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. 2019 Aug 20;9:12132. doi: 10.1038/s41598-019-48430-8

Figure 2.

Figure 2

Antiviral activities of GHE on influenza A/PR/8/34, A/PR/8/34-GFP, and H1N1 viruses in MDCK cells. MDCK cells were treated with GHE (100 and 200 µg/mL) prior to influenza A virus (A/PR/8/34, A/PR/8/34-GFP, and H1N1) infection, and cells were incubated with medium alone, or 100 or 200 µg/mL GHE prior to infection with A/PR/8/34 and H1N1 (multiplicity of infection = 1). (A) Determination of the antiviral activity of GHE 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 after viral infection. (B) GFP expression levels and (C) reduction in viral replication using flow cytometry were assessed at 24 h after viral infection in GHE-treated MDCK cells. (D) Effects of GHE treatment on A/PR/8/34 infection and viral growth in plaques. The mixtures of A/PR/8/34 and GHE were incubated for 72 h after viral infection at 37 °C with 5% CO2. Effect of GHE components on influenza A virus mRNA synthesis and protein levels. Effect of treatment with GHE (100 or 200 µg/mL) on influenza A/PR/8/34 (multiplicity of infection = 1)-infected MDCK cells and the relative mRNA levels of influenza A/PR/8/34 HA (E) and NS-1 (F) were analyzed using quantitative real-time polymerase chain reaction and normalized to β-actin mRNA levels. Bar graph (mean ± SEM) statistics were determined by three experiments’ data using one-way ANOVA with Tukey’s post-hoc test, ***P < 0.001, compared with the CON (GHE untreated) samples. ###P < 0.001, compared with the cell only sample. GHE reduced the expression of influenza A virus (H1N1) proteins NP (nuclear protein) and NA in infected MDCK cells. The reduction of NP (G) and NA (H) 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). Influenza H1N1 virus protein levels (NP, PA, M1, M2, PB1, PB2, HA, and NA) in MDCK cell lysates were detected using Western blotting, and β-actin was analyzed as an internal control (I). Full-length blots are presented in Supplementary Fig. 2.