Quantitation of Viruses
المؤلف:
Stefan Riedel, Jeffery A. Hobden, Steve Miller, Stephen A. Morse, Timothy A. Mietzner, Barbara Detrick, Thomas G. Mitchell, Judy A. Sakanari, Peter Hotez, Rojelio Mejia
المصدر:
Jawetz, Melnick, & Adelberg’s Medical Microbiology
الجزء والصفحة:
28e , p424
2025-10-21
89
A. Physical Methods
Quantitative nucleic acid-based assays such as the polymerase chain reaction can determine the number of viral genome copies in a sample. Both infectious and noninfectious genomes are detected. Virus sequence variation may reduce virus detection and quantitation by this method.
A variety of serologic tests such as radioimmunoassays and enzyme-linked immunosorbent assays can be standardized to quantitate the amount of virus in a sample. These tests do not distinguish infectious from noninfectious particles and sometimes detect viral proteins not assembled into particles.
Certain viruses contain a protein (hemagglutinin) that has the ability to agglutinate red blood cells of humans or some animal. Hemagglutination assays provide a method for quantitating the infective and noninfective particles of these types of viruses.
Virus particles can be counted directly in the electron microscope by comparison with a standard suspension of latex particles of similar small size. However, a relatively concentrated preparation of virus is necessary for this procedure, and infectious virus particles cannot be distinguished from noninfectious ones.
B. Biologic Methods
End point biologic assays depend on the measurement of ani mal death, animal infection, or cytopathic effects in tissue culture at a series of dilutions of the virus being tested. The titer is expressed as the 50% infectious dose (ID50 ), which is the reciprocal of the dilution of virus that produces infection in 50% of the cells or animals inoculated. The ratio of the number of infectious particles to the total number of virus particles varies widely, from near unity to less than one per 1000, but often is one per several hundred. Precise assays require the use of a large number of replicates.
A widely used assay for infectious virus is the plaque assay, although it can only be used for viruses that grow well in tissue culture. Monolayers of host cells are inoculated with suitable dilutions of virus and after adsorption are overlaid with medium containing agar or carboxymethyl cellulose to prevent virus spreading throughout the culture. After several days, the cells initially infected have produced virus that spreads only to surrounding cells. Multiple cycles of replication and cell killing produce a small area of infection, or plaque. The length of time from infection to when plaques can be visualized for counting depends on the replication cycle of the virus and can range from a few days (eg, poliovirus) to 2 weeks or more (eg, SV40). Under controlled conditions, a single plaque can arise from a single clonal infectious virus particle, termed a plaque-forming unit. The cytopathic effect of infected cells within the plaque can be distinguished from uninfected cells of the monolayer. A more rapid method of assay is based on determination of the number of infected cells producing a viral antigen, such as by immunofluorescence.
Certain viruses (eg, herpes and vaccinia) form pocks when inoculated onto the chorioallantoic membrane of an embryonated egg. Such viruses can be quantitated by relating the number of pocks counted to the viral dilution inoculated.
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