Treatment with human recombinant Gal-1 inhibits DENV-1 in vitro infection.(A) Biotinylated-hrGal-1 (20 µg/mL) was incubated with ECV-304 cells in presence or absence of 40 mM lactose or sucrose for 1 hour at 4°C. The binding of biotinylated-hrGal-1 to ECV-304 cells surfaces was detected by staining with streptavidin-FITC and measured by flow cytometry. The analysis was performed using a Diva software (Becton Dickson) and results are expressed as mean mean fluorescence intensity (MFI)±SD. Tests were performed in triplicates. (B) HMVEC-L, Vero-E6 and ECV-304 cells were incubated with 10 µM hrGal-1 or only medium for 1 hour at 37°C. Following, cells were inoculated with DENV-1 at a MOI of 0.5 and cultivated for 72 hours. At 72 hours postinfection the supernatants were collected and the viral RNA amounts were quantified by Real-Time PCR. Results are shown as Viral RNA amounts equivalent to PFU/ml±SD from 3 assays performed in triplicates. (C) ECV-304 cells were treated with hrGal-1 and infected with DENV-1 as described in (B) for 120 hours. The supernatants were collected at the indicated times postinfection and the viral loads were quantified by Real-Time PCR as described in (B). (N = 3) (D) ECV-304 cells were incubated with increased concentrations of monomeric-Gal-1 (hrGal-1 m), dimeric-Gal-1 (hrGal-1 d) or galectin-3 (hrGal-3) for 1 hour at 37°C before inoculation with DENV-1 (MOI 0.5). Cells were cultivated for 72 hours at 37°C and viral load was quantified as described in (A) (N = 3). (E) ECV-304 cells were treated with hrGal-1 (10 µM) in the presence of 40 mM lactose (LAC) or 40 mM sucrose (SUC) and then infected with DENV-1 as described in (B), for 72 hours. The viral load was quantified as described in (B). Data are representative from three independent experiments. *p<0.01; **p<0.001; ***p<0.0001.
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