Rescue of Bovine Respiratory Syncytial Virus from Cloned cDNA: Entire Genome Sequence of BRSV Strain A51908

Infectious bovine respiratory syncytial virus (BRSV) was produced by intracellular co-expression of five plasmid borne cDNAs, each under the control of a T7 RNA polymerase promoter. These separately encoded a full-length, genetically-marked copy of BRSV antigenome along with either BRSV or human respiratory syncytial virus (HRSV) support plasmids, which express N, P, L and M2-1 proteins. HEp2 cells were used in transfection and recombinant vaccinia virus (MVA-T7) provided T7 RNA polymerase to drive the transcription. The recovery of recombinant BRSV (rBRSV) was confirmed by immunological staining of plaques, restriction enzyme digestion and nucleotide sequencing of PCR fragments carrying the genetic markers from the rescued virus. The rBRSV was indistinguishable from its parental wild-type virus in its growth characteristics in cell culture. The present work has completed the entire genome sequence of BRSV strain A51908 (15,140 nt) and has also identified changes in sequence and growth characteristics in cell culture from the original BRSV strain A51908 laboratory isolate.