Hemagglutinin-dependent tropism of H5N1 avian influenza virus for human endothelial cells

Abstract

Although current H5N1 highly pathogenic avian influenza viruses (HPAIV) are inefficiently transmitted to humans, infected individuals can suffer from severe disease, often progressing rapidly to acute respiratory distress syndrome and multiorgan failure. This is in contrast with the situation with human influenza viruses, which in immunocompetent individuals usually cause only a respiratory disease which is less aggressive than that observed with avian H5N1 viruses. While the biological basis of inefficient transmission is well documented, the mechanisms by which the H5N1 viruses cause fatal disease remain unclear. In the present study, we demonstrate that human pulmonary microvascular endothelial cells (hPMEC) had a clearly higher susceptibility to infection by H5N1 HPAIV than to infection by human influenza viruses. This was measurable by de novo intracellular nucleoprotein production and virus replication. It was also related to a relatively higher binding capacity to cellular receptors. After infection of hPMEC, cell activation markers E-selectin and P-selectin were upregulated, and the proinflammatory cytokines interleukin-6 and beta interferon were secreted. H5N1 virus infection was also associated with an elevated rate of cell death. Reverse genetics analyses demonstrated a major role for the viral hemagglutinin in this cell tropism. Overall, avian H5N1 viruses have a particular receptor specificity targeting endothelial cells that is different from human influenza viruses, and this H5N1 receptor specificity could contribute to disease pathogenesis.

FIG. 1.

Infection and replication of influenza A viruses in hPMEC. (A and B) hPMEC/MDCK cell infectivity ratios. hPMEC and MDCK cells were infected in parallel with different dilutions of virus, and at 6 h p.i., cells were harvested and stained for NP. Virus dilutions giving 30 to 90% NP⁺ MDCK cells were selected. This corresponded to MOIs of 0.3 to 0.9 IU/cell (determined with MDCK cells) (A) Side scatter/NP dot plots from a representative experiment with the percentage of NP⁺ cells are shown (MOIs selected for the graphs: H1N1 NC, 0.9 IU/cell; H5N1, 0.7 IU/cell). (B) Infectivity ratios, determined by dividing the percentage of NP⁺ hPMEC by the percentage of NP⁺ MDCK cells. Means and standard deviations of data from three independent experiments are shown. The differences between all human isolates and the H5N1 viruses were statistically significant (P < 0.02). HP H5N1 T/T also differed significantly from NIBRG23 and H7N1 HP (P < 0.05). (C) hPMEC were infected at an MOI of 1 IU/cell and analyzed at 30 h p.i. Box plots showing median values (black line) and 25th and 75th percentiles of the percentage of NP⁺ cells were calculated from five independent experiments. P values calculated for human and all avian viruses and P values for avian H7N1 and H5N1 viruses were below 0.01, indicating statistically significant differences between these groups. The P value for HP H5N1 T/T and NIBRG23 was above 0.05. (D) Supernatants of infected hPMEC were collected at different time points after infection and then titrated on MDCK cells to determine TCID₅₀/ml. The error bars represent the standard deviations from two independent experiments. (E) hPMEC were infected at low MOIs (0.01 to 0.5 IU/cell, depending on the virus) to enable the visualization of infectious foci. At 24 h p.i., cells were stained for NP.

FIG. 2.

SA expressed on hPMEC serves as a receptor for infection. (A) hPMEC were harvested with PBS-EDTA and then stained for SA using MAL II (left) and SNA (right) lectins. As a control, hPMEC were treated with 250 mU NA/ml for 3.5 h before being harvested (dotted lines). (B) hPMEC were treated overnight with the indicated amount of NA/ml. Then, cells were infected with HP H5N1 T/T for 1 h at 4°C (MOI of 5 IU/cell). After being washed, the cells were incubated for 6 h at 37°C, harvested, and stained for intracellular NP. (C) hPMEC were treated overnight with 250 mU NA/ml cells and then stained for AnnexinV and PI to determine cell viability. Error bars represent the standard deviations of two independent experiments.

FIG. 3.

Role of H5 for infection of hPMEC. (A) Comparison of the R1/R2 virus pair and impact of insertion of the HA of R1, R2, and H5N1 Yamaguchi (Y) within the LP H5N1 Vac backbone on infectivity. Bar graphs with infectivity ratios determined as described for Fig. 1A and B and standard deviations from three independent experiments are shown. R1 differed significantly from R2 (P < 0.01) but not from LP H5N1 Vac, Vac-R1_HA, and Vac-R2_HA (P > 0.05). Vac-Y_HA differed from all other viruses (P < 0.01). (B) To quantify viral particles containing viral RNA attached to the cells, hPMEC and MDCK cells were incubated for 1 h at 4°C with virus (MOI of 1 IU/cell), and M1 RNA was quantified. The ratios of viral RNA bound to hPMEC compared to MDCK cells are shown. P values were calculated with the Mann-Whitney rank sum test and showed significant differences between H1N1 NC and HP H5N1 T/T (P = 0.005) and between LP H5N1 Vac and Vac-Y_HA (P = 0.010).

FIG. 4.

Cell death and apoptosis in infected hPMEC. (A) Microphotographs of hPMEC and MDCK cells, infected at an MOI of 1 IU/cell at indicated time points. The cells were treated with 10 μg/ml mitomycin C to prevent cell division. (B) Bar graphs of AnnexinV and NP staining, showing the percentage of AnnexinV⁺ NP⁺ (dark gray) and AnnexinV⁺ NP⁻ (light gray) cells for the hPMEC cultures shown in panel A.

FIG. 5.

hPMEC activation by influenza virus. (A) E/P-selectin upregulation in hPMEC infected at an MOI of 1 IU/cell, measured 21 h p.i. by flow cytometry. (B to D) Cytokines in supernatants harvested 24 h p.i. (B) IL-6 response determined by ELISA. Standard deviations of two independent experiments are shown. (C) IFN type I tested by bioassay. (D) IFN-β analyzed by ELISA. For panels C and D, error bars represent standard deviations of triplicates of one representative experiment.