基于NanoLuc荧光素酶的重组克里米亚-刚果出血热病毒的构建及体外抗病毒药物快速筛选模型研究

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral infectious disease, and its causative agent, Crimean-Congo hemorrhagic fever virus (CCHFV), is classified to Biosafety level 4 (BSL-4) agent. Currently, there is no licensed vaccine or antiviral drug for CCHF. Due to the range for its tick vector turns larger, and many regions belong to China's one belt one road strategy are the epidemic or high risk areas for CCHF, the possibility for the re-outbreak of CCHF in China is increasing. So it's necessary to strengthen the study for the CCHFV and developing anti-CCHF drugs. The recombinant virus with reporter signal is widely used for the antiviral drug screen, viral replication and life cycle, and viral live imaging, however there is no related report for recombinant CCHFV.In this study, it will be the first time for NanoLuciferase(NanoLuc), a new reporter introducing to the M segment for CCHFV through In-Fusion cloning and reverse genetic technology, which after the coding region for mucin, gp38, Gn, and Gc respectively. Then different infectious clones for CCHFV were rescued and screened. Finally, a recombinant CCHF virus which can stable express NanoLuc signal and has the same infection feature with parent virus will be acquired, then used to develop a rapid in vitro antiviral screening model and applied to the assessment the anti-CCHF efficiency for 4 broad-spectrum antiviral agents. The result will improve the research platform for CCHF and make a new way for the investigating of anti-CCHF drugs.

克里米亚-刚果出血热（CCHF），是一种蜱传病毒性疾病，无针对性治疗药物或疫苗，其病原属生物安全四级危害病原。随着其媒介蜱活动范围扩大，以及我国一带一路战略必经之地多为CCHF高发和易感区，极大增加了CCHF在我国再爆发的风险，因此加强对其研究和药物研发及其重要。带报告基因的重组病毒被广泛用于药物筛选、病毒复制循环和活体成像等研究，但未见有重组CCHF病毒的相关报道。本项目拟通过In-Fusion克隆和反向遗传技术，首次将NanoLuc荧光素酶，一种新型报告基因引入CCHF病毒基因组M节段上mucin、gp38、Gn和Gc编码框后, 分别拯救筛选感染性克隆，获得可稳定表达NanoLuc并与亲本感染特征保持一致的重组CCHF病毒，进一步用于体外抗病毒药物快速筛选模型的建立并评估4种广谱抗病毒抑制剂的体外抗CCHF病毒药效，这将有助于完善CCHF的研究平台并为其药物的研发提供新的途径。

克里米亚-刚果出血热（CCHF），是一种蜱传病毒性疾病，无针对性治疗药物或疫苗，其病原属生物安全四级危害病原。随着其媒介蜱活动范围扩大，以及我国一带一路战略必经之地多为CCHF高发和易感区，极大增加了CCHF在我国再爆发的风险，因此构建带报告基因的重组CCHFV病毒可用于药物筛选、病毒复制循环和活体成像等研究。本项目通过In-Fusion克隆和反向遗传技术，首次将NanoLuc荧光素酶引入CCHF病毒基因组M节段,成功获得1株可稳定分泌表达NanoLuc荧光素酶并与亲本感染特征保持一致的重组CCHFV（rCCHFV-mucin_Nluc），感染细胞上清中，表示NanoLuc活性的相对光单位（relative light unit, RLU）与病毒的半数组织感染剂量（TCID50）呈显著正相关性（cor=0.998，p=0.001）。并使用利巴韦林和Furin抑制剂评估表明该系统可用于体外抗病毒药物的快速初筛。