四川省猪流行性腹泻病毒SNJ-P株的分离鉴定与遗传进化分析

Isolation,Identification and Genetic Evolution Analysis of Porcine Epidemic Diarrhea Virus SNJ-P Strain in Sichuan Province

【目的】探究四川省猪流行性腹泻病毒(PEDV)毒株流行变异情况,并分析疫苗免疫效果不佳的原因。【方法】从四川某猪场采集仔猪肠道样本进行PCR检测、病毒纯化、病毒滴度测定以及病毒感染等试验;测定分离株全基因组序列,将分离株S基因序列与其他地区毒株及疫苗株进行比对并构建进化树;比较分离株与经典疫苗株CV777 S蛋白氨基酸位点变异情况。【结果】成功分离获得1株PEDV毒株,并命名为PEDV SNJ-P,该毒株病毒滴度为1×107.5/100μL。动物感染试验表明:分离株能引起仔猪出现腹泻和脱水等症状并导致死亡。全基因组测序及遗传进化分析表明:PEDV SNJ-P株全基因共28003 bp,基因型为S non-INDEL型;与同型的中国分离株PEDV-WS、CH/JLDH/2016和CH/HNLH/2015等毒株亲缘关系较近,与同型的疫苗株亲缘关系相对较远,与经典疫苗株亲缘关系较远。与经典疫苗株CV777相比,SNJ-P株的S蛋白存在多处氨基酸突变、缺失和插入现象。【结论】由于毒株出现变异,SNJ-P株与疫苗株亲缘关系较远,当前所用疫苗无法提供有效保护。本研究结果以期为国内PEDV毒株的研究及疫苗开发提供借鉴。

[Purpose]To investigate the epidemic variation of porcine epidemic diarrhea virus(PEDV)strains in Sichuan Province,and to analyze the causes of poor vaccination effect.[Methods]Piglet intestinal samples were collected from a pig farm in Sichuan Province for PCR detection,virus purification,determination of virus titer and virus infection experiments.Whole genome sequencing of isolated strains was determined.The S gene sequence of the isolated strain was compared with the strains from other regions and vaccine strains,and the phylogenetic tree was established.The amino acid site variation of S protein between the isolated strain and the classical vaccine strain CV777 was compared.[Results]A PEDV strain was successfully isolated and named as PEDV SNJ-P.The de-termination of virus titer was 1×107.5/100μL.Animal infection experiments showed that the isolated strain could cause diarrhea,dehydration and other symptoms and lead to death in piglets.Genome sequ-encing and phylogenetic tree analysis showed that the whole gene of PEDV SNJ-P strain was 28003 bp,and the genotype of the strain was S non-INDEL type.The strains were closely related to the strains of PEDV-WS,CH/JLDH/2016 and CH/HNLH/2015 isolated from China,and were relatively distant with the same type vaccine strain,and were far from the classical vaccine strain.Compared with the classical vaccine strain CV777,the S protein of SNJ-P strain had multiple amino acid mutations,dele-tions and insertions.[Conclusion]Due to the continuous variation of strains,SNJ-P strain is far from the vaccine strain,and the current vaccines cannot provide effective protection.The results of this study are expected to provide reference for the study of PEDV strains and vaccine development in China.