Generation of an artificially attenuated fowl adenovirus 4 viral vector using the reverse genetics system based on full-length infectious clone.

Fowl adenovirus serotype 4 (FAdV-4) is a non-enveloped double-stranded DNA virus with a 43-45 kb genome. This characteristic makes it a promising viral vector for expressing other antigens in developing multi-valent or multi-series vaccines in the poultry industry. To create an easy-to-use reverse genetics system for manipulating FAdV-4 genomic DNA, a full-length infectious clone of FAdV-4 was constructed using lambda Red-mediated recombination in Escherichia coli DH10B. Viable viruses were successfully rescued after the transfection of linearised infectious clones into LMH cells. The rescued viruses showed the same cytopathic effect and growth kinetics as wild-type FAdV-4 viruses. Based on the FAdV-4 infectious clone, the hexon coding sequence of the high-pathogenicity FAdV-4 was replaced by that of the nonpathogenic FAdV-4 using lambda Red-mediated recombination combined with rpsL counter selection without leaving extra sequences after engineering. The rescued recombinant virus was highly attenuated and showed low pathogenicity to 21-day-old SPF chickens. Hereto, the easy-to-use reverse genetics system for FAdV-4 was successfully established. With this platform, the genomic DNA of FAdV-4 can be manipulated and purified in DH10B, making it quicker and easier to generate a recombinant FAdV-4 virus to develop multi-valent/multi-series vaccines.