CONSTRUCTION OF OVINE ADENOVIRUS RECOMBINANTS BY GENE INSERTION OR DELETION OF RELATED TERMINAL REGION SEQUENCES

An ovine adenovirus which may be the prototype for a new group of aden oviruses has been engineered as a gene transfer vector. One recombinan t containing a 0.95-kb insertion expressed a sheep parasite antigen fr om the ovine adenovirus major late promoter and tripartite leader sequ ences. It was shown that insertions of at least 4.3 kb were tolerated at either one of two sites in the genome without the introduction of a compensating deletion. The unique structure of this viral genome was further emphasized by the discovery that four open reading frames at t he right hand end show significant identity to each other but not to o ther sequences in the databases. Two other unrelated open reading fram es were also present. RT-PCR analysis identified two transcripts in th is region which were derived from a promoter which was located very cl ose to, or within the ITR sequence. Splicing removed all but the first and last of the ORFs from these RNAs, suggesting that some sequences might be nonessential for replication in vitro. A similar to 2-kb dele tion, which removed or truncated the internal reading frames was intro duced into the region without affecting virus viability. The carrying capacity of OAV recombinants should therefore be at least 6.3 kb. The relative packaging capacity of OAV (114%) therefore exceeds that of Ad 5 (105%), although a comparison of virus particle sizes by electron mi croscopy showed that OAV was smaller than Ad5. These studies improve t he potential utility of OAV as a gene transfer vector. (C) 1997 Academ ic Press.