The infection of chicken tracheal epithelial cells with a H6N1 avian influenza virus

Abstract

Sialic acids (SAs) linked to galactose (Gal) in α2,3- and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. We demonstrate that chicken tracheal ciliated cells express α2,3-linked SA, while goblet cells mainly express α2,6-linked SA. In addition, the plant lectin MAL-II, but not MAA/MAL-I, is bound to the surface of goblet cells, suggesting that SA2,3-linked oligosaccharides with Galβ1-3GalNAc subterminal residues are specifically present on the goblet cells. Moreover, both α2,3- and α2,6-linked SAs are detected on single tracheal basal cells. At a low multiplicity of infection (MOI) avian influenza virus H6N1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the H6N1 virus. At a MOI of 1, ciliated, goblet and basal cells are all permissive to the AIV infection. This result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry.

Figure 1. The SAα2–3Gal expression of primary CTE cells.

The ciliated cells, goblet cells and basal cells were revealed by their specific markers: the β-tubulin (A–C), mucin (D–F) and K14 (G, H), respectively. The cellular distribution of SAα2–3Gal was characterized by the double-staining of biotin-labeled MAA (EY Laboratories, 1∶500) (A, B, D, G), MAL-1 (E) or MAL-II (both from Vector laboratories, 1∶500) (C, F, H). Scale bar, 50 µm.

Figure 2. The SAα2–6Gal expression of primary CTE cells.

The mucin (A), K14 (B) and β-tubulin (C and D) were the markers for goblet cells, basal cells and ciliated cells, respectively. The cellular distribution of SAα2–6Gal was shown by the double-staining of biotin-labeled SNA (Vector Laboratories, 1∶500) (A–C) and SNA-1 (EY laboratories, 1∶500) (D). Scale bar, 50 µm.

Figure 3. The SAα2–3Gal and SAα2–6Gal expression on basal cells.

The tracheal basal cell was identified by the expression of K14 (1∶100, Convance) (A). Panels (A, B, C) with two fluorescent tags are shown in juxtaposition, illustrating the triple immunocytostaining result in a same field. The arrows in A, B and C indicate cells that are triple-positive for K14, MAA and SNA. The cell nuclei were stained with DAPI (blue). Scale bar, 50 µm.

Figure 4. The SA expression on AIV H6N1 infected cells.

A total of 5×10⁴ CTE cells were infected with 0.5 µl of AIV H6N1 2838V (viral stock, EID₅₀ = 10⁸/ml) at a MOI of 1 for 1 h at 37°C. At 6 h.p.i., the expression of viral H6N1 proteins was detected by chicken serum against the AIV (1∶500, red) (A, B). The ratio of MAA or SNA expression on the infected cells was manually counted from five individual fields (C). Scale bar in panel A, 100 µm; in panel B, 50 µm.

Figure 5. The tropism of AIV H6N1 for CTE cells.

A total of 5×10⁴ CTE cells were infected with AIV H6N1 2838V at a MOI of 0.1 (A–C) or 1 (D–E) for 1 h at 37°C. At 6 h.p.i., infection by H6N1 was detected by chicken H6N1 immune-serum (1∶500, red). The ciliated cells, goblet cells and basal cells were revealed by the expression of β-tubulin (A, D), mucin (B, E) and K14 (C, F), respectively. Scale bar, 50 µm.

Figure 6. A speculated diagram of the SA expression and AIV infection in human and chicken tracheal epithelial cells.

The structure of the pseudostratified tracheal epithelial cells (A) and the human (A) and chicken (B) expression profiles of SAα2–3Gal (red dots) and SAα2–6Gal (blue dots) on the ciliated cells (green), goblet cells (yellow) and basal cells (pink) are illustrated. Infection of chicken tracheal epithelial cells by AIV at a MOI of 0.1and 1 are illustrated in C and D, respectively.