Darren S. Miller

Kinetics of Hepadnavirus Loss from the Liver during Inhibition of Viral DNA Synthesis

ABSTRACT Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-β-l-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of ∼33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.

Entecavir Therapy Combined with DNA Vaccination for Persistent Duck Hepatitis B Virus Infection

ABSTRACT This study was designed to test the efficacy of antiviral treatment with entecavir (ETV) in combination with DNA vaccines expressing duck hepatitis B virus (DHBV) antigens as a therapy for persistent DHBV infection in ducks. Ducks were inoculated with 109 DHBV genomes at 7 days of age, leading to widespread infection of the liver and viremia within 7 days, and were then treated orally with either ETV (0.1 mg/kg of body weight/day) or distilled water from 21 days posthatch for 244 days. Treatment with ETV caused a 4-log drop in serum DHBV DNA levels within 80 days and a slower 2- to 3-log drop in serum DHBV surface antigen (DHBsAg) levels within 120 days. Following withdrawal of ETV, levels of serum DHBV DNA and DHBsAg rebounded to match those in the water-treated animals within 40 days. Sequential liver biopsy samples collected throughout the study showed that ETV treatment reduced DHBV DNA replicative intermediates 70-fold in the liver, while the level of the stable, template form, covalently closed circular DNA decreased only 4-fold. ETV treatment reduced both the intensity of antigen staining and the percentage of antigen-positive hepatocytes in the liver, but the intensity of antigen staining in bile duct cells appeared not to be effected. Intramuscular administration of five doses of a DNA vaccine expressing the DHBV presurface, surface, precore, and core antigens, both alone and concurrently with ETV treatment, on days 50, 64, 78, 127, and 141 did not result in any significant effect on viral markers.

Identification and Characterization of Avihepadnaviruses Isolated from Exotic Anseriformes Maintained in Captivity

ABSTRACT Five new hepadnaviruses were cloned from exotic ducks and geese, including the Chiloe wigeon, mandarin duck, puna teal, Orinoco sheldgoose, and ashy-headed sheldgoose. Sequence comparisons revealed that all but the mandarin duck viruses were closely related to existing isolates of duck hepatitis B virus (DHBV), while mandarin duck virus clones were closely related to Ross goose hepatitis B virus. Nonetheless, the S protein, core protein, and functional domains of the Pol protein were highly conserved in all of the new isolates. The Chiloe wigeon and puna teal hepatitis B viruses, the two new isolates most closely related to DHBV, also lacked an AUG start codon at the beginning of their X open reading frame (ORF). But as previously reported for the heron, Ross goose, and stork hepatitis B viruses, an AUG codon was found near the beginning of the X ORF of the mandarin duck, Orinoco, and ashy-headed sheldgoose viruses. In all of the new isolates, the X ORF ended with a stop codon at the same position. All of the cloned viruses replicated when transfected into the LMH line of chicken hepatoma cells. Significant differences between the new isolates and between these and previously reported isolates were detected in the pre-S domain of the viral envelope protein, which is believed to determine viral host range. Despite this, all of the new isolates were infectious for primary cultures of Pekin duck hepatocytes, and infectivity in young Pekin ducks was demonstrated for all but the ashy-headed sheldgoose isolate.

Effect of antiviral treatment with entecavir on age- and dose-related outcomes of duck hepatitis B virus infection.

ABSTRACT Entecavir (ETV), a potent inhibitor of the hepadnaviral polymerases, prevented the development of persistent infection when administered in the early stages of duck hepatitis B virus (DHBV) infection. In a preliminary experiment, ETV treatment commenced 24 h before infection showed no significant advantage over simultaneous ETV treatment and infection. In two further experiments 14-day-old ducks were inoculated with DHBV-positive serum containing 104, 106, 108, or 5 × 108 viral genomes (vge) and were treated orally with 1.0 mg/kg of body weight/day of ETV for 14 or 49 days. A relationship between virus dose and infection outcome was seen: non-ETV-treated ducks inoculated with 104 vge had transient infection, while ducks inoculated with higher doses developed persistent infection. ETV treatment for 49 days did not prevent initial infection of the liver but restricted the spread of infection more than ∼1,000-fold, a difference which persisted throughout treatment and for up to 49 days after withdrawal. Ultimately, three of seven ETV-treated ducks resolved their DHBV infection, while the remaining ducks developed viremia and persistent infection after a lag period of at least 63 days. ETV treatment for 14 days also restricted the spread of infection, leading to marked and sustained reductions in the number of DHBV-positive hepatocytes in 7 out of 10 ducks. In conclusion, short-term suppression with ETV provides opportunity for the immune response to successfully control DHBV infection. Since DHBV infection of ducks provides a good model system for HBV infection in humans, it seems likely that ETV may be useful in postexposure therapy for HBV infection aimed at preventing the development of persistent infection.

Covalently Closed Circular DNA Is the Predominant Form of Duck Hepatitis B Virus DNA That Persists following Transient Infection

ABSTRACT 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.