Horseradish peroxidase conjugated anti mouse secondary antibody

Cells were lysed in lysis buffer (25 mM Tris HCl pH 8.8, 50 mM NaCl, 0.5% Nonidet P-40 and 0.1% sodium dodecyl sulphate supplemented with cocktails of protease and phosphatase inhibitors). Debris were removed by centrifugation at 16,000 g for 20 min at 4°C. Total protein concentration was then determined using a Micro BCA^TM Protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amount of protein lysate was heat-treated in loading sample buffer containing β-mercaptoethanol and analysed by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to nitrocellulose membrane (Hybond-ECL, Amersham, GE healthcare LifeScience, Pittsburgh, PA, USA). Membranes were covered with blocking buffer (PBS containing 5% dry milk and 0.05% Tween-20) for one hour and then incubated with a mouse anti-HEV ORF2 antibody (MAB8002, Merck Millipore, Darmstadt, Germany) or a mouse anti-actin antibody (clone AC-40, Sigma-Aldrich, Saint-Louis, MO, USA) diluted in blocking buffer (1/500 and 1/2000 dilution, respectively). After several washes in PBS containing 0.05% Tween-20, a horseradish peroxidase-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) was added (dilution 1/5000 in blocking buffer). An enhanced luminol-based chemiluminescent detection system was used to detect bound antibodies.

TBEV, LGTV, and ZIKV were titrated using focus-forming assays as previously described [4 (link), 29 (link)]. In brief, VeroB4 cells were infected with 10-fold serial dilutions of TBEV, LGTV, WNV, JEV, or ZIKV. 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 or flavivirus envelope protein (TBEV, 1493 1:1000 [33 (link)], LGTV 1786 1:1000 [33 (link)], ZIKV, WNV, JEV HB112 ATCC 1:1000 [34 (link), 35 (link)]) followed by staining with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:2000, Thermo Fisher Scientific). Viral foci were detected using TrueBlue peroxidase substrate (KPL, Gaithersburg, MD). For in vitro infections, monolayers of cells were infected with 0.1 multiplicity of infection (MOI) of TBEV, LGTV, WNV, JEV, and ZIKV. After 1 h of infection, the inoculum was removed and replaced with growth medium. Experiments with TBEV, WNV, and JEV were performed in the biosafety level 3 facility at Umeå University.

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).