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目的比较昆明市住院与门诊病例来源的10株EV71病毒分离株3D蛋白酶基因核苷酸及编码氨基酸序列的差异性与病毒毒力之间的关系。方法对分离自昆明市的10株EV71病毒3D蛋白酶基因核苷酸测序,利用Bioedit及Mega 6软件对测序结果进行序列比对及遗传特性分析。选取6株(住院病例分离株、门诊病例分离株各3株)绘制病毒增殖动力学曲线,取其中2株(住院及门诊病例来源各1株)病毒接种至Vero细胞进行空斑形成试验,比较不同病毒株形成的空斑形态大小。结果对10株EV71分离病毒株的3D蛋白酶基因进行核苷酸测序并构建进化树,5株住院病例分离株间遗传距离较近,在进化树上单独成为一簇;住院病例病毒分离株与门诊病例病毒分离株的氨基酸序列在140、197、299位点存在差异;住院病例病毒株在较短时间达到增殖高峰,空斑试验中形成的蚀斑直径大于门诊病例分离株。结论不同来源的EV71病毒分离株3D蛋白酶基因核苷酸序列及编码氨基酸序列存在差异,该差异与EV71病毒的增殖特性及毒力具有一定相关性。
Objective To compare the differences in the nucleotide and amino acid sequences of 3D protease genes of 10 EV71 isolates from hospital and outpatient cases in Kunming and the virulence of the virus. Methods The nucleotide sequences of 10 EV71 virus 3D protease genes isolated from Kunming were sequenced. The sequencing results and sequence analysis were performed by Bioedit and Mega 6 software. Viral cell proliferation kinetics curves were drawn from 6 isolates (outpatient isolates and outpatient isolates), and 2 of them (1 inpatient and outpatient cases) were inoculated into Vero cells for plaque formation test. The formation of different strains of plaque size. Results The nucleotide sequences of the 3D protease gene of 10 EV71 isolates were sequenced and the phylogenetic tree was constructed. The genetic distances among the 5 inpatient isolates were close, and they were clustered separately in the phylogenetic tree. The amino acid sequences of the case virus isolates differed at 140,197,299; in-patient virus strains reached the peak of proliferation in a relatively short period of time, and the plaque diameter formed in the plaque test was larger than that of outpatient isolates. Conclusion The nucleotide and amino acid sequences of 3D protease gene of EV71 isolates from different origins are different. The difference has some correlation with the proliferation and virulence of EV71 virus.