Baloxavir Susceptibility Assay in Humanized Cells

Baloxavir susceptibilities were determined by using a focus reduction assay as previously described [1 (link)] in humanized MDCK cells (i.e., hCK cells), which express high levels of α2,6-sialoglycans and very low levels of α2,3-sialoglycans [15 (link)]. hCK cells were kindly provided by Dr. Yoshihiro Kawaoka (University of Wisconsin–Madison). hCK cells in 96-well plates were infected with 1000 focus-forming units (FFU)/well of viruses. Virus adsorption was carried out for 1 h at 37 °C and then an equal volume of 1.2% Avicel RC-581 (DuPont Nutrition USA, Wilmington, DE, USA) in culture medium containing serial dilutions (0.025–2500 nM) of baloxavir was added to each well in triplicate. The cells were incubated for 24 h at 34 °C and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against influenza A virus nucleoprotein (Merck KGaA, Darmstadt, Germany), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare Life Sciences, Milford, MA, USA). The infected cells were stained with TrueBlue Substrate (SeraCare Life Sciences) and then washed with distilled water. After cell drying, the focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology, Cleveland, OH, USA). The results are expressed as IC₅₀ values.