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. 2006 Aug;80(16):8060-8.
doi: 10.1128/JVI.00384-06.

Infection of human airway epithelium by human and avian strains of influenza a virus

Catherine I Thompson  1 , Wendy S Barclay, Maria C Zambon, Raymond J Pickles
Affiliations

Infection of human airway epithelium by human and avian strains of influenza a virus

Catherine I Thompson et al. J Virol. 2006 Aug.

Abstract

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.

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Figures

FIG. 1.
FIG. 1.
Histological cross sections of HAE and excised human airway tissue samples were probed with anti-β-tubulin IV (red) and the lectin (green) SNA for the human influenza virus receptor (α-2,6-linked sialic acid) or MAA for the avian influenza virus receptor (α-2,3-linked sialic acid). SNA was visible on the apical surface of nonciliated (A) and ciliated cells (B). SNA on ciliated and nonciliated cells was also observed in excised human airway tissue samples (C). MAA was visible on the apical surface of ciliated cells and, to a lesser degree, on nonciliated cells (D). MAA was observed at the apical surface of ciliated cells in paraffin-embedded, excised human airway tissue (E). Arrows, ciliated cells; arrowheads, nonciliated cells.
FIG. 2.
FIG. 2.
Viral proteins were detected in ciliated (A and B) and nonciliated (C and D) cells in histological cross sections of HAE infected with recent influenza A H3N2 virus strain A/England/26/99 and fixed at 6 h postinfection with 4% paraformaldehyde. Sections were costained with anti-influenza A H3N2 rabbit polyclonal antibody (green) and anti-β-tubulin IV (red) to identify ciliated cells. Cell nuclei were visualized with DAPI staining in VectorShield mounting medium. Representative fluorescent photomicrographs of viral proteins localized in the cell nuclei and cytoplasm are shown costained with β-tubulin IV and DAPI (A and C), and the same images are shown with FITC staining only (B and D).
FIG. 3.
FIG. 3.
(A) HAE cultures were infected with human influenza A viruses isolated in England from 1969 to 2000 (A/England/878/69, A/ England/26/99, and A/England/24/00 [H3N2]) and with avian influenza A viruses (A/Duck/England/62 [H4N6], A/Duck/Ukraine/63 [H3N8], and A/Duck/Singapore/5/97 [H5N3]) (MOI, 0.1). Histological cross sections of HAE were immunostained with anti-influenza A H3N2 rabbit polyclonal antibody or anti-avian influenza nucleoprotein rabbit polyclonal antibody and counterstained for β-tubulin IV to identify ciliated cells. Viral proteins were localized at 6 h (human viruses) postinfection in both ciliated and nonciliated cell types, and the proportion of infected ciliated and nonciliated cells for the older virus (1969) and more-recent (1999 to 2000) virus strains was determined. Viral protein from avian strains was found only at 24 h postinfection almost exclusively in ciliated cells. Two independent experiments were performed. The proportion of ciliated and nonciliated cell types with respect to the total number of infected cells in three separate cross sections (10 to 100 infected cells/section) was determined. The number of infected cell types as a proportion of the total number of infected cells from a representative experiment is displayed. The lower table indicates the total number of potential glycosylation site motifs on HA for each virus of the H3 subtype. (B) Three-dimensional structure model of H3 HA (Protein Data Bank identification number 1HGF) (17, 33) produced using Protein Explorer (18), indicating the position of five new potential glycosylation sites (dark gray spacefill) on the globular head of the HA molecule in the vicinity of the receptor binding site. Numbers indicate Asn residues in the first position of the glycosylation site motif. Potential glycosylation site motifs at positions 126 and 246 were found in A/Victoria/3/75-like and A/Mississippi/1/85-like viruses, respectively. Motifs at positions 122 and 133 were found in viruses isolated from 1999 onward; the motif at position 144 was sometimes found in viruses isolated in 1999 and was absent from subsequent strains.
FIG. 4.
FIG. 4.
HAE cultures were treated with neuraminidase from Vibrio cholerae for 3 h at 37°C (A and B). Control cultures (C and D) were incubated with serum-free DMEM only. Cells were infected via the apical surface with human H3N2 influenza virus A/England/26/99 or PIV3-GFP and fixed at 24 h postinfection. Influenza virus-infected cultures were stained with mouse anti-influenza A nucleoprotein antibody, followed by goat anti-mouse IgG conjugated to Alexa Fluor 488. Representative photomicrographs of neuraminidase-treated HAE cultures infected with influenza virus (A) and PIV3-GFP (B) and control cultures infected with influenza virus (C) and PIV3-GFP (D) are shown.
FIG. 5.
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.
FIG. 6.
FIG. 6.
HAE cultures were infected with human H3N2 influenza A viruses isolated in England between 1969 and 2000 (A/England/878/69, A/England/26/99, and A/England/24/00) and with avian H5N3 influenza A strain A/Duck/Singapore/5/97 at an MOI of 0.1. Virus released from the apical surface of infected cultures at 6 and 24 h postinfection was quantified by a TCID50 assay of MDCK cells. Representative results from two independent experiments are shown. Viral titers are expressed as log TCID50/100 μl.
FIG. 7.
FIG. 7.
HAE cultures were infected with avian (A/Duck/Singapore/5/97) and human (A/England/26/99) influenza A viruses at an MOI of 0.1 and fixed at 12 h postinfection with perfluorocarbon-osmium tetroxide that preserves the air-surface microenvironment of HAE. Sections were processed for electron microscopy and analyzed. For the avian influenza strain, only one small cluster of budded virus particles was visible after many fields were scanned (A). Released human influenza virus particles were readily detected above the surfaces of cells. The released virus morphology was predominantly spherical (B), and virions were found to be accumulated at the level of microvillar tips (C). Scale bars are shown for each panel. Arrows, microvilli; small arrowheads, virus particles; large arrowhead in panel C, cilia.
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