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

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ILAR Journal V42(2) 2001
Animal Models of Hepatitis

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New Models

New Animal Models of Hepatitis B and C
Mark A. Feitelson and Jonathan D. Larkin

Mark A. Feitelson, Ph.D., is Professor in the Department of Pathology, Anatomy, and Cell Biology and in the Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania. Jonathan D. Larkin, Ph.D., was a graduate student with Dr. Feitelson and is now a postdoctoral fellow at Eli Lilly & Co., Indianapolis, Indiana.

Abstract

The narrow host range of infection and lack of suitable tissue culture systems for the propagation of hepatitis B and C viruses are limitations that have prevented a more thorough understanding of persistent infection and the pathogenesis of chronic liver disease. With hepatitis B virus (HBV), this lack of knowledge has been partially overcome by the discovery and characterization of HBV-like viruses in wild animals. With hepatitis C virus (HCV), related flaviviruses have been used as surrogate systems for such studies. Other laboratories have developed transgenic mice that express virus gene products and/or support virus replication. Some HBV transgenic mouse models develop fulminant hepatitis, acute hepatitis, or chronic liver disease after adoptive transfer, and others spontaneously develop hepatocellular carcinoma (HCC), as in human infections. Among HCV transgenic mice, most develop no disease, but acute hepatitis has been observed in one model, and HCC in another. Although mice are not susceptible to HBV and HCV, their ability to replicate these viruses and to develop liver diseases characteristic of human infections provides new opportunities to study pathogenesis and develop novel therapeutics.

Key Words: animal models; hepatitis B virus; hepatitis C virus; pathogenesis; transgenic mice

Introduction

More than 350 million people worldwide are chronic carriers of hepatitis B virus (HBV¹), and approximately 170 million are chronically infected with hepatitis C virus (HCV¹) (Delwaide and Gerard 2000; Wild and Hall 2000). These people are at high risk for the development of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC¹). HCC is one of the most frequent tumor types worldwide, with a yearly incidence of up to one million (Esquivel et al. 1999). Both cirrhosis and HCC are major causes of mortality within 20 to 40 yr after infection (Alter 1996). Although screening tests have now nearly eliminated these viruses from the blood supply (Gerlich and Caspari 1999), significant transmission still occurs sexually (for HBV) (Mast et al. 1998) and through intravenous drug abuse (for HBV and HCV) (Maddrey 2000; Smyth et al. 1995). Thus, both viruses remain very important public health threats worldwide.

Treatment for chronic infections is limited, with interferon resulting in a sustained response in only about 20% of HBV or HCV patients (Lindsay 1997; Torresi and Locarnini 2000). New therapies have recently been adopted with the introduction of lamivudine for HBV (Dusheiko 1999) and ribavirin for HCV (Reichard et al. 1997). Lamivudine is highly effective against HBV in most patients, while the combination of interferon plus ribavirin typically shows a sustained virological and histopathological response in up to 50% of HCV-infected patients after the end of treatment. Prolonged treatment with interferon has considerable side effects (Hoofnagle and Lau 1996), and lamivudine-resistant HBV has been detected in up to 20% of patients after 1 yr of treatment (Honkoop et al. 1997; Tipples et al. 1996). Given the half billion or more people chronically infected with these viruses, there is a strong mandate to develop relevant animal models to test new drug candidates to combat both of them and their associated liver diseases.

Natural Hosts for HBV and HCV

Part of the difficulty in developing new therapeutic approaches stems from a fundamental lack in understanding the pathogenesis of chronic liver disease (CLD¹) associated with these viral infections (Figures 1 and 2). Chimpanzees have been invaluable for studying the transmission and natural history of infection, have provided important information about pathogenesis, and have been used to conduct vaccine studies (Prince and Brotman 1998; Tabor et al. 1983). In the case of HCV, chimpanzees were central to the discovery of the virus (Alter et al. 1978; Bradley et al. 1979). However, their availability is limited due to their expense and endangered status. In addition, the liver disease in experimentally infected chimpanzees is characteristically mild, which makes it impossible to study the pathogenesis of cirrhosis or HCC. For these reasons, it would not be practical to use chimpanzees for the preclinical evaluation of new therapeutic approaches on a regular basis, or for the evaluation of combination therapies.

Recently, the tree shew (Tupaia) has been shown to be susceptible to HBV and HCV infections (Xie et al. 1998; Yan et al. 1996a,b). Transmission has been readily achieved using HBV-infected animals and has been prevented by prior immunization of susceptible animals with the HBV vaccine. Chronically infected animals developed a low incidence of HCC, as did uninfected animals treated with aflatoxin B1, although the frequency of HCC was more than 50% among infected animals given aflatoxin. Among HCV-infected animals, HCV viremia was transient or intermittent in about one third of the infected animals, and for a more extended time in half the animals that were immunosuppressed by irradiation before infection. Although this small animal model holds promise as being susceptible to infection by these viruses, the model is relatively uncharacterized, and only future work will determine whether it will provide any significant advantages over the chimpanzee.

Hepadnaviruses

Hepadnaviruses (Robinson et al. 1982) include HBV and a growing number of agents that naturally infect selected hosts in the wild. The three best studied are the ground squirrel hepatitis virus, which infects ground squirrels (Marion et al. 1980), the woodchuck hepatitis virus, which infects woodchucks (Summers et al. 1978), and the duck hepatitis B virus (DHBV¹), which infects Pekin ducks (Mason et al. 1980). Mammalian hepadnaviruses have a genetic organization similar to that of HBV (Figure 3), while DHBV lacks the X gene. Infected ducks have been very useful in elucidating the replication scheme of hepadnaviruses (Summers and Mason 1982), and primary duck hepatocytes are readily infected with DHBV (Pugh and Summers 1989). This makes them particularly useful for the evaluation of drugs that inhibit virus replication in a fully permissive in vitro system (Colledge et al. 1997; Schultz and Chisari 1999). Infected ducks and woodchucks have also been exploited in the area of preclinical antiviral drug development (Hurwitz et al. 1998; Mason et al. 1994), having become standard in vivo models for preclinical evaluation of nucleoside analogs with potential activity against HBV. Infected woodchucks have also been used in basic studies focused on the natural history of infection (Korba et al. 1989, 1990), which is similar to that in chronic human infections (Figure 1). Chronically infected woodchucks have also been useful in studying the pathogenesis and molecular mechanisms of CLD and HCC (Fourel et al. 1994; Hsu et al. 1988; Popper et al. 1987; Snyder et al. 1982). Even with these strengths, the lack of inbred ducks and/or woodchucks, as well as the lack of duck- and woodchuck-specific reagents to analyze the immune cells important to the pathogenesis of acute and chronic infections, remain formidable obstacles in understanding the molecular and cellular basis of disease. In addition, neither infected ducks nor woodchucks develop cirrhosis, nor do infected ducks develop HCC. In addition, given that the pathogenesis of infection with these viruses is immune mediated, immunological reagents that might have therapeutic efficacy against the woodchuck or duck viruses would probably not be useful against HBV (e.g., a monoclonal antibody), and that for human trials, HBV-specific reagents would have to be created and tested. Hence, despite the wealth of knowledge gained from studying hepadnaviruses in their natural hosts, the types of experiments that could be devised to elicidate the mechanisms of pathogenesis have been limited.

Hepaciviruses

The family Flaviviridae contains three genera. Hepaciviruses contain exclusively HCV. Flaviviruses contain a number of insect-borne agents, including yellow fever virus, dengue fever virus, as well as Japanese and tick-borne encephalitis viruses. Pestiviruses are exemplified by border disease and swine fever viruses, as well as the bovine viral diarrhea virus, which is used as a surrogate model for some aspects of HCV biology. HCV is related to these genera in terms of genome structure, the production of a polyprotein, the structure and function of the corresponding polypeptides, and their likely mode of replication (Chambers et al. 1990; Neyts et al. 1999) (Figure 4). Unlike HBV, there have been no reports of closely related HCV-like hepaciviruses that naturally infect wild animals or that can be used to experimentally infect laboratory animals. This reality restricts the available animal model systems that could be used to understand pathogenesis and to evaluate putative antiviral compounds or other novel therapeutic approaches.

Mechanisms in the Pathogenesis of CLD: Cytopathic Effects and/or Immune-mediated Disease?

Direct cytopathic effects (CPEs¹) occur when the action of virus gene products or virus replication is toxic to the infected cell, resulting in cellular damage, and sometimes cell death. With the exception of the CPE observed in cell lines or transgenic mice that constitutively overexpress HBV antigens (Chisari et al. 1989; Dunsford et al. 1990; Yoakum et al. 1983), there is little evidence to suggest that the pathogenesis of HBV infection is mediated by virus-induced CPE. In support of this idea, HBV carriers and transgenic mice with sustained, high levels of virus replication have no evidence of liver disease (Guidotti et al. 1995; Hoofnagle et al. 1987). In addition, tissue culture systems that replicate HBV do not develop CPE (Sells et al. 1987; Sureau et al. 1986).

Strong evidence exists that the pathogenesis of HBV infection is immune mediated. For example, liver and peripheral blood lymphocytes demonstrate proliferative and cytotoxic activities against HBV antigens (Feitelson 1996; Ferrari et al. 1987; Marinos et al. 1995). In addition, HBV-infected patients under immunosuppressive therapy often have widespread virus gene expression in the liver and high levels of virus in the serum without liver disease; however, they often develop severe liver disease when immunosuppressive therapy is terminated and antiviral immune responses reappear (Bianchi and Gudat 1979). Furthermore, hepatitis B surface antigen (HBsAg¹) or HBV transgenic mice adoptively transferred with virus specific cytotoxic T lymphocytes (CTLs¹) have been shown to develop either acute or fulminant hepatitis (Ando et al. 1993; Moriyama et al. 1990), as in human infections (Figure 1). In addition, HBV transgenic severe combined immunodeficient mice adoptively transferred with normal, syngeneic splenocytes develop either acute or chronic hepatitis (Larkin et al. 1999). Moreover, strong and rapid cell-mediated immune responses to multiple HBV antigens are characteristic of acute infections that resolve, whereas weak immune responses to few HBV antigens are characteristic of infections that become chronic (Chisari and Ferrari 1995; Feitelson 1989, 1996) (Figure 1). Hence, the pathogenesis of HBV is immune mediated, although the virus antigens and corresponding immune responses responsible for acute versus chronic liver disease, and that permit the persistence of virus during chronic infections, have not been clearly identified.

Flaviviruses tend to mediate pathology by CPE (Chambers et al. 1990), although the case is not so clear cut with HCV. The finding of severe cholestatic hepatitis in a subset of HCV-infected liver transplant patients (Collier and Heathcote 1998), and that HCV-associated CLD appears to be more severe in human immunodeficiency virus-infected patients compared with those without dual infections (Thomas et al. 1996), suggest CPE. This is because both types of patients are immunosuppressed. On the cellular level, the rounding and shrinkage of hepatocytes, alterations in cellular membranes, and the development of pyknotic nuclei in HCV-infected livers are also consistent with CPE (Omata et al. 1981). However, lack of liver pathology and elevated liver enzymes (transaminases) in the blood of acutely infected chimpanzees during the incubation period of infection, and the persistence of virus in chimpanzees and patients in the absence of liver disease (Bradley 2000), suggest that HCV is not directly cytopathic. Moreover, the development of lymphoid aggregates and follicles in the liver, bile duct damage, and activated sinusoidal inflammatory cells (Bronkhorst and ten Kate 1998), suggest that the lesions associated with chronic HCV infection are mediated by immune responses against virus-infected hepatocytes. Additional work has shown that T helper cells and CTLs are important effector cells in the pathogenesis of HCV (Diepolder et al. 1998), further supporting the likelihood of immune-mediated pathogenesis. However, the balance of the data for HCV does not exclude that some aspects of pathogenesis could be due to CPE while other aspects may be immune mediated (Figure 2). These features are important not only for the design of relevant animal models but also for the selection of single and multiple therapeutic approaches that are likely to target the underlying causes of CLD.

Transgenic Mice for HBV

The development of transgenic mouse technology, which permitted insertion of any gene(s) of interest into fertilized mouse ova, made possible the construction of mice that expressed the inserted sequences so that the function and consequences of transgene expression could be studied in vivo (Palmiter and Brinster 1985). This development provided opportunities to create easily manipulated laboratory animal models of HBV and HCV gene expression, replication, and disease. In 1985, two research groups created transgenic mice that produced the HBV envelope or HBsAg particles (Babinet et al. 1985; Chisari et al. 1985) encoded by the HBV surface antigen gene (Figure 3). These subviral particles lacked viral DNA and had the same morphology, diameter, density, and polypeptide composition as HBsAg particles from infected human serum samples. Moreover, mice were tolerant to HBsAg so that, despite consistently high levels of circulating HBsAg, no liver disease was detected. Independently derived lines of HBsAg producing transgenic mice revealed preferential expression in the liver and kidneys (Burk et al. 1988). Other lines of transgenic mice were made and shown to fully support HBV replication; in all cases, no liver disease was observed (Araki et al. 1989; Farza et al. 1988; Guidotti et al. 1995). This further suggested that HBV was not directly cytopathic. However, overexpression of HBsAg polypeptides containing preS sequences (Figure 3) resulted in their retention and accumulation in the liver of transgenic mice (Chisari et al. 1986). As mice aged and HBsAg continued to accumulate, it became toxic to the hepatocytes, causing chronic liver injury and inflammation, regenerative hyperplasia, transcriptional deregulation, aneuploidy, and finally the appearance of HCC (Chisari et al. 1989; Dunsford et al. 1990). Although there is no evidence that the overexpression of HBsAg causes toxic liver injury in human carriers, these studies highlighted the importance of chronic liver cell injury and hepatocellular regeneration to the pathogenesis of HCC. In this sense, these mice provide an explanation for the epidemiological findings that the most important risk factors for HCC development are the HBsAg carrier state and the presence and progression of CLD (Beasley et al. 1981) (Figure 1). HCC also developed in transgenic mice with sustained high levels of hepatitis B x antigen (HBxAg) in the liver (Kim et al. 1991; Koike et al. 1994; Ueda et al. 1995) in the absence of inflammatory liver disease, but not in transgenic mice with low or undetectable levels of HBxAg (Lee et al. 1990; Reifenberg et al. 1997). These data suggest that HBxAg is oncogenic. Mechanistic studies have shown that HBxAg binds to and inactivates the tumor suppressor p53 (Ueda et al. 1995), which contributes importantly to stepwise carcinogenesis. In addition, HBxAg appears to stimulate the production of transformation growth factor beta 1 (Yoo et al. 1996), which may contribute to pathogenesis through the triggering of increased hepatocellular apoptosis and the promotion of fibrogenesis.

The findings that persistently high levels of HBsAg in human carriers and transgenic mice do not result in the development of liver disease (Hoofnagle et al. 1987), and that immunosuppression ameliorates chronic hepatitis (Cote et al. 1991), suggest that liver cell damage may be immune mediated (Feitelson 1996). This is also implied by the finding that HBV replicates without triggering CPE in tissue culture cells (Sells et al. 1987; Sureau et al. 1986). To test whether liver disease is immune mediated, clones of HBsAg-specific CTLs were generated and then used for adoptive transfer into HBsAg transgenic mice. The results showed a clearance of HBsAg from blood and a spike of alanine transaminase within a few days after adoptive transfer. Liver cell injury was confirmed histologically and was major histocompatibility complex class I restricted (Moriyama et al. 1990), suggesting that the adoptive transfer of HBsAg-primed CTLs resulted in an immune-mediated bout of acute hepatitis. Liver disease was dependent on the production of interferon gamma (IFN-g ) by the CTL, the intrahepatic levels of HBsAg expression, and the number of HBsAg positive hepatocytes within the liver. A more detailed characterization of events over time revealed that initially, injected CTLs were associated with an increase in apoptosis among scattered hepatocytes. This was followed by the recruitment of antigen-nonspecific inflammatory cells, and then by the development of necroinflammatory foci that extended beyond the region where CTLs were present (Chisari and Ferrari 1995). Among mice that secreted HBsAg into blood, the disease was transient, nonfatal, and destroyed no more than 5% of the hepatocytes. However, when adoptive transfer was performed in transgenic mice that retained HBsAg, the adoptively transferred CTLs were activated to produce IFN-g, which in turn activated intrahepatic macrophages. These and other antigen nonspecific cells that were recruited into the liver displayed a delayed type of hypersensitivity response that resulted in the rapid development of fulminant hepatitis and the death of many animals (Ando et al. 1993). Immune-mediated acute hepatitis was also independently observed in rats transfected with a replication-competent clone of HBV DNA. When parallel experiments were conducted in normal rats, HBV DNA appeared in serum within a few days, followed by clearance of virus, a transient elevation of alanine transaminase in blood, and histopathological evidence of acute hepatitis in the liver. When the same experiment was conducted in T cell-deficient nude rats, no clearance of virus or development of liver disease was observed, suggesting that T lymphocytes play a central role in liver cell injury and the clearance of HBV (Takahashi et al. 1995). These findings independently confirm the immune-mediated nature of fulminant and acute hepatitis (Figure 1) and provide information regarding the putative mechanisms of disease.

Although this work established models of acute and fulminant hepatitis, the major clinical problem resides among carriers with CLD (Figure 1). Accordingly, adoptive transfer experiments were carried out in HBsAg-overproducing transgenic mice that did not secrete antigen. To overcome tolerance to HBsAg, these mice were irradiated and thymectomized, and reconstituted with T cel-depleted bone marrow before adoptive transfer. The results showed that the inflammatory response set up after adoptive transfer accelerated hepatocellular turnover and the appearance of HCC (Nakamoto et al. 1998), suggesting that even the development of HCC has an immune-mediated component. However, there is little evidence to suggest that cell-mediated immune responses against HBsAg are central because it is not clear that HBsAg is an immunological target in chronic human infections. For example, only a small percentage of HBsAg carriers develop CLD and HCC. Furthermore, there is no evidence that HBsAg is overexpressed in chronically infected human livers to the levels observed in the HBsAg trangenic mice (Ray 1979). In addition, there is no correlation between intrahepatic HBsAg expression and inflammatory cells (Feitelson 1989). Hence, although immune-mediated liver disease contributes importantly to the development of HCC, these results do not reflect the range of immune responses that develop in chronically infected people who develop CLD and HCC.

There is increasing evidence from the HBsAg and HBV transgenic mouse systems that the clearance of virus gene expression and replication could be accomplished by noncytolytic mechanisms that involve the appropriately timed production of cytokines (Figure 1). Early observations showed that multiple HBsAg-specific CTL clones stimulated in vitro with plate bound anti-CD3e monoclonal antibodies produced IFN-g, tumor necrosis factor alpha (TNF-a), and to a lesser extent TNF-beta mRNAs in RNA protection assays (Ando et al. 1993; Guidotti et al. 1994a). The presence of these cytokine mRNAs correlated with the development of acute hepatitis after adoptive transfer, suggesting that they are made in vivo. Interestingly, the administration of monoclonal antibodies against IFN-g or TNF-a before adoptive transfer largely prevented the CTL-mediated reduction in HBsAg expression, suggesting a cytokine-mediated noncytopathic inhibition of virus gene expression. These cytokines have also been shown to inhibit virus replication in transgenic mice (Guidotti et al. 1996b). Interestingly, virus clearance has been observed without extensive hepatocellular necrosis in WHV-infected animals even though the great majority of hepatocytes in the woodchuck liver become infected (Kajino et al. 1994). The latter study implies noncytolytic clearance of virus in a natural infection. Such clearance is consistent with the idea that there are not enough virus-specific CTL precursors in the body to mediate direct cytotoxicity with every infected hepatocyte and that cytokines are needed to amplify antigen specific responses. In addition, the fact that HBV clearance is associated with clinical recovery in people, and not death, also suggests a noncytolytic mechanism of immunologically mediated antiviral defense. In noncytolytic clearance, cytokines cause reduced steady state levels of most viral mRNAs among infected hepatocytes (Tsai et al. 1995) by triggering the binding of three cellular proteins to the viral RNAs, which then results in HBV RNA destabilization and degradation (Heise et al. 1999). In HBsAg transgenic mice, reduced levels of intrahepatic HBV RNA were observed after treatment with TNF->a (Gilles et al. 1992) or interleukin 2 (Guidotti et al. 1994b, Guilhot et al. 1993). Moreover, administration of interleukin-12, which induces T and natural killer cells to produce IFN-g, inhibits virus replication and gene expression in transgenic mice that support virus production (Cavanaugh et al. 1997). Finally, when the latter mice were infected with lymphocytic choriomeningitis virus, the cytokines triggered by that virus activated macrophages in the liver (e.g., TNF-a and IFN-a/b) effectively suppressed HBV replication (Guidotti et al. 1996a). This result not only suggests that the extensive network of intrahepatic macrophages may play an important role in viral clearance, but also may partially explain the suppression of HBV replication in many HCV-coinfected patients (Liaw 1995).

The development of CLD in HBV carriers (Figure 1) is a major target for therapeutics, yet none of the HBV transgenic models available develop CLD because all transgenic mice are tolerant to the products of the transgene. In contrast, people who are acutely infected with HBV (or HCV) are immunologically naive to the virus. So the question becomes: Can one devise a transgenic system that supports virus replication but is not tolerant to the virus so that the development and progression of CLD can be studied? In a recent report (Larkin et al. 1999), transgenic mice supporting HBV replication were constructed using severe combined immunodeficient mice hosts that lacked mature T and B cells (Schuler et al. 1986). Because the T and B cell compartments account for the bulk of specific antiviral immunity, these transgenic mice were not tolerant to HBV. A single adoptive transfer of 10 million unprimed, syngeneic splenocytes resulted in the development of chronic hepatitis, whereas a similar transfer of 50 million cells resulted in a bout of acute, resolving hepatitis. Hepatitis was accompanied by mononuclear infiltrates resembling acute and chronic hepatitis in humans and with the clearance of virus gene expression and replicative forms from the liver as well as clearance of HBsAg and virus DNA from the blood. Although this model confirms the immune-mediated nature of HBV associated acute and chronic liver disease, it will permit dissection of the immune components and virus antigen targets that contribute to pathogenesis.

Transgenic Mice for HCV

The narrow host range and lack of suitable tissue culture systems for HCV have provided significant barriers to studying the basic biology of host/virus interactions (Figure 2), including pathogenesis, and for testing putative antiviral compounds. As a consequence, a number of groups have embarked on the development of transgenic models of HCV gene expression and replication. In one study, transgenic mice that had been made using the HCV envelope 1 (E1) and 2 (E2) genes (Koike et al. 1995) expressed the HCV envelope proteins in many organs, including the liver, but did not develop any liver disease in mice up to 16 mo of age. These findings suggested that the envelope proteins of HCV were not cytopathic (Koike et al. 1995). However, these mice developed sialadenitis resembling Sjögren's syndrome (Koike et al. 1997), an autoimmune disease affecting the salivary glands and associated with HCV infection in humans (Haddad et al. 1992). However, independent work revealed that transgenic mice expressing high levels of E2 developed no evidence of liver disease (Pasquinelli et al. 1997). Likewise, HCV core transgenic mice did not develop evidence of liver disease or HCC (Pasquinelli et al. 1997), suggesting that core was not directly cytopathic. Another transgenic mouse expressing E1, E2 plus core also showed no evidence of histopathology when evaluated at 6 mo of age (Kawamura et al. 1997). In contrast, a different lineage of transgenic mice making the envelope and core proteins of HCV developed spontaneous focal inflammation, hepatocellular necrosis and degeneration, and altered foci with mitotic figures by 10 mo of age, suggesting CPE (Honda et al. 1999). Alternatively, the independent production of core transgenic mice resulted in the development of steatosis (fatty liver) in mice older than 3 mo of age (Moriya et al. 1997). Steatosis is found in infected human liver and may reflect CPE. Surprisingly, when these mice were held beyond 16 mo of age, they developed adenomas containing fat droplets in the cytoplasm of hepatocytes, followed by the development of poorly differentiated HCC within the adenomatous nodules (Moriya et al. 1998). HCV core was detected in most adenomas and in HCCs in 25 to 30% of the animals from two separate lines of transgenic mice, suggesting that it is directly oncogenic. Although the differences between these and other core transgenic mice are not clear, the mice that develop liver pathology express sustained high levels of core protein. In the life cycle of HCV, core protein is found almost exclusively in the cytoplasm of infected cells. However, its location in the nuclei of hepatocytes in transgenic mice that develop tumors suggests that HCV core may act as a transcriptional regulator that promotes tumor development (Chang et al. 1998, Ray et al. 1995).

Attempts were made to put these observations into context with the construction of multiple transgenic lines that carried the full-length HCV cDNA and supported HCV replication (Matsuda et al. 1998). Although HCV mRNA and HCV core protein were detected in the liver of one line, the levels of expression were low. In addition, no consistent histological changes were observed in the liver, suggesting that HCV replication is not directly cytopathic in vivo. In this context, the question still remains as to whether the likely immune-mediated pathogenesis of HCV, as suggested by the clinical literature (Figure 2), can be reproduced in a transgenic model. To address this question, conditional expression of the HCV envelope and core genes was carried out in mice using the Cre/loxP system (Wakita et al. 1998). When transgene expression was turned on, the expression of both envelope and core proteins was detected in the liver within 1 wk after transgene activation. A transient influx of T cells into the liver was accompanied by a peak of transaminases. At that time, core was also detected in the serum, suggesting it was released by damaged hepatocytes. When transgene induction was repeated in T cell-depleted mice, there was normal histology and transaminase levels, suggesting the inflammatory response and hepatocellular damage were T cell mediated. By day 14 after transgene activation, core was cleared from serum and replaced by anti-core (Wakita et al. 1998). These observations provide strong evidence that the pathogenesis of HCV infection is immune mediated, which provides a starting point from which the host-virus relationship can be studied in more detail.

Other Rodent Models of HBV and HCV

HBV and HCV gene expression, replication, and liver disease have also been studied in a number of nontransgenic rodent systems. For example, HBsAg expression has been demonstrated in the liver of adult rats intrahepatically injected with liposomes that carry an expression plasmid encoding the preS plus S polypeptides (Figure 3) (Kato et al. 1991). Within 2 days of injection, HBsAg was detected in serum and preS antigen in the liver. Because these antigens became undetectable within a few days, animals were given injections again with liposomes containing recombinant plasmid on day 7. Animals seroconverted to anti-HBs and, by day 14, developed mononuclear cell infiltrates within the liver around regions of focal necrosis. Hence, this approach could be used to trigger a bout of acute hepatitis in rats. Using a similar approach, cationic liposomes were used to deliver full-length HCV cDNA under control of the beta-actin promoter (Takehara et al. 1995). Two days after intrabiliary administration of liposomes, full-length HCV RNA was detected in the liver by reverse transcriptase/polymerase chain reaction, and HCV core was observed by immunohistochemical staining in a few percent of scattered hepatocytes. Alternatively, an HCV core/E1 expression vector has been introduced into rats and mice as a conjugate that is recognized by the asialoglycoprotein receptor on hepatocytes (Yamamoto et al. 1995). After chloroquine treatment (to increase transfection efficiency) and intraportal vein injection, receptor-mediated uptake resulted in the accumulation of most radiolabeled expression plasmid within hepatocytes, indicating successful liver targeting. HCV core RNA was detected by reverse transcriptase/polymerase chain reaction, and core protein was observed by immunohistochemical staining. The presence of HCV core protein in a few percent of the hepatocytes is similar to what has been observed in some human infections. These findings suggest that the continued development of this approach may lead to the development of a model that will permit the dissection of host/virus interactions in more detail.

Immunodeficient mice have also been used for transplantation of normal or infected human hepatocytes with the intent of providing a fully permissive system in vivo both to study infection and to evaluate antiviral compounds. In early work, a trimera was made from beige/nude/X-linked immunodeficient (BNX) mice that were total body irradiated and repopulated with bone marrow cells from severe combined immunodeficient mice (Galun et al. 1995). HCV-infected liver fragments from infected patients were then transplanted under the kidney capsule. HCV RNA was detected in the sera of up to half of these mice for 10 to 50 days after transplantation of liver tissue by reverse transcriptase/polymerase chain reaction. Although this model clearly demonstrated that human hepatocytes and HCV production could be maintained in vivo, the short duration and low levels of viremia limit the application of this model. Independent efforts have shown that when human hepatocytes are mixed with Matrigel, which provides a three-dimensional matrix that supports hepatocellular survival, and the mixture is transplanted into nonobese diabetic/severe combined immunodeficient mice, long-term survival is observed (Ohashi et al. 2000). These cells were capable of being infected with HBV in vivo, as demonstrated by positive staining for core and surface antigens, and supported virus replication, as shown by the presence of viral replicative forms over several months. In an alternative approach, when human hepatocytes immortalized by simian virus 40 large T antigen were stably transfected with HBV DNA and then transplanted into the spleen of Rag-2 immunodeficient mice (which lacked both T and B cells), human cells migrated to and fully integrated into the mouse liver parenchyma. These cells remained nontumorigenic, survived for at least 5 mo in the mouse liver, and produced high levels of viral DNA and detectable HBsAg in blood during that time. This model has provided a larger window for antiviral drug screening than other models that rely on transplantation of human hepatoma cells (Condreay et al. 1994) or woodchuck hepatocytes (Petersen et al. 1998) into immunodeficient mice, in which the hosts are killed by the outgrowth of tumor. However, none of the hepatocellular transplantation models are constructed to permit the development of immune-mediated liver pathogenesis, which remains a significant limitation.

Conclusions

There is considerable evidence that the pathogenesis of HBV and HCV infections is immune mediated. The challenge has been to develop easily manipulated animal models to study these human virus infections and, in particular, to understand the pathogenesis of disease as well as development of HCC. Although progress has been made in this direction with the development of several transgenic mouse models as well as several nontransgenic systems, the generation of additional models that encompass a broader range of host-virus interactions is required, especially with regard to the pathogenesis of CLD. Such models should be very useful for understanding the mechanisms of pathogenesis and for evaluating new therapeutic approaches aimed at both the virus and associated chronic liver diseases.

Acknowledgments

This work was supported by National Institutes of Health grants CA79512 and AI-99-001.

¹Abbreviations used in this article: CLD, chronic liver disease; CPE, direct cytopathic effects; CTL, cytotoxic T lymphocytes; DHBV, duck hepatitis B virus; HBsAg, hepatitis B virus surface antigen; HBxAg, hepatitis B x antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IFN-g: interferon gamma; TNF-a: tumor necrosis factor alpha.

References

Alter HJ, Purcell RH, Holland PV, Popper H. 1978. Transmissible agent in non-A, non-B hepatitis. Lancet 1:459-463.

Alter MJ. 1996. Epidemiology and disease burden of hepatitis B and C. Antiviral Ther 1(Suppl 1):9-14.

Ando K, Moriyama T, Guidotti LG, Wirth S, Schreiber RD, Schlicht HJ, Huang SN, Chisari FV. 1993. Mechanisms of class 1 restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med 178:1541-1554.

Araki K, Miyazaki J, Hino O, Tomita N, Chisaka O, Matsubara K, Yamamura K. 1989. Expression and replication of hepatitis B virus genome in transgenic mice. Proc Natl Acad Sci U S A 86:207-211.

Babinet C, Farza H, Morello D, Hadchouel M, Pourcel C. 1985. Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice. Science 230:1160-1163.

Beasley RP, Hwang LY, Lin CC, Chien CS. 1981. Hepatocellular carcinoma and HBV: A prospective study of 22,707 men in Taiwan. Lancet 2:1129-1133.

Bianchi L, Gudat F. 1979. Immunopathology of hepatitis B. In: Popper H, Schaffner F, editors. Progress in Liver Disease. Vol. 4. San Francisco: Grune and Stratton. p. 371-392.

Bradley DW. 2000. Studies of non-A, non-B hepatitis and characterization of the hepatitis C virus in chimpanzees. Curr Top Microbiol Immunol 242:1-23.

Bradley DW, Cook EH, Maynard JE, McCaustland KA, Ebert, JW, Dolana GH, Petzel RA, Kantor RJ, Heilbrunn A, Fields HA, Murphy BL. 1979. Experimental infection of chimpanzees with anti-hemophilic (factor VIII) materials. Recovery of virus-like particles associated with non-A, non-B hepatitis. J Med Virol 3:253-269.

Bronkhorst CM, ten Kate FJW. 1998. Liver histology in hepatitis C. In: Reesink HW, ed. Hepatitis C Virus. Curr Stud Hematol Blood Transf. Basel: Karger. p 119-134.

Burk RD, DeLoia JA, ElAwady MK, Gearhart JD. 1988. Tissue preferential expression of the hepatitis B virus (HBV) surface antigen gene in two lines of HBV transgenic mice. J Virol 62:649-654.

Cavanaugh VJ, Guidotti LG, Chisari FV. 1997. Interleukin-12 inhibits hepatitis B virus replication in transgenic mice. J Virol 71:3236-3243.

Chambers TJ, Hahn CS, Galler R, Rice CM. 1990. Flavivirus genome organization, expression, and replication. Ann Rev Microbiol 44

:649-688.

Chang J, Yang SH, Cho YG, Hwang SB, Hahn YS, Sung YC. 1998. Hepatitis C virus from two different genotypes has an oncogenic potential but is not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene. J Virol 72:3060-3065.

Chisari FV, Ferrari C. 1995. Hepatitis B virus immunopathogenesis. Ann Rev Immunol 13:29-60.

Chisari FV, Filippi P, McLachlan A, Milich DR, Riggs M, Lee S, Palmiter RD, Pinkert CA, Brinster RL. 1986. Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol 60:880-887.

Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, Sell S, Pinkert CA, Brinster RL, Palmiter RD. 1989. Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59:1145-1156.

Chisari FV, Pinkert CA, Milich DR, Filippi P, McLachlan A, Palmiter RD, Brinster RL. 1985. A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science 230:1157-1160.

Colledge D, Locarnini S, Shaw T. 1997. Synergistic inhibition of hepadnaviral replication by lamivudine in combination with penciclovir in vitro. Hepatology 26:216-225.

Collier J, Heathcote J. 1998. Hepatitis C viral infection in the immunosuppressed patient. Hepatology 27:2-6.

Condreay LD, Jansen RW, Powdrill TF, Johnson LC, Selleseth DW, Paff MT, Daluge SM, Painter GR, Furman PA, Ellis MN. 1994. Evaluation of the potent anti-hepatitis B virus agent (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine in a novel in vivo model. Antimicrob Agents Chemother 38:616-619.

Cote PJ, Korba BE, Steinberg H, Ramirez-Mejia C, Baldwin B, Hornbuckle WE, Tennant BC, Gerin JL. 1991. Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity. J Immunol 146:3138-3144.

Delwaide J, Gerard C. 2000. Evidence-based medicine treatment of chronic hepatitis C. Liege Study Group on Viral Hepatitis. Revue Medicale de Liege 55:337-340.

Diepolder HM, Hoffmann RM, Gerlach JT, Zachoval R, Jung MC, Pape GR. 1998. Immunopathogenesis of HCV infection. In: Reesink HW, ed. Hepatitis C Virus. Curr Stud Hematol Blood Transf. Basel: Karger. p 135-151.

Dunsford HA, Sell S, Chisari FV. 1990. Hepatocarcinogenesis due to chronic liver cell injury in hepatitis B virus transgenic mice. Cancer Res 50:3400-3407.

Dusheiko G. 1999. Lamivudine therapy for hepatitis B infection. Scand J Gastroenterol 34(Suppl 230):76-81.

Esquivel CO, Keeffe EB, Garcia G, Imperial JC, Millan MT, Monge H, So SK. 1999. Resection versus transplantation for hepatocellular carcinoma. J Gastroenterol Hepatol 14(Suppl):37-41.

Farza H, Hadchouel M, Scotto J, Tiollais P, Babinet C, Pourcel C. 1988. Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome. J Virol 62:4144-4152.

Feitelson MA. 1989. Hepatitis B virus gene products as immunological targets in chronic infection. Mol Biol Med 6:367-393.

Feitelson MA. 1996. Hepatocellular injury in hepatitis B and C virus infections. In: Feitelson MA, Zern MA, ed. Clinics in Laboratory Medicine: Hepatitis and Chronic Liver Disease. Philadelphia: WB Saunders. p 307-324.

Ferrari C, Penna A, Giuberti T, Tong MJ, Ribera E, Fiaccadori F, Chisari FV. 1987. Intrahepatic, nucleocapsid antigen-specific T cells in chronic active hepatitis B. J Immunol 139:2050-2058.

Fourel G, Couturier J, Wei Y, Apiou F, Tiollais P, Buendia MA. 1994. Evidence for long-range oncogene activation by hepadnavirus insertion. EMBO J 13:2526-2534.

Galun E, Burakova T, Ketzinel M, Lubin I, Shezen E, Kahana Y, Eid A, Ilan Y, Rivkind A, Pizov G, Shouval D, Reisner Y. 1995. Hepatitis C virus viremia in SCIDà BNX mouse chimera. J Infect Dis 172:25-30.

Gerlich WH, Caspari G. 1999. Hepatitis viruses and the safety of blood donations. J Viral Hepat 6(Suppl 1):6-15.

Gilles PN, Fey G, Chisari FV. 1992. Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice. J Virol 66:3955-3960.

Guidotti LG, Ando K, Hobbs MV, Ishikawa T, Runkel L, Schreiber RD, Chisari FV. 1994a. Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice. Proc Natl Acad Sci U S A 91:3764-3768.

Guidotti LG, Borrow P, Hobbs MV, Matzke B, Gresser I, Oldstone MBA, Chisari FV. 1996a. Viral cross talk: Intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc Natl Acad Sci U S A 93:4589-4594.

Guidotti LG, Guihot S, Chisari FV. 1994b. Interleukin-2 and interferon alpha/beta downregulate hepatitis B virus gene expression in vivo by tumor necrosis factor dependent and independent pathways. J Virol 68:1265-1270.

Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, Chisari FV. 1996b. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 4:25-36.

Guidotti LG, Matzke B, Schaller H, Chisari FV. 1995. High level hepatitis B virus replication in transgenic mice. J Virol 69:6158-6169.

Guilhot S, Guidotti LG, Chisari FV. 1993. Interleukin-2 downregulates hepatitis B virus gene expression in transgenic mice by a post-transcriptional mechanism. J Virol 67:7444-7449.

Haddad J, Deny P, Munz-Gotheil C, Ambrosini JC, Trinchet JC, Pateron D, Mal F, Callard P, Beaugrand M. 1992. Lymphocytic sialadenitis of Sjögren's syndrome associated with chronic hepatitis C virus liver disease. Lancet 339:321-323.

Heise T, Guidotti LG, Cavanaugh VJ, Chisari FV. 1999. Hepatitis B virus RNA binding proteins associated with cytokine induced clearance of viral RNA from the liver of transgenic mice. J Virol 73:474-481.

Honda A, Arai Y, Hirota N, Sato T, Ikegaki J, Koizumi T, Hatano M, Kohara M, Moriyama T, Imawari M, Shimotohno K, Tokuhisa T. 1999. Hepatitis C virus structural proteins induce liver cell injury in transgenic mice. J Med Virol 59:281-289.

Honkoop P, Niesters HGM, De Man RAM, Osterhaus ADME, Schalm SW. 1997. Lamivudine resistance in immunocompetent chronic hepatitis B---Incidence and patterns. J Hepatol 26:1393-1395.

Hoofnagle JH. 1997. Hepatitis C: The clinical spectrum of disease. Hepatology. 26(Suppl 1):15-20.

Hoofnagle JH, Lau D. 1996. Chronic viral hepatitis---Benefits of current therapies. N Engl J Med 334:1470-1471.

Hoofnagle JH, Shafritz DA, Popper H. 1987. Chronic type B hepatitis and the "healthy" HBsAg carrier state. Hepatology 7:758-763.

Hsu T, Moroy T, Etiemble J, Louise A, Trepo C, Tiollais P, Buendia MA. 1988. Activation of c-myc by woodchuck hepatitis virus insertion in hepatocellular carcinoma. Cell 55:627-635.

Hurwitz SJ, Tennant BC, Korba BE, Gerin JL, Schinazi RF. 1998. Pharmacodynamics of (-)-b -2',3'-dideoxy-3'-thiacytidine in chronically virus-infected woodchucks compared to its pharmacodynamics in humans. Antimicrob Agents Chemother 42:2804-2809.

Kajino K, Jilbert AR, Saputelli J, Aldrich C, Cullen J, Mason WS. 1994. Woodchuck hepatitis virus infections: Very rapid recovery after a prolonged viremia and infection of virtually every hepatocyte. J Virol 68:5792-5803.

Kato K, Kaneda Y, Sakurai M, Nakanishi M, Okada Y. 1991. Direct injection of hepatitis B virus DNA into liver induced hepatitis in adult rats. J Biol Chem 268:22071-22074.

Kawamura T, Furusaka A, Koziel MJ, Chung RT, Wang TC, Schmidt EV, Liang TJ. 1997. Transgenic expression of hepatitis C virus structural proteins in the mouse. Hepatology 25:1014-1021.

Kim CM, Koike K, Saito I, Miyamura T, Jay G. 1991. HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351:317-320.

Koike K, Moriya K, Iino S, Yotsuyanagi H, Endo Y, Miyamura T. Kurokawa K. 1994. High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology 19:810-819.

Koike K, Moriya K, Ishibashi K, Matsuura Y, Suzuki T, Saito I, Iino, S, Kurokawa K, Miyamura T. 1995. Expression of hepatitis C virus envelope proteins in transgenic mice. J Gen Virol 76:3031-3038.

Koike K, Moriya K, Ishibashi K, Yotsuyanagi H, Shintani Y, Fujie H, Kurokawa K, Matsuura Y, Miyamura T. 1997. Sialadenitis histologically resembling Sjögren syndrome in mice transgenic for hepatitis C virus envelope genes. Proc Natl Acad Sci U S A 94:233-236.

Korba BE, Brown TL, Wells FV, Baldwin B, Cote PJ, Steinberg H, Tennant BC, Gerin JL. 1990. Natural history of experimental woodchuck hepatitis virus infection: Molecular virologic features of the pancreas, kidney, ovary and testis. J Virol 64:4499-4506.

Korba BE, Cote PJ, Wells FV, Baldwin B, Popper H, Purcell RH, Tennant BC, Gerin JL. 1989. Natural history of woodchuck hepatitis virus infection during the course of experimental viral infection: Molecular virologic features of the liver and lymphoid tissues. J Virol 63:1360-1370.

Larkin J, Clayton MM, Sun B, Perchonock CE, Morgan JL, Siracusa LD, Michaels F, Feitelson MA. 1999. Hepatitis B virus transgenic SCID mouse model of chronic liver disease. Nat Med 5:907-912.

Lee TH, Finegold MJ, Shen RF, DeMayo JL, Woo SL, Butel JS. 1990. Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice. J Virol 64:5939-5947.

Liaw YF. 1995. Role of hepatitis C virus in dual and triple hepatitis virus infection. Hepatology 22:1101-1108.

Lindsay KL. 1997. Therapy of hepatitis C: Overview. Hepatology 26(Suppl 1):71-77.

Maddrey WC. Hepatitis B: An important public health issue. 2000. J Med Virol 61:362-366.

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

Marinos G, Torre F, Chokshi S, Hussain M, Clarke BE, Rowlands DJ, Eddleston ALWF, Naoumov NV, Williams R. 1995. Induction of T-helper cell response to hepatitis B core antigen in chronic hepatitis B: A major factor in activation of the host immune response to the hepatitis B virus. Hepatology 22:1040-1049.

Marion PL, Oshiro LS, Regnery DC, Scullard GH, Robinson WS. 1980. A virus in Beechey ground squirrels that is related to hepatitis B virus of humans. Proc Natl Acad Sci U S A 77:2941-2945.

Mason WS, Cullen J, Saputelli J, Wu TT, Liu C, London WT, Lustbader E, Schaffer P, O'Connell AP, Fourel G, Aldrich C, Jilbert AR. 1994. Characterization of the antiviral effects of 2' carbodeoxyguanosine in ducks chroncally infected with duck hepatitis B virus. Hepatology 19:398-411.

Mason WS, Seal G, Summers J. 1980. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J Virol 36:829-836.

Mast EE, Williams IT, Alter MJ, Margolis HS. 1998. Hepatitis B vaccination of adolescent and adult high-risk groups in the United States. Vaccine 16(Suppl):27-29.

Matsuda J, Suzuki M, Nozaki C, Shinya N, Tashiro K, Mizuno K, Uchinuno Y, Yamamura K. 1998. Transgenic mouse expressing a full length hepatitis C virus cDNA. Jpn J Cancer Res 89:150-158.

Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T, Ishibashi K, Matsuura Y, Kimura S, Miyamura T, Koike K. 1998. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 4:1065-1067.

Moriya K, Yotsuyanagi H, Shintani Y, Fujie H, Ishibashi K, Matsuura Y, Miyamura T, Koike K. 1997. Hepatitis C virus core protein induced hepatic steatosis in transgenic mice. J Gen Virol 78:1527-1531.

Moriyama T, Guilhot S, Klopchin K, Moss B, Pinkert CA, Palmiter RD, Brinster RL, Kanagawa O, Chisari FV. 1990. Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science 248:361-364.

Nakamoto Y, Guidotti LG, Kuhlen CV, Fowler P, Chisari FV. 1998. Immune pathogenesis of hepatocellular carcinoma. J Exp Med 188:341-350.

Neyts J, Leyssen P, De Clercq E. 1999. Infections with flaviviridae. Verhandelingen-Koninklijke Academie voor Geneeskunde van Belgie 61:661-699.

Ohashi K, Marion PL, Nakai H, Meuse L, Cullen JM, Bordier BB, Schwall R, Greenberg HB, Glenn JS, Kay MA. 2000. Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses. Nature Med 6:327-331.

Omata M, Iwama S, Sumida M, Ito Y, Okuda K. 1981. Clinicopathological study of acute non-A, non-B post transfusion hepatitis: Histological features of liver biopsies in acute phase. Liver 1:201-208.

Palmiter RD, Brinster RL. 1985. Transgenic mice.

Cell 41:343-345.

Pasquinelli C, Shoenberger JM, Chung J, Chang KM, Guidotti LG, Selby M, Berger K, Lesniewski R, Houghton M, Chisari FV. 1997. Hepatitis C virus core and E2 protein expression in transgenic mice. Hepatology 25:719-727.

Petersen J, Dandri M, Supta S, Rogler CE. 1998. Liver repopulation with xenogeneic hepatocytes in B and T cell deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma. Proc Natl Acad Sci U S A 95:310-315.

Popper H, Roth L, Purcell RH, Tennant BC, Gerin JL. 1987. Hepatocarcinogenicity of the woodchuck hepatitis virus. Proc Natl Acad Sci U S A 84:866-870.

Prince AM, Brotman B. 1998. Biological and immunolgical aspects of hepatitis C virus infection in chimpanzees. In: Reesink HW, ed. Hepatitis C Virus. Curr Stud Hematol Blood Transf. Basel: Karger. p 250-265.

Pugh JC, Summers J. 1989. Infection and uptake of duck hepatitis B virus by duck hepatocytes maintained in the presence of dimethyl sulfoxide. Virology 172:564-572.

Ray MB. 1979. Hepatitis B Virus Antigens in Tissues. Baltimore: University Park Press.

Ray RB, Lagging LM, Meyer K, Steele R, Ray R. 1995. Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Res 37:209-220.

Reichard O, Schvarcz R, Weiland O. 1997. Therapy of hepatitis C: Alpha interferon and ribavirin. Hepatology 26(Suppl 1):108-111.

Reifenberg K Lohler J, Pudollek HP, Schmitteckert E, Spindler G, Kock J. Schlicht HJ. 1997. Long-term expression of the hepatitis B virus core-e- and X-proteins does not cause pathologic changes in transgenic mice. J Hepatol 26:119-130.

Robinson WS, Marion P, Feitelson MA, Siddiqui A. 1982. The hepadna virus group: Hepatitis B and related viruses. In: Szmuness W, Alter HJ, Maynard JE, eds. Viral Hepatitis, 1981 International Symposium, Philadelphia: Franklin Institute Press. p. 57-68.

Schuler W, Weiler IJ, Schuler A, Phillips RA, Rosenberg N, Mak TW, Kearney JF, Perry RP, Bosma MJ. 1986. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell 46:963-972.

Schultz U, Chisari FV. 1999. Recombinant duck interferon gamma inhibits duck hepatitis B virus replication in primary hepatocytes. J Virol 73:3162-3168.

Sells MA, Chen ML, Acs G. 1987. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 84:1005-1009.

Smyth R, Keenan E, Dorman A, O'Connor J. 1995. Hepatitis C infection among injecting drug users attending the National Drug Treatment Centre. Israel J Med Sci 164:267-268.

Snyder RL, Tyler G, Summers J. 1982. Chronic hepatitis and hepatocellular carcinoma associated with woodchuck hepatitis virus. Am J Pathol 107:422-425.

Summers J, Mason WS. 1982. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29:403-415.

Summers J, Smolec JM, Snyder R. 1978. A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci U S A 75:4533-4537.

Sureau C, Romet-Lemonne JL, Mullins JI, Essex M. 1986. Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 47:37-47.

Tabor E, Purcell RH, Gerety RJ. 1983. Primate animal models and titered inocula for the study of human hepatitis A, hepatitis B, and non-A, non-B hepatitis. J Med Primatol 12:305-318.

Takahashi H, Fujimoto J, Hanada S, Isselbacher KJ. 1995. Acute hepatitis in rats expressing human hepatitis B virus transgenes. Proc Natl Acad Sci U S A 92:1470-1474.

Takehara T, Hayashi N, Miyamoto Y, Yamamoto M, Mita E, Fusamoto H, Kamada T. 1995. Expression of the hepatitis C virus genome in rat liver after cationic liposome-mediated in vivo gene transfer. Hepatology 21:746-751.

Thomas DL, Shih JW, Alter HJ, Vlahov D, Cohn S, Hoover DR, Cheung L, Nelson KE. 1996. Effect of human immunodeficiency virus on hepatitis C virus infection among injection drug users. J Infect Dis 174:690-695.

Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DLJ. 1996. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 24:714-717.

Torresi J, Locarnini S. 2000. Antiviral chemotherapy for the treatment of hepatitis B virus infections. Gastroenterology 118(Suppl):83-103.

Tsai LV, Guidotti LG, Ishkawa T, Chisari FV. 1995. Posttranscriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocytes-activated hepatocytes. Proc Natl Acad Sci U S A 92:12398-12402.

Ueda H, Ullrich SJ, Gangemi JD, Kappel CA, Ngo L, Feitelson MA, Jay G. 1995. Functional inacativation but not structural mutation of p53 causes liver cancer. Nat Genet 9:41-17.

Wakita T, Taya C, Katsume A, Kato, J, Yonekawa H, Kanegae Y, Saito I, Hayashi Y, Koike M, Kohara M. 1998. Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system. J Biol Chem 273:9001-9006.

Wild CP, Hall AJ. 2000. Primary prevention of hepatocellular carcinoma in developing countries. Mutation Res 462:381-393.

Xie ZC, Riezu-Boj JI, Lasarte JJ, Guillen J, Su JH, Civeira MP, Prieto J. 1998. Transmission of hepatitis C virus infection to tree shrews. Virology 244:513-520.

Yamamoto M, Hayashi N, Miyamoto Y, Tekehara T, Mita E, Seki M, Fusamoto H, Kamada T. 1995. In vivo transfection of hepatitis C virus complementary DNA into rodent liver by asialoglycoprotein receptor mediated gene delivery. Hepatology 22:847-855.

Yan RQ, Su JJ, Huang DR, Gan YC, Yang C, Huang GH. 1996a. Human hepatitis B virus and hepatocellular carcinoma. I. Experimental infection of tree shrews with hepatitis B virus. J Cancer Res Clin Oncol 122:283-288.

Yan RQ, Su JJ, Huang DR, Gan YC, Yang C, Huang GH. 1996b. Human hepatitis B virus and hepatocellular carcinoma. II. Experimental induction of hepatocellular carcinoma in tree shrews exposed to hepatitis B virus and aflatoxin b1. J Cancer Res Clin Oncol 122:289-295.

Yoakum GH, Korba BE, Lechner JF, Tokiwa T, Gazdar AF, Seeley T, Siegel M, Leeman L, Autrup H, Harris CC. 1983. High frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells. Science 222:385-389.

Yoo YD, Ueda H, Park K, Flanders KC, Lee YI, Jay G, Kim SJ. 1996. Regulation of transforming growth factor-beta 1 expression by the hepatitis B virus (HBV) X transactivator. Role in HBV pathogenesis. J Clin Invest 97:388-395.

Figure 1 Pathogenesis and outcomes of hepatitis B virus (HBV) infection. Upon acute exposure to HBV, the majority of adults (~65%) develop a subclinical infection that is indicated only by the appearance of one or more viral antibodies. Another roughly 25% of infected adults develop a strong T helper type 1 (TH1) cytokine and CTL response, which results in a bout of acute hepatitis followed by resolution and recovery. Rarely, an acute infection develops rapidly into life-threatening fulminant hepatitis. Approximately 10% of acutely infected adults develop a chronic infection, which is characterized by the persistance of HBsAg and (in some patients) virus in blood. Although most chronic carriers remain asymptomatic for years or decades, they are at great risk for the development of chronic hepatitis, cirrhosis, and hepatocellular. Among those who develop liver disease, progression is variable, with some patients developing end-stage liver disease or hepatocellular within a few years, while others undergo regression of histological lesions in the liver at any stage of chronic liver disease.

Figure 2 Pathogenesis and outcomes of hepatitis C virus (HCV) infection. Acute HCV infection has two major outcomes. Patients who develop strong, rapid, multispecific T cell responses often experience either a clinical or subclinical bout of acute hepatitis, followed by resolution, or no liver disease at all. However, the majority of acutely infected patients develop persistent viremia, frequently characterized by the appearance of chronic hepatitis. The progression or development of cirrhosis during chronic infection depends on the characteristics of the immune responses that develop, with more vigorous sustained immune responses associated with increased liver damage and disease progression. Alternatively, the triggering of weak cell-mediated immune responses after acute infection, or during the course of chronic infection, would favor the development of virus escape mutants that would persist. CTL, cytotoxic T lymphocyte; HCC, hepatocellular carcinoma. Modified from Hoofnagle JH. 1997. Hepatitis C: The clinical spectrum of disease. Hepatology 26(Suppl 1):15-20.

Figure 3 The genome of hepatitis B virus (HBV). As it exists in the virion, the 3.2-kb-long genome of HBV is a partially double-stranded structure. The long or minus strand is uniform in length and has a nick at a unique site. The short or plus strand has a unique 5' end but a variable 3' end that is different in individual virus particles. The minus strand encodes all of the virus proteins from four overlapping open reading frames (ORFs). One ORF encodes the major surface antigen (HBsAg) polypeptide (226 amino acids long; sometimes called small S) and two larger but minor HBsAg polypeptides. Among the latter, one contains the preS2/S sequences (middle S) and the other contains preS1/preS2/S sequences (large S). All of these polypeptides are found in the envelope of the virus and in subviral particles. A second ORF encodes the major nucleocapsid polypeptide, which has HBV core antigen (HBcAg) activity. A proteolytic fragment of the core polypeptide is the hepatitis B e antigen (HBeAg), which is a soluble polypeptide in blood that is a surrogate marker of virus replication. A third ORF encodes the polymerase, which is responsible for virus replication. Minus strand synthesis begins at direct repeat 1 (DR1) and plus strand at DR2. A fourth ORF encodes hepatitis B ´ antigen, which regulates virus gene expression and replication and is involved in the development of hepatocellular carcinoma.