Figure 1.
Production and characterization of the H7-pseudotyped particle system.
(A) Western blot analysis was performed using lysates from 293T cells transfected with the recombinant pCAGGS plasmids expressing H7 (A/Anhui/1/2013), H5 (A/Anhui/1/2005), H3 (A/Brisbane/10/2007), H109 (A/California/04/2009) and H107 (A/Brisbane/59/2007). Specific bands corresponding to HA0 and HA1 are indicated on the right. (B) TPCK-treated trypsin treatment is required for H7N9pp infection. The infectivities of H7N9pp and H5N1pp were determined after treatment with different concentrations of TPCK-treated trypsin (0, 2, 4, 6, 8, 10 µg/ml). Bald virus refers to culture supernatants collected from 293T cells co-transfected with 1.5 µg pNL-4.3-Luc-R-E- and 0.5 µg pCAGGS-H7 in the absence of pCAGGS-N9 and was used as a control. (C) Optimization of H7N9pp packaging. 293T cells were co-transfected with pNL-4.3-Luc-R-E-, pCAGGS-HA and pCAGGS-NA constructs. pCAGGS-HA was maintained at 0.5 µg, while pCAGGS-NA was varied at 0.1 µg, 0.25 µg, 0.5 µg, 1 µg or 1.5 µg, and the reverse was also performed. The infectivity of 293T culture supernatants collected from different times (24 h, 36 h, 48 h) post-transfection with 1.5 µg pNL-4.3-Luc-R-E-, 0.5 µg pCAGGS-HA and 0.5 µg pCAGGS-NA constructs was determined as described above. Bald/H7 refers to culture supernatants collected from 293T cells co-transfected with 1.5 µg pNL-4.3-Luc-R-E- and the indicated amount of pCAGGS-HA (In ‘NA(ug)’, Bald/H7 refers to culture supernatants collected from 293T cells co-transfected with 1.5 µg pNL-4.3-Luc-R-E- and 0.5 µg pCAGGS-HA). Bald/N9 refers to culture supernatants collected from 293T cells co-transfected with 1.5 µg pNL-4.3-Luc-R-E- and the indicated amount of pCAGGS-NA (In ‘HA(ug)’, Bald/N9 refers to culture supernatants collected from 293T cells co-transfected with 1.5 µg pNL-4.3-Luc-R-E- and 0.5 µg pCAGGS-NA). Both Bald/H7 and Bald/N9 were included as negative controls. (D) Antibody neutralization assays. H7N9pp was incubated with serially diluted monoclonal antibodies against H1, H3, H5 or H7 for 1 h at 37°C before inoculation onto MDCK cells. The cells were lysed, and luciferase activity was determined at 48 h post-infection. Viral entry in the absence of antibody (0 Ab) was set at 100%. Isotype IgG was included as a negative control.
Figure 2.
Cell permissiveness to H7N9pp and N9-mediated H7N9pp superinfection exclusion.
(A) Different subtypes of HANApp with equal amounts of p24 antigen were incubated with various cells derived from different species and tissues, and the transduction efficiencies were measured and normalized to that of MDCK cells. (B) N9 expression specifically inhibits H7N9pp infection. 293T cells were transfected with various amounts of pCAGGS-N9 (0 µg, 0.0375 µg, 0.075 µg and 0.15 µg) and then infected with equal amounts of H7N9pp at 24 h post-transfection. Pseudovirus infection was determined by a luciferase activity assay and normalized to that of cells transfected with vector alone. VSVGpp was included as negative control. (C) N9-mediated H7N9pp inhibition can be partially blocked with NA inhibitor Zanamivir (ZA). N9-expressing 293T cells were treated with the indicated concentrations of ZA and then infected with H7N9pp. Pseudovirus infection was determined by a luciferase activity assay and normalized to that of cells transfected with vector alone. VSVGpp was included as a negative control.
Figure 3.
Inhibition of H7N9 virus by peptides.
(A) Amino acid sequences of the indicated synthetic peptides. (B) Location of P155-185. Structure of HA. HA1, green; HA2, red; fusion peptide, yellow; P155-185, cyan. (C) Characterization of the antiviral activity of the synthetic peptides. Viral entry without peptide treatment was set at 100%. A scrambled peptide was included as a negative control. Cytotoxicity of the peptides to MDCK cells was measured by a MTT assay. (D) The inhibitory effect of P155-185-chol against H7N9 influenza virus in combination with neuraminidase inhibitors. P155-185-chol 2 µM; P155-185 2 µM; scrambled peptide 2 µM; ZA, 100 nM Zanamivir hydrate; OS, 200 nM Oseltamivir carboxylate. (E) The same experiments as panel D were performed. H7N9-infected MDCK cells were fixed at 36 h after the indicated treatments and processed for immunostaining and observed under confocal microscope. Fixed cell monolayers were immunostained with an anti-influenza A nucleoprotein (NP) murine monoclonal antibody (MAB8251; Millipore) and Alexa 594 conjugated secondary antibody (red). Nuclei were stained with DAPI (blue). (F) The same experiments as panel D were performed. The cytopathic effect of H7N9-infected MDCK cells in the presence or absence of the indicated treatments was visually inspected under an inverted microscope at 48 h post-infection.
Figure 4.
Inhibition of H7N9 virus by chemicals.
(A) Inhibition of HA-mediated infection by dynasore. Dynasore's concentrations range from 0 to 80 µM in the absence (left) or presence (right) of 10% fetal bovine serum (FBS). MDCK cells were kept at 37°C in Opti-MEM I Reduced Serum Medium with or without 10% FBS at all times. Dynasore was added 1 h prior to HANApp infection. Pseudovirus infection was determined by a luciferase activity assay 48 h post-infection. Viral entry in the 0 µM treatment control group was set at 100%. (B) The inhibitory effect of bafilomycin A1. MDCK cells were pretreated with bafilomycin A1 (concentration range 0 nM to 10 nM) for 1 h at 37°C followed by infection with HANApp. Pseudovirus infection was determined by a luciferase activity assay at 48 h post-infection. Viral entry in the 0 nM treatment control group was set at 100%. (C) Antiviral effects of bafilomycin A1, dynasore and chloroquine. Viral entry in the DMSO treated control group was set at 100%. The cytotoxicity of the chemicals at indicated concentrations for MDCK cells was also measured by a MTT assay.