Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

中文翻译：

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流行性出血性疾病病毒基于DNA的反向遗传学系统的开发和优化。

流行性出血性疾病病毒（EHDV）是呼肠孤病毒科Orbivirus的成员，其基因组由10个线性双链（ds）RNA片段组成。目前用于工程化EHDV基因组的反向遗传学系统（RGS）依赖于体外合成的带帽病毒RNA转录本的使用。为了获得用于EHDV的更高效，更简单的RGS，我们开发了完全基于DNA（质粒或PCR扩增子）的RGS用于病毒抢救。在与代表EHDV基因组的七个辅助病毒蛋白表达质粒和10个cDNA拯救质粒或PCR扩增子共转染后，该RGS能够从BSR-T7细胞中拯救感染性EHDV。此外，我们优化了基于DNA的系统，并确认了一些辅助表达质粒对于感染性EHDV的恢复不是必需的。从而，