猪流行性腹泻病毒JX16株的分离鉴定及体外传代对其毒力的影响

doi:10.16431/j.cnki.1671-7236.2019.06.026

《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (6): 1783-1791.doi: 10.16431/j.cnki.1671-7236.2019.06.026

猪流行性腹泻病毒JX16株的分离鉴定及体外传代对其毒力的影响

唐融志^1,2, 陈智^2,3, 焦安琪^1,2, 赵鲁^2,4, 于江^2,3, 张玉玉^2,3, 孙文博^2,3, 张树金², 曾昊², 彭军⁴, 吴家强^1,2,3,4

1. 青岛农业大学动物医学院, 青岛 266109;
2. 山东省畜禽疫病防治与繁育重点实验室, 济南 250100;
3. 山东省农业科学院畜牧兽医研究所, 济南 250100;
4. 山东农业大学动物科技学院, 泰安 271018

收稿日期:2018-12-19 出版日期:2019-06-20 发布日期:2019-06-19
通讯作者: 彭军, 吴家强 E-mail:jpeng@sdau.edu.cn;wujiaqiang2000@sina.com
作者简介:唐融志(1993-),男,山东平度人,硕士生,研究方向:动物病毒学与免疫学,E-mail:729390096@qq.com;陈智(1982-),男,山东济南人,博士,研究方向:动物病毒学与免疫学,E-mail:charleschenzhi@163.com
基金资助:
国家重点研发计划项目（2017YFD0500602）；泰山学者工程经费（ts201511069）；山东省现代农业产业技术体系（SDAIT-08-06）；山东省农业科学院科技创新工程（CXGC2016B14、CXGC2018E11、CXGC2016A10）；山东省农业重大应用技术创新项目；青年科学基金项目（31602076）

Isolation and Identification of Porcine Epidemic Diarrhea Virus JX16 Strain and Effect of Passage Culture in vitro on Its Virulence

TANG Rongzhi^1,2, CHEN Zhi^2,3, JIAO Anqi^1,2, ZHAO Lu^2,4, YU Jiang^2,3, ZHANG Yuyu^2,3, SUN Wenbo^2,3, ZHANG Shujin², ZENG Hao², PENG Jun⁴, WU Jiaqiang^1,2,3,4

1. College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China;
2. Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan 250100, China;
3. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China;
4. College of Animal Science and Technology, Shandong Agricultural University, Tai'an 271018, China

Received:2018-12-19 Online:2019-06-20 Published:2019-06-19

摘要/Abstract

摘要：

2010年中国新出现了以新生仔猪高发病率、高死亡率为主要特征的变异株。为了明确山东地区猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）变异株特征，本试验利用Vero细胞对莒县某猪场的腹泻病料进行了病毒分离和体外传代培养。通过光学显微镜和细胞超薄切片观察、RT-PCR检测、间接免疫荧光试验、S基因序列进化分析、滴度测定和致病力试验进行病毒序列及特征分析，并探究接毒后细胞脱落时间的变化。光学显微镜观察发现，该病毒感染Vero细胞后能引起典型的合胞体病变；细胞超薄切片观察发现，病毒粒子多呈致密核芯，能引起宿主细胞线粒体肿胀，溶解；RT-PCR检测和间接免疫荧光试验结果证实其为PEDV，最终通过测序证实该毒株为变异毒株。体外传代培养至100代过程中，随着病毒代次升高，其对细胞适应性不断增强。接毒后细胞脱落时间由48 h逐渐缩短至17 h，病毒滴度由6代时的10^5.3TCID₅₀/mL逐渐升高至100代的10^7.5TCID₅₀/mL。致病力试验结果显示，病毒毒力随代次升高逐渐下降，40代时对仔猪已无致病力。本试验结果为PEDV突变株致病基因的确定及疫苗的研发奠定了基础。

关键词: 猪流行性腹泻病毒(PEDV); 传代培养; 致病力

Abstract:

In order to clarify the characteristics of the porcine epidemic diarrhea virus (PEDV) mutant strain in Shandong,Vero cells were used for virus isolation and in vitro subculture of diarrhea materials from a pig farm in Juxian.The virus sequence and characteristic analysis were performed by optical microscopy and ultrathin section observation,RT-PCR detection,indirect immunofluorescence assay,S gene sequence evolution analysis,titer determination and pathogenicity test,and explored the change of cell shedding time after poisoning.Optical microscopy showed that the virus infected with Vero cells could cause typical syncytium lesions.Ultrathin sections of the cells showed that the virions were mostly dense cores,which caused the mitochondria of the host cells to swell and dissolve.The results of RT-PCR and indirect immunofluorescence assay confirmed that the isolated virus of JX16 belonged to PEDV.The strain was finally confirmed to be a variant strain by sequencing. In vitro subculture to 100 generations,as the virus generation increased,its adaptability to cells continued to increase.The cell shedding time was gradually shortened from 48 to 17 h after virus infected.The virus titer was gradually increased from 10^5.3TCID₅₀/mL at the 6th generation to 10^7.5TCID₅₀/mL at the 100th generation.The virulence test showed that the virulence of the virus gradually decreased with the increase of the generation,and the 40th generation had no pathogenicity to the piglets.This article built the foundation for the identification of the pathogenic genes and the development of vaccines of PEDV mutants.

Key words: porcine epidemic diarrhea virus(PEDV); passage culture; pathogenicity

中图分类号:

S852.6

唐融志, 陈智, 焦安琪, 赵鲁, 于江, 张玉玉, 孙文博, 张树金, 曾昊, 彭军, 吴家强. 猪流行性腹泻病毒JX16株的分离鉴定及体外传代对其毒力的影响[J]. 《中国畜牧兽医》, 2019, 46(6): 1783-1791.

TANG Rongzhi, CHEN Zhi, JIAO Anqi, ZHAO Lu, YU Jiang, ZHANG Yuyu, SUN Wenbo, ZHANG Shujin, ZENG Hao, PENG Jun, WU Jiaqiang. Isolation and Identification of Porcine Epidemic Diarrhea Virus JX16 Strain and Effect of Passage Culture in vitro on Its Virulence[J]. , 2019, 46(6): 1783-1791.

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猪流行性腹泻病毒JX16株的分离鉴定及体外传代对其毒力的影响

Isolation and Identification of Porcine Epidemic Diarrhea Virus JX16 Strain and Effect of Passage Culture in vitro on Its Virulence

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