Isolation and Infection of Avian Primary Cells

Primary cell cultures were isolated from lungs of 4-week-old broiler chickens and 4-week-old Pekin ducks as previously described [20 (link)]. Cells were grown in collagen coated cell culture flasks (Costar, Corning, UK) in Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F12 (1:1) supplemented with 2% chicken embryo extract (Biosera, Uckfield, UK), 5% fetal bovine serum, 1% insulin-transferrin selenium (Life Technologies, Paisley, UK) and antibiotics. Monolayers of primary cells in 6 well cell culture plates (Costar) were infected with LPAI or HPAI viruses at multiplicity of infection (MOI) of 1.0. Three wells of avian cells were used for each virus infection. Mock infections were performed without virus in triplicate wells for each cell type. Cells were rinsed with phosphate buffered saline (PBS) and infected with appropriate amount of the virus in serum free infection medium comprising 2% Ultroser G (Pal Biosepra, Cedex, France), 500 ng/mL TPCK trypsin (Sigma-Aldrich, Dorset, UK) and antibiotics in Ham’s F12 medium. After 2 h incubation with the virus, the cells were washed three times with PBS and fresh medium was added.