单宁酸对猪流行性腹泻病毒增殖的体外抑制作用

Inhibition of Tannic Acid on Proliferation of Porcine Epidemic Diarrhea Virus in vitro

【目的】探究单宁酸(tannic acid)对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)复制的影响,为临床治疗PEDV感染提供新的参考依据。【方法】通过检测不同浓度单宁酸对细胞活性的影响,评估单宁酸对Vero和LLC-PK1细胞的毒性;通过实时荧光定量PCR和间接免疫荧光试验检测单宁酸对PEDV YN13毒株复制的影响;将PEDV YN13毒株更换为PEDV DR13-GFP毒株、Vero细胞系换成LLC-PK1细胞系分别进行试验,检测单宁酸对PEDV复制的影响;在病毒感染的不同时间点添加单宁酸,通过实时荧光定量PCR、Western blotting和半数组织培养感染剂量(50%tissue culture infection dose,TCID 50)法检测单宁酸对PEDV复制的影响;通过荧光共振能量转移试验检测单宁酸在细胞外对PEDV 3C样蛋白酶活性的影响。【结果】低浓度的单宁酸对细胞几乎无毒性;单宁酸能够有效抑制PEDV YN13毒株的复制,且单宁酸的浓度越高抑制效果越好;在更换毒株和细胞系后,各浓度单宁酸对PEDV的复制过程仍有较好的抑制作用;单宁酸可以通过抑制PEDV吸附或入侵过程及病毒的早期复制发挥抗病毒活性;单宁酸对PEDV 3C样蛋白酶活性具有抑制作用,且呈现明显的剂量依赖性。【结论】单宁酸可以在体外通过影响病毒吸附或入侵过程抑制PEDV的感染,也可能通过抑制病毒的3C样蛋白酶的活性抑制病毒的复制过程。

【Objective】This study was aimed to explore the effect of tannic acid on the replication of Porcine epidemic diarrhea virus(PEDV),and provide a new reference for clinical treatment of PEDV infection.【Method】The toxicity of tannic acid on Vero and LLC-PK1 cells were assessed by detecting the effects of different concentrations of tannic acid on cell activity.The effect of tannic acid on the replication of PEDV YN13 strain was detected through Real-time quantitative PCR and indirect immunofluorescence assay.The effect of tannic acid on PEDV replication was tested by replacing the PEDV YN13 strain with the PEDV DR13-GFP strain and the Vero cell line to the LLC-PK1 cell line.The tannic acid was added at different time points of virus infection,and the influence of tannic acid on PEDV replication were detected by Real-time quantitative PCR,Western blotting and 50%tissue culture infectious dose(TCID 50),respectively.The extracellular effect of tannic acid on PEDV 3C-like proteases activity was examined by fluorescence resonance energy transfer test.【Result】Low concentrations of tannic acid was almost nontoxic to the cells.Tannic acid could inhibit the replication of PEDV YN13 strain in a concentration dependent manner,all concentrations of tannic acid could inhibit PEDV replication after changing PEDV strain and cell lines.Tannic acid could inhibit PEDV infection by inhibiting PEDV adsorption or invasion process and viral early replication,and also inhibit PEDV 3C-like protease activity in a concentration dependent manner.【Conclusion】Tannic acid could inhibit PEDV infection in vitro by affecting the viral adsorption or invasion process,and it might also inhibit the viral replication process by inhibiting the viral 3C-like protease.