上海巴斯德所建立可视化的埃博拉病毒药物评价动物模型

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图A：包裹有荧光素酶蛋白的埃博拉病毒样颗粒的电镜照片；图B：对照小鼠（左）和病毒样颗粒感染小鼠（右）的体内荧光信号。

7月20日，国际学术期刊《病毒学杂志》（Journal of Virology）在线发表了中国科学院上海巴斯德研究所黄忠课题组的研究成果An Ebola virus-like particle-based reporter system enables evaluation of antiviral drugs in vivo under non-BSL-4 conditions（《一种基于病毒样颗粒的报告系统可用于非生物安全4级条件下抗埃博拉病毒药物的体内评价》），该研究成功建立了一种安全有效的埃博拉病毒药物评价动物模型。

埃博拉病毒是迄今为止已知最致命的病毒之一。2013-2015年，西非爆发了历史上最大规模的埃博拉疫情。据世界卫生组织统计，此次疫情直接导致超过28,000人感染，超过11,000人死亡，引起了国际社会的广泛关注。然而，至今尚无商业化的抗埃博拉病毒药物或疫苗可供使用。由于埃博拉病毒的高致死性，操作感染性埃博拉病毒必须在生物安全4级（BSL-4）条件下进行。目前，全球仅有30多个BSL-4实验室，其有限的空间和昂贵的运营成本，不能满足候选药物和疫苗高通量筛选及保护效果评价的需求，从而严重限制了抗埃博拉药物和疫苗的研发。

为创建不依赖于BSL-4条件的埃博拉病毒药物评价动物模型，上海巴斯德所博士研究生李大鹏、硕士研究生陈探等在研究员黄忠的指导下，利用基因工程手段，制备了包装报告蛋白荧光素酶的埃博拉病毒样颗粒。这种报告病毒样颗粒无传染性，但具有类似于天然埃博拉病毒的丝状结构，能够在体外模拟埃博拉病毒入侵细胞，并将所携带的荧光素酶传递进入靶细胞中，与底物作用后产生可直接检测的荧光信号。活体实验显示，注射了报告病毒样颗粒的小鼠在局部产生荧光，可通过活体成像技术对早期侵染进行动态追踪。进一步研究发现，保护性单克隆抗体13C6可显著减弱报告病毒样颗粒在小鼠体内所产生的荧光，证明了该系统作为BSL-4实验室外抗病毒药物和疫苗保护性评价模型的可行性和有效性。该动物模型具有安全、高效及可视化等优点，将对全球范围内抗埃博拉药物和疫苗的研发产生积极影响。

该研究得到了上海巴斯德所疫苗中心金侠和周东明的科研支持，获得了中科院分子病毒与免疫重点实验室埃博拉病毒疫情防控应急项目的经费支持。

来源：上海巴斯德研究所

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上海巴斯德所博士研究生李大鹏、硕士研究生陈探等在研究员黄忠的指导下完成

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An Ebola virus-like particle-based reporter system enables evaluation of antiviral drugs in vivo under non-BSL-4 conditions

Dapeng Li1, Tan Chen1, Yang Hu1, Yu Zhou1, Qingwei Liu1, Dongming Zhou1, Xia Jin1 and Zhong Huang1*

ABSTRACT
Ebola virus (EBOV) is a highly contagious lethal pathogen. As a biosafety level four (BSL-4) agent, however, EBOV is restricted to costly BSL-4 labs for experimentation, thus significantly impeding the evaluation of EBOV vaccines and drugs. Here, we reported an EBOV-like particle (EBOVLP) based luciferase reporter system that enables the evaluation of anti-EBOV agents in vitro and in vivo outside the BSL-4 facilities. Co-transfection of HEK293T cells with four plasmids encoding the VP40, NP, and GP proteins of EBOV, and the firefly luciferase (Fluc), respectively, resulted in the production of Fluc-containing filamentous particles that morphologically resemble authentic EBOV. The reporter EBOVLP was capable of delivering Fluc into various cultured cells in a GP-dependent manner, and recognized by a conformation-dependent anti-EBOV monoclonal antibody (mAb). Significantly, inoculation of mice with the reporter EBOVLP led to the delivery of Fluc protein into target cells and rapid generation of intense bioluminescence signals that can be blocked by the administration of EBOV neutralizing mAbs. This BSL-4-free reporter system should facilitate high-throughput screening for anti-EBOV drugs targeting viral entry and efficacy testing of candidate vaccines.


IMPORTANCE
Ebola virus (EBOV) researches were limited in costly biosafety level four (BSL-4) facilities due to the lack of animal model independent of BSL-4 lab. In this study, we revealed that a firefly luciferase bearing EBOV-like particle (EBOVLP) with typical filamentous EBOV morphology was capable of delivering the reporter protein into murine target cells both in vitro and in vivo. Moreover, we demonstrated that the reporter delivery could be inhibited both in vitro and in vivo by a known anti-EBOV protective monoclonal antibody 13C6. Our work provides a BSL-4-free system that can facilitate the in vivo evaluation of anti-EBOV antibodies, drugs and vaccines. The system may also be useful for mechanistic study of the viral entry process.

http://jvi.asm.org/content/early/2016/07/14/JVI.01239-16.abstract