Viruses are unique infectious microorganisms that have several special properties. Originally, their small size distinguished them from other infectious agents; they pass through filters that retain bacteria. Viruses are distinguishable also by their obligate intracellular parasitism, simple organization, mode of replication and nucleic acid content.
Viruses grow in other cells because virus particles lack energy-synthesizing organelles, enzyme systems, and protein-synthesizing machinery. Viruses reproduce by assembling subunits into infectious particles, not by dividing, as other microorganisms do. It is these special properties which make laboratory techniques involving viruses so different.
Because viruses are obligate intracellular parasites, viral cultivation requires live hosts. Several species of laboratory animals, embryonated chicken eggs, cultures of bits of differentiated tissue, and cell cultures are widely used, however the suitability of each host depends on the virus to be studied.
Small laboratory animals are particularly useful for examining the pathogenicity of viruses. Animals that exhibit symptoms of viral infection are killed and dissected to obtain tissue for histologic examination which may reveal characteristic effects of certain viruses.
The embryonated chicken egg has long been widely used as a sensitive host for cultivation of influenza viruses. Compared with laboratory animals, embroynated eggs offer several advantages:
Influenza viruses are isolated by inoculating specimen into the allantoic and amniotic cavities of chicken eggs, which are then incubated for three days. Although the viral infection may be fatal to the embryo, it is not examined for specific pathologic changes. Instead, fluids from the amniotic and allantoic cavities are collected and tested for the presence of viral hemagglutinins. If the chorioallantoic membrane is used as the inoculation site, quantitative assay of viruses that form lesions on the CAM is feasible. Experienced virologists can distinguish lesions produced by different viruses.
Certain viruses that are very difficult to isolate or propagate in cell cultures are successfully grown and recovered in so-called organ cultures. Organ cultures do not grow appreciably while they are maintained in the laboratory and have a limited lifetime. Because initiation of each culture requires fresh human or animal tissue, organ cultures are not suited for routine use, but they are important for research and special clinical studies.
For most virology laboratories, cell cultures are the least expensive and most convenient means of isolation and cultivation of viruses from clinical specimen. Most of the more commonly encountered viruses grow and produce recognizable changes in cell cultures derived from human, monkey or small laboratory animal tissues. The cells are grown in monolayer cultures and the viruses are introduced after a specified density of cells is obtained.
Detection of viral infection of a tissue monolayer depends upon the type of virus. Characteristic morphologic changes produced by the virus replication process (cytopathic effect - CPE) can often be used to identify types of viruses. Related viruses tend to produce similar CPE, that can be distinguished from CPE produced by other virus groups. Some viruses that readily infect and grow in cell cultures do not produce CPE and these viruses must be detected and identified by other means.
Necrosis of target cells in viral infections may serve as a useful clue to the etiologic agent. For example, in poliomyelitis, the anterior horn cells of the spinal cord characteristically become necrotic; in genital herpes simplex infection, only mucosal squamous epithelium is destroyed.
Several types of inclusion bodies characterize particular viral infection. For example, infections with herpesvirus, adenovirus and papovaviruses produce intranuclear inclusions, but infections with rabiesvirus, poxviruses and cytomegalovirus produce cytoplasmic inclusions. Syncytia, that appear microscopically as masses of multinucleate giant cells, are characteristic of measles, herpes simplex, and the parainfluenza viruses.
Confirmation of the identification of viruses can be obtained by immunospecific staining. Immunofluorescence or immunoperoxidase methods can be applied both to the identification of a virus isolated in cell culture, and to the detection of viral antigens in cells obtained directly from clinical specimen.
Enzyme-linked immunoassay methods are suitable for the detection of free viral antigens (e.g., Hepatitis B carriers) and provide for rapid diagnosis of many viral diseases.
Electron microscopy has a limited but important role in the detection of viruses. It is the best method for detecting previously undetected viruses, especially those that do not grow in the usual laboratory hosts. It can also be used clinically to identify causative viral agents. Classification and identification of viruses in clinical specimen are based largely on virion morphology and substructure.
B.Viral Assay Techniques
C.Stained Slides Showing Various CPE
D.Mosquitoes and Ticks Endemic to New Orleans