Name: viral concentration determination through plaque assays: using traditional and novel overlay systems
Uploaded: 2014-11-04T14:00:00
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 border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">6-well CellStar plate for tissue culture</td>
			<td style="width:127px;">Greiner Bio-One</td>
			<td style="width:129px;">658 160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">12-well CellStar plate for tissue culture</td>
			<td style="width:127px;">Greiner Bio-One</td>
			<td style="width:129px;">665 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">96-well CellStar plate for tissue culture</td>
			<td style="width:127px;">Greiner Bio-One</td>
			<td style="width:129px;">655 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">96-2ml deep well plate</td>
			<td style="width:127px;">USA Scientific</td>
			<td style="width:129px;">C15046314</td>
			<td>For serial dilutions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">DMEM</td>
			<td style="width:127px;">Quality Biologicals</td>
			<td style="width:129px;">112 013 101</td>
			<td>Cell Media/Diluent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">2X EMEM for plaques</td>
			<td style="width:127px;">Quality Biologicals</td>
			<td style="width:129px;">115 073 101</td>
			<td>Warm to 37 oC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">MEM 100X&#160;</td>
			<td style="width:127px;">Cellgro</td>
			<td style="width:129px;">25 025 Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sodium Pyruvate&#160;</td>
			<td style="width:127px;">Cellgro</td>
			<td style="width:129px;">25 000 Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Penicillin Streptomycin</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:129px;">15140 122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">L-Glutamine</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:129px;">25030 081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:269px;">HyClone FBS&#160;</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:129px;">SH30910.03</td>
			<td>Heat inactivate before use at 62 oC for 30 min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin</td>
			<td style="width:127px;">Sigma Aldritch</td>
			<td style="width:129px;">A7030-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Trypsin TPCK</td>
			<td style="width:127px;">Sigma Aldritch</td>
			<td style="width:129px;">T1426-50mg</td>
			<td>Make aliquots/Avoid freeze thawing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">HEPES&#160;</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:129px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DEAE-Dextran</td>
			<td style="width:127px;">Sigma Aldritch</td>
			<td style="width:129px;">D9885-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Crystal Violet</td>
			<td style="width:127px;">Sigma Aldritch</td>
			<td style="width:129px;">C3886-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Formaldehyde Solution</td>
			<td style="width:127px;">Sigma Aldritch</td>
			<td style="width:129px;">F8775-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Ethanol denatured Reagent Grade</td>
			<td style="width:127px;">Sigma Aldritch</td>
			<td>362808-1L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Avicel RC-591 NF</td>
			<td style="width:127px;">FMC BioPolymer USA</td>
			<td style="width:129px;">RC-591 NF</td>
			<td>Shake vigorously when reconsitiuting&#160; for &#62;30 min to homogenize</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">UltraPure Agarose&#160;</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:129px;">16500-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Carboxymethyl cellulose, Sodium Salt Medium Viscosity&#160;</td>
			<td style="width:127px;">Sigma Aldrich</td>
			<td style="width:129px;">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 ...

viral-concentration-determination-through-plaque-assays-using

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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 ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...