Neuraminidase Inhibition Assay for Influenza Viruses

The NI assay was performed using an NA-Fluor™ Influenza Neuraminidase Assay Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions with slight modifications [28 (link), 29 (link)]. RVSE was added to assay buffer in 96-well plates at concentrations of 0–400 μg/mL for A/PR/8/34, H3N2, H1N1, and influenza type B viruses. A/PR/8/34, H1N1, H3N2, or influenza type B in assay buffer was added to RVS- and RVSE-containing wells and incubated at 37°C. Oseltamivir was considered a positive control in the assay. After 30 min, NA-Fluor Substrate was added to each well and incubated for additional 2 h, followed by recording fluorescence (excitation, 365 nm; emission, 415–445 nm) with a fluorescence spectrophotometer (Promega, Madison, WI, USA). Samples treated with only RVSE or its components were used as negative controls. Further, NA activities for 10 μM concentrations of RVSE components were evaluated by the same method described earlier. NA activity after incubation with oseltamivir carboxylate was measured in a range of 0–10,000 nM as a positive control. NI assay results are presented as mean ± SEM of three independent experiments.