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. doi: 10.1016/j.virs.2022.03.007
Citation: Tongwei Ren, Xiangling Min, Qingrong Mo, Yuxu Wang, Hao Wang, Ying Chen, Kang Ouyang, Weijian Huang, Zuzhang Wei. Construction and characterization of a full-length infectious clone of Getah virus in vivo [J].VIROLOGICA SINICA, 2022, 37(3) : 348-357.  http://dx.doi.org/10.1016/j.virs.2022.03.007

盖他病毒全长感染性克隆的构建与鉴定

  • 通讯作者: 韦祖樟, zuzhangwei@gxu.edu.cn
  • 收稿日期: 2021-09-02
    录用日期: 2022-03-02
  • 盖他病毒(GETV)是披膜病毒科甲病毒属的一种蚊媒病毒,近年来,它已经在动物中引起了几次暴发。GETV致病的分子基础尚不清楚,因此,需要通过反向遗传操作系统获得基因修饰的病毒,以研究病毒的复制及其致病机制。本研究中,我们在先前分离的GETV毒株GX201808的基础上,构建基于CMV启动的全长感染性cDNA克隆(pGETV-GX)。将pGETV-GX转染到BHK-21细胞中,可以拯救出重组病毒(rGETV-GX),该病毒具有与亲本病毒相似的生长特征。用亲代病毒或重组病毒感染三日龄的小鼠的试验表明,与亲本病毒相比,重组病毒在小鼠体内具有较温和的致病力。在已建立的CMV启动的感染性cDNA克隆的基础上,将亚基因组启动子和两个限制性内切酶位点(BamHI和EcoRI)引入E1蛋白和3’UTR之间,然后将绿色荧光蛋白(GFP)、红色荧光蛋白(RFP)和改良光氧电压(iLOV)蛋白基因插入这两个限制性内切酶位点之间。将携带报告基因的重组质粒转染到BHK-21细胞中,可以拯救救出表达报告蛋白的重组病毒。综上所述,建立GETV反向遗传系统为研究病毒生命周期的提供了有益工具,并有助开发GETV作为表达外源基因载体的发展。

Construction and characterization of a full-length infectious clone of Getah virus in vivo

  • Corresponding author: Zuzhang Wei, zuzhangwei@gxu.edu.cn
  • Received Date: 02 September 2021
    Accepted Date: 02 March 2022
  • Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK- 21 cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 30UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.

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    Construction and characterization of a full-length infectious clone of Getah virus in vivo

      Corresponding author: Zuzhang Wei, zuzhangwei@gxu.edu.cn
    • Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, 530005, China

    Abstract: Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK- 21 cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 30UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.

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