猪轮状病毒的分离鉴定及其致病性分析

doi:10.11843/j.issn.0366-6964.2025.05.024

摘要/Abstract

摘要：

旨在分离与鉴定猪轮状病毒(PoRV)的流行毒株，对其基因组序列的遗传变异以及病毒致病性进行分析。选择PoRV阳性的腹泻仔猪小肠样品，处理后接种于MA104细胞进行分离培养，通过病毒核酸检测、血清学检测和形态学观察对细胞培养物进行鉴定。利用RT-PCR和测序获得全基因组序列，比较分析其遗传进化关系，并对该分离株的致病性进行评估。结果表明，成功分离出了1株可在体外培养的PoRV毒株，将其命名为HLJ/2021，其基因型为G5-P[7]-I5-R1-C1-M1-A1-N1-T1-E1-H1型。致病性试验结果表明，该分离株能够导致仔猪出现水样腹泻、消瘦等症状，在感染后60~174 h，可在仔猪粪便中检测到病毒；剖检观察到感染仔猪肠壁变薄，内容物呈水样，病理组织学检测可见感染仔猪回肠肠绒毛高度萎缩；肠组织中病毒载量检测结果显示，回肠、盲肠、结肠和直肠中均可检测到病毒，其中回肠中病毒载量最高。综上所述，本研究成功分离1株G5P[7]型PoRV毒株，其对仔猪具有致病性。该结果为了解PoRV的流行情况提供了重要信息。

关键词: 猪轮状病毒, 分离鉴定, 遗传进化分析, 致病性分析

Abstract:

This study aims to isolate and identify a strain of PoRV, and analyze the genetic variation of its genome sequence and the pathogenicity of the virus. Small intestinal samples of diarrheal piglets were selected and processed, then inoculated into MA104 cells for isolation and culture. The cell cultures were identified through viral nucleic acid test, serological detection and morphological observation. RT-PCR and sequencing were used to obtain the whole genome sequence of the isolate, genetic evolutionary relationship and pathogenicity were evaluated. The results showed that a PoRV strain were successfully isolated and can be cultured in vitro, named HLJ/2021, and its genotype is G5-P[7]-I5-R1-C1-M1-A1-N1-T1-E1-H1 type. Pathogenicity experimental results showed that the isolate can cause watery diarrhea, emaciation and other symptoms in piglets. Virus shedding can be detected in piglet feces 60~174 h after infection. Necropsy revealed that the intestinal wall of the infected piglets became thinner and the contents were watery. Histopathological examination showed that the intestinal villi of the ileum of the infected piglets were highly atrophied. Viral load detection results in intestinal tissues showed that virus can be detected in the ileum, cecum, colon and rectum, with the highest viral load in the ileum. In summary, this study successfully isolated a G5P[7] type PoRV strain, which is pathogenic to piglets. The results provide important information for understanding the prevalence of PoRV.

Key words: porcine rotavirus, isolation and identification, genetic evolution analysis, pathogenicity analysis

中图分类号:

S852.659.4

王亚楠, 郭雅茹, 姜艳平, 崔文, 李佳璇, 李一经, 王丽. 猪轮状病毒的分离鉴定及其致病性分析[J]. 畜牧兽医学报, 2025, 56(5): 2259-2269.

WANG Yanan, GUO Yaru, JIANG Yanping, CUI Wen, LI Jiaxuan, LI Yijing, WANG Li. Isolation, Identification and Pathogenicity Analysis of Porcine Rotavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2259-2269.

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序号 Number	引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增长度/bp Fragment length
1	PoRV-VP1-F1 PoRV-VP1-R1	ATGGGGAAGTACAATCTAATCTT CTCGTTCTGACGGAAATAATC	2 012
2	PoRV-VP1-F2 PoRV-VP1-R2	AGATTATTTCCGTCAGAACG TTTGCGTATATATGTGATCGTAG	941
3	PoRV-VP2-F1 PoRV-VP2-R1	ACGCGTCGACCATGGCGTACA GCATAGTTGGAAACTGTTGTC	1 574
4	PoRV-VP2-F2 PoRV-VP2-R2	GGATGCATTATAGAAATGGTG CCGCTCGAGAATTACAGTTCG	1 112
5	PoRV-VP3-F PoRV-VP3-R	GGCTWTTAAAGCARTATTAGTAGTG GGT CAC ATC ATG ACT AGT GTG	2 508
6	PoRV-VP4-F PoRV-VP4-R	ATGGCTTCGCTCATTTATAGACAACTA ACTACTTACAGTCTACATTGCATGATT	2 337
7	PoRV-VP6-F PoRV-VP6-R	GGCTTTWAAACGAAGTCTTC GGTCACATAATCTCACT	1 356
8	PoRV-VP7-F PoRV-VP7-R	GCCTTTAAAAGCGAGAATT GGTCACATCATACAACTCTA	1 062
9	PoRV-NSP1-F PoRV-NSP1-R	CCATGGCTACTTTTAAGGATGCT CTAGGCGCTACTCTAGTGCAGGG	1 517
10	PoRV-NSP2-F PoRV-NSP2-R	GGCTTTTAAAGCGTCTCAG GGTCACATAAGCGCTTTC	1 059
11	PoRV-NSP3-F PoRV-NSP3-R	GGCTTTTAATGCTTTTCAGTG GGTCACATAACGCCCCTATAG	1 073
12	PoRV-NSP4-F PoRV-NSP4-R	GGWYACRYTAAGACCRTTCC GGCTTTTAAAGCGCTACAG	747
13	PoRV-NSP5-F PoRV-NSP5-R	GGCTTTTAAAGCGCTACAG GGTCACAAAACGGGAGT	664

基因 Gene	长度/bp Length	基因型 Genotype	参考序列名称 Reference sequence name	参考序列序列号 Reference strains accession numbers	同源性/% Homology
VP1	3 295	R1	RVA/Pig-tc/USA/OSU/1975/G5P9[7]	GU199514.1	99.76
VP2	2 686	C1	RVA/Pig-tc/USA/LS00006_OSU/1975/G5P[X]	KR052758.1	99.85
VP3	2 563	M1	RVA/Pig-tc/USA/LS00006_OSU/1975/G5P[X]	KR052759.1	99.88
VP4	2 337	P7	RVA/Pig-tc/ESP/OSU-C5111/2010/G5P[7]	KJ450845.1	99.75
VP6	1 356	I5	RVA/Pig-tc/ESP/OSU-C5111/2010/G5P[7]	KJ450847.1	99.85
VP7	1 062	G5	RVA/porcine/CH/2020/OSU/serotype 5	OR091159.1	99.81
NSP1	1 569	A1	RVA/Pig-tc/USA/LS00006_OSU/1975/G5P[X]	KR052752.1	99.61
NSP2	1 059	N1	RV/KOR/C-1	PP100168.1	99.72
NSP3	1 075	T1	RVA/KOR/OSU/G5P7	JX971586.1	100.00
NSP4	744	E1	RVA/KOR/174-1/G8P7	KF500216.1	99.73
NSP5	664	H1	RVA/Porcine-tc/KOR/K71/2006/G5P[7]	MF940468.1	99.55

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