Immunological responsiveness of Pekin ducks to core antigen of duck hepatitis B virus.

Cellular immune responses to the HBc or HBe antigen of hepatitis B viruses contribute to the pathogenesis of hepatitis B and to the elimination of the virus. Insufficient cytotoxic immune reactions against the core antigen may be one major reason for viral persistence in the absence of severe clinical symptoms. We attempted to stop viral persistence in the animal model of congenitally infected ducks by injection of recombinant DHBc particles, together with the strong immunostimulator Freund's adjuvant. However, the duck HBc antigen (DHBcAg)-treated ducks did not develop detectable liver disease, nor was the virus persistence affected. The congenitally infected ducks did not contain antibodies against DHBcAg before injection despite continuous production of duck hepatitis B virus, and developed only a weak transient antibody response after injection of recombinant DHBcAg together with Freund's adjuvant. Noninfected ducks developed, in contrast, a strong antibody response to the injected DHBcAg. We conclude that congenitally infected ducks are immunotolerant to DHBcAg and cannot be cured by immunotherapy with exogenous recombinant DHBcAg.

The half-life of duck hepatitis B virus supercoiled DNA in congenitally infected primary hepatocyte cultures.

The transcriptional template for duck hepatitis B virus (DHBV) replication is believed to be the supercoiled covalently closed circular (CCC) molecule. DHBV CCC DNA can be amplified at least 50-fold in acutely and congenitally infected hepatocyte cultures but is normally maintained at a constant copy number in vivo infections. Here we describe experiments to determine the half-life of DHBV CCC DNA in congenitally infected hepatocyte cultures using both direct and indirect labeling of DHBV CCC DNA with the DNA labeling agent 5-bromo 2-deoxyuridine (BrUdR). Direct labeling of DHBV CCC DNA with BrUdR generated a very stable molecule with no calculable half-life. For indirect labeling experiments, hepatocytes were first cultured for 5 days in the absence of BrUdR to generate a pool of unlabeled DHBV CCC DNA, in then BrUdR was added to the culture medium. By following the fate of the pool of unlabeled CCC DNA in the cultures over time we calculated the half-life of DHBV CCC DNA to be 3 and 5 days in two separate experiments. This result suggests that there is a requirement for continuous amplification of DHBV CCC DNA to maintain a persistent chronic infection.

Alterations in intrahepatic expression of duck hepatitis B viral markers with ganciclovir chemotherapy.

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the guanosine analogue, ganciclovir, and the effect on serum and intrahepatic expression of DHBV DNA and viral proteins was examined. After 21 days of ganciclovir treatment, a substantial reduction in viraemia occurred; in contrast, the level of circulating DHBV surface antigen was unchanged. Ganciclovir therapy also substantially reduced the level of DHBV DNA replicative intermediates and the expression of viral core and surface antigen in hepatocytes. However, despite the antiviral treatment some liver cells, including the bile duct epithelial cells and putative oval cells, maintained their intense staining for the viral proteins. Furthermore, DHBV-infected cells in extrahepatic sites such as the pancreas, kidney and spleen were also unaffected by ganciclovir treatment. These results suggest that monotherapy with nucleoside analogues is unlikely to eliminate chronic hepadnaviral infection, and antiviral programs should be designed to target all cell populations infected by the virus.