绵羊源哺乳动物正呼肠孤病毒的分离鉴定及全基因组分析

doi:10.11843/j.issn.0366-6964.2024.12.027

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (12): 5641-5650.doi: 10.11843/j.issn.0366-6964.2024.12.027

绵羊源哺乳动物正呼肠孤病毒的分离鉴定及全基因组分析

李霞¹^,²(), 何艺³, 蔡旭航²^,⁴, 罗润波¹, 郭容利², 索朗斯珠¹^,*(), 毛立¹^,²^,*(), 李彬¹^,²

1. 西藏农牧学院, 林芝 860000
2. 江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室, 南京 210014
3. 阜康市城关镇农业(畜牧业)发展服务中心, 阜康 831500
4. 西北农林科技大学, 咸阳 712100

收稿日期:2024-02-04 出版日期:2024-12-23 发布日期:2024-12-27
通讯作者: 索朗斯珠,毛立 E-mail:2858768331@qq.com;xzslsz@163.com;mao-li@live.cn
作者简介:李霞(1999-), 女, 四川三台人, 硕士生, 主要从事预防兽医学研究, E-mail: 2858768331@qq.com
基金资助:
国家重点研发计划(2023YFD1801302);江苏省重点研发计划(BE2022330);兽医生物技术国家重点实验室开放基金(SKLVBF202106)

Isolation, Identification and Whole Genome Analysis of Mammalian Orthoreovirus from Sheep

LI Xia¹^,²(), HE Yi³, CAI Xuhang²^,⁴, LUO Runbo¹, GUO Rongli², SUOLANG Sizhu¹^,*(), MAO Li¹^,²^,*(), LI Bin¹^,²

1. Tibet Agricultural and Animal Husbandry University, Linzhi 860000, China
2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Engineering Technology of Veterinary Biological Products, Ministry of Agriculture and Rural Affairs, Nanjing 210014, China
3. Chengguan Agriculture (Animal Husbandry) Development Service Center of Fukang City, Fukang 831500, China
4. Northwest A&F University, Xianyang 712100, China

Received:2024-02-04 Online:2024-12-23 Published:2024-12-27
Contact: SUOLANG Sizhu, MAO Li E-mail:2858768331@qq.com;xzslsz@163.com;mao-li@live.cn

摘要/Abstract

摘要：

为了解绵羊源哺乳动物正呼肠孤病毒(mammalian orthoreovirus, MRV)的生物学特性及全基因组特征，本研究从绵羊腹泻病料中分离了一株MRV，命名为MRV-XJ23株，经RT-PCR、电镜观察和间接免疫荧光试验(IFA)鉴定后，扩增病毒全基因组进行同源性及遗传进化分析。研究结果表明，MRV-XJ23分离株可在MDBK细胞稳定传代，产生特异的细胞病变，病毒滴度为10⁶ TCID₅₀·mL^-1。MRV感染细胞经IFA检测可观察到特异荧光，电镜可观察到直径约70 nm的无囊膜病毒粒子。经RT-PCR扩增和测序获得MRV全基因组，该毒株共10个节段，全长23 534 bp。序列对比及遗传进化分析结果显示，MRV-XJ23株为MRV-1血清型，其在韩国蝙蝠源BatMRV2/SNU1/Korea/2021毒株基础上，与日本的人源Homo sapiens/Osaka2005株L3、M2、S4基因、印度牛源C/bovine/Indiana/MRV00304/2014株S1基因重配，形成一株新的MRV。本研究首次从腹泻绵羊肛拭子中分离获得一株MRV-1型重配毒株，证明绵羊可感染MRV，拓宽了病毒感染宿主谱，且感染毒株为蝙蝠源、人源和牛源MRV重配毒株，证明这些毒株跨物种感染后发生了复杂的重配。本研究揭示了MRV在各物种中循环传播对公共卫生的风险，提升了深入研究MRV在绵羊乃至家畜中的流行病学、重配规律和致病性的重要性。

关键词: 哺乳动物正呼肠孤病毒, 绵羊, 分离鉴定, 重配, 进化分析

Abstract:

In order to understand the biological and genome characteristics of mammalian orthoreovirus (MRV) from sheep, a MRV strain named MRV-XJ23 was isolated from sheep suffering diarrhea, and identified by RT-PCR, electron microscopy and indirect immunofluorescence assay (IFA), respectively. The whole genome of the isolated strain was amplified by RT-PCR and the homology and genetic evolution were analyzed further. The results showed that the MRV-XJ23 strain cultured stably in MDBK cells and performed specific cytopathic effect with a virus titer of 10⁶ TCID₅₀·mL^-1. Specific fluorescence was observed in MRV-infected MDBK cells by IFA detection, and electron microscope detection showed that the virus was purified by ultracentrifugation with a diameter of about 70 nm. The whole MRV genome was obtained by RT-PCR, MRV-XJ23 contained 10 segments with the length of 23 534 bp. Nucleotide sequence alignments and genetic evolution analysis showed that the MRV-XJ23 isolate was belonged to MRV-1 serotype. Results of sequence and phylogenetic trees indicate that the novel MRV isolate was a novel reassortant among three MRV strains of BatMRV2/SNU1/Korea/2021, C/bovine/Indiana/MRV00304/2014 strain and Homo sapiens/Osaka2005. In this study, a reassorting strain of MRV-1 type originated from sheep was isolated and identified for the first time, which proved important evidence that sheep could be infected with MRV and broadened the host species. Moreover, the reassortant strain was derived from bat, bovine and homo sapiens-MRV, which indicated that these strains were infected the new host and reassortment was present after possible cross-species infection. This study revealed the risk of MRV transmission circle in various species to public health, and the importance to study the epidemiology, reassortment and pathogenicity of MRV in sheep and even livestock.

Key words: mammalian orthoreovirus, sheep, isolation and identification, reassortment, evolutionary analysis

中图分类号:

S852.659.4

李霞, 何艺, 蔡旭航, 罗润波, 郭容利, 索朗斯珠, 毛立, 李彬. 绵羊源哺乳动物正呼肠孤病毒的分离鉴定及全基因组分析[J]. 畜牧兽医学报, 2024, 55(12): 5641-5650.

LI Xia, HE Yi, CAI Xuhang, LUO Runbo, GUO Rongli, SUOLANG Sizhu, MAO Li, LI Bin. Isolation, Identification and Whole Genome Analysis of Mammalian Orthoreovirus from Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5641-5650.

图/表 7

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引物名称 Primer name	序列(5’→3’) Sequence	片段大小/bp Fragment size
MRV-F	TTCACTCAGGCATTATCCGA	559
MRV-R	TCCGCTTCTGACTCCTGA	559
MRVL1-F	GCTACACGTTCCACGACAATGTCA	3 854
MRVL1-R	GATGAGTTRACGCRCCACGRC	3 854
MRVL2-F	GCTATTGGCGCRATGGCGAAC	3 915
MRVL2-R	GATGAATTAGGCRCGCTYACGA	3 915
MRVL3-F	GCTAATCGTCAGGATGAAGCGGA	3 901
MRVL3-R	GATGARTCGGCCCAACTAGCATY	3 901
MRVM1-F	GCTATTCGCGGTCATGGCTTACAT	2 304
MRVM1-R	GATGAAGCGCGTACGTAGTCYTAG	2 304
MRVM2-F	GCTAATCTGCTGACCGTYACTCT	2 203
MRVM2-R	GATGATKTGCCTGCATCCCTTAACC	2 203
MRVM3-F	ATGGCTTCATTCAAGGGAT	2 166
MRVM3-R	TTACAGCTCATCRGTTGGAAC	2 166
MRVS1-F	GCTATTCGCGCCTATGGATGCR	1 466
MRVS1-R	GATGATTGACCCCTTGTGCCGA	1 466
MRVS2-F	GCTATTCGCTGGTCAGTTATGGCT	1 331
MRVS2-R	GATGAATGTGTGGTCAGTCGYGAG	1 331
MRVS3-F	GCTAAAKTCACGCCTGTYGTCGT	1 198
MRVS3-R	GATGATTAGGCGYCACCCACCA	1 198
MRVS4-F	GCTATTTTTGCCTCTTCCYAAACG	1 196
MRVS4-R	GATGAATGRAGCCTGTCCCACGT	1 196

参考株 Reference strains	宿主或来源 Host or origin	国家 Country
SD-14	水貂Mink	中国China
WIV3	蹄蝠(未定种)Hipposideros sp.	中国China
WIV4	蹄蝠(未定种)Hipposideros sp.	中国China
ZJ2013	猪Pig	中国China
YNSZ/V207/2016	牛Cattle	中国China
HLJYC2017	猪Swine	中国China
MRV3/TS/China/2012	树鼩Tree shrew	中国China
BtMRV-QAPpC66	伏翼蝠(XJC66)Pipistrellus pipistrellus XJC66	中国China
GD-1	猪Porcine	中国China
SC-A	猪腹泻粪样Diarrhea feces of pigs	中国China
HB-A	水貂Mink	中国China
THK0617	废水Waste water	日本Japan
Osaka2005	人Homo sapiens	日本Japan
BatMRV2/SNU1/Korea/2021	草原带须蝙蝠Myotis aurascens	韩国South Korea
17-EF40	大棕蝠Eptesicus fuscus	美国USA
C/bovine/Indiana/MRV00304/2014	牛Bovine	美国USA
SHR-A	猪Porcine	中国China
Ndelle	西非小家鼠Mus musculoides	喀麦隆Cameroon
MRV3/TS/China/2012	树鼩Tree shrew	中国China

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绵羊源哺乳动物正呼肠孤病毒的分离鉴定及全基因组分析

Isolation, Identification and Whole Genome Analysis of Mammalian Orthoreovirus from Sheep

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