Neuraminidase Inhibition Assay Protocol

Inhibition of NA enzyme activity of the 4 NA inhibitors was assessed in the fluorescence-based NA inhibition assay, using the NA-Fluor Influenza Neuraminidase Assay kit (Applied Biosystems, ThermoFisher) as previously described^{47 (link)}. IC₅₀ values, defined as the concentration of drug required to reduce enzyme activity by 50%, were calculated using GraphPad prism5 software. Interpretation of IC₅₀ was performed using the WHO Antiviral Working Group (WHO-AVWG) criteria^{47 (link)–49} : the testing virus was compared with the drug-sensitive reference virus, for influenza A viruses, a < tenfold increase in IC₅₀ represents normal inhibition, and a ten to 100-fold increase represents reduced inhibition, while a > 100-fold increase is highly reduced inhibition.