目的  构建乙型脑炎病毒减毒株SA14-14-2 囊膜蛋白第315位氨基酸（E315）回复突变株，并分析该位点回复突变后在小鼠体内的神经毒力变化。方法  采用重叠PCR扩增含基因组第1921核苷酸位点回复突变的基因片段，替换SA14-14-2全长克隆的相应区域，获得回复突变株M315。通过测定突变株的蚀斑大小和一步生长曲线并与SA14-14-2进行比较，分析M315的增殖特征；采用小鼠模型测定该突变株的脑内神经毒力、皮下感染入脑能力、腹腔感染入脑能力和乳鼠传代返祖，分析疫苗株E315位点回复突变后毒力的变化。结果  成功构建了回复突变株M315，其蚀斑大小和增殖特征与疫苗株相似。M315对17~19日龄小鼠无皮下或腹腔入脑神经毒力，鼠脑病毒有很低但可检测的脑内神经毒力（小于3.0 lg蚀斑形成单位（plaque forming unit，PFU） / 0.03 ml），皮下注射不发病。3~5日龄乳鼠脑内接种后发病，但脑内病毒毒力低于3.0 lgPFU / 0.03 ml。结论  在分子水平上证明了E315位点是影响乙型脑炎疫苗安全性的关键位点之一，但影响较弱。E315位点的回复突变虽使SA14-14-2的小鼠脑内神经毒力和乳鼠传代返祖能力增强，但没有超出中国药典规定的毒力范围。

Objective  To construct envelope 315 site (E315) revertant of Japanese encephalitis vaccine strain SA14-14-2 through reverse genetics technology and analyze the virulence change of the revertant in mice. Methods  The gene fragment containing the nucleotide site 1921 revert mutation in genome was amplified by overlap-PCR and replace corresponding fragment of SA14-14-2 to construct the revertant named M315. The plaque size and one-step growth curve of M315 was measured and compared with those of SA14-14-2 to exam the replication characteristics of M315.Neurovirulence by the intracerebral (i.c.) route and neuroinvasiveness by the subcutaneous (s.c.) route and intraperitoneal (i.p.) route of M315 and the ability of atavism in mice were tested. Results  The revertant M315 was constructed. The plaque size and growth characteristic of M315 were similar with the vaccine strain. M315 had no neuroinvasiveness by the s.c. and i.p. routes. The neurovirulence of virus isolated from diseased mice by i.c. route was less than 3.0 lg plaque forming unit (PFU) / 0.03 ml in 17-19 d old mice, no neuroinvasiveness by s.c. route. All 3-5 d old suckling mice developed disease after brain inoculation, but the neurovirulence was lower than 3.0 lgPFU / 0.03 ml. Conclusions  E315 site is proved at molecular level to be one of the key amino acid residues affecting the safety of Japanese encephalitis vaccine, but the effect is weak. Although the reverse mutation at E315 site enhances the neurovirulence of SA14-14-2 in i.c. inoculated 17-19 days old mice and the ability of atavism in suckling mice, it does not exceed the criterion specified in the China pharmacopoeia.