福建省首例人感染H7N9禽流感病例实验室诊断和病毒序列分析

Confirmation of first case of human infection with influenza A (H7N9) virus in Fujian Province : laboratory diagnosis and viral sequence analysis

目的应用real-time RT-PCR和病毒序列测定等方法对福建省首例人感染H7N9禽流感病例开展实验室诊断。方法采集人感染H7N9禽流感病例呼吸道标本并提取RNA,分别采用甲乙型流感通用引物和探针、季节性流感(包括H3N2、H1N1、H1N1pdm)特异性引物和探针以及H7N9特异性引物和探针进行real-time RT-PCR检测。利用自行设计的引物扩增病毒基因组节段,测定并分析病毒基因序列。结果 real-time RT-PCR结果表明,应用甲型通用引物扩增结果阳性,乙型及季节性流感(包括H3N2、H1N1以及H1N1pdm)扩增结果均为阴性,特异性H7N9亚型流感病毒扩增结果阳性。序列测定获得的病毒4个节段的序列与已公布的人感染H7N9禽流感病毒序列高度一致。结论病例呼吸道标本中存在人感染H7N9禽流感病毒,病毒的基因与近期国内流行的人感染H7N9禽流感病毒高度类似。

In this study, we confirmed the first case of human infection with influenza A （HTNg） virus in Fujian Province by viral specific nuclear acid detection and sequencing the four segments of viral genome. The respiratory specimen （throat swab） was collected from a case with pneumonia of unknown cause reported in Longyan City, Fujian Province. Viral RNA was extracted from the specimen and applied for real-time reverse transcript PCR with universal primer/probe sets for influenza A B, specific primer/probe sets for seasonal influenza A virus （including H3N2 subtype, HIN1 subtype and H1Nlpdm）, specific primer/probe sets for newly emerging H7N9 virus, respectively. The four near full length segments of viral genome were sequenced by using two step RT PCR methods with a set of self-designed primers. Positive results obtained with universal influenza A virus and specific HTN9 subtype virus primer/probe sets, whereas negative results with the other primer/probe sets. Four segments of viral genomic sequence were obtained and compared with the published N7N9 viral genomic sequences. The results of real-time RT-PCR and viral segments sequencing revealed that presence of H7N9 subtype influenza A virus in the specimen from the case with pneumonia of unknown cause. Viral sequence was highly identical to that of HTN9 subtype viruses currently epidemic in China's Mainland. It should conclude that the patient was the first case of human infection with novel H7N9 subtype virus in Fujian Province.