Traces of hepatitis B virus (HBV) genome can persist for years following recovery from hepatitis B. To determine overall duration, molecular characteristics, and pathological implications of this serologically undetectable form of hepadnaviral carriage, we have analyzed the expression of transcriptionally active virus genomes, their infectivity, and examined liver alterations during the natural lifespan of woodchucks convalescent from acute infection with HBVrelated woodchuck hepatitis virus (WHV). In this study, we document lifelong persistence of scanty amounts of replicating virus both in the liver and lymphatic system after spontaneous resolution of an episode of experimental hepadnaviral hepatitis. Antibodies to virus nucleocapsid (core) were found to be the most reliable immunovirological marker coexisting with occult infection. In the majority of convalescent woodchucks, serial liver biopsies showed protracted minimal to mild necroinflammation with periods of normal morphology; however, hepatocellular carcinoma (HCC) ultimately developed in 2 of 9 animals studied. Inocula derived from lymphoid cells of convalescent animals induced classical acute hepatitis in virus-naive woodchucks that progressed to chronic hepatitis and HCC in 1 of the animals, demonstrating infectivity and pathogenic competence of the carried virus. Our results reveal that low levels of infectious WHV and residual hepatic inflammation usually continue for life after resolution of hepatitis and that this recovery does not avert HCC development. They also demonstrate that, in addition to the liver, the lymphatic system is the site of the occult lifelong maintenance of replicating hepadnavirus. (HEPATOLOGY 1999;29:928-938.)Current evidence indicates that carriage of minute quantities of hepatitis B virus (HBV) genome can continue for years following complete clinical and serological recovery from acute hepatitis B. 1-5 In these convalescent patients, HBV DNA have been detected both in serum and in circulating lymphomononuclear cells, often despite the presence of neutralizing antibodies to virus envelope (hepatitis B surface antigen) 2,3,5 and a vigorous polyclonal HBV-specific cytotoxic T-cell response. 3,4,6 In addition, traces of hepatitis B surface antigen and HBV-DNA-reactive particles with physicochemical properties of intact virions have been detected in the circulation of some of the recovered individuals. 2 These findings imply that HBV eradication does not coincide with the rise of antivirusspecific humoral and cellular immune responses and with clinical resolution of acute hepatitis.It is expected that this serologically undetectable persistence of HBV may have important epidemiological and pathogenic implications, whose range has yet to be established. This assumption is based on a growing number of observations indicating that HBV infection can appear in recipients of organs from HBV serologically negative donors, [7][8][9][10][11][12] and that hepatocellular carcinoma (HCC) with integrated HBV genomic sequences can arise in ...

The importance of effective immune responses in recovery from acute hepadnaviral hepatitis has been demonstrated. However, there is no conclusive delineation of virological and immunological events occurring in the liver immediately after hepadnavirus invasion and during the preacute phase of infection. These very early events might be of primary importance in determining the recovery or progression to chronic hepatitis and the intrinsic hepadnaviral propensity to persist. In this study, applying the woodchuck model of acute hepatitis B, the hepatic kinetics of hepadnavirus replication and activation of genes encoding cytokines, cytotoxicity effectors, and immune cell markers were quantified in sequential liver biopsies collected from 1 h postinoculation onward by sensitive real-time cDNA amplification assays. The results revealed that hepadnavirus replication is established in the liver as early as 1 hour after infection. In 3 to 6 h, significantly augmented intrahepatic transcription of gamma interferon and interleukin-12 were evident, suggesting activation of antigen-presenting cells. In 48 to 72 h, NK and NKT cells were activated and virus replication was transiently but significantly reduced, implying that this early innate response is at least partially successful in limiting virus propagation. Nonetheless, T cells were activated 4 to 5 weeks later when hepatitis became histologically evident. Collectively, our data demonstrate that virus replication is initiated and the innate response activated in the liver soon after exposure to a liver-pathogenic dose of hepadnavirus. Nevertheless, this response is unable to prompt a timely adaptive T-cell response, in contrast to infections caused by other viral pathogens.

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.

Antibodies against virus nucleocapsid (anticore) normally accompany hepadnaviral hepatitis but they may also occur in the absence of symptoms and other serological indicators of the infection. This situation can be encountered following a clinically and serologically unapparent exposure to hepatitis B virus (HBV) or after recovery from hepatitis B. In this study, woodchucks inoculated with woodchuck hepatitis virus (WHV) were investigated to determine the relationship between anticore detection and the molecular status of virus replication in a primary WHV surface antigen (WHsAg)-negative infection or long-after resolution of WHV hepatitis. Serial, parallel samples of sera, peripheral blood mononuclear cells (PBMC) and liver tissue, collected for more than 5 years after inoculation with virus, were examined for WHV DNA by highly sensitive polymerase chain reaction (PCR)/nucleic acid hybridization assays. Sera were also tested for WHV DNA after DNase treatment and for WHV DNA and WHsAg after concentration in sucrose. Liver and PBMC were examined for WHV covalently closed circular DNA and viral RNA transcripts by PCR-based techniques to assess virus replication status. The study showed that anticore antibodies existing in the absence of other serological markers are a reliable indicator of occult WHV infection. This state can be accompanied by traces of circulating particles behaving as intact virions and by intermittent minimal-to-mild liver inflammation. In conclusion, the long-term presence of anticore antibodies alone is a consequence of sustained restimulation of the immune system by virus nucleocapsid produced during low-level hepadnaviral assembly. ( A ntibodies to hepatits B virus (HBV) core antigen (anti-HBc) coexist with HBV surface antigen (HBsAg) in symptomatic infection and can persist along with antibodies to HBsAg (anti-HBs) or without them for life after recovery from hepatitis B. 1,2 They may also occur in the pre-acute phase of hepatitis prior to the appearance of HBsAg. 3 Absence of anti-HBc is rare in HBV infection and has been attributed to aberrant immunological response to the virus or to infection with viral variants. The detection of anti-HBc as the only serological marker of HBV exposure ("isolated anti-core" or "anti-core alone") has been found in up to 10%-20% of blood donors in endemic areas. 4,5 Since anti-HBc-like reactivity was occasionally seen after HBV vaccination, this result has raised doubts as to the reliability of antiHBc testing. 6,7 However, individuals with isolated antiHBc showed the highest rates of HBV DNA detection by specific polymerase chain reaction (PCR) assays, 5,8 -11 suggesting ongoing low-level HBV infection despite the absence of classical serological markers. This outcome was consistent with the earlier findings of anti-HBc along with HBV DNA in serum and peripheral blood mononuclear cells (PBMC) 12 and a vigorous cytotoxic T-lymphocyte response to HBV antigens in apparently completely healthy individuals years after recovery from

Hepatitis B virus (HBV) and the closely related woodchuck hepatitis virus (WHV) are potent carcinogens that trigger development of primary hepatocellular carcinoma (HCC). The initial sites of hepadnavirus–host genome integration, their diversity and kinetics of formation can be central to virus persistence and the initiation and progression of HCC. To recognize the nature of the very early virus–host interactions, we explored de novo infection of human hepatocyte-like HepaRG cells with authentic HBV and naive woodchucks with WHV. HepaRG were analyzed from several minutes post exposure to HBV onwards, whereas woodchuck liver biopsies at 1 or 3 h and 6 weeks post infection with WHV. Inverse PCR and clonal sequencing of the amplicons were applied to identify virus–host genomic junctions. HBV and WHV DNA and their replication intermediates became detectable in one hour after virus exposure. Concomitantly, HBV DNA integration into various host genes was detected. Notably, junctions of HBV X gene with retrotransposon sequences, such as LINE1 and LINE2, became prominent shortly after infection. In woodchucks, insertion of WHV X and preS sequences into host genome was evident at 1 and 3 h post infection (h.p.i.), confirming that hepadnavirus under natural conditions integrates into hepatocyte DNA soon after invasion. The HBV and WHV X gene enhancer II/core promotor sequence most often formed initial junctions with host DNA. Moreover, multiple virus–virus DNA fusions appeared from 1 h.p.i. onwards in both infected hepatocytes and woodchuck livers. In summary, HBV DNA integrates almost immediately after infection with a variety of host’s sequences, among which tandemly repeating non-coding DNAs are common. This study revealed that HBV can engage mobile genetic elements from the beginning of infection to induce pro-oncogenic perturbations throughout the host genome. Such swift virus insertion was also evident in natural hepadnaviral infection in woodchucks.

Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, ␤ 2 -microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cellmediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.

Hepatitis C virus (HCV) and hepatitis B virus (HBV) frequently coinfect and persist long after clinical resolution. We assessed the incidence of low-level (occult) HCV infection (OCI) after sustained virological response (SVR) to standard anti-HCV therapy in individuals with or without past exposure to HBV to recognize whether HBV could influence the prevalence of OCI, HCV level and hepatic histology. Plasma and peripheral blood mononuclear cells (PBMC) were collected from 24 individuals at 6- to 12-month intervals for up to 72 months after SVR. Liver histology was available for nine patients. HCV and HBV genomes were detected with sensitivity <10 genome copies/mL. In individuals without HBV exposure (n = 15), comprehensive analyses of sequential plasma and PBMC samples revealed HCV RNA in all 15 cases (75% plasma and 61% PBMC). In the group with HBV exposure (n = 9), evidenced by circulating anti-HBc and/or HBV DNA detection by a highly sensitive assay, HCV RNA was identified in all cases (83% plasma and 59% PBMC), at levels similar to those in HBV nonexposed individuals. In both groups of patients, most liver biopsies included those reactive for viral genomes displayed low-grade inflammation (8 of 9) and fibrosis (7 of 9). Sequence polymorphisms at the 5`-UTR between PBMC and liver or plasma, as well as circulating HCV virion-like particles, were observed in patients with or without HBV exposure. In conclusion, the prevalence of OCI after SVR is comparable in individuals with or without past exposure to HBV. HCV loads and liver alterations in OCI appear to be unaffected by low-level HBV DNA carriage.

The extent of association between woodchuck hepatitis virus surface antigen and host hepatocyte plasma membrane in chronic hepatitis was studied. Purified membranes containing the antigen were treated with various agents which perturb plasma membrane constituents to elute woodchuck hepatitis virus surface antigen. The products from disrupted membranes were analyzed by sedimentation in sucrose gradients and tested to identify the antigen reactivity. The results indicated that membrane-bound woodchuck hepatitis virus surface antigen was partially released by 4 M potassium chloride, potassium thiocyanate and guanidine, 6 M urea or 0.1 N sodium hydroxide (pH 13.5), but not in the presence of low concentrations of these reagents or by 10% 2-mercaptoethanol and 1% sodium dodecyl sulfate. No more than 15% of the total membrane-associated woodchuck hepatitis virus surface antigen was eluted by 0.1 N NaOH, which was found to be the most effective eluent among tested agents at the antigen removal. The remaining woodchuck hepatitis virus surface antigen was resistant to further extraction with sodium hydroxide, as expected for an integral membrane protein. Treatment of the infected membranes with 1% Triton X-100 or 50 mM deoxycholic acid, that solubilize the membrane lipid bilayer releasing most of the integral membrane proteins, resulted in the sedimentation of almost all detectable woodchuck hepatitis virus surface antigen reactivity with the detergent-insoluble membrane residues, suggesting a firm interaction of the antigen with the plasma membrane matrix.(ABSTRACT TRUNCATED AT 250 WORDS)