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Influenza A virus infection of human middle ear cells in vitro
1Department of Otolaryngology, University of Miami School of Medicine, Florida, USA. cbuchman@med.miami.edu
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.
Insights
Human middle ear cells can be cultured to support influenza A virus (INF A) infection. These cell models are valuable for studying virus-caused otitis media pathogenesis.
Area of Science:
- Otorhinolaryngology
- Virology
- Cell Biology
Background:
- Otitis media is a common condition, often linked to viral infections.
- Understanding viral pathogenesis in the middle ear is crucial for effective treatment.
Purpose of the Study:
- To establish and characterize human middle ear mucosal cell cultures.
- To investigate the susceptibility of these cultures to influenza A virus (INF A) infection.
- To develop models for studying virus-mediated otitis media.
Main Methods:
- Harvesting and in vitro culturing of human middle ear mucosal cells.
- Characterization using microscopy and RT-PCR.
- Exposure to INF A to assess infection and replication.
Main Results:
- Successful establishment of primary human middle ear epithelial cell cultures.
- Identification of two distinct cell types (epithelial and possibly fibroblasts/dedifferentiated cells).
- Demonstrated cellular infection and viral replication of INF A in both cell types for up to 72 hours.
Conclusions:
- Human middle ear cell cultures can be established and support INF A infection.
- Developed cell models are suitable for studying the pathogenesis of virus-induced otitis media.