Primary human bronchial epithelial cells

The immortalized cell line, 16HBE14o-, was derived from bronchial surface epithelial cells [14] (link). Standard culture techniques were used as described previously [1] (link), [13] (link). In brief, cells were maintained in Minimum Essential Medium with Earle’s salts supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured on plastic flasks coated with fibronectin and collagen (BD Biosciences, San Jose, CA, USA) and were incubated in humidified 95% air-5% CO₂ at 37°C. Primary human bronchial epithelial (HBE) cells were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and maintained in Bronchial Epithelial Cell Medium (ScienCell Research Laboratories). All other general laboratory reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), and all cell culture reagents were obtained from Invitrogen (Carlsbad, CA, USA). Before the use of UDP, this nucleotide (10 mM) was incubated (1 h at 37°C) in HEPES-buffered saline containing hexokinase (10 IU/ml; Boehringer, Mannheim, Germany) and 22 mM D-glucose in order to remove contaminating nucleotide triphosphates. The resulting solution was then aliquoted and stored at −20°C [15] (link).

Human lung adenocarcinoma epithelial (A549) cells and human embryonic kidney (293T) cells were propagated and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Invitrogen, Carlsbad, CA) containing 10% foetal calf serum (FCS; Omega Scientific, Tarzana, CA) and 1% antibiotics (penicillin/streptomycin, Gibco-Invitrogen). Madin-Darby canine kidney (MDCK) cells were propagated and maintained in Eagle’s minimal essential medium (EMEM; Lonza, Allendale, NJ) supplemented with 5% FCS and 1% antibiotics. Primary human bronchial epithelial (HBE) cells were purchased from ScienCell Research Laboratories (Carlsbad, CA) and were differentiated as previously described55 (link). Cells were incubated at 37 °C in 5% CO₂ until use.
A/Mallard duck/Korea/W452/2014 (W452, H5N8) and A/Environment/Korea/W468/2015 (W468, H5N8), both HPAI viruses, were isolated from wild bird faecal samples during the first and second waves of an H5N8 outbreak in South Korea, respectively31 (link). The viruses were propagated in specific pathogen–free (SPF) 10-day-old embryonated chicken eggs and stored at –80 °C until use. Stock viral titres were determined by plaque assay in MDCK cells and are expressed as PFU/mL. All experiments with HPAI H5N8 viruses were conducted in an enhanced biosafety level three (BSL3+) laboratory approved by the Korea Center for Diseases Control.