Quantifying SARS-CoV-2 and TBEV Viral Titers

SARS-CoV-2 was diluted in ten-fold dilutions and added to VeroE6 cells followed by 1 h incubation. The inoculum was replaced with an overlay containing DMEM, 2% FBS, 1% PEST, and 1.2% Avicel. After 24 h of infection cells were fixed in 4% formaldehyde for 30 min, permeabilized in PBS 0.5% trition-X-100 and 20 mM glycine. Viral foci were detected using primary monoclonal rabbit antibodies directed against SARS-CoV-2 nucleocapsid (Sino Biological Inc., 40143-R001), and secondary anti-rabbit HRP conjugated antibodies (1:2000, Thermo Fisher Scientific). Viral foci were then revealed by incubation with TrueBlue peroxidase substrate for 30 min (KPL, Gaithersburg, MD). TBEV was titrated as previously described^{116 (link)}. VeroB4 cells were infected with 10-fold serial dilutions of TBEV. After 48 h of infection, cells were fixed with 4% formaldehyde and permeabilized in PBS containing 0.5% Triton X-100 and 20 mM glycine. Viral foci were detected using primary mouse antibodies directed against TBEV followed by staining with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:2000, Thermo Fisher Scientific).