Baloxavir acid

Oseltamivir acid, peramivir trihydrate, and zanamivir hydrate were purchased from Biosynth Carbosynth (Berkshire, UK). Laninamivir was provided by Daiichi Sankyo (Tokyo, Japan). Baloxavir acid was purchased from MedChemExpress (Monmouth Junction, NJ, USA), and favipiravir was purchased from Cayman Chemical (Ann Arbor, MI, USA). Oseltamivir, peramivir, zanamivir, Laninamivir, and favipiravir were dissolved in distilled water, and baloxavir was dissolved in dimethyl sulfoxide.

Cells in 96-well plates were washed once with PBS prior to infections with the different viruses at the appropriate MOI in PBS supplemented with 1 mM Ca²⁺ and Mg²⁺, 0.3% bovine serum albumin (BSA), and 1% P/S. After 1 h at 37°C, the inoculum was removed, cells were washed once with PBS, and postinfection medium was added: DMEM supplemented with 0.1% FBS, 0.3% BSA, 20 nM HEPES, 1% P/S, and 1 μg/ml TPCK-treated trypsin, together with 6 μM Renilla luciferase substrate (EnduRen live-cell substrate; Promega). Where noted, the virus inoculum was preincubated with the indicated concentrations of monoclonal anti-H5 antibody (no. 200-301-976; Rockland) or polyclonal anti-H5 serum (no. 2705; BEI Resources) at 4°C for 1 h before infection of cells. Also where noted, the indicated concentrations of baloxavir acid (no. HY-109025A; MedChemExpress) or S20 fusion inhibitor (58 (link)) (ChemBridge) were added to the cells following infection. Real-time luminescence measurements were taken at various time points using an EnVision multilabel reader (Perkin Elmer) or a Dynex MLX luminometer (Dynex Technologies). All experiments included infection of multiple wells for technical replicates, and each experiment was independently performed at least 3 times. Mock-infected wells, otherwise treated identically, acted as negative controls for background luminescence from the reagents.