Propagation and Maintenance of Influenza Viruses

Influenza viruses A/California/04/2009 (H1N1), A/Anhui/1/2005 (AH1, H5N1), A/CK/BJ/FH620-L1/17 (H7N9) and A/Jiangxi/262/05 (H3N2) were maintained in our laboratory. Viruses were propagated in 10-day-old specific-pathogen-free embryonated eggs (Merial, Beijing, China). HEK293T cells (human embryonic kidney cell line), A549 cells (human lung adenocarcinoma epithelial cell line) and HeLa cells were obtained from the National Infrastructure of Cell Line Resource (Shanghai, China). All cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Life Technologies), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under a humidified atmosphere containing 5% CO₂. NHBE cells (Lonza, Allendale, NJ, USA) were cultured in bronchial epithelial cell growth medium (Lonza) at the air–liquid interface, as previously described [49 (link)]. p53^˗/˗ HCT116 cells were kindly provided by Dr Jun Tang (CAU, Beijing, China). All experiments with live viruses were performed in a biosafety level 3 laboratory.