Fig 1.
Niclosamide treatment reduces viral protein expression and viral release in DENV serotype 2 PL046 (DENV2)-infected cells.
(A) LDH assays showing cytotoxicity in niclosamide (Niclo)-treated A549 (48 h) and BHK-21 (24 h) cells at various concentrations. (B) Representative Western blots showing viral NS3 and capsid protein expression. β-actin served as the internal control. The relative ratios of the measured proteins compared to β-actin are also shown. (C) Plaque assays showing viral release in DENV2 (MOI = 1)-infected A549 (48 h) and BHK-21 (24 h) cells in the presence of niclosamide (Niclo). (D) Plaque assays showing the production of infectious particles for a kinetic treatment (pre-, co-, and post-treatment) of niclosamide (Niclo) in DENV2 (MOI = 1)-infected BHK-21 (24 h) cells. Virus particles are shown as the desired pfu amount for infection and as calculated as the percentage (%) of inhibition. DMSO was used as a solvent control. The quantitative data are depicted as the mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01. ns, not significant.
Fig 2.
Niclosamide treatment neither hinders the endocytosis of DENV nor enhances IFN-β production.
(A) LDH assay showing cytotoxicity in niclosamide (Niclo)-treated Neuro-2a cells for 48 h with various concentrations. (B) Representative Western blot showing viral NS3 protein expression in DENV2 (MOI = 1)-infected Neuro-2a cells for 48 h with or without niclosamide (Niclo) pretreatment. β-actin served as the internal control. The relative ratios of the measured proteins compared to β-actin are also shown. Confocal microscopy (C) and flow cytometry (D) were used to measure the positive Neuro-2a cells carrying Alexa-594 labeled (red) DENV2 (MOI = 1) 2 h post-infection in the presence of niclosamide (Niclo). E: ELISA analysis showing IFN-β production in DENV2 (MOI = 1)-infected A549 cells for 48 h with or without niclosamide (Niclo) pretreatment. DMSO was used as a solvent control. The quantitative data are depicted as the mean ± SD of three independent experiments. ** P < 0.01. ns, not significant.
Fig 3.
Niclosamide treatment does not repress firefly luciferase activity in BHK-D2-Fluc-SGR-Neo-1 cells.
(A) Luciferase activity and (B) LDH assay in niclosamide (Niclo, 1 μM)-treated parental BHK-21 and BHK-D2-Fluc-SGR-Neo-1 cells (replicons) 24 h post-treatment. DMSO was used as a solvent control. Quantitative data are depicted as the mean ± SD of three independent experiments. ns, not significant.
Fig 4.
Niclosamide treatment reduces DENV infection independent of the mTOR, STAT3, and NF-κB signaling pathways.
Representative Western blots showing the expression of the indicated proteins in (A) niclosamide (Niclo, 1 μM)- and (B) mTOR inhibitor rapamycin (200 ng/mL)-treated Neuro-2a cells for the indicated times. With or without rapamycin (Rap, 200 ng/mL), STAT3 inhibitor Cucurbitacin I (Cu I, 0.01 μM), and NF-κB inhibitor BAY 11–7082 (BAY, 1 μM) pretreatment, (C and E) representative Western blots showing the expression of the indicated proteins and (D and F) plaque assays showing viral release in DENV2 (MOI = 1)-infected Neuro-2a cells 48 h post-infection. DMSO was used as a solvent control. β-actin served as the internal control. The relative ratios of the measured proteins compared to those for total proteins and β-actin are also shown. The quantitative data are depicted as the mean ± SD of three independent experiments. *** P < 0.001. ns, not significant.
Fig 5.
Niclosamide treatment causes endosomal deacidification to abolish DENV2 infection.
BHK-21 cells were inoculated with DENV2 (MOI = 1) for 2 h in the presence of niclosamide (Niclo, 5 μM), the protonophores CCCP (50 μM) and FCCP (5 μM), the V-ATPase inhibitor (Baf A1, 100 nM) or rapamycin (Rap, 200 ng/mL). (A) Representative ratiometric live cell imaging of acridine orange (AO) staining showing acidic compartments (red). Nuclei were stained with Hoechst 33258 (blue). (B) Representative Western blot showing viral E protein expression in DENV2 (MOI = 1)-infected BHK-21 cells for the indicated times. The relative ratio to β-actin is shown. (C) Representative immunostaining and the relative mean fluorescence intensity (MFI) of viral dsRNA (green) at 6 h post-infection. (D) Plaque assay showing the level of viral replication 24 h post-infection. DMSO was used as a solvent control. For all images, representative data were selectively obtained from three individual experiments. Quantitative data are depicted as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001. ns, not significant.
Fig 6.
Niclosamide treatment reduces viral replication and increases the survival rate in suckling mice during DENV infection.
(A) With or without niclosamide (Niclo; 2 mg/kg or 5 mg/kg) co-treatment, seven-day-old ICR suckling mice were inoculated with DENV2 by concurrent intracranial (2.5 × 105 pfu) and intraperitoneal (7.5 × 105 pfu) injections. (B) Western blot analysis of viral NS3 and NS1 protein expression. (C) Plaque assay of viral replication in the brains at 9 days post-infection. β-actin served as the internal control. The relative ratios of the measured proteins compared to β-actin are also shown. The quantitative data are depicted as the mean ± SD of three independent experiments. ** P < 0.01 and *** P < 0.001. Additionally, time-kinetic changes in (D) body weights, (E) clinical scores, and (F) survival rates were measured. P value is shown.