A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis
Open accessAbstract
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.
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- Collins PL Crowe JE Jr Knipe DM Howley PM 2007 Respiratory Syncytial Virus and Metapneumovirus Fields Virology 2 1601 1646 Lippincott, Williams, and Wilkins Philadelphia, PA
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- Authors
- Hotard, A. L., Shaikh, F. Y., Lee, S., Yan, D., Teng, M. N., Plemper, R. K., Crowe, J. E., & Moore, M. L.
- Affiliations
- Emory University (US)
- Vanderbilt University (US)
- University of South Florida (US)
- Corresponding author
- Moore, Martin L.
- ORCID
- Venue
- Virology
- Publisher
- Elsevier BV
- Published
- 2012-12
- Volume
- 434
- Issue
- 1
- Pages
- 129-136
- ISSN
- 0042-6822 (ISSN-L)
- PMC
- PMC3492879
- OpenAlex
- W1990354697
- Cited by
- 158
- References
- 32
- Citations by year
- 2026: 15 · 2025: 15 · 2024: 14 · 2023: 9 · 2022: 12 · 2021: 13 · 2020: 8 · 2019: 6 · 2018: 8 · 2017: 11 · 2016: 17 · 2015: 12 · 2014: 14 · 2013: 4
- FWCI
- 7.18
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Virology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- NIH (1R01AI087798, 1U19AI095227, R01 AI071002)
- Children's Healthcare of Atlanta (CHOA) Center for Immunology and Vaccines
- Vanderbilt Medical Scientist Training Program
- NIGMS/NIH (T32 GM007347)
- March of Dimes
- NIAID (5R01AI081977)
- Topics
- Health Sciences › Medicine › Epidemiology › Respiratory viral infections research
- Health Sciences › Medicine › Epidemiology › Virology and Viral Diseases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- respiratory syncytial virus, reverse genetics, bacterial artificial chromosome, negative-strand RNA viruses, deletion mutants, point mutants, pneumovirus, reporter virus, helper plasmid
- MeSH terms
- Red Fluorescent Protein, Animals, Cell Line, Genetic Vectors, Humans, Luciferases, Luminescent Proteins, Recombination, Genetic, Respiratory Syncytial Viruses, Mutagenesis, Genes, Reporter, Artificial Gene Fusion, Chromosomes, Artificial, Bacterial, Reverse Genetics
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