Superinfection with woodchuck hepatitis virus strain WHVNY of livers chronically infected with strain WHV7.

UNLABELLED: The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superinfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superinfection. The results are consistent with the occurrence of continuous but inefficient hepadnavirus cell-to-cell spread and superinfection during chronic infection and suggest that the replication space occupied by the superinfecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection. IMPORTANCE: This study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus superinfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous but limited hepadnavirus spread and superinfection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.

Characterization of woodchuck apolipoprotein A-I: a new tool for drug delivery and identification of altered isoforms in the woodchuck chronic hepatitis model.

Apolipoprotein A-I (ApoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum, and participates in the reverse transport of cholesterol from tissues to the liver for excretion. The natural HDL tropism to the liver and cancer cells has been used extensively to target encapsulated drugs. The alteration of the plasmatic isoforms of ApoA-I is a hallmark of chronic hepatitis and hepatocarcinoma in mice and humans. Woodchucks infected with the woodchuck hepatitis virus (WHV) represent the best animal model for the study of chronic viral hepatitis B and viral induced hepatocarcinoma (HCC). WHV-infected woodchuck represents a clinically relevant animal model under which new treatment strategies can be evaluated and optimized. Therapeutic efficacy in this model is likely to be translated into a successful therapy for patients infected with HBV. The present study describes, for the first time, the cloning and characterization of woodchuck ApoA-I. The open reading frame (ORF) of the woodchuck ApoA-I is 795 bp long, coding for 264 amino acids. Unexpectedly, phylogenetic analysis revealed that the closest sequences are those of human and macaque. Woodchuck HDLs were isolated successfully from sera by density gradient ultracentrifugation. A commercial antibody that recognized the woodchuck ApoA-I was also identified. Finally, taking advantage of the techniques and tools developed in this study, two potential applications of woodchuck HDLs are illustrated: drug delivery to a woodchuck hepatocarcinoma cell line and the use of isoelectrofocusing to identify ApoA-I isoforms.

Combining antiangiogenic therapy with immunotherapy exerts better therapeutical effects on large tumors in a woodchuck hepatoma model.

Cytokine and antiangiogenic gene therapies have proved effective in implanted hepatocellular carcinoma (HCC) models in which small tumor burdens were established in small rodents. These models, however, may not reflect human HCCs, which are frequently detected at a stage when tumors are large and multifocal. In addition, HCC in patients is often associated with viral hepatitis. To investigate the effectiveness of a mixture type of gene therapy strategy on large tumor burdens, we used the woodchuck model in which woodchuck hepatitis virus-induced HCCs are large and multifocal, simulating the conditions in humans. Adenoviruses encoding antiangiogenic factors (pigment epithelium-derived factor and endostatin) or cytokines (GM-CSF and IL-12) were delivered via the hepatic artery separately or in combination into woodchuck livers bearing HCCs. Our results showed that the mixture type of strategy, which contained two cytokines and two antiangiogenic factors, had better antitumor effects on large tumors as compared with monotherapy either with antiangiogenic or cytokine genes. The immunotherapy recruited significant levels of CD3(+) T cells that infiltrated the tumors, whereas the antiangiogenesis-based therapy significantly reduced tumor vasculature. The mixture type of gene therapy achieved both effects. In addition, it induced high levels of natural killer cells and apoptotic cells and reduced the levels of immunosuppressive effectors in the tumor regions. Hence, antiangiogenic therapy may provide the advantage of reducing immune tolerance in large tumors, making them more vulnerable to the immune reactions. Our study implies that in the future, the combination therapy may prove effective for the treatment of patients with advanced HCC.

Repeated passage of wild-type woodchuck hepatitis virus in lymphoid cells does not generate cell type-specific variants or alter virus infectivity.

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus, infects the liver but also invariably establishes persistent infection in the lymphatic system. Although the dose of invading virus appears to be the main factor in determining whether WHV infection is restricted to the lymphatic system or also engages the liver, the nature of WHV lymphotropism remains unclear and a role for a specific lymphotropic variant was not excluded. The availability of woodchuck lymphocyte and hepatocyte cultures susceptible to WHV infection allows investigation of this issue in vitro. We hypothesized that repeated passage of wild-type WHV in lymphoid cells should lead to enrichment of a lymphotropic virus variant, if in fact such a variant exists. For this purpose, wild-type WHV with a homogeneous sequence was used as the inoculum, while lymphoid cells from a single healthy woodchuck donor and a normal woodchuck WCM-260 hepatocyte line served as infection targets. The serial passage of the wild-type virus repeated up to 13 times for both cell types did not lead to the emergence of cell type-specific WHV variants, as revealed by sequence analysis of the virus envelope and the core and X gene sequences. Moreover, the virus passaged in both cell types remained infectious for naive woodchucks, produced infection profiles that depended upon virus dose but not on virus cellular origin, and retained its initial DNA sequence. These results imply that WHV lymphotropism is a natural propensity of the wild-type virus and is not a consequence of infection with a viral variant.

Suppression of lamivudine-resistant B-domain mutants by adefovir dipivoxil in the woodchuck hepatitis virus model.

Adult woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus (WHV) were treated orally with lamivudine (15 mg/kg per day) for 57 weeks. After 20 weeks of treatment a 2-3 log reduction in serum WHV DNA was detected. Serum titers of WHV then increased gradually, in the presence of lamivudine treatment, reaching pre-treatment values by week 40. Viral recrudescence was associated with development of mutations in the B domain of the WHV polymerase gene. Mutations observed in the highly conserved FLLA motif of the B domain were L564V, L565M, and A566T, with A566T being the most frequently observed. Beginning on week 57 of lamivudine treatment, one group (n = 3) was treated orally with adefovir dipivoxil at a dose of 15 mg/kg per day plus lamivudine, and a second group (n = 3) was treated with H2O placebo plus lamivudine. In woodchucks treated with adefovir dipivoxil, two had the A566T mutation, and one had both A566T and L565V. In the group maintained on lamivudine monotherapy, A566T alone was present in one animal, another carried both A566T and L565V, and in the third, no B-domain mutations were detected. There was a 4.5 log reduction in serum WHV DNA after 12 weeks of treatment with the adefovir/lamivudine combination, while in the lamivudine monotherapy controls, WHV DNA decreased by only 0.83 log (P > 0.001). A slight recurrence in serum titers of WHV DNA was observed one week after withdrawal of adefovir treatment but no further increase in viral load was observed during the remainder of the 12-week post-treatment follow-up period. The results demonstrate that supplemental adefovir dipivoxil treatment is effective in suppressing replication of lamivudine-resistant B-domain mutants in the woodchuck model of hepatitis B virus infection.

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.

Kinetics of viremia and acute liver injury in relation to outcome of neonatal woodchuck hepatitis virus infection.

The kinetics of serum viral responses and acute liver injury were studied during neonatal woodchuck hepatitis virus (WHV) infection in relation to the chronic or resolved outcome. The mean concentrations of serum WHV DNA and surface antigen were significantly higher by week 10 post infection in chronic infections compared to resolving infections, and diverged even further by the time of peak viral load development in serum (week 12). After week 12, these viral markers were detected less frequently with time and at lower concentrations in the resolved outcome. In both outcomes, mean serum activities of hepatic enzymes became increased significantly above baseline by weeks 10-12, peaked at week 14, and normalized by weeks 20-22, thus indicating transient acute liver injury. The increasing liver injury responses were comparable between outcomes at week 12, when serum viral load was markedly higher in the developing chronic infections. This suggested a deficiency in early non-cytolytic control of infection in the chronic outcome. At week 14, liver injury was significantly greater in the resolved outcome and associated with higher mean Fas ligand (FasL) and perforin messenger RNAs (mRNAs) in liver compared to the chronic outcome. This indicated greater immune-mediated killing of infected hepatocytes during resolution. Thus, chronicity as an outcome of neonatal WHV infection develops relatively early during the acute phase of infection due to reduced immune-mediated clearance of infected hepatocytes by both cytolytic and non-cytolytic processes.

Korean medicinal plant extracts exhibit antiviral potency against viral hepatitis.

OBJECTIVES: Investigation of natural ethnopharmacologic extracts exhibiting antiviral potential may lead to the discovery of new therapeutics for the treatment of chronic viral hepatitis infections. Traditional Korean medicinal herbs have been identified that exhibit potency against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Research on the antiviral potential of naturally derived extracts is facilitated through the use of appropriate animal and liver cell culture models for these hepatotrophic pathogens. Objectives of this study were to demonstrate antiviral activity of an aqueous extract of herbal formulation KYH-1 in surrogate in vitro assays for HBV and HCV and identify mechanisms of action. METHODS: Antiviral potency of KYH-1 was measured in tissue culture systems that support replication of the woodchuck hepatitis virus (WHV), and the bovine viral diarrhea virus (BVDV). These assays serve as surrogate models for HBV and HCV, respectively. A recombinant HBV polymerase gene expression assay was used to define a molecular target. RESULTS: KYH-1 exhibited potent antiviral activity against WHV and to a lesser extent against BVDV. KYH-1 and its constituent components inhibited HBV polymerase priming in vitro. Additionally, KYH-1 suppressed HBV replication in a human hepatoblastoma cell line. CONCLUSION: Evaluation of naturally derived products for antiviral activity against HBV and HCV in standardized surrogate assays provides a scientific basis for potential use as complementary or alternative medicines. This study provides significant results justifying preclinical evaluation of KYH-1 as an antiviral therapy for HBV infections.

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.

Emergence of drug-resistant populations of woodchuck hepatitis virus in woodchucks treated with the antiviral nucleoside lamivudine.

Lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] reduces woodchuck hepatitis virus (WHV) titers in the sera of chronically infected woodchucks by inhibiting viral DNA synthesis. However, after 6 to 12 months, WHV titers begin to increase toward pretreatment levels. Three WHV variants with mutations in the active site of the DNA polymerase gene are present at this time (W. S. Mason et al., Virology 245:18-32, 1998). We have asked if these mutant viruses were responsible for the lamivudine resistance and if their emergence caused an immediate rise in virus titers. Cell cultures studies implied that the mutants were resistant to lamivudine. Emergence of mutant WHV was not always associated, however, with an immediate rise in virus titers in the serum. One of the three types of mutant viruses became prominent in serum up to 7 months before titers in serum actually began to increase, at a time when wild-type virus was still predominant in the liver. The two other mutants did not show this behavior but were detected in serum and liver later, just at the time that virus titers began to rise. A factor linking all three mutants was that a similar duration of drug administration preceded the rise in titers, irrespective of which mutant ultimately prevailed. A simple explanation for these results is that the increase in virus titers following emergence of drug-resistant mutants can occur only as the preexisting wild-type virus is cleared from the hepatocyte population, allowing spread of the mutants. Thus, prolonged suppression of virus titers in the serum may sometimes be a measure of the stability of hepatocyte infection rather than of a successful therapeutic outcome.

Apparent helper-independent infection of woodchucks by hepatitis delta virus and subsequent rescue with woodchuck hepatitis virus.

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.

Characterization and immortalization of woodchuck hepatocytes isolated from normal and hepadnavirus-infected woodchucks (Marmota monax).

Primary woodchuck (Marmota monax) hepatocytes from normal woodchucks and woodchucks with chronic woodchuck hepatitis virus (WHV) infection were cultured in either a conventional serum-containing medium or a serum-free medium. The de novo synthesis of the plasma proteins albumin, transferrin, fibrinogen, and complement C3 were identical under both conditions. However, expression of the WHV and the synthesis of nitric oxide were diminished under serum-free conditions. Primary woodchuck hepatocytes cultured in conventional, serum-containing medium were immortalized utilizing the simian virus 40 T antigen oncogene. Immortalized hepatic cell lines retained differentiated functions of nitric oxide synthesis and expression of complement C3. The woodchuck hepatocyte culture model will supplement current experimental methods, allowing investigation of hepadnaviral pathogenesis, including hepatocarcinogenesis in vitro.

Woodchuck hepatitis virus X protein is required for viral infection in vivo.

The X gene of the mammalian hepadnaviruses is believed to encode a protein of 17 kDa which has been shown to transactivate a wide range of viral and cellular promoters. The necessity for X gene expression during the viral life cycle in vivo has recently been suggested (H.-S. Chen, S. Kaneko, R. Girones, R. W. Anderson, W. E. Hornbuckle, B. C. Tennant, P. J. Cote, J. L. Gerin, R. H. Purcell, and R. H. Miller, J. Virol. 67:1218-1226, 1993). We have independently constructed two variants of woodchuck hepatitis virus (WHV) with mutations in the X coding region. Transient transfection of two different hepatoma cell lines showed that these WHV X gene mutants were competent for virus replication in vitro. To determine whether X expression was required for viral replication in vivo, we injected mutant and wild-type genomes into the livers of susceptible woodchucks. While the wild-type WHV genomes were infectious in all animals examined, the mutant genomes did not initiate a WHV infection in woodchucks. These results indicate that the X gene of the hepadnaviruses plays a major role in viral replication in vivo.

Monitoring of early events of experimental woodchuck hepatitis infection: studies of peripheral blood mononuclear cells by cytofluorometry and PCR.

The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated: two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37% +/- 25 and 17% +/- 15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.