an in vitro assay for screening interferons as potent inhibitors of zika virus growth

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

Six hours after seeding a 12-well plate with na&#239;ve 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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Universidad San Sebastian

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1. Screening Cytokine Library against Zika Virus Infection
<ol>
	<li>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.</li>
	<li>At 12 hr post-treatment, perform Zika viral infection (MOI of 0.1 in 400 &#956;l per well). Include negative control wells without infection (mock).</li>
	<li>At 4 hr post-infection, replace the viral inoculum with cytokine-treated media (2 ml). Incubate the cells at 37 &#176;C for additional 44 hr.</li>
	<li>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).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vero Cell Line</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Zika viral strain PRVABC59</td>
			<td>Centers for Disease Control and Prevention (CDC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>Peprotech</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1 alpha</td>
			<td>Peprotech</td>
			<td>200-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-alpha</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon alpha A</td>
			<td>R &#38; D Systems</td>
			<td>11100-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon beta</td>
			<td>Peprotech</td>
			<td>300-02BC</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma</td>
			<td>Peprotech</td>
			<td>300-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415R</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Contreras, D. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54767">Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/22274/22274_Figure1.jpg" /> 
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 &#60;0.05). (B and ...

1. Screening Cytokine Library against Zika Virus Infection

	Seed naive Vero cells at 1 x 105 cells per well using a 12-well plate. Once the cells are ...

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&#39;s antiviral activity against the Zika virus.

21 visualizações. Um ensaio in vitro para triagem de interferons como inibidores potentes do crescimento do vírus Zika

Vídeo: Um ensaio in vitro para triagem de interferons como inibidores potentes do crescimento do vírus Zika - Experimento

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Avaliação das respostas de células T Zika vírus específicos em órgãos Immunoprivileged de infectados Ifnar1- / - ratos

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

JoVE Journal

Imunologia e Infecção

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

Isolamento e quantificação do vírus Zika de múltiplos órgãos em um mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Cell-Based Electrical Impedance Platform to Evaluate Zika Virus Inhibitors in Real Time

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture plate wells embedded with electrodes. The technology can be used to monitor the consequences of Zika virus (ZIKV) infection and adherent cell replication in real time, as this virus is highly cytopathogenic. It is a straightforward assay that does not require the use of labels or invasive methods and has the benefit of providing real-time data. The kinetics of ZIKV infection are highly dependent on the employed cell line, virus strain, and multiplicity of infection (MOI), which cannot be easily studied with conventional endpoint assays. Furthermore, the CEI assay can also be used for the evaluation and characterization of antiviral compounds, which can also have dynamic inhibitory properties over the course of infection. This methods article gives a detailed explanation of the practical execution of the CEI assay and its potential applications in ZIKV research and antiviral research in general.

Here, we demonstrate the use of cell-based electrical impedance (CEI) as a very easy and straightforward method to study Zika virus infection and replication in human cells in real time. Furthermore, the CEI assay is useful for the evaluation of antiviral compounds.

Plataforma de impedância elétrica baseada em células para avaliar inibidores do vírus Zika em tempo real

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture ...

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

Transcrição

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