ILAR Journal Online, Volume 42(2) 2001: Animal Models of Hepatitis

Online Issues

<< All Back-issues

<< This Issue's Table of Contents

ILAR Journal V42(2) 2001
Animal Models of Hepatitis

View/Download Brochure (PDF):
Hepatitis C

Hepatitis C: A Brief Clinical Overview
Doris B. Strader and Leonard B. Seeff

Doris B. Strader, M.D., is Assistant Chief of Gastroenterology/Hepatology at the Veterans Affairs Medical Center, Washington, D.C. Leonard B. Seeff, M.D., is a Senior Scientist for Hepatitis C Research at the National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland.

Abstract

Hepatitis C has emerged as an important public health problem that has affected 3.9 million Americans and 170 million people worldwide and is currently the most common indication for orthotopic liver transplantation. The disease, characterized by asymptomatic onset, is often discovered incidentally through blood tests obtained during routine physical examination or before blood donation. Spontaneous recovery occurs in about 20% of patients. Among those who remain chronically infected, an equal percentage progress to cirrhosis within 20 yr, have stable nonprogressive disease, or progress more slowly over 40 to 60 yr. At present, combination therapy with interferon plus ribavirin is the treatment of choice for hepatitis C-infected patients identified as appropriate candidates for therapy. Unfortunately, sustained response rates are only modest, with a lesser response among African Americans, and treatment is associated with a number of side effects. Research studies attempting to improve the response to current therapy, to identify alternative treatments or treatment strategies, and to develop an effective vaccine are ongoing and will hopefully provide us with the ability to better understand and manage hepatitis C virus.

Key Words: cirrhosis; combination therapy; fibrosis; genotype; hepatitis C; hepatocellular carcinoma; interferon; quasispecies

Introduction

Hepatitis C is one of the most common causes of chronic liver disease in the United States, where it has emerged as a significant public health problem in recent years. Approximately 3.9 million Americans have antibody to hepatitis C virus (anti-HCV¹), and 2.7 million (74%) of those individuals have the virus HCV RNA in their serum, which indicates ongoing infection (Alter 1995a). An estimated 20% of persons with chronic hepatitis C virus (HCV¹) infection develop cirrhosis over the course of 25 yr, and some will progress to end-stage liver disease and hepatocellular carcinoma (HCC¹) (Alter et al. 1992). In the United States, HCV causes an estimated 10,000 deaths annually and is the most common indication for orthotopic liver transplantation (Pessoa and Wright 1997).

Hepatitis C was initially referred to as non-A, non-B hepatitis and was believed to be a relatively mild disease because the clinical manifestations noted among those infected with hepatitis A or B were rarely observed (Dienstag 1983). In the late 1970s, reports of infection of primates after exposure to HCV-infected human serum followed by a subsequent report of HCV infection after a needlestick injury resulted in the identification of hepatitis C as a distinct and transmissible hepatitis virus (Seeff 1991; Tabor et al. 1978, 1981). Early epidemiological studies led to the observation that one half of the individuals infected with HCV had persistently elevated aminotransferase levels despite the absence of symptoms, 20% of patients had histological findings on liver biopsy that included moderate to marked fibrosis and cirrhosis, and 75% of persons failed to resolve their infection (Alter 1989; Alter et al. 1999). An association between HCV and HCC was subsequently observed (Gilliam et al. 1984). The asymptomatic nature of acute HCV infection and the high percentage of persistence to chronic infection accompanied by the potential to progress to end-stage liver disease or hepatocellular carcinoma indicate that HCV is a disease of considerable importance.

Virology

Hepatitis C is a member of the Flaviviridae family of viruses, which includes the pestiviruses, flaviviruses, and hepacivirus (Rice 1996). It is a small, enveloped RNA virus with a positive-sense single-stranded genome of approximately 9600 nucleotides. The viral nucleocapsid consists of the core protein and the viral genomic RNA enveloped by a bilayer of two glycoproteins E1 and E2; however, the detailed structure of the assembled virion is unknown.

The HCV genome contains a single open reading frame (ORF¹) flanked at each end by untranslated regions (Major and Feinstone 1997). Translation of the ORF produces a polyprotein precursor, which is cleaved into at least 10 proteins. The three structural proteins are cleaved within the endoplasmic reticulum (ER¹) (Hijikata et al. 1991; Reed and Rice 1998; Rice 1996). The core protein is highly conserved, contains 191 amino acids, and has a molecular mass of 21 kD (Bukh 1994; Forns and Bukh 1999; Hijikata et al. 1991). Binding of the core to the 5' untranslated region may be important for encapsidation and particle assembly (Hwang et al. 1995). In addition, the C terminus of the core contains a protein that acts to transport E1 into the ER. In vitro models of oncogenesis suggest that HCV core protein can react with cellular proto-oncogenes and may be responsible for the development of steatohepatitis and hepatocellular carcinoma (Ray et al. 1996).

The E1 and E2 envelope proteins are N-glycosylated proteins with molecular masses of 31 kD and 70 kD, respectively (Reed and Rice 1998; Rice 1996). Two forms of the E2 protein exist; E2 and E2-p7 (Lin et al. 1994; Mizushima et al. 1994). The function of the small, hydrophobic p7 protein, which is incompletely cleaved from E2, is unknown. The E1 and E2 proteins contain C-terminal hydrophobic domains and are thought to function as heterodimers that are stabilized within the ER (Dubuisson et al. 1994). It has been suggested that retention of HCV E1 and E2 glycoproteins in the ER is indicative of HCV budding and subsequent enveloping within this compartment (Rice 1996). The E1 and E2 proteins of different HCV isolates are highly heterogeneous. The N-terminus of the E2 protein contains two hypervariable regions, one of which may represent a neutralization domain. Although the mechanism by which HCV gains entry into the hepatocyte is unknown, it has been postulated that E1 and E2 proteins may be involved in receptor binding and fusion (Forns and Bukh 1999).

Seven nonstructural (NS¹) proteins are encoded by the ORF of HCV. The NS2 gene encodes a hydrophobic protein, which forms a zinc-dependent, cysteine protease (together with the N-terminal third of the NS3 protein) (Hijikata et al. 1993; Reed and Rice 1998). This protease is responsible for the cleavage at the NS2/3 junction. The NS3 gene encodes a 70-kD protein with many functions. The N-terminal of NS3 has serine-protease activity, which cleaves NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions (De Francesco et al. 1998; Hijikata et al. 1993; Honda et al. 1994). NS4A functions as an essential cofactor in the processing of NS3/4A and NS4B/5A and forms a stable complex with the serine-protease of NS3 (Reed and Rice 1998). The three-dimensional crystal structure of the NS3 protease domain and the NS3 protease/NS4A cofactor complex have been described recently (Kim et al. 1996; Love et al. 1996). The enzyme contains a zinc atom and assumes a chymotrypsin-like folding pattern. It was hoped that understanding the structure might make it possible to develop specific inhibitors of viral replication, but this development has proved difficult. An RNA helicase and NTPase are located at the C-terminus of the NS3 protein. The helicase is responsible for unwinding the RNA during replication using the energy generated by NTPase hydrolysis (Susuki et al. 1993; Tai et al. 1996).

The function of the NS4B and NS5A proteins is unknown, although it has been postulated that the phosphorylated NS5A may regulate protein-protein and protein-nucleic acid interactions (Kaneko et al. 1994). A region of the NS5A protein containing amino acids 2209 to 2248 has been suggested as the area determining the host IFN-mediated antiviral response (Enomoto et al. 1995). Mutations in this area correlate with sensitivity of HCV genotype 1b viruses to interferon (IFN¹) therapy. NS5B is a 68-kD RNA-dependent RNA polymerase. It is able to bind and copy different RNA templates without use of an exogenous priming mechanism; rather, it uses the 3' nucleotide of the template to prime synthesis of the complementary strand. It is believed that the HCV RNA polymerase might be a target for inhibition of viral replication (Bartholomeusz et al. 1994).

Genotype

HCV exists as multiple genotypes that reflect differences as high as 35% (Okamoto et al. 1992). Sequence analysis of the E1 gene and the core gene identified six major genotypes with 12 subtypes (Bukh et al. 1993). Analysis of the NS5B gene has identified the same six major genotypes and 11 subtypes; however, analyses of the full-length ORF confirmed the original classification (Bukh et al. 1998; Simmonds et al. 1993). By analyzing isolates from around the world, more than 100 subtypes have been described within genotypes 1, 2, 3, 4, and 6.

Although all genotypes can be found worldwide, clear geographic distributions of genotype exist (Bukh et al. 1995; Forns and Bukh 1998; Maertens and Stuyver 1997). Genotypes 1a, 1b, 2a, 2b, 2c, and 3a account for >90% of HCV infections in North and South America, Europe, Russia, China, Japan, Australia, and New Zealand. Genotypes 1a and 1b are responsible for 80% (each 40%) of the HCV isolates in the United States. Genotype 1b accounts for approximately two thirds of HCV isolates in Southern Europe, China, Russia, and Japan. Genotype non-3a subtypes are highly prevalent in Nepal, Bangladesh, India, and Pakistan. Genotype 4a is most prevalent in Egypt. Genotype 5a is isolated in 50% of HCV-infected persons in South Africa. Genotype 6 is found in Southeast Asia.

Quasispecies

By definition, the term "quasispecies" refers to the genetic heterogeneity within an individual that results from infection with a heterogeneous virus population or the accumulation of mutations that occur over the course of infection (Forns and Bukh 1999). Because all viral species contained within a viral inoculum may not have the capacity to cause infection equally, a new dominant species may result from a mutation or from the selection of a pre-existing minor viral species (Domingo et al. 1994; Eigen 1996). Sequence analysis of HCV in cases infected 8 to 13 yr suggests that HCV has a relatively high mutation rate---1.44 to 1.92 ´ 10^-3 nucleotide substitutions/genome site/yr (Ogata et al. 1991; Okamoto et al. 1992). Farci and colleagues (1996) performed an extensive sequence analysis of the viral genome population in the acute-phase plasma of a patient with post-transfusion HCV. A total of 104 molecular clones were sequenced and at least 19 viral strains were present simultaneously, the most prevalent making up 69% of the population. Analysis of subgenomic regions revealed that heterogeneity existed throughout the entire genome, and sequence analysis of 18 full-length HCV ORFs failed to identify any identical strains. Similar results were obtained by separate investigators who analyzed genome sequences in chimpanzees experimentally infected with HCV (Yanagi et al. 1998). These data suggest that studying quasispecies within a short subgenomic region may be problematic because heterogeneity exists throughout the genome. In addition, it has been noted that polymerase mistakes introduced during RNA amplification may result in minor false quasipecies.

The quasispecies composition of HCV within an infected individual may have great clinical significance inasmuch as several studies have suggested that quasispecies complexity might influence the severity of HCV-associated liver disease and the response to interferon therapy. Two studies have demonstrated a direct correlation between quasispecies diversity and the progression of liver disease, and a third has suggested that the degree of quasispecies hetereogeneity at the onset of HCV infection can be used to predict long-term outcome (Farci et al. 2000; Honda et al. 1994; Koizumi et al. 1995). By contrast, studies evaluating the effect of HCV quasispecies on response to therapy have noted a reciprocal relation between diversity and IFN response: the less divergent the species, the greater the IFN response (Gonzalez-Peralta et al. 1996; Okada et al. 1992; Polyak et al. 1997). In addition, HCV quasispecies may play a role in viral persistence because it appears that selective pressure allows the emergence of new dominant strains to escape the most recent immune response (Forns and Bukh 1998). The relative ease with which HCV is able to escape immune pressure makes it difficult to formulate neutralizing antibodies that could be used in vaccine development. Efforts are currently under way to identify an appropriate cell-culture system that would provide a greater understanding of HCV molecular virology and assist in the development of therapeutic agents.

Diagnosis

Hepatitis C is transmitted through contact with infected whole blood, blood products, or body fluids (Alter et al. 1989; Conrad et al. 1995; Makris et al. 1993; Pereira et al. 1992). In the United States before 1991, the leading mode of transmission was receipt of HCV-contaminated blood transfusion. However, after the introduction of routine blood screening in 1990 and direct testing for HCV in 1992, transfusion-related HCV infection has declined dramatically (Alter 1995b; Schreiber et al. 1996). At present, the most common mode of transmission of HCV in the United States is the sharing of needles by users of illicit drugs. Case control studies have identified other possible risk factors for the transmission of HCV, including health care employment, unprotected sexual intercourse with multiple partners, low socioeconomic status, exposure to household members with a history of HCV, and perinatal transfer, particularly in human immunodeficiency virus-positive coinfected mothers (Alter 1997). Although HCV infection has been reported with other percutaneous exposures such as body piercing, tattooing, commercial barbering, and folk medicine practices, such transmission has not been documented in the United States.

Data derived from sentinel surveillance studies have suggested that the number of newly acquired acute HCV infections in the United States declined between 1984 and 1995, largely due to the decline in transfusion-associated infections (Alter 1997). The diagnosis of acute hepatitis C is difficult to make because it is rarely accompanied by symptoms, occurs most frequently in the absence of jaundice, and lacks early markers of infection. Disconcertingly, acute HCV infection progresses to chronic hepatitis in 75 to 80% of cases, although recent data have challenged these numbers suggesting that in some groups, up to 45% of patients may spontaneously recover from HCV infection (Alter 1993; Alter and Seeff 2000). Most patients with chronic HCV are asymptomatic and are identified only after routine blood work reveals the presence of elevated aminotransferases, which prompts the physician to obtain hepatitis serologies.

Although the diagnosis of hepatitis C requires the identification of HCV RNA in the serum, serological testing for anti-HCV using the enzyme immunoassay (EIA¹) is recommended as the initial test of choice (NIH 1997). The more sensitive and specific second version of the HCV EIA test, EIA-2, contains HCV antigens from the core, NS3, and NS4 regions of the genome. By increasing the number of targeted antigens, the EIA-2 test improves the detection of antibody in infected individuals and allows for earlier detection of HCV antibody (Okamoto et al. 1992). A negative EIA excludes the diagnosis of hepatitis C except in immunocompromised individuals. Patients with a positive EIA (and immunocompromised patients) should have the diagnosis of HCV confirmed by documenting the presence of HCV RNA in the serum.

Assays using polymerase chain reaction (PCR¹) or branched-DNA (bDNA¹) technology are widely used to measure HCV viral load (Gretch 1997). A positive PCR or bDNA test identifies HCV infection. A negative HCV RNA by PCR or b-DNA assay usually excludes the diagnosis of HCV except on rare occasions, such as in low-risk (normal alanine transaminase), anti-HCV, positive patient blood donors. Under these circumstances, a recombinant immunoblot assay (RIBA¹) is needed to determine whether the positive anti-HCV test represents a true positive (Atrah et al. 1995). The appearance of bands of different densities from at least two of the four viral proteins on the immunoblot is considered a positive RIBA test and is independent evidence that antibody to HCV is present in the serum. A negative RIBA test indicates that the positive anti-HCV assay by EIA is a false-positive result. Patients who are positive for anti-HCV by EIA, a negative HCV RNA, and a positive RIBA are considered to have recovered from previous HCV infection (Morishima and Gretch 1999).

Natural History

The natural history of any illness refers to the uninterrupted course of that disease from its onset to the development of end-stage organ damage, which may or may not result in the death of the affected patient. Implicit within the definition of natural history is the assumption that the onset of the disease is known, that the symptoms are obvious and the progression is easy to follow, and that attempts to cure or alter the course of the disease have not taken place. Hepatitis C is characterized by silent onset and asymptomatic progression, which may take 20 to 40 yr to reach its endpoint (DiBisceglie et al. 1991). In addition, the majority of patients in whom HCV has been diagnosed before end-stage disease has developed have received some form of therapy. Clearly, these characteristics combine to make a precise assessment of the natural history of hepatitis C extremely difficult.

Early retrospective studies evaluating patients referred to tertiary care centers suggested a high rate of progression to cirrhosis and HCC (Kiyosawa et al. 1990). Subsequent prospective studies of patients who developed post-transfusion hepatitis C demonstrated that progression to cirrhosis and cancer was not inevitable. Combining the data from five such trials with follow-up ranging from 4 to 16 yr suggests that chronic hepatitis develops in 62 to 79% of patients and cirrhosis in 1 to 20%, and the development of HCC is rare (Alter and Seeff 2000). Liver-related mortality in each of the studies was <4%. Extrapolation of these data to the general population is hampered by the relatively short follow-up period and the fact that these patients received blood transfusions for some underlying disease that may have led to their demise before end-stage HCV might have been expected to occur.

A recent study evaluating blood samples in a repository of 10,000 military recruits in the Air Force between 1948 and 1954 has permitted a heretofore unprecedented 50-yr follow-up of persons infected with HCV (Seeff et al. 2000). Data could be ascertained from 8,568 individuals. Only 17 of the 8,568 (0.2%) samples in the repository were confirmed to be anti-HCV positive; 11 of the 17 were also HCV RNA positive. Outcome using Veterans Affairs and Medicare files and Social Security and National Death Index tapes revealed a mortality rate of 41% (7/17) in the HCV-positive cohort compared with 26% in the HCV-negative group. Only one of the seven deaths in the HCV-positive group, however, was due to liver disease. Hepatocellular carcinoma was the cause of death in 0.1% of the HCV-negative group, and no HCV-infected patient died of HCC.

Two separate studies of women who inadvertently received HCV-contaminated Rh immune globulin 17 to 20 yr ago also suggest that the natural history of hepatitis C is less progressive than originally thought, at least among young women. The first study of 62,667 Irish women receiving contaminated Rh immune globulin in 1977 identified 704 anti-HCV positive women, of whom 390 (55%) were also HCV RNA positive (Kenny-Walsh 1999). Mean age at infection with HCV was 28 yr, all women had genotype 1, 30% had other HCV risk factors, and 5% were alcoholic. Liver biopsy data performed on 363 subjects revealed no fibrosis in 49%, periportal or portal fibrosis in 34%, portal to portal or central to portal bridging in 15%, and cirrhosis in 2%. In the second study of 917 women in Germany who also received HCV-contaminated Rh immune globulin, no evidence of chronic hepatitis was found, and only 0.4% of patients had overt cirrhosis 20 yr after infection (Wiese et al. 2000). These data, as well as data on children (Vogt et al. 1999), indicate that only a small percentage of young people who are infected progress to severe liver disease during the first 20 yr.

A recent effort has been made to review the world literature in order to assess long-term outcome by using the accumulated data on treatment, histological progression to fibrosis and HCC, and factors responsible for progression of disease (Alter and Seeff 2000). First, it was recognized that spontaneous clearance of the virus was more common than originally presumed. Second, four projections of potential outcome were presented. In Projection I, progression of HCV was considered to be linear so that over a lifetime, approximately 50% of patients would develop severe liver disease. In Projections II and III, progression of HCV was modeled to accelerate after the first 20 yr with every patient progressing to severe liver disease over the course of 40 to 60 yr. Projection IV assumed that if severe disease had not occurred after 20 yr of infection, it would never occur. Based on the accumulated literature regarding outcome of HCV and the diverse outcomes reported, the authors concluded that HCV progression lies somewhere between Projection I and Projection IV. As a result, of 100 acutely infected HCV patients, approximately 20% would spontaneously recover and 80% would develop persistent infection. Of these 80% with persistent infection, one third would progress to cirrhosis or HCC, one third would have stable disease with no progression, and one third would have an unpredictable outcome.

At present it is not possible to predict accurately in a given individual whether the liver disease will remain stable or whether it will advance to cirrhosis or HCC. If the outcome projection mentioned above is correct, the dilemma is to identify the patients most likely to benefit from therapy and the appropriate dose and duration of antiviral treatment necessary to prevent the severe sequelae of HCV infection.

Management

Many treatment trials have been conducted over the past decade to determine the benefit of monotherapy and, subsequently, combination therapy in the management of HCV infection. The early studies of interferon (IFN¹) monotherapy (6-mo duration) revealed an end-of-treatment response (ETR¹) of approximately 50%, but a sustained response (SR¹), defined as continued loss of HCV RNA 6 mo after cessation of therapy, of only 15% (Ahmed and Keeffe 1999). Studies of ribavirin monotherapy were less promising. More recently, two large randomized controlled trials (one in the United States and one in Europe) have been conducted to compare the effect of IFN monotherapy and combination therapy using IFN plus ribavirin for 24 or 48 wk in treatment-naive patients (McHutchinson et al. 1998; Poynard et al. 1998). Both studies used similar patient evaluation, monitoring, and endpoints. Among patients receiving IFN monotherapy, ETR was achieved in 29% regardless of duration of therapy, whereas SR was achieved in 6% of those treated for 24 wk and in 16% of those receiving 48 wk of therapy. By contrast, patients receiving combination therapy for 24 and 48 wk had ETR rates of approximately 50% and SR rates that increased from 33% in those treated for 24 wk to 41% in those receiving 48 wk of therapy.

A subsequent 6-mo trial that compared the efficacy of combination treatment with that of IFN plus placebo in patients with HCV who had relapsed after IFN monotherapy revealed similar results (Davis et al. 1998). Patients who received combination treatment had a SR of 49% compared with a SR of 5% in patients receiving IFN plus placebo. Adverse events related to ribavirin were responsible for dose reduction in 12% of the combination therapy group compared with dose reduction in only 3% of the IFN plus placebo group. These data suggest that combination therapy with IFN plus ribavirin is superior to IFN alone or IFN plus placebo in treatment-naïve patients or those who relapsed after IFN monotherapy.

HCV genotype and HCV RNA levels before treatment strongly influenced response to therapy (McHutchinson et al. 1998; Poynard et al. 1998). The response rate in patients with genotype 1 was much lower than in patients with genotypes 2 or 3, regardless of duration of therapy or treatment type (although response rates were improved in all genotypes with combination therapy). Interestingly, patients with genotypes 2 and 3 who received combination therapy had similar SR rates (65%) whether treated for 24 or 48 wk. The SR rate among recipients of combination therapy was low among those with both genotype 1 and HCV RNA level >2 million regardless of treatment duration. In patients with genotypes 2 or 3, HCV RNA level appeared to have no effect on response to therapy. Factors found to be predictive of response to therapy were genotypes 2 or 3, low initial serum HCV RNA level, young age (<40 yr), low fibrosis score, and female sex.

Adverse events occur with the use of IFN alone and with combination therapy. The most common side effect of ribavirin use is a dose-related hemolysis of red cells leading to severe anemia (Bodenheimer et al. 1997). The hemolysis is noted within 1 to 4 wk of beginning therapy and can be precipitous, resulting in fatigue, shortness of breath, angina, and palpitations. The most common side effect of IFN use is often a self-limited flu-like syndrome that can be partially obviated by pretreatment with acetaminophen (Davis et al. 1989). Other less common but more serious adverse events associated with IFN use include bone marrow suppression, resulting in serious decreases in platelet and absolute neutrophil counts, and neuropsychiatric disorders ranging from loss of concentration and memory to irritability and depression and, in rare cases, suicide attempts. These data have led to the recommendation that IFN alone should be used to treat HCV in patients with anemia or those with significant cardiac disease. In addition, severe thrombocytopenia and severe psychiatric illness are considered contraindications to treatment with IFN.

Studies with pegylated IFN (i.e., IFN covalently attached to polyethylene glycol) have been performed in response to suggestions that the poor response rates achieved in the treatment of HCV are due to the short half-life of IFN (Glue et al. 2000). Alpha IFN has a half-life of 4 to 6 hr, which necessitates thrice weekly dosing, thereby leading to erratic serum concentrations and intermittent peaks and troughs. The covalent attachment of polyethylene glycol to the IFN molecule increases the half-life to 96 hr, thus creating level antiviral concentrations and permitting once-a-week dosing. A multicenter, randomized, controlled trial of pegylated IFN versus alpha IFN in the therapy of previously untreated HCV-positive patients revealed improved SR rates with pegylated IFN but a similar adverse event profile (Shiffman et al. 1999).

These preliminary data now receive support from two recently reported randomized, controlled trials designed to evaluate the efficacy of a 40-kD pegylated interferon (peginterferon alfa-2a) in comparison with standard interferon alfa-2a (Heathcote et al. 2000; Zuezem et al. 2000). In one study that targeted patients with chronic hepatitis C, almost 90% of whom had neither portal fibrosis nor cirrhosis, recipients of the pegylated interferon (180 m g once a week for 48 wks) had an ETR of 69% and an SR of 39%, whereas recipients of conventional interferon, treated for the same length of time, had an ETR of 28% and an SR of 19% (Zeuzem et al. 2000). Factors associated with a sustained response included young age, no cirrhosis or fibrosis, and an HCV genotype other than 1. There was no significant difference in the frequency of adverse reactions between the two groups. In the second study, using the same therapeutic regimen, treatment was directed at patients with chronic hepatitis C and cirrhosis (Heathcote et al. 2000). A sustained response, as defined by loss of HCV RNA, was achieved in 30% of recipients of pegylated interferon and in 8% of those treated with conventional interferon. Histological improvement was noted in 54% of those who received pegylated interferon and in 31% of those who received conventional interferon. Interestingly, the histology improved even among those who did not develop a sustained virologic response. Adverse effects included neutropenia and thrombocytopenia, requiring dose modification in some instances. Thus, pegylated interferon is clearly superior to conventional interferon. Studies are currently ongoing to compare the relative efficacies of combined pegylated interferon and ribavirin with the current "gold standard" of the combination of conventional interferon and ribavirin.

Patients who relapse (i.e., individuals whose initial ETR is followed by reappearance of HCV RNA during the 6 mo after cessation of therapy) may respond to retreatment. One such study was undertaken using consensus IFN. Consensus IFN is a genetically engineered molecule containing the most common amino acids from each of several naturally occurring alpha-IFNs. The study, a multicenter open-label trial, involved patients who had received previous treatment with alpha-IFN or low-dose (9-m g) consensus IFN and either had relapsed or had failed to respond to treatment. The patients were then retreated with high dose (15-m g) consensus IFN for 24 or 48 wk (Heathcote et al. 1998). Among the relapsed patients, sustained response rates occurred in 28% at 24 wk and 58% at 48 wk. Among previous nonresponders, the sustained response rates were 5% at 24 wk and 13% at 48 wk. Consensus IFN was well tolerated, and adverse events were comparable with those occurring in recipients of standard alpha IFN. Based on these data, it appears that consensus IFN may be effective therapy in selected patients who relapse after IFN monotherapy, but it is of no benefit in treating nonresponders.

There are no current published data regarding pegylated IFN for persons who have been treated with current regimens and have relapsed, but such studies are in progress. Similarly, studies are being conducted to determine the efficacy of pegylated IFN for nonresponders.

Currrent Approach to Therapy

Analysis of the data described above indicates that combination therapy is superior to IFN monotherapy and that 48 wk of treatment is preferable to 24 wk in patients with genotype 1. HCV RNA levels at the onset of therapy for female patients aged <40 yr who have a low fibrosis score appear to be correlated with response to therapy. However, the results of other studies (not shown here) suggest that additional factors, including the presence of concurrent infections with HIV or HBV, end-organ damage, and major, uncontrolled depressive illness, may also affect response to treatment or susceptibility to the side effects of treatment (NIH 1997).

Guidelines currently exist regarding treatment with combination therapy. These guidelines will undoubtedly change as more data on pegylated IFN, with or without ribavirin, become available.

HCV-infected patients who are considered appropriate candidates for treatment include those with moderate to severe fibrosis (Ishak score 3,4) on liver biopsy, with biopsy-proven cirrhosis that is clinically compensated (Ishak score 5,6), with modest to severe inflammation even without cirrhosis, and with extrahepatic manifestations of HCV (cryoglobulinemia) without end-organ damage (AASLD 2001). The benefit of treatment is more controversial in patients with persistently normal aminotransferase levels, younger than 18 or older than 60 yr, and with depression while on current therapy (NIH 1997). Therapy is not recommended for HCV-infected patients with decompensated cirrhosis, those who are currently drinking alcohol, and those with major depressive illness or severe concurrent medical problems including renal failure, cytopenias, poorly controlled hypertension, diabetes, or coronary heart disease (NIH 1997). Because of data suggesting teratogenic effects in the offspring of IFN-treated rats, as well as fetal loss, HCV-infected pregnant females and any candidates for therapy unwilling or unable to comply with adequate contraception should be excluded from treatment.

The decision to treat should not be based on the presence or absence of symptoms, the mode of acquisition of the virus, the genotype, or the level of HCV RNA in the serum. Before initiating therapy, patients should be informed of the likelihood of response to treatment, the potential for developing side effects, and the importance of contraception during and 6 mo after cessation of therapy to avoid the risk of fetal abnormalities if a pregnancy occurs. A liver biopsy obtained within the previous 1 yr is considered current but is not absolutely required before initiation of therapy. After a history, physical examination, and baseline blood work are obtained to rule out possible contraindications to treatment, combination therapy is begun with subcutaneous injections of IFN at 3 ´ 10⁶ million units thrice weekly and 1200 mg of ribavirin (or 1000 mg if weight <75kg) daily in two divided doses. Patients are followed weekly for the first month with a history and physical examination, evaluation for the development of any side effects, and a complete blood count (CBC¹) obtained to monitor the hemoglobin, platelet count, and absolute neutrophil count. Follow up at monthly intervals begins after week 4 and includes obtaining a hepatic function panel in addition to the CBC. Thyroid-stimulating hormone levels should be measured every 3 mo. Adverse events are managed by either halving the dose of the offending medication, holding the medication for 2 wk and restarting at one half the original dose, or discontinuing the therapy entirely. Adjustments in medication dose are made at the physician's discretion, but the patient retains the right to discontinue therapy at any time upon proper notification of his/her physician.

Six months after initiation of therapy, a CBC, a liver function panel, and an assay for HCV RNA should be assessed in all patients. Therapy should be discontinued in any patient with detectable HCV RNA in the serum, regardless of genotype, because its presence indicates lack of response. In contrast, ETR is indicated if HCV RNA cannot be detected in the serum of patients with genotypes 2 or 3. At this point, the patient should be evaluated after treatment at monthly intervals for a total of 6 mo. Patients with genotype 1 and undetectable HCV RNA in the serum should continue therapy for an additional 6 mo. HCV RNA should be reassessed at 12 mo in patients with genotype 1 who are receiving a full year of therapy, and after 6 mo of follow-up in all patients who achieved an ETR, regardless of genotype. Reappearance of HCV RNA at the 6-mo-follow-up visit in any patient with a previous ETR is considered relapsed, whereas undetectable HCV RNA 6 mo after cessation of therapy is indicative of a sustained response.

At present, few options exist for patients who relapse after or fail to respond to a full course of combination therapy. Studies are under way to determine whether the combination of pegylated IFN plus ribavirin would be of benefit in such patients.

Areas for Future Research

Despite recent medical advances that have enhanced our understanding of hepatitis C virology and slightly improved our ability to manage hepatitis C, it is clear that several important dilemmas remain. First, improvement of the relatively poor response to treatment must be addressed. At present, combination therapy with IFN and ribavirin is the treatment of choice for patients infected with HCV, but the response rates in patients with genotype 1, the most commonly encountered genotype worldwide, are only 30 to 45%. In addition, combination therapy is associated with a number of side effects that occasionally necessitate dose reduction or discontinuation, further affecting overall response. As noted above, results of treatment trials of pegylated IFN plus ribavirin should lead to modification of treatment guidelines.

A variety of drugs have been investigated alone or in combination with IFN in the hope of maximizing antiviral effect, but results have been disappointing. The use of complementary and alternative medicine for the treatment of a number of chronic illnesses, including hepatitis C, has increased approximately1.5-fold since the early 1990s (Eisenberg et al. 1998). The herb

silybum marinarum, or milk thistle, has been used extensively in European and Asian clinics and reported to be of benefit in some patients. However, it will be important to conduct randomized, placebo-controlled trials to evaluate the safety and efficacy of milk thistle, and other herbs, either alone or in combination with standard HCV therapy before any recommendation can be made regarding use.

Finally, studies of phlebotomy before initiation of HCV therapy have recently gained attention as a means of maximizing virological response. It is well known that increased hepatic iron concentration, whether primary or secondary, is toxic to the liver and can lead to progression or fibrosis and cirrhosis. However, a recent study of iron reduction in HCV-infected patients before and during a 6-month course of interferon monotherapy did not demonstrate improvement in virological SR and Knodell histological activity index (DiBiscegli et al. 2000).

A second issue of some concern in the United States is the extremely poor response to treatment noted among African Americans infected with HCV. Epidemiological data reveal that African Americans and Caucasians have a similar incidence of acute disease but a disparate prevalence of HCV infection (3.2% in African Americans vs. 1.5% in Caucasians), which is not significant after adjusting for socioeconomic conditions or risk factors (Alter 1999). However, higher rates of viremia and infection with genotype 1b among African Americans have been reported (Wiley et al. 1999). Despite the higher prevalence and higher rate of viremia, it appears that African Americans do not have an accelerated rate of progression of disease. In fact, data obtained in 93 African American and 196 non-African American patients early in the course of HCV infection reveal a lower hepatic activity index, a lower baseline alanine transaminase level, and a lower percentage of cirrhosis among African Americans compared with the non-African American cohort (Wiley et al. 1999). In contrast, an increasing body of data suggests that the SR rate in response to combination therapy among Caucasians may be as high as 45%, whereas the SR rate among African Americans is <5% (McHutchinson et al. 1999). A recent study has challenged these data, but the study included an extremely small number of African Americans. Despite the small number, differences appear not to be related to HCV RNA levels, mode of acquisition of the virus, compliance with medication regimen, or genotype. A major study, currently in development through support of the National Institutes of Health, will soon begin to determine whether racial/ethnic differences in immune recognition of HCV-infected hepatocytes or antiviral kinetics may play a role in the apparent viral resistance among African Americans.

The development of an HCV vaccine continues to be the focus of intensive investigation. Recent advances in biotechnology have improved the understanding of hepatitis C virus immunology and brought to light several new challenges. An effective HCV vaccine must be able to prevent infection or the progression of infection, protect against the predominant genotypes within the geographic region of use, and be safe and effective for widespread administration. The asymptomatic nature of HCV infection, the relatively high mutation rate of HCV, the geographic differences in predominant HCV genotype, and financial resources are a few of the obstacles that must be surmounted to develop an efficacious vaccine. Alternative treatment strategies include extracellular neutralization of virus, inhibition of viral entry and uncoating, inhibition of host or viral enzymes leading to impairment of intracellular replication, and control of hepatic inflammation. Certainly the development of tissue culture systems and viable animal models of HCV infection will greatly enhance future research in this area.

¹Abbreviations used in this article: anti-HCV, antibody to hepatitis C virus; BDNA, branched chain deoxyribonucleic acid; CBC, complete blood count; EIA, enzyme immunoassay; ER, endoplasmic reticulum; ETR, end of treatment response; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IFN, interferon; NS, nonstructural; ORF, open reading frame; PCR, polymerase chain reaction; RIBA, recombinant immunoblot assay; SR, sustained response.

References

AASLD [American Association for the Study of Liver Diseases]. 2001. AASLD Practices Guidelines: Diagnosis, management and treatment of hepatitis C (HCV) infection. Hepatology (In Press).

Ahmed A, Keeffe EB. 1999. Overview of interferon therapy for chronic hepatitis C. Clin Liver Dis 3:757-773.

Alter HJ. 1989. Chronic consequences of non-A, non-B hepatitis. In: Seeff LB, Lewis JH, eds. Hepatology: Festschrift for Hyman J Zimmerman, MD. New York: Plenum Press. p 83-97.

Alter MJ. 1993. The detection, transmission, and outcome of hepatitis C virus infection. Infect Agents Dis 2:55-66.

Alter MJ. 1995a. Epidemiology of hepatitis C in the West. Semin Liver Dis 15:5-14.

Alter MJ. 1995b. Residual risk of transfusion-associated hepatitis. NIH Consensus Development Conference on Infectious Disease Testing for Blood Transfusions. Bethesda: NIH. p 23-27.

Alter MJ. 1997. The epidemiology of acute and chronic hepatitis C. Clin Liver Dis 1:559-568.

Alter MJ. 1999. Epidemiology of hepatitis C in African Americans. (Abstract). Proceedings of the December 1999 NIH Conference on Hepatitis C in African Americans. p 24.

Alter MJ, Kruszon-Moran D, Nainan OV, McQuillan GM, Gao F, Moyer LA, Kaslow RA, Margolis HS. 1999. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 341:556-562.

Alter MJ, Margolis HS, Krawczynski K Judson FN, Mares A, Alexander WJ, Hu PY, Miller JK, Gerber MA, Sampliner RE, Meeds EL, Beach MJ. 1992. The natural history of community-acquired hepatitis C in the United States. N Engl J Med 327:1899-1905.

Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo DL, Kuo G. 1989. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 321:1494-1500.

Alter HJ, Seeff LB. 2000. Recovery, persistence, and sequelae in hepatitis C virus infection: A perspective on long-term outcome. Semin Liv Dis 20:17-35.

Atrah HI, Hutchinson F, Gough D, Ala FA, Ahmed MM. 1995. Hepatitis C virus seroconversion rate in established blood donors. J Med Virol 46:329-333.

Bartholomeusz A, Tomlinson E, Wright PJ, Birch C, Locarcini S, Weingold H, Marcoccio S, Polan G. 1994. Use of a flavivirus RNA-dependent RNA polymerase assay to investigate the antiviral activity of selected compounds. Antiviral Res 24:341-350.

Bodenheimer HC Jr, Lindsay KL, Davis GL, Lewis JH, Thong SN, Seeff LB. 1997 Tolerance and efficacy of oral ribavirin treatment of chronic hepatitis C: A multicenter trial. Hepatology 26:473-477.

Bukh J, Apgar CL, Engle R, Gouindarajan S, Hegerich PA, Tellier R, Wong DC, Elkins R, Kew MC. 1998. Experimental infection of chimpanzees with hepatitis C virus of genotype 5a: Genetic analysis of the virus and generation of a standardized challenge pool. J Infect Dis 178:1193-1197.

Bukh J, Miller RH, Purcell RH. 1995. Genetic heterogeneity of hepatitis C virus: Quasispecies and genotypes. Semin Liver Dis 15:41-63.

Bukh J, Prucell RH, Miller RH. 1993. At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide. Proc Natl Acad Sci U S A 90:8234-8238.

Bukh J, Purcell RH, Miller RH. 1994. Sequence analysis of the core gene of 14 hepatitis C virus genotypes. Proc Natl Acad Sci U S A 91:8239-8243.

Conrad EU, Gretch DR, Obermeyer KR, Moogk MS, Sayers M, Wilson JJ, Strong DM. 1995. Transmission of the hepatitis C virus by tissue transplantation. J Bone Joint Surg 77:214-224.

Davis GL, Balart LA, Schiff ER, Lindsay K, Bodenheimer HC Jr, Perrillo RP, Carey W, Jacobson M, Payne J, Dienstag JL, Van Thiel DH, Tamborro C, Lefkowitch J, Albrecht J, Meschievitz C, Ortego TJ, Gibas A, and the Hepatitits Interventional Therapy Group.. 1989. Treatment of chronic hepatitis C with recombinant interferon alfa: A multicenter randomized, controlled trial. N Engl J Med 321:1501-1506.

Davis GL, Esteban-Mur R, Rustgi V, Hoefs J, Gordon SC, Trepo C, Shiffman ML, Zeuzem S, Crax A, Ling MH, Albrecht J. 1998. Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. N Engl J Med 339:1493-1499.

De Francesco R, Pessi A, Steinkuhler C. 1998. The hepatitis C virus NS3 proteinase: Structure and function of a zinc-containing serine proteinase. In: Schinazi RF, Sommadossi J-P, Thomas HC, eds. Therapies for Viral Hepatitis. London: International Medical Press. p 225.

DiBiscegli AM, Bonkovsky HL, Chopra S, Flamm S, Reddy RK, Grace N, Killenberg P, Hunt C, Tamburro C, Tavill AS, Ferguson R, Krawitt E, Banner B, Bacon BR. 2000. Iron reduction as adjuvant to interferon therapy in patients with chronic hepatitis C who have previously not responded to interferon: a multicenter, prospective, randomized, controlled trial. Hepatology 32:135-138.

DiBisceglie A, Goodman ZD, Ishak KG, Hoofnagle JH, Melpolder JJ, Alter HJ. 1991. Long-term clinical and histologic follow up of chronic post-transfusion hepatitis. Hepatology 14:969-974.

Dienstag JL. 1983. Non-A, non-B hepatitis. I. Recognition, epidemiology and clinical features. Gastroenterology 85:439-462.

Domingo E, Holland J, Biebricher C, et al. 1994. Quasispecies: The concept and the world. In: Gibbs A, Calisher C, Garcia-Arenal F, eds. Molecular Evolution of Viruses. Cambridge: Cambridge University Press. p 181.

Dubuisson J, Hsu HH, Cheung RC, Greenberg HB, Russell DG, Rice CM. 1994. Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and sindbis viruses. J Virol 68:6147-6160.

Eigen M. 1996. On the nature of virus quasispecies. Trends Microbiol 4:216-218.

Eisenberg DM, Davis RB, Ettner SL, Appel S, Wilkey S, Van Rompay M, Kessler RC. 1998.Trends in alternative medicine use in the United States, 1990-1997. Results of a follow-up national survey. JAMA 280:1569-1575.

Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Izumi N, Maromo F, Sato C. 1995. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virs 1b: Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest 96:224-230.

Farci P, Shimoda A, Coiana A, Diaz G, Peddis G, Melpolder JC, Strazzera A, Chien DY, Munoz SJ, Balistrieri A, Purcell RH, Alter HJ. 2000. The outcome of acute hepatitis C predicted by the evolution of viral quasispecies. Science 288:339-344.

Farci P, Shimoda A, Wong D, Cabezon T, DeGioannis D, Strazzera A, Shimizo Y, Shapiro M, Albrecht J, Purcell RH. 1996. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci U S A 93:15394-15399.

Forns X, Bukh J. 1998. Methods for determining the hepatitis C virus genotype. Viral Hepatitis Rev 4:1.

Forns X, Bukh J. 1999. The molecular biology of the hepatitis C virus: Genotypes and quasispecies. Clin Liver Dis 3:693-716.

Gilliam JH III, Geisinger DR, Richter JE. 1984. Primary hepatocellular carconima after chronic non-A, non-B post-transfusion hepatitis. Ann Intern Med 101:794.

Glue P, Rouzier-Panis R, Raffanel C, Sabo R, Gupta SK, Salfi M, Jacobs S, Clement RP. 2000. A dose-ranging study of pegylated interferon a -2b and ribavirin in chronic hepatitis C. The hepatitis C Intervention Therapy Group. Hepatology 32:647-653.

Gonzalez-Peralta RP, Qian K, She JY, Davis GI, Ohno T, Mizikami M, Lau JY. 1996. Clinical implications of viral quasispecies heterogeneity in chronic hepatitis C. J Med Virol 49:242-247.

Gretch DR. 1997. Use and interpretation of HCV diagnostic tests in the clinical setting. Clin Liver Dis 1:543-557.

Heathcote EJ, Keeffe EB, Lee SS, Feinman SV, Tong MJ, Reddy RK, Albert DG, Witt K, Blat LM. 1998. Retreatment of chronic hepatitis C with consensus interferon. Hepatology 27:1136-1142.

Heathcote EJ, Shiffman ML, Cooksley GE, Dusheiko GM, Lee SM, Balart L, Reindollar R, Reddy RK, Wright TL, Lin A, Hoffman J, De Pamphilis J. 2000. Peginterferon alfa-2a in patients with chronic hepatitis C and cirrhosis. N Engl J Med 343:1673-1680.

Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Ohkoshi S, Shimotohno K. 1991. Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 175:220-228.

Hijikata M, Mizushima H, Akagi T, Mori S, Kakiuchi N, Kato N, Tanaka T, Kimura K, Shimotohno K. 1993. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67:4665-4675.

Hijikata M, Mizushima H, Tanji Y, Komoda Y, Hirowatari Y, Akagi T, Kato N, Kimura K, Shimotohno K. 1993. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A 90:10773-10777.

Honda M, Kaneko S, Sakai A, Unoura M, Murakami S, Kobayashi K. 1994. Degree of diversity of hepatitis C virus quasispecies and progression of liver disease. Hepatology 20:1144-1151.

Hwang S-B, Lo S-Y, Ou J-H, et al. 1995. Detection of cellular proteins and viral core protein interacting with the 5' untranslated region of hepatitis C virus RNA. J Biomed Sci 2:227.

Kaneko T, Tanji Y, Satoh S, Hijikata M, Asabe S, Kumura K, Shimotohno K. 1994. Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem Biophys Res Commun 205:320-326.

Kenny-Walsh E for the Irish Hepatology Research Group. 1999. Clinical outcomes after hepatitis infection from contaminated anti-globulin. N Engl J Med 340:1228-1233.

Kim JL,, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP, Markland W, Pepre CA, O'Malley ET, Harveson SL, Rice CM, Mureko MA, Caron PR, Thomson JA. 1996. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87:343-355.

Kiyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Nakano Y, Furuta S, Akahane Y, Nishioka K, Purcell RH, Alter JH. 1990. Interrelationship of blood transfusion, non-A,non-B hepatitis and hepatocellular carcinoma: Analysis by detection of antibody to hepatitis C virus. Hepatology 12:671-675.

Koizumi K, Enomoto N, Kurosaki M, Murakumi T, Izomi N, Marumo F, Sato C. 1995. Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment. Hepatology 22:30-35.

Lin CM, Lindenbach BD, Pragai BM, Mc Court DW, Rice CM. 1994. Processing in the hepatitis C virus E2-NS2 region: Identification of p7 and two distinct E2-specific products with different C termini. J Virol 68:5063-5073.

Love RA, Parge HE, Wickersham JA, Hostomsky Z, Haboka N, Mooman EW, Adachi T, Hostomska Z. 1996. The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and structural zinc binding site. Cell 87:331-342.

Maertens G, Stuyver L. 1997. Genotypes and genetic variation of hepatitis C virus. In: Harrison TJ, Zuckerman A, eds. The Molecular Medicine of Viral Hepatitis (Medical Science Series). Chichester, England: John Wiley & Sons. p 183.

Major ME, Feinstone SM. 1997.The molecular virology of hepatitis C. Hepatology 25:1527-1538.

Makris BM, Garson CJ, Ring PW, et al. 1993. Hepatitis C viral RNA in clotting factor concentrates and the development of hepatitis in recipients. Blood 81:1898-1902.

McHutchinson JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, Rustgi VK, Goodman ZD, Ling MH, Cort S, Albrecht JK. 1998. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 339:1485-1492.

McHutchinson JG, Poynard T, Pianko S, Gordon SC, Reid AE, Dienstag J, Morgan T, Yao R, Albrecht J for the International Hepatitis Interventional Therapy Group. 2000. The impact of interferon plus ribavirin on response to therapy in black patients with chronic hepatitis C. Gastroenterology 119:1317-1323.

Mizushima H, Hijikata M, Asabe S, Hirota M, Kumora K, Shimotohno K. 1994. Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol 68:6215-6222.

Morishima C, Gretch DR. 1999. Clinical use of hepatitis C virus tests for diagnosis and monitoring during therapy. Clin Liver Dis 3:717-740.

NIH [National Institutes of Health]. 1997. NIH Concensus Development Conference on the Management of Hepatitis C. Bethesda: NIH.

Ogata N, Alter HJ, Miller RH, Purcell RH. 1991. Nucleotide sequence and mutation rate fo the H strain of hepatitis C virus. Proc Natl Acad Sci U S A 88:3392-3396.

Okada S-I, Akahane Y, Suzuki H, Okamoto H, Mishiro S. 1992. The degree of variability in the amino terminal region of the E2/NS1 protein of hepatitis C virus correlates with responsiveness to interferon therapy in viremic patients. Hepatology 16:619-624.

Okamoto H, Dojima M, Okada S-I, Yoshizawa H, Iizuka H, Tamaka T, Muchmore EE, Peterson DA, Ho Y, Mishiro S. 1992. Genetic drift of hepatitis C virus during a 8.2-year infection in a chimpanzee: Variability and stability. Virology 190:894-899.

Okamoto H, Kurai K, Okada S-I, Yamamoto K, Lizuka H, Tamaka T, Fukuda S, Tsuda F, Mishio S. 1992. Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: Comparative study of four distinct genotypes. Virology 188:331-341.

Pereira BJ, Milford EL, Kirkman RL, Quan S, Sayne KR, Johnson PJ, Wilkes JC, Levey AS. 1992. Prevalence of hepatitis C virus RNA in organ donors positive for hepatitis C antibody and in the recipients of their organs. N Engl J Med 327:910-915.

Pessoa MG, Wright TL. 1997. Hepatitis C infection in transplantation. Clin Liver Dis 1:663-690.

Polyak SJ, Faulkner G, Carithers RL, Corey L, Gretch DR. 1997. Assessment of hepatitis C virus quasispecies heterogeneity by gel shift analysis: Correlation with response to interferon therapy. J Infect Dis 175:1101-1107.

Poynard T, Marcellin P, Lee S, Niederau C, Minuk GS, Ideo G, Bain V, Heathcote J, Zeuzem S, Trepo C, Albrecht J. 1998. Randomized trial of interferon alfa-2b and ribavirin for 48 weeks or for 24 weeks versus interferon alfa-2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 353:1426-1432.

Ray RB, Lagging LM, Meyer K, Ray R. 1996. Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. J Virol 70:4438-4443.

Reed KE, Rice CM. 1998. Molecular characterization of hepatitis C virus. Curr Stud Hematol Blood Transfus 62:1.

Rice CM. 1996. Flaviviridae: The viruses and their replication. In: Fields BN, Knippe DM, Howley PM, eds: Fields Virology, ed 3. Philadelphia, Lippincott-Raven. p 931.

Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. 1996. The risk of transfusion-transmitted viral infections. N Engl J Med 334:1685-1690.

Seeff LB. Hepatitis C from a needlestick injury. 1991. Ann Intern Med 115:411.

Seeff LB, Miller RN, Rabkin CS, Buskell-Bales Z, Straley-Eason DK, Smoak BL, Johnson LD, Lee SR, Kaplan EL. 2000. 45-year follow-up of hepatitis C infection in healthy young adults. Ann Intern Med 132:105-111.

Shiffman M, Pockros PJ, Reddy R, Wright TL, Reindollar R, Fried MW, Purdum PP III, Everson G, Pedder S. 1999. A controlled, randomized, multicenter, descending dose phase II trial of pegylated interferon alfa-2A (PEG) versus standard interferon alfa-2A (IFN) for treatment of chronic hepatitis C. (Abstract). Gastroenterology 116(Suppl):L0418.

Simmonds P, Holmes EC, Cha T-A, Chan SW, McOmish F, Irvine B, Beall E, Yapp PL, Kolberg J, Ordea MS. 1993. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS5 region. J Gen Virol 74:2391-2399.

Suzuki JA, Tamura JK, Palmer-Hill F, Warrener P, Grakoui A, Rice CM, Feinstone SM, Collett MS. 1993. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with related pestivirus and flavivirus enzymes. J Virol 67:6152-6158.

Tabor E, Gerety RJ, Drucker JA, Seeff LB, Hoofnagle JH, Jackson DR, April M, Barker LF, Pineda-Tainondong G. 1978. Transmission of non-A, non-B hepatitis from man to chimpanzee. Lancet 1:463-466.

Tabor E, Purcell RH, Gerety FJ. 1981. Primate animal models and titered inocula for the study of human hepatitis A, B, and non-A, non-B hepatitis. J Med Primatol 1:305-318.

Tai C-L, Chi W-K, Chen D-S, Hwang LH. 1996. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J Virol 70:8477-8484.

Vogt M, Lang T, Frosner G, Klingler C, Sendl AF, Zeller A, Wiebecke B, Langer B, Meisner H, Hess J. 1999. Prevalence and clinical outcome of hepatitis C infection in children who underwent cardiac surgery before the implementation of blood-donor screening. N Engl J Med 341:866-870.

Wiese M, Berr F, Lafrenz M, Porst H, Oesen U. 2000. Low frequency of cirrhosis in a hepatitis C genotype 1b single-source outbreak in Germany: A 20-year multicenter study. Hepatology 32:91-96.

Wiley TE, Brown JL, Chan J. 1999. The natural history of hepatitis C in African Americans. (Abstract). Proceedings of the December 1999 NIH Conference on Hepatitis C in African Americans. Bethesda: NIH. p 26-27.

Yanagi M, St. Claire M, Shapiro M, Emerson SU, Purcell RH, Bukh J. 1998. Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244:161-172.

Zeuzem S, Feinman V, Rasenack J, Heathcote EJ, Lai M-Y, Gane E, O'Grady J, Reichen J, Diago M, Lin A, Hoffman J, Brunda MJ. 2000. Peginterferon alfa-2a in patients with chronic hepatitis C. N Engl J Med 343:1666-1672.