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目的了解北京地区婴幼儿急性呼吸道感染是否与一种新发现的冠状病毒———HCoV-NL63相关。方法选取2003年12月—2004年3月,从首都儿科研究所附属儿童医院收集的245份因急性呼吸道感染就诊的门诊患儿的咽拭子标本以及住院患儿的鼻咽洗液标本,进行HCoV-NL63的筛查。这些标本已经过间接免疫荧光法和病毒分离检测,常见的七种呼吸道病毒(包括RSV,甲、乙型流感病毒,Ⅰ、Ⅱ、Ⅲ型副流感病毒和腺病毒)检测均为阴性;同时还经逆转录-聚合酶链反应(RT-PCR)方法检测人偏肺病毒(hMPV)也为阴性。首先用位于HCoV-NL631b基因的两对引物用巢式PCR方法进行筛查,阳性者再用位于HCoV-NL631a基因的两对引物扩增进行复核。对用HCoV-NL631a基因扩增的长片段产物进行测序并与GenBank中相关序列进行比较分析。结果用HCoV-NL631b基因的引物经巢式PCR方法从245份标本中检测到3份阳性标本,阳性率为1.2%。3份阳性标本用HCoV-NL631a基因的两对引物经巢式PCR方法进行复核均得到阳性结果,这3份标本均取自住院患儿,患儿年龄分别为4个月、1岁和1岁5个月,临床诊断分别为毛细支气管炎、喉炎和支气管炎,男女比例为2∶1。对其中基因扩增产量较高的BJ3140和BJ3787的1a基因长片段扩增产物(838bp)进行序列测定和分析的结果显示,这两株HCoV-NL63与GenBank公布的不同地区的HCoV-NL63的1a基因序列同源性最高,达到98%~99%。基于部分1a基因序列的系统进化分析显示,BJ3140和BJ3787属于HCoV-NL63的第一簇(group1)。结论结果提示北京地区部分婴幼儿的急性呼吸道感染与HCoV-NL63相关。
Objective To understand whether infant acute respiratory infections in Beijing are associated with a newly discovered coronavirus, HCoV-NL63. Methods From December 2003 to March 2004, 245 throat swab specimens of outpatients with acute respiratory infection and nasopharyngeal wash samples from hospitalized children were collected from Children’s Hospital affiliated to Capital Institute of Pediatrics HCoV-NL63 screening. These specimens have been tested by indirect immunofluorescence and virus isolation. The seven common respiratory viruses (including RSV, Influenza A and B viruses, parainfluenza virus types I, II, and III) and adenovirus were all negative. At the same time, Human metapneumovirus (hMPV) was also negative by reverse transcription-polymerase chain reaction (RT-PCR). First, two pairs of primers located in the HCoV-NL631b gene were screened by nested PCR. The positives were screened by two pairs of primers located in the HCoV-NL631a gene. The long fragment amplified by HCoV-NL631a gene was sequenced and compared with the related sequences in GenBank. Results Three positive samples were detected in 245 samples by nested PCR using primers of HCoV-NL631b gene. The positive rate was 1.2%. Three positive samples were positive for nested PCR using two pairs of primers of HCoV-NL631a gene. All three samples were obtained from inpatients with children aged 4 months, 1 year and 1 year old Five months, the clinical diagnosis of bronchiolitis, laryngitis and bronchitis, male to female ratio of 2: 1. The results of sequencing and analysis of the amplified fragment of 1a gene (838bp) in BJ3140 and BJ3787 with high yield of gene amplification showed that the two strains of HCoV-NL63 and GenBank published in different regions of HCoV-NL63 1a Gene sequence homology highest, reaching 98% to 99%. Phylogenetic analysis based on the partial 1a gene sequence showed that BJ3140 and BJ3787 belonged to the first cluster of HCoV-NL63. Conclusion The results suggest that acute respiratory infections in some infants and young children in Beijing are related to HCoV-NL63.