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.     

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 human hepatitis delta virus in primary cultures of woodchuck hepatocytes.

We obtained two lines of evidence that monolayer cultures of primary woodchuck hepatocytes support replication of the genome of human hepatitis delta virus (HDV). (i) From a Northern (RNA blot) analysis of the HDV-related RNA in infected cultures, both genomic and antigenomic 1.7-kilobase RNA species were detected at 11 days after infection. The ratio of genomic RNA to antigenomic RNA was 2:1 to 10:1, comparable to that previously reported in studies of experimentally infected chimpanzees and woodchucks. (ii) Replication in culture was also demonstrated by in situ hybridization with a strand-specific probe. Such studies showed that only a small fraction of the cultured cells supported replication and that in such cells the relative and absolute levels of the HDV RNAs were comparable to those in liver cells infected in vivo. Furthermore, as with the in vivo studies, the HDV RNAs were predominantly localized to the nucleus. In summary, we demonstrated that cultured cells supported both the early events of HDV adsorption and penetration and the intermediate events of genome replication.     

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.     

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.     

Tissue culture system for infection with human hepatitis delta virus.

An in vitro culture system was developed for assaying the infectivity of the human hepatitis delta virus (HDV). Hepatocytes were isolated from chimpanzee liver and grown in a serum-free medium. Cells were shown to be infectible by HDV and to remain susceptible to infection for at least 3 weeks in culture, as evidenced by the appearance of RNA species characteristic of HDV replication as early as 6 days postinfection. When repeated experiments were carried out on cells derived from an animal free of hepatitis B virus (HBV), HDV infection occurred in a consistent fashion but there was no indication of infection with the HBV that was present in the inoculum. Despite numerous attempts with different sources of HBV inocula free of HDV, there was no evidence that indicated susceptibility of these cells to HBV infection. This observation may indicate that HBV and HDV use different modes of entry into hepatocytes. When cells derived from an HBV-infected animal were exposed to HDV, synthesis and release of progeny HDV particles were obtained in addition to HBV replication and production of Dane particles. Although not infectible with HBV, primary cultures of chimpanzee hepatocytes are capable of supporting part of the life cycle of HBV and the entire life cycle of HDV.     

Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies.

Hepatitis delta virus (HDV) is a defective RNA virus which is dependent on hepatitis B virus for essential helper functions. Only a single highly basic phosphoprotein, HDV antigen (HDAg), is expressed by the HDV genome during infection in humans. Antibody directed to HDAg is important in the diagnosis of HDV infection, and it is likely but not yet proven that the immune response to HDAg provides significant protection against subsequent exposures to HDV. In an effort to map the antigenic domains of HDAg, 209 overlapping hexapeptides, spanning the entire 214 amino acid residues of the protein, were synthesized on polyethylene pins and probed by enzyme-linked immunosorbent assay with sera containing high titers of anti-HD antibodies. Domains recognized by antibodies present in serum from human chronic carriers of this virus included residues 2 to 7, 63 to 74, 86 to 91, 94 to 100, 159 to 172, 174 to 195, and 197 to 207. Antibody from an acutely superinfected woodchuck recognized similar epitopes, as well as a domain spanning residues 121 to 128. Together, residues in these antigenic domains constitute 41% of the HDAg molecule. Oligopeptides 15 to 29 residues in length and representing epitopes of HDAg found to be dominant in humans (residues 2 to 17, 156 to 184, and 197 to 211) were synthesized in bulk and found to possess significant antigenic activity by microdilution enzyme-linked immunosorbent assay. The reactivity of peptide 197-211 with human sera confirms that the entire 214 amino acids of HDAg are expressed during infection in vivo. In addition, these results suggest that synthetic peptides may be useful reagents for development of new and improved diagnostic tests for HDV infection.     

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.     

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.     

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.     

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.     

Purinergic receptor functionality is necessary for infection of human hepatocytes by hepatitis delta virus and hepatitis B virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are major sources of acute and chronic hepatitis. HDV requires the envelope proteins of HBV for the processes of assembly and infection of new cells. Both viruses are able to infect hepatocytes though previous studies have failed to determine the mechanism of entry into such cells. This study began with evidence that suramin, a symmetrical hexasulfated napthylurea, could block HDV entry into primary human hepatocytes (PHH) and was then extrapolated to incorporate findings of others that suramin is one of many compounds that can block activation of purinergic receptors. Thus other inhibitors, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) and brilliant blue G (BBG), both structurally unrelated to suramin, were tested and found to inhibit HDV and HBV infections of PHH. BBG, unlike suramin and PPADS, is known to be more specific for just one purinergic receptor, P2X7. These studies provide the first evidence that purinergic receptor functionality is necessary for virus entry. Furthermore, since P2X7 activation is known to be a major component of inflammatory responses, it is proposed that HDV and HBV attachment to susceptible cells, might also contribute to inflammation in the liver, that is, hepatitis.     

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.     

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 against woodchuck hepatitis virus (WHV) infection by gene gun coimmunization with WHV core and interleukin-12

Woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) are closely similar with respect to genomic organization, host antiviral responses, and pathobiology of the infection. T-cell immunity against viral nucleocapsid (HBcAg or WHcAg) has been shown to play a critical role in viral clearance and protection against infection. Here we show that vaccination of healthy woodchucks by gene gun bombardment with a plasmid coding for WHcAg (pCw) stimulates proliferation of WHcAg-specific T cells but that these cells do not produce significant levels of gamma interferon (IFN-gamma) upon antigen stimulation. In addition, animals vaccinated with pCw alone were not protected against WHV inoculation. In order to induce a Th1 cytokine response, another group of woodchucks was immunized with pCw together with another plasmid coding for woodchuck interleukin-12 (IL-12). These animals exhibited WHcAg-specific T-cell proliferation with high IFN-gamma production and were protected against challenge with WHV, showing no viremia or low-level transient viremia after WHV inoculation. In conclusion, gene gun immunization with WHV core generates a non-Th1 type of response which does not protect against experimental infection. However, steering the immune response to a Th1 cytokine profile by IL-12 coadministration achieves protective immunity. These data demonstrate a crucial role of Th1 responses in the control of hepadnavirus replication and suggest new approaches to inducing protection against HBV infection.     

Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence.

The complete nucleotide sequence of a woodchuck hepatitis virus genome cloned in Escherichia coli was determined by the method of Maxam and Gilbert. This sequence was found to be 3,308 nucleotides long. Potential ATG initiator triplets and nonsense codons were identified and used to locate regions with a substantial coding capacity. A striking similarity was observed between the organization of human hepatitis B virus and woodchuck hepatitis virus. Nucleotide sequences of these open regions in the woodchuck virus were compared with corresponding regions present in hepatitis B virus. This allowed the location of four viral genes on the L strand and indicated the absence of protein coded by the S strand. Evolution rates of the various parts of the genome as well as of the four different proteins coded by hepatitis B virus and woodchuck hepatitis virus were compared. These results indicated that: (i) the core protein has evolved slightly less rapidly than the other proteins; and (ii) when a region of DNA codes for two different proteins, there is less freedom for the DNA to evolve and, moreover, one of the proteins can evolve more rapidly than the other. A hairpin structure, very well conserved in the two genomes, was located in the only region devoid of coding function, suggesting the location of the origin of replication of the viral DNA.