Influenza A Virus Infection of Human Middle Ear Cells In Vitro

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Abstract
Hypothesis Human-derived normal middle ear mucosal cells can be harvested and cultured and will support influenza A virus (INF A) infection. Study Design Protocols for the collection and in vitro culture of middle ear mucosal cells were developed and used to investigate the effects of INF A infection as it relates to the pathogenesis of otitis media. Materials and Methods Middle ear mucosa was harvested during surgeries that opened the normal middle ear. Middle ear mucosal cells were plated and grown in collagen-coated dishes. Cells were characterized before and after INF A exposure using phase-contrast and immunofluorescence microscopy as well as reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 18 gene expression and INF A. Results Primary cultures of human middle ear epithelial cells were established. Prolonged growth of middle ear cells yielded a second cell type that failed to stain for cytokeratin on immunofluorescence but continued to produce positive RT-PCR results on cytokeratin 18 analysis. After INF A exposure, cytological changes and immunofluorescence staining showed cellular infection. RT-PCR analysis using INF A–specific primers showed positive results for up to 72 hours after viral exposure. Conclusions Primary cultures of human middle ear mucosal cells have been established. Two distinctly different cell culture systems have been developed: 1) middle ear epithelial cells and 2) either dedifferentiated epithelial cells or fibroblasts. Exposure of both cell types to INF A demonstrates that each can support cellular infection and viral replication. These models should be useful for studies of the pathogenesis of virus-mediated otitis media.