Quantifying Infectious Coronaviruses via IPA

The titer values of infectious HCoV-OC43 virions were determined with IPA as previously reported (4 (link)– (link)9 (link)). Briefly, HCT-8 cells were seeded in 96-well plates and infected with each sample serially diluted from 1.0 × 10⁰- to 1.0 × 10⁷-fold. Cells were incubated for 4 days, and then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. A monoclonal mouse antibody specific for the HCoV-OC43 nucleoprotein (MAB9013; Sigma-Aldrich) and horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG antibody (Vectastain Elite ABC mouse IgG kit; Vector, Burlingame, CA, USA) were used as the primary and secondary antibodies, respectively. Immune complexes were detected with 3,3-diaminobenzidine tetrahydrochloride (DAB substrate kit; vector) and 0.01% hydrogen peroxide in PBS, and the viral titers were calculated with the Karber method.