An In Vitro Assay for Screening Interferons as Potent Inhibitors of Zika Virus Growth

Abstract

Source: Contreras, D. et al., Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons. J. Vis. Exp. (2016)

The video demonstrates an in vitro assay for screening interferons with antiviral activity against the Zika virus, a human pathogen. Within the host cell, the Zika virus undergoes multiplication upon internalization, infecting neighboring cells and creating distinct zones or viral plaques. Type I interferon, acting as an antiviral agent, hinders virus multiplication, thereby preventing the formation of viral plaques in the host cell monolayer.

Protocol

1. Screening Cytokine Library against Zika Virus Infection

1. Seed naive Vero cells at 1 x 10⁵ cells per well using a 12-well plate. Once the cells are attached (6 hr post-seeding), add each cytokine at
   indicated concentrations in biological duplicates (2 ml volume/well). Include vehicle (PBS) alone control.
2. At 12 hr post-treatment, perform Zika viral infection (MOI of 0.1 in 400 μl per well). Include negative control wells without infection (mock).
3. At 4 hr post-infection, replace the viral inoculum with cytokine-treated media (2 ml). Incubate the cells at 37 °C for additional 44 hr.
4. At 48 hr post-infection, count viral plaques using a phase contrast microscope and acquire representative images of infected cells at 40X magnification (Figure 1).

Representative Results

Figure 1: Interferons demonstrate potent anti-Zika viral activity. (A) Graph showing modulation of Zika viral growth by various cytokines compared to that of vehicle control. Mean values and standard deviation are shown in the bar graph. Interferons showed statistically significant inhibition of Zika virus replication (p value <0.05). (B and C) Phase contrast images of Zika virus-infected cells with or without cytokine treatment. Viral plaques formed by infected cells can be seen as foci. Note that interferon-treated wells do not have viral plaques. Mock control without ZIKV infection included as negative control. (Scale bar = 50 µm).

Materials

Vero Cell Line	ATCC	CCL-81
Zika viral strain PRVABC59	Centers for Disease Control and Prevention (CDC)
IL-6	Peprotech	200-06
IL-1 alpha	Peprotech	200-01A
TNF-alpha	Peprotech	300-01A
Interferon alpha A	R & D Systems	11100-1
Interferon beta	Peprotech	300-02BC
Interferon gamma	Peprotech	300-02
Centrifuge 5415R	Eppendorf	5415R
Centrifuge 5810R	Eppendorf	5810R
Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI	Nikon