RSV production

Human respiratory syncytial virus (RSV, ATCC, VR-1540) stock was ordered from ATCC. Hela cells were used to produce RSV under biosafety level II conditions [87]. Briefly, Hela cells were plated in 75 cm² culture flask and incubated at 37 °C degree in DMEM + 10%FBS until they were approximately 50% confluent. The cells were then washed and infected with RSV stock under multiplicity of infection (MOI) of 0.1. After 3 h infection, the cells were washed and replaced with fresh medium (DMEM + 10%FBS) and incubated for 4 days at 37ºC, 5% CO₂. The cells and the virus suspension were collected when the cytopathology appeared, with scraping and vortexing of the cells to release more viral particles. The virus suspension was centrifuged for 10 min at 1800×g to remove the cell debris. The virus suspension without cell debris were either frozen immediately and stored at − 80 °C as seeding stock and concentrated before use with Ultra15 Amicon 100 kDa cutoff filters. The produced viruses were titrated using the median tissue culture infectious dose (TCID₅₀) method in HEK293T cells [88].