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Immunofluorescent Detection of Mouse Epithelial Cells Infected with Reovirus

  • Authors: David R. Wessner 1, Karen Hasty 2, Mallory L. West 3
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Biology, Davidson College, Davidson, NC, 28035; 2: Davidson College, Davidson, NC, 28035; 3: Davidson College, Davidson, NC, 28035
  • Citation: David R. Wessner, Karen Hasty, Mallory L. West. 2008. Immunofluorescent detection of mouse epithelial cells infected with reovirus.
  • Publication Date : October 2008
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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.   

FIG. 5. Uninfected mouse L cells viewed with epifluorescence at 200x magnification. Cell density is approximately equal to that of Fig. 1.
 
  Methods
Monolayers of permissive mouse L929 cells (approximately 2 x 105 cells/well) were inoculated with various dilutions of reovirus strain  type 3 Dearing (T3D). Following inoculation, cells were incubated at 37°C in Joklik's modified Eagle's medium. Twenty-four hours postinoculation, the medium was aspirated and the cells were fixed with ice-cold methanol. The cells then were rinsed with phosphate-buffered saline and incubated with a rabbit anti-reovirus T3D primary antibody. After removing unbound primary antibody, Alexa fluor 488-conjugated chicken anti-rabbit immunoglobulin G (excitation: 494 nm, emission: 519 nm) was added to the monolayer of cells. Following an incubation, the cells again were rinsed and then visualized with a Nikon Eclipse TS100 inverted microscope at 200x magnification under brightlight or epifluorescence. Images were captured using Image-Pro Plus. 

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.

In this particular experiment, serial 10-fold dilutions of reovirus T3D of an unknown concentration were used to inoculate mouse L929 cells. As seen in Fig. 3 and 4, a 10-fold dilution of the virus results in a roughly 10-fold decrease in the number of fluorescent (and, thus, infected) cells. By counting the number of fluorescent cells, one can determine the concentration of infectious particles in the original sample. Quantifying the amount of infectious virus present in a sample can be important for many different applications. 

References

1. Barton, E. S., J. L. Connolly, J. C. Forrest, J. D. Chappell, and T. S. Dermody.2001. Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening.  J. Biol. Chem. 276:2200–2211.  

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.  

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