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 shown in Box 2.6, with results expressed as 50% infectious dose (ID50) per milliliter. This type of assay is also suitable for high-throughput applications.
When the end-point dilution assay is used to assess the virulence of a virus or its capacity to cause disease (Volume II, Chapter 1), the result can be expressed in terms of 50% lethal dose (LD50) per milliliter or 50% paralytic dose (PD50) per milliliter, end points of death and paralysis, respectively. The 50% end point determined in an animal host can be related to virus titer, determined separately by plaque assay or other means. In this way, the effects of the route of inoculation or specific mutations on viral virulence can be quantified.
Efficiency of Plating
Efficiency of plating is defined as the infectious virus titer (in PFU/ml) divided by the total number of virus particles in the sample. The particle–to–plaque-forming-unit (PFU) ratio, a term more commonly used today, is the inverse value (Table 2.1). For many bacteriophages, the particle-to-PFU ratio approaches 1, the lowest value that can be obtained. However, for animal viruses, this value can be much higher, ranging from 1 to 10,000. These high values have complicated the study of animal viruses. For example, when the particle-to-PFU ratio is high, it may not be clear that properties measured biochemically are in fact those of the infectious particle or those of the noninfectious component.
Figure 2.9 Transformation assay. Chicken cells transformed by two different strains of Rous sarcoma virus are shown. Loss of contact inhibition causes cells to pile up rather than grow as a monolayer. One focus is seen in panel A and three foci are seen in panel B at the same magnification. Courtesy of H. Hanafusa, Osaka Bioscience Institute.
METHODS
End-point dilution assays
End-point dilution assays are usually carried out in multiwell plastic plates (see the figure above). In the example shown in the adjacent table above, 10 monolayer cell cultures were infected with each virus dilution. After the incubation period, plates that displayed cytopathic effect were scored +. At high dilutions, none of the cell cultures are infected because no infectious particles are delivered to the cells; at low dilutions, every culture is infected. The end point is the dilution of virus that affects 50% of the test units. This number can be calculated from the data and expressed as 50% infectious dose (ID50) per milliliter. Fifty percent of the cell cultures displayed cytopathic effect at the 10–5 dilution, and therefore, the virus stock contains 105 TCID50 (tissue culture infectious dose) units.
| Virus dilution | Cytopathic effect | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 10–2 | + | + | + | + | + | + | + | + | + | + |
| 10–3 | + | + | + | + | + | + | + | + | + | + |
| 10–4 | + | + | – | + | + | + | + | + | + | + |
| 10–5 | – | + | + | – | + | – | – | + | – | + |
| 10–6 | – | – |
–
|