蓝舌病毒14型的分离与鉴定
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摘要
为了解我国新疆地区羊蓝舌病的流行情况,对某羊场疑似发生蓝舌病的羊病料进行了采集,并进行病毒分离和鉴定。应用间接ELISA试剂盒检测血清抗体,抗凝血样品接种敏感的BHK21细胞进行连续盲传,对细胞病变呈阳性的样品进行间接免疫荧光鉴定,同时针对VP7片段和VP2片段设计引物进行RT-PCR检测鉴定,并对其L2基因进行系统发育树分析。结果显示,血清中BTV抗体检测均为阳性,接种BHK21细胞后均产生了特异性细胞病变,并与FITC标记的抗BTV单抗特异性结合,针对VP7扩增出1 156 bp片段,针对VP2分别扩增出850 bp和1 581 bp片段,经测序和序列分析确定为BTV-14型。结果表明,我国新疆地区存在蓝舌病毒14型,这也是我国境内首次分离到蓝舌病毒14型,为进一步防控我国蓝舌病疫情提供了科学依据。
In order to understand the prevalence of sheep bluetongue disease in Xinjiang,suspected bluetongue samples on a sheep farm were collected and subjected to virus isolation and identification.To this end,several experiments were performed,including indirect ELISA to determine BTV antibodies in sera,isolation of BTV by continuous blind passage on sensitive BHK21 monolayer cells which were inoculated with blood with anticoagulant and subsequently indirect immunofluorescence to examine the cytopathic positive samples,RT-PCR was used to detect and identify BTV VP7 and VP2 genes with respective primers,and phylogenetic tree analysis of L2 gene was carried out to determine the BTV serotype.In result,BTV antibodies in sera were positive,the suspicious samples inoculated on BHK21 cells produced specific cytopathic effects by blind passage and were specifically recognized by FITC labeled anti-BTV monoclonal antibody.A 1 156 bp fragment was amplified for VP7,and 850 bp and 1 581 bp fragments were amplified for VP2,respectively,and identified as BTV-14 by sequencing and sequence analysis.We concluded that this is the first time to isolate a serotype 14 bluetongue virus in Xinjiang,China,which provide scientific basis for further prevention and control of bluetongue disease in China.
In order to understand the prevalence of sheep bluetongue disease in Xinjiang,suspected bluetongue samples on a sheep farm were collected and subjected to virus isolation and identification.To this end,several experiments were performed,including indirect ELISA to determine BTV antibodies in sera,isolation of BTV by continuous blind passage on sensitive BHK21 monolayer cells which were inoculated with blood with anticoagulant and subsequently indirect immunofluorescence to examine the cytopathic positive samples,RT-PCR was used to detect and identify BTV VP7 and VP2 genes with respective primers,and phylogenetic tree analysis of L2 gene was carried out to determine the BTV serotype.In result,BTV antibodies in sera were positive,the suspicious samples inoculated on BHK21 cells produced specific cytopathic effects by blind passage and were specifically recognized by FITC labeled anti-BTV monoclonal antibody.A 1 156 bp fragment was amplified for VP7,and 850 bp and 1 581 bp fragments were amplified for VP2,respectively,and identified as BTV-14 by sequencing and sequence analysis.We concluded that this is the first time to isolate a serotype 14 bluetongue virus in Xinjiang,China,which provide scientific basis for further prevention and control of bluetongue disease in China.
引文
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[12]张念祖,张开礼,李志华,等.绵羊蓝舌病的调查研究报告[J].云南畜牧兽医,1989(4):3-13.ZHANG Nian-zu,ZHANG Kai-li,LI Zhi-hua,et al.Ivestigation of sheep bluetongu[J].Yunnan Journal of Animal Science and Veterinary Medicine,1998(4):3-13.(in Chinese)
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[14]张玲,王玉,谷文喜,等.蓝舌病毒蛋白结构与疫苗研究进展[J].草食家畜,2015(3):17-23.ZHANG Ling,WANG Yu,GU Wen-xi,et al.Research progress of protein structures and vaccines of bluetongue virus[J].Grass-Feeding Livestock,2015(3):17-23.(in Chinese)
[15]吕敏娜,杨恒,孙铭飞,等.蓝舌病毒7型毒株的分离与分子鉴定[J].畜牧兽医学报,2017,48(7):1281-1287.L譈Min-na,YANG Heng,SUN Ming-fei,et al.Isolation and identification of bluetongue virus serotype 7 field isolate[J].Acta Veterinaria et ZootechnicaSinica,2017,48(7):1281-1287.(inChinese)
[2]SAEGERMAN C,BERKVENS D,MELLOR P S.Bluetongue epidemiology in the European Union[J].Emerging Infectious Diseases,2008,14(4):539-544.
[3]World Organisation for Animal Health.Terrestrial Animal Health Code[M].13th ed.Paris:Office Internation aldes Epizooties,2004.
[4]HASSAN S S,ROY P L.Expression and functional characterization of bluetongue virus VP2 protein:Role in cell entry[J].Journal of Virology,1999,73(12):9832-9842.
[5]WHITE J R,EATON B T.Conformation of the VP2 protein of bluetongue virus(BTV)determines the involvement in virus neutralization of highly conserved epitopes within the BTV serogroup[J].Journal of General Virology,1990,71(6):1325-1332.
[6]BHATTACHARYA B,NOAD R J,ROY P.Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress[J].Virology Journal,2007,4(1):7.
[7]ZHANG X,BOYCE M,BHATTACHARYA B,et al.Bluetongue virus coat protein VP2 contains sialic acid-binding domains,and VP5 resembles enveloped virus fusion proteins[J].Proceedings of the National Academy of Sciences,2010,107(14):6292-6297.
[8]何宇乾,吴海燕,徐琼.蓝舌病的流行现状[J].中国兽医科学,2012,42(5):537-540.HE Yu-qian,WU Hai-yan,XU Qiong.Epidemiological status of bluetongue[J].Chinese Veterinary Science,2012,42(5):537-540.(in Chinese)
[9]MAAN S,RAO S,MAAN N S,et al.Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses[J].Journal of Virological Methods,2007,143(2):132-139.
[10]扈荣良.现代动物病毒学[M].北京:中国农业出版社,2014:931.HU Rong-liang.Modern Animal Vrology[M].Beijing:China Agricultural Press,2014:931.(in Chinese)
[11]GB/T18089-2008蓝舌病毒分离、鉴定及血清中和抗体检测技术[S].2008.GB/T18089-2008 micro-serum neutralization test,virus isolation and identification for Bluetongue[S].2008.(in Chinese)
[12]张念祖,张开礼,李志华,等.绵羊蓝舌病的调查研究报告[J].云南畜牧兽医,1989(4):3-13.ZHANG Nian-zu,ZHANG Kai-li,LI Zhi-hua,et al.Ivestigation of sheep bluetongu[J].Yunnan Journal of Animal Science and Veterinary Medicine,1998(4):3-13.(in Chinese)
[13]YANG H,XIAO L,WANG J,et al.Phylogenetic characterization genome segment 2 of bluetongue virus strains belonging to serotypes 5,7 and 24 isolated for the first time in China during 2012 to 2014[J].Transboundary&Emerging Diseases,2017,64(4):1317-1321.
[14]张玲,王玉,谷文喜,等.蓝舌病毒蛋白结构与疫苗研究进展[J].草食家畜,2015(3):17-23.ZHANG Ling,WANG Yu,GU Wen-xi,et al.Research progress of protein structures and vaccines of bluetongue virus[J].Grass-Feeding Livestock,2015(3):17-23.(in Chinese)
[15]吕敏娜,杨恒,孙铭飞,等.蓝舌病毒7型毒株的分离与分子鉴定[J].畜牧兽医学报,2017,48(7):1281-1287.L譈Min-na,YANG Heng,SUN Ming-fei,et al.Isolation and identification of bluetongue virus serotype 7 field isolate[J].Acta Veterinaria et ZootechnicaSinica,2017,48(7):1281-1287.(inChinese)