Journal
VIROLOGY
Volume 434, Issue 1, Pages 129-136Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.virol.2012.09.022
Keywords
RNA virus; Reverse genetics; Recombination-mediated mutagenesis; Bacterial artificial chromosome; Respiratory syncytial virus
Categories
Funding
- NIH [1R01AI087798, 1U19AI095227, R01 AI071002]
- Children's Healthcare of Atlanta (CHOA) Center for Immunology and Vaccines Pilot Grant
- Vanderbilt Medical Scientist Training Program NIGMS/NIH [T32 GM007347]
- March of Dimes
- NIAID [5R01AI081977]
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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-line19F Delta NS1/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. (C) 2012 Published by Elsevier Inc.
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