Hemagglutination assay was performed using 0.75% guinea pig and 0.5% turkey RBCs (Lampire Biological Labs) (WHO, 2011 ). Viruses were two-fold serially diluted in PBS with or without oseltamivir (20 nM) and incubated at room temperature for 1h. RBCs were then added, and titers (hemagglutination units/50 μL, HAU/50 μL) were recorded after 1h incubation at room temperature.
NA inhibition (NI) assay was performed using the NA-Fluor™ kit (Applied Biosystems), as previously described (Okomo-Adhiambo et al., 2010b (link)). Briefly, normalized virus preparations were incubated in the presence of NA inhibitors for 45min prior to the addition of 2-(4-(methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA) substrate. After 1h incubation at 37°C, the reaction was terminated by the addition of the NA-Fluor™ stop solution and the fluorescence (excitation = 360 nm; emission = 460 nm) produced by 4-metheylumbelliferone (4-MU), a product of MUNANA cleaved by NA, was measured using Cytation 7 (Agilent-BioTek). Curve-fitting and determination of IC50 (the drug concentration required to inhibit NA activity by 50%) were done as previously described (Okomo-Adhiambo et al., 2010b (link)). Mean and standard deviation was calculated from results collected from at least three independent tests.
NA inhibition (NI) assay was performed using the NA-Fluor™ kit (Applied Biosystems), as previously described (Okomo-Adhiambo et al., 2010b (link)). Briefly, normalized virus preparations were incubated in the presence of NA inhibitors for 45min prior to the addition of 2-(4-(methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA) substrate. After 1h incubation at 37°C, the reaction was terminated by the addition of the NA-Fluor™ stop solution and the fluorescence (excitation = 360 nm; emission = 460 nm) produced by 4-metheylumbelliferone (4-MU), a product of MUNANA cleaved by NA, was measured using Cytation 7 (Agilent-BioTek). Curve-fitting and determination of IC50 (the drug concentration required to inhibit NA activity by 50%) were done as previously described (Okomo-Adhiambo et al., 2010b (link)). Mean and standard deviation was calculated from results collected from at least three independent tests.