2.1. Cells and viruses
Human lung carcinoma cell lines (A549 cells) (ATCC, Manassas, VA, USA)
were grown at the condition of 37 °C with 5% CO2 after adding High
glucose Dulbecco’s modified Eagle’s medium (DMEM) Nutrient Mix (10%
FBS, 1% penicillin and streptomycin). Influenza A virus (IAV)
(A/PR/8/34) was obtained from the Institute of Virology (Wuhan, China)
and propagated in the allantoic cavity of 9-day-old embryonated eggs for
48 h at 37 °C and then for 12 h at 4 °C, after which allantoic fluid was
collected. The hemagglutination titer of the virus in the allantoic
fluid was detected by the hemagglutination method, and the allantoic
fluid with a high hemagglutination titer was filtered and stored. Dilute
the virus stock solution with DMEM (10-1,
10-2, 10-3, 10-4,
10-5), add it to the A549 cells that grow into a
monolayer, and incubate at 37 ℃ for 2 h. At the end of the infection,
the viral fluid was replaced by the maintenance medium containing DMEM,
100 U/mL penicillin and streptomycin, 1.2% BSA, and 1 µg/mL trypsin
(used to activate the virus). After continued incubation for 72 h, the
TCID50 (median tissue culture infective dose) of A/PR/8/34 was
calculated by the Reed-Muench method. A viral titer of 100
plaque-forming units (PFU) (PFU= 0.7×TCID50) was used in subsequentin vitro experiments. All virus experiments were conducted in the
Biosafety Level 2 laboratory of the Central Laboratory of Yichang
Central People’s Hospital.