非典型肺炎病例标本中新型冠状病毒的分离与鉴定 - 非典型肺炎病原体的.doc

PAGE PAGE 8 非典型肺炎病例标本中新型冠状病毒的分离与鉴定 祝庆余* 秦鄂德 王翠娥 于曼 司炳银 范宝昌 常国辉 彭文明 杨保安 姜涛 李豫川 邓永强 刘洪 甘永华 〔摘要〕 目的 对非典型肺炎的病原体进行分离鉴定，为该病的诊断、预防和治疗提供依据。方法 采用细胞培养和乳鼠接种法，从非典型肺炎病例标本中分离致病病原体，通过电镜形态学、血清学和动物致病性观察及RT-PCR扩增与部分基因序列分析，对分离的病原体进行鉴定。结果 成功地从死亡病例尸解的肺组织标本和患者鼻咽拭子标本中采用细胞培养法分离出病原体。通过电镜在病变细胞及其培养上清中观察到大量冠状病毒样颗粒。免疫荧光染色检测非典型肺炎患者血清，表明分离的病毒与此次流行的非典型肺炎密切相关。分离的病毒可对乳鼠致病，并在发病乳鼠的肺组织标本中通过电镜同样观察到冠状病毒样颗粒。从非典型肺炎病例尸解肺组织、传代发病小鼠肺组织及分离物感染的细胞培养物中，通过RT-PCR可分别扩增出冠状病毒的cDNA片段，测序结果显示其核苷酸序列与已知冠状病毒的同源性在60％左右。结论 从非典型肺炎病例标本中已成功分离出一种新的冠状病毒，它与此次流行的非典型肺炎密切相关，很可能是此次流行的非典型肺炎的主要病原体。 〔关键词〕非典型肺炎；病原体；冠状病毒；分离鉴定；序列分析 Isolation and identification of a novel coronavirus from patients with SARS Zhu Qing-yu, Qin E-de, Wang Cui-E, Yu Man, Si Bing-yin, Fan Bao-Cang, Chang Guo-hui, Peng Wen-ming, Yang Bao-an, Jiang Tao, Li yu-chuan, Deng yong-qiang, Liu Hong, Gan Yong-hua Institute of Microbiology and Epidemiology, Beijing, 〔Abstract〕 Objective To isolate and identify the microbial pathogen of Severe Acute Respiratory Syndrome (SARS ), thus to lay the foundation for the diagnosis, prevention and treatment of the SARS. Methods Cell Cultures and suckling mice were inoculated by the samples from patients with SARS and autoptical tissues. The isolates were identified by morphology, serology, animal experiment, and RT-PCR amplification and partial sequence analysis. The causative association between the isolates and SARS was determined. Results A possible pathogen of SARS was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with SARS. The isolated microbial agents could cause cytopathic effects on Vero-E6 cells and a large quantity of coronavirus-like particles could be observed in the infected cells and supernatants under transmission electron microcope (TEM). Twenty-three of 26 serum samples from patients with SARS were tested positive by immunofluorescence assay, while 31 of the control serum kept all negative, indicating that the isolated virus was clearly associated with current SARS ep