CAJ | 학술논문

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。
목적：대래자북경인동의원적량빈인관상병독NL63（human coronavirus NL63， HCoV-NL63）양성양본진행병독전기인조서렬측정화분석。방법이하란주（NL63_Amsterdam 1）위삼고주，설계18대포함중첩구역적、복개전장기인조적인물，채용병독RNA제취、RT-PCR분단극륭측서적방법획득NL63국내주전장기인조서렬；통과여기타독주적비대분석，구건기계통발생수。결과NL63국내주기인조전장27538 bp，여삼고주핵감산상사성위99．1％，기중재1a구유련속15개감기적결실。계통발생분석표명，NL63목전가분위A형、B형、C형화D형공4개기인형，이국내주처재신적기인형（ D형）。결론본연구수차획득료량주NL63국내분리주적전기인조서렬，진일보천명료기인조적결구특정，대기유전진화작료계통분석，위국내NL63병독적분자류행병학연구제공삼고。
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 .