猪流行性腹泻病毒HB/HEBEU/2020株全基因组遗传变异与重组分析

doi:10.16431/j.cnki.1671-7236.2022.08.030

摘要/Abstract

摘要： 【目的】了解猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的基因组特征及变异规律。【方法】以3只发病仔猪小肠病料作为模板,采用RT-PCR技术检测病原。以检测阳性的肝脏组织RNA为模板,进行RT-PCR,分段扩增PEDV全基因组,使用DNAStar软件对PEDV基因组测序结果进行剪辑、拼接和相似性分析,利用Mega X 10.0.5软件对PEDV基因组和S基因进行遗传进化分析;利用RDP4软件对PEDV基因组进行重组分析。【结果】PCR检测结果表明,3只病仔猪的小肠组织均检测到PEDV阳性。采用RT-PCR分段扩增成功获得1株PEDV全基因组序列,命名为HB/HEBEU/2020株,基因组大小为28 038 bp,含有7个开放阅读框,从5'到3'依次为复制酶1a基因、复制酶1b基因、S基因、ORF3基因、E基因、M基因和N基因。相似性分析结果显示,HB/HEBEU/2020与SNJ-P、USA/Colorado/2013和HB2018等变异毒株全基因组和S基因的相似性均较高,分别为97.8%~99.3%和95.7%~98.8%,在所有市售疫苗株中,与变异型疫苗株AJ1102毒株全基因组和S基因的相似性分别为98.3%和97.3%;遗传进化分析结果显示,21株PEDV可划分为G1群和G2群2个分支,G1群进一步分化为G1a亚群和G1b亚群,G2群进一步分化为G2a亚群和G2b亚群,HB/HEBEU/2020属于G2a亚群。在G2群内,所有流行株均为2010年后出现,HB/HEBEU/2020与市售疫苗株AJ1102遗传距离较近,其次是LW/L,与CV777和attenuated CV777株遗传距离较远;经重组分析,HB/HEBEU/2020基因组可能为重组毒株,存在3个潜在的重组事件,重组事件1~3的断点分别为15 918—22 119、100—734和2 214—2 729 bp,其中重组事件1和2发生概率较大。【结论】HB/HEBEU/2020为1株变异型PEDV,与以CV777为代表的经典毒株亲缘关系较远,与2010年后中国发生的变异型毒株亲缘性较近,并且存在潜在重组事件。本研究结果可为调研中国当前PEDV进化和变异提供重要资料,为遏制PEDV蔓延与进化、制定合理防控方案提供理论和现实依据。

关键词: 猪流行性腹泻病毒; 全基因组; 遗传变异; 重组分析

Abstract: 【Objective】 This study was aimed to investigate the genome characteristics and variation of Porcine epidemic diarrhea virus (PEDV).【Method】 The small intestine tissues of 3 diarrhea piglets were used as templates,the pathogen was detected by RT-PCR.Using the RNA of the positive intestine tissue,RT-PCR technology was used to amplify the whole genome of PEDV. DNAStar software was used to edit and splice of the amplified sequence and perform similarity alignment.Mega X 10.0.5 software was used to construct phylogenetic trees of the whole genome and S gene of PEDV strain. RDP4 software was used to analyze the potential recombination events of PEDV strain.【Result】 PCR results showed that PEDV was detected in all the three sick piglets.The whole genome of PEDV strain was successfully obtained by RT-PCR fragmented amplification,named HB/HEBEU/2020.The virus genome size was 28 038 bp and it contained 7 open reading frames (ORF) which encoded replicase polyprotein 1a,replicase polyprotein 1b,spike (S),ORF3,envelope (E),membrane (M),and nucleoprotein (N) gene in 5' to 3' orientation.The similarity alignment of the complete genome as well as S gene between HB/HEBEU/2020 and PEDV variant strains,such as SNJ-P,USA/Colorado/2013 and HB2018 were 97.8%-99.3% and 95.7%-98.8%,respectively.Among all the commercially available vaccines,AJ1102 shared the highest similarity with HB/HEBEU/2020 strain,the nucleotide similarity between the complete genome as well as S gene of HB/HEBEU/2020 and AJ1102 were 98.3% and 97.3%,respectively.Genome-wide evolution results showed that 21 PEDV strains were classified into G1 genogroup and G2 genogroup,G1 genogroup was divided into G1a subgroup and G1b subgroup,G2 genogroup was divided into G2a subgroup and G2b subgroup,HB/HEBEU/2020 belonged to G2a subgroup.All the PEDV strains that belonged to G2 genogroup in the study were emerged after 2010.Among all the commercially available vaccines,AJ1102 had the nearest genetic relation with HB/HEBEU/2020,LW/L was second,the genetic distances between HB/HEBEU/2020 and CV777 as well as attenuated CV777 were far.3 possible recombination events were identified in the genome of HB/HEBEU/2020 strain.The recombinant regions of the three possible recombination events were 15 918-22 119,100-734 and 2 214-2 729 bp,respectively.The first two recombination events had high probability of occurrence.【Conclusion】 HB/HEBEU/2020 was a variant recombinant PEDV,which harbored distant phylogenetic relationships with classic PEDV strain CV777,but was closed related to the variant strains occurred in China after 2010.The results would provide theoretical and practical reference for postponing natural selection and evolution as well as formulating the vaccination procedure of PEDV.

Key words: Porcine epidemic diarrhea virus; whole genome; genetic variation; recombination analysis

中图分类号:

S855.3

翟新国, 郑培培, 苏金辉, 李庆阳, 焦贺静, 张若冰, 李朋辉. 猪流行性腹泻病毒HB/HEBEU/2020株全基因组遗传变异与重组分析[J]. 中国畜牧兽医, 2022, 49(8): 3140-3150.

ZHAI Xinguo, ZHENG Peipei, SU Jinhui, LI Qingyang, JIAO Hejing, ZHANG Ruobing, LI Penghui. Genetic Variation and Recombination Analysis of Whole Genome of Porcine Epidemic Diarrhea Virus HB/HEBEU/2020 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3140-3150.

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