CAJ | 학술논문

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.
목적 관찰5'비편마구(5'UTR)밀정부집구변이대B조가살기병독1형(CVB1)독력화치병성적영향.방법 정점유변장CVB1기인조5'UTR제563～573감기밀정부집구5개밀정치환위표령,획득돌변주CVB1/m563-573.공반순화병독후,이용세포병변효응(CPE)시험、공반형성시험、일보생장곡선화반수치사량(LD50)등방법비교CVB1/m563-573여기원형주CVB1/wt적독력.결과 측서결과현시,CVB1/m563-573적5'UTR밀정부집구발생C565A、U567C、U568A、U570A、U572G돌변,여예기일치.CPE시험표명,CVB1/m563-573적감염성교CVB1/wt초약(A 490분별위0.710±0.074、0.812±0.092),단차이무통계학의의(t=-2.204,P>0.05).공반형성시험현시,46、58 h시간점CVB1/m563-573형성공반적수량위(6.40±1.52)×103、(11.60±2.19)×103 pfu(공반형성단위)/L,직경위(2.00±0.35)、(2.47±0.41)mm,CVB1/wt적공반수량위(8.40±2.51)×103、(11.80±1.92)×103 pfu/L,직경위(1.80±0.27)、(2.85±0.44)mm,이자비교차이무통계학의의(t치분별위8.000、0.985、10.000、9.000,P균>0.05).일보생장곡선법현시,경과3、5、7 h적확증,CVB1/m563-573소득적활병독과립수(×103 pfu/L)적상용대수(lg)분별위2.10±0.09、4.28±0.03、7.44±0;CVB1/wt적활병독과립수(×103 pfu/L)적lg치분별위2.80±0.02、4.77±0.02、8.55±0.01.CVB1/m563-573주재3개시간점균교CVB1/wt복제현저만(t치분별위-13.151、-24.319、-47.714,P균<0.01).CVB1/m563-573화CVB1/wt적LD50분별위3.10×109、1.26×107 pfu/L,CVB1/m563-573치병력교CVB1/wt명현감약.결론 감소5'UTR밀정부집구적밀정감기수량가치CVB1적감염화독력감약,해위점돌변가능시연발CVB감독활역묘적책략지일.
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/m563-573 was significantly weakened compared to that of CVB1/wt.Conclusions The infectivity and virulence of CVB1 are weakened by substitution of pyrimidines with purines in the pyrimidine-rich tract of CVB1 5'UTR.Site-directed mutagenesis in the pyrimidine-rich tract may be a strategy for developing attenuated CVB vaccine.