Mutational analysis of the left end region of fowl adenovirus 9 genome
The role of early genes during adenovirus infection has not been much studied for fowl adenoviruses (FAdVs) as opposed to that of human adenoviruses. The open reading frame (ORF) organization and early gene functions of the right end of the FAdV genome have been more studied than those of the left end region. This study, therefore, was aimed to uncover the features of the left end region of FAdV serotypes representing species 'C, D' and 'E' through nucleotide sequencing and examine the involvement of this region in virus replication through deletions using FAdV-9 as a model. Homologues to FAdV-1 and -9 left end ORFs with some variations in their content were found in the analyzed FAdV genomes. Two clusters consisting of ORFs with rightward (ORFs 1, 1A, 1B, 1C and 2) and leftward (ORFs 24, 14, 13 and 12) orientations were found as reported for the FAdV-1 and -9 left end regions. ORFs 1 and 2 were the most conserved among the analyzed FAdVs suggesting their importance during virus replication. Deletions at nucleotides (nts) 400-2782 at the left end of the FAdV-9 genome generated viable viruses (FAdV-9[Delta]s) upon transfection: FAdV-9[Delta]1 ([Delta]1,194-2,342), -9[Delta]2 ([Delta]854-2,782), -9[Delta]4 ([Delta]491-2,782), and -9~7 ([Delta]400-2,782). In contrast, viral genomes with deletions upstream nt 400 and/or downstream nt 2782 were non-viable. The putative packaging motifs VI and VII and the rightward ORFs, missing in FAdV-9[Delta]7, were dispensable for virus replication 'in vitro'. Recombinant viruses carrying the enhanced-green fluorescent protein (EGFP), FAdV-9[Delta]1-EGFP and -9[Delta]4-EGFP (rFAdV[Delta]s), were generated from FAdV-9[Delta]1 and -9[Delta]4, respectively. FAdV-9[Delta]s and rFAdV-9[Delta]s had wild-type growth kinetics and titres 'in vitro'. However, FAdV-9[Delta]4 or FAdV-9[Delta]4-EGFP, selected for 'in vivo' studies, seemed to replicate less efficiently in the host based on significantly lower antibody response, titres in the feces and viral genome copy number in the analyzed tissues compared to those from chickens inoculated with the wild-type virus. This is the first study demonstrating that the left end region of the viral genome is required for efficient virus replication 'in vivo' and thus further research should be conducted to understand the role of each ORF.