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 inactivated fetal calf serum (FCS) and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin).</li>
		<li>After thawing, passage MDCK cells at least 2x before infecting them to ensure complete recovery (but less than 30 passages to avoid any drift in cell phenotypes).</li>
		<li>Seed 75 cm2 tissue culture flask(s) containing 30 mL of sterile 1x MEM with 7.5 x 106 MDCK cells and incubate at 37 &#176;C in humidified 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>Preparation of impedance monitoring equipment
	<ol>
		<li>Place the instrument in the incubator at 35 &#176;C and allow it to warm up for at least 2 h.</li>
		<li>Connect it to the control unit placed outside the incubator.</li>
		<li>Proceed to cleaning procedures as recommended by the manufacturer before starting the remainder of the experiment.</li>
	</ol>
	</li>
	<li>Production of IAV stocks on MDCK cells
	<ol>
		<li>To propagate and amplify H1N1 viruses, seed 7.5 x 106 MDCK cells on two 75 cm2 tissue culture flasks and incubate for 24 h at 37 &#176;C to reach 90%&#8211;100% confluence.</li>
		<li>Decant the cell culture medium from the cell monolayer in 75 cm2 flasks. Wash the cells with 5 mL of sterile 1x PBS.</li>
		<li>Remove PBS and add 5 mL of 1x PBS to wash the cells again.</li>
		<li>Label one flask as the control and remove PBS before adding 15 mL of virus propagation media (1x MEM with 0% FCS) carefully on the monolayer. Incubate this flask in an incubator maintained at 35 &#176;C with 5% CO2. Use it for comparison after 3 days of propagation.</li>
		<li>Thaw one vial of IAV stock at RT. Dilute the virus to the appropriate concentration in a 1.5 mL tube containing virus propagation media (1x MEM with 0% FCS).</li>
		<li>Remove 1x PBS from the 75 cm2 flask and infect MDCK cells at a multiplicity of infection (MOI) of 1 x 10-3 or 1 x 10-4 plaque forming units per cell (pfu/cell) by adding 1 mL of the diluted virus to the cell monolayer.</li>
		<li>Adsorb virus to MDCK cells for 45 min at RT by stirring the flask regularly every 15 min.</li>
		<li>Gently remove the inoculum and add 15 mL of virus propagation media per flask containing 1 &#181;g/mL TPCK-trypsin (trypsin/L-1-tosylamide-2-phenylethyl chloromethyl ketone), to cleave the viral hemagglutinin HA0 into HA1 and HA2 subunits (an event that is required for HA fusion with the endosomal membrane and release of the viral genome)22.</li>
		<li>Incubate the flasks at 35 &#176;C and 5% CO2 for at least 3 days to replicate the virus.</li>
		<li>Observe MDCK cells under a microscope at 40x magnification and look for cytopathic effects (CPE) on the cells (by comparing to the cell control flask). If CPE is not complete (i.e., around 80% of the cells are detached from the substrate), put the flasks back in the incubator for an additional 24 h.</li>
		<li>When CPE is complete, decant the cell culture supernatant and centrifuge at 300 x g for 10 min to pellet the cellular debris.</li>
		<li>Transfer the clarified supernatant to a 15 mL tube and aliquot progeny viruses to single-use sterile cryogenic vials. Immediately place the cryotubes at -80 &#176;C to freeze and stock viruses.</li>
	</ol>
	</li>
</ol>
2. Determination of appropriate cell quantity for infecting cells
NOTE: During all experiments, keep the plates on non-electrostatic surfaces at all times, such as the paper wraps from the packaging. Follow section 2 below to determine the most appropriate concentration of cells to be seeded in the electronic microtiter plate (designed as E-Plate).
<ol>
	<li>Prepare MDCK cells in 75 cm2 flasks to obtain freshly split cells (approximately 80% confluence) 24 h before the experiment.</li>
	<li>Wash cells with 5 mL of 1x PBS and detach them by adding 3 mL of 0.25% Trypsin-EDTA solution.</li>
	<li>Add 7 mL of fresh cell culture medium and count cells using an automated cell counter with trypan blue staining.</li>
	<li>Adjust the cell concentration to 400,000 cells/mL with cell culture media. Perform two-fold serial dilutions in additional tubes to obtain cell densities of 200,000; 100,000; 50,000; 25,000; 12,500; and 6,250 cells/mL. Adjust the dilution range of the cells according to the cell type and their growth behavior.</li>
	<li>Leave the E-plate (Table of Materials) at RT for several minutes and add 100 &#181;L of cell culture media to each well using a multi-channel pipette. Do not touch the electrodes of the E-Plate.</li>
	<li>Unlock the cradles and insert the plate front end into the cradle pocket of the impedance measuring instrument (Table of Materials). Close the door of the incubator.</li>
	<li>Open the software.
	<ol>
		<li>In &#8220;Default experiment pattern setup&#8221;, choose the selected cradle(s) and double-click on the top page, then enter the name of the experiment. Click &#8220;Layout&#8221; and enter the necessary sample information for each selected well of the plate; then, click &#8220;Apply&#8221; when finished. Click &#8220;Schedule&#8221; | &#8220;Steps&#8221; | &#8220;Add a step&#8221;. The software automatically adds a step of 1 s to measure the background impedance (CI).</li>
		<li>Click on &#8220;Start/Continue&#8221; in the &#8220;Execute&#8221; tab. Click on &#8220;Plot&#8221;, add all samples by selecting the appropriated wells, and ensure CI is between -0.1 and 0.1 before proceeding to the next step.</li>
	</ol>
	</li>
	<li>Remove the plate from the cradle.</li>
	<li>Add 100 &#181;L of each cell suspension from step 2.4 in duplicate to the appropriate wells and 100 &#181;L of cell media in wells used as controls. Leave the E-plate in the laminar flow hood for 30 min at RT to allow for uniform distribution of the cells at the bottoms of the wells.</li>
	<li>Insert the E-plate into the cradle pocket. Click &#8220;Schedule&#8221; | &#8220;Add step&#8221; in the software and enter values to monitor cells every 30 min for 200 repetitions. Then, select &#8220;Start/Continue&#8221;.</li>
	<li>Check and plot the CI data by clicking on the &#8220;Plot&#8221; button in the software. Select the concentration of cells that are just before the stationary phase 24 h after seeding on the plate, in order to obtain cells that are still in a growing phase during viral infection. Stationary phase is reached when CI is at its maximum.</li>
</ol>
3. Correlation between CIT50 values and multiplicity of infection
<ol>
	<li>Add 100 &#181;L of sterile 1x MEM culture medium in each well of the E-plate. Insert E-plate into the cradle pocket of the instrument at 35 &#176;C. Measure the background as described in step 2.7.</li>
	<li>Remove the E-plate from the cradle.</li>
	<li>Seed 3 x 104 of freshly split MDCK cells on each well of the electronic microtiter plate and grow them for 24 h at 35 &#176;C with 5% CO2 so that they are in a replicative phase during IAV infection.</li>
	<li>Infect MDCK cells with different 10-fold dilutions of a known 6 log10 TCID50/mL titer of H1N1 virus, using reverse pipetting for reproducibility by following the steps below:
	<ol>
		<li>Rinse MDCK cells 2x with 100 &#181;L of MEM with no FCS (virus propagation media). Be aware of removing all media after the second wash to avoid further dilution of the inoculum.</li>
		<li>Add 100 &#181;L of viral suspension in each well using single channel pipette. To avoid contamination, proceed by starting from left-to-right then top-to-bottom in the plate, while covering the remaining wells with the lid.</li>
		<li>Insert the plate into the cradle pocket of the instrument at 35 &#176;C. Be gentle to avoid sudden movements potentially leading to contaminations.</li>
		<li>Start to monitor cell impedance every 15 min during at least 100 h as described in step 2.10.</li>
		<li>After the two cycles of measurements (i.e., 30 min), pause the apparatus by clicking on &#8220;Pause&#8221; in the &#8220;Execute&#8221; tab and remove the E-plate from the cradle.</li>
		<li>Add 1 &#181;g/mL TPCK-trypsin to the virus propagation media to cleave the viral hemagglutinin.</li>
		<li>Add 100 &#181;L of virus propagation media containing TPCK-trypsin into each well and insert the E-plate into the cradle pocket.</li>
		<li>Click &#8220;Start/Continue&#8221; in the &#8220;Execute&#8221; tab. 
		NOTE: Do not forget to create a negative control corresponding to mock-infected cells by replacing viral suspension with virus propagation media.</li>
	</ol>
	</li>
</ol>
4. IAV survival kinetics
<ol>
	<li>Expose IAV to saline distilled water at 35 &#176;C and test for their infectivity over time by measuring cell impedance decrease.
	<ol>
		<li>Prepare saline distilled water by adding NaCl to a final concentration of 35 g/L in distilled water. Add 900 &#181;L of saline water into 2 mL cryotubes.</li>
		<li>Add 100 &#181;L of viral stock in saline water and place the cryotubes in an incubator (35 &#176;C, 5% CO2) for 1 h, 24 h, or 48 h.</li>
		<li>Seed 100 &#181;L (containing 3 x 104) of freshly split MDCK cells on a 16 well microtiter plate and grow for 24 h at 37 &#176;C and 5% CO2.</li>
		<li>Infect cells with 100 &#181;L of exposed viruses (previously diluted 10x in culture media) using reverse pipetting method for reproducibility by repeating section 3.4.</li>
	</ol>
	</li>
	<li>Monitor cell impedance every 15 min for at least 100 h.</li>
</ol>
5. Evaluation of loss of infectivity
<ol>
	<li>Determination of CIT50 values to quantify CI decrease due to virus-induced cytopathic effects with the CIT50 value.
	<ol>
		<li>Click &#8220;Plot&#8221; and add all the samples by clicking &#8220;Add all&#8221;. Export the results to a spreadsheet by clicking on &#8220;Export Experiment Info&#8221;. Consider initial CI as cellular impedance value measured 5 h after cell infection by exposed viruses (i.e., 24 h after seeding of MDCK cells on the microtiter E-plate).</li>
		<li>Calculate CIT50 value corresponding to the necessary time to measure a 50% decrease from the initial CI. To calculate the CIT50 value, note the CI value at 5 hpi for each sample. Then, find the timepoint at which the CI value is equal to one-half the CI value at 5 hpi by using the index and match functions in the spreadsheet.</li>
	</ol>
	</li>
	<li>Calculation of the mean inactivation slope
	<ol>
		<li>Determine CIT50 values for each exposed viral suspension, at different exposure times (in days).</li>
		<li>Calculate a linear regression slope from CIT50 plotted values, referred to as the inactivation slope and expressed in CIT50.day-1.</li>
	</ol>
	</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abiotic+factors+affecting+the+persistence+of+avian+influenza+virus+in+surface+waters+of+waterfowl+habitats.">Abiotic factors affecting the persistence of avian influenza virus in surface waters of waterfowl habitats.</a> Applied and Environmental Microbiology. 80 (9), 2910-2917 (2014).</li><li>Stallknecht, D. E., Kearney, M. T., Shane, S. M., Zwank, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pH,+temperature,+and+salinity+on+persistence+of+avian+influenza+viruses+in+water.">Effects of pH, temperature, and salinity on persistence of avian influenza viruses in water.</a> Avian Diseases. 34 (2), 412-418 (1990).</li><li>Poulson, R. L., Tompkins, S. M., Berghaus, R. D., Brown, J. D., Stallknecht, D. 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The authors have nothing to disclose.

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 ...

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Immunology and Infection

2.9K Views.  London School of Hygiene and Tropical Medicine. 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.

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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...