WOODCHUCK VIRUS--MOLECULAR BIOLOGICAL STUDIES

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The woodchuck hepatitis virus (WHV) is taxonomically and serologically related to the hepatitis B virus (HBV). Infection with each of these viruses is associated with acute and chronic hepatitis and hepatic cell carcinoma in their respective hosts; these associations appear to be etiological in nature. Thus, the WHV/woodchuck system provides a relevant and convenient model for understanding HBV infections of humans. The following experiments were successfully completed: (1) Tests specific for the WHV antigen-antibody systems were developed. (2) Recombinant WHV DNA was transfected into the liver of susceptible woodchucks and acute and chronic WHV infections produced. (3) Genetically altered WHV genomes, with mutations in the virus X gene sequence, were constructed by recombinant DNA techniques. Mutant virus genomes were non-infectious in woodchucks, which implied that these X gene sequences were essential for virus replication. (4) Supercoiled WHV, from the nucleus of infected liver cells, was successfully cloned and the complete nucleotide sequence determined. The sequence of this replicative intermediate is identical to that of the partially double stranded WHV genome found in virions. (5) The complete nucleotide sequence of three additional WHV genomes was determined. It appears that virus subtypes of WHV are evolving, analogous to the surface antigen subtypes of HBV. (6) The in vivo fidelity of the WHV polymerase was determined. The mutation rate for WHV replication was found to be less than 2 X 10-4 changes per genome per year, which is lower than the mutation rate of retroviruses and other single strand RNA viruses. (7) Two transcripts specific for the X gene region of the WHV genome were discovered. One transcript, 0.65 kb in length, was localized to the nucleus of infected liver cells, and found to lack a poly(A) tail at its 3' end. The second transcript, 0.80 kb in length, was shown to be the poly-adenylated version of the 0.65 kb transcript.