A rapid and efficient reverse genetics method for generating RNA viruses

Introduction: Pathogenic RNA viruses pose a significant threat to global health due to their high rates of morbidity, mortality, and substantial economic impact. The rapid development of antiviral drugs, therapeutics, and vaccines during an outbreak requires adequate virus samples for efficacy evaluation. However, obtaining viruses or clinical samples during outbreaks is challenging. Reverse genetics offers a powerful solution to this problem by enabling the rapid and efficient production of viruses. This technique enhances containment and response strategies, thereby accelerating the development of preventive measures. Materials & Methods: We established a reverse genetics system for RNA viruses, focusing on vesicular stomatitis virus (VSV), a prototypical negative-sense RNA virus. Using a rapid single-tube method, we assembled the full-length cDNA clone of the VSV antigenome into a single plasmid. This involved amplifying the antigenome as five overlapping fragments, excluding the G protein genes, and integrating regulatory elements (T7 promoter, ribozyme, and T7 terminator) into a plasmid vector (pLinker). To ensure virus rescue, we cloned minigenome plasmids encoding essential accessory proteins (N, P, and L) with regulatory elements. Co-transfection of plasmids encoding VSV antisense genome, minigenome, and T7 polymerase into BHK-21 cells facilitated virus rescue. Virus titration was performed by infecting Vero-E6 cells with the collected supernatant. Results: To construct functional VSV full-length genome and minigenome plasmids, we integrated regulatory elements like leader, trailer, and ribozyme sequences (