Figure 3

HSM and oseltamivir have additive inhibitory effects. MDCK cells were layered with PSM or HSM at 1,500, 3,200, 12,000 pmol Sia/well or with PBS buffer as control. The cells were challenged for 1h at 37°C with 10⁹ TCID₅₀ of (A) A/PR/8/34(H1N1), (B) A/SD/1/2009(H1N1), (C) A/SD/17/2008(H1N1), or (D) A/Aichi/2/68(H3N2) in the presence (gray bars) or absence (black bars) of 1 μM oseltamivir. Infected cells were identified by staining with anti-NP antibodies, and quantified in twenty randomly selected images from each sample. Experiments were repeated three times, for each experiment the number of infected cells in the PBS-coated sample was set to 1, and the relative number of infected cells for each treatment was calculated. Lower number of infected cells was observed in HSM coated monolayers compared to PBS-coated monolayers for all tested virus strains (A-D, P<0.05). Dose effects of Sia content in HSM-coated samples were observed for three IAV strains (A-B, D). Significant reduction in the number of infected cells in PSM-coated monolayers was observed only for one strain (C, P<0.05). With exception of the A/SD/1/2009(H1N1) strain, oseltamivir did not inhibit infection of cell coated with either PBS or PSM (A, C-D, gray bars). In contrast, addition of oseltamivir to HSM-coated samples further reduced infection of A/PR/8/34(H1N1), A/SD/1/2009(H1N1) strains (A-B). Both of the oseltamivir-insensitive strains, A/SD/17/2008(H1N1) and A/Aichi/2/68(H3N2), were not affected by addition of the drug (C-D). Data was analyzed by 3-way ANOVA, corrected for multiple comparisons using Tukey’s HSD (see Additional file 2 for complete statistical analysis data). Error bars represent standard deviation. *P<0.05, **P<0.005.