Growth of viruses in cell culture is usually detected by visualizing morphological changes in the cells, known as cytopathic effect (CPE). Cell cultures are typically viewed microscopically to detect CPE every one to two days for the first week of incubation. When necessary, confirmation can be achieved by scraping the infected cells from the walls of the tube or vessel in which they are growing and preparing a fluorescent antibody stain with use of monoclonal antibodies specific for the virus, or a neutralization test using specific antisera. The shell vial culture method, first developed for cytomegalovirus (CMV), dramatically decreases the time required for detection of viruses in cell culture. The method involves centrifugation of the specimen onto the cell culture monolayer grown on a cover slip, which is incubated for one or two days, followed by fluorescent antibody staining of the cell culture, regardless of whether CPE is visible or not. In addition to detection of CMV, shell vial cultures have also been used to speed the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), respiratory viruses, and the enteroviruses. A recent modification of the traditional cell culture involves the use of genetically engineered cell lines such as the L 20 B, which is used for the isolation of polio viruses, and Vero cells for isolation of measles and rubella viruses. Another example of genetically engineered cell line uses genes that are transfected into indicator cell lines so that the cell line responds to a specific viral protein present in the specimen. Activation of the promoter triggers a reporter enzyme, such as ß - galactosidase, which acts on a substrate to indicate the presence of the virus being sought. This approach has been most widely used for HSV and HIV. Detection of virus antigens Methods of antigen detection include fluorescent antibody (FA) staining, immunoperoxidase staining and EIA. Of these, FA staining is the most widely used in diagnostic virology. This method was widely adopted by clinical laboratories during the 1980s, particularly for detection of respiratory viruses. The commercial availability of specific monoclonal antibodies and simultaneous staining with
Guidelines on establishment of virology laboratory in developing countries Page 21 multiple different antibodies labeled with different fluorescent labels has enabled rapid detection of a variety of viruses. Antigen detection methods are particularly useful for viruses that grow slowly or are labile, making recovery in culture difficult. The most important targets have been detection of viruses in respiratory specimens; cutaneous specimens; stool specimens; and cereberospinal fluid (CSF). Viruses such as the enteroviruses and the rhinoviruses that have extensive antigenic heterogeneity and cross-reacting antigens are not suitable for antigen-detection techniques. The advantages of antigen-detection techniques are rapidity (results can be available within hours of receipt of the specimen in the laboratory) and lack of requirement for viral viability
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- Microbiology, Virology Laboratory