畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (5): 823-828.doi: 10.11843/j.issn.0366-6964.2013.05.022

• 研究简报 • 上一篇    

牛肠道病毒2型的分离与鉴定

彭小薇1,董浩1,吴丹2,张鹤晓2,吴清民1,吕艳丽1*   

  1. (1. 中国农业大学动物医学院,农业部动物流行病和人畜共患病重点实验室,北京 100193;2. 北京出入境检验检疫局,北京 100026)
  • 收稿日期:2012-10-18 出版日期:2013-05-23 发布日期:2013-05-23
  • 通讯作者: 吕艳丽,副教授,E-mail:luyanli@cau.edu.cnI
  • 作者简介:彭小薇(1985-),女,福建宁德人,博士生,主要从事布鲁氏菌蛋白功能研究,E-mail: girlflytosky@163.com, Tel: 010-62733901

Isolation and Identification of Bovine Enterovirus Type 2

PENG Xiao-wei1, DONG Hao1, WU Dan2, ZHANG He-xiao2, WU Qing-min1, LYU Yan-li1*   

  1. (1. Key Laboratory of Zoonosis and Animal Epidemiology of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;2. Beijing Entey-exit Inspection and Quarantine Bureau of the Peoples Republic of China, Beijing 100026, China
  • Received:2012-10-18 Online:2013-05-23 Published:2013-05-23

摘要:

本研究从北京某牛场发生严重腹泻的泌乳奶牛采集直肠拭子,接种MDBK细胞,分离获得1株病毒,命名为BJ001。理化特性鉴定结果表明,该病毒大小约30 nm,为无囊膜的RNA病毒;用随机PCR方法对氯化铯梯度离心后的各区带扩增,扩增产物凝胶电泳后,回收“呈彗尾样拖带现象”最明显的区带2的500~1 500 bp的片段进行序列测定与分析,结果显示,扩增片段与GenBank中已知的牛肠道病毒2型相似性较高;用BEV2型特异性引物对病毒进行RT-PCR扩增,并将扩增产物进行序列测定与分析,结果表明,其与BEV2相似性最高可达93%。故该病毒被确定为牛肠道病毒2型。将该病毒接种2头成年健康奶牛,均未出现肉眼可见的临床症状,但病毒可在接种奶牛体内持续存在,并产生较高滴度抗体。

Abstract:

In this study, an unknown virus was isolated from rectal swabs of cattle with severe diarrhea and hemorrhagic dysentery in Beijing. The unknown virus was identified as non-enveloped particles about 30 nm in diameter and containing RNA genome by physicochemical characteristics analysis. All of four layers which were purified by cesium chloride density gradient centrifugation were amplified with random polymerase chain reaction (PCR), and the products from layer 2 ranged mainly from 500 to 1 500 bp, showing comet tail-shaped entrainment phenomena, were purified and sequenced. The sequence blast results showed that the virus shared high gene homology with bovine enterovirus type 2. Therefore, specific primers of bovine enterovirus type 2 was used further to identify the virus, and the unknown virus was finally verified exactly belonging to BEV2 (93% nucleotide identity). Two healthy adult cattle were inoculated with the virus as experiment animals, but no clinical symptoms appeared, although antigen and serum antibody were positive after challenge.

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