乙型脑炎病毒野毒株SA14包膜蛋白K279M突变对其神经毒力的影响  

The effect on K279M mutations in the envelope protein on the neurovirulence of Japanese encephalitis virus strain SA14

作　　者：冯亚岚 刘超越 李玥珂 黄荣[1] 袁磊 唐丽萍 杨健[1] FENG Ya-lan;LIU Chao-yue;LI Yue-ke;HUANG Rong;YUAN Lei;TANG Li-ping;YANG Jian(School of Basic Medical Sciences,North Sichuan Medical College Hospital,Nan-chong,Sichuan,China 637000;Infectious Diseases,North Sichuan Medical College Hospital,Nan-chong,Sichuan,China 637000)

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感染科,四川南充637000

出　　处：《中国病原生物学杂志》2019年第9期1029-1033,共5页Journal of Pathogen Biology

基　　金：南充市川北医学院校地合作项目(No.NSMC20170406)

摘　　要：目的构建乙型脑炎病毒野毒株SA14包膜蛋白K279M突变株感染性克隆并拯救病毒,探索K279M突变对其毒力的影响。方法应用重叠延伸PCR和分子克隆技术构建含有K279M突变的乙脑突变病毒全长cDNA质粒,经酶切鉴定后,以其为模板体外转录获得RNA,电转染BHK21细胞,得到恢复病毒JEV SA14(K279M)。分别用野毒株JEV SA14和JEV SA14(K279M)接种BHK21细胞和脑内接种昆明鼠,比较突变株与野毒株的蚀斑大小、生长特点以及对昆明鼠的神经毒力。结果酶切鉴定表明,突变病毒感染性克隆成功构建。病毒蚀斑试验显示,JEV SA14(K279M)的蚀斑直径约为2~3 mm,大于野毒株蚀斑直径的1~2 mm。突变株病毒LD50为0.21PFU,大于野毒株病毒LD50的0.05PFU。结论乙脑包膜蛋白K279M突变增大了病毒的蚀斑,但降低了病毒的脑内神经毒力。Objectives To construct an infectious clone of the SA14 wild strain of Japanese encephalitis virus(JEV)with a K279 M mutation in its envelope protein,to rescue the virus,and explore the effect of the K279 M mutation on its virulence.Methods A full-length cDNA plasmid of a mutant JEV containing the K279 M mutation was constructed using overlap extension PCR and molecular cloning.After restriction enzyme digestion and identification,the plasmid was used as a template to obtain viral RNA in vitro,followed by electrotransfection into BHK21 cells to obtain the recovered virus JEV SA14(K279 M).The mutant virus was detected with sequencing and a plaque assay.To compare the plaque size,growth characteristics,and neurovirulence of the virus,BHK21 cells and Kunming mice were inoculated with SA14 or JEV SA14(K279 M).Results Enzyme digestion verified that the infectious clone of the mutant virus was successfully constructed.The plaque assay indicated that the plaque diameter of JEV SA14(K279 M)was about 2-3 mm,which was larger than that of SA14(1-2 mm).The growth curve indicated that the viral titer of JEV SA14(K279 M)peaked 48 h after inoculation,while that of SA14 peaked 60 h after inoculation.The LD50 value of the mutant virus was 0.21 PFU when injected intracranially,which was greater than that of SA14(0.05 PFU).Conclusion A K279 M mutation in the envelope protein of JEV increases the plaque formed by the virus but it reduces the neurovirulence of the virus in the brain.

关 键 词：乙脑病毒 正向突变 神经毒力 

分 类 号：R373.31[医药卫生—病原生物学]