Mdck cells

Madin-Darby canine kidney (MDCK) cells were purchased from the Riken BioResource Center Cell Bank (Ibaragi, Japan) and cultured in minimal essential medium (MEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and standard antibiotics (6 (link), 41 (link)). IAV (PR8; A/Puerto Rico/8/1934; H1N1) was cultured in MDCK cells and stored as a working stock at −80°C. Viral titers were measured by focus-forming assays in MDCK cells and are expressed as the number of focus-forming units (FFU)/ml (21 (link)).

Human bronchial epithelial (Calu-3) cells and chicken fibroblast (DF-1) cells were obtained from the American Type Culture Collection, and MDCK cells and 293T cells were obtained from the RIKEN BioResource Center Cell Bank. The cells were maintained in Dulbecco’s Modified Eagle’s Medium with 10% FCS. Influenza virus A/chicken/Egypt/CL42/2013 (EG/2013) (H9N2) is a representative strain of the G1-A/B reassortant that has been prevalent in Egypt and has been described previously [20 (link)]. All recombinant H9N2 viruses were propagated once in 10-day-old embryonated eggs. The sequences of the EG/2013 gene segments have been deposited in the GenBank database (accession nos. LC379963-LC379970).

MDCK cells were obtained from RIKEN BioResource Center (Tsukuba, Japan), and were maintained in DMEM supplemented with 10% fetal calf serum (FCS). The medium contained 2 mM glutamine, 100 μg/mL streptomycin and 100 U/mL penicillin (FCS-DMEM). Cells were cultured at 37 °C in a 5% CO₂ atmosphere. For toxin cytotoxicity assays, cells were seeded on 48-well culture plates, reaching confluence 24 h later. Cells were then treated with the various inhibitors or toxin. Cytotoxicity, as evidenced by cell rounding, was assessed 4 h after toxin treatment, as described previously [10 (link)].