Name: viral concentration determination through plaque assays: using traditional and novel overlay systems
Uploaded: 2014-11-04T14:00:00.000+00:00
Duration: 9 min 28 s
Description: Watch this Scientific Journal Video about Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems at JoVE.com

We would like to thank FMC BioPolymer USA for product samples of Avicel. This work was supported through the Defense Threat Reduction Agency grant HDTRA1-13-1-0005 to KK and the NIH research grant 1R15AI100001-01A1to KK.

1. Preparation of Cells and Reagents
<ol>
	<li>The day prior to the assay, plate appropriate host cells for the virus in question (Table 1 and 2) at 90 - 100% confluency.</li>
	<li>Prepare fixing solution of 10% formaldehyde in dH2O. In example, mix&#160;5.56 ml of 36% stock formaldehyde with 14.44 ml distilled H2O (dH2O). 
	NOTE: Use appropriate safe handling practices and ventilation when using formaldehyde.</li>
	<li>Prepare crystal violet stain: 1% Crystal Violet (CV) in 20% ethanol and dH2O.</li>
	<li>Prepare 2x plaque media (cell type dependent at 2x concentration; see Table 2). Filter using a 0.2 &#181;m filter if any reagents are not sterile.</li>
	<li>Preparation of immobilizing overlays (Table 3)
	<ol>
		<li>For liquid overlays, make a sterile 2.4% solution of Avicel in dH2O. To prevent clumping, add the powder to a flask containing water that is rapidly mixing with a stir bar, pour Avicel in slowly and stir rapidly at room temperature (RT). When the solution proves too viscous for a stir bar to homogenize, switch to a flask shaker for &#62;30 min with heavy agitation to ensure even homogenization. 
		NOTE: Stock solutions can be made at lower concentrations, but the solutions may separate and will need to be remixed to ensure homogenization. Working solutions of Avicel can be used ranging from 0.6-3%.</li>
		<li>After homogenization, autoclave the solution and store sealed at RT.</li>
		<li>For agarose and carboxymethyl cellulose (CMC) overlays, prepare a stock solution in dH2O of 2% CMC or 0.6% agarose. Mix with a stir bar and autoclave or microwave to bring into solution.</li>
	</ol>
	</li>
</ol>
2. Dilutions and Infections
<ol>
	<li>The day after plating, visually check the confluency and viability of the cells prior to starting the assay. Ensure standard cellular morphology and a ~90% confluent monolayer is present.</li>
	<li>Perform a tenfold serial dilution of the infectious samples. Use the cellular growth media for viral propagation as the diluent (Table 2). Vary the number of dilutions required based on the expected titer of the virus in question, and always use an uninfected control sample to independently ensure cellular viability and aid in plaque identification.</li>
	<li>From the serial dilutions, infect the cells for 45 min to 1 hr (Table 1). Use a sufficient volume of inoculum to cover the cells, while keeping the volume as low as possible to maximize viral contact with the monolayer (Table 4). Gently rock plates every 20 min to ensure even coverage and prevent the cellular monolayer from drying.</li>
	<li>After the infection, overlay an appropriate volume of immobilizing medium directly to the inoculums in the well (Table 2) using a 1:1 mix of 2x plaque media and the immobilizing overlay of choice (CMC, agarose or Avicel). Gently rock to mix.
	<ol>
		<li>For liquid overlays, mix 1:1 with warmed 2x plaque media and 1.2 - 2.4% RT stock Avicel to obtain a working solution of 0.6-1.2% Avicel overlay medium.</li>
		<li>For an agarose overlay,&#160;use a 1:1 mixture of warmed 2x plaque media and a stock solution of heated 0.6% agarose, place in a 56 &#176;C water bath for 30 min to equilibrate the temperature obtaining a final agarose/overlay concentration of 0.3%.</li>
		<li>For CMC, prepare a 2% stock solution and treat as described for the agarose overlay.</li>
		<li>When applying the overlay to the monolayer, always equilibrate hot agarose or CMC in a 56 &#176;C water to prevent damage to the monolayer. Ensure the solution is warm, but not hot to the touch to prevent cell death and reduced viral titers. Most agarose&#160;overlays will begin to solidify below 42 &#176;C, work quickly and/or prepare small batches to prevent solidification while handling.</li>
	</ol>
	</li>
	<li>After addition of the overlay, incubate plates to produce distinct plaques that are clearly countable. Plaque formation can take 2&#8211;14 days depending on the virus being analyzed, Table 1. 
	NOTE: Once liquid overlaid plates are placed in the incubator, do not move them. Movement of the liquid overlays during the incubation period will result in smeared plaques. Agarose and CMC overlays are semisolid and can be moved or checked periodically under a light microscope to monitor plaque development.</li>
</ol>
3. Fixing and Staining Cells
<ol>
	<li>To fix the cells, pour off or aspirate the Avicel overlay, and fix cells using the 10% formaldehyde solution for 30 min to overnight (&#60;1 ml per well for a 6 well plate).
	<ol>
		<li>For agarose or CMC, directly add the formaldehyde solution to the overlay for 1 hr to overnight. 
		NOTE: Samples can be kept for extended periods of time in the fixative provided it does not evaporate and dry out as this can distort the monolayer.</li>
	</ol>
	</li>
	<li>Prior to staining and after fixation, discard the formaldehyde and remove the semisolid plugs for the agarose and CMC with either running water or manually with a spatula. Rinse Avicel plaques with water to remove residual overlay/fixative prior to staining.</li>
	<li>For staining, cover the cells with a minimal amount of crystal violet solution for ~15 min. Rock plates if necessary to ensure even coverage.</li>
	<li>Gently wash off the crystal violet stain with water. Once fixed, stained, and dried, store plaques indefinitely for future analysis.</li>
</ol>
4. Determining Viral Titers
<ol>
	<li>Count the plaques in each well, taking the average for any technical replicates of the same dilution. For large plate formats, discount wells with fewer than 5 or greater than 100 plaques. Take note of plaque size and morphology. The negative control should have a uniform monolayer and can be used as a reference control.</li>
	<li>Determine the viral titer of the stock sample by taking the average number of plaques for a dilution and the inverse of the total dilution factor.</li>
</ol>
<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/52065/52065eq1.jpg" width="300" />
NOTE: As an example, 30 and 32 plaques counted for replicates of the 1 x 10-7 dilution [31 (average)/10-7 (dilution) x 0.4 ml (inoculum)] would yield a titer of 7.75 x 108 pfu/ml.

<ol>
	<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>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 Harb. Symp. Quant. Biol. 18, 273-279 (1953).</li><li>Hartley, J. W., Rowe, 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=Tissue+culture+cytopathic+and+plaque+assays+for+mouse+hepatitis+viruses.">Tissue culture cytopathic and plaque assays for mouse hepatitis viruses.</a> Proc. Soc. Exp. Biol. Med. Soc. Exp. Biol. Med. N. Y. 113, 403-406 (1963).</li><li>Juarez, D., Long, K. C., Aguilar, P., Kochel, T. J., Halsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+plaque+assay+methods+for+alphaviruses.">Assessment of plaque assay methods for alphaviruses.</a> J. Virol. Methods. 187, 185-189 (2013).</li><li>Shurtleff, A., Keuhne, A., Biggins, J., Keeney, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+alternative+overlays+in+plaque+assays.">Use of alternative overlays in plaque assays.</a> , (2011).</li><li>Shurtleff, A. C., 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=Standardization+of+the+Filovirus+Plaque+Assay+for+Use+in+Preclinical+Studies.">Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies.</a> Viruses. 4, 3511-3530 (2012).</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> Virol. J. 3, 63 (2006).</li><li>Klebe, R. J., Harriss, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technically+simple+&#8216;non-lethal&#8217;+vital+staining+procedure+for+viral+plaque+and+cell+transformation+assays.">A technically simple &#8216;non-lethal&#8217; vital staining procedure for viral plaque and cell transformation assays.</a> Arch. Virol. 81, 359-362 (1984).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells.">Replication and plaque assay of influenza virus in an established line of canine kidney cells.</a> Appl. Microbiol. 16, 588-594 (1968).</li><li>Abiko, C., 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=Outbreak+of+Human+Metapneumovirus+Detected+by+Use+of+the+Vero+E6+Cell+Line+in+Isolates+Collected+in+Yamagata,+Japan,+in+2004+and+2005.">Outbreak of Human Metapneumovirus Detected by Use of the Vero E6 Cell Line in Isolates Collected in Yamagata, Japan, in 2004 and 2005.</a> J. Clin. Microbiol. 45, 1912-1919 (2004).</li><li>Baer, 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=Induction+of+DNA+damage+signaling+upon+Rift+Valley+fever+virus+infection+results+in+cell+cycle+arrest+and+increased+viral+replication.">Induction of DNA damage signaling upon Rift Valley fever virus infection results in cell cycle arrest and increased viral replication.</a> J. Biol. Chem. 287, 7399-7410 (2012).</li><li>Austin, D., 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=p53+Activation+following+Rift+Valley+Fever+Virus+Infection+Contributes+to+Cell+Death+and+Viral+Production.">p53 Activation following Rift Valley Fever Virus Infection Contributes to Cell Death and Viral Production.</a> PLoS ONE. 7, (2012).</li><li>B&#246;ttcher-Friebertsh&#228;user, E., Stein, D. A., Klenk, H. -. D., Garten, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+influenza+virus+infection+in+human+airway+cell+cultures+by+an+antisense+peptide-conjugated+morpholino+oligomer+targeting+the+hemagglutinin-activating+protease+TMPRSS2.">Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2.</a> J. Virol. 85, 1554-1562 (2011).</li><li>Youil, 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=Comparative+study+of+influenza+virus+replication+in+Vero+and+MDCK+cell+lines.">Comparative study of influenza virus replication in Vero and MDCK cell lines.</a> J. Virol. Methods. 120, 23-31 (2004).</li><li>Tobita, K., Sugiura, A., Enomoto, C., Furuyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plaque+assay+and+primary+isolation+of+influenza+a+viruses+in+an+established+line+of+canine+kidney+cells+(MDCK)+in+the+presence+of+trypsin.">Plaque assay and primary isolation of influenza a viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin.</a> Med. Microbiol. Immunol. (Berl.). 162, 9-14 (1975).</li></ol>

The authors have nothing to disclose and no competing or financial interests in the protocol or products described.

The most critical factor for a successful plaque assay lies in the optimization of the protocol for the particular viral culture in question as conditions can vary significantly. Key points to take into account are: host cellular compatibility with the virus in question, appropriate viral growth conditions, sufficient dilution ranges in order to clearly differentiate plaques, and correct overlay selection and staining for the cells and virus in question.
While VEEV and RVFV both grow under very similar conditions utilizing many of the same host cell types, differences in plaque morphology and growth kinetics vary significantly. When using Vero cells for plaque assays, which have traditionally been used as a propagation and indicator cell line for hemorrhagic fever viruses and alphaviruses, VEEV typically grows to higher titers than RVFV and demonstrates increased replication kinetics, developing large uniform plaques at 48 hpi4,10&#8211;12. In contrast to VEEV, RVFV requires 72 hpi and demonstrates plaques that are typically much smaller and variable in size.
In opposition to RVFV and VEEV, the influenza virus is highly host cell specific and can be difficult to propagate through tissue culture. For the influenza virus, viral entry and fusion is normally initiated by the binding of the cell surface receptor with the viral glycoprotein hamagglutinin (HA), which mediates entry into the target cell through binding with the host cell&#8217;s &#945;-sialic acid surface receptor. HA is a trimeric glycoprotein that is present in the membrane envelope of all Influenza viruses and requires cleavage into the subunits HA1 and HA2 by a specific host cell protease. To further complicate the issue, these cleavage sites can often vary between viral strains13. As the expression of proteases capable of cleaving HA is restricted to specific tissues, proteases are often added to cell culture media in order to facilitate viral fusion and entry into the selected host cell13. TPCK-trypsin is one example of a commonly used protease that is used in cell culture with both MDCK and Vero cells: facilitating multi-cycle replication (in the absence of any trypsin inactivating serum) through the proteolytic activation of viral HA14,15.
As demonstrated in this study and others, differences in viral life cycles can influence overlay selections, plate formats, and collection times, with significant variances among differing classes and species of viruses. In our study, agarose overlays demonstrated clearer plaques at higher titers than either CMC or liquid overlays for both RVFV and Influenza B virus, reinforcing its utility among a wide range of viruses and cell types. Using a low final concentration of agarose greatly aided in removing the solid plugs and simplified staining. CMC overall demonstrated the poorest efficacy as an overlay for all three viruses tested and produced very small and indistinct plaques for Influenza B virus. Although overall CMC demonstrated the least desirable characteristics, its use in rapidly growing and extremely virulent viruses could prove advantageous, as it did appear to reduce plaque size with only a minimal decrease in titer. The liquid overlay proved to be the most versatile in that it was extremely simple to prepare, increased ease of use, and was comparable to agarose across all of the viruses selected. In a higher throughput 96-well plate format, application and removal was not inhibited due to solidification as with traditional overlays, and provided accurate and consistent results. A consideration when using liquid overlays lies in the opaque coloring and inability to monitor plaque formation as the plates cannot be moved until the point of collection, limiting this adaptation to viruses with previously characterized replication kinetics.
Minor modifications in plaque assay conditions can significantly alter results, and the evaluation and performance of any new system or technique is always warranted. While an optimized and standardized plaque assay protocol does not exist for every situation, our report demonstrate the versatility and ease of use in traditional as well as novel liquid overlays for plaque assays, while providing a sufficient background for further user modifications.

The accurate isolation and quantification of viable viral samples has consistently been an ongoing research goal in virology. It was not until the advent of the plaque assay in 1952 that a means to quantitatively and qualitatively calculate animal viral titers was first developed1,2. This technique was first adapted and modified from phage assays, which had previously been used to calculate titers of stock bacteriophages in plant biology1,2. While alternative means for viral quantification have since been developed and adapted, such as immunoassays, fluorescence and transmission electron microscopy, tunable resistive pulse sensing (TRPS), flow cytometry, recombinant reporter systems, and quantitative reverse transcription polymerase chain reaction (qRT-PCR), these methods fail to identify and quantitate replication competent virons1,3. While advances in technologies and techniques continue to refine and alter the landscape; plaque assays continue to represent the gold standard in determining viral concentrations for infectious lytic virons1,4.
During a plaque assay, a confluent monolayer of host cells is infected with a lytic virus of an unknown concentration that has been serially diluted to a countable range, typically between 5-100 virions. Infected monolayers are then covered with an immobilizing&#160;overlay medium to prevent viral infection from indiscriminately spreading through either the mechanical or convectional flow of the liquid medium during viral propagation. While solid or semisolid overlays such as agarose,&#160;methyl cellulose or carboxymethyl cellulose (CMC) have traditionally been used, liquid overlays have become an increasingly attractive alternative with the development of novel liquid overlays such as Avicel5&#8211;7. Plaque assays utilizing liquid versus traditional overlays have several advantages as the overlay can be applied at room temperature, and application and removal is significantly easier. As liquid overlays do not require warming, delicate and heat labile viruses may also prove easier to plaque.
After the initial infection and application of the immobilizing overlay, individual plaques, or zones of cell death, will begin to develop as viral infection and replication are constrained to the surrounding monolayer. Infected cells will continue the replication-lysis-infection cycle, further propagating the infection, resulting in increasingly distinct and discrete plaques. Depending on the viral growth kinetics and host cell used, a visible plaque will normally form within 2-14 days. Cellular monolayers may then be counted with a standard bright field microscope, or more typically fixed and counterstained by neutral red or crystal violent in order to readily identify plaques with the naked eye. There are a wide variety of plaque counter stains available, each offering their specific advantages and disadvantages. Crystal violet is typically added at the point of collection and after the fixation/removal of the overlay, providing a rapid and distinct counter stain which allows for the identification of very small plaques when mixed morphology is present. Neutral red has the advantage of early application and constant contact with the overlay, allowing for the live monitoring of developing plaque formation, which is particularly useful when working with an unknown virus or replication kinetics. We have however found that staining is typically not as distinct when using neutral red. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) offers several advantages, as the yellow colored dye stains live cells dark blue and viral plaques can be counted without removal of the overlay as with neutral red. The high contrast between live and dead cells afforded by MTT also permits the detection of small plaques at an earlier time point post infection, although storage would still require removal of the overlay8. As crystal violet can be simply made in a solution of water and alcohol, and provides a high degree of sensitivity for mixed plaque morphology, we have chosen it as a preferred and simplified counter stain for the protocol demonstrated as we are utilizing several families of well characterized viruses.
After fixing and staining the infected cellular monolayer, plaques are counted in order to titer viral stock samples in terms of plaque forming units (pfu) per milliliter. A log drop should be noted between serial dilutions and, depending on plate size, between 5-100 plaques counted, with a negative control used as a reference. Statistically samples will vary by 10% for every 100 plaques counted when comparing sample replicates3. The advantage of using plaque assays to determine viral titers lies in their ability to quantitate the actual number of infectious viral particles within the sample. As multiple virions could potentially infect a single cell, the terminology of units versus virons is used during plaque titrations1,2.
Plaque morphology can vary dramatically under differing growth conditions and between viral species. Plaque size, clarity, border definition, and distribution should all be noted as they can provide valuable information on the growth and virulence factors of the virus in question.
Basic plaque assay principles can also be adapted and modified in a number of different ways, such as in the use of&#160;focus forming assays (FFAs). FFAs do not rely on cell lysis and counterstaining to detect plaque formation, but rather employ immunostaining techniques to directly detect intracellular viral proteins through tagged antibodies. Increased sensitivity, decreased incubation times after infection, and most importantly the ability to quantitate non-lytic viruses are all distinct advantages when employing FFAs. While widely used, the critical limiting factors in FFAs versus a traditional plaque assay lies in the need for appropriate antibodies and the ability to only probe for viral protein subunits versus actual infectious virons4.
For the purpose of this study, we will limit our discussion to classical plaque assays and describe the use of traditional solid and semisolid overlays (agarose and CMC), along with novel liquid microcrystalline cellulose overlays.

<table><tbody><tr><td>6-well CellStar plate for tissue culture</td><td>Greiner Bio-One</td><td>658 160</td><td></td></tr><tr><td>12-well CellStar plate for tissue culture</td><td>Greiner Bio-One</td><td>665 180</td><td></td></tr><tr><td>96-well CellStar plate for tissue culture</td><td>Greiner Bio-One</td><td>655 180</td><td></td></tr><tr><td>96-2ml deep well plate</td><td>USA Scientific</td><td>C15046314</td><td>For serial dilutions</td></tr><tr><td>DMEM</td><td>Quality Biologicals</td><td>112 013 101</td><td>Cell Media/Diluent</td></tr><tr><td>2x EMEM for plaques</td><td>Quality Biologicals</td><td>115 073 101</td><td>Warm to 37 &amp;#176;C</td></tr><tr><td>MEM 100x&amp;nbsp;</td><td>Cellgro</td><td>25 025 Cl</td><td></td></tr><tr><td>Sodium Pyruvate&amp;nbsp;</td><td>Cellgro</td><td>25 000 Cl</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140 122</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030 081</td><td></td></tr><tr><td>HyClone FBS&amp;nbsp;</td><td>Thermo Scientific</td><td>SH30910.03</td><td>Heat inactivate before use at 62 &amp;#176;C for 30 min</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldritch</td><td>A7030-50G</td><td></td></tr><tr><td>Trypsin TPCK</td><td>Sigma Aldritch</td><td>T1426-50mg</td><td>Make aliquots/Avoid freeze thawing</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>DEAE-Dextran</td><td>Sigma Aldritch</td><td>D9885-10G</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma Aldritch</td><td>C3886-25G</td><td></td></tr><tr><td>Formaldehyde Solution</td><td>Sigma Aldritch</td><td>F8775-500ml</td><td></td></tr><tr><td>Ethanol denatured Reagent Grade</td><td>Sigma Aldritch</td><td>362808-1L</td><td></td></tr><tr><td>Avicel RC-591 NF</td><td>FMC BioPolymer USA</td><td>RC-591 NF</td><td>Shake vigorously when reconsitiuting&amp;nbsp; for &gt;30 min to homogenize</td></tr><tr><td>UltraPure Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16500-100</td><td></td></tr><tr><td>Carboxymethyl cellulose, Sodium Salt Medium Viscosity&amp;nbsp;</td><td>Sigma Aldrich</td><td>C4888</td><td></td></tr></tbody></table>

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.

The ability of plaque assays to accurately assess viral titers relies upon numerous factors: appropriate host cell selection, proper media and growth conditions for cellular and viral viability, immobilized viral propagation, and an accurate determination of the viral incubation period to allow adequate time for distinct and countable plaque formation.
For this study, viruses from three representative families were chosen to demonstrate differences in: overlay selection, incubation periods, and plaque morphology across differing sample types. Venezuelan Equine Encephalitis (VEEV) was selected as a (+)ssRNA viral model, which can cause significant disease in equine species and humans and represents the Togaviridae family. Influenza B Taiwan strain, a segmented (-)ssRNA virus primarily infecting humans, represents the Orthomyxoviridae family. Rift Valley fever virus (RVFV), a (-)ssRNA arthropod born virus primarily infecting arthropods, ruminants and humans, was selected as a representative of the Bunyaviridae family.
For RVFV (Figure 1), titers were determined from a stock solution of a recombinant live attenuated MP12 strain of RVFV in a 12 well plate format utilizing CMC, agarose, or Avicel overlays which were incubated side-by-side for 72 hr post infection (hpi). A representative plate showing dilutions ranging from 10-4 to 10-7 can be seen in Panel A. Plaques using CMC and agarose overlays showed small, clear, and distinct plaques with a well-defined circular border. Plaques with a liquid overlay were slightly more abundant and larger in comparison to agarose and CMC plaques, and provided a less distinct border. Viral titers were compared in Panel B with all of the overlays performing comparably.
In order to obtain a clearer visual comparison among overlays for MP12 along with a larger sample size to determine reproducibility, a 6 well plate was also trialed in triplicate (Figure 2). In the 6 well plate format, the use of a CMC overlay showed smaller plaques than either agarose or liquid overlays, which were comparable in size to each other. While viral titers were similar between all three overlays (Panel D), plaques formed in agarose and liquid overlays proved easier to count due to their increased size.
In contrast with RVFV, VEEV titers and plaque morphology among the differing overlays varied markedly (Figure 3A). Plaques formed in CMC overlays demonstrated a clear and distinct morphology when using a 12 well plate format, at the expense of plaque size and sensitivity (Panel B). In contrast to CMC, the use of agarose and liquid overlays resulted in significantly larger plaques, indicating lower viral inhibition and increased sensitivity to VEEV replication. This was previously confirmed when solely comparing agarose versus CMC in a paper published by Juarez et al.4. While agarose and liquid overlays produced larger plaques than CMC, the plaques had poorly defined borders and were difficult to count in a 12 well format, with liquid overlays providing the greatest border diffusion. When plaques were trialed in 6 well plates (Figure 4), the larger 6 well format negated the issue of overtly large plaques that were difficult to differentiate in the 12 well format, with agarose and liquid overlays proving superior to the CMC overlays in terms of plaque definition and sensitively (Figure 4D).
In comparison to RVFV or VEEV, influenza provides several unique challenges when plaquing, such as the requirement of an external protease. The sensitivity of influenza virus to differing overlay selections has also been well documented in the past as significant changes have been noted when modifications as minor as differing brands of agarose have been used9.
Interestingly for the Influenza B Taiwan strain, the use of CMC as an overlay resulted in markedly smaller plaques that were difficult to count and proved difficult to reliably score (Figure 5A). The use of an agarose overlay provided the best plaques (Panel C), and resulted in a darker background stain (likely due to increased monolayer viability), and showed clearer and sharper plaques in direct comparison to the use of the liquid overlay (Panel B).
A distinct advantage of liquid polymers over solid and semisolid overlays, such as agarose and CMC, lies in the ease of removal and application. Semisolid overlays require heating, and solidification can prove problematic when handling and removing. In order to capitalize on these advantages and to determine the practicality of utilizing Avicel in a high-throughput manner for RVFV, a 96 well plate format was trialed at varying overlay concentrations (Figure 6). For RVFV MP-12, dilutions were performed in quadruplicate at both 0.6 and 1.2% final concentrations of Avicel. Overlay application and removal proved simple, with no apparent differences among replicates or between concentrations noted, demonstrating a high degree of reproducibility. When scoring, plaques were distinct and countable to the naked eye, demonstrating the feasibility of utilizing liquid overlays in a high throughput manner for RVFV.
<img alt="Figure 1" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52065/52065fig1highres.jpg" /> 
Figure 1: RVFV plaque overlay comparisons utilizing 12 well plates. Veros were plated at 2.5 x 105 cells in 12 well plates and infected with 200 &#181;l using the same serially diluted starting sample of MP12. After infection 1.5 ml overlays of 0.3% agarose,0.6% Avicel, or 1% CMC (final concentrations), were applied in order to directly compare the overlays as demonstrated in Panel A. Plaques were counted and titered in Panel B .
<img alt="Figure 2" src="/files/ftp_upload/52065/52065fig2highres.jpg" /> 
Figure 2: RVFV plaque overlay comparisons utilizing 6 well plates. Veros were plated at 5 x 105 cells in 6 well plates and infected with 400 &#181;l using the same serially diluted starting sample of MP12. Three ml overlays of 0.3% agarose, 0.6% Avicel, or 1% CMC, were applied in order to directly compare the overlays as demonstrated in Panel A, B, and C. Separate experiments were carried out identically as described for Panels A-C, with plaques counted and titered in Panel D (N = 3).
<img alt="Figure 3" src="/files/ftp_upload/52065/52065fig3highres.jpg" /> 
Figure 3: VEEV plaque overlay comparisons utilizing 12 well plates. Veros were plated at 2.5 x 105 cells in 12 well plates and infected with 200 &#181;l using the same serially diluted starting sample of the VEEV TC-83 vaccine strain. After infection 1.5 ml overlays of 0.3% agarose, 0.6% Avicel, or 1% CMC, were applied in order to directly compare the overlays as demonstrated in Panel A. Plaques were counted and titered in Panel B.
<img alt="Figure 4" src="/files/ftp_upload/52065/52065fig4highres.jpg" /> 
Figure 4: VEEV plaque overlay comparisons utilizing 6 well plates. Veros were plated at 5 x 105 cells in 6 well plates and infected with 400 &#181;l using the same serially diluted starting sample of VEEV TC-83. After infection, 3 ml overlays of 0.3% agarose, 0.6% Avicel, or a 1% CMC, were applied in order to directly compare the overlays as demonstrated in Panels A, B and C. Separate experiments were carried out identically as described for Panels A - C, with plaques counted and titered in Panel D (N = 3).
<img alt="Figure 5" src="/files/ftp_upload/52065/52065fig5highres.jpg" /> 
Figure 5: Influenza plaque overlay comparisons. MDCK cells were plated at 5 x 105 cells in 6 well plates and infected with 400 &#181;l of inoculum using the same serially diluted starting sample of Influenza B Taiwan. No fetal bovine serum (FBS) was used in the growth media or overlays, as FBS can inhibit Influenza propagation through inhibition of certain proteases which are required for viral fusion. TPCK-trypsin was added to all of the overlays prior to application in order to facilitate viral fusion and entry with the host cells. After infection, 3 ml overlays of 0.3% agarose, 0.6% Avicel, or 1% CMC were applied in order to directly compare the overlays as demonstrated in Panel A , B and C. Separate experiments were carried out identically as described in Panels A - C, with plaques counted and titered in Panel D (N = 3). While an average was taken for CMC plaques they proved difficult to reliably count as they demonstrated diffuse borders and very small plaque sizes.
<img alt="Figure 6" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52065/52065fig6highres.jpg" /> 
Figure 6: High throughput plaque overlays. A 96 well plate of Veros plated at 3 x 104 cells per well, were infected with 50 &#181;l of inoculum using the same serially diluted starting sample of RVFV MP12 for 1 hr, in quadruplicate. For the overlays, 0.6 and 1.2% final concentrations of Avicel were trialed in order to determine the feasibility and reproducibility of utilizing liquid overlays in a high-throughput manner, Panels A and B.


<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>RVFV</td>
			<td>VEEV</td>
			<td>Influenza B</td>
		</tr>
		<tr>
			<td>Cell type</td>
			<td>Vero</td>
			<td>Vero</td>
			<td>MDCK</td>
		</tr>
		<tr>
			<td>Infection period</td>
			<td>1 hr</td>
			<td>1 hr</td>
			<td>45 min</td>
		</tr>
		<tr>
			<td>Incubation time</td>
			<td>3 days</td>
			<td>2 days</td>
			<td>3 days</td>
		</tr>
	</tbody>
</table>

Table 1: Plaque Assay Inoculation Conditions and Cell Types
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>RVFV</td>
			<td>VEEV</td>
			<td>Influenza B</td>
		</tr>
		<tr>
			<td>Cell Type</td>
			<td>Vero</td>
			<td>Vero</td>
			<td>MDCK</td>
		</tr>
		<tr>
			<td>Growth Type</td>
			<td>DMEM1</td>
			<td>DMEM1</td>
			<td>DMEM2</td>
		</tr>
		<tr>
			<td>Plaque Media</td>
			<td>2xEMEMA</td>
			<td>2xEMEMA</td>
			<td>2xEMEMB</td>
		</tr>
	</tbody>
</table>
1. Dulbecco&#39;s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum, 1% L-Glutamine, 1% Penicllin/Streptomycin. 
2. Dulbecco&#39;s Modified Eagle Medium supplemented with 1% L-Glutamine, 1% Penicllin/Streptomycin, 0.2% Bovine Serum Albumin, 0.025% HEPES, DEAE-Dextran 50ug/ml. 
A. 2x Minimal Essential Media (500 ml) supplemented with 5% FBS (25 ml), 1% Minimum Essential Amino Acids (5 ml), 1% Sodium Pyruvate (5 ml), 1% L-Glutamine (5 ml), 2% Pen/Strep (10 ml). 
B. 2x Minimal Essential Media (500 ml) supplemented with 0.2% Bovine Serum Albumin, 1% Minimum Essential Amino Acids (5 ml), 50ug/ml, 0.025% HEPES, DEAE-Dextran, Trypsin-TPCK* 
*Just prior to preparation, add 1 &#181;l per 25 ml of 2 &#181;g/ml stock of TPCK-Trypsin to the aliquot you will be using to mix with agarose for plaques.

Table 2: Plaque and Viral/Cellular Growth Medias
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>RVFV</td>
			<td>VEEV</td>
			<td>Influenza B</td>
		</tr>
		<tr>
			<td>Cell type</td>
			<td>Vero</td>
			<td>Vero</td>
			<td>MDCK</td>
		</tr>
		<tr>
			<td>Infection period</td>
			<td>1 hr</td>
			<td>1 hr</td>
			<td>45 min</td>
		</tr>
		<tr>
			<td>Incubation time</td>
			<td>3 days</td>
			<td>2 days</td>
			<td>3 days</td>
		</tr>
	</tbody>
</table>
Overlay solutions will not expire when made so long as sterility is maintained.

Table 3: Overlays Stock 
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>6 well</td>
			<td>12 well</td>
			<td>96 well</td>
		</tr>
		<tr>
			<td># of cells/well</td>
			<td>5 x 105</td>
			<td>2.5 x 105</td>
			<td>3 x 104</td>
		</tr>
		<tr>
			<td>Volume of innoculum (&#956;l)</td>
			<td>400</td>
			<td>200</td>
			<td>50</td>
		</tr>
		<tr>
			<td>volume of overlay (ml)</td>
			<td>3</td>
			<td>1.5</td>
			<td>0.100</td>
		</tr>
	</tbody>
</table>
Table 4: Plate Formats&#160;

Watch this Scientific Journal Video about Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems at JoVE.com

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

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 ...

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111.6K Views.  George Mason University. 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.

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

Plaque Assay for Murine Norovirus

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

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

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

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. ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

Biology

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

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.

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 ...

Virus Propagation and Cell-Based Colorimetric Quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barr&#233; syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

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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Microbiology

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

Source: Yadavalli, T., et al. <a href="https://app.jove.com/v/62195">Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals.</a> J. Vis. Exp. (2021)
This video demonstrates the method to perform a plaque assay using a virus obtained from infected porcine cornea. The porcine cornea is used to test the antiviral efficacy of experimental drugs.

1. Virus infection quantification
NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.

	To ...