Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses

• Published in: Journal of virological methods Vol. 143; no. 2; pp. 132 - 139
• Main Authors: Maan, Sushila, Rao, Shujing, Maan, Narender Singh, Anthony, Simon John, Attoui, Houssam, Samuel, Alan Richard, Mertens, Peter Paul Clement
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.08.2007; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Bluetongue virus; Bluetongue virus - genetics; cDNA; DNA, Complementary - chemistry; DNA, Complementary - genetics; dsRNA virus; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; FLAC; Fundamental and applied biological sciences. Psychology; Genome, Viral - genetics; Microbiology; Rapid sequencing; Reovirus; RNA Viruses - genetics; RNA, Double-Stranded - genetics; RNA, Viral - genetics; Sequence Analysis, DNA - methods; Techniques used in virology; Virology; dsRNA virus; FLAC; cDNA; Rapid sequencing; Reovirus; Bluetongue virus; Microbiology; Method; Reoviridae; Virology; Virus; Rapid technique; Complementary DNA; Orbivirus; Genetics; Veterinary
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.02.016

The genetic study of double-stranded (ds) RNA viruses by sequence analyses of full-length genome segments, or entire viral genomes, has been restricted by the technical difficulties involved in analyses of dsRNA templates. This paper describes improved methods for sequence-independent synthesis of full-length cDNA copies of dsRNA genes and associated sequencing strategies. These methods include an improved version of the ‘Single Primer Amplification Technique’ (SPAT – [Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., de Micco, P. and de Lamballerie, X., 2000. Strategies for the sequence determination of viral dsRNA genomes. J. Virol. Methods 89, 147–158]), which is described here as ‘Full-Length Amplification of cDNAs’ (FLAC). They also include the development of direct sequencing methods (without cloning) for the resulting full-length cDNAs. These techniques, which are applicable to any viruses with segmented dsRNA genomes and conserved RNA termini, make it possible to generate sequence data rapidly from multiple isolates for molecular epidemiology studies.