Construction of avian adenovirus CELO recombinants in cosmids

The avian adenovirus CELO is a Promising vector for gene transfer applicati ons. In order to study this potentiality, we developed an improved method f or construction of adenovirus vectors in cosmids that was used to engineer the CELO genome. For all the recombinant viruses constructed by this method , the ability to produce infectious particles and the stability of the geno me were evaluated in a chicken hepatocarcinoma cell line (LMH cell line). O ur aim was to develop a replication-competent vector for vaccination of chi ckens, so we first generated knockout point mutations into 16 of the 22 una ssigned CELO open reading frames (ORFs) to determine if they were essential for virus replication. As the 16 independent mutant viruses replicated in our cellular system, we constructed CELO genomes with various deletions in the regions of these nonessential ORFs, An expression cassette coding for t he enhanced green fluorescent protein (eGFP) was inserted in place of these deletions to easily follow expression of the transgene and propagation of the vector in cell monolayers. Height-distinct GFP-expressing CELO vectors were produced that were all replication competent in our system. We then re tained the vector backbone with the largest deletion (i.e,, 3.6 kb) for the construction of vectors carrying cDNA encoding infectious bursal disease v irus proteins. These CELO vectors could be useful for vaccination in the ch icken species.