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PCR-based reverse genetics strategy for bluetongue virus recovery.

ABSTRACT: BACKGROUND: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. 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. RESULT:In 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. CONCLUSION:In 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.

SUBMITTER: Xu Q

PROVIDER: S-EPMC6896262 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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PCR-based reverse genetics strategy for bluetongue virus recovery.

Xu Qingyuan Q Ge Jinying J Li Maolin M Sun Encheng E Zhou Yawei Y Guo Yunze Y Wu Donglai D Bu Zhigao Z

Virology journal 20191205 1

<h4>Background</h4>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. 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 ...[more]

PMID: 31805959

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Project description:BackgroundBluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames.ResultsPlasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods.ConclusionsA limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.

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PCR-based reverse genetics strategy for bluetongue virus recovery.

Publications

PCR-based reverse genetics strategy for bluetongue virus recovery.

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