Principles of Virology. Jane Flint

Principles of Virology - Jane Flint


Скачать книгу
animal models are surrogates for the events that occur during viral infections of humans.

      There are two main types of assay for detecting viruses: biological and physical. Because viruses were first recognized by their infectivity, the earliest assays focused on this most sensitive and informative property. However, biological assays such as the plaque assay and end-point titration methods do not detect noninfectious particles. In contrast, all particles are accounted for with physical assays such as electron microscopy or by immunological methods. Knowledge of the number of noninfectious particles is useful for assessing the quality of a virus preparation.

      One of the most important procedures in virology is measuring the virus titer, the concentration of infectious virus particles in a sample. This parameter is determined by inoculating serial dilutions of virus into host cell cultures, chicken embryos, or laboratory animals and monitoring for evidence of virus multiplication. The response may be quantitative (as in assays for plaques, fluorescent foci, infectious centers, or abnormal growth and morphology) or all-or-none, in which the presence or absence of infection is measured (as in an end-point dilution assay). Please note that “titer” is not a verb.

image

       Plaque Assay

image

       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).


Скачать книгу