一株牛肠道病毒的分离与鉴定
|
摘要
本研究从内蒙古某牛场患呼吸道病牛鼻拭子中分离到1株病毒,命名为NM575,并对其进行了鉴定。分离的病毒能够抵抗100μg/m L的DNA抑制剂5-溴脱氧尿苷,确定为RNA病毒。电镜观察可见无囊膜球形的病毒粒子,直径约30 nm,与小RNA病毒形态学特征相符。理化特性鉴定结果表明分离病毒对乙醚、氯仿及胰酶具有一定抵抗力,耐酸,对热敏感,与牛肠道病毒特性相符。应用牛肠道病毒P1基因的特异性引物,通过RT-PCR可从病毒细胞培养物中扩增出特异性片段,对其进行序列测定并与Gen Bank中登录的牛肠道病毒参考毒株及其他种属的肠道病毒参考株相应的序列进行比对及系统进化分析,结果显示分离毒株NM575与牛肠道病毒BHV-261株的核苷酸同源率为80%,进一步说明分离株为牛肠道病毒,且基因分型为EV-F1。病毒的体外细胞培养嗜性试验结果显示,NM575株可感染牛、仓鼠、猪、犬和鸡等多种动物细胞,尤其在牛源细胞中复制滴度最高,为10~(7.6) TCID_(50)/m L。综合以上结果分析确定所分离的病毒株NM575为牛肠道病毒,这是我国首次从患呼吸道病牛鼻拭子中分离获得。
A virus strain designated NM575 was isolated from a nasal swab of cattle with respiratory disease from a cattle farm in Inner Mongolia and analyzed in this study. The results showed that the virus could replicate in MDBK cell cultures containing 100 μg/m L DNA inhibitor(5-bromo-2-deoxyuridine, BRDU) and was identified as RNA type. Nonenveloped spherical virions were observed under electron microscope with a diameter of 30 nm, and in agreement with the characteristics of picornaviruses. Physiochemical analysis showed that the isolated virus had certain resistance to ether, chloroform, trypsin and acid. The high temperature could destroy the virus and even inactivate the virus, but 1 mol/L MgSO_4 could significantly improve the virus resistance to heat. These physiochemical properties were in agreement with those of enteroviruses. A fragment could be amplified with the specific primers for P1 gene of bovine enterovirus with RT-PCR. Then the amplified fragment was cloned and sequenced, and its nucleotide sequence was blasted in Gen Bank. The blast results showed that the nucleotide sequence of NM575 P1 gene had a 80% homology with that of enterovirus strain BEV-261. The phylogenetic tree indicated that the NM575 was an enterovirus and typed as EV-F1. The results of growth characteristics of NM575 in cell cultures showed that the virus could replicate in cell cultures originated from cattle, hamster, pig, dog and chicken, and especially in MDBK cell culture with a titer of 10~(7.6) TCID_(50)/m L. In conclusion, the results presented in this study demonstrated that the NM575 strain was an enterovirus. So far, this was the first report for isolating bovine enterovirus from the nasal swab of cattle in China.
A virus strain designated NM575 was isolated from a nasal swab of cattle with respiratory disease from a cattle farm in Inner Mongolia and analyzed in this study. The results showed that the virus could replicate in MDBK cell cultures containing 100 μg/m L DNA inhibitor(5-bromo-2-deoxyuridine, BRDU) and was identified as RNA type. Nonenveloped spherical virions were observed under electron microscope with a diameter of 30 nm, and in agreement with the characteristics of picornaviruses. Physiochemical analysis showed that the isolated virus had certain resistance to ether, chloroform, trypsin and acid. The high temperature could destroy the virus and even inactivate the virus, but 1 mol/L MgSO_4 could significantly improve the virus resistance to heat. These physiochemical properties were in agreement with those of enteroviruses. A fragment could be amplified with the specific primers for P1 gene of bovine enterovirus with RT-PCR. Then the amplified fragment was cloned and sequenced, and its nucleotide sequence was blasted in Gen Bank. The blast results showed that the nucleotide sequence of NM575 P1 gene had a 80% homology with that of enterovirus strain BEV-261. The phylogenetic tree indicated that the NM575 was an enterovirus and typed as EV-F1. The results of growth characteristics of NM575 in cell cultures showed that the virus could replicate in cell cultures originated from cattle, hamster, pig, dog and chicken, and especially in MDBK cell culture with a titer of 10~(7.6) TCID_(50)/m L. In conclusion, the results presented in this study demonstrated that the NM575 strain was an enterovirus. So far, this was the first report for isolating bovine enterovirus from the nasal swab of cattle in China.
引文
[1]Hyypia T,Hovi T,Knowles N J,et al.Classification of enteroviruses based on molecular and biological properties[J].J Gen Virol,1997,78(Pt1):1-11.
[2]Mc Ferran J B.Bovine enteroviruses[J].Ann N Y Acad Sci,1962,101(1):436-443.
[3]Moll T,Ulrich M I.Biologic characteristics of certain bovine enteric viruses[J].Am J Vet Res,1963,24:545-550.
[4]Mc Clurkin A W.Probable role of viruses in calfhood diseases[J].J Dairy Sci,1977,60(2):278-282.
[5]Tsuchiaka S,Masuda T,Sugimura S,et al.Development of a novel detection system for microbes from bovine diarrhea by real-time PCR[J].J Vet Med Sci,2016,78(3):383-389.
[6]Moll T,Finlayson A V.Isolation of cytopathogenic viral agent from feces of cattle[J].Science,1957,126(3270):401-402.
[7]Moll T,Davis A D.Isolation and characterization of cytopathogenic enteroviruses from cattle with respiratory disease[J].Am J Vet Res,1959,20:27-32.
[8]Zhu L,Xing Z,Gai X,et al.Identification of a novel enterovirus E isolates HY12 from cattle with severe respiratory and enteric diseases[J].PLo S One,2014,9(5):e97730.
[9]Huck R A,Cartwright S F.Isolation and classification of viruses from cattle during outbreaks of Mucosal or respiratory disease and from herds with reproductive disorders[J].J Comp Pathol,1964,74:346-365.
[10]Gür S,Yapki?O,Yilmaz A.Serological survey of bovine enterovirus type 1 in different mammalian species in Turkey[J].Zoonoses Public Health,2008,55(2):106-111.
[11]李英利,常继涛,于力,等.牛肠道病毒的分离鉴定[J].中国预防兽医学报,2011,33(5):388-390.
[12]Peng X W,Dong H,Wu Q M,et al.Full genome sequence of a bovine enterovirus isolated in China[J].Genome Announc,2014,2(3):pii:e00620-14.
[13]King A M Q,Adams M J,Carstens E B,et al.Virus taxonomy.Ninth report of the international committee on taxonomy of viruses[M].San Diego:Elsevier Academic Press,2011.
[14]Tsuchiaka S,Rahpaya S S,Otomaru K,et al.Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein[J].BMC Microbiol,2017,17(1):18.
[15]邢泽黎,朱利塞,王新平,等.从吉林省肉牛场新发疫病分离出E种肠道病毒[J].中国兽医学报,2014,34(3):390-394.
[16]Zhang H,Liu H,Bao J,et al.Characterization of an Enterovirus species E isolated from naturally infected bovine in China[J].Virus Res,2014,191:101-107.
[2]Mc Ferran J B.Bovine enteroviruses[J].Ann N Y Acad Sci,1962,101(1):436-443.
[3]Moll T,Ulrich M I.Biologic characteristics of certain bovine enteric viruses[J].Am J Vet Res,1963,24:545-550.
[4]Mc Clurkin A W.Probable role of viruses in calfhood diseases[J].J Dairy Sci,1977,60(2):278-282.
[5]Tsuchiaka S,Masuda T,Sugimura S,et al.Development of a novel detection system for microbes from bovine diarrhea by real-time PCR[J].J Vet Med Sci,2016,78(3):383-389.
[6]Moll T,Finlayson A V.Isolation of cytopathogenic viral agent from feces of cattle[J].Science,1957,126(3270):401-402.
[7]Moll T,Davis A D.Isolation and characterization of cytopathogenic enteroviruses from cattle with respiratory disease[J].Am J Vet Res,1959,20:27-32.
[8]Zhu L,Xing Z,Gai X,et al.Identification of a novel enterovirus E isolates HY12 from cattle with severe respiratory and enteric diseases[J].PLo S One,2014,9(5):e97730.
[9]Huck R A,Cartwright S F.Isolation and classification of viruses from cattle during outbreaks of Mucosal or respiratory disease and from herds with reproductive disorders[J].J Comp Pathol,1964,74:346-365.
[10]Gür S,Yapki?O,Yilmaz A.Serological survey of bovine enterovirus type 1 in different mammalian species in Turkey[J].Zoonoses Public Health,2008,55(2):106-111.
[11]李英利,常继涛,于力,等.牛肠道病毒的分离鉴定[J].中国预防兽医学报,2011,33(5):388-390.
[12]Peng X W,Dong H,Wu Q M,et al.Full genome sequence of a bovine enterovirus isolated in China[J].Genome Announc,2014,2(3):pii:e00620-14.
[13]King A M Q,Adams M J,Carstens E B,et al.Virus taxonomy.Ninth report of the international committee on taxonomy of viruses[M].San Diego:Elsevier Academic Press,2011.
[14]Tsuchiaka S,Rahpaya S S,Otomaru K,et al.Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein[J].BMC Microbiol,2017,17(1):18.
[15]邢泽黎,朱利塞,王新平,等.从吉林省肉牛场新发疫病分离出E种肠道病毒[J].中国兽医学报,2014,34(3):390-394.
[16]Zhang H,Liu H,Bao J,et al.Characterization of an Enterovirus species E isolated from naturally infected bovine in China[J].Virus Res,2014,191:101-107.