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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Hepatitis C Viruses: Genomes and Molecular Biology.

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Introduction

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When the term emerging infectious diseases is loosely applied, then chronic hepatitis C is recognized as one of the most important new diseases afflicting man. The term paradigm is useful when describing this disease, since the discovery, diagnosis and initial treatments of hepatitis C virus infection are all perfect examples of the increasing impact molecular biology is currently having on disease management throughout the globe. The discovery of HCV in the late 1980s occurred without the aid of conventional tissue culture or classical virological methods other than the essential reliance of the chimpanzee model for propagation and initial definition of the infectious agent as an enveloped RNA virus. Reverse transcription and PCR amplification of a subgenomic fraction of the HCV genome not only lead to the initial genetic characterization of HCV as a putative member of the Pestivirus family. It also paved the way for development of the first diagnostic test, an enzyme linked immunoassay that utilized recombinant HCV protein fragments to capture HCV antibodies from patient serum and thus provide serological evidence of infection. This critical step was a major accomplishment for molecular medicine since it provided the first opportunity to positively identify individuals with this highly prevalent yet clinically silent disease.

Even though it was well established from epidemiological studies that non-A, non-B (NANB) hepatitis was efficiently transmitted by blood transfusion, and that screening blood products for anti-hepatitis B core antibody and ALT significantly reduced the incidence of post-transfusion NANB hepatitis, development of the first generation HCV antibody screening assay had an impact far greater than many medical scientists in the field had anticipated. Results of early studies indicated that up to 10% of all units of blood transfused in the U.S. prior to the discovery of HCV had lead to transmission of the infectious agent to recipients, accounting for the vast majority of cases of post-transfusion NANB hepatitis, a fact that may have been as surprising as it was fortuitous. However, this was not the whole iceberg; world wide population-based studies revealed a global seroprevalence of well over 100 million individuals, with current estimates being frequently quoted as 170 million HCV infections today. Initial studies reported that approximately 40% of HCV infections in the U.S. were “community acquired”, with no known risk factors for acquisition. Subsequent epidemiological studies have suggested that many of these cases were actually associated with the most important risk factor for HCV acquisition today, namely intravenous drug use. Such studies have also led to the identification of other previously unknown risk factors, so the term community acquired hepatitis C is no longer in vogue. Thus, cloning of a portion of the HCV RNA genome and development of an effective diagnostic test for HCV antibodies unveiled the insidious disease that is so heavily researched today; this would never have occurred without the use of molecular tools.

A second major accomplishment of molecular medicine was development and standardization of tests that efficiently detect and characterize HCV nucleic acids in patient blood. Use of the reverse transcription polymerase chain reaction (RT-PCR) assay in epidemiological studies revealed that of all patients acquiring post-transfusion hepatitis C, over 80% became chronically infected with persistent viremia for decades if not for life. Being able to define HCV viremia in a patient with a risk factor for infection or an asymptomatic individual with serological evidence of exposure to HCV has become a hallmark tool for hepatitis C management in the clinical setting from several perspectives. Confirmation of viremia equals confirmation of active disease. Since most patients with hepatitis C are asymptomatic, a large percentage has normal ALT levels in the blood, and up to 20% of infections spontaneously resolve, knowing HCV infection status is critical for defining subsequent management. Determining whether the disease is mild, moderate or severe still requires a liver biopsy unless the patient has clinical evidence of liver decompensation.

The ability to detect, quantify and genetically characterize HCV RNA in patients had an irreplaceable impact on our understanding of hepatitis C disease long before the molecular studies described in the following chapters began to unravel the complex mysteries associated with this truly unique virus-host relationship. Studies of HCV molecular epidemiology indicated that six distinct genotypes have evolved over centuries throughout the world. From clinical studies we learned that HCV persistently replicates in humans for decades, maintaining remarkably constant serum titers that often exceed 1 million viral genomes per milliliter of serum. Pharmacodynamic studies indicated that the HCV production rate exceeds one trillion new virions per day in the face of active immune responses, which is remarkable because this level of virus production is often without overt detriment to the infected host. However, HCV continuously evolves within the host as a pool of genetic variants termed viral quasispecies, presumably as an adaptation to host pressure. How host pressures shape these viral quasispecies without causing significant perturbations in HCV RNA titers is also a mystery, as is the mechanistic relationship between host pressure, viral evolution and disease progression. Again, development of HCV nucleic acid-based assays was an essential contribution of molecular medicine in terms of furthering our understanding of the fundamental virology of HCV infection in humans, and defining the mysteries of viral pathogenesis that may never be approachable for study by in vitro models.

An additional point to touch on with regards to the contribution of molecular medicine to hepatitis C disease pertains to recombinant human interferon, a drug that was engineered from the human genome many decades ago as new wonder drug for the treatment of cancer. Although the utility of interferon in treating cancer should not be understated, it was the astute observations of clinical investigators in the pre-hepatitis C era that interferon lead to normalization of serum ALT levels in about 50% of subjects treated for NANB hepatitis, a remarkable finding even before the discovery of the etiological agent. Long-term studies indicated that although many of these patients relapsed after completion of interferon treatment, several patients continue to have durable and sustained responses with long-term clearance of HCV RNA from blood. Thus, it is not unreasonable to assume that exogenous interferon alone can lead to a cure of this highly efficient virus from the infected host. We now know that HCV genotype and viral load are independent predictors of response to interferon, and other viral factors have also been implicated in influencing treatment outcome.

Molecular testing also played an essential role in the optimization of therapy for hepatitis C. Sentinel studies of HCV RNA dynamics following acute interferon dosing not only revealed a rapid dose response effect that was not previously recognized, they also lead directly to the understanding that thrice weekly dosing of interferon was not optimal. At the same time came the serendipitous discovery that the more traditional antiviral agent ribavirin potentiates long-term response to interferon by greatly reducing post-therapy relapse. The end result: the development and licensing of a much more effective combination therapy for hepatitis C, including a pegylated interferon compound with extended half-life, plus ribavirin. Today combination therapy gives clinicians the ability to achieve sustained clearance of HCV and subsequent improvements in liver disease in over 50% of their treated patients. This is an outstanding accomplishment when one considers the relatively poor prognosis for durable sustained remissions in other insidious chronic diseases in humans. Optimization of therapy through traditional clinical trial research without the use of molecular analysis of HCV RNA may never have lead to such a dramatic improvement in hepatitis C treatment outcome. It is at this point that present research takes over with the clear goal of developing new therapies capable of improving long-term response rates in those patients who remain resistant to the best available conventional therapies.

It is this problem combined with the perplexing molecular clinical biology of chronic hepatitis C that has fueled the enormous surge in basic research that is the topic of the following chapters. Over a decade of research in the chimpanzee model has provided much relevant information with respect to HCV infection and immunity in the host, and small animal models have been developed which should become important tools for further characterizing HCV biology in the near future. Aside from the ever growing body of knowledge related to basic HCV virology, several key interactions between HCV proteins and the host cell regulatory pathways have now been described, including some which have exciting potential in terms of designing new approaches to therapy. Development of the HCV replicon provided for the first time a highly efficient system for studying HCV protein function during viral replication and the effects of experimental drugs on specific aspects of the viral life cycle. However, one important limitation of the HCV replicon is the fact that infectious virus is not produced; thus it falls short of the ideal. Although the lack of a robust tissue culture system has been a major impediment to HCV research in the past, productive infection of culture cells by a unique HCV isolate has very recently been reported. It is the hope of investigators that this system will now provide the opportunity to study for the first time several essential steps in the HCV life cycle. However, it is also essential that more flexible and even more robust infection models be continuously developed.

In summary, both the intensity and breadth of HCV research are growing at a remarkable pace, and exciting new discoveries are becoming almost commonplace in the literature. The following chapters were written to provide in-depth reviews of several of the most critical areas of HCV molecular research today. However, it is the goal of this Introduction to remind readers and investigators that hepatitis C disease is highly complex and very likely involves multiple poorly defined viral-host interactions that still cannot be and may never be recapitulated in any animal model or in vitro system. For this reason, molecular research into other Pestivirus animal disease models should be pursued with renewed vigor. Finally, continuous research in the human disease model is essential for defining the most important questions for in vitro study, as is the continuous development of new molecular tools for dissecting the intriguing biopathogenesis of chronic hepatitis C in man. Just as the progress on this disease to date has been phenomenal, so too will be the future progress in furthering our understanding of HCV infection, replication, and molecular biology, and in improving the treatment of hepatitis C. The present state of progress and unanswered questions currently facing molecular investigators are both very well summarized in the following chapters. As for molecular medicine, hepatitis C may long remain the essential paradigm of how new technologies can impact in a very real manner existing problems afflicting man.

Copyright © 2006, Horizon Bioscience.
Bookshelf ID: NBK1634

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