Oseltamivir carboxylate oseltamivir

The NA inhibitors oseltamivir carboxylate (oseltamivir; Hoffman-La Roche) and zanamivir (GlaxoSmithKline) were prepared in sterile distilled water and stored in aliquots at − 30 °C until use.
Inhibition of NA enzyme activity by the 2 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 [15 (link)]. IC₅₀ values, defined as the concentration of drug required to reduce enzyme activity by 50%, were calculated using GraphPad Prism 5 software. The IC₅₀ values reported are the means (±SD) of IC₅₀ values measured for at least 2 independent tests with 2 duplicates for each sample. Interpretation of IC₅₀ values (obtained by comparing the test virus with the drug-sensitive reference virus) was performed using the WHO AVWG criteria for influenza A viruses: a < 10-fold increase in IC₅₀ represents normal inhibition, a 10~100-fold increase represents reduced inhibition, while a > 100-fold increase is highly reduced inhibition.

The NA inhibitors (NAIs) oseltamivir carboxylate (oseltamivir; Hoffmann-La Roche, Basel, Switzerland), zanamivir (GlaxoSmithKline, Research Triangle Park, NC, USA) and peramivir (BioCryst Pharmaceuticals, Birmingham, AL, USA) were used to test the sensitivity of the Bangladeshi H9N2 viruses. The NA activity was determined by a modified fluorometric assay using the fluorogenic substrate 2′-(4-methylumberlliferyl)-α-d-N-acetylneuraminic acid (Sigma-Aldrich, St Louis, MO, USA) as described previously.^{30 (link), 34 (link), 35 (link)} A/Fukui/20/2004 (E119) (H3N2) (wild-type virus) and A/Fukui/45/2004 (E119V) (H3N2) were used as reference viruses and are part of the World Health Organization's NAI-susceptibility panel.