Screening for Anti-Influenza Host Factors

siRNA treatment procedure, cell viability, and virus titer determination are described in detail in Watanabe et al. (32 (link)). Briefly, two siRNAs per candidate gene were selected from a predesigned genome-wide human siRNA library (FlexTube siRNA; Qiagen). AllStars Negative Control siRNA (Qiagen) was used as a negative control. The siRNA against the NP gene of WSN virus (GGA UCU UAU UUC UUC GGA GUU) purchased from Sigma-Aldrich was used as a positive control. HEK293 cells were transfected twice with 25 nM (final concentration, 50 nM) siRNA duplexes using RNAiMAX (Invitrogen). At 24 h after the second transfection, cell viability was determined using the CellTiter-Glo assay system (Promega) following the manufacturer’s instructions. To assess influenza virus replication, two parallel sets of siRNA-transfected cells were infected with 50 plaque-forming units (PFU) of WSN virus per well of a 24-well tissue culture plate at 24 h after the second siRNA transfection. At 48 h postinfection, supernatants were harvested, and virus titers were determined by plaque assay in MDCK cells.