深圳市首例人感染H5N1病毒病例的实验室诊断及分子特点分析

Laboratory Diagnosis and Molecular Characterization Analysis of the H5N1 Influenza Virus Isolated from the First Human Case in Shenzhen,China

对深圳首例疑似人禽流感病人的标本,进行了RT-PCR、Real-timePCR检测及病毒分离培养、血清中和试验、抗原比检测及发病早期不同病程多份标本的病毒载量分析;对分离物进行了HA基因、NA基因及M基因的核酸检测。结果表明:患者气管吸出物的H5N1亚型和A型流感病毒的特异核酸均呈阳性,并通过细胞培养分离到禽流感病毒A/Guangdong/2/06(H5N1)株。气管吸取物病毒载量随着病程延长逐渐减少,而血清中和抗体水平逐渐上升达到1:160之后又缓缓下降。A/Guangdong/2/06株8个片段的核苷酸序列显示,其与2005～2006年中国南部的禽流感分离株高度同源,与越南、泰国、印度尼西亚等分离到的禽流感分离株存在明显的差异。

The tracheal aspirates and serum samples of a suspected human case of high-pathogenic avian influenza （firstly found in Shenzhen, China） were collected and tested by a series of assays. The results showed that the RNA extracted from the tracheal aspirate specimens of the patient was confirmed positive for H5N1 avian influenza virus by Real-time PCR. The H5N1 avian influenza virus was isolated from patient＇s tracheal aspirates on MDCK cell and was named A/Guangdong/2/06（H5N1）. The viral load of tracheal aspirates collected at different time points were detected by Real-time PCR. The virus microneutralization and the antigenic ratio of human H5N1 isolated were also assayed. It was found that when the virus load decreased gradually after the disease onset, the serum neutralizing antibody titer in the patient increased to 1：160 and subsequently decreased gradually. By molecular analysis, the eight gene segments of A/Guangdong/2/06 revealed to be similar to that of H5N1 avian influenza viruses isolated from south China in 2005-2006. However, there were obvious differences in the gene sequence of the detected H5N1 viral RNA as compared with that of the strains isolated from Vietnam, Thailand and Indonesia.