Figure 1.
In situ ELISA assay workflow.
Table 1.
Marine seaweeds selected for antiviral screening, location of collection and cytotoxicity activity in Huh7.5 cells.
Figure 2.
(A) Effect of different blocking buffers (skim milk and FBS) and fixatives (methanol:acetone and paraformaldehyde followed by triton X-100) for the assay. (B) Cells were seeded in a range of concentration and the influence for the assay performance was evaluated. (C) The ideal MOI for all DENV serotypes was evaluated 48 hours post-infection and (D) 72 hours post-infection. Values represent mean ± SD of three independent experiments performed in triplicate.
Figure 3.
In situ ELISA assay validation.
(A) Correlation between the in situ ELISA and the foci-forming assay, where Huh7.5 was infected with DENV-1, DENV-2, DENV-3 and DENV-4 at a range of MOI (4–0.01). After 72 hours of infection the supernatant was used for the foci-forming assay and the cells submitted to the ELISA assay. Data from one representative experiment, analyzed using Pearson correlation test. (B) Comparison of the DRC and the IC50 for IFN-α 2A and DENV-4 infection, obtained with the in situ ELISA, foci-forming assay and a commercial NS1 antigen capture ELISA assay. Mean ± SD of three independent experiments, analyzed by sigmoidal dose-response curve (variable slope), the dashed line represents the detection limit of the foci-forming assay.
Figure 4.
Seaweed extracts antiviral screening.
(A) Huh7.5 were seeded in a 96-well plate and infected with DENV-1, (B) DENV-2 and (C) DENV-3 with an MOI of 4, and (D) DENV-4 with an MOI of 0.1. Interferon-α 2A (100 IU/ml) was used as a positive control and after 72 hours post-infection the ELISA was performed. Data were analyzed using one-way ANOVA followed by Tukey test. Values are mean ± SD of three independent experiments. *p<0.05.
Figure 5.
DENV infection inhibition for different strains/serotypes.
Huh7.5 was seeded in a 96-well plate and infected with each strain with a MOI of 4 for 1 h30 min, then the seaweed extracts were added and after 72 hours the ELISA was performed. Data were analyzed using one-way ANOVA followed by Dunnett test. Values are mean ± SD of three independent experiments,*p<0.05.
Figure 6.
Time of addition studies with seaweed extracts A1, A3, A8 and A12.
(A) Huh 7.5 cells were infected with DENV-4 with an MOI of 0.1 following each treatment (before the infection, −1.5 h; during the infection, 0 h; and after the infection, +1.5 h) at the MNTD, the cells were submitted to the ELISA assay (B) and the supernatant for the foci-forming assay. The dashed line represents the detection limit of the foci-forming assay.
Figure 7.
Dose response curve for the seaweed extracts A1, A3, A8 and A12.
(A) Huh 7.5 cells were infected with DENV-4 and treated during the infection in a range of concentration for each seaweed extract. (B) CC50, IC50 and SI were calculated. Mean ± SD of three independent experiments, analyzed by sigmoidal dose response curve (variable slope).
Figure 8.
Effect of the seaweed extracts A1, A3, A8 and A12 on DENV-4 entry process.
(A) Binding inhibition assay. (B) Internalization inhibition assay. Data were analyzed using one-way ANOVA followed by Dunnett test. Values are mean ± SD of three independent experiments,*p<0.05.