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

Abstract

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.

PEDF Reduces the Severity of Herpetic Simplex Keratitis in Mice

“…Samples were assayed by plaque assay as previously described. 31 The infectious specimens were performed in a 10-fold serial dilution and cultured for 6 days. The number of PFUs in each well was counted after the crystal violet staining.…”

Clone Purification, Characterization and Standardization of LaSota Strain for Developing a Live Vaccine against Newcastle Disease Virus

“…Here, we focused to generate vaccine strain which would induces less post-vaccination reactions through the clone selection procedure and then produce an efficacious live ND vaccine from it. Some markers include plaque morphology, elution time of the virus from hemagglutinates, heat stability of the hemagglutinin, and the ability of NDV strains to hemagglutinate mammalian erythrocyte have been introduced to differentiate NDV strains (20)(21)(22). Among them plaque morphology represents virus virulence feature .…”

“…Based on the morphological properties of the plaques, subpopulations from the ND strain were defined. The heterogenicity of the plaques and the presence of large plaques have been associated with pathogenic indices (10,22). Each selected plaque derived from one single virus particle was verified for pathogenicity.…”

Protein Phosphatase-1 regulates Rift Valley fever virus replication

“…Okadaic acid was used at a final concentration of 5 nM. Plaque assays were performed as previously described 27,28 .…”