Reverse Genetics of RNA Viruses

Tobias Nolden, Stefan Finke, Nolden, Tobias, Finke, Stefan

Daniel R. Perez

Methods to recover recombinant negative strand RNA viruses (rNSVs) from cloned cDNAs have been significantly improved in more than two decades of NSV reverse genetics . In particular, for non-segmented negative strand RNA viruses (NNSVs ) like rhabdoviruses , time-consuming generation of reverse genetics systems by stitching PCR subfragments of genomic rhabdovirus cDNAs using ligase-based conventional cloning approaches limited the number of available recombinant virus cDNA clones. As genetic variability is considered an intrinsic feature of RNA viruses, it is thus reasonable to conclude that reverse genetics approaches to investigate natural virus functions and pathogenesis require improved systems that reflect the complexity of naturally occurring wild-type viruses, and that largely exclude adaption to cell culture conditions.In order to allow rapid cloning of wild-type NSV genome populations into reverse genetics vector plasmids, we developed a system in which cDNA copies of complete rhabdovirus populations are inserted into a plasmid bank by linear-to-linear homologous RecE/T recombination (LLHR ). Limited requirements for sequence information a priori, high cloning efficiencies, and the possibility to directly generate recombinant viruses from individual cDNA clones now offer novel opportunities to combine forward genetic dissection of natural rhabdovirus populations and downstream reverse genetics approaches.