Mab9013

The assay was adapted for hCoV OC43 from a protocol described elsewhere [9 (link)]. In short, the virus was preincubated with a semilogarithmic dilution series of polymers control solutions for 30 minutes before it was added to a monolayer of Vero cells for infection. After an infection period of 45 minutes at RT the inoculum was removed; cells were overlaid with medium containing test substance and then cultured at 37°C, hereby maintaining the same concentrations of active agent as in the prophylactic treatment. Staining was performed on fixed cells using an antibody directed against the hCoV-OC43 nucleoprotein as a primary antibody (Merck #MAB9013) followed by a horse radish peroxidase labeled detection antibody (Thermo Scientific #31430) and TMB as substrate. For detection, a BMG Fluostar Microplate reader was utilized. To enable direct comparison of the effectiveness of the test substances, the IC₅₀ value of each sample was calculated for a sigmoidal dose–response model with XLfit Excel add-in version 5.3.1.

DMSO (D1435, ≥99.5%), crystal violet (C0775, dye content ≥90%), and methylcellulose (#M0387) were purchased from Sigma-Aldrich (St. Louis, MO, United States); remdesivir (GS-5734) (S8932, 99.3%, HPLC) and cyclosporine A (cyclosporine) (S2286, 99.6%, HPLC) from Selleckchem (Houston, TX, United States); Goat anti-human IgG-Alexa Fluor 488 (A11013), Hochest 333258 (H3569), and DAPI (D1306) from Invitrogen (Thermo Fisher Scientific, Waltham, MA, United States); and 10% formaldehyde solution from Marcon™ Chemicals (#H121-08). The antibody against nucleocapsid (N) protein of HCoV-OC43 (Mab9013) was purchased from Merck Millipore (Burlington, MA, United States), and fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin (#55499) from MP Biomedicals (Irvine, CA, United States). Anti-SARS-CoV-2 N protein antibodies were provided by Dr An-Suei Yang of the Genomics Research Center, Academia Sinica.

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.

serial two-fold sample dilutions (1:8 up to 1:16,384) in DMEM supplemented with 5% FBS, 100 Units/ml penicillin and 100 µg/ml streptomycin were incubated for 1 h at 37°C with 500 TCID₅₀/well of HCoV-OC43 in 96-well plates. HRT-18 cells were added at 20,000 cells/well and incubated for 4 days at 33°C. Plates were washed with PBS, fixed, and IPMA staining was performed as described above, using an antibody directed against the HCoV-OC43 nucleocapsid (mAb9013, Sigma-Aldrich; 1:1,000) and peroxidase-labeled goat anti-mouse IgG (Dako, P0447; 1:1,000).

RD cells were grown in eight-well chambered slides and grown overnight to a 70% confluency. We pre-treated with the peptides indicated for 1 hr followed by infection with HuCoV-OC43 (at MOI of 0.1) for 1 hr. The cells were washed with media without serum and suspended in medium containing 2% FBS with the same amount of peptides and incubated further for 48 hrs. We then fixed cells with 4% paraformaldehyde for 30 min, followed by permeabilization with PBS containing 1% Triton X-100 for 30 min at room temperature. Cells were blocked in 10% normal goat serum in PBS with 0.5% Triton X-100 for 30 min at room temperature, followed by two washing in PBS with 0.2% Tween-20 (wash buffer), for 10 min each. Mouse monoclonal antibody to HuCoV-OC43 nucleoprotein (Sigma, cat no. MAB9013) was added at a dilution of 1:500 in PBS with 0.2% Tween-20 for 2 hrs at room temperature. Cells were washed 4 times with wash buffer. We incubated with Texas red conjugated secondary anti-mouse antibody (for pJAK2 treatment) and DAPI for 30 min. Antibody treatment was followed by four washings and the addition of mounting media (Southern Biotechnology), and covering with a cover slip. When cells were treated with IFNα−C, we used AlexFluor 488 conjugated anti-mouse antibody (Invitrogen) and DAPI for staining. We imaged cells using a Keyence BZ-X700 fluorescence microscope.