Determination of OC43 Infectivity in HCT-8 Cells

The infectivity of OC43 was determined by focus‐forming assay in HCT‐8 cells and expressed as focus‐forming units (FFU) per milliliter [15 (link)]. Briefly, confluent monolayers of HCT‐8 cells were incubated at 33°C for 2 h with 10‐fold serial dilutions of virus. The cells were then washed and overlaid with infection medium containing 1% carboxymethyl cellulose (CMC). After 5 days of incubation at 33°C, the cells were fixed with 10% formalin, permeabilized with 0.1% Triton X‐100, and blocked with PBS containing 1% BSA and 0.1% Tween‐20. OC43 antigen was stained with antibodies (primary: mouse anti‐OC43 nucleoprotein antibody [MAB9013; Millipore, Burlington, MA, USA], secondary: goat anti‐mouse horseradish peroxidase‐conjugated antibody [Sigma‐Aldrich, St. Louis, MO, USA]), visualized with SIGMAFast reagent (Sigma‐Aldrich), and FFU were visually quantified. Values are the means of at least three independent determinations (with three replicates within each experiment).