Immunofluorescent Detection of Mouse Epithelial Cells Infected with Reovirus
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Infected mouse L cells viewed with bright-field microscopy at 200x magnification (Enlarged view)
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Infected mouse L cells viewed with bright-field microscopy (Labeled view)
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Infected mouse L cells viewed with epifluorescence at 200x magnification (Enlarged view)
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Infected mouse L cells viewed with epifluorescence at 200x magnification (Labeled view)
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Infected mouse L cells infected with a 1/10 viral dilution and viewed with epifluorescence at 200x magnification (Enlarged view)
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Infected mouse L cells infected with a 1/10 viral dilution and viewed with epifluorescence at 200x magnification (Labeled view)
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Infected mouse L cells infected with a 1/100 viral dilution and viewed with epifluorescence at 200x magnification (Enlarged view)
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Infected mouse L cells infected with a 1/100 viral dilution and viewed with epifluorescence at 200x magnification (Labeled view)
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Uninfected mouse L cells viewed with epifluorescence at 200x magnification (Enlarged view)
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Uninfected mouse L cells viewed with epifluorescence at 200x magnification (Labeled view)
Introduction
FIG. 1. Infected mouse L cells viewed with bright-field microscopy at 200x magnification. Following virion attachment to cell surface receptors, reovirus particles enter the host cell through receptor-mediated endocytosis. Proteolysis of the outer-capsid proteins occurs via a two-step disassembly process and replication then occurs in the cytoplasm. After assembly of new viral particles, host cells will be lysed to release new infectious viral particles. Maximal viral production within an infected cell occurs about 24 hours postentry.
FIG. 2. Infected mouse L cells viewed with epifluorescence at 200x magnification. Cells infected with virus are first tagged with a rabbit anti-reovirus antibody and subsequently tagged with a secondary anti-rabbit antibody, which is fluorescently labeled. A majority of the cells are infected and display a distinct cytoplasmic fluorescence. This image shows the same field of view as Fig. 1.
FIG. 3. Mouse L cells infected with a 1/10 viral dilution and viewed with epifluorescence at 200x magnification. The 10-fold viral dilution reduces the amount of infectious virus present, thereby reducing the number of infected mouse L cells and providing a field of view with a countable number of fluorescing cells. Cell density is approximately equal to that of Fig. 1.
FIG. 4. Mouse L cells infected with a 1/100 viral dilution and viewed with epifluorescence at 200x magnification. Cell density is approximately equal to that of Fig. 1.
Discussion
A fluorescent focus assay uses indirect immunofluorescence to quantify the amount of infectious viral particles present in a given sample. Because virions are too small to visualize using a light microscope, alternative methods of detection are necessary. Plaque assays often are used to detect and quantify infectious viral particles. Often, though, this assay requires a lengthy incubation period (3). Additionally, not all viruses form plaques in cell culture. The fluorescent focus assay, conversely, is more rapid and does not require that the virus form plaques. For these reasons, it has been used frequently with various viruses (1, 2, 4). The fluorescent focus assay not only allows detection of infectious viral particles, but also allows one to quantify the amount of infectious virus present in an inoculum by counting the number of fluorescent cells. The viral titer then can be expressed in terms of fluorescent focus units per milliliter.
References
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2. Crawford, S. E., D. G. Patel, E. Cheng, Z. Berkova, J. M. Hyser, M. Ciarlet, M. J. Finegold, M. E. Conner, and M. K. Estes. 2006. Rotavirus viremia and extraintestinal viral infection in the neonatal rat model. J. Virol. 80:4820–4832.
3. Furlong, D. B., M. L. Nibert, and B. N. Fields. 1988. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 62:246–256.
4. Payne, A. F., I. Binduga-Gajewska, E. B. Kauffman, and L. D. Kramer. 2006. Quantitation of flaviviruses by fluorescent focus assay. J. Virol. Methods 134:183–189.