Establishment of the Bat Adenovirus WIV9 Vector System Using Restriction–Assembly

Bat adenoviruses hold promise for development as vaccine vectors and oncolytic viral vectors. However, research on the construction and modification of their vector systems remains very limited. In this study, an infectious clone plasmid, pA-WIV9, containing the full-length genome of bat adenovirus WIV9, was constructed using DNA assembly technology. Subsequently, an intermediate plasmid, pKWIV9BstBIM-GFP, carrying a partial deletion of the E3 region and incorporating a GFP reporter gene, was obtained. Following digestion, this intermediate plasmid was assembled with the BstBI-linearized pA-WIV9 plasmid to generate the recombinant adenoviral plasmid pAWIV9MGFP. After transfecting the linearized pAWIV9MGFP into Vero E6 cells, the recombinant virus WIV9MGFP was successfully rescued. Its replication characteristics, plaque formation ability, and infectivity profile in different cell lines were systematically characterized. Our results demonstrated that WIV9 could transduce not only Vero E6 cells but also human embryonic kidney cells (HEK293) and chicken hepatocellular carcinoma cells (LMH). Compared to the wild-type virus, the E3-deleted virus formed smaller plaques. In this study, a recombinant bat adenovirus vector system was successfully constructed using DNA assembly combined with restriction enzyme digestion, which enables convenient replacement of the gene of interest in the E3 region, laying the foundation for the development and application of bat adenovirus vectors.