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

Abstract

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

CLC Number:

S852.659.4

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.

Figures/Tables 14

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Table 2

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

References 28

1	FUKUSHO A , SHIMIZU Y , ITO Y . Isolation of cytopathic porcine rotavirus in cell roller culture in the presence of trypsin[J]. Arch Virol, 1981, 69 (1): 49- 60. doi: 10.1007/BF01315265
2	VIASOVA A N , AMIMO J O , SAIF L J . Porcine rotaviruses: epidemiology, immune responses and control strategies[J]. Viruses, 2017, 9 (3): 48. doi: 10.3390/v9030048
3	THEUNS S , DESMARETS L M , HEYLEN E , et al. Porcine group A rotaviruses with heterogeneous VP7 and VP4 genotype combinations can be found together with enteric bacteria on Belgian swine farms[J]. Vet Microbiol, 2014, 172 (1-2): 23- 34. doi: 10.1016/j.vetmic.2014.04.002
4	TAO R , CHANG X , ZHOU J , et al. Molecular epidemiological investigation of group A porcine rotavirus in East China[J]. Front Vet Sci, 2023, 10, 1138419. doi: 10.3389/fvets.2023.1138419
5	QIAO M , LI M , LI Y , et al. Recent molecular characterization of porcine rotaviruses detected in China and their phylogenetic relationships with human rotaviruses[J]. Viruses, 2024, 16 (3): 453. doi: 10.3390/v16030453
6	DOERKSEN T , CHRISTENSEN T , LU A , et al. Assessment of porcine Rotavirus-associated virome variations in pigs with enteric disease[J]. Vet Microbiol, 2022, 270, 109447. doi: 10.1016/j.vetmic.2022.109447
7	MATTHIJNSSENS J , CIARLET M , RAHMAN M , et al. Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments[J]. Arch Virol, 2008, 153 (8): 1621- 1629. doi: 10.1007/s00705-008-0155-1
8	NURDIN JA , KOTAKI T , KAWAGISHI T , et al. N-Glycosylation of Rotavirus NSP4 protein affects viral replication and pathogenesis[J]. J Virol, 2023, 97 (1): e0186122. doi: 10.1128/jvi.01861-22
9	ANGEL J , TANG B , FENG N , et al. Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea[J]. J Infect Dis, 1998, 177 (2): 455- 458. doi: 10.1086/517374
10	WANG J , ZHOU J , ZHU X , et al. Isolation and characterization of a G9P[23] porcine rotavirus strain AHFY2022 in China[J]. Microb Pathog, 2024, 190, 106612. doi: 10.1016/j.micpath.2024.106612
11	ZHANG F , LUO Y , LIN C , et al. Epidemiological monitoring and genetic variation analysis of pathogens associated with porcine viral diarrhea in southern China from 2021 to 2023[J]. Front Microbiol, 2024, 15, 1303915. doi: 10.3389/fmicb.2024.1303915
12	谷拉强, 陶然, 程曦, 等. 2022—2023年我国部分地区猪A群轮状病毒分子流行病学调查分析[J]. 南方农业学报, 2023, 54 (10): 3083- 3091.
	GU L Q , TAO R , CHENG X , et al. Molecular epidemiological survey of group A porcine rotavirus in selected areas of China during 2022-2023[J]. Journal of Southern Agriculture, 2023, 54 (10): 3083- 3091.
13	甸子芩, 樊茂, 蒋红君, 等. 2015-2017年云南省轮状病毒感染情况及基因分型分析[J]. 中国病原生物学杂志, 2019, 14 (01): 78-82, 87.
	DIAN Z Q , FAN M , JIANG H J , et al. Analysis of the prevalence of rotavirus infection and genotyping of rotavirus isolated in Yunnan Province from 2015 to 2017[J]. Journal of Pathogen Biology, 2019, 14 (01): 78-82, 87.
14	MEBUS C A . Reovirus and rotavirus infections[J]. Proc Annu Meet U S Anim Health Assoc, 1975 (79): 345- 349.
15	WOODE G N . Pathogenic rotaviruses isolated from pigs and calves[J]. Ciba Found Symp, 1976 (42): 251- 271.
16	黄小波, 徐璐, 曹三杰, 等. 猪轮状病毒OSU株的培养特性与致病性研究[J]. 中国人兽共患病学报, 2012, 28 (02): 120-123, 147.
	HUANG X B , XU L , CAO S J , et al. Culture characteristics and pathogenicity of porcine rotavirus OSU strain[J]. Chinese Journal of Zoonoses, 2012, 28 (02): 120-123, 147.
17	宋菲菲, 郝洪吉, 张艳红, 等. Vero细胞培养轮状病毒关键条件的优化[J]. 中国生物制品学杂志, 2014, 27 (10): 1345- 1347.
	SONG F F , HAO H J , ZHANG Y H , et al. Optimization of key condition for culture of rotavirus in Vero cells[J]. Chin J Biologicals, 2014, 27 (10): 1345- 1347.
18	MIAO Q , PAN Y , GONG L , et al. Full genome characterization of a human-porcine reassortment G12P[7] rotavirus and its pathogenicity in piglets[J]. Transbound Emerg Dis, 2022, 69 (6): 3506- 3517. doi: 10.1111/tbed.14712
19	LAGAN P , MOONEY M H , LEMON K . Genome analyses of species A rotavirus isolated from various mammalian hosts in Northern Ireland during 2013-2016[J]. Virus Evol, 2023, 9 (2): vead039. doi: 10.1093/ve/vead039
20	GAO L , SHEN H , ZHAO S , et al. Isolation and pathogenicity analysis of a G5P[23] porcine rotavirus strain[J]. Viruses, 2023, 16 (1): 21. doi: 10.3390/v16010021
21	LI Q , WANG Z , JIANG J , et al. Outbreak of piglet diarrhea associated with a new reassortant porcine rotavirus B[J]. Vet Microbiol, 2024, 288, 109947. doi: 10.1016/j.vetmic.2023.109947
22	樊高, 申翰钦, 王连想, 等. 猪轮状病毒的分离鉴定和全基因组序列分析[J]. 中国兽医杂志, 2023, 59 (6): 47- 54.
	FAN G , SHEN H Q , WANG L X , et al. Isolation, identification and whole genome sequence analysis of porcine rotavirus[J]. Chinese Journal of Veterinary Medicine, 2023, 59 (06): 47- 54.
23	刘小兰, 刘昌锦, 余文洋, 等. 猪轮状病毒江西株AY01的分离鉴定[J]. 中国畜牧兽医, 2022, 49 (8): 3151- 3162.
	LIU X L , LIU C J , YU W Y , et al. Isolation and identification of porcine rotavirus Jiangxi strain AY01[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49 (08): 3151- 3162.
24	乔成鹏. 2015~2016年中国部分地区猪轮状病毒感染检测及病毒分离与鉴定[D]. 大庆: 黑龙江八一农垦大学, 2019.
	QIAO C P. Detection, Isolation and identification of porcine rotavirus infection in some areas of China from 2015 to 2016[D]. Daqing: Heilongiiang Bayi Agricultural University, 2019. (in Chinese)
25	WANG Z , LV C , XU X , et al. The dynamics of a Chinese porcine G9P[23] rotavirus production in MA-104 cells and intestines of 3-day-old piglets[J]. J Vet Med Sci, 2018, 80 (5): 790- 797. doi: 10.1292/jvms.17-0657
26	KIM H H , PARK J G , MATTHIJNSSENS J , et al. Pathogenicity of porcine G9P[23] and G9P[7] rotaviruses in piglets[J]. Vet Microbiol, 2013, 166 (1-2): 123- 137. doi: 10.1016/j.vetmic.2013.05.024
27	何晓明, 田小艳, 王东东, 等. 2021—2022年我国部分地区猪轮状病毒分子流行病学调查[J]. 畜牧与兽医, 2024, 56 (3): 77- 85.
	HE X M , TIAN X Y , WANG D D , et al. Molecular epidemiology investigation of porcine rotavirus in some regions of China in 2021-2022[J]. Animal Husbandry & Veterinary Medicine, 2024, 56 (3): 77- 85.
28	陈小飞, 张斌, 张春红, 等. 猪A群轮状病毒SCJY-13株的分离鉴定及致病性分析[J]. 中国畜牧兽医, 2022, 49 (2): 660- 668.
	CHEN X F , ZHANG B , ZHANG C H , et al. Isolation, identification and pathogenicity analysis of porcine rotavirus group A strain SCJY-13[J]. China Animal Husbandry &, Veterinary Medicine, 2022, 49 (2): 660- 668.

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

[1]	LI Ting, ZHANG Chengcheng, WANG Xiuling, ZHANG Xiaorong, WU Yantao. Isolation and Identification of a Novel Duck Reovirus Strain and the Sequence Analysis of Its σC Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2520-2524.
[2]	LIU Jian, YU Zehai, ZHANG Meiyu, LI Dan, WANG Jun, LIU Fangqin, ZHANG Qun, XU Shouzhen. Full-genome Analysis of a Bovine Enterovirus Type F and the Establishment of an Indirect ELISA Method for Antibody Detection [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 814-825.
[3]	DU Qingjie, WU Liping, ZHANG Fan, DAI Pengxiu, FENG Xiancheng, ZHANG Xinke. Difference Analysis of Oral Flora in Dogs with Periodontitis and Drug Resistance of Oral Porphyromonas [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 934-942.
[4]	Liguo GAO, Hanqin SHEN, Yiquan CHEN, Sheng CHEN, Wencheng LIN, Feng CHEN. Prokaryotic Expression of Recombinant VP6* Protein of Porcine Rotavirus and Establishment of Indirect ELISA Detection Method [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4021-4028.
[5]	Shan ZHANG, Dahu LIU, Baojing LIU, Lin LIANG, Ruiying LIANG, Xinming TANG, Xusheng QIU, Chan DING, Jiabo DING, Shaohua HOU. Isolation, Identification and Pathogenicity Analysis of a Pigeon Paramyxovirus-1 Strain [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4051-4060.
[6]	Bilin XIE, Zhimin LIN, Binbin LIN, Yijuan XU, Fengqiang LIN, Lu YAN, Huini WU, Cuiting LI, Haiou ZHOU, Zhaolong LI. Isolation, Identification and Pathogenicity Analysis of Riemerella anatipestifer Strain LC1 and CX1 [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4196-4203.
[7]	Ning PENG, Yaxu LIANG, Fei LONG, Dongming YU, Xiang ZHONG. Inhibitory Effect of Resveratrol on Rotavirus-infected Porcine Intestinal Epithelial Cells IPEC-J2 [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4213-4225.
[8]	Cheng YANG, Ye LIU, Ning CHENG, Kaiyue WANG, Xinlei LI, Jiuying SUN, Junping HAN, Wenjun LI, Huanhuan WANG, Xiao SHAO, Xuejiao CHENG, Yingfeng SUN. Genomic Characterization of a Recombinant Strain of PRRSV-2 between Lineages 1.8 and 1.5 [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3570-3578.
[9]	Yue LI, Changchun ZHANG, Guangyu LIU, Mengyuan GAO, Chaojun FU, Jiabao XING, Sijia XU, Qiyuan KUANG, Jing LIU, Xiaopeng GAO, Heng WANG, Lang GONG, Guihong ZHANG, Yankuo SUN. Application and Analysis of Meta-transcriptomics Sequencing Technology in the Diagnosis of Viral Diarrhea Diseases in Piglets [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3579-3589.
[10]	Huanqin ZHENG, Xiaomin JIANG, Hong YUE, Baoyan WANG, Yang LIU, Xingxiao ZHANG, Jianlong ZHANG, Hongwei ZHU. Isolation, Identification and Partial Biological Characteristics Analysis of Feline Herpesvirus-1 [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3040-3048.
[11]	Jitong LI, Tong ZHU, Junfeng LÜ, Yuehua GAO, Feng HU, Kexiang YU, Minxun SONG, Jianlin WANG, Yufeng LI. Isolation and Identification of Novel Picornavirus from Ducks and Whole Genome Sequence Analysis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3075-3084.
[12]	Bohua LIU, Hanyu FU, Yuheng WANG, Suolangsizhu, Jiaqiang NIU, Yuhua BAO, Jiakui LI, Yefen XU. Isolation, Identification and Genome Analysis of Type B Pasteurella multocida Isolated from Yak in Tibetan Nakchu City [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3105-3118.
[13]	ZHENG Rui, LIU Zishi, ZHANG Kangyou, YAN Yong, WEI Ling, ZEREN Wengmu, DINGZE Demi, HUANG Jianjun, WANG Li, WEI Yong. Isolation, Identification and Biological Characterization of Colletotrichum jasminigenum in Stems of Peanuts [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2206-2213.
[14]	HU Zeqi, LI Runcheng, TAN Zuming, XIE Xiuyan, WANG Jiangping, QIN Lejuan, LI Rong, GE Meng. Establishment and Preliminary Application of PEDV, PoRVA and PDCoV TaqMan Triple RT-qPCR Assay [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2267-2272.
[15]	TIAN Rui, XU Sixiang, XIE Feng, LIU Guangjin, WANG Gang, LI Qingxia, DAI Lei, XIE Guoxin, ZHANG Qiongwen, LU Yajing, WANG Guangwen, WANG Jinxiu, ZHANG Wei. Bioinformatics Analysis of the Genome of Clostridium perfringens Isolated from Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1707-1715.

Isolation, Identification and Pathogenicity Analysis of Porcine Rotavirus

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 14

References 28

Related Articles 15

Recommended Articles

Metrics

Comments