猪流行性腹泻病毒CH-HeNXX-2017株全基因组序列测定与分析
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摘要
为了解猪流行性腹泻病毒在河南流行毒株全基因组遗传进化水平,应用RT-PCR方法从疑似猪流行性腹泻病毒感染的病料中扩增出全基因组序列,并将PCR产物纯化后连接pMD20-T载体,转化JM109感受态细胞,挑选单克隆进行测序,获得的序列经过DNA STAR中Edit Sequence软件对全基因组序列进行拼接。结果表明,猪流行性腹泻病毒CH-He NXX-2017株全长28 038 nt(除去poly A);进化树分析显示,该毒株与IA1,USA Colorado2013,MN和PC22A等毒株亲缘关系较为接近,与国内外早期分离株如CV777,LZC,DR13等毒株亲缘关系较远。揭示猪流行性腹泻病毒一直处于不断的进化中,需要一直密切监控病毒进化趋势,为临床诊断以及疫苗研发等提供理论参考。
To understand the evolution of porcine epidemic diarrhea virus circulating in Henan province from full genome sequence level,RT-PCR was employed to amplify the full genome sequence from porcine epidemic diarrhea suspicious samples.The PCR products were purified and ligated with pMD20-T simple vector,transformed into competent cell JM109.The sequences were then abbembled using Edit Seq of DNA STAR software.The results showed that the full length of CH-He NXX-2017 was 28 038 nt in length.Phylogenetic analysis revealed that CH-He NXX-2017 clustered most closely to IA1,USA Colorado 2013,MN and PC22 A strain,far away to early isolated strain,such as CV777,LZC,DR13.The results indicated that PEDV has been evolving under immune press,attention should be paid to monitor the trend of PEDV evolving,thus providing information for clinical diagnosis and cvaccine development.
To understand the evolution of porcine epidemic diarrhea virus circulating in Henan province from full genome sequence level,RT-PCR was employed to amplify the full genome sequence from porcine epidemic diarrhea suspicious samples.The PCR products were purified and ligated with pMD20-T simple vector,transformed into competent cell JM109.The sequences were then abbembled using Edit Seq of DNA STAR software.The results showed that the full length of CH-He NXX-2017 was 28 038 nt in length.Phylogenetic analysis revealed that CH-He NXX-2017 clustered most closely to IA1,USA Colorado 2013,MN and PC22 A strain,far away to early isolated strain,such as CV777,LZC,DR13.The results indicated that PEDV has been evolving under immune press,attention should be paid to monitor the trend of PEDV evolving,thus providing information for clinical diagnosis and cvaccine development.
引文
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[7]宣华,邢德坤,王殿瀛,等.应用猪胎肠单层细胞培养猪流行性腹泻病毒的研究[J].兽医大学学报,1984(4):202-208.
[8]徐引弟,张青娴,李建林,等.PEDV、TGEV、RV多重RT-PCR检测方法的建立与应用[J].中国兽药杂志,2014,48(11):10-13.
[9]毛雅元,张桂红,葛俊伟,等.猪流行性腹泻病毒地方株LJB/03分离及培养特性[J].病毒学报,2010,26(6):483-488.
[10]HOFMANN M,WYLER R.Propagation of the virus of PED in cell culture[J].J Clin Microbiol,1988,26:2235-2239.
[11]WANG X M,NIU B B,YAN H,et al.Genetic properties of endemic Chinese porcine epidemic diarrhea virus strains isolated since2010[J].Arch Virol,2013,158(12):2487-2494.
[12]SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of porcine epidemic diarrhea in suckling piglets,China[J].Emerg Infect Dis,2012,18(1):161-163.
[13]赵丽,韩昊莹,张鸿鑫,等.河南省猪流行性腹泻病毒S和ORF3基因的克隆与序列分析[J].华北农学报,2018,33(4):25-30.
[14]刘文俊,王娟萍,姚敬明,等.猪流行性腹泻病毒山西分离株ORF3基因序列分析[J].华北农学报,2017,32(4):103-106.
[15]高翔,李栋梁,赵景义,等.猪流行性腹泻病毒SD2014株全基因组序列测定与遗传进化分析[J].中国兽医杂志,2016,52(8):12-15.
[2]PIJPERS A,VAN NIEUWSTADT A P,TERPSTRA C,et al.Porcine epidemic diarrhea virus as a cause of persistent diarrhea in a herd of breeding and finishing pigs[J].Vet Rec,1993,132(6):129-131.
[3]NAGY B,NAGY G,MEDER M,et al.Enterotoxigenic Escherichia coli,rotavirus,porcine epidemic diarrhea virus,adenovirus and calici-like virus in porcine postweaning diarrhea in Hungary[J].Acta of Vet Hung,1996,44(1):9-19.
[4]TAKAHASHI K,OKADA K,OHSHIMA K,et al.An outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in Japan[J].Japan J Vet Sci,1983,45(6):829-832.
[5]CHAE C,KIMO,CHOI C,et al.Prevalence of porcine epidemic diarrhea virus and transmissible gastroenteritis virus infection in Korean pigs[J].Vet Rec,2000,147(21):606-608.
[6]LEE S,LEE C.Outbreak-related porcine epidemic diarrhea virus strains similar to US strains,South Korea[J].Emerg Infect Dis,2013,20(7):1223-1226.
[7]宣华,邢德坤,王殿瀛,等.应用猪胎肠单层细胞培养猪流行性腹泻病毒的研究[J].兽医大学学报,1984(4):202-208.
[8]徐引弟,张青娴,李建林,等.PEDV、TGEV、RV多重RT-PCR检测方法的建立与应用[J].中国兽药杂志,2014,48(11):10-13.
[9]毛雅元,张桂红,葛俊伟,等.猪流行性腹泻病毒地方株LJB/03分离及培养特性[J].病毒学报,2010,26(6):483-488.
[10]HOFMANN M,WYLER R.Propagation of the virus of PED in cell culture[J].J Clin Microbiol,1988,26:2235-2239.
[11]WANG X M,NIU B B,YAN H,et al.Genetic properties of endemic Chinese porcine epidemic diarrhea virus strains isolated since2010[J].Arch Virol,2013,158(12):2487-2494.
[12]SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of porcine epidemic diarrhea in suckling piglets,China[J].Emerg Infect Dis,2012,18(1):161-163.
[13]赵丽,韩昊莹,张鸿鑫,等.河南省猪流行性腹泻病毒S和ORF3基因的克隆与序列分析[J].华北农学报,2018,33(4):25-30.
[14]刘文俊,王娟萍,姚敬明,等.猪流行性腹泻病毒山西分离株ORF3基因序列分析[J].华北农学报,2017,32(4):103-106.
[15]高翔,李栋梁,赵景义,等.猪流行性腹泻病毒SD2014株全基因组序列测定与遗传进化分析[J].中国兽医杂志,2016,52(8):12-15.