Influenza A virus infection of human middle ear cells in vitro

Hypothesis: Human-derived normal middle ear mucosal cells can be harvested and cultured and will support influenza A virus (INF A) infection. Study De sign: Protocols for the collection and in vitro culture of middle ear mucos al cells were developed and used to investigate the effects of INF A infect ion as it relates to the pathogenesis of otitis media. Materials and Method s: Middle ear mucosa was harvested during surgeries that opened the normal middle ear. Middle ear mucosal cells were plated and grown in collagen-coat ed dishes, Cells were characterized before and after INF A exposure using p hase contrast and immunofluorescence microscopy as well as reverse transcri ptase-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 cel l type that failed to stain for cytokeratin on immunofluorescence but conti nued to produce positive RT-PCR results on cytokeratin 18 analysis. After I NF A exposure, cytological changes and immunofluorescence staining showed c ellular infection. RT-PCR analysis using INF A-specific primers showed posi tive results for up to 72 hours after viral exposure. Conclusions: Primary cultures of human middle ear mucosal cells have been established. Two disti nctly different cell culture systems have been developed: 1) middle ear epi thelial cells and 2) either dedifferentiated epithelial cells or fibroblast s. 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.