METHODS IN MOLECULAR BIOLOGY978-1-4939-8678-1/1.pdfmodels, in vivo imaging, antigenic evolution, immunology and mathematical modeling. The book opens with an introduction to influenza

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ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire AL10 9AB, UK

For further volumes:http://www.springer.com/series/7651

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Influenza Virus

Methods and Protocols

Edited by

Yohei Yamauchi

School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK

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EditorYohei YamauchiSchool of Cellular and Molecular MedicineUniversity of BristolBristol, UK

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-4939-8677-4 ISBN 978-1-4939-8678-1 (eBook)https://doi.org/10.1007/978-1-4939-8678-1

Library of Congress Control Number: 2018949617

© Springer Science+Business Media, LLC, part of Springer Nature 2018This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Preface

In 1918 the Spanish Flu pandemic killed an estimated 40 million people around the world.One hundred years later, in 2018, influenza virus still maintains a significant presence in themedical and veterinary spheres. Since the first isolation of a human influenza virus strain in1933, influenza has been researched intensively and remarkable progress has been made: therecent discovery of bat influenza, decoding of the heterotrimeric influenza A polymerasestructure, and identification of the host gene responsible for avian virus polymerase adapta-tion in mammals, are a few examples. The development of influenza virus reverse genetics inthe late 1990s furthered our understanding of viral pathogenesis and viral protein functions.It also promoted the generation of novel vaccine strategies. In the early 2000s, genome-wide siRNA screening, systems biology and bioinformatics were applied to influenza andother major human viruses, revealing global virus-host interaction networks and identifyingnew host genes and processes that regulate infection. Recent technological breakthroughs insuper-resolution imaging and cryo electron microscopy have opened new avenues of detect-ing molecular interactions and viral structures at very high resolution. However, influenzavirus continues to elude our persistent efforts to develop a flawless vaccine or antiviral, andthe current knowledge of influenza virus entry, replication and pathogenesis is incomplete.

This book will equip researchers – from newcomers to experienced – with varioustechniques applicable to influenza biology research in the age of big data. The protocolscontain plenty of visual aids to help the reader replicate an experiment in his/her laboratory.The chapters are weighted on molecular biology, viral cell biology and imaging approachesthat facilitate mechanistic investigation of virus-host interactions. Leading scientists – whosebios and photos are presented at the end of the book – have been brought together to covera broad range of topics such as influenza diagnosis, virus propagation, proteomics, haploidand lentiviral screening, virus entry, single-molecule RNA imaging, correlative light andelectron microscopy (CLEM), EM, light-sheet microscopy, biochemistry, viral transcrip-tion, physiological infection models (bacterial co-infections, aerosol infection), animalmodels, in vivo imaging, antigenic evolution, immunology and mathematical modeling.The book opens with an introduction to influenza and closes with four perspective chaptersthat discuss the challenges of clinical vaccine trials, debates on the gain-of-function experi-ments with pathogens of pandemic potential, and how mathematical modeling providesmechanistic insights into infection. This book will hopefully help researchers performexperiments that fill in the gaps of our knowledge and improve the overall understandingof influenza.

Bristol, UK Yohei Yamauchi

v

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Acknowledgments

I would like to thank members of my laboratory, Alina Rozanova, Caitlin Simpson, and ShoIketani for their editorial assistance.

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Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vAcknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1 Understanding Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Edward C. Hutchinson and Yohei Yamauchi

2 Clinical Diagnosis of Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Yoshinori Ito

3 Influenza A Virus Genetic Tools: From Clinical Sampleto Molecular Clone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Stephanie Anchisi, Ana Rita Goncalves, Beryl Mazel-Sanchez,Samuel Cordey, and Mirco Schmolke

4 Propagation and Titration of Influenza Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Umut Karakus, Michel Crameri, Caroline Lanz, and Emilio Yanguez

5 Purification and Proteomics of Influenza Virions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Edward C. Hutchinson and Monika Stegmann

6 Haploid Screening for the Identification of Host Factorsin Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Evelyn Fessler and Lucas T. Jae

7 Phenotypic Lentivirus Screens to Identify Antiviral SingleDomain Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139Florian Ingo Schmidt

8 Deciphering Virus Entry with Fluorescently Labeled Viral Particles . . . . . . . . . . . 159Anja B. Hoffmann, Magalie Mazelier, Psylvia Leger,and Pierre-Yves Lozach

9 Quantitative RT-PCR Analysis of Influenza Virus Endocytic Escape. . . . . . . . . . . 185Wen-Chi Su and Michael M.C. Lai

10 Single-Molecule Sensitivity RNA FISH Analysis of InfluenzaVirus Genome Trafficking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Yi-ying Chou and Timothee Lionnet

11 3D Electron Microscopy (EM) and Correlative Light ElectronMicroscopy (CLEM) Methods to Study Virus-Host Interactions . . . . . . . . . . . . . 213Ines Romero-Brey

12 Correlative Light and Electron Microscopy of InfluenzaVirus Entry and Budding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237Lorna Hodgson, Paul Verkade, and Yohei Yamauchi

13 Influenza Virus-Liposome Fusion Studies Using FluorescenceDequenching and Cryo-electron Tomography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261Long Gui and Kelly K. Lee

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14 Metal-Tagging Transmission Electron Microscopy and ImmunogoldLabeling on Tokuyasu Cryosections to Image Influenza A VirusRibonucleoprotein Transport and Packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281Martin Sachse, Isabel Fernandez de Castro, Guillaume Fournier,Nadia Naffakh, and Cristina Risco

15 Live Imaging of Influenza Viral Ribonucleoproteins UsingLight-Sheet Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303Amar R. Bhagwat, Valerie Le Sage, and Seema S. Lakdawala

16 Purification of Unanchored Polyubiquitin Chains from Influenza Virions. . . . . . 329Yasuyuki Miyake, Patrick Matthias, and Yohei Yamauchi

17 Assays to Measure the Activity of Influenza Virus Polymerase . . . . . . . . . . . . . . . . 343Aartjan J.W. te Velthuis, Jason S. Long, and Wendy S. Barclay

18 In Vitro Models to Study Influenza Virus and Staphylococcus aureusSuper-Infection on a Molecular Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375Christin Bruchhagen, Andre van Kruchten, Carolin Klemm,Stephan Ludwig, and Christina Ehrhardt

19 Infection of Cultured Mammalian Cells with Aerosolized Influenza Virus . . . . . 387Hannah M. Creager, Terrence M. Tumpey, Taronna R. Maines,and Jessica A. Belser

20 Animal Models in Influenza Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401Johanneke D. Hemmink, Catherine J. Whittaker, and Holly A. Shelton

21 Measuring Influenza Virus Infection Using BioluminescentReporter Viruses for In Vivo Imaging and In Vitro Replication Assays . . . . . . . . 431Erik A. Karlsson, Victoria A. Meliopoulos, Vy Tran, Chandra Savage,Brandi Livingston, Stacey Schultz-Cherry, and Andrew Mehle

22 Selection of Antigenically Advanced Variants of Influenza Viruses. . . . . . . . . . . . . 461Gabriele Neumann, Shufang Fan, and Yoshihiro Kawaoka

23 Assessment of Influenza Virus Hemagglutinin Stalk-SpecificAntibody Responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487Wen-Chun Liu, Raffael Nachbagauer, Florian Krammer,and Randy A. Albrecht

24 Analyses of Cellular Immune Responses in Ferrets FollowingInfluenza Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513Anthony T. DiPiazza, Katherine A. Richards, Wen-Chun Liu,Randy A. Albrecht, and Andrea J. Sant

25 Parameter Estimation in Mathematical Models of ViralInfections Using R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531Van Kinh Nguyen and Esteban A. Hernandez-Vargas

26 Software for Characterizing the Antigenic and Genetic Evolutionof Human Influenza Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551Susanne Reimering and Alice C. McHardy

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27 Clinical Trials of Influenza Vaccines: Special Challenges . . . . . . . . . . . . . . . . . . . . . 567Adam Finn

28 The Silver Lining in Gain-of-Function Experimentswith Pathogens of Pandemic Potential. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575Michael J. Imperiale, Don Howard, and Arturo Casadevall

29 Why Do Exceptionally Dangerous Gain-of-FunctionExperiments in Influenza?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589Marc Lipsitch

30 How Computational Models Enable MechanisticInsights into Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609Ivo F. Sbalzarini and Urs F. Greber

About the Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659

Contents xi

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Contributors

RANDYA. ALBRECHT � Department of Microbiology, Icahn School of Medicine at Mount Sinai,New York, NY, USA; Global Health and Emerging Pathogens Institute, Icahn School ofMedicine at Mount Sinai, New York, NY, USA

STEPHANIE ANCHISI � Faculty of Medicine, CMU, Department of Microbiology and MolecularMedicine, University of Geneva, Geneva, Switzerland

WENDY S. BARCLAY � Faculty of Medicine, Division of Infectious Disease, Imperial CollegeLondon, London, UK

JESSICA A. BELSER � Influenza Division, National Center for Immunization and RespiratoryDiseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

AMAR R. BHAGWAT � Department of Microbiology and Molecular Genetics, University ofPittsburgh School of Medicine, Pittsburgh, PA, USA

CHRISTIN BRUCHHAGEN � Institute of Virology Muenster (IVM), Westfaelische Wilhelms-University Muenster, Muenster, Germany

ARTURO CASADEVALL � Department of Molecular Microbiology and Immunology, JohnsHopkins Bloomberg School of Public Heath, Baltimore, MD, USA

YI-YING CHOU � Program in Cellular and Molecular Medicine, Department of Cell Biology,Harvard Medical School, Boston Children’s Hospital, Boston, MA, USA

SAMUEL CORDEY � Laboratory of Virology, Infectious Diseases Service, University Hospitals ofGeneva, Geneva, Switzerland

MICHEL CRAMERI � Institute of Medical Virology, University of Zurich, Zurich, SwitzerlandHANNAH M. CREAGER � Influenza Division, National Center for Immunization and

Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USAISABEL FERNANDEZ DE CASTRO � Centro Nacional de Biotecnologia, CNB-CSIC, Cell

Structure Lab, Madrid, SpainANTHONY T. DIPIAZZA � Department of Microbiology and Immunology, David H. Smith

Center for Vaccine Biology and Immunology, University of Rochester Medical Center,Rochester, NY, USA

CHRISTINA EHRHARDT � Institute of Virology Muenster (IVM), Westfaelische Wilhelms-University Muenster, Muenster, Germany

SHUFANG FAN � Department of Pathobiological Sciences, Influenza Research Institute, Schoolof Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA

EVELYN FESSLER � Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universit€at Munchen, Munich, Germany

ADAM FINN � Bristol Children’s Vaccine Centre, Schools of Cellular & Molecular Medicineand of Population Health Sciences, University of Bristol, Bristol, UK

GUILLAUME FOURNIER � Departement de Virologie, Institut Pasteur, Unite de Genetique Moleculaire des Virus a ARN, Paris, France; CNRS, UMR 3569, Paris, France; UniversiteParis Diderot, Sorbonne Paris Cite, Paris, France

ANA RITA GONCALVES � Laboratory of Virology, Infectious Diseases Service, UniversityHospitals of Geneva, Geneva, Switzerland

URS F. GREBER � Department of Molecular Life Sciences, University of Zurich, Zurich,Switzerland

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LONG GUI � Department of Medicinal Chemistry, University of Washington, Seattle, WA,USA; Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA

JOHANNEKE D. HEMMINK � Roslin Institute and R(D)SVS, University of Edinburgh,Midlothian, UK; International Livestock Research Institute (ILRI), Nairobi, Kenya

ESTEBAN A. HERNANDEZ-VARGAS � Frankfurt Institute for Advanced Studies, Frankfurt amMain, Germany

LORNA HODGSON � School of Biochemistry, University of Bristol, Bristol, UKANJA B. HOFFMANN � From CellNetworks Cluster of Excellence and Department of Infectious

Diseases, Virology, University Hospital Heidelberg, Heidelberg, GermanyDON HOWARD � Department of Philosophy, University of Notre Dame, Notre Dame, IN,

USAEDWARD C. HUTCHINSON � MRC-University of Glasgow Centre for Virus Research, Glasgow,

UKMICHAEL J. IMPERIALE � Department of Microbiology and Immunology, University of

Michigan, Ann Arbor, MI, USAYOSHINORI ITO � Department of Pediatrics, Nagoya University Graduate School of Medicine,

Nagoya, Aichi, JapanLUCAS T. JAE � Gene Center and Department of Biochemistry, Ludwig-Maximilians-

Universit€at Munchen, Munich, GermanyUMUT KARAKUS � Institute of Medical Virology, University of Zurich, Zurich, SwitzerlandERIK A. KARLSSON � Department of Infectious Diseases, St Jude Children’s Research Hospital,

Memphis, TN, USA; Virology Unit, Institut Pasteur du Cambodge, Phnom Penh,Cambodia

YOSHIHIRO KAWAOKA � Department of Pathobiological Sciences, Influenza Research Institute,School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA;Division of Virology, Department of Microbiology and Immunology, University of Tokyo,Tokyo, Japan; International Research Center for Infectious Diseases, Institute of MedicalScience, University of Tokyo, Tokyo, Japan

CAROLIN KLEMM � Institute of Virology Muenster (IVM), Westfaelische Wilhelms-UniversityMuenster, Muenster, Germany

FLORIAN KRAMMER � Department of Microbiology, Icahn School of Medicine at Mount Sinai,New York, NY, USA

PSYLVIA LEGER � From CellNetworks Cluster of Excellence and Department of InfectiousDiseases, Virology, University Hospital Heidelberg, Heidelberg, Germany

MICHAEL M. C. LAI � China Medical University, Taichung, Taiwan; Research Center forEmerging Viruses, China Medical University Hospital, Taichung, Taiwan; Institute ofMolecular Biology, Academia Sinica, Taipei, Taiwan

SEEMA S. LAKDAWALA � Department of Microbiology and Molecular Genetics, University ofPittsburgh School of Medicine, Pittsburgh, PA, USA

CAROLINE LANZ � Institute of Medical Virology, University of Zurich, Zurich, SwitzerlandVALERIE LE SAGE � Department of Microbiology and Molecular Genetics, University of

Pittsburgh School of Medicine, Pittsburgh, PA, USAKELLY K. LEE � Department of Medicinal Chemistry, University of Washington, Seattle, WA,

USA; Biological Physics Structure and Design Program, University of Washington, Seattle,WA, USA

TIMOTHEE LIONNET � Department of Cell Biology, Institute for Systems Genetics, LangoneMedical Center, New York University, New York, NY, USA

xiv Contributors

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MARC LIPSITCH � Departments of Epidemiology and Immunology and Infectious Diseases,Center for Communicable Disease Dynamics, Harvard TH Chan School of Public Health,Boston, MA, USA

WEN-CHUN LIU � Department of Microbiology, Icahn School of Medicine at Mount Sinai,New York, NY, USA

BRANDI LIVINGSTON � Department of Infectious Diseases, St Jude Children’s ResearchHospital, Memphis, TN, USA

JASON S. LONG � Faculty of Medicine, Division of Infectious Disease, Imperial CollegeLondon, London, UK

PIERRE-YVES LOZACH � From CellNetworks Cluster of Excellence and Department ofInfectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany

STEPHAN LUDWIG � Institute of Virology Muenster (IVM), Westfaelische Wilhelms-UniversityMuenster, Muenster, Germany

TARONNA R. MAINES � Influenza Division, National Center for Immunization andRespiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

PATRICK MATTHIAS � Friedrich Miescher Institute for Biomedical Research (FMI), Basel,Switzerland

MAGALIE MAZELIER � From CellNetworks Cluster of Excellence and Department of InfectiousDiseases, Virology, University Hospital Heidelberg, Heidelberg, Germany

BERYL MAZEL-SANCHEZ � Faculty of Medicine, CMU, Department of Microbiology andMolecular Medicine, University of Geneva, Geneva, Switzerland

ALICE C. MCHARDY � Department for Computational Biology of Infection Research,Helmholtz Centre for Infection Research, Braunschweig, Germany; German Centre forInfection Research (DZIF), Braunschweig, Germany

ANDREW MEHLE � Department of Medical Microbiology and Immunology, University ofWisconsin-Madison, Madison, WI, USA

VICTORIA A. MELIOPOULOS � Department of Infectious Diseases, St Jude Children’s ResearchHospital, Memphis, TN, USA

YASUYUKI MIYAKE � Department of Virology, Nagoya University Graduate School of Medicine,Nagoya, Japan; School of Cellular and Molecular Medicine, University of Bristol, Bristol,UK

RAFFAEL NACHBAGAUER � Department of Microbiology, Icahn School of Medicine at MountSinai, New York, NY, USA

NADIA NAFFAKH � Departement de Virologie, Institut Pasteur, Unite de Genetique Moleculaire des Virus a ARN, Paris, France; CNRS, UMR3569, Paris, France; UniversiteParis Diderot, Sorbonne Paris Cite, Paris, France

GABRIELE NEUMANN � Department of Pathobiological Sciences, Influenza Research Institute,School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA

VAN KINH NGUYEN � Frankfurt Institute for Advanced Studies, Frankfurt am Main,Germany

SUSANNE REIMERING � Department for Computational Biology of Infection Research,Helmholtz Centre for Infection Research, Braunschweig, Germany

KATHERINE A. RICHARDS � Department of Microbiology and Immunology, David H. SmithCenter for Vaccine Biology and Immunology, University of Rochester Medical Center,Rochester, NY, USA

CRISTINA RISCO � Centro Nacional de Biotecnologia, CNB-CSIC, Cell Structure Lab,Madrid, Spain

Contributors xv

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INES ROMERO-BREY � Department of Infectious Diseases, Molecular Virology, University ofHeidelberg, Heidelberg, Germany

MARTIN SACHSE � Institut Pasteur, Ultrapole, Paris, FranceANDREA J. SANT � Department of Microbiology and Immunology, David H. Smith Center for

Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY,USA

CHANDRA SAVAGE � Animal Resource Center, St Jude Children’s Research Hospital, Memphis,TN, USA

IVO F. SBALZARINI � Faculty of Computer Science, TU Dresden, Dresden, Germany; MaxPlanck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

FLORIAN INGO SCHMIDT � Institute of Innate Immunity, University Hospital, University ofBonn, Bonn, Germany

MIRCO SCHMOLKE � Faculty of Medicine, CMU, Department of Microbiology and MolecularMedicine, University of Geneva, Geneva, Switzerland

STACEY SCHULTZ-CHERRY � Department of Infectious Diseases, St Jude Children’s ResearchHospital, Memphis, TN, USA

HOLLY A. SHELTON � The Pirbright Institute, Wiltshire, UK; The Pirbright Institute, Woking,UK

MONIKA STEGMANN � University of Oxford Advanced Proteomics Facility, Oxford, UKWEN-CHI SU � China Medical University, Taichung, Taiwan; Research Center for Emerging

Viruses, China Medical University Hospital, Taichung, TaiwanAARTJAN J. W. TE VELTHUIS � Division of Virology, Department of Pathology, University of

Cambridge, Cambridge, UKVY TRAN � Department of Medical Microbiology and Immunology, University of Wisconsin-

Madison, Madison, WI, USATERRENCE M. TUMPEY � Influenza Division, National Center for Immunization and

Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USAANDRE VAN KRUCHTEN � Institute of Virology Muenster (IVM), Westfaelische Wilhelms-

University Muenster, Muenster, GermanyPAUL VERKADE � School of Biochemistry, University of Bristol, Bristol, UK; Wolfson

Bioimaging Facility, University of Bristol, Bristol, UKCATHERINE J. WHITTAKER � The Pirbright Institute, Wiltshire, UKYOHEI YAMAUCHI � School of Cellular and Molecular Medicine, University of Bristol, Bristol,

UKEMILIO YANGUEZ � Institute of Medical Virology, University of Zurich, Zurich, Switzerland

xvi Contributors