寨卡病毒包膜蛋白D393E突变对乙脑/寨卡嵌合病毒小鼠脑内神经毒力的影响

Effect of D393E mutation in envelope protein of Japanese encephalitis/Zika chimeric virus on the intracerebral neurovirulence in mice

目的构造乙脑病毒(Japanese encephalitis virus,JEV)/寨卡病毒(Zika virus,ZIKV)嵌合株JEV/ZIKV包膜蛋白D393E突变的病毒感染性克隆并进行突变株的拯救,探讨D393E突变对病毒小鼠脑内神经毒力的影响情况。方法利用分子克隆和重叠延伸PCR技术构造含D393E突变的嵌合病毒质粒,并用线性化处理后的质粒片段进行转录,取得病毒RNA,电转染入细胞,获得突变株。用突变病毒、源病毒分别感染细胞和小鼠,对比两种病毒的复制效率、蚀斑特点以及脑内神经毒力差异。结果酶切结果证实:突变株JEV/ZIKV(D393E)感染性克隆成功地构建。病毒蚀斑实验:JEV/ZIKV(D393E)的蚀斑直径为0.7±0.2 mm,JEV/ZIKV蚀斑直径为1.3±0.2 mm。病毒小鼠脑内毒力检测:JEV/ZIKV(D393E)的LD50为31.51PFU,JEV/ZIKV的LD50为2.21PFU。结论ZIKV D393E突变使嵌合病毒对小鼠的脑内神经毒力减弱。

Objective To explore the effect of D393E mutation on its intracerebral neurovirulence in mice after infectious clone of the chimeric Japanese encephalitis virus(JEV)/Zika virus(ZIKV)containing envelope protein D393E mutant was built and the virus was rescued.Methods The full-length cDNA plasmid of JEV/ZIKV chimeric virus containing D393E mutation in the envelope protein was built by SOE PCR and gene cloning technology.The linearized fragment of the plasmid was used for in vitro transcription to obtain viral RNA,followed by electrotransfection into BHK-21 cells to rescue the mutant virus JEV/ZIKV(D393E).The plaque size and growth characteristics of JEV/ZIKV(D393E)and JEV/ZIKV were compared after BHK-21 cells were inoculated with the viruses.The intracerebral neurovirulence of the viruses was calculated after the viruses were inoculated in mice intracerebrally(i.c.).Results Restriction enzyme digestion evaluation confirmed that the infectious clone of mutant virus was correctly built.The plaque assay demonstrated that the plaque diameter of JEV/ZIKV(D393E)was about 0.7±0.2 mm,which was smaller than that of JEV/ZIKV(1.3±0.2 mm).The LD50 value of the mutant was 31.51 PFU when injected in mice via i.c.route,which was greater than 2.21 PFU of JEV/ZIKV.Conclusions The D393E mutation of ZIKV envelope protein in JEV/ZIKA chimeric virus decreases the neurovirulence of the virus in mice.