Antiviral Methods and Protocols

Methods in Molecular Biology 1030 Edwin Yunhao Gong Editor Antiviral Methods and Protocols Second Edition M M B ™ ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat ? eld, Hertfordshire, AL10 9AB, UK For further volumes: h ttp://www.springer.com/series/7651 Antiviral Methods and Protocols Second Edition Edited by Edwin Yunhao Gong Janssen Infectious Diseases-Diagnostics BVBA, Johnson & Johnson Corporation, Beerse, Belgium Editor Edwin Yunhao Gong Janssen Infectious Diseases-Diagnostics BVBA Johnson & Johnson Corporation Beerse, Belgium ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-483-8 ISBN 978-1-62703-484-5 (eBook) DOI 10.1007/978-1-62703-484-5 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2013940287 © Springer Science+Business Media, LLC 2 013 This work is subject to copyright. 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In the last well, only one virion is present at that drug concentration (the test compound at that concentration totally inhibits virus replication), and one can count the number of white dots to calculate the drug effect. This image represents duplicate wells for each drug test concentration. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Preface Viruses are the leading cause of disease and death worldwide, and antiviral drugs are one of the most effective ways, in addition to vaccines, to control and manage viral infections. Although antiviral drugs have been successfully developed for some viral diseases, there remains a clear unmet medical need to develop novel antiviral agents for the control and management of many viruses that currently have no or limited treatment options as well as a need to overcome the limitations associated with the existing antiviral drugs, such as adverse effects and emergence of drug-resistant mutations. The ? rst edition of A ntiviral Methods and Protocols was published in the M ethods in Molecular Medicine series (discontinued some years ago) in 2000. This new edition, pub- lished in the Methods in Molecular Biology series, re? ects the major technical advances for the last 13 years in antiviral research and discovery. Although it is ? agged as the second edition, all chapters are completely new, and none of the chapters is simply updated from the previous edition. This edition focuses on many important human viruses that cause major problems in public health, such as human immunode? ciency virus type 1 (HIV-1), hepatitis viruses (hepatitis B and C viruses), herpes viruses, and in? uenza virus. Many new chapters describing antiviral methods and protocols against respiratory viruses, such as human respiratory syncytial virus (4 chapters), and some important emerging viruses, such as dengue virus (7 chapters), West Nile virus (1 chapter), and chikungunya virus (2 chapters), are included. In addition, as the in vivo evaluation of antiviral ef? cacy and modes of action using animal models is very important to antiviral drug discovery and development, several chapters describing animal models for evaluation of antiviral agents in vivo are also presented. Consistent with the approach of the M ethods in Molecular Biology series, each chapter contains detailed cutting-edge research techniques that are currently used in the ? eld of antiviral research. Many methods and protocols presented in this edition are fully validated, high-throughput antiviral assays that can be used for screening compound libraries in phar- maceutical companies and research institutions. Overall, this new edition of Antiviral Methods and Protocols will serve as a laboratory reference for pharmaceutical and academic biologists, medicinal chemists, and pharmacologists as well as for virologists in the ? eld of antiviral research and drug discovery. I would like to thank Professor John Walker, chief editor of the series, for his advice. I also wish to express my gratitude to the editors from Springer, especially Patrick Marton, David Casey, Anne Meagher and Paul Wehn, for making this new edition possible, and to Sundaramoorthy Balasubramanian, Project Manager at SPi Global, India, for his valuable contribution to edit this book, and to all the chapter authors for their valuable contribu- tions to this book. Beerse, Belgium Edwin Yunhao Gong v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi PART I HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) 1 A Fluorescence-Based High-Throughput Screening Assay to Identify HIV-1 Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Peggy Geluykens, Koen Van Acker, Johan Vingerhoets, Christel Van den Eynde, Marnix Van Loock, and Géry Dams 2 A Homogeneous Time-Resolved Fluorescence Assay to Identify Inhibitors of HIV-1 Fusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Liesbet Smeulders, Lieve Bunkens, Inge Vereycken, Koen Van Acker, Pascale Holemans, Emmanuel Gustin, Marnix Van Loock, and Géry Dams 3 Identification of HIV-1 Reverse Transcriptase Inhibitors Using a Scintillation Proximity Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Bertrand Van Schoubroeck, Marnix Van Loock, Tania Ivens, Pascale Dehertogh, Dirk Jochmans, and Géry Dams 4 Biochemical Screening Assays to Identify HIV-1 Integrase Inhibitors . . . . . . . 25 Marleen Clynhens, Alexandra Smets, Inge Vereycken, Marnix Van Loock, Reginald Clayton, Geert Meersseman, and Olivia Goethals 5 HIV-1 Genotyping of the Protease-Reverse Transcriptase and Integrase Genes to Detect Mutations That Confer Antiretroviral Resistance . . . . . . . . . . . . . . . 37 Peter Van den Eede, Liesbeth Van Wesenbeeck, Yvan Verlinden, Maxim Feyaerts, Veerle Smits, Ann Verheyen, Leen Vanhooren, Alain Deloof, Jorge Villacian, and Theresa Pattery PART II HEPATITIS VIRUSES 6 In Vitro Kinetic Profiling of Hepatitis C Virus NS3 Protease Inhibitors by Progress Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . 59 Rumin Zhang and William T. Windsor 7 A Novel Hepatitis C Virus NS5B Polymerase Assay of De Novo Initiated RNA Synthesis Directed from a Heteropolymeric RNA Template . . . . . . . . . . 81 Eric Ferrari and Hsueh-Cheng Huang 8 Selecting and Characterizing Drug-Resistant Hepatitis C Virus Replicon. . . . . 93 Inge Vliegen, Leen Delang, and Johan Neyts vii viii Contents 9 A Cellular Replicon-Based Phenotyping Assay to Determine Susceptibility of Hepatitis C Virus Clinical Isolates to NS3/4A Protease Inhibitors. . . . . . . . 105 Leen Vijgen, Jannick Verbeeck, Barbara Van Kerckhove, Jan Martin Berke, Diana Koletzki, Gregory Fanning, and Oliver Lenz 10 Expression and Purification of Hepatitis C Virus Protease from Clinical Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Liesbet van der Helm 11 Surface Plasmon Resonance as a Tool to Select Potent Drug Candidates for Hepatitis C Virus NS5B Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Erna Cleiren, Benoit Devogelaere, and Katleen Fierens 12 Amplification and Sequencing of the Hepatitis C Virus NS3/4A Protease and the NS5B Polymerase Regions for Genotypic Resistance Detection of Clinical Isolates of Subtypes 1a and 1b . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Diana Koletzki, Theresa Pattery, Bart Fevery, Leen Vanhooren, and Lieven J. Stuyver 13 A Southern Blot Assay for Detection of Hepatitis B Virus Covalently Closed Circular DNA from Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Dawei Cai, Hui Nie, Ran Yan, Ju-Tao Guo, Timothy M. Block, and Haitao Guo 14 In Vitro Phenotyping of Recombinant Hepatitis B Virus Containing the Polymerase/Reverse Transcriptase Gene from Clinical Isolates. . . . . . . . . . 163 Yang Liu and Kathryn M. Kitrinos PART III FLAVIVIRUSES 15 Cell-Based Antiviral Assays for Screening and Profiling Inhibitors Against Dengue Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Edwin Yunhao Gong, Marleen Clynhens, Tania Ivens, Pedro Lory, Kenny Simmen, and Guenter Kraus 16 A Duplex Real-Time RT-PCR Assay for Profiling Inhibitors of Four Dengue Serotypes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Edwin Yunhao Gong, Alexandra Smets, Nick Verheyen, Marleen Clynhens, Emmanuel Gustin, Pedro Lory, and Guenter Kraus 17 High-Throughput Screening Using Dengue Virus Reporter Genomes . . . . . . 205 Wolfgang Fischl and Ralf Bartenschlager 18 Fluorimetric and HPLC-Based Dengue Virus Protease Assays Using a FRET Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Christoph Nitsche and Christian D. Klein 19 Expression and Purification of Dengue Virus NS5 Polymerase and Development of a High-Throughput Enzymatic Assay for Screening Inhibitors of Dengue Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 Edwin Yunhao Gong, Hannah Kenens, Tania Ivens, Koen Dockx, Katrien Vermeiren, Geneviève Vandercruyssen, Benoit Devogelaere, Pedro Lory, and Guenter Kraus Contents ix 20 Detection and Quantification of Flavivirus NS5 Methyl-T ransferase Activities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Siew Pheng Lim, Christophe Bodenreider, and Pei-Yong Shi 21 Testing Antiviral Compounds in a Dengue Mouse Model . . . . . . . . . . . . . . . . 269 Wouter Schul, Andy Yip, and Pei-Yong Shi 22 Construction of Self-Replicating Subgenomic West Nile Virus Replicons for Screening Antiviral Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 Sofia L. Alcaraz-Estrada, Erin Donohue Reichert, and Radhakrishnan Padmanabhan PART IV HERPES VIRUSES 23 Methods for Screening and Profiling Inhibitors of Herpes Simplex Viruses . . . 303 Edwin Yunhao Gong 24 In Vivo Evaluation of Antiviral Efficacy Against Genital Herpes Using Mouse and Guinea Pig Models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Frances Valencia, Ronald L. Veselenak, and Nigel Bourne 25 A Fluorescence-Based High-Throughput Screening Assay for Identifying Human Cytomegalovirus Inhibitors. . . . . . . . . . . . . . . . . . . . . 327 Christel Van den Eynde, Ellen Van Damme, Tania Ivens, and Edwin Yunhao Gong PART V RESPIRATORY VIRUSES 26 A Fluorescence-Based High-Throughput Antiviral Compound Screening Assay Against Respiratory Syncytial Virus . . . . . . . . . . . . . . . . . . . . 337 Leen Kwanten, Ben De Clerck, and Dirk Roymans 27 Screening and Evaluation of Anti-respiratory Syncytial Virus Compounds in Cultured Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Anna Lundin, Tomas Bergström, and Edward Trybala 28 Evaluation of Antiviral Efficacy Against Human Respiratory Syncytial Virus Using Cotton Rat and Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Joke Van den Berg, Leen Kwanten, and Dirk Roymans 29 In Vivo Evaluation of Antiviral Compounds on Respiratory Syncytial Virus Using a Juvenile Vervet Monkey (Chlorocebus pygerythrus) Infection Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Lieselot Houspie, Hans Stevens, Maina Ngotho, Els Keyaerts, Gabriela Ispas, René Verloes, Marc Van Ranst, and Piet Maes 30 Methods to Determine Mechanism of Action of Anti-i nfluenza Inhibitors. . . . 383 Angela Luttick, Stephanie Hamilton, and Simon P. Tucker 31 Methods for Evaluation of Antiviral Efficacy Against Influenza Virus Infections in Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 Donald F. Smee and Dale L. Barnard