Figure 3.

Seasonal, high- and low-pathogenicity avian, porcine, and pandemic 2009 viruses do not infect type I pneumocytes. A, Human lung tissue was infected with Pan/99(H3N2) for 24 hours. Slices were incubated with fluorescently labeled anti–influenza A virus antibody (green channel) to detect virus-infected cells (white arrowheads) and anti-EMP2 antibody (red channel) to detect type I pneumocytes. B, Lung explants were not infected (mock) or were infected with the strains NC/99(H1N1), Thai/04(H5N1), Dk/Alb(H12N5), Sw/Wis(H1N1), and Bay/09(H1N1pdm) for 24 hours and processed as described in A. Micrographs show fields with merged red and green channels. Representative pictures of at least 3 independent experiments are shown. C, Detection of human (α-2,6SA) and avian (α-2,3SA) influenza virus receptors on type I and type II pneumocytes. Sections of uninfected human lung tissue were incubated with alkaline phosphatase–conjugated lectin from Maackia amurensis (MAA; α-2,3SA specificity) or Sambucus nigra agglutinin (SNA; α-2,6SA specificity) and developed by Vector Red staining (red channel), followed by incubation with anti–caveolin 1 (green channel) to detect type I pneumocytes or anti–prosurfactant protein C (green channel) to detect type II pneumocytes. Nuclei were stained with DAPI (blue channel). The slides were analyzed by confocal microscopy by spectral imaging, utilizing the fluorescent properties of the Vector Red alkaline phosphatase substrate and unmixing the different spectra of Alexa 488, tissue autofluorescence, and Vector Red (objective 63 × , Plan-Apochromat, NA 1.4).