CAJ | 학술논문

目的鉴别日本脑炎疫苗株ML-17的两个大斑变异株(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的两个大斑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,分析引起斑形改变的基因突变,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10 977个,与ML-17和JaOH0566相比较,分别有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1×106FFU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,可能引起了斑形的改变;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.
목적 감별일본뇌염역묘주ML-17적량개대반변이주(ML-17L2화ML-17L4)적존재,병통과전기인조서렬측정분석인기반형화독력개변적기인돌변.방법 수선대역묘주ML-17재전대과정중출현적다반ML-17sh적량개대반변이주진행순화화감정,재대량개대반변이주진행전기인조서렬측정화전편마안기산서렬추도,병여소반역묘주ML-17급기대반친대야독주JaOH0566적전핵감산서렬화전편마안기산서렬진행비교,분석인기반형개변적기인돌변,병응용소백서시험검사ML-17변이전후적신경침습력화독력변화. 결과 전기인조서렬분석현시량개대반변이주적핵감산총수균위10 977개,여ML-17화JaOH0566상비교,분별유33화34개핵감산변이산재분포우ML-17L2화ML-17L4적전기인조중,기중우분별유12화13개핵감산도치료안기산적개변.재ML-17L2화ML-17L4적의각단백구몰유안기산변이,단재포막단백구각유일개안기산변이;재5'단비편마구몰유핵감산변이,단재3'단비편마구유3～4개핵감산변이,차각증가료일개액외적10699핵감산.ML-17화ML-17sh적소서LD50분별위대우1×106FFU화481FFU,변이후독력명현증강. 결론 발생재량개대반변이주적제5비결구단백구화기타구적안기산반조변이(변회여JaOH0566상동)화신변이,가능인기료반형적개변;이전부5개안기산반조변이화2～3개안기산신변이가능여기독력개변유관.본차연구위ML-17L2화ML-17L4적반형화독력개변제공료분자유전학기출.
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 - 17and ML- 17sh were more than 1 × 106 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 Ml - 17L4.