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 Video 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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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. 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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 Video at JoVE.com

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis Video (Video) | JoVE

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.

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 ...

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