Antiviral activity of clevudine [L-FMAU, (1-(2-fluoro-5-methyl-beta, L-arabinofuranosyl) uracil)] against woodchuck hepatitis virus replication and gene expression in chronically infected woodchucks (Marmota monax)

L: -FMAU [1-(2-fluoro-5-methyl-beta,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of hepatitis B virus (HBV) and duck hepatitis B virus replication in cell culture and duck hepatitis B virus replication in acutely infected Peking ducks. The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of woodchuck hepatitis virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection.     

Animal models of hepatocellular carcinoma: hepadnavirus-induced liver cancer in woodchucks.

Woodchuck hepatitis virus (WHV), a member of the Hepadnaviridae, is closely related to hepatitis-B virus (HBV) in its virus structure, genetic organization, and mechanism of replication. As with HBV in man, persistent WHV infection is common in natural woodchuck populations and is associated with chronic hepatitis and hepatocellular carcinoma (HCC). Experimental studies have established that WHV causes HCC in woodchucks. Chronic WHV carriage as an outcome of experimental infection is a function of animal age at time of exposure, virus dose, and, possibly, virus strain. Almost all (97%) chronic carriers developed histologically confirmed HCC within 3 years while no HCC developed in uninfected animals held concurrently in the same laboratory setting. The model has application in the study of underlying mechanisms of hepadnavirus-induced hepatocarcinogenesis and to the development of prophylactic and therapeutic strategies of disease control.     

Treatment of chronic woodchuck hepatitis virus infection in the Eastern woodchuck (Marmota monax) with nucleoside analogues is predictive of therapy for chronic hepatitis B virus infection in humans

The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been established as a model of hepatitis B virus (HBV)-induced disease. Several published studies have used this experimental animal model system to demonstrate potential antiviral therapies for chronic HBV infections. However, there has been little comparative information available on compounds used in clinical anti-HBV studies in WHV-infected woodchucks, thereby making interpretations of the potential relative effectiveness of new antiviral agents in humans more difficult. In this report, using a series of placebo-controlled studies, we compared the relative effectiveness of several nucleoside analogues that have been used in clinical trials for the treatment of chronic HBV infection against WHV replication in chronically infected woodchucks. Adenine-5'-arabinoside monophosphate (Ara-AMP [vidarabine]), ribavirin, (-)beta-L-2',3'-dideoxy-3'-thiacytidine (3TC [lamivudine]), and famciclovir (oral prodrug of penciclovir) induced depressions in viremia and intrahepatic WHV-DNA replication that were consistent with their relative effectiveness in anti-HBV human clinical trials. As observed in HBV-infected patients, 3' azido-3'-deoxythymidine (AZT [zidovudine]) had no effect on WHV replication in these studies. These experimental results more firmly establish chronic WHV infection in woodchucks as an accurate and predictive model for antiviral therapies against chronic HBV infection in humans and provide a baseline for comparative antiviral effects of other experimental antiviral agents in the WHV/woodchuck model system.     

The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection

INTRODUCTION Infection of adult humans with the hepatitis B virus (HBV) results characteristically in self-limited hepatic disease with recovery based on serological and clinical parameters. Progression to chronic HBV infection occurs infrequently in infe…     

Modulation of regulatory T-cell activity in combination with interleukin-12 increases hepatic tolerogenicity in woodchucks with chronic hepatitis B

Regulatory T cells (Treg) play a critical role in the modulation of immune responses to viral antigens in chronic viral hepatitis. Woodchucks (Marmota monax) infected with the woodchuck hepatitis virus (WHV) represent the best animal model for chronic hepatitis B virus (HBV) infection. Examination of intrahepatic and peripheral Treg in uninfected and WHV chronically infected woodchucks showed a significant increase of intrahepatic Treg numbers in chronically infected animals, whereas no differences were found in peripheral blood. In agreement with these data, higher expression levels of Forkhead box P3 (Foxp3), interleukin (IL)-10, transforming growth factor beta (TGF-β) were detected in the liver of chronic WHV carriers in comparison to uninfected animals. Furthermore, treatment of WHV-infected animals with an adenovirus encoding IL-12 failed to reduce viral load, a finding that was associated with lymphocyte unresponsiveness to IL-12 stimulation in vitro. We observed that TGF-β and Treg play a major role in the lack of lymphocyte response to IL-12 stimulation, as TGF-β inhibition and Treg depletion allowed recovery of T-cell responsiveness to this cytokine. Based on these results, woodchucks were treated with IL-12 in combination with a TGF-β inhibitory peptide or Treg depletion. However, no antiviral effect was achieved and, instead, an enhancement of the intrahepatic tolerogenic environment was observed. CONCLUSION: Our data show that TGF-β inhibition or Treg depletion had no added benefit over IL-12 therapy in chronic WHV infection. IL-12 immunostimulation induces a strong immunosuppressive reaction in the liver of chronic WHV carriers that counteracts the antiviral effect of the treatment.     

Frequent amplification of c-myc in ground squirrel liver tumors associated with past or ongoing infection with a hepadnavirus.

Persistent infection with hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC) in humans. HCC has also been observed in animals chronically infected with two other hepadnaviruses: ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV). A distinctive feature of WHV is the early onset of woodchuck tumors, which may be correlated with a direct role of the virus as an insertional mutagen of myc genes: c-myc, N-myc, and predominantly the woodchuck N-myc2 retroposon. In the present study, we searched for integrated GSHV DNA and genetic alterations of myc genes in ground squirrel HCCs. Viral integration into host DNA was detected in only 3/14 squirrel tumors and did not result in insertional activation of myc genes, despite the presence of a squirrel locus homologous to the woodchuck N-myc2 gene. This suggests that GSHV may differ from WHV in its reduced ability to induce mutagenic integration events. However, the high frequency of c-myc amplification (6/14) observed in ground squirrel HCCs indicates that myc genes might be preferential effectors in the tumorigenic processes associated with rodent hepadnaviruses, a feature not reported so far in HBV-induced carcinogenesis. Together with previous observations, our results suggest that hepadnaviruses, despite close genetic and biological properties, may use different pathways in the genesis of liver cancer.     

Animal models of hepadnavirus-associated hepatocellular carcinoma

Animal models of hepatitis B virus infection have been valuable for determining the mechanisms of hepadnavirus replication, for studies of pathogenesis, and for investigations of viral hepatocarcinogenesis. The woodchuck model also seems to be useful in the discovery and development of antiviral drugs to treat HBV infection and for testing new forms of immunotherapy. In particular, the woodchuck seems to be ideal for studying the effect of antiviral treatment and immunotherapy on the outcome of hepadnavirus infection and on survival. The median life expectancy of experimentally infected, chronic WHV carriers is approximately 29 months, and almost all develop HCC. New types of prophylaxis or therapy can be evaluated under controlled experimental conditions, in a relevant animal model, and within a reasonable time frame.     

Hepatocarcinogenicity of the woodchuck hepatitis virus.

During investigations of the evolution of experimental laboratory infections of woodchucks (Marmota monax) with the woodchuck hepatitis virus (WHV), eight hepatocellular carcinomas (HCC) were observed, six in newborns and two in young adult animals, all within 17-36 months after infection. The absence of an external cocarcinogenic effect in the well-monitored woodchucks indicates the carcinogenicity of WHV and suggests the same for the genetically and biologically similar human hepatitis B virus (HBV). Laboratory infections of woodchucks with two strains of WHV, not reported here in detail, resembled human and chimpanzee HBV infections histologically and serologically. In these studies, eight woodchucks became carriers of surface antigen of WHV for greater than 1 year. All eight woodchucks developed HCC, indicating a 100% risk of HCC in experimentally infected chronic WHV antigen carriers, which is analogous to the high risk of HCC in human hepatitis B surface antigen carriers. Histologically, the absence of cirrhosis in the examined pericarcinomatous tissue permits recognition of gradual transition from normal parenchyma to neoplastic nodules to HCC of rising anaplasia, indicating a continuum of increasingly more malignant neoplastic stages, as known for chemical carcinogenesis. The HCC developed in carrier woodchucks infected as newborns with only minor, if any, hepatitic changes but is associated with antigen-carrying hepatocytes and sometimes with hyperplastic nodules. This stage was preceded in infected adults by an early, acute, weeks-long hepatitis coinciding with the appearance of surface antigen. These findings are also analogous to typical HBV infection in human newborns and young adults, respectively. At the time of HCC development in all animals with adequate histologic material, an acute recent necroinflammation appeared around the tumor, associated with abnormal hematopoietic cells around and within the tumor. A promoting role in carcinogenesis of this necroinflammation of yet unestablished pathogenesis is being postulated, to be confirmed by determination of the status of the WHV DNA in the HCC and by prospective histologic study of the inflammatory reaction.     

Therapeutic vaccination in chronic hepatitis B: preclinical studies in the woodchuck model

Summary.  Interferon-α and nucleoside analogues are available for the treatment of chronic hepatitis B virus (HBV) infection but do not lead to a satisfactory result. New findings about the immunological control of HBV during acute infection suggest the pivotal role of T-cell mediated immune responses. Several preclinical and clinical trials were undertaken to explore the possibility of stimulating specific immune responses in chronically infected animals and patients by vaccination. However, vaccination with commercially available HBV vaccines in patients and immunization in woodchucks with core or surface proteins of woodchuck hepatitis virus (WHV) did not result in effective control of HBV and WHV infection, suggesting that new formulations of therapeutic vaccines are needed. Some new approaches combining antiviral treatments with nucleoside analogues, DNA vaccines and protein vaccines were tested in the woodchuck model. It could be shown that therapeutic vaccinations are able to stimulate specific B- and T-cell responses and to achieve transient suppression of viral replication. These results suggest the great potential of therapeutic vaccination in combination with antivirals to reach an effective and sustained control of HBV infection.     

Effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection

Acute hepadnavirus infections either resolve or progress to chronicity. Factors that influence chronicity as an outcome of hepatitis B virus (HBV) infection in humans can be studied experimentally in the woodchuck model. Accordingly, several woodchuck hepatitis virus (WHV) inocula were characterized. Representative inocula had high titers of infectious virus (approximately 10(7.7)-10(9.5) woodchuck 50% infectious doses per milliliter [WID(50%)/mL] by subcutaneous inoculation), with 1 WID(50%) ranging between 21 and 357 physical virion particles. WHV7P1 (standard high dose, 5 x 10(6) WID(50%)) produced a 72% chronicity rate (i.e., percent chronic of total infected) in neonatal woodchucks (1-3 days old). Comparable doses of WHV8P1 resulted in a lower chronicity rate in neonates (34% chronic) indicating that it represented a strain different from WHV7P1. Neonatal woodchucks were more susceptible to chronic infection by high doses of WHV7P1 (range, 65%-75% chronic) compared with 8-week-old weanlings (33% chronic) and adult woodchucks (0% chronic; i.e., all resolved). High doses of cloned wild-type viruses also induced high rates of chronicity in neonates (70%-80% chronic). Chronicity rates in neonates were decreased for low doses of WHV7P1 (500 WID(50%), 9% chronic) and for high doses of a precore WHeAg-minus mutant WHV8 clone (17% chronic). Thus, both age and viral determinants can influence chronicity as an outcome of experimental WHV infection. Standardized inocula will enable the study of mechanisms that initiate and maintain chronic hepadnavirus infection and also provide a means for developing WHV carriers for therapeutic studies.     

Immunosuppression reactivates viral replication long after resolution of woodchuck hepatitis virus infection

Resolution of hepatitis B virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with woodchuck hepatitis virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such woodchucks resulted in transient reactivation of WHV replication. Serum of 1 woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual virus. CONCLUSION: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection.     

土拨鼠肝炎病毒和乙型肝炎病毒的核心蛋白存在共同线性B淋巴细胞表位

目的寻找并证实土拨鼠肝炎病毒核心抗原（WHcAg）和HBcAg存在共同的B淋巴细胞表位。方法用变性重组WHcAg和HBcAg免疫Balb/c小鼠以制备单克隆抗体；用单克隆抗体对WHcAg和HBcAg进行ELISA、Western blot及免疫组织化学对比分析,同时用该抗体对WHcAg和HBcAg进行表位分析,并在氨基酸水平进行比对。结果经过ELISA、Western blot、免疫组织化学对比分析,制备的单克隆抗体中有2株与HBcAg和WHcAg有交叉反应,而且灵敏度和特异性相似；针对的抗原表位分别位于HBcAg和WHcAg的1～8位氨基酸和125～140位氨基酸。结论WHcAg和HBcAg存在2个共同的线性B淋巴细胞表位,分别位于1～8位和125～140位氨基酸。     

Evidence against a requisite role for defective virus in the establishment of persistent hepadnavirus infections.

The factors involved in the establishment of persistent hepadnavirus infection are poorly understood. Recent findings demonstrate that the sequence of the genome of hepatitis B virus (HBV) is variable in infected individuals and that, in some cases, virus mutants predominate. Our objectives in the present study were to analyze the variability of woodchuck hepatitis virus (WHV) genomes in an infected animal and to determine whether sequence heterogeneity played a critical role in the ability of WHV to induce chronic infection. We cloned and determined the complete nucleotide sequence of three supercoiled genomes from an animal that became infected after inoculation with a standardized WHV serum pool (i.e., the WHV7 virus pool). We found that there were four nucleotide substitutions among the three genome sequences as well as a 73-nucleotide deletion in one of the recombinants. DNA transfection experiments revealed that only one of the three recombinants was capable of independent replication. These data suggest that a significant proportion of replicative templates in woodchucks that are infected with WHV are defective virus genomes. Next, we compared the outcome of acute infection after inoculation with a serum pool containing a uniform population of replication competent virus (i.e., the WHV7R pool) with a serum pool composed of WHV genomes of variable sequence. The WHV7R serum pool originated from a woodchuck that became a chronic carrier after in vivo transfection of the liver with the infectious WHV7 recombinant. Neonatal woodchucks were inoculated with 5 x 10(6) WHV genome equivalents of either the WHV7 pool or the WHV7R pool. All animals in the study became acutely infected with WHV. Of the animals infected with the WHV7 serum pool, 65% became chronic carriers, while 80% of the animals infected with the WHV7R serum pool developed chronic infection. Thus, infection of woodchucks with a serum pool containing defective virus resulted in a rate of chronic WHV infection that was similar to, or even lower than, a rate from a pool containing only wild-type virus. This suggests that the presence of defective virus in the inoculum is not a prerequisite for the establishment of persistent hepadnavirus infections.     

Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks

Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus-induced hepatocellular carcinoma (HCC). Woodchucks (Marmota monax) that were chronically infected with HBV-related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery the WHV carrier woodchucks were superinfected with WHV-enveloped HDV (wHDV). Six weeks later the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real-time polymerase chain reaction assay, and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues. CONCLUSION: Cells of WHV-induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Because others previously hypothesized that hepadnavirus-induced HCCs are resistant to reinfection with a hepadnavirus in vivo, our data suggest that if such a resistance exists it likely occurs via a block at the post-entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection.     

乙型肝炎病毒核心相关抗原的监测及临床意义

监测乙型肝炎病毒核心相关抗原(HBcrAg),能有效预测核苷(酸)类药物抗病毒治疗后产生的病毒学反弹及耐药风险,具有重要的临床实用价值。     

The role of the woodchuck model in the treatment of hepatitis B virus infection

Experimental studies of animals with chronic hepadnavirus infection could provide valuable insight into optimal therapeutic strategies for individuals with chronic HBV infection. In this review, we focus on the contributions of the woodchuck model to our understanding of HBV biology and on its role in the development of antiviral drug. Furthermore, we consider the implications of studies focusing on the natural history of WHV infection for the management of HBV and the capacity of treatment to prevent complications of chronic hepatitis B infection.     

Induction of antibodies to the PreS region of surface antigens of woodchuck hepatitis virus (WHV) in chronic carrier woodchucks by immunizations with WHV surface antigens

BACKGROUND/AIMS: One goal of therapeutic vaccinations against chronic hepatitis B virus infection is to stimulate the B-cell responses to viral surface antigens in chronic carriers. Here we investigated the induction of antibody responses to hepadnaviral surface antigens in the woodchuck model, with emphasis on the vaccination of woodchucks chronically infected with woodchuck hepatitis virus (WHV). METHODS: Naive and chronically WHV-infected woodchucks were immunized with plasma-derived WHV surface antigens (p-WHsAg) containing the S and PreS sequences. Antibody responses to WHsAg and the WHV PreS region and viral load in immunized woodchucks were monitored. RESULTS: After repeated immunizations with WHsAg, 17 of 18 chronic WHV carriers developed a persistent antibody response to WHsAg. These antibodies were mainly directed to epitopes within the PreS region and detectable by Western blotting. However, neither WHV DNA nor WHsAg concentrations in these woodchucks changed significantly by immunizations and during the follow up. Sequence analysis of WHV genomes showed that no WHV mutants emerged after the induction of anti-WHs/anti-WHpreS antibodies. No immunopathological changes in livers of immunized animals were recognized thus far. CONCLUSIONS: Our study demonstrated that the immunological unresponsiveness of chronically WHV-infected woodchucks to WHsAg can be partially overcome by repeated immunizations with WHsAg.