Propagation of Influenza A Virus

IAV strain A/WSN/33 (WSN) was grown on Madin-Darby canine kidney (MDCK) cells, and IAV reporter virus WSN-Ren was propagated on MDCK-HA cells overexpressing the HA protein of strain A/WSN/33 (20 (link), 36 (link)). Cells were infected at the indicated multiplicity of infection (MOI) in PBS for infection (PBSi) (Dulbecco’s phosphate-buffered saline [DPBS; Thermo Fisher Scientific] supplemented with 0.3% bovine serum albumin [BSA] [Sigma-Aldrich], 1 mM Ca²⁺ Mg²⁺, 100 units/mL penicillin, and 100 μg/mL streptomycin [Thermo Fisher Scientific]) for 1 h at 37°C or for 1 h on ice for synchronized infection. After removing the inoculum and washing the cells with DPBS, they were overlaid with postinfectious DMEM (piDMEM) (DMEM [Thermo Fisher Scientific] supplemented with 0.1% FBS [Thermo Fisher Scientific], 0.3% BSA [Sigma-Aldrich], 20 mM HEPES [Thermo Fisher Scientific], 100 units/mL penicillin, and 100 μg/mL streptomycin [Thermo Fisher Scientific] containing 1 μg/mL TPCK [tosylsulfonyl phenylalanyl chloromethyl ketone]-treated trypsin [Sigma-Aldrich]) for the indicated times. For WSN-Ren infections, piDMEM was supplemented with 6 μM Renilla luciferase substrate (EnduRen live-cell substrate; Promega) and real-time luminescence measurements were taken at the indicated time points using an EnVision multilabel reader (Perkin Elmer) or a Dynex MLx luminometer (Dynex Technologies).