Neuraminidase

Nystatin, filipin III, and oseltamivir inhibit the labeling of trophozoites by CTXB and GM1 antibodies. (A) Trophozoites were treated with nystatin (27 μM), filipin III (7.6 μM), and oseltamivir (20 μM) for 30 min before labeling with Alexa Fluor-conjugated CTXB antibody (images a to d) and GM1 antibody (images a′ to d′). Myriocin (27 μM), a sphingolipid inhibitor, was used as a negative control (images e and e′). N, nucleus; PM, plasma membrane; F, flagella; VD, ventral disc. Bars, 5 μm. (B and C) Changes in intensity of control and inhibitor-treated trophozoites. For intensity measurements, cells were randomly selected from 10 to 15 fields from 3 to 5 separate experiments. Approximately 50 cells were considered for each condition and were analyzed using Zeiss Zen 2009 confocal software. One-way ANOVAs were performed to evaluate differences (means ± SDs) between the treatment and control groups. Mean intensities from 3 to 5 separate experiments are shown in panels B and C. Statistical significance was calculated using a one-way ANOVA test, followed by the Tukey (B) and Holm-Šídák (C) methods. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

Dose-response curve of the SH5190, SH5190 R292K and the A(H1N1)pdm09 (2167) viruses in the presence of peramivir (C) using a chemiluminescent (NA-star) assay. The NA activity of the H7N9 or H1N1 viruses under increasing concentrations (0.03-1000 nM) of the NAIs was determined. Each data point represents the normalized mean ± SEM neuraminidase activity from three replicated wells. The experiments were repeated twice.

Dose-response curve of the SH5190, SH5190 R292K and the A(H1N1)pdm09 (2167) viruses in the presence of oseltamivir zanamivir (B) using a chemiluminescent (NA-star) assay. The NA activity of the H7N9 or H1N1 viruses under increasing concentrations (0.03-1000 nM) of the NAIs was determined. Each data point represents the normalized mean ± SEM neuraminidase activity from three replicated wells. The experiments were repeated twice.

Nystatin, filipin III, and oseltamivir inhibit the labeling of trophozoites by CTXB and GM1 antibodies. (A) Trophozoites were treated with nystatin (27 μM), filipin III (7.6 μM), and oseltamivir (20 μM) for 30 min before labeling with Alexa Fluor-conjugated CTXB antibody (images a to d) and GM1 antibody (images a′ to d′). Myriocin (27 μM), a sphingolipid inhibitor, was used as a negative control (images e and e′). N, nucleus; PM, plasma membrane; F, flagella; VD, ventral disc. Bars, 5 μm. (B and C) Changes in intensity of control and inhibitor-treated trophozoites. For intensity measurements, cells were randomly selected from 10 to 15 fields from 3 to 5 separate experiments. Approximately 50 cells were considered for each condition and were analyzed using Zeiss Zen 2009 confocal software. One-way ANOVAs were performed to evaluate differences (means ± SDs) between the treatment and control groups. Mean intensities from 3 to 5 separate experiments are shown in panels B and C. Statistical significance was calculated using a one-way ANOVA test, followed by the Tukey (B) and Holm-Šídák (C) methods. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

Dose-response curve of the SH5190, SH5190 R292K and the A(H1N1)pdm09 (2167) viruses in the presence of peramivir (C) using a chemiluminescent (NA-star) assay. The NA activity of the H7N9 or H1N1 viruses under increasing concentrations (0.03-1000 nM) of the NAIs was determined. Each data point represents the normalized mean ± SEM neuraminidase activity from three replicated wells. The experiments were repeated twice.

Dose-response curve of the SH5190, SH5190 R292K and the A(H1N1)pdm09 (2167) viruses in the presence of oseltamivir zanamivir (B) using a chemiluminescent (NA-star) assay. The NA activity of the H7N9 or H1N1 viruses under increasing concentrations (0.03-1000 nM) of the NAIs was determined. Each data point represents the normalized mean ± SEM neuraminidase activity from three replicated wells. The experiments were repeated twice.