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Improved strategies for sequence-independent amplification and sequencing of viral dsRNA genomes

¹ Onderstepoort Veterinary Institute;
² National Institute for Communicable Diseases;
³ Tib Molbiol GmbH;
⁴ North-West University

⁵ E-mail: potgieterc{at}arc.agric.za

This paper reports significant improvements in the efficacy of sequence-independent amplification and quality of sequencing of viruses with segmented double-stranded RNA (dsRNA) genomes. We demonstrate that most remaining bottlenecks in dsRNA virus genome characterisation have now been eliminated. Both the amplification and sequencing technologies used require no previous sequence knowledge of the viral dsRNA, there is no need to separate genome segments or amplicons any more and the sequence-determined bias observed in cloning has been overcome. Combining very efficient genome amplification with pyrophoshate-based 454 (GS20/FLX) sequencing enabled sequencing of complete segmented dsRNA genomes and accelerated the sequence analysis of the amplified viral genomes. We report the complete consensus sequence of seven viruses from four different dsRNA virus groups which include the first complete sequence of the genome of equine encephalosis virus (EEV), the first complete sequence of an African horsesickness virus (AHSV) genome determined directly from a blood sample and a complete human rotavirus genome determined from faeces. We also present the first comparison between the complete consensus sequence of a virulent and an attenuated strain of AHSV1. Ultra-deep sequencing (> 400 fold coverage) of the AHSV1 reference and attenuated strains revealed different ratios of reassortants in the reference strain and allowed quasi-species detection in the plaque purified attenuated strain of AHSV1. This approach amounts to a paradigm shift in dsRNA virus research, since it is sensitive and specific enough for comprehensive investigations of the evolution and genetic diversity in dsRNA virus populations.

Received 3 December 2008; accepted 18 February 2009.