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目的研究EV71济南分离株的基因特点,寻找潜在的EV71毒力决定位点。方法将6株EV71济南分离株(JN200803、JN200804、Jinan1002、Jinan1004、Jinan1005和Jinan1006株)分别接种1d龄BALB/c小鼠,确定其对小鼠的致病性程度。对6株EV71全基因组核苷酸、氨基酸序列进行比较,对非编码区进行RNA二级结构的预测和分析,对VP1区进行遗传进化分析。结果通过动物实验可将6株EV71济南分离株分为强、中、弱毒力株。小鼠感染强毒株(JN200804、Jinan1002、Jinan1004、Jinan1005株)的主要症状为后肢麻痹并死亡,感染弱毒株(Jinan1006株)的主要症状为后肢麻痹但能自愈,感染无毒株(JN200803株)未引起明显症状。不同毒力EV71分离株全基因组序列比对发现,共有40个核苷酸位点的差异,导致编码区8个氨基酸突变,其中第937位氨基酸(位于2A片段)在强毒株、弱毒株和无毒株间发生了连续突变(937aa:S→C→G)。非编码区RNA二级结构预测表明,5′UTR第115位核苷酸突变对内部核糖体进入位点(IRES)的结构有较大影响。遗传进化分析显示,与国内2003年以后的流行毒株相同,6株EV71济南分离株均属C4亚型的C4a进化分支。结论全基因组序列比较分析发现不同毒力EV71济南分离株之间存在差异位点,为采用反向遗传学方法鉴定EV71毒力决定位点奠定了基础。
Objective To study the genetic characteristics of EV71 Jinan isolate and to find potential virulence sites for EV71. Methods Six EV71 Jinan isolates (JN200803, JN200804, Jinan1002, Jinan1004, Jinan1005 and Jinan1006) were inoculated with 1-day-old BALB / c mice to determine their pathogenicity to mice. The nucleotide and amino acid sequences of 6 EV71 genomes were compared. The RNA secondary structure of the non-coding region was predicted and analyzed, and the VP1 region was analyzed by genetic evolution. Results Six animal strains of EV71 Jinan could be divided into strong, medium and weak virulent strains. The main symptoms of mice infected with virulent strains (JN200804, Jinan1002, Jinan1004 and Jinan1005) were paralysis and death of the hind limbs. The main symptoms of the attenuated attenuated strain (Jinan1006) were paralysis of the hind limbs but self-healing and infection of non-toxic strains (strain JN200803 ) Did not cause significant symptoms. The genome-wide sequence alignment of different virulent EV71 isolates revealed a total of 40 nucleotide differences that led to a mutation of 8 amino acids in the coding region. The 937th amino acid (located in 2A fragment) was highly virulent, virulent and Non-toxic strains occurred between the continuous mutation (937aa: S → C → G). RNA secondary structure prediction of non-coding region showed that the 115th nucleotide mutation of 5’UTR has a greater impact on the structure of the internal ribosome entry site (IRES). Genetic evolution analysis showed that, with the same epidemic strains in China after 2003, all six strains of EV71 Jinan belong to the C4a evolutionary branch of the C4 subtype. Conclusion The comparative analysis of whole genome sequence revealed that there are differences between different virulence EV71 Jinan isolates, which laid the foundation for the identification of EV71 virulence sites by reverse genetics.