Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

Reverse genetics is a crucial tool for studying virus replication and pathogenesis in negative strand RNA viruses (NSVs). However, its application has been limited by a scarcity of infectious cDNA clones from virulent field strains, as most available clones originate from cell-adapted or attenuated viruses, hindering pathogenesis research. To overcome this, a novel high-throughput system was developed for directly cloning complete NSV genomes into reverse genetics vectors via linear-to-linear RedE/T recombination (LLHR). This method demonstrated reliability by rapidly cloning multiple rabies virus (RABV) full-length genomes, achieving 73% to 88% insertion rates identical to field virus consensus. Recombinant RABVs were successfully recovered, exhibiting comparable growth kinetics, preserved cell type-specific replication, and virulence in mouse models. The system’s flexibility was also confirmed by efficiently cloning an orthobunyavirus L genome segment. This technological advance significantly improves the analysis of virus variability and phenotypical characterization of recombinant viruses at a clonal level boosting future pathogenesis research.
(MKO)