Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus

Abstract

We describe here the successful establishment of a reverse genetics system for rotavirus (RV), a member of the Reoviridae family whose genome consists of 10-12 segmented dsRNA. The system is based on the recombinant vaccinia virus T7 RNA polymerase-driven procedure for supplying artificial viral mRNA in the cytoplasm. With the aid of helper virus (human RV strain KU) infection, intracellularly transcribed full-length VP4 mRNA of simian RV strain SA11 resulted in the rescue of the KU-based transfectant virus carrying the SA11 VP4 RNA segment derived from cDNA. In addition to the rescued transfectant virus with the authentic SA11 VP4 gene, three more infectious RV transfectants, into which silent mutation(s) were introduced to destroy both or one of the two restriction enzyme sites as gene markers in the SA11 VP4 genome, were also rescued with this method. The ability to artificially manipulate the RV genome will greatly increase the understanding of the replication and the pathogenicity of RV and will provide a tool for the design of attenuated vaccine vectors.