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.     

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.     

Enrichment of a precore-minus mutant of duck hepatitis B virus in experimental mixed infections

A precore-deficient mutant of duck hepatitis B virus (DHBV) produced by site-directed mutagenesis was tested for its ability to compete with wild-type virus in a mixed infection of 3-day-old ducklings. The mutation was shown to produce a cis-acting defect, resulting in a replication rate that was about one-half that of wild-type virus. Accordingly, wild-type virus was rapidly selected during the spread of infection. During the chronic phase of the infection, however, two selection patterns were seen. In 4 of 10 ducks, the wild-type virus slowly replaced the precore mutant. In another four ducks, the precore mutant virus slowly replaced the wild-type virus. In the remaining two ducklings, ratios of wild-type and precore mutant virus fluctuated, with wild-type virus slowly predominating. The replacement of wild-type virus was not due to the emergence of a rapidly replicating variant of the precore mutant, since genomes cloned from the infected ducks retained their original replication defect. Replacement of wild-type virus, however, correlated with elevated anti-core antibody titers, which continued to increase with time. The selection of a precore-negative strain of DHBV may be analogous to the selection for precore mutants of HBV during chronic hepatitis in humans.     

Correlation of Virus and Host Response Markers with Circulating Immune Complexes during Acute and Chronic Woodchuck Hepatitis Virus Infection

Woodchuck hepatitis virus (WHV) is an established model for human hepatitis B virus. The kinetics of virus and host responses in serum and liver during acute, self-limited WHV infection in adult woodchucks were studied. Serum WHV DNA and surface antigen (WHsAg) were detected as early as 1 to 3 weeks following experimental infection and peaked between 1 and 5 weeks postinfection. Thereafter, serum WHsAg levels declined rapidly and became undetectable, while WHV DNA levels became undetectable much later, between 4 and 20 weeks postinfection. Decreasing viremia correlated with transient liver injury marked by an increase in serum sorbitol dehydrogenase (SDH) levels. Clearance of WHV DNA from serum was associated with the normalization of serum SDH. Circulating immune complexes (CICs) of WHsAg and antibodies against WHsAg (anti-WHs) that correlated temporarily with the peaks in serum viremia and WHs antigenemia were detected. CICs were no longer detected in serum once free anti-WHs became detectable. The detection of CICs around the peak in serum viremia and WHs antigenemia in resolving woodchucks suggests a critical role for the humoral immune response against WHsAg in the early elimination of viral and subviral particles from the peripheral blood. Individual kinetic variation during WHV infections in resolving woodchucks infected with the same WHV inoculum and dose is likely due to the outbred nature of the animals, indicating that the onset and magnitude of the individual immune response determine the intensity of virus inhibition and the timing of virus elimination from serum.     

Primary Seronegative but Molecularly Evident Hepadnaviral Infection Engages Liver and Induces Hepatocarcinoma in the Woodchuck Model of Hepatitis B

Hepadnavirus at very low doses establishes in woodchucks asymptomatic, serologically undetectable but molecularly evident persistent infection. This primary occult infection (POI) preferentially engages the immune system and initiates virus-specific T cell response in the absence of antiviral antibody induction. The current study aimed to determine whether POI with time may culminate in serologically identifiable infection and hepatitis, and what are, if any, its pathological consequences. Juvenile woodchucks were intravenously injected with inocula containing 10 or 100 virions of woodchuck hepatitis virus (WHV) to induce POI and followed for life or up to 5.5 years thereafter. All 10 animals established molecularly detectable infection with virus DNA in serum (<100–200 copies/mL) and in circulating lymphoid cells, but serum WHV surface antigen and antibodies to WHV core antigen remained undetectable for life. By approximately 2.5–3.5 years post-infection, circulating virus transiently increased to 10³ copies/mL and virus replication became detectable in the livers, but serological markers of infection and biochemical or histological evidence of hepatitis remained undetectable. Nonetheless, typical hepatocellular carcinoma (HCC) developed in 2/10 animals. WHV DNA integration into hepatic and lymphatic system genomes was identified in 9/10 animals. Virus recovered from the liver virus-negative or virus-positive phases of POI displayed the wild-type sequence and transmitted infection to healthy woodchucks causing hepatitis and HCC. In summary, for the first time, our data demonstrate that an asymptomatic hepadnaviral persistence initiated by very small amounts of otherwise pathogenic virus, advancing in the absence of traditional serological markers of infection and hepatitis, coincides with virus DNA integration into the host''s hepatic and immune system genomes, retains liver pro-oncogenic potency and is capable of transmitting liver pathogenic infection. This emphasizes the role for primary occult hepatitis B virus infection in the development of seemingly cyptogenic HCC in seronegative but virus DNA reactive patients.     

Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.     

Mutations of the woodchuck hepatitis virus polymerase gene that confer resistance to lamivudine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil.

Administration of either lamivudine (2'-deoxy-3'-thiacytidine) or L-FMAU (2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947-1954, 1999; Y. Zhu et al., J. Virol. 75:311-322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to L-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.     

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

Abstract 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.     

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.     

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

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

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.     

Coadministration of gamma interferon with DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen enhances the specific immune response and protects against WHV infection

DNA vaccinations are able to induce strong cellular immune responses in mice and confer protection against infectious agents. However, DNA vaccination of large animals appears to be less effective and requires repeated injections of large amounts of plasmid DNA. Enhancement of the efficiency of DNA vaccines may be achieved by coapplication of cytokine-expressing plasmids. Here we investigated, with woodchucks, whether coadministration of an expression plasmid for woodchuck gamma interferon (IFN-gamma), pWIFN-gamma, can improve DNA vaccination with woodchuck hepatitis virus core antigen (WHcAg). Animals were immunized with pWHcIm (a plasmid expressing WHcAg) alone or with a combination of pWHcIm and pWIFN-gamma using a gene gun. Six weeks postimmunization, all animals were challenged with 10(5) genome equivalents of woodchuck hepatitis virus (WHV). The antibody and lymphoproliferative immune responses to WHV proteins were determined after immunization and after challenge. Vaccination with pWHcIm and pWIFN-gamma led to a pronounced lymphoproliferative response to WHcAg and protected woodchucks against subsequent virus challenge. Two of three animals vaccinated with pWHcIm alone did not show a detectable lymphoproliferative response to WHcAg. A low-level WHV infection occurred in these woodchucks after challenge, as WHV DNA was detectable in the serum by PCR. None of the pWHcIm-vaccinated animals showed an anti-WHcAg antibody response after DNA vaccination or an anamnestic response after virus challenge. Our results indicate that coadministration of the WIFN-gamma gene with pWHcIm enhanced the specific cellular immune response and improved the protective efficacy of WHV-specific DNA vaccines.     

Failure to detect polyalbumin-binding sites on the woodchuck hepatitis virus surface antigen: implications for the pathogenesis of hepatitis B virus in humans.

Binding sites for polymerized albumin on hepatitis B virus components were reported in human hepatitis B virus chronic carriers predominantly with active viral replication (HB e antigen positive). The presence of comparable albumin-binding sites in the woodchuck hepatitis virus (WHV) model was examined on WHV components obtained from woodchucks with active viral replication (DNA polymerase positive). Binding sites for polymerized woodchuck serum albumin were not detected on the intact WHV virion, on 22-nm woodchuck hepatitis surface antigen (WHsAg), or on WHsAg polypeptides. Woodchuck albumin was not detected in purified 22-nm WHsAg, and anti-albumin antibodies were not detected in WHV chronic-carrier woodchucks. Our results in the WHV model argue against a role for viral polyalbumin-binding sites in tissue- and host-specific virus infectivity.     

Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.     

Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.     

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.     

Expression of the precore region of an avian hepatitis B virus is not required for viral replication.

The core-antigen-coding region of all hepadnaviruses is preceded by a short, in-phase open reading frame termed precore whose expression can give rise to core-antigen-related polypeptides. To explore the functional significance of precore expression in vivo, we introduced a frameshift mutation into this region of the duck hepatitis B virus (DHBV) genome and examined the phenotype of this mutant DNA by intrahepatic inoculation into newborn ducklings. Animals receiving mutant DNA developed DHBV infection, as judged by the presence in hepatocytes of characteristic viral replicative intermediates; molecular cloning and DNA sequencing confirmed that the original mutation was present in the progeny genomes. Infection could be efficiently transmitted to susceptible ducklings by percutaneous inoculation with serum from mutant-infected animals, indicating that infectious progeny virus was generated. These findings indicate that expression of the precore region of DHBV is not essential for genomic replication, core particle morphogenesis, or intrahepatic viral spread.