The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis.

Residual hepatitis B virus (HBV) DNA can be detected following the resolution of acute HBV infection. Our previous work using duck hepatitis B virus (DHBV) infected ducks, indicated that ~80% of residual DHBV DNA in the liver is in the covalently closed circular DNA (cccDNA) form, suggesting that viral DNA synthesis is suppressed. The current study asked more directly if maintenance of residual DHBV cccDNA is dependent upon ongoing viral DNA synthesis. Ducks that recovered from acute DHBV infection were divided into 2 groups and treated with the antiviral drug, Entecavir (ETV), or placebo. No major differences in the stability of cccDNA or levels of residual cccDNA were observed in liver biopsy tissues taken 95 days apart from ETV treated and placebo control ducks. The data suggest that residual DHBV cccDNA is highly stable and present in a cell population with a rate of turnover similar to normal, uninfected hepatocytes.

Covalently closed circular DNA is the predominant form of duck hepatitis B virus DNA that persists following transient infection.

Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.