Emerging and re-emerging viruses from wild animals have seriously threatened the health of humans and domesticated animals in recent years.Herein,we isolated a new mammalian orthoreovirus(MRV),Pika/MRV/GCCDC7/2019(PMR...Emerging and re-emerging viruses from wild animals have seriously threatened the health of humans and domesticated animals in recent years.Herein,we isolated a new mammalian orthoreovirus(MRV),Pika/MRV/GCCDC7/2019(PMRV-GCCDC7),in the Qinghai-Tibet Plateau wild pika(Ochotona curzoniae).Though the PMRVGCCDC7 shows features of a typical reovirus with ten gene segments arranged in 3:3:4 in length,the virus belongs to an independent evolutionary branch compared to other MRVs based on phylogenetic tree analysis.The results of cellular susceptibility,species tropism,and replication kinetics of PMRV-GCCDC7 indicated the virus could infect four human cell lines(A549,Huh7,HCT,and LoVo)and six non-human cell lines,including Vero-E6,LLCMK2,BHK-21,N2a,MDCK,and RfKT cell,derived from diverse mammals,i.e.monkey,mice,canine and bat,which revealed the potential of PMRV-GCCDC7 to infect a variety of hosts.Infection of BALB/c mice with PMRVGCCDC7 via intranasal inoculation led to relative weight loss,lung tissue damage and inflammation with the increase of virus titer,but no serious respiratory symptoms and death occurred.The characterization of the new reovirus from a plateau-based wild animal has expanded our knowledge of the host range of MRV and provided insight into its risk of trans-species transmission and zoonotic diseases.展开更多
Genome replication of reovirus occurs in cytoplasmic inclusion bodies called viral factories or viroplasms. The viral nonstructural protein uNS, encoded by genome segment M3, is not a component of mature virions, but ...Genome replication of reovirus occurs in cytoplasmic inclusion bodies called viral factories or viroplasms. The viral nonstructural protein uNS, encoded by genome segment M3, is not a component of mature virions, but is expressed to high levels in infected cells and is concentrated in the infected cell factory matrix. Recent studies have demonstrated that uNS plays a central role in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly.展开更多
目的根据构建的人致病性纳尔逊海湾病毒(NBVs)M3基因片段质粒,纯化融合蛋白制备NBVs M3多克隆抗体。方法利用构建的纳尔逊海湾病毒(NBVs)M3基因质粒Pris His MB-M3,转化至大肠埃希菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半...目的根据构建的人致病性纳尔逊海湾病毒(NBVs)M3基因片段质粒,纯化融合蛋白制备NBVs M3多克隆抗体。方法利用构建的纳尔逊海湾病毒(NBVs)M3基因质粒Pris His MB-M3,转化至大肠埃希菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,产物经过镍柱亲和层析法获得Pris His MB-M3融合蛋白;将纯化的融合蛋白作为抗原免疫家兔,获得NBVs M3多克隆抗体;蛋白免疫印迹检测(Western blot)蛋白准确性以及多克隆抗体抗性。结果 SDS-PAGE电泳考马斯亮蓝染色检测显示:25℃、IPTG浓度为0.3 mmol/L,诱导12 h,Pris His MB-M3融合蛋白表达量最高;在咪唑浓度为MCAC-20、MCAC-40条件时洗脱下目的蛋白。用纯化的融合蛋白免疫家兔制备抗体,Western blot验证成功制备出NBVs M3多克隆抗体。结论成功获得了灵敏性及特异性较高的纳尔逊海湾病毒(NBVs)M3多克隆抗体,为进一步研究该病毒的致病性提供了很高的应用价值。展开更多
基金the National Key Research and Development Program of China(2021YFC0863400,2022YFC2604105).
文摘Emerging and re-emerging viruses from wild animals have seriously threatened the health of humans and domesticated animals in recent years.Herein,we isolated a new mammalian orthoreovirus(MRV),Pika/MRV/GCCDC7/2019(PMRV-GCCDC7),in the Qinghai-Tibet Plateau wild pika(Ochotona curzoniae).Though the PMRVGCCDC7 shows features of a typical reovirus with ten gene segments arranged in 3:3:4 in length,the virus belongs to an independent evolutionary branch compared to other MRVs based on phylogenetic tree analysis.The results of cellular susceptibility,species tropism,and replication kinetics of PMRV-GCCDC7 indicated the virus could infect four human cell lines(A549,Huh7,HCT,and LoVo)and six non-human cell lines,including Vero-E6,LLCMK2,BHK-21,N2a,MDCK,and RfKT cell,derived from diverse mammals,i.e.monkey,mice,canine and bat,which revealed the potential of PMRV-GCCDC7 to infect a variety of hosts.Infection of BALB/c mice with PMRVGCCDC7 via intranasal inoculation led to relative weight loss,lung tissue damage and inflammation with the increase of virus titer,but no serious respiratory symptoms and death occurred.The characterization of the new reovirus from a plateau-based wild animal has expanded our knowledge of the host range of MRV and provided insight into its risk of trans-species transmission and zoonotic diseases.
基金National Basic Research Program of China (973 Program) ( 2009CB118701)National Natural Scientific Foundation of China (30671615, 30871940)Innovation project of the Chinese Academy of Sciences(KSCX2-YW-N-021 to QF).
文摘Genome replication of reovirus occurs in cytoplasmic inclusion bodies called viral factories or viroplasms. The viral nonstructural protein uNS, encoded by genome segment M3, is not a component of mature virions, but is expressed to high levels in infected cells and is concentrated in the infected cell factory matrix. Recent studies have demonstrated that uNS plays a central role in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly.
文摘目的根据构建的人致病性纳尔逊海湾病毒(NBVs)M3基因片段质粒,纯化融合蛋白制备NBVs M3多克隆抗体。方法利用构建的纳尔逊海湾病毒(NBVs)M3基因质粒Pris His MB-M3,转化至大肠埃希菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,产物经过镍柱亲和层析法获得Pris His MB-M3融合蛋白;将纯化的融合蛋白作为抗原免疫家兔,获得NBVs M3多克隆抗体;蛋白免疫印迹检测(Western blot)蛋白准确性以及多克隆抗体抗性。结果 SDS-PAGE电泳考马斯亮蓝染色检测显示:25℃、IPTG浓度为0.3 mmol/L,诱导12 h,Pris His MB-M3融合蛋白表达量最高;在咪唑浓度为MCAC-20、MCAC-40条件时洗脱下目的蛋白。用纯化的融合蛋白免疫家兔制备抗体,Western blot验证成功制备出NBVs M3多克隆抗体。结论成功获得了灵敏性及特异性较高的纳尔逊海湾病毒(NBVs)M3多克隆抗体,为进一步研究该病毒的致病性提供了很高的应用价值。
