日本脑炎ML-17两个变异株的基因组序列及突变分析(英文)

Analysis of complete nucleotide sequences and mutations of 2 variants of attenuated Japanese encephalitis live vaccine strains ML-17

目的鉴别日本脑炎疫苗株ML-17的两个大宽变异株（ML-17L2和ML-17L4）的存在，并通过全基因组序列测定分析引起斑形和毒力改变的基因突变。方法首先对疫苗株ML-17在传代过程中出现的多斑ML-17sh的两个大宽变异株进行纯化和鉴定，再对两个大斑变异株进行全基因组序列测定和全编码氨基酸序列推导。并与小斑疫苗株ML-17及其大斑亲代野毒株JaOH0566的全核苷酸序列和全编码氨基酸序列进行比较，分析引起宽形改变的基因突变。并应用小白鼠试验检查ML-17变异前后的神经侵袭力和毒力变化。结果全基因组序列分析显示两个大斑变异株的核苷酸总数均为10977个，与ML-17和JaOH0566相比较，分剐有33和34个核苷酸变异散在分布于ML-17L2和ML-17L4的全基因组中，其中又分别有12和13个核苷酸导致了氨基酸的改变。在ML-17L2和ML-17L4的衣壳蛋白区没有氨基酸变异，但在包膜蛋白区各有一个氨基酸变异；在5’端非编码区没有核苷酸变异，但在3’端非编码区有3～4个核苷酸变异，且各增加了一个额外的10699核苷酸。ML-17和ML-17sh的小鼠LD50分别为大于1×10^6FFU和481FFU。变异后毒力明显增强。结论发生在两个大斑变异株的第5非结构蛋白区和其它区的氨基酸返祖变异（变回与JaOH0566相同）和新变异，可能引起了宽彤的改变；而全部5个氨基酸返祖变异和2—3个氨基酸新变异可能与其毒力改变有关。本次研究为ML-17L2和ML-17L4的斑形和毒力改变提供了分子遗传学基础。

Objective To identify the two large plaque variants （ML - 17L2 and ML - 17L4） of vaccine strain ML - 17 and clarify the gene mutations which might cause plaque morphological and virulent changes through the full - length genome sequencing of the 2 ML- 17 variants. Methods The large plaque variants of ML- 17 from ML- 17sh （mixed plaque sizes） were purified and confirmed, the mutations which caused the different plaque morphology through genome sequencing was analized, deducing amino acid （AA） sequence and with attenuated ML- 17（small plaque） and its wild- type strain JaOH0566 （large plaque） wmpared, and the neuroinvasiveness/neurovirulence using animal model was checked. Results The genomic sequence analysis revealed that the 2 virion RNA molecules were 10 977 nucleotides（NT） long,compared with attenuated vaccine strain ML- 17 and its parental wildtype JaOH0566, 33 and 34 NT substitutions were found to be scattered all over the genomes of ML- 17L2 and ML- 17L4, respectively, of these, correspondingly, 12 and 13 resulted in AA changes within viral proteins. No AA mutation was found in C protein region of each variant. But in E protein region, one AA change in each of ML- 17L2 and ML - 17L4. No NT change in the 5＇ - terminal untranslated regions of the 2 variants. But there were 3 - 4 NT changes in the 3＇ - terminal untranslated regions of the 2 variants, moreover, there was an additional NT change at the same position （NT 10699） of each variant. The mouse LD50 of ML - 17 and ML - 17sh were more than 1 × 10^6 FFU and 481 FFU, respectively. Conclusion All these reverse （ back to JaOH0566） and new mutations in NS5 and other regions of the 2 variants might be resposible for the plaque morphological changes. The virulence of 2 large plaque variants might also be changed, because 5 AA reverse mutations and 2, 3 AA new changes in ML - 17L2, ML - 17L4, respectively. These data and comparative results could provide a molecular basis for plaque morphological and virulent changes of ML- 17L2 and M1 - 17L4.