Objective To sequence and analyze the complete genome of two human coronavirus NL63 (HCoV-NL63) strains collected from Beijing Children Hospital .Methods Eighteen pairs of primers were designed according to the gene sequences of HCoV-NL63 reference strain ( HCoV-NL63_Amsterdam 1) and used to amplify the target fragments covering the complete genome of HCoV-NL63 strains.Rapid ampli-fication of cDNA ends ( RACE) and RT-PCR assays were used to amplify the full length genome of HCoV-NL63 strains.Phylogenetic analysis was conducted by using Mega 5.0 software.Results The complete ge-nome sequences of the two HCoV-NL63 strains were 27 538 bp in length, showing a homology of 99.1%in nucleotide sequences .There were 15 consecutive bases deleted from 1a region.The systematic phylogenetic analysis demonstrated that four genotypes of NL 63 virus including A , B, C and D have been identified , and two domestic strains were belonged to the new genotype D .Conclusion The complete genome sequences of two domestic HCoV-NL63 isolates were identified for the first time .This study provided evidence for further investigation on molecular epidemiology of HCoV-NL63 in China .%目的:对来自北京儿童医院的两份人冠状病毒NL63(human coronavirus NL63, HCoV-NL63)阳性样本进行病毒全基因组序列测定和分析。方法以荷兰株(NL63_Amsterdam 1)为参考株,设计18对包含重叠区域的、覆盖全长基因组的引物,采用病毒RNA提取、RT-PCR分段克隆测序的方法获得NL63国内株全长基因组序列;通过与其他毒株的比对分析,构建其系统发生树。结果NL63国内株基因组全长27538 bp,与参考株核苷酸相似性为99.1%,其中在1a区有连续15个碱基的缺失。系统发生分析表明,NL63目前可分为A型、B型、C型和D型共4个基因型,而国内株处在新的基因型( D型)。结论本研究首次获得了两株NL63国内分离株的全基因组序列,进一步阐明了基因组的结构特征,对其遗传进化作了系统分析,为国内NL63病毒的分子流行病学研究提供参考。
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