人偏肺病毒在传代细胞和人呼吸道上皮细胞中分离和鉴定

Isolation and Identification of the Human Metapneumovirus in Continuous Cells and Human Airway Epithelium Cells

人偏肺病毒(Human metapneumovirus,HMPV)是2001年鉴定出的新发呼吸道病毒,婴幼儿、老人和免疫抑制人群易感,引起上呼吸道和下呼吸道感染,目前尚无疫苗和特异性治疗方案。为获得北京地区HMPV临床流行毒株,本研究将经荧光定量PCR检测为HMPV阳性的鼻咽抽吸物样本分别接种LLC-MK2、Vero-E6和分化良好的人呼吸道上皮细胞(Human Airway Epithelium,HAE),观察细胞病变、检测免疫荧光、电镜观察病毒形态、测定病毒滴度及分析复制特点,对分离获得的HMPV分离株进行鉴定。结果表明,HMPV感染LLC-MK2细胞可形成合胞体,但在Vero-E6中多呈单个细胞感染;经免疫荧光检测,HAE、LLC-MK2和Vero-E6细胞均可见绿色荧光;电镜结果可见病毒为近似球型的颗粒,有包膜和刺突,直径约在150nm~200nm之间;HMPV在HAE和LLC-MK2两种细胞上的复制特点基本相同。本研究成功建立了临床样本在LLC-MK2、Vero-E6和HAE分离培养HMPV的方法,分离并鉴定了HMPV临床分离株,为HMPV感染机制的研究奠定基础。

The human metapneumovirus(HMPV)discovered in 2001 is a common cause of respiratory-tract infections in children,elderly,and immunocompromised patients. The predominant clinical scenario is an infection in the upper and/or lower respiratory tract. However,licensed vaccine or specific treatment for HMPV infection are lacking. We wished to isolate HMPV strains from clinical samples in Beijing,China. Positive nasopharyngeal aspirates were detected by HMPV-specific polymerase chain reaction,and inoculated into human airway epithelium(HAE),LLC-MK2 or Vero-E6 cells,respectively,to isolate the HMPV. The cytopathic effect,indirect immunofluorescence,morphology using transmission electron microscopy(TEM),virulence,and replication property were determined in isolated strains to identify the HMPV. Syncytia were observed in LLC-MK2 cells infected with the HMPV. A single-cell infection focus was observed in Vero-E6 cells. Green fluorescence was found in HAE, LLC-MK2 and Vero-E6 cells according to an immunofluorescence assay. The HMPV was an irregular sphere with an envelope and spikes and diameter of150 nm–200 nm according to TEM. The replication property of the HMPV in HAE and LLC-MK2 cells was almost identical. In summary,we established a method for the isolation and culture of the HMPV in LLC-MK2,Vero-E6 and HAE cells,isolated and identified one strain of the HMPV,and provided a theoretical basis for control and prevention of HMPV infection.