猪脑心肌炎病毒的分离与鉴定

畜牧兽医学报 ›› 2007, Vol. 38 ›› Issue (1): 59-65.

猪脑心肌炎病毒的分离与鉴定

盖新娜;杨汉春;郭鑫;吕艳丽;王芳;陈艳红;查振林

中国农业大学农业部预防兽医学重点开放实验室，北京 100094

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-01-25 发布日期:2007-01-25

Isolation and Characterization of Porcine Encephalomyocarditis Virus

Key Laboratory of Preventive Veterinary Medicine of Ministry of Agriculture, China Agricultural University

Received:1900-01-01 Revised:1900-01-01 Online:2007-01-25 Published:2007-01-25

摘要/Abstract

摘要： 从规模化猪场疑似病例组织病料中分离到5株脑心肌炎病毒（EMCV），对其中的2株（EMCV BJC3和 EMCV HB1）进行了系统鉴定。结果表明，病毒粒子大小约为27 nm，呈圆形；2株病毒均不耐酸，对氯仿不敏感，对胰蛋白酶敏感性有所不同，60 ℃加热1 h可被灭活，二价阳离子对病毒没有明显的保护作用。用EMCV多克隆抗体作间接免疫荧光，分离毒株感染的BHK21细胞的胞浆中可见特异性荧光。对分离毒株进行了VP1和3D基因的扩增和序列测定，结果表明，2个毒株VP1基因的核苷酸序列同源性为99.49%，氨基酸序列的同源性为98.46%，与GenBank中EMCV毒株的核苷酸序列同源性介于81.61%～99.59%，氨基酸序列同源性在95.5%以上；3D基因扩增片段序列与其它毒株的核苷酸与氨基酸序列同源性均为100%。基于VP1基因的系统进化树表明，2株分离毒均位于进化树的主干上，变异度不大。由此表明，EMCV感染已在我国猪场存在。

关键词: 猪脑心肌炎病毒, 分离, 鉴定

Abstract: Five isolates of encephalomyocarditis virus were isolated from clinical tissue samples originated from suspected cases of intensive pig farms. Two isolates of them (BJC3 and HB1) were systematically identified. The results showed that round virus particles with diameter of 27 nm were observed by immune electron microscopy. The physicochemical character tests indicated that the isolates BJC3 and HB1 were not able to endure acid and insensitive to chloroform. However, the two isolates had different sensitivity to trypsin treatment. Exposed under 60 ℃ for 1 h, the viruses could be inactivated and protection of bivalent cation at temperature of 50 ℃ was negligible. The specific fluorescence was observed in the cytoplasm of BHK21 cells infected with the isolates by indirect fluorescence assay using polyclonal antibody against EMCV. The VP1 gene and partial segment of 3D gene of the two isolates were amplified by RTPCR and sequenced. Sequence analysis revealed that the nucleotide identity of VP1 gene was 99.49% and the predicted amino acid identity was 98.46% among the two isolates. The nucleotide identity of VP1 genes between EMCV strains in GenBank database and the isolates ranged from 81.61% to 99.59%. Correspondingly, the amino acid identity was above 95.5%. The identity of 3D gene amplified of both the nucleotide and amino acid was 100% among the two isolates and other EMCV strains.Phylogenetic analysis based on the nucleotide sequences of VP1 gene of EMCV showed that the two isolates were located on the backbone of the phylogenetic tree, with lesser variation. These results suggest that EMCV infection exist in pig farms in China.

Key words: EMCV, isolation, characterization

盖新娜;杨汉春;郭鑫;吕艳丽;王芳;陈艳红;查振林. 猪脑心肌炎病毒的分离与鉴定[J]. 畜牧兽医学报, 2007, 38(1): 59-65.

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