Department of Pathobiology, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1 Canada.
Virus Res. 2010 May;149(2):167-74. doi: 10.1016/j.virusres.2010.01.014. Epub 2010 Feb 2.
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.
本研究的目的是证明禽类腺病毒 9（FAdV-9）基因组左端的非必需区域可用于产生重组病毒，研究其体外生长特性，并确定其转导非禽类细胞的能力。本研究生成了携带增强型绿色荧光蛋白（EGFP）基因的三种 FAdV-9 载体（rFAdV-9）：FAdV-9inEGFP、FAdV-9 Delta 1-EGFP 和 FAdV-9 Delta 4-EGFP。FAdV-9inEGFP 携带 EGFP 盒插入非必需区域而不进行缺失，导致基因组大小增加到野生型的 103.7%。FAdV-9 Delta 1-EGFP 和 FAdV-9 Delta 4-EGFP（rFAdV-9 Delta s）携带 EGFP 盒取代核苷酸 1194-2342 和 491-2782 处的非必需序列。所有 rFAdV-9 均具有野生型的生长动力学和蚀斑形态。rFAdV-9 Delta s 在 CH-SAH 细胞中复制的滴度与野生型病毒相同。rFAdV-9 Delta s 和 wt FAdV-9 在感染后 36 和 48 小时（h.p.i.）的 FAdV-9inEGFP 滴度约低 1 个对数级。感染 rFAdV-9s 的禽类和哺乳动物细胞均表达 EGFP。基于分光荧光光度法，在感染 FAdV-9inEGFP 的鸡肝癌细胞中 EGFP 的表达明显高于感染 rFAdV-9 Delta s 的细胞，在 18 和 24 h.p.i 时，这表明一些或所有非必需 ORF 在异源基因表达中发挥了功能作用。本研究证明了非必需区域作为外源基因插入/替换位点的适用性，可用于生成基于 FAdV-9 的载体，该载体可作为家禽的重组疫苗或哺乳动物系统的基因传递载体。
