SARS-CoV-2 isolate Fin/25/20 (Gisaid: EPI_ISL_412971) from lineage B.1 was isolated from the nasopharyngeal sample of COVID-19 patient in Finland in February 2020. Swab sample in transport medium was inoculated onto African green monkey kidney epithelial VeroE6 cells at +37 °C and 5% CO2 in culture medium (Eagle’s minimum essential medium (EMEM) supplemented with 2% fetal bovine serum (FBS), 0.6 μg/mL penicillin, 60 μg/mL streptomycin, 2 mM l -glutamine, 20 mM HEPES). Virus was propagated in VeroE6 cells for a total of three times. Subsequently, a VeroE6 clone expressing TMPRSS2, a serine protease essential for SARS-CoV-2 spike protein integrity, was generated (VeroE6-TMPRSS2-H10)44 . Fin/25/20 was further propagated twice in VeroE6-TMPRSS2-H10 cell line (virus isolate named FIN-25). Another 2020 isolate, SR121 from lineage B.1.463, isolated from a patient in Finland in September 2020, was isolated and propagated only in VeroE6-TMPRSS2-H10 cells44 . Variants 85HEL of B.1.1.7 lineage and HEL12-102 of B.1.351 lineage were isolated from patients in Finland as described23 and further propagated only in VeroE6-TMPRSS2-H10 cells. VeroE6-TMPRSS2-H10 cells were maintained in D-MEM (Lonza) supplemented with 10% FBS, 2 mM l -glutamine (Gibco), and penicillin/streptomycin. For virus propagation in VeroE6-TMPRSS2-H10 cells, D-MEM supplemented with 2% FBS, 2 mM l -glutamine, and penicillin/streptomycin was used. Supernatants containing viruses were harvested, cell debris removed with centrifugation at 500×g for 5 min, and aliquots stored at −80 °C.
Fifty-percent tissue culture infective dose (TCID50) of virus stocks was determined with endpoint dilution assay in VeroE6-TMPRSS2-H10 cells. Briefly, 50,000 cells per well were plated on 96-well tissue culture plates (Sarstedt), and the next day media was changed to infection media (2% FBS). Ten-fold virus dilutions in infection media were applied onto cells, and the plates were incubated for 3 days at +37 °C and 5% CO2. Cells were fixed with 4% formaldehyde and stained with crystal violet. Virus dilution resulting in 50% cell death was determined to represent TCID50 value of the stock virus. Virus propagations and end-point dilution assays were done in BSL-3 laboratory conditions.
Fifty-percent tissue culture infective dose (TCID50) of virus stocks was determined with endpoint dilution assay in VeroE6-TMPRSS2-H10 cells. Briefly, 50,000 cells per well were plated on 96-well tissue culture plates (Sarstedt), and the next day media was changed to infection media (2% FBS). Ten-fold virus dilutions in infection media were applied onto cells, and the plates were incubated for 3 days at +37 °C and 5% CO2. Cells were fixed with 4% formaldehyde and stained with crystal violet. Virus dilution resulting in 50% cell death was determined to represent TCID50 value of the stock virus. Virus propagations and end-point dilution assays were done in BSL-3 laboratory conditions.
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