We thank members of the Wobus laboratory for critical comments and suggestions. Work in the laboratory of CEW was funded by start-up funds from the University of Michigan, a career development grant from the NIH/NIAID Regional Center of Excellence for Bio-defense and Emerging Infectious Diseases Research (RCE) Program, Region V 'Great Lakes' RCE (NIH award 1-U54-AI-057153) and NIH R01 AI080611. M.B.G.-H. was funded by the Experimental Immunology (NIH T32 A1007413-16) and the Molecular Mechanisms in Microbial Pathogenesis (NIH T32 A1007528) training grants to the University of Michigan. J.B.C. was funded by Coordena&ccedil;&atilde;o de Aperfei&ccedil;oamento de Pessoal de N&iacute;vel Superior (CAPES), Brasilia, Brazil.

1. Culturing of the Macrophage Cell Line RAW 264.7
<ol>
	<li>Maintain RAW 264.7 cells (ATCC, catalog # TIB-71) in DMEM-10 media, which consists of high glucose DMEM with 10% (v/v) low-endotoxin fetal bovine serum (&lt; 10 EU/ml), 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM non-essential amino acids, 2 mM L-glutamine. Cells are typically maintained in 175 cm2 tissue culture flasks containing 35 ml of media per flask and incubated at 37 °C and 5% CO2 in a tissue culture incubator. However, any size flask can be used with a volume of media that is appropriate for the size of the flask.</li>
	<li>To split cells: aspirate off the old media, add 10 ml of fresh DMEM-10 media to the cells, and then scrape the cells from the bottom of the flask by using a cell scraper. Next, resuspend the cells into a homogenous solution by drawing up cells into a 10 ml pipette and forcefully squeezing the cells through the pipette tip pressed against the bottom of the flask. Repeat this action at least 3 times so the cells no longer clump together. Verify by light microscopy that a single cell suspension was generated. Then transfer 1 ml (1:10 dilution or ~1x107 cells) - 2 ml (1:5 dilution or ~2x107 cells) of the cell suspension to a new 175 cm2 flask, and bring the final volume of media up to 35 ml.</li>
	<li>Split cells when they are nearly confluent (~1x108 cells total/175 cm2 flasks): every three days if starting with a 1:10 dilution, or every two days if starting with a 1:5 dilution. Use light microscopy to check cell morphology before splitting cells. Most of the cells should look round and not activated. Activated cells have granules and/or extended, spindly morphology with appendages. Do not let cells overgrow as those cells do not typically form plaques. Keep track of the passage number and frequently start over by thawing a lower passage aliquot of cells. (We use passage 30 as a cut-off).</li>
</ol>
2. Infect RAW 264.7 Cells with MNV Inoculum
<ol>
	<li>Seed RAW 264.7 cells into 6-well plates (3.5 cm diameter) at a density of 1x106 viable cells/ml in DMEM-10 media, and add 2 ml of this suspension to each well. It is important to distribute cells evenly in wells either by rocking plates by hand at least 10 times or by using a rocking apparatus for ~10 min. Do not swirl the plates as this will cause the cells to cluster in the center of the well. Place plates into a tissue culture incubator (at 37 °C and 5% CO2). Allow cells to attach overnight or for at least 4 hr at 37 °C. Cells should be 60 - 80% confluent for the plaque assay and distributed evenly throughout the well.</li>
	<li>The next day, prepare the virus inoculum, which can be from MNV-infected cells in tissue culture or from homogenized tissues or fecal samples of MNV-infected mice. When using tissue samples, pea-sized pieces of tissue are homogenized in 2 ml screw-cap tubes containing sterile silica beads in 1 ml of DMEM-10 using a tissue homogenizer (e.g. MagnaLyser; Roche). For fecal samples, no more than 3 fecal pellets should be homogenized in 1 ml media. All samples are then frozen (at -80 °C) and thawed once before performing the plaque assay.</li>
	<li>Prepare 10-fold dilutions of the virus inoculum in complete DMEM-5 medium, which consists of DMEM/High glucose, 5% (v/v) low-endotoxin fetal bovine serum (&lt; 10 EU/ml), 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM non-essential amino acids, 2 mM L-glutamine.</li>
	<li>Ten-fold serial dilutions are prepared in 24-well plates: A repeater pipette is used to dispense 1.35 ml media into multiple wells, the 10-1 dilution is made by mixing 1.35 ml of media and 0.15 ml of virus-containing sample, and then 0.15 ml of the 10-1 dilution is added to 1.35 ml of media to make the 10-2 dilution and so on. It is important to change tips each time you make a new dilution. A multichannel pipette can be used to make the dilutions of multiple samples at a time with two tips fitting into one well of a 24-well plate transferring a total volume of 0.15 ml per well (see Figure 3A).</li>
	<li>A typical dilution range for tissue homogenates and fecal contents is 10-1 to 10-3. However, plaques from these samples tend to be smaller compared to those from tissue culture samples. Furthermore, in some cases a 1:100 dilution of fecal samples is needed to sufficiently dilute out any toxic components of the feces that may disrupt the cell monolayer, thus hindering the ability to count plaques. The dilution range of tissue culture lysates depends on the time point of interest during the viral life cycle. Dilutions that go up to 10-9 may be needed at the peak of infection.</li>
	<li>After the serial dilutions are prepared, label the 6-well plates containing RAW 264.7 monolayers (from section 2.1) with the sample name and dilutions being plated. One plate at a time, remove all media by flicking it out or aspirating it. Immediately afterwards add 0.5 ml of a diluted sample to a well, then repeat with a duplicate well, before proceeding to the next dilution. Once all 3 dilutions are added to one plate, tilt plate back and forth by hand to ensure all cells have been covered. Handle one plate at a time to ensure that cells will not dry out.</li>
	<li>After adding 0.5 ml of the dilutions to each well, stack plates upright and incubate them for 1 hr at room temperature. Because the volume added to each well is not sufficient to cover the monolayer completely, the plates need to be gently tilted back and forth by hand every 10-15 min or placed on a rocking apparatus (~18 oscillations per min). This prevents cells from drying out.</li>
</ol>
3. Low Melting Point Agarose (SeaPlaque) Overlay Preparation
Note: it is advisable to have several bottles with autoclaved SeaPlaque agarose prepared ahead of time. Agarose can be re-melted in a microwave before use.
<ol>
	<li>Calculate the amount of overlay required for the total volume of plates before the 1 hr incubation is complete. The volume needed is 2 ml/well or 12 ml/6-well plate. Prepare agarose (see section 3.2) and media (see section 3.3) separately.</li>
	<li>To prepare the agarose, suspend 3 g of SeaPlaque agarose in a total volume of 100 ml of distilled water (3% w/v) in a glass bottle. Autoclave for 20-30 min. (If agarose was already prepared before-hand, re-melt agarose in microwave.) It is important to equilibrate SeaPlaque agarose to 42 °C in a water bath before use because if the agarose is too hot, it will kill the cells. Make sure water level is equal to or above the level of the agarose to avoid undesired solidification.</li>
	<li>To prepare the media: make 100 ml of 2x MEM media, which consists of 2x MEM, 10% (v/v) low-endotoxin fetal bovine serum (&lt; 10 EU/ml), 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM L-glutamine. Equilibrate media to 37 °C in a water bath.</li>
	<li>Mix both the SeaPlaque agarose and the 2x MEM media together in a sterile bottle at a 1:1 ratio immediately before overlaying the infected cell monolayers. If more than 200 ml overlay is needed, split volume into multiple bottles and keep in 37 °C water bath until ready to use.</li>
	<li>At the end of the 1 hr incubation (see section 2.7), aspirate the inoculum off of each well. Slowly add 2 ml of overlay to the edge of each well by placing the pipette tip against the wall of each well. Up to 5 plates can be handled simultaneously without cells drying out.</li>
	<li>Allow the overlay to solidify for approximately 10 min at room temperature before placing plates upright into the tissue culture incubator. Incubate plates for 48 hr at 37 °C in 5% CO2.</li>
	<li>After the incubation period, plaques are faintly visible to the naked eye, so check unstained plates for presence of plaques. If no plaques are visible, incubate for an additional 4 hr and check again. However, the maximum incubation time should not exceed 72 hr.</li>
</ol>
4. Visualization of Plaques by Neutral Red Staining
<ol>
	<li>To visualize plaques, the neutral red staining solution is prepared by adding 3 ml of neutral red (0.33% w/v in DPBS; Sigma, catalog # N2889) to every 97 ml of 1x PBS (tissue culture grade, Mg2+-, Ca2+-free; Gibco, catalog # 10010). Calculate the volume of neutral red staining solution needed for the experiment: 12 ml neutral red staining solution are required for each 6-well plate. Then, add 2 ml to each well. Although some plaque assay protocols require the agarose plug to be removed from the wells, in this protocol the neutral red staining solution is added directly onto the overlay.</li>
	<li>After a one hr incubation at 37 °C, check if plaques are visible with neutral red staining solution still in wells. If plaques are not readily apparent, allow the staining to continue for another hr. Continue incubating until plaques are visible. (Note: Staining for more than 3 hr is not optimal and if no plaques are visible in the positive control sample after 3 hr of staining, the plaque assay did not work properly.) After the staining is complete, aspirate the neutral red staining solution, ensuring the agarose plug is not disturbed, and then proceed to counting the plaques.</li>
	<li>Count plaques by placing plate upside down on a light box and marking a dot on counted plaques to avoid duplicate counts. Choose the dilution to count plaques in wells where plaques are clearly separated (i.e. no visual evidence of plaques fusing together). If possible, count plaques at two dilutions. It is important to note that plaque size may vary between MNV strains, virus inoculum, and depends on the condition of the RAW 264.7 cells during the plaque assay.</li>
	<li>If no plaques are visible in a well, either there was no virus present in the sample or the amount of virus was under the limit of detection of the plaque assay. In this case, the wells stain red with a similar color as other plaque-containing wells. Alternatively, the absence of plaques is also observed when there are too many viral particles present in a given dilution. This leads to lysis of the entire monolayer and wells appear orange/yellow in color.</li>
	<li>Calculate viral titers. Add the number of plaques in both wells at a single dilution and multiply by the dilution factor (i.e. 1 ml if 2 wells are infected with 0.5 ml). This will yield the amount of plaque forming units (PFU) in your inoculum volume of 1 ml. For example, in Figure 4 at the 10-2 dilution, one well (marked "II") has 14 plaques and the other well (marked "V") has 17 plaques. Thus, the viral titer will be 14x102 + 17x102 = 3,100 (3.1x103) pfu/ml.</li>
</ol>
5. Representative Results
Infectious MNV-1 particles can be quantified using a plaque assay as outlined schematically in Figure 1. Figure 2A shows a well with a monolayer of RAW 264.7 cells just prior to infection, while Figure 2B shows three visible plaques indicated by roman numbers I, II and III in a well. Individual steps of the assay are depicted in Figures 3A through F. Figure 3A shows the preparation of the 10-fold dilution series of a virus-containing sample. Figure 3B shows the transfer of dilutions to duplicate wells of a 6-well plate. Figure 3C shows the rocking apparatus used to incubate RAW 264.7 cells with the inoculum at room temperature for 1 hr. Figure 3D shows cells being overlaid with the SeaPlaque:MEM mixture. Figure 3E shows a plate at room temperature to allow the overlay to solidify, while Figure 3F shows cells being stained with a 0.01% neutral red solution 48 hr later. After staining cells for 1-3 hr and aspirating the neutral red staining solution, plaques are visible and can be counted (Figure 4).
<img alt="Serial dilution process diagram showing bacterial culture concentration reduction steps with assay results." src="/files/ftp_upload/4297/4297fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
 Figure 1. Schematic of the MNV plaque assay protocol.
<img alt="Cell cultures under microscope; bacterial growth analysis, microscopic image, sample A and B comparison." src="/files/ftp_upload/4297/4297fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
 Figure 2. Representative images of a well of a monolayer before infection and after formation of plaques. A) RAW 264.7 cells were cultured overnight and imaged under a light microscope at 20x magnification. B) Cells were stained with a 0.01% neutral red solution after 48 hr of infection and visualized under a light microscope at 4x magnification. Roman numbers I, II, and III indicate three visible plaques.
<img alt="Cell culture experiment; using multi-well plates in laboratory setup for biological analysis method." src="/files/ftp_upload/4297/4297fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
 Figure 3. Representative images of the different plaque assay steps. A) MNV-1 inoculum is prepared in 10-fold dilutions. B) Inoculum is added to cell monolayers in duplicate wells. C) Cells and inoculum are incubated by rocking for 1 hr at room temperature. D) Cells are overlaid with a 1:1 mixture of SeaPlaque agarose and 2x MEM media. E) Plates are incubated for 10 min at room temperature to allow the overlay to solidify. F) Staining of cells with the neutral red staining solution 48 hr post-infection.
<img alt="Plaque assay results; dilution series; virus quantification; six-well plate; viral titer measurement." src="/files/ftp_upload/4297/4297fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
 Figure 4. MNV-1 forms plaques in cell monolayers. Shown here is a representative plaque assay plate 48 hr post-infection, showing plaques stained with neutral red staining solution after 1 hr of incubation. The plate shows duplicate wells of three 10-fold dilutions. Wells labeled with roman numbers I and IV correspond to the 10-1 dilution; II and V correspond to the 10-2 dilution; III and VI correspond to the 10-3 dilution. The viral titer of the sample is indicated below (see Section 4.5 for details of the calculation).

<ol>
	<li>Wobus, C. E. <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+Norovirus+in+cell+culture+reveals+a+tropism+for+dendritic+cells+and+macrophages.">Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.</a> PLoS Biol. 2, e432 (2004).</li><li>Wobus, C. E., Thackray, L. B., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+norovirus:+a+model+system+to+study+norovirus+biology+and+pathogenesis.">Murine norovirus: a model system to study norovirus biology and pathogenesis.</a> Journal of virology. 80, 5104-5112 (2006).</li><li>Condit, R. C., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Fields Virology. 1, 25-58 (2007).</li><li>Thackray, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+noroviruses+comprising+a+single+genogroup+exhibit+biological+diversity+despite+limited+sequence+divergence.">Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence.</a> Journal of virology. 81, 10460-10473 (2007).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+estimating+50%+endpoints.">A simple method for estimating 50% endpoints.</a> American Journal of Hygiene. 27, 493-497 (1932).</li><li>Chachu, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+is+critical+for+the+clearance+of+murine+norovirus+infection.">Antibody is critical for the clearance of murine norovirus infection.</a> Journal of virology. 82, 6610-6617 (2008).</li><li>Barron, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+murine+norovirus+strains+isolated+from+asymptomatic+mice+of+different+genetic+backgrounds+within+a+single+U.S.+research+institute.">Diversity of murine norovirus strains isolated from asymptomatic mice of different genetic backgrounds within a single U.S. research institute.</a> PLoS ONE. 6, e21435 (2011).</li><li>Cox, C., Cao, S., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection+and+study+of+murine+norovirus-1+using+a+more+efficient+microglial+cell+line.">Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line.</a> Virology journal. 6, 196 (2009).</li><li>Cooper, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plaque+assay+of+animal+viruses.">The plaque assay of animal viruses.</a> Adv. Virus Res. 8, 319-378 (1961).</li><li>Hyde, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+norovirus+replication+is+associated+with+virus-induced+vesicle+clusters+originating+from+membranes+derived+from+the+secretory+pathway.">Mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway.</a> Journal of virology. 83, 9709-9719 (2009).</li><li>Simmonds, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatic+and+functional+analysis+of+RNA+secondary+structure+elements+among+different+genera+of+human+and+animal+caliciviruses.">Bioinformatic and functional analysis of RNA secondary structure elements among different genera of human and animal caliciviruses.</a> Nucleic acids research. 36, 2530-2546 (2008).</li></ol>

No conflicts of interest declared.

The plaque assay method for MNV-1 presented here is a way of quantifying infectious MNV particles. By following the assay steps illustrated in Figure 3, one can obtain reproducible viral titers. The limit of detection of the assay depends on the starting dilution used. When starting with a 1:10 dilution of sample as described above, the limit of detection of the plaque assay is 10 pfu (i.e., 1 plaque visible at the 10-1 dilution). Since each plaque represents a single virus, the plaque assay can also be used to purify clonal populations of MNV by picking isolated plaques and propagating them as described previously 1. In addition, plaque purifications can also be used to separate an individual virus population from mixed virus populations. A limitation of using a plaque assay for the detection of MNV infection is that not all MNV strains form plaques 4. However, it may be possible to overcome the inability of some MNV strains, isolated from animals, to form plaques by serially passaging these viruses in tissue culture 7. An alternative to the plaque assay is to measure infectious particles via the TCID50 technique 3, 4. This assay quantifies the amount of virus required to produce CPE in 50% of inoculated tissue culture cells following endpoint dilutions and takes 1 week to complete for MNV 4. In addition to being slower than a plaque assay, the TCID50 assay is also not as sensitive (limit of detection = 200 TCID50/ml) due to the toxicity of tissue samples to RAW 264.7 cells 4.
Although critical steps within the protocol have been described throughout the protocol, the following section provides a summary to facilitate trouble-shooting. The most critical step in the protocol is to ensure that RAW 264.7 cells remain viable throughout the assay to support virus replication. This can be monitored at each stage of the assay via light microscopy. Cell viability is ensured in two ways. First, care should be taken not to let cells dry out while handling plates. Thus, plates are inoculated one at a time, rocked during the infection period, and should remain closed whenever they are not being handled. Second, solutions added onto cells should be equilibrated to ~37 &deg;C. Furthermore, it is vital for the overall health of the RAW 264.7 cells to maintain them in media containing low endotoxin serum (&lt; 10 EU/ml), which limits activation of cells. In addition, we have observed a higher failure rate of the plaque assay when using cells from passage 30 or higher. Although this will likely vary from lab to lab, it is important to include a positive control (e.g., a sample with a known viral titer) to ensure reproducible titers, especially when using higher passage RAW 264.7 cells. To limit use of higher passage cells, it is advisable to freeze vials of early passage cells upon receipt of RAW 264.7 cells and start a new culture from the frozen vials frequently. Starting over with low passage cell cultures will also be helpful when cells exhibit altered characteristics, such as failure to adhere, changes in cell morphology (e.g. from round to spindly and spread out), or when mycoplasma contamination has been detected. Another important point to pay attention to is to ensure that pipette tips are changed between samples and during dilutions. This will ensure accurate serial dilutions and prevent cross-contamination between samples. The one step in the protocol where the same pipette tip can be used again is when serial dilutions of the same sample are added to wells. In that case, one should start from the most diluted inoculum to the least, and vigorously pipette up and down when drawing up a new dilution.
The plaque assay protocol is amendable to several modifications. One modification that can be made when there are not enough cells for inoculating wells in duplicate is to inoculate only a single well for each dilution. However, since the inoculum volume is 0.5 ml, the number of plaques then needs to be multiplied by a factor of 2 to normalize to pfu/ml. The plaque assay can also be adapted for use with any other adherent cell line that is able to support replication of MNV, and this has been described for the murine microglial BV-2 cell line 8. Other modifications that can be implemented are adaptations that have been described for plaque assay protocols developed for other viruses. In case of MNV, the following modifications have already been implemented successfully; the use of methyl cellulose instead of Sea Plaque agarose 9, and staining of cells with crystal violet or methylene blue instead of neutral red 10, 11.
Overall, this protocol can easily be adapted as needed to quantify other plaque-forming viruses or used for other viruses that cause lytic infections in RAW 264.7 cells, making it a useful tool to quantify infectious viral particles in general.

<table><tbody><tr><td>DMEM/ High glucose</td><td>Hyclone</td><td>SH30243.02</td><td></td></tr><tr><td>2x MEM</td><td>Gibco</td><td>11935</td><td></td></tr><tr><td>100x Penicillin and streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>10 mM Non-essential amino acids</td><td>Hyclone</td><td>SH30238.01</td><td></td></tr><tr><td>1M HEPES</td><td>Hyclone</td><td>SH30237.01</td><td></td></tr><tr><td>200 mM (100x) L-glutamine</td><td>Hyclone</td><td>SH30034.01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco, Hyclone</td><td>10437, SH30070.02</td><td></td></tr><tr><td>Sea Plaque Agarose</td><td>Lonza</td><td>50100</td><td></td></tr><tr><td>Neutral Red 0.33%</td><td>Sigma</td><td>N2889</td><td></td></tr><tr><td>1x PBS</td><td>Gibco</td><td>10010</td><td></td></tr><tr><td>1.0 mm Zirconia/Silica beads</td><td>BioSpec Products</td><td>11079110z</td><td></td></tr><tr><td>Model 35 Speed Rocker</td><td>Labnet</td><td>S2035</td><td></td></tr><tr><td>Magna Lyser Instrument</td><td>Roche</td><td>03358968001</td><td></td></tr><tr><td>Raw 264.7 cell line</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>Tissue culture incubator</td><td>Sanyo</td><td>MCO-18AIC</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Plaque Assay for Murine Norovirus at JoVE.com

Plaque Assay for Murine Norovirus

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 ...

Nanyang Technological University

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JoVE Journal

Immunology and Infection

54.5K Views.  University of Michigan, Ann Arbor. Hier beschrijven we een methode om infectieuze deeltjes van murine norovirus (MNV), de enige norovirus die efficiënt repliceert in celkweek kwantificeren. De plaque assay maakt gebruik van tropisme MNV voor murine macrofagen en kan worden aangepast voor gebruik met biologische of milieumonsters met MNV.

Video: Plaque Assay for Murine Norovirus

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 ...

Environment

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting 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. ...

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 ...

Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 &#176;C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Here we present two protocols, one for measuring the specific growth rate and the other for the cell-binding ability of rotavirus using the plaque assay and RT-qPCR. These protocols are available for confirming the differences in phenotypes between rotavirus strains.

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known ...

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 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 ...

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Viruses that infect prokaryotic organisms, called bacteriophages or simply phages, were identified in the early 20th century by Twort (1) and d&#39;H&#233;relle (2) independently. Phages have since been widely recognized for their therapeutic value (3) and their influence on human (4), as well as global, ecosystems (5). Current concerns have fueled a renewed interest in the use of phages as an alternative to modern antibiotics in treatment of infectious disease (6). Essentially all phage research relies on the ability to purify and quantify viruses, also known as a viral titer. Initially described in 1952, this was the purpose of the plaque assay (7). Decades and multiple technological advancements later, the plaque assay remains one of the most reliable methods for determination of viral titer (8).
Bacteriophages subsist by injecting their genetic material into host cells, hijacking the machineries for production of new phage particles, and eventually causing the host to release numerous progeny virions through cell lysis. Because of their minute size, bacteriophages cannot be observed using solely light microscopy; therefore, scanning electron microscopy is required (Figure 1). Additionally, phages cannot be cultivated on nutritional agar plates like bacteria, as they need host cells to prey on.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10514/10514fig1.jpg" /> 
Figure 1: The morphology of a bacteriophage, here exemplified by an E. coli phage, can be studied using scanning electron microscopy. Most bacteriophages belong to Caudovirales (tailed bacteriophages). This particular phage has a very short tail structure and an icosahedral head, placing it in the family of Podoviruses.
The plaque assay (Figure 2) is based on incorporation of host cells, preferentially in log-phase growth, into the medium. This creates a dense, turbid layer of bacteria able to sustain viral growth. An isolated phage can subsequently infect, replicate within, and lyse one cell. With each lysed cell, multiple adjacent ones become immediately infected. Several cycles in, a clear zone (a plaque) can be observed in the otherwise turbid plate (Figure 2B/Figure 3A), indicating the presence of what was initially a single bacteriophage particle. The number of plaque forming units per volume (i.e. PFU/mL) of a sample, can thus be determined from the number of plaques generated.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10514/10514fig2.jpg" /> 
Figure 2: Testing for plaque forming units (PFU) is a common method for determining the number of bacteriophages in a sample. (A) The base of a sterile Petri dish is covered with an appropriate solid nutrient medium, followed by a mixture of soft media, susceptible host cells and a dilution of the original bacteriophage sample. Note that the phage-suspension could, in some cases, also be evenly spread-out across the surface of already solidified soft agar. (B) Growth of the host bacteria forms a lawn of cells in the top agar layer. Bacteriophage replication generates clear zones, or plaques, caused by host cell lysis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10514/10514fig3.jpg" /> 
Figure 3: Results from PFU-testing show multiple plaques generated by bacteriophages. Due to lysis of susceptible host cells, plaques can be seen as clearing zones in the bacterial lawn, either with (A) full clearance, or (B) partial re-growth caused by generation of resistant bacteria (or possibly by temperate phages in the lysogenic cycle).
Certain temperate phages can adopt what is referred to as a lysogenic lifecycle, in addition to the formerly described lytic growth. In lysogeny, the virus assumes a latent state through incorporation of its genetic material into the genome of the host cell (9), often conferring resistance to further phage infections. This is sometimes revealed through a slight clouding of the plaque (Figure 3B). It is worth noting however, that plaques can also appear blurred due to re-growth of bacteria that have evolved resistance to the phage independent of previous phage infections.
Viruses can attach, or adsorb, to only a limited range of host bacteria (10). Host ranges are further limited by intracellular anti-viral strategies such as the CRISPR-Cas system (11). The resistance/sensitivity towards specific phages displayed by bacterial subgroups has historically been used to categorize bacterial strains into different phage types (Figure 4). Although the effectiveness of this method has now been surpassed by novel sequencing techniques, phage typing can still provide valuable information about bacteria-phage interactions, for instance, facilitating the design of a phage cocktail for clinical usage.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10514/10514fig4.jpg" /> 
Figure 4: Phage sensitivity of different bacterial strains. Soft agar plates with Cutibacterium acnes strain (A) AD27 and (B) AD35, were spotted with 21 different C. acnes bacteriophages. Only phage 11 were able to infect and kill AD27 while strain AD35 showed sensitivity towards all phages. This technique, termed phage typing, can be used to divide bacterial species and strains into different subgroups based on phage susceptibility.

Determining Viral Titer as Plaque Forming Units PFU

Source: Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 ...

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An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

Source: Madrigal, J. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61048">Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry.</a> J. Vis. Exp. (2020).
This video demonstrates an assay for quantifying human norovirus virus-like particles (VLPs) binding to human intestinal bacteria. The bacterial suspensions are mixed with the VLPs and incubated to allow the formation of VLP-bacteria complexes. The complexes are then labeled with fluorophore-tagged VLP-specific antibodies, followed by flow cytometry-based visualization and quantification to identify the percentage of VLP-bound bacteria.

1. 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 ...

Plaque Assay for Murine Norovirus

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