Abstract:
ABSTRACT
Recombinant adenoviruses are currently under intense investigation as potential
gene delivery and gene expression vectors with applications in human and veterinary
medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based
vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open
reading frames (ORFs) were found with the potential to encode polypeptides that were
at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide
sequences were found to share homology to already identified adenovirus proteins.
The arrangement of the genes revealed that the BAV2 genomic organization closely
resembles that of well-characterized human adenoviruses.
In the course of this study, continuous propagation of BAV2 over many
generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in
which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses
produced concordant results that precisely located the deletion and revealed that its
size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further
propagated in cell culture. It appeared that the replication of such a virus lacking a
portion of the E3 region was not affected, at least in cell culture.
Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin
resistance gene in the E3 region yielded a candidate as verified with extensive
Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not
successful, suggesting that such recombinant BAV2 was helper-dependent.
Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector
were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after
extended incubation period. This may be due to limitations of various transfection
methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.
