乙脑/寨卡病毒嵌合减毒活疫苗的构建与评价  

Construction and Preliminary Evaluation of Japanese Encephalitis/Zika Virus Chimeric Attenuated

作　　者：罗月 黄荣 陈岚 贺雅静 陈晨 谭宁 杨航 袁磊 杨健[1,2] LUO Yue;HUANG Rong;CHEN Lan;HE Ya-jing;CHEN Chen;TAN Ning;YANG Hang;YUAN Lei;YANG Jian(School of Basic Medicine and forensic medicine,North Sichuan Medical College,Nanchong 637000;Key Laboratory for metabolic drugs and biologicals of Nanchong,Nanchong 637000;Laboratory Department of Zigong Third People′s Hospital,Zigong 643000)

机构地区：[1]川北医学院基础医学与法医学院,四川南充637000 [2]川北医学院代谢病药物与生物制品南充市重点实验室,四川南充637000 [3]自贡市第三人民医院检验科,四川自贡643000

出　　处：《微生物学杂志》2025年第1期96-105,共10页Journal of Microbiology

基　　金：南充市川北医学院校地合作项目(18SXHZ0497,20SXQT0338,20SXQT0239)。

摘　　要：为构建乙脑/寨卡嵌合病毒减毒活疫苗候选株并评估其安全性及免疫保护作用,将寨卡病毒FSS13025株和PRVABC59株的prM/E基因分别替换至乙脑病毒疫苗株的相应区域,构建乙脑寨卡嵌合病毒全长感染性克隆,体外转录获得病毒RNA,并电转染入BHK-21细胞拯救获得ChimZIKV(13025)和ChimZIKV(59)两株嵌合病毒。酶切、免疫荧光及测序结果显示,感染性克隆构建正确且嵌合病毒拯救成功;蚀斑实验发现与亲本株相比,嵌合病毒的蚀斑直径变小(P<0.01);生长曲线显示,嵌合病毒在BHK-21和C6/36细胞中的增殖能力弱于乙脑疫苗株;细胞毒性检测发现嵌合病毒对传代细胞的毒性弱于乙脑疫苗株(P<0.01);嵌合病毒对小鼠表现出低神经侵袭力与较高的脑内神经毒力;ChimZIKV(13025)和ChimZIKV(59)免疫小鼠可产生较高的中和抗体效价,免疫雌鼠后,新生乳鼠对寨卡野毒株PRVABC59的攻击保护率分别为95%和89%,且在乳鼠体内检测不到病毒血症。本研究结果表明嵌合病毒ChimZIKV(13025)和ChimZIKV(59)具有较好的免疫原性和较低的神经侵袭力,免疫雌鼠后可对新生乳鼠提供较强的保护力,但对小鼠仍有一定的脑内神经毒力。To construct an attenuated live vaccine candidate for Japanese encephalitis(JE)/Zika chimeric virus and to assess its safety and immune protection effects.The prM/E genes of Zika virus strains FSS13025 and PRVABC59 were respectively replaced into the corresponding regions of the JE vaccine strain to construct full-length infectious clones of the JE/Zika chimeric virus.Viral RNA was transcribed in vitro,and electroporated into BHK-21 cells to rescue the two chimeric viruses,ChimZIKV(13025)and ChimZIKV(59).Results from restriction enzyme digestion,immunofluorescence,and sequencing showed that the infectious clones were correctly constructed,and the chimeric viruses were successfully rescued.Plaque assays indicated that the plaque diameters of the chimeric viruses were smaller compared to the parental strains(P<0.01).Growth curves showed that the chimeric viruses proliferated less efficiently in BHK-21 and C6/36 cells compared to the JE vaccine strain.Cytotoxicity assays revealed that the chimeric viruses exhibited lower toxicity to passage cells compared to the JE vaccine strain(P<0.01).In mice,the chimeric viruses exhibited low neuroinvasiveness and higher neurovirulence in the brain.Immunization of mice with ChimZIKV(13025)and ChimZIKV(59)induced higher neutralizing antibody titers.After immunizing female mice,the protection rates of newborn suckling mice against the wild-type Zika virus strain PRVABC59 were 95% and 89%,respectively,with no detectable viremia in the suckling mice.The results of this study indicate that the chimeric viruses ChimZIKV(13025)and ChimZIKV(59)possess good immunogenicity,low neuroinvasiveness,and provide strong protection to newborn suckling mice following immunization with the chimeric viruses.However,the chimeric viruses still exhibit some degree of neurovirulence in the mouse brain.

关 键 词：嵌合病毒 乙型脑炎病毒 寨卡病毒 免疫原性 神经毒力 神经侵袭力 

分 类 号：Q939.93[生物学—微生物学]