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A quantitative infection assay for human type I, II, and III interferon antiviral activities.

Voigt E, Inankur B, Baltes A, Yin J - Virol. J. (2013)

Bottom Line: Upon virus infection, cells secrete a diverse group of antiviral molecules that signal proximal cells to enter into an antiviral state, slowing or preventing viral spread.These paracrine signaling molecules can work synergistically, so measurement of any one antiviral molecule does not reflect the total antiviral activity of the system.Further, it eliminates cell fixation, rinsing, and staining steps, and is inexpensive to implement.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biological Engineering, University of Wisconsin, Madison, USA.

ABSTRACT

Background: Upon virus infection, cells secrete a diverse group of antiviral molecules that signal proximal cells to enter into an antiviral state, slowing or preventing viral spread. These paracrine signaling molecules can work synergistically, so measurement of any one antiviral molecule does not reflect the total antiviral activity of the system.

Results: We have developed an antiviral assay based on replication inhibition of an engineered fluorescent vesicular stomatitis virus reporter strain on A549 human lung epithelial cells. Our assay provides a quantitative functional readout of human type I, II, and III interferon activities, and it provides better sensitivity, intra-, and inter-assay reproducibility than the traditional crystal violet based assay. Further, it eliminates cell fixation, rinsing, and staining steps, and is inexpensive to implement.

Conclusions: A dsRed2-strain of vesicular stomatitis virus that is sensitive to type I, II, and III interferons was used to develop a convenient and sensitive assay for interferon antiviral activity. We demonstrate use of the assay to quantify the kinetics of paracrine antiviral signaling from human prostate cancer (PC3) cells in response to viral infection. The assay is applicable to high-throughput screening for anti-viral compounds as well as basic studies of cellular antiviral signaling.

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Comparison of live/dead cell stains and fluorescent-protein expressing assay virus. A549 cells were incubated under serial 2-fold dilutions of recombinant human IFNβ for 24 hours, then infected with either wild-type or recombinant VSV as indicated at a multiplicity of 5 pfu/cell. After 24 hours of infection, assay plates were stained, imaged, and quantified as discussed in the Methods section. Positive signal indicates cell survival for the crystal violet assay, virus replication for the fluorescent virus assays, and cell death in the Sytox assay. Positive control wells are cells untreated by antivirals and infected. Negative control wells are untreated, uninfected cells.
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Figure 2: Comparison of live/dead cell stains and fluorescent-protein expressing assay virus. A549 cells were incubated under serial 2-fold dilutions of recombinant human IFNβ for 24 hours, then infected with either wild-type or recombinant VSV as indicated at a multiplicity of 5 pfu/cell. After 24 hours of infection, assay plates were stained, imaged, and quantified as discussed in the Methods section. Positive signal indicates cell survival for the crystal violet assay, virus replication for the fluorescent virus assays, and cell death in the Sytox assay. Positive control wells are cells untreated by antivirals and infected. Negative control wells are untreated, uninfected cells.

Mentions: We compared the traditional virus-induced cytopathology measurement (crystal violet after 28-hour infection) with a more recently developed fluorescent dead cell stain (Sytox®) and our two fluorescent VSV reporter strains. To do so, we incubated A549 cells under 2-fold dilutions of an IFNβ standard solution in media for 24 hours. The cells were then infected with either wild-type or one of the fluorescent reporter VSV strains as indicated in Figure 2, at an MOI of 5 pfu/cell, and the infection was allowed to progress for 28 hours. WtVSV-infected plates were rinsed and stained as indicated. Assay results were quantified by fluorescent scanning at the appropriate wavelengths and subsequent normalization to positive (no IFN) and negative (no virus) controls, as shown in Figure 2.


A quantitative infection assay for human type I, II, and III interferon antiviral activities.

Voigt E, Inankur B, Baltes A, Yin J - Virol. J. (2013)

Comparison of live/dead cell stains and fluorescent-protein expressing assay virus. A549 cells were incubated under serial 2-fold dilutions of recombinant human IFNβ for 24 hours, then infected with either wild-type or recombinant VSV as indicated at a multiplicity of 5 pfu/cell. After 24 hours of infection, assay plates were stained, imaged, and quantified as discussed in the Methods section. Positive signal indicates cell survival for the crystal violet assay, virus replication for the fluorescent virus assays, and cell death in the Sytox assay. Positive control wells are cells untreated by antivirals and infected. Negative control wells are untreated, uninfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716869&req=5

Figure 2: Comparison of live/dead cell stains and fluorescent-protein expressing assay virus. A549 cells were incubated under serial 2-fold dilutions of recombinant human IFNβ for 24 hours, then infected with either wild-type or recombinant VSV as indicated at a multiplicity of 5 pfu/cell. After 24 hours of infection, assay plates were stained, imaged, and quantified as discussed in the Methods section. Positive signal indicates cell survival for the crystal violet assay, virus replication for the fluorescent virus assays, and cell death in the Sytox assay. Positive control wells are cells untreated by antivirals and infected. Negative control wells are untreated, uninfected cells.
Mentions: We compared the traditional virus-induced cytopathology measurement (crystal violet after 28-hour infection) with a more recently developed fluorescent dead cell stain (Sytox®) and our two fluorescent VSV reporter strains. To do so, we incubated A549 cells under 2-fold dilutions of an IFNβ standard solution in media for 24 hours. The cells were then infected with either wild-type or one of the fluorescent reporter VSV strains as indicated in Figure 2, at an MOI of 5 pfu/cell, and the infection was allowed to progress for 28 hours. WtVSV-infected plates were rinsed and stained as indicated. Assay results were quantified by fluorescent scanning at the appropriate wavelengths and subsequent normalization to positive (no IFN) and negative (no virus) controls, as shown in Figure 2.

Bottom Line: Upon virus infection, cells secrete a diverse group of antiviral molecules that signal proximal cells to enter into an antiviral state, slowing or preventing viral spread.These paracrine signaling molecules can work synergistically, so measurement of any one antiviral molecule does not reflect the total antiviral activity of the system.Further, it eliminates cell fixation, rinsing, and staining steps, and is inexpensive to implement.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biological Engineering, University of Wisconsin, Madison, USA.

ABSTRACT

Background: Upon virus infection, cells secrete a diverse group of antiviral molecules that signal proximal cells to enter into an antiviral state, slowing or preventing viral spread. These paracrine signaling molecules can work synergistically, so measurement of any one antiviral molecule does not reflect the total antiviral activity of the system.

Results: We have developed an antiviral assay based on replication inhibition of an engineered fluorescent vesicular stomatitis virus reporter strain on A549 human lung epithelial cells. Our assay provides a quantitative functional readout of human type I, II, and III interferon activities, and it provides better sensitivity, intra-, and inter-assay reproducibility than the traditional crystal violet based assay. Further, it eliminates cell fixation, rinsing, and staining steps, and is inexpensive to implement.

Conclusions: A dsRed2-strain of vesicular stomatitis virus that is sensitive to type I, II, and III interferons was used to develop a convenient and sensitive assay for interferon antiviral activity. We demonstrate use of the assay to quantify the kinetics of paracrine antiviral signaling from human prostate cancer (PC3) cells in response to viral infection. The assay is applicable to high-throughput screening for anti-viral compounds as well as basic studies of cellular antiviral signaling.

Show MeSH
Related in: MedlinePlus