猪流行性腹泻病毒变异株GDqy2017的分离及S和ORF3基因序列分析  

Isolation of Porcine Epidemic Diarrhea Virus Strain GDqy2017 and Sequence Analysis of Its S and ORF3 Genes

作　　者：楚品品 蒋智勇 林德锐 徐志宏 李艳 勾红潮 卞志标 李春玲 蔡汝健 CHU Pin-pin;JIANG Zhi-yong;LIN De-rui;XU Zhi-hong;LI Yan;GOU Hong-chao;Bian Zhi-biao;LI Chun-ling;CAI Ru-jian(Institute of Animal Heath,Guangdong Academy of Agricultural Sciences,Key Laboratory of Livestock Disease Prevention of Guangdong Province,Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture,Guangzhou,Guangdong,510640,China;Guangdong Winsun Bio-pharmaceutical Co.,Ltd.,Guangzhou,Guangdong,511356,China)

机构地区：[1]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业部兽用药物与诊断技术广东科学观测实验站,广东广州510640 [2]广东永顺生物制药股份有限公司,广东广州511356

出　　处：《动物医学进展》2018年第9期10-17,共8页Progress In Veterinary Medicine

基　　金："十三五"国家重点研发计划项目(2016YFD0500709);产学研协同创新民生科技重大专项(201604020138);广东省科技基础条件建设领域项目(2017A030303043);广州市科技计划项目(201508020055)

摘　　要：2017年广东某猪场送检腹泻死亡仔猪小肠样本,经实验室检测、细胞分离及RT-PCR鉴定,确定分离到1株猪流行性腹泻病毒,命名为GDqy2017,克隆并测定其S基因与ORF3基因序列,并对序列进行分析。结果显示,该分离株对Vero细胞高度适应,第1代即出现细胞病变(CPE),第4代起1d内即出现CPE,且CPE时间和形态稳定。其S基因长4 158nt,与经典毒株CV777、中国疫苗株CV777及韩国疫苗株attenuated DR13核苷酸同源性为94.2%、94.1%、94.2%,氨基酸同源性为93.4%、92.7%、93.0%,存在2处插入、2处缺失及98处突变(与经典毒株CV777相比),属于不同进化群,与国内2010年后参考株(除CHXBC-01-2015、CH/GDQY/2011)在同一进化群,S蛋白的主要抗原表位存在突变,其中有1处新突变出现(640位F→S),其ORF3全长675nt,不存在疫苗株中的大片段缺失及表达截短现像,但存在突变,进化树分析显示与经典毒株CV777、疫苗株属于不同进化群,亲缘关系较远。研究结果为后续PEDV分子生物学分析、流行病学调查及研制更为有效的疫苗提供基础数据。A virus strain was isolated from the small intestine samples of dead piglets with diarrhea from a pig farm in Guangdong province in 2017.The strain was determined to be PEDV and was named GDqy2017 by laboratory detection, Vero cell isolation and RT-PCR identification. The S gene and ORF3 gene of GDqy2017 were amplified, cloned and sequenced, subsequently, sequence analysis was performed. The re- sults showed that the isolate was sensitive to Vero cells,owing to the CPE could be observed from the first generation and occurred within first day from the fourth passage, and the time and morphology were sta- ble.The S gene was 4 158 nucleotides in length.Identity analysis and phylogenetic analysis based on the S gene revealed that GDqy2017 shared nucleotide sequence and amino acid sequence identities of 93.4%, 92.7%,93.0% and 93.4%,92.7%,93.0% with the classic strain CV777,the vaccine strain CV777 used in China and the vaccine strain attenuated DR13 used in Korea,had some insertions（2）,deletions（2） and mu- tations（98） （compared with the classic strain CV777）,and belonged to different cluster with above strains. However,it shared high identities and belonged to the same cluster with other Chinese reference strains since 2010（except CH-XBC-01-2015,CH/GDQY/2011）.Some mutations were found in the major epitopes of S protein,and particularly occurred a novel mutation at position 640（F→S）.The ORF3 was 675 nt,had some mutations,but no large fragment deletion and reading frame-shift occurred in the vaccine strain.Fur- thermore, phylogenetic analysis revealed that the relationship between GDqy2017 and the classic strain CV777,the vaccine strains belonged to different cluster and was distant.This study provided the basis for the following PEDV molecular biology analysis, epidemiological investigation and development for the more effective vaccines.

关 键 词：猪流行性腹泻病毒 分离 S基因 ORF3基因 序列分析 

分 类 号：S852.659.6[农业科学—基础兽医学] Q786[农业科学—兽医学]