乙脑/寨卡嵌合病毒包膜蛋白T120A位点突变对小鼠脑内神经毒力的影响

Effect of T120A Mutation in Envelope Protein of Japanese Encephalitis/Zika Chimeric Virus on Virulence in Mice Intracelebral Nerve

为探索包膜蛋白T120A突变对乙脑/寨卡嵌合病毒JEV/ZIKV小鼠脑内神经毒力的影响,应用重叠延伸PCR和分子克隆技术构建含T120A突变的乙脑/寨卡嵌合病毒全长cDNA质粒,经酶切、测序鉴定后,以其为模板体外转录制备RNA,电转染导入BHK21细胞,收获培养液上清获得突变病毒株JEV/ZIKV(T120A)。分别用JEV/ZIKV和JEV/ZIKV(T120A)感染BHK21细胞,绘制病毒增殖曲线,比较蚀斑大小;脑内接种昆明鼠,比较病毒小鼠脑内神经毒力差异。突变病毒感染性克隆经酶切鉴定表明成功构建,增殖曲线发现:突变病毒高峰滴度为5.59 log10pfu/mL,低于JEV/ZIKV的峰值(6.8 log10pfu/mL),JEV/ZIKV(T120A)的小鼠脑内神经毒力LD50为1.38 PFU(0.03 mL),JEV/ZIKV病毒LD50为2.21 PFU(0.03 mL)。研究表明寨卡病毒包膜蛋白T120A突变降低了乙脑/寨卡嵌合病毒的复制能力,但却轻微增强了病毒小鼠脑内神经毒力。

The effect of T120A mutation on envelope protein of Japanese Encephalitis/Zika chimeric virus(JEV/ZIKV)on virulence in mice intracelebral nerve was investigated.The full-length cDNA plasmid of chimeric virus containing T120A mutation was constructed with overlap extension PCR and molecular cloning techniques.After identification with restriction enzyme digestion and sequencing,RNA was prepared with in vitro transcription using the plasmid as a template,and electro-transfected into BHK21 cells,and the mutant strain JEV/ZIKV(T120A)was harvested in culture supernatant.BHK21 cells were infected with JEV/ZIKV and JEV/ZIKV(T120A)respectively,the plaque size was compared,and the viral proliferation curve was drawn;Kunming mice were inoculated intracerebrally,and the difference of intracerebral neurovirulence in mice was compared.Through clone digestion characterization of the mutant virus infectiousness indicated that the construction was a success.The proliferation curve found that the peak titer of mutant virus was at 5.59 log10pfu/mL,lower than that of the peak value of JEV/ZIKV(6.8 log10pfu/mL).And the LD50 of JEV/ZIKV(T120A)intracerebral virulence in mice was 1.38 PFU(0.03 mL)(plaque forming units),LD50 of JEV/ZIKV was 2.21 PFU(0.03 mL).This study showed that the mutation of envelope protein T120A mutation of Zika virus weakened the viral replication ability of JEV/ZIKV,but enhanced the intracerebral neurovirulence of the virus in mice lightly.