Efficient bunyavirus rescue from cloned cDNA.

Bunyaviruses are trisegmented, negative-sense RNA viruses. Previously, we described a rescue system to recover infectious Bunyamwera virus (genus Orthobunyavirus) entirely from cloned cDNA (Bridgen, A. and Elliott, R.M. (1996) Proc. Nat. Acad. Sci. USA 93, 15400-15404) utilizing a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase to drive intracellular transcription of transfected T7 promoter-containing plasmids. Here we report efforts to improve the efficiency of the system by comparing different methods of providing T7 polymerase. We found that a BHK-derived cell line BSR-T7/5 that constitutively expresses T7 RNA polymerase supported efficient and reproducible recovery of Bunyamwera virus, routinely generating >10(7) pfu per rescue experiment. Furthermore, we show that the virus can be recovered from transfecting cells with just three plasmids that express full-length antigenome viral RNAs, greatly simplifying the procedure. We suggest that this procedure should be applicable to viruses in other genera of the family Bunyaviridae and perhaps also to arenaviruses.