Considerable information about the structural features of the HCV-encoded RdRp is now available from the crystal structure of the enzyme (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999) (Ch. 16). However, the lack of a highly productive tissue culture system that consistently supports virus replication (Ch. 11) has made it difficult to isolate replication complexes in order to study NS5B function in its appropriate context. For this reason, a number of laboratories have isolated recombinant HCV NS5B for functional studies as well as for drug screening (Behrens et al., 1996; Al et al., 1997, 1998; Lohmann et al., 1997; Yamashita et al., 1998; Hagehorn et al., 2000). Some of the initial attempts to isolate NS5B from recombinant insect cells or E. coli showed that a large percentage was insoluble. To solve this problem, NS5B was isolated under denaturing conditions and then refolded into active enzyme (Yuan et al., 1997), where it was shown to replicate HCV RNA as well as globin mRNA, poly(A), poly(C) and other templates in the absence of additional virus or host factors (Behrens et al., 1996; Al et al., 1997, 1998; Lohmann et al., 1997). Secondary structural analysis of HCV NS5B showed that it had a hydrophobic carboxy-terminus, which may explain its hydrophobicity and membrane association in HCV replication complexes. Removal of the hydrophobic 21 carboxy-terminal amino acid residues from NS5B facilitated purification and increased the yield of functional enzyme (Yamashita et al., 1998).

Much of what is known about the clinical course of HCV infection comes from retrospective studies of patients with acquired NANB PTH and from prospective studies of those who were acutely exposed to HCV (Prince et al., 1974; Alter et al., 1975; Feinstone et al., 1975; Knodell et al., 1975; Esteban et al., 1990; Tremolada et al., 1991). Overall, the clinical presentation of acute hepatitis is similar in patients infected with HAV, HBV or HCV, although acute HCV infection tends to be milder than acute HBV infection. In fact, 70–95% of HCV-infected people do not recall an episode of acute hepatitis, suggesting a subclinical course is very common (Meyers et al., 1977; Koretz et al., 1993b; Hoofnagle, 1997). Apart from the fact that the average incubation period for acute HCV infection (Seeff, 1991; Koretz et al., 1993b) is between that of HAV and HBV (Table 1.1), acute cases of symptomatic HCV infection are indistinguishable from severe acute HBV or HAV infections. Symptoms of acute HCV infection, which vary in duration and intensity, span 2–12 weeks (Ch. 4) and often include malaise, nausea, and upper right quadrant pain, followed by jaundice and dark urine (Hoofnagle, 1997). Loss of appetite may also occur (CDC, 1998). These symptoms occur weeks after the appearance of HCV RNA in blood and overlap in time with the elevation of transaminases (Fig. 4.1). ALT elevations often reach a peak 10-fold or more above normal, independent of whether patients develop symptoms.

Is hepatitis C cytopathic?

There are a number of mechanisms whereby HCV could trigger liver cell injury during infection (Table 5.1). Injury could be triggered directly by the virus (i.e., a cytopathic effect) and/or by immune responses against virus-infected cells. HCV-associated liver cell injury could also result from the interactions of HCV with a variety of environmental agents, some of which are mentioned below. Flaviviruses are often cytopathic (Chambers et al., 1990) in that they damage or destroy the cells in which they are growing; consequently it was initially thought that the related HCV might also be cytopathic (Table 5.2). Some evidence has supported such a cytopathic effect whereas other studies have suggested that HCV is not cytopathic. Cell damage was observed with a minimal inflammatory response in monkeys experimentally infected with selected flaviviruses (Monath, 1990). Early studies with NANBH showed cell rounding, shrinkage, and nuclear pyknosis, which are characteristic, cytopathic effects. Damage was also observed in the membranes of mitochrondria and ER (Omata et al., 1981; Dienes et al., 1982). Following the discovery of HCV, it was shown that HCV RNA in the liver, but not in serum, was associated with severe inflammation (Lau et al., 1993). The likelihood that HCV maturation and replication is membrane associated may provide an explanation for the membrane alterations in HCV infection.

The anatomy of the normal liver is such that hepatocytes are closely accessible to the bloodstream without intervening basement membranes (Gumuncio & Berkowtiz, 1992). Combined with the fact that the liver is a major organ of detoxifcation, and that it takes up foodstuffs, hormones, chemicals, asialoglycoproteins, metal ions, and a wide variety of other molecules, it is no surprise that the liver is a good candidate for the development of antiviral gene therapy against HCV. The goal of antiviral gene therapy is to make cells resistant to viral infection and/or block the virus life cycle at multiple steps. Antiviral gene therapy could supplement immunotherapy and/or the administration of small molecule inhibitors of NS3, NS5B, or other viral targets. However, the lack of a good animal model in which to study HCV pathogenesis (Chs. 13 and 15) and evaluate various forms of antiviral gene therapy represents a major challenge (see related question 21 in Ch. 24).

Despite these limitations, some progress has been made. For example, HCV-specific ribozymes against the 5' UTR and core region have been shown partially to inhibit virus translation in vitro and in cells transfected with a reporter gene cloned downstream from the HCV 5' UTR and core sequences (Sakamoto, Wu & Wu, 1996; Welch et al., 1996). In these experiments, ribozyme-mediated cleavage of the viral RNA was responsible for the decrease in the levels of virus gene expression.

NS3 has protease, helicase and NTPase activities. NS3 protease assays have been developed using full-length NS3 or its serine protease domain (residues 1–181) (Section 2.4). Polypeptides made by in vitro translation (Bouffard et al., 1995; Hahm et al., 1995; Lin & Rice, 1995), and expressed in E. coli (Yamada et al., 1998), mammalian (Bartenschlager et al., 1993; Eckart et al., 1993; Grakoui et al., 1993b) or insect cells (Hirowatari et al., 1993; Overton et al., 1995) have been shown to be active. For the in vitro assays, translated NS3 was mixed with radiolabeled synthetic peptide containing the desired cleavage site, and the products assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis or reversed phase high pressure liquid chromatography. For some cleavage reactions, in vitro translated NS4A cofactor and commercially available microsomal membranes were added along with the synthetic substrate. Purified protease from E. coli has also been tested against radiolabeled substrates made by solid-phase peptide synthesis or by in vitro translation (Bianchi et al., 1996; Shimizu et al., 1996; Sudo et al., 1996). In some studies, recombinant NS3 contained a hexahistadine tag, which permitted easy purification by affinity chromatography using a nickel (Ni2+) column that tightly bound the tag sequence (D'Souza et al., 1995; Kakiuchi et al., 1995).

An active female business executive, aged 39 years and a mother of three young children, visited her family doctor for an annual check-up. She had never been seriously ill. A few days after her visit, she received a phone call from the doctor's office, informing her that her liver enzymes were slightly elevated. Since this result may reflect an underlying problem in the liver, she was asked to come back in for more tests. She tested positive for antibodies against a recently discovered hepatitis virus that establishes a chronic, asymptomatic infection in most people, which is often accompanied by an underlying liver disease that is serious and often progresses toward cirrhosis. Cirrhosis, or end-stage liver disease, is accompanied by considerable morbidity and mortality. She left the doctor's office with the knowledge that within 10–15 years the virus infection could destroy her liver. She was treated with antiviral therapy, which was expensive, had considerable side effects, and was not effective beyond the period of treatment. She was then informed that her best chances for survival may eventually be a liver transplant. This is the clinical face that is often seen in chronic hepatitis C virus (HCV) infection. This book will present the basic features of the virus and associated diseases as well as discuss present and future treatment options, in the hope that it will stimulate further research and provide a brighter future for those infected with HCV.

In order to study the natural history and pathogenesis of acute and chronic HCV infections in more detail, infectious clones of HCV RNA have been constructed and used for experimental infection of chimpanzees. A number of lessons have been learned from these experiences. Initially, a full-length viral RNA was constructed from fragments of liver-derived material, but when a full-length cDNA was used for intrahepatic injection of chimpanzees, it was not infectious (Major & Feinstone, 2000). Since the intrahepatic viral RNA originally cloned for this work may have been shorter than a genome length replication intermediate, cloning was done again using a patient's serum as starting material. Compared with the liver-derived material, the serum-derived clone had an additional 98 bp at the 3′ end, within an UTR that was highly conserved among HCV isolates at the primary and secondary structural levels (Tanaka et al., 1995; Kolykhalov et al., 1996), suggesting that these sequences may be important for replication. This was confirmed by showing that some mutations within the 3′ 98 bp untranslated region rendered an otherwise infectious clone uninfectious in chimpanzees (Yanagi et al., 1999). Another striking observation was that RT/PCR amplification of full-length clones from serum were also noninfectious, in all probability because of point mutations introduced during the amplification procedures. Based upon this assumption, a “consensus” clone was made, and only this proved to be infectious. Experimental infection of two chimpanzees with the “consensus” RNA by intrahepatic infection resulted in the establishment of chronic infections with mild (periportal) hepatitis.

Therapeutic decisions in chronic HCV infection have been made difficult by the subclinical nature of most acute and chronic infections. This is because, in many cases, infections are not even detected unless anti-HCV or HCV RNA tests are performed (Section 1.6 and Ch. 19). This does not lessen the danger of chronic HCV, which often does not become symptomatic until the development of cirrhosis some 20 years following infection. Given the high morbidity and mortality associated with cirrhosis, combined with the insidious nature and variable progression of CLD, it is not surprising that so many chronically infected patients are not treated until liver disease is fairly advanced and considerable permanent damage is done. For example, patients that become symptomatic and then present with cirrhosis may already be in end-stage liver disease. Such patients are also at very high risk for the development of HCC (Chs. 4 and 6). Given these considerations, HCV is often not diagnosed for a long period after virus exposure, since patients are ‘well’ and do not seek medical help. This represents one of the major challenges in treating HCV infection.

IFN-α is one of a family of naturally occurring glycoproteins that have both direct antiviral and immunomodulatory activities. Initially, IFN-α was obtained from leukocyte cultures in very limited quantities, but the availability of recombinant IFN-α in the 1980s finally permitted large-scale, controlled trials of its utility.

Infection with the RNA-containing hepatitis C virus, which was isolated little more than 10 years ago, causes damage to the liver resulting in cirrhosis, liver failure and cancer of the liver. In the majority of individuals, hepatitis C infection progresses slowly over many years and approximately 85% of those who contract the disease remain chronically infected, with the virus replicating throughout their lifetime. Lifelong measures are, therefore, required to limit the spread of infection to others. The importance of the disease is clear from the fact that 170 million people are estimated to be chronically infected worldwide. In the USA, where liver failure from chronic hepatitis C infection is one of the most common reasons for liver transplants, some 4 million people are infected by the virus. Hepatitis C is probably also the most common cause of primary liver cancer in the developed world.

There is no doubt that important advances have been made in recent years. As detailed in this book, the development and deployment of assays that specifically detect anti-HCV antibodies and HCV RNA in infected patients have reduced the risk of acquiring HCV from contaminated blood to almost zero. However, there is currently no cure for liver disease caused by hepatitis C, and interferon therapy is of limited efficacy and has significant side-effects.

The development of HCV subgenomic replicons that produce high levels of one or more HCV polypeptides (Lohmann et al., 1999; Blight et al., 2000), in contrast to the very low and inconsistent levels of wild-type virus produced in tissue culture cells infected with HCV (Table 11.1 and Ch. 11), suggests that there are properties of wild-type HCV that normally attenuate virus gene expression and replication, and that when these constraints are removed, much higher levels of expression could be achieved. One of these constraints may be the complex secondary structural features within the 5' and 3' UTRs of the virus. Many laboratories are developing additional self-replicating replicons that are capable of persisting, and some at high copy number, within transfected or infected cells. These include replicons made from alphavirus (Garoff & Li, 1998; Ying et al., 1999), pestivirus (Moser et al., 1999), other flavivirus (Varnavski & Khromykh, 1999), and coronavirus (Thiel, Siddell & Herold, 1998) vectors. Whether the entire HCV polyprotein could be expressed from such constructs remains to be seen. In addition, since the HCV would be produced from artificial templates, it is not clear whether the sensitivity of virus gene expression and replication to putative antiviral agents would be the same or different to that of virus made from native HCV templates in an infected cell.

Prevalence and risk factors

The association between HCV and HCC predates the discovery of the virus, since early on it had been shown that there was a close relationship between NANBH and the development of HCC (Kiyosawa et al., 1982, 1984, 1990; Sakamoto et al., 1988). However, once HCV was discovered (Choo et al., 1989) and serological assays became available for its detection (Kuo et al., 1989) (Sections 1.4 and 1.5), a close association was established between anti-HCV and HCC all over the world (Bruix et al., 1989; Colombo et al., 1989; Hasan et al., 1990; Kew et al., 1990; Saito et al., 1990; Vargas, Costella & Esteban, 1990; Yu et al., 1990; Sulaiman et al., 1991). In southern Europe and Japan, for example, more than half of the reported HCC has been associated with HCV (Bruix et al., 1989; Colombo et al., 1989) whereas the prevalence of anti-HCV in Alaskan patients with HCC is nearly zero. Intermediate frequencies of anti-HCV have been reported among patients from Greece, Austria, and Taiwan (Jeng & Tsai, 1991; Baur et al., 1992; Hadziyannis et al., 1993), indicating geographic variation in this association. Worldwide, there are an estimated 170 million people who are chronically infected with HCV (Bradley et al., 1983; Choo et al., 1989; Houghton, 1996; Delwaide & Gerard, 2000; Wild & Hall, 2000) (Section 3.1), and the annual incidence rate of newly diagnosed HCC associated with HCV infection ranges from 1 to 7% in different populations (Di Bisceglie, 1997).

There are several fundamentally different methods that have been developed to detect HCV RNA in blood. These include RT/PCR, branched chain signal amplification (bDNA), nucleic acid sequence-based amplification, ligase chain reaction, transcription-mediated amplification, and various postamplification hybrid capture systems (Hodinka, 1998; Allain, 2000). Northern blot analysis, in situ hybridization and RT/PCR have also been used to detect HCV RNA in tissue samples (Negro, 1998). However, RT/PCR has evolved as the most common method for viral RNA detection and quantification in clinical samples (Ch. 18). Briefly, total RNA is extracted and purified from clinical samples. The extraction procedure may involve pronase digestion in the presence of chaotrophic agents (e.g., guanidinium chloride) to release the RNA from the virion and with phenol extraction and ethanol precipitation to separate nucleic acids from protein. Alternatively, the clinical samples could be “lysed” by addition of detergent, and the released RNA purified and recovered from “mini-spin” columns, which bind nucleic acid and permit the removal of denatured proteins and potential inhibitors of PCR prior to RNA recovery. The HCV RNA in the resulting material is converted to DNA in a two-step procedure. Since HCV RNA is plus stranded, it is converted to minus strand DNA by addition of a complementary primer and by RT, which synthesizes the remaining minus strand. After minus strand synthesis, some protocols call for extraction and purification of the minus strand DNA followed by addition of plus and minus strand primers together with Taq polymerase and then finally by standard PCR.

Soon after the discovery of HCV, high frequencies of anti-HCV (>50%) were reported among patients with autoimmune hepatitis (Esteban et al., 1989; Lenzi et al., 1990) and, later, among patients with other autoimmune diseases (Table 7.1 and Section 1.5). Further work among patients with HCV-associated autoimmune hepatitis showed that many anti-HCV results were caused by false positives generated by hypergammaglobulinemia (McFarlane et al., 1990) and by immune complexes (McFarlane et al., 1983), which interfered with first-generation anti- HCV ELISAs. False-positive anti-HCV results were also noted in patients with other autoimmune diseases (Fusconi et al., 1990), although the frequency of anti-HCV correlated directly with the levels of gammaglobulin in sera (Schwarcz, von Sydow & Weiland, 1990) (Section 1.5). The introduction of second-generation anti-HCV testing and RT/PCR, however, still showed that 20–40% of patients with autoimmune hepatitis had detectable markers of HCV infection (Cassani et al., 1992; Nishiguchi et al., 1992). Independent work showed that the frequency of anti-HCV among patients with primary biliary cirrhosis or primary sclerosing cholangitis was low (<5%) and similar to that of the general population (Lohse et al., 1995), suggesting that HCV infection is not closely linked with autoimmune diseases. Likewise, anti-HCV was found in 3–5% of patients with liver–kidney microsomal autoantibodies (Abuaf et al., 1993), a non-organ specific marker of autoimmunity, although the titers in patients with HCV-associated hepatitis are usually much lower than in patients with autoimmune hepatitis (Strassburg et al., 1996).