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目的将乙型脑炎病毒减毒活疫苗株SA14-14-2经乳鼠脑内回传毒力回复后,对回复后病毒测其神经毒力和全长基因序列并与减毒株SA14-14-2和母株SA14进行核苷酸和氨基酸序列比较。方法将回传一代SA14-14-2 M1和SA14-14-2 M3病毒在幼鼠脑内测神经毒力和全基因组序列。运用RT-PCR方法,分段扩增出覆盖基因组全长的基因片段,PCR产物纯化后直接测序,针对5′和3′端的片段TA克隆后进行测序。经核苷酸序列测定后,利用DNAStar软件,进行序列比对分析。结果实验结果表明SA14-14-2 M1病毒毒力未见增强,SA14-14-2 M3病毒的脑内致病力明显增强,但皮下接种仍无致病性。通过序列比对分析发现,M1和M3病毒均在非结构区的NS3-343、NS4A-57位发生氨基酸回复突变,但只有M3病毒在E区毒力相关的E107、E138位点发生回复突变。结论在SA14-14-2减毒株病毒结构基因中E蛋白区E107、E138位氨基酸是与毒力密切相关的重要位点,应该是神经毒力减弱的重要分子基础,非结构区的个别位点对神经毒力减弱也可能起一定的协同作用。
OBJECTIVE: To determine the virulence and full-length gene sequence of SA14-14-2 attenuated live attenuated strain of Japanese encephalitis virus (JEV) 14-2 and parental strain SA14 for nucleotide and amino acid sequence comparison. Methods The neurovirulence and whole genome sequences of the SA14-14-2 M1 and SA14-14-2 M3 viruses were returned to the brains of young rats. The full-length genomic DNA fragment was amplified by RT-PCR. The purified PCR products were directly sequenced and sequenced after TA cloning of the 5 ’and 3’ ends. After nucleotide sequence determination, DNAStar software was used for sequence alignment analysis. Results The experimental results showed that the virulence of SA14-14-2 M1 virus was not enhanced and the pathogenicity of SA14-14-2 M3 virus was significantly increased in the brain. However, the pathogenicity of SA14-14-2 M1 virus was still non-pathogenic. Sequence alignment revealed that the M1 and M3 viruses all mutated amino acids at the NS3-343 and NS4A-57 in the non-structural domain, but only the M3 virus showed a back-mutation in the virulence-associated E107 and E138 sites in the E region. Conclusion The amino acids E107 and E138 of E protein region in SA14-14-2 attenuated strain are important molecular sites closely related to virulence and should be the important molecular basis for the decrease of virulence. Decreased neurovirulence may also play a synergistic effect.