The authors thank Cl&#233;ment Heymann and Gorrit Lootsma for proofreading the manuscript. The study was supported by internal grants of the Laboratory of Virology and Chemotherapy (Rega Institute, KU Leuven).

All steps described are carried out under sterile conditions in a laminar flow cabinet of a Biosafety level 2 laboratory. All used viruses were obtained and used as approved, according to the rules of a Belgian institutional review board (Departement Leefmilieu, Natuur en Energie, protocol SBB 219 2011/0011) and the Biosafety Committee at the KU Leuven.
1. Cell culture maintenance
NOTE: This protocol is performed with a selection of cell lines, but can of course be adapted to any adherent cell line, only requiring cell seeding density optimization.
<ol>
	<li>Grow the A549 human lung adenocarcinoma cell line, the U87 human malignant glioblastoma cell line, and the HEL 299 human embryonal lung fibroblast cells in T75 culture flasks at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
	<li>Subculture the cells at 80%-95% confluency. All reagents should be at room temperature (RT) before culturing the cells.</li>
	<li>Remove the conditioned culture medium from the cells. Use 5 mL of Dulbecco&#39;s phosphate-buffered saline (DPBS) to wash the cell monolayer once.</li>
	<li>Distribute 1 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) evenly over the cell monolayer. Then, incubate for up to 3 min at 37 &#176;C until the cells start to detach.</li>
	<li>Add 9 mL of fresh complete growth medium (see Table of Materials for details). Pipet gently up and down to resuspend the cells.</li>
	<li>Transfer the desired volume of cell suspension to a new T75 culture flask and add an appropriate volume of fresh growth medium to obtain a total volume of 15 mL. 
	&#8203;NOTE: The in vitro cell lines used in this protocol are subcultured in 1:4 to 1:10 dilutions, depending on the specific cell line, to allow ideal growth conditions. Subcultures are performed every 3-4 days, so the desired cell suspension volume can also vary based on the number of incubation days.</li>
</ol>
2. Preparation of CEI plate
<ol>
	<li>Take a 96-well plate with interdigitated electrodes (see Table of Materials) and fill all the wells with 100 &#181;L of growth culture medium.</li>
	<li>Fill the spaces between the wells with DPBS. 
	NOTE: This is done to reduce the edge effect due to evaporation that is observed when culturing cells in 96-well plates.</li>
	<li>Incubate the plate for 4 h at 37 &#176;C in an incubator with 5% CO2.</li>
</ol>
3. Seeding of cells
<ol>
	<li>Detach the cells from the culture flask, as described in section 1, and resuspend them in fresh growth medium in a 50 mL tube.</li>
	<li>Count the number of viable cells using the method of choice. 
	NOTE: In the case of A549 cells, viability generally reaches ~100%. To determine the cell number, we use an automated cell analysis system based on acridine orange/propidium iodide staining (see Table of Materials). Other cell counting methods work equally well.</li>
	<li>Resuspend the cells in fresh growth medium to obtain the desired cell density. In this study, the optimal A549 cell density is 0.15 &#215; 106 (viable) cells/mL.</li>
	<li>Take the 96-well plate with interdigitated electrodes out of the incubator and remove the medium with a multichannel pipet.</li>
	<li>Dispense 100 &#181;L of the cell suspension (corresponding to 15 &#215; 103 cells in this case) per well in the 96-well plate.</li>
	<li>Allow the cells to equilibrate for 15 min at room temperature before placing them in the CEI device.</li>
</ol>
4. Setting up and running the CEI assay
<ol>
	<li>Place the incubator that houses the CEI device plate holder at 37 &#176;C and 5% CO2.</li>
	<li>Place the 96-well plate in the device.</li>
	<li>Open the device&#39;s software and set up a new experiment by clicking Setup in the Collect Data pane. Wait for the laptop to connect to the ECIS device and configure the plate by checking the wells&#39; impedances. Check whether all the wells are correctly configured, as indicated by a green color. If not, repeat step 4.2 until all the wells are green.</li>
	<li>In the Well Configuration pane, select the array type, according to the used culture ware.</li>
	<li>Select Multiple Frequency/Time mode. 
	NOTE: In this mode, the device measures impedance changes at a range of frequencies.</li>
	<li>Start the measurement by clicking Start. 
	&#8203;NOTE: Real-time impedance can be monitored on the software&#39;s interface. Select the desired frequency to be shown. In this example, 16 kHz is chosen.</li>
</ol>
5. Compound preparation and incubation
NOTE: As an example of an antiviral compound, labyrinthopeptin A1 is used (See Table of Materials). Since this protocol can be generalized to all compounds with potential antiviral activity, we refer to it as &#39;the compound&#39;.
<ol>
	<li>Allow complete cell culture medium without the addition of fetal bovine serum (FBS) (referred to as &#39;assay buffer&#39;) to equilibrate at RT.</li>
	<li>Keep the compounds on ice.</li>
	<li>Prepare a 10x concentrated dilution of each compound in assay medium in 5 mL polypropylene tubes.</li>
	<li>Serially dilute the compounds in assay medium in 5 mL polypropylene tubes to obtain the desired 10x concentrations.</li>
	<li>Pause the CEI device by clicking Pause in the Data Collection Setup pane. Wait for the current time point to be completed and take out the 96-well plate.</li>
	<li>Verify the adherence and monolayer formation of the cells under a light microscope.</li>
	<li>Remove 20 &#181;L from each well with a multichannel pipet.</li>
	<li>Add 20 &#181;L of compound solution or vehicle in the desired wells. Prepare a layout with the specific conditions for proper pipetting.</li>
	<li>Incubate the plate for 15 min at 37 &#176;C with 5% CO2. 
	NOTE: A normal cell incubator is used for this incubation step instead of a CEI device. Cell control (CC) wells are vehicle-treated wells.</li>
</ol>
6. Virus preparation and infection
<ol>
	<li>Thaw a vial of virus stock. In this example, ZIKV MR766 and ZIKV PRVABC59 are used.</li>
	<li>Prepare virus dilutions at the desired MOI or dilutions in assay medium in a 15 mL polypropylene tube. 
	NOTE: In this representative example, MOI 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.005, and 0.00005 are used.</li>
	<li>Add 100 &#181;L of virus dilution in the assigned wells of the 96 well plate. Add assay medium to the CC wells.</li>
	<li>Decontaminate the employed virus containers.</li>
	<li>Put the plate back in the CEI device and continue the measurements for 6 consecutive days by clicking Resume Experiment. 
	NOTE: Virus control (VC) wells are vehicle-treated infected cells, whereas in CC wells, assay medium is added instead of virus dilution. If the cytotoxicity of the compounds is evaluated, add 100 &#181;L of assay buffer instead of virus dilution.</li>
</ol>
7. Data analysis
<ol>
	<li>End the experiment by clicking Finish and add optional summary comments, such as information about the specific experimental conditions in the appearing window. Click OK when data collection is complete.</li>
	<li>Select the desired frequency in the Graph pane where the largest impedance difference between CC and VC is measured at the end of the experiment.
	<ol>
		<li>To determine the best frequency, run a pilot experiment with uninfected and infected cells and choose the frequency that-at the time point where the impedance of the infected cells has dropped to its baseline level-leads to the largest observed impedance difference between the uninfected and infected cells. In the case of A549 cells infected with ZIKV, this is 16 kHz.</li>
	</ol>
	</li>
	<li>To export all the data in a spreadsheet format, make sure all wells are selected in the Well Configuration pane and click File | Export data | Graph data.</li>
	<li>Normalize the data by subtracting the average VC impedance value measured at the end of the experiment from all the data points and dividing this by the average CC impedance value of the last time point measured before infection.</li>
	<li>Plot the normalized data in the function of time to obtain CEI profile graphs.</li>
	<li>Calculate the normalized area under the curve (AUCn) of each condition to determine parameters such as 50% inhibitory concentrations (IC50).</li>
	<li>Calculate other useful parameters, such as CIT50, to, for example, compare the infectivity of various virus strains.</li>
</ol>

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The authors confirm that there is no conflict of interest.

This article describes the use of the CEI assay in ZIKV antiviral research. The assay has the benefit of real-time monitoring and can therefore be used to evaluate the kinetics of ZIKV infection and antiviral inhibition with selective compounds. The data obtained with this method allows for an objective and visual observation of the virus-induced CPE and the potency of a potential antiviral compound.
Since CEI is a very sensitive method, great care must be taken when performing this cellular assay. It is crucial that precise pipetting is performed to avoid pipetting variation as much as possible. Moreover, other factors that might influence experimental outcomes, such as cell number and used viral stock, must be kept constant between experiment repetitions. These are factors that highly influence the kinetics of cell growth and infection and could therefore lead to variation in calculated CIT50 values and/or other parameters.
When performing the CEI assay in the presence of (potential) antiviral compounds, it is important to determine if they influence the impedance of the cells in the absence of a virus. The technique is so sensitive that impedance changes might be measured well below the compound&#39;s cytotoxic concentrations19.
Although only ZIKV data are shown in this article, the assay can be easily applied to other cytopathogenic (flavi)viruses, with only minimal optimization effort, such as optimal CEI monitoring frequency determination. Adaptations of the CEI assay have been employed with various human and animal viruses, such as chikungunya, influenza A, and human and equine herpesviruses25,26,27,28. Here, it is also used as a simple method for the quantification of viral titers or for the identification of antiviral compounds. These studies used real-time cell analysis, an alternative CEI method.
The use of the CEI technology is limited to adherent cell types, as the assay&#39;s principle relies on the properties of adherent cells to spread across the electrode-embedded wells. Well surface coatings such as fibronectin can be used to improve cell attachment29. The methodology has some other drawbacks, the first related to its cost. Apart from the investment in the CEI monitoring device and accompanying hard- and software, the consumables are also somewhat expensive. Further, since the protocol requires monitoring viral infection for several days, one 96-well plate per week can be evaluated. Together with the high cost, this limits the use to low- to medium-throughput settings.
Because of these throughput issues, the technology is unsuitable for antiviral screening settings. However, it is highly appealing in initial experimental phases, for example, when evaluating cell susceptibility for the study of ZIKV infection in a certain disease-related setting. The technology is also useful with transfected or knockout cell lines to easily observe changes in infection kinetics, for example, in the presence or absence of certain entry receptors. This is interesting when investigating the involvement of cellular factors in ZIKV infection. Further, in later preclinical phases, when a certain lead candidate has been selected, the CEI assay can be used for further in-depth characterization of a selected compound. CEI biosensors can also be modified by immobilizing certain biorecognition elements, such as virus-specific antibodies, for the detection of viruses18. Altogether, this highlights the versatile use of CEI in a virology setting.

Zika virus (ZIKV) outbreaks are associated with serious disease complications, such as microcephaly and Guillain-Barre syndrome1,2,3. Although future epidemics are plausible due to various risk factors such as spreading mosquito vector distribution and increasing urbanization, to date, no vaccine or antiviral drug has made it to market yet3,4. Therefore, traditional ZIKV research methods should be supplemented with novel tools to study this virus and its potential antiviral compounds. Antiviral research is often based on phenotypic assays, where the endpoint is the presence of a specific parameter, such as the appearance of a virus-induced cytopathic effect (CPE) or production of a particular virus-induced protein by means of a reporter gene5,6,7. However, these methods have endpoint readouts, are labor-intensive, and may require complex analysis. Therefore, impedance-based methods offer an attractive alternative.
Cell-based electrical impedance (CEI) is defined as the resistance to current flow from one electrode to another, caused by an adherent cell layer seeded on electrode-containing wells.The established CEI technology employed in this methodology is Electric Cell-substrate Impedance Sensing (ECIS), originally developed by Giaever and Keese8,9. This is used in a broad field of biological research domains, such as cancer metastasis, toxicology, and wound healing10,11,12. Its principle relies on an electric field that is generated by a continuous sweeping of alternating current (AC) voltages over a range of frequencies13. The cells are exposed to these noninvasive electric fields, and at predetermined intervals, the impedance changes caused by cell growth or changes in cell adherence or morphology are measured14. Furthermore, altered cell viability also leads to impedance changes, rendering the technology a useful tool to monitor infection with cytopathogenic viruses15,16,17. As morphological changes of the cell layer will be detected at the nanoscale range, the technology offers a highly sensitive detection tool. The CEI assay described in this article is an easy, noninvasive, real-time, label-free method to measure the impedance changes of the cell layer over time.
Although CEI has not been used to evaluate the course of ZIKV infection or its potential antivirals, its use as a diagnostic tool has been investigated before18. In a recent study, the use of the CEI assay to determine the antiviral activity of several ZIKV inhibitors in A549 cells was validated for the first time19. This methods article describes this CEI assay in more detail and expands it to various adherent cell lines, as well as a variety of ZIKV strains at various multiplicities of infection (MOI). Hereby, the versatile use of this method in flavivirus antiviral research is demonstrated. The method has the crucial benefit of real-time cell monitoring, which allows the detection of important infection time points and the dynamic inhibiting activity by potential antiviral compounds. Taken together, the CEI technology offers a powerful and valuable tool to complement the current range of antiviral methodologies.

<table><tbody><tr><td>A549 cells</td><td>American Type Culture Collection (ATCC)</td><td>CCL-185</td><td>These cells are cultured in MEM Rega-3 supplemented with 10% FBS, 2 mM L-glutamine, 0.075% sodium bicarbonate.</td></tr><tr><td>Acridine Orange/Propidium Iodide Stain</td><td>Logos Biosystems</td><td>F23001</td><td>Live/dead stain</td></tr><tr><td>Cellstar polypropylene tubes, 15 mL</td><td>Greiner bio-one</td><td>1,88,271</td><td></td></tr><tr><td>Cellstar polypropylene tubes, 50 mL</td><td>Greiner bio-one</td><td>2,27,261</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Essential Medium (DMEM)</td><td>Gibco, Thermo Fisher Scientific</td><td>41965-039</td><td>Cell culture medium for U87 and HEL 299 cells</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Gibco, Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>ECIS cultureware 96W20idf</td><td>Applied BioPhysics</td><td>96W20idf PET</td><td>Assay plate for CEI device</td></tr><tr><td>Electric cell-substrate impedance sensing (ECIS) Z array station</td><td>Applied BioPhysics</td><td></td><td>CEI device, including 96 well station, external control module and laptop with control, acquisition and display software. The necessary software is installed before shipment. NOTE: this device is currently out of production and has been replaced by the more advanced ECIS Z-Theta device</td></tr><tr><td>Falcon polystyrene tubes, 5 mL</td><td>Corning</td><td>352054</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Cytiva</td><td>SV30160.03</td><td>Cell culture medium additive for all used cells</td></tr><tr><td>HEL 299 cells</td><td>ATCC</td><td>CCL-137</td><td>These cells are cultured in DMEM supplemented with 10% FBS and 0.01 M HEPES.</td></tr><tr><td>HEPES buffer, 1 M</td><td>Gibco, Thermo Fisher Scientific</td><td>15630-056</td><td>Cell culture medium additive for U87 cells</td></tr><tr><td>Labyrinthopeptin A1</td><td>Kind gift from Prof. Dr. Roderich S&amp;uuml;ssmuth, Technische Universit&amp;auml;t Berlin, Germany</td><td></td><td>Antiviral compound used as an example in this protocol. It is dissolved in DMSO.</td></tr><tr><td>L-Glutamine, 200 mM</td><td>Gibco, Thermo Fisher Scientific</td><td>25030-024</td><td>Cell culture medium additive for A549 cells</td></tr><tr><td>Luna cell counting slides</td><td>Logos Biosystems</td><td>L12001</td><td></td></tr><tr><td>Luna-FL dual fluoresence cell counter</td><td>Logos Biosystems</td><td>L20001</td><td>Cell viability counter</td></tr><tr><td>Minimum Essential Medium Rega-3 (MEM Rega-3)</td><td>Gibco, Thermo Fisher Scientific</td><td>19993013</td><td>Cell culture medium for A549 cells</td></tr><tr><td>Sodium Bicarbonate, 7.5%</td><td>Gibco, Thermo Fisher Scientific</td><td>25080-060</td><td>Cell culture medium additive for A549 cells</td></tr><tr><td>Tissue culture flask T75</td><td>TPP</td><td>Y9076</td><td></td></tr><tr><td>Trypsin-EDTA, 0.25%</td><td>Gibco, Thermo Fisher Scientific</td><td>25200-056</td><td>Dissociation reagent</td></tr><tr><td>U87 cells</td><td>ATCC</td><td>HTB-14</td><td>These cells are cultured in DMEM supplemented with 10% FBS and 0.01 M HEPES.</td></tr><tr><td>&amp;nbsp;Virkon Rely+On tablets</td><td>Lanxess</td><td>115-0020</td><td>Disinfectant sollution</td></tr><tr><td>Zika virus (ZIKV) MR766</td><td>ATCC</td><td>VR-84</td><td>ZIKV prototype strain, isolated from sentinel Rhesus monkey in Uganda in 1968</td></tr><tr><td>ZIKV PRVABC59</td><td>ATCC</td><td>VR-1843</td><td>ZIKV strain isolated from patient in Puerto Rico in 2015</td></tr></tbody></table>

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.

In this article, the use of the CEI assay in ZIKV research is described in detail. The workflow of this assay is illustrated in Figure 1. We demonstrate the convenience of real-time monitoring of ZIKV infection in a desired cell type, as well as the evaluation of infection inhibition by an antiviral compound. As an antiviral example, we use the well-described peptide labyrinthopeptin A1 (Laby A1); we refer to this as &#39;the compound&#39;, since its specific properties are beyond the scope of this methods article and are discussed elsewhere20,21. Of note, the reproducibility of the method is not discussed in the results section, since we want to focus on the individual impedance profiles obtained using the methodology. However, this has been described previously19.
In the first set of experiments, we demonstrate the importance of cell density optimization when performing CEI infection assays (Figure 2). When too many cells are seeded, the cell monolayer will have fully grown and be unable to further spread across the CEI electrodes. This leads to a smaller difference between CC and VC, and thus a lower resolution, decreasing the detection window of antiviral activity. This is well exemplified in Figure 2E. For certain larger cell types, such as U87 cells, seeding cells too densely might even lead to complete detachment of the cell monolayer when manipulating the plate, as demonstrated here (Figure 2F). This is also observed microscopically.
Figure 2&#160;also demonstrates that the assay is very useful for performing cell susceptibility studies. It is clear from Figure 2A,B that the infection kinetics are highly dependent on the cell type. Figure 2C demonstrates that U87 cells are only susceptible to ZIKV infection at high MOIs.
In the second set of experiments, the versatile use of the CEI infection assay is highlighted using various ZIKV strains at different MOIs and in the presence of a well-described antiviral compound (Figure 3). These experiments are performed with A549 cells, a model commonly used in flavivirus research22,23. This cell line has also been used in other studies that have demonstrated its suitability in CEI assays24. First, infection kinetics by a representative ZIKV strain of the African lineage MR766, are compared to those of the Asian lineage PRVABC59. Figure 3A demonstrates that both strains have comparable CEI patterns, with PRVABC59 having slightly slower CPE-inducing properties. This is also reflected by the CIT50 values (Figure 3B). This interesting parameter was first introduced by Fang et al.16 and takes into account the kinetics of CEI measurements. It is defined as the time needed to reduce impedance by 50% as compared to the cell control.
Next, the cells were infected with three different MOIs of either ZIKV MR766 or PRVABC59, in the presence or absence of various compound concentrations, and the impedance profiles were monitored. As can be observed from Figure 3C-H, certain compound concentrations inhibit or delay the impedance drop caused by ZIKV infection. This is also reflected when the AUC values are calculated. The CEI assay results also demonstrate visually that the antiviral activity depends on the MOI and on the time point of potency evaluation. AUCn calculations can be used to determine IC50 values, as demonstrated in Figure 3I. Finally, Figure 3H shows that CIT50 values can also be used to determine and compare compound potencies. Inactive compounds or compound concentrations are characterized by CEI profiles, AUC, and CIT50 values comparable to those of VC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65149/65149fig01.jpg" /> 
Figure 1: Schematic overview of the assay&#39;s workflow. The timeline and different handling and incubation steps of the CEI assay are depicted here. Abbreviations: CEI = cell-based electrical impedance; ZIKV = Zika virus; D = days. <a href="https://www.jove.com/files/ftp_upload/65149/65149fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65149/65149fig02.jpg" /> 
Figure 2: Normalized CEI profiles of ZIKV-infected cells at different densities. (A,D) A549, (B,E) HEL 299, or (C,F) U87 cells were seeded at (A-C) 15,000-20,000 (&#34;optimal&#34;) or (D-F) 75,000 (&#34;suboptimal&#34;) cells per well in a 96-well CEI plate. After 24 h of impedance monitoring, the cells were infected with tenfold MOI dilutions of ZIKV MR766. Impedance was further monitored for 5 consecutive days. CEI profiles (mean &#177; range of two technical replicates) of a representative experiment are shown. Abbreviations: CEI = cell-based electrical impedance; MOI = multiplicity of infection; ZIKV = Zika virus; Norm = normalized. <a href="https://www.jove.com/files/ftp_upload/65149/65149fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65149/65149fig03.jpg" /> 
Figure 3: Normalized CEI profiles of A549 cells after infection with two ZIKV strains and inhibition by an antiviral compound. (A) Adherent A549 cells were infected with various MOIs of either ZIKV MR766 or ZIKV PRVABC59, and impedance was monitored continuously. Mean &#177; SD of three technical replicates of a representative experiment is shown. (B) CIT50 values were calculated based on the CEI profile of A and compared. Mean &#177; SD of three technical replicates performed is shown. (C-H) Adherent A549 cells were treated with various compound concentrations and subsequently infected with a specific MOI of either ZIKV MR766 (C-E) or ZIKV PRVABC59 (F-H). Impedance was continuously monitored. Mean &#177; range of two technical replicates of a representative experiment is shown. (I) To compare compound inhibition against different ZIKV strains and dilutions, the AUCn was calculated, and inhibitory percentages were determined by subtracting all conditions by the CC AUCn and dividing by the VC AUCn. (J) The CIT50 values of the experiment shown in C-H were calculated to compare the different ZIKV strains and MOI. Mean &#177; range of two technical replicates is shown. Abbreviations: CEI = cell-based electrical impedance; MOI = multiplicity of infection; ZIKV = Zika virus; Norm = normalized; CIT50 = time at which impedance decreased by 50% relative to untreated control;&#160;AUC = area under the curve; CC = cell control; VC = virus control. <a href="https://www.jove.com/files/ftp_upload/65149/65149fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell-Based Electrical Impedance Platform to Evaluate Zika Virus Inhibitors in Real Time at JoVE.com

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

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.

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

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Immunology and Infection

765 Views.  KU Leuven. Virale infecties zijn zeer dynamisch en traditionele eindpunttests in de virologie hebben een beperking dat een gekozen eindpunt kan leiden tot gemiste informatie. Het belangrijkste voordeel van deze techniek is dat het met celgebaseerde elektrische impedantie mogelijk is om een virale infectie en de activiteit van antivirale verbindingen in realtime te volgen. Eef Meyen, een laborant van ons laboratorium, demonstreert deze procedure.Om te beginnen bereidt u de CEI-plaat voor door een ...

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

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

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.

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

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

Monitoring Changes in Human Umbilical Vein Endothelial Cells upon Viral Infection Using Impedance-Based Real-Time Cell Analysis

Endothelial cells line the inner surface of all blood and lymphatic vessels, creating a semi-permeable barrier regulating fluid and solute exchange between blood or lymph and their surrounding tissues. The ability of a virus to cross the endothelial barrier is an important mechanism that facilitates virus dissemination in the human body. Many viruses are reported to alter endothelial permeability and/or cause endothelial cell barrier disruption during infection, which is able to cause vascular leakage. The current study describes a real-time cell analysis (RTCA) protocol, using a commercial real-time cell analyzer to monitor endothelial integrity and permeability changes during Zika virus (ZIKV) infection of the human umbilical vein endothelial cells (HUVECs). The impedance signals recorded before and after ZIKV infection were translated to cell index (CI) values and analyzed. The RTCA protocol allows the detection of transient effects in the form of cell morphological changes during a viral infection. This assay could also be useful for studying changes in the vascular integrity of HUVECs in other experimental setups.

The current study describes a protocol to monitor the changes in human umbilical vein endothelial cells (HUVECs) during viral infection using a real-time cell analysis (RTCA) system.

Endothelial cells line the inner surface of all blood and lymphatic vessels, creating a semi-permeable barrier regulating fluid and solute exchange ...

Biology

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

Source: Contreras, D. et al., <a href="https://app.jove.com/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.

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 ...

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Immunology

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Immunogenics and Vaccine Development

Cellular Impedance-Based Analysis for Monitoring of Cellular Processes in Real-Time

Source: Labadie, T., et al., <a href="https://app.jove.com/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the process of real-time monitoring of cellular events, including the proliferation of adherent cells, via a cellular impedance assay. Here, the adherent cells act as insulators, restricting the current flow between the electrodes and increasing the impedance.

Source: Labadie, T., et al., Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis. J. Vis. Exp. (2021)
In this video, we ...

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Cell-based assays

Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival

Source: Labadie, T. et al., <a href="https://app.jove.com/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021).
This video describes a cellular impedance-based technique to study the survival kinetics of viruses that induce cytopathic effects. The method measures the electrical impedance of virus-infected adherent cells pre-treated with saline.

Source: Labadie, T. et al., Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis. J. Vis. Exp. (2021).
This video describes ...

An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection

Source: Valiant, W. G., et al. <a href="https://app.jove.com/v/57371">A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to measure antibody-dependent enhancement (ADE) of Dengue virus infection using Zika Virus Convalescent Serum. The assay uses Dengue reporter viral particles (RVPs) encoding a green fluorescent protein. Serum antibodies against the Zika Virus cross-react with RVPs to form a complex. The bound antibodies enhance RVP uptake by cells expressing the Fc receptor, which is detected using flow cytometry.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Day 1
NOTE: Perform all the steps ...

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

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