Figure 4.

Antiviral activities of GHE components on influenza A/PR/8/34, A/PR/8/34-GFP, H1N1, influenza B viruses. Measurement of the antiviral activity of GN using neuraminidase (NA) inhibition assay. Influenza A viruses including A/PR/8/34 (A), H3N2 (B), H1N1 (C), and influenza type B (D) were added to the indicated concentrations of GN and oseltamivir carboxylate (OTC). Fluorescence was measured using fluorescence spectrophotometry (excitation, 365 nm and emission, 415–445 nm). Antiviral activities of GHE components on influenza A H1N1, H3N2, A/PR/8/34 and A/PR/8/34-GFP viruses in MDCK cells. MDCK cells were treated with constituents of GHE (100 μM) prior to influenza A virus infection, and cells were incubated with medium alone, 100 μM constituents of GHE prior to infection with A/PR/8/34 and H3N2 (multiplicity of infection = 1). The antiviral activity of the constituents of GHE was determined in MDCK cells. The viability of MDCK cells was assessed using an MTS assay after treatment with the indicated concentrations of GHE for 48 h against (E) H1N1 and (F) H3N2. (G) GFP expression levels and (H) reduction in viral replication using flow cytometry were assessed at 24 h after viral infection in geraniin (GN)-treated MDCK cells (GN: 10 and 100 μM). (I) Effects of GN treatment on A/PR/8/34 infection and viral growth in plaques. The mixtures of A/PR/8/34 and GN were incubated for 72 h at 37 °C with 5% CO₂. GN reduced the expression of influenza A virus (H1N1) proteins NP and NA in infected MDCK cells to virus (J). The reduction of NP and NA proteins in MDCK cells were observed with fluorescence microscopy using the influenza A viral proteins NP- and NA-specific antibodies. MDCK cells were also stained with DAPI (blue), and the merged images represent NP and NA (red). Bar graph (mean ± SEM) statistics were determined by three experiments’ data using one-way ANOVA with Tukey’s post-hoc test, ***P < 0.001; *P < 0.05, compared with the CON (GHE untreated) samples. ^###P < 0.001, compared with the cell only sample.