2016年~2022年福建地区猪流行性腹泻病毒S基因的遗传变异分析

Analysis of genetic variation of porcine epidemic diarrhea virus S gene in Fujian Province from 2016 to 2022

为了解2016年~2022年福建地区猪流行性腹泻病毒(PEDV)流行现状及遗传变异情况,本研究对从福建地区采集的231份疑似猪流行性腹泻(PED)发病仔猪病料样品经RT-PCR进行PEDV的检测,选取不同地区22份PEDV阳性样品经PCR扩增S基因并测序,采用MegAlign分析PEDV S基因及其编码氨基酸序列的相似性,采用MEGA7.0软件构建其系统发育树,采用MegAlign分析S基因编码氨基酸序列的分子特征,分别利用RDP4、SimPlot、MEGA7.0软件进行S基因的重组分析,并利用BEAST软件进行该基因分歧时间估算。结果显示,福建地区PEDV总阳性率为51.51%(119/231),福建5个不同地区PEDV阳性率为40.00%~63.16%。从22份PEDV阳性样品中获得19条PEDV S基因序列,全长4149 bp~4161 bp,共编码1382 aa~1386 aa,19株PEDV S基因序列及其编码氨基酸序列之间相似性分别为94.4%~100%和93.8%~100%。19株PEDV S基因系统发育分析结果显示,其中18株PEDV属于GIIb亚型,1株属于GIb亚型。S蛋白氨基酸序列分析结果显示,与经典疫苗株CV777相比,18株GIIb亚型PEDV S蛋白的氨基酸序列在aa55~aa56、aa135~aa136和aa155~aa156处存在插入与缺失,具有典型的PEDV变异株的分子特征;其中14株还出现了独特的aa1193缺失。与GII型疫苗株AJ1102相比,19株PEDV S1区氨基酸突变率为60.00%~83.82%,其中18株GIIb亚型PEDV S1区氨基酸突变位点中有10个位于S蛋白重要结构域;基因重组分析结果显示,有3株PEDV S基因存在重组事件,其中FJLY01-2018株由GD-B株和CH-S株重组而来,FJLY02-2018株由CH/HNPJ/2017株和PEDV4-S-3株重组而来,FJLY03-2022株由PEDV-1931-1-Valladolid-Molpeceres株和HUA-M17株重组而来;S基因分歧时间估算结果显示,福建地区GIIa亚型、GIIb亚型、GIa亚型及GIb亚型PEDV的最早分歧时间约为1995年、2009年、2010年和2011年。上述结果表明福建地区PEDV流行率较高,田间流行株基因型具有多样性,且存在重组现象,临床上应予以高度重视。本研究为福建地区PED的流行病学调查及免疫防控提供了参考。

In order to understand the epidemic status and genetic variation of porcine epidemic diarrhea virus(PEDV)in Fujian Province from 2016 to 2022,231 samples of piglets suspected of PED disease were collected from Fujian Province.PEDV genome was detected by RT-PCR,afterwards,22 PEDV-positive samples were selected for PCR amplification and sequencing of S gene.MegAlign was used to analyze the similarity of PEDV S gene sequences and its encoded amino acid sequences.MEGA7.0 software was used to construct phylogenetic tree.MegAlign was then used for the recombination analysis,and BEAST software was used to estimate the divergence time.The results showed that the total positive rate of PEDV in Fujian was 51.51%(119/231),and the positive rates of PEDV in 5 different regions of Fujian ranged from 40.00%to 63.16%.19 PEDV S gene sequences were obtained from 22 positive samples.The total length of PEDV S gene sequences was 4149bp-4161bp,encoding a total of 1382-1386 amino acids.The similarity between the PEDV S gene sequences and their encoded amino acid sequences was 94.4%-100%and 93.8%-100%,respectively.Phylogenetic analysis of 19 PEDV S genes showed that 18 PEDV strains belonged to GIIb subtype and 1 to GIb subtype.Amino acid sequence analysis of S protein showed that compared with the classic vaccine strain CV777,18 GIIb subtype PEDV S fragments had insertion or deletion at three loci of aa55-aa56,aa135-aa136 and aa155-aa156,showing the molecular characteristics of typical PEDV variants.Compared with GII vaccine strain AJ1102,the amino acid mutation rate in S1 region of 19 PEDV strains ranged from 60.00%-83.82%,among which 10 amino acid mutation sites were located in the key domain of S protein.The results of gene recombination showed that three PEDV S genes were recombinant,among which FJLY01-2018 was originated from GD-B strain and CH-S strain,while FJLY02-2018 was recombined from CH/HNPJ/2017 strain and PEDV4-S-3 strain.FJLY03-2022 was derived from PEDV-1931-1-Valladolid-Molpeceres strain and HUA-M17 strain.The results of divergence time estimation showed that the earliest divergence time of the GIIa subtype,GIIb subtype,GIa subtype and GIb subtype PEDV in Fujian was about in 1995,2009,2010 and 2011,respectively.The above results indicated that the detection rate of PEDV in Fujian is relatively high,and the genotypes of endemic strains in the field are diverse and recombination exists,which should be paid great attention.This study provides a reference for the epidemiological investigation and immunization prevention and control of porcine epidemic diarrhea in Fujian Province.