concentration of the virus particles in the sample to be determined by comparison.
Hemagglutination
Members of the Adenoviridae, Orthomyxoviridae, and Paramyxoviridae, among others, contain proteins that bind to erythrocytes (red blood cells); these viruses can link multiple cells, resulting in formation of a lattice. This property is called hemagglutination. For example, influenza viruses contain an envelope glycoprotein called hemagglutinin (HA), which binds to N-acetylneuraminic acid-containing glycoproteins on erythrocytes. In practice, 2-fold serial dilutions of the virus stock are prepared, mixed with a known quantity of red blood cells, and added to small wells in a plastic tray (Fig. 2.10). Unlinked red blood cells tumble to the bottom of the well and form a sharp dot or button. In contrast, agglutinated red blood cells form a diffuse lattice that coats the well. Because the assay is rapid (30 min), it is often used as a quick indicator of the relative quantities of virus particles. However, it is not sufficiently sensitive to detect small numbers of particles.
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.
Figure 2.10 Hemagglutination assay. (Top) Samples of different influenza viruses were diluted, and a portion of each dilution was mixed with a suspension of chicken red blood cells and added to the wells. After 30 min at 4°C, the wells were photographed. Sample A does not contain virus. Sample B causes hemagglutination until a dilution of 1:512 and therefore has a hemagglutination titer of 512. Elution of the virus from red blood cells at the 1:4 dilution is caused by neuraminidase in the virus particle. This enzyme cleaves N-acetylneuraminic acid from glycoprotein receptors and elutes bound viruses from red blood cells. (Bottom) Schematic illustration of hemagglutination of red blood cells by influenza virus. Top, Courtesy of C. Basler and P. Palese, Mount Sinai School of Medicine of the City University of New York.
It would not be wrong to state that every virology laboratory is in possession of at least one centrifuge and probably has access to more. The uses of the centrifuge in virology are manifold: from low-speed separation of virus particles from infected cell debris in cell culture medium to fractionation of infected cells to isolate nuclei, cytoplasm, or ribosomes, and to purification of virus particles.
Differential centrifugation is used to separate viruses, organelles, or subcellular structures from cells. Preformed gradients of sucrose are often used because particles that move with various velocities can be separated differentially in the increasing viscosity of the solution. One application of sucrose gradients is the purification of virus particles. Another is polysome profiling, an analysis of the mRNAs associated with ribosomes (Fig. 2.11). Because mRNAs undergoing translation can be associated with different numbers of ribosomes, they can be separated on a sucrose gradient. A more modern use of the polysome profile is to extract the RNA from each fraction and determine which mRNAs are being actively translated.
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.
Figure 2.11 Polysome analysis. To study the association of mRNAs with ribosomes, cell lysates are prepared and separated by centrifugation through sucrose gradients. Fractions are collected and their optical density measured to locate mRNAs bound to one or more ribosomes. The graph shows the optical density of fractions from the top (left) to the bottom (right) of the gradient. The slower-moving materials at the top of the gradient are ribosomal subunits, while mRNAs associated with one or more ribosomes move faster in the sucrose gradient.
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 in eggs or animals.
Some neutralizing antibodies define type-specific antigens on the virus particle. For example, the three serotypes of poliovirus are distinguished on the basis of neutralization tests: type 1 poliovirus is neutralized by antibodies to type 1 virus but not by antibodies to type 2 or type 3 poliovirus. The results of neutralization tests were once used for virus classification, a process now accomplished largely by comparing viral genome sequences. Nevertheless, the detection of antiviral antibodies in animal sera is still extremely important for identifying infected hosts. These antibodies may also be used
