Vero e6

HEK293T, Vero E6, and MDCK cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies). The human lung adenocarcinoma cell line H1975 was generously provided by Prof. Chih-Hsin Yang (Graduate Institute of Oncology, Cancer Research Center, National Taiwan University) and was maintained in RPMI1640 containing 10% FBS. H1975 cells were transduced with lentivirus encoding full-length ACE2 before being used as target cells (H1975-ACE2). The expression of ACE2 in H1975-ACE2 cells was described in more details in another recently submitted manuscript by our group^{56 (link)}. All adherent cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and 20% O₂. Sputum specimens obtained from SARS-CoV-2-infected patients were propagated in Vero E6 cells in DMEM supplemented with 2 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich). Culture supernatant was harvested when CPE were seen in more than 70% of cells, and viral titers were determined by a plaque assay. The virus isolates used in the current study were summarized in Table 1. The experimental protocols were approved by the NTUH Research Ethics Committee (202002002RIND by Dr. Jann-Tay Wang), and the informed consent was obtained from all subjects.

Henipavirus isolates were obtained from the Special Pathogens Branch of the Centers for Disease Control and Prevention, Atlanta, GA or Public Health Agency, Winnipeg, Canada. NiV Bangladesh (GenBank no. AY988601), NiV Malaysia (GenBank no. AF212302), and HeV (GenBank no. AF017149) have been passaged three, four, and three times in VeroE6 cells respectively. All virus propagation in this manuscript was performed in VeroE6 cells in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 2% fetal bovine serum (Gibco), 1 mM L-glutamine (Gibco), 50 U/ml penicillin (Gibco), and 50 μg/ml streptomycin (Gibco) (2% DMEM). VeroE6 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1 mM L glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin.

Vero CCL-81 (ATCC, Manassas, VA, USA) and Vero E6 (CRL-1586, ATCC, Manassas, VA, USA) cells were maintained in medium 199 (Gibco, Billings, MA, USA), supplemented with 5% and 10% fetal bovine serum (FBS; Gibco, Billings, MA, USA), respectively, and 40 µg/mL of gentamicin sulfate (Santisa, Bauru, SP, Brazil). For Vero E6, the concentration of L-glutamine (Sigma-Aldrich, Saint Luis, MO, USA) was adjusted to 2 mM. Both cell lines were kept under 5% CO₂ at 37 °C.

All cell lines were obtained from American type culture collection (ATCC). Human embryonic kidney cells (293T), African green monkey kidney epithelial cells (Vero E6), and Madin-Darby canine kidney cells (MDCK) were maintained in Dulbecco's modified eagle medium (DMEM)-high glucose (Sigma Aldrich) supplemented with 10% low endotoxin fetal bovine serum (FBS) (Cegrogen Biotech) and penicillin–streptomycin.

The African green monkey kidney cell line Vero E6 (ATCC, Rockville, MD) was cultured as monolayers in a 5% CO₂ atmosphere at 37°C in DMEM (Sigma-Aldrich, Argentina) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Argentina), 100 U/mL penicillin and 100 μg/mL streptomycin.
Vero E6 cells were seeded at a concentration of 8 × 10⁴ cells/well in 24-well plates and incubated with supernatants from frozen tissues that were mechanically disaggregated as described in point 2.2. The supernatants were diluted 1/4 in DMEM and incubated with the cells for 4 h at 37°C under a 5% CO₂ atmosphere. The cells were then washed six times with DMEM, and the last wash was stored at −70°C to detect any remaining SARS-CoV-2 genomic RNA. The cells were incubated in DMEM supplemented with 2 mM L-glutamine, 2% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cytopathic effect, evidenced by the destruction of the cell monolayer observed under a light microscope at 20× magnification, was monitored every 24 h. After 7 days, the supernatants were inspected for the presence of SARS-CoV-2 RNA using RT-qPCR, as described in point 2.3.
Live SARS-CoV-2 manipulation was performed in biosafety level 3 facilities at the INBIRS.