Abstract
BackgroundBluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription.ResultIn this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others.ConclusionIn this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.
Highlights
Bluetongue virus (BTV) is a species in the genus Orbivirus which is the largest of 15 genera within the family of Reoviridae
Preparation of helper plasmids and T7 RNA polymerasebased PCR product Based on a previous report, helper plasmids is necessary for rescue of BTV using reverse genetics (RG) system, making the system much more versatile and generally applicable [16]
With consideration that the VP7 stabilizes the BTV replication complex, seven helper plasmids were applied in this RG system VP7 plasmid can be omitted [16]
Summary
Bluetongue virus (BTV) is a species in the genus Orbivirus which is the largest of 15 genera within the family of Reoviridae. The distribution of BTV has BTV genome is composed of 10 linear segments of double-stranded RNA (dsRNA) encoding seven structural proteins (VP1–VP7) and four non-structural proteins (NS1, NS2, NS3/NS3a and NS4) [7,8,9]. Bluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. The target genes must be cloned into a specific vector to prepare templates for RNA transcription
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