STRUCTURE AND FUNCTION OF THE ADV GENOME

点击翻译按钮获取中文摘要

The purpose of this project is the study of genome structure and function of the Aleutian disease parvovirus (ADV). Further studies have included detailed physical mapping of genomic segments from there strains of ADV. These clones were derived by molecular cloning of replicative forms prepared from Hirt supernatants of infected cell cultures. The results indicated that the three viruses were very similar, but that discreet differences could be detected in the portion of the genome coding for viral structural genes. An alternate strategy was employed to study DNA from viruses that do not grow in cell culture. Single stranded virion DNA was prepared from virus purified from the organs of mink infected with either Utah I ADV or Pullman ADV. This DNA was converted to duplex molecules in vitro, cleaved with restriction enzymes and cloned. Molecular clones were derived that expressed viral antigens in E. coli. It will now be possible to compare the genomic structure and function of ADV strains without concern for possible selection factors exerted by cell culture adaptation. Detailed analysis of these molecular clones is currently underway. We have also constructed a full length molecular clone of ADV-G, developed by tailing of intact replicative form. Preliminary analysis indicates that DNA from this recombinant plasmid produces viral antigen when transfected into cultures of cells permissive for ADV replication. Finally, transcription of the ADV genome is also under study and the results to date suggest that ADV encodes at least three mRNA species ranging in size from 2-4 kb. One of these, the 4.0 kb RNA, can be detected prior to the onset of detectable DNA replication. This mRNA may represent the nonvirion protein thought by some to have a role in modulating viral DNA replication.