Name: quantifying human norovirus virus-like particles binding to commensal bacteria using flow cytometry
Uploaded: 2020-04-29T14:00:00
Description: Watch this Scientific Journal Video about Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry at JoVE.com

We would like to thank Sutonuka Bhar and Chanel Mosby-Haundrup for their critical review of the written manuscript, as well as Alfonso Carrillo for assistance with generating bacterial standard curves. This work is funded in part by a grant from the National Institute of Health (R21AI140012) and by a seed grant from the University of Florida, Institute of Food and Agricultural Sciences.

NOTE: The bacterial growth conditions outlined in the protocol are standard culture conditions for Enterobacter cloacae and Lactobacillus gasseri. To perform the virus:bacteria attachment assay with other bacterial species, the chosen bacteria should be cultured under standard conditions appropriate for the bacterium.
1. Preparing Bacterial Growth Medium
<ol>
	<li>Enterobacter cloacae growth media
	<ol>
		<li>Prepare liquid medium by dissolving 10 g of tryptone, 5 ...

<ol>
	<li>Hall, A. J., Glass, R. I., Parashar, U. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+global+burden+of+noroviruses+and+opportunities+for+prevention.">New insights into the global burden of noroviruses and opportunities for prevention.</a> Expert Review of Vaccines. 15 (8), 949-951 (2016).</li><li>Jones, M. K., 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=Enteric+bacteria+promote+human+and+mouse+norovirus+infection+of+B+cells.">Enteric bacteria promote human and mouse norovirus infection of B cells.</a> Science. 346 (6210), 755-759 (2014).</li><li>Baldridge, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+norovirus+binding+to+select+bacteria+representative+of+the+human+gut+microbiota.">Human norovirus binding to select bacteria representative of the human gut microbiota.</a> PLOS One. 12 (3), (2017).</li><li>Robinson, C. M., Woods Acevedo, M. A., McCune, B. T., Pfeiffer, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Related+enteric+viruses+have+different+requirements+for+host+microbiota+in+mice.">Related enteric viruses have different requirements for host microbiota in mice.</a> Journal of Virology. , (2019).</li><li>Ettayebi, K., 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=Replication+of+human+noroviruses+in+stem+cell-derived+human+enteroids.">Replication of human noroviruses in stem cell-derived human enteroids.</a> Science. 353 (6306), 1387-1393 (2016).</li><li>Van Dycke, 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=A+robust+human+norovirus+replication+model+in+zebrafish+larvae.">A robust human norovirus replication model in zebrafish larvae.</a> PLOS Pathogens. 15 (9), 1008009 (2019).</li><li>Li, D., Breiman, A., le Pendu, J., Uyttendaele, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+to+histo-blood+group+antigen-expressing+bacteria+protects+human+norovirus+from+acute+heat+stress.">Binding to histo-blood group antigen-expressing bacteria protects human norovirus from acute heat stress.</a> Frontiers in Microbiology. 6, 659 (2015).</li><li>Almand, E. A., Moore, M. D., Jaykus, L. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+norovirus+binding+to+gut-associated+bacterial+ligands.">Characterization of human norovirus binding to gut-associated bacterial ligands.</a> BMC Research Notes. 12 (1), 607 (2019).</li><li>Debbink, K., 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=Within-host+evolution+results+in+antigenically+distinct+GII.4+noroviruses.">Within-host evolution results in antigenically distinct GII.4 noroviruses.</a> Journal of Virology. 88 (13), 7244-7255 (2014).</li><li>Harrington, P. R., Lindesmith, L., Yount, B., Moe, C. L., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+Norwalk+virus-like+particles+to+ABH+histo-blood+group+antigens+is+blocked+by+antisera+from+infected+human+volunteers+or+experimentally+vaccinated+mice.">Binding of Norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice.</a> Journal of Virology. 76 (23), 12335-12343 (2002).</li><li>Harrington, P. R., Vinje, J., Moe, C. L., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+capture+with+histo-blood+group+antigens+reveals+novel+virus-ligand+interactions.">Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions.</a> Journal of Virology. 78 (6), 3035-3045 (2004).</li><li>Mallory, M. L., Lindesmith, L. C., Graham, R. L., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GII.4+Human+Norovirus:+Surveying+the+Antigenic+Landscape.">GII.4 Human Norovirus: Surveying the Antigenic Landscape.</a> Viruses. 11 (2), (2019).</li><li>Prasad, B. V., 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=X-ray+crystallographic+structure+of+the+Norwalk+virus+capsid.">X-ray crystallographic structure of the Norwalk virus capsid.</a> Science. 286 (5438), 287-290 (1999).</li><li>Baric, R. 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The ability to quantify binding of enteric viruses to bacteria is a critical first step for elucidating the mechanisms by which these bacteria alter viral infection. The methods described herein have been optimized to measure human norovirus VLP interactions with both E. cloacae (gram-negative bacterium) and L. gasseri (a gram-positive bacterium), but can be adapted for use with any mammalian virus and bacterium of interest. While VLPs are an ideal alternative to live vi...

Human noroviruses (HuNoVs) are the leading cause of gastrointestinal illness worldwide, responsible for 685 million infections and over 200,000 deaths each year1. As with other enteric viruses, the presence of commensal bacteria has been shown to enhance infection of this pathogen as well as its surrogate virus, murine norovirus2,3. There are also conflicting reports that bacteria may inhibit infection by human norovirus4,5,6. For several viruses, direct interaction between the virus and bacteria appear to underlie the mechanisms that impact viral infection2,7,8,9,10, and it has been shown through electron microscopy that human noroviruses bind directly to the surfaces of bacteria11,12. Therefore, characterizing these interactions has become critical to determining the mechanisms by which bacteria impact viral infection. This characterization has classically begun with quantifying viral binding to an array of bacterial species that are components of the host microbiome7,12,13. These attachment assays not only reveal the amount of virus bound to bacteria, but also aid in determining the impact of this interaction on viral fitness and survival.
To quantify viral attachment, traditionally employed methods include PCR-based assays which quantify viral genomes12 or the generation of radiolabeled virus and the use of scintillation counting to quantify viral particles7,8,9,13. The use of these methods generally require access to high-titer virus stocks and in vitro cultivation techniques with which to generate them. While several culture systems for human norovirus now exist2,14,15, none support the robust replication required to generate these highly concentrated stocks which restricts or eliminates the use of PCR and scintillation counting to quantify human norovirus/bacterial interactions.
To circumvent this issue, virus-like particles (VLPs) can be used as a surrogate to live virus to investigate interactions between human norovirus and bacteria16,17. VLPs are non-infectious particles that closely resemble the virus from which they are derived. In the case of human norovirus, these particles are generated from the expression of the VP1 (and sometime the VP2) protein, which self-assemble to create intact viral capsids lacking genetic material (i.e., RNA for noroviruses). These VLPs have been well characterized, are structurally and antigenically similar to the wild-type viruses from which they are derived18,19,20,21,22,23. Therefore, VLPs serve as an ideal surrogate for investigating the surface interactions between human norovirus and commensal bacteria. Given that VLPs lack genetic material, PCR-based assays cannot be used to quantify viral binding. An antibody-based flow cytometry method was previously described and able to detect low levels of VLP binding to bacteria in a semi-quantitative manner16. This method was optimized to allow for accurate quantification of human norovirus VLP binding to both gram-negative and gram-positive commensal bacteria16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5ml Polystrene Round-Bottom Tubes with Cell-Strainer Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>After antibody staining, sample are transferred into tubes for flow cytometry analysis.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A7002</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>AnaeroPack</td>
			<td>Thermo Scientific</td>
			<td>R681001</td>
			<td>Anaerobic gas pack used for culture of Lactobacillus gasseri</td>
		</tr>
		<tr>
			<td>BD FacsDiva software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR Fortessa flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1605</td>
			<td>Used for flow cytometry</td>
		</tr>
		<tr>
			<td>Flow Cytometry Stain Buffer (FCSB)</td>
			<td>BD Biosciences</td>
			<td>554657</td>
			<td>Used for flow cytometry</td>
		</tr>
		<tr>
			<td>Mouse IgG2b kappa Isotype Control (eBMG2b), PE, eBioscience</td>
			<td>Thermo Fisher Scientific</td>
			<td>12473281</td>
			<td>Isotype control. This antibody is purchased in the conjugated form from the manufacturer.</td>
		</tr>
		<tr>
			<td>MRS Powder</td>
			<td>BD Biosciences</td>
			<td>288130</td>
			<td>Used for media preparation and to culture Lactobacillus gasseri.</td>
		</tr>
		<tr>
			<td>Norovirus capsid G2 Monoclonal Antibody (L34D)</td>
			<td>Thermo Fisher Scientific</td>
			<td>MA5-18241</td>
			<td>Norovirus GII antibody. This antibody is only available in the unconjugated form and thus must be fluorescently conjugated prior to use in the outlined flow cytometry assays. In this protocol, PE was the chosen fluor, however, other fluorescent molecules can be chosen as best suits the flow cytometer being used by the researcher.</td>
		</tr>
		<tr>
			<td>Norovirus GII.4 VLP</td>
			<td>Creative Biostructure</td>
			<td>CBS-V700</td>
			<td>human norovirus virus like particle, VLPs were generated using the baculovirus system and resuspended in phosphate buffered saline with 10% glycerol. The authors performed independent nanosight tracking analysis to determine the particle concentration of the VLPs. The concentration is approximately 1011 VLPs per milliliter. Based on the protein concentration of the VLPs, approximately 200 particles are added per bacterium in VLP attachment assays.</td>
		</tr>
		<tr>
			<td>PBS 10X</td>
			<td>Fisher Bioreagents</td>
			<td>BP665</td>
			<td>Dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>SiteClick R-PE Antibody Labeling Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>S10467</td>
			<td>Conjugation kit used for labling of unconjugated antibody.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>Tube Revolver</td>
			<td>ThermoFisher Scientific</td>
			<td>88881001</td>
			<td>Used in virus:bacterium attachment assay. Set to max speed (40 rpm).</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td>Used for media preparation</td>
		</tr>
	</tbody>
</table>

quantifying human norovirus virus-like particles binding to commensal bacteria using flow cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The gating strategies used to quantify human norovirus VLP binding to commensal bacteria are shown in Figure 1. Representative density dot provides an overview of how samples were gated to eliminate cellular debris and cell clumps so VLP attachment was determined on singlet populations (Figure 1A). Representative histograms demonstrate low levels of anti-norovirus antibody signal in bacteria only samples lacking ...

Watch this Scientific Journal Video about Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry at JoVE.com

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Virus-bacterial interactions can be important for successful viral infection and stimulation of a host immune response. But currently, highly concentrated and purified stock of human norovirus cannot be produced in the lab. This technique can be used with any virus that binds to bacteria, for which either virus-like particles or live virus are available.It enables us to quantify interactions between these viruses and bacteria. Inoculate five milliliters of liquid medium with a single isolated colony of Enterobacter cloacae from an agar plate directly from frozen glycerol stock. Grow the bacteria overnight.The following day, transfer 1.3 milliliter aliquots of the culture into two separate 1.5 milliliter centrifuge tubes. Centrifuge the tubes at 10, 000 x g for five minutes. Remove the supernatants, then wash the samples twice using one milliliter of sterile 1X PBS for each wash.Centrifuge the samples again at 10, 000 x g for five minutes. Remove the supernatant and resuspend the pellet in 1.3 milliliters of sterile 1X PBS. Beginning with 0.5 milliliters of washed culture, serially dilute the bacteria in 1X PBS from 10 to the minus one to 10 to the minus four.Using a spectrophotometer, measure the optical density of the washed undiluted culture and each of the four dilutions at 600 nanometers. Perform 10-fold serial dilutions in 1X PBS for each of the previous dilutions. Spread 100 microliters of the last three dilutions for each series onto agar plates to determine the number of colony forming units per milliliter for each sample.Allow the plates to dry at room temperature for five minutes. Invert the plates and incubate them in the incubator. After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL-2 biosafety cabinet.Virus-like particles for human norovirus are listed as BSL-2 pathogens and all work perform using VLP should be conducted in a certified biosafety cabinet. Add 10 micrograms of human norovirus VLPs to each tube of bacteria and mix thoroughly by pipetting. Incubate the tubes for one hour at 37 degree Celsius with constant rotation.After incubation, centrifuge the tubes at 10, 000 x g for five minutes. Aspirate and discard the supernatant and resuspend the bacterial pellet in one milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10, 000 X g for five minutes.Discard the supernatant and resuspend the VLP-bacteria pellet in 150 microliters of 5%blocking buffer. A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment and add one treatment all at once to the tubes.For each bacterial sample, prepare 50 microliters of diluted human norovirus GII antibody. Using 5%blocking buffer, dilute the antibody one to 125 for E.cloacae samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios.Divide each attachment assay sample into 350 microliter aliquots by transferring them into clean 1.5 milliliter centrifuge tubes. To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the GII antibody dilution to the second set of aliquots.Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots. Incubate all the samples on ice and in the dark for 30 minutes. Then centrifuge all the samples at 10, 000 X g for five minutes.Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10, 000 X g for five minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB.After centrifuging the samples one final time and discarding the supernatants, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degree Celsius until they are analyzed by flow cytometry.VLP attachment was measured using flow cytometry. First, density plots were used to gate out cellular debris, followed by subsequent dot plot gating to remove bacterial doublets and cellular clumps. Representative histograms demonstrate a lack of PE signal in bacterial only samples and a shift in PE signal intensity in VLP:bacterium samples compared to unstained and isotype controls.After one hour of incubation with 10 micrograms of VLP, flow cytometry detected particle binding to both E.colacae and L.gasseri. To determine the limit of binding quantification for this assay, a dilution series of the VLPs was generated prior to addition to the bacteria. Reductions in the amount of VLP resulted in corresponding reductions to the percent bacteria bound by VLP.Knowing the virus:bacterium ratio and keeping the ratio consistent between experiments is important in interpreting results. Bacterial and viral mutants can be generated to specifically determine the microbial surface structures that mediate this interaction. This technique allows researchers to answer questions regarding circumstances and conditions that impact norovirus-bacterial interactions, including how changes in virus genotype, bacterial growth conditions, and changes in bacterial surface structures alter viral binding.

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Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...