Plaque assay

Some of these tests will now be described. As the number of possible viruses and tests is endless, those chosen simply reflect those with which I am familiar. 

The method depends on infecting a small number of cells in a confluent monolayer. The virus produced in the infected cells will move laterally to infect adjacent cells and various techniques are used to prevent further spreading. The degenerative effect on the cells spreads until a visible area of dead cells (a plaque) is apparent. Staining of the cell sheet makes the colourless plaque more easy to see. 

Usually cell cultures are infected with different dilutions of the viral preparation covering a range of 104. Thus a viral stock will be diluted in 10-fold increments. 

Set up a series of tubes containing 0.9 ml BSS or PBS or Eagle s medium without serum and to the first tube add 0.1 ml virus stock. Mix the contents and remove 0.1 ml to the second tube and so on. A solution is required which contains 200-400 infectious units per ml and usually the 10 5 to 10 9 dilutions are assayed. 

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Methods to Detect and Quantitate Viral Agents in Fluids. In order to assess the effectiveness of membrane filtration the abihty to quantitate the amount of vims present pre- and post-filtration is critical. There are a number of techniques used. The method of choice for filter challenge studies is the plaque assay which utilizes the formation of plaques, localized areas in the cell monolayer where cell death caused by viral infection in the cell has occurred on the cell monolayer. Each plaque represents the presence of a single infectious vims. Vims quantity in a sample can be determined by serial dilution until the number of plaques can be accurately counted. The effectiveness of viral removal may be determined, as in the case of bacterial removal, by comparing the vims concentration in the input suspension to the concentration of vims in the effluent. 

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

Plaque assays, at present, apply to only a very limited number of viruses, e.g. poliovirus, herpes virus, human rotavirus. The principle ofthese assays is as follows test virus is dried on to coverslips which are immersed in various concentrations oftest disinfectant... 

Fig. 11.7 A, diagrammatic representation of plaque assay for evaluating virucidal activity and B, monolayers of baby hamster ki(hiey (BHK) cells C, virus tte untreated virus (o represents a plaque-forming unit, pfu, in BHK cells) D, virus titre disinfectant-treated virus (before plating onto BHK, die disinfectant must be neuh alized in an appropriate manner). Note the greatly reduced nimiber of pfu in D, indicative of fewer iminactivated virus particles than in C. 

Yellow fevert Aqueous homogenate of chick embryos infected with attenuated yellow fever virus 170 1 Centrifugation to remove cell debris 2 Freeze drying Infectivity-titration in cell cultures by plaque assay Tests to exclude extraneous viruses... 

Baert, L., Wobus, C. E., Van Coillie, E., Thackray, L. B., Debevere, J., and Uyttendaele, M. (2008c). Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat exposure. Appl. Environ. Microbiol. 74, 543-546. 

Plaque assay When a virus particle initiates an infection upon a layer or lawn of host cells which is growing spread out on a flat surface, a zone of lysis or growth inhibition may occur which results in a clearing of the cpll growth. This clearing is called a plaque, and it is assumed that each plaque has originated from one virus particle. 

The plaque procedure also permits the isolation of pure virus strains, since if a plaque has arisen from one virus particle, all the virus particles in this plaque are probably genetically identical. Some of the particles from this plaque can be picked and inoculated into a fresh bacterial culture to establish a pure virus line. The development of the plaque assay technique was as important for the advance of virology as was Koch s development of solid media for bacteriology. 

Efficiency of plating One important concept in quantitative virology involves the idea of efficiency of plating. Counts made by plaque assay are always lower than counts made with the electron... 

Figure 5.8 Quantification of bacterial virus by plaque assay using the agar overlay technique.

Immunogenicity can be identified by identifying potential antibodies in plasma. These usually involve ELISA methods. T-cell-dependent antibody responses can be evaluated, and plaque assays involving IgM antibody responses are available. 

Protocol 1.7) and normally generate high-titre stocks. The viral titre is determined by plaque assay (Protocol 1.8) so that known amounts of the recombinant virus are used in subsequent virus amplification experiments (Protocol 1.9) to produce large viral stocks. 

Protocol 1.8 Purification of recombinant virus and determination of viral titre by plaque assay... 

Determine the recombinant viral titre by plaque assay. 

Maeda, S. (1984) A plaque assay and cloning of Bombyx mori nuclear polyhedrosis virus. J. Seric. Sci.Jpn. 53, 547-548. 

Hink WF, Vail PV (1973), A plaque assay for titration of alfalfa lopper nuclear polyhedrosis virus in a cabbage lopper (TN 368) cell line, J. Inverteb. Pathol. 22 168-174. 

The assay for interferon involves incubating cells overnight with increasing dilutions of interferon and then challenging the cells with, say, vesicular stomatitis virus (VSV) at 20 p.f.u. per cell. Twenty hours later the culture fluids are harvested and assayed for VSV using a plaque assay ( 14.3.1) on mouse cells. The greatest dilution of interferon which inhibits virus yield by 3.2 fold (0.5 log10) contains 1 unit of interferon (Baron, 1969). 

Fig. 14.1. Time course of SV40 infection. Monkey cells in monolayer culture, infected with SV40 at 1-10 p.f.u. per cell. T antigen may be detected by immunofluorescence, viral DNA synthesis by labelling with [yH]thymidine followed by separation of viral DNA (by Hirt extraction, SDS gradient centrifugation or agarose gel electrophoresis) and mature virions by infectivity using a plaque assay. (Data from Tooze, 1973 Girard et al., 1975 and Basilico and Zouzias, 1976.)...

Castano JP, Kineman RD, Frawley LS (1994) Dynamic fluctuations in the secretory activity of individual lactotropes as demonstrated by a modified sequential plaque assay. Endocrinology 755 1747-1752. 

The traditional method for determining anti-viral drug activity involves conducting a plaque assay. A preliminary mass based screening can be helpful in reducing the number of drug candidates that need to be screened by more... 

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