广西部分地区猪群PEDV N基因克隆及序列分析
|
摘要
【目的】了解广西区猪流行性腹泻病毒(PEDV)的感染及病毒的遗传变异特点,为更好防控猪流行性腹泻提供科学依据。【方法】应用RT-PCR方法从2012-2016年广西地区25个猪场收集的PEDV病料中扩增出86株PEDV的N基因,并进行序列比对及遗传进化分析。【结果】86株PEDV的N基因核苷酸同源性为94.2%~100.0%,与参考毒株核苷酸同源性为94.1%~100.0%。N基因遗传进化分析显示广西的PEDV可分为2个群,Cluster2由韩国株DR13、日本株83P-5、中国株CH/S以及本研究的GP35-8、LC37-22、LC107-5、LC107-19和LC107-16毒株组成,其他81株毒株属于Cluster 3,包括中国株DX、LJB-03、Hu N、JS-2004-2;Cluster 1和Cluster 4则由其它参考毒株组成。【结论】PEDV是引起广西规模化猪场新生仔猪腹泻的主要病原,广西地区的PEDV流行毒株可分为2个群且其N基因仍然保守。
【Objective】To provide a scientific basis for better prevention and control of PEDV,the infection of PEDV and its current characteristics of genetic evolution in Guangxi were studied.【Method】86 PEDV field strains were isolated from 25 farms in Guangxi during 2012-2016. N gene was amplified from the 86 PEDV field strains by RT-PCR,cloned,sequenced and compared with the other reference strains in Gen Bank. 【Result】Sequence analyses of the N genes were performed,and the results showed that 86 strains had nucleotide homology of94. 2 %-100. 0 % and 94. 1 %-100. 0 % homology with reference strains. Phylogenetic analysis of N gene indicated that Guangxi strain could be divided into two groups. Cluster 2 consisted of the Korean strains DR13,Japanese strain 83 P-5 and six strains from our study(GP35-8,LC037-22,LC107-5,LC107-5,LC107-19,LC107-16). Other 81 strains and Chinese strain DX,LJB-03,Hu N,JS-2004-2 belonged to cluster 3 while cluster 1 and cluster 4 were composed of other reference strains.【Conclusion】PEDV was the main pathogen that caused diarrhea of neonatal piglets in Guangxi. The PEDV-N genes were divided into two series,and the N genes of the current PEDV were still relatively conservative.
【Objective】To provide a scientific basis for better prevention and control of PEDV,the infection of PEDV and its current characteristics of genetic evolution in Guangxi were studied.【Method】86 PEDV field strains were isolated from 25 farms in Guangxi during 2012-2016. N gene was amplified from the 86 PEDV field strains by RT-PCR,cloned,sequenced and compared with the other reference strains in Gen Bank. 【Result】Sequence analyses of the N genes were performed,and the results showed that 86 strains had nucleotide homology of94. 2 %-100. 0 % and 94. 1 %-100. 0 % homology with reference strains. Phylogenetic analysis of N gene indicated that Guangxi strain could be divided into two groups. Cluster 2 consisted of the Korean strains DR13,Japanese strain 83 P-5 and six strains from our study(GP35-8,LC037-22,LC107-5,LC107-5,LC107-19,LC107-16). Other 81 strains and Chinese strain DX,LJB-03,Hu N,JS-2004-2 belonged to cluster 3 while cluster 1 and cluster 4 were composed of other reference strains.【Conclusion】PEDV was the main pathogen that caused diarrhea of neonatal piglets in Guangxi. The PEDV-N genes were divided into two series,and the N genes of the current PEDV were still relatively conservative.
引文
[1]Timoney J F,Gillespie J H,Scott F W,et al.Microbiology and infectious disease of domestic animals[M].Ithaca:Cornell University Press,1988.
[2]孔园园,范京惠,左玉柱,等.猪流行性腹泻病毒S基因的研究进展[J].猪业科学,2016,11(33):102-105.
[3]宣华,邢德冲.应用猪胎肠单层细胞培养猪流行性腹泻病毒的研究[J].中国人民解放军兽医大学学报,1984,3(4):202-208.
[4]孙东波,冯力,时洪艳,等.猪流行性腹泻病毒分子生物学研究进展[J].动物医学进展,2006,27(10):11-14.
[5]Rodak L,Valicek L,Smid B,et al.Z.An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces[J].Veterinary Microbiology,2005,105(1):9-17.
[6]Van der Meer Y,Snijder E J,Dobbe J C,et al.Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication[J].Journal of Virology,1999,73:7641-7657.
[7]Denison M R,Spaan W J,Van der Meer Y,et al.The putative helicase of the coronavirus mouse atitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis[J].Journal of Virology,1999,73(8):6862-6871.
[8]Ndifuna A,Waters A K,Zhou M,et al.Recombinant nucleocapsid protein is potentially an inexpensive,effective serodiagnostic reagent for infectious bronchitis virus[J].Journal of Virology,1998,70(1):37-44.
[9]Seah J N,Yu L,Kwang J.Localization of linear B-cell epitopes on infectious bronchitis virus nucleocapsid protein[J].Veterinary Microbiology,2000,75(1):11-16.
[10]Mackay I M,Arden K E,Nitsche A.Survey and summary real-time PCR in virology[J].Nucleic Acids Research,2002,30(6):1292-1305.
[11]郭旋,陈静,刘欢,等.广西仔猪腹泻病毒病原流行病学调查[J].南方农业学报,2013,44(1):155-160.
[12]卢冰霞,秦毅斌,何颖,等.2011-2014年广西猪流行性腹泻病毒检测及其M基因的序列分析[J].中国畜牧兽医,2015,42(3):549-557.
[13]罗六龙,黄冬妮,黄良宗,等.广东省猪流行性腹泻病毒S1基因的克隆与序列分析[J].中国兽医杂志,2016,52(3):19-21.
[14]毛黎红,张双翔,周碧君,等.6株猪流行性腹泻病毒贵州株S基因序列分析[J].中国畜牧兽医,2017,44(6):1621-1629.
[15]Lee H K,Yeo S G.Cloning and sequence analysis of the nucleocapsid gene of Porcine epidemic diarrhea virus Chinju99[J].Virus Genes,2003,26(2):207-212.
[16]吴玉璐,朱建平,杨莘,等.猪流行性腹泻病毒N基因的表达及抗原性分析[J].中国预防兽医学报,2013,35(4):299-303.
[17]Chen J F,Liu X Z,Lang H W,et al.Genetic variation of nucleocapsid genes of porcine epidemic diarrhea virus field strains in China[J].Archives of Virology,2013,158(6):1397-1401.
[2]孔园园,范京惠,左玉柱,等.猪流行性腹泻病毒S基因的研究进展[J].猪业科学,2016,11(33):102-105.
[3]宣华,邢德冲.应用猪胎肠单层细胞培养猪流行性腹泻病毒的研究[J].中国人民解放军兽医大学学报,1984,3(4):202-208.
[4]孙东波,冯力,时洪艳,等.猪流行性腹泻病毒分子生物学研究进展[J].动物医学进展,2006,27(10):11-14.
[5]Rodak L,Valicek L,Smid B,et al.Z.An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces[J].Veterinary Microbiology,2005,105(1):9-17.
[6]Van der Meer Y,Snijder E J,Dobbe J C,et al.Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication[J].Journal of Virology,1999,73:7641-7657.
[7]Denison M R,Spaan W J,Van der Meer Y,et al.The putative helicase of the coronavirus mouse atitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis[J].Journal of Virology,1999,73(8):6862-6871.
[8]Ndifuna A,Waters A K,Zhou M,et al.Recombinant nucleocapsid protein is potentially an inexpensive,effective serodiagnostic reagent for infectious bronchitis virus[J].Journal of Virology,1998,70(1):37-44.
[9]Seah J N,Yu L,Kwang J.Localization of linear B-cell epitopes on infectious bronchitis virus nucleocapsid protein[J].Veterinary Microbiology,2000,75(1):11-16.
[10]Mackay I M,Arden K E,Nitsche A.Survey and summary real-time PCR in virology[J].Nucleic Acids Research,2002,30(6):1292-1305.
[11]郭旋,陈静,刘欢,等.广西仔猪腹泻病毒病原流行病学调查[J].南方农业学报,2013,44(1):155-160.
[12]卢冰霞,秦毅斌,何颖,等.2011-2014年广西猪流行性腹泻病毒检测及其M基因的序列分析[J].中国畜牧兽医,2015,42(3):549-557.
[13]罗六龙,黄冬妮,黄良宗,等.广东省猪流行性腹泻病毒S1基因的克隆与序列分析[J].中国兽医杂志,2016,52(3):19-21.
[14]毛黎红,张双翔,周碧君,等.6株猪流行性腹泻病毒贵州株S基因序列分析[J].中国畜牧兽医,2017,44(6):1621-1629.
[15]Lee H K,Yeo S G.Cloning and sequence analysis of the nucleocapsid gene of Porcine epidemic diarrhea virus Chinju99[J].Virus Genes,2003,26(2):207-212.
[16]吴玉璐,朱建平,杨莘,等.猪流行性腹泻病毒N基因的表达及抗原性分析[J].中国预防兽医学报,2013,35(4):299-303.
[17]Chen J F,Liu X Z,Lang H W,et al.Genetic variation of nucleocapsid genes of porcine epidemic diarrhea virus field strains in China[J].Archives of Virology,2013,158(6):1397-1401.