Persistence of isolated antibodies to woodchuck hepatitis virus core antigen is indicative of occult infection

Abstract

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

Primary Occult Hepadnavirus Infection Induces Virus-Specific T-Cell and Aberrant Cytokine Responses in the Absence of Antiviral Antibody Reactivity in the Woodchuck Model of Hepatitis B Virus Infection

“…2, group A), indicating that these samples were acquired prior to the acute phase of infection. Following clearance of WHsAg, minimal to moderate intermittent liver inflammation remained evident in all animals, similar to what has been observed in previous studies (11,26,53). Challenging animal 2/M with 50 vge and animal 4/M with 1.1 ϫ 10 6 vge did not induce the reappearance of WHs antigenemia or increase WHV DNA load in serum or hepatic tissue (Fig.…”

“…In woodchucks, this infection persists for life, and virus replicative intermediates, including WHV covalently closed circular DNA and mRNA, are detectable by highly sensitive assays employing PCR combined with identification of the resulting amplicons by nucleic acid hybridization (NAH), i.e., PCR/NAH (10,53). Moreover, the virus assembled during SOI is infectious, can induce hepatitis and HCC, and is transmissible from mothers to offspring (10,11,26,53). Interestingly, SOI can be reactivated following treatment with an immunosuppressive agent, cyclosporine A, leading to the reappearance of serum WHsAg-positive infection (46).…”

Repeated Exposure to Trace Amounts of Woodchuck Hepadnavirus Induces Molecularly Evident Infection and Virus-Specific T Cell Response in the Absence of Serological Infection Markers and Hepatitis

“…WHV-naïve, adult woodchucks (Marmota monax) were housed in the Woodchuck Hepatitis Research Facility at Memorial University, St. John's, Newfoundland, Canada. Their WHV-naïve status was verified by the absence of WHV DNA, as analyzed by highly sensitive WHV-specific nested PCR-nucleic acid hybridization (PCR/NAH) assays (sensitivity, Ͻ10 vge/ml or Ͻ10 vge/g of total DNA) in randomly selected liver, peripheral blood mononuclear cell (PBMC), and serum samples, and by repeated serum negativity for WHsAg and anti-WHc antibodies as reported previously (10,13,15,39). Experimental protocols were approved by the Institutional President's Committee on Animal Bioethics and Care.…”

“…Serial serum samples were examined for WHsAg, anti-WHc, and antibodies to WHsAg (anti-WHs) using specific enzyme-linked immunosorbent assays reported previously (10,12,13). WHV DNA in serum and liver samples was evaluated by nested PCR/NAH assays with WHV core (C), surface (S), and X gene-specific primers (sensitivity, Ͻ10 vge/ml or Ͻ10 vge/g of total DNA) (13,15).…”

“…The data acquired from the woodchuck model of hepatitis B showed that exposure to a singular low dose (i.e., Ͻ1,000 virions) of woodchuck hepatitis virus (WHV), which is a close relative of HBV (7)(8)(9), establishes serologically undetectable infection in which the virus genome and its replication are detectable when nucleic acid amplification assays of enhanced sensitivity are applied (10)(11)(12)(13). This molecularly evident but immunovirologically silent infection was designated primary occult infection (POI) (12,14).…”