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首页> 中文期刊> 《国际生物制品学杂志》 >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

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

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摘要

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 neurovirulence 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 JD1-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.%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(dengue virus,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t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1 000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.

著录项

  • 来源
    《国际生物制品学杂志》 |2018年第4期|157-161|共5页
  • 作者

    何婷; 范凤鸣; 牟建超; 杨欢; 刘莉娜; 刘俐; 汪伟; 曾献武; 杨会强;

  • 作者单位

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

    610023 成都生物制品研究所有限责任公司病毒疫苗研究室;

  • 原文格式 PDF
  • 正文语种 chi
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  • 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;

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