Table Of ContentAntiviral Methods and Protocols
M E T H O D S IN M O L E C U L A R M E D I C I N E "
John M. Walker, SERIES EDITOR
42. Vaccine Adjuvants: Preparation Methods 24. Antiviral Methods and Protocols, edited
and Research Protocols, edited by Derek by Derek Kinchington and Raymond F.
T. O Hagan, 2000 Schinazi, 2000
41. Celiac Disease: Methods and Protocols, 23. Pepti dominie tics Protocols, edited by
edited by Michael N. Marsh, 2000 Wieslaw M. Kazmierski, 1999
40. Diagnostic and Therapeutic Antibodies, 22. Neurodegeneration Methods and
edited by Andrew J. T. George and Protocols, edited by Jean Harry
Catherine E, Urch, 2000 and Hugh A. Tilson, 1999
39, Ovarian Cancer: Methods and Protocols, 21. Adenovirus Methods and Protocols,
edited by John M. S Bartiett, 2000 edited by William S. M. Wold, 1998
38. Aging Methods and Protocols, edited by 20, Sexually Transmitted Diseases: Methods
Yvonne A. Barneti and Christopher P. and Protocols, edited by Rosanna Peeling
Burnett, 2000 and P. Frederick Sparling, 1999
37. Electrically Mediated Delivery of 19. Hepatitis C Protocols, edited by Johnson
Molecules to Cells, edited by MarkJ. Yin-Nam Lau, 1998
Jaroszeski, Richard Heller, and Richard I 8. Tissue Engineering, edited byJeffrey R.
Gilbert, 2000 Morgan and Martin L. Yartnush, 1999
36. Septic Shock Methods and Protocols, 17, HIV Protocols, edited by Nelson Michael
edited by Thomas J. Evans, 2000 and Jerome H. Kim. 1999
35. Gene Therapy of Cancer: Methods and 16. Clinical Applications of PCR, edited by
Protocols, edited by Wolfgang Walther Y. M. Dennis Lo, 1998
and Ulrike Stein, 2000 15. Molecular Bacteriology: Protocols and
34. Rotavirus Methods and Protocols, edited Clinical Applications, edited by Neil
by James Gray and Ulrich Dessetberger. Woodford and Alan Johnson, 1998
2000 14. Tumor Marker Protocols, edited by
33. Cytotnegalovirus Protocols, edited by Margaret Hanausek and Zbigniew
John Sinclair, 2000 Walaszek, 1998
32, Alzheimer's Disease: Methods and 13. Molecular Diagnosis of infectious
Protocols, edited by Nigel M. Hooper. Diseases, edited by Udo Reischl, 1998
1999 12. Diagnostic Virology Protocols, edited by
31. Hemostasis and Thrombosis Protocols: John R. Stephenson and Alan Warnes,
Methods in Molecular Medicine, edited 1998
by David J. Perry and K. John Pasi, 1999 1 1. Therapeutic Application of Ribozymes,
3D. Vascular Disease: Molecular Biology and edited by KevinJ. Scanlon, 1998
Gene Therapy Protocols, edited by 10. Herpes Simples Virus Protocols, edited
Andrew!-!. Baker. 1999 by S. Moira Brown and Alasdair
29. DNA Vaccines: Methods and Protocols, MacLean, 1998
edited by Douglas B. Lowrie and Robert 9. Leettn Methods and Protocols, edited by
Whaien. 1999 Jonathan M. Rhodes and Jeremy D.
2E. Cytotoxic Drug Resistance Mechanisms, Milton, 1998
edited by Robert Brown and Via Bbgvr- 8. Helicobacter pylori Protocols, edited by
Brown, 1999 Christopher L. Clayton and Harry L. T.
27 Clinical Applications of Capillary Mobley, 1997
Elect rophoresis, edited by Stephen M. 7. Gene Therapy Protocols, edited by Paul
Palfrey. 1999 D. Rohbins, 1997
26. Quantitative PCR Protocols, edited by 6. Molecular Diagnosis of Cancer, edited by
Bernd Koctianowski and Udo Reischl, 1999 Finbarr Cotter, 1996
25. Drug Targeting, edited by G, B. Francis 5. Molecular Diagnosis of Genetic Diseases,
and Cristina Delgado, 1999 edited by Rob Eiles, 1996
M E T H O D S I N M O L E C U L A R M E D I C I N E™
Antiviral Methods
and Protocols
Edited by
Derek Kinchington
St. Bartholomew's and the Royal London School of Medicine
and Dentristry, London, UK
and
Raymond F. Schinazi
Emory University, Decatur, GA
Humana Press Totowa, New Jersey
© 2000 Humana Press Inc.
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Cover illustration: Figure 1 from Chapter 11, “Antiviral Screening Assays for Human Herpesviruses 6 and
7,” by Jodi B. Black and Philip E. Pellett.
Cover design by Patricia F. Cleary.
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Main entry under title:
Methods in molecular medicine™.
Antiviral methods and protocols/edited by Derek Kinchington and Raymond F. Schinazi.
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ISBN 0-89603-561-1 (alk. paper)
1. Antiviral agents—Laboratory manuals. I. Kinchington, Derek. II. Schinazi, Raymond F. III.
Series: Methods in molecular medicine; 24.
[DNLM: 1.Hepatitis, Viral, Human—drug therapy. 2. Antiviral Agents—therapeutic use. 3.DNA
Virus Infections—drug therapy. 4. RNA Virus Infections—drug therapy. WC 536 A633 1999]
RM411.A575 1999
616.9'25061—dc21
DNLM/DLC
for Library of Congress 99-25099
CIP
Preface
This latest addition to the Methods in Molecular Medicineseries,Antivi-
ral Methods and Protocols, is opportune because there is an increasing inter-
est in discovering compounds that are effective against both chronic and acute
viral infections. A number of the methods described in the volume are unpub-
lished and their inclusion indicates the speed at which this field is moving.
This volume is not a review but each chapter contains methods validated by
the experts who have spent time in developing the protocols.
The hallmark of this series is the comprehensive way in which the meth-
ods are described, which includes a list of all the reagents needed for each
protocol. Of importance is the section on tips and pitfalls that the authors have
discovered while developing their protocols. The manual itself is designed to
be used by researchers in universities and industry who are familiar with a
range of biological techniques but who want to set up quickly a novel assay
system. We encourage a dialog between readers and authors, which may also
result in useful collaborations.
The first chapter gives an overview of the change in methodologies that
have been used for discovering active compounds. Safety considerations have
also been reviewed because there are some significant differences in contain-
ment requirements between the United States and the United Kingdom, for
example. Routine quality testing of cell cultures has also been highlighted
because there are many stories of results being invalidated when target cells
have been contaminated by micro-organisms, which, even at low levels, may
significantly alter antiviral endpoints.
For each virus group the chapters that describe the assays start with cell-
based systems and progress through to those of biochemical and molecular
design. Those associated with hepatitis viruses predominantly describe proto-
cols for HBV, but a relatively new method is described for a high throughput
screen for compounds against HVC. In the herpesvirus section, cell-based
assays are described for HSV 1 and 2, CMV, VZV, and EBV. A chapter
details the methodology for monitoring drug resistance in clinical herpesvi-
ruses, which is now mandatory in immunocompromised patients. The section
on HIV methods contains a wide variety of methods ranging from those evalu-
ating the activity of topical virucidal agents, an area that may prove to be
v
vi Preface
important in reducing the rates of infection, through cell-based assays and
molecular techniques used in identifying agents active against reverse tran-
scriptase, protease, and integrase. Of importance is the chapter describing the
methods that identify the phenotypic and genotypic properties of resistant clini-
cal isolates. This methodology undoubtedly will become more automated, but
in nondiagnostic settings it will be necessary to carry out such studies on a
small scale. The two assays on human papillomaviruses describe a relatively
simple method for identifying E6 and E7 proteins, responsible for cellular
immortalization, in cell lysates and a molecular approach for identifying the
E1 and E2 proteins by in vitro translation. Finally, some chapters describe the
virological and biochemical assays that measure the resistance of viruses against
one of the new generations of neuraminidase inhibitors.
Production of volumes such as this are always flawed by the passage of
time. However, we hope that it will provide a snapshot of the valuable and
exciting work that continues in this area of science and be a significant aid to
those involved in discovering new compounds.
Derek Kinchington
Raymond F. Schinazi
Contents
Preface .............................................................................................................v
List of Contributors..........................................................................................xi
1 Changing Methods for Discovering Antiviral Drugs
Philip S. Jones.......................................................................................1
2 Laboratory Safety Considerations
Paul M. Feorino, John D. Williamson,
and Raymond F. Schinazi...............................................................13
3 Routine Quality Control Testing of Cell Cultures:
Detection of Mycoplasma
Alison Stacey and Glyn Stacey..........................................................27
PART I. HEPATITIS VIRUS ..................................................................................41
4 Hep AD38 Assay: A High-Throughput, Cell-Based Screen
for the Evaluation of Compounds Against Hepatitis B Virus
Robert W. King and Stephanie K. Ladner.........................................43
5 Hepatitis B Virus Cell Culture Assays for Antiviral Activity
Karen Schmidt and Brent Korba........................................................51
6 A Rapid Microtiter Assay for Duck Hepatitis Virus Reverse
Transcriptase
Michael J. Otto and Richard A. Whitaker.........................................69
7 Assays for Measuring Hepadnaviral Supercoiled DNA
Stephen A. Locarnini and Gilda M. Civitico.....................................77
8 Assays for Hepatitis B Virus DNA- and RNA-Dependent DNA
Polymerase Activities
Timothy Shaw and Stephen A. Locarnini.........................................87
9 Development of a Hepatitis C Virus RNA Helicase High Throughput
Assay
Ann D. Kwong and Christine Risano................................................97
PART II. HERPESVIRUS.....................................................................................117
10 Determination of Viral Infectivity: HSV, VZV, and CMV
David R. Harper..................................................................................119
11 Antiviral Screening Assays for Human Herpesviruses 6 and 7
Jodi B. Black and Philip E. Pellett...................................................129
vii
viii Contents
12 Strategies for Evaluation of Antiviral Agents Against Epstein-Barr
Virus in Culture
Jung-Chung Lin.................................................................................139
13 Monitoring Drug Resistance for Herpesviruses
Graciela Andrei, Pierre Fiten, Erik De Clercq, Robert Snoeck,
and Ghislain Opdenakker.............................................................151
14 Herpesvirus Protease Assays
Peter Ertl, Linda Russell, and Jane Angier....................................171
PART III.HUMAN IMMUNODEFICIENCY VIRUS ........................................................183
15 Testing Compounds for Antiviral Activity in Cell Cultures Infected
with HIV
Donna Devine, Naomi Mathews, and Derek Kinchington.............185
16 Evaluation of Compounds That Prevent Reactivation of HIV-1
in OM-10.1 Cells
Salvatore T. Butera............................................................................201
17 Assessment of Activity of Topical Virucidal Agents
Timothy O’Connor.............................................................................207
18 Assays That Measure Selective Killing of Virus-Infected Cells
Sharon Randall...................................................................................213
19 Evaluating Clinical Isolates for Their Phenotypic and Genotypic
Resistance Against Anti-HIV Drugs
Anne-Mieke Vandamme, Myriam Witvrouw,
Christophe Pannecouque, Jan Balzarini, Kristel Van Laethem,
Jean-Claude Schmit, Jan Desmyter, and Erik De Clercq...........223
20 Line Probe Assay for Detecting Mutations in HIV-1 Reverse
Transcriptase
Lieven Stuyver, Ann Wyseur, Gonda Verpooten,
and Rudi Rossau...........................................................................259
21 RT-PCR for Amplification of Specific Fragments of HIV-1 Genome
Marie-Pierre de Béthune and Kurt Hertogs....................................269
22 Evaluation of Compounds Against Recombinant HIV Reverse
Transcriptase
Jan Balzarini.......................................................................................283
23 Ultrasensitive Detection of Reverse Transcriptase Activity
by the Amp-RT Assay: Applications to the Measurement
of Virus Loads and Phenotypic Drug Resistance
J. Gerardo García Lerma and Walid Heneine.................................291
24 Reverse Transcriptase Assay Based on Product Enhancement
for Assessing the Drug Susceptibility of Retroviruses
Jürg Böni and Jörg Schüpbach.......................................................301
Contents ix
25 Identifying and Characterizing HIV Protease Inhibitors
Eric S. Furfine....................................................................................313
26 Retroviral Integrase: A Novel Target in Antiviral Drug Development
and Basic In Vitro Assays with the Purified Enzyme
Abhijit Mazumder, Nouri Neamati, Sanjay Sunder,
Joshua Owen, and Yves Pommier..............................................327
PART IV. HUMAN PAPILLOMA VIRUS..................................................................339
27 Papillomavirus DNA Replication
Maureen C. Ferran and Alison A. McBride.....................................341
28 Quantitative Immunodetection of Viral Oncoprotein Levels
in Human Papillomavirus-Transformed Human Cells
Jeffery A. Johnson and J. David Gangemi.....................................361
PART V.INFLUENZA VIRUSES.............................................................................365
29 Biochemical Methods for the Characterization of Influenza Viruses
with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en
Richard C. Bethell, Graham J. Hart, Tony J. Blick,
Anjali Sahasrabudhe, and Jennifer L. McKimm-Breschkin.....367
30 Virological Methods for the Generation and Characterization
of Influenza Viruses with Reduced Sensitivity
to 4-Guanidino-Neu5Ac2en
Jennifer L. McKimm-Breschkin, Anjali Sahasrabudhe,
Tony J. Blick, Amanda J. Jowett, and Richard C. Bethell.......375
Index............................................................................................................383
Contributors
GRACIELA ANDREI • Rega Institute for Medical Research, Katholieke
Universiteit, Leuven, Belgium
JANE ANGIER • Glaxo-Wellcome Medicines Research Centre, Stevenage, UK
JAN BALZARINI • Rega Institute for Medical Research, Katholieke
Universiteit, Leuven, Belgium
RICHARD C. BETHELL • Discovery Biology Unit, Pfizer Ltd., Kent, UK
JODI B. BLACK • Centers for Disease Control and Prevention, Atlanta, GA
TONY J. BLICK • Biomolecular Research Institute, Parkville, Australia
JÜRG BÖNI • Swiss National Center for Retroviruses, Zurich, Switzerland
SALVATORE T. BUTERA • Retrovirus Disease Branch, Centers for Disease
Control and Prevention, Atlanta, GA
GILDA M. CIVITICO • Victorian Infectious Diseases Reference Laboratory,
Fairfield Hospital, Fairfield, UK
MARIE-PIERRE DE BÉTHUNE • Tobotec, Mechelen, Belgium
ERIC DE CLERQ • Rega Institute for Medical Research, Katholieke
Universiteit, Leuven, Belgium
JAN DESMYTER • Rega Institute for Medical Research, Katholieke
Universiteit, Leuven, Belgium
DONNA DEVINE • Celltech, Slough, UK
PETER ERTL • Glaxo-Wellcome Medicines Research Centre, Stevenage, UK
PAUL M. FEORINO • Department of Medical Microbiology, Section of
Virology and Cell Biology, Imperial College of Science, Technology, and
Medicine, London, UK
MAUREEN C. FERRAN • Laboratory of Viral Diseases, NAIAID, National
Institutes of Health, Bethesda, MD
PIERRE FITEN • Rega Institute for Medical Research, Katholieke Universiteit,
Leuven, Belgium
ERIC S. FURFINE • Molecular Biochemistry Department, Glaxo-Wellcome,
Inc., Research Triangle Park, NC
J. DAVID GANGEMI • Department of Microbiology and Molecular Medicine,
Clemson University, Clemson, SC
J. GERARDO GARCIA LERMA • Retrovirus Disease Branch, Centers for
Disease Control, Atlanta, GA
xi
