Principles of Virology. Jane Flint

Principles of Virology - Jane Flint


Скачать книгу
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.

image

       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

       End-Point Dilution Assay

      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.

image

      METHODS

       End-point dilution assays

image

      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
Скачать книгу
Librs.Net