Amino acid changes responsible for attenuation of virus neurovirulence in an infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus
Amino acid changes responsible for attenuation of virus neurovirulence in an infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus
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DOI：
10.1099/vir.0.19668-0
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发表时间：
2004-04-01
影响因子：
3.8
通讯作者：
Takashima, I
中科院分区：
文献类型：
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作者：
Hayasaka, D;Gritsun, TS;Takashima, I
采用高保真长链RT-PCR和一步法克隆技术，获得了森林脑炎病毒大岛株（远东亚型）的稳定全长感染性cDNA克隆。感染性克隆（O-IC）有四个氨基酸取代，并产生较小的空斑时，与亲本大岛5-10株。使用定点诱变，使取代回复以恢复亲本病毒序列（O-IC-pt）。虽然基因相同，但亲本病毒Oshima 5-10和从O-IC-pt回收的病毒表现出一些生物学差异，这可能是由于原始病毒种群中存在具有不同毒力特征的准种。这些观察结果可能对基于修饰的感染性克隆的疫苗有影响。结果表明，E糖蛋白40位氨基酸E-S-40-->P的取代为。导致空斑尺寸减小、细胞培养物中感染性病毒产量降低和小鼠神经毒力降低。此外，非结构（NS）5蛋白（病毒RNA依赖性RNA聚合酶）NS 5-V-378 -->A和NS 5-R-674-->K中的两个氨基酸取代也有助于减弱小鼠中的毒力，但在幼仓鼠肾细胞培养物中没有表现出明显的生物学效应。比较神经毒力测试揭示了单个突变（E-S-40-->P、NS 5-V-378-->A和NS 5-R-674-->K）的积累如何导致病毒的减毒。
A stable full-length infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus (Far-Eastern subtype) was developed by a long high-fidelity RT-PCR and one-step cloning procedure. The infectious clone (O-IC) had four amino acid substitutions and produced smaller plaques when compared with the parent Oshima 5-10 strain. Using site-directed mutagenesis, the substitutions were reverted to restore the parent virus sequence (O-IC-pt). Although genetically identical, parent virus Oshima 5-10 and virus recovered from O-IC-pt demonstrated some biological differences that are possibly explained by the presence of quasispecies with differing virulence characteristics within the original virus population. These observations may have implications for vaccines based on modified infectious clones. It was also demonstrated that the amino acid substitution E-S-40-->P at position 40 in the envelope (E) glycoprotein was. responsible for plaque size reduction, reduced infectious virus yields in cell culture and reduced mouse neurovirulence. Additionally, two amino acid substitutions in the non-structural (NS)5 protein (virus RNA-dependent RNA polymerase) NS5-V-378 -->A and NS5-R-674-->K also contributed to attenuation of virulence in mice, but did not demonstrate a noticeable biological effect in baby hamster kidney cell culture. Comparative neurovirulence tests revealed how the accumulation of individual mutations (E-S-40-->P, NS5-V-378-->A and NS5-R-674-->K) can result in the attenuation of a virus.