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.
Lowen
Anice C
AC
Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 5JR, Scotland, UK.
Noonan
Carol
C
McLees
Angela
A
Elliott
Richard M
RM
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D016851:Orthobunyavirus / Q000235:genetics* / Q000502:physiology*; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins; D014773:Virology / Q000379:methods*; D014779:Virus Replication
D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases