寨卡病毒包膜蛋白Y158H突变对乙脑/寨卡嵌合病毒感染小鼠脑内神经毒力的影响  

The effect of a mutant envelope protein Y158H of a chimeric Japanese encephalitis/Zika virus on intracerebral neurovirulence in mice

作　　者：李玥珂 冯亚岚 任阳 陈岚 黄荣[1] 周仲辉[2] 杨健[1] LI Yue-ke;FENG Ya-lan;RENG Yang;CHEN Lan;HUANG Rong;ZHOU Zhong-hui;YANG Jian(School of Basic Medical Sciences,North Sichuan Medical College,Nanchong 637000,Sichuan,China;Department of Infectious Disease^Affiliated Hospital to North Sichuan Medical College)

机构地区：[1]川北医学院基础医学院,四川南充637000 [2]川北医学院附属医院传染科

出　　处：《中国病原生物学杂志》2021年第6期655-660,共6页Journal of Pathogen Biology

基　　金：南充市川北医学院校地合作项目(No.18SXHZ0495)。

摘　　要：目的研究乙脑/寨卡嵌合病毒(Japanese encephalitis/Zika chimeric virus,JE/ZIKV)包膜蛋白(envelope protein,E蛋白)第158位氨基酸Y158H突变对感染小鼠脑内神经毒力的影响。方法运用重叠延伸PCR技术和基因克隆技术构建含Y158H突变全长cDNA的质粒,单酶切形成线性化DNA分子后体外转录获得RNA,电转染BHK21细胞完成重组病毒包装。通过病毒基因测序、间接免疫荧光试验、生长曲线及蚀斑试验鉴定病毒,通过动物试验评价病毒的小鼠脑内神经毒力。结果基因测序和酶切证实,成功构建含突变位点Y158H全长cDNA质粒。蚀斑试验和病毒基因测序、间接免疫荧光试验证实成功完成病毒的拯救,JE/ZIKV蚀斑直径(1.7±0.2)mm,JE/ZIKV(Y158H)蚀斑直径(1.6±0.1)mm,两种病毒的蚀斑大小差异无统计学意义(P>0.05)。两种病毒增殖规律相似,但整个过程中JE/ZIKV(Y158H)的增殖效率略低于母本病毒。分别用两种病毒进行动物试验,JE/ZIKV(Y158H)的LD50为58.93 PFU/0.03 ml,JE/ZIKV为2.21 PFU/0.03 ml,JE/ZIKV(Y158H)的毒力低于JE/ZIKV。结论ZIKV E蛋白Y158H位点突变可降低嵌合病毒对小鼠脑内神经毒力,E158是影响E蛋白神经毒力的重要氨基酸之一。Objective To study the effect of a Y158 H mutation of an envelope protein(E protein)of a chimeric Japanese encephalitis/Zika virus(JE/ZIKV)on its neurovirulence in mice.Methods A full-length cDNA plasmid containing the Y158 H mutation was constructed using overlap extension PCR and gene cloning technology.The plasmid was digested with a single enzyme to form a linearized DNA molecule.RNA transcribed in vitro was electro-transfected into BHK21 cells to rescue the recombinant virus.The virus was identified using virus gene sequencing,indirect immunofluorescence,a growth curve,and a plaque assay.The neurovirulence of the virus was evaluated in an animal experiment involving mice.Results Gene sequencing and enzyme digestion verified that a full-length cDNA plasmid containing the Y158 H mutation was successfully constructed.The plaque assay,gene sequencing,and indirect immunofluorescence verified that the virus was successfully rescued.The plaque diameter of JE/ZIKV was 1.7±0.2 mm and that of JE/ZIKV(Y158 H)was 1.6±0.1 mm.Plaque size did not differ significantly between the two viruses(P>0.05).The two viruses proliferate similarly,However,the proliferation efficiency of JE/ZIKV(Y158 H)was slightly lower than that of mother virus throughout the whole period.The LD50 of JE/ZIKV(Y158 H)was 58.93 PFU/0.03 mL and that of JE/ZIKV was 2.21 PFU/0.03 mL when the two viruses were tested in animals,indicating that the neurovirulence of JE/ZIKV(Y158 H)was weaker than that of JE/ZIKV.Conclusion The Y158 H mutation in the Zika virus E protein can reduce the neurovirulence of a chimeric virus.E158 is a key amino acid that affects the neurovirulence of the E protein.

关 键 词：乙脑/寨卡嵌合病毒 E蛋白 突变 神经毒力 

分 类 号：R373.3[医药卫生—病原生物学]