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Title: A primary chicken tracheal cell culture system for the study of infection with avian respiratory viruses

Author
item Zaffuto, Kristin
item Estevez, Carlos
item Afonso, Claudio

Submitted to: Avian Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/2/2007
Publication Date: 1/17/2008
Citation: Zaffuto, K.M., Estevez, C., Afonso, C.L. 2008. Primary chicken tracheal cell culture system for the study of infection with avian respiratory viruses. Avian Pathology. 37(1):25-31.

Interpretive Summary: A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through the tracheal cells of the respiratory tract. A cell-culture system for tracheal epithelial cells from chicken embryos is described here, as well as its use in studies to examine the effect of low virulence AIV and NDV on the cells. The cells cultured from chicken embryos were confirmed to be epithelial by different staining techniques, observed conformation via microscopy, and analysis of cellular gene expression. Actual infection of the epithelial cell cultures with low virulence AIV and NDV was demonstrated using fluorescent microscopy, and the cells were found to be capable of supporting the growth of both viruses. This cell-culture system will be useful to study the mechanisms of viral infection and early host responses because it mimics the route of natural infection.

Technical Abstract: A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through cells of the airway epithelium. Here we describe the development and optimization of conditions for culture of tracheal epithelial cells from chicken embryos as well as their use in studies of low virulence avian respiratory viruses. Positive immunostaining for cytokeratin, the presence of cilia and microvilli, and microarray analysis of transcribed RNA demonstrated that the isolated cells were epithelial in nature. Infection of the epithelial cell cultures with low virulence AIV and NDV was demonstrated using immunofluorescence or green fluorescence protein fluorescence microscopy, respectively. Growth curves of AIV and NDV in tracheal epithelial cell (TEC) cultures revealed that TECs can fully support the growth of AIV and NDV and viral spread. This system, which mimics that of the natural infection, will be useful to study the mechanisms of viral infection and the early host transcriptional responses.