猪流行性腹泻病毒JS2017株的分离鉴定及S基因序列分析
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摘要
为了解江苏省某规模化猪场猪流行性腹泻病毒(PEDV)遗传变异情况,本试验采集腹泻仔猪小肠组织及肠道内容物,通过RT-PCR进行PEDV鉴定。将检测为阳性的病料组织经20μg/mL胰酶处理后,接种到Vero细胞中进行培养,将细胞培养物进行反复冻融后,收获病毒液,进行RT-PCR鉴定。采用分段重叠策略对其S基因进行全基因序列扩增,并在此基础上分析该毒株S基因的核苷酸同源性及遗传进化关系。结果显示,经RT-PCR鉴定,病料组织呈PEDV阳性,阳性病料接种Vero细胞盲传4代后,出现明显的细胞病变,表现为细胞合胞体病变,空泡化,最终裂解脱落。收获的病毒液经RT-PCR鉴定后,确认分离到1株PEDV,命名为JS2017株。核苷酸序列分析表明,JS2017株S基因序列与美国变异株OH1414、加拿大变异株ON-007、中国变异株PEDV-LYG和CH/PDS/2015位于同一进化分支,其核苷酸同源性高达99.0%以上;其中与中国变异株YC2014亲缘关系最近,核苷酸同源性为100.0%;与经典株CV777、DR13株同源性较低,亲缘关系较远。结果表明,本研究分离获得的JS2017毒株是一株PEDV地方流行变异毒株,与2014年中国江苏分离株(YC2014株)亲缘关系最密切,与当前猪流行性腹泻疫苗株亲缘关系较远。
In order to understand the genetic variation of porcine epidemic diarrhea virus(PEDV)in a large-scale pig farm in Jiangsu,the intestinal content of piglets with watery diarrhea were collected and detected for PEDV by RT-PCR.The diseased tissue detected as positive was treated with 20μg/mL trypsin,and then inoculated into Vero cells for culture.After the cell culture was repeatedly frozen and thawed,the virus solution was harvested and identified by RT-PCR.The S gene was amplified by a segmentation overlap strategy,and the nucleotide homology and genetic evolution of Sgene was analyzed.The results showed that the pathological tissue was positive for PEDV by RT-PCR.After 4 generations of inoculation with Vero cells,the positive pathogens showed obvious cytopathic changes,which showed cell syncytosis,vacuolization and finally lysis.After the harvested virus solution was identified by RT-PCR,it was confirmed that one PEDV was isolated and designated as JS2017 strain.Sequence analysis showed that the Sgene sequence of JS2017 strain was in the same evolutionary branch as the American variant strain OH1414,Ca-nadian variant strain ON-007,Chinese variant strain PEDV-LYG and CH/PDS/2015,and its nucleotide homology was as high as 99.0%.Among them,the Chinese mutant strain YC2014 had the closest relationship,and its nucleotide homology was 100.0%;JS2017 strain had low homology with the classical strains CV777 and DR13,and had a distant relationship.The results showed that the JS2017 strain isolated in this study was a PEDV local epidemic strain,which was closely related to the 2014 Jiangsu isolate(YC2014 strain),and had a far-reaching relationship with the current swine epidemic diarrhea vaccine strain.
In order to understand the genetic variation of porcine epidemic diarrhea virus(PEDV)in a large-scale pig farm in Jiangsu,the intestinal content of piglets with watery diarrhea were collected and detected for PEDV by RT-PCR.The diseased tissue detected as positive was treated with 20μg/mL trypsin,and then inoculated into Vero cells for culture.After the cell culture was repeatedly frozen and thawed,the virus solution was harvested and identified by RT-PCR.The S gene was amplified by a segmentation overlap strategy,and the nucleotide homology and genetic evolution of Sgene was analyzed.The results showed that the pathological tissue was positive for PEDV by RT-PCR.After 4 generations of inoculation with Vero cells,the positive pathogens showed obvious cytopathic changes,which showed cell syncytosis,vacuolization and finally lysis.After the harvested virus solution was identified by RT-PCR,it was confirmed that one PEDV was isolated and designated as JS2017 strain.Sequence analysis showed that the Sgene sequence of JS2017 strain was in the same evolutionary branch as the American variant strain OH1414,Ca-nadian variant strain ON-007,Chinese variant strain PEDV-LYG and CH/PDS/2015,and its nucleotide homology was as high as 99.0%.Among them,the Chinese mutant strain YC2014 had the closest relationship,and its nucleotide homology was 100.0%;JS2017 strain had low homology with the classical strains CV777 and DR13,and had a distant relationship.The results showed that the JS2017 strain isolated in this study was a PEDV local epidemic strain,which was closely related to the 2014 Jiangsu isolate(YC2014 strain),and had a far-reaching relationship with the current swine epidemic diarrhea vaccine strain.
引文
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[18]SATO T,OROKU K,OHSHIMA Y,et al.Efficacy of genogroup 1based porcine epidemic diarrhea live vaccine against genogroup 2field strain in Japan[J].Virology Journal,2018,15:28.
[19]WANG X,CHEN J,SHI D,et al.Immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes G1and G2[J].Archives of Virology,2016,161(3):537-547.
[20]秦毅斌,卢冰霞,何颖,等.猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析[J].中国畜牧兽医,2018,45(1):178-188.QIN Y B,LU B X,HE Y,et al.Isolation,identification and complete genome sequence analysis of porcine epidemic diarrhea virus CH/GX/2015/750A[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):178-188.(in Chinese)
[21]LIN H,CHEN L,GAO L,et al.Epidemic strain YC2014of epidemic diarrhea virus could provide piglets against homologous challenge[J].Virology Journal,2016,13:68.
[22]BAEK P S,CHOI H W,LEE S,et al.Efficacy of an inactivated genotype 2bporcine epidemic diarrhea virus in neonatal piglets[J].Veterinary Immunology and Immunopathology,2016,174:45-59.
[23]董建国,王瑞,曲哲会,等.2014-2015年豫南地区猪流行性腹泻病毒S基因变异分析[J].畜牧兽医学报,2018,49(4):859-864.DONG J G,WANG R,QU Z H,et al.Genetic variations of Sgene of porcine epidemic diarrhea virus in Sourthern Henan province from 2014to 2015[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(4):859-864.(in Chinese)
[2]PENANERT M B,DE BOUCK P.A new coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,1978,58(3):243-247.
[3]XUAN H,XING D,WANG D,et al.Study on the culture of porcine epidemic diarrhea virus adapted to fetal porcine primary cell monolayer[J].Chinese Journal of Animal and Veterinary Sciences,1984,4(3):202-208.
[4]王淞,陈智,焦安琪,等.猪流行性腹泻病毒变异株的分离鉴定及其S基因序列分析[J].内蒙古大学学报,2017,48(2):174-180.WANG S,CHEN Z,JIAO A Q,et al.Isolation and identification of a variant strain of porcine epidemic diarrhea virus and the sequence analysis of its Sgene[J].Journal of Inner Mongolia University(Natural Science Edition),2017,48(2):174-180.(in Chinese)
[5]MASTER P S.The molecular biology of coronaviruses[J].Advances in Virus Research,2006,66:193-202.
[6]刘文俊,姚敬明,吴忻,等.猪流行性腹泻病毒山西分离株S基因遗传变异分析[J].中国畜牧兽医,2017,44(7):1982-1989.LIU W J,YAO J M,WU X,et al.Genetic variation analysis of Sgene of porcine epidemic diarrhea virus isolated in Shanxi provine[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1982-1989.(in Chinese)
[7]LIN C N,CHUNG W B,CHANG S W,et al.US-like strain of porcine epidemic diarrhea virus outbreaks in Taiwan,2013-2014[J].Journal of Veterinary Medicine Science,2014,76(9):1297-1299.
[8]LEE D,PARK C,KIM S,et al.Heterogeneity in spike protein genes of porcine epidemic diarrhea virus isolated in Korea[J].Virus Research,2010,149(2):175-182.
[9]朱海侠,陈雷,曾亮明,等.鸡传染性支气管炎病毒SX01株的分离鉴定及M基因序列分析[J].中国畜牧兽医,2014,41(12):89-93.ZHU H X,CHEN L,ZENG L M,et al.Isolation,identification and sequence analysis of Mgene of avian infectious bronchitis virus SX01strain[J].China Animal Husbandry&Veterinary Medicine,2014,41(12):89-93.(in Chinese)
[10]朱海侠,曾亮明,傅星源,等.猪流行性腹泻疫苗株与野毒株RT-PCR检测方法的建立与应用[J].福建畜牧兽医,2016,38(3):14-16.ZHU H X,ZENG L M,FU X Y,et al.Establishment and application of RT-PCR assay for detection of PEDV vaccine and wild strains[J].Fujian Journal of Animal Husbandry and Veterinary Medicine,2016,38(3):14-16.(in Chinese)
[11]王楠楠,王辉暖,高慎阳,等.猪流行性腹泻病毒LN-2015-1株分离鉴定及其S基因的变异分析[J].中国兽医学报,2017,37(8):1457-1462.WANG N N,WANG H N,GAO S Y,et al.Isolation and identification of porcine epidemic diarrhea virus strain LN-2015-1and its Sgene mutation analysis[J].Chinese Journal of Veterinary Science,2017,37(8):1457-1462.(in Chinese)
[12]HOFMANN M,WYLER R.Propagation of the virus of porcine epidemic diarrhea in cell culture[J].Journal of Clinical Microbiology,1998,26(11):2235-2239.
[13]陶洁,熊年年,张春玲,等.PEDV流行株的分离鉴定及其遗传进化分析[J].病毒学报,2016,32(5):603-609.TAO J,XIONG N N,ZHANG C L,et al.Identification and characterization of the porcine epidemic diarrhea virus(PEDV)isolates[J].Chinese Journal of Virology,2016,32(5):603-609.(in Chinese)
[14]朱俊,高又文,张杰,等.猪流行性腹泻病毒GD-A株的分离鉴定及S基因序列分析[J].华南农业大学学报,2015,36(4):21-25.ZHU J,GAO Y W,ZHANG J,et al.Isolation and identification of GD-A strain of porcine epidemic diarrhea virus and phylogenetic tree analyses based on Sgene[J].Journal of South China Agricultural University,2015,36(4):21-25.(in Chinese)
[15]CHEN Q,LI G,STASKO J,et al.Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013disease outbreak among swine in the United States[J].Journal of Clinical Microbiology,2014,52(1):234-243.
[16]TIAN Y,YU Z,CHENG K,et al.Molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in Gansu,China in2012[J].Viruses,2013,5(8):1991-2004.
[17]孙莹.基因2b亚型猪流行性腹泻病毒变异株的分离鉴定和疫苗研制[D].扬州:扬州大学,2017.SUN Y.Isolation and identification of an variant subtype GⅡb porcine epidemic diarrhea virus and development of an inactivated vaccine[D].Yangzhou:Yangzhou University,2017.(in Chinese)
[18]SATO T,OROKU K,OHSHIMA Y,et al.Efficacy of genogroup 1based porcine epidemic diarrhea live vaccine against genogroup 2field strain in Japan[J].Virology Journal,2018,15:28.
[19]WANG X,CHEN J,SHI D,et al.Immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes G1and G2[J].Archives of Virology,2016,161(3):537-547.
[20]秦毅斌,卢冰霞,何颖,等.猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析[J].中国畜牧兽医,2018,45(1):178-188.QIN Y B,LU B X,HE Y,et al.Isolation,identification and complete genome sequence analysis of porcine epidemic diarrhea virus CH/GX/2015/750A[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):178-188.(in Chinese)
[21]LIN H,CHEN L,GAO L,et al.Epidemic strain YC2014of epidemic diarrhea virus could provide piglets against homologous challenge[J].Virology Journal,2016,13:68.
[22]BAEK P S,CHOI H W,LEE S,et al.Efficacy of an inactivated genotype 2bporcine epidemic diarrhea virus in neonatal piglets[J].Veterinary Immunology and Immunopathology,2016,174:45-59.
[23]董建国,王瑞,曲哲会,等.2014-2015年豫南地区猪流行性腹泻病毒S基因变异分析[J].畜牧兽医学报,2018,49(4):859-864.DONG J G,WANG R,QU Z H,et al.Genetic variations of Sgene of porcine epidemic diarrhea virus in Sourthern Henan province from 2014to 2015[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(4):859-864.(in Chinese)