Neuraminidase assay.

Neuraminidase activity was assayed in the presence of the substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) (65, 66). The viruses were serially 2-fold diluted from 2 × 10⁷ EID₅₀/ml, and the copy number of the M gene was quantitated by real-time PCR to ensure accurate virus dose as described by Yen et al. (56). Briefly, 50 μl of 200 μM MUNANA was mixed with 50 μl of virus dilution and incubated at 37°C for 10 min; the reaction was stopped by adding 100 μl of 1 M Na₂CO₃. The fluorescence was measured at excitation and emission wavelengths of 365 nm and 450 nm, respectively. The assay was performed in triplicate three times.