Hepatitis C Protocols

601 Pages·1999·41.75 MB·English

by  Michael P. Manns

Hepatitis C Protocols
METHODS IN MOLECULAR MEDICINE
TM
John M. Walker, SEIRES ROTIDE
.91 Hepatitis C Protocols, edited by Johnson Yiu-Nam Lau, 1998
.81 Tissue Engineering, edited by Jeffrey R. Morgan
and Martin L. Yarmush, 1998
.71 HIV Protocols, edited by Nelson Michael and Jerome H. Kim,
8991
.61 ClinicalApplications of PCR, edited by Y M. Dennis Lo, 1998
.51 Molecular Bacteriology: Protocols and Clinical Applications,
edited by Neil Woodford and Alan Johnson, 8991
.41 Tumor Marker Protocols, edited by Margaret Hanausek
and Zbigniew Walaszek, 1998
13. Molecular Diagnosis of Infectious Diseases, edited by
Udo Reischl, 1998
.21 Diagnostic Virology Protocols, edited by John R. Stephenson
and Alan Warnes, 1998
.11 Therapeutic Application of Ribozymes, edited by
Kevin J. Scanlon, 1998
.01 Herpes Simplex Virus Protocols, edited by .S Moira Brown
and Alasdair MacLean, 1998
9. Lectin Methods and Protocols, edited by
Jonathan M. Rhodes and Jeremy D. Milton, 1998
.8 Helicobacterpylori Protocols, edited by
Christopher L. Clayton and Harry L. .T Mobley, 1997
.7 Gene Therapy Protocols, edited by Paul D. Robbins, 1997
.6 Molecular Diagnosis of Cancer, edited by Finbarr Cotter, 1996
5. Molecular Diagnosis of Genetic Diseases, edited by
Rob Elles, 1996
.4 Vaccine Protocols, edited by Andrew Robinson,
Graham H. Farrar, and Christopher N. Wiblin, 1996
.3 Prion Diseases, edited by Harry .F Baker
and Rosalind M. Ridley, 1996
.2 Human Cell Culture Protocols, edited by Gareth E. Jones, 6991
.1 Antisense Therapeutics, edited by Sudhir Agrawal, 1996
Hepatitis C Protocols
Edited by
Johnson Yiu-Nam kau, DM
Current address: ICN Pharmaceuticals, Costa Mesa, AC
sdroweroF by
.T .lake Liang
Gary L. Davis
Michael Houghton, .Q L. Choo, and George Kuo
anamuH sserP ~
Totowa, weN Jersey
© 1998 Humana Press Inc.
999 Riverview Drive, Suite 208
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Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in molecular medicine",.
Hepatitis C protocols/edited by Johnson Y. N. Lau; forewords by Q. L. Choo, Gary L. Davis, Michael
Houghton, George Kuo, T. Jake Liang.
p. cm.--(Methods in molecular medicine; 19)
Includes index.
ISBN 0-89603-521-2 (alk. paper)
.1 Hepatitis C virus--Research--Methodology. .1 Lau, Johnson Y. N. (Johnson Yiu-Nam) .11
Series.
:MLND[ t. Hepatitis C-~tiagnosis. 2. Hepatitis C -genetics. 3. Hepatitis C Antibodies. 4. Hepa-
titis C Antigens. WC 536 H53363 1998]
QR201.H46H447 1998
616.3'623~c2 l
DNLM/DLC
for Library of Congress 98-24896
CIP
Foreword
The identification of hepatitis C virus by Michael Houghton and his col-
leagues at the Chiron Corporation nearly 01 years ago represented a technical
tour de force of modern molecular medicine. This breakthrough not only
unearthed the causative agent of non-A non-B hepatitis that had eluded the
best of scientists for more than 20 years, but also was symbolic of another
chapter in the changing paradigm of modern science and medicine. The revo-
lutionary concept of identifying a pathogen without actually visualizing or
detecting it will forever redefine the way we approach pathogenesis of dis-
eases whose cause is unknown. It is benefitting that this discovery treads on
the heels of the human immunodeficiency pandemic, and parallels the alarm-
ing emergence of various microbial pestilences in the world. Despite the rapid
advances in our understanding of the virus, there is much remaining to learn
about it. This book highlights some of the important areas yet to be unraveled.
As illustrated in this book, the marriage between basic science and clinical
medicine is essential in our quest for these unknowns. The last decade has
focused on the fundamentals of the virus; the next decade must underscore
translational research, bridging clinical medicine and basic science with real-
ization of fundamental knowledge to improve prevention, diagnosis, and treat-
ment of viral hepatitis C. Undoubtedly in the next millenium, we will develop
a much better strategy to combat this virus and have a much brighter outlook
on the disease for the millions of people infected with the virus.
T. Jake Liang
Liver Diseases Section
National Institute of Diabetes and Digestive
and Kidney Diseases
National Institutes of Health
Bethesda, MD
Foreword
After agreeing to write the foreword to this volume of highly technical
reviews of the basic science of hepatitis C virus (HCV) research, I found myself
wondering what ! had gotten myself into. Why would a clinician and clinical
researcher be asked to provide an introduction to such a "high tech" reference
book? However, as I read these excellent chapters and reflected on how I might use
the information in my own practice, the answer became clear.
Throughout most of our history, physicians have been comforters and, until
relatively recently, they offered little to the patient that the family could not pro-
vide. Thus, physicians had a very limited role in the practice of medicine, such as it
was at that time. The widespread marriage of science and medicine occurred later
than one might think. The discoveries of Pasteur, Koch, Ehrlich, and others begin-
ning in the 1860s fueled the public's view that science could be utilized for the
public good and offer at least the potential of a meaningful treatment for the
patient. But science was not something that the family and community could pro-
vide without the services of a physician. However, physicians did not achieve sig-
nificant credibility in society until they began to apply science in their training and
practice. Licensing of physicians did not start in earnest until 1877. Before this
time, there were few prerequisites to establishing a medical practice. Johns Hopkins,
the prototype of modem medical schools, was not established until 1893. The
application of science was directly responsible for the legitimization of medicine
in the United States; technology provided substance and value to the practice.
The impact of science on the practice of medicine is even more evident
today and becomes increasingly important each year. Yet it has become such an
integral part of our practices that many of us take the evolution for granted. It is
simply astounding to stop and consider the role that technology plays in the daily
office and hospital practices ofclinicans today. Little of the technology that we use
in our practices today was available 20, or even ,01 years ago. Diagnostic tests
based on molecular hybridization and amplification, magnetic resonance imaging,
cytokine therapies, modem synthetic and recombinant pharmaceuticals, and com-
puter-based informatics are just a few examples.
The impact of science is obvious in the diagnosis and treatment of viral dis-
eases. This is perhaps most apparent in the case of HIV, where major research
discoveries have occurred at an incredible pace. Recent developments have led to
vi
Foreword vii
the ability to diagnose infection when even low levels of viremia are present, to charac-
terize the virus, to design therapeutic tools directed at specific targets in the virus, and to
predict and monitor the outcome of treatment. The understanding of the hepatitis C virus
has also grown at a rapid pace since it was identified in 1989. However, despite the fact
that nearly 4 million people in the United States have this infection, research funding has
been limited and we have not yet come as far as we have with HIV. Political issues aside,
the chapters in this volume reflect the state-of-the-art in the research and understanding
of the virology of the hepatitis C virus. It is these tools and this knowledge that will allow
us, as scientists, to better understand the inner working of this agent and design better
ways to diagnose and treat the infection. This science of today will become the clinical
practice of tomorrow.
Several of the chapters review issues that are immediately valuable to gastro-
enterologists, hepatologists, infectious disease physicians, and those working in
laboratory medicine. They tell us where we are and where we are going in this
field. First, the chapters dealing with different methodologies and quality control
for the detection and quantitation of HCV are particularly timely. Qualitative and
quantitative tests have already become commercially available, even though most
lack standardization and we as clinicians are still struggling with how to use them.
These articles point out the benefits and limitations of the current methodology and
tell us how improved sensitivity and standardization can be accomplished. This is
essential before the assays can be widely applied in clinical practice. Second, the
discussion of viral heterogeneity is quite relevant. Viral heterogeneity will cer-
tainly be important in unraveling the interaction between the virus and the host.
Although information on heterogeniety is currently most useful in basic research
in immunodiagnostics and immuno-pathogenesis, epidemiologic surveys, and
designing virus-targeted treatments, it may also have a role in clinical practice. It is
now evident that viral genotype and the degree of viral heterogeneity have an im-
pact on the response to interferon therapy. Genotype assays are already commer-
cially available, but once again, the role of these tests in the clinic is not yet clear.
Finally, the chapters on the biological characterization of the virus, in vitro culture
techniques, and animal models provide us with a glimpse of the future of HCV
research, particularly with respect to therapeutics. As with HIV, future therapies
for HCV are likely to target the replicative machinery of the virus. Some of the
targets of these agents, e.g., proteases, can currently be isolated, inserted, and
expressed in artificial systems in order to screen potential new agents.
Today's high technology will become tomorrow's work horse. The incred-
ible science described in this book today will provide the basis for the design of the
tools that we will use in the diagnosis, management, and treatment of our patients
with HCV infection tomorrow.
Gary L. Davis, MD
ytisrevinU of Florida College of Medicine
,ellivseniaG FL
Foreword
Almost 01 years have now passed since the application of molecular clon-
ing methods resulted in the identification and characterization of the hepatitis
C virus. During this time, significant medical advances have been made in the
areas of immunodiagnosis and blood screening as well as methods determin-
ing viral load, which have facilitated the clinical management of patients. In
addition, valuable information has been obtained concerning genome organi-
zation, viral polyprotein processing, and viral heterogeneity and its role in dis-
ease progression and sensitivity to interferon treatment. However, many
important objectives still remain. Most importantly, given the high disease
burden associated globally with the hepatitis C virus and the continued high
incidence of infection in many countries, the development of more effective
antivirals as well as the introduction of vaccines represent key goals. In addi-
tion, a clearer understanding of those factors influencing the progression of
liver disease as well as nonhepatic diseases remains very important. Apart from
these key medical objectives, a number of fundamental areas of viral biology
also remain to be defined. These include viral replication strategies, virion struc-
ture, and mechanisms of virion assembly and release. In particular, since this
virus has a well-known propensity to cause chronic, persistent infections and
disease, the identification of those mechanisms responsible for such persis-
tence and evasion of the host immune response represents a substantial but
most intriguing challenge.
It is with these issues in mind that the current volume has been assembled
from many of the leading researchers in the field and describes state-of-the-art
methods and tools that are being used to address these important medical and
scientific questions. We can look forward, therefore, over the next
decades of hepatitis C research to acquiring a greater knowledge of this unique
RNA virus and its interactions with the host as well as, hopefully, the develop-
ment of specific therapeutics and vaccines.
Michael Houghton
Qui-Lim Choo
George Kuo
VIII
Preface
Since the report on the cloning of the hepatitis C virus (HCV) genome in
1989, an explosion of information on the epidemiology and natural history of the
virus load has evolved. HCV infection afflicts 170 million people worldwide.
Most people who acquire HCV progress to develop chronic infection and hepati-
tis; a proportion of them will have their liver disease advance to liver cirrhosis
and end-stage liver disease in one to three decades. At present, HCV-related end-
stage liver disease is a major indication for liver transplantation. The health
impact of HCV worldwide is enormous.
With the use of molecular biology, immunology, and cell biology techniques,
important information has been gathered on the virologic and immunologic
aspects of HCV infection. Certainly, much more data are needed to further our
knowledge on the clinical, virologic, and immunopathogenic aspects of HCV
infection. This information may provide the foundation for the development of
new therapies and better therapeutic strategies for patients with HCV infection.
Hepatitis C Protocols contains a collection of research techniques used for the
study of HCV. The authors were chosen based on their expertise in their respec-
tive areas. The chapters are written in a fashion that allows investigators to use
them as manuals. A few reviews were included in some specialized areas. There
is also a chapter that discusses the use of liver transplant recipients with HCV
infection as a model for the study ofpathogenesis. It is our hope that researchers
will find this manual useful in their pursuit of HCV research, and clinical investi-
gators will find this book helpful for their understanding of the principles
involved in laboratory research on HCV.
! would like to take this opportunity to extend my gratitude to my teachers
and friends: Professor P. C. Wu, Professor C. L. Lai, Dr. H. J. Lin, Professor
Roger Williams, Dr. Graeme J. M. Alexander, Professor James E. McGuigan,
Professor Philip P. Toskes, Professor Gerold Schiebler, Professor Richard W.
Moyer, Professor Gary L. Davis, and Professor Sum Lee. Without their guidance
and support, I would not have the opportunity of presenting this highly practical
manual to you today.
Johnson Yiu-Nam Lau
ix