CAJ | 학술논문

目的：分析柯萨奇病毒B3（ CVB3） KM06/2009全基因序列，并对其分子变异、进化特点和毒力特征进行分析。方法设计针对CVB3引物，提取病毒RNA，RT-PCR扩增和产物直接测序获得序列。利用Mega 4.1、RDP3和SimPlot3.5.1等软件分析全基因序列。结果 CVB3 KM06/2009病毒株全基因组全长7401 bp个核苷酸（nt），5′端和3′端分别为743 nt和100 nt的非编码区，5′和3′非编码区间为一个6558 nt长的开放读码框，编码一个含2185个氨基酸的多聚蛋白，编码区内未见核苷酸的插入或缺失。与GenBank 库中CVB3 Nancy 原型株比较，其全基因组序列核苷酸同源性为81.4％，氨基酸同源性为95.7％；与无心肌毒力的临床分离株CVB3 GA比较，其全基因组序列核苷酸同源性为80.7％，氨基酸同源性为96.4％；与历史同期国内临床分离株Beijing0811、SSM、GZ0803株比较，整个基因组的核苷酸同源性分别为98.1％、89.7％和88.4％，氨基酸同源性分别为99.4％、98.1％和98.1％。基于全基因组和全长VP1核酸序列的进化树图均显示，CVB3病毒株具有明显的时空聚簇分布特征。 CVB3心肌毒力相关区域5′非编码区颈环结构Ⅱ RNA二级折叠结构分析结果表明，CVB3 KM06/2009病毒株具有明显的心肌毒力倾向。结论基于基因分子生物信息学分析，与CVB3临床分离株比较，KM06/2009病毒株具有明显的心肌毒力倾向。
목적：분석가살기병독B3（ CVB3） KM06/2009전기인서렬，병대기분자변이、진화특점화독력특정진행분석。방법설계침대CVB3인물，제취병독RNA，RT-PCR확증화산물직접측서획득서렬。이용Mega 4.1、RDP3화SimPlot3.5.1등연건분석전기인서렬。결과 CVB3 KM06/2009병독주전기인조전장7401 bp개핵감산（nt），5′단화3′단분별위743 nt화100 nt적비편마구，5′화3′비편마구간위일개6558 nt장적개방독마광，편마일개함2185개안기산적다취단백，편마구내미견핵감산적삽입혹결실。여GenBank 고중CVB3 Nancy 원형주비교，기전기인조서렬핵감산동원성위81.4％，안기산동원성위95.7％；여무심기독력적림상분리주CVB3 GA비교，기전기인조서렬핵감산동원성위80.7％，안기산동원성위96.4％；여역사동기국내림상분리주Beijing0811、SSM、GZ0803주비교，정개기인조적핵감산동원성분별위98.1％、89.7％화88.4％，안기산동원성분별위99.4％、98.1％화98.1％。기우전기인조화전장VP1핵산서렬적진화수도균현시，CVB3병독주구유명현적시공취족분포특정。 CVB3심기독력상관구역5′비편마구경배결구Ⅱ RNA이급절첩결구분석결과표명，CVB3 KM06/2009병독주구유명현적심기독력경향。결론기우기인분자생물신식학분석，여CVB3림상분리주비교，KM06/2009병독주구유명현적심기독력경향。
Objective To analyze the complete genome sequence of a coxsackievirus B 3 (CVB3) strain KM06/2009 and its genetic variation , evolution and cardiovirulence .Methods Eight clones with overlapped gene fragments covering the complete viral genome ( excluding the poly-A tail) were obtained by RT-PCR and sequenced .The nucleotide ( nt ) and amino acid ( aa ) sequences of the eight clones were aligned with sequences of other known CVB 3 clinical strains .Phylogenetic and pairwise alignment analyses based on the genome and complete VP 1 regions were conducted by using Mega 4.1,RDP3 and SimPlot3.5.1 softwares.The RNA secondary structure of CVB3 stem loopⅡ (SLⅡ) was determined by using Mfold web server.Results The complete genome of CVB3 strain KM06/2009 was 7401 nt in length, consisting of 743 nt and 100 nt on 5′untranslated region (UTR) and 3′UTR,respectively.The entire open reading frame contained 6558 nt, encoding a 2185 aa polyprotein.There was no nucleotide deletion or insertion in the coding region,but some changes of amino acid were unique .KM06/2009 strain showed 81.4%and 95.7%identities in nucleotide and amino acid sequences respectively as compared with CVB 3 prototype Nancy strain.It shared 88.4%-98.1%nucleotide and 98.1%-99.4%amino acid homology with the other Chinese clinical strains isolated at the same period .KM06/2009 strain and CVB3 GA strain without cardiovirulence shared 80.7%homology in nucleotide and 96.4% in amino acid.The phylogenetic analysis indicated that the significant spatial and temporal clustering was detected in CVB 3 isolate.CVB3 KM06/2009 strain showed a strong cardiovirulence tendency as indicated by the RNA secondary structure of CVB 3 SL Ⅱ. Conclusion CVB3 KM06/2009 isolate showed a strong cardiovirulence tendency in comparison with other CVB3 clinical isolates based on the bioinformatics analysis .