中国蓝舌病病毒血清5型毒株的分离与鉴定
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摘要
为掌握我国云南省蓝舌病病毒(BTV)的流行情况,笔者在云南省师宗县设定哨兵动物,定期从哨兵动物上采集血液进行BTV分离。2012年笔者通过"鸡胚-C6/36细胞-BHK细胞"接种的方式从哨兵牛上分离出1株BTV(毒株号:V084/YN/2012),电镜观察显示,病毒粒子无囊膜,直径约80 nm,表面分布有纤突。血清中和试验表明,分离的病毒为蓝舌病病毒血清5型(BTV-5),使用抗BTV-5型的多克隆抗体为一抗进行间接免疫荧光染色,进一步证实分离毒株为BTV-5型。设计特异性引物对V084/YN/2012毒株的Segment 2 (Seg-2)与Segment 3 (Seg-3)的ORF区进行RT-PCR扩增与克隆测序,序列分析结果显示,V084/YN/2012与BTV-5型参考毒株(RSArrrr/05)的Seg-2核酸序列相似度为95.4%,在系统发育树上聚为一簇,属Seg-2基因E型;Seg-3在系统发生树上属Eastern topotype型,与中国BTV-4型YTS4毒株聚为一簇,核酸序列相似度高达97.5%。病毒蚀斑与增殖曲线测定试验表明,V084/YN/2012与实验室保存的BTV-5型参考毒株(RSArrrr/05)在BHK21细胞上可形成形态大小相近的病毒蚀斑,二者在BHK21细胞上的增殖特性也基本一致。本研究确认了BTV-5型V084/YN/2012毒株在我国的分离,掌握了病毒Seg-2与Seg-3基因的遗传特征以及病毒在BHK21细胞上的增殖特性。研究结果为进一步开展中国BTV-5型的基因组测序、流行病学调查与致病性研究奠定了基础。
In order to study the prevalence of bluetongue virus(BTV) in Yunnan province of China,we set sentinel animals in Shizong county,Yunnan province and blood samples of the sentinel animals were regularly collected for the isolation of BTV.In 2012,a BTV strain(V084/YN/2012) was isolated from the sentinel cattle by inoculation with "chicken embryo-C6/36 cells-BHK cells".Virion was 80 nm in diameter,without envelope and distributed spikes on the surface under electron microscope.The serum neutralization test showed that V084/YN/2012 strain belonged to serotype of BTV-5.Using the polyclonal antibody against BTV-5 as the primary antibody,indirect immunofluorescence staining confirmed that the isolated strain belonged to serotype of BTV-5.Specific primers were designed to amplify the ORF regions of Segment 2(Seg-2) and Segment 3(Seg-3) of V084/YN/2012 and subsequently cloning and sequencing.The sequence analysis demonstrated that the Seg-2 of V084/YN/2012 strain showed 94.3% nucleotide identity to BTV-5 reference strain(RSArrrr/05),and clustered together in the phylogenetic tree,belonged to the Seg-2 nucleotype E.Seg-3 of V084/YN/2012 belonged to Eastern topotype and clustered with the Chinese BTV-4 strain(YTS4) in the phylogenetic tree,and the nucleotide sequence identity was as high as 97.5%.The results of plaque formation and proliferation curve assays indicated that V084/YN/2012 strain could form similar size plaques as the BTV-5 reference strain on the BHK-21 cells and the proliferation characteristics of the both virus strains was very close too.We confirmed the isolation of BTV-5 strain(V084/YN/2012) in China,elucidated the evolutionary characteristics of the virus Seg-2 and Seg-3 genes and the proliferation characteristics of the virus on BHK-21 cells.The results laid a foundation for further researches on the genome sequencing,epidemiological investigation and pathogenicity of BTV-5 in China.
In order to study the prevalence of bluetongue virus(BTV) in Yunnan province of China,we set sentinel animals in Shizong county,Yunnan province and blood samples of the sentinel animals were regularly collected for the isolation of BTV.In 2012,a BTV strain(V084/YN/2012) was isolated from the sentinel cattle by inoculation with "chicken embryo-C6/36 cells-BHK cells".Virion was 80 nm in diameter,without envelope and distributed spikes on the surface under electron microscope.The serum neutralization test showed that V084/YN/2012 strain belonged to serotype of BTV-5.Using the polyclonal antibody against BTV-5 as the primary antibody,indirect immunofluorescence staining confirmed that the isolated strain belonged to serotype of BTV-5.Specific primers were designed to amplify the ORF regions of Segment 2(Seg-2) and Segment 3(Seg-3) of V084/YN/2012 and subsequently cloning and sequencing.The sequence analysis demonstrated that the Seg-2 of V084/YN/2012 strain showed 94.3% nucleotide identity to BTV-5 reference strain(RSArrrr/05),and clustered together in the phylogenetic tree,belonged to the Seg-2 nucleotype E.Seg-3 of V084/YN/2012 belonged to Eastern topotype and clustered with the Chinese BTV-4 strain(YTS4) in the phylogenetic tree,and the nucleotide sequence identity was as high as 97.5%.The results of plaque formation and proliferation curve assays indicated that V084/YN/2012 strain could form similar size plaques as the BTV-5 reference strain on the BHK-21 cells and the proliferation characteristics of the both virus strains was very close too.We confirmed the isolation of BTV-5 strain(V084/YN/2012) in China,elucidated the evolutionary characteristics of the virus Seg-2 and Seg-3 genes and the proliferation characteristics of the virus on BHK-21 cells.The results laid a foundation for further researches on the genome sequencing,epidemiological investigation and pathogenicity of BTV-5 in China.
引文
[1] MACLACHLAN N J,DREW C P,DARPEL K E,et al.The pathology and pathogenesis of bluetongue[J].Journal of Comparative Pathology,2009,141(1):1-16.
[2] MACLACHLAN N J.Bluetongue:history,global epidemiology,and pathogenesis[J].Preventive Veterinary Medicine,2011,102(2):107-111.
[3] SAEGERMAN C,BERKVENS D,MELLOR P S.Bluetongue epidemiology in the European Union[J].Emerging Infectious Diseases,2008,14(4):539-544.
[4] World Organisation for Animal Health.Terrestrial animal health code[M].13th ed.Paris:Office International des Epizooties,2004.
[5] ROY P.Bluetongue virus proteins[J].Journal of General Virology,1992,73(Pt12):3051-3064.
[6] ROY P.Orbivirus structure and assembly[J].Virology,1996,216(1):1-11.
[7] HASSAN S S,ROY P.Expression and functional characterization of bluetongue virus VP2 protein:role in cell entry[J].Journal of Virology,1999,73(12):9832-9842.
[8] 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(Pt6):1325-1332.
[9] NOMIKOU K,DOVAS C I,MAAN S,et al.Evolution and phylogenetic analysis of full-length VP3 genes of eastern mediterranean bluetongue virus isolates[J].PLoS One,2009,4(7):e6437.
[10] BELBIS G,ZIENTARA S,BREARD E,et al.Bluetongue virus:from BTV-1 to BTV-27[J].Advances in Virus Research,2017,99:161.
[11] BENELLI G,BUTTAZZONI L,CANALE A,et al.Bluetongue outbreaks:looking for effective control strategies against culicoides vectors[J].Research in Veterinary Science,2017,115:263-270.
[12] VENTER G J.Culicoides spp.(Diptera:Ceratopogonidae)as vectors of bluetongue virus in South Africa-a review[J].Veterinaria Italiana,2015,51(4):325-333.
[13] ZHANG N,MACLACHLAN N J,BONNEAU K R,et al.Identification of seven serotypes of bluetongue virus from the People’s Republic of China[J].Veterinary Record,1999,145(15):427-429.
[14] ZHU J B,YANG H,LI H C,et al.Full-genome sequence of bluetongue virus serotype 1(BTV-1)strain Y863,the first BTV-1 isolate of eastern origin found in China[J].Genome Announcements,2013,1(4):142.
[15] YANG H,ZHU J B,LI H C,et al.Full genome sequence of bluetongue virus serotype 4 from China[J].Journal of Virology,2012,86(23):13122-13123.
[16] CLAVIJO A,HECKERT R A,DULAC G C,et al.Isolation and identification of bluetongue virus[J].Journal of Virological Methods,2000,87(1):13-23.
[17] GB/T 18636-2017.蓝舌病诊断技术国家标准[S].2017.GB/T 18636-2017.National standard for diagnosis of bluetongue disease[S].2017.(in Chinese)
[18] MOHL B P,ROY P.Bluetongue virus capsid assembly and maturation[J].Viruses,2014,6(8):3250-3270.
[19] MAAN S,MAAN N S,SAMUEL A R,et al.Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes[J].Journal of General Virology,2007,88(Pt2):621-630.
[20] HEMADRI D,MAAN S,CHANDA M M,et al.Dual infection with bluetongue virus serotypes and first-time isolation of serotype 5 in India[J].Transboundary&Emerging Diseases,2017,64(6):1912-1917.
[21]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.The prevalence of bluetongue[J].Chinese Veterinary Science,2012,42(5):537-540.(in Chinese)
[22] PURSE B V,MELLOR P S,ROGERS D J,et al.Climate change and the recent emergence of bluetongue in Europe[J].Nature Reviews Microbiology,2005,3(2):171-181.
[23] YANG H,LV M N,SUN M F,et al.Com plete genome sequence of the first bluetongue virus serotype 7isolate from China:evidence for entry of Africanlineage strains and reassortment between the introduced and native strains[J].Archives of Virology,2016,161(1):223-227.
[2] MACLACHLAN N J.Bluetongue:history,global epidemiology,and pathogenesis[J].Preventive Veterinary Medicine,2011,102(2):107-111.
[3] SAEGERMAN C,BERKVENS D,MELLOR P S.Bluetongue epidemiology in the European Union[J].Emerging Infectious Diseases,2008,14(4):539-544.
[4] World Organisation for Animal Health.Terrestrial animal health code[M].13th ed.Paris:Office International des Epizooties,2004.
[5] ROY P.Bluetongue virus proteins[J].Journal of General Virology,1992,73(Pt12):3051-3064.
[6] ROY P.Orbivirus structure and assembly[J].Virology,1996,216(1):1-11.
[7] HASSAN S S,ROY P.Expression and functional characterization of bluetongue virus VP2 protein:role in cell entry[J].Journal of Virology,1999,73(12):9832-9842.
[8] 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(Pt6):1325-1332.
[9] NOMIKOU K,DOVAS C I,MAAN S,et al.Evolution and phylogenetic analysis of full-length VP3 genes of eastern mediterranean bluetongue virus isolates[J].PLoS One,2009,4(7):e6437.
[10] BELBIS G,ZIENTARA S,BREARD E,et al.Bluetongue virus:from BTV-1 to BTV-27[J].Advances in Virus Research,2017,99:161.
[11] BENELLI G,BUTTAZZONI L,CANALE A,et al.Bluetongue outbreaks:looking for effective control strategies against culicoides vectors[J].Research in Veterinary Science,2017,115:263-270.
[12] VENTER G J.Culicoides spp.(Diptera:Ceratopogonidae)as vectors of bluetongue virus in South Africa-a review[J].Veterinaria Italiana,2015,51(4):325-333.
[13] ZHANG N,MACLACHLAN N J,BONNEAU K R,et al.Identification of seven serotypes of bluetongue virus from the People’s Republic of China[J].Veterinary Record,1999,145(15):427-429.
[14] ZHU J B,YANG H,LI H C,et al.Full-genome sequence of bluetongue virus serotype 1(BTV-1)strain Y863,the first BTV-1 isolate of eastern origin found in China[J].Genome Announcements,2013,1(4):142.
[15] YANG H,ZHU J B,LI H C,et al.Full genome sequence of bluetongue virus serotype 4 from China[J].Journal of Virology,2012,86(23):13122-13123.
[16] CLAVIJO A,HECKERT R A,DULAC G C,et al.Isolation and identification of bluetongue virus[J].Journal of Virological Methods,2000,87(1):13-23.
[17] GB/T 18636-2017.蓝舌病诊断技术国家标准[S].2017.GB/T 18636-2017.National standard for diagnosis of bluetongue disease[S].2017.(in Chinese)
[18] MOHL B P,ROY P.Bluetongue virus capsid assembly and maturation[J].Viruses,2014,6(8):3250-3270.
[19] MAAN S,MAAN N S,SAMUEL A R,et al.Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes[J].Journal of General Virology,2007,88(Pt2):621-630.
[20] HEMADRI D,MAAN S,CHANDA M M,et al.Dual infection with bluetongue virus serotypes and first-time isolation of serotype 5 in India[J].Transboundary&Emerging Diseases,2017,64(6):1912-1917.
[21]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.The prevalence of bluetongue[J].Chinese Veterinary Science,2012,42(5):537-540.(in Chinese)
[22] PURSE B V,MELLOR P S,ROGERS D J,et al.Climate change and the recent emergence of bluetongue in Europe[J].Nature Reviews Microbiology,2005,3(2):171-181.
[23] YANG H,LV M N,SUN M F,et al.Com plete genome sequence of the first bluetongue virus serotype 7isolate from China:evidence for entry of Africanlineage strains and reassortment between the introduced and native strains[J].Archives of Virology,2016,161(1):223-227.