title: Development of a reverse genetics system for epizootic hemorrhagic disease virus and evaluation of novel strains containing duplicative gene rearrangements.
abstract: Epizootic haemorrhagic disease is a non-contagious infectious viral disease of wild and domestic ruminants caused by epizootic hemorrhagic disease virus (EHDV). EHDV belongs to the genus Orbivirus within the family Reoviridae and is transmitted by insects of the genus Culicoides. The impact of epizootic haemorrhagic disease is underscored by its designation as a notifiable disease by the Office International des Epizooties. The EHDV genome consists of 10 linear dsRNA segments (Seg1-Seg10). Until now, no reverse genetics system (RGS) has been developed to generate replication-competent EHDV entirely from cloned cDNA, hampering detailed functional analyses of EHDV biology. Here, we report the generation of viable EHDV entirely from cloned cDNAs. A replication-competent EHDV-2 (Ibaraki BK13 strain) virus incorporating a marker mutation was rescued by transfection of BHK-21 cells with expression plasmids and in vitro synthesized RNA transcripts. Using this RGS, two additional modified EHDV-2 viruses were also generated: one that contained a duplex concatemeric Seg9 gene and another that contained a duplex concatemeric Seg10 gene. The modified EHDV-2 with a duplex Seg9 gene was genetically stable during serial passage in BHK-21 cells. In contrast, the modified EHDV-2 with a duplex Seg10 gene was unstable during serial passage, but displayed enhanced replication kinetics in vitro when compared with the WT virus. This RGS provides a new platform for the investigation of EHDV replication, pathogenesis and novel EHDV vaccines.
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Jikai
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Qingyuan
authors.initials: Q
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Encheng
authors.initials: E
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Junping
authors.initials: J
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Lv
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Yufei
authors.initials: Y
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Haixiu
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Donglai
authors.initials: D
authors.lastname: Wu
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology*; D020440:Gene Duplication; D015321:Gene Rearrangement; D018164:Hemorrhagic Disease Virus, Epizootic / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D010802:Phylogeny; D012088:Reoviridae Infections / Q000662:veterinary* / Q000821:virology; D059386:Reverse Genetics / Q000379:methods*; D014779:Virus Replication