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Journal of Virology, October 2003, p. 11201-11211, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11201-11211.2003

Genetic Recombination during Coinfection of Two Mutants of Human Respiratory Syncytial Virus

Kirsten M. Spann, Peter L. Collins,* and Michael N. Teng{dagger}

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892

Received 24 April 2003/ Accepted 15 July 2003

Recombination between coinfecting viruses had not been documented previously for a nonsegmented negative-strand RNA virus (mononegavirus). We investigated the potential of intermolecular recombination by respiratory syncytial virus (RSV) by coinfecting HEp-2 cells with two recombinant RSV (rRSV) mutants lacking either the G gene ({Delta}G/HEK) or the NS1 and NS2 genes ({Delta}NS1/2). These viruses replicate inefficiently and form pinpoint plaques in HEp-2 cells. Therefore, potential recombined viruses with a growth and/or plaque formation advantage should easily be identified and differentiated from the two parental viruses. Further identification of potential recombinants was aided by the inclusion of point mutation markers in the F and L genes of {Delta}G/HEK and the design of reverse transcription-PCR (RT-PCR) primers capable of detecting these markers. Independent coinfections and control single infections by these two rRSV mutants were performed. In one of six coinfections, an RSV variant was identified that produced plaques slightly larger than those of wild-type RSV in HEp-2 cells. RT-PCR and sequencing provided evidence that this variant was a recombined RSV (rec-RSV). The rec-RSV appeared to have been generated by a polymerase jump from the {Delta}G/HEK genome to that of {Delta}NS1/2 and back again in the vicinity of the SH-G-F genes. This apparently involved nonhomologous and homologous recombination events, respectively. The recombined genome was identical to that of the {Delta}G/HEK mutant except that all but the first 12 nucleotides of the SH gene were deleted and replaced by an insert consisting of the last 91 nucleotides of the G gene and its downstream intergenic region. This insert could have come only from the coinfecting {Delta}NS1/2 virus. This resulted in the formation of a short chimeric SH:G gene. Northern and Western blot analysis confirmed that the rec-RSV did not express the normal SH and G mRNAs and proteins but did express the aberrant SH:G mRNA. This provides an experimental demonstration of intermolecular recombination yielding a viable, helper-independent mononegavirus. However, the isolation of only a single rec-RSV under these optimized conditions supports the idea that RSV recombination is rare indeed.

* Corresponding author. Mailing address: NIAID, Building 50, Room 6503, 50 South Dr., MSC 8007, Bethesda, MD 20892-8007. Phone: (301) 594-1590. Fax: (301) 496-8312. E-mail: pcollins{at}niaid.nih.gov.

{dagger} Present address: Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA 16802.

Journal of Virology, October 2003, p. 11201-11211, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11201-11211.2003




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