High glucose

HEK 293 T cells were seeded into 25-cm² flasks (1 × 10⁶ cells/flask) and incubated at 37°C with 5% CO₂. When cells were sub-confluent, the culture medium was removed, and the cells were transfected with pCMV-ORF45HA or pEGFP-C1 (a mock control) using polyethylenimine (PEI) transfection reagent (Polysciences, Inc.). Briefly, 7.5 μg of DNA were mixed with 18.75 μg of PEI (1 mg/ml; ratio 1:2.5 DNA:PEI) in 500 μl of Dulbecco's modified essential medium (DMEM) with high glucose (Euroclone) without serum. After 15 min of incubation at room temperature, 2,000 μl of medium without serum was added, and the transfection solution was transferred to the cells (monolayer) and left for 6 h at 37°C with 5% CO₂, in a humidified incubator. The transfection mixture was then replaced with fresh cEMEM medium with 10% FBS and incubated for 24 h at 37°C with 5% CO₂. To analyze the subcellular localization of BoHV-4 ORF45, HEK 293 T cells were also transfected with pEGFP-ORF45-HA or pEGFP-C1 as a mock control. The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Scientific) and observed with a confocal microscope (Leica Microsystems) 24 h after the transfection.

VERO E6 cells (ATCC—CRL 1586) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), High Glucose (Euroclone, Pero, Italy), supplemented with 2 mM L-glutamine (Lonza, Milano, Italy), 100 units/mL penicillin–streptomycin mixture (Lonza, Milano, Italy) and 10% fetal bovine serum (FBS) (Euroclone, Pero, Italy), in a 37 °C and 5% CO₂ humidified incubator.
Adherent subconfluent cell monolayers of VERO E6 were prepared in growth medium (DMEM High Glucose containing 2% FBS, 2 mM L-glutamine, 100 units/mL penicillin–streptomycin) in 175 cm² flasks or 96-well plates for propagation or titration and neutralization tests of SARS-CoV-2, respectively.
Cells were seeded in a 175 cm² flask at a density of 1 × 10⁶ cells/mL. After 18–20 h, the subconfluent cell monolayer was washed twice with sterile Dulbecco’s phosphate-buffered saline (DPBS). After the DPBS was removed, cells were infected with 3.5 mL of DMEM 2% FBS containing the SARS-CoV-2 virus at a multiplicity of infection (MOI) of 0.01. After 1 h of incubation at 37 °C in a humidified atmosphere with 5% CO₂, 50 mL of DMEM containing 2% FBS was added. The flasks were observed daily, and the virus was harvested when 80–90% of the cells manifested cytopathic effect (CPE). The culture medium was centrifuged at +4 °C and 469× g for 8 min, aliquoted, and stored at −80 °C.