The overall objective of this project is to study molecular mechanisms and events in the development of persistent hepatitis B virus (HBV) infection in relation to development and progression of liver disease. Such studies will be carried out in both human and chimpanzee chronic hepatitis B virus carriers, the only known hosts for HBV. Specific areas to be studied include (1) molecular events of viral replication in persistent carrier states with regard to the possible existence of a reverse transcripatase (RNA intermediate) mechanism, (2) the development and temporal progression of viral integration into the host hepatic genome with specific analysis of viral genomic organization, characterization of genomic integration sites and control of the integration mechanisms. The chimpanzee chronic carrier model will be examined for the presence of integrated HBV sequence by creating and screening a genomic library from chimpanzee liver DNA and hybridization of this library with HBV probes, (3) comparison of genomic integration sites in chronic HBV carriers to those present in coexisting primary liver cancer. Such studies will involve the development of probes for determination of flanking regions and will examine the proximity of such regions to known oncogenic sequences, (4) the relationship between molecular events of viral persistence and progression of liver histopathology and expression of viral genomic products. Such studies will involve not only the sensitive techniques of Southern blot analysis but also the development of in situ HBV DNA hybridization to permit direct comparison of histopathology, HBV DNA and viral genomic products identified by immunofluorescence methods, and (5) development of a tissue culture system from chronically infected chimpanzee hepatocytes to facilitate the study of HBV persistence. Such approaches will be expected to enhance our knowledge of pathophysiological mechanisms of viral persistence at various stages in the HBV life cycle, will hopefully elucidate mechanisms for viral integration into the hepatocellular genome, and may therefore lead to development of strategies to eradicate the carrier state following HBV infection.
