In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.     

Low doses of hepadnavirus induce infection of the lymphatic system that does not engage the liver

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus and is considered to be principally hepatotropic, invades the host's lymphatic system and persists in lymphoid cells independently of whether the infection is symptomatic and serologically evident or concealed. In this study, we show, with the woodchuck model of hepatitis B, that hepadnavirus can establish an infection that engages the lymphatic system, but not the liver, and persists in the absence of virus serological markers, including antiviral antibodies. This primary occult infection is caused by wild-type virus invading the host at a quantity usually not greater than 10(3) virions. It is characterized by trace virus replication progressing in lymphatic organs and peripheral lymphoid cells that, with time, may also spread to the liver. The infection is transmissible to virus-naive hosts as an asymptomatic, indefinitely long, occult carriage of small amounts of biologically competent virus. In contrast to residual silent WHV persistence, which normally endures after the resolution of viral hepatitis and involves the liver, primary occult infection restricted to the lymphatic system does not protect against reinfection with a large, liver-pathogenic WHV dose; however, the occult infection is associated with a swift recovery from hepatitis caused by the superinfection. Our study documents that the lymphatic system is the primary target of WHV infection when small quantities of virions invade a susceptible host.     

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.     

Quantitative detection of hepadnavirus-infected lymphoid cells by in situ PCR combined with flow cytometry: implications for the study of occult virus persistence

The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean +/- standard error of the mean, 9.6% +/- 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean +/- standard error of the mean, 4.8% +/- 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.     

Natural history of woodchuck hepatitis virus infections during the course of experimental viral infection: molecular virologic features of the liver and lymphoid tissues.

In this study, the kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the liver and the five primary components of the lymphoid system (peripheral blood lymphocytes, lymph nodes, bone marrow, spleen, and thymus). 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. Infection by WHV was not limited to the liver but involved the major components of the lymphoid system during all stages of virus infection. A complex series of kinetic patterns was observed for the appearance of WHV DNA in the different lymphoid compartments and the liver during the entire course of viral infection. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was also observed. Lymphoid cells of the bone marrow were the first cells in which WHV DNA was detected, followed in order by the liver, the spleen, peripheral blood lymphocytes, lymph nodes, and finally the thymus. Several differences were observed in the cellular WHV DNA patterns between woodchucks that developed chronic WHV infections and those that serologically recovered from acute WHV infections. The observations compiled in this study indicate that the host lymphoid system is intimately involved in the natural history of hepadnavirus infections from the earliest stages of virus entry.     

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.     

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.     

The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.     

Nucleotide sequence of the woodchuck hepatitis virus surface antigen mRNAs and the variability of three overlapping viral genes

A cDNA library was constructed from the liver of a woodchuck chronically infected with woodchuck hepatitis virus (WHV). A clone, pWS23, encompassing the entire surface and X genes of WHV was isolated. Comparison of the complete nucleotide (nt) sequence of pWS23 with those of genomic DNAs from two different WHV isolates showed that it contained a nearly full-length copy of the major mRNA encoding the viral surface antigen (5 mRNA). It was colinear with the WHV genome over 1858 nt and terminated 22 nt downstream from the variant polyadenylation signal within the core gene. Evidence for heterogeneity of the 5′ -terminal region of the S mRNA came from direct sequencing of the 5′ extremities of 20 cDNA inserts, similar to that of pWS23, isolated from a second cDNA library of the same woodchuck liver. In agreement with previous mapping studies of hepadnaviruses, two main initiation regions of S mRNA were localized 27–30 nt upstream and 22–49 nt downstream from the pre-S2 initiation codon. Further analysis of the amino acid sequences of the surface, polymerase and X genes of WHV showed a high conservation among three WHY isolates and a similar distribution of conserved and variable regions in woodchuck and human hepatitis B viruses.     

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.     

Inhibition of cellular proteasome activities enhances hepadnavirus replication in an HBX-dependent manner

The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.     

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.     

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.     

荧光定量PCR检测土拨鼠肝炎病毒核酸方法的建立

目的建立土拨鼠肝炎病毒（woodchuck hepatitis virus,WHV）核酸的荧光定量PCR（Real-time PCR）检测方法,应用于土拨鼠肝炎病毒模型的研究。方法分别根据土拨鼠肝炎病毒核心抗原（WHcAg）和表面抗原（WHsAg）的DNA序列设计13对扩增引物,从中筛选无非特异性扩增及引物二聚体且灵敏度高的引物,用于土拨鼠血清中WHV DNA的Real-time PCR检测。建立感染土拨鼠肝炎病毒的土拨鼠血清中WHV核酸的Real-timePCR检测方法。结果根据WHsAg基因的5＇端设计的一对引物WHVSF1与WHVSR1,检测灵敏度可达1×101拷贝/μL,病毒拷贝数与Real-time PCR Ct值的标准曲线的R2值为0.997,且电泳未见明显非特异性条带及引物二聚体。结论建立了土拨鼠血清中WHV DNA的Real-time PCR检测方法,该方法为进一步研究土拨鼠肝炎病毒模型奠定了基础。     

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.     

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.