Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus
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ORIGINAL ARTICLE
Development and optimization of a DNA‑based reverse genetics systems for epizootic hemorrhagic disease virus Yunze Guo1,2 · Jakobus M. Pretorius3 · Qingyuan Xu1 · Donglai Wu1 · Zhigao Bu1 · Jacques Theron3 · Encheng Sun1 Received: 18 December 2019 / Accepted: 8 February 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract 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.
Introduction Epizootic hemorrhagic disease (EHD) is an infectious, noncontagious disease of wild and domestic ruminants caused by infection with epizootic hemorrhagic disease virus (EHDV). EHDV is primarily transmitted by several species of biting midges of the genus Culicoides. EHDV infection is widespread among members of the family Cervidae (i.e., white-tailed deer) and is associated with a high mortality rate [1–3]. Although most EHDV serotypes rarely cause disease in livestock, EHDV-2 (Ibaraki virus) is an exception Handling Editor: Zhenhai Chen. * Encheng Sun [email protected] 1
The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
2
Department of Veterinary Pathology, Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture, College of Veterinary Medicine, Inner Mongolia Agricultural University, Zhaowuda Road 306, 010018 Hohhot, Inner Mongolia, China
3
Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria 0002, South Africa
and has caused widespread disease in cattle in Japan [4, 5]. Recently, outbreaks of EHDV in cattle have been reported in several countries around the world, including Israel, Japan, Canada, Turkey, Morocco, and Réunion [6–12]. Therefore, EHDV represents an important threat to livestock, requiring that greater attention be paid to this economically important viral pathogen. EHDV is a member of the genus Orbivirus of the family Reoviridae, and its genome contains 10
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