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On-line Access: 2021-04-08

Received: 2020-08-28

Revision Accepted: 2020-12-06

Crosschecked: 2021-03-11

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Sun-jian Lyu

https://orcid.org/0000-0001-7862-3097

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Journal of Zhejiang University SCIENCE B 2021 Vol.22 No.4 P.295-304

http://doi.org/10.1631/jzus.B2000504


Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis


Author(s):  Sunjian LYU, Xuemei YUAN, Li LIU, Haiqi ZHANG, Zhe YU, Xiaoying HANG, Weida SHI, Yinglei WU

Affiliation(s):  Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture, Key Laboratory of Fish, Health and Nutrition of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China; more

Corresponding email(s):   Liuli6655@hotmail.com, zmk407@126.com

Key Words:  Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV), Replicase, Virus localization, Immune genes


Sunjian LYU, Xuemei YUAN, Li LIU, Haiqi ZHANG, Zhe YU, Xiaoying HANG, Weida SHI, Yinglei WU. Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis[J]. Journal of Zhejiang University Science B, 2021, 22(4): 295-304.

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author="Sunjian LYU, Xuemei YUAN, Li LIU, Haiqi ZHANG, Zhe YU, Xiaoying HANG, Weida SHI, Yinglei WU",
journal="Journal of Zhejiang University Science B",
volume="22",
number="4",
pages="295-304",
year="2021",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B2000504"
}

%0 Journal Article
%T Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis
%A Sunjian LYU
%A Xuemei YUAN
%A Li LIU
%A Haiqi ZHANG
%A Zhe YU
%A Xiaoying HANG
%A Weida SHI
%A Yinglei WU
%J Journal of Zhejiang University SCIENCE B
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%P 295-304
%@ 1673-1581
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%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2000504

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T1 - Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis
A1 - Sunjian LYU
A1 - Xuemei YUAN
A1 - Li LIU
A1 - Haiqi ZHANG
A1 - Zhe YU
A1 - Xiaoying HANG
A1 - Weida SHI
A1 - Yinglei WU
J0 - Journal of Zhejiang University Science B
VL - 22
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SP - 295
EP - 304
%@ 1673-1581
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B2000504


Abstract: 
Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV) is an arterivirus newly discovered in Chinese softshell turtles. Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles. In this study, a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system, and its polyclonal antibody was generated. The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay (dot-ELISA). The distribution of TSHSV in different organs of T. sinensis was examined by immunohistochemistry (IHC) and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed, and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles. The IHC assay indicated that the virus was highly localized in various cells, including intestinal lymphocytes, enterocytes, kidney epithelial cells, spleen cells, lung macrophages, and cardiomyocytes. The qRT-PCR analysis revealed that TSHSV was detected in all organs tested, including the lungs, liver, kidneys, spleen, and heart. The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group. The interferon-stimulated genes (ISGs), myxovirus resistance protein 2 (MX2) and radical S-adenosyl methionine domain containing 2 (RSAD2) were highly upregulated in all groups of infected turtles. Antibody-dependent enhancement (ADE) seemed to occur after stimulation by the polyclonal antibody, because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group. Overall, these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

基于重组复制酶的中华鳖出血综合征病毒定位及对中华鳖抗病毒基因的影响评估

目的:探究中华鳖出血综合征病毒在不同组织细胞的定位及复制酶的多克隆抗体对病毒及中华鳖免疫基因的影响。
创新点:基于获得的中华鳖出血综合征病毒复制酶的多克隆抗体,确定了该病毒在不同组织细胞中的定位,并发现该病毒存在抗体依赖增强效应,即该多克隆抗体促进了病毒复制。
方法:基于中华鳖出血综合征病毒的基因组信息,通过RT-PCR技术从中华鳖出血综合征病毒的阳性样品中克隆得到TSHSV-HP4(复制酶)的部分序列。利用pET-30a载体构建中华鳖出血综合征病毒复制酶的原核表达系统(pET30a-TSHSV-661)并表达该蛋白。利用该蛋白免疫兔子获得兔多克隆抗体,并利用聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹法(western blot)和斑点杂交确定该多克隆抗体的特异性。进一步用免疫组化(IHC)分析该病毒在不同组织细胞内的定位。注射单一病毒、单一抗体和病毒抗体混合液于中华鳖体内,并利用荧光定量聚合酶链反应(qRT-PCR)分析各组病毒、MX2RSAD2在不同组织的差异,确定抗体对病毒和免疫基因的影响。
结论:构建的pET30a-TSHSV-661原核表达系统成功表达了中华鳖出血综合征病毒复制酶,且制备的兔多克隆抗体能够特异性结合病毒蛋白。免疫组化结果显示该病毒能够感染多种细胞,包括肠淋巴细胞、肠上皮细胞、肾上皮细胞、脾细胞、肺巨噬细胞和心肌细胞。qRT-PCR结果显示病毒抗体组中华鳖的肺和心脏组织中的病毒拷贝数明显高于病毒组。另外,MX2RSAD2在病毒组和病毒抗体组均呈高表达,且后者表达量要高于前者。推测该病毒存在抗体依赖增强效应。

关键词:中华鳖出血综合征病毒;复制酶;病毒定位;免疫基因

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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