氯喹抑制寨卡病毒在vero细胞中感染复制的实验研究

Inhibition of Zika virus replication by Chloroquine(CQ)in vero cells

目的探究氯喹对寨卡病毒在vero细胞上感染复制的抑制效果,寻找一种可预防和治疗寨卡病毒感染的药物。方法寨卡病毒分别感染用不同浓度(0、5、10和20μmol/L)的氯喹预处理的对数生长期vero细胞,然后通过显微镜观察、免疫荧光、免疫印迹分析等方法检测氯喹对寨卡病毒基因表达和复制的抑制作用。结果显微镜观察发现,与无寨卡病毒感染的细胞相比,分别加入5、10和20μmol/L的氯喹处理寨卡病毒感染的vero细胞72 h后发现,细胞脱落、死亡等现象逐渐消失,寨卡病毒的致细胞病变效应明显减弱。间接免疫荧光结果显示,感染复数(MOI)=1的寨卡病毒感染vero细胞48 h后,几乎所有细胞都被染成红色,显示E抗原阳性,随着氯喹浓度的增加,荧光强度逐渐减弱,当加入20μmol/L的氯喹后,荧光基本消失了,氯喹很好地抑制了寨卡病毒E蛋白的表达,且这种抑制效应具有剂量依赖性。免疫印迹法检测显示,用MOI=1的寨卡病毒感染vero细胞48 h后,在细胞中能够检测到NS5蛋白的表达,而随着氯喹加入浓度的增加,寨卡病毒NS5蛋白的表达逐渐降低,特别是当氯喹的浓度达到20μmol/L后,抑制效果更明显。qRT-PCR结果显示,5μmol/L的氯喹即可抑制上清中病毒产量的90%(P<0.01)。结论氯喹能够抑制寨卡病毒在vero细胞中的感染和复制,表明氯喹可作为一种治疗寨卡病毒感染的潜在药物。

Objective To investigate the effect of Chloroquine(CQ)on preventing and treating Zika virus infection.Method After vero cells were pretreated with different dose of CQ and then infected with Zika virus,the gene expression and replication of Zika virus were detected.Results Vero cells were infected with Zika virus for 72 h after pretreated with different doses(5,10 and 20μmol/L)of CQ for 2 h.Microscopy observation found that infected cells floated from the bottom of the flask in the medium and cell death casused by Zika virus disappeared.It indicated that CQ could protect vero cells from cytopathic effect(CPE)induced by Zika virus.IFA showed that all the cells were fluorescence-stained after infected with 1 multiplicity of infection(MOI)per well at 48 hours,and fluorescence was significantly decreased as increasing in CQ concentration.It showed that CQ could inhibit Zika virus E protein expression and this inhibition was concentration-dependent.The expression of NS5 protein could be detected after infected with 1 MOI per well at 48 hours.After vero cells were pretreated with CQ for 2 h and then infected with Zika virus(MOI=1)for 48 h,the expression of NS5 protein was down-regulated as increasing in CQ concentration,especially when the concentration of CQ was 20μmol/L.The qRT-PCR results demonstrated that CQ(5μmol/L)could effectively inhibit 90%of the virion production in vero cells(P<0.01).Conclusion Antimalarial drug CQ could suppress gene expression and replication of Zika virus,and these studies suggest that CQ might be an effective drug against Zika virus infection.