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.

中文翻译：

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基于PCR的蓝舌病毒恢复的反向遗传学策略。

背景技术蓝舌病病毒（BTV）是一种新兴的昆虫载体介导的既影响野生反刍动物又影响家畜的病原体，其基因组由10个线性双链RNA基因组片段组成。BTV对世界许多地区的农业产生了严重的经济影响。当前挽救BTV的逆向遗传学（RG）策略主要依赖于在辅助质粒的帮助下从对应于病毒基因组片段的克隆互补DNA（cDNA）的RNA转录本的体外合成。RNA合成是一项艰巨的工作，需要昂贵的试剂和精细的操作程序，这使工作更加复杂。另外，必须将靶基因克隆到特定载体中以制备用于RNA转录的模板。结果在这项研究中，我们开发了一种基于PCR的BTV RG系统，该系统易于进行两步转染。在BSR细胞上用7个辅助质粒进行第一次转染，然后用10种PCR产物进行第二次转染后，回收了活病毒。此外，通过间接免疫荧光测定（IFA）和基因测序对回收的病毒进行了表征。并且还将这些病毒的增殖特性与野生型BTV进行了比较。有趣的是，我们发现含有重配BTV基因组第2段的病毒比其他病毒的生长速度稍慢。结论在本研究中，建立了一个方便的基于PCR的BTV RG平台，该策略可以有效替代原有的BTV救援方法。此外，此RG策略可能适用于其他Orbiviruses。