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Journal of Virology, March 2007, p. 2179-2186, Vol. 81, No. 5
0022-538X/07/$08.00+0     doi:10.1128/JVI.01819-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Recovery of Infectious Bluetongue Virus from RNA{triangledown}

Mark Boyce and Polly Roy*

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom

Received 21 August 2006/ Accepted 22 November 2006

Bluetongue virus (BTV) is an insect-vectored emerging pathogen of ruminants with the potential for devastating economic impact on European agriculture. BTV and many other members of the Reoviridae have remained stubbornly refractory to the development of methods for the rescue of infectious virus from cloned nucleic acid (reverse genetics). Partially disassembled virus particles are transcriptionally active, synthesizing viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. With the goal of generating a reverse-genetics system for BTV, we examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (single-stranded RNA [ssRNA]) synthesized in vitro using BTV core particles. Following transfection, viral-protein synthesis was detected by immunoblotting, and confocal examination of the cells showed a punctate cytoplasmic distribution of inclusion bodies similar to that seen in infected cells. Viral double-stranded RNA (dsRNA) was isolated from ssRNA-transfected cells, demonstrating that replication of the ssRNA had occurred. Additionally, infectious virus was present in the medium of transfected cells, as demonstrated by the passage of infectivity in BSR cells. Infectivity was sensitive to single-strand-specific RNase A, and cotransfection of genomic BTV dsRNA with transcribed ssRNA demonstrated that the ssRNA species, rather than dsRNA, were the active components. We conclude that it is possible to recover infectious BTV wholly from ssRNA, which suggests a means for establishing helper virus-independent reverse-genetics systems for members of the Reoviridae.

* Corresponding author. Mailing address: London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 44 020-79272324. Fax: 44 020-79272839. E-mail: polly.roy{at}lshtm.ac.uk.

{triangledown} Published ahead of print on 6 December 2006.

Journal of Virology, March 2007, p. 2179-2186, Vol. 81, No. 5
0022-538X/07/$08.00+0     doi:10.1128/JVI.01819-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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