中文摘要：

目的 观察5＇非编码区（5＇UTR）嘧啶富集区变异对B组柯萨奇病毒1型（CVB1）毒力和致病性的影响.方法 定点诱变将CVB1基因组5＇UTR第563～573碱基嘧啶富集区5个嘧啶置换为嘌呤,获得突变株CVB1/m563-573.空斑纯化病毒后,利用细胞病变效应（CPE）试验、空斑形成试验、一步生长曲线和半数致死量（LD50）等方法比较CVB1/m563-573与其原型株CVB1/wt的毒力.结果 测序结果显示,CVB1/m563-573的5＇UTR嘧啶富集区发生C565A、U567C、U568A、U570A、U572G突变,与预期一致.CPE试验表明,CVB1/m563-573的感染性较CVB1/wt稍弱（A 490分别为0.710±0.074、0.812±0.092）,但差异无统计学意义（t=-2.204,P〉0.05）.空斑形成试验显示,46、58 h时间点CVB1/m563-573形成空斑的数量为（6.40±1.52）×103、（11.60±2.19）×103 pfu（空斑形成单位）/L,直径为（2.00±0.35）、（2.47±0.41）mm,CVB1/wt的空斑数量为（8.40±2.51）×103、（11.80±1.92）×103 pfu/L,直径为（1.80±0.27）、（2.85±0.44）mm,二者比较差异无统计学意义（t值分别为8.000、0.985、10.000、9.000,P均〉0.05）.一步生长曲线法显示,经过3、5、7 h的扩增,CVB1/m563-573所得的活病毒颗粒数（×103 pfu/L）的常用对数（lg）分别为2.10±0.09、4.28±0.03、7.44±0 CVB1/wt的活病毒颗粒数（×103 pfu/L）的lg值分别为2.80±0.02、4.77±0.02、8.55±0.01.CVB1/m563-573株在3个时间点均较CVB1/wt复制显著慢（t值分别为-13.151、-24.319、-47.714,P均〈0.01）.CVB1/m563-573和CVB1/wt的LD50分别为3.10×109、1.26×107 pfu/L,CVB1/m563-573致病力较CVB1/wt明显减弱.结论 减少5＇UTR嘧啶富集区的嘧啶碱基数量可致CVB1的感染和毒力减弱,该位点突变可能是研发CVB减毒活疫苗的策略之一.

英文摘要：

Objective To evaluate the infectivity and virulence variation caused by mutations in the 5＇ untranslated region（5＇UTR）pyrimidine-rich tract of coxsackievirus B1（CVB1）genome.Methods Five pyrimidines in the 5＇UTR pyrimidine-rich tract（nt563-nt573）of CVB1 genome were substituted with purines by site-directed mutagenesis.The mutant,CVB1/m563-573,was purified by plaque assay,and subjected to infectivity and virulence assessments by means of cytopathic effect（CPE）,plaque forming,one-step growth curve,and 50% lethal dose（LD50）assays.Results Sequencing data revealed that the sequence of pyrimidine-rich tract in the 5＇UTR of CVB1/m563-573 mutant was exactly identical to our design（C565A,U567C,U568A,U570A,and U572G）.CPE assay showed that the infectivity of CVB1/m563-573 was weaker than that of its prototype CVB1/wt（A490=0.710±0.074,0.812±0.092）though no significant difference could be observed（t=-2.204,P〉0.05）.Plaque forming assay showed that the plaque quantities of CVB1/m563-573 were（6.40±1.52）×103,（11.60±2.19）×103 pfu/L and the plaque diameters of CVB1/m563-573 were（2.00±0.35）,（2.47±0.41）mm at 46 and 58 hours pestinfection,respectively.The plaque quantities of CVB1/wt were（8.40±2.51）×103,（11.80±1.92）×103 pfu/L and the plaque diameters of CVB1/wt were（1.80±0.27）,（2.85±0.44）mm,respectively.There was no significant difference between the plaque quantities and sizes of CVB1/m563-573 and CVB1/wt（t=8.000,0.985,10.000,9.000,all P〉0.05）.One-step growth curve demonstrated that the numbers（lg）of CVB1/m563-573 progenies at time-points of 3,5,7 h postinfection were 2.10±0.09,4.28±0.03,7.44±0 and that of CVB1/wt progenies were 2.80±0.02,4.77±0.02,8.55±0.01,respectively.The replication of CVB1/m563-573 was significantly slower than that of CVB1/wt at all three time-points（t=-13.151,-24.319,-47.714,all P〈0.01）.The LD50 of CVB1/m563-573（3.10×109 pfu/L）and CVB1/wt（1.26×107 pfu/L）indicated that the virulence of CVB1/m56