The authors thank all the participants involved. We acknowledge the Victorian Government's Operational Infrastructure Support Program. PVL is a NHMRC Career Development Fellowship recipient.

NOTE: All steps have to be performed in a BSL2 hood unless stated differently. Viral titration is required in advance of a PRN assay to determine the optimal RSV concentration used in the PRN assay. It is recommended to aliquot the virus stocks in a small volume that will be thawed once and used for each NAb assay. Using the same viral stock for all NAb assays performed for all samples from one study is also recommended. Make sure culture media and phosphate-buffered saline (PBS) is warmed at 37 &#176;C before adding to cell plates.
1. RSV Viral Titration
NOTE: Depending on the number of virus stocks and the number of duplicates, the assay plate can be set up according to Figure 1. Each virus stock should be titrated in triplicate down the assay plate, starting at the highest viral concentration (i.e., 1:10). Serial titrations can be typically 1:10. A549 cell culture and maintenance as well as RSV culture procedure are done using standard procedures and are not included in this protocol.
<ol>
	<li>Seeding plates (Day 1) 
	<ol>
		<li>Resuspend A549 cells in Dulbecco&#8217;s Modified Eagles medium (DMEM) + 10% fetal calf serum (FCS) + 1000 IU penicillin/streptomycin (pen/strep) at 4 x 105/mL. Seed 96-well flat-bottom sterile plates with 100 &#956;L/well containing 4 x 104 A459 cells.</li>
		<li>Incubate plates overnight at 37 &#176;C, 5% CO2 (cells will be in the log-phase growth). 
		NOTE: Use new A549 cell vial after 23 passages. It is recommended to not start the experiments when a new cell vial is still at the first three passages.</li>
	</ol>
	</li>
	<li>Virus infection (Day 2) 
	<ol>
		<li>Preparation of virus serial dilution
		<ol>
			<li>Rapidly thaw a single frozen vial of RSV in a 37 &#176;C water bath until almost completely thawed and place immediately on ice. Prepare 3 replicates for each RSV virus stock to be assayed, use an initial virus stock dilution of 1:10 with 100 &#956;L of diluted virus/well and reserve at least one column for negative control. 
			NOTE: Figure 1 shows negative control in triplicates.</li>
			<li>Add 100 &#956;L of each diluted virus at 1:10 in triplicate of row A of a 96-well U-bottom sterile plate. Add 90 &#956;L/well of DMEM + 1000 IU pen/strep without FCS to rows B to H.</li>
			<li>Perform serial 1:10 dilutions down the plate by transferring 10 &#956;L from row A to row B. Mix solution by pipetting content up and down 5 times and continue the ten-fold dilutions until row H. Discard the final 10 &#956;L so that the final volume in each well is 90 &#956;L. 
			NOTE: It is important to use new tips for each dilution.</li>
		</ol>
		</li>
		<li>Virus inoculation to A549 cells
		<ol>
			<li>Retrieve A549 cell plate(s) prepared on the previous day. Ensure that A549 cell monolayers in the 96-well plates are ~80% confluent. Discard media by gently inverting plate and lightly blotting on sterile absorbent paper towel.</li>
			<li>Wash all wells with 100 &#956;L of PBS twice, discard excess PBS by gently inverting plate and lightly blotting on sterile absorbent paper towel after each wash. Do not allow plates to dry.</li>
			<li>Transfer the virus dilutions from the viral titration plate (Figure 1) to corresponding wells on the A549 cell plate. Incubate the plate for 1 h at 37 &#176;C, 5% CO2. After 1 h incubation, decant supernatants using a pipette (to avoid cross contamination). Take out 90 &#956;L/well.</li>
			<li>Add 100 &#956;L of 1x medium 199 (M199, see Table of Materials) + 1.5% carboxymethylcellulose sodium salt (CMC) low viscosity + 2% FCS containing pen/strep to each well. 
			NOTE: 2x M199 + 4% FCS + 2% pen/strep and 3% CMC solutions need to be prepared in advance and stored at 4 &#176;C for future use. Make sure the solution is warmed at 37 &#176;C before adding to plates.
			<ol>
				<li>Prepare 2x M199 solution: 1 sachet/500 mL distilled water + 20 mL FCS + 10 mL pen/strep (to make up 2x MI99). Filter the solution using a 0.22 &#956;m filter unit.</li>
				<li>For preparation of 3% CMC, add 15 g of CMC slowly into 500 mL of distilled water. Dissolve the CMC in water by using metallic stirrer heater at 50 &#176;C, which takes about 1 h of preparation. Mix well to make 3% CMC and autoclave the solution. 
				NOTE: The CMC solid must be added to the water in order to be dissolved; adding water to the dry solid produces a &#8220;clump&#8221; of solid that is very difficult to dissolve.</li>
				<li>Prepare 1x M199 + 1.5% CMC low viscosity + 2% FCS containing pen/strep by mixing 1:1 the 2x M199 and 3% CMC prepared in step 1.2.2.4.2.</li>
			</ol>
			</li>
			<li>Incubate plate for 3 days at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Developing assay plate and analysis (Day 5)
	<ol>
		<li>Fixation and developing assay plate
		<ol>
			<li>Discard M199 + CMC + 2% FCS containing pen/strep by gently inverting the plate. Add slowly (in order to avoid disturb the cell layer) 200 &#956;L of fixation buffer (80% acetone, 20% PBS, stored at -20 &#176;C) to fix cells. Incubate at -20 &#176;C for 20 min.</li>
			<li>Discard the fixation buffer and gently blot on absorbent paper towel. Leave plate face down to dry for 10 min. 
			NOTE: From this step onwards, assay can be performed outside the BSL2 hood.</li>
			<li>Prepare 5% milk diluent blocking solution in filtered PBS containing 0.05% polysorbate 20 (PBS-polysorbate, see Table of Materials). For one plate, calculate the volume needed for blocking based on 200 /well and 110 wells: 200/well x 110 wells = 22 mL (1.1 mL concentrated milk diluent in 20.9 mL filtered PBS-polysorbate). At this stage, also make up sufficient blocking solution for primary and secondary antibodies. For one plate, calculate the volume needed for each antibody solution based on 50 /well and 110 wells: 50 /well x 110 wells = 5.5 mL. Therefore, the total volume of milk diluent blocking solution required for a single plate assay is 33 mL.</li>
			<li>Add 200 /well of blocking solution. Incubate plate at room temperature (RT) for 30 min. Discard the solution by gently inverting the plate and blot on absorbent paper towel.</li>
			<li>Prepare 1:500 Goat X RSV antibody (mAb &#8211; primary antibody) diluted in blocking solution. Add 50 /well of the mAb solution and incubate at 37 &#176;C for 1 h. Wash plate 3 times with filtered PBS-polysorbate.</li>
			<li>Prepare 1:5,000 Alexa-Fluor donkey anti-goat IgG (secondary antibody) diluted in blocking solution. Add 50 /well of the secondary antibody solution and incubate at 37 &#176;C for 1 h. Wash plate 5 times with filtered PBS-polysorbate.</li>
			<li>Read plate on an automated spots reader using the fluorescein isothiocyanate (FITC) channel and count settings as shown in Figure 2. Wrap plate in foil and store at 4 &#176;C. Calibrate the instrument using an unused 96-well culture plate and inputting count settings before assay. 
			NOTE: Re-scanning within a few days is possible if required. The calibration and count setting can be saved and used for the future scanning if the assay uses the same type of plate used for the calibration.</li>
			<li>Check the images of wells for artefact plaques or disrupted cell monolayers (Figure 3). Exclude wells with disrupted cell monolayers. 
			NOTE: Depending on the size of the artefact plaques, manual counts may need to be performed for those wells or those wells may need to be discarded from data analysis. Criteria for a valid result include no disrupted cell monolayer or artefact plaques detected in more than one replicate well and no PFU detected in negative wells (wells without added virus).</li>
		</ol>
		</li>
		<li>Determine viral concentrations 
		<ol>
			<li>Choose the last two dilutions where spots can be counted clearly. Calculate the average number of spots from replicate wells at the same dilution. Calculate the viral titer (PFU per mL) of the stock sample using the formula below: 
			PFU/mL = Average number of plaques/(Dilution factor &#215; Volume of diluted virus added to the well) 
			NOTE: The virus concentration determined for each RSV stock can now be used in the PRN assay (step 2).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. RSV Neutralization Assay
<ol>
	<li>Seeding plates (Day 1)
	<ol>
		<li>Resuspend A549 cells in DMEM + 10% FCS + 1000 IU pen/strep at 4 x 105/mL. Seed 96-well flat-bottom sterile plates with 100 &#956;L/well containing 4 x 104 A459 cells and incubate plates overnight at 37 &#176;C, 5% CO2 (cells will be in the log-phase growth). 
		NOTE: Use new A549 cell vial after 23 passages. It is not recommended to use A549 cells before passage number 3.</li>
	</ol>
	</li>
	<li>Virus and serum preparation (Day 2) 
	NOTE: Depending on the sample type, there are required steps for pre-processing samples before the NAb assay. It is recommended to use the RSV international standard sera that are now available upon request from the National Institute for Biological Standards and Controls (NIBSC).
	<ol>
		<li>Preparation of serum dilution
		<ol>
			<li>Thaw the human RSV reference antiserum (reference serum, REF) and serum test samples at RT. Heat-inactivate serum samples and reference antiserum in a water bath at 56 &#176;C for 30 min prior to use. Prepare dilutions as per the plate template (Figure 4).</li>
			<li>Prepare 1:100 dilution of the REF. Prepare a minimum of 110 &#956;L of REF diluted in DMEM + pen/strep without FCS per assay plate (e.g., 1.5 &#956;L of REF in a total volume of 150 &#956;L). Add 110 &#956;L of 1:100 diluted REF in well A12 of a 96-well U-bottom sterile plate.</li>
			<li>Add 55 &#956;L of DMEM + pen/strep without FCS to wells B12 to H12 in column 12. Perform serial 1:2 dilutions down the plate by transferring 55 &#956;L from row A to row B, mixing solution by pipetting content up and down 5 times and continuing until row H. Discard the final 55 &#956;L of media so that the final volume in each well is 55 &#956;L. Use new tips for each dilution.</li>
			<li>Prepare 1:100 dilution of serum test samples. As all serum samples are assayed in triplicate, prepare a minimum volume of 110 &#956;L/well x 3 = 330 &#956;L per serum test sample.</li>
			<li>Add 110 &#956;L of each diluted serum test sample (1:100; S1 = serum 1, S2 = serum 2, etc.) to corresponding wells A1 to A9 and also E1 to E9 of the 96-well sterile plate according to Figure 4.</li>
			<li>Add 55 &#956;L of DMEM + pen/strep without FCS to all wells labelled X. Perform serial 1:2 dilutions as per step 2.2.1.3 until row D (for samples 1, 2, and 3). Discard the final 55 &#956;L of media so that the final volume in each well is 55 &#956;L. Similarly, perform the serial 1:2 dilutions for samples 4, 5, and 6 for rows E to H. 
			NOTE: It is important to use new tips for each dilution.</li>
			<li>Add 55 &#956;L of DMEM + pen/strep without FCS to wells in columns 10 and 11.</li>
		</ol>
		</li>
		<li>Preparation of virus
		<ol>
			<li>Rapidly thaw a single frozen vial of RSV in a 37 &#176;C water bath until almost completely thawed and place immediately on ice.&#8232;</li>
			<li>Dilute virus in DMEM + pen/strep without FCS based on the concentration of the RSV aliquot determined in step 1.3.2. Apply a dilution that gives approximately 200 PFU/well. Prepare a total volume of 5.5 mL (for 100 wells).</li>
		</ol>
		</li>
		<li>Preparation of virus-serum mixture
		<ol>
			<li>Add 55 of the diluted virus to all wells of columns 1-9 and wells of rows E-H of columns 10 and 11 (positive control) according to the plate template (Figure 4).</li>
			<li>Add 55 of DMEM + pen/strep without FCS to all wells of rows A-D of columns 10 and 11 (negative control). Incubate the plate for 1 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Virus inoculation to A549 cells
		<ol>
			<li>Prior to the completion of incubation period, retrieve A549 cell plate(s) prepared in step 2.1.1. Ensure that A549 cell monolayers in the 96-well plates are ~80% confluent.</li>
			<li>Discard media by gently inverting plate and lightly blotting on sterile absorbent paper towel. Wash all wells with 100 &#956;L of PBS twice, discard excess PBS by gently inverting plate and lightly blotting on sterile absorbent paper towel after each wash. Do not allow plates to dry.</li>
			<li>Transfer 100 /well of the virus-serum mixture to the corresponding wells on the A549 cell plate following the plate template Incubate the plate for 1 h at 37 &#176;C, 5% CO2.</li>
			<li>After 1 h, using a pipette, take out 90 /well and add 100 of prewarmed 1x M199 + 1.5% CMC + 2% FCS + pen/strep. Incubate plate for 3 days at 37 &#176;C, 5% CO2. 
			NOTE: Make sure the solution is warmed at 37 &#176;C before adding to plates.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Developing assay plate and analysis (Day 5) 
	<ol>
		<li>Develop fixation and assay plate following steps 1.3.1.1 to 1.3.1.8.</li>
		<li>Determine the 50% neutralization titer &#8232;
		<ol>
			<li>Determine the 50% neutralization titer by calculating the proportional distance between the reciprocal serum dilutions above and below 50% of the &#8216;no serum&#8217; control wells (positive virus control - column 10, rows E-H).</li>
			<li>Count the number of PFU (i.e., plaques) arising from individual viral infections in serum wells and no serum control wells. Calculate the mean number of PFU of the no serum control wells and determine the 50% value.</li>
			<li>Identify serum dilutions with counts which are immediately above and below the 50% value of the no serum control wells.Using a semi-log graph or spreadsheet template, plot the number of PFU on the x-axis (linear scale) and the reciprocal serum dilution on the y-axis (log10 scale). Draw a line between the two points and read the 50% neutralization titers of the test serum samples from this line. 
			NOTE: The RSV PRN assay worksheet (Supplementary Worksheet) is used to determine the 50% neutralization titer.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Shi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global%2C+regional%2C+and+national+disease+burden+estimates+of+acute+lower+respiratory+infections+due+to+respiratory+syncytial+virus+in+young+children+in+2015%3A+a+systematic+review+and+modelling+study%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study.</a> Lancet. 390 (10098), 946-958 (2017).</li>
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</ol>

The authors have nothing to disclose.

We have developed and optimized a simple and efficient RSV micro-neutralization assay that can be readily adapted in most laboratories. This assay is able to measure viral infection ability as well as measuring the inhibition of viral infection by NAb at the cellular level using computerized image scanning. The use of an imaging-based platform and specific antibody-based systems has increased the specificity and sensitivity of spot detection compared to traditional plaque detection methods6,<...

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in the pediatric population worldwide1. Despite its high burden, there is still no vaccine or treatment available. Since 2013, the World Health Organization (WHO) has declared RSV vaccine development as a major research priority, with annual WHO consultation meetings2,3. The WHO has agreed on using RSV neutralizing antibody (NAb) measurement to monitor vaccine immunogenicity, as this is recognized as the major serological marker of protection4. NAbs have been shown to protect against severe RSV infection in a number of studies as well as clinical trials of the anti-RSV monoclonal antibody palivizumab, currently the only prophylactic strategy available4.
There are multiple NAb assay formats used by laboratories worldwide, including cell-based and molecular-based assays, which have made standardization efforts challenging5,6,7,8. However, the conventional plaque-reduction neutralization (PRN) assay that measures the number of reduced plaque forming units (PFU) by the presence of an RSV-specific antibody still remains the gold standard9. Here, we report an improved, simplified, and high-throughput PRN protocol that can be used on numerous cell lines, for different RSV strains and with increased assay throughput. This protocol has been tested using clinical samples from different settings as well as on samples from animal model experiments.

<table><tbody><tr><td>&lt;strong&gt;Cell line&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>provided by Dr Keith Chappell, University of Queensland</td></tr><tr><td>&lt;strong&gt;Viral strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RSV A2</td><td>ATCC</td><td>VR-1540</td><td>lot number 60430286</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td>Merck</td><td>1000142511</td><td></td></tr><tr><td>Alexa-Fluor donkey anti-goat IgG (stored at 4 &amp;deg;C)</td><td>Life Technologies</td><td>A11055</td><td></td></tr><tr><td>CMC sodium salt powder</td><td>Sigma-Aldrich</td><td>C5678-500G</td><td></td></tr><tr><td>DMEM (no serum, 3.7 g/L NaHC, P/S) (stored at 4 &amp;deg;C)</td><td>Scientific Services &amp;ndash; Tissue Culture</td><td></td><td>MCRI in house supply</td></tr><tr><td>Foetal calf Serum (stored in 50 mL aliquots at -20 &amp;deg;C)</td><td>Interpath</td><td>SFBS-F</td><td></td></tr><tr><td>Goat X RSV antibody</td><td>Merck</td><td>AB1128</td><td></td></tr><tr><td>human polyclonal antiserum to respiratory syncytial virus (RSV) (stored in 45 &amp;micro;L aliquots at -20 &amp;deg;C)</td><td>BEI Resources</td><td>NR-4022</td><td>Free order through BEI Resources upon registration. This serum belong to a panel of human antiserum and immune globulin to RSV (NR-32832)</td></tr><tr><td>M199 powder</td><td>Life Technologies</td><td>31100035</td><td></td></tr><tr><td>Milk diluent blocking solution (stored at 4 &amp;deg;C)</td><td>Australian Biosearch</td><td>50-82-01</td><td></td></tr><tr><td>Penicillin/Streptomycin (stored in 6mL aliquots at -20 &amp;deg;C)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>s.d.H&lt;sub&gt;2&lt;/sub&gt;O from Milli-Q dispenser</td><td>Merck</td><td></td><td>In-house dispensation</td></tr><tr><td>Sterile 1x PBS for culture (stored at 4 &amp;deg;C)</td><td>Scientific Services &amp;ndash; Tissue Culture</td><td></td><td>MCRI in house supply</td></tr><tr><td>Tween 20 polysorbate</td><td>Sigma-Aldrich</td><td>9005-64-5</td><td></td></tr><tr><td>&lt;strong&gt;General Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Conical Falcon tubes (50 mL)</td><td>Invitro Technologies</td><td>FAL352070</td><td></td></tr><tr><td>Filter unit 0.22 &amp;mu;m (500 mL)</td><td>Thermo Fisher</td><td>NAL5660020</td><td></td></tr><tr><td>Sterile Eppendorf tubes (1.5 mL)</td><td>Australia PL</td><td>AM12400</td><td></td></tr><tr><td>Sterile flat-bottom plates (96-well with lid)</td><td>Interpath</td><td>655180</td><td></td></tr><tr><td>Sterile U-bottom plates (96-well with lid)</td><td>Interpath</td><td>650180</td><td></td></tr><tr><td>5 mL serological pipette</td><td>Sigma-Aldrich</td><td>CLS4487-200EA</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Interpath</td><td>607180</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Sigma-Aldrich</td><td>CLS4251-200EA</td><td></td></tr><tr><td>Tip Pipette 1-200 &amp;micro;L Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960</td><td>Fisher Biotec</td><td>TF-200-L-R-S</td><td></td></tr><tr><td>Tip Pipette 5-20 &amp;micro;L Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960</td><td>Fisher Biotec</td><td>TF-20-L-R-S</td><td></td></tr><tr><td>Tip Pipette 100-1,000 &amp;micro;LClear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1000</td><td>Fisher Biotec</td><td>TF-1000-L-R-S</td><td></td></tr><tr><td>Tip Pipette 1-10 &amp;micro;L Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1001</td><td>Fisher Biotec</td><td>TXLF-10-L-R-S</td><td></td></tr><tr><td>&lt;strong&gt;Equipments and softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ELISpot reader system</td><td>AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany</td><td></td><td></td></tr><tr><td>AID ELISpot software version 5.0</td><td>AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany</td><td></td><td></td></tr><tr><td>Microsoft Excel 2007</td><td></td><td></td><td></td></tr></tbody></table>

an improved and high throughput respiratory syncytial virus (rsv) micro-neutralization assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

The titration of a virus stock was performed from 1:10 to 1:108 dilution to determine the virus stock concentration prior to the PRN assay (representative results shown in Figure 5). From Figure 5, PFU can be counted reliably at dilutions of 1:104 and 1:105. The average number of PFU from triplicate wells at the same dilution was calculated. Since the average number of spots at 1:105 di...

Watch this Scientific Journal Video about An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay at JoVE.com

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

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10.8K Views.  Murdoch Children's Research Institute. This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

Video: An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Immunology and Infection

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

An Assay to Measure the Neutralizing Antibody Titer Specific for Respiratory Syncytial Virus

Source: Do, L. A. H., et al., <a href="https://app.jove.com/v/59025">An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates an assay for measuring neutralizing antibodies against respiratory syncytial virus (RSV). Pre-incubation with serum containing RSV-neutralizing antibodies neutralizes the virus particles, reducing cellular infection and plaque formation. Virus-infected cells are then subjected to fixation, blocking, and fluorophore-conjugated antibody labeling for plaque visualization. This process enables the identification of the highest serum dilution that neutralizes fifty percent of RSV, representing the neutralization titer.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE:&#160;All steps must be ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

Source: Gross, F. L. et al., <a href="https://app.jove.com/v/56448">Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing &#945;2,6-linked sialic acid in human sera. This assay is often used for influenza human serology

All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on ...

Immunodiagnostics

Specialized Assays

Assessing Functional SARS-CoV-2 Neutralizing Antibodies

This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and luminescence-based quantification, revealing the neutralization titer.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pseudotype-based neutralization ...

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Summary

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