NanoLuc标记的重组克里米亚-刚果出血热病毒及其初步用于抗病毒药物筛选

摘要

目的 为了构建可分泌表达NanoLuc荧光素酶的重组克里米亚-刚果出血热病毒(Crimean-Congo hemorrhagic fever virus,CCHFV)并探讨其用于抗病毒药物高通量快速初筛的可行性.方法 将NanoLuc基因与CCHFV M节段上的mucin编码框融合,拯救重组病毒,并以利巴韦林为阳性对照,评估Furin抑制剂在体外对重组病毒的抑制效果.结果 成功获得可分泌表达NanoLuc荧光素酶的重组CCHFV(rCCHFV mucin NLuc),且在感染细胞上清中,表示NanoLuc活性的相对光单位(relative light unit,RLU)与病毒半数组织感染剂量(TCID50)呈显著正相关性(cor=0.998,P=0.001).10μmol/L浓度下,Furin抑制剂处理后1～3 d感染细胞上清中NanoLuc荧光素酶活力与利巴韦林处理组无明显差异(P>0.1),但第4天Furin抑制剂处理组上清中RLU值显著高于利巴韦林处理组(P=0.001),且与未处理病毒对照组无明显差异(P>0.1).结论 成功构建1株NanoLuc标记的rCCHFV并可用于抗病毒药物的体外快速初筛.%Objective To construct the recombinant Crimean-Congo hemorrhagic fever virus (CCHFV) which can express the secreted nanoluciferase (NanoLuc) and investigate its potential application for rapid antiviral compounds screening. Methods The ORF of NanoLuc and the mucin encoded by the M segment of CCHFV were merged, and the recombinant CCHFV ( rCCHFV) was rescued through reverse genetic system. Then rCCHFV was used to evaluate the antiviral effect for ribavirin and Furin inhibitor in vitro. Results The rCCHFV mucin NLuc with NanoLuc reporter was obtained, and the relative light unit (RLU) which can reflect NanoLuc activity was positively correlated with median tissue culture infective dose (TCID50 ) in the infected cell supernatant ( cor = 0. 998, P = 0. 001). When the concentration for the compounds was 10 μmol/ L, there was no significant difference for the NanoLuc activity in the infected cell supernatant between Furin inhibitor and ribavirin (P > 0. 1) from day 1 to 3 after treatment. But at day 4, the NanoLuc activity in Fruin inhibitor treated group was significantly higher than that of ribavirin treated group (P = 0. 001), and no significant difference was found between the Furin inhibitor and untreated group (P > 0. 1). Conclusions The rCCHFV with NanoLuc reporter was recovered successfully and it could be used for the primary rapid screening of antiviral compounds in future.