Crl 1586

Vero E6 (ATCC® CRL-1586™) and Vero-STAT1 knockout cells (ATCC® CCL-81-VHG™) were cultured in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 units/ml), and 10 mM HEPES. Calu-3 cell (ATCC 184HTB-55) were cultured in Eagle’s 188 Minimum Essential Medium (EMEM) (ATCC 30–2003) containing 10% FBS. SARS-CoV-2 isolates USA-WI1/2020 (BEI; cat# NR-52384), USA-WA1/2020 (BEI; cat# NR-NR-52281) were passaged in Vero-STAT1 knockout cells, whereas hCoV-19/USA/PHC658/2021 (Delta Variant) (BEI; cat# NR-55672) was passaged in Calu-3 cells. The viral titer was determined using the plaque assay as described previously ^{65 (link)}. In brief, Vero E6 cells (2.510 ^5) were seeded in 6-well plates and incubated for 24 h. After 24 h, cells were washed with sterile 1X PBS, and the virus stock was ten-fold serially diluted in serum-free OptiMEM media and then added to the cells in duplicate. The plates were incubated at 37°C for 1 h with slight shaking every 15 minutes. Then, 2 ml of 0.5% agarose in minimal essential media (MEM) containing 5% FBS and antibiotics were added to each well and incubated at 37°C for 72 h. The cells were fixed with 4% paraformaldehyde overnight, followed by removing the overlay and staining with 0.2% crystal violet to visualize plaque-forming units (PFU). All assays were performed in a BSL-3 laboratory setting.

The IBV strain rYN and 82-nt deletion strain rYN-Δ5a were constructed and rescued using a vaccinia virus-based reverse genetic system in a previous study and preserved at –80°C for further use (10 (link)). African green monkey kidney cells (Vero) (CRL-1586; ATCC) and human embryonic kidney (HEK) 293T cells (CRL-11268; ATCC) were preserved in our laboratory. CEK cells were prepared from 18-day-old SPF chicken embryos as previously described (42 (link)). SPF embryonated eggs were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd., (Beijing, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (catalog no. 12100-046; Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

Human colon adenocarcinoma cell line Caco-2 (ATCC HTB-37, ATCC, Manassas, VA, USA) and monkey kidney epithelial cell line Vero E6 (ATCC CRL-1586, ATCC, Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) FBS, 1% (v/v) sodium pyruvate and 1% (v/v) penicillin/streptomycin (all from Gibco-Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in humidified incubator containing 5% CO₂. SARS-CoV-2 was isolated from a patient at the Microbiology Unit, University Hospital of Padua. The viral strain was propagated in Vero E6 cells and characterized by whole genome sequencing. LF (Globoferrina/Transferrin, bovine lactoferrin, SOFAR SpA, Trezzano Rosa, MI, Italy) was resuspended in antibiotic-free medium and used at a final concentration of 100 µg/mL.

VeroE6 cells (Chlorocebus sabaeus; sex: female, kidney epithelial) obtained from ATCC (CRL-1586™) and from Ralph Baric (University of North Carolina at Chapel Hill), and Caco-2 cells (Homo sapiens; sex: male, colon epithelial) obtained from the ATCC (HTB-37™) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% nonessential amino acids (NEAA) and 10% fetal bovine serum (FBS) at 37°C and 5% CO₂. All cell lines tested negative for contamination with mycoplasma.

The IBV strain rYN and 82-nt deletion strain rYN-Δ5a were constructed and rescued using a vaccinia virus-based reverse genetic system in a previous study and preserved at –80°C for further use (10 (link)). African green monkey kidney cells (Vero) (CRL-1586; ATCC) and human embryonic kidney (HEK) 293T cells (CRL-11268; ATCC) were preserved in our laboratory. CEK cells were prepared from 18-day-old SPF chicken embryos as previously described (42 (link)). SPF embryonated eggs were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd., (Beijing, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (catalog no. 12100-046; Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

Liver tissues were homogenized^{32 (link)} and 250 μl of the homogenized tissue solution were used to infect Vero cells (CRL-1586; ATCC). Growth was confirmed by RT–qPCR of cell culture supernatants²⁸ .