无小鼠神经毒力的乙型脑炎病毒／登革病毒1型嵌合病毒的筛选与序列分析  

Screening and sequence analysis of Japanese encephalitis virus/dengue virus type 1 chimeric virus without neurovirulence in mice

作　　者：何婷[1] 范凤鸣 牟建超[1] 杨欢 刘莉娜[1] 刘俐[1] 汪伟[1] 曾献武[1] 杨会强[1] He Ting;Fan Fengming;Mou Jianchao;Yang Huan;Liu Lina;Liu Li;Wang Wei;Zeng Xianwu;Yang Huiqiang(Viral Vaccine Research Laboratory,Chengdu Institute of Biological Products Co.,Ltd.,Chengdu610023,China.)

机构地区：[1]成都生物制品研究所有限责任公司病毒疫苗研究室,610023

出　　处：《国际生物制品学杂志》2018年第4期157-161,共5页International Journal of Biologicals

基　　金：国家高技术研究发展计划（863计划）（2012AA02A401）

摘　　要：目的采用蚀斑纯化法筛选对小鼠无神经毒力的乙型脑炎病毒（Japanese encephalitis virus，JEV）／登革病毒（denguevirus，DENV）1型嵌合病毒（JD1）纯化株，分析影响JD1小鼠神经毒力的相关位点。方法将JD1在乳地鼠。肾细胞中进行3轮蚀斑纯化，检测JD1纯化株对小鼠的脑内神经毒力。然后提取无神经毒力纯化株的基因组RNA，用逆转录PCR分段扩增全基因组序列并进行序列测定。将测序结果分别与原DENV-1的前膜-包膜蛋白和JEV骨架病毒株SA-14-14-2的基因序列进行比对。选取2个无神经毒力纯化株进行小鼠免疫保护实验。采用单侧￡检验对结果进行比较。结果经过3轮蚀斑纯化后，获得大、中、小3种蚀斑形态的6个JD1纯化株。3个纯化株对小鼠无神经毒力，小鼠脑内注射后全部存活；1株神经毒力较弱，70％以上小鼠存活；2株具有强神经毒力，小鼠全部死亡。测序结果显示，对小鼠无神经毒力的3个纯化株存在2处相同的氨基酸突变。将其中2个纯化株JD1-PP-61和JD1-PP-31腹腔免疫小鼠后，再用1000半数致死量DENV-1鼠脑适应株进行脑内攻击。JD1-PP-61能较好地保护小鼠抵抗病毒攻击，仅20．0％小鼠死亡（t=7．237，P〈0．001）；JD1-PP-31保护效果稍弱，41．9％小鼠死亡（t=4．752，P〈0．01）。结论通过蚀斑纯化筛选出对小鼠无神经毒力的JD1纯化株，并发现了可能与神经毒力减弱相关的位点。Objective To screen Japanese encephalitis virus （JEV）/dengue virus type 1 （DENV- 1） chimeric virus （JD1） purified strains without neurovirulence in mice by plaque purification method and analyze neurovirulence-related sites. Methods The purified JD1 strains were obtained by 3 rounds of plaque purifications in neonatal hamster kidney cells, and detected for intracerebral neurovirulence in mice. Genomic RNAs of non-neurovirulent JD1 strains were extracted, and the whole genome sequence was fragmentally amplified by reverse transcription PCR and sequenced. The sequencing results were compared with the gene sequences of premembrane-envelope protein in parental DENV-1 and backbone JEV SA-14-14-2 strain. Two JD1 strains without neurovirulence were selected and tested for protective efficacy in mice. The results were analyzed using one-tailed t-test. Results After 3 rounds of plaque purifications, 6 purified JD1 strains with big, middle or small plaque morphology were obtained. Of which, 3 strains showed no neurovirulenee with all mice survived, 1 strain had slight neurovirulence with 70% mice survived, and the rest 2 had strong neurovirulence with all mice died. Sequencing results indicated that the 3 non-neurovirulent strains had 2 identical amino acid mutations. Two of the above 3 strains, JD1-PP-61 and JDI-PP-31 ,were intraperitoneally injected in mice followed by intracerebral challenge with 1 000 50% lethal dose of a DENV-1 mouse brain-adapted strain. JD1-PP-61 protected mice against DENV-1 infection, with only 20. 0% mice died （t = 7. 237, P 〈0. 001）. JD1-PP-31 showed weak protection, with 41.9% mice died （t = 4. 752, P 〈0. 01）. Conclusions The purified JD1 strains without neurovirulenceare are screened out by plaque purification. The sites potentially related to reduced neurovirulence are found.

关 键 词：登革热病毒 脑炎病毒 日本 疫苗 减毒 蚀斑纯化 神经毒力 

分 类 号：R373[医药卫生—病原生物学]