The measurement of virus titers by plaque assay was first developed for bacteriophages by d’Herelle in 1917 and then modified for animal viruses by Renato Dulbecco in 1952. In this procedure, monolayers of cultured cells are incubated with a preparation of virus to allow adsorption to cells. After removal of the inoculum, the cells are covered with nutrient medium containing a supplement, most commonly agar, which forms a gel. When the original infected cells release new progeny particles, the gel restricts their spread to neighboring uninfected cells. As a result, each infectious particle produces a circular zone of infected cells, a plaque. If the infected cells are damaged, the plaque can be distinguished from the surrounding monolayer. In time, the plaque becomes large enough to be seen with the naked eye (Fig. 2.7). Only viruses that cause visible damage of cultured cells can be assayed in this way. A movie that depicts the microscopic development of a plaque can be found at this link: http://bit.ly/Virology_VZVGFP.
For the majority of animal viruses, there is a linear relationship between the number of infectious particles and the plaque count (Fig. 2.8). One infectious particle is therefore sufficient to initiate infection, and the virus is said to infect cells with one-hit kinetics. Some examples of two-hit kinetics, in which two different types of virus particle must infect a cell to ensure replication, have been recognized. An example is the genomes of some (+) strand RNA viruses of plants that consist of two RNA molecules which are encapsidated separately. Both RNAs are required for infectivity. The dose-response curve in plaque assays for these viruses is therefore parabolic rather than linear (Fig. 2.8).
Figure 2.8 The dose-response curve of the plaque assay. The number of plaques produced by a virus with one-hit kinetics (red) or two-hit kinetics (blue) is plotted against the relative concentration of the virus. In two-hit kinetics, there are two classes of uninfected cells, those receiving one particle and those receiving none. The Poisson distribution can be used to determine the proportion of cells in each class: they are e–m and me–m (Box 2.12). Because one particle is not sufficient for infection, P(0) = e–m(1 + m). At a very low multiplicity of infection, this equation becomes P(i) = (1/2)m2 (where i = infection), which gives a parabolic curve.
The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter (Box 2.5). The plaque assay may also be used to prepare clonal virus stocks. When one infectious virus particle initiates a plaque, the viral progeny within the plaque are biological clones, and virus stocks prepared from a single plaque are known as plaque purified. The tip of a small pipette is plunged into the overlay above the plaque, and the plug of agar containing the virus is recovered. The virus within the agar plug is eluted into buffer and used to prepare virus stocks. To ensure purity, this process is usually repeated at least one more time.
Fluorescent-Focus Assay
The fluorescent-focus assay, a modification of the plaque assay, can be done more rapidly and is useful in determining the titers of viruses that do not form plaques. The initial procedure is the same as in the plaque assay. However, after a period sufficient for adsorption and gene expression, cells are made permeable and incubated with an antibody raised against a viral protein. A second antibody, which recognizes the first, is then added. This second antibody is usually conjugated to a fluorescent molecule. The cells are then examined under a microscope at an appropriate wavelength. The titer of the virus stock is expressed in fluorescent-focus-forming units per milliliter. When the gene encoding a fluorescent protein is incorporated into the viral genome, foci may be detected without the use of antiviral antibodies.
METHODS
Calculating virus titer from the plaque assay
To calculate the titer of a virus in plaque-forming units (PFU) per milliliter, 10-fold serial dilutions of a virus stock are prepared in a buffer, and suitable aliquots are inoculated onto susceptible cell monolayers which are covered with an agar overlay (see figure). After a suitable incubation period, the monolayers are stained and the plaques are counted. To minimize error in calculating the virus titer, only plates containing between 10 and 100 plaques are counted, depending on the area of the cell culture vessel. Plates with >100 plaques are generally not counted because the plaques may overlap, causing inaccuracies. According to statistical principles, when 100 plaques are counted, the sample titer varies by ±10%. For accuracy, each dilution is plated in duplicate or triplicate (not shown in the figure). In the example shown, 10 plaques are observed on the plate produced from the 10–6 dilution. Therefore, the 10–6 dilution tube contains 10 PFU per 0.1 ml, or 100 PFU per ml, and the titer of the virus stock is 100 × 106 or 1 × 108 PFU/ml.
Infectious-Centers Assay
Another modification of the plaque assay, the infectious-centers assay, is used to determine the fraction of cells in a culture that are infected with a virus. Monolayers of infected cells are suspended before progeny viruses are produced. Dilutions of a known number of infected cells are then plated on monolayers of susceptible cells, which are covered with an agar overlay. The number of plaques that form on the indicator cells is a measure of the number of cells infected in the original population. The fraction of infected cells can therefore be determined. A typical use of the infectious-centers assay is to measure the proportion of virus-producing cells in persistently infected cultures.
Transformation Assay
The transformation assay provides a method for determining the titers of some retroviruses that do not form plaques. For example, when Rous sarcoma virus transforms chicken embryo cells, the cells lose their contact inhibition (the property that governs whether cells in culture grow as a single monolayer [see Volume II, Chapter 6]) and become heaped up on one another. The transformed cells form small piles, or foci, that can be distinguished easily from the rest of the monolayer (Fig. 2.9). Infectivity is expressed in focus-forming units per milliliter.
End-Point Dilution Assay
The end-point dilution assay provided a means to determine virus titer before the development of the plaque assay. It is still used for measuring the titers of certain viruses that do not form plaques or for determining the virulence of a virus in animals. Serial dilutions of a virus stock are inoculated into replicate test units (typically 8 to 10), which can be cell cultures, eggs, or animals. The number of test units that have become infected is then determined for each virus dilution. In cell culture, infection may be determined by the development of cytopathic effect; in eggs or animals, infection may be gauged by virus titer, death, or disease. An example of an end-point dilution assay using cell cultures is
