Name: monitoring influenza virus survival outside the host using real-time cell analysis
Uploaded: 2021-02-20T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis at JoVE.com

Handle all influenza viruses according to appropriate biosafety level requirements (BSL-2 or higher depending on the subtype). Use IAV strains with a low passage history (less than 5x on MDCK cells) to insure low variation between experiments.
1. Preparation of reagents and starting materials
<ol>
	<li>Preparation of MDCK cells and sterile cell culture medium
	<ol>
		<li>Cultivate Madin-Darby Canine Kidney (MDCK) cells in modified Eagle&#8217;s medium (MEM) supplemented with 10% heat inactiv...

<ol>
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D., Swayne, D. E., Cooper, R. J., Burns, R. E., Stallknecht, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+H5+and+H7+avian+influenza+viruses+in+water.">Persistence of H5 and H7 avian influenza viruses in water.</a> Avian Diseases. 51, 285-289 (2007).</li><li>Dublineau, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+the+2009+pandemic+influenza+A+(H1N1)+virus+in+water+and+on+non-porous+surface.">Persistence of the 2009 pandemic influenza A (H1N1) virus in water and on non-porous surface.</a> PLoS ONE. 6 (11), 28043 (2011).</li><li>Titration of Influenza Viruses. Springer Nature Experiments Available from: <a target="_blank" href="https://experiments.springernature.com/articles/10.1007/978-1-4939-8678-1_4">https://experiments.springernature.com/articles/10.1007/978-1-4939-8678-1_4</a> (2020)</li><li>Szretter, K. J., Balish, A. L., Katz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza:+Propagation,+Quantification,+and+Storage.">Influenza: Propagation, Quantification, and Storage.</a> Current Protocols in Microbiology. 3 (1), 1-22 (2006).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+Plaque+Assay+of+Influenza+Virus+in+an+Established+Line+of+Canine+Kidney+Cells.">Replication and Plaque Assay of Influenza Virus in an Established Line of Canine Kidney Cells.</a> Applied Microbiology. 16 (4), 588-594 (1968).</li><li>Witkowski, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+impedance+measurement+as+a+new+tool+for+poxvirus+titration,+antibody+neutralization+testing+and+evaluation+of+antiviral+substances.">Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances.</a> Biochemical and Biophysical Research Communications. 401 (1), 37-41 (2010).</li><li>Lebourgeois, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Real-Time+Cell+Analysis+(RTCA)+Method+as+a+Fast+and+Accurate+Method+for+Detecting+Infectious+Particles+of+the+Adapted+Strain+of+Hepatitis+A+Virus.">Development of a Real-Time Cell Analysis (RTCA) Method as a Fast and Accurate Method for Detecting Infectious Particles of the Adapted Strain of Hepatitis A Virus.</a> Frontiers in Cellular and Infection Microbiology. 8, 335 (2018).</li><li>Teng, Z., Kuang, X., Wang, J., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+cell+analysis--a+new+method+for+dynamic,+quantitative+measurement+of+infectious+viruses+and+antiserum+neutralizing+activity.">Real-time cell analysis--a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.</a> Journal of Virological Methods. 193 (2), 364-370 (2013).</li><li>Fang, Y., Ye, P., Wang, X., Xu, X., Reisen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+flavivirus+induced+cytopathogenesis+using+cell+electric+impedance+technology.">Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology.</a> Journal of Virological Methods. 173 (2), 251-258 (2011).</li><li>Charretier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+real-time+cell+analysis+method+for+determining+viral+infectious+titers+during+development+of+a+viral+vaccine+production+process.">Robust real-time cell analysis method for determining viral infectious titers during development of a viral vaccine production process.</a> Journal of Virological Methods. 252, 57-64 (2018).</li><li>Tian, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Cell+Analysis+for+Measuring+Viral+Cytopathogenesis+and+the+Efficacy+of+Neutralizing+Antibodies+to+the+2009+Influenza+A+(H1N1)+Virus.">Real-Time Cell Analysis for Measuring Viral Cytopathogenesis and the Efficacy of Neutralizing Antibodies to the 2009 Influenza A (H1N1) Virus.</a> PLoS ONE. 7 (2), 31965 (2012).</li><li>Teng, Z., Kuang, X., Wang, J., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+cell+analysis--a+new+method+for+dynamic,+quantitative+measurement+of+infectious+viruses+and+antiserum+neutralizing+activity.">Real-time cell analysis--a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.</a> Journal of Virological Methods. 193 (2), 364-370 (2013).</li><li>Thieulent, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+evaluation+of+antiviral+compounds+against+Equid+alpha-herpesviruses+using+an+impedance-based+cellular+assay.">Screening and evaluation of antiviral compounds against Equid alpha-herpesviruses using an impedance-based cellular assay.</a> Virology. 526, 105-116 (2019).</li><li>Labadie, T., Bat&#233;jat, C., Manuguerra, J. -. C., Leclercq, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+Virus+Segment+Composition+Influences+Viral+Stability+in+the+Environment.">Influenza Virus Segment Composition Influences Viral Stability in the Environment.</a> Frontiers in Microbiology. 9, 1496 (2018).</li><li>Wiley, D. C., Skehel, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+function+of+the+hemagglutinin+membrane+glycoprotein+of+influenza+virus.">The structure and function of the hemagglutinin membrane glycoprotein of influenza virus.</a> Annual Review of Biochemistry. 56, 365-394 (1987).</li></ol>

RTCA is an impedance-based technology that is increasingly used for real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. In this study, the capacity of this technology to assess IAV survival outside the host is demonstrated by measuring virus inactivation slope. Fastidious techniques such as TCID50 and plaque assays are replaced by objective real-time assessment of cell viability, thus reflecting cytopathic effects induced by the virus. Similar to the TCID...

The transmission of a virus relies on the combination of several factors. For a virus secreted in the environment, its transmission also depends on the ability to persist in conditions outside of the host. Studying viral inactivation in general is, therefore, a crucial step in helping national health authorities and policy makers implement control and biosafety measures.
Knowledge about virus persistence in natural and laboratory settings has increased considerably over the last decade. In the case of influenza A viruses (IAV), their transmission routes submit viral particles to a wide range of environmental conditions. Specifically, they can be transmitted via 1) fecal-oral routes through water (i.e., avian viruses), or 2) direct or indirect contact by contaminated fomites, as well as aerosols and respiratory droplets (i.e., poultry and mammalian viruses)1. In any case, IAV are submitted to various physicochemical parameters (i.e., pH, salinity, temperature, and humidity), which more or less rapidly affects their infectivity2,3,4,5,6,7,8,9. It is of great importance, especially regarding zoonotic and pandemic viruses, to assess the potential of environmental factors to affect virus dynamics and the risks of exposure and cross-species transmission.
So far, traditional virology techniques (i.e., viral titer determination through plaque assays or 50% tissue culture infectious dose estimation) have been used to assess IAV infectivity over time; but these techniques are time-consuming and require many supplies10,11,12. Measuring infected cells impedance over time with microelectrodes serves as a useful tool to monitor IAV survival in different environmental conditions, as well as viral inactivation in general. This method provides objective, real-time data that replaces subjective human observation of cytopathic effects. It can be used to determine virus titration, thus replacing traditional measurements with lower confidence intervals and avoiding labor-intensive endpoints assays.
A linear correlation exists between titration results obtained by measurement of cell impedance and by classical plaque assay or TCID50 methods. Therefore, data obtained with the impedance-based titration method can be easily transformed in TCID50 or pfu values by creating a standard curve with serial dilution of the virus13,14,15,16,17. Detection, quantification, and efficacy of neutralizing antibodies present in serum samples can also be achieved using this experimental approach18,19. More recently, impedance-based cellular assays have been used to screen and evaluate antiviral compounds against Equid alphaherpesviruses20.
This technology has been used to evaluate the persistence of IAVs in saline water at different temperatures and to identify mutations in the hemagglutinin of IAV that increase or decrease IAV persistence in the environment21. Such screening would require extensive work if using traditional titration methods. However, this methodology can be used for any virus that has an impact on cell morphology, cell number, and cell surface attachment strength. It can also be used to monitor persistence in various environmental conditions (i.e., in the air, in water, or on surfaces).
The protocol described here applies IAV survival in water as an example. Human influenza viruses are exposed to different physicochemical parameters during extended periods. Saline (35 g/L NaCl) water at 35 &#176;C was chosen as the environmental model based on previous results9. Residual infectivity of exposed viruses is quantified at different timepoints through cell infection. MDCK cells, the reference cell type for IAV amplification, are seeded on 16 well microtiter plates coated with microelectrode sensors and infected by exposed viruses 24 h later. Cell impedance is measured every 15 min and expressed as an arbitrary unit called the cell index (CI). Cytopathic effects induced by the influenza virus, whose rate of onset directly depends on the number of infectious viral particles inoculated to the cells culture, leads to CI decrease, which is subsequently quantified as the CIT50 value. This value corresponds to the time necessary to measure a 50% reduction from the initial CI (i.e., before virus addition). CIT50 values calculated for several environmental exposure times allow for deduction of the inactivation slope of a virus after linear regression of CIT50 values.

<table>
	<tbody>
		<tr>
			<td>0.25%Trypsin</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flask</td>
			<td>Falcon</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Plate 16 (6 plates)</td>
			<td>ACEA Biosciences, Inc</td>
			<td>5469830001</td>
			<td>E-plates are avalible in different packaging</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life technologies (gibco)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM 1X</td>
			<td>Life technologies (gibco)</td>
			<td>31095029</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Life technologies (gibco)</td>
			<td>14040091</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life technologies (gibco)</td>
			<td>11548876</td>
			<td></td>
		</tr>
		<tr>
			<td>TPCK-Trypsin</td>
			<td>Worthington</td>
			<td>LS003740</td>
			<td></td>
		</tr>
		<tr>
			<td>xCELLigence Real-Time Cell Analysis Instrument S16</td>
			<td>ACEA Biosciences, Inc</td>
			<td>380601310</td>
			<td>The xCELLigence RTCA S16 instruments are available in different formats (16-well, 96-well, single or multi-plate)</td>
		</tr>
	</tbody>
</table>

monitoring influenza virus survival outside the host using real-time cell analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Raw data obtained after 120 h with different concentrations of MDCK cells, from 15,000 to 120,000 cells per well, is shown in Figure 1. After 24 h, CI measures show that cells in wells seeded with 30,000 cells were still in the exponential phase of growth, and this cell concentration was used for further experiments. Figure 2 illustrates the linear correlation between CIT50 values and the initial multiplicity of infection. MDCK cells are cultured for ...

Watch this Scientific Journal Video about Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis at JoVE.com

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Methods for various particle quantification represent a critical aspect of most biology studies. This protocol allows a precise quantification of the viral data in real time. This technique replaces the traditional measurement of viral data.By objective realtime data, we are below our confidence interval, avoiding labor-intensive endpoints assessed. This method can be used for all viruses that induce a cytopathic effect. Demonstrating the procedure will be Quentin Grassin, a laboratory technician from our laboratory.To propagate and amplify H1N1 viruses, seed 7.5 times 10 to the 6 MDCK cells on two 75-square-centimeter tissue culture flasks according to standard protocols, and incubate the cells for 24 hours at 37 degrees Celsius to reach 90 to 100%confluence. The next day, wash the cells two times with five milliliters of sterile PBS per flask per wash, and label one flask as the control. Next, dilute a thawed vial of influenza A virus stock to the appropriate experimental concentration in a 1.5-milliliter tube containing fresh virus propagation medium.Use one milliliter of the diluted stock to infect the MDCK cells at a multiplicity of infection of 1 times 10 to the negative 3, or 1 times 10 to the negative 4 plaque-forming units per cell, and add one milliliter of virus propagation medium to the control flask. Adsorb the virus to the cells for 45 minutes at room temperature, with stirring every 15 minutes. At the end of the incubation, remove the inoculum and replace it with 15 milliliters of virus propagation medium supplemented with one microgram per milliliter of TPCK trypsin to facilitate cleavage of viral hemagglutinin HA0 into HA1 and HA2 subunits.Then place the flask in the 35-degree-Celsius incubator for three days. At the end of the incubation, compare the cells in each culture under a 40x objective on a light microscope to assess the cytopathic effects on the cells. When the cytopathic effect is complete, add the cell culture supernatants to a 15-millimeter tube and collect the cellular debris from each culture by centrifugation.Then, transfer the clarified virus culture supernatant to a 15-milliliter tube, and aliquot the progeny viruses to single-use sterile cryogenic vials for minus 80 degrees Celsius storage. To determine the appropriate cell quantity for cell infection, prepare 24-hour approximately 80%confluent MDCK cell cultures as demonstrated before washing the cells with PBS and harvesting them with a 45-minute incubation in three milliliters of 0.25%Trypsin-EDTA per flask at 37 degrees Celsius. When the cells have detached, stop the reaction with seven milliliters of fresh culture medium per flask, and count the cells from each culture.Dilute the cells to 4 times 10 to the 5th cells per milliliter of cell culture medium and perform twofold serial dilutions of the cells as indicated. Place an E-plate at room temperature for several minutes before adding 100 microliters of cell culture medium to each well without touching the electrodes of the E-plate. Unlock the cradles and insert the plate front end into the cradle pocket of the impedance measuring instrument.Close the door of the incubator and open the software. In the Default Experiment Pattern Setup, highlight the selected cradles and double-click on the top page to enter the name of the experiment. Click Layout and enter the necessary sample information for each selected well of the plate.Click Schedule, Steps and Add a Step. The software will automatically add a one-second step to measure the background impedance Click Execute and Start, Continue. Click Plot and Add to select the appropriate wells, confirming that the background impedance is between negative 0.1 and 0.1 before proceeding to the next step.Next, remove the plate from the cradle and add 100 microliters of each cell suspension to the appropriate wells in duplicate. Leave the E-plate in the laminar flow hood for 30 minutes at room temperature to allow uniform distribution of the cells onto the bottoms of the wells before loading the E-plate into the cradle pocket. Click Schedule and Add Step and enter values to monitor the cells every 30 minutes for 200 repetitions before selecting Start, Continue.To check and plot the cell impedance data, click Plot and select the concentration of cells that is just before the stationary phase 24 hours after seeding to obtain cells that are still in a growing phase during the viral infection. To determine the correlation between the CIT50 values and the multiplicity of infection, after culturing 3 times 10 to the 4th freshly-split MDCK cells into each well of the electronic microtiter plate for 24 hours, wash the cells two times with 100 microliters of fresh virus propagation medium per well, per wash, and use a single-channel pipette to add 100 microliters of viral suspension to each well. When all of the virus dilutions have been added, gently load the plate into the cradle pocket of the instrument at 35 degree Celsius, and begin monitoring the cell impedance every 15 minutes for at least 100 hours, as demonstrated.After two cycles of measurements, click to pause the apparatus, and remove the E-plate from the cradle. Add 100 microliters of virus propagation medium supplemented with TPCK trypsin to each well and return the E-plate into the cradle pocket. Then, start the analysis.To assess the influenza A virus survival kinetics, add sodium chloride to a final concentration of 35 grams per liter in distilled water, and add 900 microliters of the resulting saline solution to two-milliliter cryo-tubes. Add 100 microliters viral stock to each cryo-tube and place the tubes in the 35-degree-Celsius incubator for the appropriate experimental time period. The day before the end of the incubation, seed 3 times 10 to the 4th freshly split MDCK cells and 100 microliters of virus propagation medium per well in a 16-well microtiter plate in repeat steps, allowing measurement of the background impedance and uniform distribution of the cells onto the bottom of the wells.Then grow the cells for 24 hours at 37 degree Celsius and 5%carbon dioxide. The next day, wash the cells twice, then infect the cells with 100 microliters of the saline-distilled virus diluted 10 times in fresh culture medium, as demonstrated, and monitor the cell impedance every 15 minutes for at least 100 hours. Here, raw data obtained after 120 hours with different concentrations of MDCK cells are shown.After 24 hours, cell index measurements revealed that cells in wells seeded with 3 times 10 to the 4th cells were still in the exponential phase of growth, therefore, this cell concentration was used for subsequent experiments. MDCK cell cultures demonstrate a clear linear relationship between the CIT50 and the initial multiplicity of infection by influenza virus. The results from a typical survival kinetic experiment show a decrease in the cell index due to virus-induced cytopathic effect.The CIT50 can be used to calculate the viral inactivation slope for each virus in each condition for the determination of which virus had the greatest stability in the studied environment. The stability of the virus indirectly correlates to the inactivation slope, allowing, for example, amino acid residues in the hemagglutinin glycoprotein that are involved in influenza A virus survival outside the host to be identified. This technique is very sensitive to small variations.Therefore, using an automatic cell counter is encouraged in order to obtain reproducible results between experiments. This method can also be used to compare the replication of different viruses, to investigate the virus tropism for several cell lines at a time, or to study specific steps of the virus cycle.

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