甲磺酸瑞波西汀体外颉颃塞内卡病毒的研究

doi:10.16431/j.cnki.1671-7236.2024.01.027

中国畜牧兽医 ›› 2024, Vol. 51 ›› Issue (1): 268-277.doi: 10.16431/j.cnki.1671-7236.2024.01.027

甲磺酸瑞波西汀体外颉颃塞内卡病毒的研究

巩有权¹, 周晓翠³, 郑辉^2,5, 陈峰⁴, 曹振山⁴, 沙洲^2,5, 张慧^2,5, 崔进^2,5, 武瑞^1,6, 尼博^2,5

1. 黑龙江八一农垦大学动物科技学院, 大庆 163319;
2. 中国动物卫生与流行病学中心, 青岛 266032;
3. 中国兽药监察所, 北京 100081;
4. 山东省动物疫病预防与控制中心, 济南 250100;
5. 农业农村部动物生物安全风险预警及防控重点实验室(南方), 青岛 266032;
6. 佳木斯大学, 佳木斯 154000

收稿日期:2023-06-21 出版日期:2024-01-05 发布日期:2023-12-27
通讯作者: 武瑞, 尼博 E-mail:fuhewu@126.com;nibo@cahec.cn
作者简介:巩有权,E-mail:1216548980@qq.com。
基金资助:
“十四五”国家重点研发计划"口蹄疫病毒的分子流行病学与传播机制"（2021YFD1800300）；中国动物卫生与流行病学中心创新基金（DW2021009）

Study on the Antagonism of Reboxetine Mesylate Against Senecavirus in vitro

GONG Youquan¹, ZHOU Xiaocui³, ZHENG Hui^2,5, CHEN Feng⁴, CAO Zhenshan⁴, SHA Zhou^2,5, ZHANG Hui^2,5, CUI Jin^2,5, WU Rui^1,6, NI Bo^2,5

1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China;
2. China Animal Health and Epidemiology Center, Qingdao 266032, China;
3. China Institute of Veterinary Drug Supervision, Beijing 100081, China;
4. Shandong Animal Disease Prevention and Control Center, Jinan 250100, China;
5. Key Laboratory of Animal Biosafety Risk Prevention and Control (South), Ministry of Agriculture and Rural Affairs, Qingdao 266032, China;
6. Jiamusi University, Jiamusi 154000, China

Received:2023-06-21 Online:2024-01-05 Published:2023-12-27

摘要/Abstract

摘要： 【目的】从含有127种化合物的FDA批准上市药物库中筛选具有颉颃A型塞内卡病毒（Senecavirus A，SVA）活性的化合物并研究其作用途径。【方法】利用荧光素酶重组塞内卡病毒（rSVA-NLuc）结合荧光素酶高通量筛选技术，建立抗SVA化合物体外筛选平台。从FDA批准上市药物库中筛选浓度为10 μmol/L时具有抑制荧光素酶活性效应的化合物，并进一步利用实时荧光定量RT-PCR验证其抑制活性，通过反映乳酸脱氢酶（LDH）释放水平的细胞毒性试验进而确定其最大无毒浓度。针对病毒感染周期的吸附、入胞、复制和组装释放等4个主要过程，采用不同的细胞处理方法和实时荧光定量RT-PCR、间接免疫荧光（indirect immunofluorescence assay，IFA）、蛋白免疫印迹（Western blotting）和病毒滴度测定（TCID₅₀）等技术进行化合物分子颉颃机制研究。【结果】从包含127种分子的FDA获批上市药物库中筛选出8种候选抗SVA活性分子，通过实时荧光定量RT-PCR及细胞毒性检测，鉴定出1种安全、有效的化合物——甲磺酸瑞波西汀。在病毒感染细胞36 h内甲磺酸瑞波西汀可使SVA VP3蛋白表达量和病毒滴度均极显著下降（P<0.01）。甲磺酸瑞波西汀处理金黄仓鼠肾细胞（BSR-T7/5）可减少SVA的吸附和入胞；实时荧光定量RT-PCR也显示甲磺酸瑞波西汀能抑制SVA的组装阶段，但它对SVA的复制和释放阶段没有影响。【结论】本试验从FDA批准上市药物库中筛选出了1种细胞毒性较低且颉颃SVA效果优异的化合物——甲磺酸瑞波西汀，该化合物通过抑制SVA的吸附、入胞和组装阶段来对抗SVA感染。本研究为抗SVA药物的进一步研发提供重要参考。

关键词: A型塞内卡病毒(SVA); FDA药物库; 高通量筛选

Abstract: 【Objective】 The aim of this study was to screen compounds with antagonistic Senecavirus A (SVA) activity from an FDA-approved drug library containing 127 compounds and studying their pathways of action.【Method】 An in vitro active compounds screening platform for SVA was established using luciferase recombinant Senecavirus (rSVA-NLuc) combined with Fluc high-throughput screening technology.The compounds were screened from the FDA-approved drug library for their luciferase inhibitory activity at a concentration of 10 μmol/L.The inhibitory activity was further verified by Real-time quantitative RT-PCR, and the maximum non-toxic concentration was determined by a cytotoxicity assay which was measured by the released lactate dehydrogenase (LDH) from cell.According to the four main processes of the virus infection cycle, such as adsorption, endocytosis, replication, assembly and release, different cell treatment methods and Real-time quantitative RT-PCR, indirect immunofluorescence assay(IFA), Western blotting and viral titer assay (TCID₅₀) were used to study the antagonistic mechanism of the screeded compound.【Result】 Eight candidate anti-SVA active molecules were screened from the FDA drug library containing 127 molecules, and one safe and effective compound, reboxetine mesylate, was identified by Real-time quantitative RT-PCR and cytotoxicity assay.Within 36 hours of virus infection in cells, reboxetine mesylate significantly reduced the expression of SVA VP3 protein and the SVA titer (P<0.01).The treatment of golden hamster kidney cells (BSR-T7/5) with reboxetine mesylate could reduce the adsorption and entry of SVA.Real-time quantitative RT-PCR also showed that reboxetine mesylate could inhibit the assembly stage of SVA, but it had no effect on the replication and release stage of SVA.【Conclusion】 This experiment screened a compound with low cytotoxicity and excellent SVA antagonistic effect from the FDA-approved drug library:Riboxetine mesylate.This compound fighted SVA infection by inhibiting the adsorption, entry, and assembly stages of SVA.This study provided important references for the further development of anti-SVA drugs.

Key words: Senecavirus A (SVA); FDA-approved drug library; high-throughput screening

中图分类号:

S853.74

巩有权, 周晓翠, 郑辉, 陈峰, 曹振山, 沙洲, 张慧, 崔进, 武瑞, 尼博. 甲磺酸瑞波西汀体外颉颃塞内卡病毒的研究[J]. 中国畜牧兽医, 2024, 51(1): 268-277.

GONG Youquan, ZHOU Xiaocui, ZHENG Hui, CHEN Feng, CAO Zhenshan, SHA Zhou, ZHANG Hui, CUI Jin, WU Rui, NI Bo. Study on the Antagonism of Reboxetine Mesylate Against Senecavirus in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(1): 268-277.

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甲磺酸瑞波西汀体外颉颃塞内卡病毒的研究

Study on the Antagonism of Reboxetine Mesylate Against Senecavirus in vitro

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