猪繁殖与呼吸综合征病毒变异株的分离鉴定及遗传变异分析

doi:10.7668/hbnxb.2014.01.042

华北农学报 ›› 2014, Vol. 29 ›› Issue (1): 232-238. doi: 10.7668/hbnxb.2014.01.042

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猪繁殖与呼吸综合征病毒变异株的分离鉴定及遗传变异分析

王小敏¹, 何孔旺¹, 周忠涛^1,2, 茅爱华¹, 俞正玉¹, 汪伟¹, 倪艳秀¹, 温立斌¹, 张雪寒¹, 郭容利¹, 李彬¹, 于洋¹

1. 江苏省农业科学院兽医研究所, 农业部兽用生物制品工程技术重点实验室国家兽用生物制品,工程技术研究中心.江苏南京 210014;
2. 南京农业大学动物医学院,南京 210095

收稿日期:2013-08-09 出版日期:2014-02-28
作者简介:王小敏(1983-)，女，江苏盐城人，助理研究员，硕士，主要从事动物病毒学研究.
基金资助:
江苏省自主创新专项(CX(11)2060)

Isolation,Identification and Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus Variant

WANG Xiao-min¹, HE Kong-wang¹, ZHOU Zhong-tao^1,2, MAO Ai-hua¹, YU Zheng-yu¹, WANG Wei¹, NI Yan-xiu¹, WEN Li-bin¹, ZHANG Xue-han¹, GUO Rong-li¹, LI Bin¹, YU Yang¹

1. Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China;
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2013-08-09 Published:2014-02-28

摘要/Abstract

摘要： 从江苏某猪繁殖与呼吸综合征(PRRS)发病猪场采集的血清样本中分离到1株猪繁殖与呼吸综合征病毒(PRRSV)毒株,对该毒株的生物学特性进行了鉴定,并对其Nsp2和ORF5序列进行测定和序列比对分析。利用细胞传代的方法对PRRSV检测阳性,PPV和PRV检测阴性的猪血清进行病毒分离。通过RT-PCR、CPE和IFA对PRRSV分离毒株进行鉴定,并测定其TCID₅₀。测序获得Nsp2和ORF5基因,并利用DNAStar软件进行序列同源性和进化分析。成功分离获得1株PRRSV毒株,该毒株能够在Marc-145细胞上增殖并产生细胞病变,免疫荧光检测可观察到特异性荧光,TCID₅₀为10^5.5。Nsp2序列分析表明,该分离株具有HP-PRRSV 30个氨基酸缺失的基因特征,同时495~516位也存在缺失。分离株ORF5基因与其他参考毒株氨基酸同源性为57.1%~99.5%,与NJ-1106的同源性最高,为99.5%。系统进化树分析表明,该分离株属于北美型,与WUH4、NJ-1106等HP-PRRSV毒株属于同一分支。本试验分离得到一株HP-PRRSV变异毒株,为分析我国PRRSV的遗传变异和防控奠定了基础。

关键词: 猪繁殖与呼吸综合征病毒, 分离与鉴定, 遗传变异

Abstract: A porcine reproductive and respiratory syndrome virus(PRRSV) strain was isolated from the serum of a PRRSV infected pig in Jiangsu Province. The biological characteristics of this isolate was identified and the genome of Nsp2 and ORF5 were determined and analyzed. The serum which were detected PRRSV positive and PPV, PRV negative was inoculated and passaged on Marc-145 cells. This isolate was identified by RT-PCR and CPE,and confirmed by immunofluorescence assay(IFA). The TCID ₅₀ of this isolate was measured and the genome of Nsp2 and ORF5 were sequenced and its homology and evolutionary relationship were analyzed by DNAStar software. The results indicated that the PRRSV strain was successfully isolated and it could induce cytopathogenic effect(CPE) on Marc-145 cells. The specific fluorescence could be observed and the TCID ₅₀ of this strain was 10^{5. 5}. Comparison analysis of the Nsp2 genome of the isolate 2010JS02 and other strains revealed that the isolate has a discontinuous deletion of 30 amino acids and another deletion at the position of 495- 516 amino acid's site. According to the se-quences analysis of GP5,the isolate 2010JS02 showed identity of 57. 1%- 99. 5% among other strains,a high identity of 99. 5% with NJ-1106. The phylogenetic tree was combined and showed that 2010JS02 belonged to NA and classified into HP-PRRSV like WHU4,NJ-1106 and other HP-PRRSV strains. We successfully isolated a HPPRRSV variant and named it as 2010JS02. These results provided foundations for further studying the genetic variation and the prevention of PRRSV.

Key words: Porcine reproductive and respiratory syndrome virus(PRRSV), Isolation and identification, Genet-is vanatron

中图分类号:

S432.4

王小敏, 何孔旺, 周忠涛, 茅爱华, 俞正玉, 汪伟, 倪艳秀, 温立斌, 张雪寒, 郭容利, 李彬, 于洋. 猪繁殖与呼吸综合征病毒变异株的分离鉴定及遗传变异分析[J]. 华北农学报, 2014, 29(1): 232-238. doi: 10.7668/hbnxb.2014.01.042.

WANG Xiao-min, HE Kong-wang, ZHOU Zhong-tao, MAO Ai-hua, YU Zheng-yu, WANG Wei, NI Yan-xiu, WEN Li-bin, ZHANG Xue-han, GUO Rong-li, LI Bin, YU Yang. Isolation,Identification and Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus Variant[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(1): 232-238. doi: 10.7668/hbnxb.2014.01.042.

http://www.hbnxb.net/CN/Y2014/V29/I1/232

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猪繁殖与呼吸综合征病毒变异株的分离鉴定及遗传变异分析

Isolation,Identification and Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus Variant

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