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J. Virol. doi:10.1128/JVI.01819-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Recovery of Infectious Bluetongue Virus from RNA

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

^* To whom correspondence should be addressed. Email: polly.roy{at}lshtm.ac.uk.

	Abstract

Bluetongue virus (BTV), is an insect vectored emerging pathogen of ruminants, with the potential for causing devastating economic impact on European agriculture. BTV and many other Reoviridae members 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, synthesising viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. Towards the generation of a reverse genetics system for BTV we have examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (ssRNA) synthesised 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 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 component. 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.

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