猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析

doi:10.16431/j.cnki.1671-7236.2018.01.023

《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (1): 178-188.doi: 10.16431/j.cnki.1671-7236.2018.01.023

猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析

秦毅斌, 卢冰霞, 何颖, 段群棚, 李斌, 陈忠伟, 梁家幸, 周英宁, 苏乾莲, 蒋冬福, 赵武

广西兽医研究所, 广西兽医生物技术重点实验室, 南宁 530001

收稿日期:2017-06-09 出版日期:2018-01-20 发布日期:2018-01-20
通讯作者: 赵武 E-mail:zhaowu168866@163.com
作者简介:秦毅斌(1983-),男,广西临桂人,硕士,助理研究员,研究方向:动物传染病病原与分子生物学,E-mail:qinyibin5188@163.com
基金资助:
广西水产畜牧科技项目（桂渔牧科（201633041、201633044、201633034））；广西自然科学基金项目（2017GXNSFBA198092）；广西基本科研业务费专项（桂科专项17-2）；广西兽医生物技术重点实验室开放基金课题（16-380-45-B-3）；广西南宁市西乡塘区科技项目（201710317）

Isolation, Identification and Complete Genome Sequence Analysis of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A

QIN Yibin, LU Bingxia, HE Ying, DUAN Qunpeng, LI Bin, CHEN Zhongwei, LIANG Jiaxing, ZHOU Yingning, SU Qianlian, JIANG Dongfu, ZHAO Wu

Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China

Received:2017-06-09 Online:2018-01-20 Published:2018-01-20

摘要/Abstract

摘要：

本研究旨在获得可在细胞培养中稳定、有效生长增殖的猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）分离毒株，并对其全基因组序列进行测定分析。应用Vero细胞从广西腹泻仔猪肠道内容物中进行病毒分离，通过细胞病变和RT-PCR对细胞培养物进行鉴定，应用下一代测序技术对分离毒株全基因组序列进行测定。结果显示，成功分离到1株PEDV，命名为CH/GX/2015/750A。该毒株可稳定有效地在Vero细胞生长增殖，并引起典型的细胞病变；已在Vero细胞连续传代25代，病毒滴度随着传代次数的增加逐渐提高并稳定在10^7.50TCID₅₀/mL。该毒株全基因组序列长28 038 bp；与22个参考毒株的全基因序列比对显示，核苷酸同源性为96.8%~99.8%，其中与YC2014株同源性最高，为99.8%。全基因组和S基因系统进化分析显示，PEDV CH/GX/2015/750A分离毒株属于Ⅱa亚群，与YC2014、PEDV-WS等变异毒株亲缘关系密切。结果表明，本研究分离获得的CH/GX/2015/750A毒株是PEDV地方流行变异毒株。

关键词: 猪流行性腹泻病毒(PEDV); 分离鉴定; 全基因; 序列分析

Abstract:

In order to obtain a porcine epidemic diarrhea virus (PEDV) isolate that could grow and propagate efficiently in cell culture, and to know the genome sequence of the isolate, virus isolation was conducted on Vero cells through inoculating the intestinal content of piglets with watery diarrhea from swine farms in Guangxi. The strain was identified by cytopathic effects (CPE) and RT-PCR. The complete genome sequence of the isolate was determined by the next generation sequencing technology. One PEDV strain named as CH/GX/2015/750A was successfully isolated from the intestinal content of a piglet with watery diarrhea. The isolate could grow efficiently in cell culture, it had been serially propagated for over 25 passages and typical CPE was observed. The infectious titer of the virus gradually increased and kept at 10^7.50 TCID₅₀/mL. The complete genome sequence of CH/GX/2015/750A was 28 038 bp in length. The nucleotide homologies with 22 reference strains were 96.8% to 99.8%, and with 99.8% nucleotide homology to YC2014 PEDV strain. The whole genome and S gene phylogenetic analysis revealed that PEDV CH/GX/2015/750A isolate belonged to Ⅱa subgroup, and closely related with YC2014 and PEDV-WS mutant strains. The results showed that the PEDV strain obtained in the present study was a variant strain.

Key words: porcine epidemic diarrhea virus (PEDV); isolation and identification; complete genome; sequence analysis

中图分类号:

S852.65

秦毅斌, 卢冰霞, 何颖, 段群棚, 李斌, 陈忠伟, 梁家幸, 周英宁, 苏乾莲, 蒋冬福, 赵武. 猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析[J]. 《中国畜牧兽医》, 2018, 45(1): 178-188.

QIN Yibin, LU Bingxia, HE Ying, DUAN Qunpeng, LI Bin, CHEN Zhongwei, LIANG Jiaxing, ZHOU Yingning, SU Qianlian, JIANG Dongfu, ZHAO Wu. Isolation, Identification and Complete Genome Sequence Analysis of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A[J]. , 2018, 45(1): 178-188.

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猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析

Isolation, Identification and Complete Genome Sequence Analysis of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A

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