我国两株森林脑炎病毒的生物学性质及E蛋白基因核苷酸序列的比较

目的：比较我国1953年从患者脑组织分离的森张株和2001年从患者血清中分离的MDJ01株森林脑炎病毒(TBE)的生物学特性及E蛋白基因核苷酸序列的变化，分析TBE病毒E蛋白基因的变化与功能的关系，从分子水平上探讨我国TBE的传播、流行规律及致病机制。方法：两株病毒同时接种乳鼠和，BHK／21细胞，观察两株病毒对乳鼠和BHK／2l细胞的致病性；从两株病毒感染的BHK／2l细胞中提取病毒的RNA，对E蛋白基因进行RT-PCIR扩增，扩增产物纯化后与pGEM-T载体连接，分别进行克隆测序。结果：森张株对乳鼠和BHK／21的致病性均比MDJ01株明显；两株病毒E蛋白基因长为l488nt，编码496个氨基酸，核苷酸的变异为33个，导致了2个氨基酸的变化，第一个氨基酸的变异发生在113位(MDJ01株由Ⅰ变成Ⅴ)，第二个氨基酸的变异发生在163位(MDJ01由P变成A)，两株病毒核苷酸序列的同源性为97．8％，推测的氨基酸序列的同源性为99．6％。与国外远东株比较，森张株核苷酸序列的同源性稍高于MDJ01株，与欧洲株、西伯利亚株的同源性基本一致。结论：新分离株MDJ01对细胞和乳鼠的致病性稍低于1953年分离的森张株，E蛋白基因的核苷酸序列分析表明两株病毒同属于远东亚型，推测MDJ01株可能是由森张株在自然界长期演变而来。

Comparison of biology characteristics and nucleotide sequences of the E protein genes of two TBE viruses isolated from China

Objective:To compare the biological function and variation of E protein gene nucleotide of two strains of tick_borne encephalitis(TBE) viruses isolated in 1953 ( Senzhang strain) and 2001 (MDJ01 strain) in China.Methods:The suspensions of mouse brains infected by Senzhang and MDJ01 strains were inoculated into suckling mice and BHK/21 cells. Total RNA was extracted from BHK/21 cells infected by Senzhang and MDJ01 strains. The E protein gene was amplified by RT_PCR and the amplified fragment was cloned in pGEM_T easy vector. The nucleotide sequence was determined by PRISM TM ABI377automated sequencer.Results:Two virus strains were different in virulence to suckling mice and BHK/21 cells. The E gene of both strains was 1?488 nucleotides in length. nucleotide sequence homology was 97.2% and deduced amino acid sequence homology was 99.6%. Comparison of nucleotide sequences of Senzhang and MDJ01 strains with far_eastern subtype respectively found that homology of Senzhang strain was higher than that of MDJ01 strain. Homology of the two strains was similar in comparison with Europe and Siberia subtypes.Conclusion:The virulence of the two virus strains in suckling mice and BHK/21 cells was different with MDJ01 strain being weaker than Senzhang strain. Nucleotide sequence analysis of the E gene of two virus strains showed that they belonged to Far_eastern subtype of tick_borne encephalitis virus. MDJ01 strain may be a variant of Senzhang strain. [