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 g of yeast extract and 10 g of sodium chloride (NaCl) in 1 L of de-ionized (DI) water (see Table of Materials). Mix all media thoroughly and sterilize by autoclaving for 30 min.</li>
		<li>Prepare solid medium by dissolving 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 1 L of DI water. Mix all media thoroughly and sterilize by autoclaving for 30 min.</li>
	</ol>
	</li>
	<li>Lactobacillus gasseri growth media
	<ol>
		<li>Prepare liquid medium by dissolving 55 g of De Man, Rogosa and Sharpe (MRS; see Table of Materials) powder in 1 L of DI water. Use medium within one month of preparation. To sterilize, heat medium for 15 min until boiling followed by autoclaving for 15 min.</li>
		<li>Prepare solid medium by dissolving 55 g of MRS powder and 15 g of agar in 1 L of DI water. To sterilize, heat medium for 15 min until boiling followed by autoclaving for 15 min.</li>
	</ol>
	</li>
</ol>
2. Establishing a Standard Curve Correlating Optical Density (OD) and Bacteria Concentration
<ol>
	<li>Inoculate 5 mL of appropriate liquid medium with a single, isolated colony from an agar plate (E. cloacae) or directly from frozen glycerol stock (L. gasseri).</li>
	<li>Grow the bacterium overnight, under proper atmospheric conditions (Enterobacter cloacae: 37 &#176;C with aerobic shaking at 200 rpm; L. gasseri: 37 &#176;C water bath with no shaking in an airtight container).</li>
	<li>Transfer 2 x 1.3 mL aliquots of overnight culture into two separate 1.5 mL centrifuge tubes and centrifuge bacteria at 10,000 x g for 5 min.</li>
	<li>Remove the supernatant and wash samples with 1 mL of sterile 1x phosphate buffered saline (PBS). Repeat this wash step for a total of 2 washes.</li>
	<li>Centrifuge the samples again at 10,000 x g for 5 min, remove the supernatant and resuspend in 1.3 mL of sterile 1x PBS.</li>
	<li>Beginning with 0.5 mL of washed culture, serially dilute the bacteria in 1x PBS from 10-1 to 10-4.</li>
	<li>Using a spectrophotometer, measure the optical density of the washed, undiluted culture and each of the four dilutions at 600 nm (OD600).</li>
	<li>Perform 10-fold serial dilutions in 1x PBS for each dilution from step 2.6. Spread plate 100 &#181;L of the last 3 dilutions for each series onto appropriate solid medium to determine the number of colony forming units per milliliter (CFU/mL) of each sample. Plate each dilution in triplicate.</li>
	<li>Allow the plates to dry at room temperature for 5 min. Invert the plates and incubate under the appropriate atmospheric conditions (E. cloacae: aerobic at 37 &#176;C, incubate plates overnight; L. gasseri: anaerobic at 37 &#176;C, incubate plates for 48 h in an airtight container with anerobic air sachets (see Table of Materials)). 
	NOTE: Use 1 anaerobic air sachet per 2.5 L jar or 3 sachets per 7.0 L jar (see Table of Materials).</li>
	<li>Use plate counts to determine CFU/mL for each of the 5 samples (i.e., undiluted, 10-1, 10-2, 10-3, 10-4). Generate a standard curve comparing OD600 vs CFU/mL. The equation for this line will be used in VLP-bacteria attachment assays to determine CFU/mL based on OD600.</li>
</ol>
3. VLP-bacteria Attachment Assay
CAUTION: Human norovirus VLPs are a biosafety level (BSL)-2 hazard and all work involving VLPs should be performed in a biosafety cabinet. Preparation of the bacterial cultures, prior to the attachment assay, should be performed using safety conditions appropriate for the organism.
<ol>
	<li>Reagent preparation
	<ol>
		<li>1x phosphate buffered saline (PBS)
		<ol>
			<li>Prepare 1 L of 10x PBS by dissolving an entire packet in 1 L of DI water.</li>
			<li>Autoclave solution for 30 min.</li>
			<li>Prepare a 1x solution by diluting the above solution 1:10 in DI water.</li>
			<li>Filter sterilize the solution by passing it through a 0.22 &#956;m filter and store at room temperature.</li>
		</ol>
		</li>
		<li>5% BSA
		<ol>
			<li>Add 5 g of bovine serum albumin (BSA) powder to 100 mL of PBS to generate a 5% (w/v) solution.</li>
			<li>Mix solution by vortexing or on a stir plate using a metal stir bar until clumps have dissolved.</li>
			<li>Filter sterilize using a 0.22 &#956;m filter.</li>
			<li>Store the solution at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Flow cytometry stain buffer (FCSB)
		<ol>
			<li>Filter purchased FCSB (see Table of Materials) through a 0.22 &#956;m filter.</li>
			<li>Store remaining solution at 4 &#176;C.</li>
		</ol>
		</li>
		<li>5% Blocking Buffer (BB) 
		NOTE: Prepare fresh each time.
		<ol>
			<li>Add 0.5 g of BSA to 10 mL of sterile FCSB.</li>
			<li>Vortex to fully mix sample and leave on ice until use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Antibody conjugation
	<ol>
		<li>Conjugate human norovirus GII antibody (see Table of Materials) with r-phycoerythrin (PE) using an R-PE antibody labeling kit per the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Store the conjugated antibody in the dark at 4 &#176;C for future use in VLP-bacterium attachment assay analysis. 
		NOTE: The primary antibody should be titrated prior to use in the VLP-bacteria attachment assay to determine the proper concentration necessary for flow cytometry analysis. Antibody titration should be performed every time the conjugation reaction is performed and for each bacterium used in the attachment assay.</li>
	</ol>
	</li>
	<li>Antibody titration
	<ol>
		<li>Dilute the conjugated anti-human norovirus GII antibody 100-fold, with a starting dilution of 1:100 and an ending dilution of 1:600 (six dilutions in total).</li>
		<li>Perform a virus attachment assay as described below with the following exception: in step 3.5.7 resuspend the bacterial pellet in 350 &#181;L of 5% BB.</li>
		<li>Divide the sample into seven 50 &#181;L aliquots.</li>
		<li>Add 50 &#181;L of each antibody dilution to one tube.</li>
		<li>Add 50 &#181;L of 5% BB to the seventh tube as an unstained control.</li>
		<li>Perform flow cytometry on all samples as described below.</li>
		<li>Compare diluted antibody samples to unstained controls and to each other. The lowest antibody concentration that does not result in a decrease or loss of positive signal should be chosen for use in subsequent assays.</li>
	</ol>
	</li>
	<li>Preparation of bacteria
	<ol>
		<li>Grow the bacteria in 40 mL of appropriate liquid medium under appropriate atmospheric conditions until the bacteria are in stationary phase.
		<ol>
			<li>Grow E. cloacae cultures aerobically overnight in Luria Broth at 37 &#176;C, shaking at 200 rpm to reach stationary phase.</li>
			<li>Grow L. gasseri cultures in MRS broth in black screw cap tubes without shaking in a water bath set to 37 &#176;C for 18 h to reach stationary phase.</li>
		</ol>
		</li>
		<li>Transfer stationary phase cultures to clean 50 mL conical tubes and centrifuge samples at 2,288 x g for 10 min to pellet the bacteria.</li>
		<li>Remove the supernatant and resuspend in 13 mL of sterile 1x PBS. Repeat this wash step for a total of two washes.</li>
		<li>Centrifuge the samples again, remove the supernatant and resuspend samples in 20 mL of 1x PBS. 
		NOTE: If the cell pellet is small, the resuspension volume can be decreased.</li>
		<li>Measure the OD600 of the culture.</li>
		<li>Using the previously prepared standard curve, determine the CFU per mL of the washed culture.</li>
		<li>Dilute the bacteria as needed with 1x PBS to adjust culture concentration to 1 x 108 CFU/mL.</li>
		<li>Transfer 1 mL of the 1 x 108 CFU/mL bacterial culture to the required number of 1.5 mL centrifuge tubes.</li>
		<li>Centrifuge the tubes at 10,000 x g for 5 min.</li>
		<li>Resuspend the bacterial pellet in 1 mL of sterile 5% BSA.</li>
		<li>Incubate the tubes for 1 h at 37 &#176;C with constant rotation.</li>
	</ol>
	</li>
	<li>Virus attachment
	<ol>
		<li>Working inside a BSL-2 biosafety cabinet, add 10 &#181;g of HuNoV VLPs (see Table of Materials) to each tube containing bacteria resuspended in PBS and mix thoroughly by pipetting. 
		NOTE: VLP concentration differs with each VLP preparation. Therefore, the volume added to bacteria will vary between preparation batches and should be adjusted accordingly so that 10 &#181;g is added to each tube in the experiment. For bacteria only controls (e.g., samples without VLP), add a volume of PBS that equals the volume of VLP added to experimental samples.</li>
		<li>Incubate tubes for 1 h at 37 &#176;C with constant rotation. 
		NOTE: A tube revolver (see Table of Materials) set to 40 rpm is used during incubation.</li>
		<li>After incubation, centrifuge the samples at 10,000 x g for 5 min.</li>
		<li>Discard supernatant and resuspend bacterial pellet in 1 mL of PBS.</li>
		<li>Repeat the wash steps 3.5.3 and 3.5.4.</li>
		<li>Centrifuge the samples at 10,000 x g for 5 min.</li>
		<li>Discard supernatant and resuspend bacterial pellet in 150 &#181;L of 5% BB.</li>
	</ol>
	</li>
	<li>Antibody staining
	<ol>
		<li>Prepare fresh 5% BB. 
		NOTE: All work using the fluorescently tagged antibody should be performed in the dark and the antibody, BB and FCSB should be kept on ice.</li>
		<li>Dilute human norovirus GII antibody 1:125 for E. cloacae samples and 1:150 for L. gasseri samples in 5% BB, preparing 50 &#181;L of diluted antibody per sample.</li>
		<li>Dilute the isotype antibody in the same way the human norovirus GII antibody was diluted for each bacterium in 5% BB, preparing 50 &#181;L of diluted isotype control per sample.</li>
		<li>Divide each attachment assay sample from step 3.3.7 into three 50 &#181;L aliquots by transferring them into clean 1.5 mL centrifuge tubes.</li>
		<li>To the first set of samples, add 50 &#181;L of BB. This set will be the unstained (Uns) controls.</li>
		<li>To the second set, add 50 &#181;L of the GII antibody dilution. This results in a final antibody concentration of 1:250 for E. cloacae and 1:300 for L. gasseri. This sample set will be the stained (AB) samples.</li>
		<li>To the third set, add 50 &#181;L of the diluted isotype antibody. This set will be the isotype controls (IC). Mix well by pipetting. Incubate the samples on ice and in the dark for 30 min.</li>
		<li>Centrifuge all samples at 10,000 x g for 5 min.</li>
		<li>Discard the supernatant and resuspend the samples in 100 &#181;L of FCSB.</li>
		<li>Centrifuge all samples at 10,000 x g for 5 min. Discard the supernatant and resuspend the samples in 100 &#181;L of FCSB.</li>
		<li>Centrifuge all samples at 10,000 x g for 5 min.</li>
		<li>Discard the supernatant and resuspend the samples in 150 &#181;L of FCSB.</li>
		<li>Transfer each sample to a FCSB tube containing 400 &#181;L of FCSB buffer for a total volume of 550 &#181;L.</li>
		<li>Keep samples at 4 &#176;C until they are analyzed by flow cytometry. 
		NOTE: Flow cytometry is performed within 4 h of antibody staining.</li>
	</ol>
	</li>
</ol>
4. Flow Cytometry
NOTE: The voltage settings described below are based on the flow cytometer and software listed in the Table of Materials and will likely vary with different flow cytometers. Settings should be optimized for each bacterium. Ensure that the axes for all graphs are in biexponential phase.
<ol>
	<li>Setting up the workspace 
	NOTE: The set-up for plots used to establish the workspace differ from those used in gating and data analysis. The purpose of setting up the workspace is to visualize the cell population, ensure there is no excessive clumping of the bacterial cells that might impact downstream analysis and to distinguish between stained and unstained cells while data is being collected.
	<ol>
		<li>In order to ensure bacteria are not clumping together, set up a series of density plots.
		<ol>
			<li>Generate a forward scatter area (FSC-A) versus side scatter area (SSC-A) to visualize the total population.</li>
			<li>Set up plots to evaluate single cells versus cell clumps by creating two graphs that compare both forward (FSC-W) and side scatter width (SSC-W) to forward (FSC-H) and side scatter height (SSC-H), respectively.</li>
			<li>Create a plot that shows only the PE positive population (PE-A vs. SSC-A).</li>
		</ol>
		</li>
		<li>Set baseline voltage to 500 for E. cloacae and 340 for L. gasseri. These will be further adjusted later.</li>
		<li>Set the total events counted to 10,000 events.</li>
	</ol>
	</li>
	<li>Running samples
	<ol>
		<li>Create three separate histogram plots that show count plotted against SSC-A, FSC-A or PE-A.</li>
		<li>Place an unstained sample on the flow cytometer and acquire the sample events, ensuring that the maximum peak on the histogram for SSC-A is within 102 to 103 and the maximum peak on the histogram for FSC-A is between 104 to 105 on the x-axis. If not, peaks do not fall within the specified ranges, adjust the FSC and SSC voltages accordingly.</li>
		<li>Place a stained sample (AB) on the flow cytometer and adjust the voltage PE to make the maximum peak past 103 on the x-axis.</li>
		<li>Based on these measurements, set the positive PE gate and set the gate on the PE-A versus SSC-A density graph.</li>
		<li>Run all samples under the determined settings at low or medium speed.</li>
	</ol>
	</li>
</ol>

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The authors do not have any conflicts of interest.

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 virus for use in attachment assays and these particles can be readily quantified using flow cytometry, P particles have also been used to examine interactions between human noroviruses and bacteria24. In this study, the amount of virus bound to the bacteria was quantified as opposed to the bacterial population, as is reported here. P particles provide an advantage over VLPs in that they are easier to produce while providing antigenic similarity to both VLPs and wild-type virus24,25. However, human norovirus VLPs are commercially available providing a means for laboratories lacking the capacity to generate VLPs or P particles. P particles differ from VLPs in that, while VLPs maintain the size of a wild-type viral particle, P particles are smaller and have tetrahedral rather than icosahedral symmetry25. The impact of these characteristics on interactions with bacteria have not been explored and P particles can serve as a viable alternative to VLPs in characterizing surface interactions between human norovirus and bacteria.
As mentioned previously, the assay described above can be used to further characterize interactions between human norovirus VLPs and bacteria. Investigations into how growth conditions and changes in bacterial surface structure expression alter viral binding can be explored using this technique. In addition, this assay can also be used to determine specific bacterial structures bound by the virus through competitive inhibition assays using bacterial proteins or glycans, enzymatic treatment to remove specific surface structures, or incubation with mutant bacterial strains deficient in particular a structure. This assay can also be employed to investigate the ability of other norovirus strains to interact with commensal bacteria.
Because this assay quantifies the proportion of the bacterial population bound by norovirus VLP, it is critical to accurately determine the correlation between CFU/mL and OD600 so bacterial culture concentration can be measured. Fluctuations in the VLP:bacteria ratio alters the percent of the bacteria population bound by VLPs and can lead to variability in results. Care should also be taken to add sufficient quantities of viral particles, as the limit of detection of this assay approaches 0.1 &#181;g of VLP/108 CFU of bacteria. Ratios below this limit consistently yielded percent attachment values of 13&#8211;19%; thus observed population attachment at or below these percentages may not be real.
Antibody titrations were performed for each newly conjugated antibody and against each bacterial strain prior to use in VLP:bacteria attachment experiments. Antibody concentrations required for each bacterium were similar ranging from 1:250 for E. cloacae and 1:300 for L. gasseri. The small size of bacteria, relative to the size of eukaryotic cells, requires both voltage adjustment as well as the use of a binomial distribution during data collection to adequately separate bacteria from debris that may be found in samples or circulating within the instrument. After data collection, proper gating can be used to further remove larger debris particles and bacterial clumps so only single cell populations are analyzed. It is also critical to establish unique voltage settings for each bacterial species tested as these fluctuate widely, particularly between gram-negative and gram-positive bacteria.
Inclusion of proper controls including bacteria only and isotype controls are also critical for accurate analysis of the data. Both types of controls inform regarding levels of non-specific antibody binding. Our results demonstrate that the antibodies used to do not bind non-specifically to the bacterial species tested, but non-specific binding could change with changes in bacterial species or antibody.
The method presented here quantifies human norovirus particle binding to both gram-positive and gram-negative bacteria and is useful in characterizing virus:bacterial interactions. Furthermore, this base protocol can be easily optimized for use with other genotypes of human norovirus, as well as other mammalian viruses and bacteria.

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><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 10&lt;sup&gt;11&lt;/sup&gt; 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 norovirus VLP and low background signal of VLP-bacteria samples stained with the isotype control antibody (Figure 1B). Isotype control peaks also overlap with unstained samples while staining of the same samples with anti-human norovirus GII antibody results in significant shift in peak.
The Overton method of histogram subtraction was used to compare the PE-positive signal in anti-human norovirus GII antibody stained samples to the PE-positive signal of the corresponding isotype control and determine the percent of the bacterial population bound by human norovirus VLPs (Figure 1B). After 1 h of incubation with 10 &#181;g of VLP, flow cytometry detected particle binding to both E. cloacae and L. gasseri (Figure 2). In fact, high levels of binding occurred under these conditions for both bacteria; binding to L. gasseri occurred at slightly higher, but significant (p &#60; 0.0001), levels compared to E. cloacae. These assays demonstrate that flow cytometry can be used to detect human norovirus binding to both Gram-positive and Gram-negative bacteria.
To determine the limit of binding quantification for this assay, a dilution series of the VLPs was generated prior to addition to the bacteria (Figure 3). For both genera of bacteria, reductions in the amount of VLP added to the bacterial culture resulted in corresponding reductions in the percent of bacteria bound by VLP. Changes in the percent of E. cloacae bound by the particle were more gradual compared to L. gasseri, but percent attachment for both bacteria leveled off despite further reductions in VLP concentration. Specifically, 0.1 &#181;g or less (data not shown) of VLP in 108 CFU of bacteria resulted in a plateau of percent attachment averaging between 13&#8211;19% for both bacteria, indicating that this percentage is the limit of quantification for this assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61048/61048fig1a.jpg" /> 
Figure 1: Representative flow cytometric analysis of human norovirus VLP binding. (A) Representative flow cytometry gating strategy to quantify VLP binding to bacteria. Density plots were used to gate out cellular debris, followed by subsequent dot plot gating to remove bacterial doublets and cellular clumps. (B) 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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61048/61048fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61048/61048fig2b.jpg" /> 
Figure 2: VLP attachment to E. cloacae and L. gasseri. Overnight cultures of E. cloacae (n = 6) and L. gasseri (n = 6) were diluted to 1 x 108 CFU/mL in PBS. The bacteria were incubated with 10 &#181;g of GII.4 VLPs for 1 h then VLP attachment was measured using flow cytometry. A representative plot can be found in Figure 1B. Percent attachment was determined using the Overton method of histogram subtraction comparing the PE-positive signal of the GII Human Norovirus stained samples to the PE-positive signal of the corresponding isotype control. Statistical analysis was performed using an unpaired Student&#39;s t-test (p &#60; 0.0001). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61048/61048fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61048/61048fig3b.jpg" /> 
Figure 3: VLP dilution series for E. cloacae and L. gasseri. 10 &#181;g of GII.4 VLPs was serially diluted and the VLP dilutions were each added to 1 x 108 bacteria in a final volume of 1 mL (n = 3 for both bacteria). Samples were incubated for one hour at 37 &#176;C. VLP attachment to the bacteria was measured using flow cytometry. Percent attachment was determined using the Overton method of histogram subtraction comparing the PE-positive signal of the GII Human Norovirus stained samples to the PE-positive signal of the corresponding isotype control. Reducing input amounts of VLP resulted in stepwise reductions in percent attachment for both (A) E. cloacae and (B) L. gasseri. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61048/61048fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

quantifying-human-norovirus-virus-like-particles-binding-to-commensal

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

7.7K Views.  University of Florida. 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.

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Biology

Quantitative Analysis of Microbial Autoaggregation Using Imaging Flow Cytometry

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Author Spotlight: Advancing Research in Microbial Autoaggregation Using Imaging Flow Cytometry

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

Source: Singh, B. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54653">Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the detection of vitronectin&#39;s interaction with bacterial surface proteins. The flow-cytometry analysis measures the fluorescence signal of fluorescently labeled antibodies bound to vitronectin, confirming its presence at the bacterial surface. This technique provides insights into host-pathogen interactions and potential bacterial infection therapies.

Wykrywanie wiązania witronektyny z powierzchniami bakteryjnymi za pomocą cytometrii przepływowej

1. Analysis of Vitronectin, Vn as a Bacterial Surface Protein-Ligand

	Detection of Vn-binding at the bacterial surface using flow cytometry
	NOTE: In ...

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

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EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

Plaque Assay for Murine Norovirus

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Imaging Flow Cytometry to Study Microbial Autoaggregation

Wykrywanie wiązania witronektyny z powierzchniami bakteryjnymi za pomocą cytometrii przepływowej