Principles of Virology, Volume 1. Jane Flint

such as a fluorescent dye or an enzyme (Fig. 2.12). A more sensitive approach is indirect immunostaining, in which a second antibody is coupled to the indicator. The second antibody recognizes a common region on the virus-specific antibody.

Figure 2.12 Direct and indirect methods for antigen detection. (A) The sample (tissue section, smear, or bound to a solid phase) is incubated with a virus-specific antibody (Ab). In direct immunostaining, the antibody is linked to an indicator such as fluorescein. In indirect immunostaining, a polyclonal antibody, which recognizes several epitopes on the virus-specific antibody, is coupled to the indicator. Mab, monoclonal antibody. (B) Use of immunofluorescence to visualize pseudorabies virus replication in neurons. Superior cervical ganglion neurons were grown in culture and infected with a recombinant virus that produces green fluorescent protein (GFP) fused to the VP26 capsid protein. Neurons were stained with AF568-phalloidin, which stains actin red, and anti-GM130 to stain the Golgi blue. GFP-VP26 is visualized by direct fluorescence. Courtesy of L. Enquist, Princeton University.

      Multiple second-antibody molecules bind to the first antibody, resulting in an increased signal from the indicator compared with that obtained with direct immunostaining. Furthermore, a single indicator-coupled second antibody can be used in many assays, avoiding the need to purify and couple an indicator to multiple first antibodies.

      In practice, virus-infected cells (unfixed or fixed with acetone, methanol, or paraformaldehyde) are incubated with polyclonal or monoclonal antibodies (Box 2.7) directed against viral antigen. Excess antibody is washed away, and in direct immunostaining, cells are examined by microscopy. For indirect immunostaining, the second antibody is added before examination of the cells by microscopy. Commonly used indicators fluoresce on exposure to UV light. Filters are placed between the specimen and the eyepiece to remove blue and UV light so that the field is dark, except for cells to which the antibody has bound, which emit light of distinct colors (Fig. 2.12). Today’s optics are much better at keeping the wavelengths separated, permitting the use of different colors to detect various components in the same specimen. Antibodies can also be coupled to molecules other than fluorescent indicators, including enzymes such as alkaline phosphatase, horseradish peroxidase, and β-galactosidase, a bacterial enzyme that in a test system converts the chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) to a blue product. In these instances, excess antibody is washed away, a suitable chromogenic substrate is added, and the presence of the indicator antibody is revealed by the development of a color that can be visualized.

      Immunostaining has been applied widely in the research laboratory for determining the sub-cellular localization of cellular and viral proteins (Fig. 2.12), monitoring the synthesis of viral proteins, determining the effects of mutation on protein production, localizing the sites of viral genome replication in animal hosts, and determining the effect of infection on structure of the tissue. It is the basis of the fluorescent-focus assay.

      Immunostaining of viral antigens in smears of clinical specimens may be used to diagnose viral infections. For example, direct and indirect immunofluorescence assays with nasal swabs or washes can detect a variety of viruses, including influenza virus and measles virus. Viral proteins or nucleic acids may also be detected in infected animals by immunohistochemistry. In this procedure, tissues are embedded in a solid medium such as paraffin, and thin slices are produced using a microtome. Viral antigens can be detected within the cells in the sections by direct and indirect immunofluorescence assays.

Enzyme immunoassay. Detection of viral antigens or antiviral antibodies can be accomplished by solid-phase methods, in which an antiviral antibody or protein is adsorbed to a plastic surface (Fig. 2.13A). To detect antibodies to viruses, viral protein is first linked to the plastic support, and then the specimen is added (Fig. 2.13B). Like other detection methods, enzyme immunoassays are used in both experimental and diagnostic virology. In the clinical laboratory, enzyme immunoassays are used to detect a variety of viruses, including rotavirus, herpes simplex virus, and human immunodeficiency viruses. A modification of the enzyme immunoassay is the lateral fow immunochromatographic assay, which has been used in rapid antigen detection test kits (Fig. 2.14). The lateral fow immunochromatographic assay does not require instrumentation and can be read in 5 to 20 min in a physician’s office or in the field. Commercial rapid antigen detection assays are currently available for influenza virus, respiratory syncytial virus, and rotavirus.

Figure 2.13 Detection of viral antigen or antibodies against viruses by enzyme-linked immunosorbent assay (ELISA). (A) To detect viral proteins in serum or clinical samples, antibodies specific for the virus are immobilized on a solid support such as a plastic well. The sample is placed in the well, and viral proteins are “captured” by the immobilized antibody. After washing to remove unbound proteins, a second antibody against the virus is added, which is linked to an indicator. The second antibody will bind if viral antigen has been captured by the first antibody. Unbound second antibody is removed by another washing, and when the indicator is an enzyme, a chromogenic molecule that is converted by the enzyme to an easily detectable product is then added. The enzyme amplifies the signal because a single catalytic enzyme molecule can generate many product molecules. Another wash is done to remove unbound second antibody. If viral antigen has been captured by the first antibody, the second antibody will bind and the complex will be detected by the indicator. (B) To detect antibodies to a virus in a sample, viral antigen is immobilized on a solid support such as a plastic well. The test sample is placed in the well, and antiviral IgG antibodies present in the sample will bind the immobilized antigen. After washing to remove unbound components in the sample, a second antibody, directed against a general epitope on the first antibody, is added. Unbound second antibody is removed by another wash. If antibodies against the virus are present in the specimen, the second antibody will bind to them and the complex will be detected via the indicator attached to the second antibody, as described in (A).

       Fluorescent Proteins

The discovery of green fluorescent protein revolutionized the study of the cell biology of virus infection. This protein, isolated from the jellyfish Aequorea victoria, is a convenient reporter for monitoring gene expression, because it is directly visible in living cells without the need for fixation, substrates, or coenzymes. Mutagenesis of the gene encoding this protein has led to the development of new fluorescent probes ranging in color from blue to yellow (Fig. 2.15A). Additional fluorescent proteins emitting in the red, deep red, cyan, green, yellow, and orange spectral regions have been isolated from other marine species. Codon optimization for maximum translation in specific cell types and improved stability and brightness are other modifications that have broadened the utility of these proteins.

       Fluorescence Microscopy

      Fluorescence microscopy allows virologists to study all steps of virus reproduction, including cell surface attachment, cell entry, trafficking, replication, assembly, and egress. Single virus particle tracking can be achieved by inserting the coding sequence for a fluorescent protein into the viral genome, often