Viruses.

The following virus isolates were used (Tables ​(Tables11 and ​and2):2): A/NewCaledonia/20/99 (H1N1) and A/Wisconsin/67/05/9187 (H3N2) (kindly obtained from Werner Wunderli, National Reference Center for Influenza, University Hospital, Geneva, Switzerland), A/PR/8/34 (H1N1) (kindly obtained from Georg Kochs, University of Freiburg, Freiburg, Germany), and HPAIV isolates A/cygnus olor/Italy/742/06 (H5N1), A/turkey/Turkey/1/2005 (H5N1), and A/ostrich/Italy/2332/2000 (H7N1) (kindly provided by William Dundon, IZSV Istituto Zooprofilattico Sperimentale delle Venezie, Venice, Italy). The following viruses created by reverse genetics were used: NIBRG23, with the HA (polybasic cleavage site of HA removed) and NA from H5N1 A/turkey/Turkey/1/2005 and the remaining genes from H1N1 A/PR/8/34 (kindly obtained from Jim Robertson, National Institute for Biological Standards and Control, United Kingdom); R1, with the HA and NA derived from A/Hong Kong/68 (H3N2) and the remaining genes from A/WS/33 (H1N1); R2, identical to R1 but modified for α-2,3-SA binding specificity (22); A/duck/Hokkaido/Vac-1/04 (LP H5N1 Vac), composed of H5 from a low-pathogenic avian influenza virus (LPAIV), isolate A/duck/Mongolia/54/01 (H5N2), and N1 from isolate A/duck/Mongolia/47/01 (H7N1); and Yamaguchi (Y), generated from an HPAIV, A/chicken/Yamaguchi/7/04 (H5N1) (20, 34) (Table ​(Table1).1). All viruses were propagated in 10-day-old embryonated, specific-pathogen-free chicken eggs. We followed the protocol of the WHO Manual on Animal Influenza Diagnosis and Surveillance with some minor changes. Instead of 0.1 ml we inoculated 0.2 ml of different dilutions of virus. The inoculated eggs were incubated for 24 h (HPAIV) or 48 h (other influenza viruses).

Reverse genetics.

293T and MDCK cells were mixed (1:1) in MDCK medium at a concentration of 1.5·10⁵ cells/ml and plated in six-well plates at 2 ml/well. The set of eight plasmids (kindly provided by R. Webster, St. Jude Children's Research Hospital, Memphis, TN) (11) was used to create the LP H5N1 Vac virus (LPAI H5N1 A/duck/Hokkaido/Vac-1/04) (34). In this set, the HA and NA were exchanged with those from an HPAIV, H5N1 A/chicken/Yamaguchi/7/04, and those of the R1 and R2 viruses described above (Table ​(Table1).1). For plasmid DNA (1 μg) transfection, we used Lipofectamine (Invitrogen) according to the manufacturer's instructions. Before transfection, cells were washed and 1.5 ml fresh medium containing 5% FBS was added per well. At 6 h after transfection, the medium was removed and replaced by MEM containing 1× nonessential amino acids, 1 mM sodium pyruvate, 1 mM HEPES, 0.125% bovine serum albumin (BSA), and 2 μg/ml l-1-tosylamide-2-phenylethyl chloromethyl ketone trypsin (Sigma-Aldrich, Buchs, Switzerland). Supernatants were harvested 72 h later and tested for virus by an HA test and titration on MDCK cells. For HA tests, supernatants were serially diluted 1:2 in 96-well plates. Then, 100 μl of supernatants were mixed with 100 μl 2% chicken erythrocytes.

Virus titrations.

Calculations of multiplicities of infection (MOI) were based on numbers of infectious units (IU) on MDCK cells, determined using a protocol adapted from Matrosovich et al. (21). At 6 h after titration, cells were washed with phosphate-buffered saline (PBS), and then the plates were dried and frozen at −20°C overnight. The next day, cells were fixed with 4% paraformaldehyde (Sigma) solution in PBS for 15 min at room temperature (RT), washed, and permeabilized with 100 μl/well of PBS containing 0.5% Triton X-100 and 20 mM glycine. Cells were immunostained for viral nucleoprotein (NP) by incubating for 1 h with monoclonal antibody HB65 (ATCC) diluted in PBS supplemented with 10% horse serum (Häst) and 0.05% Tween 80. Cells were washed five times with PBS-0.05% Tween 80, followed by 1 h of incubation with peroxidase-labeled rabbit anti-mouse antibodies (Dako, Zug, Switzerland) diluted 1:1,000 in PBS supplemented with 10% horse serum and 0.05% Tween 80. Cells were washed again five times with PBS-0.05% Tween 80 and then incubated for 15 min with TrueBlue substrate (KPL). Plates were washed with tap water and then dried. Single infected cells were counted under the microscope to determine numbers of IU/ml. Viral titers in cell culture supernatants were determined as 50% tissue culture infectious doses (TCID₅₀)/ml. To this end, supernatants were titrated on MDCK cells in 96-well plates in the presence of 1 μg/ml l-1-tosylamide-2-phenylethyl chloromethyl ketone trypsin (Sigma). At 72 h postinfection (p.i.), cells were washed and stained with crystal violet. The TCID₅₀/ml was calculated according to the Reed-Muench formula (WHO Manual on Animal Influenza Diagnosis and Surveillance).

Infection of EDC.

hPMEC and MDCK cells were seeded out in 12-well plates the day before infection at a concentration of 10⁵ cells/well. For infection, growth medium was removed and replaced by RPMI supplemented with 0.125% BSA (Intergen), 1 mM HEPES (Invitrogen), and 1× GlutaMax (Invitrogen). Cells were infected with an MOI of 1 IU/cell. Alternatively, hPMEC/MDCK cell infectivity ratios were determined by parallel infection of the two cell types with three different 10-fold dilutions starting with a 1:10 dilution of the original virus stock, and after 1 h of incubation at 4°C, the medium was removed and replaced by growth medium. Then, cells were incubated for 6 h at 37°C and 5% CO₂. To calculate the infectivity ratios, the conditions giving 30 to 90% NP⁺ MDCK cells were used.

To follow the infection over longer time periods, hPMEC were seeded out in 12-well plates the day before infection at a concentration of 10⁵ cells/well. For infection, growth medium was removed and replaced by RPMI supplemented with 0.125% BSA (Intergen), 1 mM HEPES (Invitrogen), and 1× GlutaMax (Invitrogen). After infection at an MOI of 1 IU/cell, the cells were incubated at 4°C to let the virus attach. As a negative control for infection, hPMEC were incubated with control allantoic fluid. After 1 h, cells were washed extensively with PBS (Invitrogen) to remove all unbound viruses. Then, growth medium was added and cells were incubated at 37°C and 5% CO₂ until harvesting. As a positive control for activation, we added 10 μg/ml poly(IC) (Sigma) to hPMEC when shifting cells from 4 to 37°C.

Virus inactivation.

A 1 M stock solution of 2-bromoethylamine hydrobromide (BEI) was prepared and pH controlled to ensure the BEI circularization into the active form at pH 9. The solution was incubated for 1 h at 37°C, and BEI was added to virus preparations at a final concentration of 3 mM. After 24 h at RT, BEI was neutralized by the addition of 10% (vol/vol) 1 M Na₂S₂O₃ for 1 h at RT. Inactivation was controlled on MDCK cells checked for cytopathogenic effects after 72 h of culture.

Virus binding assay.

MDCK cells and hPMEC were seeded out in 24-well plates at 10⁵ cells/well. After 24, the medium was replaced with RPMI (4°C) supplemented with 0.125% BSA (Intergen, NY), 1 mM HEPES (Invitrogen), and 1× GlutaMax (Invitrogen). Cells were kept on ice and infected with H1N1 NC, HP H5N1 T/T, LP H5N1 Vac, and Vac-Y_HA at an MOI of 1 IU/cell. After 1 h of incubation at 4°C, cells were washed five times with PBS (4°C) and lysed with 400 μl lysis buffer containing a defined amount of enhanced green fluorescent protein (EGFP) RNA (Machete-Nagel kit nucleospin multi 96-virus). The RNA extraction was done by a Tecan Freedom EVO Roboter, and M1 and EGFP RNA were amplified by real-time reverse transcriptase PCR. Primers and probe with the following sequences were used: 5′-TCAGGCCCCCTCAAAGCCGA-3′ (probe), 5′-AGATGAGYCTTCTAACCGA-3′ (forward primer), and 5′-GCAAAGACATCTTCAAGTYTC-3′ (reverse primer). RNA was amplified using the following protocol: 30 min at 48°C, 10 min at 95°C, 15 s at 95°C, 60 s at 54°C, and 60 s at 70°C. The last three steps were repeated 45 times in total. Threshold cycle (C_T) values were corrected to the amount of EGFP RNA. To compare the virus binding, the dilutions at which virus binding to the cells was not saturated and with C_T values below 30 were chosen. The relative amount of viral RNA was calculated by the ΔC_T method, and the amount of viral RNA relative to EGFP RNA was expressed as 2^−C_T (18). RNA levels detected on MDCK cells were set to 1 and compared to the RNA levels bound to hPMEC.

Cytokine analysis.

Supernatants of infected hPMEC were analyzed using the interleukin-6 (IL-6) duo kit reagents from R&D Systems (United Kingdom) and a beta interferon (IFN-β) enzyme-linked immunosorbent assay (ELISA) kit from PBL (R&D Systems) following manufacturers' instructions. Alternatively, IFN-α/β bioactivity was quantified using a porcine kidney cell line expressing firefly luciferase under the control of the murine Mx1 promoter (9). To this end, SK-6 cells (14) were transfected with a mixture of plasmids pGL3-Mx1P-Luc (kindly provided by Georg Kochs, Department of Virology, Institute of Medical Microbiology and Hygiene, Freiburg, Germany) (13) and pCI-neo (Promega) at a 20:1 (wt/wt) ratio using FuGENE 6 transfection reagent (Roche). After 48 h, the cell culture medium was replaced with fresh complete medium supplemented with 500 μg/ml G418-sulfate (Calbiochem-Novabiochem). Three rounds of end point dilution were applied to isolate G418-resistant colonies from single cells. The clones with the highest rates of porcine IFN-β-mediated luciferase induction and lowest background activity were selected. The purified SK6-MxLuc clones were maintained under selection with 250 μg/ml G418-sulfate. For IFN-α/β bioactivity, cells from an SK6-MxLuc clone were seeded in 24-well plates at 3·10⁵ cells/well. The next day, test supernatants of hPMEC infected at an MOI of 1 IU/cell were collected and inactivated by UV treatment to avoid direct stimulation of the luciferase transcription by the virus. Supernatant (500 μl) was added to one well of a six-well plate. Six-well plates were put on ice for UV treatment with 9,999 mJ UV. The standard was treated the same way. SK6-MxLuc cells were washed, and 150 μl of fresh medium was added, together with 150 μl of supernatants. Eighteen hours later, supernatants were removed and cells were lysed with 200 μl cell culture lysis reagent from the Promega luciferase assay kit (Promega). Luciferase activity was measured in triplicate with the luminometer.

Flow cytometry.

For E/P-selectin staining, cells were harvested with PBS-A (Invitrogen)-0.94 mM EDTA, washed with Cell Wash (Becton Dickinson), and incubated with CD62E/P antibody (Serotec) for 20 min at 4°C. Cells were washed and then incubated with fluorescein isothiocyanate-conjugated goat-anti mouse immunoglobulin G1 (IgG1) antibody (Southern Biotech) for 15 min at 4°C before a final wash. For NP detection, cells prepared as described above were fixed and permeabilized with Fix & Perm cell permeabilization kit (An der Grub) according to the manufacturer's instructions and labeled using anti-NP clone HB65 (ATCC). Then, the cells were washed and incubated with R-phycoerythrin-conjugated goat-anti mouse IgG2a antibody (Southern Biotech) for 10 min at RT. After a final washing step, the cells were resuspended in 200 μl Cell Wash. The percentage of NP⁺ hPMEC was divided by the percentage of NP⁺ MDCK cells to obtain the infectivity ratio. Apoptotic cells were quantified using the AnnexinV/PI kit from BenderMed following the manufacturer's instructions. To fix and permeabilize AnnexinV-stained cells for intracellular NP staining, cells were fixed for 10 min at RT in 4% paraformaldehyde diluted in AnnexinV staining buffer (140 mM NaCl, 2.5 mM CaCl₂, 10 mM HEPES in 500 ml double-distilled water). After fixation, cells were permeabilized by washing with 0.3% saponin in AnnexinV buffer. NP antibody was diluted in 0.6% saponin in AnnexinV buffer and incubated with the cells for 20 min on ice. Cells were washed with 0.3% saponin in AnnexinV buffer, and then R-phycoerythrin-conjugated goat anti-mouse IgG2a antibody was added to the cells diluted in 0.6% saponin in AnnexinV buffer. After 15 min on ice, cells were washed in 0.3% saponin in AnnexinV buffer and then once more with AnnexinV binding buffer before acquisition. The expression of α-2,3-SA and α-2,6-SA receptors on hPMEC was detected using the biotinylated lectins Maackia amurensis lectin II (MAL II) (Vector Laboratories) and Sambucus nigra lectin (SNA) (Vector Laboratories). The cells were harvested with PBS-EDTA and then washed with 10 mM Tris (pH 7.5), 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, and 0.5% BSA in double-distilled water. Cells were incubated with lectins at 20 μg/ml in the above buffer for 20 min on ice, washed, and stained with streptavidin-SpectralRed conjugated (Southern Biotech) for 15 min on ice. After a final washing step, fluorescence was measured by flow cytometry. All data were acquired with a FACSCalibur (Becton Dickinson) and analyzed with FlowJo software (Treestar Inc., Ashland, OR).

For enzymatic removal of SA, 10⁶ cells were incubated with NA from Vibrio cholerae (Sigma) at 37°C at the indicated time points and concentrations.

Infection of hPMEC is SA dependent.

Using lectin staining, we determined that hPMEC express avian and human type SA receptors for influenza virus. Both α-2,3-SA and α-2,6-SA (stained by MAL II and SNA, respectively) were homogenously expressed on hPMEC (Fig. ​(Fig.2A).2A). After treatment with NA, the staining intensity decreased, confirming the lectin-staining specificity for SA.

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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.

In order to demonstrate that HPAIV infection of hPMEC was SA dependent, we treated hPMEC with different concentrations of NA. This treatment reduced the infection of hPMEC by HP H5N1 T/T virus in a dose-dependent manner (Fig. ​(Fig.2B).2B). With a high dose of NA, which did not influence the viability of hPMEC (Fig. ​(Fig.2C),2C), the infection was almost abrogated.

H5 plays a major role in conferring infectivity of influenza virus for hPMEC.

In order to investigate the contribution of the viral surface glycoproteins HA and NA in the infection of hPMEC relative to the internal genes, the role of HA was studied further by infecting hPMEC with a pair of viruses possessing specific mutations in the HA. These mutations alter the receptor binding site specificity from α-2,6-SA (R1 virus) to α-2,3-SA (R2 virus) (22). R1 virus behaved like other human viruses, and infection led to a low ratio of infectivity (Fig. ​(Fig.3A).3A). In contrast, R2 was significantly more efficient in infecting hPMEC, resulting in a ratio of infectivity similar to that of the H7N1 virus shown in Fig. ​Fig.1B1B.

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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).

This analysis was elaborated using a different set of viruses composed of genes from the avian isolates A/duck/Mongolia/54/01 (H5N2) and A/duck/Mongolia/47/01 (H7N1) (LP H5N1 Vac). This LP H5N1 Vac virus containing an HA from an LPAIV isolate of a duck was relatively inefficient at infecting hPMEC (Fig. ​(Fig.3A).3A). However, exchanging the HA of the LP H5N1 Vac virus with the HA of R1 and R2 did reduce the capacity of the reassortant to infect hPMEC, compared to the parent LP H5N1 Vac virus (Fig. ​(Fig.3A,3A, Vac-R1_HA, Vac-R2_HA). In contrast, when the HA of the LP H5N1 Vac was exchanged with an HA from the HP H5N1 A/chicken/Yamaguchi/7/04 isolate (Fig. ​(Fig.3A,3A, Vac-Y_HA), a high infectivity ratio similar to that found with the HP H5N1 742 and T/T isolates (shown in Fig. ​Fig.1B)1B) was obtained.

To determine if the receptor binding of the HA was responsible for the observed differences between avian and human viruses, we investigated viral attachment to hPMEC. Binding of human H1N1 NC and avian HP H5N1 T/T virus and reverse genetics viruses LP H5N1 Vac and Vac-Y_HA to hPMEC and MDCK cells was measured by reverse transcriptase PCR (Fig. ​(Fig.3B).3B). The results showed that H1N1 NC virus did bind less efficiently to hPMEC than did HP H5N1 T/T. The same observation was made for the two reverse genetics viruses LP H5N1 Vac and Vac-Y_HA. Therefore, receptor binding specificity of the HA represents a crucial factor in determining tropism of H5N1 for EDC.

Virus-induced hPMEC apoptosis.

Despite the large NP amounts detected in hPMEC infected with HPAIV, we could not observe a strong increase in cell death by light microscopy. The cell monolayer remained intact during the observation period (data not shown). Since it appeared that dead cells were replaced by healthy, dividing cells, hPMEC and MDCK cells were treated with 10 μg/ml mitomycin C overnight in order to inhibit cell proliferation. Cells were then infected at an MOI of 1 IU/cell and observed by microscopy and AnnexinV/NP double staining over a time period of 64 h. Already after 23 h p.i., the MDCK cell monolayer was almost destroyed after infection with H3N2 WS67 or HP H5N1 (Fig. ​(Fig.4A).4A). In contrast, infected hPMEC did not show any sign of virus-induced cytopathogenicity at this time point. At 64 h p.i., a reduction of viable cells was detectable under all conditions, including mock, most probably due to the cytotoxic effects of the mitomycin C. By AnnexinV/NP staining it was possible to detect virus-induced cytopathogenic effects in the HP H5N1 T/T-infected cells already at 23 h p.i. (Fig. ​(Fig.4B).4B). Around 40 to 50% of the AnnexinV positive cells were NP⁻ during the whole observation period, indicating that apoptosis was also induced in uninfected cells. AnnexinV staining in H3N2 WS67-infected cells did not differ from that in mock controls at 23 h p.i., and only a small percentage of AnnexinV-positive cells were also positive for NP. These results indicate that HP H5N1 virus infection does induce apoptosis in hPMEC although at relatively low levels and late time points considering the high percentage of infected cells and in comparison to MDCK cells.

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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.

HPAIV induces E/P-selectin upregulation in hPMEC.

EDC play an important role in inflammatory responses through regulation of leukocyte migration, which is mediated by an activation-dependent expression of adhesion molecules such as E/P-selectin. Therefore, we were interested in the expression patterns of E/P-selectin following infection of hPMEC. After stimulation of hPMEC with poly(IC) as a positive control, E/P-selectin expression was upregulated (Fig. ​(Fig.5A).5A). Infection with H1N1 NC virus induced only a minor upregulation (Fig. ​(Fig.5A).5A). In contrast, HPAIV H7N1 virus was more potent at inducing E/P-selectin expression, and the highest rate of induction was found with H5N1 virus infection, where most of the hPMEC population showed an increased amount of E/P-selectin expression (Fig. ​(Fig.5A).5A). Double staining showed that not all E/P-selectin-expressing cells were NP⁺, indicating a role for indirect effects such as cytokines. Nevertheless, the addition of IL-6 or IFN-β alone to the cell culture was not sufficient to induce E/P-selectin upregulation in hPMEC (data not shown).

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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.

HPAIV-induced cytokine responses in hPMEC.

EDC also participate in inflammatory responses through secretion of cytokines and chemokines, for example IL-6. Therefore, we analyzed the supernatants of infected hPMEC for cytokines. HPMEC secreted IL-6 in response to poly(IC). Compared with mock controls, the viruses efficiently infecting hPMEC also induced increased levels of IL-6. This included the H5N1 viruses NIBRG23, T/T, and 742, as well as the H7N1, LP H5N1 Vac, and R2 viruses (Fig. ​(Fig.5B).5B). When we inactivated NIBRG23 virus with BEI before infection, induction of IL-6 secretion by hPMEC was abolished, showing that viral replication is essential in order to induce IL-6 secretion.

This work was in part funded by BVET grants 1.05.q and 1.07.i and the EU FP6 projects Panfluvac and Flupath.

We thank Heidi Gerber for help with cell culture. We are also very grateful to Jim Robertson, NIBCS, United Kingdom, for providing NIBRG23 virus; William Dundon, IZSV, Italy, for providing H7 and H5 isolates; Werner Wunderli, National Reference Center for Influenza, Switzerland, for the human IAV isolates; Georg Kochs, University of Freiburg, Germany, for the H1N1 PR8 virus; and Robert Webster, St. Jude Children's Research Hospital, United States, for plasmids for reverse genetics.