Chicken red blood cells

Ninety-six-well V-bottom plates (Costar, Corning, NY, USA) were used for the HI assay as described previously [15 (link)]. Receptor-destroying enzyme-treated serum samples were serially diluted two-fold with PBS and then incubated with an equal volume of 4 hemagglutination units (4HA) of WSN or H3N2 Php for 30 min. After incubation, an equal volume of 0.5% chicken red blood cells (Innovative Research, Novi, MI, USA) were added to the wells and incubated for 30 min at room temperature, and HI titers were measured.

Dimethyl sulfoxide, and human and mouse IFN-β were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Chicken red blood cells were obtained from Innovative Research Inc. (Southfield, MI, USA). Pro-Prep protein extraction solution was purchased from Intron Biotechnology (Seoul, Korea). NP, NA, HA, M2, and PB1 antibodies were obtained from GeneTex (Irvine, CA, USA). CH was purchased from the Korea Apicultural Agriculture Cooperative (Anseong, Korea) in 2020. The honey sample was stored at room temperature under dark conditions until the experiments.

On days 1 through 10 after infection, mice (n = 4) were euthanized, lungs were collected, homogenized in phosphate buffered saline (supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL of amphotericin B), and centrifuged, and the supernatants were used to infect MDCK cells in quadruplicate. Cells were incubated for 72 hours at 37°C and 5% CO₂. After incubation, cell culture supernatants were tested for the presence of virus by hemagglutination assay using 0.5% (v/v) chicken red blood cells (Innovative Research, MI, USA). Viral loads were determined as the reciprocal of the dilution at which 50% of the wells positive for viral infection were expressed as TCID50 per mL in logarithmic scale.