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Journal of Virology, September 2008, p. 8339-8348, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00808-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of Reverse Genetics Systems for Bluetongue Virus: Recovery of Infectious Virus from Synthetic RNA Transcripts{triangledown}

Mark Boyce, Cristina C. P. Celma, and Polly Roy*

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

Received 15 April 2008/ Accepted 30 May 2008

Bluetongue virus (BTV), an insect-vectored emerging pathogen of both wild ruminants and livestock, has had a severe economic impact in agriculture in many parts of the world. The investigation of BTV replication and pathogenesis has been hampered by the lack of a reverse genetics system. Recovery of infectious BTV is possible by the transfection of permissive cells with the complete set of 10 purified viral mRNAs derived in vitro from transcribing cores (M. Boyce and P. Roy, J. Virol. 81:2179-2186, 2007). Here, we report that in vitro synthesized T7 transcripts, derived from cDNA clones, can be introduced into the genome of BTV using a mixture of T7 transcripts and core-derived mRNAs. The replacement of genome segment 10 and the simultaneous replacement of segments 2 and 5 encoding the two immunologically important outer capsid proteins, VP2 and VP5, are described. Further, we demonstrate the recovery of infectious BTV entirely from T7 transcripts, proving that synthetic transcripts synthesized in the presence of cap analogue can functionally substitute for viral transcripts at all stages of the BTV replication cycle. The generation of BTV with a fully defined genome permits the recovery of mutations in a defined genetic background. The ability to generate specific mutants provides a new tool to investigate the BTV replication cycle as well as permitting the generation of designer vaccine strains, which are greatly needed in many countries.

* Corresponding author. Mailing address: London School of Hygiene and Tropical Medicine, Keppel St., London, WC1E 7HT, United Kingdom. Phone: 44 20 79272324. Fax: 44 20 79272839. E-mail: polly.roy{at}lshtm.ac.uk

{triangledown} Published ahead of print on 18 June 2008.

Journal of Virology, September 2008, p. 8339-8348, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00808-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Grigoras, I., Timchenko, T., Katul, L., Grande-Perez, A., Vetten, H.-J., Gronenborn, B. (2009). Reconstitution of Authentic Nanovirus from Multiple Cloned DNAs. J. Virol. 83: 10778-10787 [Abstract] [Full Text]  
  • Matsuo, E., Roy, P. (2009). Bluetongue Virus VP6 Acts Early in the Replication Cycle and Can Form the Basis of Chimeric Virus Formation. J. Virol. 83: 8842-8848 [Abstract] [Full Text]  
  • Celma, C. C. P., Roy, P. (2009). A Viral Nonstructural Protein Regulates Bluetongue Virus Trafficking and Release. J. Virol. 83: 6806-6816 [Abstract] [Full Text]  


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