Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay

1. Viral Inactivation Assay

Note: Examples of incubation period and viral dose for various viruses are listed in Figure 1A. Higher concentrations of the virus can also be tested by increasing the MOI/PFU.

1. Seed Huh-7.5 cells in a 96-well plate (1 × 10⁴ cells per well) and incubate at 37 °C in a 5% CO₂ incubator O/N to obtain a monolayer.
2. Incubate the test compounds or controls (final concentrations are: CHLA = 50 μM; PUG = 50 μM; heparin = 1,000 μg/ml; DMSO = 1%) with the HCV particles at 37 °C (Figure 1A, 'Long-Term') in a 1:1 ratio. For example, to a 100 μl virus inoculum containing 1 x 104 FFU, add 100 μl of a 100 μM CHLA working dilution; this yields CHLA treatment at a final concentration of 50 μM.
3. Dilute the virus-drug mixture to a "sub-therapeutic" (ineffective) concentration of the test compounds. For example, the ineffective concentration of CHLA and PUG against HCV is at 1 μM31; therefore, this requires a 50-fold dilution of the virus-drug mixture which can be accomplished with 9.8 ml of basal medium (cell culturing medium with 2% FBS).
   Note: The dilution to sub-therapeutic concentration prevents significant interaction between the test compounds and the host cell surface and allows examination of the treatment effect on the cell-free virions. Note that this dilution is dependent on the antiviral dose response of the test compounds against the particular viral infection and is determined prior to performing this particular assay.
4. For comparison, mix the virus with the test compounds and immediately dilute (no incubation period) to sub-therapeutic concentration prior to infection (Figure 1A, 'Short-Term').
5. Add 100 μl of the diluted HCV-drug mixture onto the Huh-7.5 cell monolayer (the amount of virus is now at 1 x 10² FFU; final MOI = 0.01) and incubate for 3 hr at 37 °C to allow viral adsorption.
6. Following the infection, remove the diluted inocula and gently wash the wells with 200 μl of PBS twice.
   Note: Perform the washes gently to avoid lifting the cells.
7. Apply 100 μl of basal medium to each well and incubate at 37 °C for 72 hr.
8. Analyze the resulting infection by assaying the supernatant for luciferase activity.