Use of fluorescence resonance energy transfer for rapid detection of enteroviral infection in vivo

Abstract

Enteroviruses can be easily transmitted through the fecal-oral route and cause a diverse array of clinical manifestations. Recent outbreaks associated with enteroviral contamination in aquatic environments have called for the development of a more efficient and accurate virus monitoring system. To develop a simple, rapid, and direct method for identifying enteroviral infections, we generated a fluorescent reporter system in which genetically engineered cells express a hybrid fluorescent indicator composed of a linker peptide, which is exclusively cleaved by the 2A protease (2A(pro)), flanked with a cyan fluorescent protein (CFP) and a yellow fluorescent protein undergoing fluorescence resonance energy transfer. The covalent linkage between two fluorophores is disrupted due to 2A(pro) activity upon viral infection, which results in an increase in CFP intensity. This allows the rapid (within 7.5 h) detection of very low numbers (10 PFU or fewer) of infectious enteroviruses.

FIG. 1.

Schematic representation of fluorescent indicator for monitoring 2A^pro-mediated proteolytic processing. (A) In the presence of 2A^pro, the linker peptide is cleaved via the protease and results in the increased ECFP signal. (B) The amino acid sequence of the linker peptide is shown, and the cleavage site between two amino acids is shown in boldface.

FIG. 2.

The fluorimetric assay of the FRET substrate with PV 2A^pro in vitro. Each experiment was performed in triplicate. Western blot analysis of GFP variants shows that the upper band represents the conjugated FRET substrate with a molecular mass of approximately 60 kDa, and the lower band represents the proteolytic products with a molecular mass of approximately 30 kDa. AFU, artificial fluorescent unit.

FIG. 3.

Fluorescence microscopy analysis of the reporter cells. Fluorescence intensity of ECFP was monitored via a 440-nm/480-nm filter set (A) and a 480-nm/530-nm filter set for EYFP (B), and the superimposed image is shown in panel C.

FIG. 4.

Fluorescence microscopy analysis of the reporter cells challenged with two different concentrations of PV. Control cells (A) show no sign of increased ECFP signal, while cells challenged with either 100 PFU (B) or 1,000 PFU (C) of virus exhibit increased ECFP intensity.

FIG. 5.

The fluorimetric assay of the FRET substrate with various 2A proteases in vitro. Each experiment was performed in triplicate, and 100 μg of each protease was added to the reaction. Western blotting of total cell lysate from the bioreporter cells using anti-GFP variant monoclonal antibody shows the same result as the in vitro assay, in which the upper band indicates the intact FRET substrate and the lower bands are the cleaved products. AFU, artificial fluorescent unit.