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.     

Electroporation enhances immunogenicity of a DNA vaccine expressing woodchuck hepatitis virus surface antigen in woodchucks

The development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection.     

Hepatic lesions in woodchucks (Marmota monax) seronegative for woodchuck hepatitis virus

Livers from 128 captive woodchucks (Marmota monax) that were negative for serological markers of woodchuck hepatitis virus (WHV) were examined grossly and histologically. Livers from 19 additional seronegative woodchucks were examined only grossly. The most common finding (61% of histological specimens) was mononuclear cells in portal areas. Moderate to severe portal infiltrates in association with similar scattered mononuclear cell aggregates and extramedullary hematopoiesis, were present in woodchucks that had extrahepatic inflammatory lesions. We concluded that these changes represent a response to a variety of chronic infections and are not specific for WHV infection. Other findings included parasitic granulomas, focal and diffuse lipidosis, bile duct proliferation, lesions secondary to cardiovascular compromise, and vasculitis. Two woodchucks had neoplastic lesions.     

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.     

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks

Woodchuck hepatitis virus (WHV), like the related hepatitis B virus, induces in its natural host hepatocellular carcinomas that contain integrated viral sequences. As a first step in determining whether and how the integrated sequences contribute to formation of the tumors in which they are found, we have cloned two such integrations of WHV and have determined their structure by restriction mapping and heteroduplex electron microscopy. The identity of the cloned sequences was confirmed by comparison of restriction sites in the clones with those located by Southern blot analysis of tumor DNA. Viral sequences in both integrations are extensively rearranged, and in neither were all parts of the viral genome represented. In this respect, the behavior of WHV in vivo is similar to that of other DNA tumor viruses that have been studied in vitro. We discuss the implications of these results in relation to possible mechanisms for tumor induction by WHV.     

Antigenic analysis of woodchuck hepatitis virus surface antigen with site-specific radioimmunoassays

Monoclonal antibodies to five nonoverlapping antigenic domains of woodchuck hepatitis virus surface antigen (WHsAg) were used to develop site-specific radioimmunoassays. The assays were based on the solid-phase sandwich principle in which different combinations of individual domain-specific antibodies were used as immunoadsorbents and radioiodinated probes. Over 85% of the combinations tested were able to detect serum WHsAg, including those using the same antibody as immunoadsorbent and probe. The limits for antigen detection in one site-specific system ranged between 16 and 80 ng of WHsAg per ml. The antigenic similarity of serum WHsAg from 13 colony woodchucks was shown with several combination assay systems. WHsAg was equally immunoreactive in these assay systems whether obtained by immunoaffinity chromatography or standard rate zone centrifugation methods. Further site-specific analysis demonstrated that Formalin treatment of purified antigen did not affect the immunoreactivity of these WHsAg sites.     

Genetic variation between woodchuck populations with high and low prevalence rates of woodchuck hepatitis virus infection

Woodchuck hepatitis virus (WHV) infection is known to be endemic in areas of the mid-Atlantic states but is apparently absent from populations in New York and much of New England. Blood samples of 40 woodchucks (Marmota monax) from New York and from Delaware were examined by starch gel electrophoresis, and 18 monomorphic and six polymorphic protein-coding genetic systems were identified. Mendelian inheritance of variants of the six polymorphic systems was confirmed in 52 laboratory offspring of the original samples. Average heterozygosity of 0.066 in New York woodchucks and 0.039 in Delaware woodchucks were high values for mammals, although similar to those of other sciurids. Significant heterogeneity between samples from New York and Delaware woodchucks was observed at two loci (peptidase with glycyl leucine-4 and phosphogluconate dehydrogenase), suggesting that these populations were genetically distinct. Whether there are genetically determined differences in response to WHV infection remains to be determined experimentally.     

Immunization with the gene expressing woodchuck hepatitis virus nucleocapsid protein fused to cytotoxic-T-lymphocyte-associated antigen 4 leads to enhanced specific immune responses in mice and woodchucks

A number of options are available to modify and improve DNA vaccines. An interesting approach to improve DNA vaccines is to fuse bioactive domains, like cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), to an antigen. Such fusion antigens are expressed in vivo and directed to immune cells by the specific bioactive domain and therefore possess great potential to induce and modulate antigen-specific immune responses. In the present study, we tested this new approach for immunomodulation against hepadnavirus infection in the woodchuck model. Plasmids expressing the nucleocapsid protein (WHcAg) and e antigen (WHeAg) of woodchuck hepatitis virus (WHV) alone or in fusion to the extracellular domain of woodchuck CTLA-4 and CD28 were constructed. Immunizations of mice with plasmids expressing WHcAg or WHeAg led to a specific immunoglobulin G2a (IgG2a)-dominant antibody response. In contrast, fusions of WHcAg to CTLA-4 and CD28 induced a specific antibody response with comparable levels of IgG1 and IgG2a. Furthermore, the specific IgG1 response to WHcAg/WHeAg developed immediately after a single immunization with the CTLA-4-WHcAg fusion. Woodchucks were immunized with plasmids expressing WHeAg or the CTLA-4-WHcAg fusion and subsequently challenged with WHV. CTLA-4-WHcAg showed an improved efficacy in induction of protective immune responses to WHV. In particular, the anti-WHsAg antibody response developed earlier after challenge in woodchucks that received immunizations with CTLA-4-WHcAg, consistent with the hypothesis that anti-WHs response is dependent on a Th cell response to WHcAg. In conclusion, the use of fusion genes represents a generally applicable strategy to improve DNA vaccination.     

Characterization of T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.     

Nonoverlapping antigenic sites of woodchuck hepatitis virus surface antigen and their cross-reactivity with ground squirrel hepatitis virus and hepatitis B virus surface antigens.

Five nonoverlapping antigenic sites (sites I through V) of woodchuck hepatitis virus surface antigen were identified with competitive binding assays involving monoclonal antibodies. Site I contributed to cross-reactions among surface antigens of hepatitis B-like viruses infecting woodchucks, ground squirrels, and humans. At least three distinct sites (sites I, II, and III) are responsible for cross-reactions between woodchuck and ground squirrel hepatitis virus surface antigens. Sites IV and V of woodchuck hepatitis virus surface antigen are not major cross-reactive sites, suggesting that these elicit virus-specific antibodies. There were no cross-reactions with duck hepatitis B virus surface antigen.     

Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen.

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.     

Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV)

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8 + memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection.The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RT-PCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model.     

Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV)

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8+memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection. The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RT-PCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model.     

Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV)

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.     

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.     

Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity.

Immunosuppression is known to influence the state of chronic hepatitis B virus infection, and is thought to increase the risk of developing chronic infection in newly exposed individuals. Cyclosporin A (CsA), an immunosuppressive agent that inhibits Th cell function, was administered to woodchucks chronically infected with woodchuck hepatitis virus (WHV), and resulted in a decreased severity of chronic hepatitis and an increased viremia during the treatment. Adult woodchucks inoculated with WHV and given CsA for 14 wk had increased viremias, decreased acute phase liver injury, and developed chronic infections at a higher rate compared with immunocompetent woodchucks given virus alone (chronicity in seven of seven WHV + CsA + vs zero of nine WHV + CsA-; p less than 0.001). These results in a relevant animal model of hepatitis B virus infection indicate: 1) that liver injury in acute hepadnavirus infections is immune-mediated and not a direct cytopathic effect of virus replication; 2) that Th cells function in the inflammatory response and in the immunologic control of hepadnavirus infection; and 3) that suppression of Th cell function in acute hepadnavirus infection decreases liver injury but alters the outcome of infection in favor of chronicity. These results also suggest continued challenges in the application of CsA in liver transplantation for hepatitis B virus-induced diseases.     

Serological relationship of woodchuck hepatitis virus to human hepatitis B virus.

Two antigenic systems of the woodchuck hepatitis virus have been identified. The relationship between viral antigens of the woodchuck hepatitis virus and the human hepatitis B virus was determined by using immunoprecipitation, hemagglutination, and immune electron microscopy techniques. Antigens found on the cores of the two viruses were cross-reactive. Lack of cross-reactivity between the surface antigens of the two viruses in immunodiffusion experiments suggested that the major antigenic determinants of the viral surfaces are different; however, results of passive hemagglutination tests indicated that there are common minor determinants. Nucleic acid homology, as measured by liquid hybridization, was found to be 3 to 5% of the viral genomes. The results of this study provide further evidence that woodchuck hepatitis virus is the second member of a new class of viruses represented by human hepatitis B virus. Since virus-infected woodchucks may acquire chronic hepatitis and hepatocellular carcinoma, these antigens and their respective antibodies will be useful markers for following the course of virus infection in investigations of the oncogenic potential of this class of viruses. The nucleocapsid antigen described may be a class-specific antigen of these viruses and, thus, may be useful in discovering new members of the group.