基孔肯雅病毒单轮感染性病毒样颗粒的制备及其免疫效果评价

In recent years, the re-emerging Chikungunya fever represents an important mosquito-borne infectious disease, posing significant threats to public health in tropical and subtropical regions. Currently there are no vaccines or antiviral therapies to treat or prevent Chikungunya fever in humans. In our previous study, we isolated a Chikungunya virus (CHIKV) during an outbreak in Dongguan, China, and constructed a chimeric CHIKV using Venezuelan Equine Encephalitis virus (VEEV) replicon as backbone by standard reverse genetic technology. Based on these results, we aim to establish a platform to produce single-round infectious virus-like particles (SRIPs) of CHIKV for vaccine development. Two necessary components will be developed: a stable mammalian cell line that constitutively expresses the capsid of CHIKV and a capsid defective VEE/CHIKV. This trans-complementation system will be developed by transfection of capsid defective VEE/CHIKV RNA into the capsid-expressing cells to produce the SRIPs. Then the biological characterizations of SRIPs will be tested by IFA, WB assays and electron microscopic analysis, together with the analysis on the infectivity and thermal stability of SRIPs. Subsequently, the immunogenicity and protection capacity of SRIPs will be evaluated in BALB/c mice and an A129 mouse model of Chikungunya virus infection. These results will lay the foundation for the development of efficacious vaccines against CHIKV infection. The trans-complementation system for SRIPs developed in this project will also be useful for many aspects of CHIKV research such as viral replication and particle assembly.

基孔肯雅热是一种典型的新突发虫媒传染病，严重威胁热带和亚热带地区公共卫生安全，临床上尚无有效的疫苗和抗病毒药物。最近，我们自东莞基孔肯雅热患者体内分离获得了一株基孔肯雅病毒，并成功构建了感染性全长cDNA克隆，拯救获得了委马/基孔肯雅嵌合病毒株。在此基础上，本研究将分别构建衣壳蛋白缺陷型委马/基孔肯雅嵌合病毒和稳定表达基孔肯雅病毒衣壳蛋白的细胞系，通过反式互补的方法，获得基孔肯雅病毒单轮感染性病毒样颗粒（SRIP）；通过免疫荧光、WB、电镜和稳定性试验等鉴定其生物学特征，在不同细胞中观察其单轮感染特征；进一步免疫小鼠后测定其诱导的细胞和体液免疫反应，并利用A129小鼠感染模型评估其免疫保护效果。本研究将能够建立基孔肯雅病毒SRIP的高效包装系统，为新型基孔肯雅热疫苗的研制奠定基础，对病毒复制和装配机制的研究也具有重要意义。

基孔肯雅热是一种典型的新突发虫媒传染病，严重威胁热带和亚热带地区公共卫生安全，临床上尚无有效的疫苗和抗病毒药物。本课题对我国基孔肯雅病毒分离株进行了分析，构建了感染性全长cDNA 克隆，建立了必要的小动物感染模型，并分别利用委马/基孔肯雅嵌合病毒、基孔肯雅/乙脑、基孔肯雅/黄热、基孔肯雅/腺病毒等不同的嵌合表达系统，对不同组合的基孔肯雅病毒结构蛋白E3-E2-E1的表达及其免疫原性和免疫保护进行了观察，结果发现表达E3-E2或E3的乙脑或黄热的嵌合重组病毒具有良好的减毒特性和免疫原性。本研究的摸索，为完善基孔肯亚病毒样颗粒类型疫苗研究提供重要科学依据和指导。

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