Determination of Baloxavir Antiviral Activity

The antiviral activity of baloxavir was also determined by using a focus reduction assay (Tilmanis et al., 2017 (link)) under Avicel overlays (Matrosovich et al., 2006 (link)). Confluent monolayers of MDCK cells in 96-well plates were inoculated with 100 μl of 1000 focus-forming unit (FFU)/well of viruses. Virus adsorption was carried out for 1 h at 37°C and then 100 μl of 1.2% Avicel RC-581 (FMC BioPolymer) in culture medium containing serial dilutions (0.025–2500 nM) of baloxavir acid was added to each well in triplicate. The cells were incubated for 24 h and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against influenza A or B virus nucleoprotein (Merck; MAB8251, MAB8661), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare) as previously described (Tilmanis et al., 2017 (link)). The infected cells were stained with TrueBlue Substrate (SeraCare) and then washed with distilled water. After drying, the number of FFU was quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (CTL) as previously described (Tilmanis et al., 2017 (link); van Baalen et al., 2017 (link)). The IC₅₀ values were calculated by using GraphPad Prism.