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

Abstract

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.

Transplacental infection in goats experimentally infected with a European strain of bluetongue virus serotype 8

“…For conventional RT-PCR, selected blood and tissue samples were extracted several times by using Trizol. Viral double stranded RNA (dsRNA) was thereafter precipitated from total RNA (Potgieter et al, 2009) and concentrated by passing the dsRNA from several extraction replicates through one QIAquick™ PCR purification column (QIAGEN) prior to washing and elution.…”

Fungal negative-stranded RNA virus that is related to bornaviruses and nyaviruses

“…RNA samples, either from virus particles or from mycelia mass, were used for cDNA cloning and terminal determination in accordance with the procedures described by Xie et al (14). The terminal sequences of the viral genome were confirmed in accordance with methods described by Potgieter et al (37) and Briese et al (38). Primers and other oligonucleotides used for in this study are listed in Table S3.…”

Mobuck virus genome sequence and phylogenetic analysis: identification of a novel Orbivirus isolated from a white-tailed deer in Missouri, USA

“…The 'anchor-primer' PC3-T7 loop, similar to that outlined by Maan et al (2007) and previously designed by Potgieter et al (2009), was used for ligation. The primer PC3-T7 loop (59-GGATCCCGGGAATTCGGTAATACGACTCACTATATTTTTA-TAGTGAGTCGTATTA-OH-39) was ordered from Integrated DNA Technologies (IDT).…”