NA inhibition assay

By quantifying the fluorescent product upon the cleavage of 4-methylumbelliferyl- a-d-N-acetylneuraminic acid (MUNANA; Sigma, St Louis, MO, USA) by NA, IAV NA activity was assessed [28, 31]. The reaction system contains 20 μl of sample, 20 μl of enzyme and 60 μl of substrate buffer mix (20 μM MUNANA, 33 mM MES buffer (pH 3.5), 4 mM CaCl₂, and double distilled water). Briefly, neuraminidases derived from influenza A viruses were incubated with diluted drug samples for 60 min at room temperature and then 60 μl neuraminidases substrate was added. The luminescence signal indicating neuraminidase activity was determined on Enspire (Perkin Elmer, Waltham, MA, USA) with excitation wavelength 355 nm and emission wavelength 460 nm before and after incubating for 15 min at 37 °C. The inhibition ratio was calculated according to the equation.