Figure 5.

Construction and Characterization of Recombinant miR124-Regulated EV-A71

(A) Schematic representation of miRT EV-A71. Three tandem copies of sequences complementary to miRNAs were inserted into the viral genome. The brain-specific miR124-target sequence (miR124T, blue) and Caenorhabditis elegans-derived miR39-target sequence (CelTCON, red) are shown, with spacer elements in black. (B) Replication kinetics of EV-A71 BrCr, EV-A71-miR124T, or EV-A71-CelTCON in Vero cells (MOI = 1). (C) The EV-A71-miR124T virus titers were determined after transfection with increasing doses of miR124 mimics (MOI = 20). (D) Vero cells transfected with 200 nM miRNA mimics were subsequently infected with EV-A71 BrCr, EV-A71-miR124T, or EV-A71-CelTCON (MOI = 20), and the cytotoxicity was determined at 24 h postinfection by an LDH assay. (E) Viral titers in the supernatants of the miRNA mimic-treated cells were determined. Bars represent the mean ± standard deviations of three independent experiments. (F) Suckling mice were injected intracerebrally with vehicle or 10⁷ TCID50s of EV-A71 BrCr or EV-A71-miR124T. The overall survival of mice was assessed. (G) Viral titers within the brain at 2 days postinjection were determined. Individual viral load values are plotted along with the mean viral load (solid line). (H–J) Primary human neurons (HNs) or mouse neurons (MNs) were infected with EV-A71 BrCr, EV-A71-miR124T at an MOI of 10 for 48 h or mock-treated, respectively. (H) Viral antigen (red) and β-tubulin III (green) were detected using IFA. Cell nuclei (blue) were counterstained with Hoechst 33342. Scale bar, 20 μm. The results of one representative experiment out of three are shown. (I) Virus titers in the culture were determined by the TCID50 assay. (J) Cytotoxicity was assessed by an LDH assay. The results are presented as the mean ± standard deviations obtained from three independent experiments. ∗∗∗p < 0.001.