从北京地区急性呼吸道感染患儿标本中检测到新型冠状病毒NL63基因

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-NL63 1a基因的两对引物扩增进行复核。对用HCoV—NL63 1a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-NL63 1b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-NL63 1a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（group 1）。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。

Human coronavirus-NL63 was detected in specimens from children with acute respiratory infection in Beijing, China

Objective A new human coron av irus, HCoV-NL63, was identified recently from two Dutch children with acute res piratory infection (ARI) by two scientists in the Netherlands in 2004. To inves tigate if this newly discovered virus is associated with acute respiratory infec tions in pediatric patients in Beijing, tests were developed to detect HCoV-NL6 3 gene fragments from throat swab and nasopharyngeal aspirates collected from ch ildren in outpatient and inpatient departments with ARI in Beijing from Dec. 20 03 to Mar. 2004.Methods A total of 245 clinical samples , which were negative either for diagnostic tests of human respiratory syncytial virus, influenza virus A and B, adenovirus, parainfluenza virus 1, 2 and 3 by i ndirect immunofluoresence assay or human metapneumovirus by RT-PCR, were screen ed for HCoV-NL63 by nested PCR amplifying gene fragments located on the 1b and 1a genes. Amplicon of PCR from 1a gene of HCoV-NL63 was sequenced and the sequ ences were compared with those in GenBank nucleotide sequence database. Results Three (1.2%) out of the 245 samples were positive for HC oV-NL63 by nested-PCR using primers on 1b gene. These three samples also show ed positive results on nested PCR in which primers were designed with sequences complementary to 1a gene segments. These positive samples were collected from hospitalized children under 2 years of age with pneumonia, bronchiolitis and bro nchitis, respectively. The partial 1a gene sequences from two positive samples (BJ3140 and BJ3787) of HCoV-NL63 showed 100% homology between each other and hi gh homology (98%~99%) with the sequences of 1a gene of HCoV-NL63 reported from different countries in GenBank. Phylogenetic analysis showed that BJ3140 and BJ 3787 fell into the same genetic cluster (group 1).Conclusions These data suggest that some of acute respiratory infections in young chil dren in Beijing area are related to the newly identified HCoV-NL63.