我国16省份猪流行性腹泻病毒临床检测及基于S基因的遗传进化分析

Clinical detection and genetic evolution analysis of porcine epidemic diarrhea virus based on S gene in 16 provinces of China

为了解猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)在国内的流行与变异情况,本研究应用RT-PCR方法对2020—2021年采集的16省市共732份猪腹泻病料进行PEDV核酸检测,并对PEDV阳性样本进行S基因扩增、测序以及遗传进化分析。结果显示,2020—2021年采集的732份样本中489份PEDV阳性,阳性率为66.80%。获得76株PEDV S基因序列,遗传进化和核苷酸一致性分析显示71株为GⅡb型、5株为S INDEL型,毒株间核苷酸序列一致性为93.9%~100%。对贵州铜仁2020年2株和2021年3株S基因序列比对结果显示,2021年3株S基因序列一致性为100%,且与2020年的1株序列一致性为99.9%;对江苏盐城2020年2株和2021年2株S基因序列比对分析,其核苷酸一致性为97.6%~99.2%。对获得的76株S基因编码蛋白的氨基酸序列比对分析,与疫苗株CV777相比,71株GⅡb型毒株S基因序列与GII型代表株具有高度相似的遗传标记,包括在58~59位氨基酸之间存在4个氨基酸的插入,在139~140位氨基酸间存在1个氨基酸的插入;5株S基因序列除具有与S-INDEL型代表株相同的遗传标记外,在139~140位氨基酸间存在1个氨基酸的缺失,且在S蛋白N端存在多处连续突变;此外,ZJLS10-2021株、GXNN14-2021株与其他基因型相比分别在1267~1268位氨基酸之间存在1个氨基酸的插入,在379~383位氨基酸之间存在3个氨基酸的插入。研究结果表明,我国不同省市PEDVS基因具有易突变特性,同一地域主要流行毒株具有相对遗传稳定性。本研究为进一步监测和分析引起我国猪群腹泻的发生、流行、追踪和溯源及研究PEDV传播的途径和遗传变异规律提供了有力的数据支持,同时为猪腹泻病的防控和疫苗研发提供理论和技术参考。

In order to understand the prevalence and variation of porcine epidemic diarrhea virus(PEDV),RT-PCR was used,in this study,to detect PEDV in 732 pig diarrhea samples collected from 16 provinces in a period from 2020 to 2021;and PEDV positive samples were analyzed by S gene amplification,sequencing and genetic evolution.The results showed that 489 PEDV samples were positive in 732 samples collected,at a positive rate of 66.80%.PEDV S gene sequences of 76 strains were obtained and phylogenetic trees were constructed.The results showed that 71 strains belonged to GⅡb and 5 strains belonged to S INDEL.S gene sequence alignment of the 76 strains showed that the sequence identity was 93.9%-100%.The S gene sequence alignment of 2 strains in 2020 and 3 strains in 2021 in Tongren,Guizhou Province,showed that the S gene sequence identity of the 3 strains in 2021 was 100%,and the sequence identity of the 1 strain obtained in 2020 was 99.9%.The nucleotide identity of the S gene sequences of 2 strains in 2020 and 2 strains in 2021 in Yancheng,Jiangsu Province was 97.6%-99.2%.The amino acid sequence alignment analysis of the S protein encoded by the S gene of the 76 strains obtained showed that,compared with the vaccine strain CV777,the S gene sequence of 71 GⅡb strains had highly similar genetic markers with the represent⁃ative strain of GⅡ,including the presence of four amino acid insertions between amino acids at positions 58 and 59.There was an amino acid insertion between amino acids 139-140.The S gene sequences of the 5 strains had the same genetic markers as those of the representa⁃tive strain of S INDEL,but there was a deletion of one amino acid between 139-140 and several continuous mutations at the N-terminal of S protein.In addition,compared with other genotypes,ZJLS10-2021 and GXNN14-2021 had one amino acid insertion between positions 1267 and 1268,and 3 amino acid insertions between positions 379 and 383,respectively.The above results indicated that the S gene of PEDV was mutated easily in different provinces of China,and the main epidemic strains in the same region had relative genetic stability.These results were important data support for further monitoring and analyzing the occurrence and prevalence of porcine diarrhea in China,for tracing the disease,and for studying the transmission routes and genetic variation rules of PEDV.The results would also serve as theoretical and technical reference for the prevention and control of porcine diarrhea and for research on and development of vaccines.