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

Overview

This video demonstrates the viral inactivation assay, showcasing the evaluation of the impact of antiviral agents on viral particles.

Protocol

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.

Representative Results

Figure 1. Inactivation of viral infections by the test compounds CHLA and PUG. Different viruses were treated with the test compounds for a long period (incubated for 1.5 – 3 hr before titration; light gray bars) or short period (immediately diluted; dark gray bars) at 37 °C before a dilution to sub-therapeutic concentration and subsequent analysis of infection on the respective host cells. (A) Schematics of the experiment (shown on the left) with the final virus concentration (PFU/well or MOI), long-term virus-drug incubation period (i), and subsequent incubation time (ii) indicated for each virus in the table on the right. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results are plotted against the DMSO negative control treatment for virus infection and the data shown are the means ± standard errors of the mean (SEM) from three independent experiments. This figure has been modified from the reference.

Materials

Name	Company	Catalog Number	Comments
DMEM	GIBCO	11995-040
FBS	GIBCO	26140-079
Penicillin-Streptomycin	GIBCO	15070-063
Amphotericin B	GIBCO	15290-018
DMSO	Sigma	D5879
In vitro toxicology assay kit, XTTbased	Sigma	TOX2
PBS pH 7.4	GIBCO	10010023
Microplate reader	Thermo Scientific	89087-320
Microcentrifuge	Thermo Scientific	75002420
BioLux Gaussia luciferase assay kit	New England Biolabs	E3300L
Luminometer	Promega	GloMax-20/20
Sodium citrate, dihydrate	Sigma	71402
Potassium chloride	Sigma	P5405