Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay
Transcription
To begin the viral inactivation assay, first, plate 1 times 10 to the fourth cells per well in a 96-well plate. Incubate the cells at 37 degrees Celsius with 5% carbon dioxide overnight for attachment. For infection, prepare Gaussia luciferase reporter-tagged hepatitis C virus or HCV particles, as referenced in the text protocol.
In a sterile tube, mix 100 microliters of 100 micromolar CHLA or PUG with 100 microliters of 10 to the fourth focus-forming units or FFU of HCV. For a positive control, mix HCV with heparin at a final concentration of 1,000 micrograms per milliliter. Incubate at 37 degrees Celsius for three hours.
After three hours, dilute the virus compound mixture 50-fold with 9.8 milliliters of basal medium at room temperature. Then, prepare a new virus compound mixture, and immediately dilute this mixture in basal medium for the zero-hour incubation sample. After removing the incubation medium from the cells, add 100 microliters of the diluted virus drug solution per well in triplicate.
The solution now contains 10 to the second FFU per well. Incubate at 37 degrees Celsius and 5% carbon dioxide for three hours to allow viral infection of the cells. After incubation, remove the viral suspension from the wells and gently wash the cells with 200 microliters of PBS twice.
Incubate the cells in 100 microliters of basal medium for 72 hours for viral replication and release of luciferase reporter into the supernatant. Then, collect the supernatants from the cultures in microtubes, and centrifuge at 17,000 times g for 5 minutes at 4 degrees Celsius to remove cellular debris. Mix 20 microliters of each supernatant with 50 microliters of Gaussia luciferase assay reagent, and measure the luminescence from the luciferase reporter activity in a luminometer.
