PCR-based reverse genetics strategy for bluetongue virus recovery

文献类型： 外文期刊

第一作者： Xu, Qingyuan

作者： Xu, Qingyuan;Ge, Jinying;Sun, Encheng;Guo, Yunze;Wu, Donglai;Bu, Zhigao;Li, Maolin;Zhou, Yawei

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关键词： Bluetongue virus; Reverse genetics; T7 RNA polymerase; Genome modification; Reassortment

期刊名称：VIROLOGY JOURNAL （ 影响因子：4.099； 五年影响因子：3.719 ）

ISSN：

年卷期： 2019 年 16 卷 1 期

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收录情况： SCI

摘要： 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.

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