Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

Sustained efficacy and seroconversion with the Toll-like receptor 7 agonist GS-9620 in the Woodchuck model of chronic hepatitis B.

BACKGROUND & AIMS: New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV). METHODS: After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n = 7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules. RESULTS: GS-9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8(+) T cell, NK cell, B cell and interferon response transcriptional signatures. CONCLUSIONS: The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients.

Semliki forest virus expressing interleukin-12 induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma.

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with >or=1.5-log(10) reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.

Kinetics of WHV-HDV replication in acute fatal course of woodchuck hepatitis.

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.

Detection of hepatitis B and woodchuck hepatitis viral DNA in plasma and mononuclear cells from heparinized blood by the polymerase chain reaction.

Amplification by the polymerase chain reaction (PCR) of hepatitis B virus (HBV) DNA extracted from parallel samples of serum and heparinized plasma gave contradictory results, indicating that heparin inhibits virus detection. Similarly, analysis of PCR products of woodchuck hepatitis virus (WHV) DNA showed that heparinization of blood abolished WHV DNA amplification, while anticoagulation with sodium EDTA or acid citrate dextrose did not. Amplification of recombinant WHV and HBV DNA in the presence of increasing concentrations of sodium heparin progressively inhibited and finally abolished virus genome detection. The inhibitory effect of heparin was reversed by treatment of either plasma or isolated DNA with heparinase (5 U/reaction, 1 h at 28 degrees C) prior to PCR. In contrast, heparin did not influence the detection of hepadnavirus in peripheral blood mononuclear cells (PBMC), even after prolonged incubation of the cells with heparin in culture. These findings confirm that heparin exerts a dramatic inhibitory effect on hepadnaviral DNA detection by PCR and they demonstrate that this effect can be reversed by heparinase. The findings also show that extensively washed PBMC derived from heparinized blood can be a reliable source of nucleic acids for amplification of hepadnavirus genome. These results imply that previous data should be reassessed if samples of heparinized plasma were found hepadnavirus DNA nonreactive by PCR or when these samples were used as a starting material for PCR quantitation of viral genome.

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.

Enhanced levels of DNA adducts in the liver of woodchucks infected with hepatitis virus.

Liver DNA specimens from woodchucks kept in captivity, 10 naturally infected with hepatitis virus (WHV) and five WHV-free, were examined for the presence of carcinogen-DNA adducts by 32P-postlabeling. The number of adducts was significantly higher in WHV carriers than in uninfected animals, and the total amounts of adducts per 10(9) nucleotides were also considerably enhanced by WHV infection, when using both butanol extraction (22.2 +/- 7.1 vs. 12.6 +/- 2.8, means +/- S.D.) and nuclease P1 enrichment (8.5 +/- 5.9 vs. 2.8 +/- 1.7). Two individual adducts were also significantly higher in WHV carriers. No significant variation occurred as related to age, sex or time length of captivity. These findings are consistent with our previous studies supporting an enhanced metabolism of chemical hepatocarcinogens in both human and woodchuck hepadnavirus infections. Several significant and remarkable correlations were pointed out by relating DNA adduct data to more than 30 virological, histopathological and metabolic parameters which had been previously evaluated in the same animals. For instance, numbers and/or levels of adducts were positively related to the amounts of virus present in hepatocytes, to cell damage (gamma-glutamyltranspeptidase activity), to the severity of the liver histopathological picture, and to monooxygenase activities, while they were inversely related to cellular glutathione concentrations and to detoxification of the direct-acting mutagen 4-nitroquinoline 1-oxide. The major adduct significantly correlated with the metabolic activation of the aromatic amine 2-aminofluorene and of the heterocyclic amines 3-amino-1-methyl-5H-pyrido(4,3)indole (Trp-P-2) and 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ), whereas another adduct significantly correlated with the metabolic activation of the mycotoxin aflatoxin B1. Thus, the enhanced metabolism of chemical hepatocarcinogens and the increased formation of carcinogen-DNA adducts in the liver of WHV carriers appear to represent one of the mechanisms contributing to the association between chronic hepadnavirus infection and development of primary hepatocellular carcinoma.

Hepatic cirrhosis occurring in a young woodchuck (Marmota monax) due to vertical transmission of woodchuck hepatitis virus (WHV).

Histologic and clinicopathologic findings of a woodchuck (Marmota monax) vertically infected with woodchuck hepatitis virus (WHV) are presented. The liver exhibits marked cirrhotic changes, which is characteristic of the pre-transformation phase of WHV. At necropsy, the woodchuck exhibited ascites and the liver had a grossly nodular appearance. Microscopically, focal hepatocyte necrosis and inflammatory cells were observed in midzonal and periportal areas in the liver. In Macchiavellos stained sections, cytoplasmic inclusion bodies appeared reddish granular materials. We believe that this may represent a new suitable and cost-effective cirrhotic model for the disease processes associated with hepadnaviruses in a number of other species, most notably Hepatitis B virus infection in man.

Nucleoside analogues alone or combined with vaccination prevent hepadnavirus viremia and induce protective immunity: alternative strategy for hepatitis B virus post-exposure prophylaxis.

OBJECTIVES: The current strategies for hepatitis B virus (HBV) post-exposure prophylaxis (PEP) are not generally available in remote and rural areas of developing countries and/or carry potential risks for infection with blood-borne transmitted pathogens. Nucleotide analogues (NAs) are successfully used for human immunodeficiency virus PEP, and maybe effective for HBV PEP. In this study, we tested the NA-based strategies for HBV PEP using the Chinese woodchuck model. METHODS: Chinese woodchucks were inoculated intravenously with different doses of woodchuck hepatitis virus (WHV). A deoxyguanosine analogue entacavir (ETV), a DNA vaccine pWHcIm, or ETV plus pWHcIm were applied to the infected animals 24h later. Twenty weeks later, the animals were re-challenged with WHV to test for the presence of immunity against WHV. RESULTS: Inoculation with different WHV doses had a strong influence on the course of WHV infection; NA alone or in combination with a DNA vaccine completely prevented viremia after a high dose of WHV inoculation in Chinese woodchucks and induced partial or complete protective immunity, respectively. CONCLUSIONS: NA-based PEP strategies (NA alone or in combination with vaccine) may be an alternative of HBV PEP, especially in those living in the remote and rural areas of the developing countries and the non-responders to the current vaccine, and may be valuable in the PEP of HBV and HIV co-infection after occupational and non-occupational exposure. Further clinical studies are warranted to confirm the valuable of NA-based strategies in HBV PEP.

Hexokinase and glucose-6-phosphatase activity in woodchuck model of hepatitis virus-induced hepatocellular carcinoma.

2-Deoxy-2-[(18)F]fluoro-D-glucose ([(18)F] FDG) is used for PET imaging of woodchuck (Marmota monax) model of hepatocellular carcinoma (HCC). The usefulness of FDG on this animal model needs to be validated according to the hypothesized mechanisms. In this study, two key enzymes involved in glucose or [(18)F] FDG metabolism, hexokinase (HK) and glucose-6-phophatase (G6Pase), were examined for their enzymatic activities in the woodchuck models of HCC, which has not been studied before. After dynamic PET scans, woodchuck liver tissue samples were harvested and the homogenate was centrifuged. The supernatant was used for HK activity assay and the microsomal pellet was used for G6Pase assay. HK and G6Pase activities were measured by means of colorimetric reactions via kinetic and end-point assays, respectively. Total protein content was measured by the Bradford method and used to normalize all enzyme activities. HK and G6Pase activities in woodchuck HCC will be used to correlate with in vivo PET imaging data. The woodchuck model of HCC had significantly increased levels of HK in the livers compared to the age-matching healthy woodchuck (7.96 +/- 1.27 vs. 2.74 +/- 0.66 mU/mg protein, P < 0.01) and significantly decreased levels of G6Pase compared to healthy woodchuck (40.35 +/- 19.28 vs. 237.01 +/- 17.32 mU/mg protein, P < 0.01), reflecting an increase in glycolysis. In addition, significant differences were found in HK and G6Pase activities between HCC liver region (HK: 7.96 +/- 1.27 mU/mg protein; G6Pase: 40.35 +/- 19.28 mU/mg protein) and surrounding normal liver region (HK: 2.98 +/- 0.92 mU/mg protein; G6Pase: 140.87 +/- 30.62 mU/mg protein) in the same woodchuck model of HCC (P < 0.01). Our study demonstrated an increased HK activity and a decreased G6Pase activity in liver of the woodchuck models of HCC as compared to normal woodchuck liver.

A pre-S/S CHO-derived hepatitis B virus vaccine protects woodchucks from WHV productive infection.

We evaluated whether a non-adjuvanted vaccine derived from Chinese hamster ovary cells was capable of providing protection against woodchuck hepatitis virus (WHV). Three woodchucks were vaccinated with four 50-microg doses and challenged with a previously characterized virus isolate (WHV197). In all three animals, titre levels of antibodies against hepatitis B surface antigens (anti-HBs) exceeded 10 mIU/ml, peaking at 150 mIU/ml. Challenge resulted in productive acute infection in the two non-vaccinated woodchucks yet in none of the vaccinated woodchucks. In the vaccinated animals, there was evidence of abortive infection. The results demonstrate that a human vaccine is able to protect woodchucks from WHV infection.

A TaqMan PCR assay using degenerate primers for the quantitative detection of woodchuck hepatitis virus DNA of multiple genotypes.

Woodchuck hepatitis virus (WHV) is a valuable animal model system for studies of hepatitis B virus infection and accurate assessments of WHV viral load are necessary in these studies. Wild-captured woodchucks that are naturally infected with WHV are sometimes used in these studies, however, the sequence variation in WHV isolates generally precludes the use of TaqMan PCR. To facilitate this, we have created a real-time TaqMan PCR assay for WHV using degenerate primers with inosine residues employed at the locations of known sequence heterogeneity. This TaqMan assay has a dynamic range of 10-10(8) genomic equivalents (ge) of WHV DNA per reaction and the assay is robust and reproducible in the 10(2)-10(7) ge WHV DNA per reaction range (intra-assay coefficient of variation (CV) <2.1%, inter-assay CV <2.9%). During our assay validation, we cloned and analyzed a series of six naturally occurring virus variants that contained sequence heterogeneity in the TaqMan primer sequence region. We showed that the presence of some of these sequence variations prevented the PCR amplification of the target when regular primer sequences were used, while degenerate primer sequences were able to efficiently amplify all tested sequences equally well.

Recurrence of WHV integration in the b3n locus in woodchuck hepatocellular carcinoma.

Frequent occurrence of woodchuck hepatitis virus DNA (WHV DNA) integration into or in proximity to myc oncogenes and in the win locus of cellular genome in woodchuck hepatocellular carcinomas (HCC) has been described by several authors. We report a further cellular locus as a recurrent target for WHV integration in woodchuck HCCs. A WHV DNA integration and its cellular flanking regions were cloned from a HCC developed in a chronically WHV-infected woodchuck. Sequence analysis showed integration of rearranged C, PreS1, and 5' truncated X regions of the WHV genome, located in a cellular locus previously described for WHV integration in another woodchuck HCC. The two integration sites are only about 0.5 kb apart. In addition to Alu-like repeats and a gag-like coding region, previously described, we found several features of MAR (matrix attachment region) chromosomal sequences in the normal cellular locus, leading us to predict that part of it might be a previously unrecognized MAR.

Titration of recombinant woodchuck hepatitis virus DNA in adult woodchucks.

In vivo transfection of Eastern woodchucks (Marmota monax) with recombinant woodchuck hepatitis virus (WHV) DNA is effective in inducing virus infection for the study of replication, pathogenicity, and oncogenicity of wild-type and mutated WHV. The one drawback to this procedure is the need for preparation of large amounts of WHV DNA. Reduction of the amount of WHV DNA in the transfection protocol necessary to induce infection would save considerable time and resources. Therefore, we conducted a titration of WHV DNA, ranging from 50 micrograms to 50 pg of DNA, in adult woodchucks to determine the minimum infectious dose of recombinant WHV DNA. As little as 50 ng of transfected WHV DNA induced productive infection in adult woodchucks. Thus, transfection with large amounts of recombinant WHV DNA appears to be unnecessary.

Apoptosis and regeneration of hepatocytes during recovery from transient hepadnavirus infections.

It is well known that hepatitis B virus infections can be transient or chronic, but the basis for this dichotomy is not known. To gain insight into the mechanism responsible for the clearance of hepadnavirus infections, we have performed a molecular and histologic analysis of liver tissues obtained from transiently infected woodchucks during the critical phase of the recovery period. We found as expected that clearance from transient infections occurred subsequent to the appearance of CD4(+) and CD8(+) T cells and the production of interferon gamma and tumor necrosis factor alpha in the infected liver. These events were accompanied by a significant increase in apoptosis and regeneration of hepatocytes. Surprisingly, however, accumulation of virus-free hepatocytes was delayed for several weeks following this initial influx of lymphocytes. In addition, we observed that chronically infected animals can exhibit levels of T-cell accumulation, cytokine expression, and apoptosis that are comparable with those observed during the initial phase of transient infections. Our results are most consistent with a model for recovery predicting replacement of infected hepatocytes with regenerated cells, which by unknown mechanisms remain protected from reinfection in animals that can be cured.

Hepatocellular carcinoma in black-tailed prairie dogs (Cynomys ludivicianus): tumor morphology and immunohistochemistry for hepadnavirus core and surface antigens.

From 1994 to 2002, tissues from 61 prairie dogs were submitted to Northwest ZooPath for histopathology. Of these, 12 (20%) had hepatocellular carcinoma (HCC). Three were pets submitted from private veterinary practices. The others were submitted from zoos in the United States. All were adults, ranging from young adult to 7 years of age, with average age of 5.1 years. The most common clinical signs were weight loss, lethargy, palpable abdominal mass, and respiratory difficulty. All tumors were well-differentiated HCCs in which four histologic patterns were recognized. The trabecular pattern was predominant in nine tumors, and the pseudoglandular pattern was predominant in two tumors. The pelioid pattern was also represented in eight tumors. A papillary pattern was present in one case. In seven cases vacuolar change resembling lipidosis was present in the neoplastic hepatocytes of both primary and metastatic tumors. Anaplasia was mild to moderate in most tumors, but a marked degree of anaplasia was noted in the metastatic foci of the case with papillary differentiation. Metastasis to lung was noted in five cases. One of these also had metastasis to the spleen, and another had metastasis to heart and mediastinum. In two cases there was concurrent hepatitis and in two cases, cirrhosis. All tumors and nonneoplastic liver stained negatively for woodchuck hepatitis virus surface and core antigens, and orcein and Victoria blue positive staining of hepatocytes typical of hepadnavirus infection in humans and woodchucks was not present. HCC is apparently common in captive prairie dogs. The hepatic neoplasia observed in prairie dogs was similar to that associated with hepadnaviral infection in humans, woodchucks, and ground squirrels, but no direct evidence of hepadnaviral infection was detected. The rate of metastasis in captive prairie dogs was higher than that reported in woodchucks.

Pathogenesis of experimental neonatal woodchuck hepatitis virus infection: chronicity as an outcome of infection is associated with a diminished acute hepatitis that is temporally deficient for the expression of interferon gamma and tumor necrosis factor-alpha messenger RNAs.

Surgical biopsies of the liver were obtained from woodchuck hepatitis virus (WHV)-infected neonatal woodchucks at 2 time points before the self-limited or chronic outcomes became obvious by serologic criteria. Following segregation of outcomes, livers were analyzed for intrahepatic type 1 cytokine messenger RNAs (mRNAs) (interleukin 2 [IL-2], interferon gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha]) and leukocyte inflammatory phenotype (IgG+ plasma cells, lysozyme+ macrophages, CD3+ T cells). Baselines were assessed using age-matched uninfected control livers. At week 8 (early acute phase), intrahepatic type 1 cytokine mRNAs were similarly low in both outcome settings and no different from age-matched uninfected controls. This was consistent with the minimal initial viral loads and lack of histologic inflammation at this time. At week 14 (mid-acute phase), changes in viral load between outcome groups related inversely to the intrahepatic inflammatory responses. Animals that eventually became resolved had increased intrahepatic expression of IFN-gamma and TNF-alpha mRNAs and robust inflammation by CD3+ T cells, plasma cells, and macrophages. At the same time point of infection, animals that eventually became chronic carriers had an acute hepatitis involving the same cell types, but at diminished levels, and markedly deficient intrahepatic expression of IFN-gamma and TNF-alpha mRNAs. IL-2 mRNA remained at baseline control levels in both outcome groups. These cotemporal comparisons map a critical deviation in host response to the acute stage of an evolving chronic infection. They strongly suggest that increasing viral load and chronicity as an outcome of neonatal WHV infection result from a temporal deficiency in the acute intrahepatic effector mechanisms mediated by IFN-gamma and TNF-alpha.

Serial transmission of spongiocytic hepatitis to woodchucks (possible association with a specific delta strain).

BACKGROUND/AIMS: Outbreaks of severe hepatitis have been reported from Africa and South America. Description of the cases has shown the histological hallmark to be the presence of ballooning hepatocytes with fat drops surrounding the nucleus (spongiocytes or morula cells). METHODS: Experimental reproduction of this syndrome for the verification of a possible role of a specific HDV strain was performed by the inoculation of serum and liver extracts from African patients (Bangui-Central African Republic), who died with this syndrome, into American woodchuck carriers of WHV (WC 231,144), the results of which were then compared with animals inoculated with a reference wild HDV strain (WC 300,173,154), and those which received material from a European fulminant HDV case (WC 88,93). RESULTS: Following the initial inoculation, the animals receiving African inocula had a delayed anti-HDV seroconversion, high mortality and showed the presence of spongiocytes, while the other animals had a classical evolution of HDV superinfection in woodchucks. Furthermore, the African inocula caused less inhibition of WHV replication, as well as a predominant cytoplasmic expression of HDAg, in contrast to the animals which received the other inocula. The second passage experiments gave similar results. CONCLUSIONS: We conclude that this peculiar form of HDV fulminant hepatitis can be experimentally reproduced and might be specifically related to a more pathogenic strain.