Principles of Virology. Jane Flint

Principles of Virology - Jane Flint


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assays are usually more rapid and easier to carry out than those for infectivity, which may be slow, cumbersome, or impossible. Assays for subviral components also provide information on particle number if the amount of these components in each virus particle is known.

       Electron Microscopy

      With few exceptions, virus particles are too small to be observed directly by light microscopy. However, they can be seen readily in the electron microscope. If a sample contains only one type of virus, the particle count can be determined. A virus preparation is mixed with a known concentration of latex beads, and the numbers of virus particles and beads are then counted, allowing the concentration of the virus particles in the sample to be determined by comparison.

       Hemagglutination

       Centrifugation

      The use of centrifugal force to separate particles from solution according to size, shape, or density has been a staple of virology. The instrument used for such separations is called a centrifuge, which can range from small tabletop devices that accommodate small tubes to large floor models with greater capacity and to ultracentrifuges that can achieve revolutions per minute in excess of 70,000. The ultracentrifuge was invented by Theodor Svedberg in 1925, and it is the first initial of his last name that is used to describe the sedimentation coefficient of a particle as measured by centrifugation, e.g., the 16S ribosomal subunit.

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      Another method for purifying viruses is by isopycnic centrifugation, which separates particles solely on the basis of their density. A virus preparation is mixed with a compound (e.g., cesium chloride) that forms a density gradient during centrifugation. Virus particles move down the tube until they reach the point at which their density is the same as the gradient medium. Structural studies of virus particles often require highly purified preparations which can be made by differential or isopycnic centrifugation.

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       Measurement of Viral Enzyme Activity

      Some animal virus particles contain nucleic acid polymerases, which can be detected by mixing permeabilized particles with precursors and measuring their incorporation into nucleic acid. This type of assay is used most frequently for retroviruses, many of which neither transform cells nor form plaques. The reverse transcriptase incorporated into the virus particle is assayed by mixing cell culture supernatants with a mild detergent (to permeabilize the viral envelope), an RNA template and primer, and a radioactive nucleoside triphosphate. If reverse transcriptase is present, a radioactive product will be produced by priming on the template. This product can be detected by precipitation or bound to a filter and quantified. Because enzymatic activity is proportional to particle number, this assay allows rapid tracking of virus production in the course of an infection. Many of these assays have been modified to permit the use of safer, nonradioactive substrates. For example, when nucleoside triphosphates conjugated to biotin are used, the product can be detected with streptavidin (which binds biotin) conjugated to a fluorochrome. Alternatively, the reaction products may be quantified by quantitative real-time PCR (see “Detection of Viral Nucleic Acids” below).

       Serological Methods

      The specificity of the antibody-antigen reaction has been used to design a variety of assays for viral proteins and antiviral antibodies. These techniques, such as immunostaining, immunoprecipitation, immunoblotting, and the enzyme-linked immunosorbent assay, are by no means limited to virology: all these approaches have been used extensively to study the structures and functions of cellular proteins.

      Virus neutralization. When a virus preparation is inoculated into an animal, an array of antibodies is produced. These antibodies can bind to virus particles, but not all of them can block infectivity (neutralize), as discussed in Volume II, Chapter 4. Virus neutralization assays are usually conducted by mixing dilutions of antibodies with virus, incubating them, and assaying for remaining infectivity in cultured cells, eggs, or animals. The end point is defined as the highest dilution of antibody that inhibits the development of cytopathic effect in cells or virus reproduction


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