Source: Tai, C. et al., Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds. J. Vis. Exp. (2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.
NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the biosafety level of the samples being handled. For the purpose of describing the protocols, Gaussia luciferase reporter-tagged hepatitis C virus (HCV) is used as a model virus. In the context of the representative results, the compounds chebulagic acid (CHLA) and punicalagin (PUG) are used as candidate antivirals that target viral glycoprotein interactions with cell surface glycosaminoglycans during the early viral entry steps. Heparin, which is known to interfere with the entry of many viruses, is used as a positive control treatment in such a context. For basic background on virology techniques, propagation of viruses, determination of virus titer, and expression of infectious dose in plaque forming units (PFU), focus forming units (FFU), or multiplicity of infection (MOI), the reader is referred to reference. For prior examples and optimized conditions used for viruses shown in the representative results, the reader is referred to references as well as details listed in Table 1, Figure 1A.
1. Cell Culture, Compound Preparation, and Compound Cytotoxicity
2. Readout of Viral Infection
NOTE: The readout of viral infection depends on the virus system used and can involve methods such as plaque assays or measuring reporter signals from reporter-tagged viruses. The method for detecting reporter-HCV infection based on the luciferase reporter activity is described below.
3. Viral Attachment Assay
NOTE: Examples of incubation period and viral dose for various viruses are listed in Figure 1A, 'Attachment'. Higher concentrations of the virus can also be tested by increasing the MOI/PFU.
Table 1: Host cell type for viral infection. The cell type used for each viral infection system described in the representative results is indicated. Additional details regarding the cells can be found in the reference.
Virus | Cell Type |
HCMV | HEL |
HCV | Huh-7.5 |
DENV-2 | Vero |
MV | CHO-SLAM |
RSV | HEp-2 |
Figure 1. Evaluation of antiviral activities of the test compounds CHLA and PUG against virus attachment and entry/fusion. (A) The experimental procedure, virus concentration (PFU/well or MOI), and the time of addition and treatment with the test compounds (i, ii, iii) are presented for each virus in the schematics and the associated tables. In virus attachment analysis (light gray bars), monolayers of different cell types were pre-chilled at 4 °C for 1 hr, then co-treated with the respective viruses and test compounds at 4 °C (1.5 – 3 hr; i) before washing off the inoculates and test compounds for subsequent incubation (37 °C; ii) and examination of virus infection. In virus entry/fusion analysis (dark gray bars), seeded cell monolayers were pre-chilled at 4 °C for 1 hr and then challenged with the respective viruses at 4 °C for 1.5 – 3 hr (i). Cells were then washed and treated with the test compounds for an additional incubation period (ii) during which the temperature was shifted to 37 °C to facilitate the viral entry/fusion event. At the end of the incubation, extracellular viruses were removed by either citrate buffer (pH 3.0) or PBS washes, and the cells were further incubated (iii) for analysis of virus infection. Results for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Data are plotted against the DMSO negative control treatment of virus infection and are presented as means ± SEM from three independent experiments.
The authors have nothing to disclose.
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, XTT-based | Sigma | TOX2 | |
PBS pH 7.4 | GIBCO | 10010023 | |
Microplate reader | Bio-Tek Instrument, Inc. | ELx800 | |
Microcentrifuge | Thermo Scientific | 75002420 | |
BioLux Gaussia luciferase assay kit | New England Biolabs | E3300L | |
Luminometer | Promega | GloMax-20/20 |