牛源流行性出血病病毒(EHDV)血清10型毒株在我国的分离鉴定
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摘要
我国存在多种血清型流行性出血热病毒(Epizootic haemorrhagic disease virus,EHDV)的流行,但尚未有关于EHDV-10型毒株的分离报道。为了解云南省EHDV的流行情况,2012～2015年,本研究在云南省设立江城、师宗、芒市三个监控点,定期采集监控动物血液,接种幼仓鼠肾细胞(Baby hamster kidney cell,BHK-21)进行病毒分离;通过PCR检测、血清中和试验、琼脂糖凝胶电泳和电镜观察等方法对分离病毒进行鉴定;对分离毒株的Seg-2/VP2与Seg-3/VP3基因节段进行克隆、测序与序列分析。2013年在云南省师宗县的哨兵牛上分离出一株EHDV毒株(YNSZ-V277-2013),病毒可引起BHK-21细胞出现圆缩、裂解的细胞病变(Cytopathic effect,CPE);电镜下病毒粒子呈球形,无囊膜,表面有大量纤维突,直径在70～80nm之间;病毒基因组dsRNA的琼脂糖凝胶电泳显示分离毒株与其他血清型EHDV一致,呈现"3-3-3"的电泳带型;序列分析显示YNSZ-V277-2013毒株的Seg-2/VP2与Seg-3/VP3序列与日本EHDV-10型毒株(ON-4/N/98)相似度最高,分别为97.5%/98.5%与98.1%/99.8%,证实分离毒株为EHDV-10型;系统发育分析显示YNSZ-V277-2013毒株的Seg-2与日本EHDV-10型毒株(ON-4/N/98)的亲缘关系最近,Seg-3与分离至日本和澳大利亚的EHDV毒株同属Eastern型。本研究首次报道了EHDV-10型毒株在我国的分离以及分离毒株的Seg-2与Seg-3基因序列特征,为进一步开展中国EHDV-10型的流行病学调查与致病性研究提供了基础。
There are many serotypes of the epizootic hemorrhagic disease virus(EHDV) prevalent in China,but there is no report about the isolation of EHDV-10 strains. We wished to investigate the epidemiology of EHDV in Yunnan Province, China. Monitoring points were established at Jiangcheng, Shizong and Mangshi Counties within Yunnan Province from 2012 to 2015. For virus isolation, blood samples were collected regularly from sentinel herds and blind passed on baby hamster kidney cells(BHK-21 cells). The isolates were identified by polymerase chain reaction(PCR) detection, serum neutralization test, agarose-gel electrophoresis and electron microscopy observation. Seg-2/VP2 and Seg-3/VP3 genes of the isolated virus were cloned and sequenced. In 2013, an EHDV strain named YNSZ-V277-2013 was isolated from sentinel cattle at Shizong County. The isolate induced a cytopathic effect in BHK-21 cells that involved "rounding up" and exfoliation. Electronmicroscopic observation showed that the virus particles were spherical, without envelopes, and had fibers distributed on their surface,with a diameter of 70 nm ～80 nm. Agarose-gel electrophoresis of the genomic doublestranded RNA showed that the YNSZ-V277-2013 strain was consistent with other serotypes of EHDV, and showed a "3-3-3" type of migration pattern. Sequencing analyses of Seg-2/VP2 and Seg-3/VP3 showed that the YNSZ-V277-2013 isolate shared the highest nucleotide and amino-acid identity with the Japanese EHDV-10 strain ON-4/N/98(97.5%/98.5% and 98.1%/99.8%, respectively), which indicated that the isolated strain belonged to EHDV serotype 10. Phylogenetic analysis of Seg-2/VP2 revealed that the YNSZ-V277-2013 isolate displayed the greatest similarity with the Japanese EHDV-10 strain ON-4/N/98. Seg-3/VP3 of the isolate belonged to Eastern topotype with EHDV strains isolated from Japan and Australia. In the present study, the isolation of an EHDV-10 strain with sequence characteristics of Seg-2/VP2 and Seg-3/VP3 in China was reported for the first time, which could build a foundation for further epidemiological investigation and pathogenicity research of EHDV-10 in China.
There are many serotypes of the epizootic hemorrhagic disease virus(EHDV) prevalent in China,but there is no report about the isolation of EHDV-10 strains. We wished to investigate the epidemiology of EHDV in Yunnan Province, China. Monitoring points were established at Jiangcheng, Shizong and Mangshi Counties within Yunnan Province from 2012 to 2015. For virus isolation, blood samples were collected regularly from sentinel herds and blind passed on baby hamster kidney cells(BHK-21 cells). The isolates were identified by polymerase chain reaction(PCR) detection, serum neutralization test, agarose-gel electrophoresis and electron microscopy observation. Seg-2/VP2 and Seg-3/VP3 genes of the isolated virus were cloned and sequenced. In 2013, an EHDV strain named YNSZ-V277-2013 was isolated from sentinel cattle at Shizong County. The isolate induced a cytopathic effect in BHK-21 cells that involved "rounding up" and exfoliation. Electronmicroscopic observation showed that the virus particles were spherical, without envelopes, and had fibers distributed on their surface,with a diameter of 70 nm ～80 nm. Agarose-gel electrophoresis of the genomic doublestranded RNA showed that the YNSZ-V277-2013 strain was consistent with other serotypes of EHDV, and showed a "3-3-3" type of migration pattern. Sequencing analyses of Seg-2/VP2 and Seg-3/VP3 showed that the YNSZ-V277-2013 isolate shared the highest nucleotide and amino-acid identity with the Japanese EHDV-10 strain ON-4/N/98(97.5%/98.5% and 98.1%/99.8%, respectively), which indicated that the isolated strain belonged to EHDV serotype 10. Phylogenetic analysis of Seg-2/VP2 revealed that the YNSZ-V277-2013 isolate displayed the greatest similarity with the Japanese EHDV-10 strain ON-4/N/98. Seg-3/VP3 of the isolate belonged to Eastern topotype with EHDV strains isolated from Japan and Australia. In the present study, the isolation of an EHDV-10 strain with sequence characteristics of Seg-2/VP2 and Seg-3/VP3 in China was reported for the first time, which could build a foundation for further epidemiological investigation and pathogenicity research of EHDV-10 in China.
引文
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[8] Clavijo A, Sun F, Lester T, Jasperson D C, Wilson W C. An improved real-time polymerase chain reaction for the simultaneous detection of all serotypes of epizootic hemorrhagic disease virus[J]. J Vet Diagn Invest2010, 22(4):588-593.
[9] Shirafuji H,Kato T,Yamakawa M,Tanaka T,Minemori Y, Yanase T. Characterization of genome segments 2, 3 and 6 of epizootic hemorrhagic disease virus strains isolated in Japan in 1985-2013:identification of their serotypes and geographical genetic types[J]. Infect Genet Evol, 2017, 53, 38-46.
[10] Allison A B, Goekjian V H, Potgieter A C, Wilson W C, Johnson D J, Mertens P P C, Stallknechtaf D E. Detection of a novel reassortant epizootic hemorrhagic disease virus(EHDV)in the USA containing RNA segments derived from both exotic(EHDV-6)and endemic(EHDV-2)serotypes[J]. J Gen Virol, 2010, 91(Pt2), 430.
[11]Allison A B,Holmes E C,Potgieter A C,Wright I M,Sailleau C,Breard E, Ruder M G,Stallknechtaf D E.Segmental configuration and putative origin of the reassortant orbivirus,epizootic hemorrhagic disease virus serotype 6, strain Indiana[J]. Virology, 2012, 424(1):67-75.
[12]张怡轩,林俊,曹颖颖,朱建波,杜毅超,杨振兴,姚俊,李华春,吴健敏.广西鹿流行性出血热病毒血清型调查及分布影响分析[J].上海畜牧兽医通讯,2016(4):19-21.
[13]朱建波,杨振兴,肖雷,高林,苗海生,何于雯,孟锦昕,杨恒,李华春.流行性出血病多克隆抗体C-ELISA检测方法的建立[J].畜牧兽医学报,2018(7):1440-1450.
[14]杨恒,肖雷,李占鸿,孟锦昕,杨振兴,吕敏娜,林栩慧,廖德芳,牛保生,李华春.2012-2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析[J].畜牧兽医学报,2018, 49(4):784-793.
[15] Maan S,Rao S,Maan N S,Anthony S J,Attoui H,Samuel A R, Mertens P P C. Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses[J]. J Virol Methods,2007,143(2):132-139.
[16] Nomikou K,Dovas C I,Maan S,Anthony S J,Samuel A R,Papanastassopoulou M. Maan N S,Mangana O,Mertens P P C. Evolution and phylogenetic analysis of full-length vp3 genes of eastern mediterranean bluetongue virus isolates[J/OL]. PLoS One, 2009, 4(7):e6437.
[17]吕敏娜,朱建波,李娟,杨振兴,林栩慧,陈琴苓,张健騑,廖申权,戚南山,吴彩艳,陈志虹,李华春,孙铭飞.广东一株牛源流行性出血病病毒的分离鉴定[J].中国预防兽医学报,2017,39(1):67-70.
[18]曹颖颖,吴健敏,林俊,朱建波,杨振兴,覃绍敏,蓝显利.广西首例牛源鹿流行性出血热病毒的分离鉴定[J].中国预防兽医学报,2015, 37(10):746-750.
[19] Attoui H, Mohd J F, Belhouchet M, Aldrovandi N,Tao S, Chen B, Guodong L, Robert B T, Philippe M,Xavier L. Yunnan orbivirus,a new orbivirus species isolated from culex tritaeniorhynchus mosquitoes in China[J]. J Gen Virol,2005,86(Pt 12):3409-3417.
[20] Lei W W, Guo X F, Fu S H, Feng Y, Nie K, Song J D, Li Y, Ma X J, Liang G D, Zhou H N. Correction:isolation of Tibet orbivirus, TIBOV, from Culicoides collected in Yunnan, China[J/OL]. PLoS One, 2015,10(8):e0136257.
[21] Wang J L,Li C H,He Y W, Yang Z,Meng J X,Zhu W Y,CHEN H Y,Liao D F,MAN Y P. Isolation andgenetic characterization of mangshi virus:a newly discovered seadornavirus of thereoviridae family found in Yunnan province, China[J/OL]. PLoS One, 2015, 10(12):e0143601.
[22] Liu H,LI M H,Zhai Y G,Meng W S,Sun X H,Cao Y X,Fu S H,Wang H Y,Xu L H,Tang Q,Liang G D. Banna virus, China,1987-2007[J]. Emerg Infect Dis,2010, 16(3):514-517.
[23] Liao Y K,Inaba Y,Li N J,Chain C Y,Lee S L,Liou P P. Epidemiology of bovine ephemeral fever virus infection in Taiwan[J]. Microbiol Res, 1998, 153(3):289-295.
[24] Lee F, Ting L J, Lee M S, Chang W M, Wang F I.Genetic analysis of two taiwanese bluetongue viruses[J].Vet Microbiol, 2011, 148(2):140-149.
[25] Eagles D, Deveson T, Walker P J, Zalucki M P, Durr P. Evaluation of long-distance dispersal of culicoides midges into northern Australia using a migration model[J].Med Vet Entomol, 2012,26(3):334-340.
[26] Eagles D,Walker P J,Zalucki M P,Durr P. Modelling spatio-temporal patterns of long-distance Culicoides dispersal into northern Australia[J]. Prev Vet Med 2013,110(3-4):312-322.
[2] Yadin H, Brenner J, Bumbrov V, Oved Z, Stram Y,Klement E, Perl S, Anthony S, Maan S, BattenC,Mertens P P. Epizootic haemorrhagic disease virus type7 infection in cattle in Israel[J]. Vet Rec, 2008, 162(2):53-56.
[3] Mutlu T E,Kadir Y,Carrie B,Sezgin S,Maan N S,Clement M P P, Batmaz H. Epizootic hemorrhagic disease in cattle, Western Turkey[J]. Emerg Infect Dis,2009, 15(2):317-319.
[4] Corbel M J,Macmillan A P. Office International des Epizooties OIE Manual of standards for diagnostic tests and vaccines Paris:Office International des Epizooties O.I.E 2008[M]. Manual of standards for diagnostic tests and vaccines, 1996.
[5] Mertens P P C,Maan S,Samuel A,Attoui H. Orbiviruses, Reoviridae. In:Fauquet CM,Mayo MA,Maniloff J,Desselberger U and Ball LA. Virus Taxonomy Eighth Report of the International Committee on Taxonomy of Viruses[M]. London:Elsevier/Academic Press, 2005:466-483.
[6] Anthony S J, Maan S, Maan N, Kgosana L, Bachanekbankowska K, Batten C, Darpel K E, Sutton G, Attoui H, Mertens P P C. Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus(EHDV):comparison of genetic and serological data to characterise the ehdv serogroup[J]. Virus Res, 2009, 145(2):200-210.
[7] Anthony S J, Maan N S. Genetic and phylogenetic analysis of the core proteins VP1, VP3, VP4, VP6 and VP7 of epizootic haemorrhagic disease virus(EHDV[J]. Virus Res,2009, 145(2):187-199.
[8] Clavijo A, Sun F, Lester T, Jasperson D C, Wilson W C. An improved real-time polymerase chain reaction for the simultaneous detection of all serotypes of epizootic hemorrhagic disease virus[J]. J Vet Diagn Invest2010, 22(4):588-593.
[9] Shirafuji H,Kato T,Yamakawa M,Tanaka T,Minemori Y, Yanase T. Characterization of genome segments 2, 3 and 6 of epizootic hemorrhagic disease virus strains isolated in Japan in 1985-2013:identification of their serotypes and geographical genetic types[J]. Infect Genet Evol, 2017, 53, 38-46.
[10] Allison A B, Goekjian V H, Potgieter A C, Wilson W C, Johnson D J, Mertens P P C, Stallknechtaf D E. Detection of a novel reassortant epizootic hemorrhagic disease virus(EHDV)in the USA containing RNA segments derived from both exotic(EHDV-6)and endemic(EHDV-2)serotypes[J]. J Gen Virol, 2010, 91(Pt2), 430.
[11]Allison A B,Holmes E C,Potgieter A C,Wright I M,Sailleau C,Breard E, Ruder M G,Stallknechtaf D E.Segmental configuration and putative origin of the reassortant orbivirus,epizootic hemorrhagic disease virus serotype 6, strain Indiana[J]. Virology, 2012, 424(1):67-75.
[12]张怡轩,林俊,曹颖颖,朱建波,杜毅超,杨振兴,姚俊,李华春,吴健敏.广西鹿流行性出血热病毒血清型调查及分布影响分析[J].上海畜牧兽医通讯,2016(4):19-21.
[13]朱建波,杨振兴,肖雷,高林,苗海生,何于雯,孟锦昕,杨恒,李华春.流行性出血病多克隆抗体C-ELISA检测方法的建立[J].畜牧兽医学报,2018(7):1440-1450.
[14]杨恒,肖雷,李占鸿,孟锦昕,杨振兴,吕敏娜,林栩慧,廖德芳,牛保生,李华春.2012-2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析[J].畜牧兽医学报,2018, 49(4):784-793.
[15] Maan S,Rao S,Maan N S,Anthony S J,Attoui H,Samuel A R, Mertens P P C. Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses[J]. J Virol Methods,2007,143(2):132-139.
[16] Nomikou K,Dovas C I,Maan S,Anthony S J,Samuel A R,Papanastassopoulou M. Maan N S,Mangana O,Mertens P P C. Evolution and phylogenetic analysis of full-length vp3 genes of eastern mediterranean bluetongue virus isolates[J/OL]. PLoS One, 2009, 4(7):e6437.
[17]吕敏娜,朱建波,李娟,杨振兴,林栩慧,陈琴苓,张健騑,廖申权,戚南山,吴彩艳,陈志虹,李华春,孙铭飞.广东一株牛源流行性出血病病毒的分离鉴定[J].中国预防兽医学报,2017,39(1):67-70.
[18]曹颖颖,吴健敏,林俊,朱建波,杨振兴,覃绍敏,蓝显利.广西首例牛源鹿流行性出血热病毒的分离鉴定[J].中国预防兽医学报,2015, 37(10):746-750.
[19] Attoui H, Mohd J F, Belhouchet M, Aldrovandi N,Tao S, Chen B, Guodong L, Robert B T, Philippe M,Xavier L. Yunnan orbivirus,a new orbivirus species isolated from culex tritaeniorhynchus mosquitoes in China[J]. J Gen Virol,2005,86(Pt 12):3409-3417.
[20] Lei W W, Guo X F, Fu S H, Feng Y, Nie K, Song J D, Li Y, Ma X J, Liang G D, Zhou H N. Correction:isolation of Tibet orbivirus, TIBOV, from Culicoides collected in Yunnan, China[J/OL]. PLoS One, 2015,10(8):e0136257.
[21] Wang J L,Li C H,He Y W, Yang Z,Meng J X,Zhu W Y,CHEN H Y,Liao D F,MAN Y P. Isolation andgenetic characterization of mangshi virus:a newly discovered seadornavirus of thereoviridae family found in Yunnan province, China[J/OL]. PLoS One, 2015, 10(12):e0143601.
[22] Liu H,LI M H,Zhai Y G,Meng W S,Sun X H,Cao Y X,Fu S H,Wang H Y,Xu L H,Tang Q,Liang G D. Banna virus, China,1987-2007[J]. Emerg Infect Dis,2010, 16(3):514-517.
[23] Liao Y K,Inaba Y,Li N J,Chain C Y,Lee S L,Liou P P. Epidemiology of bovine ephemeral fever virus infection in Taiwan[J]. Microbiol Res, 1998, 153(3):289-295.
[24] Lee F, Ting L J, Lee M S, Chang W M, Wang F I.Genetic analysis of two taiwanese bluetongue viruses[J].Vet Microbiol, 2011, 148(2):140-149.
[25] Eagles D, Deveson T, Walker P J, Zalucki M P, Durr P. Evaluation of long-distance dispersal of culicoides midges into northern Australia using a migration model[J].Med Vet Entomol, 2012,26(3):334-340.
[26] Eagles D,Walker P J,Zalucki M P,Durr P. Modelling spatio-temporal patterns of long-distance Culicoides dispersal into northern Australia[J]. Prev Vet Med 2013,110(3-4):312-322.
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