ventura, T., Monteiro, A. C., & Moreira, M. E. L. (2019). Neurogenic bladder in the settings of congenital Zika syndrome: A confirmed and unknown condition for urologists. Journal of Pediatric Urology. https://doi.org/10.1016/ j.jpurol.2019.04.018. Costa Monteiro, L. M., Cruz, G. N. D. O., Fontes, J. M., Saad Salles, T. R. D., Boechat, M. C. B., Monteiro, A. C., & Moreira, M. E. L. (2018). Neurogenic bladder findings in patients with congenital Zika syndrome: A novel condition. PLoS One, 13(3). https://doi.org/10.1371/journal.pone.0193514, e0193514. Costa Monteiro, L. M., Cruz, G. O., Fontes, J. M., Vieira, E. T. R. C., Santos, E. N., Arau´jo, G. F., & Ramos, E. G. (2017). Early treatment improves urodynamic prognosis in neurogenic voiding dysfunction: 20 years of experience. Jornal de Pediatria, 93(4), 420–427. https://doi.org/10.1016/ j.jped.2016.11.010. Cugola, F. R., Fernandes, I. R., Russo, F. B., Freitas, B. C., Dias, J. L. M., Guimara˜es, K. P., … Beltra˜o-Braga, P.C.B. (2016). The Brazilian Zika virus strain causes birth defects in experimental models. Nature. https://doi.org/10.1038/nature18296. D’Ancona, C. A. L., Gomes, M. J., & Rosier, P. F. W. M. (2017). ICS teaching module: Cystometry (basic module). Neurourology and Urodynamics, 36 (7), 1673–1676. https://doi.org/10.1002/nau.23181. Delaney, A., Mai, C., Smoots, A., Cragan, J., Ellington, S., Langlois, P., … Honein, M.A. (2018). Population-based surveillance of birth defects potentially related to Zika virus infection—15 states and U.S. territories, 2016. MMWR. Morbidity and Mortality Weekly Report, 67(3), 91–96. https://doi.org/ 10.15585/mmwr.mm6703a2. Dik, P., Klijn, A. J., van Gool, J. D., de Jong-de Vos van Steenwijk, C. C. E., & de Jong, T. P. V. M. (2006). Early start to therapy preserves kidney function in spina bifida patients. European Urology, 49(5), 908–913. https://doi.org/10.1016/j.eururo.2005.12.056. Drzewiecki, B. A., & Bauer, S. B. (2011). Urodynamic testing in children: Indications, technique, interpretation and significance. The Journal of Urology, 186(4), 1190–1197. https://doi.org/10.1016/j.juro.2011.02.2692. Dyer, O. (2015). Zika virus spreads across Americas as concerns mount over birth defects. BMJ, h6983. https://doi.org/10.1136/bmj.h6983. Einspieler, C., Utsch, F., Brasil, P., Panvequio Aizawa, C. Y., Peyton, C., Hydee Hasue, R., … For the GM Zika Working Group. (2019). Association of infants exposed to prenatal zika virus infection with their clinical, neurologic, and developmental status evaluated via the general movement assessment tool. JAMA Network Open, 2(1). https://doi.org/10.1001/jamanetworkopen.2018.7235, e187235. Filler, G., Gharib, M., Casier, S., L€odige, P., Ehrich, J. H. H., & Dave, S. (2012). Prevention of chronic kidney disease in spina bifida. International Urology and Nephrology, 44(3), 817–827. https://doi.org/10.1007/s11255-010-9894-5. Guerra, L., Leonard, M., & Castagnetti, M. (2014). Best practice in the assessment of bladder function in infants. Therapeutic Advances in Urology, 6(4), 148–164. https://doi.org/10.1177/1756287214528745. Gulland, A. (2016a). Zika virus is a global public health emergency, declares WHO. BMJ, i657. https://doi.org/10.1136/bmj.i657. Gulland, A. (2016b). First case of Zika virus spread through sexual contact is detected in UK. BMJ, i6500. https://doi.org/10.1136/bmj.i6500. Health Surveillance Secretariat j Ministry of Health (Ed.). (2019). Sı´ndrome cong^ enita associada a` infecc¸a˜o pelo vı´rus Zika situac¸a˜o epidemiolo´gica, ac¸o˜es desenvolvidas e desafios de 2015 a 2019 Secretaria de Vigil^ancia em Sau´de, Ministerio da Sau´de (Boletim epidemiologico special issue Nov 2019; congenital Zika syndrome) https://documentcloud.adobe.com/link/track?uri¼urn%3Aaaid%3Ascds%3AUS%3A3a5d2319-d281-4f81af6c-aef3f0a3f084.

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Horovitz, D. D. G., da Silva Pone, M. V., Moura Pone, S., Dias Saad Salles, T. R., & Bastos Boechat, M. C. (2016). Cranial bone collapse in microcephalic infants prenatally exposed to Zika virus infection. Neurology, 87(1), 118–119. https://doi.org/10.1212/WNL.0000000000002814. Kari, J. A. (2006). Neuropathic bladder as a cause of chronic renal failure in children in developing countries. Pediatric Nephrology, 21(4), 517–520. https://doi.org/10.1007/s00467-006-0034-5. Lamb, L. E., Bartolone, S. N., Tree, M. O., Conway, M. J., Rossignol, J., Smith, C. P., & Chancellor, M. B. (2018). Rapid detection of Zika virus in urine samples and infected mosquitos by reverse transcription-loop-mediated isothermal amplification. Scientific Reports, 8(1). https://doi.org/10.1038/ s41598-018-22102-5. Lebov, J. F., Arias, J. F., Balmaseda, A., Britt, W., Cordero, J. F., Galva˜o, L. A., … Zorrilla, C. (2019). International prospective observational cohort study of Zika in infants and pregnancy (ZIP study): Study protocol. BMC Pregnancy and Childbirth, 19(1). https://doi.org/10.1186/s12884-019-2430-4. Lehnert, T., Weisser, M., Till, H., & Rolle, U. (2012). The effects of long-term medical treatment combined with clean intermittent catheterization in children with neurogenic detrusor overactivity. International Urology and Nephrology, 44(2), 335–341. https://doi.org/10.1007/s11255-011-0030-y. Liu, T., Tang, L., Tang, H., Pu, J., Gong, S., Fang, D., … Huang, X. (2019). Zika virus infection induces acute kidney injury through activating NLRP3 inflammasome via suppressing Bcl-2. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.01925. McKibben, M. J., Seed, P., Ross, S. S., & Borawski, K. M. (2015). Urinary tract infection and neurogenic bladder. Urologic Clinics of North America, 42(4), 527–536. https://doi.org/10.1016/j.ucl.2015.05.006. Melo, A. S., de, O., Aguiar, R. S., Amorim, M. M. R., Arruda, M. B., Melo, F. D. O., … Tanuri, A. (2016). Congenital zika virus infection: Beyond neonatal microcephaly. JAMA Neurology. https://doi.org/10.1001/jamaneurol.2016.3720. ^ ^ ^ Ministerio da Saude. (2017a). Orientac¸o˜es integradas de vigilancia e atenc¸a˜o a` sau´de no ambito da Emerg^ encia de Sau´de Pu´blica de Importancia Nacional. Ministerio da Saude. http://bvsms.saude.gov.br/publicacoes/orientacoes_emergencia_gestacao_infancia_zika.pdf. Ministerio da Saude. (2017b). Protocolo de atenc¸a˜o a` sau´de e resposta a` ocorr^ encia de microcefalia relacionada a` infecc¸a˜o pelo vı´rus zika. www.saude. gov.br/sas. Musso, D., Ko, A. I., & Baud, D. (2019). Zika virus infection—After the pandemic. New England Journal of Medicine, 381(15), 1444–1457. https://doi. org/10.1056/NEJMra1808246. Neveus, T., & Sillen, U. (2013). Lower urinary tract function in childhood; normal development and common functional disturbances. Acta Physiologica, 207(1), 85–92. https://doi.org/10.1111/apha.12015. Olandoski, K. P., Koch, V., & Trigo-Rocha, F. E. (2011). Renal function in children with congenital neurogenic bladder. Clinics, 66(2), 189–195. https:// doi.org/10.1590/S1807-59322011000200002. Oliveira-Szejnfeld, P. S. D., Levine, D., Melo, A. S. D. O., Amorim, M. M. R., Batista, A. G. M., Chimelli, L., … Tovar-Moll, F. (2016). Congenital brain abnormalities and Zika virus: What the radiologist can expect to see prenatally and postnatally. Radiology, 281(1), 203–218. https://doi.org/10.1148/ radiol.2016161584. Rasmussen, S. A., Jamieson, D. J., Honein, M. A., & Petersen, L. R. (2016). Zika virus and birth defects—Reviewing the evidence for causality. New England Journal of Medicine, 374(20), 1981–1987. https://doi.org/10.1056/NEJMsr1604338. Reynolds, M. R., Jones, A. M., Petersen, E. E., Lee, E. H., Rice, M. E., Bingham, A., … Zaki, S. (2017). Vital signs: Update on Zika virus-associated birth defects and evaluation of all U.S. infants with congenital Zika virus exposure—U.S. Zika Pregnancy Registry, 2016. MMWR. Morbidity and Mortality Weekly Report, 66(13), 366–373. https://doi.org/10.15585/mmwr.mm6613e1. Rodrı´guez-Ruiz, M., Somoza, I., & Curros-Mata, N. (2016). Study of kidney damage in paediatric patients with neurogenic bladder and its relationship with the pattern of bladder function and treatment received. Actas Urologicas Espan˜olas, 40(1), 37–42. https://doi.org/10.1016/j.acuro.2015.06.002. Schuler-Faccini, L., Ribeiro, E. M., Feitosa, I. M. L., Horovitz, D. D. G., Cavalcanti, D. P., Pessoa, A., … Brazilian Medical Genetics Society–Zika Embryopathy Task Force. (2016). Possible association between Zika virus infection and microcephaly—Brazil, 2015. MMWR. Morbidity and Mortality Weekly Report, 65(3), 59–62. https://doi.org/10.15585/mmwr.mm6503e2. Vasco Aragao, M.d. F., van der Linden, V., Brainer-Lima, A. M., Coeli, R. R., Rocha, M. A., Sobral da Silva, P., … Valenca, M. M. (2016). Clinical features and neuroimaging (CT and MRI) findings in presumed Zika virus related congenital infection and microcephaly: Retrospective case series study. BMJ. https://doi.org/10.1136/bmj.i1901, i1901. Verpoorten, C., & Buyse, G. M. (2008). The neurogenic bladder: Medical treatment. Pediatric Nephrology, 23(5), 717–725. https://doi.org/10.1007/ s00467-007-0691-z. Vigil, H. R., & Hickling, D. R. (2016). Urinary tract infection in the neurogenic bladder. Translational Andrology and Urology, 5(1), 72–87. https://doi.org/ 10.3978/j.issn.2223-4683.2016.01.06. ´ lvarez, A ´ ., Baldrich-Gomez, O., Posso, H., … Rodrı´guez-Morales, A. J. (2019). Urinary bladder Villamil-Go´mez, W. E., Padilla-Ruiz, D., Mendoza, A., A agenesis and renal hypoplasia potentially related to in utero Zika virus infection. International Journal of Infectious Diseases, 85, 54–56. https://doi. org/10.1016/j.ijid.2019.05.021.

Chapter 26

Zika virus and impact on male fertility Jocelyne Piret and Guy Boivin Research Center in Infectious Diseases, Laval University, Quebec City, QC, Canada

List of abbreviations AdC7 BTB E IFN IFNAR ISGs MRT prM TAM ZIKV

chimpanzee adenovirus type 7 blood-testis barrier envelope interferon interferon-α/β receptor interferon-stimulated genes male reproductive tract precursor of membrane/membrane Tyro3, Axl, and Mer Zika virus

Introduction Zika virus (ZIKV) is a reemerging arbovirus that belongs to the family Flaviviridae. The majority of patients infected with ZIKV (50%–80%) remain asymptomatic. After an incubation period that ranges from 2 to 12 days, clinical symptoms and signs of ZIKV infection are typically mild and most often include a maculopapular rash, fever, arthralgia, myalgia, and conjunctival hyperemia that resolve within 1 week. The main mechanism of ZIKV spread is through mosquito bites, primarily of the Aedes genus (Musso, Ko, & Baud, 2019). The transmission of the ancestral African lineage was restricted between nonhuman primates and Aedes africanus mosquitos. During the first 50 years after its discovery in the Zika forest of Uganda, only sporadic infections in humans were reported in Africa and Southeast Asia. In contrast, the transmission of the Asian lineage of ZIKV was extended to human-adapted Aedes mosquitos. In urban areas, Aedes aegypti is the major vector for horizontal transmission of ZIKV to humans with a lower contribution of Aedes albopictus. Two outbreaks were then reported in the Micronesian Yap Islands (in 2007) and French Polynesia (in 2013) concomitant with more severe clinical manifestations such as Guillain-Barre Syndrome (an autoimmune disease causing acute or subacute flaccid paralysis) in adult patients. Thereafter, a new American subclade of ZIKV emerged from the Asian lineage and spread in a human population that was not previously exposed to the virus in Brazil (in 2015). During this outbreak, an association was established between ZIKV infection and congenital malformations such as microcephaly, cerebral malformations as well as ophthalmological and hearing defects in the newborns. There is no vaccine and antiviral drug approved for the prevention or treatment of ZIKV infection. Prevention strategies consist of individual protection against mosquitos’ bites and vector control methods. During the pandemic, the high infection rate led to the identification of ZIKV-associated neurological disorders. However, it is still unknown if these complications resulted from an increased viral fitness or tropism or additional routes of viral transmission. For instance, other alternative modes of ZIKV transmission have been described in humans such as blood transfusion, intrauterine, intrapartum, and sexual transmission.

Human cases of ZIKV sexual transmission There is no means to distinguish between vector-borne and sexual transmission in regions where ZIKV transmission is endemic. Therefore, travel-associated cases represent an opportunity to identify sexual transmission events. The majority of suspected cases of ZIKV sexual transmission occurred from a symptomatic male to a female (Table 1). A case of male-tomale sexual transmission that occurred within 1 week after one of the partners came back from a ZIKV endemic region has Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00026-2 Copyright © 2021 Elsevier Inc. All rights reserved.

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TABLE 1 Suspected male-to-female Zika virus sexual transmission reports. Country of original Zika virus infection (partner)

Country of suspected sexual transmission

Senegal (man)

Viral load in semen

Time between onset of symptoms for the 2 partners

References

USA

Not available

Within 1 week

Foy et al. (2011)

Thailand (man)

Italy

Not available

Within 2 weeks

Venturi et al. (2016)

Caribbean (man)

USA

Not available

Within 1 week

Hills et al. (2016)

Central America (man)

USA

Not available

Within 1 week

Hills et al. (2016)

Central America (man)

USA

Not available

Within 2 weeks

Hills et al. (2016)

Brazil (man)

France

2.9
10 copies/mL and 3.5
109 copies/mL on days 18 and 24, respectively Infectious virus isolated

Within 2 weeks

D’Ortenzio et al. (2016)

Martinique (man and woman)

France

Not available

Within 5–6 weeks

Turmel et al. (2016)

Puerto Rico (man)

Germany

6.0
104 copies/mL on day 45

Within 2 weeks

Frank et al. (2016)

Martinique (man and woman)

France

Positive on day 39 after return

Within 3 and 5 weeks

Freour et al. (2016)

Samoa (man)

New Zealand

Cycle threshold of 25, 29 and 35 on days 23, 35 and 76, respectively

Within 2 weeks

Harrower et al. (2016)

Dominican Republic (man)

USA

Negative on day 31 after return

Within 2 weeks

Brooks et al. (2016)

Maldives (man and woman)

Spain

Positive by RT-PCR on days 69 and 96 Positive by culture on day 69

Within 2 weeks

Arsuaga, Bujalance, Diaz-Menendez, Vazquez, and Arribas (2016)

Columbia, Costa Rica, El Salvador, Haiti, Puerto Rico and Suriname (man)

USA (9 cases)

1 patient negative by RT-PCR on day 42 1 other patient positive by RT-PCR on days 28 and 39, equivocal on day 46 and negative on day 60

Between 2 and 3 weeks

Russell et al. (2017)

Dominican Republic (man)

Italy

4.5
104 copies/mL on day 24

Within 2 weeks

Grossi et al. (2018)

8

also been reported (Deckard et al., 2016). Furthermore, a female-to-male transmission event could have occurred within a week after the female partner had returned from a region with ongoing transmission of ZIKV (Davidson, Slavinski, Komoto, Rakeman, & Weiss, 2016). Importantly, an asymptomatic man was reported to transmit the virus to his female partner (Brooks et al., 2016). A male-to-female sexual transmission in an asymptomatic couple has also been detected during virological testing in the context of assisted reproduction therapy as the male partner presented with nonobstructive azoospermia (Freour et al., 2016). As up to 80% of infected individuals are asymptomatic, this suggests that the risk of ZIKV sexual transmission can be underestimated. Probable sexual transmission of ZIKV from a vasectomized man to his female partner was also reported (Arsuaga et al., 2016). This suggests that cells in prostate and seminal vesicles could be potentially infected with ZIKV and could contribute to viral shedding in semen. Most cases of probable male-to-female sexual transmission occurred within a period of 2 weeks after symptom onset. However, late sexual transmission of the virus could have occurred 5–6 weeks after the onset of illness in two couples (Freour et al., 2016; Turmel et al., 2016). However, in both cases, the two partners traveled in regions of active ZIKV transmission and it is therefore difficult to conclude which mode of transmission was involved. Mathematical models predicted that the contribution of sexual

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transmission to the spread of ZIKV was 3.0%–4.8% (Gao et al., 2016; Maxian, Neufeld, Talis, Childs, & Blackwood, 2017). By comparing the incidence of ZIKV and dengue virus infections in Rio de Janeiro in 2015–2016, it was suggested that women of reproductive age were 90% more likely to acquire ZIKV than men (Coelho et al., 2016). The occurrence of maleto-female sexual transmission of ZIKV could thus increase the risk of pregnant women to acquire the virus in endemic and nonendemic areas. For instance, a case of congenital ZIKV syndrome after sexual transmission of the virus from a man who traveled to Haiti to his partner who did not travel to ZIKV endemic areas has been described during assisted reproductive therapy and pregnancy (Yarrington, Hamer, Kuohung, & Lee-Parritz, 2019). The male reproductive tract (MRT) may thus constitute a reservoir of ZIKV with a potential risk for sexual transmission of the virus, especially during pregnancy.

Prolonged shedding of ZIKV in semen The identification of ZIKV in body fluids relies mainly on the detection of RNA by RT-PCR whereas the isolation and culture of the infectious virus using permissive cell lines are not typically performed. ZIKV infection is divided into two phases, the acute or viremic phase that lasts approximately 5 days after symptom onset and the convalescent phase when viremia has disappeared. The duration of the excretion of ZIKV RNA and infectious virus in body fluids after the onset of symptoms was evaluated in several studies and reported in Table 2. Results showed that the mean duration of detection of viral RNA in serum, urine and semen were 11, 18, and 39 days after the onset of illness, respectively. Successful isolation of infectious virus has been performed on day 3 in serum and on day 4 in urine, whereas it was still detectable after a mean of 30 days postillness in semen. The length of time that ZIKV persists in semen varies according to different studies and ranges from days to months after symptom onset. Viral RNA and infectious virus were respectively detected in semen for as long as 370 days (Barzon et al., 2018) and 69 days (Arsuaga et al., 2016) after the onset of illness. Two weeks after onset of symptoms, the viral load in semen was 100,000-fold higher than those in serum and urine (Mansuy et al., 2016) suggesting that viral replication could occur in the MRT. More importantly, ZIKV RNA was also detected in the semen of asymptomatic patients (Brooks et al., 2016; Freour et al., 2016). The long-lasting excretion of ZIKV in semen

TABLE 2 Detection of viral RNA and infectious Zika virus in different body fluids. Body fluid

Median and range of detection of viral RNA after symptom onset

References

Isolation of infectious virus after symptom onset

Serum

Until day 6

Jeong et al. (2017)

Day 3

Until day 10

Rossini, Gaibani, Vocale, Cagarelli, and Landini (2017)

Medina et al. (2019)

Day 11.5 [6–24 days]

Barzon et al. (2018)

Day 15 [14–17 days]

Paz-Bailey, Rosenberg, and Sharp (2019)

Until day 14

Jeong et al. (2017)

Day 4

Until day 21

Rossini et al. (2017)

Zhang, Li, Deng, Tong, and Qin (2016)

Day 24 [17–34 days]

Barzon et al. (2018)

Day 11 [9–12 days]

Paz-Bailey et al. (2019)

Until day 30

Mead et al. (2018)

Day 30

Mead et al. (2018)

Up to 38 days

Medina et al. (2019)

Until 58 days

Jeong et al. (2017)

Days 15–38

Day 25 [14–29 days]

Barzon et al. (2018)

Medina et al. (2019)

Day 42 [35–50 days]

Paz-Bailey et al. (2019)

Days 15–38

Paz-Bailey et al. (2019)

Urine

Semen

References

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B Effects on neurological and body systems

suggests that the MRT is a target for viral replication and constitutes a potential reservoir for the virus. However, the cell types that sustain productive infection of ZIKV in the MRT need to be identified.

Replication of ZIKV in vitro The testis is divided into the interstitial space and the seminiferous tubules. The interstitial space contains blood vessels, immune cells, and Leydig cells which produce and secrete the testosterone. The immune-privileged seminiferous tubules contain peritubular cells and developing germ cells that are protected by the blood-testis barrier (BTB). The BTB is composed of Sertoli cells maintained together by tight and adherens junctions. Sertoli cells are involved in the nutrition of developing germ cells, in maintaining immune homeostasis in the seminiferous tubules and in providing the structural integrity of the BTB. Primary human Sertoli cells demonstrate high levels of ZIKV replication and shedding for long periods of time suggesting that these cells could constitute a reservoir for the virus in the testis (Kumar et al., 2018). In contrast, Leydig cells are relatively resistant to ZIKV infection (Kumar et al., 2018) (Fig. 1). ZIKV was shown to modulate cellular proliferation pathways and host antiviral response to establish infection and persistence in primary human Sertoli cells (Strange, Green, Siemann, Gale Jr., & Verma, 2018). Axl is a TAM (Tyro3, Axl, Mer) receptor tyrosine kinases that plays a pivotal role in maintaining an immunosuppressive environment in the testis through interaction with the interferon (IFN)-α/β receptor (IFNAR). Axl is highly expressed in human Sertoli cells compared to Leydig cells and was identified as an entry receptor for the contemporary ZIKV strain PRVABC59 (Strange et al., 2019). Furthermore, the entry of ZIKV into Sertoli cells through Axl is associated with a reduced expression of interferon-stimulated genes (ISGs) that promotes robust and persistent viral replication. By using an in vitro model of human Sertoli cells barrier, cell-free ZIKV was shown to migrate through these cells to the lumen side even if the barrier permeability and the expression of junction proteins are not altered (Siemann, Strange, Maharaj, Shi, & Verma, 2017). In addition, ZIKV-infected Sertoli cells increase their expression of cell adhesion molecules (such as vascular cell adhesion molecule 1 and intracellular adhesion molecule 1) that facilitate the adhesion of naive immune cells to Sertoli cells leading to a disruption of the Sertoli cells barrier permeability. The inflammatory response mediated by ZIKV-infected macrophages may further increase the permeability of the Sertoli cells barrier. Overall, these data suggest that the virus may reach the site of development of germ cells in the testis. In human testicular explants, contemporary Asian ZIKV strains (MRS_OPY_MartiniquePaRi-2015 and H/PF/2013) infect a broad range of testicular cell types including resident macrophages and the germ cell lines (Matusali et al., 2018). The tissue architecture and histology of human testis explants is not affected by ZIKV infection. ZIKV triggers a broad antiviral response without upregulation of IFN synthesis and a minimal proinflammatory response in human testicular tissue. ZIKV

FIG. 1 Zika virus-infected cells in the male reproductive tract and the testis.

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infection does not alter the production of basal testosterone and inhibin B levels. Finally, ZIKV infects, replicates, and produces infectious virus in human prostate mesenchymal stem cells, epithelial cells and organoids made of a combination of these cells (Spencer et al., 2018). In organoids, stromal cells are more permissive to ZIKV infection than epithelial cells indicating a viral tropism for stem cells. These data suggest that the prostate and seminal vesicles (Fig. 1) could contribute to the viral shedding in semen and could explain the detection of ZIKV RNA in the semen of vasectomized men.

Testis damage induced by ZIKV in animal models Due to immune antagonism, wild-type mice cannot sustain ZIKV infection (Grant et al., 2016). This limitation has required the administration of an anti-IFNAR antibody to wild-type mice or the use of type I IFN receptor-deficient A129 mice or type I and II IFN receptor-deficient AG129 mice to allow ZIKV replication and pathogenicity. Wild-type C57BL/6 mice treated with an anti-IFNAR monoclonal antibody were infected subcutaneously with a mouse-adapted African ZIKV strain Dakar 41,519 or an Asian ZIKV strain H/PF/2013 (Govero et al., 2016). ZIKV replicated and persisted in the testis and epididymis resulting in tissue injury. ZIKV RNA was detected in spermatogonia, primary spermatocytes, Sertoli cells and to a lesser extent in Leydig cells. On day 14 postinfection, the fluid from epididymis and mature sperm exhibited high levels of ZIKV RNA and the total and motile sperm counts were reduced. The structure of the seminiferous tubules in the testis was damaged and the BTB was altered. This was associated with an infiltration of inflammatory cells in the seminiferous tubules. The weight and size of the testes were markedly decreased. In contrast to human testicular explants, the basal levels of testosterone and inhibin B were reduced in mice. In another study, IFNAR/ mice were infected intraperitoneally with a contemporary ZIKV strain SMGC-1 (Ma et al., 2017). ZIKV was detected in the testes and epididymides but not in the prostate and seminal vesicles and this correlated with the detection of Axl receptor which is proposed to be involved in ZIKV entry into cells. The testes were severely damaged as evidenced by the necrosis of germ and Leydig cells. An important infiltration of leukocytes in the seminiferous tubules and interstitial space resulted in acute orchitis. Another study compared the outcome of A129 and AG129 mice infected subcutaneously with ZIKV strain PRVABC59 (Clancy, Van Wettere, Siddharthan, Morrey, & Julander, 2018). ZIKV was detected in the testes and epididymides of both mouse strains whereas it was present in the prostate and seminal vesicles of AG129 mice only. Both mouse strains exhibited signs of epididymitis followed by orchitis with more severe inflammation in AG129 mice. The lack of inflammatory response in prostatic epithelial cells could favor a sustained low-level viral replication in the prostate and seminal vesicles. Another group developed a nonlethal model of ZIKV infection in immunodeficient mice that induced high viremia without causing the death of animals (Uraki et al., 2017). IFNAR/ mice were infected subcutaneously with the contemporary ZIKV strain MEX2–81. After a viremic phase, the virus disappeared from the blood but was detected in Leydig cells and sperm cells present in the lumen of the epididymal ducts. On day 21 postinfection, the weight and size of the testes were reduced. A significant decrease in testosterone levels was observed on days 5 and 21 postinfection. It was suggested that viral replication in Leydig cells could decrease the production of testosterone and ultimately lead to testicular atrophy. Rhesus and cynomolgus macaques infected subcutaneously with Thai and Puerto Rican strains of ZIKV demonstrated viral RNA in the testes, prostate and seminal vesicles that could persist for at least 28 days postinfection (Osuna et al., 2016).

Sexual transmission of ZIKV and reduced male fertility in animal models Sexual transmission of ZIKV from male AG129 mice infected intraperitoneally with ZIKV strain PRVABC59 to naı¨ve female AG129 mice led to intrauterine transmission to the fetus (Duggal, McDonald, Ritter, & Brault, 2018). Infectious virus was detected in semen between 7 and 21 days and ZIKV RNA for 58 days postinfection. Vasectomized mice shed lower levels of infectious virus in semen but sexual transmission to AG129 female mice still occurred. By using a nonlethal model in which male A129 mice infected subcutaneously with ZIKV strain MEX2–81 were mated with naı¨ve female A129 mice, it was shown that the weight of pups was significantly reduced and that some fetuses exhibited ocular malformations (Uraki et al., 2017). Intravaginal infection of rhesus and cynomolgus monkeys with ZIKV strain ArD 41,525 (prepared from Aedes africanus mosquitos) resulted in a detectable viremia in 50% of animals suggesting that sexual transmission of the virus could occur (Haddow et al., 2017). Infection of nonhuman primates with Asian/American lineage of ZIKV by the subcutaneous or intraamniotic/intravenous route early in gestation led to fetal demise later in gestation without the onset of clinical signs (Dudley et al., 2018) suggesting that sexual transmission of the virus during pregnancy could be a risk for the fetus. Mating of naı¨ve wild-type C57BL/6 female mice treated with an anti-IFNAR antibody with male counterparts infected subcutaneously with ZIKV strain Dakar 41,519 led to a reduced rate of pregnancy and number of viable fetuses compared to mating with uninfected males (Govero et al., 2016). A sublethal model consisting in older IFNAR/ mice infected

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FIG. 2 Fertility of male IFNAR/ mice mated with female C57BL/6 mice on days 28, 75, and 207 postinfection. Percentage of female mice that became pregnant after mating with mock animals (black), mice infected subcutaneously with 6
105 plaque forming units of Zika virus (ZIKV; red) and mice that received a DNA-based vaccine (GLS-5700) 2 weeks before infection with Zika virus (blue). Results are the mean  SD of 6–9 mice per group. *P < 0.05; **P < 0.01. dpi, days postinfection. (Data are from de La Vega, M. A., Piret, J., Griffin, B. D., Rheaume, C., Venable, M. C., Carbonneau, J., et al. (2019). Zika-induced male infertility in mice is potentially reversible and preventable by deoxyribonucleic acid immunization. The Journal of Infectious Diseases, 219, 365–374, with permission from the Publishers.)

subcutaneously with ZIKV strain PRVABC59, that exhibited viremia without clinical signs of disease, was developed to evaluate male fertility (de La Vega et al., 2019). Male IFNAR/ mice infected with ZIKV were mated with naı¨ve female C57BL/6 mice. Although the total sperm and progressively motile sperm counts as well as microscopic appearance of the testes were similar in uninfected and ZIKV-infected mice, 33% of mated males were infertile on day 28 postinfection (Fig. 2). On days 75 and 207 postinfection, a partial recovery of fertility was observed in 66% of previously infertile males. It was suggested that sperm cells may be structurally and/or functionally altered following their infection with ZIKV.

Effects of ZIKV on sperm cells Germ cells isolated from testes of wild-type C57BL/6/Sv129 mice are more susceptible to infection with ZIKV strain MR766 compared to Sertoli cells, Leydig cells, and peritubular myoid cells (Robinson et al., 2018). A reduced expression of interferon-induced protein 44 in germ cells infected with ZIKV allows high levels of viral replication. Another study using electron microscopy revealed that ZIKV virions were bound to developing and mature sperm cells within mouse testes (Uraki, Jurado, et al., 2017). Sperm cells of animals infected with ZIKV strain MEX2–81 were also shown to contain more residual bodies than uninfected controls. These bodies or cytoplasmic droplets have been associated with reduced fertility in several animals species (Cooper, 2005). A study in a cohort of 15 ZIKV-infected men in Guadeloupe over a 6-month period showed a reduction in the median values of total sperm counts and total motile sperm counts on day 30, with a statistically significant decrease on day 60 after the onset of symptoms suggesting that ZIKV could affect male fertility ( Joguet et al., 2017). Recovery to normal values was seen on days 90 and 120 postillness. The proportion of ZIKV-positive spermatozoa detected by immunofluorescence analysis was estimated to be 3.5% on three smears of semen sample obtained from a patient (Mansuy et al., 2016). The virus was detected in the head of spermatozoa by the use of confocal and stimulated emission depletion microscopy. ZIKV was shown to bind to the mid-piece of motile and viable human spermatozoa but not to nonmotile or dead sperm cells (Bagasra et al., 2017). Tyro3 receptors, which are mainly expressed on the mid-piece of spermatozoa, could play a role in the binding and entry of ZIKV in these cells (Fig. 3).

Experimental drugs against ZIKV infection of the MRT Treatment of multicellular human testicular organoids with R428, an Axl kinase inhibitor, reduced ZIKV replication by increasing the expression of ISGs (Strange et al., 2019). Furthermore, incubation of ZIKV-infected Sertoli cells with R428 reduces the phosphorylation of signal transducer and activator of transcription 1 and the levels of suppressor of cytokine signaling 1 and 3 leading to an upregulation of ISGs. It is suggested that R428 disrupts the Axl-IFNAR complex. This results in an increased expression of IFN-dependent ISGs and thereby to a reduced viral replication. Compound NITD008, a nucleoside analogue targeting the viral polymerase, inhibits ZIKV replication in primary human Sertoli cells at concentrations of 3–5 μM as determined by a plaque reduction assay (Siemann et al., 2017). Compounds that could inhibit ZIKV

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FIG. 3 Zika virus infection of spermatozoa through the Tyro3 receptor.

infection in germ cells were identified by a small-scale repurposing screen of molecules reported to inhibit other flaviviruses or target pathways important for ZIKV infection (Robinson et al., 2018). Among the hit compounds, berberine chloride (a plant-derived alkaloid) demonstrated a strong inhibitory effect against ZIKV with a 50% inhibitory concentration of 2 μM. The amphipathic Z2 peptide, derived from the envelope (E) protein region of ZIKV, inhibits viral replication in vitro and in vivo (Si et al., 2019). Z2 peptide demonstrated antiviral activity against ZIKV strain MR766, SZ01 and FLR (the latter two from Asian lineage) in TM4 cells, a murine Sertoli cell line. After intraperitoneal administration, Cy5-labeled Z2 peptide accumulated in the testes and epididymides of A129 mice. Treatment with Z2 peptide reduced viral RNA copies in testes, epididymides, and sperm cells after intraperitoneal challenge with ZIKV. Z2 peptide prevented histopathological damage to the testes, the reduction in their weight and size as well as the decrease in total sperm counts and motility. The nucleoside analogue, 7-deaza-20 -C-methyladenosine given the day of viral challenge with ZIKV strain MR766 for 7 days reduced viremia and detection of viral RNA and infectious virus in testes but not in epididymides ( Jacobs, Delang, Verbeken, Neyts, & Kaptein, 2019). Finally, the efficacy of recombinant type 1 IFNs was evaluated in BALB/c mice that received dexamethasone to mimic the situation prevailing in immunosuppressed individuals (Chan et al., 2016). After intraperitoneal injection of ZIKV strain PRVABC59, these mice exhibited viremia and disseminated viral infection. Treatment with recombinant pegylated IFN-α2B or IFN-β1B was associated with improved outcome with no mortality, reduced viral load and inflammatory response in various tissues including the testes/epididymides and prostate.

Experimental vaccines against ZIKV infection of the MRT The precursor of membrane/membrane (prM) and E proteins form a heterodimer (prM/E) in immature virions that is cleaved during ZIKV maturation. The prM/E complex was shown to induce high antibody titers in several studies and is the most frequently used immunogen in ZIKV vaccine programs. Intramuscular injection followed by electroporation of a synthetic DNA vaccine encoding the prM/E antigenic regions prior to subcutaneous challenge with ZIKV strain PRVABC59 completely protected IFNAR/ mice against damage to the testes and abnormalities in total sperm counts and motility induced by the virus (Griffin et al., 2017). ZIKV persistence in the testes, epididymides, and sperm cells was also prevented by the synthetic DNA vaccine. The vaccine efficacy was further evaluated against ZIKVinduced fertility loss in older IFNAR/ male mice (de La Vega et al., 2019). This study demonstrated that the loss of fertility seen after mating with C57BL/6 female mice could be prevented by immunization with the prM/E DNA vaccine (Fig. 2). Another vaccine based on recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins (AdC7-M/E) conferred protective immunity after challenge with ZIKV strain MR766 in both immunocompetent and immunodeficient mice characterized by a rapid and sustained humoral and T cell responses (Xu et al., 2018). Furthermore, AdC7-M/E vaccine completely prevented viral burden in testes and ZIKV-induced testicular damage.

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Conclusions In humans, the long-lasting shedding of ZIKV in semen and the occurrence of male-to-female sexual transmission could increase the risk of transmission to the fetus during pregnancy. Furthermore, germ cells can sustain ZIKV replication in vitro and in vivo. Human sperm cells were shown to be infected with the virus and a reduction in total sperm counts and motile sperm counts has been reported. In immunodeficient mice, ZIKV infection has been shown to induce important testis damage, testicular atrophy as well as fertility loss. Thus, the impact of ZIKV on male fertility in humans cannot be excluded and should be carefully investigated.

Policy and procedures Recommendations for the prevention of sexual transmission of ZIKV in pregnant women can be found at https://www.cdc. gov/zika/prevention/sexual-transmission-prevention.html. Pregnant women should adopt safe sex practices during the entire pregnancy. In regions with no ZIKV transmission, safe sex practices (correct and consistent use of male or female condom) or abstinence for a period of 3 and 2 months are, respectively, recommended for men and women returning from a region of ongoing ZIKV transmission. Male partner of a pregnant woman returning from a region with active ZIKV transmission should adopt safe sex practices or abstinence during the rest of the pregnancy. Before attempting conception, men should allow a delay of 3 months after the onset of symptoms or possible exposure to the virus whereas a delay of 2 months is recommended for women.

Mini-dictionary of terms Epididymitis: Inflammation of the epididymis, a duct behind the testis that is connected to the vas deferens. Inhibin B: Hormone produced by Sertoli cells in the testis and associated with spermatogenesis. Nonobstructive azoospermia: Semen which contains no sperm due to a failure in spermatogenesis. Orchitis: Inflammation of one or both testicles. Sperm motility: Ability of sperm cells to move. Testosterone: Male sex hormone produced by Leydig cells in the testis.

Key facts of the impact of Zika virus on male fertility l l l l l

Zika virus may persist in semen of symptomatic and asymptomatic men for months. Human cases of male-to-female transmission of ZIKV have occurred. Congenital Zika virus syndrome has occurred after sexual transmission in humans. Zika virus infection may cause fertility loss in immunodeficient male mice. Effect of Zika virus on male fertility should be investigated in humans.

Summary points l l l l l l

There is a long-lasting Zika virus shedding in semen compared to blood and urine. The majority of sexual transmission of Zika virus is from male-to-female. Human Sertoli cells and germ cells can sustain Zika virus replication. Testicular injury is observed in immunodeficient mice infected with Zika virus. Infection of immunodeficient male mice with Zika virus leads to fertility loss. Experimental drugs and vaccines could prevent the effects of Zika virus on the male reproductive tract.

References Arsuaga, M., Bujalance, S. G., Diaz-Menendez, M., Vazquez, A., & Arribas, J. R. (2016). Probable sexual transmission of Zika virus from a vasectomised man. The Lancet Infectious Diseases, 16, 1107. Bagasra, O., Addanki, K. C., Goodwin, G. R., Hughes, B. W., Pandey, P., & McLean, E. (2017). Cellular targets and receptor of sexual transmission of Zika virus. Applied Immunohistochemichemistry Molecular Morphology, 25, 679–686.

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Barzon, L., Percivalle, E., Pacenti, M., Rovida, F., Zavattoni, M., Del Bravo, P., … Baldanti, F. (2018). Virus and antibody dynamics in travelers with acute Zika virus infection. Clinical Infectious Diseases, 66, 1173–1180. Brooks, R. B., Carlos, M. P., Myers, R. A., White, M. G., Bobo-Lenoci, T., Aplan, D., … Feldman, K. A. (2016). Likely sexual transmission of Zika virus from a man with no symptoms of infection - Maryland, 2016. Morbidity and Mortality Weekly Report, 65, 915–916. Chan, J. F., Zhang, A. J., Chan, C. C., Yip, C. C., Mak, W. W., Zhu, H., … Yuen, K. Y. (2016). Zika virus infection in dexamethasone-immunosuppressed mice demonstrating disseminated infection with multi-organ involvement including orchitis effectively treated by recombinant type I interferons. eBioMedicine, 14, 112–122. Clancy, C. S., Van Wettere, A. J., Siddharthan, V., Morrey, J. D., & Julander, J. G. (2018). Comparative histopathologic lesions of the male reproductive tract during acute infection of Zika virus in AG129 and Ifnar(/) mice. The American Journal of Pathology, 188, 904–915. Coelho, F. C., Durovni, B., Saraceni, V., Lemos, C., Codeco, C. T., Camargo, S., … Armstrong, M. (2016). Higher incidence of Zika in adult women than adult men in Rio de Janeiro suggests a significant contribution of sexual transmission from men to women. International Journal of Infectious Diseases, 51, 128–132. Cooper, T. G. (2005). Cytoplasmic droplets: The good, the bad or just confusing? Human Reproduction, 20, 9–11. Davidson, A., Slavinski, S., Komoto, K., Rakeman, J., & Weiss, D. (2016). Suspected female-to-male sexual transmission of Zika virus - new York City, 2016. Morbidity and Mortality Weekly Report, 65, 716–717. de La Vega, M. A., Piret, J., Griffin, B. D., Rheaume, C., Venable, M. C., Carbonneau, J., … Boivin, G. (2019). Zika-induced male infertility in mice is potentially reversible and preventable by deoxyribonucleic acid immunization. The Journal of Infectious Diseases, 219, 365–374. Deckard, D. T., Chung, W. M., Brooks, J. T., Smith, J. C., Woldai, S., Hennessey, M., … Mead, P. (2016). Male-to-male sexual transmission of Zika virus-Texas, January 2016. Morbidity and Mortality Weekly Report, 65, 372–374. D’Ortenzio, E., Matheron, S., Yazdanpanah, Y., de Lamballerie, X., Hubert, B., Piorkowski, G., … Leparc-Goffart, I. (2016). Evidence of sexual transmission of Zika virus. The New England Journal of Medicine, 374, 2195–2198. Dudley, D. M., Van Rompay, K. K., Coffey, L. L., Ardeshir, A., Keesler, R. I., Bliss-Moreau, E., … O’Connor, D. H. (2018). Miscarriage and stillbirth following maternal Zika virus infection in nonhuman primates. Nature Medicine, 24, 1104–1107. Duggal, N. K., McDonald, E. M., Ritter, J. M., & Brault, A. C. (2018). 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Prevention and control of Zika as a mosquito-borne and sexually transmitted disease: A mathematical modeling analysis. Scientific Reports, 6, 28070. Govero, J., Esakky, P., Scheaffer, S. M., Fernandez, E., Drury, A., Platt, D. J., … Diamond, M. S. (2016). Zika virus infection damages the testes in mice. Nature, 540, 438–442. Grant, A., Ponia, S. S., Tripathi, S., Balasubramaniam, V., Miorin, L., Sourisseau, M., … Garcia-Sastre, A. (2016). Zika virus targets human STAT2 to inhibit type I interferon signaling. Cell Host & Microbe, 19, 882–890. Griffin, B. D., Muthumani, K., Warner, B. M., Majer, A., Hagan, M., Audet, J., … Kobinger, G. P. (2017). DNA vaccination protects mice against Zika virus-induced damage to the testes. Nature Communications, 8, 15743. Grossi, P. A., Percivalle, E., Campanini, G., Sarasini, A., Premoli, M., Zavattoni, M., … Rovida, F. (2018). An autochthonous sexually transmitted Zika virus infection in Italy 2016. New Microbiology, 41, 80–82. Haddow, A. D., Nalca, A., Rossi, F. D., Miller, L. J., Wiley, M. R., Perez-Sautu, U., … Nasar, F. (2017). High infection rates for adult macaques after intravaginal or intrarectal inoculation with Zika virus. Emerging Infectious Diseases, 23, 1274–1281. Harrower, J., Kiedrzynski, T., Baker, S., Upton, A., Rahnama, F., Sherwood, J., … Pulford, D. (2016). Sexual transmission of Zika virus and persistence in semen, New Zealand, 2016. Emerging Infectious Diseases, 22, 1855–1857. Hills, S. L., Russell, K., Hennessey, M., Williams, C., Oster, A. M., Fischer, M., & Mead, P. (2016). Transmission of Zika virus through sexual contact with travelers to areas of ongoing transmission - continental United States, 2016. Morbidity and Mortality Weekly Report, 65, 215–216. Jacobs, S., Delang, L., Verbeken, E., Neyts, J., & Kaptein, S. J. F. (2019). A viral polymerase inhibitor reduces Zika virus replication in the reproductive organs of male mice. International Journal of Molecular Sciences, 20, 2122. Jeong, Y. E., Cha, G. W., Cho, J. E., Lee, E. J., Jee, Y., & Lee, W. J. (2017). Viral and serological kinetics in Zika virus-infected patients in South Korea. Virology Journal, 14, 70. Joguet, G., Mansuy, J. M., Matusali, G., Hamdi, S., Walschaerts, M., Pavili, L., … Bujan, L. (2017). Effect of acute Zika virus infection on sperm and virus clearance in body fluids: A prospective observational study. The Lancet Infectious Diseases, 17, 1200–1208. Kumar, A., Jovel, J., Lopez-Orozco, J., Limonta, D., Airo, A. M., Hou, S., … Hobman, T. C. (2018). Human Sertoli cells support high levels of Zika virus replication and persistence. Scientific Reports, 8, 5477. Ma, W., Li, S., Ma, S., Jia, L., Zhang, F., Zhang, Y., … Gao, G. F. (2017). Zika virus causes testis damage and leads to male infertility in mice. Cell, 168, 542. Mansuy, J. M., Dutertre, M., Mengelle, C., Fourcade, C., Marchou, B., Delobel, P., … Martin-Blondel, G. (2016). 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Mansuy, J. M., Suberbielle, E., Chapuy-Regaud, S., Mengelle, C., Bujan, L., Marchou, B., … Martin-Blondel, G. (2016). Zika virus in semen and spermatozoa. The Lancet Infectious Diseases, 16, 1106–1107. Matusali, G., Houzet, L., Satie, A. P., Mahe, D., Aubry, F., Couderc, T., … Dejucq-Rainsford, N. (2018). Zika virus infects human testicular tissue and germ cells. The Journal of Clinical Investigation, 128, 4697–4710. Maxian, O., Neufeld, A., Talis, E. J., Childs, L. M., & Blackwood, J. C. (2017). Zika virus dynamics: When does sexual transmission matter? Epidemics, 21, 48–55. Mead, P. S., Duggal, N. K., Hook, S. A., Delorey, M., Fischer, M., Olzenak McGuire, D., … Hinckley, A. F. (2018). Zika virus shedding in semen of symptomatic infected men. The New England Journal of Medicine, 378, 1377–1385. Medina, F. A., Torres, G., Acevedo, J., Fonseca, S., Casiano, L., De Leon-Rodriguez, C. M., … Munoz-Jordan, J. L. (2019). Duration of the presence of infectious Zika virus in semen and serum. The Journal of Infectious Diseases, 219, 31–40. Musso, D., Ko, A. I., & Baud, D. (2019). Zika virus infection—After the pandemic. The New England Journal of Medicine, 381, 1444–1457. Osuna, C. E., Lim, S. Y., Deleage, C., Griffin, B. D., Stein, D., Schroeder, L. T., … Whitney, J. B. (2016). Zika viral dynamics and shedding in rhesus and cynomolgus macaques. Nature Medicine, 22, 1448–1455. Paz-Bailey, G., Rosenberg, E. S., & Sharp, T. M. (2019). Persistence of Zika virus in body fluids - final report. The New England Journal of Medicine, 380, 198–199. Robinson, C. L., Chong, A. C. N., Ashbrook, A. W., Jeng, G., Jin, J., Chen, H., … Chen, S. (2018). Male germ cells support long-term propagation of Zika virus. Nature Communications, 9, 2090. Rossini, G., Gaibani, P., Vocale, C., Cagarelli, R., & Landini, M. P. (2017). Comparison of Zika virus (ZIKV) RNA detection in plasma, whole blood and urine - case series of travel-associated ZIKV infection imported to Italy, 2016. Journal of Infection, 75, 242–245. Russell, K., Hills, S. L., Oster, A. M., Porse, C. C., Danyluk, G., Cone, M., … Brooks, J. T. (2017). Male-to-female sexual transmission of Zika virusUnited States, January-April 2016. Clinical Infectious Diseases, 64, 211–213. Si, L., Meng, Y., Tian, F., Li, W., Zou, P., Wang, Q., … Lu, L. (2019). A peptide-based virus inactivator protects male mice against Zika virus-induced damage of testicular tissue. Frontiers in Microbiology, 10, 2250. Siemann, D. N., Strange, D. P., Maharaj, P. N., Shi, P. Y., & Verma, S. (2017). Zika virus infects human Sertoli cells and modulates the integrity of the in vitro blood-testis barrier model. Journal of Virology, 91, e00623-17. Spencer, J. L., Lahon, A., Tran, L. L., Arya, R. P., Kneubehl, A. R., Vogt, M. B., … Rico-Hesse, R. R. (2018). Replication of Zika virus in human prostate cells: A potential source of sexually transmitted virus. The Journal of Infectious Diseases, 217, 538–547. Strange, D. P., Green, R., Siemann, D. N., Gale, M., Jr., & Verma, S. (2018). Immunoprofiles of human Sertoli cells infected with Zika virus reveals unique insights into host-pathogen crosstalk. Scientific Reports, 8, 8702. Strange, D. P., Jiyarom, B., Pourhabibi Zarandi, N., Xie, X., Baker, C., Sadri-Ardekani, H., … Verma, S. (2019). Axl promotes Zika virus entry and modulates the antiviral state of human Sertoli cells. MBio, 10, e01372-19. Turmel, J. M., Abgueguen, P., Hubert, B., Vandamme, Y. M., Maquart, M., Le Guillou-Guillemette, H., & Leparc-Goffart, I. (2016). Late sexual transmission of Zika virus related to persistence in the semen. Lancet, 387, 2501. Uraki, R., Hwang, J., Jurado, K. A., Householder, S., Yockey, L. J., Hastings, A. K., … Fikrig, E. (2017). Zika virus causes testicular atrophy. Science Advances, 3, e1602899. Uraki, R., Jurado, K. A., Hwang, J., Szigeti-Buck, K., Horvath, T. L., Iwasaki, A., & Fikrig, E. (2017). Fetal growth restriction caused by sexual transmission of Zika virus in mice. The Journal of Infectious Diseases, 215, 1720–1724. Venturi, G., Zammarchi, L., Fortuna, C., Remoli, M. E., Benedetti, E., Fiorentini, C., … Bartoloni, A. (2016). An autochthonous case of Zika due to possible sexual transmission, Florence, Italy, 2014. Euro Surveillance, 21, 30148. Xu, K., Song, Y., Dai, L., Zhang, Y., Lu, X., Xie, Y., … Gao, G. F. (2018). Recombinant chimpanzee adenovirus vaccine AdC7-M/E protects against Zika virus infection and testis damage. Journal of Virology, 92, e01722-17. Yarrington, C. D., Hamer, D. H., Kuohung, W., & Lee-Parritz, A. (2019). Congenital Zika syndrome arising from sexual transmission of Zika virus, a case report. Fertilility Research and Practice, 5, 1. Zhang, F. C., Li, X. F., Deng, Y. Q., Tong, Y. G., & Qin, C. F. (2016). Excretion of infectious Zika virus in urine. The Lancet Infectious Diseases, 16, 641–642.

Chapter 27

Testicular cell types and infection by Zika virus Luwanika Mleraa and Marshall E. Bloomb a

BIO5 Institute, University of Arizona, Tucson, AZ, United States, b Biology of Vector-Borne Viruses Section, Laboratory of Virology, National Institute

of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States

List of abbreviations DC-SIGN DPI IFN MOI MRT RNA TAM TBB TJP ZIKV

dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin days postinfection interferon multiplicity of infection male reproductive tract ribonucleic acid Tyro3, AXL, Mer testis-blood barrier tight junction proteins Zika virus

Introduction Surprisingly, the human testis has emerged as a critically important organ in the biology of Zika virus (ZIKV) infection. In the recent and ongoing ZIKV outbreak in Latin America, it is apparent that the mosquito-borne flavivirus was crossing international and interpersonal borders and being transmitted sexually even among asymptomatic individuals without the more usual vector-borne transmission (Brooks et al., 2016; Fr
eour et al., 2016; Nicastri et al., 2016; Sakkas, Bozidis, Giannakopoulos, Sofikitis, & Papadopoulou, 2018). The earliest recording of sexual transmission of ZIKV was described when two American scientists acquired ZIKV while working in Senegal in 2008 and inadvertently donated blood meals to mosquitoes (Foy et al., 2011). The 36-year old patient 1 had hematospermia, which was noted by his wife who developed afebrile symptoms 4 days after the onset of the husband’s symptoms. Patient 1 was afebrile, but presented swollen ankles, extreme fatigue, headache, and maculopapular rash on the torso (Foy et al., 2011). Most reported sexual transmissions of ZIKV have been male-to-female, but a suspected female-to-male case was also documented (Davidson, Slavinski, Komoto, Rakeman, & Weiss, 2016). Studies involving vaginal inoculation of rhesus macaques and mice showed that ZIKV preferentially replicates in the female reproductive tract (Carroll et al., 2017; Haddow et al., 2017; Tang et al., 2016; Yockey et al., 2016), but the reported higher number of male-to-female cases of ZIKV transmission in humans is compelling evidence that sexual transmission is primarily dependent on the male reproductive system. In this chapter, we review the structure and cells of the testis and highlight current research on ZIKV infection of these cells. We also discuss the pathology associated with testicular ZIKV infection and the pathways that are specifically affected by infection with this virus.

Structural and functional anatomy of the testis The testis is an external part of the male reproductive tract (MRT), and it is a gonad encapsulated by a fibrous tunica albuginea. Inside the tunica albuginea are fine coiled tubes known as seminiferous tubules. These tubules are internally lined by germ cells that develop into spermatozoa. Sertoli cells nourish and support the germ cells and they also form tight junctions that make up the testis-blood-barrier (TBB). Sertoli cells are responsible for the production of inhibins, hormones which Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00027-4 Copyright © 2021 Elsevier Inc. All rights reserved.

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inhibit follicle-stimulating hormone (Hedger, 2015; Le Gac & de Kretser, 1982; Steinberger, 1979). A collection of 2–4 seminiferous tubules make up a lobule, and there are 200–300 lobules in each testis. Leydig cells are in the interstitium, which surrounds seminiferous tubules, and they function in the production of the hormone testosterone. The interstitial tissue also contains the vasculature, lymphatic system and nerves of the testis (Hedger, 2015). Detailed descriptions of the structure and function of the testis are available in basic anatomy and histology textbooks.

Zika virus infection of testicular cells ZIKV entry into different cells is mediated by different receptors, and molecules such as DC-SIGN and heparan sulfate which have been proposed to function as receptors for mosquito-borne flaviviruses may also play a role in ZIKV infection (Sirohi & Kuhn, 2017). In neural stem cells, the TAM (Tyro3, AXL Mer) tyrosine kinase AXL was proposed to be important for ZIKV entry (Nowakowski et al., 2016). Blocking the expression of AXL with antibodies or specific RNAi-mediated silencing in skin fibroblasts severely and negatively affected ZIKV entry (Hamel et al., 2015). Testicular cells, particularly Sertoli and Leydig cells also express high levels of AXL, and it was shown that this kinase is also important for ZIKV entry into Sertoli cells (Kumar et al., 2018; Ma et al., 2016; Strange et al., 2019). Other TAM receptors like Mer and Tyro3 or TIM receptors, such as TIM1 and TIM4 did not have a significant impact on ZIKV infection of Sertoli cells because there was no change in virus replication when they were blocked with antibodies (Kumar et al., 2018). However, Tyro3 was important for ZIKV entry into spermatozoa and the midpiece of these cells expresses high levels of Tyro3 (Bagasra et al., 2017). Although Leydig cells express AXL, infection in these cells is generally lower compared to Sertoli cells, suggesting that there is a different layer of resistance to ZIKV infection in these cells. The role of other receptors in ZIKV entry into cells of the testes needs further investigation. Animal studies have been instrumental for an understanding of how ZIKV infects cells of the testes. The pathway by which ZIKV ends up in the different cells of the testes is likely complex and not completely understood. A study in mice by Tsetsarkin and colleagues suggested that ZIKV infection of the testes starts in the interstitial compartment, which contains blood and lymphatic vessels (Tsetsarkin et al., 2018). One of the conclusions in that paper was that ZIKV initially disseminates to the testes via hematogenous/lymphogenous spread from the systemic circulation. Although a single suspected case of female-to-male and male-to-male transmission have been reported (Deckard et al., 2016), cases of male to female transmission are more numerous (Lazear & Diamond, 2016; Mansuy et al., 2016; Sirohi & Kuhn, 2017). Further, males are more likely to transmit the virus because the virus may be transported along with semen or seminal fluids. Once the blood/lymph brings ZIKV to the interstitial compartment of the testis, the virus can then spread to infect other cells in the testis. In an IFNAR
/
mouse model, immunohistochemistry showed that the NS1 protein was mainly distributed to interstitial cells of the testes where Leydig cells reside (Uraki et al., 2017). In the same report, abundant viral antigen was detected in the epididymal duct lumen. Because epididymis duct conveys sperm, the identification of ZIKV in this anatomical site is consistent with sexual transmission by males. Although the NS1 antigen is a surrogate marker for ZIKV infection, it does not unequivocally define active virus replication as in situ hybridization does. Dr. Michael Diamond’s group used in situ hybridization to determine which cells in the testis are targets for ZIKV infection. Using this approach, they demonstrated ZIKV RNA in spermatogonia, primary spermatocytes and the trophic, as well as in the inhibin-producing Sertoli cells (Fig. 1) (Govero et al., 2016). In this study, Leydig cells were relatively spared by ZIKV infection, an observation echoed by Kumar and colleagues (Kumar et al., 2018). The disparity between the Diamond Lab and Uraki’s report of Leydig cell staining may be because of ZIKV strain differences or timing of analyses. At any rate, ZIKV can disseminate into all the cells of the testis, albeit to different degrees. ZIKV infection of Sertoli cells has been studied better than other testicular cells. ZIKV targeting of Sertoli cells may be because these cells express high levels of AXL, which was reported to play an important role in neuronal stem cell and endothelial cell infection by ZIKV (Liu, DeLalio, Isakson, & Wang, 2016; Nowakowski et al., 2016). The study by Kumar and colleagues indicated that Leydig cells were relatively spared although the cells also express AXL. The ZIKV infection also likely starts in the interstitial cells having been brought in by the circulatory system. Taken together, these observations indicate a need for additional research into the dynamics of ZIKV infection in Leydig cells. Gene expression profiling of these cells in comparison to Sertoli cells may shed light into host cell restriction factors that may inhibit ZIKV replication.

Testicular pathology associated with ZIKV infection A prominent feature of ZIKV infection of the testes in mice is testicular atrophy in comparison to uninfected controls (Fig. 2) (Govero et al., 2016; Ma et al., 2016; Uraki et al., 2017). Testicular injury following ZIKV infection was evident as shown by infiltration of inflammatory cells (Govero et al., 2016). Infected testes in mice undergo degeneration and sloughing of germ cells by 11 DPI (Duggal et al., 2017). Histological examinations of testes from mice infected with ZIKV

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FIG. 1 Zika virus infects different cells of the testes in a mouse model. Left panel: Low- and high-magnification images showing histological and in situ hybridization staining of mock- or ZIKV-infected cells at 7 DPI. In situ hybridization with a ZIKV-specific probe (brown color) identifies ZIKV RNA in infected cells. Green arrows indicate Sertoli cells, whereas red arrows indicate spermatogonia and spermatocytes. Right panel: Immunofluorescence microscopy of ZIKV-infected cells in the testes at 7 DPI. The different cells are identified using cell-specific markers CD45 (pan-leukocyte), TRA98 (germ cells), ETV5 (BTB), and GATA4 (Sertoli cells). White arrows indicate staining for leukocytes in the interstitium, orange arrows for germ cells, Sertoli cells are indicated by magenta arrows and the green ones indicate the BTB. White lines outline tubules of the seminiferous epithelium. Infiltration of leukocytes is evident in ZIKV-infected testis. The scale bar represents 50 μm. (Reproduced from Govero, J., Esakky, P., Scheaffer, S.M., Fernandez, E., Drury, A., Platt, D.J., ... Diamond, M.S. (2016). Zika virus infection damages the testes in mice. Nature, 540, 438, doi:10.1038/nature20556, with permission from the publisher.)

FIG. 2 Zika virus infection of cells of mouse testes leads to their destruction to cause testicular atrophy. Left panel: Immunofluorescence microscopy of mock- and ZIKV-infected cells at 21 DPI. White lines demarcate tubules in the seminiferous epithelium. Infiltration of leukocytes and macrophages is evident from CD45- and F4/80-stained cell populations (white and cyan arrows, respectively) in ZIKV-infected testes, but these are absent in mockinfected testes. Germ cells (orange arrows) are lost in ZIKV-infected cells in comparison to mock-infected cells as indicated by a lack of TRA98 staining. Sertoli cells (magenta arrows) and the BTB (green arrows) degenerate in ZIKV-infected cells while intact in mock-infected and macrophages (cyan). The ZIKV-mediated destruction of cells of the testes leads to testicular atrophy, depicted in the right panel. Testicular atrophy appears to be sow because the size of the ZIKV-infected testis at 7 DPI is comparable to mock-infected at the same time. Scale bars in the left panel represent 50 μm and those in the right panel represent 2 mm. (Reproduced from Govero, J., Esakky, P., Scheaffer, S.M., Fernandez, E., Drury, A., Platt, D.J., ... Diamond, M.S. (2016). Zika virus infection damages the testes in mice. Nature, 540, 438, doi:10.1038/nature20556, with permission from the publisher.)

revealed destruction of the seminiferous epithelium. An obvious result of testicular atrophy is infertility as demonstrated in male mouse models (Ma et al., 2016). Interestingly, a study looking at human males reported that the sperm counts were not negatively affected in ZIKV-infected males although progressive sperm motility was negatively affected in the range 2.4%–22% when the normal reference range is >32% (Avelino-Silva et al., 2018). A limitation with this study was that the sample size on which seminograms were performed was small. However, these results were similar to those observed in

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male mice infected by ZIKV in which sperm motility, inhibin B, and testosterone levels were reduced 10-fold compared to normal controls (Govero et al., 2016). An interesting observation in humans is the persistent shedding of ZIKV RNA in semen for more than 6 months following the acute infection (Mead et al., 2018). In rhesus macaques, viral RNA was detected in semen as late as 28 dpi (Osuna et al., 2016). This has been shown to be true for other animal models where viral RNA persisted for 21 days in testes of infected mice (Duggal et al., 2017; Govero et al., 2016). It is unclear how long ZIKV can remain viable in semen. However, one study reported that seminal amyloid does not affect ZIKV infection of anogenital cells, but semen and seminal plasma markedly suppressed infection of these cells by blocking viral attachment (M€uller et al., 2018). However, this study spiked ZIKV into liquified semen obtained from donors (M€uller et al., 2018), and addresses only the free ZIKV particles in semen. In addition to free particles, ZIKV in semen may be associated with cells and spermatozoa (da Silva, 2018). Thus, the cell-associated particles may be protected from the inhibitory effects of semen or seminal plasma. In addition, the inhibitory effect of semen, which prevents the virus from attaching to target cells may be neutralized by vaginal fluids in vivo to facilitate transmission and infection in females. However, careful studies are required to elucidate the viability of ZIKV in semen and seminal fluids. Although the presence of the viral RNA in semen does not precisely indicate the exact source, it may suggest that the viral RNA persists in the testis. It is also still unclear which cells of the mouse testes harbor the RNA because the organs were homogenized to extract total RNA for PCR detection. However, the viral RNA persistence was not accompanied by the presence of infectious virus particles. The continued harboring of viral RNA, even in the absence of infectious particles is intriguing. The ZIKV RNA is (+)ssRNA, meaning that it is potentially infectious inside cells. Why this is not the case, or what prevents the viral RNA from being translated into virus proteins that would be packaged into infectious particles remains unknown. Is the viral genome silenced by epigenetic machinery? In addition, a careful look for viral protein expression from the persistent RNA has not been reported yet. Because the RNA is foreign, it may also continue to be sensed by host cell pathogen recognition receptors, such as RIG-I and a consequence of such signaling could be chronic inflammation. More detailed studies aimed at understanding this phenomenon are required to elucidate the pathology associated with persistent ZIKV RNA. The Sertoli cell barrier in the testes is comprised of tight junction proteins (TJPs) including ZO-1, occludins (Ocln), and claudins (Cldn) (Cheng & Mruk, 2012). ZIKV affects the integrity of tight junctions as was shown by a decrease in the expression of Ocln and Cldn family members (Sheng et al., 2017). ZO-1 appeared unaffected in this study and these data were generated using a mouse model (Sheng et al., 2017). A different study evaluated the expression of the tight junction proteins in Sertoli cells in culture and the authors reported no changes in TJPs in ZIKV-infected Sertoli cells compared to mock-infected cells (Siemann, Strange, Maharaj, Shi, & Verma, 2017). Compromised tight junctions may cause leakage of cellular and/or organ contents into new environments they are not normally present and this can lead to serious pathological problems. In summary, ZIKV infection of the testis causes gross anatomical changes and may lead to complex pathologies such as infertility and inflammation.

Persistent ZIKV infection of testicular cells As related to in earlier sections, several studies have reported the detection of ZIKV RNA in urine or semen for variable lengths of time after the initial infection in males. In one study involving 1327 semen samples from 184 males, 33% (60/ 184) had detectable ZIKV RNA in semen (Mead et al., 2018). ZIKV RNA was detected at 61% in men who submitted semen samples within 30 days of onset of illness and the rate dropped to 7% if the semen sample was submitted more than 90 days postillness onset (Mead et al., 2018). In one case, viral RNA was still detectable by 281 days. Only 4% (7/1327) of the men shed ZIKV RNA in the urine, indicating that the testis and surrounding anatomical sites were most likely the sources of the virus being shed. Lower recovery rates of viral RNA in urine have been reported and this is possible because ZIKV maybe unprotected in urine samples, compared to semen where the virus could be protected by being inside cells or the semen protein matrix. It was demonstrated that ZIKV can be isolated from semen and not urine 10 weeks after the initial onset of symptoms (Musso et al., 2015). Regardless, it is important to appreciate which cells of the testis harbor the infection in a persistent state. To determine which testicular cells harbor persistent infection, we and others infected testicular cell lines with ZIKV and examined cultures over time. We infected primary Sertoli cells, primary testicular fibroblasts (Hs1.Tes) and two seminoma cell lines representing germ cells. While both the lab-adapted ZIKV MR766 and a clinical isolate ZIKV Paraiba infected all the cells, ZIKV Paraiba established a persistent infection in all cells tested (Mlera & Bloom, 2019). ZIKV MR766 established a persistent infection only in Sertoli cells and the fibroblast Hs1.Tes cells. Persistent

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ZIKV infection was demonstrated for up to five passages prior to complete cell death, possibly due to the limited life span of primary cells. In a different study, Kumar and colleagues showed that ZIKV replicated to high titers in Sertoli cells, but Leydig cells were relatively resistant to infection (Kumar et al., 2018). In this study, both American and African strains persisted in Sertoli cells for up to 6 weeks, and the authors concluded that these cells were a major ZIKV reservoir in the human testes.

Testicular cell pathways affected by ZIKV infection In earlier sections, we discussed how ZIKV infects, persists in, and destroys cells of the testes. All these different biological aspects of ZIKV infection are possible by evasion of immune signaling pathways, blockage of cell death pathways, and hijacking of broad host-cell pathways that facilitate ZIKV replication. ZIKV causes major gene dysregulation as exemplified by >9000 genes being differentially expressed 48 h postinfection in Sertoli cells (Kumar et al., 2018). The number of genes perturbed varied according to the MOI used because a lower MOI of 1 resulted in the differential expression of 647 genes in the same cells (Strange, Green, Siemann, Gale Jr, & Verma, 2018). Apoptotic cell death pathways were highly activated in nontesticular cells, such as the human neuroblastoma cell line SK-N-SH cells and A549 cell, but ZIKV induces a limited change in these pathways in Sertoli cells (Kumar et al., 2018; Offerdahl, Dorward, Hansen, & Bloom, 2017). The lack of cytopathic effects in Sertoli cells infected by ZIKV from 3 to 9 dpi was also reported by others (Siemann et al., 2017; Strange et al., 2018). Kumar and colleagues attributed the lack of cytopathic effect in ZIKV-infected Sertoli cells to an inability to mount an effective antiviral response (Kumar et al., 2018). However, a different study immunoprofiling the response of Sertoli cells to ZIKV suggested that Sertoli cells do mount a robust antiviral response to ZIKV (Strange et al., 2018). These variant conclusions may be reconciled by the fact that Kumar et al. were comparing the response of Sertoli cells to that of A549 cells in which ZIKV induces a very strong innate immune response. Immunoprofiling in the paper by Strange et al. (2018), was done comparing responses at each time postinfection to mock-infected cells of the same cell type. In both cases, however, the type I interferon signaling pathway is demonstrated to be activated. The pathways affected in ZIKV-infected Sertoli cells depend on infection state. When Sertoli cells were persistently infected, their transcriptome profiles at 6 weeks closely resembled those of mock-infected cells (Kumar et al., 2018). Only 42 genes were dysregulated during viral persistence, compared to >9000 genes at 48 hpi (Kumar et al., 2018). Among the upregulated genes were IFN-stimulated genes associated with an antiviral response, such as OAS and IFN-inducible tetratricopeptides. Downregulated were cramped chromatin regulator homolog 1 (an RNA polymerase II-specific transcription factor) and a splicing factor SWAP homolog (SFSWAP) (Kumar et al., 2018). The specific changes in the virus or infected cells to allow the cells to tolerate the persistent virus infection remain to be precisely identified. The Sertoli cell studies mentioned earlier were performed in culture and it can be challenging to make conclusions from cell culture models when they lack the surrounding tissue/cells that make up a complete anatomical organ. Mouse models have also been very useful, but pathological changes and lethality can only be observed in animals with compromised immunity, such as AG129 mice, which lack IFNs α, β, and γ. This makes conclusions drawn from these models relatively limited. In vivo studies in humans are complicated by ethical considerations as well as the difficulty to acquire tissue or biopsies for laboratory studies. A research group in France was recently able to use ex vivo organotypic testes culture obtained from human donors. The donations were either obtained at autopsy or from prostate cancer patients who had not received any hormonal therapy and samples were processed within 2 h. Infection of the ex vivo organotypic cultures with ZIKV paralleled observations that the virus infects Sertoli cells, macrophages and to a lesser extent Leydig cells (Matusali et al., 2018). The authors also noted that the level of activation of the caspase-dependent apoptosis pathway in ZIKV-infected ex vivo cultures was like that in mock-infected cultures. This further supports observations in Sertoli cell cultures that ZIKV does not induce the apoptosis pathway. Matusali and colleagues next examined if ZIKV induced innate immune response pathways in the human testicular explants. They reported that ZIKV triggered a very broad antiviral response, but IFN-β was not upregulated and the virus also induced a minimal proinflammatory response although CXCL10 was highly upregulated (Matusali et al., 2018). These results were consistent with earlier reports that the IFN response in Sertoli cells was minimal compared to A549 cells (Kumar et al., 2018). The testes are considered an immune-privileged organ and we speculate that there may be an inherent inability of the cells to avoid detrimental activation of proinflammatory and apoptotic pathways unless this is deemed extremely critical. Thus, ZIKV maybe exploiting this testicular cell phenotype or “sensing” the cellular environment to replicate with minimal damage to host cells. This subsequently enables the virus to persist in cells of the testes.

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Policy and procedures: Diagnosis of testicular infection by ZIKV, sexual transmission, and prevention ZIKV infection of cells of the testes is a problem for sexual transmission of the virus and has the potential to cause in utero infections. ZIKV acquired in utero has been shown to cause microcephaly with devastating outcomes for neonates. ZIKV infection of the testes can be demonstrated by the detection of viral RNA by PCR in semen or urine. The Centers for Disease Control and Prevention has provided guidelines specifically for the prevention of sexual transmission in couples seeking to conceive naturally or by in vitro fertilization. These guidelines are available at https://www.cdc.gov/zika/prevention/ sexual-transmission-prevention.html. France is an example of a European country, which requires couples seeking in vitro fertilization or assisted conception to be tested for ZIKV infection. These guidelines are particularly important because sexual transmission may occur in asymptomatic individuals.

Mini-dictionary of terms l l

l l

l

Vector-borne transmission: Transmission of ZIKV to humans by mosquitoes. Sexual transmission: ZIKV transmission is usually by mosquito vectors, but sexual transmission represents human-tohuman transmission via sexual contact. Hematogenous/lymphogenous spread: Systemic spread of ZIKV through the circulatory and lymphatic systems Viral persistence: An infection state in which cells are unable to clear the virus. This may be characterized by productive virus replication or dormancy where the viral genome is maintained. Persistent shedding: Continued release of viral particles or RNA in genital fluids/secretions

Key facts of testicular cell types and infection by ZIKV l l l l l

Testicular cell types are varied and include Sertoli cells and Leydig cells ZIKV infection of testicular cells is mediated by hematogenous/lymphogenous spread from the systemic circulation ZIKV infection is mainly limited to Sertoli cells ZIKV infection of testicular cells does not lead to overt and robust antiviral gene signaling pathways The most notable consequences of ZIKV infection of cells of the testes include (i) viral persistence, (ii) testicular atrophy over time, and (iii) sexual transmission of virus

Summary points l

l l l

l

l

l

l

l l l

ZIKV is a mosquito-borne flavivirus, which causes devastating pathologies particularly in neonates who acquire the virus in utero. Apart from vectored transmission, it became apparent that the virus can also be transmitted sexually. Systemic ZIKV infection in males leads to infection of cells of the testis via the hematogenous/lymphogenous routes. Infection of testicular cells is mediated by the AXL expressed on the Sertoli and Leydig cells, but infection of spermatozoa requires the expression of Tyro3. In vitro studies have demonstrated that ZIKV infection of testicular cells, especially Sertoli cells, leads to a persistent infection. In vivo, ZIKV persistence has been indicated by the continued shedding of ZIKV RNA in semen, but the specific cells harboring the infection requires to be elucidated. Once infected, testicular cells are not activated to robustly express apoptosis-inducing gene products, but slow degeneration of testicular cells ensues. The relatively low induction of apoptosis in testicular cells infected with ZIKV may be part of the reason why a persistent infection is established and sustained. The resulting atrophy of the testes is associated with low-level hormone secretion, which may reduce fertility in males. Obviously persistent ZIKV infection of the testis facilitates sexual transmission. Additional studies are required to delineate the biology associated with persistent ZIKV infection of the testes.

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Acknowledgment This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases/National Institutes of Health.

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e, D., Aubry, F., Couderc, T., … Dejucq-Rainsford, N. (2018). Zika virus infects human testicular tissue and germ cells. The Journal of Clinical Investigation, 128(10), 4697–4710. https://doi.org/10.1172/JCI121735. Mead, P. S., Duggal, N. K., Hook, S. A., Delorey, M., Fischer, M., Olzenak McGuire, D., … Hinckley, A. F. (2018). Zika virus shedding in semen of symptomatic infected men. The New England Journal of Medicine, 378(15), 1377–1385. https://doi.org/10.1056/NEJMoa1711038. Mlera, L., & Bloom, M. E. (2019). Differential Zika virus infection of testicular cell lines. Viruses, 11(1), 42. https://doi.org/10.3390/v11010042. M€ uller, J. A., Harms, M., Kr€uger, F., Groß, R., Joas, S., Hayn, M., … M€unch, J. (2018). Semen inhibits Zika virus infection of cells and tissues from the anogenital region. Nature Communications, 9(1), 2207. https://doi.org/10.1038/s41467-018-04442-y.

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Chapter 28

Proteome alterations promoted by Zika virus infection Juliana Miranda Tataraa, Luc
elia Santib, and Walter Orlando Beys-da-Silvab,c a

Post-Graduation Program in Cellular and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, b Faculty of Pharmacy and

Post-Graduation Program in Cellular and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, c Experimental Research Center, Clinical Hospital of Porto Alegre, Porto Alegre, Brazil

Abbreviations AKT ALDOA APP CKB DCX FN HCV HEK293T/17 HFF1 hMSC hNPC IFN iPS ISG LC-MS/MS LLC-MK2 MAP2 MOI MS mTOR MudPIT NSC PARK7 PDIA3 PFU PI3K SK-N-BE2 TGFB1 U251 U937 UPLC VERO WHO ZIKV

protein kinase B aldolase A amyloid precursor protein creatine kinase B-type doublecortin fibronectin hepatitis C virus human kidney epithelial cell line human skin fibroblast cell line human mesenchymal stem cell human neuroprogenitor cell interferon induced pluripotent stem cells interferon stimulated gene liquid chromatography with tandem mass spectrometry kidney epithelial cell line from Rhesus monkey microtubule-associated protein 2 multiplicity of infection mass spectrometry mammalian target of rapamycin multidimensional protein identification technology neuronal stem cell Parkinson’s disease protein 7 protein disulfide-isomerase A3 plaque-forming unit phosphatidylinositol-3-kinase human neuroblastoma cell line transforming growth factor beta-1 human glioblastoma astrocytoma cell line human histiocytic lymphoma cell line ultraperformance liquid chromatography kidney epithelial cell line from African green monkey World Health Organization Zika virus

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00028-6 Copyright © 2021 Elsevier Inc. All rights reserved.

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Introduction The Zika virus (ZIKV) is a Flavivirus with an RNA strand, which encodes a polyprotein comprising three structural genes for capsid and envelope, and seven nonstructural genes, expressing NS1, NS2A, NS2B, NS3, NS4A, and NS4B E NS5 (Noor & Ahmed, 2018). This virus was firstly identified in 1947 in Uganda, Africa, and for decades have not presented clinical relevance until several outbreaks happened worldwide in the last few years. In fact, only after its association with a microcephaly outbreak in Brazil, the research community focused on ZIKV describing other neurological issues linked to the infection and other aspects toward this virus (Platt & Miner, 2017). The first ZIKV outbreak was reported in 2007 on Yap Island, Micronesia (Hendrixson & Newland, 2018) while the last and major one happened in Brazil in 2014–15. In 2016, the World Health Organization classified ZIKV as a public health emergency of international concern (WHO, 2016), due to the significant increase in infants with congenital microcephaly born from mothers who were infected during pregnancy. In addition, the onset of some other neurological diseases, such as Guillain-Barr
e syndrome, was further associated with ZIKV (Poland et al., 2018). Viruses have multiple mechanisms to enter host cells, resulting in a successful infection. As a consequence of the infection process, molecular alterations are promoted triggering pathological effects linked to the pathogenic process and/or host response, which are still poorly characterized (Beltran, Federspiel, Sheng, & Cristea, 2017). For instance, host cells have its machinery modulate by ZIKV infection that manipulates the cell system to support its replication (Xin et al., 2017). Transcriptomic analyses are important tools to investigate the molecular mechanisms related to virus infection such as virus replication, host cell defenses, and metabolic alterations, among other processes. However, poor correlation between RNA level and proteins, the real players in cellular machinery, has been reported and discussed (Edfors et al., 2016; Maier, Guell, & Serrano, 2009; Payne, 2015; Schwanh€ausser et al., 2011; Vogel & Marcotte, 2012). In this case, proteomic analyses allow a more significant profiling of molecular alterations promoted by host-pathogen interaction process during the infection (Lum & Cristea, 2016). Advances in mass spectrometry (MS) technologies and bioinformatics are supporting the proteomic characterization of biological phenomena, including a deeper understanding of pathogenic effect in microbial infections (Lum & Cristea, 2016; Willard & Ginsburg, 2012), such as ZIKV. The first proteomic study regarding the ZIKV was reported in 2017 (Garcez et al., 2017), and until this moment, only 11 papers have been published on this topic, showing a lack of proteomic studies compared to transcriptomics (Rosa et al., 2019), even proteins being more biologically significant in gene expression measurements. These studies presented different methodological approaches applying different ZIKV strains infecting several host cells. The resulting differential proteomes presented thousands of different proteins found differentially expressed and consequently hundreds of pathways altered after the infection. Overall, the results described in those papers improved the poorly characterized molecular scenario of ZIKV infection, including the identification of potential biomarkers for neurological disorders associated with ZIKV. In this article, the different methodological approaches applied in these proteomic surveys, as well as the main results found, are reviewed and discussed.

Strains and infection conditions Using phylogenetic analyses, two main lineages of ZIKV have been identified—the Asian and the African strain (Gong, Xu, & Han, 2017). The Asian is further divided among the Malaysian and Micronesian strains, while the African is divided among the Nigerian and MR766 strains. Recently, many ZIKV strains have been isolated from different outbreaks worldwide, being the majority related to Asian lineage although the standard ZIKV reference strain being the African MR766 (Shrivastava et al., 2018). Phylogenetic analyses showed that the Brazilian isolates and French Polynesian isolate—KJ776791 share 99% sequence homology and being derivate from the Asian strain (Calvet et al., 2016). Currently, the infection proteomes reported have used ZIKV strains of different origins: Brazilian (three studies), Asian (five studies, being one Thai and one Cambodian strain), French Polynesian (two studies), and African (one study) (Table 1). Only one proteomic survey evaluates the effect of an African strain (MR766) using an animal model (Garcez et al., 2018). In this work, proteome profile array for angiogenesis proteins were used to characterize the defects found in vasculature during development. Concerning Asian strains, ZIKV differential infection proteome was reported using VERO cell line, kidney epithelial cells from African green monkey (Glover et al., 2019), human fetal neural progenitor cells (hNPCs) ( Jiang et al., 2018) and glioblastoma astrocytoma cells (U-251) (Sher et al., 2019). A fourth work applied another Asian strain, reported specifically as a Thai strain, infecting Rhesus monkey kidney epithelial cell line (LLC-MK2) (Diteepeng et al., 2019). The main reason to apply nonneural cells such as VERO and LLC-MK2 in ZIKV studies is the fact that these cell lines are frequently used to virus replication due to the success of the virus to infect and replicate

Zika infection proteomics Chapter

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TABLE 1 Overview of the methodological approaches of proteomic studies performed with ZIKV infection.

ZIKV strain

Number of proteins differentially expressed

Days postinfection (DPI)

Host model

Proteomic approach

NSC (neurosphere)

Nano-UPLC-MS/ MS

453

hMSC

MudPIT

hNPC and neuronsb

MOIa

Reference

4

0.25– 0.0025

Garcez et al., 2017

1075

2

10

Beys-da-Silva et al., 2019

Reversed-phase nano-LC-MS/MS

897 and 1767

4

1

Rosa-Fernandes et al., 2019

Fetal hNPC

Reversed-phase nano-LC-MS/MS

458

1

3

Jiang et al., 2018

VERO cell line

SOMAscan

84

0.5, 1, 2

3

Glover, Gao, Zahedi-Amiri, & Coombs, 2019

U251 cell line

SOMAscan

170

0.5, 1, 2

3

Sher, Glover, & Coombs, 2019

Thai/ Thailand

LLC-MK2 cell line

Reversed-phase nano-LC-MS/MS

7

2

2

Diteepeng, Khongwichit, Paemanee, Roytrakul, & Smith, 2019

African

Mouse

Immunoassay (Proteome Profiler™ Mouse Angiogenesis Antibody Array kit)

47

3

PFUc

Garcez et al., 2018

French Polynesian

hNPC and SK-N-BE2 cell line

Reversed-phase LCMS/MS

386

2

3

Scaturro et al., 2018

HFF1 and U937 cell lines

SILAC; nano LCMS/MS

16

2

1

Wichit et al., 2019

HEK293T/17 cell line

2D-gel; LC-MS/MS

7

2

2

Tangsongcharoen, Roytrakul, & Smith, 2019

Brazilian

Asian

Cambodiad

Representative studies from different host models: NSC ¼ neuronal stem cell. hMSC ¼ human mesenchymal stem cell. hNPC ¼ human neuroprogenitor cell. VERO ¼ kidney epithelial cell line from African green monkey. LLC-MK2 ¼ kidney epithelial cell line from Rhesus monkey. U251 ¼ human glioblastoma astrocytoma cell line. SK-N-BE2 ¼ human neuroblastoma cell line. HFF1 ¼ human skin fibroblast cell line. U937 ¼ human histiocytic lymphoma cell line. HEK293T/17 ¼ human kidney epithelial cell line. DPI in bold: more significant protein expression. a MOI: multiplicity of infection. b Neurons derived from induced pluripotent stem cells. c Plaque-forming units (PFU). d Only NS2B-NS3 protease.

in those host models. Although these cells can be criticized due to its nonrelated status regarding ZIKV main clinical consequences, they also can be considered as good host models (ones able to be easily infected). In this way, these hosts still can be successfully used to evaluate the gene expression impact and cellular alterations to be further tested and surveyed in clinics and other models. Also, VERO still is the most commonly used cell line to test a number of antiviral drugs (Sher et al., 2019). Diteepeng et al. (2019) tested two different ZIKV strains, African and Thai, in different cell lines of human and monkey. However, the proteomic analysis was made only with the Thai strain in LLC-MK2, using a gel-based proteomics approach

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that identified only seven proteins differentially expressed in ZIKV-infected cells. This work did not apply high-throughput proteomics, and consequently did not allow a global view of the infection molecular effects, limiting a more consistent understanding of the dramatic change on host gene expression. Another work used a Cambodian strain of ZIKV (Tangsongcharoen et al., 2019), but the experimental design was built to identify new host cell protein targets of ZIKV NS2B-NS3 protein, based on a 2D gel-based approach. Accordingly, they found two proteins, protein disulfide-isomerase A3 (PDIA3) and aldolase A (ALDOA), whose expression in HEK293T/17 cells was modulated by ZIKV NS2B-NS3 protease. These protein targets are mostly implicated in the endoplasmic reticulum stress response and glycolysis, respectively, previously reported to be critical in pathogenesis, neurodegeneration and microbial infections (Lin, Walter, & Yen, 2008; Rosa et al., 2019; Tangsongcharoen et al., 2019). Glover et al. (2019), using VERO cells to analyze the host response after infection of Asian ZIKV strain, identified several activated biological processes potentially altered, after 2 days postinfection (DPI), such as the complement cascade and JAK/STAT pathways. In a second paper, the same group infected human astrocytic cells (U-251), and also after two DPI, found altered several cellular functions and signaling pathways (Sher et al., 2019). Among those molecular alterations are proteins related to synaptic control, protein translation, and cell differentiation. For both works, the authors used a recently described proteomic technique called multiplexed aptamer-based technique (SOMAScan) that is not mass spectrometry-based. Instead, this technique is an aptamer-based proteomics assay capable of measuring up to 1305 human proteins from different biological samples with high sensitivity and specificity (Candia et al., 2017; Gold et al., 2010). Also analyzing the host differential proteomics after ZIKV infection on primary hNPCs, Jiang et al. (2018) revealed a potential alteration on molecular processes such as cell proliferation, differentiation, and migration, as also found in U-251 cells (Sher et al., 2019). The research group further used fetal mouse brain as target organ to validate the proteomic results. ZIKV strains from French Polynesia, where the first outbreaks and associated neurological alterations were described, were also tested. Scaturro et al. (2018) used hNPCs and the neuronal cell line SK-N-BE2 to characterize molecular responses after infection at the proteome and phosphoproteome level. Further, they also apply affinity proteomics to identify cellular targets of ZIKV proteins. Accordingly, they identified hundreds of ZIKV-interacting proteins and host markers of neuronal development, retinal defects, and infertility. They observed profound modulation of fundamental signaling pathways, providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. In other work, Wichit et al. (2019) reported the differential expression of interferon-stimulated proteins and defense response proteins for either ZIKV and Chikungunya virus after infection of human skin fibroblast. As the skin is a major viral entry site for ZIKV, the evaluation of how these cells respond to viral infection is interesting.

Brazilian strains of ZIKV Since the most dramatic clinical outcomes, such as microcephaly, were first described in Brazil (Schuler-Faccini et al., 2016), the use of clinical strains from the Brazilian outbreak is obviously important in order to measure experimentally the molecular and cellular effects that might be implicated in those differential neurological impairments. Garcez et al. (2017) characterized the molecular alterations in human neurospheres derived from induced pluripotent stem cells (iPS) using a ZIKV strain isolated during the Brazilian outbreak. Using transcriptomics combined with shotgun proteomics, they identified over 500 genes and proteins differentially expressed after infection. Potential interference on cell cycle, RNA processing, splicing, and neuronal differentiation were identified as the main processes impacted by ZIKV and are linked to biological mechanisms implicated in brain malformations. Rosa-Fernandes et al. (2019) also used human iPS infected with both African and Brazilian ZIKV strains to derive neuroprogenitor cells (NPCs) and neurons. They evaluate cellular and molecular alterations on these differentiate cells. Through shotgun proteomics pathways associated with neurological diseases were found altered, as well as routes involved in cell death and embryonic development. Interestingly, these differential alterations were obtained from the comparison of cells infected with the Brazilian strain (isolated from the 2015 outbreak) vs an African strain of ZIKV. They observed that only the Brazilian strain induced alterations in NPCs, showing a potential adaptation that might explain the differential clinical outcomes happened during the outbreak in Brazil. It is also in accordance with the previous phylogenetic data, where strains isolated from recent outbreaks are phylogenetically related to Asian lineage (Shrivastava et al., 2018). Another neural cell tested as host to evaluate the impact on gene expression was the human mesenchymal stem cells (hMSC) that can differentiate to NPCs, neurons, and other nervous system-related cells. Using a clinical strain of ZIKV, 17SM, isolated in the southeast of Brazil in 2015, hundreds of proteins were identified differentially expressed (Beys-da-Silva et al., 2019). Several pathways were potentially found impacted after infection. Among these proteins identified, several molecular markers of neurological and brain diseases, including already described as ZIKV clinical phenotype (microcephaly and epilepsy), and others that might happen as potential later implications, such as Alzheimer’s disease, Autism spectrum disorder, and Parkinson’s disease were identified.

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Proteomic techniques Several high-throughput techniques were applied for the proteomics characterizations of ZIKV infection (Table 1). The techniques applying shotgun mass spectrometry-based proteomics are applied due to their enormous analytical advantages, allowing the analysis of complex samples from its digested peptide mixtures (either gel-based—proteins digested directly from gel electrophoresis) or gel-free (in solution) (Abdallah, Dumas-Gaudot, Renaut, & Sergeant, 2012; Beys-da-Silva et al., 2019; Garcez et al., 2017; Sher et al., 2019). MudPIT, multidimensional protein identification technology, developed by Yates team in 2001 (Washburn, Wolters, & Yates 3rd., 2001) and applied by Beys-da-Silva et al. (2019), is used for large-scale proteomes analysis, as well as other LC–MS/MS approaches that eliminates the 2D-gel separation. Besides label-free shotgun proteomics, Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based mass spectrometry was applied to identify proteins differentially expressed of fibroblasts cultured in medium supplemented with heavy or light isotopes (Wichit et al., 2019). A workflow of a proteomic pipeline, with alternatives between high-throughput and gelbased techniques, can be seen in Fig. 1. The alternative and recent multiplexed aptamer-based technique, SOMAScan, was also used for proteomic analyses in two works (Glover et al., 2019; Sher et al., 2019). The authors could detect and measure specifically 1322 proteins simultaneously in 92 different samples. The called SOMAmers, from the SOMAScan, are made from small DNAs strands, which binds to specific proteins. After a purification step, the SOMAmers are identified in the SOMAscan datasets through bioinformatics. This technique is very interesting for protein biomarkers discovery (Raychoudhuri et al., 2011). For the in vivo study, the molecular technique performed was an immunoassay using the Proteome Profiler Mouse Angiogenesis Antibody Array kit (R&D Systems). Thiss technique can identify 53 angiogenesis-related proteins and it was specific for the host (mouse) infected with ZIKV (Garcez et al., 2018). Other nonhigh-throughput and gel-based techniques were also used (Diteepeng et al., 2019; Tangsongcharoen et al., 2019), but as discussed above, limit a wide overview of the molecular alterations promoted after ZIKV exposure.

Main molecular alterations after ZIKV infection Evaluating all proteomics studies published until now, different pathways were identified heavily altered by ZIKV infection. Among those pathways are mainly: (i) signaling pathways; (ii) immune system; (iii) neurogenesis; and (iv) metabolic processes. The impact of these alterations is discussed as follows.

FIG. 1 Overview of the general proteomic pipeline used to evaluate the ZIKV infection.

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Signaling pathways are affected by ZIKV infection Cell signaling is fundamental for any cell and involves several molecules, resulting in a cell response to an initial stimulus. It involves several controlling factors that are responsible to regulate the effective cellular response, as observed during viral infection. ZIKV can modulate the cell signaling response, dysregulating some important pathways. For example, proteins from PI3K/AKT/mTOR signaling pathway, important in regulating main cellular functions, such as cell survival and proliferation, cellular trafficking, protein translation, and RNA processing (Wang, Yang, Fikrig, & Wang, 2017) were inhibited in hMSC (Beys-da-Silva et al., 2019) and human glioblastoma astrocytoma cells (Sher et al., 2019). This pathway has been described regulating the differentiation and proliferation of NPCs (Peltier, O’Neill, & Schaffer, 2007) and triggering inflammatory responses in neural cells, through NF-kB (Beys-da-Silva et al., 2019). One glycoprotein with multifunctions, fibronectin (FN), which can also activate the PI3K/AKT pathway via integrins (Matsuo, Sakurai, Ueno, Ohtani, & Saiki, 2006), was affected by ZIKV infection in several models (Glover et al., 2019; Jiang et al., 2018; Rosa-Fernandes et al., 2019; Sher et al., 2019). The AKT/mTOR signaling pathway has been reported to be inhibited in hMSC infected with Brazilian ZIKV strain (Beys-da-Silva et al., 2019) and neuroblastoma cells infected with a French Polynesian ZIKV strain (Scaturro et al., 2018). The dysregulation of mTOR pathway is related to several human diseases, such as depression and some types of cancer (Beevers, Li, Liu, & Huang, 2006). This pathway is also known to be suppressed by two of the nonstructural proteins of ZIKV, NS4A, and NS4B, leading the cells to autophagy (Liang et al., 2016). Therefore, the deregulation of PI3K/AKT/mTOR pathway may cause an impairment in cell proliferation, differentiation, and inflammatory response, which could explain changes observed in ZIKV infection, both in vitro and in vivo. Interestingly, some brain malformations (e.g., microcephaly) have been reported related to dysregulated PI3K/AKT/mTOR pathway (Melo et al., 2017). Besides, fibronectin is involved in astrocytic cell growth, migration, and actin polymerization, and its dysregulation might disrupt the astrocytic function, leading to microcephaly (Sher et al., 2019).

The immune cell response for viral infection The first host cell defense against viral infection is the innate immune response, which includes secretion of type I interferons (IFNs), other cytokines and specific immune cells (Ye, Zhu, Fu, Chen, & Cao, 2013). Once released, type I IFN leads to the expression of proteins that will prevent viral replication. Interferons can also regulate IFN-induced proteins, such as IFIT family, which plays a role in antiviral activity and has been reported to inhibit replication of several viruses in mammalian cells through multiple mechanisms (Raychoudhuri et al., 2011; Wichit et al., 2019). Interestingly, one of these proteins, IFIT1 was found upregulated in different cell models infected with Brazilian, French Polinesian, and Asian ZIKV strains (Beys-da-Silva et al., 2019; Jiang et al., 2018; Rosa-Fernandes et al., 2019; Wichit et al., 2019). These results suggest that cells are fighting against viral infection, where IFIT1 exerting antiviral activity, probably by engaging the viral 50 RNA, inhibiting replication (Pichlmair, 2011; Wichit et al., 2019). Contrarily IFIT1 protein, the JAK-STAT pathway, one of the three major pathways that can be activated by INFα/β, was found as the most downregulated pathway in VERO cells infected with Asian strain (Glover et al., 2019). This pathway plays an important role in immune response (Kiu & Nicholson, 2016), inducing the expression of interferon-stimulated genes (ISGs), promoting an antiviral state (Takaoka & Yanai, 2006). The downregulation of this pathway in VERO cells is in accordance with previous results, where ZIKV inhibit JAK-STAT signaling by promoting the degradation of Jak1 and reduction of apoptosis virus-induced through NS2B3 (Wu et al., 2017). Therefore, the virus could modulate the host immune response via its nonstructural proteins, deregulating signaling cascades. Other viruses, such as hepatitis C virus (HCV), can also modulate the JAK-STAT pathway and induce viral replication (Cheng, da Silva, Huang, Jung, & Gao, 2018). However, the effect may vary among different cell types and viral strains. Altogether, the results observed for IFIT1 and JAK-STAT pathway suggest these ZIKV-mediated proteomic alterations are different for each infected cell type. These similarities and differences are fundamental to evaluate strategies adopted by viruses to modulate the immune system when studying antiviral drugs (Sher et al., 2019).

Neurological-associated molecular alterations caused by ZIKV Impairments in molecular pathways responsible for neuronal cell differentiation can lead to brain malformations and abnormal conditions like microcephaly. Some works used neuronal cells to evaluate the impact of ZIKV infection. In neurospheres, Garcez et al. (2017) shown that ZIKV changes the molecular pattern of neural stem cells, activating responses to viral replication, cell death, triggering the immune system, and downregulation of neuronal development and

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differentiation. In the same way, hNPCs, and neurons infected with Brazilian ZIKV strain showed increased cell death, activation of viral transcription machinery, impairment in neuronal migration and differentiation, disruption of synaptic formation and stability, leading to disabilities observed in congenital Zika syndrome (CZS) (Rosa-Fernandes et al., 2019). Astrocytes are very abundant cells in the CNS, controlling axonal guidance, synaptic signaling, neuronal transmission, and maintenance of neurons and synaptic plasticity (Schiweck, Eickholt, & Murk, 2018). Once glial cells seem to be the preferred target for ZIKV (Retallack et al., 2016), Sher et al. (2019) showed that an Asian strain of ZIKV impacts the proteome of glioblastoma astrocytoma cells. The majority of the differentially expressed proteins belong to specific astrocytic functions, including inhibition of differentiation, cell growth deregulation, apoptosis, and cell reactivity. These alterations observed during ZIKV infection could be linked with disruption in neuronal support and synaptic functioning, two crucial functions of normally differentiated astrocytes (Li et al., 2012). The microtubule-associated protein doublecortin (DCX) has been suggested as a biomarker for neurogenesis, due to its well-known expression in developing neurons (Ayanlaja et al., 2017). DCX is involved in neuronal migration through the cortex. In hNPC infected with Asian ZIKV strain, two nonstructural ZIKV proteins, NS4A and NS5, were suggested to be involved in DCX downregulation ( Jiang et al., 2018). DCX was also found as downregulated in hMSC infected with Brazilian strain (Beys-da-Silva et al., 2019). However, this protein was upregulated in neuroblastoma cells (Scaturro et al., 2018) and in neurons differentiated from NPC and neurospheres (Rosa-Fernandes et al., 2019). Another protein involved in neuronal migration and integrity, transforming growth factor beta-1 (TGFB1), was identified as downregulated in neurons (Rosa-Fernandes et al., 2019). The reduced TGFB1 expression may lead to neurodegenerative process with increased number of apoptotic neurons and reduced neocortical presynaptic integrity (Brionne, Tesseur, Masliah, & Wyss-Coray, 2003). Other proteins involved in neuronal maturation and differentiation were also affected by ZIKV in different models. These proteins include amyloid precursor protein (APP), microtubule-associated protein 2 (MAP2) and Parkinson’s disease protein 7 (PARK7). Therefore, ZIKV infection deregulates neuronal migration, triggering premature differentiation associated with neurogenesis impairment, contributing to a neurodegenerative phenotype. In the same way, several molecular markers of different neurodegenerative diseases were found differentially expressed after ZIKV infection in hMSC (Beys-da-Silva et al., 2019). Altogether, these proteomic data show the neurodegenerative potential associated with ZIKV infection.

Alteration of metabolic processes as a consequence of ZIKV infection A global proteomic view of hMSC ZIKV-infected identified many metabolic pathways being most impacted, such as lipid, nucleotide, and energy metabolism (Beys-da-Silva et al., 2019). These pathways could modulate autophagy by generating increased energy production through fatty acid metabolism and B-oxidation (Heaton & Randall, 2011). The downregulation of lipid metabolism utilizing hypolipidemic agents has been hypothesized as a factor able to reduce ZIKV infection with a good pharmacological potential (Martı´n-Acebes, Jim
enez de Oya, & Saiz, 2019). In addition, proteins involved in energy production such as gluconeogenesis and glycolysis were upregulated in monkey kidney cells (Diteepeng et al., 2019; Glover et al., 2019), and downregulated in astrocytic cells (Sher et al., 2019), indicating that ZIKV can potentially modulate autophagy as previously described (Heaton & Randall, 2011; Liang et al., 2016). Interestingly, human embryonic kidney cells expressing the NS2B-NS3 protease of a Thai ZIKV strain showed differential expression of aldolase A (ALDOA) and protein disulfide-isomerase A3 (PDIA3) (Tangsongcharoen et al., 2019). Aldolases contribute to energy production, acting as a key enzyme in glycolysis process, and as a regulator for tumor cells growth (Huang et al., 2018) and was found downregulated in embryonic kidney cells (Tangsongcharoen et al., 2019) and neurons differentiated from NPCs (Rosa-Fernandes et al., 2019). PDI proteins are involved in the regulation of protein folding, and PDIA3 was reported upregulated in human embryonic kidney cells (Tangsongcharoen et al., 2019) and neurospheres (Rosa-Fernandes et al., 2019). These alterations in the energy metabolism of infected cells could increase the available energy for virus replication and virion production while increasing infected cell survival as previously reported (Sanchez & Lagunoff, 2015). Another enzyme that plays an important role in cellular homeostasis is creatine kinase B-type (CKB), which was differentially regulated in several cell types infected with ZIKV (Beys-da-Silva et al., 2019; Garcez et al., 2017; Glover et al., 2019; Sher et al., 2019). This protein is involved in astrocyte growth and migration and is crucial to maintain ion homeostasis in the brain and glutamate uptake (Steen, Wilczak, Hoogduin, Koch, & De Keyser, 2010). Therefore, the ZIKVinduced differential expression of CKB could disrupt the astrocyte function, as well as energy induction, culminating in deleterious effects like microcephaly and other disorders (Warren, Aicher, Fessel, & Konradi, 2017).

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Concluding remarks In this review, it was possible to observe that each cell type presents specific responses that may be influenced by the ZIKV strain used as the infectious agent. Anyway, some pathways affected by the infection also show a pattern regarding host response, highlighting alterations in cell cycle progression and apoptosis. In addition, signaling pathways were significantly affected by ZIKV, and provided more explanation of how this virus can cause impairment in cell proliferation and differentiation. These altered signaling pathways as well as the impact on immune-response related proteins and metabolic processes alterations contribute to explain the neurological impairments associated with ZIKV. The molecular processes and markers linked to ZIKV clinical phenotypes and neurodegenerative diseases found in the proteomic data published up to date reinforce the potential of proteomics in explaining how the infection can lead to these clinical outcomes, known and potential ones. However, since the evident lack of proteomic studies on ZIKV issue, new approaches in order to characterize the differential expression of proteins promoted by ZIKV in different host models (cellular and animal) are urgently needed. The understanding of how different cells and tissues respond to ZIKV infection may help to develop therapies to avoid the most dramatic clinical consequences of ZIKV infection.

Policy and procedures Measuring of the head circumference The measurement of the baby’s head is made 24 h after birth and every month during the first year. Tables and charts of head circumference for different ages during childhood can be found here: https://www.who.int/childgrowth/standards/hc_for_ age/en/.

Sample preparation for mass spectrometry Proteins can be extracted directly from cell cultures or tissue lysates, using different protocols. Samples must be prepared with extreme care avoiding contamination. If detergents are used for sample preparation and/or for protein extraction, make sure to remove it. Always wear gloves and highest purity buffers and other reagents. Protocols basely follow a general pipeline (Fig. 1). Usually, the initial buffer contains urea in high concentration, which increases the protein denaturation and solubilization. Reduce disulfide bonds with a reduction agent, i.e., DTT or TCEP and alkylate with iodoacetamide. Proteins can be digested with modified trypsin or other proteases, such as Lys-C. Different protocols can be found in this site: http://proteomicswiki.com/wiki/index.php/Lab_Protocols.

Mini-dictionary of terms Proteome. Set of all proteins produced in one cell, tissue or organ, in a specific moment and condition. Shotgun proteomics. Bottom-up proteomics applied on a mixture of proteins. MudPIT. Multidimensional protein identification technology, a LC-MS/MS technique for measuring global changes in protein expression. Microcephaly. Malformation where a baby’s head circumference is smaller than normal. Neurosphere. Culture system based on neural stem cells.

Key facts Key facts of Zika virus l l l l

Zika virus was firstly identified in 1947, in Zika forest in Uganda, Africa, with no clinical relevance. Since 2007, several outbreaks were caused by ZIKV. The major outbreak happened in 2015 in Brazil and was associated with a congenital microcephaly outbreak. Since the major outbreak, neurological impairments and other associations were related to ZIKV.

Key facts of microcephaly l

Microcephaly is a rare condition in which a baby’s head size is smaller than normal 24 h after birth (congenital) or the head stops growing after few months (acquired).

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Microcephaly was defined by the Brazilian Ministry of Health as a head circumference less than 32 cm for full-term babies. The measurement of head size is important to monitor a child’s brain development. The number of microcephalic babies arose from 12 in 2014 to 988 in 2015 in only one Brazilian state (Pernambuco), due to ZIKV infection.

Key facts of neurodegenerative diseases and virus l

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Recently, other viruses, such as cytomegalovirus, varicella-zoster, and West Nile virus have been associated with different neuropathological conditions. HIV was associated with dementia and herpesviruses with Alzheimer’s disease and multiple sclerosis.

Key facts of proteomics l

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The term was used for the first time in 1994, by Marc Wilkins, and describe the study of a set of proteins produced by cells or tissue in a specific moment and condition. It can be used to identify pathogens, search biomarkers for different diseases or conditions, and evaluate the progress of a specific biological alteration. Contrarily genome, the proteome is highly variable.

Key facts of infection protocols l l l

Different virus strains can lead to different outcomes and host alterations. Different hosts (cells or organisms) can be differentially susceptible or resistant to infection. Controls of ZIKV infection experiments can be prepared using noninfected host, inoculum with attenuate or inactivate virus or other viruses that do not produce the same clinical outcomes.

Summary points l l l l l

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Proteomic analyses of ZIKV-infected cells lead to differential expression of several proteins related to signaling. The PI3K-AKT-mTOR signaling pathway is the most affected pathway during ZIKV infection. ZIKV can modulate the host immune response, such as JAK-STAT pathway, via its nonstructural proteins. Infected cells fight against ZIKV infection, where IFIT1 seems to be a key protein with antiviral activity. ZIKV cause increased neuronal cell death and downregulation of development and differentiation, leading to disabilities like microcephaly. The upregulation of energy metabolism identified in ZIK-infected cells is important for viral replication. Proteomic analyses have shown that ZIKV might be potentially associated with neurological disorders beyond already described clinical phenotypes.

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Sanchez, E. L., & Lagunoff, M. (2015). Viral activation of cellular metabolism. Virology, 479–480, 609–618. Scaturro, P., Stukalov, A., Haas, D. A., Cortese, M., Draganova, K., Płaszczyca, A., et al. (2018). An orthogonal proteomic survey uncovers novel Zika virus host factors. Nature, 561(7722), 253–257. Schiweck, J., Eickholt, B. J., & Murk, K. (2018). Important shapeshifter: Mechanisms allowing astrocytes to respond to the changing nervous system during development, injury and disease. Frontiers in Cellular Neuroscience, 12, 261. Schuler-Faccini, L., Ribeiro, E. M., Feitosa, I. M., Horovitz, D. D., Cavalcanti, D. P., Pessoa, A., et al. (2016). Possible association between Zika virus infection and microcephaly—Brazil, 2015. MMWR—Morbidity and Mortality Weekly Report, 5(3), 59–62. Schwanh€ausser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., … Selbach, M. (2011). Global quantification of mammalian gene expression control. Nature, 473(7347), 337–342. Sher, A., Glover, K., & Coombs, K. (2019). Zika virus infection disrupts astrocytic proteins involved in synapse control and axon guidance. Frontiers in Microbiology, 10, 596. Shrivastava, S., Puri, V., Dilley, K. A., Ngouajio, E., Shifflett, J., Oldfield, L. M., et al. (2018). Whole genome sequencing, variant analysis, phylogenetics, and deep sequencing of Zika virus strains. Scientific Reports, 8, 15843. Steen, C., Wilczak, N., Hoogduin, J. M., Koch, M., & De Keyser, J. (2010). Reduced creatine kinase B activity in multiple sclerosis normal appearing white matter. PLoS One, 5(5), e10811. Takaoka, A., & Yanai, H. (2006). Interferon signalling network in innate defence. Cellular Microbiology, 8, 907–922. Tangsongcharoen, C., Roytrakul, S., & Smith, D. R. (2019). Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression. Biochimica et Biophysica Acta, Proteins and Proteomics, 1867(2), 89–97. Vogel, C., & Marcotte, E. M. (2012). Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Reviews Genetics, 13(4), 227–232. Wang, L. L., Yang, L., Fikrig, E., & Wang, P. H. (2017). An essential role of PI3K in the control of West Nile virus infection. Scientific Reports, 7, 3724. Warren, E. B., Aicher, A. E., Fessel, J. P., & Konradi, C. (2017). Mitochondrial DNA depletion by ethidium bromide decreases neuronal mitochondrial creatine kinase: Implications for striatal energy metabolism. PLoS One, 12(12), e0190456. https://doi.org/10.1371/journal.pone.0190456. Washburn, M. P., Wolters, D., & Yates, J. R., 3rd. (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnology, 19(3), 242–247. WHO-World Health Organization. (2016). Zika situation report. March 4, 2016 http://www.who.int/emergencies/zika-virus/situation-report/4-march2016/en/. Wichit, S., Hamel, R., Zanzoni, A., Diop, F., Cribier, A., Talignani, L., et al. (2019). SAMHD1 enhances Chikungunya and Zika virus replication in human skin fibroblasts. International Journal of Molecular Sciences, 20(7), E1695. Willard, H. F., & Ginsburg, G. S. (2012). Genomic and personalized medicine (2nd ed.). MA: Elsevier/Academic Press. Wu, Y., Liu, Q., Zhou, J., Xie, W., Chen, C., Wang, Z., … Cui, J. (2017). Zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro. Cell Discovery, 3, 17006. Xin, Q. L., Deng, C. L., Chen, X., Wang, J., Wang, S. B., Wang, W., et al. (2017). Quantitative proteomic analysis of mosquito C6/36 cells reveals host proteins involved in Zika virus infection. Journal of Virology, 91(12), e00554-17.43. Ye, J., Zhu, B., Fu, Z. F., Chen, H. C., & Cao, S. B. (2013). Immune evasion strategies of flaviviruses. Vaccine, 31, 461.

Chapter 29

SOMAscan proteomics of Zika-infected cells Kevin M. Coombs Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada

Abbreviations CPE hpi IPA MOI PBS PBS-EDTA SOMA ZIKV

cytopathic effect hours postinfection Ingenuity Pathway Analyses multiplicity of infection phosphate buffered saline PBS with EDTA (ethylene diamine tetra acetic acid) slow off-rate modified aptamer Zika virus

Introduction All viruses are obligate intracellular parasites. Thus, they require host cell processes and machinery to carry out their replication and induce pathogenesis. With the exception of the “giant” fungal viruses, most viruses, especially those known to infect humans, are composed of a few to as many as a few hundred genes and proteins. By contrast, eukaryotic cells consist of tens of thousands of genes that encode, via alternative splicing and variable posttranslational modifications, many orders of magnitude more protein permutations. Thus, while cellular gene regulation has been successfully probed during the past few decades by “gene array” transcriptomics, and more recently by RNA-Seq, delineating the host protein repertoire (proteome) has been more challenging. Transcriptomics has provided abundant important information (e.g., Baas et al., 2006; Geiss et al., 2002; Kobasa et al., 2007) about gene expression changes, but, since most effector molecules are proteins, a newer frontier in biomedical research has been to attempt to elucidate complex protein profiles and how they may be modified by stressful perturbations, including those brought about by viral infection.

Proteomics Researchers have had tools for many years to study individual proteins. However, these tools, including immunological ones such as immunoblotting, only allow the analysis of one or a few proteins at a time. Proteomics is the simultaneous identification of large numbers of proteins. Quantitative proteomics allows the identification and measurement, whether absolute or relative, of much larger numbers of proteins, currently approaching tens of thousands of proteins, which allows rapid screening of a significant proportion of the cellular proteome. Thus, quantitative proteomics is an ideal tool to determine rapidly which proteins in a cell may be affected by virus infection compared to noninfected cells. One of the most common quantitative proteomic methods employs mass spectrometry (reviewed in Coombs, 2011; Yates, Ruse, & Nakorchevsky, 2009). Comparative mass spectrometry-based proteomics has been used by us (e.g., Berard, Coombs, & Severini, 2015; Coombs et al., 2010; Kroeker, Ezzati, Coombs, & Halayko, 2013) and many other researchers (e.g., Dove et al., 2012; Jiang et al., 2018; Vester, Rapp, Gade, Genzel, & Reichl, 2009) to determine which cellular proteins are downregulated by virus infection, which proteins are upregulated, and which are unaffected. These nonbiased strategies rapidly measure 1000s of cellular proteins and allow determination of dysregulated cellular pathways, many of which had been identified by earlier individual targeted approaches. However, mass spectrometry-based nonbiased global screens suffer a few limitations. They usually identify only the more abundant proteins, and many critical cellular regulators are low abundant proteins. In addition, the stochastic nature of identification leads to less than 100% concurrence between Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00029-8 Copyright © 2021 Elsevier Inc. All rights reserved.

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any two mass spectrometry runs. Thus, despite performing biologic or technical replicates, some proteins may only be identified a single time, preventing them from being statistically analyzed. In addition, only a single set of samples can be analyzed at a time by mass spectrometry. Depending on labeling strategies, as many as 16 samples may be mixed together, but larger numbers of samples require more runs. Furthermore, depending on the desired depth of analysis and numbers of separation steps used prior to injection into the mass spectrometer, it may take a few days to complete a single run; thus, comparisons of multiple samples may take many days to weeks, or even longer.

SOMAscans as an alternate proteomic strategy In contrast to nonbiased screening methods, such as mass spectrometry, an alternate approach would be to use targeted multiplex approaches. Immunoblotting is one such targeted approach, but as indicated earlier, is limited to one, or a few closely related cross-reacting proteins. Luminex provides a means to identify, and measure as many as 100 proteins, and the same 100 proteins can be accurately measured time after time in as many samples as desired. While Luminex strategies offer two orders of magnitude more identification than individual protein-targeted immunoblotting, it still represents a tiny fraction of the cellular proteome. Several companies offer antibody arrays that target multiple proteins. For example, RayBiotech offers antibody arrays that target hundreds of proteins. SomaLogics, Inc., (Denver, Colorado, USA) selected and characterized a panel of aptamers. Aptamers are short nucleotide sequences with the capacity to bind to specific proteins. The company chemically modified these aptamers to produce “slow off-rate modified aptamers” (SOMAmers), each of which was selected to bind stably with high specificity to individual human proteins although many also have the capacity to bind to cognate proteins from other species including monkey, feline, canine, and murine (SOMAscan, 2017). Because the aptamers are nucleotides, they, in turn, may be measured on standard “DNA chips,” such as those marketed by Affymetrix. In a typical “SOMAscan,” protein samples of

FIG. 1 Generalized SOMAscan technique. The basic components (depicted in the dashed region); left: individual 96-well wells, each of which contains a sample of interest; right: general SOMAmer (Slow off-rate Modified Aptamer) structure with 1 of 1305 specific aptamers indicated by pink rhomboid; central cleavable linker region; and rightmost yellow biotin moiety. Reaction steps are (A) mixing each sample with thousands of copies of each SOMAmer; (B) binding of SOMAmer/protein complexes to Streptavidin (SA) beads; (C) attaching more biotin to SA-captured proteins; (D) SOMAmer linker cleavage with wash step to release SOMAmer-bound proteins; (E) capture of biotinylated proteins on fresh SA beads; and (F) SOMAmer release. In this cartoon, green protein 1 is recognized by one of the SOMAmers whereas gray protein 2 is not.

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interest are mixed with a panel of SOMAmers in a 96-well plate format (Fig. 1). This allows the simultaneous analysis of up to 88 samples (8 wells are also used as internal controls). The first generation SOMAscans had 1128 SOMAmers and the next generation SOMAscan could identify and measure 1307 individual proteins, along with about a dozen proprietary nonprotein control markers that are added to each well to ensure postanalysis protein sample normalization. The latest generation SOMAscan can now measure > 4500 proteins. A typical SOMAscan involves mixing the pool of SOMAmers with three different dilutions of each protein sample. This is reported to allow accurate measurement across eight orders of magnitude (SOMAscan, 2017). The mixture is incubated to allow SOMAmers to bind to cognate proteins (Fig. 1A). The SOMAmers, which contain a biotin moiety, are then captured on Streptavidin beads and nonbound proteins removed by washing (Fig. 1B). After washing to remove proteins that are not recognized by the panel of SOMAmers, proteins that are now bound to the Streptavidin beads are biotinylated (Fig. 1C) to provide a selection marker for later steps. The newly biotinylated SOMAmer/protein complexes are then released from the first set of Streptavidin beads by photolysis of the cleavable linker (Fig. 1D) and recaptured on fresh Streptavidin beads (Fig. 1E). After additional wash steps to remove residual nonspecific nonbiotinylated proteins, the SOMAmers are released from the SOMAmer/protein complexes (Fig. 1F). The relative quantities of each SOMAmer remaining after all these steps is in direct proportion to the amount of each protein in the original sample, and the quantities of each SOMAmer are determined by hybridization/amplification on a standard Affymetrix DNA chip, with these quantities reported as relative fluorescence units (RFU).

SOMAscan-determined protein dysregulations in cancer and neurological diseases Many of the SOMAmers were selected because of their capacity to selectively bind to proteins of great interest. These proteins were identified as important biomarkers in a variety of diseases. Thus, SOMAscans have been used to identify and measure cancer biomarkers (Mehan et al., 2012; Qiao, Pan, Parlayan, Ojima, & Kondo, 2017; Xiong et al., 2019). They also have been used to identify biomarkers of Alzheimer’s disease (Sattlecker et al., 2014; Westwood et al., 2017), of Duchenne muscular dystrophy (Hathout et al., 2015, 2019), of multiple sclerosis (Masvekar et al., 2019), and biomarkers associated with postsurgery inflammatory responses (Fong et al., 2019). Marion and colleagues used SOMAscan to measure proteomic alterations in the nasal secretions of patients suffering from influenza A virus infection (Marion et al., 2016).

SOMAscan-determined ZIKV-induced protein dysregulation The capacity to simultaneously measure 1307 human proteins in as many as 88 different samples allowed us to design experiments to assess how ZIKV affects cellular proteins in multiple cell types and across multiple time points postinfection in an unprecedented manner. Using this platform, we assessed ZIKV-induced host protein dysregulation in African green monkey Vero (Glover, Gao, & Coombs, 2019), human U-251 astrocytoma (Sher, Glover, & Coombs, 2019), and human Sertoli (Rashid et al., 2020) cells. Vero cells are highly susceptible to ZIKV infection and are the preferred cell type for measuring ZIKV infectivity (Anfasa et al., 2017; Chan et al., 2016). U-251 are a transformed cell line that models astrocytes, a major brain cell population that is now considered an essential first site of infection in the developing brain (Potokar, Jorgacevski, & Zorec, 2019; van den Pol, Mao, Yang, Ormaghi, & Davis, 2017). ZIKV RNA has been detected in the semen of previously infected but asymptomatic patients for months after initial infection (Atkinson et al., 2017; Matheron et al., 2016) and the virus can persist in Sertoli cells without causing significant pathology (Kumar et al., 2018; Rashid et al., 2020), suggesting these cells may serve as a reservoir leading to long-term ZIKV sexual transmission (Borges et al., 2019). Our analyses of these various cell types showed that ZIKV infection induced greater host cell protein dysregulation in all tested cell types as infection progressed (Glover et al., 2019; Rashid et al., 2020; Sher et al., 2019). For example, no proteins were significantly upregulated, and only four proteins were downregulated >1.5-fold, in Vero cells prior to 48 h postinfection (hpi), but 24 cellular proteins were significantly upregulated >1.5-fold and 17 were significantly downregulated >1.5-fold at 48 hpi, the last time point examined (Fig. 2) (Glover et al., 2019). Similarly, in U-251 astrocytoma cells, two cell proteins were significantly upregulated >1.5-fold, and 17 were significantly downregulated >1.5-fold prior to 48 hpi, but 18 cellular proteins were significantly upregulated >1.5-fold and 151 were significantly downregulated >1.5-fold at 48 hpi, the last time point examined (Sher et al., 2019). Protein dysregulation was very different in Sertoli cells. Instead of assaying these cells at 12, 24, and 48 hpi, as was done for the Vero and U-251 cells, we measured host protein dysregulation at 24, 72, and 120 hpi (1, 3, and 5 days postinfection; dpi) to assess differences during the acute viral infection stage vs later persistent stages. For the Sertoli cells, 19 cell proteins were significantly upregulated >1.5-fold, and 51 were significantly

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FIG. 2 Heatmaps of cellular proteins dysregulated by ZIKV infection. Heatmap of significantly dysregulated proteins in each cell type at indicated times postinfection. Only the 347 proteins significantly dysregulated >1.50-fold in either direction by Zika virus (ZIKV) in any cell, and the Ingenuity Pathway Analysis (IPA)-determined overall functions of each, are shown. Red depicts upregulated, blue indicates downregulated, and black indicates not significantly dysregulated >1.50-fold. Note the increase in numbers of dysregulated proteins as time progresses, and that many Sertoli cell proteins switch from being downregulated at 3 dpi to upregulated at 5 dpi. Values determined from 2 or more biological replicates.

downregulated >1.5-fold prior to 3 dpi, but, conversely, 157 cellular proteins were significantly upregulated >1.5-fold and only 12 were significantly downregulated >1.5-fold at 5 dpi (Rashid et al., 2020).

ZIKV infection induces similar and dissimilar protein dysregulation in different cells Comparisons of the specific host proteins dysregulated at later infection time points indicated generally poor correlation between any two sets of cells (Fig. 3). For example, a small number of proteins were similarly up or downregulated in U251 and Vero cells at 48 hpi. Cytokines CCL5 and CXCL11, and transcription regulators HMGN1 and SSRP1 were significantly upregulated in both cell types. Peptidase Complement C4A and transporters IGFBP5, IGFBP7, and SNX4 were significantly downregulated in both cell types. Two proteins, kinases EPHA3 and PRKACA were significantly upregulated in Vero but significantly downregulated in U251 cells. There were no observed proteins significantly upregulated >1.5-fold in U251 cells but downregulated in Vero cells. Larger numbers of proteins were similarly and dissimilarly dysregulated in the two different human cells we tested. For example, comparison of proteins dysregulated in U251 astrocytoma cells at

FIG. 3 Pairwise comparisons of protein dysregulation. U251 astrocytoma and Vero cell proteins dysregulated at 48 hpi (hours postinfecetion) are compared to each other and to Sertoli cell proteins dysregulated at 3 and 5 dpi (days postinfection), with R2 values indicated in the upper right of each plot. A few significantly dysregulated proteins are indicated and described in the text.

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48 hpi to those dysregulated in Sertoli cells at 3 dpi showed several similarly upregulated and more that were similarly downregulated. For example, interferon-stimulated gene ISG15 was significantly upregulated in both U251 and in Sertoli at both 3 and 5 dpi, and several peptidases, including CFI, Cathepsins CTSA and CTSH (and other molecules) were significantly downregulated in both U251 at 48 hpi and in Sertoli at 3 dpi. One protein, kinase CSK, was significantly upregulated in Sertoli at 3 dpi but significantly downregulated in U251, and no proteins were significantly upregulated in U251 but downregulated in Sertoli at 3 dpi. Three Sertoli cell proteins, cytokine CXCL8, ISG15, and transcription regulator STAT1, were similarly significantly upregulated at 3 dpi compared to 5 dpi, and no Sertoli proteins were similarly significantly downregulated at 3 dpi compared to 5 dpi. However, 19 Sertoli cell proteins were dissimilarly significantly dysregulated, with 5, including peptidase CASP3 and transcription regulators ING1 and NACA being upregulated at 3 dpi but downregulated at 5 dpi, and 14, including peptidases CTSA and HTRA2, cytokine TIMP1 and enzymes CHI3L1 and IDUA being downregulated at 3 dpi but upregulated at 5 dpi (Fig. 3).

Different biological functions are activated and inhibited by ZIKV in a cell-dependent manner Advances in bioinformatics now allow analyses of multiple proteins and placement of them into canonical pathways, protein networks, and predictions of affected biological functions. Upstream regulators and predicted inhibitors may also be considered. Several platform tools are available for these analyses, including STRING, InnateDB, DAVID, and Ingenuity Pathway Analyses (IPA). The Vero cell analysis revealed that IGF-1 signaling, PI3K/Akt signaling, NF-κB signaling, and Tec kinase signaling were activated, whereas PTEN signaling, IL-6 signaling, and growth hormone signaling were predicted to be inhibited (Glover et al., 2019). Although this analysis was performed in monkey kidney cells, we noted that Tec kinase signaling was predicted to activate neuronal and motor neuron cell death, and apoptosis of neuroglia and oligodendrocytes, hallmarks of microencephalopathy, which led us to also examine astrocytes. SOMAscan analysis of U-251 cells, followed by IPA analysis, predicted that many astrocytic functions associated with synapse control and axon guidance, including axonal guidance signaling, FGF signaling, STAT3 signaling, AMPK signaling, and ERK/MAPK signaling were impaired by ZIKV infection (Sher et al., 2019). A large number of Sertoli cells proteins were affected by ZIKV infection (Rashid et al., 2020). These proteins play key roles in important Sertoli cell functions, including innate immunity, glycolysis, and spermatogenesis. MicroRNAs are critically important for male fertility, and bioinformatics analyses of dysregulated Sertoli cell proteins suggested ZIKV infection could downregulate some of these key miRNAs (Rashid et al., 2020). Collectively, concurrent analyses of the impact ZIKV has on multiple cell types at different times postinfection indicated a few common proteins are affected by the virus, but that many unique proteins are affected in a cell-specific, and time-dependent, manner. These proteins tie into numerous important cellular pathways and biological functions and help explain some of the phenotypic effects ZIKV has on these cells and organs.

Future perspectives The capacity to rapidly measure large numbers of proteins in large numbers of samples makes comparative multiomic analyses more feasible. This allows not only analyses of a single infectious agent in multiple cell types, as described above, but also allows comparative analyses of multiple infectious agents within multiple cell types, including cells that may be differentially treated with various pharmacologic agents. This will allow, for example, rapid therapeutic screening. Such a strategy that uses aptamers capable of binding malachite green has already been reported to screen for rifampin-resistant mycobacterium (Scharf, Molodtsov, Kontos, Murakami, & Garcia, 2017). SOMAscan was used to examine disease biomarkers in aplastic anemia before and after immunosuppressive therapies (Giudice et al., 2018). These quantitative proteomic platforms will become more powerful as the numbers of reagents are increased. SomaLogics, Inc. plans to more than triple the numbers of aptamers their system employs to detect more proteins than is currently feasible. Other companies also are developing more powerful detection methods. For example, RayBiotech has produced larger and larger antibody arrays. Alternative detection strategies also are being developed. For example, OLink has developed an antibody-based proximity extension assay system in which proteins are recognized by pairs of antibodies bound to oligonucleotides. The paired system increases stringency and provides a substrate for oligonucleotide amplification, which is reported to enhance quantitation. OLink currently has >1000 such paired detection/quantitation antibodies, with 548 that overlap with the SOMAscan v.1.3 system, divided into biologic categories, also with plans to increase the numbers of detectable proteins. This system has been used to examine ovarian cancer (Finkernagel et al., 2019).

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Policy and procedures: Measuring host protein alterations The SOMAscan procedure requires a minimum of 70 μL of protein extract at a concentration of 200 μg/mL (SOMAscan, 2013, 2017). Thus, it is convenient to use sets of 10 cm dishes of appropriate cells. For every infected sample, a timematched mock (sham-infected) sample must be included. This allows comparison and normalization. Thus, as an example, to determine the kinetics of host protein dysregulation at three time points (arbitrarily: early, middle, and late), use appropriate tissue culture aseptic technique (Kruse & Patterson, 1973) to set up appropriate cells in 6 10 cm dishes and allow cells to grow to
80% confluency. Once cells achieve sufficient confluency, three dishes should be infected with ZIKV (or other viruses of choice) at a multiplicity of infection (MOI) ratio 3 infectious viruses per cell to ensure, as determined by Poisson calculation (Fox & Glynn, 1988), that >95% of cells are initially infected so protein effects are synchronous. Treat the other three dishes with diluent only, to serve as mock-infected controls. Harvest sets of one infected and one mockinfected dish at each time point (e.g., 24, 48, and 72 hpi), by washing the cells with ice-cold PBS-EDTA and then soaking them in 5 mL ice-cold PBS-EDTA to loosen cells, then scraping cells off the plastic substrate with a sterile cell scraper into conical centrifuge tubes. Wash the cells 3  with ice-cold PBS to remove serum in the media and transfer cells after two washes into sterile clean microcentrifuge tubes. Carefully remove all liquid after third wash and resuspend cells in 200 μL of MPER mammalian cell culture reagent supplemented with 1  HALT protease inhibitor (Pierce). Incubate cell lysate for 100 , and then remove insoluble material at 14,000  g for 120 . Transfer supernatants to fresh microcentrifuge tubes and determine sample protein concentrations (e.g., micro-BCA assay). Adjust protein concentrations to 200 μg/mL using MPER/1  HALT. For statistical rigor and experimental reproducibility, perform three biological replicates of the above sets of mock-infected and infected samples. Submit the 18 samples (three replicates of three sets of mock and infected samples) to SomaLogics for analysis. The SOMAscan consists, as described earlier, of a series of reactions and washes which ultimately yield protein values expressed as RFU. For analysis, use Excel or other spreadsheet programs to convert these RFU to Log2 values. Subtract the relevant mock Log2 values from the time-matched and biological replicate-matched infected sample values to determine Log2 fold-change values. Continue using the spreadsheet program to perform t-tests and/or Z-score analyses (Coombs et al., 2010).

Mini-dictionary of terms Aptamer A short nucleotide sequence of
50 bases which is capable of folding upon itself and which, as a result of its folding, is capable of binding to a particular protein with high specificity. Cytopathic effect (CPE) Cytopathology, usually induced by virus infection, characterized by rounding up of cells, detachment from a substrate and eventual death. Multiplicity of infection (MOI) In virology, the ratio of infectious virus particles to cells. Because of statistical probabilities, described more fully by the “Poisson distribution,” every cell in a population will not be infected with equivalent amounts of added virus. An MOI of 1 results in about 65% of cells being infected, whereas an MOI of 3 is needed to result in >95% of cells being infected. Proteomics The study of multiple proteins within a complex sample. May consist of biologic fluids (i.e., plasma and urine), tissues, or in vitro samples. Sertoli cells A specialized male reproductive cell responsible for spermatogenesis. These cells support Zika virus replication but are resistant to virus-induced cytopathology, thus may serve as a long-term virus reservoir.

Key facts of Zika virus-induced proteomic responses l l l l l l

Zika virus infects many cells in vitro. Zika dysregulates numerous cellular proteins. Cellular protein dysregulation is dependent on cell type. Cellular protein dysregulation also is dependent on time postinfection. Proteins generally affected by Zika virus belong to numerous classes. Sertoli cells support Zika virus and dysregulate different cellular proteins; most measured proteins were downregulated during early acute infection, but most were upregulated during a subsequent chronic infection.

Summary points l

Quantitative proteomic tools (i.e., Mass spectrometry and SOMAscans) identify and measure 1000s of cellular proteins, and identify 100s that are significantly up- or downregulated after virus infection.

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Proteins generally affected by Zika virus belong to cell death, enzymes including kinases and peptidases, immune modulatory, and transcription and translation initiation factor classes. Different cellular proteins are dysregulated, depending on whether the cells are monkey kidney, human astrocytes, or human reproductive cells. Sertoli cells support Zika virus and dysregulate different cellular proteins depending on whether an early acute infection or a prolonged chronic infection.

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M., Dang, U. J., et al. (2019). Disease-specific and glucocorticoid-responsive serum biomarkers for Duchenne muscular dystrophy. Scientific Reports, 9, 12167. Jiang, X., Dong, X., Li, S. H., Zhou, Y. P., Rayner, S., Xia, H. M., et al. (2018). Proteomic analysis of Zika virus infected primary human fetal neural progenitors suggests a role for Doublecortin in the pathological consequences of infection in the cortex. Frontiers in Microbiology, 9, 1067. Kobasa, D., Jones, S. M., Shinya, K., Kash, J. C., Copps, J., Ebihara, H., et al. (2007). Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature, 445, 319–323. Kroeker, A. L., Ezzati, P., Coombs, K. M., & Halayko, A. J. (2013). Influenza A infection of primary human airway epithelial cells up-regulates proteins related to purine metabolism and ubiquitin-related signaling. Journal of Proteome Research, 12, 3139–3151. Kruse, P. F. J., & Patterson, M. K. J. (1973). In P. F. J. Kruse, & M. K. J. Patterson (Eds.), Tissue culture: Methods and applications. New York: Academic Press. Kumar, A., Jovel, J., Lopez-Orozco, J., Limonta, D., Airo, A. M., Hou, S. M., et al. (2018). Human Sertoli cells support high levels of Zika virus replication and persistence. Scientific Reports, 8, 5477. Marion, T., Elbahesh, H., Thomas, P. G., DeVincenzo, J. P., Webby, R., & Schughart, K. (2016). Respiratory mucosal proteome quantification in human influenza infections. PLoS One, 11, e0153674. Masvekar, R., Wu, T. X., Kosa, P., Barbour, C., Fossati, V., & Bielekova, B. (2019). Cerebrospinal fluid biomarkers link toxic astrogliosis and microglial activation to multiple sclerosis severity. Multiple Sclerosis and Related Disorders, 28, 34–43. Matheron, S., d’Ortenzio, E., Leparc-Goffart, I., Hubert, B., de Lamballerie, X., & Yazdanpanah, Y. (2016). Long-lasting persistence of Zika virus in semen. Clinical Infectious Diseases, 63, 1264. Mehan, M. R., Ayers, D., Thirstrup, D., Xiong, W., Ostroff, R. 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Potokar, M., Jorgacevski, J., & Zorec, R. (2019). Astrocytes in flavivirus infections. International Journal of Molecular Sciences, 20, E691. Qiao, Z. W., Pan, X. Q., Parlayan, C., Ojima, H., & Kondo, T. (2017). Proteomic study of hepatocellular carcinoma using a novel modified aptamer-based array (SOMAscan (TM)) platform. Biochimica et Biophysica Acta, Proteins and Proteomics, 1865, 434–443. Rashid, M. U., Zahedi-Amiri, A., Glover, K. K. M., Ang, G., Nickol, M. E., Kindrachuk, J., et al. (2020). Zika virus dysregulates human Sertoli cell proteins involved in spermatogenesis with little effect on blood-testes tight junctions. PLoS Neglected Tropical Diseases, 14(6), e0008335. Sattlecker, M., Kiddle, S. J., Newhouse, S., Proitsi, P., Nelson, S., Williams, S., et al. (2014). Alzheimer’s disease biomarker discovery using SOMAscan multiplexed protein technology. Alzheimer’s & Dementia, 10, 724–734. Scharf, N. T., Molodtsov, V., Kontos, A., Murakami, K. S., & Garcia, G. A. (2017). Novel chemical scaffolds for inhibition of rifamycin-resistant RNA polymerase discovered from high-throughput screening. Slas. Discov., 22, 287–297. Sher, A. A., Glover, K. K. M., & Coombs, K. M. (2019). Zika virus infection disrupts astrocytic proteins involved in synapse control and axon guidance. Frontiers in Microbiology, 10, 596. SOMAscan. (2013). Technical white paper, doc# SSM-002, DCN 13-038. 24 pp. SOMAscan. (2017). Proteomic assay technical white paper, SSM-002, Rev. 4. van den Pol, A. N., Mao, G., Yang, Y., Ormaghi, S., & Davis, J. N. (2017). Zika virus targeting in the developing brain. The Journal of Neuroscience, 37, 2161–2175. Vester, D., Rapp, E., Gade, D., Genzel, Y., & Reichl, U. (2009). Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines. Proteomics, 9, 3316–3327. Westwood, S., Liu, B., Baird, A. L., Anand, S., Nevado-Holgado, A. J., Newby, D., et al. (2017). The influence of insulin resistance on cerebrospinal fluid and plasma biomarkers of Alzheimer’s pathology. Alzheimer’s Research & Therapy, 9, 31. Xiong, H. J., Yan, J. H., Cai, S. D., He, Q. Y., Peng, D. M., Liu, Z. B., et al. (2019). Cancer protein biomarker discovery based on nucleic acid aptamers. International Journal of Biological Macromolecules, 132, 190–202. Yates, J. R., Ruse, C. I., & Nakorchevsky, A. (2009). Proteomics by mass spectrometry: Approaches, advances, and applications. Annual Review of Biomedical Engineering, 11, 49–79.

Chapter 30

Zika virus as an oncolytic therapy against brain tumors Carolini Kaida, Matt Sherwoodb, Thiago Mitsugia, and Mayana Zatza a

Human Genome and Stem-Cell Center (HUG-CELL), Biosciences Institute, University of S. Paulo, Sa˜o Paulo, SP, Brazil, b School of Biological Sciences,

Faculty of Environmental and Life Sciences, University of Southampton, Southampton, United Kingdom

Abbreviations AT/RT BBB CNS CSC CZS ECT GSC HPI ICD IFN MB NPC TME ZIKV

atypical teratoid rhabdoid tumor blood-brain barrier central nervous system cancer stem cells congenital Zika syndrome embryonal CNS tumor glioblastoma stem cells hours postinfection immunogenic cell death interferon medulloblastoma neural progenitor cells tumor microenvironment ZIKA virus

Introduction The ZIKA virus (ZIKV) emerged as an international concern in 2015, leading to intensive worldwide investigation aiming to understand the virus pathology and the cause of congenital Zika syndrome (CZS). The tropism of ZIKV toward the brains of the developing fetus and the mother infected during pregnancy only displayed adverse effects on the fetal brain, sparking the interest of researchers to consider ZIKV as a potential virotherapy. Could ZIKV have a bright side as a potential oncolytic virus against pediatric and adult brain tumors? The confirmation of this unconventional idea would turn ZIKV into a powerful weapon against brain tumors, which are responsible for a yearly average of over 200,000 deaths worldwide (GLOBOCAN 2019). However, there are still safety concerns and questions regarding the ZIKV mechanism of action, viral tropism, and its potential against specific brain tumors, which need to be addressed are discussed in this chapter.

Brain tumors Cancer is a complex and multifactorial group of diseases that depends on environmental and lifestyle factors, as well as genetic and epigenetic backgrounds. As opposed to other types of cancer, where environmental conditions are known to increase the risk of developing it significantly (e.g., 70% of lung cancer cases are linked to smoking), the causes of brain cancer remain unclear. Brain tumors are usually classified by histology, which cell type they arise from, and according to molecular features and aggressiveness (clinical grades). Low-grade tumors are considered nonmalignant, while high-grade tumors are malignant and cancerous. However, most diagnosed brain tumors are secondary tumors, that is, a metastatic site from a tumor that originated outside of the cranium and migrated to the brain. The UK Cancer Research Organization predicts Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00030-4 Copyright © 2021 Elsevier Inc. All rights reserved.

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that only 3% of brain cancers are preventable, a considerably low number if compared to most other types of cancer. Genetic and epigenetic factors influence the risk of developing cancer, but reported studies show that they contribute to less than 10% of the total cases of cancer (Lichtenstein et al., 2000). Previous studies have identified pathways and molecular features that are often altered in a large number of primary brain cancers. Unraveling the aberrant molecular features in such cases is crucial for the improved diagnosis, treatment, and management of brain cancer (Park et al., 2017). The probability of developing brain cancer increases with age; however, some tumors are more frequent in children and teenagers. The most common primary brain tumors are gliomas, either in adults or children. Gliomas arise from glial cells and can be classified as ependymomas, oligodendrogliomas, and astrocytomas (or glioblastomas when highly aggressive) (Louis et al., 2016). Glioblastomas are the prevailing malignant brain cancers in adults and are responsible for the greatest number of brain cancer-related deaths in humans. In children and teenagers, despite gliomas being more common, the embryonal CNS tumors (ECTs) represent the most frequent malignant primary brain tumors (Dobrovoljac, Hengartner, Boltshauser, & Grotzer, 2002). Some intracranial tumors arise from nonneuronal and nonglial cells, such as Meningiomas and Hemangioblastomas, which originate from the meninges and the brain’s blood vessels, respectively. Meningiomas are the most common intracranial tumors, but despite their occurrence directly affecting the brain, histologically they are not brain tumors because the meninges are external to the brain itself.

Brain tumor treatment challenges From the clinical and therapeutic perspectives, brain tumors present a multitude of challenges. Prognosis can vary due to numerous variables, including patient’s age, cancer stage at diagnosis, tumor localization, and aggressiveness. Patients usually experience symptoms once the tumor has a substantial volume, and these symptoms can be very broad and generic (e.g., headaches, nausea, and dizziness). Therefore, efforts have focused on developing target-specific therapies and strategies for early diagnosis, which will allow more effective treatments and an improved prognosis (Kaid, Assoni, et al., 2020). The available classical treatment strategy aims to remove or destroy all the tumor cells via a multimodal approach of surgical resection, chemotherapy, and radiotherapy. Unfortunately, these are particularly invasive and frequently ineffective procedures. Some tumor cells are resistant to classical therapies and can cause tumor recurrence following surgical resection (Yang et al., 2020). These resistant and highly aggressive cells, named cancer stem cells (CSC), commonly possess some level of stemness and show molecular similarities to progenitor cells. CSCs give rise to cells which are highly proliferative and due to their stemness can contribute to intratumoral heterogeneity, metastasis and poor patient prognosis (Meacham & Morrison, 2013), thus complicating the specific therapeutic targeting of only cancer cells. Another challenge to brain cancer treatment is the unique feature of the blood-brain barrier (BBB), which acts as a physiological barrier between the circulatory system and the brain. The BBB protects healthy neuronal cells from potentially harmful agents circulating in the blood. However, it also prevents a diversity of drugs and cell therapies from accessing the brain via the bloodstream (Steeg, Camphausen, & Smith, 2011). Primary brain tumors commonly arise beyond the BBB and are therefore protected by it. Nevertheless, the unorganized growth of brain tumors can lead to disruption of the BBB, exposing the tumor and the brain to agents in the bloodstream. The BBB and the unique physiology of CNS tissues generate a complex, and still not fully understood, tumoral microenvironment (TME) in brain cancers. The TME and neurophysiology are essential to consider when developing and optimizing therapies to target brain cancer cells with minimal neurotoxicity (Quail & Joyce, 2017). Despite our growing knowledge of neuro-oncology, brain tumors remain one of the deadliest cancers. Constant academic efforts and progress are made to tackle the obstacles faced by brain cancer research and translate this scientific progress into clinical therapies (Aldape et al., 2019).

ZIKA virotherapy The ZIKA virus (ZIKV) was considered an epidemic of international concern by the WHO in 2015 following its outbreak in Brazil and its association with microcephaly and other associated malformations in newborns, classified as CZS (Franc¸a et al., 2016). ZIKV was isolated in 1948 from a rhesus monkey in Africa and was first described capable of infecting humans in 1954. Since then, a few cases in Africa and Asia have been reported with mild symptoms. The epidemic ZIKV strain emerged from the Asian viral lineage through genetic mutations that enabled ZIKV to infect human neural progenitor cells (NPC). Several studies have shown an incidence of 4%–6% for fetal CZS when women become infected with ZIKV during pregnancy (Musso & Gubler, 2016). The very severe and sometimes fatal neurological complications observed in CZS revealed two important viral features: the direct neurotropism and the potential to infect and induce death of a specific

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FIG. 1 The two-pronged mechanisms of action of ZIKV oncolytic virotherapy. (A) Virus infects normal cells through specific cell membrane receptors, is unable to replicate due to the activated Interferon antiviral response, which leads to cell growth impairment and apoptosis. (B) Virus infects cancer cell and can replicate freely due to the absence of effective antiviral responses. The intense viral replication results in oncolysis (direct cell death), releasing additional virions into the microenvironment to infect neighboring tumor cells. (C) An antitumoral immune response (indirect cell death) is raised against the presence of the virus, the infected cells and the intracellular components released by the oncolysed cancer cells. (D) Complementarity of these two mechanisms for induction of tumor remission by an oncolytic virus.

neural stem-like cell type (McGrath et al., 2017). These two unique ZIKV characteristics drew scientists’ attention and raised the hypothesis that the virus may be able to act as an oncolytic agent against brain tumors. Viral-based cancer therapy is a recent approach that gained momentum in 2015 after the FDA approved the first oncolytic virotherapy for the treatment of advanced Melanoma; T-VEC, a genetically modified herpes simplex virus, type 1 (Chen et al., 2017). The benefits of employing virotherapy against a tumor depend on the effectiveness, rapid response, direct targeting, minimal side effects, and fewer clinical interventions. ZIKV may solve the main therapeutic difficulties that contribute to a poor prognosis for brain tumor patients: the BBB physiological isolation, the presence of aggressive stem-like tumor cells resistant to conventional therapy, and the immune-privileged status of the brain (Foreman, Friedman, Cassady, & Markert, 2017). Before translating this potential virotherapy to humans with CNS tumors, it is imperative to understand the two-pronged mechanism of action through which oncolytic viruses yield their therapeutic properties: the cytotoxic and immunogenic effects (Fig. 1). This concept highlights the unique advantages of oncolytic virotherapy since patients in whom the oncolytic effect is not prominent, the indirect antitumoral immune response can act and induce tumor remission (Fig. 1D).

ZIKV and brain tumors ZIKV against glioblastoma Glioblastomas are the most lethal brain cancers in adults. Most patients succumb to fatal tumor recurrence. Although glioblastomas rarely spread outside of the CNS they are highly aggressive and challenging to treat due to the presence of Glioblastoma stem-like cells (GSC), the BBB physiologic isolation and the brain’s immune-privileged status. ZIKV preferentially infects GSCs which reduces cell growth, induces differentiation, and cell death (Iannolo et al., 2019; Zhu et al., 2017). Initial studies are starting to elucidate the molecular mechanisms that ZIKV utilize to infect and kill GSCs, which is fundamental for establishing oncolytic virotherapy. A recently proposed mechanism to resolve the unique sensitivity of GSCs to ZIKV infection implicates the GSC markers, integrin αvβ5, and focal adhesion kinase (FAK) signaling (Wang et al., 2020; Zhu et al., 2020). According to the authors, both mechanisms could be targeted for the treatment of patients during a pathological infection with ZIKV. Importantly, their use of GSCs to model neural stem cells directly implicates FAK signaling in the virus oncolytic effect in GSCs.

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A common consequence of neoplastic transformation is the suppression of innate antiviral responses, which in GSCs, is achieved through the aberrant expression of the neural stem cell marker, SOX2 (Zhu et al., 2020). Due to insufficient antiviral responses, ZIKV replicates in SOX2+ GSCs hijacking the GSC marker and RNA-binding protein, MSI1 (Chavali et al., 2017; Zhu et al., 2020). ZIKV additionally disrupts host protein expression through upregulation of miR34c, which leads to reduced viability and stemness of GSCs (Iannolo et al., 2019). Interestingly, a recent genome-wide CRISPR-Cas9 screen identified 92 host genes specific to ZIKV infection of GSCs (Wang et al., 2020). Analysis of four of ZIKV’s seven nonstructural proteins (NS1, NS3, NS4B, & NS5) showed that ZIKV’s largest protein, NS5, possessed tumor-suppressive properties against human Glioblastoma in vitro and mouse Glioblastoma in vivo (Li et al., 2019). The mechanisms involving GSC host proteins and the nonstructural ZIKV proteins in oncolysis are currently unclear. However, we know ZIKV to disrupt Glioblastoma cells through inducing oxidative stress, metabolic alterations, the inflammasome and the reduction of peroxisomes (Dabaja et al., 2018; Tricarico, Caracciolo, Crovella, & D’Agaro, 2017; Wong et al., 2019). Previous studies have shown that although ZIKV is an effective oncolytic agent against GSCs, the remaining tumor has cells resistant to ZIKV infection and oncolysis (Zhu et al., 2017). The advantage of oncolytic virotherapy is that it could target these remaining cells indirectly by inducing the production of cytokines from ZIKV-infected human Glioblastoma cells and an antitumoral immune response (Chen et al., 2018).

ZIKV against pediatric brain tumors Pediatric brain cancers are the most common class of malignant cancer, accounting for a significant portion of the cancerrelated death and morbidity, among children (Fry, Perrow, & Paul, 2014). Early diagnosis improves patient outcomes since greater tumor resection is achieved, lower drug doses can be administered and, the brain is subject to less neurotoxicity (Brinkman et al., 2016;). Unfortunately, pediatric brain tumors are frequently diagnosed at advanced stages due to their aggressive characteristic and the lack of awareness for childhood cancers (Magge & DeAngelis, 2015). The relatively short overall survival of these patients, and the long-term sequelae of survivors as a result of current medical intervention, highlights the urgent necessity to develop more effective and less harmful treatments. The most common form of malignant pediatric brain tumor is medulloblastoma (MB) (Fry et al., 2014) classified as an ECT grade IV. MB is thought to arise from NPCs transformed during brain development, which retain a degree of stemness and consequently generate highly aggressive tumors. The progenitor-like characteristics of these tumor cells correlate with aggressiveness, but also makes these cells targetable by ZIKV. Gliomas also affect children but are often diagnosed as benign. The ability to cure Neuroblastoma, the second most common childhood tumor, has significantly improved due to recent progress in treatment strategies. However, cured patients continually suffer from the damage caused by the numerous therapy-associated toxicities (Tsubota & Kadomatsu, 2018). Interestingly a recent study showed a variable response to ZIKV infection in vitro. Six human neuroblastoma cell lines were infected with ZIKV with a high MOI of 10. Four days after infection, only two cell lines presented a reduction in cell viability to below 50%. The variability became most evident 10 days postinfection, when some cell lines demonstrated strong resistance, while two lines proved sensitive to infection, resulting in near-total cell death (Mazar et al., 2018). Such results may be explained by neuroblastomas significant intertumor heterogeneity and differences between the subtypes of neuroblastoma cell lines, namely neuroblastic (neural-type), nonneuronal Schwann cell-like (glial-type) and morphologically intermediate cells (Van Groningen et al., 2017). ZIKV’s tropism for neuronal cells suggests that the virus may present an oncolytic effect against just the neuroblastic subtype of neuroblastoma cells; a concept supported by IMR-32, a neuroblastic neuroblastoma cell line, demonstrating high sensitivity to ZIKV (Mazar et al., 2018). In this case, the effectiveness of ZIKV virotherapy against neuroblastoma would be limited when compared to tumors of known neural origin with CSCs, such as ECTs.

ZIKV and embryonal CNS tumors ECTs are highly malignant, undifferentiated tumors and are the leading cause of death associated with pediatric brain tumors (Shih & Koeller, 2018). ECT includes MB, Atypical teratoid/rhabdoid tumor (AT/RT), the rare primitive neuroectodermal tumor and additional tumors with embryonal histology (Louis et al., 2016). These poorly differentiated tumors originate from the progenitor neuroepithelial lineage found in the cerebellum, brainstem, and spinal cord (Shih & Koeller, 2018). Both MB and AT/RT originate from NPC’s, with high expression of stem-like markers (Azzarelli, Simons, & Philpott, 2018). Recently, the ZIKV oncolytic effect was tested by our group and others in different pediatric brain tumors, ECT (MB and AT/RT) (Kaid et al., 2018a, 2018b), ependymoma, pontine glioma (Zhu et al., 2020) and also neuroblastoma (Mazar et al., 2018), which all presented differing degrees of susceptibility to ZIKV infection.

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FIG. 2 Comparison of ZIKV oncolytic effect between embryonal CNS tumors (ECT) and Glioblastoma. Relative cell number of glioblastoma cell lines (U138MG, U343MG, and U251MG, unpublished data) and ECT cell lines (Daoy, USP13MED, and USP7ATRT) infected by ZIKV at MOI 2 (A), MOI 1 (B), and MOI 0.1 (C) at 24, 48, and 72 hpi. (D) Immunofluorescence staining of ZIKV in infected USP7ATRT cell line (MOI 0.1) at 48 hpi. Scale bar, 10 μm.

The first preclinical study with human ECT showed a high ZIKV oncolytic effect against MB and AT/RT. Intratumoral injection of only 2000 ZIKV particles in mice bearing human ECT was sufficient to induce complete tumor remission and effectively inhibit metastatic spread (Kaid et al., 2018a, 2018b). ZIKV infection showed significant death of ECT cells using low MOI (0.01–2) in vitro at 72 h postinfection (hpi), with MOI 1 resulting in complete cell death by 72 hpi. The level of ZIKV-induced oncolysis at low MOI was notably greater for ECT cell lines than those observed in both glioblastoma and neuroblastoma cells, even at higher MOIs. When we considered ECT and glioblastoma cell lines under identical ZIKV infection conditions, glioblastoma proliferation was impaired but total ECT cell death occurred at MOI 1 and 2, 72 hpi (Fig. 2A–C). In USP7ATRT, we observed ZIKV internalization and cell death even at MOI 0.1 (Fig. 2C and D), this sensitivity can be explained by the phenotype similarity between AT/RT and NPC lines when compared to MB and cells of different neural developmental stages (Kaid et al., 2018a, 2018b). Due to the common NPC origin and high expression of stem-like factors in ECTs, the oncolytic effect of ZIKV is stronger than in glioblastoma or neuroblastoma. Accordingly, pediatric patients with ECTs should be key priority for a ZIKV clinical trial, especially when considering the lack of effective therapies for these patients and their poor overall survival.

ZIKV and ependymoma Ependymoma is a metastatic CNS tumor from the ependymal cells that line the wall of the ventricular system. They are responsible for 10% of pediatric brain tumors associated with a survival of only 23%–69% (Vitanza & Partap, 2016).

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Ependymoma can originate from specific NPCs called embryonic radial glial cells, which express the stem-like factors CD133 and NESTIN (Poppleton & Gilbertson, 2007). Gene expression signatures of over 100 ependymomas showed a prevalence of many factors known to regulate NPCs, including NOTCH signaling, essential for normal neural stem cell maintenance (Hitoshi et al., 2002). This NPC similarity and the presence of CSCs make ependymoma a possible candidate for ZIKV virotherapy. Since a patient-derived ependymoma cell line (USP21-EPE) established in our laboratory expressed the stem cell markers SOX2 and NESTIN (Fig. 3A and B), we questioned whether ZIKV could act as an oncolytic agent against aggressive ependymoma. Monolayer culture of USP21-EPE showed MOI-dependent cell death 72 hpi for the four MOI’s tested: 0.01, 0.1, 1, and 2. MOI 0.1 resulted in 50% reduction of cell viability, while higher MOIs resulted in massive cell death by 72 hpi (Fig. 3C and E). Furthermore, ZIKV significantly disrupted ependymoma tumorspheres, reinforcing its oncolytic and therapeutic potential against such tumors (Fig. 3D and F). A subsequent study infecting EP1 ependymoma cells with ZIKV supports our observations (Zhu et al., 2020) reinforcing ZIKV potential against another form of pediatric tumor.

FIG. 3 Oncolytic effects of ZIKV against ependymoma in-house cell line, unpublished data. (A) Ependymoma cell line (USP21-EPE) immunofluorescence staining of the nucleus (DAPI) and cytoskeleton (vimentin). Scale bar, 20 μm. (B) Abnormal expression of the pluripotency markers NESTIN and SOX2 by flow cytometry analysis of USP21-EPE (n ¼ 3 replicates). Representative phase contrast images of USP21-EPE cell line in 2D (C) and 3D (D) culture after ZIKV infection in different MOIs at 0, 24, 48, and 72 hpi. Scale bar, 10 μm. (E) Total cell number mean and SEM of ependymoma cell line infected by ZIKV at different MOI conditions at 24, 48, and 72 hpi (****P < .01, two-way ANOVA compared with respective day mock condition, n ¼ 5 replicates per cell line). (F) Area quantification mean and SEM of USP21-EPE tumorspheres after 24 h, 48, and 72 hpi (****P < .0001, two-way ANOVA always compared with respective mock condition, n ¼ 30 tumorsphere per cell line).

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ZIKV against non-CNS and prostate tumors One characteristic of ZIKV is its primary tropism toward the brain. Therefore, despite being a potential alternative therapy for brain tumors, ZIKV would not be expected to be effective against non-CNS tumors since the cells would not be susceptible to the virus. Indeed, when we effects in five different human tumors (MB, AT/RT, Breast, Prostate, and Colon rectal) as compared to normal NPCs and neurons in vitro, we demonstrated that ZIKV’s oncolytic effects are specific to CNS tumors (Kaid et al., 2018a, 2018b) (Fig. 4). The virus was incapable of infecting breast and colon rectal tumor cell lines (MCF-7 and HCT8, respectively). However, the virus infected and impaired prostate cancer cell growth (Du-145) at low levels. Our observation of prostate tumor cell susceptibility to ZIKV has been confirmed by a study that tested the effect of ZIKV infection in the PC3 Prostate cancer line (Delafiori et al., 2019). The study showed antiproliferative activity of ZIKV against these cells, associated with metabolic alterations in inflammatory mediators (Delafiori et al., 2019). Many studies have reported the ZIKV tropism and replication in the male reproductive system of mice and humans. In rodents, the virus was found in the testicles and has been shown to cause infertility in mice. In humans, ZIKV persisted in semen for up to 117 days, causing transient testicle inflammation (Oliveira et al., 2018). Following viral clearance, the reproductive system of affected patients presented no damage, and they were able to generate healthy babies (Oliveira et al., 2018). Although the virus cannot oncolyse prostate tumor cells, the ZIKV tropism for male testes and the immune system signaling highlights the potential of ZIKV therapy against prostate tumors via the indirect antitumoral immune response.

Virotherapy is an immunotherapy As discussed previously, the second mechanism of virus-mediated tumor cell death is via immune cell recruitment, also known as immunogenic cancer cell death (ICD) (Krysko et al., 2012). Consequently, many researchers consider virotherapy an immunotherapy and sometimes refer to it as “oncolytic immunotherapy” (Tsun, Miao, Wang, & Yu, 2016). The ICD action, derived from cancer cells infected with a virus, involves both innate and adaptive immune responses with infiltration of immune cells into the tumor mass, offering long-term immunologic protection against tumors. Current oncolytic clinical trials employing an adenovirus against recurrent glioblastoma prolonged patient median overall survival for 3.5 years and identified macrophage/CD8 T-cell infiltration into the resected tumor site as well as inflammatory

FIG. 4 Oncolytic effects of ZIKV against CNS and non-CNS tumors. Immunofluorescence representative images staining of nucleus (blue), α-tubulin (green) and ZIKV (red) in CNS tumors, including MB and AT/RT cell lines, and non-CNS tumors (prostate, colon rectal, and breast cancer) cell lines. Scale bar, 10 μm.

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cytokines in serum that promoted cell-mediated immunity (Omuro et al., 2018). These observations are surprising since glioblastoma is known as a “cold tumor,” with low ratios of tumor-infiltrating T-cells and local immunosuppression, due to the naturally isolated brain microenvironment (Han et al., 2016). For this reason, many immunotherapy clinical trials failed when tested against glioblastoma (Martikainen & Essand, 2019). However, as observed in many other oncolytic immunotherapy studies against glioblastoma, ZIKV could have the capability of “heating” the immune-suppressed TME by inducing ICD and inflammation (Koks et al., 2015; Lang et al., 2018). Recent studies demonstrated that ZIKV infection activates IFN signaling in GSCs (Zhu et al., 2017). In placental macrophage and microglia, ZIKV infection results in the production of type I IFN, proinflammatory cytokines, and upregulation of IFN-stimulated genes (Chen, Zhong, & Li, 2019; Quicke et al., 2016). These immune cells may play important roles in the ICD of ZIKV virotherapy. We showed for the first time that ZIKV intrathecal injections in dogs bearing spontaneous brain tumors induced expression of IL-8, KC-like, and MCP-1 in both blood serum and cerebrospinal fluid. These results suggest a prevalent cytokine/chemokine cluster of an inflammatory response that drives and controls leukocyte migration (Zlotnik & Yoshie, 2012). The postmortem tumor histological analysis from the treated dogs showed extensive necrosis, cellular debris in addition to macrophage, lymphocyte, and plasma cell infiltration (Kaid, Azevedo dos Santos Madi, et al., 2020). The dogs presented local inflammation, due to tumor cell ZIKV infection and intense local necrosis, leading to a temporary increase in brain pressure and subsequent reappearance of neurological symptoms. To avoid potential brain damage and patient deterioration, small doses of antiinflammatory drugs were administered to regulate the swelling and inflammation, and the dogs presented immediate clinical improvement. The observed immune modulation following ZIKV injection in this veterinary clinical trial, alongside the ZIKV-induced production of type I IFN, proinflammatory cytokines, and expression of IFN-stimulated genes, are important considerations for a future human clinical trial. Collectively, these results demonstrate the oncolytic immunotherapy potential of ZIKV against brain tumors known to be unresponsive to conventional therapies and current immunotherapy approaches.

Safety, virus delivery, and conclusion ZIKV virotherapy safety The primary concern regarding any clinical trial is the safety of the proposed drug or therapeutic approach. Manipulation of ZIKV to increase its safety profile may be required by regulatory authorities prior to future clinical application. Recent studies showed that ZIKV’s safety profile could be improved through either an E218A mutation in NS5 or a specific 10-nucleotide deletion in the 30 UTR of the viral genome, both of which sensitize the virus to type I IFN inhibition (Chen et al., 2018; Zhu et al., 2017). While specificity for GSCs was retained, showing promise for the clinic, both manipulations reduced ZIKV’s oncolytic capability, and their effect on the ZIKV-induced ICD is unknown (Chen et al., 2018; Zhu et al., 2017).

ZIKV virotherapy delivery and outcome in a canine model In a recent study, we injected ZIKV intrathecally in three dogs bearing intracranial brain tumors at advanced stages. No lasting clinical adverse side effects were observed following ZIKV injections. No viral particles were identified in blood, cerebrospinal fluid, or urine samples 14 days following the initial injection. Two dogs that were followed presented with a brief clinical decline, due to tumor pseudo-progression. However, after the second administration of ZIKV, in a 10-fold higher concentration, tumor regression and remarkable clinical improvement were observed (Kaid, Azevedo dos Santos Madi, et al., 2020). These observations in a canine model address some of the clinical concerns regarding ZIKV’s future application as virotherapy; ZIKV safety, intracranial tumor remission, ability to invoke an immune system response and recruit immune cells to the tumor site. Ahead of employing ZIKV as an alternative therapy in humans bearing brain tumors, virus delivery routes must be investigated. One possibility may be direct delivery into the tumor environment to target remaining cancerous cells following surgical resection. Another approach could be systemic administration and transport via blood or cerebrospinal fluid. Direct administration into the resected tumor site should avoid clearance of the virus by the immune system before reaching the target tumor site. Importantly, the well-known ZIKV neurotropism could allow a systemic delivery, thus circumventing the requirement for neurosurgery. Pediatric CNS tumors frequently metastasize before detection and surgical resection is particularly troublesome for children. Systemic delivery approaches are theoretically more applicable for these patients as the broad distribution of the virotherapy throughout the body can target metastases while being administered in a noninvasive, and possibly, regular manner.

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Conclusion Studies are continually showing promising results for the future development of a ZIKV-based oncolytic therapy against both pediatric and adult CNS tumors. Nevertheless, continued research is essential to enhance our comprehension of both ZIKV’s oncolytic and ICD mechanisms of action. Regarding safety, it is important to note that the 2015 ZIKV epidemic in Brazil, which affected thousands, showed that the vast majority of infected individuals developed little or no symptoms at all. These observations are extremely relevant for obtaining approval from regulatory authorities, such as the FDA, regarding the use of a live virus as a therapy. It is feasible to propose that a multimodal approach, encompassing ZIKV-based virotherapy with other therapeutic agents such as chemotherapy, may result in significant benefit to both adult and pediatric brain tumor patients. Tumor recurrence in adult glioblastoma patients is often due to the presence of GSCs and resistance to chemotherapy (Stupp et al., 2009). Since ZIKV preferably infects adult GSCs, ZIKV administration with chemotherapies could improve adult patient disease-free survival (Zhu et al., 2017). This approach should also be explored in pediatric patients, where ZIKV could be administered to the resected site following surgery, in combination with lower doses of chemotherapy and radiotherapy. High doses of chemotherapy impair a child’s neurodevelopment, consequently, this multimodal approach is likely to increase therapy efficiency and reduce the morbidity which is common in pediatric patients. Furthermore, elucidating the mechanisms through which ZIKV triggers the antitumoral immune response, recruits immune cells and induces tumor cell death, could lead to the development of a therapy that achieves these outcomes in the absence of the virus. In short, taking into account research to date, the observed safety in humans during the 2015 outbreak and in dogs bearing brain tumors, ZIKV virotherapy appears as a viable and promising approach to combat currently untreatable brain tumors.

Policy and procedures In vitro assays, cell line establishment, human samples, and ZIKA virus strain The ZIKA viral particles used for the studies are provided by the Instituto Butantan, Sa˜o Paulo, Brazil. ZIKA virus strain used was donated by Dr. Pedro Vasconcelos, Instituto Evandro Chagas, Brazil (Faria et al., 2016). Viral particles were produced in VERO cells, cultured in serum-free medium (VP SFM, Thermo scientific) after two serial passages, at a multiplicity of infection of 0.05 and harvesting supernatants after 72 h. Here we present unpublished data with a patient-derived ependymoma cell line (USP21-EPE) established from a 21 months-old girl diagnosed with grade II ependymoma and characterized by the Medical School Hospital of the University of Sa˜o Paulo. The procedure was approved by the Internal Review Board (CEP-IB No.121/2011), and informed consent was obtained from the patient’s parents. The cell line establishment method employed is described in previous publications of the laboratory (Kaid et al., 2018a, 2018b; Silva et al., 2016). In vitro generated samples were obtained through culturing and infecting cell lines in a cell culture facility in the Research Center of the Human Genome and Stem Cells at the University of Sa˜o Paulo, Brazil. Briefly, cells (4.2  103 cells/cm2) in 2D (monolayer) were exposed to ZIKA virus for 1 h at 37°C, washed with culture medium, and maintained up to 72 h. Tumorspheres (3D), generated and characterized as previously reported (Silva et al., 2016), were infected with viral exposure for 2 h to ensure complete sphere infection. Cell viability and tumorspheres area were evaluated using ImageJ software. The immunofluorescence images were generated by using fixed tumor cells (3.7% formaldehyde for 10 min) treated for 30 min with 0.1% Triton X-100 in 1X PBS, prior to 2 h incubation in 5% bovine serum albumin in 1X PBS, and then stained with the primary ZIKV-NS2B (GTX133308, GeneTex) and Alpha-tubulin (WB100-690S, Sigma-Aldrich) antibodies at 4°C. Cell nuclei were stained with 1 μg/mL DAPI for 2 min. Tumor cells were mounted on glass slides and coverslips with VectaShield. All images were taken in a confocal microscope (Zeiss LSM 800).

Mini-dictionary of terms Oncolysis: Tumor cell membrane disruption and death in response to intracellular viral replication. Multimodal: Strategy to combine treatments to improve clinical outcomes. Interferon: Molecules produced by host cells to increase antiviral responses in response to viral infections. Cytotoxicity: Quality of an agent being toxic to the cell, causing cell death. Neoplastic transformation: Transition of a healthy cell into a tumor cell.

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ZIKA virus nonstructural proteins: Seven ZIKA virus proteins that are mainly in charge of metabolic and viral replicative functions in host cells. Multiplicity of Infection (MOI): Ratio between the number of viral particles added to infect one host cell. Placental transmigration: Transposition of the selective maternal-fetal barrier.

Key facts l

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Worldwide incidence of brain tumors in 2018 was 296,851 cases, with around 80% of these cases resulting in mortality (GLOBOCAN 2018). The most common malignant adult brain tumors are glioblastomas, constituting 15.6% of all brain tumor cases. Among the brain tumors, gliomas (glioblastomas, ependymomas, astrocytomas, oligodendrogliomas, and oligoastrocytomas) represent 28% of cases. When considering only malignant tumors, gliomas represent 80% and embryonic central nervous system tumors are in second place with an incidence of 3.3%. The most common brain tumors in children (0–4 years) are the malignant embryonic central nervous system tumors. Virotherapy is a recent approach that gained momentum in 2015 after the FDA approved the first oncolytic virotherapy for the treatment of advanced Melanoma.

Summary points l

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Aggressive brain tumors: Brain tumors are challenging and frequently aggressive tumors that remain poorly treated by classical procedures (chemotherapy, radiotherapy, and surgical resection). Brain tumor therapy: Current brain tumor treatment options are invasive and can lead to long-term sequelae, especially among children. Oncolytic virus: Virotherapy is a modern strategy that employs viruses to specifically target tumor cells. ZIKA virus virotherapy: ZIKA virus has shown to be a promising virotherapy against brain tumors. ZIKA virus antitumoral effect: ZIKA virus acts directly, with an oncolytic effect to kill tumor cells, and indirectly, activating the immune system to expose tumoral cells to the antitumoral immune response.

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Chapter 31

Zika virus in Brazil Andrezza Nascimentoa, Alberto Jos e da Silva Duarteb, and Sabri Saeed Sanabania,b,∗ a

Laboratory of Medical Investigation LIM-56, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, b Laboratory of Medical Investigation LIM-3, Hospital das Clinicas da FMUSP, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil ∗ (Laboratory of Medical Investigation LIM-3/56, Hospital das Clinicas da FMUSP, Faculty of Medicine, University of Sa˜o Paulo, Av. Dr. Eneas de Carvalho Aguiar, 470 3º andar, Sa˜o Paulo 05403 000, Brazil. Phone: + 5511 3061 7194 ext:218.), e-mail: [email protected]

Abbreviations BMH CZS PAHO WHO ZIKV

Brazilian Ministry of Health congenital Zika syndrome Pan American Health Organization World Health Organization Zika virus

Historical perspective Zika virus (ZIKV) is an emerging arbovirus that belongs to the family Flaviviridae and genus Flavivirus, a group of RNA viruses transmitted by arthropods. The virus name originates from the Zika Forest of Uganda, where it was first isolated in 1947 from an infected rhesus monkey (Dick, Kitchen, & Haddow, 1952). It was later detected in humans in 1952 in Uganda and the United Republic of Tanzania. Since then, reports of ZIKV sporadic infections came from Asia and Africa, until April–July 2007, when the Yap Island in the Federated States of Micronesia reported the first known outbreak typically accompanied by mild illness (Duffy et al., 2009). After 7 years, the virus appeared in French Polynesia and larger outbreaks were reported in New Caledonia, the Cook Islands, and Easter Island, whereby coincident with the French Polynesia ZIKV outbreak, an increased incidence of neurological complications were observed (Roth et al., 2014). Infection with ZIKV has been reported among travelers returning from nonendemic countries, including Japan, Germany, Italy, Canada, Australia, and the United States (Kutsuna et al., 2014; Kwong, Druce, & Leder, 2013; Tappe et al., 2014) and from endemic countries such as Brazil (Zammarchi et al., 2015), thus the virus spread to other countries in North and South America (http://wwwnc.cdc.gov/travel/notices; Centers for Disease Control and Prevention (CDC), 2016). As a consequence, ZIKV was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016 (http://www.who.int/mediacentre/news/statements/2016/1st-emergencycommittee-zika/en/; World Health Organization (WHO), 2016). Brazil, the world’s fifth largest country, occupies roughly half of the South American continent landmass (>8,500,000 km2) and has the world’s longest tropical coast, with extensive forests in the Amazon Region, as well as forests in the east, southeast, and south coast. It also has a large swamp region (Pantanal) in the Midwest, a savannah region (Cerrado) in the central plateau area, and a dry region (Caatinga) in the northeastern interior. Thus, the country is a suitable territory for the existence of the vector and, therefore, for the occurrence of arboviruses. Over the last few decades, Brazil has experienced several mosquito-borne arboviral disease outbreaks including Dengue, Chikungunya virus-associated arthralgia, and most recently Zika and the yellow fever virus-associated diseases (Figueiredo, 2015). Circulation of other emerging viruses like West Nile virus, St. Louis encephalitis, Mayaro virus, and Hantavirus have been reported in the country (Fonseca, Oliveira, & Duarte, 2018; Leao, Gueiros, Lodi, Robinson, & Scully, 2017). In late 2014, a few months after the Football World Cup in Brazil, several urban health centers reported an unusual increase in the number of cases of undetermined exanthematous syndrome in northeastern Brazil. From February 2015 onward, other municipalities began to notify the Brazilian Ministry of Health (BMH) about similar cases. At the end of the same month, the BMH began to monitor the increase in the number of exanthem in the affected region and, in March 2015, began to investigate the potential cause and source of the outbreak, with the hypothesis that reported symptoms were

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caused by dengue, rubella, or Chikungunya virus. However, the preliminary results excluded infection by these pathogens (Heukelbach, Alencar, Kelvin, de Oliveira, & Pamplona de Goes Cavalcanti, 2016). Also, in March of the same year, ZIKV was isolated in Natal, the capital of Rio Grande do Norte, and the following month, the circulation of the virus was confirmed by laboratory tests. In May 2015, the Brazilian Ministry of Health issued a warning to the population of the autochthonous transmission of ZIKV (Brito et al., 2018; Esposito, de Moraes, & Antonio Lopes da Fonseca, 2018; Heukelbach et al., 2016), declared a state of emergency, and deployed teams of experts to the affected states to support surveillance and response (http://portalsaude.saude.gov.br/index.php/cidadao/principal/agencia-saude/20629; Ministerio da Sau´de (Brazil), 2015). Subsequently, the Pan American Health Organization (PAHO) issued an epidemiological alert describing the infection and providing recommendations to make maximum use of the existing surveillance systems for dengue and Chikungunya to improve their sensitivity to make the diagnosis in patients with suspected ZIKV infection (PAHO; World Health Organization (WHO) and Pan American Health Organization (PAHO), 2015). The PAHO alert also included details on laboratory testing, case management, and prevention and control measures including recommendations to travelers. By December 2015, 18 of the 26 Brazilian states had confirmed the autochthonous transmission of ZIKV (Musso & Gubler, 2016). In 2016, the virus spread to almost all states except the remote areas in the Amazon region and the southernmost part of Brazil where the climate is not favorable for the vector (Lowe et al., 2018). At the beginning of the outbreak, no great concern was given because no severe or lethal case had been previously documented by the scientific community about ZIKV infection. However, this calmness did not last for long as the health authorities in Pernambuco, Bahia, Rio Grande do Norte, and Maranha˜o announced an unusual increase in cases of severe neurological complications in patients who had previously complained of exanthematous disease shortly after (SchulerFaccini et al., 2016). On 17 November, the Brazilian Ministry of Health confirmed the molecular detection of ZIKV in amniotic fluid samples collected from two fetuses with microcephaly in the northeast (Calvet et al., 2016). Both pregnant women presented symptoms of ZIKV infection during early pregnancy. Besides the microcephalic neonates who died shortly after birth, two adults and one newborn with neurological disorders associated with ZIKV died in late November in Brazil (Musso & Gubler, 2016). Thus, the outbreak in Brazil became associated with a surge in congenital microcephaly and accordingly it was considered a global public health emergency by the WHO. With the accumulation of cases with neuropathological conditions as a result of ZIKV intrauterine transmission, the Brazilian scientists have coined the term congenital Zika syndrome (CZS) to designate a spectrum of congenital neuropathological anomalies including microcephaly, complex brain malformations, and ocular injury (Kleber de Oliveira et al., 2016; Melo et al., 2016; Moore et al., 2017). A report of a follow-up study of a series of 13 Brazilian infants with laboratory evidence of congenital ZIKV infection with normal head size revealed that most of these infants had poor head growth with microcephaly developing after birth (van der Linden et al., 2016). It has been estimated that a maternal ZIKV infection may result in 5%–13% fetal anomalies and this rate rises to its highest level, which the infection reaches, early in pregnancy (Hoen et al., 2018; Sanz Cortes et al., 2018; Shiu et al., 2018). The results from a follow-up study of a Brazilian cohort of 116 pregnant women from Rio de Janeiro with laboratoryconfirmed ZIKV infections have reported that 42% of fetuses had CZS including epilepsy, dysphagia with feeding difficulties, visual and hearing deficits, spasticity, and hypertonicity (Brasil et al., 2016). The same study suggested that intrauterine growth restriction may occur in approximately 9% of pregnancies. A profile of disproportionate fetal growth has also been reported in other Brazilian case studies of pregnant women with laboratory evidence of ZIKV infections (Oliveira Melo et al., 2016; Silva et al., 2018).

Introduction of ZIKV to Brazil There are many hypotheses about the timing and origin of the ZIKV that caused the outbreak in Brazil, but the routes of entry of this virus in the Americas are still not entirely clear (Campos et al., 2018). The first theory assumed that the virus was introduced during the World Cup soccer competition hosted in 2014, but the teams from the Pacific countries in which ZIKV was circulating had not engaged in this event. Then, it was suggested that the virus may have been introduced by the athletic teams from Pacific countries who participated in the World Spring Canoe championship in Rio de Janeiro in August 2014. The first strong phylogenetic evidence with a reliable number of samples provided by Pessoa et al., (2016a) demonstrated that sequences from Brazilian ZIKV were very close to the Chilean ZIKV strains and proposed that the Brazilian ZIKV had its origin in Chile. Of note, the Chilean ZIKV strains were associated with the outbreak on Easter Island that began in January 2014 (Tognarelli et al., 2016). Nevertheless, the same study by Pessoa et al., (2016a) revealed a high degree of sequence similarity among the 14 partial sequences of the ZIKV NS5 gene and thus suggested a single introduction of the virus into Brazil (Fig. 1). These results were further supported by the Bayesian phylogeographic analysis conducted by Lednicky et al. (2016) that show the Asian origin of the South American viral lineages, the close phylogenetic

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FIG. 1 Phylogenetic tree constructed using a maximum-likelihood method from a partial NS5 region of 14 samples during the peak of the epidemic in the city of Tuparetama, Pernambuco, northeast of Brazil, (indicated by red circles), 51 sequences from Chile and three other Asiatic lineages (indicated by blue circle), and the 48 Zika virus sequences originated from Africa (indicated by green square). The tree was obtained from the previously published study by Pessoa et al., (2016a).

relationship among the strains circulating in Haiti, Brazil, Suriname, and Puerto Rico, and the clustering of these strains within a larger clade of isolates from Easter Island. Further support was provided by Faria et al. (2016) who used various approaches including the phylogenetic, epidemiological, and mobility data to quantify ZIKV evolution and explore the introduction of the virus to the Americas. Their results obtained from the phylogenetic analysis of ZIKV complete coding genomes revealed that all viral strains sampled in the Americas, including those from Brazil, strongly positioned in a monophyletic cluster within the Asian genotype and share a common ancestor with the ZIKV strain that circulated in French Polynesia in November 2013. However, the recent study by Delatorre and colleagues (Delatorre, Fernandez, & Bello,

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2018) did not agree with the hypothesis that assumes the role of Easter Island in Migration of ZIKV from South Pacific to Americas. The findings from this study indicated that French Polynesia was the main source of virus dissemination, from where it spread to the Americas and to other Pacific areas via two simultaneous introductions. A recently published phylogenetic study by da Silva Pessoa Vieira (Vieira, Machado, Pena, de Morais Bronzoni, & Wallau, 2019) of ZIKV viral complete genomes extracted from two samples obtained during the epidemic in 2015 in Sinop, a city located in the southern border of the Amazonian forest, in the state of Mato Grosso, Midwest region of Brazil revealed two lineages of the Asian ZIKV genotype circulating in the region and that the last common ancestor of the isolates containing such lineages has different time estimates.

ZIKV phylogenetics The rapid emergence of ZIKV could be a consequence of genetic mutations that contribute to its evolution of virulence thus causing adverse pregnancy outcomes including fetal loss, developmental abnormalities, and neurological damage ( Johansson, Mier-y-Teran-Romero, Reefhuis, Gilboa, & Hills, 2016; Melo et al., 2016; Nguyen et al., 2017). Based on the accumulation of the nucleotide changes in the viral genomes over time and geographical space, researchers have classified the ZIKV into two main lineages by the phylogenetic tree constructed from various partial or entire viral genomes; the African and Asian lineages. The African lineage was initially characterized in 1947 and is considered the older of the two lineages. Historically, no clinical evidence was provided in the literature implicating ZIKV in CZS. This could be due to multiple reasons including the potential pathogenicity of one lineage over the other, or because of preexisting host humoral and cellular immunity against flaviviruses, differences in species of mosquitoes vectors, or lack or paucity epidemiological studies in affected countries or with poor resources (Rossi, Ebel, Shan, Shi, & Vasilakis, 2018). A recent study by Pompon et al. (2017) used oral infection of mosquitoes collected in Singapore to identify the vector species, to quantify the blood infection threshold and to compare transmissibility between the Polynesia 2013 (H/PF13) and Brazil 2015 (BE H 815744) ZIKV strains. These results suggest that the Brazilian strains are more efficiently transmitted than the Asian strains, which raises concerns about the introduction of American strains in Asia. A comparison of ZIKV complete genomes sampled from a deceased newborn with CZS (BeH823339) and one from a fatal adult case with lupus and rheumatoid disease (BeH818305) collected from the northeastern estate Ceara´ and Maranha˜o, respectively with other Brazilian isolates revealed various nucleotide changes at different sites along the genomes between the isolates (Faria et al., 2016). The detailed results revealed that the circulating isolates in Sa˜o Paulo had 32 nucleotide changes compared with the microcephaly case, and 34 compared with the fatal case from Maranha˜o. Isolates BeH819966 (from Belem), BeH815744 (from Paraı´ba), and BeH18995 (from Belem) had a maximum of five nucleotide changes.

ZIKA vectors in Brazil ZIKV is transmitted through the bite of an infected Aedes mosquito, mainly Aedes aegypti. Aedes albopictus has also been associated with the transmission of ZIKV (Marcondes & Ximenes Mde, 2016). Both vectors are found in the peridomestic environment where they preferentially feed on humans and domestic animals and usually breed in indoor and outdoor settings in a variety of natural and artificial water-holding containers (de Souza et al., 2011; Figueiredo et al., 2013; Vilela et al., 2010). Other studies that aimed to evaluate the vector competence for transmission of ZIKV revealed that the fieldcaught Culex quinquefasciatus in Northeast Brazil had detectable ZIKV in the midgut and salivary glands (Franca, Neves, Ayres, Melo-Neto, & Filho, 2016; Guedes et al., 2017). However, this vector has not been incriminated for transmitting ZIKV in Rio de Janeiro (Fernandes et al., 2016) and it has largely been discounted as a vector by other studies (Amraoui et al., 2016; Diagne et al., 2015). Between the 1950s and 1960s, there was a sharp decline in the spread of dengue fever in Latin America including Brazil as a result of a rigorous eradication program organized by PAHO that aimed at eliminating A. aegypti (http://www.ncbi. nlm.nih.gov/pubmed/9128110, 1997). However, the failure to continue the eradication campaign led to the return of the mosquito to the countries from which it had been previously eliminated (Diaz-Nieto, Macia, Perotti, & Beron, 2013). In Brazil, since the early 1980s, the dengue transmission has been linked to A. aegypti (Lambrechts, Scott, & Gubler, 2010; Martins et al., 2012), which is currently present in all Brazilian states and 4834 of the 5570 municipalities (Lowe et al., 2018). A. albopictus was first observed in Brazil in 1986, in the states of Espı´rito Santo, Minas Gerais, Rio de Janeiro and Sa˜o Paulo (Ayres, Romao, Melo-Santos, & Furtado, 2002; Forattini, 1986). Currently, this species is found in all the Brazilian states (Fares, Souza, Anez, & Rios, 2015). Factors such as the climate, population density in major cities, precarious socioeconomic status, educational attainment, poor housing, and lack of sanitation infrastructure have largely contributed

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FIG. 2 Number of ZIKV cases reported by the Brazilian Ministry of Health (BMH) in 2019 distributed by geographical region. The numbers were obtained from Brazilian Ministry of Health website accessed at http://portalsaude.saude.gov.br (last accessed on Oct 09, 2019).

to the proliferation of these vectors and thus supported the chance of transmitting arboviruses in Brazil (Fares et al., 2015; Gomez-Dantes & Willoquet, 2009; Martins et al., 2012).

ZIKA epidemiology update During the period Dec 2018–Aug 2019, according to the BMH, 9.813 probable cases of ZIKV disease had been reported in 2019 as of week 34 compared to 6.669 cases reported over the same period in 2018 (Fig. 2) (Surveillance SoH & Ministry of Health of Brazil MoHoB., 2019). The Northeast, Midwest, and North had the highest incidence rates in the country with 6.7, 6.2, and 4.9 cases per 100.000 inhabitants, respectively. From a total of 27 federal cities and districts in Brazil, Tocantins, Rio Grande do Norte, Alagoas, and Espı´rito had the highest ZIKV disease cases, reporting 32.3, 27, 18, and 15.7 cases. The data also revealed 1649 pregnant women with suspected ZIKV infection, of whom 447 had confirmed the diagnosis. It is noteworthy that 42.95% (192) of the confirmed cases were registered in Rio de Janeiro, followed by Espı´rito Santo (14.77%; 66), Minas Gerais (10.51%; 47), Alagoas (7.16%; 32), Paraı´ba (3.58%, 16), and Mato Grosso do Sul (with 3.13%; 14). According to the official data from the epidemiological bulletins, two deaths due to ZIKV infection were confirmed in the state of Paraı´ba.

Conclusion ZIKV infection is primarily transmitted by the Aedes mosquito. Approximately 80% of infected people are asymptomatic and the infection is generally mild and a self-limiting illness for most patients in whom clinical manifestations develop. Although rare, serious CZS has been associated with ZIKV infection. There is no vaccine or specific antiviral drugs. Data for ZIKV infections so far in 2019 in Brazil shows a substantial plunge of cases from 205,578 probable cases in mid-April 2016 to 9813 as of October this year according to the MOH. This precipitous drop of ZIKV cases was expected because of

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herd immunity. Because 80% of infected people are asymptomatic, it is also likely that the transmission may persist at low but steady levels. The low incidence of ZIKV infection should neither make the health authorities tolerate the control measures nor lessen the need for a vaccine.

Policy and procedures The development of a structured surveillance program that includes screening before an outbreak arises, collecting baseline data, and researching the ecology of the disease is crucial to prevent an outbreak. Screening both humans and mosquitoes for the presence of the ZIKV is an important initial step to reduce the risk of a Zika epidemic in Brazil. Moreover, understanding the genetic characteristics and diversity of the virus would enable the scientific community to understand the extent of the disease. One of the greatest scientific achievements of the 21st century is the development of molecular techniques for the direct study of a specific viral genome from the clinical sample. Laboratory assays that amplify nucleic acids such as PCR, nucleic acid sequence-based amplification (NASBA), and Lawrence Livermore Microbial Detection Array (LMDA) are considered top technology for rapid detection for most known human viruses including ZIKV. For instance, we used different molecular methods to screen for ZIKV in different clinical samples including partial amplification of the NS5 region by quantitative real-time PCR (Pessoa et al., 2016a; Pessoa, Patriota, de Souza Mde, Abd El Wahed, & Sanabani, 2016b), a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay (Abd El Wahed et al., 2017), the combination of strand displacement isothermal method and nanopore sequencing technology for rapid identification of ZIKV outbreak (Hansen et al., 2020), and peptide microarrays that encompass the full amino acid sequences of ZIKV proteome from different geographical locations (Hansen et al., 2019). This essay constitutes a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.

Mini-dictionary of terms Flaviviridae: A family of arthropod-borne small enveloped viruses with positive-sense RNA genomes of approximately 9.0–13 kb. Emerging viruses: New and potentially threatening viruses that either arise de novo or of an agent from a previously unknown source. Congenital Zika syndrome: A distinct pattern of birth defects and disabilities found among fetuses and neonates infected with Zika virus during pregnancy. Autochthonous transmission: Local transmission of the viruses between cases. Mosquito-borne arboviral diseases: Diverse and serious viral diseases transmitted by insect vectors or spread as zoonoses.

Key facts of Brazil l l l l l l

Is the fifth most populous country in the world and accounts for one-third of Latin America’s population. Has 3.2 mile2 of total area and borders 10 countries in South America. Geopolitically, Brazil is divided into five large regions North, Northeast, Central-West, Southeast, and South. The climate in the majority of Brazil is tropical. An estimated average of life expectancy for the Brazilian total population in 2018 was 74.3 years. Retained its 79th position on the ranking with 189 countries organized by their Human Development Index.

Summary points l

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Zika virus, transmitted by mosquitoes belonging to the A. aegypti and other Aedes genu, was first detected in Brazil in May 2015 several months after the football World Cup and rapidly spread across the country. The outbreak of Zika virus in Brazil became associated with a surge in congenital microcephaly designated as congenital Zika syndrome. Phylogenetic studies showed ZIKV complete coding genomes sampled in the Americas, including those from Brazil, clustered within the Asian genotype and sharing a common ancestor with the ZIKV strain that circulated in French Polynesia in November 2013. According to the Ministry of Health, the up-to-date data showed a substantial plunge of cases; this could be attributed to the herd immunity or to the fact that the transmission may persist at low but steady levels, particularly, since we know that 80% of infected people are asymptomatic.

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A., Mier-y-Teran-Romero, L., Reefhuis, J., Gilboa, S. M., & Hills, S. L. (2016). Zika and the risk of microcephaly. The New England Journal of Medicine, 375(1), 1–4. https://doi.org/10.1056/NEJMp1605367. Kleber de Oliveira, W., Cortez-Escalante, J., De Oliveira, W. T., do Carmo, G. M., Henriques, C. M., Coelho, G. E., & Araujo de Franca, G. V. (2016). Increase in reported prevalence of microcephaly in infants born to women living in areas with confirmed Zika virus transmission during the first trimester of pregnancy—Brazil, 2015. MMWR—Morbidity and Mortality Weekly Report, 65(9), 242–247. https://doi.org/10.15585/mmwr.mm6509e2. Kutsuna, S., Kato, Y., Takasaki, T., Moi, M., Kotaki, A., Uemura, H., … Ohmagari, N. (2014). Two cases of Zika fever imported from French Polynesia to Japan, December 2013 to January 2014 [corrected]. Euro Surveillance, 19(4). https://doi.org/10.2807/1560-7917.es2014.19.4.20683. Kwong, J. C., Druce, J. D., & Leder, K. (2013). Zika virus infection acquired during brief travel to Indonesia. The American Journal of Tropical Medicine and Hygiene, 89(3), 516–517. https://doi.org/10.4269/ajtmh.13-0029. Lambrechts, L., Scott, T. W., & Gubler, D. J. (2010). Consequences of the expanding global distribution of Aedes albopictus for dengue virus transmission. PLoS Neglected Tropical Diseases, 4(5), e646. https://doi.org/10.1371/journal.pntd.0000646. Leao, J. C., Gueiros, L. A., Lodi, G., Robinson, N. A., & Scully, C. (2017). Zika virus: Oral healthcare implications. Oral Diseases, 23(1), 12–17. https:// doi.org/10.1111/odi.12512. Lednicky, J., Beau De Rochars, V. M., El Badry, M., Loeb, J., Telisma, T., Chavannes, S., … Morris, J. G., Jr. (2016). Zika virus outbreak in Haiti in 2014: Molecular and clinical data. PLoS Neglected Tropical Diseases, 10(4). https://doi.org/10.1371/journal.pntd.0004687, e0004687. Lowe, R., Barcellos, C., Brasil, P., Cruz, O. G., Honorio, N. A., Kuper, H., & Carvalho, M. S. (2018). The Zika virus epidemic in Brazil: From discovery to future implications. International Journal of Environmental Research and Public Health, 15(1). https://doi.org/10.3390/ijerph15010096. Marcondes, C. B., & Ximenes Mde, F. (2016). Zika virus in Brazil and the danger of infestation by Aedes (Stegomyia) mosquitoes. Revista da Sociedade Brasileira de Medicina Tropical, 49(1), 4–10. https://doi.org/10.1590/0037-8682-0220-2015. Martins, V. E., Alencar, C. H., Kamimura, M. T., de Carvalho Araujo, F. M., De Simone, S. G., Dutra, R. F., & Guedes, M. I. (2012). Occurrence of natural vertical transmission of dengue-2 and dengue-3 viruses in Aedes aegypti and Aedes albopictus in Fortaleza, Ceara, Brazil. PLoS One, 7(7), e41386. https://doi.org/10.1371/journal.pone.0041386. Melo, A. S., Aguiar, R. S., Amorim, M. M., Arruda, M. B., Melo, F. O., Ribeiro, S. T., … Tanuri, A. (2016). Congenital Zika virus infection: Beyond neonatal microcephaly. JAMA Neurology, 73(12), 1407–1416. https://doi.org/10.1001/jamaneurol.2016.3720. Ministerio da Sau´de (Brazil). (2015). Minist erio da Sau´de investiga aumento de casos de microcefalia em Pernambuco [Internet]. (Updated 11 November 2015; cited 11 November 2015) Available from: http://portalsaude.saude.gov.br/index.php/cidadao/principal/agencia-saude/20629. Moore, C. A., Staples, J. E., Dobyns, W. B., Pessoa, A., Ventura, C. V., Fonseca, E. B., … Rasmussen, S. A. (2017). Characterizing the pattern of anomalies in congenital Zika syndrome for pediatric clinicians. JAMA Pediatrics, 171(3), 288–295. https://doi.org/10.1001/jamapediatrics.2016.3982. Musso, D., & Gubler, D. J. (2016). Zika virus. Clinical Microbiology Reviews, 29(3), 487–524. https://doi.org/10.1128/CMR.00072-15. Nguyen, S. M., Antony, K. M., Dudley, D. M., Kohn, S., Simmons, H. A., Wolfe, B., … Golos, T. G. (2017). Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques. PLoS Pathogens, 13(5). https://doi.org/10.1371/journal.ppat.1006378, e1006378. Oliveira Melo, A. S., Malinger, G., Ximenes, R., Szejnfeld, P. O., Alves Sampaio, S., & Bispo de Filippis, A. M. (2016). Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: Tip of the iceberg? Ultrasound in Obstetrics & Gynecology, 47(1), 6–7. https://doi.org/10.1002/ uog.15831. Pessoa, R., Patriota, J. V., Lourdes de Souza, M., Felix, A. C., Mamede, N., & Sanabani, S. S. (2016a). Investigation into an outbreak of dengue-like illness in Pernambuco, Brazil, revealed a cocirculation of Zika, Chikungunya, and dengue virus type 1. Medicine (Baltimore), 95(12). https://doi.org/10.1097/ MD.0000000000003201, e3201. Pessoa, R., Patriota, J. V., de Souza Mde, L., Abd El Wahed, A., & Sanabani, S. S. (2016b). Detection of Zika virus in Brazilian patients during the first five days of infection—urine versus plasma. Eurosurveillance, 21(30). Pompon, J., Morales-Vargas, R., Manuel, M., Huat Tan, C., Vial, T., Hao Tan, J., … Misse, D. (2017). A Zika virus from America is more efficiently transmitted than an Asian virus by Aedes aegypti mosquitoes from Asia. Scientific Reports, 7(1), 1215. https://doi.org/10.1038/s41598-017-01282-6. Rossi, S. L., Ebel, G. D., Shan, C., Shi, P. Y., & Vasilakis, N. (2018). Did Zika virus mutate to cause severe outbreaks? Trends in Microbiology, 26(10), 877–885. https://doi.org/10.1016/j.tim.2018.05.007. Roth, A., Mercier, A., Lepers, C., Hoy, D., Duituturaga, S., Benyon, E., … Souares, Y. (2014). Concurrent outbreaks of dengue, chikungunya and Zika virus infections—An unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014. Euro Surveillance, 19(41). https://doi.org/ 10.2807/1560-7917.es2014.19.41.20929. Sanz Cortes, M., Rivera, A. M., Yepez, M., Guimaraes, C. V., Diaz Yunes, I., Zarutskie, A., … Parra Saavedra, M. (2018). Clinical assessment and brain findings in a cohort of mothers, fetuses and infants infected with ZIKA virus. American Journal of Obstetrics and Gynecology, 218(4), 440.e1–440. e436. https://doi.org/10.1016/j.ajog.2018.01.012.

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Schuler-Faccini, L., Ribeiro, E. M., Feitosa, I. M., Horovitz, D. D., Cavalcanti, D. P., Pessoa, A., … Sanseverino, M. T. (2016). Possible association between Zika virus infection and microcephaly—Brazil, 2015. MMWR—Morbidity and Mortality Weekly Report, 65(3), 59–62. https://doi.org/ 10.15585/mmwr.mm6503e2. Shiu, C., Starker, R., Kwal, J., Bartlett, M., Crane, A., Greissman, S., … Curry, C. L. (2018). Zika virus testing and outcomes during pregnancy, Florida, USA, 2016. Emerging Infectious Diseases, 24(1), 1–8. https://doi.org/10.3201/eid2401.170979. Silva, A. A., Barbieri, M. A., Alves, M. T., Carvalho, C. A., Batista, R. F., Ribeiro, M. R., … Bettiol, H. (2018). Prevalence and risk factors for microcephaly at birth in Brazil in 2010. Pediatrics, 141(2). https://doi.org/10.1542/peds.2017-0589. Surveillance (SoH), & Ministry of Health of Brazil (MoHoB). (2019). Ministry of Health of Brazil, Secretariat of Health Surveillance. Epidemiological Bulletin, 22. https://portalarquivos2.saude.gov.br/images/pdf/2019/setembro/11/BE-arbovirose-22.pdf. Tappe, D., Rissland, J., Gabriel, M., Emmerich, P., Gunther, S., Held, G., … Schmidt-Chanasit, J. (2014). First case of laboratory-confirmed Zika virus infection imported into Europe, November 2013. Euro Surveillance, 19(4). https://doi.org/10.2807/1560-7917.es2014.19.4.20685. Tognarelli, J., Ulloa, S., Villagra, E., Lagos, J., Aguayo, C., Fasce, R., … Fernandez, J. (2016). A report on the outbreak of Zika virus on Easter Island, South Pacific, 2014. Archives of Virology, 161(3), 665–668. https://doi.org/10.1007/s00705-015-2695-5. van der Linden, V., Pessoa, A., Dobyns, W., Barkovich, A. J., Junior, H. V., Filho, E. L., … Moore, C. A. (2016). Description of 13 infants born during October 2015-January 2016 with congenital Zika virus infection without microcephaly at birth—Brazil. MMWR. Morbidity and Mortality Weekly Report, 65(47), 1343–1348. https://doi.org/10.15585/mmwr.mm6547e2. Vieira, C., Machado, L. C., Pena, L. J., de Morais Bronzoni, R. V., & Wallau, G. L. (2019). Spread of two Zika virus lineages in Midwest Brazil. Infection, Genetics and Evolution, 75. https://doi.org/10.1016/j.meegid.2019.103974, 103974. Vilela, A. P., Figueiredo, L. B., dos Santos, J. R., Eiras, A. E., Bonjardim, C. A., Ferreira, P. C., & Kroon, E. G. (2010). Dengue virus 3 genotype I in Aedes aegypti mosquitoes and eggs, Brazil, 2005-2006. Emerging Infectious Diseases, 16(6), 989–992. https://doi.org/10.3201/eid1606.091000. World Health Organization (WHO). (2016). WHO statement on the frst meeting of the International Health Regulations (2005) Emergency Committee on Zika virus and observed increase in neurological disorders and neonatal malformations, February 1, 2016. http://www.who.int/mediacentre/news/ statements/2016/1st-emergency-committee-zika/en/. (Accessed 26 February 2016). World Health Organization (WHO), & Pan American Health Organization (PAHO). (2015). Epidemiological alert: Zika virus infection. 7 maio. Zammarchi, L., Tappe, D., Fortuna, C., Remoli, M. E., Gunther, S., Venturi, G., … Schmidt-Chanasit, J. (2015). Zika virus infection in a traveller returning to Europe from Brazil, March 2015. Euro Surveillance, 20(23). https://doi.org/10.2807/1560-7917.es2015.20.23.21153.

Chapter 32

Zika virus and the Middle East Eyal Meltzera,b a

The Center for Geographic Medicine, Department of Medicine C, The Chaim Sheba Medical Center, Tel Hashomer, Israel, b Sackler Faculty of Medicine

at the Tel Aviv University, Tel Aviv, Israel

Abbreviations WHO ZIKV

World Health Organization Zika virus

Introduction Throughout historical times, the Middle East had been an important hub of old-world human movement. Land and maritime trade routes, immigration, and pilgrimage routes all intersect in the region, with the Hajj and Umrah drawing millions of pilgrims annually from all around the globe. World Bank data suggests that annual tourist entries to Middle East countries approach 100,000,000, while airfreight volume approaches 40 billion ton-km (Anonymous, 2019). Tragically, the Middle East has been and still is today the scene of armed conflict, which had resulted in large-scale refugee crises. Pestilence often follows War, and the Middle East saw epidemics ranging from bubonic plague in Napoleonic times to current outbreaks of poliomyelitis and cutaneous leishmaniasis in Syria (Mbaeyi et al., 2017; Rehman et al., 2018; Youssef et al., 2019) and cholera in Yemen (Spiegel et al., 2019). The advent of the 2016 Zika virus (ZIKV) near-global outbreak led to concern for its potential spread to the Middle East. The aim of this review is to detail the data relating to this potential and to the actual status of travel-related ZIKV in the region.

Definitions The Middle East is a somewhat fluid geographical concept and its meaning changed several times. For the purpose of this review, the region studied included the Levant coast from Turkey to Egypt, the Arabian Peninsula, Syria, Iraq, and Iran. A systematic literature review was performed on the Medline database. The search strategy utilized the keywords Zika virus, Aedes Aegypti, Aedes albopictus, Middle East, and also each country of the region. The bibliography of identified articles was also evaluated for additional studies. The databases of ProMED-mail and ProMED-MENA (available at https://promedmail.org/promed-posts/) were further analyzed for reports of ZIKV in Middle Eastern countries. In Addition, email contacts requesting information on diagnosed cases of ZIKV were sent to health ministries in the included region and to the WHO’s relevant regional offices.

ZIKV vectors in the Middle East ZIKV is transmitted mainly by mosquito bite although horizontal and vertical person-to-person/mother-to-fetus transmission plays a secondary albeit important role in its epidemiology. Reproductive concerns about adverse fetal outcomes are perhaps the leading concern of infected patients. The likelihood of imported outbreaks of ZIKV hinges on the presence of competent vectors. Most of the Middle East lies in the subtropics, with the southern portion of the Arabian Peninsula being in the tropical region. Both vectors of ZIKV: A. aegypti and A. albopictus are present in the Middle East but with very different distributions (Fig. 1). Aedes aegypti: A. aegypti is the most important and the more efficient vector of ZIKV, as well as other arboviruses. It is present in several regions of the western Arabian Peninsula, including Jeddah—the largest seaport and main entry point for pilgrims in Saudi Arabia, and the holy cities of Makkah and Al-Madinah (Hassan, Kenawy, Al Ashry, & Shobrak, 2017; Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00032-8 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Aedes aegypti and Aedes Albopictus distribution in the Middle East.

Kheir, Alahmed, Al Kuriji, & Al Zubyani, 2010). The significance of this population is evidenced by outbreaks of dengue fever that had occurred in both pilgrimage destinations as well as in other regions of western Saudi Arabia (Al-Raddadi et al., 2019; Ashshi et al., 2017; Hashem et al., 2018; Khan et al., 2008; Organji, Abulreesh, & Osman, 2017). A. aegypti is also present in neighboring Yemen and was associated with an outbreak of Chikungunya virus in Hodeida in 2011, as well as with proven dengue activity in Yemen since at least 1984 and up to the present civil war (Alghazali et al., 2019; Jimenez-Lucho, Fisher, & Saravolatz, 1984; Madani et al., 2013; Malik et al., 2014; Zayed et al., 2012). As its name suggests, A. aegypti had been recognized in Egypt many years ago and was associated with large outbreaks of dengue in the early 20th century (Kamal, 1928). The vector reemerged in Egypt in recent years and is again associated with dengue outbreaks: initially in the southern Nile valley (Ghweil, Osman, Khodeary, Okasha, & Hassan, 2019) and most recently in the Red Sea region (Saifullin et al., 2018). Historical data suggests that A. aegypti had previously circulated in Iran and Iraq (Foote & Cook, 1959), however, the only Aedes species currently documented is A. caspius (Bagheri et al., 2015; Hantosh, Hassan, Ahma, & Al-fatlawy, 2012). It is important to note the evolving aspect of A. aegypti distribution in the Middle East: historically, it was to be found in most regions of the Mediterranean as well as the black sea regions, and had disappeared from most of its former range as an effect of widespread DDT use (Curtin, 1967). Reintroduction is likely to occur, laying the foundation for high efficiency transmission of arboviruses. Proof of this potential was seen in recent years in Turkey, where A. aegypti was reestablished in the Black Sea seaboard (Akiner, Demirci, Babuadze, Robert, & Schaffner, 2016; Akiner et al., 2019). A. albopictus: Whereas A. aegypti is currently present only in specific regions of the Middle East, A. albopictus is widely distributed. This mosquito was first detected in Israel in 2002 (Leshem, Bin, Shalom, Perkin, & Schwartz, 2012), and later in Syria, Lebanon, Iran, and Turkey (Ducheyne et al., 2018; Haddad, Harbach, Chamat, & BouharounTayoun, 2007). A. albopictus had been implicated in limited outbreaks of arboviruses in Europe, including two relatively large outbreaks of chikungunya (CHIKV) in Emilia-Romagna and in Lazio, Italy (Angelini et al., 2008; Lindh et al., 2019). Its potential to cause outbreaks of DENV, CHIKV, and ZIKV in the Middle East had been discussed in the Israeli, and in the wider Middle Eastern context (Ducheyne et al., 2018; Leshem et al., 2012), however, to date no such outbreaks have eventuated. In France, studies have suggested that local A. albopictus strains require high viremic blood meal in order to result in viral propagation and migration to the mosquito’s salivary glands (Vazeille et al., 2019). It is possible that the fitness of A. albopictus strains circulating in the Middle East to transmit ZIKV is similarly low, but no studies have assessed this question. Still, small-scale autochthonous “mini-outbreaks” of ZIKV transmitted by A. albopictus are possible—as had been the case in Southern France in 2019 (Giron et al., 2019).

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Travel-related ZIKV in the Middle East Current recommendations for testing returning travelers for ZIKV Although travel-related cases of ZIKV antedated the ZIKV epidemic in Latin America by many years, the magnitude of the epidemic and the recognition of the virus’s association with adverse fetal outcomes first created a high demand for ZIKV testing. The diagnostic approach was perfected with time and recommendations relating to who and how should be tested are available from American CDC (CDC, 2019), as well as health authorities in the UK (Public Health England (PHE), 2020), Israel (Ministry of Health, Israel, 2019) and elsewhere. Current recommendations from these bodies suggest testing for ZIKV all travelers with symptoms suggestive of ZIKV, including pregnant women, their spouses, and those planning reproduction. Due to insufficient specificity, all guidelines suggest against serological testing of asymptomatic returning travelers. Rather, couples are advised to delay reproduction plans (by at least 8 and 12 weeks for returning female and male travelers respectively) after travel to ZIKV endemic areas, while asymptomatic pregnant women are advised to ensure optimal antenatal follow-up. The CDC does, however, suggest that Nucleic Acid-based testing may still be considered up to 12 weeks after travel. Following is a detailed review of travel-related ZIKV in the Middle East; data are summarized in Table 1.

TABLE 1 Summary of data on Zika Virus in Middle Eastern countries.

Endemic A. aegypti

Outbreaks of A. aegypti associated arboviruses

Active refugee crisis/civil war

ZIKV cases in returning travelers

Autochthonous ZIKV cases

ND

ND

–

–

–

–

Egypt

+

++

++

–

–

–

Iran

–

–

–

–

–

–

Iraq

–

–

–

+

–

–

Israel

+

–

–

–

++

–

Jordan

+

–

–

+

–

–

Kuwait

–

–

–

–

–

–

Lebanon

+

–

–

+

–

–

Oman

+

++

++

–

–

–

Palestinian Authority

+

–

–

–

–

–

Qatar

–

–

–

–

–

–

Saudi Arabia

+

++

++

–

–

–

Syria

+

–

–

+

–

–

Turkey

+

+

–

+

+

–

United Arab Emirates

–

–

–

–

–

–

Yemen

+

++

++

+

–

–

Country

Endemic ZIKV vectors

Bahrain

ND: no data. +: anecdotal data/single report. ++: repeated reports/outbreaks.

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Israel Most data on travel-related ZIKV in the Middle East involves Israeli travelers, despite Israel’s small geographical size and the fact that its population comprises only about 2% of the total Middle Eastern population. However, Israelis travel extensively, including to ZIKV endemic and epidemic countries. The earliest ZIKV cases in Israel were diagnosed in December 2015 (Meltzer, Leshem, Lustig, Gottesman, & Schwartz, 2016). These early cases were all diagnosed in travelers to South and Central America. Often, cases were diagnosed very shortly after ZIKV was recognized in the traveled country. Some cases also provided evidence of a very high local infection density, which reflected the ascending arm of the epidemic curve. Thus, a family of four, that were exposed to ZIKV epidemic area (Girardot, Colombia) for only one weekend in January 2016, resulted in three cases of ZIKV infection, including one case of probable encephalitis (Meltzer, Leshem, et al., 2016). For a short period in 2016, ZIKV was the most frequent imported arbovirus and had remained since the most tested-for arbovirus in returning Israeli travelers. However, in 2017 the emergence of Southeast Asia as a major source of imported ZIKV was documented (Meltzer, Lustig, & Schwartz, 2019). This probably reflected the declining arm of the Latin American epidemic curve but also increased awareness and vigilance for Asian cases of ZIKV. Infected travelers included the first recognized case of ZIKV acquired in Vietnam—a country that until that time was not known to harbor the virus (Meltzer, Lustig, et al., 2016). However, most Asian cases were acquired in Thailand and the Philippines. The incidence of ZIKV in travelers to Thailand was calculated to be 0.97 cases/100,000 travel entries (Leshem, Lustig, Brosh-Nissimov, Paran, & Schwartz, 2019) which is much higher than the risk for most other travel-related diseases in travelers to Thailand, including malaria and Japanese encephalitis. On the clinical level, ZIKV was rarely asymptomatic in travelers: most reported symptoms, which in some cases included neurological/psychiatric symptoms, as well as one case of encephalitis (Meltzer, Leshem, et al., 2016). To date, there were no cases of congenital ZIKV infection, nor of Guillain-Barre syndrome in Israeli travelers.

Turkey Turkey is the only Middle Eastern country except Israel to report a case of clinical ZIKV infection in travelers, with a single case-couple imported from Cuba (Sezen, Yildirim, Kultur, Pehlivanoglu, & Menemenlioglu, 2018). Turkey is one of the largest countries in the Middle East, with a population of nearly 82 million; it is a major international tourist destination with more than 36 million tourist entries, which is about 10-times higher than Israel, however, outbound tourism is at about 0.1 trips/person*year, which is nearly 10 times lower than the Israeli average. This is perhaps what underlies the lack of more reports of ZIKV in Turkey. Concern on the potential importation of ZIKV to Turkey was raised considering the prevalence of Aedes mosquitoes in the country, however, no local transmission had been reported to date.

Travel-related ZIKV in the rest of the Middle East No other reports on ZIKV infection in Middle Eastern travelers have been found in the medical literature. A 2016 multinational collaborative review on ZIKV surveillance in the WHO’s Eastern Mediterranean Region also reported no such cases (Minh et al., 2016). Attempts by the author to enquire at each health ministry have resulted in no additional data on cases. Also, communications with authors who published on Aedes mosquitoes or Flaviviruses in several Middle Eastern countries (Iran, Saudi Arabia, and Egypt), where tests for ZIKV are available, however, none were aware of other cases (Eyal Meltzer, personal communications). It is not clear whether this “absence of evidence” should be interpreted as “evidence of absence”: it is possible that some ZIKV cases are undiagnosed due to lack of clinical awareness, and absence of or limited access to diagnostic facilities. In some conflict regions (e.g., Syria, Iraq, and Yemen) an absence of surveillance and low availability of health services make arthropod-borne disease outbreaks more likely. However, it is unlikely that a large-scale autochthonous ZIKV outbreak would have occurred and had gone unnoticed.

What can we learn from the absence of Zika in the Middle East? Even at the early phase of the ZIKV epidemic in the Americas, concern for its importation and spread to the Middle East was raised. After all, the region sees an immense volume of commerce and travel to Zika endemic/epidemic countries. However, this had not happened: what does this “absence” suggest about the epidemiology of ZIKV? It is likely that rather than considering only the prevalence of Aedes vectors, several other sociodemographic parameters should coincide to enable large-scale ZIKV outbreaks (Fig. 2 and Table 1). A high-density A. aegypti population is certainly

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FIG. 2 Suggested conditions required to create large-scale ZIKV outbreaks.

required; however, to create an epidemic, a large population living in poor conditions (due to poverty, internal displacement, or cross-border refugee crises) is required, as well as the current Asian lineage ZIKV strain. Thus, when ZIKV was introduced to the Southern United States, an area with endemic A. aegypti and A. albopictus, only a small outbreak occurred in Florida which was swiftly contained. Similarly, whereas poverty and a high vector burden are present in much of sub-Saharan Africa, an epidemic of ZIKV was only recorded there (in Angola) after the Asian strain was introduced from South America (Hill et al., 2019); other urban centers in Africa remain at high risk (Kraemer et al., 2017). In the Middle East, The Kingdom of Saudi Arabia is not a poor, densly populated country, but it does host some of the largest mass gatherings on Earth, i.e., the Hajj and Umrah. Historically, this conjunction of people from many origins was conducive to outbreaks such as cholera (Mackie, 1893). In the 21st century, several million pilgrims arrive each year from all over the world, including large contingents from Africa and Southeast Asia (although the numbers from South America are small) (Elachola, Gozzer, Zhuo, & Memish, 2016). The Hejaz, where the holy cities of Makkah and Madinah are located and where most pilgrims reside in large tent encampments, is endemic to A. aegypti and had seen outbreaks of dengue fever (Alhaeli, Bahkali, Ali, Househ, & El-Metwally, 2016; Al-Raddadi et al., 2019). That these outbreaks have not involved the pilgrim population, and that no local transmission of ZIKV had been documented in Hajjis probably reflect an investment in public health measures including vector control during the pilgrimage season. Two countries in the Middle East: Egypt and Yemen do have a conjunction of poverty and emerging A. aegypti populations. Both may therefore be at special risk for ZIKV epidemics, should an introduction event of the Asian strain virus occur.

Conclusions The Middle East was considered an area of risk for ZIKV outbreaks; however, these have not eventuated. Travelers, especially from Israel, have been diagnosed with imported ZIKV infection. Reports in travelers have closely mirrored the epidemic curve in Latin America but also led to new insight into the presence and burden of ZIKV in Southeast Asia. Whereas small-scale autochthonous transmission of ZIKV is possible in many Middle Eastern countries where A. albopictus is prevalent, it is poor countries with established/emerging A. aegypti infestation that are at risk for large-scale ZIKV outbreaks. The Red Sea region is an area of higher risk; this is especially true of Yemen, where civil war, a large-scale refugee crisis, active human trafficking from Africa, and A. aegypti all coincide.

Policy and procedures Testing returning travelers for Zika virus Consensus among various national bodies including the US CDC includes the following recommendations: l l

ZIKV testing should be performed on returning travelers with symptoms suggestive of the infection. Serological testing should not be offered to asymptomatic travelers including pregnant women.

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Nucleic acid-based tests may be considered in asymptomatic women up to 12 weeks from exposure. Delaying pregnancy for at least 8 weeks for women and 12 weeks for men who had traveled to a ZIKV endemic area. Updated list of areas at risk is maintained by the CDC: https://wwwnc.cdc.gov/travel/page/zika-information.

Mini-dictionary of terms Middle East: World region variously defined to include the countries of Southwestern Asia and Northern Africa. Hajj: An annual Islamic pilgrimage to Makkah, Saudi Arabia, during a prespecified time in the Muslim lunar calendar. Umrah: Pilgrimage to Makkah that is undertaken at any time of the year except the Hajj.

Key facts on Zika virus in travelers from the Middle East l l l

l l

Commerce, pilgrimage, and tourism make the Middle East one of the most interconnected world regions. The Zika virus vectors Aedes aegypti and Aedes albopictus are endemic to many Middle Eastern countries. As the Zika virus South American epidemic evolved, the signal among Middle Eastern travelers was almost simultaneous with the local recognition of the virus and sometimes even preceded it. Most reports of travel-related Zika involved Israeli travelers, with a single report from Turkey. No autochthonous transmission had been documented in the Middle East to date. However, countries such as Egypt and Yemen with large, impoverished or displaced populations, with endemic Aedes aegypti, and a history of recent dengue outbreaks should be considered at risk for ZIKV outbreaks.

Summary points l l

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This chapter focuses on the epidemiology of Zika virus infection in the Middle East. For the purposes of the chapter, the term Middle East relates to the Eastern Mediterranean seaboard, the Arabian Peninsula through to Iran. Reports of ZIKV in returning travelers to the Middle East began almost concomitantly with the South American outbreak. Travelers can serve as sentinels for outbreak epidemiology and recognition of cases in travelers was correlated with outbreak intensity and even preceded local recognition. The Middle East is one of the most interconnected world regions by land, sea, and air, and hosts some of the largest mass gatherings in the world, e.g., the Hajj. Most countries are endemic to one of the ZIKV mosquito vectors and are therefore at risk for ZIKV importation. However, conditions that emulate those of epidemic Latin American countries, vis large impoverished/displaced populations, poor health resources, and endemic Aedes aegypti exist in Egypt and Yemen, which should be considered areas of excess risk.

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Current and future distribution of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in WHO Eastern Mediterranean Region. International Journal of Health Geographics, 17(1), 4. https://doi. org/10.1186/s12942-018-0125-0. Elachola, H., Gozzer, E., Zhuo, J., & Memish, Z. A. (2016). A crucial time for public health preparedness: Zika virus and the 2016 Olympics, Umrah, and Hajj. Lancet, 387(10019), 630–632. https://doi.org/10.1016/S0140-6736(16)00274-9. Foote, R. H., & Cook, D. R. (1959). Mosquitoes of medical importance: Agricultural research service. U.S. Department of Agriculture. Ghweil, A. A., Osman, H. A., Khodeary, A., Okasha, A., & Hassan, M. H. (2019). Relative frequency of acute pancreatitis from dengue outbreaks as a late complication, in Egypt. Virus Disease, 30(4), 498–503. https://doi.org/10.1007/s13337-019-00556-z. Giron, S., Franke, F., Decoppet, A., Cadiou, B., Travaglini, T., Thirion, L., … Leparc-Goffart, I. (2019). 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Mosquitoes (Diptera: Culicidae) of the Western Coastal Area, Kingdom of Saudi Arabia: species composition, abundance, diversity and medical importance. Journal of the Egyptian Society of Parasitology, 47(1), 167–176. Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/30157346. Hill, S. C., Vasconcelos, J., Neto, Z., Jandondo, D., Ze-Ze, L., Aguiar, R. S., … Faria, N. R. (2019). Emergence of the Asian lineage of Zika virus in Angola: An outbreak investigation. The Lancet Infectious Diseases, 19(10), 1138–1147. https://doi.org/10.1016/s1473-3099(19)30293-2. Ministry of Health, Israel. (2019). Zika virus related disease (Hebrew). Retrieved from: https://www.health.gov.il/Subjects/disease/zika/Pages/faq.aspx. Jimenez-Lucho, V. E., Fisher, E. J., & Saravolatz, L. D. (1984). Dengue with hemorrhagic manifestations: An imported case from the Middle East. The American Journal of Tropical Medicine and Hygiene, 33(4), 650–653. https://doi.org/10.4269/ajtmh.1984.33.650. Kamal, H. (1928). The 1927 epidemic of Dengue in Egypt. British Medical Journal, 1(3521), 1104–1106. https://doi.org/10.1136/bmj.1.3521.1104-a. Khan, N. A., Azhar, E. I., El-Fiky, S., Madani, H. H., Abuljadial, M. A., Ashshi, A. M., … Hamouh, E. A. (2008). Clinical profile and outcome of hospitalized patients during first outbreak of dengue in Makkah, Saudi Arabia. Acta Tropica, 105(1), 39–44. https://doi.org/10.1016/j. actatropica.2007.09.005. Kheir, S. M., Alahmed, A. M., Al Kuriji, M. A., & Al Zubyani, S. F. (2010). Distribution and seasonal activity of mosquitoes in al Madinah Al Munwwrah, Saudi Arabia. Journal of the Egyptian Society of Parasitology, 40(1), 215–227. Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/20503600. Kraemer, M. U. G., Brady, O. J., Watts, A., German, M., Hay, S. I., Khan, K., & Bogoch, I. I. (2017). Zika virus transmission in Angola and the potential for further spread to other African settings. Transactions of the Royal Society of Tropical Medicine and Hygiene, 111(11), 527–529. https://doi.org/ 10.1093/trstmh/try001. Leshem, E., Bin, H., Shalom, U., Perkin, M., & Schwartz, E. (2012). Risk for emergence of dengue and chikungunya virus in Israel. Emerging Infectious Diseases, 18(2), 345–347. https://doi.org/10.3201/eid1802.111648. Leshem, E., Lustig, Y., Brosh-Nissimov, T., Paran, Y., & Schwartz, E. (2019). Incidence of laboratory-confirmed Zika in Israeli travelers to Thailand: 2016–2019. Journal of Travel Medicine, 26(7). https://doi.org/10.1093/jtm/taz057. Lindh, E., Argentini, C., Remoli, M. E., Fortuna, C., Faggioni, G., Benedetti, E., … Venturi, G. (2019). The Italian 2017 outbreak Chikungunya virus belongs to an emerging Aedes albopictus—Adapted virus cluster introduced from the Indian subcontinent. Open Forum Infectious Diseases, 6(1). https://doi.org/10.1093/ofid/ofy321. ofy321. Mackie, J. (1893). Cholera at Mecca and quarantine in Egypt. British Medical Journal, 2(1700), 222–223. https://doi.org/10.1136/bmj.2.1700.222. Madani, T. A., Abuelzein el, T. M., Al-Bar, H. M., Azhar, E. I., Kao, M., Alshoeb, H. O., & Bamoosa, A. R. (2013). Outbreak of viral hemorrhagic fever caused by dengue virus type 3 in Al-Mukalla, Yemen. BMC Infectious Diseases, 13, 136. https://doi.org/10.1186/1471-2334-13-136. Malik, M. R., Mnzava, A., Mohareb, E., Zayed, A., Al Kohlani, A., Thabet, A. A., & El Bushra, H. (2014). Chikungunya outbreak in Al-Hudaydah, Yemen, 2011: Epidemiological characterization and key lessons learned for early detection and control. Journal of Epidemiology and Global Health, 4(3), 203–211. https://doi.org/10.1016/j.jegh.2014.01.004. Mbaeyi, C., Ryan, M. J., Smith, P., Mahamud, A., Farag, N., Haithami, S., … Ehrhardt, D. (2017). Response to a large polio outbreak in a setting of conflict—Middle East, 2013–2015. MMWR—Morbidity and Mortality Weekly Report, 66(8), 227–231. https://doi.org/10.15585/mmwr.mm6608a6. Meltzer, E., Leshem, E., Lustig, Y., Gottesman, G., & Schwartz, E. (2016). The clinical spectrum of Zika virus in returning travelers. The American Journal of Medicine, 129(10), 1126–1130. https://doi.org/10.1016/j.amjmed.2016.04.034.

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Meltzer, E., Lustig, Y., Leshem, E., Levy, R., Gottesman, G., Weissmann, R., … Schwartz, E. (2016). Zika virus disease in traveler returning from Vietnam to Israel. Emerging Infectious Diseases, 22(8), 1521–1522. https://doi.org/10.3201/eid2208.160480. Meltzer, E., Lustig, Y., & Schwartz, E. (2019). Zika virus in Israeli travelers: emergence of Asia as a major source of infection. The American Journal of Tropical Medicine and Hygiene, 100(1), 178–182. https://doi.org/10.4269/ajtmh.18-0379. Minh, N. N., Huda, Q., Asghar, H., Samhouri, D., Abubakar, A., Barwa, C., … Malik, M. (2016). Zika virus: No cases in the Eastern Mediterranean Region but concerns remain. Eastern Mediterranean Health Journal, 22(5), 350–355. Retrieved from: http://www.ncbi.nlm.nih.gov/pubmed/27553402. Organji, S. R., Abulreesh, H. H., & Osman, G. E. (2017). Circulation of Dengue virus serotypes in the city of Makkah, Saudi Arabia, as determined by reverse transcription polymerase chain reaction. Canadian Journal of Infectious Diseases and Medical Microbiology, 2017, 1646701. https://doi.org/ 10.1155/2017/1646701. Rehman, K., Walochnik, J., Mischlinger, J., Alassil, B., Allan, R., & Ramharter, M. (2018). Leishmaniasis in Northern Syria during civil war. Emerging Infectious Diseases, 24(11), 1973–1981. https://doi.org/10.3201/eid2411.172146. Saifullin, M. A., Laritchev, V. P., Grigorieva, Y. E., Zvereva, N. N., Domkina, A. M., Saifullin, R. F., … Butenko, A. M. (2018). Two cases of Dengue fever imported from Egypt to Russia, 2017. Emerging Infectious Diseases, 24(4). https://doi.org/10.3201/eid2404.172131. Sezen, A. I., Yildirim, M., Kultur, M. N., Pehlivanoglu, F., & Menemenlioglu, D. (2018). Cases of Zika virus infection in Turkey: Newly married couple € returning from Cuba. Mikrobiyoloji Bulteni, 52(3), 308–315. https://doi.org/10.5578/mb.66991. Spiegel, P., Ratnayake, R., Hellman, N., Ververs, M., Ngwa, M., Wise, P. H., & Lantagne, D. (2019). Responding to epidemics in large-scale humanitarian crises: A case study of the cholera response in Yemen, 2016–2018. BMJ Global Health, 4(4). https://doi.org/10.1136/bmjgh-2019-001709, e001709. Public Health England (PHE). (2020). Zika virus: Sample testing advice. Retrieved from: https://www.gov.uk/guidance/zika-virus-sample-testing-advice. Vazeille, M., Madec, Y., Mousson, L., Bellone, R., Barre-Cardi, H., Sousa, C. A., … Failloux, A. B. (2019). Zika virus threshold determines transmission by European Aedes albopictus mosquitoes. Emerging Microbes and Infections, 8(1), 1668–1678. https://doi.org/10.1080/22221751.2019.1689797. Youssef, A., Harfouch, R., El Zein, S., Alshehabi, Z., Shaaban, R., & Kanj, S. S. (2019). Visceral and cutaneous Leishmaniases in a City in Syria and the effects of the Syrian Conflict. The American Journal of Tropical Medicine and Hygiene, 101(1), 108–112. https://doi.org/10.4269/ajtmh.18-0778. Zayed, A., Awash, A. A., Esmail, M. A., Al-Mohamadi, H. A., Al-Salwai, M., Al-Jasari, A., … Mnzava, A. (2012). Detection of Chikungunya virus in Aedes aegypti during 2011 outbreak in Al Hodayda, Yemen. Acta Tropica, 123(1), 62–66. https://doi.org/10.1016/j.actatropica.2012.03.004.

Chapter 33

Genetic diversity of Zika virus in Thailand: How does this compare with other countries Atchara Phumeea and Padet Siriyasatienb a

Department of Medical Technology, School of Allied Health Sciences, Walailak University, Nakhon Si Thamarat, Thailand, b Vector Biology and Vector

Borne Disease Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Abbreviations Ae. C Cx. DENV E GBS JEV Kb MAFFT NCBI NS1 NS2A NS2B NS3 NS4A NS4B NS5 ORF PCR prM RNA RT-PCR WGS WHO WNV YFV ZIKV

Aedes Capsid Culex Dengue virus Envelope Guillain-Barre syndrome Japanese encephalitis virus Kilobase Multiple Alignment using Fast Fourier Transform National Center for Biotechnology Information Nonstructural protein 1 Nonstructural protein 2A Nonstructural protein 2B Nonstructural protein 3 Nonstructural protein 4A Nonstructural protein 4B Nonstructural protein 5 Open reading frame Polymerase chain reaction Premembrane/membrane Ribonucleic acid Reverse transcription polymerase chain reaction Whole genome sequencing World Health Organization West Nile virus Yellow fever virus Zika virus

Introduction Zika virus (ZIKV) is an emerging mosquito-borne positive stranded RNA virus of the genus Flavivirus and family Flaviviridae, related to dengue (DENV), Japanese encephalitis (JEV), yellow fever (YFV), and West Nile (WNV) viruses. It is transmitted to humans through the bite of infected mosquitoes. ZIKV was first isolated from a febrile sentinel rhesus monkey, from pooled specimens of Aedes africanus mosquitoes, and from humans in Uganda in 1947, 1948, and 1952, respectively (Dick, 1952; Dick, Kitchen, & Haddow, 1952). Presently, ZIKV strains have been classified into two major lineages, known as African and Asian/American types (Barzon, Trevisan, Sinigaglia, Lavezzo, & Palu`, 2016; Musso &

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00033-X Copyright © 2021 Elsevier Inc. All rights reserved.

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Gubler, 2016). ZIKV requires vertebrate hosts and mosquito vectors for replication. However, ZIKV can also be transmitted by several routes (Grischott, Puhan, Hatz, & Schlagenhauf, 2016) such as blood transfusion (Marano, Pupella, Vaglio, Liumbruno, & Grazzini, & G., 2016), sexual transmission (Musso et al., 2015), and perinatal transmission (Petersen et al., 2016). Most of the ZIKV infections are asymptomatic, but some can present with an influenza-like illness or with neurological abnormalities (Haby, Pinart, Elias, & Reveiz, 2018). ZIKV infection in pregnancy can cause a birth defect called microcephaly. It may play a role in the etiology of Guillain-Barre syndrome (GBS) (Adachi & NielsenSaines, 2018). ZIKV had been neglected and only a little was known. We did not have good understanding about its transmission cycle between monkeys, mosquitoes, and humans. However, after a number of outbreaks in 2013 reported in French Polynesia and in other Pacific Islands (Cao-Lormeau et al., 2014), including the one back in 2007 reported in the Yap State of Micronesia (Duffy et al., 2009), and the largest of all in 2016 reported in Brazil and in the region of the Americas (Hennessey, Fischer, & Staples, 2016), researchers started to heavily investigate ZIKV around the world, and Thailand was one of them. In order to understand how the ZIKV transmits and circulates, it would be essential to study the genome sequences of the virus. A standard technique to obtain the sequence data of ZIKV is the reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR assay. Recently, with the high-throughput sequencing technologies and whole genome sequencing (WGS), the full sequence of virus genomes was obtained. In this chapter, we focus on the history and outbreak patterns of ZIKV in Thailand. The genetic diversity of ZIKV in Thailand and other countries is discussed. The data of the ZIKV sequences in mosquitoes would lead to a better understanding of the association between vector competence and ZIKV phylogeny. This will be helpful for further development of control strategies and public health measures.

Timeline of the ZIKV outbreaks in Thailand ZIKV was first identified in a sentinel of rhesus macaque monkeys in the Zika forest of Uganda back in 1947. The virus was first isolated from a pool of Ae. africanus mosquitoes collected in the same site (Dick, 1952) and was detected in humans in 1952 (Dick, Kitchen, & Haddow, 1952). During 1969–1983, there were reports on ZIKV geographical distribution in Africa and then in Asia, including Malaysia, India, Pakistan, Vietnam, the Philippines, and Indonesia (Darwish, Hoogstraal, Roberts, Ahmed, & Omar, 1983; Lanciotti et al., 2008; Marchette, Garcia, & Rudnick, 1969; Olson, Ksiazek, Suhandiman, & Triwibowo, 1981). However, ZIKV infection did not receive much attention until 2007, when the outbreak affected approximately 75% of the residents of the Yap State, Federated States of Micronesia (Duffy et al., 2009). In 2013, outbreaks of ZIKV infection occurred in many areas including French Polynesia, Cook Islands, Easter Island, Vanuatu, and the Solomon Islands (Musso, 2015). Genetic studies have been used to demonstrate the patterns of ZIKV spreading. Several reports suggested that the phylogenetic analysis of ZIKV sequences in Brazil in 2015 belonged to the Asian lineage, which were reported in the outbreaks on the Pacific Islands (Campos, Bandeira, & Sardi, 2015; Zanluca et al., 2015). Since the outbreaks of ZIKV in Brazil during 2015–2016, ZIKV has rapidly been spreading in Africa, the Americas, South-East Asia, and Western Pacific regions. ZIKV infections in pregnancy can cause microcephaly in newborns (Bogoch et al., 2016; CaoLormeau et al., 2014; WHO, 2019). ZIKV in the South-East Asia Region was first isolated from Ae. aegypti mosquitoes, which were caught in the town of Bentong, Malaysia in 1966 (Marchette, Garcia, & Rudnick, 1969) (Fig. 1). The genetic sequence of ZIKV-Malaysian isolate was compared with other isolates and it was classified as Asian lineage (Woon et al., 2019). In 1977, the first case of human ZIKV infection in South-East Asia was reported in Central Java, Indonesia (Olson, Ksiazek, Suhandiman, & Triwibowo, 1981). In 2016, there were reports of ZIKV outbreaks in Singapore (Singapore Zika Study Group, 2017). The first description of ZIKV infection in Thailand was in 1954 in sera of Thai residents. It was confirmed by using neutralizing antibodies against ZIKV (Pond, 1963). In 2010, sporadic cases of ZIKV infection in Thailand were reported (Buathong, 2018; Buathong, Loasiritaworn, Kanjanasombut, Ruchiseesarod, & Wacharaplusadee, 2019). Several ZIKV cases were found in travelers returning from Thailand, Germany (Tappe et al., 2014), Japan (Shinohara, 2014), Canada (Fonseca et al., 2014), and Taiwan (CDC, 2018). Buathong et al. (2015) described seven of the acute ZIKV infection cases from the Thai provinces of Sisaket, Phetchabun, Ratchaburi, and Lamphun, all confirmed by molecular or serological testing. Then the phylogenetic tree showed that these ZIKV belonged to Asian lineage (Buathong et al., 2015). During January 2016–December 2017, the surveillance report in Thailand investigated over 1698 confirmed cases of ZIKV infection (1121 cases in 2016 and 577 cases in 2017). The report revealed that 45% of cases were asymptomatic infections and 121 confirmed cases were found in pregnant women. The epidemic curve demonstrated a seasonal pattern with the highest incidence during rainy season. It was reported that after the peak of the ZIKV epidemic had passed, the peak of dengue outbreak followed approximately 6–8 weeks later in the two consecutive years (Buathong, 2018; Buathong, Loasiritaworn, Kanjanasombut, Ruchiseesarod, & Wacharaplusadee, 2019). In 2018, the WGS was performed on these two isolations from the Thai patients (Ellison et al., 2016). Thailand had reported two indigenous cases of ZIKV-related

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FIG. 1 ZIKV outbreak timeline. Represent the history of ZIKV worldwide (A) and Thailand 2016–18 (B).

microcephaly caused by the Asian lineage. This finding suggested that ZIKV can circulate in Asia and can cause microcephaly (Wongsurawat et al., 2018). Recently, the study of widespread long-term circulation of ZIKV in Thailand indicated that the ZIKV could have circulated since 2002 (Ruchusatsawat et al., 2019).

Vectors of ZIKV Several studies have reported the potential vectors of ZIKV. The virus was detected in mosquitoes, including Ae. africanus, Ae. aegypti, Ae. albopictus, Ae. apicocoargenteus, Ae. furcifer, Ae. vitattus, and Ae. luteocephalus, Ae. taylori, Ae. dalzieli, Ae. hirsutus, Ae. metallicus, Ae. unilinaetus, Ae. henselli, Culex perfuscus, Cx. quinquefasciatus, Armigeres sulalbatus, Mansonia uniformis, and Anopheles coustani (Diallo et al., 2014; Fagbami, 1979; Gardner, Chen, & Sarkar, 2016; Haddow, Williams, Woodall, Simpson, & Goma, 1964; Li, Wong, Ng, & Tan, 2012; McCrae & Kirya, 1982; Tawatsin et al., 2018; Wong, Li, Chong, Ng, & Tan, 2013). Ae. africanus was claimed to be the principal vector of ZIKV in Africa (Dick, Kitchen, & Haddow, 1952), whereas Ae. henselli was found to be the main vector for ZIKV outbreaks in the Yap Island (Duffy et al., 2009; Lanciotti et al., 2008), while Ae. aegypti was suspected to be the responsible vector for the outbreaks in the Americas, the Pacific region, and Asia (Musso, Nilles, & Cao-Lormeau, 2014). Although mosquitoes in the genus Aedes (Ae. aegypti as the principal vector) appear to be the predominant vector responsible for the transmission and spreading of ZIKV outbreaks worldwide, in Thailand, ZIKV detection in mosquitoes was carried out by using RT-PCR at the region of the nonstructural protein 5 (NS5) gene. ZIKV was detected in male, female, and larvae of Ae. aegypti and Cx. quinquefasciatus, and in both female and male Ar. subalbatus mosquitoes. ZIKV RNA was not detected in Ae. albopictus. The studies showed that the genetic variations of ZIKV in various geographical regions had 1%–6% sequence variations within the Asian lineage (Phumee et al., 2019; Tawatsin et al., 2018). Laboratory experiments of ZIKV infection in these mosquito species revealed that ZIKV could be vertically transmitted in Cx. quinquefasciatus and Ae. aegypti, but not in Ae. albopictus (Phumee et al., 2019). These findings could lead to the conclusion that Cx. quinquefasciatus, Ae. aegypti, and Ar. subalbatus may be the potential vectors for ZIKV transmission in Thailand.

ZIKV genetic variability in Thailand ZIKV belongs to the Flaviviridae family. The viral genome consists of a linear, positive-sense, and single-stranded RNA molecule with a single open reading frame (ORF), approximately 10–11 kilobases (Kb) in length. A polyprotein is cleaved into three structural proteins consisting of the capsid (C), premembrane/membrane (prM), envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5) (Kuno & Chang, 2007) (Fig. 2).

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FIG. 2 Schematic representation of the ZIKV genome. The genomic regions of ZIKV polyprotein: 50 -C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-2KNS4B-NS5-30 .

Genetic variations of ZIKV have been used to investigate in ZIKV how the virus spreads. Unfortunately, there is limited information about the genome sequence available on the database until now. This chapter explains the genetic variability of ZIKV in Thailand and the comparison with other countries. The NS5 phylogenetic tree of ZIKV in humans and in mosquitoes in Thailand revealed a similar finding to those from the past studies published in the Asian/American lineage (also previously reported in Thailand). However, the sequences from humans have a high sequence similarity to those from the mosquitoes in Thailand, Cambodia, the Philippines, Singapore, and Yap State of Micronesia. We need to bear in mind that our data in Thailand are limited and incomplete due to the low ZIKV genomic diversity and possible complication arising from the extensive global travels. Interestingly, there was no evidence of the African lineage reported in Thailand (Fig. 3).

Policy and procedures Phylogenetic tree construction The phylogenetic tree demonstrates the evolutionary relationships and the evolutionary history among various taxa, based on molecular biology technologies. It is constructed on the basis of the sequences in genes, full genomic data, or even amino acid sequences in different species. It is now recognized throughout biology as the best way to classify the biological diversity of organisms. In this chapter, we constructed the phylogenetic tree by using the IQ-TREE program. Firstly, we searched all ZIKV sequences based on the country and the global region in NCBI GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/). All ZIKV sequences were used to generate alignment gene sequences by using MAFFT (Tippmann, 2004), and then the alignments were manually adjusted by BioEdit Sequence Alignment Editor Version 7.2.5 (Hall, 1999). The phylogenetic trees were constructed using the maximum-likelihood method with IQ-TREE on the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) with 1000 ultrafast bootstrap replicates. The best-fit model of substitution was found using the auto function on the IQ-TREE web server (Nguyen, Schmidt, von Haeseler, & Minh, 2015). Finally, the reconstructed trees were visualized and edited using FigTree v1.4.4 software.

Mini-dictionary of terms Genetic diversity. The genetic variability or diversity present in a population of one species and in populations to adapt to the changing environments or different geographical regions. Arthropod-borne Flavivirus disease. A general term used to describe infective diseases caused by a group of Flaviviruses transmitted to humans by the bite of infected arthropod species. Lineage. A single continuous line of evolutionary descent present in the phylogenetic tree. Microcephaly. A baby’s head due to a brain abnormality or smaller than a normal head size when compared to babies of the same sex and age. Microcephaly can be caused by ZIKV infection during pregnancy. Guillain-Barre syndrome (GBS). A syndrome in which the body immune system attacks its own peripheral nervous system, leading to muscle weakness and paralysis. ZIKV may be one of the potential causes of GBS.

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FIG. 3 Phylogenetic analysis of NS5-ZIKV sequences. The maximum likelihood of the phylogenetic tree was constructed with IQ-TREE by using the maximum-likelihood method with 1000 ultrafast bootstrap replicates. The best-fit model of substitution was found using the auto function on the IQ-TREE web server. The sequences from humans and mosquitoes are indicated in blue and red, respectively.

Key facts of Zika virus (ZIKV) l

l l

l l

Zika fever, also known as ZIKV disease, is caused by a virus transmitted via mosquito bites and other routes, for example, perinatal, sexual, and blood-borne transmissions. Symptoms are generally asymptomatic or mild, but they can cause a severe birth defect such as microcephaly. The large outbreak of ZIKV infection in Brazil back in 2015 revealed its association with microcephaly and GuillainBarre syndrome. There is no vaccine or medication to treat ZIKV infection. The only way to prevent ZIKV infection is to develop and adopt effective preventative and control measures, minimizing mosquito bites.

Summary points l l l

l

l

ZIKV infection is an emerging and reemerging disease in Thailand. A large ZIKV infection outbreak in Thailand was reported during 2015–2016. The phylogenetic tree of NS5-ZIKV showed that ZIKV infection in humans and mosquitoes reported in Thailand was closely related to the Asian/American lineage. ZIKV infection in mosquitoes (Cx. quinquefasciatus, Ae. aegypti, and Ar. subalbatus) showed a higher genetic diversity than in humans. The sequencing data of ZIKV infection reported in Thailand are still limited with no report of the African lineage.

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Buathong, R., Hermann, L., Thaisomboonsuk, B., Rutvisuttinunt, W., Klungthong, C., Chinnawirotpisan, P., et al. (2015). Detection of Zika virus infection in Thailand, 2012-014. American Journal of Tropical Medicine and Hygiene, 93, 380–383. https://doi.org/10.4269/ajtmh.15-0022. Buathong, R., Loasiritaworn, Y., Kanjanasombut, H., Ruchiseesarod, C., & Wacharaplusadee, S. (2019). Zika virus serology and cross-reactive dengue antibody in Zika virus infection Thailand 2017. In Presented at 29th European congress of clinical microbiology and infectious diseases (ESCMID). Amsterdam, Noord Holland, Netherlands: RAI Amsterdam. Campos, G. S., Bandeira, A. C., & Sardi, S. I. (2015). Zika virus outbreak, Bahia, Brazil. Emerging Infectious Diseases, 21(10), 1885–1886. https://doi.org/ 10.3201/eid2110.150847. Cao-Lormeau, V. M., Roche, C., Teissier, A., Robin, E., Berry, A. L., Mallet, H. P., et al. (2014). Zika virus, French polynesia, South pacific, 2013. 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Fonseca, K., Meatherall, B., Zarra, D., Drebot, M., MacDonald, J., Pabbaraju, K., et al. (2014). First case of Zika virus infection in a returning Canadian traveler. American Journal of Tropical Medicine and Hygiene, 91, 1035–1038. https://doi.org/10.4269/ajtmh.14-0151. Gardner, L. M., Chen, N., & Sarkar, S. (2016). Global risk of Zika virus depends critically on vector status of Aedes albopictus. The Lancet Infectious Diseases, 16, 522–523. https://doi.org/10.1016/S1473-3099(16)00176-6. Grischott, F., Puhan, M., Hatz, C., & Schlagenhauf, P. (2016). Non-vector-borne transmission of Zika virus: A systematic review. Travel Medicine and Infectious Disease, 14(4), 313–330. https://doi.org/10.1016/j.tmaid.2016.07.002. Haby, M. M., Pinart, M., Elias, V., & Reveiz, L. (2018). Prevalence of asymptomatic Zika virus infection: a systematic review. Bulletin of the World Health Organization, 96(6), 402–413D. https://doi.org/10.2471/BLT.17.201541. Haddow, A. J., Williams, M. C., Woodall, J. P., Simpson, D. I., & Goma, L. K. (1964). Twelve isolations of Zika virus from Aedes (Stegomyia) africanus (Theobald) taken in and above a Uganda forest. Bulletin of the World Health Organization, 31, 57–69. Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98. Hennessey, M., Fischer, M., & Staples, J. E. (2016). Zika virus spreads to new areas—Region of the Americas, May 2015–January 2016. Morbidity and Mortality Weekly Report, 65(3), 55–58. https://doi.org/10.15585/mmwr.mm6503e1. Kuno, G., & Chang, G. J. (2007). Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses. Archives of Virology, 152, 687–696. Lanciotti, R. S., Kosoy, O. L., Laven, J. J., Velez, J. O., Lambert, A. J., Johnson, A. J., et al. (2008). Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerging Infectious Diseases, 14(8), 1232–1239. https://doi.org/10.3201/ eid1408.080287. Li, M. I., Wong, P. S., Ng, L. C., & Tan, C. H. (2012). Oral susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika virus. PLOS Neglected Tropical Diseases, 6, e1792.

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Marano, G., Pupella, S., Vaglio, S., Liumbruno, G. M., & Grazzini, & G. (2016). Zika virus and the never-ending story of emerging pathogens and transfusion medicine. Blood Transfusion, 14(2), 95–100. https://doi.org/10.2450/2015.0066-15. Marchette, N. J., Garcia, R., & Rudnick, A. (1969). Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia. American Journal of Tropical Medicine and Hygiene, 18(3), 411–415. McCrae, A. W., & Kirya, B. G. (1982). Yellow fever and Zika virus epizootics and enzootics in Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene, 76, 552–562. Musso, D. (2015). Zika virus transmission from French Polynesia to Brazil. Emerging Infectious Diseases, 21(10), 1887. https://doi.org/10.3201/ eid2110.151125. Musso, D., & Gubler, D. J. (2016). Zika virus. Clinical Microbiology Reviews, 29, 487–524. Musso, D., Nilles, E. J., & Cao-Lormeau, V. M. (2014). Rapid spread of emerging Zika virus in the Pacific area. Clinical Microbiology and Infection, 20 (10), O595–O596. https://doi.org/10.1111/1469-0691.12707. Musso, D., Roche, C., Robin, E., Nhan, T., Teissier, A., & Cao-Lormeau, V. M. (2015). Potential sexual transmission of Zika virus. Emerging Infectious Diseases, 21(2), 359–361. https://doi.org/10.3201/eid2102.141363. Nguyen, L. T., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Molecular Biology and Evolution, 32, 268–274. Olson, J. G., Ksiazek, T. G., Suhandiman, & Triwibowo. (1981). Zika virus, a cause of fever in Central Java, Indonesia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75(3), 389–393. Petersen, E. E., Staples, J. E., Meaney-Delman, D., Fischer, M., Ellington, S. R., Callaghan, W. M., et al. (2016). Interim guidelines for pregnant women during a Zika virus outbreak-United States, 2016. Morbidity and Mortality Weekly Report, 65(2), 30–33. https://doi.org/10.15585/mmwr.mm6502e1. Phumee, A., Chompoosri, J., Intayot, P., Boonserm, R., Boonyasuppayakorn, S., Buathong, R., et al. (2019). Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes. Scientific Reports, 9(1), 5257. https://doi.org/10.1038/s41598-019-41727-8. Pond, W. L. (1963). Arthropod-borne virus anti bodies in sera from residents of South-east Asia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 57, 364–371. Ruchusatsawat, K., Wongjaroen, P., Posanacharoen, A., Rodriguez-Barraquer, I., Sangkitporn, S., et al. (2019). Long-term circulation of Zika virus in Thailand: An observational study. The Lancet Infectious Diseases, 19(4), 439–446. https://doi.org/10.1016/S1473-3099(18)30718-7. Shinohara, K. (2014). Zika virus—Japan ex Thailand. ProMed. Archive no. 20140823.2716731. Available from: http://www.promedmail.org. Singapore Zika Study Group. (2017). Outbreak of Zika virus infection in Singapore: An epidemiological, entomological, virological, and clinical analysis. The Lancet: Infectious Diseases, 17(8), 813–821. https://doi.org/10.1016/S1473-3099(17)30249-9. Tappe, D., Rissland, J., Gabriel, M., Emmerich, P., Gunther, S., Held, G., et al. (2014). First case of laboratory-confirmed Zika virus infection imported into Europe, November 2013. Eurosurveillance, 19, 20685. https://doi.org/10.2807/1560-7917.ES2014.19.4.20685. Tawatsin, A., Phumee, A., Thavara, U., Sirisopa, P., Ritthison, W., Thammakosol, K., et al. (2018). High infection rate of Zika virus in mosquitoes collected from an area of active Zika virus transmission of eastern Thailand. The Thai Journal of Veterinary Medicine, 48, 551–558. Tippmann, H. F. (2004). Analysis for free: Comparing programs for sequence analysis. Briefings in Bioinformatics, 5, 82–87. Wong, P. S., Li, M. Z., Chong, C. S., Ng, L. C., & Tan, C. H. (2013). Aedes (Stegomyia) albopictus (Skuse): A potential vector of Zika virus in Singapore. PLOS Neglected Tropical Diseases, 7, e2348. Wongsurawat, T., Athipanyasilp, N., Jenjaroenpun, P., Jun, S. R., Kaewnapan, B., Wassenaar, T. M., et al. (2018). Case of microcephaly after congenital infection with Asian lineage Zika virus, Thailand. Emerging Infectious Diseases, 24(9), 1758–1761. https://doi.org/10.3201/eid2409.180416. Woon, Y. L., Lim, M. F., Tg Abd Rashid, T. R., Thayan, R., Chidambaram, S. K., Syed Abdul Rahim, S., et al. (2019). Zika virus infection in Malaysia: An epidemiological, clinical and virological analysis. BMC Infectious Diseases, 19(1), 152. https://doi.org/10.1186/s12879-019-3786-9. World Health Organization. (2019). Countries and territories with current or previous Zika virus transmission. Accessible at: https://www.who.int/ emergencies/diseases/zika/countries-with-zika-and-vectors-table.pdf. Zanluca, C., Melo, V. C., Mosimann, A. L., Santos, G. I., Santos, C. N., & Luz, K. (2015). First report of autochthonous transmission of Zika virus in Brazil. Memorias do Instituto Oswaldo Cruz, 110(4), 569–572.

Chapter 34

Zika virus in Vietnam: Biology, transmission, pathology, associated conditions, and controls Nguyen Thai Sona, Ho Huu Thob, and Dinh-Toi Chuc a

Department of Microbiology, Vietnam Military Medical University, Hanoi, Vietnam, b Institute of Biomedicine & Pharmacy, Vietnam Military

Medical University, Hanoi, Vietnam, c Center for Biomedicine and Community Health, International School, Vietnam National University Hanoi, Cau Giay, Hanoi, Vietnam

Abbreviations CNS E GBS M VMoH WHO ZIKV

central nervous system envelope Guillain-Barr
e syndrome membrane Vietnam Ministry of Health The World Health Organization Zika virus

Introduction The earliest identification of the Zika virus (ZIKV) was from a rhesus monkey of the Zika Forest in Uganda in 1947. During 1962–1963, a ZIKV case was confirmed in a man in Uganda (Wikan & Smith, 2016). In addition, it was isolated from a Nigerian man in 1968 (Wikan & Smith, 2017). Thereafter, only sporadic cases were reported in several countries around the world. However, 2007 marked the first ZIKV outbreak with 185 confirmed cases on Yap Island in the western Pacific Ocean (Duffy et al., 2009). Then, in 2015 the biggest recorded outbreak occurred in Brazil, which estimated more than half a million suspected cases (Aryal, 2019). It was extremely horrible due to the occurrence of microcephaly. As of Oct. 21, 2015, Brazil had 3174 neonates with microcephaly; in 38 cases, the child did not survive (Aryal, 2019). Meanwhile, the total deaths from the ZIKV infection worldwide reached 152 (Aryal, 2019). Other ZIKV epidemics have emerged in the Pacific regions, the Americas, and Caribbean countries (Musso & Gubler, 2016). In Asia, ZIKV was first identified in the 1960s, with the detection of ZIKV in 16.8% of human sera obtained from various districts throughout India. It is believed that ZIKV was present in Southeast Asia over the past 50 years (Haddow et al., 2012). During 2012–2014, five countries (Malaysia, the Philippines, Cambodia, Indonesia, and Thailand) reported the presence of ZIKV, which was confirmed by molecular and serological tests to detect viral nucleic acid and IgM antibodies against ZIKV, respectively (Petersen, Jamieson, Powers, & Honein, 2016). At least five cases of ZIKV infection were reported in visitors who stayed in Vietnam, Cambodia, and Laos (Dinh et al., 2019). In Vietnam, the first two ZIKV cases were in Nha Trang City (Khanh Hoa Province) and Ho Chi Minh City in 2016 (Bui et al., 2018). In this year, Vietnam saw at least 212 other cases in the Central Highlands and Southern regions, including Dak Lak, Phu Yen, Long An, Tra Vinh, Binh Duong, Ba Ria-Vung Tau Tay Ninh, and Dong Nai (Chu, Ngoc, & Tao, 2017). An analysis of viral RNA indicated that ZIKV spread in Vietnam was connected to the Asian lineage (Meltzer et al., 2016). According to the US Centers for Disease Control and Prevention (CDC), Vietnam is considered an “area with current or past transmission but no Zika outbreak” (www.cdc.gov). Although Vietnam has a history of previous ZIKV transmission, there is no evidence of an ongoing ZIKV outbreak now.

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00034-1 Copyright © 2021 Elsevier Inc. All rights reserved.

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Biology of ZIKV in Vietnam ZIKV is a flavivirus belonging to the Flaviviridae family, which includes several other mosquito-borne viruses such as dengue, yellow fever, West Nile, Japanese, and other encephalitis viruses (Hamel et al., 2015). Therefore, the biological structure of ZIKV was found to correlate with related viruses in the same genus of Flavivirus (Sirohi et al., 2016). As illus˚ and 500 A ˚ , respectively. In trated in Fig. 1, ZIKV immature and mature structures have diameters of approximately 600 A ZIKV, a positive-sense, single-stranded RNA genome of 11 kb was encapsulated within a viral capsid protein (C) and an outer host-derived lipid bilayer (Prasad et al., 2017). The enveloped icosahedral virus has a surface containing 180 copies of envelope (E) and membrane (M) glycoproteins (Hasan, Sevvana, Kuhn, & Rossmann, 2018; Murray, Jones, & Rice, 2008). The ZIKV genome is nonsegmented and consists of a long open reading frame (ORF) with two flanking noncoding regions at the 50 and 30 ends. The ZIKV ssRNA genome is 50 capped without a 30 poly (A) tail (Musso & Gubler, 2016). The ORF encodes for a polyprotein that is then proteolytically modified into three structural proteins (E, C, and precursor M) and seven nonstructural proteins, which are essential for viral replication (Fig. 2 and Table 1) (Faye et al., 2014). The invasion of ZIKV into host cells is via receptor-mediated endocytosis. Then, the fusion of the viral membrane and the host endosomal membranes causes the release of the viral RNA genome into the cytoplasm. Genome translation is initiated to form NS proteins that are responsible for the replication of viral RNA. Thereafter, multiple viral nucleocapsid proteins are formed, followed by maturation of the virion at the Golgi complex (Lee et al., 2018).

Transmission of ZIKV in Vietnam Currently, there are two modes of ZIKV infection: mosquito and nonmosquito transmission. The main route leading to ZIKV infection is mosquito transmission by several subgenera in Aedes spp., such as A. africanus, A. aegypti, A. luteocephalus, A. furcifer, and A. taylori (Diallo et al., 2014). The transmission cycle of ZIKV is from mosquito-to-humanmosquito through vector blood feeding. A. aegypti mosquitoes, which are mainly distributed in tropical regions, play an important role in ZIKV transmission (Kraemer et al., 2015). In particular, several developing and underdeveloped

FIG. 1 Structure of ZIKV. The top images (A, B) are the mature viral structure and the bottom images (C, D) are the immature viral structure. (A, C) Three-dimensional construction view. (B, D) cross-section view. (Adapted from Prasad, V. M., Miller, A. S., Klose, T., Sirohi, D., Buda, G., Jiang, W., ... ˚ resolution. Nature Structural & Molecular Biology, 24(2), 184.) Rossmann, M. G. (2017). Structure of the immature Zika virus at 9 A

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Flavivirus genome Genomic polyprotein 5’

3’-OH

C

M

Signal peptidase

E

NS1NS2ANS2B NS3

Golgi protease

NS4A NS4B

NS5

NS3 protease

FIG. 2 Genome of ZIKV. (Adapted from Igwebueze, O. I. (2016). Zika-virus-the-emerging-global-health-challenge. Diversity and Equality in Health and Care, 13, 394–401, doi:10.21767/2049-5471.100083.)

TABLE 1 Seven encoded nonstructural proteins of ZIKV. Protein code

Function

NS1

 

RNA replication Particle assembly

NS2A, NS4B

 

RNA replication Immune evasion

NS2B, NS4A



Cofactor of NS3

NS3



Major viral protease

NS5



RNA synthesis and modification

countries in Africa and Asia, where hygienic conditions and sanitation are not a strong focus, have become major areas of outbreaks of ZIKV and other mosquito-borne diseases (Al-Afaleq, 2017) (Table 2). In another way, ZIKV is considered a sexually transmitted virus. It should be noted that all ZIKV-infected males who returned from ZIKV epidemic areas transmitted the virus to their spouses a few days before or after the onset of symptoms (Foy et al., 2011; Hills et al., 2016). It is known that ZIKV RNA can be found in patient semen samples, indicating the transmission of ZIKV from person to person through sexual contact (Atkinson et al., 2016). Moreover, ZIKV can be diffused from infected women to fetuses during their pregnancy. There is much evidence revealing the presence of ZIKV RNA in the amniotic fluid of infected women as well as in fetal tissue and even the brains of newborn babies, which may lead to microcephaly (Martines et al., 2016; Mlakar et al., 2016). Other transmission routes have been revealed, including blood transfusions (Liu et al., 2019), body fluids (Bonaldo et al., 2016), laboratory exposure (Filipe, Martins, & Rocha, 1973), and animal bites (Leung, Baird, Druce, & Anstey, 2015). In Vietnam, the ZIKV epidemic was mostly transmitted to humans by mosquito vectors. The Aedes mosquitoes are widely distributed in Vietnam, especially in the Central Highlands and Southern regions. Although these mosquitoes can reproduce in the Northern regions during spring and summer, no ZIKV case has been found in those areas. The vectors transmit ZIKV through bites on humans, and they can be active all day, especially in the early morning. In the 236 cases of ZIKV infection during 2016–2017, only one case of maternal transmission to an infant was found; however, the child suffered the most serious symptom (i.e., microcephaly) (Duoc, 2017; Moi et al., 2017; Phan et al., 2019). It should be noted that no case in Vietnam has been found with ZIKV infection by other routes, such as unsafe sexual contact or a blood transfusion (Nguyen-Tien et al., 2019). However, in many cases, healthy travelers who were infected with ZIKV in Vietnam may become vectors to transmit ZIKV to people in their countries (Hashimoto et al., 2017; Katanami et al., 2017; Lim, Lim, & Yoon, 2017; Meltzer et al., 2016). Therefore, the CDC informed people to consider their destinations before travel to other countries.

Pathology of ZIKV infection in Vietnam The incubation time from mosquito bite to symptom onset is between 3 and 12 days. In an experimental model, the virus levels were found to increase around day 15 and remain high for 20–60 days (Boorman & Porterfield, 1956). In humans,

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TABLE 2 ZIKV infections in Vietnam (2016). No

Province/city

Number of cases (212)

Region

Area

1

Ho Chi Minh City

186

Southern

Urban and semiurban

2

Binh Duong

7

Southern

Urban

3

Dong Nai

4

Southern

Urban

4

Ba Ria-Vung Tau

2

Southern

Urban

5

Can Tho

1

Southern

Rural

6

Binh Phuoc

1

Southern

Semiurban

7

Tay Ninh

1

Southern

Semiurban

8

Long An

1

Southern

Urban

9

Phu Yen

1

Central

Rural

10

Khanh Hoa

6

Central

Urban and semiurban

11

Dak Lak

2

Central

Highland

Source: National Institute of Hygiene and Epidemiology; Adapted from Nguyen-Tien, T., Lundkvist, A˚., & Lindahl, J. (2019). Urban transmission of mosquito-borne flaviviruses—a review of the risk for humans in Vietnam. Infection Ecology & Epidemiology, 9(1), 1660129.

almost 80% of infected cases were asymptomatic (Duffy et al., 2009; Ioos et al., 2014). According to a CDC review that summarized data from 158 cases of travel-acquired ZIKV among children (1 month to 17 years), the most common symptom of ZIKV is a rash, corresponding to more than 82% of reported cases (Adachi & Nielsen-Saines, 2018; Buathong et al., 2015; Salehuddin et al., 2017). Maculopapular rash often covers the face and trunk, then spreads to the extremities (Simpson, 1964). The second most common symptom of ZIKV infection is a mild and nonspecific fever, accounting for more than 65% of reported cases (Arzuza-Ortega et al., 2016). Generally, ZIKV symptoms are similar to those of the Dengue virus, and the only way to distinguish between these two diseases is the grade of the fever. A mild fever (usually 38–39°C) only appears in a ZIKV infection (Chan, Choi, Yip, Cheng, & Yuen, 2016). In addition, ZIKV infection can cause arthritis or arthralgia (especially in the small joints of the hands and legs), correlating to 61.9% of reported cases (Foy et al., 2011; Musso et al., 2015). More than 50% of ZIKV cases have nonpurulent conjunctivitis, which affects both eyes (Goorhuis et al., 2016; Gourinat, O’Connor, Calvez, Goarant, & Dupont-Rouzeyrol, 2015). In some cases, gastrointestinal and constitutional problems can occur, affecting the patient’s immunity (Olson, Ksiazek, Suhandiman, & Triwibowo, 1981; Zammarchi et al., 2015). It should be noted that most infected patients can recover from all these symptoms within 7 days; however, rare cases showed longer persistence (Agumadu & Ramphul, 2018; Arzuza-Ortega et al., 2016). In Vietnam, it was reported that the symptoms of the first ZIKV-infected patients were rashes, fevers, conjunctivitis, and headaches (Chu et al., 2017). A typical case was a Japanese male infected with ZIKV in Vietnam. He developed a fever and a diffused rash on his face, arms, and legs when he returned to Japan. However, ZIKV RNA was not detected in his serum or semen samples, and his symptoms completely resolved within 7 days without treatment (Hashimoto et al., 2017). It’s possible that ZIKV has been in Vietnam for several decades; however, with poor knowledge about ZIKV and few diagnostic tests, it did not received adequate concern (Pond, 1963).

Associated conditions of ZIKV infection in Vietnam It was documented in Brazil that ZIKV infection in pregnant women resulted in an elevated number of newborn babies with microcephaly (Schuler-Faccini, 2016). Accordingly, microcephaly cases were detected in about 0.2% of live births in 2015, but only about 0.005% of live births in 2014 (Schuler-Faccini, 2016). Several reports have shown that microcephaly and the congenital central nervous system (CNS) were considered serious complications of ZIKV infection in many countries with ZIKV outbreaks (Pardy & Richer, 2019). In Vietnam, the first case of a pregnant woman with ZIKV infection was reported on March 30, 2016. Both this woman and her fetus were positive with ZIKV RNA, but there was a lack of information on microcephaly in the newborn. The second case came 3 months later when a girl was born with microcephaly in Dak Lak Province (Fig. 3). Her mother was infected with ZIKV during the second trimester (Lan et al., 2017). Few studies have

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FIG. 3 Facial features of a girl born with microcephaly. (Adapted from Lan, P. T., Quang, L. C., Huong, V. T. Q., Thuong, N. V., Hung, P. C., Huong, T. T. L. N., ... Nolen, L. D. (2017). Fetal Zika virus infection in Vietnam. PLoS Currents, 9.)

revealed the pathogenesis of microcephaly in newborns whose mothers were infected with ZIKV (Fig. 4). Accordingly, it has been hypothesized that the presence of ZIKV in humans affects the normal autophagy process and centrosome stability while also elevating the number of centrosomes in neural cells (Faizan et al., 2016;Onorati et al., 2016 ; Tetro, 2016). The abnormal function of centrosomes may affect the development of the fetal brain, in which the degradation of the fetal skull and the subsequent destruction of fetal brain tissue occur. It was also proved in a mouse model that when the number of centrosomes was increased, the mice suffered from microcephaly (Onorati et al., 2016; Tetro, 2016). In addition, ZIKV infection was found to affect the adult CNS, causing Guillain-Barre syndrome (GBS) (Cao-Lormeau et al., 2016), meningoencephalitis (Carteaux et al., 2016), and meningitis (M
echarles et al., 2016). The relationship between ZIKV contagion and GBS was verified by a French Polynesian case study in 2014 (Uncini, Shahrizaila, & Kuwabara, 2017). Especially, the increased incidence of GBS varied among regions with ZIKV epidemics, particularly in Venezuela (877%), Suriname (400%), Colombia (211%), the Bahia state of Brazil (172%), the Dominican Republic (150%), Honduras (144%), and El Salvador (100%) (Dos Santos et al., 2016). However, research on the pathogenesis of ZIKV-associated GBS

FIG. 4 Proposed mechanism for the formation of ZIKV-associated microcephaly in newborns. ZIKV infects the neural progenitor cells of the fetus through AXL receptor-mediated endocytosis. In the cells, ZIKV utilizes autophagy as a way for rapid virus replication. In addition, it abnormally elevates the number of centrosomes that play a crucial role in mitosis. As a consequence, the mitosis and viability of the cells are significantly impaired, leading to microcephaly in newborns (Onorati et al., 2016).

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is still a challenge (Uncini et al., 2017). The main clinical presentations of ZIKV-associated GBS are a high percentage of facial palsy and symmetrical limb weakness, which clearly divided into two phases, including rapid progression to a nadir (1 week) and a short plateau phase (4 days) (Uncini et al., 2017). Others ZIKV-associated nonneurologic consequences are hearing loss, hypotension, and genitourinary symptoms (there were very few cases of death related to ZIKV infection) (Al-Afaleq, 2017).

Controls of ZIKV in Vietnam According to the recommendation of the Pan American Health Organization (PAHO), there are three ways to control ZIKV infection: (a) decreasing the mosquito population; (b) preventing potential mosquito breeding sites; and (c) using suitable personal protection (Hajra, Bandyopadhyay, & Hajra, 2016). In Vietnam, the main route of ZIKV infection is mosquitoborne transmission; therefore, the prevention of ZIKV spread in Vietnam by mosquito control is of importance. Vietnam has suitable natural conditions for the development of infectious vectors. Also, poor water supply in the countryside and urban areas as well as the bad habit of accumulating rain water in unsafe containers (cans, tires, flower pots, and so on) around houses lead to favorable conditions for mosquitoes laying eggs and widely spreading. Therefore, the first task is to control and reduce mosquito populations by removing breeding sites. Any water containers should be regularly wiped or protected by lids, which would prevent the penetration of mosquitoes. In addition, larvivorous fish could be released into water containers to feed on mosquito larvae (Barrera et al., 2014). Second, while it is very difficult to remove all mosquito populations, personal protection from mosquito bites is of importance. It is recommended that people in endemic areas wear light-colored long-sleeved clothes, spray insect repellent on their skin and surrounding living area, and use suitable sleeping nets. Travelers who visit Vietnam should realize the ZIKV danger, and they should be carefully quarantined for possible virus infection before and after their journey. Especially, women who are pregnant or plan to be pregnant should be tested for ZIKV. All suspected pregnancy cases must be further examined and monitored to avoid affecting the fetus. Moreover, sexual transmission should be prevented by using condoms during sexual contact. Furthermore, infected patients should be isolated from the community to reduce ZIKV transmission through body fluids. In another aspect, the Vietnamese government plays a critical role in ZIKV surveillance and response through its collaborations and partnerships with the WHO, CDC, and other organizations. The Vietnam Ministry of Health (VMoH) should act to prepare for and respond to the threat of ZIKV, including the adaptation of existing surveillance systems to encompass ZIKV surveillance. The role of the VMoH is the creation of ZIKV response plans, including training healthcare workers, improving community knowledge of ZIKV infection, and establishing ZIKV diagnostic testing assays. Three ZIKV informational trainings have been conducted that covered all 63 provinces of Vietnam and educated a total of 637 local healthcare workers. Additionally, guidance for clinicians regarding early diagnosis, services, and care of pregnant women has been developed. So far, there is no ZIKV vaccine on the market; therefore, the prevention and treatment of ZIKV infection is still a challenge.

Policy and procedures According to the Ministry of Health, it is possible to continue to record more cases of ZIKV infection in Vietnam due to the exchange among countries as well as in the country, and Vietnam has circulating mosquitoes. Aedes aegypty has been identified as a vector of transmission in the community. Although the characteristics of the disease are usually moderate to mild, many cases do not show symptoms and victims recover without significant health effects. However, due to the potential association with ZIKV infection in pregnant women that leads to microcephaly in newborns, health monitoring for pregnant women and pregnant women infected with ZIKV should be considered for prevention. To proactively prevent a Zika virus epidemic to limit community spread and stabilize the social security of the people, the Ministry of Health recommends that localities focus on implementing the following activities: (1) Raising awareness about ZIKV and establishing a ZIKV action plan. Continuing to promote the monitoring and testing of ZIKV samples in the community for early detection of infection, then taking suitable actions to isolate patients affected by ZIKV. (2) Implementing all prevention plans to reduce the vector population, including control of mosquito larvae, prevention of mosquito bites, and training people and communities with ZIKV information. Mosquito control organizations using chemical sprayers have been used.

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(3) According to the Prime Minister’s 02/2016/QD TTg of Jan. 28, 2016, a broad multifaceted approach to control is needed in each province to take specific actions when an epidemic occurs. (4) Women who are pregnant or plan to be pregnant should be tested for ZIKV. The only way to completely prevent ZIKV infection during pregnancy is to not travel to areas with a risk of ZIKV and to use precautions or avoid sex with someone who has recently traveled to a risk area. The Ministry of Health will continuously update and share information to mass media agencies about the epidemic in the world and in the country while also posting on the websites of the Ministry of Health (moh.gov.vn) and the Department of Preventive Medicine (vncdc.gov.vn). Currently, the Wolbachia method (to prevent dengue, Zika, and some other mosquito-borne diseases) is being expanded to 12 countries, including Australia, Vietnam, Indonesia, Brazil, Colombia, Mexico, India, and others. In Vietnam, the Wolbachia method is being applied by the National Institute of Hygiene and Epidemiology (Ministry of Health) in collaboration with the Nha Trang Pasteur Institute and the Department of Health of Khanh Hoa Province. Wolbachia are extremely common bacteria that parasitize in Aedes aegypti mosquitoes. This means that when Aedes aegypti mosquitoes carry natural Wolbachia bacteria, the transmission of viruses such as dengue, Zika, chikungunya, and yellow fever is reduced.

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On April 5, 2016, the Ministry of Health of Vietnam officially confirmed two positive cases for ZIKV in Khanh Hoa and Ho Chi Minh City. By Oct. 17, 2016, there were seven cases of ZIKV infection in Khanh Hoa, Phu Yen, Binh Duong, and Ho Chi Minh City. 236 cases of ZIKV infection were reported during 2016–2017. On June 3, 2018, the project released Wolbachia mosquitoes in Vinh Luong and Nha Trang City. Although Vietnam has a history of previous ZIKV transmission, there is no evidence of an ongoing ZIKV outbreak now.

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This chapter provides information on the biology of the Zika virus as well as its transmission, pathology, and control in Vietnam. In Asia, Vietnam is the most recent country with a Zika virus epidemic. Vietnam had a total of 236 cases, mostly in the Southern region, from March 2016 to April 2017. The biological structure of the Zika virus is similar to other viruses in the same genus of Flavivirus, containing three main components: an RNA genome encapsulated within a viral capsid protein and an outer host-derived lipid bilayer. ZIKV infection involves invasion into host cells via receptor-mediated endocytosis, then viral RNA replication and finally the release of a complete structure outside the infected cells. ZIKV transmission in Vietnam is mostly via Aedes mosquito vectors, although there was one case of maternal transmission. The symptoms of ZIKV infection in Vietnam are mostly rashes, fevers, conjunctivitis, and headaches, and one case of a newborn baby with microcephaly. The Vietnamese government plays a critical role in controlling ZIKV infection by training healthcare workers, improving community knowledge, and establishing ZIKV diagnostic testing assays.

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echarles, S., Herrmann, C., Poullain, P., Tran, T.-H., Deschamps, N., Mathon, G., … Lannuzel, A. (2016). Acute myelitis due to Zika virus infection. The Lancet, 387(10026), 1481. https://doi.org/10.1016/S0140-6736(16)00644-9. Meltzer, E., Lustig, Y., Leshem, E., Levy, R., Gottesman, G., Weissmann, R., & Schwartz, E. (2016). Zika virus disease in traveler returning from Vietnam to Israel. Emerging Infectious Diseases, 22(8), 1521–1522. https://doi.org/10.3201/eid2208.160480. Mlakar, J., Korva, M., Tul, N., Popovic, M., Poljsak-Prijatelj, M., Mraz, J., … Avsic Zupanc, T. (2016). Zika virus associated with microcephaly. The New England Journal of Medicine, 374(10), 951–958. https://doi.org/10.1056/NEJMoa1600651. Moi, M. L., Nguyen, T. T. T., Nguyen, C. T., Vu, T. B. H., Tun, M. M. N., Pham, T. D., … Hasebe, F. (2017). Zika virus infection and microcephaly in Vietnam. The Lancet Infectious Diseases, 17(8), 805–806. https://doi.org/10.1016/S1473-3099(17)30412-7. Murray, C. L., Jones, C. T., & Rice, C. M. (2008). Architects of assembly: Roles of Flaviviridae non-structural proteins in virion morphogenesis. Nature Reviews. Microbiology, 6(9), 699–708. https://doi.org/10.1038/nrmicro1928. Musso, D., & Gubler, D. J. (2016). Zika Virus. Clinical Microbiology Reviews, 29(3), 487–524. https://doi.org/10.1128/CMR.00072-15. Musso, D., Roche, C., Robin, E., Nhan, T., Teissier, A., & Cao-Lormeau, V. M. (2015). Potential sexual transmission of Zika virus. Emerging Infectious Diseases, 21(2), 359–361. https://doi.org/10.3201/eid2102.141363. ˚ ., & Lindahl, J. (2019). Urban transmission of mosquito-borne flaviviruses—A review of the risk for humans in Vietnam. Nguyen-Tien, T., Lundkvist, A Infection Ecology & Epidemiology, 9(1), 1660129. Olson, J. G., Ksiazek, T. G., Suhandiman, & Triwibowo. (1981). Zika virus, a cause of fever in Central Java, Indonesia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75(3), 389–393. https://doi.org/10.1016/0035-9203(81)90100-0. Onorati, M., Li, Z., Liu, F., Sousa, A. M., Nakagawa, N., Li, M., … Tebbenkamp, A. T. (2016). Zika virus disrupts phospho-TBK1 localization and mitosis in human neuroepithelial stem cells and radial glia. Cell Reports, 16(10), 2576–2592. Pardy, R. D., & Richer, M. J. (2019). Zika virus pathogenesis: From early case reports to epidemics. Viruses, 11(10). https://doi.org/10.3390/v11100886. Petersen, L. R., Jamieson, D. J., Powers, A. M., & Honein, M. A. (2016). Zika virus. The New England Journal of Medicine, 374(16), 1552–1563. https:// doi.org/10.1056/NEJMra1602113. Phan, T. L., Luong, C. Q., Do, T. H. H., Chiu, C. H., Cao, M. T., Nguyen, T. T. T., … Le, H. N. (2019). Findings and lessons from establishing Zika virus surveillance in southern Viet Nam, 2016. Western Pacific Surveillance and Response, 10(2), 22–30. Pond, W. L. (1963). Arthropod-borne virus antibodies in sera from residents of South-East Asia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 57(5), 364–371. ˚ resolution. Prasad, V. M., Miller, A. S., Klose, T., Sirohi, D., Buda, G., Jiang, W., … Rossmann, M. G. (2017). Structure of the immature Zika virus at 9 A Nature Structural & Molecular Biology, 24(2), 184. Salehuddin, A. R., Haslan, H., Mamikutty, N., Zaidun, N. H., Azmi, M. F., Senin, M. M., … Thent, Z. C. (2017). Zika virus infection and its emerging trends in Southeast Asia. Asian Pacific Journal of Tropical Medicine, 10(3), 211–219. https://doi.org/10.1016/j.apjtm.2017.03.002. Schuler-Faccini, L. (2016). Possible association between Zika virus infection and microcephaly—Brazil, 2015. MMWR. Morbidity and Mortality Weekly Report, 65, 59–62. Simpson, D. I. H. (1964). Zika virus infection in man. Transactions of the Royal Society of Tropical Medicine and Hygiene, 58(4), 335–338. https://doi.org/ 10.1016/0035-9203(64)90200-7. Sirohi, D., Chen, Z., Sun, L., Klose, T., Pierson, T. C., Rossmann, M. G., & Kuhn, R. J. (2016). The 3.8 A resolution cryo-EM structure of Zika virus. Science, 352(6284), 467–470. https://doi.org/10.1126/science.aaf5316. Tetro, J. A. (2016). Zika and microcephaly: Causation, correlation, or coincidence. Microbes and Infection, 18(3), 167–168. Uncini, A., Shahrizaila, N., & Kuwabara, S. (2017). Zika virus infection and Guillain-Barr
e syndrome: A review focused on clinical and electrophysiological subtypes. Journal of Neurology, Neurosurgery, and Psychiatry, 88(3), 266–271.

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Wikan, N., & Smith, D. R. (2016). Zika virus: History of a newly emerging arbovirus. The Lancet Infectious Diseases, 16(7), e119–e126. https://doi.org/ 10.1016/S1473-3099(16)30010-X. Wikan, N., & Smith, D. R. (2017). Zika virus from a southeast Asian perspective. Asian Pacific Journal of Tropical Medicine, 10(1), 1–5. https://doi.org/ 10.1016/j.apjtm.2016.11.013. Zammarchi, L., Stella, G., Mantella, A., Bartolozzi, D., Tappe, D., G€unther, S., … Schmidt-Chanasit, J. (2015). Zika virus infections imported to Italy: Clinical, immunological and virological findings, and public health implications. Journal of Clinical Virology, 63, 32–35. https://doi.org/10.1016/j. jcv.2014.12.005.

Chapter 35

Zika virus in Vietnam: Epidemic, distribution, strain origin, and potential risks for community health Dinh-Toi Chua, Tiep Tien Nguyenb, and Nguyen Thai Sonc a

Center for Biomedicine and Community Health, International School, Vietnam National University Hanoi, Cau Giay, Hanoi, Vietnam, b College of

Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk, South Korea, c Department of Microbiology, Vietnam Military Medical University, Hanoi, Vietnam

Abbreviations ECDPC GBS PHEIC TCDC WHO ZIKV

European Centre for Disease Prevention Control Guillain-Barr
e syndrome Public Health Emergency of International Concern Taiwan Centers for Disease Control The World Health Organization Zika virus

Introduction The Zika virus (ZIKV), a type of arbovirus in the Flavivirus genus, was first extracted from a Uganda rhesus monkey’s serum in 1947 (Dick, Kitchen, & Haddow, 1952a, 1952b). The first report of ZIKV contagion in humans came in 1952 when scientists were looking for the epidemiology of yellow fever in Uganda and Nigeria (Macnamara, 1954). In Nigeria, ZIKV was isolated from one patient and serum ZIKV antibodies were found to be increased in the other two patients (Macnamara, 1954). For five decades thereafter, the ZIKV infection appeared in several Asian and African countries with limited cases, and the collective symptoms were mild, such as rashes, headaches, fevers, arthralgia, and conjunctivitis. The remarkable ZIKV infection event was the large epidemic on Yap Island in Micronesia in 2007, estimated
73% population of 3 year olds (Lanciotti et al., 2008). Genetic and immunologic tests from 185 patients determined 59 probable and 49 confirmed cases. This event was an alert to the potential expansion of ZIKV worldwide in the future (Kindhauser, Allen, Frank, Santhana, & Dye, 2016; Song, Yun, Woolley, & Lee, 2017). In 2008, two American scientists were infected with ZIKV when they worked in a disease-endemic area in Senegal (Foy et al., 2011). It should be noted that one of them transferred the disease to his spouse after returning to Colorado, which was subsequently concluded to be via sexual contact (Foy et al., 2011). From 2012 to 2015, there were sporadic cases of European travelers infected with ZIKV after returning from trips to some Southeast Asian countries (Korhonen et al., 2016; Kwong, Druce, & Leder, 2013; Tappe et al., 2014). Most of the ZIKV-associated clinical presentation was considered mild until the enormous epidemic in French Polynesia during 2013–2014 (ECDPC, 2014). In that occurrence, nearly 10,000 suspected cases were reported. Strikingly, ZIKV RNA was identified in >50% specimens (396/746) sent to the laboratory, and it was estimated that
11.5% of the population received medical care due to ZIKV-like symptoms. In addition, the outbreak in French Polynesia was followed by 38 cases of Guillain-Barr
e syndrome (GBS) and 25 cases of other neurological disorders, which had never been reported in the previous outbreaks (ECDPC, 2014). In early 2015, northeast Brazil saw its first outbreak of ZIKV infection (Schuler-Faccini, 2016). The report from the Brazil Ministry of Health showed that maternal transmission of ZIKV can lead to significantly increased incidents of microcephaly in newborns; this was confirmed by positive ZIKV RNA tests in both mothers and their infants with microcephaly (Schuler-Faccini, 2016). According to a recent systematic review, in the regions with ZIKV outbreaks, the incidences of neural abnormalities such as GBS and microcephaly were found to be significantly increased (Wahid, Ali, Waqar, & Idrees, 2018). Also, the number of ZIKV-infected patients with Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00035-3 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Timeline of key events of ZIKV disease in the world and in Vietnam.

hydrancephaly/hydrops fetalis, myasthenia gravis, meningoencephalities, and myelitis has been climbing (Bla´zquez & Saiz, 2016; Wahid et al., 2018). Until 2016, the number of ZIKV infections worldwide was estimated at about 3–4 million cases, with half of them occurring in Brazil (WHO, 2016a). Therefore, the WHO instituted a public health emergency of international concern (PHEIC) in 2016 (WHO, 2016a). As of July 2019, 87 countries and territories have reported evidence of ZIKV disease, and 50 regions had their first outbreaks in 2015–2016 (WHO, 2016a, 2019). In Asia, ZIKV epidemics have been found in at least 17 countries, and Vietnam has seen the highest number of infected cases (Lim, Lim, & Yoon, 2017). Given many favorable conditions for the spread of ZIKV, Vietnam has been receiving great interest for the surveillance of viral transmission. In this chapter, we will provide recent reports on ZIKV epidemics as well as discuss the distribution and strain origin of ZIKV in Vietnam. In addition, we will reveal potential risks of ZIKV to public health security. Fig. 1 shows a timeline with key events of the ZIKV disease worldwide and in Vietnam.

Epidemics of ZIKV in Vietnam The first sign of ZIKV infection in Vietnam was in 1954 when scientists attempted to explore an arthropod-borne virus in the Southeast Asian population. The report showed that one case of Tonkinese in North Vietnam was positive with antiZIKV antibodies, as shown by a serological test (Pond, 1963). Since the large ZIKV epidemics on Yap Island (2007) and in French Polynesia (2014), Vietnamese authorities have paid much more attention to this contagious disease. A retrospective study on Dengue virus-negative plasma samples collected from 2010 to 2014 revealed two more positive ZIKV cases among 5617 tested samples (Quyen et al., 2017). However, Vietnam saw peak ZIKV transmission with 236 cases in 12 provinces during 2016–2017 (Phan et al., 2019). The first event of the outbreak came in late March 2016, when two autochthonous cases were found in women in Nha Trang City and Ho Chi Minh City; the existence of ZIKV RNA in blood specimens was confirmed by the PCR technique (WHO, 2016b). The only case of congenital microcephaly was a girl born in Dak Lak Province; this was reported in June 2016 (Moi et al., 2017). Before that, her young mother had been diagnosed with ZIKV infection in the second trimester of pregnancy, but did not receive complete treatment. High levels of a ZIKVneutralizing antibody were found in the sera of the child and her close relatives when she was 3–4 months old (Moi et al., 2017). The ZIKV disease in Vietnam was observed not only in native residents but also in foreign travelers (Dinh et al., 2019). The first case was an old Israeli man who returned to his country after short visits to Ho Chi Minh City and Hong Kong during the last days of 2015 (Meltzer et al., 2016). He suffered a mild illness and was subsequently diagnosed with ZIKV

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infection (positive for both ZIKV RNA and antibodies) by the National Center for Zoonotic Viruses in Israel. Two other ZIKV-infected foreigners were from Japan and Germany, and both were in Ho Chi Minh City before being diagnosed at a hospital in Japan (Hashimoto et al., 2017; Katanami et al., 2017). The ZIKV disease was also exported from Vietnam to Korea, but the traveler’s information was not fully revealed ( Jeong et al., 2017). In 2017, the total number of ZIKV infections in Vietnam decreased to only 24 patients from January to April (Duoc, 2017). There was no report of local ZIKV transmission afterward. However, an announcement by the Taiwan Centers for Disease Control (TCDC) revealed one imported case from Vietnam to Taiwan in 2018, indicating that ZIKV still seems to be active in Vietnam (TCDC, 2018). Vietnam and four other countries (Indonesia, the Philippines, Thailand, and Cameroon) are under strict surveillance by the GeoSentinel network for exported cases of ZIKV (Wilder-Smith, Chang, & Leong, 2018).

Distribution of ZIKV in Vietnam Up to now, all ZIKV cases in Vietnam were found in residents who live in the Central Highlands and Southern regions (Fig. 2), except the single case in North Vietnam described nearly seven decades ago (Chu, Ngoc, & Tao, 2017; Duoc, 2017; Phan et al., 2019; Pond, 1963). Like Dengue virus distribution, the number of ZIKV-infected patients in Southern region takes 70–80% cases (Duoc, 2017). Among transmission sites, Ho Chi Minh City saw the highest number of ZIKV infections with 194 cases in 2016 (Phan et al., 2019). There were only sporadic cases found in other provinces, including Binh Duong, Tay Ninh, Binh Phuoc, Long An, Can Tho, Ba Ria–Vung Tau, Dong Nai, Lam Dong, Khanh Hoa, Dak Lak, and Phu Yen (Figs. 2 and 3) (Duoc, 2017). It should be noted that most of these sites have been experiencing increased urbanization, with the shift of inhabitants from rural areas to crowded cities to find jobs. The high number of ZIKV-infected

FIG. 2 Mapping distribution of cases of ZIKV infection in Vietnam’s provinces and cities during 2016–2017. (Adapted from Duoc, V. (2017). Situations of dengue and Zika virus diseases-control activities in Vietnam. National Institute of Hygiene and Epidemiology, Vietnam.)

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FIG. 3 The number of reported cases of ZIKV disease in Vietnam’s provinces and cities during 2016–2017. (Adapted from Duoc, V. (2017). Situations of dengue and Zika virus diseases-control activities in Vietnam. National Institute of Hygiene and Epidemiology, Vietnam.)

FIG. 4 Distribution of cases with ZIKV infection in different population groups in 2016. The values on the x-axis indicate population groups by age (years). The value on the top of each chart indicates the percentage of cases in each group. (Adapted from Phan, T. L., Luong, C. Q., Do, T. H. H., Chiu, C. H., Cao, M. T., Nguyen, T. T. T., ... Le, H. N. (2019). Findings and lessons from establishing Zika virus surveillance in southern Viet Nam, 2016. Western Pacific Surveillance and Response, 10(2).)

patients who are of working age (20–59 years old) may be evidence for ZIKV occurrence in cities (Fig. 4). High population density and increased activities (trade and travel) in big cities may increase the chances of rapidly spreading infectious diseases among people in a community. Several cases of foreign travelers who stayed for short times in regions with endemic ZIKV disease (Ho Chi Minh City) were typical examples for viral transmission (Dinh et al., 2019). It is notable that the ZIKV disease in Vietnam was mostly active from October to December 2016, with nearly 90% of the outbreak’s total cases (Table 1) (Duoc, 2017). The weather in Southern Vietnam, particularly in Ho Chi Minh City, is characterized by a tropical monsoon climate with a wet season and a dry season. The wet season is often longer, starting from May to November. In the last months of the year, the temperature is around 25–30 °C and the humidity is high (>70%), which ideally favors the rapid expansion of the Aedes mosquito vectors of ZIKV. Many city dwellers live in areas with poor sanitary conditions such as a lack of piped water. Therefore, they often collect rain water in several tanks or even discarded tires surrounding their house without using well-protective furniture. Such conditions provide ideal breeding grounds for mosquitoes to lay their eggs.

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TABLE 1 Distribution of ZIKV infection cases by months. Quarter, year

Month of year

Number of cases (N 5 236)

Percentages (%)

Quarter 1, 2016

March

2

0.9

Quarter 2, 2016

June

1

0.4

Quarter 3, 2016

September

3

1.3

Quarter 4, 2016

October

32

13.6

November

109

46.2

December

65

27.5

January

12

5.0

February

3

1.3

March

6

2.5

April

3

1.3

Quarter 1, 2017

Quarter 2, 2017

Adapted from Duoc, V. (2017). Situations of dengue and Zika virus diseases-control activities in Vietnam. National Institute of Hygiene and Epidemiology, Vietnam.

Strain origin of ZIKV in Vietnam Up to now, many strains of ZIKV have been identified worldwide since the initial detection (Baronti et al., 2014; Campos, Bandeira, & Sardi, 2015; Dick et al., 1952a, 1952b; A. Haddow, Williams, Woodall, Simpson, & Goma, 1964; A. D. Haddow et al., 2012; Heang et al., 2012; Weinbren & Williams, 1958). They are divided into two main branches: strains that originated from West and East Africa, and strains that originated from Asian and American countries (Yun et al., 2016). A comprehensive analysis of the differences in ZIKV RNA sequences from various geographical regions indicated their remarkable alterations in both protein and nucleotide levels over the past decades (Wang et al., 2016). It is hypothesized that this variation would lead to different clinical presentations in the outbreaks. However, the link between ZIKV RNA variation and clinical severity is still unclear (Da Silva, Cheng, & Gao, 2018). In most cases, the main symptoms were considered to be mild, including light fevers, headaches, rashes, and conjunctivitis. In fact, severe ZIKV-associated neurological complications, including GBS in adults and congenital microcephaly in infants, were not described before the large Polynesian outbreak in 2013 (Mlakar et al., 2016; Oehler et al., 2014). There was no report determining the strain diversity of the ZIKV circulating in Vietnam, although this country saw hundreds of infectious cases in recent years. However, a nucleotide sequence analysis of ZIKV samples from several cases of travelers to Vietnam showed that strains of ZIKV in this country belong to the Asian lineage (Hashimoto et al., 2017; Katanami et al., 2017; Meltzer et al., 2016). Also, no evidence has indicated the strain origin of Vietnam’s ZIKV, but it was supposed to be locally circulating strains (Chu et al., 2017). Unlike the ZIKV strains that caused severe neurological symptoms in South American outbreaks, those in Vietnam were found to be less virulent with a low possibility of microcephaly (Cao-Lormeau et al., 2016; Mlakar et al., 2016). Only the single ZIKV-associated microcephaly case of a Vietnamese infant has been reported (Moi et al., 2017), although many women were infected with ZIKV during their pregnancy (47 positive per 205 tested samples) (Phan et al., 2019).

Potential risks of ZIKV in Vietnam ZIKV is now considered one of the most dangerous diseases among flavivirus infections. Although Aedes mosquitoes are key transmission vectors, ZIKV can also infect healthy people via nonmosquito sources, including maternal transmission, sexual contact, and blood transfusions, which makes it very hard to control the epidemic (Fig. 5) (Grischott, Puhan, Hatz, & Schlagenhauf, 2016; Moreira, Peixoto, Siqueira, & Lamas, 2017; Musso et al., 2014). In fact, ZIKV RNA is known to exist in various bodily fluids, including blood, breast milk, saliva, urine, seminal fluid, and amniotic fluid (Grischott et al., 2016). Since 2018, no ZIKV cases have been found in Vietnamese residents; however, it should be realized that this virus may be still active and cause potential risks to community health. Favorable tropical weather, climate change, low public awareness, unsafe sexual intercourse, and increased travel and trade in Vietnam are risk factors that may lead to the recurrence of ZIKV in the future (Fig. 5). It would be particularly dangerous if ZIKV strains in Vietnam underwent modifications

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FIG. 5 ZIKV transmission in community: risk factors and complications. The first box indicates the initial factors (natural factors and human factors) that affect the population of mosquito vectors of ZIKV. The middle box indicates the mode of ZIKV infection in mosquito vectors and humans. The last box indicates ZIKV transmission in the human community by nonmosquito vectors and potential complications.

to be more virulent and cause neurological complications. The first case of microcephaly in a newborn in Vietnam warned us not to underestimate it. Well known as “Asia’s Leading Destination,” Vietnam has attracted about 15.5 million foreign visitors, and most of them visited the biggest cities such as Hanoi and Ho Chi Minh City (Das, 2019). Some of these visitors who carry ZIKV may threaten community health security (Wilder-Smith et al., 2018). Therefore, international cooperation needs to be well established to monitor and detect ZIKV epidemics worldwide as well as maintain health service systems (Gostin & Hodge, 2016). Also, the Vietnamese authorities should play a crucial role in controlling the spread of ZIKV by providing timely surveillance reports and raising people’s awareness about the danger of ZIKV infection as well as its prevention. Especially, an urgent public health priority should be to reduce the mosquito vector population and to prevent ZIKV infection in pregnant women (Bell, Boyle, & Petersen, 2016; Da Silva et al., 2018; Kuadkitkan, Wikan, Sornjai, & Smith, 2020).

Policy and procedures: Mosquito vector control Aedes mosquitoes such as Aedes aegypti are the main vectors for transmitting Zika virus via their bites to feed on human blood. The mosquitoes are found mostly in tropical areas and rapidly increase their populations under relatively warm and wet conditions. They prefer to bite humans to feed on blood at any time of the day. Therefore, the prevention of mosquito vector-transmitted Zika virus requires a combination of various strategies. First, it is important to reduce the number of mosquitoes in living areas. The vectors prefer to lay their eggs in static water containers, such as cans, buckets and discarded tyres, or any highly wet surfaces. Unnecessary containers should be eliminated or washed and kept well-covered or upside-down. Useful containers should be regularly checked and the water inside exchanged with fresh water. In addition, larvivorous fish could be released into water containers to feed on mosquito larvae. Moreover, bushes should be frequently cleared and treated with mosquito repellents such as methoprene or Bacillus thuringiensis israelensis to prevent vector growth. The second strategy is to prevent mosquito bites. Long jackets and pants should be worn, especially when working in areas of high mosquito activity such as forested regions. In addition, exposed skin outside should be protected with mosquito repellents that contain active ingredients such as DEET, picardin, essential oils of Corymbia citriodora, or IR3535 (reference to doi: https://doi.org/10.1155/2018/6860271). Other methods, including window and door screenings to avoid mosquitoes infiltrating a room and using fine mosquito nets when sleeping are highly recommended.

Key facts from the World Health Organization l

l

Aedes mosquitoes, mainly Aedes aegypti, are general vectors for transmitting the Zika virus, dengue virus, chikungunya, and yellow fever. Numerous people who carry the Zika virus have no clear symptoms.

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Pregnant women with Zika virus infection can give birth to babies with microcephaly or other congenital malformations. Also, Zika virus infection in the course of pregnancy can cause increased chances of preterm birth, stillbirth, and miscarriage. There is no available vaccine for Zika virus disease.

Summary points l l

l

l

l

l

This chapter provides recent data on the Zika virus outbreak in Vietnam and its potential risks to community health. As of July 2019, 87 countries and territories around the world have reported Zika epidemics with increased incidents of severe neurological problems. During 2016–2017, Vietnam saw a Zika outbreak with 236 reported cases (including one newborn with microcephaly) in the Southern regions and Central Highlands, especially in Ho Chi Minh City. Strains of Zika virus in Vietnam are the Asian lineage, according to results from a nucleotide sequence analysis of several travelers in Vietnam. Favorable tropical weather, rapid urbanization, unsafe water containers, unsafe sexual contact, and increased trade and travel are the main risk factors for Zika virus infection in Vietnam. The urgent public health priority is to reduce the mosquito vector population and to prevent ZIKV infection in pregnant women.

References Baronti, C., Piorkowski, G., Charrel, R. N., Boubis, L., Leparc-Goffart, I., & de Lamballerie, X. (2014). Complete coding sequence of zika virus from a French polynesia outbreak in 2013. Genome Announcements, 2(3), e00500–e00514. Bell, B. P., Boyle, C. A., & Petersen, L. R. (2016). Preventing Zika virus infections in pregnant women: An urgent public health priority. American Journal of Public Health, 106(4), 589. Bla´zquez, A.-B., & Saiz, J.-C. (2016). Neurological manifestations of Zika virus infection. World Journal of Virology, 5(4), 135. Campos, G. S., Bandeira, A. C., & Sardi, S. I. (2015). Zika virus outbreak, Bahia, Brazil. Emerging Infectious Diseases, 21(10), 1885. Cao-Lormeau, V.-M., Blake, A., Mons, S., Laste`re, S., Roche, C., Vanhomwegen, J., … Larre, P. (2016). Guillain-Barr
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Hashimoto, T., Kutsuna, S., Tajima, S., Nakayama, E., Maeki, T., Taniguchi, S., … Hayakawa, K. (2017). Importation of Zika virus from Vietnam to Japan, November 2016. Emerging Infectious Diseases, 23(7), 1223. Heang, V., Yasuda, C. Y., Sovann, L., Haddow, A. D., da Rosa, A. P. T., Tesh, R. B., & Kasper, M. R. (2012). Zika virus infection, Cambodia, 2010. Emerging Infectious Diseases, 18(2), 349. Jeong, Y. E., Cha, G.-W., Cho, J. E., Lee, E. J., Jee, Y., & Lee, W.-J. (2017). Viral and serological kinetics in Zika virus-infected patients in South Korea. Virology Journal, 14(1), 70. Katanami, Y., Kutsuna, S., Taniguchi, S., Tajima, S., Takaya, S., Yamamoto, K., … Kato, Y. (2017). Detection of Zika virus in a traveller from Vietnam to Japan. Journal of Travel Medicine, 24(5), tax031. Kindhauser, M. K., Allen, T., Frank, V., Santhana, R. S., & Dye, C. (2016). Zika: The origin and spread of a mosquito-borne virus. Bulletin of the World Health Organization, 94(9), 675. Korhonen, E. M., Huhtamo, E., Smura, T., Kallio-Kokko, H., Raassina, M., & Vapalahti, O. (2016). Zika virus infection in a traveller returning from the Maldives, June 2015. Euro Surveillance, 21(2), 30107. Kuadkitkan, A., Wikan, N., Sornjai, W., & Smith, D. R. (2020). Zika virus and microcephaly in Southeast Asia: A cause for concern? Journal of Infection and Public Health, 13(1), 11–15. Kwong, J. C., Druce, J. D., & Leder, K. (2013). Zika virus infection acquired during brief travel to Indonesia. The American Journal of Tropical Medicine and Hygiene, 89(3), 516–517. Lanciotti, R. S., Kosoy, O. L., Laven, J. J., Velez, J. O., Lambert, A. J., Johnson, A. J., … Duffy, M. R. (2008). Genetic and serologic properties of Zika virus associated with an epidemic, Yap state, Micronesia, 2007. Emerging Infectious Diseases, 14(8), 1232. Lim, S.-K., Lim, J. K., & Yoon, I.-K. (2017). An update on Zika virus in Asia. Infection & Chemotherapy, 49(2), 91–100. https://doi.org/10.3947/ ic.2017.49.2.91. Macnamara, F. (1954). Zika virus: A report on three cases of human infection during an epidemic of jaundice in Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 48(2), 139–145. Meltzer, E., Lustig, Y., Leshem, E., Levy, R., Gottesman, G., Weissmann, R., … Mendelson, E. (2016). Zika virus disease in traveler returning from Vietnam to Israel. Emerging Infectious Diseases, 22(8), 1521. Mlakar, J., Korva, M., Tul, N., Popovi
c, M., Poljsˇak-Prijatelj, M., Mraz, J., … Fabjan Vodusˇek, V. (2016). Zika virus associated with microcephaly. New England Journal of Medicine, 374(10), 951–958. Moi, M. L., Nguyen, T. T. T., Nguyen, C. T., Vu, T. B. H., Tun, M. M. N., Pham, T. D., … Hasebe, F. (2017). Zika virus infection and microcephaly in Vietnam. The Lancet Infectious Diseases, 17(8), 805–806. https://doi.org/10.1016/S1473-3099(17)30412-7. Moreira, J., Peixoto, T. M., Siqueira, A. M. D., & Lamas, C. C. (2017). Sexually acquired Zika virus: A systematic review. Clinical Microbiology and Infection, 23(5), 296–305. Musso, D., Nhan, T., Robin, E., Roche, C., Bierlaire, D., Zisou, K., & Broult, J. (2014). Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014. Euro Surveillance, 19(14), 20761. Oehler, E., Watrin, L., Larre, P., Leparc-Goffart, I., Lastere, S., Valour, F., … Ghawche, F. (2014). Zika virus infection complicated by Guillain-Barre syndrome—Case report, French Polynesia, December 2013. Euro Surveillance, 19(9), 20720. Phan, T. L., Luong, C. Q., Do, T. H. H., Chiu, C. H., Cao, M. T., Nguyen, T. T. T., … Le, H. N. (2019). Findings and lessons from establishing Zika virus surveillance in southern Viet Nam, 2016. Western Pacific Surveillance and Response, 10(2). Pond, W. L. (1963). Arthropod-borne virus antibodies in sera from residents of South-East Asia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 57(5), 364–371. Quyen, N. T. H., Kien, D. T. H., Rabaa, M., Tuan, N. M., Vi, T. T., Hung, N. T., … Quang, H. K. (2017). Chikungunya and Zika virus cases detected against a backdrop of endemic dengue transmission in Vietnam. The American Journal of Tropical Medicine and Hygiene, 97(1), 146–150. Schuler-Faccini, L. (2016). Possible association between Zika virus infection and microcephaly—Brazil, 2015. MMWR. Morbidity and Mortality Weekly Report, 65(3), 59–62. Song, B.-H., Yun, S.-I., Woolley, M., & Lee, Y.-M. (2017). Zika virus: History, epidemiology, transmission, and clinical presentation. Journal of Neuroimmunology, 308, 50–64. Tappe, D., Rissland, J., Gabriel, M., Emmerich, P., G€unther, S., Held, G., … Schmidt-Chanasit, J. (2014). First case of laboratory-confirmed Zika virus infection imported into Europe, November 2013. Euro Surveillance, 19(4), 20685. TCDC. (2018). Taiwan Centers for Disease Control (Taiwan CDC) announced one new imported Zika case identified in Taiwan. Retrieved from https:// www.cdc.gov.tw/En/Bulletin/Detail/xZ_NvpydeyFPi-_sxxK_pQ?typeid¼158. Wahid, B., Ali, A., Waqar, M., & Idrees, M. (2018). An updated systematic review of Zika virus-linked complications. Asian Pacific Journal of Tropical Medicine, 11(1), 1. Wang, L., Valderramos, S. G., Wu, A., Ouyang, S., Li, C., Brasil, P., … Jiang, T. (2016). From mosquitos to humans: Genetic evolution of Zika virus. Cell Host & Microbe, 19(5), 561–565. Weinbren, M., & Williams, M. (1958). Zika virus: Further isolations in the Zika area, and some studies on the strains isolated. Transactions of the Royal Society of Tropical Medicine and Hygiene, 52(3), 263–268. WHO. (2016a). Situation report: Zika virus, microcephaly, Guillain-Barr
e syndrome. Retrieved from https://apps.who.int/iris/handle/10665/250633. WHO. (2016b). Zika virus infection—Viet Nam. (Online). Retrieved from http://www.who.int/csr/don/12-april-2016-zika-viet-nam/en/. WHO. (2019). Zika epidemiology update. Retrieved from https://www.who.int/emergencies/diseases/zika/zika-epidemiology-update-july-2019.pdf? ua¼1.

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Wilder-Smith, A., Chang, C. R., & Leong, W. Y. (2018). Zika in travellers 1947–2017: A systematic review. Journal of Travel Medicine, 25(1), tay044. Yun, S.-I., Song, B.-H., Frank, J. C., Julander, J. G., Polejaeva, I. A., Davies, C. J., … Lee, Y.-M. (2016). Complete genome sequences of three historically important, spatiotemporally distinct, and genetically divergent strains of Zika virus: MR-766, P6-740, and PRVABC-59. Genome Announcements, 4 (4), e00800–e00816.

Chapter 36

Zika virus infection in Mexico: Epidemiological and clinical data Alfonso Vallejos Para´sa, Concepcio´n Grajales Mun˜izb, Teresita Rojas Mendozab, Lumumba Arriaga Nietoa, and David Alejandro Cabrera Gayta´na a

Coordination of Epidemiological Surveillance, Mexican Social Security Institute (IMSS), Mexico City, M exico, b Technical Input Control Coordination,

Mexican Social Security Institute (IMSS), Mexico City, M exico

Abbreviations IMSS InDRE PCR SCZ SGB SSA ZIKV

Mexican Social Security Institute Mexican Institute of Epidemiological Diagnosis and Reference polymerase chain reaction congenital syndrome associated with Zika Guillain-Barre syndrome Mexican Ministry of Health Zika virus

Introduction In 2015, the circulation of Zika virus (ZIKV) was identified on the American continent (Pan American Health Organization/World Health Organization, 2017b), when an epidemic outbreak was reported in Brazil (Rodriguez Morales, 2015). The ZIKV epidemic spread rapidly in Latin American countries, with the presence of confirmed cases of ZIKV in humans in 19 countries of the region outside Brazil only a few months later (Liu, 2016). It was at the end of 2015 that Mexico saw the arrival of an emerging arbovirus in its territory. However, it was considered as a disease without major complications causing rash and was an arbovirosis with minor repercussions compared to dengue and chikungunya because, before 2015, little was known about zika apart from reports of previous small outbreaks in Micronesia and French Polynesia (Pan American Health Organization/World Health Organization, 2017a). It was not until February 2016 that the World Health Organization declared ZIKV and its complications to be a public health emergency of international importance (World Health Organization, 2016). The graphic images of newborns with microcephaly appeared widely when in Mexico medical care, specialized epidemiological surveillance, and research protocols were initiated to establish the association between ZIKV and its complications. The situation was complex: a virus that shares its form of vector transmission with chikungunya and dengue fever, and with the epidemiological conditions suitable for starting an outbreak in most of the country. In Mexico, the protocol for epidemiological surveillance of vector-borne diseases was updated and amended. Laboratory diagnosis has been dynamic, according to the clinical and epidemiological information of the region and the country, as well as the findings published in the scientific literature on affections and complications in certain groups. In June 2016, a meeting was held in Mexico City, attended by more than 60 researchers and public health professionals from 14 countries of the Americas and Europe to propose standardized research protocols in Zika virus research (Van Kerkhove, 2016). In Mexico, from 2015 to 2019, 12,932 indigenous cases of ZIKV infection have been identified in 29 of Mexico’s 32 states (territories), including 7134 PCR-positive pregnant women (Secretarı´a de Salud. Direccio´n General de Epidemiologı´a, 2019a, 2019b). Cases of Guillain-Barre syndrome (SGB) associated with Zika and cases of congenital syndrome associated with Zika (SCZ) have also been documented. However, some authors suggest that the magnitude of ZIKV in Mexico is much greater. This chapter summarizes the main epidemiological and clinical aspects, focusing on population groups in Mexico. Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00036-5 Copyright © 2021 Elsevier Inc. All rights reserved.

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Background Because of its territorial extension, Mexico ranks 14th in the world, with a continental area of 1,960,189 km2 (National Institute of Statistics and Geography, 2015), and is the 11th most populous country in the world with a population of 119 million people, with an average age of 27 years and a male:female ratio of 94.4:100. Every year, just over 2 million births are registered and the average number of live births to women of reproductive age is 2.3. The gross domestic product per capita amounts to $8902.83 USD (2017), representing the second largest economy in Latin America, and the 15th largest in the world. In all, 78% of the population live in urban areas (where more than 2500 people live). The climate in the country is mainly tropical, extending from north to south from the Tropic of Cancer, along the coastal plains of the Gulf of Mexico and the Pacific Ocean, as well as in the Tehuantepec Isthmus and much of the Yucatan Peninsula. These climates are characterized by their average annual temperature being higher than 18°C and it rains from 800 to 4000 mm per year. Depending on the rainfall, there are three types of tropical climates: tropical with rainfall all year round; tropical with rainfall in summer; and tropical with monsoon rains. The mosquito Aedes aegypti has been present since the time of the conquest in 1519. And Aedes albopictus was confirmed to be present in towns established in some cities bordering the United States of America in 1993 (Iban˜ez-Bernal & Go´mez Dantes, 1995). In Mexico, the first reports of dengue fever were recorded in 1941, when 6955 cases were reported in the country. Few cases were subsequently reported, until their disappearance in 1963 thanks to the aedes eradication campaign that kept dengue fever absent for 12 years. However, in 1978, it was reintroduced in Mexico, and by 1980 cases began to increase, making dengue one of the most important public health problems of today at the national level (Torres-Galicia, 2014). After the arrival of ZIKV, all these aspects of Mexico make the perfect Epidemiological Triad where the disease is the result of the interaction between the agent, the susceptible guest, and the environment, although there is no specific treatment, no vaccine, the presence of a vector, and a climate change that favors its reproduction and dissemination.

Zika distribution and extent in Mexico In Mexico, the identification of ZIKV has been documented since January 2015 (Dı´az-Quin˜onez, 2016), although the first cases confirmed by the autochthonous circulation laboratory were registered in October 2015. The first confirmed indigenous case of Zika was in a resident of the city of Monterrey, the capital of the state of Nuevo Leo´n, a state in the north of the country (Pan American Health Organization/World Health Organization, 2017a). Since the beginning of the outbreak in 2015, an increase in the number of confirmed cases of Zika was observed until the peak was reached in 2016. Transmission continues throughout 2017, although with less intensity (Fig. 1).

FIG. 1 Epidemic curve of Zika cases in Mexico, by month of symptom onset, 2015–18. (The number of Zika cases are laboratory confirmed by PCR. Own elaboration based on data from the Ministry of Health, available at http://www.epidemiologia.salud.gob.mx/anuario/html/incidencia_enfermedad. html. The declaration of accessibility to the information is of universal access to all the people who consult it, and can be consulted at https://www.gob.mx/ accesibilidad.)

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The epidemic curve is based only on laboratory-confirmed Zika cases and may not accurately illustrate the dynamics and magnitude of the epidemic. At the beginning of the epidemic, many cases were estimated for the country; however, the magnitude of the epidemic has not been accurately determined, since the Government of Mexico only publishes confirmed cases of ZIKV and only 10% of suspected cases as well as 100% of pregnant women suspected of ZIKV are taken as laboratory samples to confirm or rule out cases. Initially, the diagnosis of ZIKV is carried out at the Mexican Institute of Epidemiological Diagnosis and Reference “Dr. Martı´nez Ba´ez” (InDRE) of the Ministry of Health of Mexico, by molecular detection (real-time RT-PCR), including internal multiplex platforms. The InDRE has also implemented genetic sequencing for viruses and molecular detection of Zika virus and other arboviruses in mosquitoes. Currently, the diagnosis is decentralized in the Major Public Health Laboratory of Mexico (25 laboratories in the country), which includes tests of competence through an external quality evaluation scheme. Diagnostic algorithms for arboviruses in Mexico have been modified to include molecular tests for chikungunya, dengue (DENV 1–4), and Zika virus. To date, 12,932 indigenous cases have been officially identified in 29 of Mexico’s 32 states (territories) by ZIKV, including 7134 positive pregnant women, in Mexico from 2015 to 2019. The states of Mexico with the highest number of laboratory-confirmed accumulated Zika cases, from 2015 to 2019, are in the Gulf of Mexico region: Nuevo Leo´n, Tamaulipas, Veracruz; in the Pacific Ocean region: Nayarit, Jalisco, Guerrero and Chiaas. And in the Caribbean region: Yucatan (Fig. 2). ´ vila, 2018). This study However, a study estimates that the epidemic had a greater magnitude and impact (Herna´ndez-A stimates that, between November 25, 2015 and September 2, 2016, there were 60,172 symptomatic cases of ZIKV infection, which is approximately 40 times higher than indicated by the corresponding incidence rate previously reported by the government, 1.66 cases per 100,000 population, 16 and almost 30 times the number of confirmed Zika cases reported for Mexico during this time.

FIG. 2 Distribution of Zika laboratory confirmed cases by accumulated State of Mexico 2015–19. (Figure made by the authors with information from the Ministry of Health available at https://www.gob.mx/salud/documentos/casos-confirmados-de-infeccion-por-virus-zika-2020. The declaration of accessibility to the information is of universal access to all the people who consult it, and can be consulted at https://www.gob.mx/accesibilidad.)

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A recent study on the insured population at the Mexican Social Security Institute (IMSS, for its acronym in Spanish), the country’s largest social security institution providing medical services in Mexico, identified 43,725 suspected cases during the 2016 epidemic with an incidence of 82 per 100,000 inhabitants and 186.1 positive cases of ZIKV per 100,000 pregnant women (Grajales-Mun˜iz, 2019). In this study, the age group with the highest incidence was registered in individuals between 20 and 24 years of age, in both sexes, with an incidence rate of 169.8 per 100,000. This value was 219.6 in women and 119.7 in men. The main clinical symptoms presented by suspected cases of ZIKV infection included rash in 41,808 (95.6%) and pruritus in 36,788 (84.1%).

Microcephaly and congenital syndrome associated with Zika Microcephaly is a neonatal malformation defined by a head circumference of less than
2 standard deviations for gestational age and sex; this should be measured at birth and confirmed at 24 h postpartum and contrasted with standardized reference tables. Microcephaly may be associated with structural brain disorders and neurological development problems (Pan American Health Organization, 2016). From outbreaks of ZIKV disease in Brazil and French Polynesia, it was observed that the number of births with microcephaly increased, with an estimated risk of microcephaly associated with maternal ZIKV infection in the first quarter of 1%–113% in Bahia Brasil in 2015–2016; and an estimated risk of microcephaly of 95 cases per 10,000 women who acquired ZIKV during the first quarter in French Polynesia in 2013–15 (Cauchemez et al., 2016). Other countries added to the reports: from October 2015 to January 2018, 27 countries and territories in the Americas have reported confirmed cases of congenital syndrome associated with ZIKV infection (Pan American Health Organization, 2018). According to the WHO, congenital syndrome associated with Zika is considered to be that newborn in which microcephaly is identified or some congenital malformation of the central nervous system and whose mother in pregnancy had a history of residence or travel to an area with the presence of the vector; or antecedent of sexual intercourse with a partner with possible exposure to ZIKV by previous residence or travel history, and confirmed by laboratory (Pan American Health Organization, 2016). This definition was adapted in Mexico for the classification of the syndrome. A study in Mexico estimated that during the period following the confirmed introduction of ZIKV to the country, there was a microcephaly incidence of 11.5 cases per 100,000 births: three times higher than the estimated incidence of micro´ vila, 2018). cephaly of 3.7 cases per 100,000 births in the period prior to the introduction of the virus (Herna´ndez-A Also, the monthly number of microcephaly cases per 100,000 live births was significantly higher after the introduction of the virus than before (incidence rate ratio, IRR 2.9; confidence interval, 95% CI: 2.3–3.6); and was even higher among babies of women living at altitudes below 2200 m above sea level (IRR: 3.4; 95% CI: 2.9–3.9). This shows the association between the positive introduction of ZIKV with a significant increase in the monthly incidence of microcephaly mainly among populations living in localities with an altitude below 2200 m above sea level, where the mosco Aedes aegypti, ´ vila, 2018). which acts as a vector, is endemic (Herna´ndez-A Based on the operational definition established in Mexico by the national surveillance system for the classification of congenital syndrome associated with Zika, and after the opinion of an experts group, 54 cases of congenital syndrome associated with Zika were confirmed between 2016 and 2019. These cases had mainly microcephaly and in some others were reported hydrocephalus, male equine foot, ventriculomegalia, holoproscencephaly, or anencephaly. However, another study found evidence that the Zika virus epidemic reversed the declining trend of microcephaly deaths among children in Mexico and that the number of microcephaly deaths associated with the Zika virus was 50% higher than that reported by the existing SCZ monitoring system. In addition, based on the 22% case fatality rate for the reported SCZ, at least 79 SCZ cases would have occurred in 2016–17. An increase in fetal death rates coded as microcephaly was also observed in 2016–17 (Ca´rdenas, 2019). The spectrum and pathogenesis of SCZ in Mexico are still being investigated, as in the case of the one reported by Valdespino-Va´zquez, where ZIKV antigens were detected in a fetus of 30 weeks’ gestation in multiple organs using the monoclonal antibody 4G2. In the brain, protein E was detected in ventricular ependymal epithelial cells and macrophages located in perivascular spaces. The strong immunoreactivity shown by peripheral macrophages toward F4/80 and the protein ZIKV E supports the hypothesis that the hematogenic spread of the virus could play a role in the development of intrauterine ZIKV infections with multiple involvement organs. The presence of viral particles in renal tissues and the isolation of infectious ZIKV from renal tissues demonstrate that the kidneys are an active site of ZIKV replication in the fetus. In addition, the renal tubular epithelium appears to be at risk for ZIKV infection (Valdespino-Va´zquez, 2019).

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Guillain-Barr e syndrome associated with Zika in Mexico According to information published by the Mexican Ministry of Health (SSA), the number of confirmed cases of SGB associated with ZIKV in Mexico since the identification of the circulation of the virus amounts to 19 people, diagnosed by PCR, which are distributed in nine federal entities: Guerrero (6), Chiapas (3), Tabasco (2), Veracruz (2), Morelos (2), Quintana Roo (1), Michoaca´n (1), Yucatan (1), and the State of Mexico (1). Of which in eight cases were reported, they are located in coastal areas (except Morelos). There are six women and 13 men, with an average age of 35.4 years (5–75 years); in the first 32.2 years (5–59 years) and in men 37 years (5–75 years). As in other countries, the phenomenon of a correlation directly proportional to the number of reported cases of ZIKV with patients with GBS was presented.

Policy and procedures In Mexico, the epidemiological surveillance guidelines for vector-borne diseases use the following operational definitions for the notification and study of patients with zika. Probable case of zika virus infection: Patient with rash and at least two or more of the following signs or symptoms: Fever, Headache, Conjunctivitis (not purulent/hyperemia), Arthralgias, Myalgia, Periarticular edema Pruritus, Retroocular pain. The presence of the vector Aedes aegypti or Aedes albopictus; or Antecedent of visit or residence in areas of transmission in the 2 weeks prior to the beginning of the clinical picture; or Existence of confirmed cases in the locality. Have a history of unprotected sexual contact within 2 weeks prior to the onset of symptoms, with a person who within 8 weeks prior to sexual contact has a history of residence or travel to an area with local VZIK transmission or the presence of vectors.

Probable case in pregnant women with Zika virus disease Any pregnant woman who presents with two or more of the following signs or symptoms: fever, rash, conjunctivitis (not purulent), headache, myalgia, arthralgia or retroocular pain, periarticular edema, pruritus, and who identifies some epidemiological association.

Confirmed case of Zika virus disease Any case likely to be positive for ZIKV based on the detection of viral RNA using real-time RT-PCR in serum samples taken in the first 5 days of onset of the clinical picture.

Discarded case of Zika virus disease Any case in which there is no evidence of the presence of any virological marker for ZIKV by laboratory techniques endorsed by the Epidemiological Reference and Diagnostic Laboratory of the Mexican Ministry of Health. The case of Guillain-Barre syndrome associated with zika virus infection is defined as any case of acute flaccid paralysis that in the 21 days prior to the onset of paralysis has met the definition of probable cases of zika virus infection.

Mini-dictionary of terms According to the guidelines for the epidemiological surveillance of zika and its complications, in Mexico the following definitions were established: Microcephaly: Neurological disorder in which the occipitofrontal circumference is smaller than the corresponding one by age, race, and sex. It is defined as a head circumference with two standard deviations below the mean for age and sex or approximately less than the second percentile. Congenital Syndrome Associated with Zika: In any newborn it is identified as a pattern of birth defects with any of the following characteristics: l l l l

Severe microcephaly in which the skull partially collapses. Decreased brain tissue with a specific pattern of brain damage. Damage to the back of the eye. Joints with limitations in movement, such as male equine foot.

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Too much muscle tone restricts body movement just after birth.

The presence of the virus in the mother or in the product through laboratory tests endorsed by the Epidemiological Diagnostic and Reference Laboratory of the Mexican Ministry of Health. The case of acute flaccid paralysis probable from Guillain-Barre syndrome associated with zika virus infection is defined as any case of acute flaccid paralysis that in the 21 days prior to the onset of paralysis has met the definition of a probable case of zika virus infection. The case of acute flacid paralysis confirmed Guillain-Barre syndrome associated with zika virus infection is defined as the case classified as SGB associated with zika virus infection and with laboratory confirmation to this virus by detecting viral RNA using real-time RT-PCR.

Key facts l

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From 2015 to 2019, 12,932 cases of Zika virus (ZIKV) infection have been identified in 29 out of 32 states (territories) of Mexico. 7134 PCR-positive pregnant women. A total of 19 cases of Guillain-Barre syndrome (SGB) associated with Zika. Overall 54 cases of congenital syndrome associated with Zika (SCZ) have also been documented in this period. In Mexico, only 10% of suspected Zika cases and 100% of suspected Zika pregnant women with suspected Zika virus infection are sampled from laboratories. The laboratory uses only the PCR technique for the confirmation of ZIKV cases and does not use serology.

Summary points l l l l l

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In Mexico, the Zika virus epidemic was greater than reported. Cases of SCZ and SGB associated with zika virus have been documented. From 2017, there is a decrease in Zika cases in Mexico. Entomological and viral surveillance should anticipate epidemiological surveillance. Up-to-date protocols for the organization of health services in view of the increased demand for medical care, including in areas not considered endemic or with high viral circulation. Research studies responding to knowledge gaps related to virology, immunology, and epidemiology.

References Ca´rdenas, V. M. (2019). Subinformacio´n del sı´ndrome de zika congenito fatal, Mexico, 2016-2017. Enfermedades infecciosas emergentes, 1560–1562. Cauchemez, S., Besnard, M., Bompard, P., Dub, T., Guillemette-Artur, P., Eyrolle-Guignot, D., et al. (2016). Association between Zika virus and microcephaly in French Polynesia, 2013-15: A retrospective study. Lancet (London, England), 387(10033), 2125–2132. Dı´az-Quin˜onez. (2016). Evidence of the presence of the Zika virus in Mexico since early 2015. Virus Genes, 52, 855–857. Grajales-Mun˜iz. (2019). Zika virus: Epidemiological surveillance of the Mexican institute of social security. PLoS One, 14, e0212114. ´ vila. (2018). Zika virus infection estimates. Mexico: Bulletin of the World Health Organization. Herna´ndez-A Iban˜ez-Bernal, S., & Go´mez Dantes, H. (1995). Los vectores del dengue en M exico: una revisio´n crı´tica. Salud Pu´blica de Mexico. Recuperado de http:// saludpublica.mx/index.php/spm/article/view/4564/5018. Instituto Nacional de Estadı´stica y Geografı´a, Mexico. (2015). Extensio´n territorial de M exico. Liu, S. (2016). Zika virus: A flavivirus caused pandemics in Latin America. Virologica Sinica, 31, 101–102. Pan American Health Organization. (2016). 69th world health assembly Zika photostory. Pan American Health Organization/World Health Organization. (2017a). Zika—Epidemiological report Mexico. Washington, DC: PAHO/WHO. Pan American Health Organization/World Health Organization. (2017b). Zika—Epidemiological report Mexico. Washington, DC: PAHO/WHO. March. Pan American Health Organization. (2018). Zika Cumulative Cases 2018. PAHO. https://www.paho.org/hq/index.php?option¼com_content& view¼article&id¼12390:zika-cumulative-cases&Itemid¼42090&lang¼en. Rodriguez Morales, A. (2015). Zika: The new arbovirus threat for Latin America. Journal of Infection in Developing Countries, 9, 684–685. Secretarı´a de Salud. Direccio´n General de Epidemiologı´a. (2019a). Casos Confirmados de Enfermedad por Virus del Zika. Secretarı´a de Salud. Direccio´n General de Epidemiologı´a. (2019b). Casos confirmados de Sı´ndrome Cong enito asociado a Zika, M exico 2016-2018. Torres-Galicia. (2014). Dengue en Mexico: ana´lisis de dos decadas. Gaceta M edica de M exico, 150, 122–127.

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Valdespino-Va´zquez. (2019). Congenital Zika syndrome and extra-central nervous system detection of Zika virus in a pre-term newborn in Mexico. Clinical Infectious Diseases, 68, 903–912. Van Kerkhove, M. D. (2016). Harmonisation of Zika virus research protocols to address key public health concern. The Lancet Global Health, 4, e911–e912. World Health Organization. (2016). https://www.who.int/news/item/18-11-2016-fifth-meeting-of-the-emergency-committee-under-the-internationalhealth-regulations-(2005)-regarding-microcephaly-other-neurological-disorders-and-zika-virus.

Chapter 37

Cytopathicity and pathogenesis of Zika virus strains Sergio P. Alpuche-Lazcanoa and Anne Gatignola,b a

Department of Medicine, Division of Experimental Medicine at McGill University, Lady Davis Institute for Medical Research, Montreal, QC, Canada,

b

Department of Microbiology and Immunology at McGill University, Lady Davis Institute for Medical Research, Montreal, QC, Canada

Abbreviations ADAMTS9 C CDC CXCL10 CZVS DENV E ER IFN IL ISG MAVS MDA5 miRNA MOI NPCs NS prM RIG-I RNAi sfRNA SG STAT2 TLR-3 TNF TRIM25 UTR XRN1 ZIKV

a disintegrin and metallopeptidase with thrombospondin motifs 9 capsid Center for Diseases Control C-X-C motif chemokine ligand congenital ZIKV syndrome dengue virus envelope endoplasmic reticulum interferon interleukin interferon stimulated gene mitochondrial antiviral signaling protein melanoma differentiation associated gene 5 micro-RNA multiplicity of infection neural progenitor cells nonstructural proteins premembrane retinoic acid-inducible gene I RNA interference subgenomic flavivirus RNAs stress granule signal transducer and activator of transcription 2 toll-like receptor 3 tumor necrosis factor tripartite motif containing 25 untranslated region 50 -30 exoribonuclease 1 Zika virus

Introduction Zika virus (ZIKV) is an emerging virus of the Flaviviridae family and Flavivirus genus, which also includes Dengue (DENV), West Nile, yellow fever, and Japanese encephalitis viruses (Heinz & Stiasny, 2017). They are primarily transmitted by Aedes mosquito bites from an infected person to another. The first infection in humans was reported in 1954 in Nigeria, and the virus spread to other African territories and reached Asia in 1966 (Marchette, Garcia, & Rudnick, 1969). Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00037-7 Copyright © 2021 Elsevier Inc. All rights reserved.

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After sporadic cases and small outbreaks, the first major outbreak took place in 2007 on Yap Island in the Federated States of Micronesia in the Pacific Ocean (Duffy et al., 2009). A massive epidemic followed in French Polynesia in 2012–13 and ZIKV reached the Americas between 2013 and 2014 (Rasmussen, Jamieson, Honein, & Petersen, 2016; Schuler-Faccini et al., 2016). The largest ZIKV outbreak occurred in Brazil, with 7000 cases between February and April 2015, followed by Paraguay, Bolivia, Ecuador, Colombia, Venezuela, all Central America, including Mexico and some regions in the USA (Kindhauser, Allen, Frank, Santhana, & Dye, 2016). Meanwhile, in 2016, another outbreak with local strains was declared in Singapore, the Philippines, Vietnam, and Thailand (Lim, Lim, & Yoon, 2017). Currently, sporadic cases all over the globe are reported. As of June 2019, the Center for Diseases Control (CDC) reported 18 new ZIKV cases in North American territories (CDC, 2019). The ZIKV origin and pandemics can be observed in (Fig. 1). In adults, ZIKV infection generally causes mild symptoms such as fever, headache, rash, conjunctivitis, and arthralgia (Du et al., 2019; Ferraris, Yssel, & Misse, 2019). After the transient symptoms, ZIKV can remain hidden mainly in the host reproductive tract and can be transmitted by intercourses (Stassen, Armitage, van der Heide, Beagley, & Frentiu, 2018). During pregnancy, ZIKV can be transmitted vertically at any stage, which leads to congenital ZIKV syndrome (CZVS). CZVS gives rise to abnormal brain development including microcephaly (Mlakar et al., 2016; Musso, Ko, & Baud, 2019). In 2015, Brazil reported an incidence of microcephaly cases 20 times higher than those observed in previous years in regions where ZIKV had been detected (Calvet et al., 2016). Various reports confirmed the ZIKV vertical transmission and the neurological abnormalities in neonates (Franca et al., 2016; Mlakar et al., 2016). The distinctive hallmarks of CZVS are associated with recent Asian/American strains but not with the African or early Asian ones. The most likely explanation is the acquisition of different mutations by the Asian lineage during decades of circulation until it reached America. Indeed, a retrospective study demonstrated that the first cases of CZVS occurred in French Polynesia during the 2013 outbreak, suggesting that ZIKV’s neurotropism emerged at some point between Asia and French Polynesia (Cauchemez et al., 2016). We and others have reported that mutations of contemporary Asian/American strains compared to early Asian ones resulted in a growth advantage causing different cytopathology (Alpuche-Lazcano et al., 2018; Barnard, Rajah, & Sagan, 2018; Kuivanen et al., 2017). Moreover, the cytopathicity and pathogenesis induced by African, early Asian, and late Asian/American strains are distinct and led us to distinguish three groups instead of the two lineages derived from phylogenetic trees, with the Asian lineage separated into the early Asian and the late Asian/ American group.

FIG. 1 ZIKV dissemination map with relevant dates. ZIKV emerged in Uganda in 1947 and disseminated to central Africa. During the 1960s, ZIKV reached Asia, causing sporadic infections and some outbreaks for more than four decades. The French Polynesia outbreak was pivotal for ZIKV to reach the Americas. Between October 2013 and March 2014, ZIKV was introduced into 11 Brazilian cities by tourists from French Polynesia (Massad et al., 2017). Shortly after, Latin America and some regions in the USA reported cases of infected people with ZIKV (Kindhauser et al., 2016).

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ZIKV genome and evolution ZIKV possesses an ssRNA (+) genome of around 11 kb that encodes a single polyprotein, which is processed by cellular and viral proteases into structural proteins (capsid (C), premembrane (prM) and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (Shi & Gao, 2017; Ye et al., 2016). The 50 UTR is capped, but in the absence of the cap, the 50 UTR can efficiently act as an internal ribosomal entry site, producing an infection in mammalian or insect cells (Song, Mugavero, Stauft, & Wimmer, 2019). ZIKV structural proteins form the nascent viral particle with a size of 50 nm (Shi & Gao, 2017). The C protein binds tightly to the genomic RNA, whereas E and prM mediate the binding, fusion, and entry in the cell (Lazear & Diamond, 2016; Mukhopadhyay, Kuhn, & Rossmann, 2005; Shi & Gao, 2017). The NS proteins have not thoroughly been studied, but the similarity with those from other flaviviruses suggests related functions. NS1 is a glycoprotein involved in the formation of the replication complex as well as the modulation of the innate immune response (Crook, Miller-Kittrell, Morrison, & Scholle, 2014; Youn, Ambrose, Mackenzie, & Diamond, 2013). Furthermore, NS1 activates the transmission from humans to mosquitoes (Nicastri et al., 2016). NS2A destabilizes the adherent junction complex impairing the cell-cell junctions (Yoon et al., 2017). NS2B is the cofactor of NS3, the ZIKV protease essential for the polyprotein cleavage (Shiryaev et al., 2017). NS4A regulates energy consumption by NS3-NS2B during the viral RNA unwinding process (Shiryaev, Chernov, Aleshin, Shiryaeva, & Strongin, 2009). NS4B has a critical role in remodeling the endoplasmic reticulum (ER) and in the formation of replication-competent membrane structures (Kaufusi, Kelley, Yanagihara, & Nerurkar, 2014). Finally, NS5 is the RNA-dependent RNA polymerase that catalyzes the RNA synthesis through the N domain, while the methyltransferase domain in the C terminus adds the cap for translation (Zhao et al., 2017). Phylogenetic analyses have divided ZIKV into African and Asian lineages. The first reported ZIKV strain corresponds to the African strain called MR766 (Dick, Kitchen, & Haddow, 1952). Most of the African strains belong to monkeys and mosquitoes and only a small number of human cases of ZIKV infections were documented in Africa, suggesting that the transmission of the African strains to humans is not very efficient (Haddow et al., 2012). P6-740, a ZIKV isolated in 1966 in Malaysia, is considered as the common ancestor of Asian strains (Beaver, Lelutiu, Habib, & Skountzou, 2018). The outbreak in Micronesia, represented by the FSM/Micronesia/2007 strain, is related to P6-740 and belongs to the early Asian lineage along with strains from Cambodia, the Philippines, and Thailand, which emerged between 2010 and 2014. In contrast, the H/PF/2013 strain (French Polynesia/2013) is more closely related to contemporary American strains and can be considered a late Asian strain. The comparison between 8 African and 25 Asian strains has shown 59 amino acid variations in the polyprotein sequence. Four hundred nucleotide changes leading to 26 amino acid substitutions occurred between P6740 and FSM as well as eight additional amino acids for a total of 34 modifications in the late Asian/American strains (Wang et al., 2016). These phylogenetic studies, the changes in amino acids sequences, as well as the correlation with the cytopathicity and pathogenesis after the French Polynesia epidemic further suggest two Asian sublineages represented by the early Asian and late Asian/American groups.

Cytopathic effects and replication capacity of ZIKV lineages Various studies in different cell types have contributed to the understanding of the cytopathicity and replication capacity of the African and Asian lineages of ZIKV during the infection. In vitro experiments using hepatocytic cell lines found that ZIKV had an average replication peak at day 3 postinfection and exhibited cytopathic effects that included loss of attachment and cytolysis (Sherman et al., 2019). The appearance of the cytopathic effects varies depending on the ZIKV strain. Plaque morphology from in vitro cell culture has been documented after infection with African, preepidemic (early Asian), and postepidemic (late Asian and American) ZIKV strains. Plaques generated by African strains are small and uniform in size, whereas plaques from early Asian isolates are larger and have indefinite borders. Furthermore, plaques from late Asian/American strains are more prominent with well-defined edges (Alpuche-Lazcano et al., 2018; Barnard et al., 2018) (Fig. 2). Electron microscopy images showed that, during the peak of ZIKV replication, cells produced vesicles or viral particles, which led to the cytopathic effect (Sherman et al., 2019). Images generated by immunofluorescence and electron tomography show that, in human hepatoma and neural progenitor cells (NPCs), these vesicles are generated from ER membrane invagination and contain viral replication factories (Cortese et al., 2017) (Fig. 3). These structures were obtained with an African (MR766) and a late Asian (H/PF/2013) strain, showing that ZIKV from different lineages modifies the organization of microtubules and intermediate filaments surrounding these replication factories, which induces cytopathicity and affects cell function. Differences also exist in studies about the replication rate that seems to be multifactorial. Indeed, African strains seem to have a faster replication rate than Asian or Asian/American strains in vitro and in vivo models (Bowen et al., 2017;

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FIG. 2 Comparison of plaque formation between ZIKV early Asian and late Asian/American strains. Plaques generated by the early Asian strain differ from the contemporary Asian/American ones. (Left) The early Asian ZIKV PLCal_ZV forms fuzzy plaques with indefinite edges with the shape of a comet. (Right) Contemporary Asian/American strains (HS-2015-BA-01) form rounded plaques with defined edges.

FIG. 3 The architecture of ZIKV replication factories. ZIKV forms vesicles within the ER membrane through invaginations where replication factories are contained. zER, zippered ER; Vi, ZIKV virions; Ve, virus-induced invaginated vesicles. (Reproduced from Fig. 4A in Cortese, M., Goellner, S., Acosta, E.G., Neufeldt, C.J., Oleksiuk, O., Lampe, M., Haselmann, U., Funaya, C., Schieber, N., Ronchi, P., Schorb, M., Pruunsild, P., Schwab, Y., Chatel-Chaix, L., Ruggieri, A., & Bartenschlager, R. (2017). Ultrastructural characterization of Zika virus replication factories. Cell Reports, 18, 2113–2123. Under the terms of Creative commons CC-BY license.)

Shao et al., 2017). Studies of cytopathicity in vitro have compared African, early Asian and American strains wherein a Brazilian strain at high MOI elicited higher replication, a significant reduction of cell viability, and elevated viral RNA accumulation. In contrast, the cytopathic effect at low MOI is significantly higher in an African strain with high RNA accumulation in distinct foci within the A549 lung cells (Barnard et al., 2018). Our work demonstrated that cytopathicity induced by an American strain from Bahia (Brazil), but not from an early Asian strain, becomes visible 24 h postinfection with an MOI of 0.1 (Alpuche-Lazcano et al., 2018). Furthermore, infection in NPCs and astrocytes revealed that an African isolate (ArB41644) had stronger infectivity than the French Polynesian strain H/PF/2013 (Simonin et al., 2016).

The innate immune cell response triggered by different ZIKV lineages contributes to their cytopathicity The time variation for the appearance of the cytopathic effects between different strains may be linked to the cell response against the infection through the innate immunity pathway by the interferon (IFN)/cytokine response (Fig. 4). For example,

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FIG. 4 ZIKV lineages trigger different innate immune cell responses that contribute to the cell death rate. (Left) Upon virus entry, the African lineage shows a high replication rate with a strong cell response characterized by a high production of proinflammatory cytokines and higher activation of innate immune receptors. (Right) Upon virus entry, the Asian lineage induces a weaker cell response than the African strains with a lower replication rate, although some American isolates have a higher replication rate. Asian lineages induce a weak activation of innate immune receptors, low production of proinflammatory cytokines, high production of antiinflammatory IL-10 and cellular factors ADAMTS9 and fibronectin.

the African ArB41644 strain upregulates genes involved in the induction of IFN, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and Toll-like receptor 3 (TLR-3), whereas the Asian strain downregulated genes such as C-X-C motif chemokine ligand (CXCL)10, caspase-1, and cathepsin-s, which modulate the cell death and the cytopathic effect rate. Interestingly, both strains downregulated CXCL8, a chemotactic factor that attracts neutrophils, basophils, and T-cells (Simonin et al., 2016). Human CD14+ blood monocytes infected with different lineages of ZIKV contribute to the classical or nonclassical monocyte polarization. Infection with MR766 induced proinflammatory M1-skewed responses with a production of cytokines like CXCL10, Interleukin (IL)-23A, IL-18, whereas H/PF/2013 infection polarizes to M2 responses associated with higher production of the antiinflammatory and immunosuppressive cytokine IL-10. Reports using the Asian/American and African strains have demonstrated that ZIKV NS5 targets signal transducer and activator of transcription 2 (STAT2), interfering with the transcription of IFN-stimulated genes (ISGs) (Grant et al., 2016; Osterlund et al., 2019). Other studies using IFN α/β receptor 1/ and STAT2/ murine models showed that African strains have a higher infectivity and mortality ratio than Asian strains correlated to a robust inflammatory response with the release of IL-6, CXCL10, tumor necrosis factor (TNF)-α, IFN, IL-1β, and T-cell infiltration markers after African ZIKV infection (Tripathi et al., 2017). Of note, Asian strains cause less severe neurological symptoms than African strains but persist in neuronal cells for a longer time. Consistent with these studies, transcriptomic analysis between African and Asian strains demonstrated that MR766 dysregulates around 90% more genes than a Brazilian strain from Paraiba (Dang, Tiwari, Qin, & Rana, 2019). Finally, the partial degradation of genomic RNA by XRN1 produces subgenomic flavivirus RNAs (sfRNAs) in most of the flaviviruses. SfRNAs contribute to viral cytopathicity by interfering with innate immune factors like the Tripartite motif containing 25 (TRIM25) or RIG-I activation, which leads to a decreased induction of IFN (Mazeaud, Freppel, & ChatelChaix, 2018). The production of sfRNAs by contemporary ZIKV strains antagonizes not only the activity of RIG-I, but the activity of RNA binding proteins involved in RNA splicing and decay as well (Donald et al., 2016; Michalski et al., 2019).

Cellular RNA interference and stress response contribute to cytopathicity RNA interference (RNAi) could also contribute to the cytopathicity of each lineage. The micro-RNA (miRNA) profile upon ZIKV entry has been linked to the dysregulation of different mRNAs and pathways such as inflammation, neurogenesis,

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metabolism, stem cell maintenance, and apoptosis (Azouz, Arora, Krause, Nerurkar, & Kumar, 2019; Dang et al., 2019; Zhang et al., 2019). The MiRNA profile analysis of neurons from mice fetuses has identified a downregulation in several miRNAs that regulates inflammatory responses, but only a few upregulated miRNAs (miR-155, miR-203, and miR29b) that have antiviral activity against other viruses (Azouz et al., 2019; Pareek et al., 2014; Thounaojam, Kaushik, Kundu, & Basu, 2014; Zhang et al., 2018). Similarly, miRNAs were mostly downregulated in neuroblastoma cell lines infected with a Brazilian ZIKV strain. Among the few upregulations, miR-145 target proteins of the cell cytoskeleton and miR-148a are likely involved in neuronal development. These miRNAs were also upregulated in the postmortem tissues of children with CZVS (Castro et al., 2019). Another study of miRNA-mRNA dynamics during ZIKV infection in human NPCs showed dysregulation of genes involved in neurogenesis, stem cell maintenance, and metabolism by miRNAs (Dang et al., 2019). The infection of NPCs with ZIKV induced a higher amount of transcribed genes with the African isolate than with the American strains. Nonetheless, more miRNAs with an activity in the attenuation of the cell response, like miR-125a involved in the inhibition of a mitochondrial antiviral signaling protein (MAVS), had a higher concentration in cells infected with the American strain than with the African one (Dang et al., 2019). Other mechanisms contribute to the cytopathicity of ZIKV. In stress conditions, eukaryotic cells phosphorylate eukaryotic translation initiation factor 2α, which arrests the RNA translation and triggers the assembly of stress granules (SGs). ZIKV American strain PRVABC59 counteracts the assembly of SG under oxidative stress conditions induced by arsenite, which facilitates virus replication (Amorim, Temzi, Griffin, & Mouland, 2017). Furthermore, peroxisomes involved in lipid metabolism and regulation of reactive oxygen species are functional to eliminate viruses through the induction of IFN. ZIKV depletes 60%–70% of peroxisomes independently of the virus lineage, through the C protein and the sequestration of peroxisomal biogenesis factor 19, an essential protein for the peroxisome biogenesis. Interestingly, the loss of peroxisomes in an astrocytic cell line was more pronounced with PRVABC59 than with MR766 (Wong et al., 2019). These data suggest that American strains dismantle cellular mechanisms more efficiently. The comparison of amino acid differences between the African and the contemporary ZIKV strains should reveal mutations involved in this long-term infection.

Contemporary Asian-American ZIKV strains contain key mutations that contribute to the pathogenesis Among a large number of amino acid sequence variations, recent Asian/American isolates have distinctive mutations associated with their pathogenesis. In 2016, a phylogenetic comparison between different ZIKV isolates discovered 28 amino acid differences between the early Asian isolate from Micronesia and contemporary ones (Fig. 5). Some of these mutations may be associated with the epidemic emergence and neuropathology of ZIKV. Most of the evidence suggests that NS1 variations could be deeply implicated (Pettersson et al., 2016). Neuropathologies triggered by ZIKV were firstly reported in French Polynesia with the appearance of the mutation S139N localized in residue 17 of the membrane protein prM (Pettersson et al., 2016). Similar protein structures in DENV suggest that this mutation could be involved in the virus maturation process or immune evasion (Screaton & Mongkolsapaya, 2017). ZIKV infection performed in mice and in NPCs confirmed that the single substitution S139N is sufficient to increase the replication rate, to induce cell apoptosis, and to contribute to severe microcephaly in fetal mice (Yuan et al., 2017). Future studies will determine whether this mutation has been crucial in virus-induced neuropathologies during the recent outbreaks. Furthermore, an American ZIKV isolate (KU527068) from a human fetal brain tissue showed a variation in NS1 (T1028A) (Wang et al., 2017). T1028A destabilizes the structure of NS1 at the dimer interface and affects

FIG. 5 ZIKV mutations that have arisen in contemporary Asian strains. ZIKV acquired mutations detected in recent isolates compared to the ancestral strain EU545988.1 are pointed out with arrows. Red arrows indicate the acquired mutations from French Polynesia isolates onward.

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its general conformation, which may increase the viral replication. Another recently acquired substitution only present in American isolates is M2634V in NS5 and contributes to viral fitness (Liu, Shi, & Qin, 2019). The Brazilian strain HS-2015-BA-01 has been studied in vitro and in vivo. It possesses the prM S139N, the NS5 M2634V, and other substitutions compared to an early Asian one (PLCal_ZV). HS-2015-BA-01 induces an increased cytopathicity and a higher RNA accumulation compared to PLCal_ZV in different cell types as measured by the induction of lytic plaques, a metabolic assay, and viral RNA quantitation. At high MOI, this isolate also had higher titers and faster replication than African and other contemporary strains in lung and astrocytic cells (Alpuche-Lazcano et al., 2018; Barnard et al., 2018). While HS-2015-BA-01 strains induced neuronal damage in immunocompromised mice, it induced a mild infection in immunocompetent mice and monkeys (Costa et al., 2017; Pardy et al., 2017; Silveira et al., 2017). Therefore, variations in specific amino acids in contemporary Asian-American strains are associated with higher cytopathicity and pathogenesis in humans.

Conclusion The differences in cytopathicity that exist between ZIKV lineages reflect not only the genome and protein expression but also the cell response that is triggered by the virus (Fig. 6). African ZIKVs are more virulent with faster replication kinetics and a higher cytopathicity. As a consequence of this efficient replication, cell response is mounted with a strong wave of proinflammatory cytokines. In vitro models allow us to observe such cytokine expression in the cell but do not preclude further outcomes in the body. Through this wave of cytokines and activation of different mechanisms to eliminate ZIKV, a human infected with an African lineage is likely to eradicate the virus rapidly. This is probably one of the reasons why African strains are not as efficient as Asian/American strains to spread among humans. In contrast, Asian strains trigger a less potent immune response and distinct studies point out that the RNAi pathway and other cellular mechanisms like SGs and peroxisomes may play a substantial role in viral replication. A weak immune response allows the virus to remain in the cells and the body for a longer period, which likely gives it enough time to cross the placental barrier and affects the development of the brain in neonates. Of note, some American strains have been observed to have higher rates of replication and RNA accumulation than African isolates, which could explain the differences in pathogenesis.

Policy and procedures Phylogenetic studies: Phylogenetic studies are used to know and understand the evolutionary relationships between viruses. To visualize these relationships, phylogenetic trees are created by protein or nucleotide sequences and the use of mathematical models that can be performed at this link: http://tree.bio.ed.ac.uk/software/figtree/.

FIG. 6 Comparison of effects of different ZIKV strains. In general, ZIKV African strains produce high cytopathicity, high cell response, and high production of viral particles. Early Asian strains produce low cytopathicity, low cell response, and low virus production. Late Asian/American strains have a better virus replication rate with a higher cytopathic effect than early Asian strains. *Some American strains have higher replication, cytopathicity, and RNA accumulation than the African lineage.

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Cytolysis measured by plaque assay: Plaque assays are commonly used to determine the titer of cytolytic viruses. The indicator cells are seeded into 6-well culture plates at a determined density. In parallel, serial 10-fold dilutions of the virus stock are prepared. After cell medium removal, dilutions of the virus are applied to the cells. After incubation, the virus dilutions are removed and replaced with a mixture containing carboxymethylcellulose in culture medium. Several days postinfection, the medium is removed, cells are fixed with paraformaldehyde and incubated with a solution of crystal violet to visualize plaques. Cells appear colored and cells lysed by the virus appear as holes or plaques. Each plaque is considered to correspond to one virus in the original stock and the amount can be estimated from the counts and the dilutions. Details can be found at this link: https://www.sciencedirect.com/topics/medicine-and-dentistry/plaque-assay. Electron microscopy: Transmission electron microscopy (TEM) is a technique that allows users to observe small structures of the size of nanometers. TEM accelerates electrons that are beamed to sample. The result allows the users to observe key features such as structures or morphologies of cells or viruses. The technique is explained in: https://pubmed.ncbi.nlm. nih.gov/28249158/. Viral RNA quantitation: To perform viral RNA quantification, the RNA is first extracted from virus-infected cells. To quantify it, the extracted RNA must be transformed into complementary DNA by reverse transcription. The newly synthesized DNA and specific primers are used to quantify the viral DNA by quantitative polymerase chain reaction (qPCR). The technique is explained in: https://pubmed.ncbi.nlm.nih.gov/29382068/.

Mini-dictionary of terms Cytopathicity: The condition of undergoing cytopathic change during cell disease. Pathogenesis: The mechanism whereby something causes a disease. Lineage: Lineal descent from a common ancestor. Phylogeny: Evolution of a genetically related group of organisms. Innate immune cell response: General immune response not specific to a particular pathogen. RNA interference: Conserved biological response to double-stranded RNA that regulates the expression of proteincoding genes.

Key facts of cytopathicity and pathogenesis l l l

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Congenital ZIKV syndrome gives rise to abnormal brain development and microcephaly. Phylogenetic analyses have divided ZIKV into African and Asian lineages. Cytopathic effects produce vesicles generated from the ER membrane invagination and contain viral replication factories. Cytopathic effects lead to cell lysis and are visible as plaques. The innate immune cell response triggered by different ZIKV lineages contributes to their cytopathicity. Cellular RNA interference and stress response contribute to cytopathicity. Asian-American ZIKV strains contain key mutations that contribute to the pathogenesis. The Asian lineage can be further separated into an early Asian and an Asian/American group for cytopathic effects correlated to pathogenicity.

Summary points of cytopathicity and pathogenesis l l l

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ZIKV has been divided into African and Asian lineages. In vitro, the African lineage leads to faster replication, generating higher cytopathicity and cell death. Early Asian strains replicate slower than African strains but most of the contemporary American/Asian isolates replicate faster. Viral replication factories induce cytopathic effects by forming vesicles generated from the endoplasmic reticulum membrane. Cytopathic effects lead to cell death. Asian strains survive longer in the host by inducing a lower immune response. The cytopathic effects correlated to the pathogenicity lead to the separation of the Asian lineage into an early Asian and an Asian/American group.

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Chapter 38

Molecular evolution and codon usage bias of Zika virus Marianoel Pereira-Go´meza,b and Juan Cristinaa a

Laboratory of Molecular Virology, Centro de Investigaciones Nucleares, Universidad de la Repu´blica, Montevideo, Uruguay, b Laboratory of

Experimental Evolution of Viruses, Institut Pasteur de Montevideo, Montevideo, Uruguay

Abbreviations + ssRNA ADAR CAI DENV ENC GC content JEV LAV NS1 RNA RSCU s/s/y tRNA WNV YFV ZIKV

single stranded positive-sense RNA RNA-dependent adenosine deaminase codon adaptation index dengue virus effective number of codons guanosine + cytosine percentage Japanese encephalitis virus live viral vaccines nonstructural protein NS1 ribonucleic acid relative synonymous codon usage substitutions/site/year transfer ribonucleic acid West Nile virus yellow fever virus Zika virus

Introduction Zika virus (ZIKV) is an enveloped virus with a linear positive sense single-stranded RNA (+ssRNA) genome of 10,794 ribonucleotides which belongs to the Flaviviridae family (genus Flavivirus). Flavivirus include several phylogenetically related viruses that are naturally transmitted by mosquitoes, such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and yellow fever virus (YFV) (Weaver & Barrett, 2004). ZIKV was originally isolated from a rhesus monkey in the Zika Forest, Uganda (Dick, 1952a), and further isolations within the same region came from the mosquito Aedes africanus (Dick, 1952a; Weinbren & Williams, 1958). The first evidence of human infection by ZIKV came from the presence of high titers of specific antibodies in human populations living near the Zika Forest (Dick, 1952b). Subsequent reports showed that ZIKV was mostly silently circulating in several countries of Africa and Asia (Herrera et al., 2017). The first ZIKV outbreak occurred in Oceania in 2007 (Duffy et al., 2009). Then the virus reached Latin America from French Polynesia (Campos, Bandeira, & Sardi, 2015). The most recent ZIKV outbreaks were related to severe clinical manifestations including congenital microcephalia in the fetuses of pregnant women infected with ZIKV (Petersen et al., 2016) and Guillain-Barre syndrome (Oehler et al., 2014). ZIKV is an arbovirus, which means that its transmission occurs in enzootic or urban cycles involving the infection of a human or nonhuman primate by an arthropod, primarily mosquito species of the genus Aedes. However, evidence of human-to-human infection by body fluids was also found (Foy et al., 2011). The fact that the viral maintenance depends on the alternation between two different cell host types subjects that ZIKV to different selective pressures that influence its molecular evolution by imposing evolutionary constraints (Coffey et al., 2008). Experimental studies have shown that Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00038-9 Copyright © 2021 Elsevier Inc. All rights reserved.

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viruses which cycle between vertebrates and invertebrates have a lower rate of evolution than related viruses which only replicate in a single host (Novella et al., 1995; Weaver, Brault, Kang, & Holland, 1999). This could be, at least in part, due to differences in the viral spontaneous mutation rate across host cells which seems to be related to the payoffs of viral transmission modes (Combe & Sanjua´n, 2014). In addition, viral vector specificity seems to be related to codon usage bias in flaviviruses ( Jenkins, Pagel, Gould, Zanotto, & Holmes, 2001). Therefore, a better understanding of the molecular evolution of ZIKV populations, including codon usage bias, is needed to shed light on the determining mechanisms of virus-host interaction, as well as viral fitness, evasion of the host’s immune response, and viral emergence. Finally, the study of the codon usage bias has practical implications in the development of ZIKV live viral vaccines.

What is codon usage bias? One of the key features of the genetic code is its redundancy. That means that most amino acids are encoded by up to six different codons, called synonymous codons. Codon usage bias refers to the fact that some synonymous codons are used more or less often than expected at random during gene translation. Molecular evidence suggests that codon usage bias is common across genomes and may contribute to genome evolution (Sharp & Matassi, 1994). However, the patterns of codon usage vary significantly between organisms and between genes within the same genome (Akashi, 2001). From a point of view of the neutral theory of molecular evolution (Kimura, 1968, 1977), synonymous codons should evolve neutrally since the protein primary sequence is not changed. Therefore, codon usage bias was initially interpreted as evidence of differential selection between alternative synonymous codons (Sharp, Stenico, Peden, & Lloyd, 1993). For example, in unicellular organisms, codon usage bias correlates with the gene expression level (Sharp et al., 1988) as a consequence of selection for translational efficiency where optimal codons are selected (Sharp & Li, 1986). However, there are additional factors that might influence codon usage such as gene length (Moriyama & Powell, 1998), GC content, and RNA stability (Carlini, Chen, & Stephan, 2001) among others.

Evolutionary theories of codon usage bias There are two main evolutionary explanations for the existence of the codon usage bias observed in organisms: the selectionist and the mutational or neutral theories. As mentioned, the selectionist theory states that codon usage bias contributes to the efficiency and/or accuracy of gene expression, and therefore codon usage bias is a consequence of the action of natural selection. In contrast, the mutational or neutral theory states that codon usage bias is a consequence of the differential mutational pattern of synonymous codons. This means that some alternative codons can suffer more mutations than others, leading to differences in codon usage (Hershberg & Petrov, 2008; Sharp et al., 1993). There is evidence that one of the most important parameters explaining codon usage bias in organisms is their genomic GC content (Chen, Lee, Hottes, Shapiro, & McAdams, 2004; Knight, Freeland, & Landweber, 2001), which is also related to the frequency of CpG dinucleotides. For example, in mammals, CpGs are prone to methylation and subsequent spontaneous deamination, which in turn produce a C (cytidine) to T (thymine) mutation resulting in TpG dinucleotides (Bird, 1980; Singal & Ginder, 1999). Therefore, these organisms show an underrepresentation of CpGs. Several viruses, including DNA and RNA viruses, also show CpG avoidance. However, since RNA viruses do not form a DNA intermediate, the methylation-deamination model is not likely. Therefore, it has been proposed that CpGs avoidance is beneficial because it allows the evasion of the host innate immunity (Cheng et al., 2013). Population genetic theory predicts that the probability of one mutation to increase or decrease its frequency in a population depends on the product of the effective population size (Ne) and the selection coefficient (s) (Ohta, 1992). Thus, if population sizes or selection coefficients are large, evolution is dominated by selection, whereas if population sizes or selection coefficients are small, evolution is driven by drift. Therefore, at present, the accepted model is the mutationselection-drift balance model of codon usage bias, also called the major codons preference model (Fig. 1), which explains that optimal codons are positively selected, whereas mutation bias and genetic drift allow the persistence of the less frequent codons (Hershberg & Petrov, 2008).

Genetic variability and molecular evolution of ZIKV Phylogenetic inference is useful to understand the epidemiological history and how the viruses emerge and spread, which potentially can enable the design of both prevention and intervention strategies (Grubaugh et al., 2017; Theze et al., 2018). Several independent phylogenetic analyses showed the existence of two main ZIKV lineages, known as the African and Asian lineages (Faria et al., 2016; Gong, Xu, & Han, 2017; Haddow et al., 2012; Liu et al., 2016; Pettersson et al., 2016;

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FIG. 1 Evolutionary forces involved in generation and maintenance of codon usage bias. Codon usage bias depends on the balance of alternative synonymous codons generated by mutation and their frequency changes by genetic drift and natural selection (See text for details).

FIG. 2 Phylogenetic relationship of ZIKV strains. Phylogenetic tree based on available ZIKV envelope gene sequences. The tree was inferred using the neighbor-joining method. Lineages are indicated in blue (African) and green (Asian). The tree is drawn to scale expressed in units of the number of base substitutions per site.

Shen et al., 2016; Wang, Valderramos, et al., 2016; Zhu et al., 2016) (Fig. 2). The Asian lineage, which diverged from the African lineage, includes French Polynesian isolates that circulated in 2013 (Campos et al., 2015), which subsequently gave rise to the epidemic strains responsible for the Latin American outbreak (Fajardo, Cristina, & Moreno, 2016; Faria et al., 2016). ZIKV outbreaks observed in Florida (USA) in 2016 seem to be the consequence of multiple introductions from the Caribbean (Grubaugh et al., 2017). Analysis of sequences from field isolates with known sampling dates indicated high rates of molecular evolution similar to those observed in other RNA viruses (Duffy, Shackelton, & Holmes, 2008). However, the Asian lineage seems to evolve faster than the African lineage, as indicated by the differences among their molecular evolution estimates (Faria et al., 2016; Liu et al., 2016; Metsky et al., 2017; Simo´n, Fajardo, Moreno, Moratorio, & Cristina, 2018; Yokoyama & Starmer, 2017). The higher molecular evolution rate observed in the Asian lineage could be the consequence of using sequences with transient polymorphisms in these studies (Duffy et al., 2008; Scholle, Ypma, Lloyd, & Koelle, 2013). Additionally, epidemiological mathematical models predict that substitution rates are expected to be higher in epidemic regions as well as in regions with high contact rates than in endemic regions or with low contact rates (Scholle et al., 2013).

Evolution of the codon usage bias in ZIKV As mentioned before, factors such as viral genome composition and codon usage can shed light on the viral adaptation process as well as pathogenesis and population spread. Because of this, after ZIKV outbreaks, several independent studies

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of ZIKV codon usage bias were performed. The main findings of some of these studies are reviewed here (For details on how to quantify codon usage from sequences, see the Policy and Procedure section). Overall, a weak general codon usage of ZIKV was found, as indicated by the low estimates of the effective number of codons (ENC) among ZIKV strains. This might be beneficial for efficient replication in both mosquito and vertebrate host cells with different codon usage preferences as it was observed in other arboviruses ( Jenkins & Holmes, 2003). In addition, it was found that synonymous codon choice was constrained by the GC content at the third codon position indicating that codon usage bias is mainly determined by mutation pressure (Cristina, Fajardo, Son˜ora, Moratorio, & Musto, 2016; Singh & Tyagi, 2017; van Hemert & Berkhout, 2016). However, there is evidence that both mutation pressure and translational selection are involved in shaping the codon usage pattern of ZIKV as shown by the correlation between the Codon Adaptation Index (CAI) and ENC values (Wang, Liu, Zhang, & Wei, 2016). Moreover, the ZIKV codon usage pattern was also found to be significantly correlated with aromaticity and hydrophobicity protein indexes, indicating that these features are also involved in ZIKV codon usage (Wang, Liu, et al., 2016). In contrast, other studies have found that selection is the major force influencing ZIKV codon usage. This was indicated by neutrality plots analysis and comparison of the relative synonymous codon usage frequency (RSCU) of ZIKV and its hosts (Fig. 3) and CAI values (Fig. 4), where ZIKV codon usage was found to be more similar to humans than mosquitoes (Butt, Nasrullah, Qamar, & Tong, 2016; Tao & Yao, 2019). This host-specific pattern was also observed in the study where Cristina et al. (2016) concluded that codon usage in ZIKV is mainly determined by mutation pressure. This is because RNA viruses tend to show a predominance of the mutational pressure compared to natural selection in their codon usage bias, as a result of their high mutation rates ( Jenkins & Holmes, 2003).

Aedes aegypti

ZIKV

Homo sapiens

Codon CCU UUA AAU CAG GGC CGG UGC CUG UCC AUU GCC AUC GUA CGA CGC CAC ACC UUC GGU UCG UGU GCG GAU ACG CGU AAG CCG UAC GAA GUU AGC UCU UAU CUC GAG CCC GUG UUU CUU ACU GAC AAA GGG GCA ACA ACG CCA CAU GCU AUA AGA UCA CAA CUA GGA AAC ACU GUC UUG

2 1.5

RSCU 1 0.5

Amino acid Ala Arg Asn Asp Cys Gln Glu Gly Hls lle Leu Lys Phe Pro Ser Thr Tyr Val

FIG. 3 Relative synonymous codon usage (RSCU) frequency of ZIKV and its hosts. Heatmap of codon usage (estimated as RSCU values) of ZIKV, Homo sapiens, and Aedes aegypti. Each column corresponds to the three different species. No scaling was applied to rows. Both rows and columns were clustered using correlation distance and average linkage. (Data was taken from Cristina, J., Fajardo, A., Son˜ora, M., Moratorio, G., & Musto, H. (2016). A detailed comparative analysis of codon usage bias in Zika virus. Virus Research, 223, 147–152.)

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FIG. 4 Codon adaptation index (CAI) estimates of ZIKV genes in relation to its host. Codon adaptation index of ZIKV expressed in relation to the reference codon usage set of Homo sapiens, Aedes aegypti, and Aedes albopictus represented as white, black, and gray bars, respectively. (Data was taken from Cristina, J., Fajardo, A., Son˜ora, M., Moratorio, G., & Musto, H. (2016). A detailed comparative analysis of codon usage bias in Zika virus. Virus Research, 223, 147–152.)

Indeed, the host-specific pattern of codon usage and nucleotide composition could also reflect large-scale mutational bias in the absence of natural selection, especially because RNA viruses use the nucleoside triphosphate pool available inside the host cell and also other host editing factors can contribute to the mutation pressure bias. For example, in silico inferences have shown that viral RNA editing by host enzymes, such as RNA-dependent adenosine deaminase (ADAR), is an important mutational and evolutionary force that affects the molecular evolution of ZIKV contributing to viral codon usage bias (Piontkivska, Frederick, Miyamoto, & Wayne, 2017). However, more experimental work is needed to assess how ADAR influences ZIKV molecular evolution and codon usage preferences in natural populations.

Vaccines development as application of the study of codon usage bias in viruses Previous studies performed in other RNA viruses have shown that optimal codons can be replaced in viral genomes with alternative synonymous codons for the development of live attenuated viral vaccines (LAV) (Carrau et al., 2019; Coleman et al., 2008; Moratorio et al., 2017). Additionally, codon usage knowledge is important to develop subunit vaccines (recombinant or chimeric vaccines expressing viral prM/E proteins) which are based on efficient viral gene expression to generate immunity (Morabito & Graham, 2017). As a consequence of the ZIKV codon bias, replacing codons toward alternative synonymous codons, which are optimal in the specific platform of vaccine production, is a key step (van Hemert & Berkhout, 2016). Vaccines are one of the most cost-effective health interventions currently available (Minor, 2015). Recombinant subunit vaccines tend to be safer compared to live attenuated viral vaccines because, unlike the latter, subunit vaccines have no infectious potential. However, subunit vaccines often show low efficacy (Tai et al., 2019). To date, there is only one published paper based on attenuation by codon deoptimization on ZIKV vaccine candidates with promising results (Li et al., 2018). This study showed that codon deoptimization of E and/or NS1 genes of ZIKV displayed an attenuated phenotype in mammalian cells yet not in insect cells. Furthermore, the attenuated phenotype was also confirmed in vivo where ZIKV vaccine candidates elicited protective immunity and prevented vertical transmission of the virus during pregnancy (Li et al., 2018) (Fig. 5). The main disadvantage of live attenuated vaccines is their likelihood to be transmitted and evolve. However, they can be rationally designed to largely reduce their evolution (Bull, Smithson, & Nuismer, 2018). The ZIKV vaccine candidate of Li and collaborators (2018) took this aspect into account introducing hundreds of synonymous substitutions to generate the codon pair-deoptimized vaccine candidates, thus making reversion to virulent wild-type phenotype very unlikely (Li et al., 2018). However, in this work, live attenuated vaccine candidates were not assessed in their transmission cycle from mosquitoes to mice and even nonhuman primates under laboratory conditions, assays which are needed for further vaccine development.

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Structural

WT

Inoculation

Non Structural

5’UTR

3’UTR

5’UTR

3’UTR

Codon De-optimized CDR

Disease development Immunity and vertical transmission blocked

FIG. 5 Codon deoptimized mutants of ZIKV are interesting vaccine candidates. ZIKV vaccine candidate constructed by codon deoptimization of a region of the structural polyprotein (keeping the same phenotype than the wild-type virus) showed protective immunity and blocked viral vertical transmission after inoculation in mice. Upper panel: wild-type (WT) virus and its effect in vivo. Bottom panel: codon deoptimized mutant and its effect in vivo. CDR, codon deoptimized region.

Conclusions and future directions ZIKV is mainly transmitted by Aedes mosquitoes, which also transmit DENV, CHIKV, and YFV, although human-tohuman transmission has also been documented. ZIKV disease usually does not develop symptoms. However, severe complications of ZIKV infection have been reported. Phylogenetic studies of ZIKV are useful because they can reveal the course of the outbreak and identify mutations that can be later experimentally assessed in order to determine their biological significance. Moreover, phylogenetic studies allow the genetic characterization of the circulating viral variants contributing to the surveillance of new antigenic variants and therefore in vaccine design. Codon usage bias refers to the fact that not all synonymous codons are used with the same probability. Molecular data indicate that codon usage bias is important in genome evolution. Codon usage bias is under the influence of natural selection as well as mutation bias and genetic drift. Currently, the accepted evolutionary model is the mutation-selection-drift balance model of codon usage bias. In general, ZIKV codon usage was found to be mainly influenced by mutational bias and nucleotide composition, although some evidence of the action of natural selection in ZIKV codon usage was also reported. Codon usage evolution of ZIKV probably results from the joint action of different evolutionary forces including mutation and natural selection to adapt its codon usage to different environments and hosts. Finally, codon usage studies also have biomedical implications. Codon usage knowledge can be used against viruses to rationally design vaccines. Live attenuated vaccines generation involves the deoptimization of viral codon usage resulting in virus attenuation, whereas subunit vaccines development involves optimization to the platform vaccine production (i.e., human cells). Currently, there is no treatment or vaccine available against ZIKV, despite several research efforts being made in different aspects of ZIKV biology. More evolutionary studies combined with both virological and genetic studies are necessary to build efficient surveillance programs in order to prevent outbreaks in the future.

Policy and procedures: Quantification of codon usage bias As mentioned, codon usage bias study is important from an evolutionary point of view. There are several methods to calculate codon usage bias. Here we summarize the most used indexes for its calculation from molecular data. One of the most commonly used indexes is the Effective Codon Usage (ENC) which measures the deviation from equal use of synonymous codons. The ENC values range from 20 to 61, where a high value indicates no codon bias and a value of 20 indicates a maximum bias (i.e., just one alternative codon is used from each synonymous codon group) (Wright, 1990). The main advantage of this index is that it does not require prior knowledge of optimal codons or a reference set. The other commonly used index is the Relative Synonymous Codon Usage (RSCU) which allows the comparison between coding sites of different sizes (Sharp & Li, 1986). The RSCU value for each codon is the observed frequency of this codon divided by the expected frequency of the synonymous codon, assuming that all synonymous codons are used at equal frequency. Therefore, synonymous codons with RSCU values higher than 1 indicate that these codons are used

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more than expected while the ones with RSCU values lesser than 1 indicate that these codons are less used than expected. The RSCU index calculations are given by
X # RSCU ij ¼ ij "  X (1) 1 ni   Xij ni j¼1 where Xij is the observed frequency of the jth codon for the ith amino acid and n is the number of synonymous codons for the ith amino acid. Another useful index is the Codon Adaptation Index (CAI) which allows to determine the degree of adaptation of the coding sequence to a biased codon usage observed in another organism (Sharp & Li, 1987). CAI is the geometric mean of the relative adaptiveness values of alternative codons. The relative adaptiveness is calculated as the ratio of the observed frequency of a codon divided by the frequency of the most used synonymous codon in a reference set of highly expressed genes. CAI values range from 0 to 1, being 1 if the frequency of codon usage by the test genes data set equals the frequency of usage in the reference sequence data set. CAI can be calculated as
X (2) CAI ¼ ij Ximax where Xij is the observed frequency of the ith codon for the jth amino acid and Ximax is the maximal value of X for codons for the same amino acid.

Mini-dictionary of terms Arbovirus. Also known as the arthropod-borne virus, it is a nontaxonomic category that includes viruses that are transmitted By hematophagous vectors and infect vertebrates. Effective population size (Ne). Size of an ideal population that would lose genetic variation through genetic drift at the same rate that is observed in the real population of study. Importantly, the effective population size is much smaller than the census population size consequence of different aspects of the population structure. Genetic drift. Random fluctuations in allele frequencies from one generation to the next, the consequence of a sampling error in a finite population. Genetic drift produces a nonadaptive evolution. Optimal codon. Refers to the use of those synonymous codons that are correlated to the tRNA pool available inside the cell. Mutation-Selection-drift balance. Refers to the equilibrium frequencies of alleles (i.e., nonoptimal codons) which are reached when the input of new variants generated by mutation is balanced by the removal by both natural selection and genetic drift. Live attenuated vaccines. Vaccines generated keeping the viability of a pathogenic organism and its ability to activate the host immune system, while reducing its virulence. These vaccines have to be rationally designed to achieve a small probability of reversion to virulence.

Key facts of codon usage in ZIKV l

l

l

l

l

l

Codon usage bias is a consequence of the degeneracy of the genetic code and refers to differences in the frequency of occurrence of alternative synonymous codons during translation. The selectionist theory states that codon usage bias contributes to the efficiency and/or accuracy of gene expression, and therefore codon usage bias is a consequence of the action of natural selection. Contrarily, the mutational or neutral theory states that codon usage bias is a consequence of the differential mutational pattern showed by alternative synonymous codons. This means that some alternative codons can suffer more mutations than others, leading to differences in codon usage. ZIKV codon usage was found to be mainly influenced by mutational bias and nucleotide composition. However, there is also evidence of the action of natural selection in ZIKV codon usage. Codon usage evolution in ZIKV may be a result from the joint action of different selective forces including mutation and natural selection to adapt its codon usage to different hosts and environments. Viral codon usage studies are important to monitoring outbreaks as well as to rationally design vaccines.

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Summary points l

l

l

l

l

l

l

l l

l

ZIKV is a small RNA virus (10 kb) which is naturally transmitted by mosquitoes and infects humans. Recent ZIKV infections were related to severe clinical manifestations including congenital microcephalia in the descendance of pregnant women and Guillain-Barre syndrome. Codon usage bias refers to the fact that some synonymous codons are more or less probably used than expected by chance. Molecular evidence suggests that codon usage bias is common across genomes and may contribute to genome evolution. Codon usage bias studies are the key to shed light on the determining mechanisms of virus-host interaction, viral fitness, evasion of the host’s immune response, viral emergence, and intervention strategies. The selectionist and the mutational or neutral theories were the main evolutionary explanations for the existence of codon usage bias. The former states that codon usage bias contributes to the efficiency and/or accuracy of gene expression, and therefore codon usage bias is a consequence of the action of natural selection, whereas the latter states that codon usage bias is a consequence of the alternative synonymous codons mutational pattern. This means that some alternative codons can suffer more mutations than others, therefore leading to differences in codon usage. Currently, the accepted model of codon usage bias in organisms is the mutation-selection-drift balance model, also called the major codons preference model, which explains that optimal codons are positively selected, whereas mutation bias and genetic drift allow the persistence of the less frequent codons. Phylogenetic analyses revealed the existence of two main ZIKV lineages: the African and Asian lineages. The latter was involved in the most recent ZIKV outbreaks. Overall, ZIKV codon usage bias was found to be low or weak, constrained by GC content at the third codon position and mainly driven by mutational bias pressure. However, there is also evidence of selection shaping ZIKV codon usage. Evolution of ZIKV codon usage probably results from the joint action of different selective forces. Knowledge of codon usage bias in viruses is also important from an applied point of view, especially in the development and production of live attenuated vaccines. Currently, there is a ZIKV vaccine candidate based on attenuation by codon deoptimization with promising results in vitro and in vivo.

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F. (2016). From discovery to outbreak: The genetic evolution of the emerging Zika virus. Emerging Microbes & Infections, 5, e111. Metsky, H. C., Matranga, C. B., Wohl, S., Schaffner, S. F., Freije, C. A., Winnicki, S. M., … Sabeti, P. C. (2017). Zika virus evolution and spread in the Americas. Nature, 546(7658), 411–415. Minor, P. D. (2015). Live attenuated vaccines: Historical successes and current challenges. Virology, 479–480, 379–392. Morabito, K. M., & Graham, B. S. (2017). Zika virus vaccine development. Journal of Infectious Diseases, 216, S957–S963. Moratorio, G., Henningsson, R., Barbezange, C., Carrau, L., Borderı´a, A. V., Blanc, H., … Vignuzzi, M. (2017). Attenuation of RNA viruses by redirecting their evolution in sequence space. Nature Microbiology, 2, 17088. Moriyama, E. N., & Powell, J. R. (1998). Gene length and codon usage bias in Drosophila melanogaster, Saccharomyces cerevisiae and Escherichia coli. Nucleic Acids Research, 26(13), 3188–3193. Novella, I. 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Chapter 39

Endosomal compartmentation and the transport route of Zika virus Katarzyna Owczarek and Krzysztof Pyr
c Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Cracow, Poland

Abbreviations C DC-SIGN E EE EEA1 ER ERC FCHo1/2 GAG Gas6 LE LY6E M prM ProS TIM ZIKV

capsid dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin envelope early endosome early endosome antigen 1 endoplasmic reticulum endocytic recycling compartment Fer/Cip4 homology domain-only proteins 1 and 2 glycosaminoglycan growth-arrest-specific protein 6 late endosome lymphocyte antigen 6 locus E membrane precursor to membrane protein S T-cell immunoglobulin and mucin domain receptor Zika virus

Introduction The first barrier to viruses needs to overcome to reach their replication site is a cell membrane. Viruses developed multiple strategies to hijack cellular entry portals, which in physiological conditions serve cells for nutrition and communication. The route of virus entry is determined not only by the size and shape of the particle but mostly by the cellular molecule and composition and charge of the host cell membrane at the attachment site. Particles abundant on the cell surface (e.g., sugar moieties) are extensively used by viruses as attachment factors, whereas membrane proteins are usually used to play the role of entry receptors. Enveloped viruses, such as the Zika virus (ZIKV), enter permissive cells by fusing their membrane with a host cell membrane (Plemper, 2011). During this process, fusion pore is formed, and the viral genome is released to the cytoplasm. Since the fusion does not occur spontaneously, activation energy needs to be provided to initiate this process. Viruses acquire the activation energy by changing the conformation of their fusogenic proteins to an energetically favorable state, in which fusion peptide of the virus is propelled toward the host cell membrane. The conformation change may be induced by low pH, receptor binding, redox changes, or proteolytic cleavage (Fig. 1). Whereas some viruses may be activated for fusion already at the cell surface, others need to be delivered to a precisely defined intracellular compartment, where pH and membrane composition are optimal. The scope of this chapter is to describe the early events during the ZIKV infection. Virus-cell interactions, entry routes, and intracellular trafficking will be discussed, alongside cellular factors indispensable for these processes. Further, inhibitors targeting these steps will be discussed. Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00039-0 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Activation of viral fusogenic proteins. Enveloped viruses fuse with a host cell membrane in order to transfer their genetic material into the cytoplasm, which may take place on the cell surface or in the endocytic compartment. The conformation change of viral fusogenic proteins to the active state can be induced by low pH, receptor binding, redox changes, or proteolytic processing.

The Zika virus Mature ZIKV particle is comprised of three structural proteins. A dense core is formed by the capsid (C) protein, together with the viral genomic RNA (gRNA). This nucleocapsid is covered with the double membrane, decorated with the remaining two structural proteins: membrane (M) and envelope (E) (Fig. 2A). Similar to other members of the Flaviviridae family, ZIKV E protein is responsible for receptor binding and fusion. As the E protein is unstable and readily undergoes irreversible conformation changes when exposed to the low pH, in the newly synthesized flaviviral particles, it needs to be shielded from the mildly acidic environment of the secretory pathways (Seksek, Biwersi, & Verkman, 1995). In order to prevent their premature activation during virus progeny release, the E protein interacts with the M protein precursor (prM). During virus maturation, the prM undergoes proteolytic processing with furin in trans-Golgi network (Heinz et al., 1994; FIG. 2 Diversity of ZIKV surface. Schematic representations of (A) mature ZIKV virion; (B) partially mature, mosaic ZIKV virion; (C) “virus breathing” phenomenon. Symbols illustrating the respective viral proteins (prM, M, E) are denoted on the right side of the panel.

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Owczarek et al., 2019). The small cleavage product remains associated with the E protein until the progeny virus leaves the invaded cell—their dissociation occurs in a more basic environment within the extracellular matrix. Interestingly, there is increasing evidence that the furin cleavage may be incomplete in different tissues and different hosts, which results in the release of partially mature, mosaic virions (Pierson & Diamond, 2012; Yu et al., 2008) (Fig. 2B). Although the progeny virions decorated with the prM-E heterotrimeric spikes not processed by furin remain fusion- and infection-incompetent, partial heterogeneity of the virion surface may be beneficial for the virus in terms of the entry and immune evasion. Interestingly, the West Nile virus was reported to interact with DC-SIGN by a single N-linked glycan at the prM protein, and this interaction alone was sufficient for the infection of human cells (Davis et al., 2006). Very high variability of the prM protein-encoding gene was reported for the ZIKV strains circulating in the Americas, compared to the ancestral Asian and African strains (Wang et al., 2016). Some speculate that these may be one of the reasons for the evolutionary success in recent years (Heinz & Stiasny, 2018). Flavivirus surface is heterogenic not only due to incomplete maturation but also due to the phenomenon called “virus breathing,” which refers to the dynamics of the flaviviral envelope. Rather than a closed shell, the ZIKV envelope is a dynamic structure that supports substantial protein movements. This implies exposure of otherwise hidden surfaces of E proteins and renders viral membrane temporarily accessible for interaction with different molecules in the host cell membrane (Kuhn, Dowd, Beth Post, & Pierson, 2015) (Fig. 2C).

Apoptotic mimicry of ZIKV ZIKV acquires its membrane during budding from the endoplasmic reticulum (ER). The luminal surface of the ER is enriched in phosphatidylserine; thus, it is reasonable to expect such enrichment of the ZIKV membrane (Kay, Koivusalo, Ma, Wohland, & Grinstein, 2012; Mukhopadhyay, Kuhn, & Rossmann, 2005). In physiological conditions, this negatively charged phospholipid is present on the outer side of the plasma membrane only if the cell is programmed for apoptosis. Recognition of phosphatidylserine by specialized lipid receptors on the neighbor cells results in signaling cascades that lead to endocytosis and degradation of apoptotic debris, as well as to the production of anti-inflammatory cytokines and suppression of inflammatory cytokines (Voll et al., 1997). The hijacking of this machinery was described as “apoptotic mimicry” and is rather common among many viral families, including alphaviruses, arenaviruses, baculoviruses, filoviruses, flaviviruses, poxviruses, and rhabdoviruses (Amara & Mercer, 2015). By the presentation of phosphatidylserine on their surface pathogens mimic apoptotic debris, which presumably elicits their endocytosis along with the inhibition of the immune response. Considering the multitude of cells that are capable of apoptotic cell clearance, apoptotic mimicry seems to be a fast track to expand the tropism of a virus without the need for fine-tuning the specificity of its receptor-binding protein (Amara & Mercer, 2015). In such a scenario, structural dynamics and heterogeneity of ZIKV might explain the ability of the virus to infect a broad range of cells, including almost all cell types in the brain (Cumberworth et al., 2017; Meertens et al., 2017; Stefanik et al., 2018), many types of cells of reproductive tract (Aagaard et al., 2017; Govero et al., 2016; Miner & Diamond, 2017; Pagani et al., 2017), cornea, optic nerves (Furtado, Espo´sito, Klein, Teixeira-Pinto, & da Fonseca, 2016), skin keratinocytes, fibroblasts, and macrophages (Hamel et al., 2015; Miner & Diamond, 2017).

Interaction between the Zika virus and the cell It is known that ZIKV enters susceptible cells via receptor-mediated endocytosis, and this process may be triggered by interaction with a number of different molecules identified as adhesion or entry receptors (Table 1). Adhesion receptors allow for virus binding to the cell surface and usually are broadly distributed on target tissues (e.g., sugar moieties). This interaction leads to the increase in virus density on the cells and increases the chance (or enables) interaction between the virus and the entry receptor. Binding to the entry receptor initiates the entry process and fusion between the viral and the cellular membranes. For the Zika virus, long anionic polysaccharides—glycosaminoglycans (GAGs)—such as chondroitin sulfate and heparan sulfate were described to serve as adhesion receptors (Kim et al., 2017). The interaction is mediated by basic residues Lys291 and Lys295 within the E protein domain III (Watterson, Kobe, & Young, 2012). Recently, it was reported that N-linked glycosylation of the E protein affects ZIKV fitness, pathogenicity, and infectivity for different host organisms (Routhu et al., 2019). Due to N-glycosylation at Asn154, the E protein of ZIKV is able to interact with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), which was found to permit ZIKV entry to cells, thus possibly serving as ZIKV entry receptor (Hamel et al., 2015). DC-SIGN is a

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TABLE 1 ZIKV attachment and entry receptors.

Attachment factors Putative entry receptors

ZIKV-interacting molecules

References

Chondroitin sulfate

(Kim et al., 2017)

Heparan sulfate DC-SIGN

(Hamel et al., 2015)

TIM1, TIM3, and TIM4

(Hamel et al., 2015; Tabata et al., 2016)

TYRO3, AXL, and MER (indirect interaction via Gas6 and ProS ligands)

(Hamel et al., 2015; Meertens et al., 2017; Strange et al., 2019)

representative of C-type (calcium-dependent) lectins, which are known to activate the immune response of the infected cell by recognizing carbohydrates on pathogens and directing them to endosomes to initiate antigen presentation (Kerrigan & Brown, 2009). It is worth mentioning that other C-type lectins have also been recognized as entry-facilitating factors for closely related flaviviruses (Chen et al., 2012; Davis et al., 2006; Miller et al., 2008). On the other hand, there are several reports suggesting that not the proteins, but the lipids that build the ZIKV membrane play a dominant role in virus entry (Tabata et al., 2016). Negatively charged aminophospholipids (phosphatidylserine and phosphatidylethanolamine) in ZIKV membrane have been shown to interact with lipid receptor families that are responsible for sensing “eat me” proapoptotic signal, probably hijacking them in the process of apoptotic mimicry, as described above. Both direct interactions with T-cell immunoglobulin and mucin domain receptors (TIM1, TIM3, and TIM4), as well as indirect interactions with TAM receptors (TYRO3, AXL, and MER) via growth-arrest-specific protein 6 (Gas6) and protein S (ProS) ligands, have been reported (Hamel et al., 2015; Meertens et al., 2017; Strange et al., 2019; Tabata et al., 2016). It is not clear which receptors are essential for the virus entry and this process seems to be cell-type dependent.

Clathrin-dependent endocytosis of Zika virus Several routes are leading through the intracellular labyrinth. The large cargos are engulfed via phagocytosis or macropinocytosis, while smaller particles (up to 500 nm in diameter) can employ endocytosis (Rejman, Oberle, Zuhorn, & Hoekstra, 2004). Following the attachment, viruses float on the cell surface until they encounter their entry receptor(s). Interaction with the receptor molecule(s) on the cell surface initiates the assembly of the endocytic machinery. The most common, and the best-described route is clathrin-dependent endocytosis. It is worth to note, however, that there is a number of alternative pathways, including caveolin-mediated endocytosis (Kiss & Botos, 2009; Nabi & Le, 2003), fast endophilinmediated endocytosis (Boucrot et al., 2015), CLIC/GEEC pathway (Lundmark et al., 2008; Sabharanjak, Sharma, Parton, & Mayor, 2002), or entosis (Overholtzer et al., 2007). These pathways will not be discussed here, as almost all known flaviviruses enter the cells by clathrin-dependent endocytosis (Kalia, Khasa, Sharma, Nain, & Vrati, 2013). For those interested in further exploration of endocytosis pathways, we may refer to some excellent reviews focused on this subject (Mayor & Pagano, 2007; Sandvig, Kavaliauskiene, & Skotland, 2018). Clathrin-dependent endocytosis is initiated by the binding of Fer/Cip4 homology domain-only proteins 1 and 2 (FCHo1/ 2) F-BAR domain to phosphatidylinositol 4,5-bisphosphates, which induces the formation of the plasma membrane curvature. Further, μHD domain of FCHo1/2 interacts with epidermal growth factor receptor substrate 15 and intersectin-1, which recruit adaptor protein 2 to the nucleation site. Clathrin subunits start to accumulate at the spot, and once the density exceeds the critical p, they polymerize and create a coat surrounding the cargo (Henne et al., 2010; Rappoport, Kemal, Benmerah, & Simon, 2006). As the invagination deepens, amphiphysin attaches to the newly formed neck and recruits dynamin, which polymerizes into a characteristic ring shape around the narrows. Dynamin is a protein that possesses a GTPase activity; upon GTP hydrolysis, the protein changes its conformation, and the ring-shaped structure shrinks, which ultimately leads to the scission of the cargo-containing vesicle from the cell surface (Takei, Slepnev, Haucke, & De Camilli, 1999). Zika virus was found to colocalize with clathrin 2–10 min postinoculation in Vero cells (Owczarek et al., 2019). Depletion of clathrin heavy chain expression by RNAi resulted in virus accumulation on the cell surface (Owczarek et al., 2019) and a significant reduction of the number of ZIKV-infected cells (Meertens et al., 2017). Moreover, inhibitors specific to clathrin-dependent endocytosis (amantadine and chlorpromazine) were reported to hamper ZIKV infection in

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FIG. 3 Clathrin-dependent endocytosis of ZIKV. Upon attachment to GAGs, ZIKV interacts with its entry receptor (yet unidentified; several candidates are listed in the figure), which induces clathrin-mediated dynamin-dependent endocytosis of the virus. Symbols illustrating the respective host cell molecules involved in ZIKV internalization are denoted on the right side of the panel.

Vero and Cf2Th epithelial cell lines (Owczarek et al., 2019; Persaud, Martinez-Lopez, Buffone, Porcelli, & Diaz-Griffero, 2018). A similar effect of infection inhibition was observed by the application of compounds that block phospholipidbinding and GTPase domains of dynamin, as well as by transient silencing of dynamin expression in Vero and CHME3 microglial cell lines, respectively (Meertens et al., 2017; Owczarek et al., 2019). Noteworthy, the internalization of the virus from the cell surface was reported to be promoted by microtubulesresembling lymphocyte antigen 6 locus E (LY6E) tubules, suggesting that a specialized pathway is required for the uptake of as large clathrin-dependent endocytosis cargoes as flaviviruses (Hackett & Cherry, 2018). The route of ZIKV internalization process is shown in Fig. 3.

Intracellular trafficking of the ZIKV Overcoming the barrier of the plasma membrane is only half the battle for ZIKV entry process—upon clathrin-dependent internalization, virus particles remain trapped within intracellular double-membrane vesicles. The next milestone is merging between the viral envelope and the vesicle membrane, which occurs due to the activity of viral fusion protein. ZIKV E is a typical class II fusion protein, which in the prefusion state forms antiparallel homodimers lying along the virus surface. Similar to other fusion proteins of this class, it becomes activated by the pH decrease, which triggers the realignment of E proteins into surface-perpendicular trimers, exposing the fusion loop peptide toward the host vesicle membrane (White, Delos, Brecher, & Schornberg, 2008; Zhang et al., 2017). In other words, virus escape from the intracellular vesicle (and RNA release to the cytoplasm) is highly dependent on the internal pH, ergo “proper” ZIKV trafficking is of critical significance for further infection development. Once the virus-containing vesicle is cut off the plasma membrane and the clathrin coat is not needed anymore, it becomes disassembled by the cooperative action of auxilin and heat shock cognate protein 70 (Xing et al., 2010). Such “stripped” vesicle is now able to undergo maturation, driven by its coalescence with other intracellular compartments and by the incorporation of specific proteins to its membrane, including small GTPases of Rab family. Colocalization studies revealed that in the timeframe between 2 and 10 min postinoculation ZIKV is present in early endosome antigen 1 (EEA1)-positive structures, indicating that the ZIKV-containing vesicles are initially transported to early endosomes (EE) (Owczarek et al., 2019). Accordingly, bioinformatics studies reported the importance of Rab5 in ZIKV infection (Esteves, Rosa, Correia, Arrais, & Barros, 2017). EEs are highly dynamic, tubular, and vacuolar structures of moderately acidic pH (6.3–6.8). From this compartment, virions are sorted out toward the degradative pathway, where gradually decreasing pH

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finally allows for the fusion. Kinetic analysis of this process showed that although ZIKV hemifusion could occur already in EEs, its efficiency increases approximately threefold within the pH range 6.9–4.6, as the endosome matures (Rawle, Webster, Jelen, Kasson, & Boxer, 2018). The ZIKV particles have been detected in late endosomes (LEs). The colocalization of viral C and E proteins with Rab7 was reported up to 15 min postinoculation. Afterward, the fusion occurs, and the C protein together with viral RNA is injected into the cytoplasm, whereas E protein follows a slow recycling to the cell surface in the Rab11-dependent manner (Owczarek et al., 2019). Although different other factors may promote or inhibit fusion within the endosomal hub [e.g., presence of negatively charged lipids in LEs, protease activity, temperature, virus surface maturation (Mukherjee et al., 2016; Zaitseva, Yang, Melikov, Pourmal, & Chernomordik, 2010)], pH drop seems to be sufficient for the activation of the ZIKV E protein. The intracellular trafficking of ZIKV within the endosomal hub transportation system is presented in Fig. 4.

FIG. 4 ZIKV intracellular trafficking. The landscape of intracellular transport routes with highlighted ZIKV entry pathway from the cell surface to the fusion site within late endosomes. Color-code for particular pH conditions is indicated in the right bottom corner of the figure. Symbols illustrating the respective host cell molecules involved in ZIKV internalization are the same as in Fig. 3.

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Early stages of virus infection and antiviral drug development Currently, there is no ZIKV vaccine or treatment available. While for the majority of cases the disease is not lifethreatening, there is an urgent need for the development of prevention and treatment strategies, as the infection is associated with the risk of neurological sequelae in adults and severe developmental disorders in a fetus (World Health Organization, 2019). Understanding of the biology of the virus entry enabled the development of novel antiviral compounds (Table 2). Among the direct-acting antivirals, epigallocatechin gallate (a polyphenol present in green tea) was found to disrupt the viral lipid envelope, significantly reducing ZIKV infection in Vero E6 cells (Carneiro, Batista, Braga, Nogueira, & Rahal, 2016). Using a novel, simulation-based approach Fernando et al. predicted sites within a three-dimensional structure of ZIKV E at which small drug-like molecules could interact. Subsequent in vitro testing showed that ZINC33683341 significantly decreases ZIKV infection by blocking the interaction of the E protein with the cellular receptor (Fernando, Fernando, Stefanik, Eyer, & Ruzek, 2016). Accordingly, glycosaminoglycans and their analogs, such as highly sulfated heparin, dextran sulfate, and approved antiparasitic drug suramin, were reported to inhibit ZIKV attachment and entry TABLE 2 ZIKV entry inhibitors. Target category Directacting antivirals

Entry inhibitor

Mechanism of action

References

Epigallocatechin gallate

Interacts and disrupts the viral lipid envelope

(Carneiro et al., 2016)

ZINC33683341

Blocks the interaction between the E protein and its cellular receptor

(Fernando et al., 2016)

Dextran sulfate

Block the interaction between the virus and its attachment receptors

(Tan et al., 2017)

25Hydroxycholesterol

Impairs the process of ZIKV fusion with the cellular membrane

(Li, Deng, et al., 2017)

Nanchangmycin

Selectively inhibits clathrin-dependent uptake of large cargos

(Rausch et al., 2017)

Ammonium chloride

Functions as proton sink and impairs acidification of the endosome; in these conditions virions are rapidly recycled back to the cell surface and never reach the fusion site

(Owczarek et al., 2019)

Bafilomycin A1

Functions as a vacuolar-type H+-ATPase inhibitor that impairs endosome acidification; in these conditions, the fusion does not occur; virions are directed for degradation in lysosomes

Chloroquine

Block endosome acidification and ZIKV internalization

Heparin sulfate Suramin Hosttargeting antivirals

Niclosamide

(Delvecchio et al., 2016; Li, Zhu, et al., 2017) (Kuivanen et al., 2017)

Obatoclax mesylate Saliphenylhalamide MYD1

Disturb the ligand-receptor interaction between the virus and Axl; impair the kinase activity of Axl

(Meertens et al., 2017)

Curcumin

Interferes with the process of virus attachment to the cell

(Mounce, Cesaro, Carrau, Vallet, & Vignuzzi, 2017)

Arbidol

Blocks ZIKV entry

(Fink et al., 2018)

R248 Unknown mechanism

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to Vero cells (Tan, Sam, Chong, Lee, & Chan, 2017). One may, however, question the possible translation of these results to the in vivo situation. On the other hand, MYD1 and R248 disturb ZIKV interaction with the Axl and kinase activity of Axl, the molecule that binds negatively charged lipids of the ZIKV membrane (Meertens et al., 2017). There are, however, some controversies concerning their application against ZIKV infections—Axl functions as a neuroprotector and helps to maintain the blood-brain barrier. Therefore, its inhibition is liable to severe adverse effects (Lee & Shin, 2019). Some drug candidates interfere with the subsequent steps of virus internalization. Nanchangmycin was reported to impede the entry of several enveloped viruses, including ZIKV, due to the selective inhibition of clathrin-dependent uptake of large cargos (Rausch et al., 2017). 25-hydroxycholesterol was described to impair the process of ZIKV fusion with the cellular membrane (Li, Deng, et al., 2017). It was also suggested that modification of the endosomal microenvironment might also be used as a strategy to hamper ZIKV infection. Application of bafilomycin A1, a vacuolar-type H+-ATPase inhibitor that blocks endosome acidification, was reported to prevent ZIKV fusion; instead, the virus is transported to lysosomes where it undergoes proteolytic degradation (Owczarek et al., 2019). Moreover, ZIKV internalization was blocked with an approved antimalaria drug chloroquine (Delvecchio et al., 2016; Li, Zhu, et al., 2017), an experimental drug for the treatment of different types of cancer obatoclax mesylate, an approved drug in the treatment of intestinal helminthiasis niclosamide, and saliphenylhalamide, all of which block endosome acidification (Kuivanen et al., 2017). Interestingly, NH4Cl, which functions as a proton sink and therefore also impairs acidification of the endosome, inhibits ZIKV entry in an utterly different manner; in the presence of the compound, virions are rapidly recycled to the cell surface and never reach the fusion site (Owczarek et al., 2019).

Final remarks In conclusion, intensive research conducted upon the ZIKV epidemic brought us a thorough insight into the mechanisms underlying ZIKV infection, and this knowledge already serves as the theoretical background for specific therapy development. Accordingly, immense progress has been made in the understanding of the complex process of ZIKV entry to the cells. Attachment factors and putative receptors have been identified; virus internalization via clathrin-mediated endocytosis has been described; and the following steps of ZIKV intracellular trafficking have been tracked up to the site of the virus fusion within LEs. The multitude of anti-ZIKV drug candidates gives us hope that in the near future a specific therapy against ZIKV infection will be developed. However, there are some questions to be answered. Which molecules play the role of the ZIKV receptor in different cell types? Is it a single molecule or more of them are needed to create an entry portal for the virus? Is apoptotic mimicry the dominant way of virus internalization? How do the proposed antivirals impact healthy tissues? Are they able to cross the placental barrier and the blood-brain barrier? Would they be safe for both the pregnant woman and her fetus? Clearly, pieces are missing from this puzzle and further research is needed to come up with the answers.

Mini-dictionary of terms Attachment receptor: A molecule on the cell surface that allows virus binding. Entry receptor: A molecule on the cell surface that initiates virus entry and fusion processes. Virus fusion: It is a process of viral membrane merging with a host cell membrane, which ultimately leads to the injection of virus genetic material into the cytoplasm. It is mediated by viral fusogenic protein and can occur on the cell surface or within the endosomal hub. Virus breathing: Dynamics of virus envelope associated with protein movements on the virion surface. This process implies exposure of otherwise hidden surfaces of viral proteins and renders viral lipid envelope temporarily accessible for interaction with different molecules in the host cell membrane. Apoptotic mimicry: The presentation of negatively charged lipids (e.g., phosphatidylserine) on pathogen’s surface. In such a way, pathogens mimic apoptotic debris, which presumably elicits their endocytosis and hampers the immune response of the host cell.

Key facts of intracellular transport compartments l

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The early endosome is the first sorting station within the endosomal hub. It is a highly dynamic structure, characterized by the presence of Rab5 and EEA1 marker proteins. pH within this compartment ranges between 6.3 and 6.8. LE is the next docking compartment for cargos selected by ESCRT machinery toward the degradative pathway (characterized by gradually decreasing pH). This Rab7-directed structure has a pH range between 4.8 and 6.0.

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The lysosome is the target compartment of the degradative pathway. This LAMP1-positive structure exhibits the lowest pH within the endosomal hub (4.5–5.0) and serves as a storage site for hydrolases. Recycling compartments target cargo back to the plasma membrane. Their pH value is maintained at 6.5. There are two major recycling routes—a fast one sending the vesicles directly to the cell surface that requires Rab4 and Rab35, and a slow one that directs vesicles first to the endocytic recycling compartment (ERC) localized near the microtubuleorganizing center at the perinuclear region of the cell. Golgi apparatus is another compartment that may take cargo on the retrograde pathway toward the cell surface, docking both Rab9-addressed endosomes and cargos that are directed in a Rab9-independent manner, by the presence of specific protein motifs. The pH value within the Golgi apparatus ranges between 6.0 and 6.7. The endoplasmic reticulum is a continuous membrane system that makes up the largest organelle in eukaryotic cells. It plays a major role in the production, processing, and transport of proteins. Its luminal pH oscillates about 7.2.

Key facts of ZIKV structure l

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ZIKV is an enveloped virus. Its lipid envelope is a Ø40–60 nm sphere, rich in negatively charged lipids such as phosphatidylserine and phosphatidylethanolamine. Mature virion comprises three structural proteins: capsid (C), membrane (M), and envelope (E). C protein recruits single strain positive-sense viral RNA to form nucleocapsid of the offspring virions. The precursor of M protein (prM) matures within the Golgi apparatus as a result of furin cleavage. It protects E protein from the premature exposition to the mildly acidic environment within this compartment. E protein is a class II fusion protein responsible for attachment factors and receptor binding on the cell surface, as well as for fusion with host cell membrane within the endosomal hub. ZIKV structure is dynamic and heterogenic, due to incomplete maturation of prM and the “virus breathing” effect.

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ZIKV attaches to the cell surface due to the interaction of basic residues within E protein domain III with long anionic glycosaminoglycans decorating host cell membrane. It is not clear which receptors are essential for the virus entry, and this process seems to be cell type dependent. The surface of ZIKV is heterogenic due to the dynamics of its envelope and incomplete cleavage of prM by furin. Apoptotic mimicry is a possible explanation of the ZIKV ability to infect a broad range of cells, including those of the neural system, reproductive tract, olfactory cells, skin keratinocytes, fibroblasts, and macrophages. ZIKV enters permissive cells by clathrin-mediated endocytosis. Virus-containing vesicles are cut off the cell surface by dynamin and internalized along LY6E tubules. The fusion between ZIKV and host cell membranes depends strictly on pH and occurs in LEs 10–15 min postinternalization; viral nucleoprotein is injected into the cytoplasm, whereas E protein follows a slow recycling pathway to the cell surface in the Rab11-dependent manner. Among the currently identified entry inhibitors, one may distinguish direct-acting antivirals that affect ZIKV lipid envelope and E protein, and host-acting antivirals that hamper acidification of endosomes, block activity, or disable interaction with putative receptors, impair clathrin-dependent endocytosis and membrane fusion process.

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Chapter 40

Location of virus antigens in murine tissues infected with Zika virus Anto´nio Pedro Alves de Matosa, Libia Z
e-Z
eb, Fa´tima Amarob, and Maria Joa˜o Alvesb a

Centre for Electron Microscopy and Histopathology, Egas Moniz Interdisciplinary Research Centre, Egas Moniz, Cooperativa de Ensino Superior CRL,

Campus Universita´rio-Quinta da Granja, Caparica, Portugal, b Centre for Vectors and Infectious Diseases Research, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge, A´guas de Moura, Portugal

Abbreviations A129 AG129 CZS FISH GBS IF IFN IFNAR ISH IUGR MAR1-5A3 NPC PBS RNA WHO WT ZIKV

type I interferon receptor deficient mice type I and type II interferon receptor deficient mice congenital Zika syndrome fluorescence in situ hybridization Guillan-Barr
e syndrome immunofluorescence interferon type I interferon receptor in situ hybridization intrauterine growth restriction anti-IFNAR1 monoclonal antibody neuronal precursor cell phosphate-buffered saline ribonucleic acid World Health Organization wild type Zika virus

Introduction The location of Zika virus (ZIKV) antigens within the organs and tissues of the hosts infected is of paramount importance to the understanding of the pathological features of the ZIKV-induced disease (Dowall et al., 2016; Li et al., 2016). The distribution of virus antigens as revealed by immunofluorescence (IF) is one of the main methods to detect virus infection of the cells in organs and tissues. However, the expression of virus antigens without virion formation can occur and can lead to pathological traits of the disease as well. This may be by alteration of the cell physiology and/or viability, or by eliciting autoimmune responses (Mackenzie & Westaway, 2001; Smatti et al., 2019). ZIKV is an emerging arbovirus of the Flaviviridae family (Wikan & Smith, 2016). The family includes about 70 species, with which ZIKV shares clinical features of the diseases induced (Brault et al., 2016; Saiz et al., 2016). Recently, Asian strains of ZIKV produced an epidemic that spread around the world displaying severe pathological traits that were not apparent in the earlier manifestations of the disease (Wikan & Smith, 2016). The association of ZIKV with large outbreaks of disease and the serious neurological complications prompted the WHO to declare Zika a global health emergency (Heymann et al., 2016). This leads to a surge of research on Zika where several models were introduced for the study of its various pathological manifestations (Bradley & Nagamine, 2017; Morrison & Diamond, 2017). Mouse models were used extensively, despite the different responses of mice and humans to infection (Fig. 1) (Morrison & Diamond, 2017). Immunofluorescence studies of the location of ZIKV antigens in the cells and tissues of these animal models contributed

Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00040-7 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Main animal models and their usefulness. Newly developed mouse and nonhuman primate models. Mice are preferred because of their prevalence a laboratory model and ease of use. Mouse studies require special experimental procedures to overcome the IFN-induced innate immune response and achieve infection. The image shows the main ZIKA-related problems that can be addressed by the use of models. (From Morrison, T. E., & Diamond, M. S. (2017). Animal models of Zika virus infection, pathogenesis, and immunity. Journal of Virology, 91, e00009-17.)

greatly to the understanding of some of the most puzzling new features (Hofer, 2016). A small number of studies used fluorescence in situ hybridization (FISH), in situ hybridization (ISH), or other variants of immunohistochemistry not based on fluorescent probes. These methods are complementary to immunofluorescence and address the same issues of virus location. Where relevant, results from these studies were included in this overview.

Mouse models of ZIKV infection Mice are often the first choice for modeling viral infections in humans: they can mimic many aspects of human disease and allowing the study of pathological features including the location of viral antigens by IF (Gorman et al., 2018; Nazerai, Christensen, & Thomsen, 2019; Wu et al., 2016), and they represent much the easiest way to provide mechanistic information (Li, Xu, et al., 2016). However, ZIKV adult mouse infections lead only to mild disease, since IFN receptor-mediated signaling renders mice resistant to infection (Lee & Ashkar, 2018), whereas in humans, the ZIKV NS5 protein binds to the STAT2 receptor leading to its degradation and effectively shutting down the innate immune response (Morrison & Diamond, 2017). Thus, mouse studies are usually done either in newborn mice that are still immunologically immature and susceptible to infection (B€ uttner, Heer, Traichel, Schwemmle, & Heimrich, 2019; Manangeeswaran, Ireland, & Verthelyi, 2016) or in genetically deficient mice. Other manipulations such as route of infection or anti-IFN antibody injections allow the virus to overcome innate immunity and other barriers to virus spread within the infected mice and reproduce at least some of the disease features (Morrison & Diamond, 2017).

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FIG. 2 Main types of mouse models used in research and their usefulness. Wild type (WT) mice are usually immature newborn mice. Pregnant mice are used for the study of vertical transmission and/or placental alterations leading to IUGR. Fetuses are important mainly for the understanding of the congenital Zika syndrome (CZS). Immunodeficient mice allow virus replication, which permits the study of pathology in adult animals. (From Dong, S., & Liang, Q. (2018). Recent advances in animal models of Zika virus infection. Virologica Sinica, 33, 125–130.)

Mice with genetic deficiencies in type I interferon signaling are affected by Zika allowing infection, although older mice are more resistant (Lazear et al., 2016; Rossi et al., 2016). Added deficiencies in type II interferon produces more severe disease (Rossi et al., 2016), with increased mortality in all age groups (Morrison & Diamond, 2017). Steroid-induced immunosuppression in mice was also used for studying virus spreading and can be useful for studying the effects of tuning of the immune response. Other immune deficiencies and approaches have been used for generating models suitable for the study of specific pathological mechanisms (Morrison & Diamond, 2017; Tsetsarkin et al., 2018). A common model of mice with an IFN I defect is the mouse strain A129, while strain AG129 shows defects in both types I and II interferon responses (Fig. 2) (Dong & Liang, 2018). Adult WT mice in spite of their resistance to infection can also provide useful models for the study of events at the inoculation site (Hayashida et al., 2019) and conditions like Guillan-Barr
e syndrome that are immune mediated (Manangeeswaran et al., 2016). Infected pregnant IFN-deficient mouse models are used for studying the transplacental infection of mouse fetuses. These models enable the study, among others, of lesions of the developing nervous system leading to microcephaly (Caine, Jagger, & Diamond, 2018). The mouse models can be infected by different strains of the virus, inducing different lesions (Noguchi et al., 2020), thus allowing important insights into the variable pathogenic manifestations of ZIKV.

Spread of ZIKV through the infected mice Spread of the virus through the infected organism has been studied first in by intracerebral inoculation of susceptible newborn mice and was found by IF to infect a large number of cell types and tissues (deMatos, Z
e-Z
e, Amaro, & Alves, 2017) (Fig. 3). These include not only the CNS but also cells of the male and female reproductive tract (da Silva, 2018), and many others (Caine et al., 2018; deMatos et al., 2017; deMatos, Z
e-Z
e, Amaro, & Alves, 2018; Richard et al., 2017) (Fig. 4). Experiments with other routes of infection such as intraperitoneal or subcutaneous in various newborn mouse strains and in immunodeficient adults confirmed the susceptibility of the mice to severe systemic infection, including CNS infection (Lazear et al., 2016; Li et al., 2018; Manangeeswaran et al., 2016; Miner & Diamond, 2017). However, different strains of mice showed different patterns of infection (Lazear et al., 2016; Li et al., 2018), and the virus strain used can also lead to different results (Morrison & Diamond, 2017). Major Zika pathological features such as microcephaly are often associated with specific viral strains and mutations of ZIKV (Noguchi et al., 2020).

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FIG. 3 Antigens in liver endothelial cells. Intracerebrally inoculated newborn mice showing antigens detected by immunofluorescence in the liver. Green fluorescence shows the ZIKV antigens in the endothelium of sinusoids and central vein. Bar ¼ 10 μm. (From deMatos, A. P., Z
e-Z
e, L., Amaro, F., & Alves, M. J. (2017). Ultrastructural and immunofluorescence studies of zika infection. Ultrastructural Pathology, 41, 105–106.)

FIG. 4 Zika virus tropism. The image shows the main organs to which ZIKV has special tropism, allowing infection by contact and generating severe features of congenital Zika syndrome. (From Miner, J. J., & Diamond, M. S. (2017). Zika virus pathogenesis and tissue tropism. Cell Host & Microbe, 21, 134–142.)

Adult immunocompetent mice infected through peripheral routes usually produce no disease (Lazear et al., 2016; Rossi et al., 2016), but immunosuppressed mice develop severe disease, and the virus disseminates through most tissues, including brain, liver, kidney, testis, spleen ovary, uterus, prostate, and pancreas (Chan et al., 2016). In immunocompetent fetuses and newborn mice with immature CNS, where immunity participates in the antiviral response the immune response induces further damage to the CNS including the medulla (Manangeeswaran et al., 2016). It is involved in the development of GBS and other neurological manifestations, which raises concerns about long-term manifestations of ZIKV infection in humans (Manangeeswaran et al., 2016; Yockey et al., 2018).

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Persistent infection of the male sexual organs with the shedding of the virus in sperm is particularly important as it is one of the main mechanisms allowing human-to-human sexual transmission of the virus (Clancy, Van Wettere, Siddharthan, Morrey, & Julander, 2018).

The congenital Zika syndrome ZIKV is the only flavivirus known to be associated with birth defects leading to the aggressive congenital Zika syndrome (CZS) (Li, Xu, et al., 2016; Noguchi et al., 2020). The syndrome can result in microcephaly, fetal growth restriction, neurosensorial disorders, calcifications, epileptic seizures, arthrogryposis, neuromotor deficits, and abortion (Saad et al., 2018). Microcephaly is one of the most dramatic manifestations. It is the hallmark of CZS (Caine et al., 2018), and arises from the virus having access to the fetal brain (Li, Xu, et al., 2016; Wu et al., 2016). The intact placenta provides a natural barrier to the transmission of the virus to the fetus but it can be overcome in the mouse models by aggressive infection methods that have no natural counterpart, or by the failure of immune responses allowing virus replication in the placenta (Miner et al., 2016; Vermillion et al., 2017). Successful infection is also influenced by the developmental stage of the fetus. Changes in the type and intensity of pathology induced between different fetal stages of mice have been demonstrated (Szaba et al., 2018; Vermillion et al., 2017). Tropism for neural precursor cells has been demonstrated by immunofluorescence in mouse models (Fig. 5) (Li et al., 2016; Li, Xu, et al., 2016; Wu et al., 2016). Besides the location of virus antigen, the location of cell type markers by immunofluorescence has also been used to identify the cell types infected or otherwise involved in the pathology (Li, Xu, et al., 2016; Wu et al., 2016). These studies showed that ZIKV infection of the fetal brain leads to the preferential infection and depletion of neuronal precursor cells (NPCs) both in the fetus and in the adult mouse through the combined action of infection, apoptosis, and inhibition of NPC replication. The location of the affected NPCs within the brain, however, is different in different models (Li, Xu, et al., 2016; Shi et al., 2018; Wu et al., 2016). These results were supported by recent studies of infection of slice cultures of the mouse hippocampus, which showed infection of several populations of neuronal cells irrespective of their maturation state (B€uttner et al., 2019) resulting in highly reduced gray matter and cortex thinning (Li, Xu, et al., 2016). These effects can extend to postnatal mice with reduction of the pool of neural precursor cells and inhibition of adult neurogenesis that can have lifelong effects (Li, Saucedo-Cuevas, et al., 2016; Morrison & Diamond, 2017). In slice cultures of the hippocampus, astrocyte, and microglia reaction indicate tissue injury and phagocytosis.

FIG. 5 Infection of brain precursor neurons by ZIKV. Immunofluorescent images of embryonic brains injected in the lateral ventricle with ZIKV. Images of coronal sections labeled with ZIKV antiserum (green). Left: mock-infected (control), middle: 1/10 diluted, right: undiluted virus. Lower panel: High magnification of the area outlined by the yellow box. Scale bar, 40 mm. (From Li, C., Xu, D., Ye, Q., Hong, S., Jiang, Y., Liu, X., et al. (2016). Zika virus disrupts neural progenitor development and leads to microcephaly in mice. Cell Stem Cell, 19, 120–126.)

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Neutrophils were also found to be infected in IFN deficient fetuses and may provide a spreading mechanism of the virus through the body (Julander et al., 2018).

Other nervous structures Besides the CNS, ZIKV, but not other flaviviruses, can also affect the spinal cord, causing hind limb weakness and paralysis both in the neonatal and adult mice (Julander et al., 2018; Lazear et al., 2016; Shi et al., 2018; Yockey et al., 2016). Spinal cord and cerebellum neuron infection were observed by IF, accompanied by apoptosis and loss of spinal cord alpha motor neurons (Julander et al., 2018; Manangeeswaran et al., 2016; Shi et al., 2018). The role of these neurons in movement control and muscle contraction, respectively, can explain the symptoms of muscle paralysis (Shi et al., 2018). Infection of newborn immunocompetent mice fails however to produce paralysis of the hind limbs and after showing movement discoordination, they recover, contrasting with immunodeficient mice. Virus antigen is restricted to the cerebellum and hippocampus in the immunocompetent animals and increased neuronal cell death due to the immune response in the cerebellum was observed (Manangeeswaran et al., 2016). Infection of fetal, neonatal, and adult immune-deficient mice leads to infection and to atrophic changes of the eye and of the optic nerve. Shedding of the virus in tears can contribute to its transmission (Miner et al., 2016; Shi et al., 2018). These studies show the infection of the retina, particularly of the ganglion cell neurons, but otherwise, there is a considerable disparity in results among different studies, which may be a consequence of different experimental protocols. A study from Miner et al. (2016) detected additional infection of the cornea, but no significant morphological or functional sensory alterations. By contrast, in the study of Shi et al. (2018), the affected eyeballs and optic nerves in surviving newborn mice born from infected mothers showed atrophic changes, and multiple layers of the retina were infected. Infection was accompanied by apoptosis with large cell losses leading to thinning and alteration of its cytoarchitecture. Massive destruction of the retinal ganglion cells is probably responsible for the alterations of the optic nerve. These profound alterations, and results from visual projection studies, demonstrate severe visual impairment in this model (Shi et al., 2018).

Infection of the placenta Infection of the fetus by the mother requires the crossing of the placental barrier and results in vertical transmission and infection of fetal brain cells leading to microcephaly and other manifestations of the CZS (Miner et al., 2016; Noguchi et al., 2020; Wu et al., 2016). In WT mice models, whose innate immune system is not disrupted by the virus, the placental barrier effectively prevents virus passage (Yockey et al., 2016). In models where the mother IFN response is compromised (Caine et al., 2018; Miner et al., 2016), or intrauterine inoculation facilitates placental infection, the placental barrier can be overcome, and vertical transmission of the fetus is facilitated (Vermillion et al., 2017). During vertical transmission, the placenta itself is infected by ZIKV (Miner et al., 2016; Yockey et al., 2016). Immunofluorescence allows the detection of ZIKV antigens in placental cells, and immunofluorescence staining of cell markers can help identify the affected cells (Vermillion et al., 2017). Immunohistochemistry combined with FISH studies disclosed a tropism of the virus for various trophoblast cell types, with preference to glycogen trophoblasts and spongiotrophoblast (Miner et al., 2016), Endothelial cells of both mother and fetus were also found infected (Miner et al., 2016; Vermillion et al., 2017) (Fig. 6). Trophoblast apoptosis, which can lead to disruption of the placental trophoblast-endothelial barrier, was also detected (Miner et al., 2016). In immunodeficient mice, these alterations can be severe with the destruction of the vasculature and induction of pathological alterations resulting in placental insufficiency (Miner et al., 2016). In pregnant WT mice with an intact immune response, ZIKV infection induces alterations that can result in placental insufficiency with several degrees of injury to the fetus, even without vertical transmission. These effects are increased in older fetuses with more developed placentas and add to the congenital effects of ZIKV infection. Although small amounts of ZIKV RNA could be found in these placentae, IF was unable to detect the expression of virus antigens, which suggests the involvement of immunopathology in these alterations (Szaba et al., 2018). Placental infection can be achieved by vaginal inoculation (Caine et al., 2018; Yockey et al., 2018). This can help sexual transmission since the virus affects the male reproductive system and is shed in semen for an extended period of time (Miner & Diamond, 2017). The vaginal epithelium supports the growth of ZIKV (Yockey et al., 2016), helping to maintain an infection focus that allows access to the uterus and placenta.

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FIG. 6 Placental infection of trophoblast and endothelial cells. (A–C) Red fluorescence—ZIKV antigen; DAPI blue fluorescence shows nuclei; (B and C) right images are magnifications of the areas marked in the left images; (A) ZIKV antibody is detected in the placenta (right image). Left—mock-infected control; (B) green fluorescence shows cytokeratin as a trophoblast marker. ZIKV antigens are detected in trophoblast; and (C) green fluorescence shows vimentin as an endothelial cell marker. ZIKV antigens are associated with endothelial cells. (From Vermillion, M. S., Lei, J., Shabi, Y., Baxter, V. K., Crilly, N. P., McLane, M., et al. (2017). Intrauterine Zika virus infection of pregnant immunocompetent mice models transplacental transmission and adverse perinatal outcomes. Nature Communications, 8, 14575.)

Infection of the reproductive system The virus reaches a high titer in the male reproductive system during infection of immunodeficient but not of WT mice (Govero et al., 2016; Ma et al., 2016; Rossi et al., 2016), and long-term infection of testis and epididymis can persist after viremia subsides (Uraki et al., 2017). This is consistent with the long-term shedding of virus in human semen and the known sexual transmission route of ZIKV (McDonald et al., 2019; Mead, Hills, & Brooks, 2018). The infection leads to cell death in the testis and epididymis with decreased sperm counts and fertility (Govero et al., 2016; Uraki et al., 2017). It is accompanied by an inflammatory response that contributes to further damage and atrophy of the testis and epididymis (Govero et al., 2016; Ma et al., 2016). Immunofluorescence studies showed the presence of viral antigen in Sertoli cells, spermatogonia, spermatocytes, and mature sperm, but also in the epididymis and the epididymis epithelium. It also extends into interstitial peritubular myoid cells and Leydig cells (Clancy et al., 2018; Govero et al., 2016; Kawiecki et al., 2017; Ma et al., 2016; Uraki et al., 2017). The distribution of the infection among the nongerminal cell types was however variable over the models studied. Contrasting with previous studies, Clancy et al. (2018), using AG129 and IFNAR / mice, disclosed a heavy infection of epithelial cells of the epididymis and also found infection of the prostate gland using immunofluorescence and immunohistochemistry methods. These studies suggest that the persistence of infection in the male reproductive tract and

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transmission of the virus through the sperm is caused by these infected cell types. The differences from other studies could be related to the particular strains of virus or mouse used (Clancy et al., 2018). A recent study using micro-RNA targeted ZIKV clones selectively inhibited the infection in either germinal cells or epididymal epithelium (Tsetsarkin et al., 2018). The results of the antigen distribution indicate that both the epididymal epithelium and the germinal cells can be infected by a hematogenous/lymphogenous route. Transmission of the virus through the sperm is enhanced by the susceptibility of the vaginal epithelium to virus replication even in WT mice (Yockey et al., 2016). Replication in the vaginal epithelium is accompanied by damping the immune response of the female reproductive tract and can result in transgenital transmission leading to disease and vertical transmission to the fetus (Khan et al., 2016; Tang et al., 2016; Yockey et al., 2016). In immunodeficient mice, the virus was found to replicate in the vaginal epithelial cells only during the progesteroneinduced diestrus-like phase. A few stromal cells and macrophage-like cells in draining lymph nodes and spleen were also infected and ZIKV was able to spread to the cerebral neurons in AG129 mice producing lethal disease (Tang et al., 2016). Replication in the vaginal epithelium in pregnant mice is able to infect the fetus even in WT mice (Yockey et al., 2016), implying a direct mechanism of fetus infection.

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Prepare frozen tissue sections of your ZIKV infected tissues. Air-dry the sections for 10 min at room temperature. Fix the tissue sections by immersing the slides in cold acetone for 10 min. Remove the fixative and evaporate acetone from the tissue sections at room temperature (less than 20 min). Rinse the slides in 10 mM phosphate-buffered saline (PBS) pH 7 3  5 min. Incubate the slides in blocking buffer (1% BSA, 0.3% Triton, in PBS) at 37°C for 30 min in a wet chamber. Remove the blocking buffer. Apply an appropriately diluted anti-ZIKV primary antibody in 0.5% bovine serum albumin in PBS and incubate overnight at 4°C in a wet chamber. Rinse the slides in PBS for 3  5 min. Apply fluorescein-conjugated secondary antibody for 1 h at 37°C (e.g., Fluorescein-Conjugated Goat antihuman IgG if the primary antibody is human). Rinse the slides in PBS for 2  5 min. Mount with ProLong Diamond antifade mounting media containing DAPI (ThermoFisher, Carlsbad, CA). Observe and photograph with a fluorescence microscope.

(General laboratory protocol adjusted to Zika according to Ma et al. (2016)). Note: Appropriate controls are essential for the interpretation of IF labeling. a) Include a negative control by incubating with buffer with no primary antibody to identify labeling due to nonspecific binding of the fluorochrome-labeled antibodies. b) Include controls where mock-infected tissue is submitted to the full staining to identify tissue antigens that can crossreact with the primary antibody.

Mini-dictionary of terms Flavivirus. Flavivirus is the only genus of the family Flaviviridae. The genome is positive-sense single-stranded RNA molecule and the viruses are transmitted by arthropods, in particular mosquitos and ticks. These include human pathogens such as West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DENV), and Zika virus (ZIKV). ZIKV is now considered a TORCH pathogen (i.e., a pathogen that can cause miscarriage, retarded growth, and other developmental abnormalities) due to its capability for vertical transmission and congenital infection induction. Innate immune response. This is the first response to the multiple layers of antipathogen protection provided by the immune system. It generates a nonspecific response that immediately restricts the ability of the pathogen to spread via phagocytosis and intracellular killing by leukocytes. It can be triggered by interferon in viral infections. STAT2. STATs are transcription factor subunits (signal transducers and activators of transcription) that migrate to the nucleus to activate IFN responsive genes. STAT2 is a subunit of the transcription factor IFN-stimulated gene factor 3, induced by IFN-α and -β, but not by IFN-γ.

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Interferon. Interferons are key players in antiviral responses, and also in antitumoral and immunoregulatory responses. There are two main types of Interferons, type I (IFN-α and IFN-β) and type II (IFN-γ), that operate through distinct but related transcription factors. They work through the induction of the synthesis of proteins in responsive cells. They are activated by specific extracellular molecules that stimulate Toll-like receptors, which play a key role in the innate immune response by activating immune cell responses. Microcephaly. Microcephaly shows as a smaller than normal head circumference and is usually caused by exposure of the fetus to drugs, toxins, or certain viruses like ZIKV. It can also be caused by genetic abnormalities. Congenital Zika syndrome. CZS is a spectrum of pathologies produced by ZIKV infection that remains incompletely defined. The hallmark of the syndrome is microcephaly that can be associated with a wide variety of alterations of the nervous system (ventriculomegaly, calcifications, malformations of cortical development, arthrogryposis, eye abnormalities, and motor abnormalities) and also with nonneurological diseases such as intrauterine growth restriction, placental insufficiency, and others. Vertical transmission. The transmission of a pathogen from a mother to the offspring before or immediately after birth. Mechanisms may involve trans-placental transmission, breastfeeding, or direct contact. Placental insufficiency. The inability of the placenta to deliver an adequate supply of oxygen or nutrients to the fetus results in failure to support the fetus development. It can result from damage induced by pathogens such as ZIKV, directly or mediated by immune mechanisms, or failure to develop normally during the growth of the fetus.

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Normal WT mice are able to control ZIKV infection, displaying only mild and transient symptoms. In mice lacking the IFN response, ZIKV is able to produce a systemic disease involving most organs and leading to death in the most severe cases. Infection of the placenta results in vertical transmission of the virus to the fetus, causing the congenital Zika syndrome. ZIKV infects neuronal precursors in the CNS and causes neuron depletion and microcephaly in fetuses and impaired neuronal production later in life. Motor and sensory defects result from the infection of medullar neurons and of the retina of developing fetuses. Infection of the male sexual system involves multiple cell types of the testis, epididymis, and accessory glands leading to the shedding of virus for long periods after viremia has subsided. The vaginal epithelium is highly susceptible to virus infection, which helps support sexual transmission, and fetus vertical infection.

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This chapter focus on the distribution of ZIKV in mice models as detected by antigen location by immunofluorescence, for detection of infected cells and their distribution. The spread of ZIKV through the infected mice is prevented by an intact innate immune response triggered by IFN I, while ZIKV is able to shut down the IFN I induced an innate immune response in humans. Several types of manipulations of the immune system, the route of infection, and others have been used to generate mice models able to develop some of the manifestations of the disease. Infection of the fetal brain causes depletion of NPCs, resulting in manifestations of the CZS. Vertical transmission of the virus to the fetus depends on crossing the placental barrier. Several placental cell types can be infected, allowing the virus to reach the fetus and induce CZS. The male reproductive system is a major target for ZIKV infection, involving cells of the germinal line and Sertoli cells, but also Leydig cells and epithelial cells from the epididymis and accessory glands. Prolonged maintenance of infection of the male reproductive tract and susceptibility to infection of the vaginal epithelium allows sexual transmission and spread of the infection.

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Chapter 41

The role of seminal Zika viral shedding: Tropism, duration, and magnitude Erin M. McDonald and Aaron C. Brault Division of Vector-borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, United States

Abbreviations BLT CGS EEC IFN IFNR ISH NHP RIG RNA SCB TLR tRNA ZIKV

bone liver thymus congenital Zika virus syndrome epididymal epithelial cell interferon interferon receptor in situ hybridization nonhuman primate retinoic acid-inducible gene ribonucleic acid Sertoli cell barrier toll-like receptor transfer ribonucleic acid Zika virus

Introduction In humans, Zika virus (ZIKV) RNA has been detected in multiple bodily fluids of infected individuals, including urine, saliva, vaginal secretions, and semen. Long-term detection of ZIKV RNA in semen is common, with approximately 50% of infected symptomatic men shedding ZIKV RNA in semen for 42–54 days following illness onset (Fig. 1). Long-term persistence of ZIKV RNA in semen has been detected as far out as 139–186 days post illness onset (Huits et al., 2017; Mead et al., 2018a; Medina et al., 2019; Paz-Bailey et al., 2018). Viral RNA shedding has been observed in seminal fluids from nonvasectomized as well as vasectomized men (Fig. 2) (Mead et al., 2018b). This long-term ZIKV RNA shedding in semen has also been demonstrated in nonhuman primate (NHP) and murine models (Duggal et al., 2017; Koide et al., 2016; Peregrine et al., 2019). In mice, the peak of sexual transmission efficiency coincides with ZIKV infection in the tail of the epididymis, specifically of epididymal epithelial cells (EECs) (McDonald, Duggal, Ritter, & Brault, 2018). The cellular reservoir that leads to viral RNA shedding in semen in the long term is unknown.

Zika virus tropism for the male reproductive tract The male reproductive tract comprises testes, epididymides, vas deferens, and accessory glands (seminal vesicle, prostate gland). Each pair of testes connects to a single, long coiled tube, known as the epididymis. The tubular epididymis transitions into the vas deferens, which then joins a seminal vesicle and forms the ejaculatory duct. The ejaculatory duct traverses through the prostate gland and empties into the urethra. The urethra is a hollow tube that runs through the penis; from the urethra, semen is ejaculated. In mice and NHP models, ZIKV RNA and/or infectious virus has been detected in the testes, epididymides, seminal vesicles, and prostate (Duggal et al., 2017; Govero et al., 2016; Hirsch et al., 2017; Koide et al., 2016; Li et al., 2016; McDonald et al., 2018, 2019; Peregrine et al., 2019). Zika Virus Biology, Transmission, and Pathways. https://doi.org/10.1016/B978-0-12-820268-5.00041-9 Copyright © 2021 Elsevier Inc. All rights reserved.

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FIG. 1 Persistence of detectable Zika virus RNA in symptomatic men. Proportion of men with seminal samples for which ZIKV RNA was detectable by RT-qPCR at different time points postsymptom onset (original image).

FIG. 2 Magnitude of detectable Zika virus RNA in symptomatic men. The magnitude of viral RNA (determined as RNA copy) was assessed from seminal samples obtained from symptomatic men at various days postsymptom onset. Vasectomized men were found to have viral RNA levels that were significantly lower in magnitude than nonvasectomized men (original image).

The epididymis and testis are sites of immune privilege, in which tissue organization and immunosuppressive activities limit immune reactions against spermatogonial stem cells and mature spermatozoa. Physical blood-tissue barriers contribute to immune privilege maintenance. Specialized tight junctions between adjacent Sertoli cells and adjacent EECs form the blood-tissue barriers for the testes and epididymides, respectively. The blood epididymis barrier is weaker than the blood-testis barrier, making it more accessible than the testis to immune cell invasion. Leukocytes and immunoglobulins can gain access directly to the epididymal lumen when breakdown occurs. Despite being immune privileged sites, the epididymis and testis respond to pathogens, toxins, and other stimulants. These organs express antimicrobial receptors, including toll-like receptors (TLRs) and RNA sensors, such as retinoic acid-inducible gene I (RIG-1). Other receptors involved in inflammation and antiviral pathways, such as the receptors involved in binding type I and type II interferons (IFN-alpha/beta and IFN-gamma receptors, respectively) and the type III interferon IFN-lambda receptor (interferon-lambda receptor 1/IL-10RB; IFNLR1), are also expressed in the male reproductive tract. These antiviral pathways and inflammatory signaling cascades may restrict ZIKV replication in the male reproductive tract, but do not eliminate infection, as evidenced by multiple animal models demonstrating ZIKV tropism for these sites.

The epididymis is critical for sexual transmission Each epididymis is a single, long, highly coiled tube. Smooth muscle and connective tissue surround the pseudostratified epithelium; the connective tissue contains resident immune cells and the inner lumen of the tube contains sperm and fluid. The epididymis is divided into three sections: the head, body, and tail. Immature spermatogonia leave the testis and transit to the head of the epididymis. As sperm move through the epididymis, they mature and become motile and capable of

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FIG. 3 Epididymal tail demonstrating ZIKV RNA within epididymal epithelial cells. In situ hybridization of murine epididymal tail epithelium. Tissues were stained with Zika virus (+)-sense RNA probe set (original image).

fertilization. Mature spermatozoa are stored in the tail of the epididymis until an ejaculation forces them into the vas deferens. Infectious ZIKV has only been isolated from human semen samples following acute symptom onset (Mead et al., 2018a) (Fig. 1). This is recapitulated in sexual transmission potential models in both immunodeficient and immunocompetent mice (Duggal et al., 2017; Duggal, McDonald, Ritter, & Brault, 2018; McDonald et al., 2019). Following subcutaneous ZIKV inoculation, the murine testis and epididymis are infected concurrently and independently of one another (Clancy, Van Wettere, Siddharthan, Morrey, & Julander, 2018b; McDonald et al., 2018). Furthermore, based on murine tissue ISH and immunohistochemistry, it is likely that infection originates in the head of the epididymis and then traverses down the length of the epididymis (Fig. 3). In an immunocompetent mouse model, infectious virus and viral RNA were detected in the epididymides days before the infectious virus was detected in the seminal vesicles or testes (McDonald et al., 2019). Lastly, ZIKV RNA has been detected in epididymides in a subset of olive baboons and Cynomolgus macaques (Koide et al., 2016; Peregrine et al., 2019). In murine models, ZIKV replicates in EECs, immature spermatids, and infiltrating leukocytes. Infection of the tail of the epididymis coincides with the peak of sexual transmission efficiency in both IFN-ɑ/β/ɣ receptor knockout (AG129) mice and immunocompetent C57Bl/6 mice (McDonald et al., 2018, 2019). After the peak of sexual transmission, mice can shed viral RNA in semen for months (Duggal et al., 2017), which parallels case studies in humans, with ZIKV RNA being detected in semen from laboratory confirmed infected symptomatic men for as long as 281 days post symptom onset (Mead et al., 2018a). Data from bone marrow, liver, thymus (BLT) humanized mice show that the testis and epididymis become ZIKV RNA positive by dpi 15 and that only the epididymis remains ZIKV RNA positive out to 224 dpi (Fig. 4; unpublished data). Lastly, AG129 mice, IFN-ɑ/β receptor knockout (A129) mice, and C57BL/mice treated with a monoclonal antibody to IFNAR1 exhibit epididymitis following subcutaneous ZIKV inoculation (Clancy, Van Wettere, Siddharthan, et al., 2018b; Duggal et al., 2017; McDonald et al., 2018). Another cell population in the epididymis that stains positive for ZIKV RNA in the epididymis is mature spermatozoa (Govero et al., 2016; McDonald et al., 2018) (Fig. 5). However, it is unlikely that mature spermatozoa are a source of infectious virus, since these cells have condensed chromatin, lack tRNAs, and have shut down RNA transcription.

ZIKV infects the testis and seminal vesicles Spermatogenesis, the production of sperm from stem cells, takes place in the seminiferous tubules of the testes. These tubules are lined with specialized epithelial cells, called Sertoli cells, whose function is twofold: to secrete nutrients to differentiate germ cells (spermatogonia and spermatocytes) and to maintain the blood-tissue barrier (via tight junctions between adjacent Sertoli cells). Between the tubules are Leydig cells, which produce male hormones. Macrophages and other resident immune cells make up the remainder of the interstitial cell population. In the murine testes, the initial cells to become infected are scattered interstitial leukocytes and peritubular myoid cells (Clancy, Van Wettere, Siddharthan, et al., 2018b; Duggal et al., 2017; McDonald et al., 2018). As infection progresses, the

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FIG. 4 BLT humanized mouse epididymis stains positive for ZIKV RNA at 224 dpi. In situ hybridization of murine testis and epididymis 224 days postinoculation (dpi). Tissues were stained with ZIKV (+) probe set. Testis did not stain positive at 224 dpi. Epithelial cells in the epididymis stained positive for ZIKV (+) RNA (red punctate staining). The slides were counterstained with Gill’s hematoxylin I (original image).

FIG. 5 Mature spermatozoa obtained from seminal fluid of an AG129 male mouse inoculated with ZIKV positive for ZIKV RNA. Mature spermatozoa viewed directly by confocal microscopy from seminal fluid obtained at 12 days post Zika virus inoculation. The green signal represents an ISH positive signal for genomic ZIKV RNA (original image).

seminiferous epithelium and seminiferous tubules become infected. In histopathological studies on the male Olive Baboon and Rhesus and Cynomolgus macaques, ZIKV antigen/RNA has been found in the spermatogonial zone and intermediate layers of the seminiferous tubules. Positive staining for ZIKV antigen/RNA in NHP testes has only been observed in a subset of animals in each study, and has been detected as late as dpi 11 in the Olive Baboon and dpi 7–28 in Cynomolgus and Rhesus macaques (Hirsch et al., 2017; Koide et al., 2016; Li et al., 2016; Peregrine et al., 2019). This is in stark contrast to the prolonged detection of ZIKV in murine testes. ZIKV replicates in a variety of primary human reproductive tract cell lines, including low passage Sertoli cells, a testicular fibroblast cell line, and two different seminoma cell lines (germ cell tumor of the testicle) (Kumar et al., 2018; Mlera & Bloom, 2019; Siemann, Strange, Maharaj, Shi, & Verma, 2017; Strange et al., 2018, 2019). The TAM (Tyro3, Axl, Mer) receptor tyrosine kinase Axl is a known receptor for ZIKV and is expressed in the male reproductive tract, specifically in Sertoli cells and Leydig cells in the testes and in glandular cells in the epididymis (Human protein atlas; https://www. proteinatlas.org/ENSG00000167601-AXL). Despite the expression of Axl by Leydig cells, primary human Leydig cells do not support ZIKV replication (Kumar et al., 2018). Multicellular human testicular organoids, composed of Sertoli cells, Leydig cells, peritubular myoid cells, and spermatogonial stem cells, have demonstrated the critical role Axl plays in ZIKV entry and replication. Organoids treated with either an antibody to anti-Axl6, the Axl ligand, Gas6, or the Axl kinase inhibitor, R428, showed reduced production of ZIKV RNA (Strange et al., 2019). Ex vivo testicular models support a critical role for Sertoli cells and interstitial macrophages in ZIKV replication and dissemination in the testes. Siemann et al. used an in vitro Sertoli cell barrier (SCB) model to demonstrate that ZIKV infection of Sertoli cells led to increased monocyte adhesion to the SCB (Siemann et al., 2017). Furthermore, they demonstrated that the supernatant from ZIKV-infected monocytes contained proinflammatory mediators and SCB models

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treated with this supernatant increased permeability of the SCB. Increased permeability of the blood-testis barrier is one potential mechanism ZIKV may take advantage of to gain access to this immune privileged site. Testicular tissue explants from healthy human donors support ZIKV replication and RNA ISH on these explants identified ZIKV RNA (+) interstitial macrophages, Sertoli cells, Leydig cells, myoid peritubular cells, and early and late germ cells (Matusali et al., 2018a). Using immunofluorescence microscopy, Matusali et al. demonstrated colabeling of germ cells and spermatogonia with ZIKV envelope/NS1 in semen cell smears from ZIKV-infected donors. Murine models demonstrate these same staining patterns (Matusali et al., 2018b). Lastly, murine models have looked at histopathology following ZIKV inoculation. After ZIKV subcutaneous inoculation of mice lacking type I and type I and II interferon receptors, A129 and AG129 mice, respectively, orchitis, and a decrease in spermatids and loss of structure in seminiferous tubules were observed (Clancy, Van Wettere, Siddharthan, et al., 2018b; Govero et al., 2016; McDonald et al., 2018). These pathological observations were more severe in AG129 compared to A129 mice (Clancy, Van Wettere, Siddharthan, Morrey, & Julander, 2018a). Similar pathological observations were made in C57BL/6 Ifnar1
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and C57BL/6 anti-Ifnar1 treated mice (Govero et al., 2016; Ma et al., 2016b). In contrast, WT C57BL/6 mice did not develop pathology in the male reproductive tract following intraperitoneal inoculation of ZIKV, suggesting a role for type I interferons in maintaining the blood-testis barrier. When the blood-testis barrier was physically bypassed by intratesticular injection of ZIKV, WT C57BL/6 mice developed acute orchitis, with leukocytic infiltration into the testis, followed by the appearance of necrotic cells and loss of seminiferous tubule structure. These effects were similar to those of intraperitoneally inoculated immunodeficient mice (Ma et al., 2016b). Following subcutaneous inoculation of ZIKV of AG129 and A129 mice, the prostate and seminal vesicles have been shown to stain positive for ZIKV antigen, ZIKV RNA, and infectious virus (Clancy, Van Wettere, Siddharthan, et al., 2018b; McDonald et al., 2018). Ifnar
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mice did not show evidence of viral antigen in the prostate or seminal vesicles (Ma et al., 2016a). The role of the Axl receptor in ZIKV entry has been established; seminal vesicles and the prostate do not express Axl (Human protein atlas; https://www.proteinatlas.org/ENSG00000167601-AXL) and this was confirmed in mouse tissues by IHC (Ma et al., 2016b).

Duration and magnitude of ZIKV shedding in semen At present, ZIKV is unique among mosquito-borne flaviviruses to be sexually transmitted. In murine models, ZIKV can be sexually transmitted via semen (Duggal et al., 2017, 2018). Semen is composed of seminal fluid, spermatozoa, and epithelial and myeloid cells from the testis and urogenital lining. The majority of seminal fluid is derived from the accessory glands: approximately 65% of seminal fluid is derived from the seminal vesicles, and 25% from the prostate. Only 10% of the fluid comes from the testes and epididymides. The remainder of seminal fluid derives from the bulbourethral glands, urethral glands, and vas deferens (Drabovich, Saraon, Jarvi, & Diamandis, 2014). In an immunodeficient mouse model, the majority of infectious ZIKV was detected in the cell-free portion of seminal fluid, thus suggesting that cell-free ZIKV may be largely responsible for sexual transmission (McDonald et al., 2018) (Fig. 6). Multiple murine studies have been published that model sexual transmission. Sexual transmission has been mimicked by intravaginal inoculation of virus (Khan et al., 2016; Tang et al., 2016; Yockey et al., 2016) or by intravaginal inoculation of female mice with homogenized accessory gland fluid and epididymal lumen fluid from ZIKV-inoculated male mice (Clancy, Van Wettere, Morrey, & Julander, 2018, 2019; Simanjuntak et al., 2018). Immunodeficient mice inoculated via these routes become viremic, demonstrate infection in the female reproductive tract, and succumb to infection. Following intravaginal inoculation of pregnant immunocompetent dams, ZIKV was shown to replicate in the vaginal mucosa and to cause intrauterine growth restriction of fetuses (Yockey et al., 2016). FIG. 6 Infectious ZIKV identified for two different viral strains from seminal plasma vs cellular seminal components. Male AG129 mice were inoculated subcutaneously with two different Asian genotype strains of Zika virus. Seminal fluids were collected, centrifuged to separate seminal plasma, and the cellular and plasma fractions tested for infectious Zika virus at different time points postinoculation (original image).

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Sexual transmission murine models via coitus have demonstrated that viral RNA is shed from both intact males and vasectomized males; also, viral RNA is shed for a longer duration compared to shedding of infectious virus in semen. Lastly, immunodeficient males can sexually transmit infectious virus to naı¨ve immunodeficient female mice, resulting in viremia, in utero transmission, and increased morbidity and mortality in the female mice (Duggal et al., 2017; Duggal et al., 2018). Based on the timing of ZIKV RNA and infectious virus in semen, ZIKV infection of and replication in immature spermatids in the testes most likely does not contribute to sexual transmission. Although ZIKV RNA positive mature spermatozoa in semen have been identified in murine models and from human clinical samples, it is unlikely that the spermatozoa are infected in the testes. Spermatogenesis takes approximately 34.5, 36, and 64 days in mice, NHPs, and humans, respectively (Barr, 1973; Heller & Clermont, 1963; Oakberg, 1956). Semen is positive for ZIKV RNA within a week following inoculation of murine and NHP models with ZIKV, and semen from humans is positive for ZIKV RNA within a week following symptom onset. Sexual transmission models have not been carried out in NHPs due to the cost of such an experiment. But ZIKV RNA has been detected in seminal fluid from dpi 6–41 of inoculated male olive baboons, Rhesus, and Cynomolgus macaques (Koide et al., 2016; Peregrine et al., 2019; Silveira et al., 2017).

Concluding remarks Sexual transmission serves as a unique transmission route for a Zika virus for which mosquito-borne transmission had been believed to be the sole transmission route. The evolutionary basis for this transmission route has not been established but could be related to the need for transmission between arboreal NHPs during time periods when mosquito populations would be too low to promote efficient transmission or possibly as an evolutionary byproduct of evolved venereal transmission in the mosquito vector. Nevertheless, the consequences of sexual transmission of ZIKV are significant as this provides a unique route to the developing fetus. Although assessments of the direct impact of sexual transmission on cases of congenital ZIKV syndrome have not been practical as the vast majority of the CGS have occurred in locations with autochthonous mosquito-borne transmission, the potential implications are clear. Animal models have shown greater impact to the developing fetus through sexual transmission as opposed to through a peripheral route (Duggal et al., 2018). Furthermore, a sexual transmission route affords the virus a potential for circulation during time periods and in geographical areas in which mosquito transmission would not be possible such as at higher elevations or during the winter or the dry season. Zika virus can clearly be sexually transmitted. Although a number of questions have been answered regarding this route of transmission such as the duration of shedding of infectious virus and viral RNA in seminal fluids from symptomatic infected men, a number of questions remain: (i) How does the duration and relative transmission risk from asymptomatic men compare to that demonstrated for symptomatic men? (ii) Is there a risk of recrudescence of men shedding viral RNA due to waning immunity or due to immunosuppression? (iii) Does sexual transmission pose an increased risk for CZS in developing fetuses as has been demonstrated in animal models?

Policy and procedures Molecular detection of Zika viral RNA in seminal fluids After receipt of samples, multiple aliquots of semen should be immediately frozen at
80°C. Prior to RNA extraction, 25 μL semen samples should be treated with 25 μL freshly prepared 10 mM dithiothreitol (DTT, Pierce Biotechnology) and mixed with 1  104 JAR human placental cells to serve as an internal control for RNA extraction efficiency (described below). RNA should be extracted from at least a 50 μL sample using the MagMAX Viral RNA isolation kit (Ambion). ZIKV RNA can be detected through the use of a Qiagen QuantiTect Probe reverse-transcription polymerase chain reaction (qRT-PCR) kit with primers Zika4481 (50 - CTGTGGCATGAACCCAATAG-30 ) and Zika4552c (50 -ATCCCATAGAGCACCACTCC-30 ) and probe Zika4507cFAM (50 -CCACGCTCCAGCTGCAAAGG-30 ) using previously described qRT-PCR conditions (Lanciotti et al., 2008; Mead et al., 2018b). Samples should be tested in duplicate and samples with equivocal results (one ZIKV positive and one ZIKV negative) should be re-extracted and re-quantified. A standard curve generated by in vitro transcription of pCDNA3.1 containing a fragment of ZIKV spanning nucleotides 3576–4631 of strain P6-740 should be included. The lower detection limit for this assay has been determined to be 2.3 log10 ZIKV RNA copies/ mL. The RNA extraction efficiency of semen specimens should be assessed by amplifying the human beta-2-microgobulin (b2m) transcript for each sample using a TaqMan primer/probe mix (Applied Biosystems). Samples demonstrating b2m internal control failure should be re-extracted and qRT-PCR repeated. A subsequent b2M internal control failure should be interpreted as a compromised sample and not be included in your analyses. Semen samples with detectable ZIKV RNA by qRT-PCR (cycle thresholds