A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis.
We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered two RSV reporter viruses, one expressing the red fluorescent protein monomeric Katushka 2 (A2-K-line19F) and one expressing Renilla luciferase (A2-RL-line19F). As proof of principle, we efficiently generated a RSV gene deletion mutant (A2-line19FΔNS1/NS2) and a point mutant (A2-K-line19F-I557V) by recombination-mediated BAC mutagenesis. Together with sequence-optimized helper expression plasmids, BAC-RSV is a stable, versatile, and efficient reverse genetics platform for generation of a recombinant Pneumovirus.
Department of Pediatrics, Emory University, Atlanta, GA 30322, USA.
Anne L
AL
Hotard
Fyza Y
FY
Shaikh
Sujin
S
Lee
Dan
D
Yan
Michael N
MN
Teng
Richard K
RK
Plemper
James E
JE
Crowe
Martin L
ML
Moore
D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D016296:Mutagenesis; D011995:Recombination, Genetic; D012136:Respiratory Syncytial Viruses / Q000235:genetics*; D059386:Reverse Genetics / Q000379:methods*; D000097573:Red Fluorescent Protein
D008156:Luciferases; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein