猪流行性腹泻病毒JS2017株的分离鉴定及S基因序列分析

doi:10.16431/j.cnki.1671-7236.2019.02.030

摘要/Abstract

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20 μg/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。

关键词: 猪流行性腹泻病毒(PEDV); S基因; 变异毒株

Abstract:

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 20 μg/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 S gene 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 S gene sequence of JS2017 strain was in the same evolutionary branch as the American variant strain OH1414,Canadian 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.

Key words: porcine epidemic diarrhea virus; S gene; variant strain

中图分类号:

S852.65

朱海侠. 猪流行性腹泻病毒JS2017株的分离鉴定及S基因序列分析[J]. 《中国畜牧兽医》, 2019, 46(2): 582-589.

ZHU Haixia. Isolation and Identification of Porcine Epidemic Diarrhea Virus Strain JS2017 and Its S Gene Sequence Analysis[J]. , 2019, 46(2): 582-589.

参考文献

[1] 施标,董世娟,朱于敏,等.中国猪流行性腹泻病毒分子流行病学研究进展[J].中国农业科学,2013,46(20):4362-4369. SHI B,DONG S J,ZHU Y M,et al.Advances in study of molecular epidemiology of porcine epidemic diarrhea virus in China[J].Scientia Agricultura Sinica,2013,46(20):4362-4369.(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 S gene[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 S gene 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 M gene of avian infectious bronchitis virus SX01 strain[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-2015-1 and its S gene 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 S gene[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 2013 disease 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 in 2012[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 1 based porcine epidemic diarrhea live vaccine against genogroup 2 field 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 G1 and 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 YC2014 of 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 2b porcine 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 S gene of porcine epidemic diarrhea virus in Sourthern Henan province from 2014 to 2015[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(4):859-864.(in Chinese)