The non-essential left end region of the fowl adenovirus 9 genome is suitable for foreign gene insertion/replacement.
The goals of this study were to demonstrate that a non-essential region at the left end of the fowl adenovirus 9 (FAdV-9) genome could be used to generate recombinant viruses, examine their in vitro growth characteristics and determine their ability to transduce non-avian cells. Three FAdV-9 vectors (rFAdV-9s) were generated carrying the enhanced-green fluorescent protein (EGFP) gene: FAdV-9inEGFP, FAdV-9 Delta 1-EGFP and FAdV-9 Delta 4-EGFP. FAdV-9inEGFP carried the EGFP cassette inserted into the non-essential region without deletion resulting in an increase of the genome size to 103.7% of the wild-type. FAdV-9 Delta 1-EGFP and FAdV-9 Delta 4-EGFP (rFAdV-9 Delta s) carried the EGFP cassette replacing the non-essential sequences at nucleotides 1194-2342 and 491-2782, respectively. All rFAdV-9s had wild-type growth kinetics and plaque morphology. The rFAdV-9 Delta s replicated in CH-SAH cells with the same titers as the wild-type virus. The FAdV-9inEGFP titers were approximately 1 log lower than those of rFAdV-9 Delta s and wt FAdV-9 at 36 and 48 h post-infection (h.p.i.). EGFP was expressed in avian and mammalian cells infected with rFAdV-9s. EGFP expression, based on spectrofluorometry, was significantly higher in chicken hepatoma cells infected with FAdV-9inEGFP than in those with rFAdV-9 Delta s at 18 and 24h.p.i, suggesting a functional role of some or all non-essential ORFs on foreign gene expression. This study demonstrated the suitability of the non-essential region as an insertion/replacement site for foreign genes to generate FAdV-9-based vectors that can be applied as recombinant vaccines for poultry or gene delivery vehicles for mammalian systems.
Department of Pathobiology, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1 Canada.
Juan Carlos
JC
Corredor
Eva
E
Nagy
D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002645:Chickens; D005347:Fibroblasts; D017930:Genes, Reporter; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D022781:Hepatocytes; D006801:Humans; D016254:Mutagenesis, Insertional; D011784:Quail; D013194:Staining and Labeling / Q000379:methods; D019076:Transgenes; D019562:Viral Load; D010948:Viral Plaque Assay
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins