Plaquing of Herpes Simplex Viruses

Summary

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.

Abstract

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.

Introduction

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 cells^5,6. 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 assays^{5,10,11,12,13,14,15,16}, 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.

Protocol

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

1. The day before initiating the plaque assay, trypsinize Vero cells and resuspend them in regular Dulbecco'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.
2. Count the cells by one's preferred method (e.g., a standard hemocytometer with Trypan Blue exclusion)¹⁹.
3. 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.
4. Allow the cells to grow overnight at 37 °C/5% CO₂ in a humidified incubator.
   ​NOTE: Humidity is maintained with a pan of distilled water containing an algicide in the bottom of the incubator.

2. Sample dilution

1. 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.
2. The day 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.
3. 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.
4. Keep all the viral dilutions on ice (no longer than 1 h) until ready to add these diluted virus samples to the cells.

3. Addition of virus to the cells

1. 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.
2. Carefully add the diluted virus sample (100-400 µL and 50-200 µL 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.
3. 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 °C to allow the virus to adsorb.
4. 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's preference dictates that decision.
5. To diminish the possibility of inadvertently counting unabsorbed inoculum, remove the virus sample from the wells with 1000 µL 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.
6. Place 2.5 mL of a methylcellulose overlay (dissolve 5% methylcellulose w/w in 100 mL of PBS, autoclave for 15 min at 121 °C and 15 psi on a liquid cycle, then add 375 mL of DMEM plus 25 mL of FBS).
7. 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.
8. Disinfect the waste beaker, typically with the addition of bleach, an iodophor, or a similar virucide, before removing it from the biosafety cabinet.

4. Staining for plaques

1. 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.
2. 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.
3. Incubate the plate with all wells filled with the stain for 30 min at 37 °C. At this point, disinfect the waste beaker as above in step 3.8.
4. 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.
5. At this point, ensure that there are approximately 10-fold differences in the number of plaques across each dilution series.
6. Allow the plates to dry overnight upside down, after which the individual plaques may be counted.

5. Counting plaques and determining virus titer

1. 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 ½-log dilutions instead of full-log dilutions.
2. 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.
3. Perform the calculations in steps 5.3.1 to 5.3.5, considering the use of mock data in Table 1.
   1. 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.
   2. 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.
   3. 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.
   4. 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 ± 0.98 x 10⁷ PFU/mL for this sample.

Results

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.

Discussion

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 assay^{5,6,10,11,12,13,14,15,16,20}. Althoug...

Materials

Name	Company	Catalog Number	Comments
12-well plates	Corning	3512
6-well plates	Corning	3516
Alpha-MEM	Lonza	12169F
Antibiotic/antimycotic	Gibco	15240096
Crystal violet	Alfa Aesar	B2193214
DMEM	Gibco	11965092
Dulbecco's PBS (no Mg++ or Ca++)	Gibco	14190144
Fetal calf serum	Millipore-Sigma	TMS-013-B
L-alanyl-L-glutamine (Glutamax)	Gibco	GS07F161BA
Hemacytometer	Thermo Fisher	02-671-54
Methylcellulose	Millipore-Sigma	27-441-0
Quaternary agent (Lysol I.C.)	Thermo Fisher	NC9645698
Trypan Blue	Corning	25900CI
Trypsin	Cytiva	SH30042.01
Vero cells	ATCC	CCL-81