Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 82

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.