Human and avian influenza viruses target different cell types in cultures of human airway epithelium

Abstract

The recent human infections caused by H5N1, H9N2, and H7N7 avian influenza viruses highlighted the continuous threat of new pathogenic influenza viruses emerging from a natural reservoir in birds. It is generally believed that replication of avian influenza viruses in humans is restricted by a poor fit of these viruses to cellular receptors and extracellular inhibitors in the human respiratory tract. However, detailed mechanisms of this restriction remain obscure. Here, using cultures of differentiated human airway epithelial cells, we demonstrated that influenza viruses enter the airway epithelium through specific target cells and that there were striking differences in this respect between human and avian viruses. During the course of a single-cycle infection, human viruses preferentially infected nonciliated cells, whereas avian viruses as well as the egg-adapted human virus variant with an avian virus-like receptor specificity mainly infected ciliated cells. This pattern correlated with the predominant localization of receptors for human viruses (2-6-linked sialic acids) on nonciliated cells and of receptors for avian viruses (2-3-linked sialic acids) on ciliated cells. These findings suggest that although avian influenza viruses can infect human airway epithelium, their replication may be limited by a nonoptimal cellular tropism. Our data throw light on the mechanisms of generation of pandemic viruses from their avian progenitors and open avenues for cell level-oriented studies on the replication and pathogenicity of influenza virus in humans.

Figures

Fig. 1.

Differential cellular tropism of human and avian influenza viruses in HTBE cultures. The cultures were infected with Mem96-M (a and c) or DkH1 (b and d) viruses at the moi of ≈1, fixed 7 h after infection, and double-immunostained for virus antigen and for cilia of ciliated cells as described in Materials and Methods.(a and b) Light microscopy [virus (red), cilia (gray)]. (c and d) Fluorescent confocal microscopy [virus (red), cilia (green)]. Virus antigen was not observed in the negative controls in which infected cultures were either immunostained with omission of the antiviral Abs or fixed 2 h after infection. (Bars = 10 μm.)

Fig. 6.

Receptor specificity and cell tropism of influenza viruses. (a) Association constants (Kass) of virus complexes with monospecific HRP-labeled fetuin, which carried either 2-3-linked or 2-6-linked sialic acids (filled and hatched bars, respectively). Higher Kass values indicate stronger binding. (b) HTBE cultures were infected, fixed 7 h after infection, and double-immunostained for light microscopy. Percentages of infected ciliated cells (filled bars) and infected nonciliated cells (hatched bars) with respect to the total amount of infected cells were determined as described in Materials and Methods. For Mem96-M and DkH1, the experiments were repeated three times with different lots of HTBE cultures. Mem96-E1 was tested in parallel with two other viruses in the first two experiments.

Fig. 2.

Human influenza virus targets secretory cells. The HTBE cultures were infected with human virus Mem96-M, fixed 7 h after infection, and immunostained for cilia (gray) and virus antigen (red). Secretory cells were identified by using Alcian blue (pH 3)-periodic acid-Schiff staining (purple). The cultures were counterstained with hematoxylin, embedded (Crystal Mount, Biomeda), and sectioned (5 μm). (Bar = 10 μm.)

Fig. 3.

Cellular tropism of human and avian influenza viruses in differentiated cultures of nasal epithelial cells. The cultures were infected with either Mem96-M (a) or DkH1 (b) viruses, fixed 7 h after infection, and immunostained for cilia (gray) and virus antigen (red). (Bars = 10 μm.)

Fig. 4.

Spread of human and avian viruses in HTBE cultures in the course of multicycle infection. Cultures were infected with Mem96-M (a and c) or DkH1 (b and d) at a moi of ≈0.02, fixed 24 h after infection, and immunostained [virus (red), cilia (gray)]. [Objectives, ×10 (a and b) and ×40 (c and d). Bars = 25 μm.]

Fig. 5.

Localization of 2-6- and 2-3-linked sialic acids on the surface of HTBE cultures. Cultures were incubated with digoxigenin-labeled lectins SNA (a and c) or MAA (b and d), followed by fixation and double-immunostaining for digoxigenin (red) and cilia (gray). (a and b) Scanned images of stained cultures (membrane diameter 12 mm). (c and d) Micrographs of mounted cultures. (Bars = 10 μm.)