The NA-Fluor Influenza Neuraminidase Assay Kit (Applied Biosystems, Foster City, CA, USA) was used to measure the NA activity according to the manufacturer's instructions. Briefly, VB was combined with an assay buffer in a 96-well plate at 0–100 μmol/L for H1N1, H3N2, and influenza B viruses. Next, H1N1, H3N2, or influenza B were added to the VB-containing wells and incubated at 37 °C. Oseltamivir was used as a positive control in the assay. After 30 min, NA-Fluor substrate was added to each well and incubated for extra 2 h followed by measurement (excitation: 365 nm; emission: 415–445 nm) with a fluorescence spectrophotometer (Promega, Madison, WI, USA).
For the NA-XTD influenza neuraminidase assay, VB or oseltamivir carboxylate were added to the assay buffer in 96-well plates at 0–100 μmol/L for oseltamivir-resistant A/PR8/34. Subsequently, oseltamivir-resistant A/PR8/34 in assay buffer was added to VB or oseltamivir carboxylate-containing wells and incubated at 37 °C. Oseltamivir carboxylate was considered as the positive control in the assay. After 20 min, NA-XTD substrate was added to each well and incubated for an additional 30 min. Next, NA-XTD accelerator was added to each well, followed by recording the luminescence using a luminescence plate reader (Promega, Madison, WI, USA).
For the NA-XTD influenza neuraminidase assay, VB or oseltamivir carboxylate were added to the assay buffer in 96-well plates at 0–100 μmol/L for oseltamivir-resistant A/PR8/34. Subsequently, oseltamivir-resistant A/PR8/34 in assay buffer was added to VB or oseltamivir carboxylate-containing wells and incubated at 37 °C. Oseltamivir carboxylate was considered as the positive control in the assay. After 20 min, NA-XTD substrate was added to each well and incubated for an additional 30 min. Next, NA-XTD accelerator was added to each well, followed by recording the luminescence using a luminescence plate reader (Promega, Madison, WI, USA).