生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (3): 69-.doi: 10.3969/j.issn.2095-1736.2023.03.069

• 研究报告 • 上一篇    下一篇

猪流行性腹泻病毒变异株分离鉴定及攻毒模型建立

焦安琪1,2， 郭效珍3， 陈　智2， 于　江2， 于克戬3， 逯晓寒3， 辛忠昊3， 韩先杰1， 吴家强1,2   

  1. 1. 青岛农业大学 动物医学院， 青岛 266109；
    2. 山东省农业科学院畜牧兽医研究所 山东省畜禽疫病防治与繁育重点实验室， 济南 250000；
    3. 山东省农业科学院家禽研究所 山东省家禽疫病诊断与免疫重点实验室， 济南 250000
  • 出版日期:2023-06-18 发布日期:2023-06-19
  • 通讯作者: 吴家强，研究员，主要从事动物微生物与免疫学研究, E-mail:wujiaqiang2000@sina.com；韩先杰，副教授，主要从事动物疫病诊断与防控技术研究，E-mail:hanxianjie2006@163.com；吴家强和韩先杰为共同通信作者
  • 作者简介:焦安琪，硕士研究生，主要从事动物病毒学研究，E-mail:937873018@qq.com；郭效珍，博士研究生，主要从事动物传染病研究，E-mail:gxz0821@163.com；焦安琪和郭效珍为共同第一作者
  • 基金资助:
    山东省现代农业产业技术体系项目（SDAIT-08-01）; 山东省农业重大应用技术创新项目（SD2019XM007）; 高层次人才工程项目;山东省农业科学院创新工程项目； 国家自然科学基金项目（32070178）; 山东省重点研发计划项目（农业良种工程）

Isolation and identification of porcine epidemic diarrhea virus variant strain and establishment of challenge model

JIAO Anqi1,2, GUO Xiaozhen3, CHEN Zhi2, YU Jiang2, YU Kejian3, LU Xiaohan3,XIN Zhonghao3, HAN Xianjie1, WU Jiaqiang1,2   

  1. 1.College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China; 2.Key Laboratory
    of Animal and Poultry Disease Control and Breeding, Shandong Province, Institute of Animal Husbandry and
    Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250000, China; 3. Key Laboratory of
    Poultry Disease Diagnosis and Immunity in Shandong Province, Poultry Research Institute, Shandong Academy of
    Agricultural Sciences, Jinan 250000, China
  • Online:2023-06-18 Published:2023-06-19

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摘要： 猪流行性腹泻（PED）是由猪流行性腹泻病毒（PEDV）引起的一种急性高度接触性肠道传染病。研究成功分离到一株PEDV，命名为PEDV-20200946。对该毒株的S基因进行测序分析，遗传进化树结果显示，该分离毒株属于G2亚群。通过Reed-Muench法测定该毒株毒价为106.1 TCID50/mL。通过动物回归试验进一步探究该分离毒株对仔猪的致病情况，将12头20日龄仔猪平均分为3组，分别为口服攻毒组、口服+肌注攻毒组和对照组。攻毒后观察临床症状，72 h后采集各组的粪便及发病猪的肠道进行RT-PCR检测和免疫组化分析。结果显示，口服+肌注组仔猪发病最快，口服攻毒组次之，对照未发病。剖检攻毒组仔猪可见其肠壁变薄、肠道膨胀，内含大量黄色液体。免疫组化试验显示，攻毒组猪小肠内可检测到大量PEDV阳性信号，而对照组未检测到。研究建立了PEDV-20200946毒株仔猪攻毒模型，为深入研究PEDV流行毒株的分子特性以及弱毒疫苗的开发奠定基础。

关键词: 猪流行性腹泻病毒, 流行毒株, 分离鉴定, 攻毒模型, 遗传进化树

Abstract: Porcine epidemic diarrhea (PED) is an acute contact intestinal infectious disease in pigs caused by the porcine epidemic diarrhea virus (PEDV). In this study, a PEDV strain was isolated and named PEDV-20200946. S protein gene was sequenced and analyzed, and the phylogenetic tree showed that the isolated strain belonged to G2 genotype. The virus titer of this strain was 106.1TCID50/mL determined by Reed-Muench method. Subsequently, the pathogenicity of the isolated strains on piglets was further explored through animal challenge. A total of twelve 20-day-old piglets were divided into three groups on average, and they were named oral group, oral and intramuscular injection group, and control group, respectively. After the viral infection, clinical symptoms were observed, and the feces of each group and the intestines of the affected pigs were collected 72 h later for RT-PCR detection and immunohistochemical analysis. The results showed that piglets in the oral and intramuscular injection group showed the fastest diarrhea occurrence, followed by the oral group, and the control did not have any clinical symptoms. Autopsy examination showed that the intestinal walls of diseased piglets got thinned, their intestines were swollen, and contained a large amount of yellow liquid. Immunohistochemical tests showed that a large number of PEDV positive signals could be detected in the small intestine of piglets in the challenge groups, while the control group showed normal. The piglet challenge model of PEDV-20200946 strain was established, which laid a foundation for in-depth study of the molecular characteristics of PEDV epidemic strains and the development of attenuated vaccines.

Key words: porcine epidemic diarrhea virus, epidemic strain, isolation and identification, challenge model, phylogenetic tree

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