This article describes an experimental setup to evaluate the antiviral activity of interferons against the Zika virus using epithelial cell monolayers. The methodology includes the use of control and test wells to assess the impact of interferons on viral replication and plaque formation.
Take wells containing virus-susceptible epithelial cell monolayers.
Add serial dilutions of a test interferon to the test wells. The control well lacks the interferon.
In test wells, interferons bind to cell receptors, triggering a signaling cascade for antiviral protein expression.
After incubation, add the Zika virus to all the wells.
The Zika virus interacts with the host cell receptors and endocytoses.
Now, remove unadsorbed viruses. Add media to the control well and media with additional interferons to the test wells.
Incubate. In the control well, internalized viruses use host cell machinery to form new viral particles.
The infected cells lyse, releasing virus particles that infect and lyse other cells, forming clear lysed zones or plaques in the cell monolayer.
In interferon-treated cells, the antiviral proteins inhibit viral replication and cell lysis, preventing plaque formation.
Using a phase-contrast microscope, count viral plaques in the wells.
A smaller number of plaques in interferon-treated wells indicates the interferon's antiviral activity against the Zika virus.
Six hours after seeding a 12-well plate with naïve Vero cells, add cytokines of interest at indicated concentrations in biological duplicates. 12 hours post-treatment, perform Zika virus infection, and include negative control wells without infection. Four hours after infection, replace the viral inoculum with cytokine-treated medium, and then incubate the cells at 37 degree Celsius for an additional 44 hours. At 48 hours post-infection, count viral plaques using a phase-contrast microscope, and acquire representative images of infected cells at 40x magnification.
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