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。

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-61and 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% of mice died (t=7.237, P<0.001). JD1-PP-31 showed weak protection, with 41.9% of mice died (t=4.752, P<0.01). Conclusions The purified JD1 strains without neurovirulence are screened out by plaque purification. The sites related to potentially reduced neurovirulence are found.