中国畜牧兽医 ›› 2019, Vol. 46 ›› Issue (12): 3725-3732.doi: 10.16431/j.cnki.1671-7236.2019.12.032

• 预防兽医 • 上一篇    下一篇

羊口疮病毒大足株的分离鉴定

李鹏飞1, 张友1, 鲜思美1,2, 李婷1, 包细明1, 张益1, 饶体宇1, 吴伯梅1   

  1. 1. 贵州大学动物科学学院, 贵阳 550025;
    2. 贵州省动物疫病研究所, 贵阳 550025
  • 收稿日期:2019-07-04 出版日期:2019-12-20 发布日期:2019-12-21
  • 通讯作者: 鲜思美 E-mail:xiansimei2005@163.com
  • 作者简介:李鹏飞(1990-),男,河南南阳人,硕士,研究方向:预防兽医学,E-mail:lpf0405@yeah.net;张友(1993-),男,贵州铜仁人,硕士生,研究方向:预防兽医学,E-mail:2855718827@qq.com
  • 基金资助:
    贵州省科技计划项目(黔科合支撑[2018]2264号);贵州省科技厅基础研究项目(黔科合基础[2019]1113号)

Isolation and Identification of Orf Virus DZ Strain

LI Pengfei1, ZHANG You1, XIAN Simei1,2, LI Ting1, BAO Ximing1, ZHANG Yi1, RAO Tiyu1, WU Bomei1   

  1. 1. College of Animal Science, Guizhou University, Guiyang 550025, China;
    2. Institute of Animal Disease Research of Guizhou, Guiyang 550025, China
  • Received:2019-07-04 Online:2019-12-20 Published:2019-12-21

摘要: 本研究旨在了解重庆地区羊口疮病毒(orf virus,ORFV)流行情况。从重庆大足等地区羊场采集18份临床疑似羊口疮病料,提取病料DNA,经PCR鉴定为ORFV阳性;将阳性病料做常规处理,接种羔羊睾丸细胞(LT),并盲传至5代,观察接毒LT细胞病变,将F5代细胞培养物进行间接免疫荧光试验(IFA)、PCR扩增、测序、同源性比对、遗传进化分析和TCID50测定。结果显示,经PCR检测,18份疑似羊口疮病料ORFV核酸阳性率为61.1%(11/18);大足株病料接种LT细胞36 h后,长梭型细胞变圆、融合、呈拉网状、皱缩,72 h后大部分细胞脱落;间接免疫荧光试验在显微镜下观察到细胞浆中出现特异性绿色荧光,且荧光绕核分布;F1至F5代细胞培养物经PCR扩增出特异性目的条带,大小为1 137 bp;将测序结果与GenBank中19株ORFV B2L基因进行同源性比对,其核苷酸同源性在97.9%~100%之间,与美国SA00分离株同源性达100%,且遗传进化分析显示处于同一进化分支,亲缘关系较近;测定F5代细胞培养物TCID50值为10-5.68/0.1 mL,在细胞中能稳定增殖。综上所述,本研究成功分离并获得1株ORFV,为后续ORFV研究提供了生物材料,同时为防控重庆地区的羊口疮奠定基础。

关键词: 羊口疮病毒(ORFV); 分离鉴定; 遗传进化分析

Abstract: This study was aimed to understand the prevalence of orf virus (ORFV) in Chongqing.18 samples of clinical suspected orf disease materials were collected from sheep farms in Dazu,Chongqing,etc,and DNA of the samples was extracted,which was identified as positive by PCR.Positive disease materials were routinely treated,inoculated with lamb testicular cells (LT),and blind transmitted to the F5 generation.Toxic LT cell lesion CPE was observed,and F5 generation cell cultures were systematically identified by indirect immunofluorescence test (IFA),PCR amplification,sequencing,homology alignment,genetic evolution analysis and TCID50 determination.The results showed that the nucleic acid positive rate of ORFV detected by PCR was 61.1% (11/18).After LT cells were inoculated with Dazu strain for 36 h,the long-spindle cells became round,fused,reticulated and shriveled,and most of the cells fell off after 72 h.IFA observed the specific green fluorescence in cytoplasm,and the fluorescence was distributed around the nucleus.Specific target bands of F1 to F5 cell cultures were amplified by PCR with a size of 1 137 bp.The sequencing results were compared with 19 strains of ORFV B2L genes in GenBank,and the nucleotide homologies were 97.9% to 100%,which was up to 100% of the homology with the SA00 isolates from the United States,and the genetic evolution analysis showed that they were in the same evolutionary branch,with close relationship.The TCID50 value of F5 generation cell culture was 10-5.68/0.1 mL,which could be stable proliferation in cells.In conclusion,this study successfully isolated and obtained an ORFV strain,provided biological materials for the follow-up ORFV research,and laid a foundation for the prevention and control of stomatitis in Chongqing.

Key words: orf virus (ORFV); isolation and identification; genetic evolution analysis

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