In vitro Studies on The Inhibition of Replication of Zika and Chikungunya Viruses by Dolastane Isolated from Seaweed Canistrocarpus cervicornis

Abstract

The lack of vaccines and antiviral treatment, along with the increasing number of cases of Zika virus (ZIKV) and Chikungunya virus (CHIKV) infections, emphasize the need for searching for new therapeutic strategies. In this context, the marine brown seaweed Canistrocarpus cervicornis has been proved to hold great antiviral potential. Hence, the aim of this work was to evaluate the anti-ZIKV and anti-CHIKV activity of a marine dolastane isolated from brown seaweed C. cervicornis and its crude extract. Vero cells were used in antiviral assays, submitted to ZIKV and CHIKV, and treated with different concentrations of C. cervicornis extract or dolastane. The crude extract of C. cervicornis showed inhibitory activities for both ZIKV and CHIKV, with EC₅₀ values of 3.3 μg/mL and 3.1 μg/mL, respectively. However, the isolated dolastane showed a more significant and promising inhibitory effect (EC₅₀ = 0.95 µM for ZIKV and 1.3 µM for CHIKV) when compared to both the crude extract and ribavirin, which was used as control. Also, the dolastane showed a very potent virucidal activity against CHIKV and was able to inhibit around 90% of the virus infectivity at 10 μM. For the ZIKV, the effects were somewhat lower, although interesting, at approximately 64% in this same concentration. Further, we observed that both the extract and the dolastane were able to inhibit the replication of ZIKV and CHIKV at different times of addition post-infection, remaining efficient even if added after 8 hours post-infection, but declining soon after. A synergistic effect using sub-doses of the extract and isolates was associated with ribavirin, inhibiting above 80% replication even at the lowest concentrations. Therefore, this work has unveiled the anti-ZIKV and CHIKV potential of C. cervicornis crude extract and an isolated dolastane, which, in turn, can be used as a preventive or therapeutic strategy in the future.

Figure 1

Chemical structure of compound 1 from Canistrocarpus cervicornis.

Figure 2

Inhibition of ZIKV or CHIKV replication by C. cervicornis extract, dolastane, and ribavirin. (A) Inhibition of ZIKV replication. (B) Inhibition of CHIKV replication. Vero cells were infected with ZIKV or CHIKV (MOI 0.1) and treated at concentrations of 0.65, 1.25, 2.5, 5, 10, or 20 μg/mL or µM. The results were evaluated from three independent experiments in triplicate. Data are presented as the percentage of virus titer when compared to control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using the Tukey test in comparison to C. cervicornis extract, dolastane, and ribavirin in each concentration: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Effect of the addition of C. cervicornis extract, dolastane, and ribavirin on ZIKV or CHIKV replication over time. (A) Inhibition of ZIKV replication. (B) Inhibition of CHIKV replication. Monolayers of Vero cells were infected with ZIKV or CHIKV at an MOI of 3 at time zero. At times indicated, extract, dolastane or ribavirin was added to a final concentration of 10 μg/mL for extract or 10 μM/mL, for dolastane or ribavirin. The data were presented by the virus titers in PFU/mL when compared to control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using the Tukey test in comparison to C. cervicornis extract, dolastane, and ribavirin in each concentration: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Virucidal effect. (A) Effect on the ZIKV. (B) Effect on the CHIKV. The viral suspension (ZIKV or CHIKV) was incubated with C. cervicornis extract, dolastane, and ribavirin at concentrations of 2.5, 5, and 10 µg/mL for extract or 10 µM for dolastane or ribavirin for 2 h and then added to Vero cells. Viral cytopathic effect was assessed after 72 h of incubation. Data are presented as the percentage of virus titer when compared to control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using the Tukey test in comparison to C. cervicornis extract, dolastane, and ribavirin in each concentration: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Evaluation of the synergistic effect in combination of C. cervicornis extract or dolastane with ribavirin on ZIKV or CHIKV replication. (A) Inhibition of ZIKV replication. (B) Inhibition of CHIKV replication. Monolayers of Vero cells were infected with ZIKV at an MOI of 0.1 and subsequently treated with extract and Ribavirin sub-doses (0.5 µg/mL and 0.5 µM, respectively) and concentrations of 10 µg/mL and 10 µM. In addition, the dose combination was also performed at both concentrations for synergism assessment. Evaluation of the synergistic antiviral effect was determined by inhibition of cytopathic effect by plaque assay. Data are presented as the percentage of virus titer when compared to control cells and are expressed as the mean of three experiments ± standard error. Statistical significance following a one-sample t-test is indicated (*p < 0.05; **p < 0.001).

Figure 6

Synergistic effects of ribavirin and extract or dolastane against ZIKV and CHIKV. Synergy distribution plots and score matrix tables calculated by Bliss and Loewe methods for combination effects of ribavirin and extract or dolastane against (A) ZIKV, and (B) CHIKV are shown. Synergy scores are represented as mean ± standard deviation (n = 3). Statistical significance following a one-sample t-test is indicated (*p < 0.05; **p < 0.001).