Principles of Virology. Jane Flint

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 to map the three-dimensional structure of neutralization antigenic sites on the virus particle (Box 2.7).

BOX 2.7

      DISCUSSION

       Neutralization antigenic sites

      Antigenic sites defined by antibodies. (A) Locations of neutralization antigenic sites on the capsid of poliovirus type 1. Amino acids that change in viral mutants selected for resistance to neutralization by monoclonal antibodies are shown in white on a model of the viral capsid. These amino acids are in VP1 (blue), VP2 (green), and VP3 (red) on the surface of the virus particle. Figure courtesy of Jason Roberts, Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Australia. (B) Conformational and linear epitopes bound to antibody molecules. Linear epitopes are made of consecutive amino acids, while conformational epitopes are made of amino acids from different parts of the protein.

      Knowledge of the antigenic structure of a virus is useful in understanding the immune response to these agents and in designing new vaccination strategies. The use of monoclonal antibodies (antibodies of a single specificity made by a clone of antibody-producing cells) in neutralization assays permits mapping of antigenic sites on a virus particle or of the amino acid sequences that are recognized by neutralizing antibodies.

      Each monoclonal antibody binds specifically to 8 to 12 residues that fit into the antibody-combining site. These amino acids are either next to one another either in primary sequence (linear epitope) or in the folded structure of the native protein (nonlinear or conformational epitope). In contrast, polyclonal antibodies comprise the repertoire produced in an animal against the many epitopes of an antigen. Antigenic sites may be identified by cross-linking a monoclonal antibody to the virus and determining which protein is the target of that antibody. Epitope mapping may also be performed by assessing the abilities of monoclonal antibodies to bind synthetic peptides representing viral protein sequences. When the monoclonal antibody recognizes a linear epitope, it may react with the protein in immunoblot analysis, facilitating direct identification of the viral protein harboring the antigenic site.

      An elegant understanding of antigenic structures has come from the isolation and study of variant viruses that are resistant to neutralization with specific monoclonal antibodies (called monoclonal antibody-resistant variants). By identifying the amino acid change(s) responsible for this phenotype, the antibody-binding site can be located and, together with three-dimensional structural data, can provide detailed information on the nature of antigenic sites that are recognized by neutralizing antibodies (see the figure).

      Hemagglutination inhibition. Antibodies against viral proteins with hemagglutination activity can block the ability of virus to bind red blood cells. In this assay, dilutions of antibodies are incubated with virus, and erythrocytes are added as outlined above. After incubation, the titer is read as the highest dilution of antibody that inhibits hemagglutination. This test is sensitive, simple, inexpensive, and rapid, and can be used to detect antibodies to viral hemagglutinin in animal and human sera. For example, hemagglutination inhibition assays were used to identify individuals who had been infected with the newly discovered avian influenza A (H7N9) virus in China during the 2013 outbreak.

Visualization of proteins. Antibodies can be used to visualize viral or cellular proteins in infected cells or tissues. In direct immunostaining, an antibody that recognizes a viral protein is coupled directly to an indicator 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