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

Abstract

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

CLC Number:

S855.3

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.

Figures/Tables 12

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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

Isolation， Identification and Genetic Evolution Analysis of PEDV Epidemic Strains in Heilongjiang Province

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