Journal of Virological Methods 143 (2007) 132–139 Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses Sushila Maan a,1 , Shujing Rao a,b,1 , Narender Singh Maan a , Simon John Anthony a , Houssam Attoui a , Alan Richard Samuel a , Peter Paul Clement Mertens a,∗ a Department of Arbovirology, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF, UK b Clemson University, 114 Long Hall, Clemson, SC 29634-0315, United States Received 22 December 2006; received in revised form 23 February 2007; accepted 27 February 2007 Available online 11 April 2007 Abstract 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Bluetongue virus; Reovirus; dsRNA virus; cDNA; Rapid sequencing; FLAC 1. Introduction The family Reoviridae is one of eight families of dsRNA viruses (Birnaviridae, Picobirnaviridae, Cystoviridae, Hypoviridae, Partitiviridae, Chrysoviridae, and Totiviridae [Mertens, 2004]). The reoviruses (a term used here to describe any member of the family Reoviridae) have 10–12 dsRNA genome segments, which range in size from ∼ 0.2 to ∼ 4.5 kbp (Mertens et al., 2005a). Most of the methods previ- ously used for sequencing individual genome segments are technically demanding, time consuming and require relatively large amounts of purified and undamaged RNA. Although some sequence data have been accumulated for a few of the reoviruses (e.g. rotavirus SA11 – Mitchell and Both, 1990) mostly by a number of laboratories working in collaboration, studies of their ∗ Corresponding author. Division of Epidemiology, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF, UK. Tel.: +44 1483 232441/231189; fax: +44 1483 232448. E-mail address: peter.mertens@bbsrc.ac.uk (P.P.C. Mertens). 1 Joint first authors. evolutionary relationships have been hampered by the lack of rapid and efficient sequencing methods. Direct sequencing of RNA molecules by enzymatic tech- niques is possible, although it usually generates only relatively short terminal sequences and the ubiquitous nature of ribonu- cleases makes it technically demanding (Donis-Keller et al., 1977; Mertens et al., 1984). Makeyev and Bamford (2001) have recently reported use of the highly processive Phi 6 RNA depen- dent RNA polymerase in RNA sequencing reactions, which may provide a basis for direct RNA sequencing methods in the future. However, the current absence of such methods has made it essen- tial to develop strategies for the synthesis of cDNA copies from RNA templates, which can then be analysed by established DNA sequencing techniques (Attoui et al., 2000; Bigot et al., 1995; Lambden et al., 1992; Potgieter et al., 2002; Vreede et al., 1998). Individual reovirus genome segments need to be separated, or selected in some way prior to sequencing, to avoid generating data from several different RNAs simultaneously, making the data unreadable. Separation can be achieved by electrophore- sis, by bacterial cloning of cDNAs (although this requires extra work/time and can generate ‘cloning artifacts’) or by synthesis 0166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2007.02.016