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 methodology19. Resuspend the trypsinized cells in 10 mL of DMEM per T-75 flask of the confluent Vero cells (~107 cells). 
	NOTE: DMEM is used for plaque assays instead of alpha-MEM because it slightly slows the cell growth rate and favors better plaque assay results.</li>
	<li>Count the cells by one&#39;s preferred method (e.g., a standard hemocytometer with Trypan Blue exclusion)19.</li>
	<li>Seed the cells at 4 x 106 cells/plate; for HSV-2, use 6-well plates with 2.5 mL of DMEM (with supplements) per well; and for HSV-1, use 12-well plates with 1.25 mL of DMEM (with supplements) per well. 
	NOTE: The number of cells should be constant regardless of the plate used, though the size of the wells matters concerning accuracy in the plaque assay. It is essential to get cells evenly distributed, which can be most effectively accomplished by moving the plate back and forth, not in a circular fashion.</li>
	<li>Allow the cells to grow overnight at 37 &#176;C/5% CO2 in a humidified incubator. 
	&#8203;NOTE: Humidity is maintained with a pan of distilled water containing an algicide in the bottom of the incubator.</li>
</ol>
2. Sample dilution
<ol>
	<li>Complete the sample dilution and the addition of virus to cells in one lab session. 
	CAUTION: HSV-1 and -2 are infectious for humans and must be handled under proper biosafety containment (BSL-2). Keep all materials that come into contact with the virus separate and disinfect with a quaternary agent at 1/256 dilution (see Table of Materials), iodophor, bleach, or strong ionic detergent (e.g., SDS) before removing them from the biosafety cabinet.</li>
	<li>The day&#160;after seeding of the Vero cells, serially dilute each virus sample to be plaqued in 1x PBS; typically use 10-fold dilutions for most precise tracking.</li>
	<li>Make these dilutions through 10-4 (for low concentrations of viruses) or through 10-9 (for expectations of higher titer samples) with at least 1 mL of each sample remaining for the plaque assay itself.</li>
	<li>Keep all the viral dilutions on ice (no longer than 1 h) until ready to add these diluted virus samples to the cells.</li>
</ol>
3. Addition of virus to the cells
<ol>
	<li>Remove the cell culture medium from one or two wells by pipette. 
	NOTE: Do not use an aspirator because it removes too much liquid from the cell monolayer and dries the cells out. One crucial consideration is ensuring that the cell monolayer remains hydrated throughout the entire course of the experiment. If the cells dry out, they will wash off the plate in the final step and generate unusable data. Therefore, process only one or two wells at a time to reduce this possibility.</li>
	<li>Carefully add the diluted virus sample (100-400 &#181;L and 50-200 &#181;L of virus sample are used for 6-well and 12-well plates, respectively) dropwise to each monolayer, adding the drops down 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. 
	NOTE: Adding the drops to the center of the well may inadvertently slough cells off the substrate.</li>
	<li>Gently rock the entire plate by hand to ensure the PBS that contains the virus covers the entire monolayer of cells in each well, then place the plate in a CO2 incubator at 37 &#176;C to allow the virus to adsorb.</li>
	<li>Every 10 min within 1 h, 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. 
	NOTE: Each experiment will dictate the number of replicates and which dilutions are used; the reader&#39;s preference dictates that decision.</li>
	<li>To diminish the possibility of inadvertently counting unabsorbed inoculum, remove the virus sample from the wells with 1000 &#181;L pipette tips and place it in a waste beaker. 
	NOTE: Again, this procedure is conducted with only one or two wells at a time to maintain hydration of the cell monolayer.</li>
	<li>Place 2.5 mL of a methylcellulose overlay (dissolve 5% methylcellulose w/w in 100 mL of PBS, autoclave for 15 min at 121 &#176;C and 15 psi on a liquid cycle, then add 375 mL of DMEM plus 25 mL of FBS).</li>
	<li>Once an entire plate contains overlay, place the plate back in the incubator to allow growth for two days. 
	NOTE: If the virus and cells are allowed to grow for three days, even in DMEM plus supplements, a substantial loss of cells in the monolayer may result, thereby compromising the final data.</li>
	<li>Disinfect the waste beaker, typically with the addition of bleach, an iodophor, or a similar virucide, before removing it from the biosafety cabinet.</li>
</ol>
4. Staining for plaques
<ol>
	<li>After the two-day incubation, remove the methylcellulose overlay by a pipette, again one or two wells at a time, and place it in a waste beaker.</li>
	<li>Add ~2 mL of 1% crystal violet (see Table of Materials) in 50% ethanol to each well to stain the plaques. 
	NOTE: It is not essential to remove every last drop of the overlay.</li>
	<li>Incubate the plate with all wells filled with the stain for 30 min at 37 &#176;C. At this point, disinfect the waste beaker as above in step 3.8.</li>
	<li>Wash the plates vigorously with tap water until the runoff is clear. 
	NOTE: A gentle stream of water is not vigorous enough; full pressure from a lab sink fixture is required.</li>
	<li>At this point, ensure that there are approximately 10-fold differences in the number of plaques across each dilution series.</li>
	<li>Allow the plates to dry overnight upside down, after which the individual plaques may be counted.</li>
</ol>
5. Counting plaques and determining virus titer
<ol>
	<li>Regardless of the approach (see NOTE below), multiply the number of plaques by the dilution factor (e.g., if the plaques were counted in the 10-4 well, then the number of plaques would be multiplied by 104). 
	NOTE: Although counting within a range of 10-100 plaques is useful5, counting wells with 30-300 plaques is somewhat more reliable and takes into account approximately &#189;-log dilutions instead of full-log dilutions.</li>
	<li>Divide this number by the volume of inoculum (as above in step 3.2), and average all wells that have plaques within the stated range to get the most accurate titer of HSV in the original sample.</li>
	<li>Perform the calculations in steps 5.3.1 to 5.3.5, considering the use of mock data in Table 1.
	<ol>
		<li>Consider only wells with 30-300 plaques (step 5.1). Hence, in Table 1, use only the 10-5 column for replicates 1, 2, and 3.</li>
		<li>Take the number of plaques at the given dilution and multiply by the reciprocal of the dilution factor. Hence, the 10-5 column of sample 1 goes from 81 plaques at the 10-5 dilution to 81 plaques times 105.</li>
		<li>Then divide that number by the volume (in mL) of the virus dilution added to the cells in that well. Assuming the data on HSV-1 in Table 1 are from a 12-well plate, 0.2 mL would be the most appropriate measurement. Hence the calculation from step 5.3.2 becomes 81 x 105 divided by 0.2 mL, or 4.0 x 107 PFU/mL of HSV-1.</li>
		<li>Repeat this process for all useful data and average. Hence conduct the calculation in 5.3.2-5.3.3 on all samples in the 10-5 column, assuming they originated from the same virus sample and the biological replicates were conducted to obtain the most accurate titer measurement. In the case of Table 1, average 4.0 x 107, 2.1 x 107, and 2.6 x 107 PFU/mL to obtain a final average titer of 2.9 x 107 &#177; 0.98 x 107 PFU/mL for this sample.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

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. Although the instructions in each published version of a plaque assay differ ever so slightly, few of these manuscripts elucidate the critical nuances involved in obtaining consistent results.
Hence, there are some parts of this protocol that are more art than science. When Vero cells are seeded in wells for the assay, they must be spread evenly throughout the well (Figure 4). All steps involving a liquid transfer (e.g., steps 3.1, 3.2, 3.5, 3.6, 4.1, and 4.2) must be done carefully to keep the Vero cell monolayer hydrated (Figure 3 and Figure 4). It is also crucial not to touch the bottom of the well with any pipette, or the cells may scrape off, further compromising the final data (Figure 4).
While methylcellulose is advantageous as an overlay material to prevent diffusion of virions across the entire well, alternatives may be used. For example, one may use microcrystalline cellulose21,22 or carboxymethyl cellulose21. Regardless, methylcellulose is one of the easiest and least expensive liquid overlay media to use, though. One may also use a different dye21 or fixing solution (e.g., paraformaldehyde)21 for the staining part of the protocol (step 4), but crystal violet in 50% ethanol is the easiest and least expensive among the options.
While it may be overtly straightforward to conduct these assays, they do require training and practice. It is well worth using samples that are not irreplaceable for learning this method so that one has mastered the technique when it matters most.
Plaque assays themselves should only be used, though, when one needs to count bona fide live virions. Plaquing is a much older methodology to measure virus particles5,10 compared to newer quantitative methods like fluorescence microscopy23, ELISA24, enzymatic activity25,26, or qPCR27. However, these latter methods generate surrogate data that suggest the presence of whole virions, yet may lead to a gross overestimate in viable particles because the user would be quantifying an indicator of infection, not real virions. Fluorescence microscopy measures infectious virions by how many cells exhibit the presence of a protein expressed by the virus but does not necessarily detect complete, replication-competent virions. ELISA similarly detects the presence of virus proteins that may exist without an utterly infectious virion. Assaying enzymatic activity, while suggestive of an intact virion, only shows that a single active enzyme is present and does not reflect the true contagious nature of a whole virion. And qPCR merely quantifies copies of a virus&#39; genome, whether that genome is defective or not. The only method comparable to a plaque assay to quantify live viruses is an endpoint dilution assay28, which still requires a statistical estimation via the Reed-Muench or Spearman-K&#228;rber methods29,30,31 to determine virion concentration.
While plaque assays are a routine method used throughout virology, there are particular cases where they are not the quantitative assay of choice. For example, plaque assays require host cells to attach to a substrate; if the best host cell for infection is non-adherent, plaques will never form32. Some viruses grow more slowly than the typical cell cycle of their hosts, and therefore any burgeoning plaques would be overgrown by the host cells themselves21. Plaques may sometimes not form well, just by the nature of the cytopathic effect, making the determination of bona fide plaques a limitation21.
The approach to plaquing herpesviruses presented here is not necessarily unique. However, this in-depth description of the minor caveats associated with a successful HSV plaque assay may diminish the difficulties experienced by many users, especially novices. Regardless, the methodology described is also broadly applicable to plaque assays with many other viruses, including but not limited to picornaviruses33, orthomyxoviruses34, flaviruses35, coronaviruses36, and many other systems in which a quantifiable amount of infectious animal viruses are required.

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><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&#039;s PBS (no Mg&lt;sup&gt;++&lt;/sup&gt; or Ca&lt;sup&gt;++&lt;/sup&gt;)</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.
However, Figure 3, bottom row, shows an entirely different result when a plaque assay is not conducted well. First, there are very few plaques visible from any dilution; hence, the data are unusable. Second, there are visible areas around the top where the Vero cells have entirely lifted from the substrate, evidence either of allowing the cells to dry out in the process or of pipetting too vigorously in that region of the well.
Table 2 also shows a set of potentially aberrant outcomes in plaque counts and requires cautions along the way. Of note, neither the top row nor the bottom row of replicates follows a regular 10-fold difference in plaque counts as one moves from one dilution to the next; these data indicate user error during the virus dilution process. Furthermore, it is not likely that a user would accurately count over 15,000 plaques, so one must call those data into question. Finally, one may note that in replicate 1, 10-6 dilution, there is a plaque count in the 30-300 range; nonetheless, given the poor nature of the rest of the data in this replicate, it is unlikely that the 42 plaque count is an accurate number. Hence, the only believable data in Table 2 are in replicate 2.
Figure 4 shows the same problems with cells drying out or being mishandled during the plaque assay. However, Figure 4 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.
<img alt="Plaque assay diagram with virus dilution, Vero cell seeding, incubation, and crystal violet staining." class="xfigimg" src="/files/ftp_upload/62962/62962fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Overall plaque assay scheme. Adapted from Biorender template, "Viral Plaque Assay Protocol," by BioRender.com. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62962/62962fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Colony formation assay in 12-well plate, dilution series 10⁻² to 10⁻⁵; cancer cell viability test." class="xfigimg" src="/files/ftp_upload/62962/62962fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Plaque assay for HSV-1 in twelve-well plates. Biological replicates were conducted as outlined in Figure 1 on one sample of HSV-1. Serial 10-fold dilutions of an HSV-1 sample (starting at the 10-2 dilution) were added to the Vero cells, incubated, then stained with crystal violet. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62962/62962fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell colony dilution analysis on agar plates, showing bacterial growth with serial dilution factors." class="xfigimg" src="/files/ftp_upload/62962/62962fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Plaque assay for HSV-2 in six-well plates. Biological replicates were conducted as outlined in Figure 1 on one sample of HSV-2. Serial 10-fold dilutions of an HSV-2 sample (starting at the 10-2 dilution) were added to Vero cells, incubated, then stained with crystal violet. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62962/62962fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Titration plate with purple wells, dilution series for compound concentration analysis, experiment setup." class="xfigimg" src="/files/ftp_upload/62962/62962fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Plaque assay for HSV-1 in twelve-well plates. Biological replicates were conducted as outlined in Figure 1 on one sample of HSV-1. Serial 10-fold dilutions of an HSV-1 sample (starting at the 10-2 dilution) were added to Vero cells, incubated, then stained with crystal violet. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62962/62962fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Dilution factor</td>
		</tr>
		<tr>
			<td>Replicate</td>
			<td>10-3</td>
			<td>10-4</td>
			<td>10-5</td>
			<td>10-6</td>
		</tr>
		<tr>
			<td>1</td>
			<td>5721</td>
			<td>635</td>
			<td>81</td>
			<td>5</td>
		</tr>
		<tr>
			<td>2</td>
			<td>4592</td>
			<td>365</td>
			<td>42</td>
			<td>0</td>
		</tr>
		<tr>
			<td>3</td>
			<td>5,519</td>
			<td>521</td>
			<td>53</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: Representative plaque counts from one successful assay. Serial dilutions from each biological replicate were conducted; representative plaque counts (starting at the 10-3 dilution) are shown for each dilution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Dilution factor</td>
		</tr>
		<tr>
			<td>Replicate</td>
			<td>10-3</td>
			<td>10-4</td>
			<td>10-5</td>
			<td>10-6</td>
		</tr>
		<tr>
			<td>1</td>
			<td>15225</td>
			<td>635</td>
			<td>450</td>
			<td>42</td>
		</tr>
		<tr>
			<td>2</td>
			<td>4592</td>
			<td>365</td>
			<td>42</td>
			<td>0</td>
		</tr>
		<tr>
			<td>3</td>
			<td>5519</td>
			<td>5400</td>
			<td>53</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 2: Representative plaque counts from one unsuccessful assay. Serial dilutions from each biological replicate were conducted; representative plaque counts (starting at the 10-3 dilution) are shown for each dilution.

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 ...

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JoVE Journal

Immunology and Infection

5.2K Views.  Towson University. 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.

Video: Plaquing of Herpes Simplex Viruses

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

Neuroscience

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer immunotherapy. OVs, including oHSV, selectively replicate in and kill cancer cells (sparing healthy/normal cells) while inducing anti-tumor immunity. Because of these unique properties, oHSV-based treatment strategies are being increasingly used for the treatment of cancer, preclinically and clinically, including FDA-approved talimogene laherparevec (T-Vec). Growth, purification, and titration are three essential laboratory techniques for any OVs, including oHSVs, before they can be utilized for experimental studies. This paper describes a simple step-by-step method to amplify oHSV in Vero cells. As oHSVs multiply, they produce a cytopathic effect (CPE) in Vero cells. Once 90-100% of the infected cells show a CPE, they are gently harvested, treated with benzonase and magnesium chloride (MgCl2), filtered, and subjected to purification using the sucrose-gradient method. Following purification, the number of infectious oHSV (designated as plaque-forming units or PFUs) is determined by a &#34;plaque assay&#34; in Vero cells. The protocol described herein can be used to prepare high-titer oHSV stock for in vitro studies in cell culture and in vivo animal experiments.

In this manuscript, we describe a simple method of growth, purification, and titration of the oncolytic herpes simplex virus for preclinical use.

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer ...

Virus Plaque Assay: A Technique to Measure Infectious Herpes Simplex Virus via Quantification of Plaques Formed in Monolayer of Virus-Infected Cells

Source: Sadowski, L. A. et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62962">Plaquing of Herpes Simplex Viruses.</a> J. Vis. Exp. (2021)
This video describes the virus plaque assay of the infectious herpes simplex virus or HSV. The diluted viral particles are inoculated to the monolayer of epithelial cells. Upon incubation, the virus forms plaques and is counted visually to determine the infectious virus titer.

Source: Sadowski, L. A. et al. Plaquing of Herpes Simplex Viruses. J. Vis. Exp. (2021)
This video describes the virus plaque assay of the infectious ...

Plaquing of Herpes Simplex Viruses

Summary

Explore More Videos

Chapters in this video

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Virus Plaque Assay: A Technique to Measure Infectious Herpes Simplex Virus via Quantification of Plaques Formed in Monolayer of Virus-Infected Cells