以乙脑减毒活疫苗SA14-14-2为骨架的重组嵌合登革4病毒的构建与鉴定  

CONSTRUCTION AND CHARACTERIZATION OF CHIMERIC DENGUE4 VIRUS USING THE JAPANESE ENCEPHALITIS LIVE VACCINE STRAIN AS BACKBONE

作　　者：解东洋 祝琴[3] 刘忠钰 赵慧 邓永强 叶青 李晓峰 秦成峰[1,2] XIE Dong-Yang;ZHU Qin;LIU Zhong-Yu;ZHAO Hui;DENG Yong-Qiang;YE Qing;LI Xiao-Feng;QIN Cheng-Feng(Anhui Medical University,Hefei,Anhui 230032,China;Department of Virology,State Key Laboratory of Pathogen and Biosecurity,Beijing Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Beijing 100071,China;Guangzhou Medical University,Guangzhou,Guangdong 510182,China)

机构地区：[1]安徽医科大学,安徽合肥230032 [2]军事科学院军事医学研究院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071 [3]广州医科大学附属广州市第八人民医院,广东广州510182

出　　处：《寄生虫与医学昆虫学报》2018年第2期80-86,共7页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家自然科学基金资助项目(No.81522025;81661148054)

摘　　要：登革4型病毒近年来在我国东南沿海地区时有流行,威胁人民健康与公共卫生安全。本研究以乙脑减毒活疫苗SA14-14-2为骨架,通过反向遗传学技术构建了携带登革4型病毒主要结构蛋白prM/E基因重组嵌合病毒(ChinDENV4);进一步通过蚀斑、生长曲线、动物实验等对其生物学特征进行了鉴定。结果显示,重组嵌合病毒Chin DENV4可高效表达登革4型病毒的结构蛋白,蚀斑大小与亲本株类似;ChinDENV4并能够在BHK-21、C6/36等细胞中高效复制,滴度分别达到1. 26×10~5和1. 58×10~6PFU/m L。更重要的是,嵌合病毒ChinDENV4在乳鼠模型中的神经毒力显著弱于亲本株。本研究成功构建了乙脑/登革4型嵌合病毒,为下一步四价嵌合登革减毒活疫苗研究奠定了重要基础。The co-circulation of dengue viruses （DENV） type 1-4 poses serious threat to public health. In this study, we rationally designed and constructed a novel recombinant chimeric dengue virus type 4 （DENV4） using the live-attenuated Japanese encephalitis virus （JEV） vaccine strain SA14-14-2 as the genetic backbone. By using the reverse genetic technology, the prM/E-coding region of JEV SA14-14-2 was replaced with the corresponding region of DENV4 strain （DENV4-GZ30） , resulting in a chimeric plasmid named pChinDENV4. Then the chimeric virus ChinDENV4 was rescued in BHK-21 cells from the in vitro transcribed RNAs derived from pChinDENV4. The chimeric virus was further characterized by standard plaque forming assay, growth curve, biochemical assay, and animal experiments. Combined assays showed that the recovered ChinDENV4 expressed the major structural protein of DENV4 and the non-structural proteins of JEV as expected, and the plaque morphology of ChinDENV4 was similar to wild type viruses. Especially, the chimeric virus replicated efficiently in both BHK-21 and C6/36 cells with a peak titers of 1.26 x 10^5 and 1.58 x 10^6 PFU/mL, respectively. More importantly, ChinDENV4 exhibited decreased neurovirulence in suckling mice compared with the parental viruses. The chimeric JEV/DENV4 virus described here has laid an important foundation for the further study of live-attenuated tetravalent chimeric DENV vaccines.

关 键 词：乙脑病毒 登革4型病毒 反向遗传学 减毒活疫苗 嵌合病毒 

分 类 号：R392[医药卫生—免疫学]