黑龙江省猪流行性腹泻病毒流行毒株分离鉴定与遗传进化分析

doi:10.16431/j.cnki.1671-7236.2026.01.034

摘要/Abstract

摘要：

目的通过对黑龙江省猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）毒株进行分离鉴定，基于PEDV分离株S、ORF3基因序列进行遗传进化分析，丰富现有PEDV材料，为猪流行性腹泻（porcine epidemic diarrhea，PED）的防治及疫苗研发提供参考依据。方法采集养猪场疑似猪流行性腹泻病料，利用Vero-E6细胞进行适应性培养获得病毒，并通过RT-PCR和间接免疫荧光试验（IFA）对毒株进行分离鉴定。通过PCR分别扩增PEDV分离毒株的S1、S2和ORF3基因，构建重组质粒并测序鉴定，获得PEDV分离株的S、ORF3基因序列。将分离株与26株参考毒株的S和ORF3基因序列进行核苷酸序列相似性比对和遗传进化分析，确定分离毒株的所属亚群。结果在Vero-E6细胞上培养分离的PEDV毒株，感染细胞出现变形圆缩、折光性降低，细胞发生融合，后期细胞脱落出现空斑等典型细胞病变；RT-PCR和IFA检测显示，扩增均出现片段大小约600 bp的特异性条带，分离株感染细胞后均可与PEDV N蛋白单克隆抗体发生特异性反应，表明分离的3株病毒株均为PEDV，分别命名为HLJ、HLJ2020和HLJ2021株。成功构建PEDV各分离株的S1、S2和ORF3基因重组质粒并测序获得相应基因序列。核苷酸序列相似性比对与遗传进化树分析结果显示，PEDV HLJ与HLJ2020株为GⅠb亚群毒株，PEDV HLJ2021株归入GⅡc亚群。结论本研究从黑龙江地区腹泻猪小肠组织中成功分离获得3株PEDV流行毒株，其中分离毒株PEDV HLJ与PEDV HLJ2020属于GⅠb亚群， PEDV HLJ2021株属于GⅡc亚群。

关键词: 猪流行性腹泻病毒（PEDV）; 分离鉴定; S基因; ORF3基因; 遗传进化分析

Abstract:

Objective The purpose of this experiment was to isolate and identify the Porcine epidemic diarrhea virus （PEDV） strains in Heilongjiang province. Phylogenetic analysis based on the S and ORF3 gene sequences of PEDV isolates， genetic evolution analysis was conducted to enrich the existing PEDV materials， providing a reference basis for the prevention and control of porcine epidemic diarrhea （PED） and the development of vaccines. Method Collect samples from suspected swine epidemic diarrhea cases in the pig farm， and use Vero-E6 cells for adaptive cultivation to obtain the virus， and isolate and identify the virus strains through RT-PCR and indirect immunofluorescence assay （IFA）. S1， S2 and ORF3 genes of PEDV isolates were respectively amplified by PCR， and recombinant plasmids were constructed and sequenced for identification. The sequences of S and ORF3 genes of PEDV isolates were obtained. The nucleotide sequence similarities of S and ORF3 genes of the isolates were compared with those of 26 reference strains， and genetic evolutionary analysis was conducted to determine the subgroup of the isolated strains. Result PEDV isolates were cultured on Vero-E6 cells， and exhibited typical cytopathic effects， deformation and rounding， decreased refractivity， cell fusion， and followed by cell detachment and plaque formation. RT-PCR and IFA tests showed that specific bands of approximately 600 bp in size were amplified. After the isolated strains infected cells， they all reacted specifically with the PEDV N protein monoclonal antibody， indicating that the isolated strains were all PEDV， and were respectively named PEDV HLJ， HLJ2020 and HLJ2021 strains. Recombinant plasmids of S1， S2， and ORF3 genes of PEDV isolates were successfully constructed and sequenced to obtain the corresponding gene sequences. The results of nucleotide sequence similarity alignment and genetic evolutionary analysis indicated that the PEDV HLJ and HLJ2020 strains belonged to GⅠb subgroup， while PEDV HLJ2021 strain was classified into GⅡc subgroup. Conclusion In this study， 3 prevalent PEDV strains were successfully isolated from the small intestinal tissues of diarrhea pigs in Heilongjiang region. Among them， the isolated strains PEDV HLJ and PEDV HLJ2020 belonged to GⅠb subgroup， while the PEDV HLJ2021 strain belonged to GⅡc subgroup.

Key words: Porcine epidemic diarrhea virus （PEDV）; isolation and identification; S gene; ORF3 gene; genetic evolution analysis

中图分类号:

S855.3

张欣欣, 张岳, 崔安, 李金儒, 张芸菁, 何丽霞, 杨桂君, 任玉东, 李广兴. 黑龙江省猪流行性腹泻病毒流行毒株分离鉴定与遗传进化分析[J]. 中国畜牧兽医, 2026, 53(1): 378-389.

ZHANG Xinxin, ZHANG Yue, CUI An, LI Jinru, ZHANG Yunjing, HE Lixia, YANG Guijun, REN Yudong, LI Guangxing. Isolation， Identification and Genetic Evolution Analysis of PEDV Epidemic Strains in Heilongjiang Province[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(1): 378-389.

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引物 Primer	引物序列 Primer sequences （5′→3′）	产物大小 Product size/bp
PEDV-ORF3-F	GCGCATGTTTCTTGGACTTTTTC	675
PEDV-ORF3-R	TCATTCACTAATTGTAGCATACTCG	675
PEDV-S1-F	GTCAACGTAAACAAATGAGG	2 367
PEDV-S1-R	ACAGGCGTGTTGTAAAGCTG	2 367
PEDV-S2-F	AAGTCAAGATTGCACCCACG	1 782
PEDV-S2-R	CCAAGAAACATCACTGCACG	1 782

毒株 Strains	基因型 Genotype	地区 Location	年份 Year	GenBank登录号 GenBank accession No.
CV777	GⅠa	瑞士	1978	AF353511.1
attenuated DR13	GⅠb	韩国	1999	JQ023162.1
LZC	GⅠa	中国甘肃	2006	EF185992.1
Italy/7239/2009	/	意大利	2009	KR061458.1
LC	GⅡb	中国广东	2011	JX489155.1
SM98	GⅠa	韩国	2011	GU937797.1
AJ1102-2012	GⅡb	中国湖北	2012	JX188454.1
AH2012	GⅡa	中国安徽	2012	KC210145.1
GD-A	GⅡb	中国广东	2012	JX112709.1
CH/YNKM-8/2013	GⅡb	中国武汉	2013	KF761675.1
OH851	GⅡc	美国	2013	KJ399978.1
USA/Iowa/18984/2013	GⅡa	美国	2013	KF804028.1
CBR2	GⅡb	泰国	2014	KR610994.1
GER/L00719/2014	GⅡc	德国	2014	LM645058.1
ON-018	GⅡa	加拿大	2014	KM189367.2
PEDV-LS	GⅡb	中国江苏	2014	KM609211.1
SC1402	GⅠb	中国广东	2014	KP162057.1
FR/001/2014	GⅡc	法国	2015	KR011756.1
PEDV/USA/Texas134/2015	GⅡa	美国	2015	KU982966.1
PC21A	GⅡa	美国	2015	KR078299.1
ZL29	GⅡc	中国武汉	2015	KU847996.1
1842/2016 ITA	GⅡc	意大利	2016	KY111278.1
85-7-C40	GⅠa	中国江苏	2017	KY486714.1
KUPE21	/	韩国	2017	MF737355.1
PEDV-SX	GⅠb	中国山西	2017	KY420075.1
TW/Yunlin550/2018	GⅡa	中国台湾	2018	MK673545.1