Chronic Woodchuck Hepatitis Virus Carriers Research Articles

The Woodchuck, a Nonprimate Model for Immunopathogenesis and Therapeutic Immunomodulation in Chronic Hepatitis B Virus Infection.

The woodchuck hepatitis virus (WHV) and its host, the eastern woodchuck, is a very valuable model system for hepatitis B virus infection. Many aspects of WHV replication and pathogenesis resemble acute and chronic hepatitis B infection in patients. Since the establishment of immunological tools, woodchucks were used to develop new therapeutic vaccines and immunomodulatory approaches to treat chronic hepadnaviral infections. Combination therapy of nucleos(t)ide analogs, with prime-boost vaccination and triple therapy, including immunomodulatory strategies by blocking the interaction of the programmed death-1 (PD-1) receptor with its ligand inducing a potent T-cell response in chronic WHV carrier woodchucks, suppression of viral replication, and complete elimination of the virus in 30% of the animals. Both strategies may be used for future therapies in patients with chronic hepatitis B.

Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.

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. 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.

Treatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.

Treatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.

Chemoimmunotherapy of Chronic Hepatitis B Virus Infection in the Woodchuck Model Overcomes Immunologic Tolerance and Restores T-Cell Responses to Pre-S and S Regions of the Viral Envelope Protein

Treatment of chronic hepatitis B virus (HBV) infection could combine potent antiviral drugs and therapeutic vaccines to overcome immunological tolerance and induce the recovery phenotype to protect against disease progression. Conventional vaccination of woodchucks chronically infected with the woodchuck hepatitis virus (WHV) elicited differential T-cell response profiles depending on whether or not carriers were treated with the potent antiviral drug clevudine (CLV), which significantly reduces viral and antigen loads. The differential T-cell responses defined both CLV-dependent and CLV-independent epitopes of the pre-S and S regions of the WHV envelope protein. Only combined treatment involving CLV and conventional vaccine therapeutically restored the T-cell response profile of chronic WHV carrier woodchucks to that seen in prophylactic vaccination and in recovery from acute WHV infection. The results have implications for mechanisms of immunological tolerance operating in chronic HBV infection and suggest that such combined chemoimmunotherapy may be useful for treatment of humans with chronic HBV infection.

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

This review describes the woodchuck and the woodchuck hepatitis virus (WHV) as an animal model for pathogenesis and therapy of chronic hepatitis B virus (HBV) infection and disease in humans. The establishment of woodchuck breeding colonies, and use of laboratory-reared woodchucks infected with defined WHV inocula, have enhanced our understanding of the virology and immunology of HBV infection and disease pathogenesis, including major sequelae like chronic hepatitis and hepatocellular carcinoma. The role of persistent WHV infection and of viral load on the natural history of infection and disease progression has been firmly established along the way. More recently, the model has shed new light on the role of host immune responses in these natural processes, and on how the immune system of the chronic carrier can be manipulated therapeutically to reduce or delay serious disease sequelae through induction of the recovery phenotype. The woodchuck is an outbred species and is not well defined immunologically due to a limitation of available host markers. However, the recent development of several key host response assays for woodchucks provides experimental opportunities for further mechanistic studies of outcome predictors in neonatal- and adult-acquired infections. Understanding the virological and immunological mechanisms responsible for resolution of self-limited infection, and for the onset and maintenance of chronic infection, will greatly facilitate the development of successful strategies for the therapeutic eradication of established chronic HBV infection. Likewise, the results of drug efficacy and toxicity studies in the chronic carrier woodchucks are predictive for responses of patients chronically infected with HBV. Therefore, chronic WHV carrier woodchucks provide a well-characterized mammalian model for preclinical evaluation of the safety and efficacy of drug candidates, experimental therapeutic vaccines, and immunomodulators for the treatment and prevention of HBV disease sequelae.

Adoptive transfer of immunity: a new strategy to interfere with severe hepatitis virus reinfection after woodchuck liver transplantation.

The effective transfer of humoral immunity to hepatitis B virus was first demonstrated after clinical and experimental bone marrow transplantation. This strategy is now evaluated in the woodchuck transplantation model, in which protection from reinfection can be tested. Animals negative for woodchuck hepatitis virus (WHV) (n = 3) were vaccinated using plasmids expressing woodchuck hepatitis virus surface (WHs), woodchuck hepatitis virus core (WHc), and woodchuck interferon-gamma in combination with a protein vaccine (WHs antigen [Ag]) three times before liver donation. Control animals (n= 4) received the liver from non-immunized donors. Chronic WHV carriers served as recipients. The viral load in serum and liver tissue was monitored pretransplant and posttransplant for up to 11 weeks by dot blot, Northern blot, Southern blot, and immunohistochemistry for WHc and WhsAg. Donor vaccination was effective, as indicated by the development of anti-WHc and anti-WHs antibodies. Transplanting the livers of these donors resulted in a reduction of viral load in two of three animals. No viral DNA was detected in repeated serum samples by dot-blot hybridization technique. However, polymerase chain reaction for viral DNA extracted from serum was always positive. WHV replication intermediates and WHV RNA were absent in repeated liver biopsies. Only few hepatocytes stained weakly positive for WHc protein and frequency, and the intensity of WHsAg positive hepatocytes was low. The third animal developed severe reinfection within 3 weeks, similar to the woodchucks in the control group. Liver transplantation from immunized donors to chronic carriers seems to be a promising strategy to reduce and delay severe reinfection, which may be applicable in clinical liver transplantation.

Integration of woodchuck hepatitis and N-myc rearrangement determine size and histologic grade of hepatic tumors.

Integrations of woodchuck hepatitis virus (WHV) DNA and rearrangements of the N-myc 2 gene have been detected frequently in hepatocellular carcinoma (HCC) of Eastern woodchucks (Marmota monax) chronically infected with WHV. Fifty-five hepatocellular neoplasms and matched nontumor hepatic tissue specimens obtained postmortem from 13 chronic WHV carriers were analyzed and the frequency of WHV DNA integrations and of N-myc rearrangements compared in tumors of different size and histologic grade. Four small tumor nodules were classified histologically as adenomas and integrated sequences of WHV DNA were detected in two of the four tumor nodules. In one of the two nodules, there was evidence of N-myc rearrangement. Fifty-one neoplasms were classified as HCC. Seven were grade 1 HCCs. WHV DNA integrations were demonstrated in 43% but none had N-myc rearrangements. Twenty grade 2 HCCs had WHV DNA integrations in 80% and in 38% N-myc rearrangements were present. Twenty-four grade 3 HCCs had integrations of WHV DNA in 79% and N-myc rearrangements in 74%. In two other grade 3 HCCs, rearrangements of N-myc were detected in the absence of WHV DNA integrations. The 12 largest tumors in the series all were grade 2 or 3 HCCs, and in 83%, both WHV DNA integrations and N-myc rearrangements were demonstrated. In conclusion, molecular changes observed in this study suggest a progression of genetic alterations providing either a significant proliferative stimulation and/or a growth advantage in hepatocarcinogenesis of woodchucks with chronic WHV 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.

The woodchuck model of hepatitis B virus infection.

The woodchuck hepatitis virus (WHV) was the first of the mammalian and avian hepadnaviruses described after discovery of the virus of hepatitis B (HBV). Woodchucks chronically infected with WHV develop progressively severe hepatitis and hepatocellular carcinoma, which present as lesions that are remarkably similar to those associated with HBV infection in humans. The initial virological studies and studies of pathogenesis utilized woodchucks that had been trapped in the wild and had acquired WHV infection naturally. Research with wild woodchucks was complicated by lack of knowledge of their backgrounds (e.g., dietary history, exposure to parasites or environmental toxins, and source and duration of WHV infection). Breeding colonies of woodchucks have been established and maintained in laboratory animal facilities, and laboratory-reared woodchucks are superior for experimental studies of pathogenesis or hepatocarcinogenesis. It is possible to infect neonatal woodchucks born in the laboratory with standardized inocula and produce a high rate of chronic WHV carriers that are useful for controlled investigations. WHV has been shown experimentally to cause hepatocellular carcinoma, supporting conclusions based on epidemiological and molecular virological studies that HBV is an important etiological factor in human hepatocarcinogenesis. Chronic WHV carrier woodchucks have become a valuable animal model for the preclinical evaluation of antiviral therapy for HBV infection, providing useful pharmacokinetic and pharmacodynamic results in a relevant animal disease model. It also has been shown that the pattern of toxicity and hepatic injury observed in woodchucks treated with certain fluorinated pyrimidines is remarkably similar to that observed in humans that were treated with the same drugs, suggesting the woodchuck has significant potential for the preclincial assessment of antiviral drug toxicity.

Enhanced antiviral benefit of combination therapy with lamivudine and famciclovir against WHV replication in chronic WHV carrier woodchucks

Cell culture studies in our laboratory and others have previously demonstrated synergistic antiviral activity for combinations of 3TC (lamivudine) and penciclovir against Hepatitis B Virus (HBV) replication and the Duck Hepatitis B Virus (DHBV). Based on these results, a study was designed to determine if an enhanced antiviral effect with combinations of 3TC and famciclovir (FCV, oral prodrug of penciclovir) could be demonstrated in vivo using the Woodchuck Hepatitis Virus (WHV)/woodchuck experimental model of chronic HBV infection. Both antiviral agents have been shown to be effective against WHV replication in WHV chronic carriers in previous studies by our laboratories. The antiviral effects of four different combinations of lamivudine and FCV were found to be greater than those observed for the corresponding monotherapies. All four combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. Two of the combination treatments produced antiviral effects that were significantly greater than that expected for additive effects, indicative of synergistic antiviral interactions. These studies demonstrate that combination therapy of chronic WHV infection has enhanced antiviral benefit over corresponding monotherapies and indicate that combination treatment of chronic HBV infection can be superior to therapies using a single antiviral agent.

Mapping of the cellular immune responses to woodchuck hepatitis core antigen epitopes in chronically infected woodchucks.

T-cell responses to hepatitis B virus nucleocapsid antigens (HBcAg and HBeAg) play an important role in disease outcome in those infected with hepatitis B virus (HBV). The woodchuck is naturally infected in the wild with woodchuck hepatitis virus (WHV), which shows a high degree of genetic homology to HBV and produces a similar pattern of infection in its natural host. Twenty-three overlapping peptides were constructed to cover the entire WHV core region and used to identify immunodominant cellular epitopes in the nucleocapsid antigen using peripheral blood lymphocytes from 12 chronic WHV carrier and 4 uninfected control animals. A peripheral blood lymphocyte response was seen in all of the chronic WHV carrier animals to at least one peptide, and in 8 of the 12 chronic carrier animals a response was observed to 5 common peptides: peptide analogues of amino acids 16-30, 38-52, 50-69, 76-90 and 91-105. Peptide 91-105 produced maximal proliferation in 5 out of 12 infected animals. In addition, a difference in response was observed between wild and laboratory infected animals; the latter appeared to have a lower response to peptides than animals infected in the wild. This study provides evidence that the woodchuck has a population of peripheral blood cells which are sensitised to epitopes within the nucleocapsid protein and provides a basis on which to develop the use of the woodchuck as an immunological model of HBV infection for testing therapeutic means of enhancing this response.

Determination of peripheral blood mononuclear cell responses to mitogens and woodchuck hepatitis virus core antigen in woodchucks by 5-bromo-2'-deoxyuridine or 2[3H]adenine incorporation.

Characterization of cellular immune response to antigens of woodchuck hepatitis virus (WHV) can contribute to the understanding of acute resolving and chronic outcome of hepadnavirus infection. Studies were limited because peripheral blood mononuclear cells (PBMCs) of woodchucks failed to incorporate [3H]thymidine sufficiently. Therefore, we established a non-radioactive proliferation assay for woodchuck PBMCs using 5-bromo-2'-deoxyuridine (BrdU) as thymidine analogue. Mitogen- and WHV core protein-(WHcAg) induced PBMC proliferation was detected by BrdU incorporation and compared to an assay using 2[3H]adenine. After stimulation with concanavalin A (ConA) and phytohaemagglutinin (PHA) we observed significant PBMC proliferation with both assays. Mitogen-induced nucleoside uptake of PBMCs into cellular DNA was confirmed by detection of 1',2'[3H]BrdU and 2[3H]adenine in extracted DNA. PBMCs obtained during the acute phase of WHV infection could be stimulated by WHcAg, whereas no WHcAg-induced proliferation of PBMCs was found in WHV-negative animals. PBMCs of chronic WHV carriers showed only a weak response to WHcAg. The established assays will be useful in determining the kinetics of cellular immune responses to different WHV antigens in the course of WHV infection and may provide an insight into mechanisms responsible for chronic outcome of hepadnavirus infection.

Treatment of woodchuck hepatitis virus infection in vivo with 2', -3'-dideoxycytidine (ddC) and 2',-3'-dideoxycytidine monophosphate coupled to lactosaminated human serum albumin (L-HSA ddCMP).

Dideoxycytidine (ddC) is a nucleoside analogue active against human immunodeficiency virus and with in vitro activity against human hepatitis B virus. We investigated the ability of ddC to inhibit one of the Hepadnaviridae, the woodchuck hepatitis virus and compared the results with the effect obtained by a conjugate of lactosaminated human serum albumin 2',-3'-dideoxycytidine monophosphate (L-HSA ddCMP). This compound specifically enters the hepatocyte via the asialoglycoprotein receptor. We treated five chronic woodchuck hepatitis virus carriers with intravenous injections of 0.5 mg/kg body weight of ddC for 5 consecutive days, and under the same protocol five woodchucks with 10.4 mg/ kg L-HSA ddCMP, a dose equivalent to 0.25 mg/kg of free ddC. A reduction of serum woodchuck hepatitis virus DNA (5-125 fold) was observed during therapy in three out of five animals receiving ddC and in two of the five animals treated with L-HSA ddCMP. In responding woodchucks, virus DNA levels rebounded immediately after stopping therapy. No signs of toxicity were observed during or after the course of therapy. These preliminary results of short-term treatment indicate that ddC has anti-viral activity against woodchuck hepatitis virus. When the dose was reduced by 50%, L-HSA ddCMP showed anti-viral activity to an even lesser degree.

Natural history of experimental woodchuck hepatitis virus infection: molecular virologic features of the pancreas, kidney, ovary, and testis

The kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the pancreas, kidneys, ovaries, and testes. Groups of woodchucks experimentally infected with a standardized inoculum of WHV were sacrificed at different times over a 65-week period beginning in the preacute phase of viral infection and continuing to the period of serologic recovery or the establishment of chronic infections and subsequent hepatocellular carcinoma (B. E. Korba, P. J. Cote, F. V. Wells, B. Baldwin, H. Popper, R. H. Purcell, B. C. Tennant, and J. L. Gerin, J. Virol. 63:1360-1370, 1989). Tissues from an additional group of long-term (2 to 3 years) chronic WHV carriers which had been infected with the same WHV inocula were also examined. Viral DNA replication intermediates were found in all four tissues during the acute phase of WHV infection. However, WHV DNA replication intermediates were observed only in the kidneys of a small proportion of the chronically infected animals. Following the acute phase of infection, WHV DNA was present only in the pancreas, kidneys, and ovaries of the chronically infected woodchucks. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was observed in these tissues. Histologic evaluation of these four tissues revealed only minimal, localized lesions which were not correlated with the state of WHV activity. The observations compiled in this study further extend the tissue tropism of WHV.

Hepatitis delta virus (HDV) and woodchuck hepatitis virus (WHV) nucleic acids in tissues of HDV-infected chronic WHV carrier woodchucks.

The molecular forms of genomic and antigenomic hepatitis delta virus (HDV) RNA and of woodchuck hepatitis virus (WHV) DNA and WHV RNA were studied in nonneoplastic liver (NL) tissues, hepatocellular carcinoma (HCC) tissues, and several extrahepatic tissues of chronic WHV carrier woodchucks acutely (two animals) and chronically (six animals) superinfected with HDV. HDV was shown to replicate in all NL and HCC tissues but not in any of the extrahepatic tissues analyzed, which included spleen, peripheral blood lymphocytes, kidney, ovary, testis, thymus, lung, and stomach. HDV RNA was present as species with molecular weights consistent with those of monomers, dimers, and trimers of both strand polarities, supporting the rolling circle model proposed for HDV RNA replication. WHV DNA levels in NL, HCC, spleens, and serum were 10- to 100-fold lower than the levels typically observed in chronic WHV carrier woodchucks not infected with HDV. WHV DNA replicative intermediates were rarely observed and only at very low levels, representing less than 10% of the total WHV DNA. By contrast, WHV RNA transcription was not significantly depressed and both primary WHV RNA transcripts, 2.3 and 3.6 kilobases, were observed in NL, HCC, spleens, and in one of the kidney tissues. In addition, a 2.6-kilobase WHV RNA transcript was found in the majority of the NL tissues.

Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: Presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections

During long-term studies of the natural history of woodchuck hepatitis virus infection, five cases of histologically confirmed, primary hepatocellular carcinoma were observed in a total of 92 woodchucks which had recovered, by analysis of viral serologic markers (WHsAg-, anti-WHc+, anti-WHs+), from experimental acute woodchuck hepatitis virus infections 20 to 30 months prior to the detection of hepatocellular carcinoma. No hepatocellular carcinoma was observed in 167 uninfected controls at least 3 years of age and held in the same laboratory environment. Southern blot hybridization analysis of liver tissue taken from four of these recovered woodchucks revealed the presence of low levels (0.1 to 0.3 copies per cell) of integrated woodchuck hepatitis virus DNA in hepatocellular carcinoma (four of four animals) and nonneoplastic tissue (three of four animals). Similarly, hepatocellular carcinoma tissue obtained from two wild-caught, naturally infected and serologically recovered woodchucks also contained low levels of integrated woodchuck hepatitis virus DNA. Liver tissues from another 27 of these 92 recovered woodchucks (without hepatocellular carcinoma) were examined for woodchuck hepatitis virus nucleic acids 13 to 31 months following experimental woodchuck hepatitis virus infection. Nonreplicating woodchuck hepatitis virus DNA was present in the liver of eight (30%) and in the peripheral blood lymphocytes from eight (30%) of these 27 animals. These results were in marked contrast to the analysis of woodchuck hepatitis virus DNA in the liver tissue of chronic woodchuck hepatitis virus carriers (20 experimentally infected and nine naturally infected). In these animals, high levels of replicating woodchuck hepatitis virus DNA (up to 2,000 copies per cell) were observed in all hepatocellular carcinoma and nonneoplastic liver tissue. Integrated woodchuck hepatitis virus DNA was found in eight of 60 individual hepatocellular carcinomas detected in 29 chronic carriers, 15 to 40 months postinfection. Integrated woodchuck hepatitis virus DNA was present in the nonneoplastic tissue from four of these 29 chronic woodchuck hepatitis virus carriers.