2006—2012年鲁豫冀地区15株猪繁殖与呼吸综合征病毒的分离鉴定及ORF5/Nsp2基因的遗传变异分析

›› 2013, Vol. 40 ›› Issue (10): 26-32.

2006—2012年鲁豫冀地区15株猪繁殖与呼吸综合征病毒的分离鉴定及ORF5/Nsp2基因的遗传变异分析

唐娜^1,2, 管宇², 魏凤², 王金良², 曲光刚², 肖跃强², 李峰², 谢金文², 张松林², 张倩³, 于新友³, 沈志强², 柳增善¹

1. 吉林大学畜牧兽医学院, 吉林长春 130062;
2. 山东省滨州畜牧兽医研究院, 山东滨州 256600;
3. 山东绿都生物科技有限公司, 山东滨州 256600

修回日期:2013-06-25 出版日期:2013-10-20 发布日期:2013-12-19
通讯作者: 柳增善
作者简介:唐娜(1982-),女,江苏人,博士,助理研究员,研究方向:预防兽医学与疫苗免疫学。
基金资助:
国家自然科学基金(31201913);山东省现代农业产业技术体系生猪创新团队项目。

Isolation, Identification and ORF5/Nsp2 Genes Genetic Diversity Analysis of 15 Porcine Reproductive and Respiratory Syndrome Virus Isolates from Shandong, Henan and Hebei Areas During 2006 to 2012

TANG Na^1,2, GUAN Yu², WEI Feng², WANG Jin-liang², QU Guang-gang², XIAO Yue-qiang², LI Feng², XIE Jin-wen², ZHANG Song-lin², ZHANG Qian³, YU Xin-you³, SHEN Zhi-qiang², LIU Zeng-shan¹

1. College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China;
2. Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China;
3. Shandong Lvdu Bio-sciences and Technology Co., Ltd., Binzhou 256600, China

Revised:2013-06-25 Online:2013-10-20 Published:2013-12-19

摘要/Abstract

摘要： 为研究鲁豫冀地区猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的遗传变异情况,对2006—2012年来自3省区发病猪场的42份样品进行PRRSV分离鉴定,并进行了生物学特性研究和PCR鉴定,结果显示先后分离到15株PRRSV。分别采用RT-PCR扩增其ORF5基因和部分Nsp2基因并测序,与GenBank中68个ORF5序列和40个Nsp2序列的推导氨基酸序列进行比对,遗传变异分析结果表明,15株分离株均属于美洲型毒株,其中13个毒株Nsp2基因推导的氨基酸序列均存在氨基酸的不连续缺失,其ORF5基因推导的氨基酸序列与JXA1株有较高的同源性(95.5%~97.5%);SDDY2007株与疫苗株RespPRRS MLV和VR2332株亲缘关系相近,处于同一个亚群中;而HN25-2009分离株Nsp2基因推导的氨基酸序列有30个氨基酸的特征性缺失,其ORF5基因推导的氨基酸序列的遗传进化分析结果显示该分离株处于VR2332所在亚群(氨基酸同源性97.5%),具有一定特殊性。本试验结果表明,2006—2012年高致病性PRRSV是鲁豫冀地区的优势流行毒株,且存在疫苗毒株,3省区流行毒株间有一定遗传差异,但无明显地域特征。

关键词: 猪繁殖与呼吸综合征病毒; 分离; 遗传变异; ORF5基因; Nsp2基因

Abstract: To study the genetic variation of porcine reproductive and respiratory syndrome virus(PRRSV)in Shandong, Henan and Hebei province of China, 42 samples from pig farms in three provinces were isolated and identified during 2006 to 2012. 15 PRRSV isolates were successfully isolated, identified by PCR and biological characteristics were studied. The complete ORF5 and partial Nsp2 genes of 15 isolates were amplified and sequenced. The deduced amino acid sequences were blasted with deduced sequences of 68 ORF5 genes and 40 Nsp2 genes in GenBank. The analysis showed that all 15 isolates belonged to American groups, 13 of them presented discontinuous amino acid deletions in Nsp2, and the homology of ORF5 deduced amino acid sequences with JXA1 were from 95.5% to 97.5%. Besides, isolate SDDY2007 showed higher similarity to vaccine strain RespPRRSV MLV(98.5%), which was located in America subgroupⅡ. A more interesting thing was that, though Nsp2 sequence of HN25-2009 contained 30 amino acids deletion, its deduced amino acid sequences showed higher similarity to VR2332(97.5%). In short, highly pathogenic PRRSV was the dominant PRRSV in Shandong, Henan and Hebei during 2006 to 2012. Although there were some genetic variations among PRRSV isolates in three provinces, no obvious regional characteristics could be found.

Key words: porcine reproductive and respiratory syndrome virus; isolation; genetic diversity; ORF5 gene; Nsp2 gene

中图分类号:

S852.65

唐娜, 管宇, 魏凤, 王金良, 曲光刚, 肖跃强, 李峰, 谢金文, 张松林, 张倩, 于新友, 沈志强, 柳增善. 2006—2012年鲁豫冀地区15株猪繁殖与呼吸综合征病毒的分离鉴定及ORF5/Nsp2基因的遗传变异分析[J]. , 2013, 40(10): 26-32.

TANG Na, GUAN Yu, WEI Feng, WANG Jin-liang, QU Guang-gang, XIAO Yue-qiang, LI Feng, XIE Jin-wen, ZHANG Song-lin, ZHANG Qian, YU Xin-you, SHEN Zhi-qiang, LIU Zeng-shan. Isolation, Identification and ORF5/Nsp2 Genes Genetic Diversity Analysis of 15 Porcine Reproductive and Respiratory Syndrome Virus Isolates from Shandong, Henan and Hebei Areas During 2006 to 2012[J]. , 2013, 40(10): 26-32.

参考文献

1 王艳艳. 山东省猪繁殖与呼吸综合征、猪流感流行病学调查[D]. 泰安:山东农业大学,2010.
2 刘飞,王开功,周碧君,等. 贵州省PRRSV分离株Nsp2基因的克隆及序列分析[J]. 中国预防兽医学报,2010,32(12):924~928.
3 郎广平, 郭敏, 韩盈盈,等. 猪繁殖与呼吸综合征诊断方法与疫苗研究进展[J]. 中国畜牧兽医,2013,40(6): 195~198.
4 童武,周艳君,肖少波,等. 2010年国内部分省份猪场常见病毒性疾病的病原学调查[J].中国动物传染病学报,2011,5:1~7.
5 Darwich L, Da I, Mateu E. Certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology[J]. Virus Research, 2010, 154: 123~130.
6 Dokland T. The structural biology of PRRSV[J].Vrius Research, 2010,154:86~97.
7 Johnson C R, Griggs T F, Gnanandarajah J, et al. Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses[J]. J Gene Virol, 2011, 92: 1107~1116.
8 Kim W I, Kim J J, Cha S H, et al. Significance of genetic variation of PRRSV ORF5 in virus neutralization and molecular determinants corresponding to cross neutralization among PRRS viruses[J].Veterinary Microbiology, 2013,162(1):10~22.
9 Liu D, Zhou R, Zhang J L, et al. Recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo[J]. Virus Research, 2011,155(2): 473~486.
10 Martin-Valls G E, Burgara-Estrella A J, Darwich L, et al. Ecombination events are common in genotype 1 and 2 PRRS virus worldwide and may generate mosaic viruses[A]. 2013 International PRRS symposium[C].2013.
11 Murtaugh M P, Stadejek T, Abrahante J E, et al. The ever-expanding diversity of porcine reproductive and respiratory syndrome virus[J]. Virus Research, 2010, 154: 18~30.
12 Rosendal T. Association between PRRSV ORF5 genetic distance and differences in space, time, ownership, and animal sources among commercial pig herds[A]. 2011 International PRRS Symposium, Chicago[C]. 2011.
13 Wang F X, Song N, Chen L Z, et al. Non-structural protein 2 of the porcine reproductive and respiratory syndrome (PRRS) virus: A crucial protein in viral pathogenesis, immunity and diagnosis[J]. Research in Veterinary Science, 2013, 95(1):1~7.
14 Xu X G, Wang Z S, Zhang Q, et al. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: Baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2[J]. J Virol Methods, 2012, 179: 359~366.
15 Zhou Z, Ni J Q, Cao Z, et al. The epidemic status and genetic diversity of 14 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) isolates from China in 2009[J]. Vet Microbiol, 2011, 150:257~269.

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