夫西地酸钠抗肠道病毒EV-A71的活性分析  

Antiviral activity of sodium fusidate against enterovirus A71

作　　者：刘健 曾施暖[1,2] 孟小斌 江鑫莹[2] 郭学敏 LIU Jian;ZENG Shinuan;MENG Xiaobin;JIANG Xinying;GUO Xuemin(Insitute of Human Virus Research, Zhongshan School of Medicine, Sun-Yet Sen University, Guangzhou, Guangdong 510080, China;The Ministry of Education Key Laboratory of Tropical Disease Prevention and Control Research,Guangzhou,Guangdong 510080, China)

机构地区：[1]中山大学中山医学院人类病毒学研究所,广东广州510080 [2]热带病防治研究教育部重点实验室,广东广州510080 [3]梅州市人民医院,广东梅州514031

出　　处：《中国热带医学》2019年第6期519-524,共6页China Tropical Medicine

基　　金：广东省引进创新科研团队计划资助(No.2009010058)

摘　　要：目的分析夫西地酸钠在细胞水平上的抗肠道病毒活性并分析其抗EV-A71的作用机制。方法通过空斑分析测定夫西地酸钠在RD细胞中抗EV-A71和CV-A16的量-效曲线;通过加入时间分析(time of addition)、病毒吸附及内吞实验、反转录-定量PCR(RT-qPCR)测定病毒RNA水平在复制周期中的变化、携带GFP的病毒RNA(EV-A71-GFP RNA)转染合并荧光观察以及双报告基因分析初步确定夫西地酸钠阻断EV-A71复制的作用机制。结果夫西地酸钠可以显著抑制EV-A71和CV-A16在RD细胞中的复制,MOI为0.001时IC50分别为3.56μmol/L和5.41μmol/L。夫西地酸钠抑制EV-A71复制主要发生在复制早期阶段,降低了病毒RNA的丰度和病毒蛋白的合成水平,但不影响病毒的吸附和内吞;与未经药物处理的对照组相比,EV-A71感染5 h和8 h时,夫西地酸钠导致EV-A71 RNA相对丰度降低了大约70%;EV-A71-GFP RNA在292A细胞中的翻译明显受到夫西地酸钠的抑制,GFP阳性细胞明显减少;夫西地酸钠对EV-A71 5′-UTR所驱动的报告基因翻译抑制程度为30%。结论夫西地钠酸具有抗肠道病毒活性,主要通过直接或间接抑制病毒基因组RNA复制以及干扰病毒蛋白合成或加工而发挥抗EV-A71活性。Objective To evaluate the in vitro antiviral activity of sodium fusidate against enteroviruses responsible for hand-foot-mouth diseases (HFMD) and to explore its potential antiviral mechanism. Methods The dose-response of sodium fusidate against enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) in vitro was determined by plague reduction assay. The antiviral mechanism of sodium fusidate inhibiting EV-A71 replication was explored by performing time of addition assay, virus attachment and entry assay, measurement of the viral RNA abundance at different time points of EV-A71 life stage by reverse transcription-quantitative PCR (RT-qPCR), viral translation assay of GFP-carrying viral genomic RNA (EV-A71-GFP RNA) combined with fluorescence microscopy observation as well as a dual-reporter assay. Results Sodium fusidate significantly inhibited the replication of EV-A71 and CV-A16 in rhabdomyosarcoma (RD) cells with the 50% inhibitory concentration (IC50) at 3.56 μmol/L and 5.41 μmol/L, respectively. The inhibitory effect occurred at the early stage of EV-A71 life cycle, most likely through interfering with the viral RNA replication and viral protein synthesis. Compared to the control without drug treatment, sodium fusidate treatment reduced the viral RNA level by 70% and the EV-A71-5’UTR-mediated luciferase translation by 30%;meanwhile, the presence of sodium fusidate also inhibited the GFP expression from EV-A71-GFP RNA. The virus attachment and entry as well as the virus release were affected weakly or not at all. Conclusion Sodium fusidate possesses antiviral activities against EV-A71 and CV-A16. The potential antiviral mechanism is related to the repression of viral RNA replication and viral protein synthesis.

关 键 词：手足口病 肠道病毒EV-A71 夫西地酸钠 抗病毒活性 

分 类 号：R512.5[医药卫生—内科学]