Development of a reverse genetic system for mononegaviruses using a circular polymerase extension reaction

Abstract

Reverse genetic systems are commonly used to study viruses; the ability to rapidly generate recombinant viruses is critical for studying the functions of viral genes and for the development of new interventions, such as antivirals or vaccines. Reverse genetic methods for mononegaviruses, viruses with non-segmented negative-strand RNA genomes, commonly incorporate a full-length viral genome cDNA into a bacterial artificial chromosomes (BACs) or plasmid DNA. However, the large size of mononegavirus genomes makes their manipulation challenging. In this study, to overcome this limitation, we adopted and optimized the circular polymerase extension reaction (CPER) method for mononegavirus reverse genetics. We segmented the genome of the mononegavirus, respiratory syncytial virus (RSV), into approximately 1.2–2.5 kb fragments and reassembled them with a linker fragment containing a T7 promotor into a circular full-length viral cDNA. Recombinant RSV was then generated by co-transfecting cells with the circular cDNA and helper plasmids carrying viral proteins that are essential to initiate viral replication. This reverse genetic system has the potential to be applied to other mononegaviruses.