应用原代人呼吸道上皮细胞（HAE）分离鉴定陈旧性呼吸道样本中流感病毒

Isolation and identification of human influenza virus from a stale respiratory tract specimen with human airway epithelial cells

目的 采用分化良好的原代人呼吸道上皮细胞（HAE）对陈旧性呼吸道样本中流感病毒H3N2分离并与MDCK细胞进行比较.方法 选择青海地区1株2010年因不明原因肺炎采集的鼻咽拭子标本,经检测流感核酸弱阳性,血凝阴性.分别接种等量的呼吸道样本于HAE和MDCK细胞并进行传代,观察细胞病变,电镜观察病毒形态,测定全基因组序列,同时测定血凝活性.结果 MDCK细胞无法分离获得病毒,但HAE可成功分离病毒：盲传3代后,可观察明显细胞病变;电镜下为典型的流感病毒形态;流感血凝抑制活性1∶16;经病毒基因组序列测定确定为近年来流行的季节性流感病毒H3N2,命名A/Qinghai/178/2010（H3N2）,其序列与南京2010年流行株A/Nanjing/1655/2010（H3N2）亲缘关系最近.结论 HAE可应用于陈旧性呼吸道样本的病毒分离鉴定,用于流感病毒分离比MDCK细胞更灵敏.

Objective To investigate the possibility of using well-differentiated human airway epithelial cells （HAE） to isolate and identify human influenza A virus from a stale respiratory tract specimen.Methods The stale specimen used in this study was a nasopharyngeal swab specimen collected from a patient with unexplained pneumonia in Qinghai in 2010.It was positive for influenza A virus （H3N2） RNA, but negative for hemagglutination.Equal amount of the specimen was inoculated on HAE and on Madin-Darby canine kidney （MDCK） cells for virus isolation and passage.Cytopathic effects were observed daily after inoculation.Hemagglutination inhibition test was performed at every passage.Electron microscope was used to observe viral morphology.Viral genome was sequenced, followed by molecular evolutionary analysis.Results No progeny virus was isolated in MDCK cells, while a influenza A virus subtype H3N2 strain [A/Qinghai/178/2010（H3N2）] was isolated in HAE with a typical morphology and cytopathic effect of influenza A infection.The hemagglutination inhibition activity was 1∶16.Results of the molecular evolutionary analysis of viral genome showed that the influenza A virus （H3N2） strain was highly homologous to the A/Nanjing/1655/2010（H3N2） strain, which was isolated during the 2010 influenza pandemic in Nanjing.Conclusion HAE can be used for isolation and identification of virus from stale respiratory tract specimens.It is more sensitive than MDCK cells with regard to human influenza virus isolation.