A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis.

Hotard AL, Shaikh FY, Lee S, Yan D, Teng MN, Plemper RK, Crowe JE, Moore ML
Virology. 2012 434 (1): 129-36

PMID: 23062737 · PMCID: PMC3492879 · DOI:10.1016/j.virol.2012.09.022

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.

Copyright © 2012 Elsevier Inc. All rights reserved.

MeSH Terms (13)

Animals Artificial Gene Fusion Cell Line Chromosomes, Artificial, Bacterial Genes, Reporter Genetic Vectors Humans Luciferases Luminescent Proteins Mutagenesis Recombination, Genetic Respiratory Syncytial Viruses Reverse Genetics

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