Figure 4.

Substitution V135A enhances PSGL1 usage by mouse-adapted EV-A71. The relative levels of mRNA of (A) SCARB2 and (B) PSGL1 in ten different tissues was quantified by means of RT-qPCR. Data were normalized to β-actin (housekeeping gene). RT-qPCR data are expressed as relative levels. Horizontal bars represent the mean. (C) SCARB2 and PSGL1 in various organs tissues assessed by means of immunohistochemistry. SCARB2 protein expression in CNS, intestine, pancreas, liver and lung. (D-E) Infection of EV-A71 #812 and #812MA in L929 cells transfected with mouse SCARB2 and PSGL1 expression plasmids; Cells were infected with #812 and #812MA 24 h post transfection and frozen at 0 (D) or 24 (E) hours p.i. Following three freeze-thaw cycles, infectious virus titres (CCID₅₀/ml) were determined by means of end-point titration on RD cells. (F) Mouse SCARB2 and PSGL1-transfected L929 cells or RD cells were infected with #812 and #812MA; L929 cells were fixed at 48 h p.i., RD cells at 24 h p.i., followed by dsRNA (green) and nuclei (blue) staining. Images shown are representative fluorescent micrographs. The data shown are mean ± SEM of two (F) or three (D-E) independent experiments each containing three (D-E) or four (F) biological replicates. P values were calculated by two-way ANOVA in GraphPad, **P < .01; ***P < .001; ns, P > .05.