猪流行性腹泻病毒SD201604株的分离鉴定及致病性研究

doi:10.16431/j.cnki.1671-7236.2018.11.030

摘要/Abstract

摘要：

本研究旨在获得猪流行性腹泻病毒（PEDV）分离株，并对其致病性进行研究。应用Vero细胞从山东某猪场腹泻病料中进行病毒分离，通过细胞病变、免疫荧光试验、电镜观察和RT-RCR进行鉴定，并对分离株的S基因序列进行分析；应用10^3.5 TCID₅₀/mL分离株口服接种3日龄仔猪并观察临床症状和病理变化；用不同滴度的分离株分别口服接种3日龄仔猪（3 mL/只），统计各组仔猪的死亡率，确定最小致死量。结果显示，成功分离到1株PEDV，命名为PEDV SD201604株。该分离毒株能在Vero细胞上增殖，产生细胞病变，传代至F6代时病毒滴度可达10^3.5 TCID₅₀/mL，免疫荧光试验和RT-RCR检测均为PEDV阳性。电镜观察可见直径大小约为100 nm的病毒粒子，有明显的囊膜和纤突，具有PEDV病毒粒子典型的形态特征，确定分离株为PEDV。S基因序列分析显示，分离株为国内流行的PEDV变异株。动物回归试验结果显示，口服感染该分离株的5头3日龄仔猪全部出现典型的PED临床症状和病理变化，其中3头死亡。最小致死量试验结果表明，口服感染3 mL病毒含量为10^4.5 TCID₅₀/mL的分离株可使仔猪全部死亡。本试验结果可为PEDV的分离鉴定及生物学研究提供参考与借鉴。

关键词: 猪流行性腹泻病毒(PEDV); 分离; 鉴定; 致病性

Abstract:

In order to obtain porcine epidemic diarrhea virus (PEDV) isolate and study its pathogenicity,virus isolation was conducted in Vero cells through inoculating diarrhea sample collected from a pig farm in Shandong province.The strain was identified by cytopathogenic effect (CPE),IFA,electron microscope observation and RT-PCR methods,and the S gene sequence of virus was analyzed.The 3-day-old piglets were inoculated orally with 10^3.5 TCID₅₀/mL virus and observed for clinical symptoms and pathological changes.The 3-day-old piglets were inoculated orally with 3 mL different titers of virus.The mortality of piglets in each group was calculated to determine the minimum lethal dose.The results indicated that a strain of PEDV was successfully isolated in the cell culture and designated PEDV SD201604.The strain replicated well in Vero cells and could cause obvious CPE.The infectious titer of the 6th passage of PEDV SD201604 was 10^3.5 TCID₅₀/mL.The strain was detected to be PEDV positive by IFA and RT-PCR methods.Electron microscope observation showed that the size of the virus particle was about 100 nm in diameter,with an obvious envelope and spikes,which was the typical morphological feature of PEDV.Then the virus was identified as a prevalent PEDV variant of China through the analysis of S gene sequence.In the animal pathogenicity experiment,five 3-day-old piglets inoculated orally with 10^3.5 TCID₅₀/mL of PEDV SD201604 strain all showed typical PEDV clinical symptoms and pathological lesions,and 3 of them died in the test.The minimum lethal dose (MLD) experiment showed that piglets all died when they were orally inoculated with 3 mL of virus with a virus content of 10^4.5 TCID₅₀/mL.Together,the experimental results provided references for PEDV isolation,identification and biological characteristics research.

Key words: porcine epidemic diarrhea virus (PEDV); isolation; identification; pathogenicity

中图分类号:

S852.65

吴白, 苏玮玮, 鞠德财, 夏铭崎, 张树成, 王炜. 猪流行性腹泻病毒SD201604株的分离鉴定及致病性研究[J]. 《中国畜牧兽医》, 2018, 45(11): 3229-3236.

WU Bai, SU Weiwei, JU Decai, XIA Mingqi, ZHANG Shucheng, WANG Wei. Isolation, Identification and Pathogenicity Evaluation of Porcine Epidemic Diarrhea Virus SD201604 Strain[J]. , 2018, 45(11): 3229-3236.

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