FIG. 5.

HAE cultures were infected apically with equal titers of two different subtypes of avian influenza virus A/Duck/Singapore/5/97 (H5N3) (A) and A/Duck/England/62 (H4N6) (B) and a human influenza virus A/England/26/99 (H3N2) (C) for 1 h at 37°C. Virus was removed, and after a further 23-h incubation, the cultures were fixed with methanol/acetone (50:50) and stained en face with anti-influenza A nucleoprotein antibody and goat anti-mouse conjugated to Alexa Fluor 488. Representative fluorescent photomicrographs of the stained cultures are shown. Avian influenza viruses A/Duck/Singapore/5/97 (D) and A/Duck/England/62 (E) were found to be localized exclusively in ciliated cells by costaining with rabbit anti-avian influenza A nucleoprotein (green) and mouse monoclonal β-tubulin IV antibody (red) to identify ciliated cells and visualization with anti-rabbit FITC and anti-mouse Texas Red. Arrows, avian virus-infected ciliated cells.