First Versatile Reverse Genetics System for DNA Viruses Using Circular Polymerase Extension Reaction

Reverse genetics enables the generation of recombinant viruses; however, conventional approaches using full-length cDNA cloned into bacterial artificial chromosomes or plasmids are time-consuming and technically challenging. While the circular polymerase extension reaction (CPER) has been applied to RNA viruses, it has not been used for DNA viruses. Here, we successfully applied CPER to generate infectious recombinant adenoviruses (AdVs) of two serotypes. The resulting viruses replicated comparably to parental strains, and replication was confirmed by immunostaining. These findings demonstrate that CPER is a rapid and efficient platform for reverse genetics of DNA viruses and may accelerate AdV research and therapeutic development.