The susceptibility of 96 avian influenza A (H5N1) viruses of clade 2.1 to zanamivir and oseltamivir were tested in a fluorescence-based enzyme inhibition assay. This assay involved a fluorogenic substrate, from Carbosynth, called 4-methylumbelli-feryl N-acetyl-α-D-neuraminic acid (MUNANA).
First, the sensitivity of the samples to the inhibitors were tested by pre-incubating them for 30 mins, followed by the addition of MUNANA for 60 mins. Fluorescence was measured at endpoint. Secondly, an IC50 kinetic assay was performed to see changes in binding or dissociation of mutants to the inhibitor. This assay involved the addition of MUNANA to the virus and inhibitor, without pre-incubation. Fluorescence was monitored at 1 min intervals.
For more information, please refer to the original paper: McKimm-Breschkin et al., 2018.