2.4. Antiviral Activity Using Plaque Reduction Assay

A plaque reduction assay was performed to confirm the antiviral activity of the three tested commercially available forms of Vitamin B12 as previously described [45]. Vero E6 cells were cultivated in 6-well plates till confluency. Viral dilution from each virus was prepared and then treated with concentrations of 10, 5, 2.5, and 1.25 µM of the tested compounds. The virus–vitamin mixes were then incubated for 1 h at 37 °C before being added to the Vero E6 cells. After 1 h of virus adsorption, the mixed inoculum was removed and fresh vitamin dilutions were added to their corresponding wells with the agarose/MEM overlay and the plates were left to solidify. Then, the plates were incubated at 37 °C for three days until the formation of viral plaques. The plates were fixed with formalin and visualized using crystal violet staining. Calculations were performed as follows:

Plaque reduction assay was also performed for commercially available mixture of vitamins B1,B2, B3, B5 and B6 (B-Com, Amoun Pharmaceutical Industries Company, El Obour city, Egypt) against SARS-CoV-2 and MERS-CoV. Vitamin mix was purchased BN:225204, manufacturing date 10/2022. Molar concentrations of each vitamin in the mixture are illustrated in Figure S1.