Quantification of Viruses

In order to obtain any significant understanding of the nature of viruses and virus replication, it is necessary to be able to quantify the number of virus particles. Virus particles are almost always too small to be seen under the light microscope. Although virus particles can be observed under the electron microscope, the use of this instrument is cumbersome for routine study. In general, viruses are quantified by measuring their effects on the host cells which they infect. It is common to speak of a virus infectious unit, which is the. smallest unit that causes a detectable effect when placed with a susceptible host. By determining the number of infectious units per 

Plaque assay When a virus particle initiates an infection upon a layer or lawn of host cells which is growing spread out on a flat surface, a zone of lysis or growth inhibition may occur which results in a clearing of the cpll growth. This clearing is called a plaque, and it is assumed that each plaque has originated from one virus particle. 

Plaques are essentially windows in the lawn of confluent cell growth. With bacterial viruses, plaques may be obtained when virus particles are mixed into a thin layer of host bacteria which is spread out as an agar overlay on the surface of an agar medium. During incubation of the culture, the bacteria grow and form a turbid layer which is visible 

The plaque procedure also permits the isolation of pure virus strains, since if a plaque has arisen from one virus particle, all the virus particles in this plaque are probably genetically identical. Some of the particles from this plaque can be picked and inoculated into a fresh bacterial culture to establish a pure virus line. The development of the plaque assay technique was as important for the advance of virology as was Koch s development of solid media for bacteriology. 

Plaques may be obtained for animal viruses by using animal cell-culture systems as hosts. A monolayer of cultured animals cells is prepared on a plate or flat bottle and the virus suspension overlayed. Plaques are revealed by zones of destruction of the animal cells. 

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Immobilized cell walls can also be used for studying and perhaps also for quantification of viruses. Thus, using columns with cell walls of Escherichia coli immobilized on kiselguhr it was possible to study the adsorption of specific phages to the cell walls as well as the DNA-injection process (46). 

Reiber HO and Lange P (1991) Quantification of virus-specific antibodies in cerebrospinal fluid and serum sensitive and specific detection of antibody synthesis in brain. Clinical Chemistry 37 1153-1160. 

CV) ranged from 17 to 39%. The %CV was highest near the quantification limit of the assay. The results from the first- and second-generation assays were highly correlated (r = 0.96), allowing meaningful comparisons of virus concentrations in specimens tested with either assay. 

Detmer, J et al. (1996). Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology. J. Clin. Microbiol. 34,901-907. 

Revets, H., etal. (1996). Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICOR-HTV Monitor, and QUAN l lPLEX HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol 34,1058-1064. 

Wilber, J. C., and Urdea, M. S. (1995). Chapter 6. In Molecular methods for virus detection. Quantification of viral nucleic acids using branched DNA signal amplification. (San Diego Academic Press). 

Figure 5.8 Quantification of bacterial virus by plaque assay using the agar overlay technique.

Animal infectivity methods Some viruses do not cause recognizable effects in cell cultures but cause death in the whole animal. In such cases, quantification can only be done by some sort of titration in infected animals. The general procedure is to carry out a serial dilution of the unknown sample, generally at ten-fold dilutions, and samples of each dilution are injected into numbers of sensitive animals. After a suitable incubation period, the fraction of dead and live animals at each dilution is tabulated and an end point dilution is calculated. This is the dilution at which, for example, half of the injected animals die. Although such serial dilution methods are much more cumbersome and much less accurate than cell culture methods, they may be essential for the study of certain types of viruses. 

J. Schulze-Horsel, Y. Genzel, U. Reichl, 2007. Quantification of intracellular accumulation of Ml and NP of influenza A virus - monitoring of infection status of production cells during vaccine production by flow cjdometry. Submitted to BioMedCentral Biotechnology. 

Successful treatment of human immunodeficiency virus (HIV-1) infection has been achieved through successful implementation of highly active antiretroviral therapy, frequently referred to as HAART. This involves simultaneous administration of both nucleoside and nonnucleoside reverse transcriptase inhibitors and one or more protease inliibitors. The common nucleoside reverse transcriptase inhibitors are the thymidine analogs didanosine (ddl), lamivudine (3TC), and zalcitabine (ddC) and the non-thymidine analogs abacavir (Ziazen), stavudine (d4T), and zidovudine (AZT). The nonnucleoside reverse transcriptase inhibitors include delavirdine, efavirenz, and nevirapine. The protease inhibitors include indinavir, nelfinavir, ritonavir, and saquinavir. Response to therapy is monitored by quantification of HIV-RNA copies (viral load) and CD-4+ T-lymphocyte count. Successful therapy is indicated when viral load is reduced to <50 copies/mL and CD-4+ count >500 per mL. 

Leb V, Stocher M, Valentine-Thon E, Holzl G, Kessler H, Stekel H, et al. Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments. J Clin Microbiol 2004 42 585-90. 

Sommer JM, Smith PH, Parthasarathy S, Isaacs J, Vijay S, Kieran J, Powell SK, McClelland A, Wright JF. Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Mol Ther 2003 7 122-128. 

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