Detection of infective poliovirus by a simple, rapid, and sensitive flow cytometry method based on fluorescence resonance energy transfer technology

Abstract

The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2A(pro)) as an indication of infection. Quantification of the infectious-virus titers was resolved by using flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2A(pro) were infected with different titers of PV1. After incubation at various time points, cells were harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.

FIG. 1.

Principle of FRET. ex, excitation wavelength; em, emission wavelength (in nanometers).

FIG. 2.

Poliovirus 1 (PV1) infection of BGM-PV cells. Confluent monolayers of BGM-PV cells (ca. 1.6 × 10⁶ cells) in 6-well plates were infected with 10-fold dilutions of PV1. After 8 h postinfection (hpi), cells were trypsinized, washed with 10% FBS followed by 1× TBS plus 3 mM EDTA (pH 8.0), resuspended in the latter, and then subjected to flow cytometry. x axis, CFP intensity; y axis, YFP intensity from CFP excitation. Each dot represents a single event (cell), and dots inside each polygon represent cells that were included in the analysis. The percentage of infected cells shown above each graph was determined by counting the number of cells without a FRET signal divided by the total number of cells counted. (A to C) MOI of 0.4 to 0.004; (D) uninfected cells.

FIG. 3.

Time course of PV1 infection of BGM-PV cells at an MOI of 0.2. Cells were grown and infected as described in the text and were harvested at different time points. Those cells that lost FRET due to the uncoupling of the CFP-YFP pair brought about by PV1 infection were counted using FC.

FIG. 4.

Direct comparison of FC-based and plaque assays. Confluent monolayers of BGM-PV cells in 12-well plates were infected with PV1 (20 min of absorption). For the FC-based assay, cells were harvested after 8 h as described in the text and then subjected to flow cytometry. For the plaque assay, infected cells were overlaid with 1% CMC in MEM (plus 2% FBS), incubated for 48 h, and then fixed/stained with 0.8% crystal violet in a 3.7% formaldehyde solution.

FIG. 5.

Comparison of FC-based assay and plaque assay for detecting poliovirus from an urban water runoff sample. The correlation was established by challenging the reporter cells with 2-fold serial dilutions of PV1. The numbers of infected cells were regressed from a calibration curve established in parallel with water analysis and plotted against the numbers of PFU determined by plaque assay. Each data point represents means of 4 replicates.