ANIMAL MODELS OF HBV: INFECTION AND DISEASE

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We propose to continue our studies of disease and infection in 2 animal models of hepatitis B virus, which is associated with hepatitis and hepatocellular carcinoma (HCC) in man. In 1 of the models, ground squirrels infected with ground squirrel hepatitis virus (GSHV), we have seen developed of HCC significantly associated with virus infection, and propose to continue study of the long term effects of virus infection in the animals remaining the colony. We will continue to study the effects of environmental factors such as aflatoxin B1 and antivirals on HCC development. We will determined what common and differentiating characteristics exist between the integrated and unintegrated forms of GSHV DNA in the 8 HCC observed in carrier squirrels and those from studies of WHV- and HBV-related HCC. We will clone and sequence single integrations of GSHV DNA in HCC and study whether c-myc or other oncogenes are rearranged or amplified. We will continue to sequence virus-virus junction areas in the "novel" DNA forms cloned from chronically- infected ground squirrels to determine if these forms reveal a mechanism for integration of viral DNA into chromosomal DNA. In the other animal model, duck hepatitis B virus (DHBV) infection of ducks, we will study what factors influence the type and amount of hepatitis that we have observed in ducklings experimentally injected with DHBV, but absent in congentially- infected animals and uninjected controls. We will follow experimentally-infected ducklings to see if these ducks develop HCC, unlike the congenitally-infected ducklings that we have studied so far. We will use DHBV infection of primary duck hepatocyte cultures to study early events in viral infection, to test monoclonal antibodies to DHBV for neutralizing ability, and to establish a rapid test for antiviral effectiveness. We will attempt to develop alternate cell culture systems for growing DHBV by culturing yolk sac endodermal cells or their precursors and by culturing hepatocytes from ducklings treated with diethylnitrosamine. We will use electroporation-aided transfection of DHBV DNA into both susceptible and non- susceptible cells to study the mechanism of host specificity.