Influenza a m1

Sf9 cells (5×10⁵ cells/well) cultured in a 6-well plate were infected with the recombinant baculoviruses (MOI=1) for 48 h at 27°C. The IFA was performed as described by Xu et al. (Xu et al., 2021 (link)). Briefly, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and washed three times with PBS for 5 min each. Next, the cells were blocked with 1% skim milk at 37°C for 30 min. After that, the cells were incubated with 500 μL AcMNPV GP64 antibody (1:1000, Sino Biological, China), SARS-CoV-2 (2019-nCoV) Spike RBD Antibody (1:1000, Sino Biological, China), or Influenza A M1 (1:1000, GeneTex, USA) at 37°C for 120 min. Then, the cells were incubated with 500 μL FITC-labeled Goat Anti-Rabbit IgG (H+L) (1:2,000, Beyotime, China) at 37°C for 40 min in the darkroom. Finally, the cells were examined using an Eclipse TE2000-V (Nikon, Japan).

Western blot was performed according to the protocol described by Xu et al. (Xu et al., 2021 (link)). Briefly, cell lysate or protein solution was loaded (1:5) and separated via 10% SDS-PAGE. Then, the protein was transferred onto the PVDF membrane, blocked with 5% skim milk for 1 h at room temperature, followed by incubating with the convalescent serum of the COVID-19 patient (1:1000) or Influenza A M1 (1:1000, GeneTex, USA) for 2 h at room temperature. After that, the membrane was incubated with HRP-labeled Goat Anti-Rabbit IgG (H+L) or HRP-labeled Goat Anti-Mouse IgG (H+L) (1:3000, Beyotime, China) for 1 h at room temperature. Subsequently, the band was developed using the GEGEGNOME XRQ enhanced chemiluminescence (ECL) (Thermo Fisher SCIENTIFIC, USA).