蓝舌病病毒血清9型毒株在我国的首次分离
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摘要
旨在分离流行于我国云南省的蓝舌病病毒(BTV),掌握分离BTV的遗传特征与感染特性。采用"鸡胚—C6/36细胞—BHK-21细胞"接种的方式,采集哨兵牛的BTV阳性血液进行病毒分离;采用血清中和试验以及Segment 2与Segment 6ORF区的克隆测序确定分离病毒的血清型;通过病毒噬斑形成和增殖曲线的测定,分析病毒在BHK-21细胞的增殖特性;通过qRT-PCR与血清中和试验分析BTV感染动物血液中病毒含量与中和抗体动态变化情况。结果显示:2013年8月,在云南芒市设定的哨兵牛中分离出一株BTV(毒株号V013/YN/2013),血清中和试验显示V013/YN/2013为BTV-9型病毒,Segment 2与Segment 6序列分析表明分离的病毒属BTV-9Eastern型,与日本毒株和澳大利亚BTV-9型毒株具有最近的亲缘关系。病毒噬斑与增殖曲线测定结果显示V013/YN/2013在BHK-21细胞上增殖能力明显强于BTV-9型参考毒株。自然感染V013/YN/2013的牛在连续5个月的监控期内未出现临床症状,感染动物虽产生了特异性中和抗体,但血液中始终能持续检测到病毒核酸。本研究首次报道了BTV-9Eastern型毒株V013/YN/2013在我国的分离,为进一步开展中国BTV-9型病毒的全基因组测序、诊断方法的建立、流行病学调查与致病性研究奠定了基础。
The study aims to isolate bluetongue virus(BTV)endemic in Yunnan Province of China and to understand the genetic and infection characteristics of the isolated BTV.Virus isolation was performed by inoculation method of"Embryos-C6/36 cells-BHK-21 cells"with BTV nucleic acid positive blood samples collected from sentinel cattle.Serotype of the isolated virus was decided by micro-neutralization test and sequencing the ORF regions of Segment 2 and Segment 6.The proliferation characteristics of isolated virus in BHK-21 cells were determined by the tests of growth curve and plaque formation.The viral load and neutralizing antibody of infected animal were analyzed by qRT-PCR and neutralization test.Results were as follows:In August 2013,BTV strain of V013/YN/2013 was isolated form sentinel cattle in Mangshi,Yunnan Province.Micro-neutralization test showed that V013/YN/2013 belonging to serotype of BTV-9.Further sequence analysis confirmed the identity of isolated virus by correlation with other isolated BTV-9 strains belonging to Eastern topotype and showed closest relationship with BTV-9 strains isolated from Japan and Australia.The results of growth curve and plaque formation showed that the proliferation capacity of V013/YN/2013 is significantly higher than BTV-9 reference strain.Although specific neutralizing antibodies are produced in infected animals,the presence of the virus in the blood can be continuously detected,suggesting that the virus can cause a long-lasting infection in cattle.This study reported for the first time the isolation of an BTV-9 strain V013/YN/2013 belonging to Eastern topotype in China,these results will provide a basis for further wholegenome sequencing,establishment of diagnostic methods,epidemiological investigation and pathogenicity study of BTV-9 in China.
The study aims to isolate bluetongue virus(BTV)endemic in Yunnan Province of China and to understand the genetic and infection characteristics of the isolated BTV.Virus isolation was performed by inoculation method of"Embryos-C6/36 cells-BHK-21 cells"with BTV nucleic acid positive blood samples collected from sentinel cattle.Serotype of the isolated virus was decided by micro-neutralization test and sequencing the ORF regions of Segment 2 and Segment 6.The proliferation characteristics of isolated virus in BHK-21 cells were determined by the tests of growth curve and plaque formation.The viral load and neutralizing antibody of infected animal were analyzed by qRT-PCR and neutralization test.Results were as follows:In August 2013,BTV strain of V013/YN/2013 was isolated form sentinel cattle in Mangshi,Yunnan Province.Micro-neutralization test showed that V013/YN/2013 belonging to serotype of BTV-9.Further sequence analysis confirmed the identity of isolated virus by correlation with other isolated BTV-9 strains belonging to Eastern topotype and showed closest relationship with BTV-9 strains isolated from Japan and Australia.The results of growth curve and plaque formation showed that the proliferation capacity of V013/YN/2013 is significantly higher than BTV-9 reference strain.Although specific neutralizing antibodies are produced in infected animals,the presence of the virus in the blood can be continuously detected,suggesting that the virus can cause a long-lasting infection in cattle.This study reported for the first time the isolation of an BTV-9 strain V013/YN/2013 belonging to Eastern topotype in China,these results will provide a basis for further wholegenome sequencing,establishment of diagnostic methods,epidemiological investigation and pathogenicity study of BTV-9 in China.
引文
[1]MACLACHLAN N J,OSBURN B I.Teratogenic bluetongue and related orbivirus infections in pregnant ruminant livestock:timing and pathogen genetics are critical[J].Curr Opin Virol,2017,27:31-35.
[2]ZIENTARA S,SNCHEZ-VIZCANO J M.Control of bluetongue in Europe[J].Vet Microbiol,2013,165(1-2):33-37.
[3]MAAN S,MAAN N S,ROSS-SMITH N,et al.Sequence analysis of bluetongue virus serotype 8from the Netherlands 2006and comparison to other European strains[J].Virology,2008,377(2):308-318.
[4]CARPENTER S,WILSON A,MELLOR P S.Culicoides and the emergence of bluetongue virus in northern Europe[J].Trends Microbiol,2009,17(4):172-178.
[5]BENELLI G,BUTTAZZONI L,CANALE A,et al.Bluetongue outbreaks:Looking for effective control strategies against Culicoides vectors[J].Res Vet Sci,2017,115:263-270.
[6]ROY P.Bluetongue virus structure and assembly[J].Curr Opin Virol,2017,24:115-123.
[7]KOCHINGER S,RENEVEY N,HOFMANN M A,et al.Vesicular stomatitis virus replicon expressing the VP2outer capsid protein of bluetongue virus serotype 8induces complete protection of sheep against challenge infection[J].Vet Res,2014,45:64.
[8]XIE S Y,SHI Y M,GONG R Y,et al.Identification of antigenic epitopes of monoclonal antibodies against the VP2protein of the 25serotype of bluetongue virus[J].Vet Microbiol,2018,219:136-143.
[9]MAAN S,MAAN N S,SAMUEL A R,et al.Analysis and phylogenetic comparisons of full-length VP2genes of the 24bluetongue virus serotypes[J].J Gen Virol,2007,88(Pt 2):621-630.
[10]ZHANG X,BOYCE M,BHATTACHARYA B,et al.Bluetongue virus coat protein VP2contains sialic acid-binding domains,and VP5resembles enveloped virus fusion proteins[J].Proc Natl Acad Sci U S A,2010,107(14):6292-6297.
[11]RUSSELL B L,PARBHOO N,GILDENHUYS S.Analysis of conserved,computationally predicted epitope regions for VP5and VP7across three orbiviruses[J/OL].Bioinform Biol Insights,2018,doi:10.1177/1177932218755348[2017-07-10].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802602/.
[12]HOFMANN M A,RENZULLO S,MADER M,et al.Genetic characterization of toggenburg orbivirus,a new bluetongue virus,from goats,Switzerland[J].Emerg Infect Dis,2008,14(12):1855-1861.
[13]MAAN S,MAAN N S,NOMIKOU K,et al.Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait[J].PLoS One,2011,6(10):e26147.
[14]SCHULZ C,BRARD E,SAILLEAU C,et al.Bluetongue virus serotype 27:Detection and characterization of two novel variants in Corsica,France[J].J Gen Virol,2016,97(9):2073-2083.
[15]MCLACHLANA N J,DREW C P,DARPEL K E,et al.The pathology and pathogenesis of bluetongue[J].J Comp Pathol,2009,141(1):1-16.
[16]KIRKLANDA P D,ZHANG N,HAWKES R A,et al.Studies on the epidemiology of bluetongue virus in China[J].Epidemiol Infect,2002,128(2):257-263.
[17]CLAVIJOA A,HECKERT R A,DULAC G C,et al.Isolation and identification of bluetongue virus[J].J Virol Methods,2000,87(1-2):13-23.
[18]李华春,朱建波,花群义,等.GB/T 18636-2017蓝舌病诊断技术[S].北京:中国标准出版社,2017.LI H C,ZHU J B,HUA Q Y,et al.GB/T 18636-2017Diagnostic techniques for bluetongue[S].Beijing:China Standard Press,2017.(in Chinese)
[19]YANG H,ZHU J B,LI H C,et al.Full genome sequence of bluetongue virus serotype 4from China[J].J Virol,2012,86(23):13122-13123.
[20]TAMURA K L,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Mol Biol Evol,2013,30(12):2725-2729.
[21]SHIRAFUJI H,YANASE T,KATO T,et al.Genetic and phylogenetic characterization of genome segments 2and 6of bluetongue virus isolates in Japan from 1985to 2008[J].J Gen Virol,2012,93(Pt 7):1465-1473.
[22]RAO P P,REDDY Y V,MEENA K,et al.Genetic characterization of bluetongue virus serotype 9isolates from India[J].Virus Genes,2012,44(2):286-294.
[23]FIRTH C,BLASDELL K R,AMOS-RITCHIE R,et al.Genomic analysis of bluetongue virus episystems in Australia and Indonesia[J].Vet Res,2017,48(1):82.
[24]BOYLE D B,BULACH D M,AMOS-RITCHIE R,et al.Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia[J].J Virol,2012,86(12):6724-6731.
[25]NOMIKOU K,HUGHES J,WASH R,et al.Widespread reassortment shapes the evolution and epidemiology of bluetongue virus following European invasion[J].PLoS Pathog,2015,11(8):e1005056.
[26]ATTOUI H,MAAN S,SIMON J,et al.Bluetongue virus,other orbiviruses and other reoviruses:Their relationships and taxonomy[M]//MELLOR P S,BAYLIS M,MERTENS P P C.Bluetongue.London,UK:Elsevier Academic Press,2009.
[27]MAAN N S,MAAN S,BELAGANAHALLI M N,et al.Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2[J].PloS One,2012,7(2):e32601.
[28]SHAFIQ M,MINAKSHI P,BHATEJA A,et al.Evidence of genetic reassortment between Indian isolate of bluetongue virus serotype 21(BTV-21)and bluetongue virus serotype 16(BTV-16)[J].Virus Res,2013,173(2):336-343.
[29]COETZEE P,VAN VUUREN M,VENTER E H,et al.A review of experimental infections with bluetongue virus in the mammalian host[J].Virus Res,2014,182(4):21-34.
[30]WILSON A,DARPEL K,MELLOR S.Where does bluetongue virus sleep in the winter?[J].PLoS Biol,2008,6(8):e210.
[2]ZIENTARA S,SNCHEZ-VIZCANO J M.Control of bluetongue in Europe[J].Vet Microbiol,2013,165(1-2):33-37.
[3]MAAN S,MAAN N S,ROSS-SMITH N,et al.Sequence analysis of bluetongue virus serotype 8from the Netherlands 2006and comparison to other European strains[J].Virology,2008,377(2):308-318.
[4]CARPENTER S,WILSON A,MELLOR P S.Culicoides and the emergence of bluetongue virus in northern Europe[J].Trends Microbiol,2009,17(4):172-178.
[5]BENELLI G,BUTTAZZONI L,CANALE A,et al.Bluetongue outbreaks:Looking for effective control strategies against Culicoides vectors[J].Res Vet Sci,2017,115:263-270.
[6]ROY P.Bluetongue virus structure and assembly[J].Curr Opin Virol,2017,24:115-123.
[7]KOCHINGER S,RENEVEY N,HOFMANN M A,et al.Vesicular stomatitis virus replicon expressing the VP2outer capsid protein of bluetongue virus serotype 8induces complete protection of sheep against challenge infection[J].Vet Res,2014,45:64.
[8]XIE S Y,SHI Y M,GONG R Y,et al.Identification of antigenic epitopes of monoclonal antibodies against the VP2protein of the 25serotype of bluetongue virus[J].Vet Microbiol,2018,219:136-143.
[9]MAAN S,MAAN N S,SAMUEL A R,et al.Analysis and phylogenetic comparisons of full-length VP2genes of the 24bluetongue virus serotypes[J].J Gen Virol,2007,88(Pt 2):621-630.
[10]ZHANG X,BOYCE M,BHATTACHARYA B,et al.Bluetongue virus coat protein VP2contains sialic acid-binding domains,and VP5resembles enveloped virus fusion proteins[J].Proc Natl Acad Sci U S A,2010,107(14):6292-6297.
[11]RUSSELL B L,PARBHOO N,GILDENHUYS S.Analysis of conserved,computationally predicted epitope regions for VP5and VP7across three orbiviruses[J/OL].Bioinform Biol Insights,2018,doi:10.1177/1177932218755348[2017-07-10].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802602/.
[12]HOFMANN M A,RENZULLO S,MADER M,et al.Genetic characterization of toggenburg orbivirus,a new bluetongue virus,from goats,Switzerland[J].Emerg Infect Dis,2008,14(12):1855-1861.
[13]MAAN S,MAAN N S,NOMIKOU K,et al.Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait[J].PLoS One,2011,6(10):e26147.
[14]SCHULZ C,BRARD E,SAILLEAU C,et al.Bluetongue virus serotype 27:Detection and characterization of two novel variants in Corsica,France[J].J Gen Virol,2016,97(9):2073-2083.
[15]MCLACHLANA N J,DREW C P,DARPEL K E,et al.The pathology and pathogenesis of bluetongue[J].J Comp Pathol,2009,141(1):1-16.
[16]KIRKLANDA P D,ZHANG N,HAWKES R A,et al.Studies on the epidemiology of bluetongue virus in China[J].Epidemiol Infect,2002,128(2):257-263.
[17]CLAVIJOA A,HECKERT R A,DULAC G C,et al.Isolation and identification of bluetongue virus[J].J Virol Methods,2000,87(1-2):13-23.
[18]李华春,朱建波,花群义,等.GB/T 18636-2017蓝舌病诊断技术[S].北京:中国标准出版社,2017.LI H C,ZHU J B,HUA Q Y,et al.GB/T 18636-2017Diagnostic techniques for bluetongue[S].Beijing:China Standard Press,2017.(in Chinese)
[19]YANG H,ZHU J B,LI H C,et al.Full genome sequence of bluetongue virus serotype 4from China[J].J Virol,2012,86(23):13122-13123.
[20]TAMURA K L,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Mol Biol Evol,2013,30(12):2725-2729.
[21]SHIRAFUJI H,YANASE T,KATO T,et al.Genetic and phylogenetic characterization of genome segments 2and 6of bluetongue virus isolates in Japan from 1985to 2008[J].J Gen Virol,2012,93(Pt 7):1465-1473.
[22]RAO P P,REDDY Y V,MEENA K,et al.Genetic characterization of bluetongue virus serotype 9isolates from India[J].Virus Genes,2012,44(2):286-294.
[23]FIRTH C,BLASDELL K R,AMOS-RITCHIE R,et al.Genomic analysis of bluetongue virus episystems in Australia and Indonesia[J].Vet Res,2017,48(1):82.
[24]BOYLE D B,BULACH D M,AMOS-RITCHIE R,et al.Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia[J].J Virol,2012,86(12):6724-6731.
[25]NOMIKOU K,HUGHES J,WASH R,et al.Widespread reassortment shapes the evolution and epidemiology of bluetongue virus following European invasion[J].PLoS Pathog,2015,11(8):e1005056.
[26]ATTOUI H,MAAN S,SIMON J,et al.Bluetongue virus,other orbiviruses and other reoviruses:Their relationships and taxonomy[M]//MELLOR P S,BAYLIS M,MERTENS P P C.Bluetongue.London,UK:Elsevier Academic Press,2009.
[27]MAAN N S,MAAN S,BELAGANAHALLI M N,et al.Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2[J].PloS One,2012,7(2):e32601.
[28]SHAFIQ M,MINAKSHI P,BHATEJA A,et al.Evidence of genetic reassortment between Indian isolate of bluetongue virus serotype 21(BTV-21)and bluetongue virus serotype 16(BTV-16)[J].Virus Res,2013,173(2):336-343.
[29]COETZEE P,VAN VUUREN M,VENTER E H,et al.A review of experimental infections with bluetongue virus in the mammalian host[J].Virus Res,2014,182(4):21-34.
[30]WILSON A,DARPEL K,MELLOR S.Where does bluetongue virus sleep in the winter?[J].PLoS Biol,2008,6(8):e210.