UConn biochemist Simon White joined the team as well. They also produced video clips of how the spike protein moves. January 13, January 12, UConn Today. News Series. Archives Contact Us. UConn University of Connecticut. Skip to content January 14, Try out PMC Labs and tell us what you think. Learn More. 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 overlays for certain heat labile viruses. 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 that a means to quantitatively and qualitatively calculate animal viral titers was first developed 1,2.
This technique was first adapted and modified from phage assays, which had previously been used to calculate titers of stock bacteriophages in plant biology 1,2.
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 virons 1,3. 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 virons 1,4.
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 virions. Infected monolayers are then covered with an immobilizing 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, 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 Avicel 5— 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 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. 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.
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 overlay 8. 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 plaques counted, with a negative control used as a reference.
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 titrations 1,2. 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 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 virons 4.
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. In example, mix 5. NOTE: Use appropriate safe handling practices and ventilation when using formaldehyde. Prepare 2x plaque media cell type dependent at 2x concentration; see Table 2.
Filter using a 0. For liquid overlays, make a sterile 2. 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. 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. Mix with a stir bar and autoclave or microwave to bring into solution. The day after plating, visually check the confluency and viability of the cells prior to starting the assay.
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.
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.
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