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. 2013 Jul 6:10:224.
doi: 10.1186/1743-422X-10-224.

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

Emily Voigt  1 , Bahar Inankur, Ashley Baltes, John Yin
Affiliations

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

Emily Voigt et al. Virol J. .

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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Figures

Figure 1
Figure 1
Genotype maps of wild-type and fluorescent reporter VSV strains used in this study. Recombinant VSV strains were created by reverse-genetics, incorporating fluorescent reporter variants of GFP(ZsGreen) and RFP(DsRed2) along with the five native VSV proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L).
Figure 2
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.
Figure 3
Figure 3
Assay time-course development. Recombinant VSV antiviral activity assays were imaged on a fluorescent biomolecular imager every four hours post-infection to monitor fluorescent signal development indicating viral replication. Mean fluorescence values were extracted from plate images, normalized to positive and negative controls and plotted. Darker markers indicate longer development times. IC50 values were calculated as described, and the coefficient of variance was calculated between four replicates at each time point.
Figure 4
Figure 4
Validation of the assay’s ability to measure multiple interferon types. The final DsRed2 form of the assay was used to measure antiviral activity of human type I (IFNα1, IFNβ), type II (IFNγ), and type III (IFNλ1, IFNλ2, IFNλ3) recombinant bioactive interferon standards. IC50 values in pg/ml of added interferon were calculated as described, and are also shown in pmol/ml for molar comparisons.
Figure 5
Figure 5
Kinetics of functional antiviral signaling and virus progeny release of PC3 cells in response to virus infection. Data points are averages of biological duplicates assayed in duplicate (titer) or quadruplicate (activity). Three antiviral activity data points (2, 4, and 6 hours post-infection) resulted in antiviral signal beneath the assay detection limit (grey). Closed symbols: antiviral activity. Open symbols: viral titer. Error bars are +/− standard deviation.
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