Determination of the 50% tissue cell infectious dose
(TCID50)
The TCID50 assay was performed as we described
previously (Peng et al., 2019). Briefly, the viruses were diluted in
DMEM without serum to a concentration of 10-1 to
10-11 and then added to MDCK cells in 96‑well plates,
respectively. After incubation at 37 °C with 5% CO2 for
1 h, the supernatants were removed. The plates were washed twice with
PBS, and then 100 µL of DMEM was added to each well. After incubation at
37°C with 5% CO2 for 72 h, the HA titers of the cell
supernatants were analyzed. The virus titers were calculated according
to the Reed-Muench formula (Reed & Muench, 1937).