We thank countless students in our labs (PJD and BJM) who have worked with us over the years refining these methods. A special thanks to Stan Person, under whose tutelage this methodology was first developed. This work was partially supported by the Towson University Fisher College of Science and Math Undergraduate Research Support fund and NIGMS Bridges to the Baccalaureate grant 5R25GM058264. This content is solely the authors&#39; responsibility and does not necessarily represent the official views of the National Institutes of Health&#39;s National Institute of General Medical Sciences.

All procedures with the Vero cells and live herpesviruses have been approved by the Towson University Institutional Biosafety Committee. A generalized scheme of these procedures is represented in Figure 1.
1. Seeding of the Vero cells
<ol>
	<li>The day before initiating the plaque assay, trypsinize Vero cells and resuspend them in regular Dulbecco&#39;s Modified Eagles Medium (DMEM) and supplement as per standard cell culture methodology<sup cla...

<ol>
	<li>Dimmock, N., Easton, A., Leppard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Moden Virology. 6th end. , (2007).</li><li>Mahy, B., Collier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topley and Wilson's Microbiology and Microbial Infections. 9th edn. 1, (1998).</li><li>Anderson, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+bacteriophage+particles:+Comparative+analysis+of+the+traditional+plaque+assay+and+real-time+QPCR-+and+nanosight-based+assays.">Enumeration of bacteriophage particles: Comparative analysis of the traditional plaque assay and real-time QPCR- and nanosight-based assays.</a> Bacteriophage. 1 (2), 86-93 (2011).</li><li>Earle, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+malignancy+in+vitro;+IV.+The+mouse+fibroblast+cultures+and+changes+seen+in+the+living+cells.">Production of malignancy in vitro; IV. The mouse fibroblast cultures and changes seen in the living cells.</a> Journal of the National Cancer Institute. 4 (2), 165-212 (1943).</li><li>Dulbecco, R., Vogt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+problems+of+animal+virology+as+studied+by+the+plaque+technique.">Some problems of animal virology as studied by the plaque technique.</a> Cold Spring Harbor Symposia on Quantitative Biology. 18, 273-279 (1953).</li><li>Enders, J. F., Weller, T. H., Robbins, F. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology. 1, 1823-1897 (2013).</li><li>Madavaraju, K., Koganti, R., Volety, I., Yadavalli, T., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+cell+entry+mechanisms:+An+update.">Herpes simplex virus cell entry mechanisms: An update.</a> Frontiers in Cellular and Infection Microbiology. 10, 617578 (2020).</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> Advances in Virus Research. 8, 319-378 (1961).</li><li>Farnham, A. 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L., Pesce Schito, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short-term+plaque+assay+for+antiviral+drug+research+on+herpes+simplex+virus+type+2.">A short-term plaque assay for antiviral drug research on herpes simplex virus type 2.</a> New Microbiologica. 19 (3), 257-261 (1996).</li><li>Sato, S., Kaneki, H., Shirai, J., Takahashi, K., Kawana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+herpes+simplex+virus+types+1+and+2+by+plaque+appearances+on+semicontinuous+rabbit+lens+epithelial+cells+in+the+clinical+laboratory.">Differentiation of herpes simplex virus types 1 and 2 by plaque appearances on semicontinuous rabbit lens epithelial cells in the clinical laboratory.</a> Kansenshogaku Zasshi. 67 (6), 561-573 (1993).</li><li>Yazaki, S., Taniguchi, S., Yoshino, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+plaque+assay+of+herpes+simplex+virus+in+HeLa+cells.">Improvement of the plaque assay of herpes simplex virus in HeLa cells.</a> Japanese Jouranl of Microbiology. 10 (3), 133-139 (1966).</li><li>Stanners, C. P., Eliceiri, G. L., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+ribosome+in+mouse-hamster+hybrid+cells.">Two types of ribosome in mouse-hamster hybrid cells.</a> Nature New Biology. 230 (10), 52-54 (1971).</li><li>Giannasca, N. J., Suon, J. S., Evans, A. C., Margulies, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-based+controlled+release+delivery+of+acyclovir+from+poly-(ethylene+co-vinyl+acetate)+rings.">Matrix-based controlled release delivery of acyclovir from poly-(ethylene co-vinyl acetate) rings.</a> Journal of Drug Delivery Science and Technology. 55, 101391 (2020).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. 7th edn. , (2016).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+plaques+in+monolayer+tissue+cultures+by+single+particles+of+an+animal+virus.">Production of plaques in monolayer tissue cultures by single particles of an animal virus.</a> Proceedings of the National Academy of Science, USA. 38 (8), 747-752 (1952).</li><li>Baer, A., Kehn-Hall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+concentration+determination+through+plaque+assays:+using+traditional+and+novel+overlay+systems.">Viral concentration determination through plaque assays: using traditional and novel overlay systems.</a> Journal of Visualized Experiments. (93), e52065 (2014).</li><li>Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+low-viscosity+overlay+medium+for+viral+plaque+assays.">New low-viscosity overlay medium for viral plaque assays.</a> Virology Journal. 3, 63 (2006).</li><li>Culley, S., Towers, G. J., Selwood, D. L., Henriques, R., Grove, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+counter:+Automated+quantification+of+in+vitro+virus+replication+by+fluorescence+microscopy.">Infection counter: Automated quantification of in vitro virus replication by fluorescence microscopy.</a> Viruses. 8 (7), 201 (2016).</li><li>Hudu, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Hepatitis+B+e+antigen:+A+better+predictor+of+Hepatitis+B+virus+DNA+than+quantitative+Hepatitis+B+surface+antigen.">Quantitative Hepatitis B e antigen: A better predictor of Hepatitis B virus DNA than quantitative Hepatitis B surface antigen.</a> Clinical Laboratory. 64 (4), 443-449 (2018).</li><li>Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-dependent+DNA+polymerase+in+virions+of+RNA+tumour+viruses.">RNA-dependent DNA polymerase in virions of RNA tumour viruses.</a> Nature. 226 (5252), 1209-1211 (1970).</li><li>Scolnick, E. M., Aaronson, S. A., Todaro, G. J., Parks, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+dependent+DNA+polymerase+activity+in+mammalian+cells.">RNA dependent DNA polymerase activity in mammalian cells.</a> Nature. 229 (5283), 318-321 (1971).</li><li>Strick, L. B., Wald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostics+for+herpes+simplex+virus:+is+PCR+the+new+gold+standard.">Diagnostics for herpes simplex virus: is PCR the new gold standard.</a> Molecular Diagnosis and Therapy. 10 (1), 17-28 (2006).</li><li>Ramakrishnan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+50%+endpoint+titer+using+a+simple+formula.">Determination of 50% endpoint titer using a simple formula.</a> World Jouranl of Virology. 5 (2), 85-86 (2016).</li><li>K&#228;rber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beitrag+zue+kollektiven+Behandlung+pharmakologischer+pharmakologischer+Reihenversuche.">Beitrag zue kollektiven Behandlung pharmakologischer pharmakologischer Reihenversuche.</a> Archiv for Experimentelle Pathologie und Pharmakologie. 162 (4), 480-487 (1931).</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+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27 (3), 493-497 (1938).</li><li>Spearman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Method+of+'Right+and+Wrong+Cases'+(Constant+Stimuli)+without+Gauss's+formula.">The Method of 'Right and Wrong Cases' (Constant Stimuli) without Gauss's formula.</a> British Journal of Psychology. 2 (3), 227-242 (1908).</li><li>Ryu, W. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Virology of Human Pathogenic Viruses. 1st edn. , (2017).</li><li>Burrill, C. P., Strings, V. 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While plaque assays are almost as old as mammalian cell culture itself, it seems that each lab has its own set of protocols to execute this basic assay5,6,10,11,12,13,14,15,16,20. Althoug...

The beginnings of virus plaque assays go back to the first discoveries of viruses in the 1890s1. Tobacco mosaic virus was first isolated and passed on tobacco leaves, where individual spots of infection could be recognized and quantified as originating from a single, live virus entity2, later identified as a virion2. Later seminal studies with bacteria and bacteriophages perfected the techniques used to plaque these viruses, including bacteria at the mid-log phase of growth, serial dilution of bacteriophage samples, and top agar with subsequent visualization of literal holes (named plaques) in the bacterial lawn3.
Plaquing of animal viruses lagged the exciting research being conducted with bacteriophages, mainly because the methods required for growing mammalian cells in culture were not developed until the 1940s4. However, the advent of growing murine cells in the absence of the entire host organism4 spawned a new era in the ability to culture and count viruses. Such work was extended for the propagation and quantitation of Western Equine Encephalomyelitis virus in chicken cells and poliovirus in human cells5,6. As the realm of culturable mammalian cells expanded, the bevy of different host cells for various viral infections gave the world a cornucopia of possibilities to study all manner of viruses7. This included the propagation and quantification of human herpesviruses, particularly herpes simplex virus-1 (HSV-1) and -2 (HSV-2), which cause mucocutaneous lesions8. Importantly, all plaque assays are dependent on the existence of live virions, which can enter host cells in a receptor-mediated fashion in a sample9. Regardless of the ubiquity and multitude of publications on the execution of plaque assays5,10,11,12,13,14,15,16, these methods for HSV-1/-2 are a mixture of both art and science; one cannot conduct the assay without proper attention to every detail in the protocol, nor can one execute a successful assay without a crticial eye for subtlety in the process. This manuscript depicts one of the most consistently reproducible methods for HSV-1/-2 plaque assays, with precise details towards the art of the assay that are seldom discussed.
This current protocol obtains live plaque-forming units (PFU) counts for HSV-1 and -2 reliably. Best results are obtained using Vero cells (transformed African green monkey kidney epithelial cells) at low passage (below passage number 155) and routinely grown in alpha-MEM17 supplemented with 10% fetal calf serum (FCS), L-alanyl-L-glutamine, and an antibiotic/antimycotic mixture18. Vero cells are standardly propagated in this medium two to three times per week at a 1/5 dilution each time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-well plates</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha-MEM</td>
			<td>Lonza</td>
			<td>12169F</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic</td>
			<td>Gibco</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Alfa Aesar</td>
			<td>B2193214</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (no Mg++ or Ca++)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Millipore-Sigma</td>
			<td>TMS-013-B</td>
			<td></td>
		</tr>
		<tr>
			<td>L-alanyl-L-glutamine (Glutamax)</td>
			<td>Gibco</td>
			<td>GS07F161BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Thermo Fisher</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Millipore-Sigma</td>
			<td>27-441-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Quaternary agent (Lysol I.C.)</td>
			<td>Thermo Fisher</td>
			<td>NC9645698</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>25900CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Cytiva</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td></td>
		</tr>
	</tbody>
</table>

plaquing of herpes simplex viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Table 1 shows an experiment that has optimal results. All 10-fold dilutions follow an approximately 10-fold decrease in plaque counts. These kinds of data can also be seen in Figure 2, an actual plaque assay where the countable number of plaques fell in the 10-4 range for all three replicates. The same can be seen in Figure 3, the top row, where the countable number of plaques was in the 10-3 dilution.
<p class="jove_co...

Watch this Scientific Journal Video about Plaquing of Herpes Simplex Viruses at JoVE.com

Plaquing of Herpes Simplex Viruses

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

The protocol helps the students identify individual infections clearly and perform the assay easily, making it more efficient to quantify viral titer. This protocol incorporates a simple fixation and staining procedure that produces reliable and reproducible results. This method can provide insight into the accuracy and effectiveness of antiviral treatments against diseases.The more difficult hurdles of this plaquing protocol are the cell counts for seeding plates, making sure that the cells do not overcrowd, dry out, or get scratched throughout the process. One may also find difficulty in the overall meticulous nature of this protocol. However, practice is a major contributor of the success of this experiment.To begin, complete the sample dilution in the addition of virus to cells in one lab session. Serially dilute each virus sample being plaqued in PBS. Typically use 10-fold dilutions for most precise tracking.Keep all the viral dilutions on ice until ready to add these diluted virus samples to the cells. Remove the cell culture medium from one or two wells using a pipette. Carefully add the diluted virus sample dropwise to each monolayer of cells dropwise through the side of each well.Repeat the process for every one or two wells until the entire plate is filled with the virus samples being plaqued. Gently rock the entire plate by hand. Then incubate the plate at 37 degree Celsius for one hour to allow the virus adsorption.After every 10 minutes, remove the plate from the incubator, gently rock it again to spread the virus more evenly across each well, and then place it back in the incubator. To diminish the possibility of inadvertently counting unabsorbed inoculum, remove the virus sample from the wells using a 1000-microliter pipette tip and discard the sample in a waste beaker. Place 2.5 milliliters of a methylcellulose overlay on the plate, and place it back in the incubator.Disinfect the waste beaker, typically with the addition of bleach, an iodophor, or a similar virucide, before removing it from the biosafety cabinet. After the two-day incubation, remove the methylcellulose overlay from one or two wells at a time using a pipette, and place it in a waste beaker. Add approximately two milliliters of 1%crystal violet in 50%ethanol to each well.Incubate the plate with all wells filled with the stain for 30 minutes at 37 degrees Celsius. At this point, disinfect the waste beaker as demonstrated previously. Wash the plates vigorously with tap water until the runoff is clear.Ensure that there are approximately 10-fold differences in the number of plaques across each dilution series. The figure depicts a 10-fold decrease in plaque counts where the countable number of plaques fell in the 10 to the minus four range for all three replicates. The first three images in the figure depict plaques in the 10 to the minus three dilution.However, the bottom row shows results when a plaque assay is not conducted well. There are very few plaques visible from any dilution in the visible areas around the top where the Vero cells have entirely lifted from the substrate. Similarly, this figure shows problems with cells drying out or mishandling during the plaque assay.However, this figure critically shows poor Vero cell seeding. Every well shows darker purple spots, indicative of cells not spread well across the well and piled on top of one another in clusters. For best results, make sure the virus is spread uniformly across the plate to avoid clumped up plaques Plaque assays are more effective than other quantitative methods because it only counts live virus.That way, true antiviral efficacy can be established. Additionally, this technique has allowed us to quantify viral presence on fomites and explore the effectiveness of drug delivery from novel medical devices within our sector of herpes virus research.

Research

JoVE Journal

Immunology and Infection

4.6K 조회수.  Towson University. 이 프로토콜은 학생들이 개별 감염을 명확하게 식별하고 분석을 쉽게 수행 할 수 있도록 도와 주므로 바이러스 역가를 정량화하는 것이 더 효율적입니다. 이 프로토콜은 신뢰할 수 있고 재현 가능한 결과를 생성하는 간단한 고정 및 염색 절차를 통합합니다. 이 방법은 질병에 대한 항 바이러스 치료의 정확성과 효과에 대한 통찰력을 제공 할 수 있습니다.이 플라킹 프로...