Vaccinia virus-free recovery of vesicular stomatitis virus.
The advent of reverse-genetics represents a powerful new approach to elucidate aspects of negative-sense RNA virus replication. The reverse-genetics system established previously for vesicular stomatitis virus (VSV) required four plasmids encoding the nucleoprotein (N), phosphoprotein (P), polymerase (L), and the full-length, anti-genomic RNA. Transcription to yield the antigenomic RNA as well as the N, P, and L, mRNAs was initiated by bacteriophage T7 polymerase expressed from a recombinant Vaccinia virus. In this report, we describe the successful recovery of infectious VSV in the absence of Vaccinia virus. The N, P, and L genes of VSV were inserted downstream of both the T7 promoter and an internal ribosomal entry site (IRES element). T7 polymerase was expressed constitutively from BSR-T7/5 cells. RTPCR was used to confirm that the recovered VSV was derived from transfected DNA. Virion protein profile, CPE in tissue culture, and virus titer of the recombinant VSV were indistinguishable from those of parental VSV. Thus, the need for Vaccinia virus is eliminated with this system, making it an attractive, alternative approach for the recovery of infectious VSV from DNA.
Harty
R N
RN
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6049, USA. rharty@vet.upenn.edu
Brown
M E
ME
Hayes
F P
FP
Wright
N T
NT
Schnell
M J
MJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D005818:Genetic Engineering; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000302:isolation & purification; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000302:isolation & purification*; D014776:Virus Cultivation
D004279:DNA, Viral