Fig. 4

Effects of Heparin on DTMUV attachment and entry. The effect of heparin on DTMUV binding was determined by incubating DEF cells with 1000TCID₅₀ DTMUV at 4 °C for 1 h with different concentrations of heparin. The unbound DTMUV was removed by washing the cells three times with PBS, after which the cells were incubated with cell culture medium at 37 °C for 24 h. To determine the effect of heparin on DTMUV entry, DEF cells were incubated with DTMUV at 4 °C for 1 h and then the unbound DTMUV was removed by washing the cells three times with PBS. Subsequently, the DEF cells were incubated with culture medium containing the different concentrations of heparin at 37 °C for 5 h. Culture medium was replaced with fresh medium without heparin. At 24 h post-infection at 37 °C, the NS5 protein levels in the DTMUV-infected DEF cells were detected by Western blotting (a) and quantification was reported as gray-scale value. b DTMUV titers in the supernatants were detected by TCID50. c DTMUV was treated with different concentration of trypsin at 37 °C for 15 min and then incubated with monolayer BHK21 or DEF cells at 4 °C for 1 h. d After treating the cells with different concentrations of trypsin at 37 °C for 15 min, the cell cytotoxicity was tested using a CCK8 kit. e 1000TCID₅₀ DTMUV was concentrated by ultracentrifugation. and then the virus treated with 50 μg/ml trypsin at 37 °C for 15 min. The virus was subjected to SDS-PAGE, the E protein was detected by anti-flavivirus group antigen antibody and quantification was reported as gray-scale value. Values shown represent means from 4 independent experiments