乙脑/寨卡嵌合病毒包膜蛋白V343A及I341V/V343A联合突变对小鼠脑内神经毒力的影响  

Effect on Intracerebral Neurovirulence in Mice of V343A and I341V/V343A Joint Mutation in Envelope Protein of Japanese Encephalitis/Zika Chimeric Virus

作　　者：陈岚 李玥珂 任阳 黄荣 冯亚岚[1] 袁磊 杨健[1] CHEN Lan;LI Yue-ke;REN Yang;HUANG Rong;FENG Ya-lan;YUAN Lei;YANG Jian(Schl.of Basic Med.Sci.&Forensic Med.,North Sichuan Med.Coll.,Nanchong 637000)

机构地区：[1]川北医学院基础医学与法医学院,四川南充637000

出　　处：《微生物学杂志》2023年第6期104-112,共9页Journal of Microbiology

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

摘　　要：为探讨乙脑/寨卡嵌合病毒JE/ZIKV(MR766)的包膜蛋白V343A突变及I341V/V343A联合突变对小鼠神经毒力的影响,分别构建了V343A和I341V/V343A联合突变的嵌合病毒全长cDNA质粒,经酶切鉴定后,体外转录获得病毒RNA,并电转染入BHK21细胞拯救病毒。利用病毒测序和免疫荧光实验鉴定病毒;蚀斑实验和生长曲线对比突变病毒与亲本株生物学特性差异;动物实验比较神经毒力差异。基因测序与免疫荧光等证明突变病毒JE/ZIKV(V343A)和JE/ZIKV(I341V/V343A)拯救成功。突变病毒JE/ZIKV(V343A)和JE/ZIKV(I341V/V343A)的蚀斑直径分别为(1.09±0.15) mm和(1.15±0.29) mm,小于亲本株JE/ZIKV(MR766)蚀斑直径(2.09±0.36) mm(P<0.001);生长曲线显示两突变病毒增殖速度均快于亲本株,且JE/ZIKV(I341V/V343A)快于JE/ZIKV(V343A);动物实验结果显示,JE/ZIKV(V343A)的LD50为5.62 pfu/0.03 mL,JE/ZIKV(I341V/V343A)的LD50为34.84 pfu/0.03 mL,均高于亲本株(LD50=2.21 pfu/0.03 mL)。研究结果表明,寨卡病毒E蛋白V343A突变使嵌合病毒小鼠脑内神经毒力减弱,I341V/V343A联合突变使病毒毒力进一步减弱,表明E蛋白的341和343位氨基酸残基参与了嵌合病毒对小鼠的脑内神经毒力的调控。In order to explore the effect of V343A mutation and I341V/V343A mutation on envelope protein(E protein)of chimeric virus JE/ZIKV(MR766)on neurovirulence in mice,full-length cDNA plasmids containing V343A and I341V/V343A joint mutations were constructed respectively.And after identification by enzyme digestion,the viruses′RNA was obtained in vitro transcription,then transferred RNA into BHK21 cells by electro-transfection to save the mutant viruses.Sequencing and immunofluorescence experiments were used to identify the viruses.Plaque experiments and growth curves were used to compare the biological characteristics difference of mutant viruses and parental strains,animal experiments were performed to compare the differences in their neurovirulence.And mutant viruses JE/ZIKV(V343A)and JE/ZIKV(I341V/V343A)were rescued successfully proved by gene sequencing and immunofluorescence.The plaque diameter of JE/ZIKV(V343A)and JE/ZIKV(I341V/V343A)were(1.09±0.15)mm and(1.15±0.29)mm respectively,and smaller than parental strain JE/ZIKV(MR766)(2.09±0.36)mm(P<0.001).The growth curves showed that both two mutant viruses proliferated faster than JE/ZIKV(MR766),and JE/ZIKV(I341V/V343A)was faster than JE/ZIKV(V343A).The LD50 of JE/ZIKV(V343A)was 5.62 pfu/0.03 mL,and the LD50 of JE/ZIKV(I341V/V343A)was 34.84 pfu/0.03 mL,all were higher than parental strain(LD50=2.21 pfu/0.03 mL).The results showed that E protein mutation of Zika virus V343A weakened the neurovirulence of chimeric virus,and the virus virulence was further weakened after joint mutation of I341V/V343A,suggested that amino acid residues 341 and 343 of E protein participated in regulating the intracerebral neurovirulence of chimeric virus in mice.

关 键 词：寨卡病毒 乙脑病毒 嵌合病毒 E蛋白 神经毒力 

分 类 号：Q939.93[生物学—微生物学]