Preparation of PEDV-PT Virus Antigen

The G2b PEDV-PT strain (GenBank: KY929405) was prepared as previously described [44 (link)]. The 6th passage of PEDV-PT (PEDV-PT-P6) was used as the antigen for the immunostainings in the present study. In brief, to prepare the virus-infected Vero cell plates with visible cytopathic effect (CPE), i.e. the presence of syncytial cells, the Vero cells were seeded onto 96-well culture plates (Thermo Fisher Scientific, Waltham, MA, USA) with a 90% confluence after 18 h. The cells were washed with PBS (Gibco, Gaithersburg, MD, USA) three times and inoculated with 1000 TCID₅₀/mL of PEDV-PT-P6 diluted in post-inoculation (PI) medium, containing DMEM (Gibco), 0.3% tryptose phosphate broth (Sigma, St. Louis, MO, USA), 0.02% yeast extract (Acumedia, Lansing, CA, USA), and 10 μg/mL of trypsin (Gibco). After 24 h of incubation, the CPE was observed, the supernatant was discarded and the PEDV-infected Vero cells with 10% area covered by cytopathic effect (CPE) were fixed with 80% acetone (Sigma) for 20 min. After removing the acetone and air-drying for 1 h, the plates were stored at − 20 °C until use.