The growth of viruses requires cell cultures because viruses replicate only in living cells, not on cell-free media the way most bacteria can. Because many viruses are inactivated at room temperature, it is important to inoculate the specimen into the cell culture as soon as possible; brief transport or storage at 4°C is acceptable.
Virus growth in cell culture frequently produces a characteristic cytopathic effect (CPE) that can provide a presumptive identification. CPE is a change in the appearance of the virus-infected cells. This change can be in such features as size, shape, and the fusion of cells to form multinucleated giant cells (syncytia). CPE is usually a manifestation of virus-infected cells that are dying or dead. The time taken for the CPE to appear and the type of cell in which the virus produces the CPE are important clues in the presumptive identification.
If the virus does not produce a CPE, its presence can be detected by several other techniques:
Hemadsorption (i.e., attachment of erythrocytes to the surface of virus-infected cells). This technique is limited to viruses with a hemagglutinin protein on their envelope, such as mumps, parainfluenza, and influenza viruses.
Interference with the formation of a CPE by a second virus. For example, rubella virus, which does not cause a CPE, can be detected by interference with the formation of a CPE by certain enteroviruses, such as echovirus or Coxsackie virus.
A decrease in acid production by infected, dying cells. This can be detected visually by a color change in the phenol red (a pH indicator) in the culture medium. The indicator remains red (alkaline) in the presence of virus-infected cells but turns yellow in the presence of metabolizing normal cells as a result of the acid produced. This technique can be used to detect certain enteroviruses.
A definitive identification of the virus grown in cell culture is made by using known antibody in one of several tests. Complement fixation, hemagglutination inhibition, and neutralization of the CPE are the most frequently used tests. Other procedures such as fluorescent antibody, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy are also used in special instances. A brief description of these tests follows. They are described in more detail in the section on immunology.
If the antigen (the unknown virus in the culture fluid) and the known antibody are homologous, complement will be fixed (bound) to the antigen–antibody complex. This makes it unavailable to lyse the “indicator” system, which is composed of sensitized red blood cells.
If the virus and antibody are homologous, the virus is blocked from attaching to the erythrocytes and no hemagglutination occurs. Only viruses that agglutinate red blood cells can be identified by this method.
If the virus and antibody are homologous, the antibody bound to the surface of the virus blocks its entry into the cell. This neutralizes viral infectivity because it prevents viral replication and subsequent CPE formation or animal infection.
Fluorescent Antibody Assay
If the virus-infected cells and the fluorescein-tagged antibody are homologous, the typical apple-green color of fluorescein is seen in the cells by ultraviolet (UV) microscopy.
If the virus and the antibody are homologous, there is less antibody remaining to bind to the known radiolabeled virus.
Enzyme-Linked Immunosorbent Assay
In the ELISA test to identify a virus, known antibody is bound to a surface. If the virus is present in the patient’s specimen, it will bind to the antibody. A sample of the antibody linked to an enzyme is added, which will attach to the bound virus. The substrate of the enzyme is added, and the amount of the bound enzyme is determined.
If the antibody is homologous to the virus, aggregates of virus–antibody complexes are seen in the electron microscope.