A Viral Attachment Assay for Screening of Antiviral Compounds

For the viral attachment assay, plate 1 x 10⁴ cells per well in a 96-well plate, and incubate overnight for cell attachment. The next morning, first, chill the 96-well plate containing the cell monolayer at 4 degrees Celsius for 1 hour.

Prepare the test compounds and virus mixture on ice. For example, HCV-CHLA, HCV – 1% DMSO, or positive control HCV-heparin solutions with 10² FFU virus for each treatment well. Aspirate the medium from the cell culture wells gently. Then, immediately add 100 microliters of virus test compound mixture in triplicate wells, taking care not to raise the temperature. Incubate the plate in a refrigerator maintained at 4 degrees Celsius for three hours.

Virus particles will attach on the cell surface at 4 degrees Celsius but will not be internalized. Next, aspirate the supernatants. Gently wash the cells twice with 200 microliters of ice-cold PBS. Add 100 microliters of basal medium to each well, and then incubate at 37 degrees Celsius for 72 hours. Finally, for quantitating the released luciferase reporter from the infected cells, collect the supernatant from each sample well and perform the Gaussia luciferase assay.