[Objective]The paper was to develop a rapid method for the detection of spring Viremia of carp virus(SVCV).[Method]The specific primers were designed by targeting the G gene of SVCV.The recombinase polymerase amplific...[Objective]The paper was to develop a rapid method for the detection of spring Viremia of carp virus(SVCV).[Method]The specific primers were designed by targeting the G gene of SVCV.The recombinase polymerase amplification(RPA)assay for detecting SVCV was estab-lished by optimizing the reaction conditions.The optimal amplification temperature of RPA assay was 30℃,and the test could be finished within 20 min.[Result]The method was specific with no cross-reaction with other common fish infectious viruses.Sensitivity test showed that the lowest detection limit of the method was 89.2 copies/μL,higher than that of traditional RT-PCR.Moreover,a total of 80 clinical samples were detected by RPA and RT-PCR,respectively.The weak positive samples tested by RT-PCR could be detectable with RPA,indicating that RPA assay could be used in clinical detection.[Conclusion]The method established is rapid,simple,specific and sensitive for testing SVCV,and it will be widely used in grassroots laboratory and on-site inspection.展开更多
Spring viremia of carp virus(SVCV)is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range.To develop effective agents to control SVCV infection,we selected 16 n...Spring viremia of carp virus(SVCV)is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range.To develop effective agents to control SVCV infection,we selected 16 naturally active small molecules to assess their anti-SVCV activity.Notably,dihydroartemisinin(DHA)(100μmol/L)and(S,S)-(+)-tetrandrine(TET)(16μmol/L)exhibited high antiviral effects in epithelioma papulosum cyprinid(EPC)cells,with inhibitory rates of 70.11%and 73.54%,respectively.The possible antiviral mechanisms were determined as follows:1.Preincubation with DHA and TET decreased viral particle infectivity in fish cells,suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted;2.Although neither had an effect on viral adhesion,TET(but not DHA)interfered with SVCV entry into host cells(>80%),suggesting that TET may have an antiviral function in early viral replication.For in vivo study,both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3%,significantly decreased viral load,and modulated the expression of antiviralrelated genes,indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection.Overall,our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication,thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.展开更多
为了揭示鲤春病毒血症病毒（spring viremia of carp virus,SVCV）糖蛋白编码基因的主要免疫原性决定区域,本研究对SVCV的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar ...为了揭示鲤春病毒血症病毒（spring viremia of carp virus,SVCV）糖蛋白编码基因的主要免疫原性决定区域,本研究对SVCV的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar 6.0软件对SVCV糖蛋白基因的跨膜区、亲水性以及抗原表位进行分析后,采用RT-PCR对该基因主要抗原决定区域编码片段进行扩增,并构建重组表达质粒p GEX-Gtr,对其进行诱导表达后获得截短的SVCV糖蛋白的重组蛋白。将表达的重组蛋白进行纯化复性后,作为免疫原制备兔抗血清,采用ELISA法检测其效价,采用免疫印迹以及间接免疫荧光技术检测该重组蛋白的免疫原性。研究结果显示,截短表达的糖蛋白编码基因长1317 bp,编码439个氨基酸（29~467）,推测分子量为49.6 k D。利用该重组蛋白制备的兔抗血清,其与重组蛋白的反应效价为1∶64000,。免疫印迹及间接免疫荧光结果显示,该兔抗血清与SVCV-HN株能发生特异性反应,表明该重组蛋白与天然的病毒表面糖蛋白的免疫原性无差异。上述研究结果表明,截短表达的重组蛋白具有很好的免疫原性,可应用于免疫诊断技术研发与基因工程疫苗的研制。展开更多
基金Supported by National Key Research and Development Program (2017YFF0211103)Scientific Research Project of General Administration of Quality Supervision,Inspection and Quarantine (2017IK232)
文摘[Objective]The paper was to develop a rapid method for the detection of spring Viremia of carp virus(SVCV).[Method]The specific primers were designed by targeting the G gene of SVCV.The recombinase polymerase amplification(RPA)assay for detecting SVCV was estab-lished by optimizing the reaction conditions.The optimal amplification temperature of RPA assay was 30℃,and the test could be finished within 20 min.[Result]The method was specific with no cross-reaction with other common fish infectious viruses.Sensitivity test showed that the lowest detection limit of the method was 89.2 copies/μL,higher than that of traditional RT-PCR.Moreover,a total of 80 clinical samples were detected by RPA and RT-PCR,respectively.The weak positive samples tested by RT-PCR could be detectable with RPA,indicating that RPA assay could be used in clinical detection.[Conclusion]The method established is rapid,simple,specific and sensitive for testing SVCV,and it will be widely used in grassroots laboratory and on-site inspection.
基金supported by the National Natural Science Foundation of China(31902410)Natural Science Foundation of Zhejiang Province(LQ22C190002)+3 种基金Natural Science Foundation of Ningbo City(2021J117)Program of Science and Technology Department of Ningbo City(2021S058)One Health Interdisciplinary Research Project of Ningbo University(HZ202201)Zhejiang Xinmiao Talents Programs(2022R405B066)。
文摘Spring viremia of carp virus(SVCV)is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range.To develop effective agents to control SVCV infection,we selected 16 naturally active small molecules to assess their anti-SVCV activity.Notably,dihydroartemisinin(DHA)(100μmol/L)and(S,S)-(+)-tetrandrine(TET)(16μmol/L)exhibited high antiviral effects in epithelioma papulosum cyprinid(EPC)cells,with inhibitory rates of 70.11%and 73.54%,respectively.The possible antiviral mechanisms were determined as follows:1.Preincubation with DHA and TET decreased viral particle infectivity in fish cells,suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted;2.Although neither had an effect on viral adhesion,TET(but not DHA)interfered with SVCV entry into host cells(>80%),suggesting that TET may have an antiviral function in early viral replication.For in vivo study,both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3%,significantly decreased viral load,and modulated the expression of antiviralrelated genes,indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection.Overall,our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication,thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.
文摘为了揭示鲤春病毒血症病毒（spring viremia of carp virus,SVCV）糖蛋白编码基因的主要免疫原性决定区域,本研究对SVCV的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar 6.0软件对SVCV糖蛋白基因的跨膜区、亲水性以及抗原表位进行分析后,采用RT-PCR对该基因主要抗原决定区域编码片段进行扩增,并构建重组表达质粒p GEX-Gtr,对其进行诱导表达后获得截短的SVCV糖蛋白的重组蛋白。将表达的重组蛋白进行纯化复性后,作为免疫原制备兔抗血清,采用ELISA法检测其效价,采用免疫印迹以及间接免疫荧光技术检测该重组蛋白的免疫原性。研究结果显示,截短表达的糖蛋白编码基因长1317 bp,编码439个氨基酸（29~467）,推测分子量为49.6 k D。利用该重组蛋白制备的兔抗血清,其与重组蛋白的反应效价为1∶64000,。免疫印迹及间接免疫荧光结果显示,该兔抗血清与SVCV-HN株能发生特异性反应,表明该重组蛋白与天然的病毒表面糖蛋白的免疫原性无差异。上述研究结果表明,截短表达的重组蛋白具有很好的免疫原性,可应用于免疫诊断技术研发与基因工程疫苗的研制。
