Zhu Ru-nan, Qian Yuan, Zhao Lin-qing, Deng Jie, Wang Fang, Liao Bin
Laboratory of Virology, Beijing Municipal Laboratory of Infection and Immunity, Capital Institute of Pediatrics, Beijing 100020, China.
Zhonghua Er Ke Za Zhi. 2006 Mar;44(3):202-5.
A new human coronavirus, HCoV-NL63, was identified recently from two Dutch children with acute respiratory infection (ARI) by two scientists in the Netherlands in 2004. To investigate if this newly discovered virus is associated with acute respiratory infections in pediatric patients in Beijing, tests were developed to detect HCoV-NL63 gene fragments from throat swab and nasopharyngeal aspirates collected from children in outpatient and inpatient departments with ARI in Beijing from Dec. 2003 to Mar. 2004.
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 indirect immunofluorescence assay or human metapneumovirus by RT-PCR, were screened 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 sequences were compared with those in GenBank nucleotide sequence database.
Three (1.2%) out of the 245 samples were positive for HCoV-NL63 by nested-PCR using primers on 1b gene. These three samples also showed 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 bronchitis, respectively. The partial 1a gene sequences from two positive samples (BJ3140 and BJ3787) of HCoV-NL63 showed 100% homology between each other and high homology (98%-99%) with the sequences of 1a gene of HCoV-NL63 reported from different countries in GenBank. Phylogenetic analysis showed that BJ3140 and BJ3787 fell into the same genetic cluster (group 1).
These data suggest that some of acute respiratory infections in young children in Beijing area are related to the newly identified HCoV-NL63.
2004年，荷兰的两位科学家从两名患有急性呼吸道感染（ARI）的荷兰儿童中发现了一种新型人类冠状病毒，即HCoV-NL63。为了调查这种新发现的病毒是否与北京儿科患者的急性呼吸道感染有关，研究人员开发了检测方法，以检测2003年12月至2004年3月期间从北京门诊和住院的ARI儿童采集的咽拭子和鼻咽抽吸物中的HCoV-NL63基因片段。
共有245份临床样本，通过间接免疫荧光法检测人类呼吸道合胞病毒、甲型和乙型流感病毒、腺病毒、副流感病毒1、2和3，或通过RT-PCR检测人偏肺病毒，结果均为阴性，采用巢式PCR扩增位于1b和1a基因上的基因片段，对这些样本进行HCoV-NL63筛查。对HCoV-NL63 1a基因的PCR扩增产物进行测序，并将序列与GenBank核苷酸序列数据库中的序列进行比较。
在245份样本中，有3份（1.2%）使用1b基因引物进行巢式PCR检测HCoV-NL63呈阳性。这三份样本在使用与1a基因片段互补序列设计引物的巢式PCR中也显示出阳性结果。这些阳性样本分别来自患有肺炎、细支气管炎和支气管炎的2岁以下住院儿童。HCoV-NL63的两个阳性样本（BJ3140和BJ3787）的部分1a基因序列彼此之间显示出100%的同源性，与GenBank中不同国家报告的HCoV-NL63 1a基因序列具有高度同源性（98%-99%）。系统发育分析表明，BJ3140和BJ3787属于同一基因簇（第1组）。
这些数据表明，北京地区幼儿的一些急性呼吸道感染与新发现的HCoV-NL63有关。
