HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplementary Material All Versions of this Article: vir.0.009381-0v1 90/6/1423    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Potgieter, A. C. Articles by van Dijk, A. A. PubMed PubMed Citation Articles by Potgieter, A. C. Articles by van Dijk, A. A. Agricola Articles by Potgieter, A. C. Articles by van Dijk, A. A.

Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes

¹ OIE reference laboratories for African horsesickness and bluetongue, Virology Division, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa
² Viral Gastroenteritis Unit, National Institute for Communicable Diseases, Private Bag X4, Sandringham 2131, South Africa
³ Diarrhoeal Pathogens Research Unit, University of Limpopo, Medunsa Campus, PO Box 173, Medunsa 0204, South Africa
⁴ Molecular Biology Division, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa
⁵ Tib Molbiol GmbH, Eresburgstrasse 22–33, D12103 Berlin, Germany
⁶ Biochemistry Division, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa

Correspondence
A. C. Potgieter
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 characterization have now been eliminated. Both the amplification and sequencing technologies used require no previous sequence knowledge of the viral dsRNA, there is no longer a need to separate genome segments or amplicons and the sequence-determined bias observed in cloning has been overcome. Combining very efficient genome amplification with pyrophosphate-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 quasispecies 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.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AM883164–AM883173, FJ011107–FJ011116, FJ196584–FJ196593 and FJ183353–FJ183393 (given in Table 2).

A supplementary figure and the full technical protocol for sequence-independent amplification of viral dsRNA genomes are available with the online version of this paper.