2.5. Neuraminidase Assay

The fluorescent substrate, 2′-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA, Sigma, M8639), for the neuraminidase (NA) of influenza viruses was used to detect the levels of NA. Briefly, the virus-containing culture supernatant was transferred to a black opaque 96 or 384 well plate (PerkinElmer, 6005270 or 6007270) and mixed with 20 µmol/L of MUNANA dissolved in MES solution (33 mmol/L 2-[N-morpholino] ethanesulfonic acid and 4 mmol/L CaCl2, pH = 6.5), followed by incubation at 37 °C for 1 h. The reaction was terminated by the addition of stop solution (0.14 mol/L NaOH in 83% ethanol). Fluorescence intensity was measured at an excitation wavelength of 355 nm and an emission wavelength of 485 nm using multi-label plate readers (Envision2103, PerkinElmer, Waltham, MA, USA).