In Vitro Antiviral Activity Against Zika Virus From a Natural Product of the Brazilian Brown Seaweed Dictyota menstrualis

Abstract

Natural products isolated from seaweeds have shown great antiviral potential against numerous viruses such as human type 1 herpes, human immunodeficiency virus, and dengue. Diterpenes produced by the brown seaweeds Dictyota and Canistrocarpus, in particular, have shown antiviral or virucidal activity. Recently, the Zika virus (ZIKV) has become a major public health concern due to its widespread dissemination throughout the Americas. Since no vaccines are available, and no drugs have effectively treated recent cases of infection, our group evaluated products from Dictyota menstrualis for their antiviral potential, alone and in combination with Ribavirin. We first evaluated the compounds’ cytotoxicity at high concentrations, and then evaluated the inhibition of ZIKV replication by crude extracts and acetylated crude extracts and their fractions at 20 μg/mL. The F-6 and FAc-2 fractions, rich in cyclic diterpenes with aldehyde groupings, inhibited ZIKV replication by >74%, with inhibition behaving in a dose-dependent manner and the 50% effective concentration (EC₅₀) values of 2.80 (F-6) and 0.81 (FAc-2) μg/mL. Regarding the mechanism of action, FAc-2 had strong virucidal potential, and F-6 inhibited viral adsorption. Associating FAc-2 with Ribavirin at suboptimal dosages produced a strong synergistic effect that completely inhibited viral replication. Our results indicate that these natural products have excellent inhibitory potential against ZIKV replication and may be promising for developing affective therapies.

Since 1996, our group has been investigating the antiviral potential of marine natural products produced by brown algae of the genera Dictyota JV Lamour, Stypopodium Kützing, and Canistrocarpus De Paula and De Clerck, all representatives of the Dictyotaceae family. The marine natural products produced by these algae are cyclic diterpenes, many exclusive to the marine environment. ^{1 -16}

The Zika virus (ZIKV) was first isolated in 1947 from the febrile rhesus monkey in the Zika Forest in Uganda. ¹⁷ The first case of human infection was originally described in 1954 in Africa after some sporadic infections in humans were recorded. A 2007 ZIKV outbreak reporting the expressive rates of neurological disturbances was described in the Yap Islands in the Pacific, which was followed by another outbreak in the French Polynesian Islands in 2013. ^{18 -20}

In 2014, ZIKV entered the Americas. It was first reported in northeast Brazil and migrated to several countries before spreading to still others, including the United States. The transmission of ZIKV has largely been observed through Aedes aegypti species; however, Aedes albopictus (Stegomyia albopicta) has also been found to harbor ZIKV. ²¹

In over 60 years of observations and descriptions of previous outbreaks, ZIKV infections were reported as asymptomatic or causing mild-to-moderate symptoms. Severe symptoms were not observed but were similar to those of a Dengue virus (DENV) infection, a febrile illness that has a self-limiting association with maculopapular rash, headache, conjunctivitis, and musculoskeletal pain. In vitro studies indicate that ZIKV can also enhance infection by antibody dependence, which has already been observed in the hemorrhagic fever caused by DENV; however, the clinical significance of this finding is uncertain. ²²

Reports of outbreaks described in Asia and the Americas since 2014 have been directly related to clinical manifestations outside the conventional standard and have high severity profiles such as Guillain-Barré syndrome in adults and congenital ZIKV syndrome in infected pregnant women, which ranges from spontaneous abortion to normal fetal growth, microcephaly, and cerebral calcifications. ^23,24

Due to a Brazilian ZIKV epidemic, our group began new studies on the antiviral potential of natural seaweed products against ZIKV. This work presents one of the first results on the antiviral activity of natural products from Brazilian seaweeds against ZIKV. Those isolated from the alga Dictyota menstrualis have shown potential antiviral activity. This is a species that has been studied by our group since 1996, so its chemistry and abundant diterpenes are well known. ^{25 -27}

We initially tested a crude extract (EDM) and an acetylated crude extract (EDMAc) from D. menstrualis. To determine the cytotoxicity of the extracts, MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) assays were performed on Vero cells using concentrations of 25, 50, 100, 200, 400, and 800 µg/mL. The results in Table 1 show that the extracts had low cytotoxicity, with 50% cytotoxic concentration (CC₅₀) values of 984 and 735 µM, respectively. To determine the antiviral potential of the extracts, tests at a constant concentration of 20 µg/mL demonstrated that the substances could inhibit ZIKV replication by 42% (EDM) and 38% (EDMAc) at the fixed concentration. Based on these results, the extracts were fractionated and tested to discover which fractions inhibited ZIKV the most. The fractions obtained from the crude extract (F-1-F-8) and acetylated crude extract (FAc-1 and FAc-2) were also tested for their cytotoxicity at the same concentrations as above, and with the exception of F-8, all had low toxicity. In addition, we determined the antiviral potential of the fractions at a constant concentration (20 µg/mL) and found that F-6 and FAc-2 could inhibit ZIKV replication by >74% at this concentration, while maintaining low cytotoxicity (Table 1).

Table 1

Cytotoxicity (CC₅₀) and the Effect of the Dictyota Menstrualis Extracts and Their Fractions on Zika Virus Replication.

Compounds	CC50, μg/mL	PI, %
EDM	984	42
EDMAc	735	38
F-1	390	38
F-2	271	30
F-2	331	38
F-4	178	32
F-5	235	49
F-6	592	74
F-7	241	51
F-8	40	39
FAc-1	124	58
FAc-2	482	98

CC₅₀, 50% cytotoxic concentration, the concentration that reduces cell viability by 50% compared to untreated controls; EDM, D. menstrualis crude extract; EDMAc, acetylated D. menstrualis crude extract; PI, percentage of inhibition.

The ¹H nuclear magnetic resonance (NMR) spectrum of the fractions indicated the presence of α,β-unsaturated dialdehyde diterpenes, terpenes derived from a xeniane skeleton and typical of this seaweed, such as 6-hydroxy-dichotoma-2,13-diene-16,17-dial, 6-acetoxydichotoma-2,13-diene-16,17-dial (dichotomane skeleton), 5-hydroxy-1,6-cycloxenia-2,13-diene-16,17-dial and 5-acetoxy-1,6-cycloxenia-2,13-diene-16,17-dial (cycloxeniane skeleton), and acetoxydictyodial (xeniane skeleton). ^{25 -27} The ¹H NMR spectra of fractions F-6 and FAc-2 were compared to spectra previously obtained for products isolated by our group. ^{25 -27} The results revealed that an acetylated cycloxeniane was the most abundant chemical component in the F-6 fraction (Figure 1, compounds 1 -2), while the FAc-2 fraction showed a mixture of acetylated diterpenes, with dichotomane diterpenes being the most abundant (Figure 1, compounds 3 -4).

Figure 1

Structures of 1 to 4.

Several studies in the literature have demonstrated the great potential of algae and natural marine products against viruses, bacteria, and fungi, among others, and they are recognized as having low toxicity and very promising antiviral activity. ^{28 -30} The outstanding inhibition results against ZIKV obtained for the F-6 and FAc-2 fractions led to further tests. To estimate their potential for reducing the production of infectious viral particles, Vero cells were infected with an African strain of ZIKV at a multiplicity of infection (MOI) of 0.1 and subsequently treated with F-6 and FAc-2 at concentrations of 0.65, 1.25, 2.5, 5.0, 10, and 20 µg/mL. The Vero cells and supernatants were collected 18 hours postinfection and used to infect fresh Vero cells, with the plaques counted 3 to 5 days later. Ribavirin (5.0 µM) was used as a control, which had a 92% inhibition rate. The compounds showed dose-dependent inhibition, with viral replication inhibited by 90% at the highest concentrations while maintaining low EC₅₀ values of 2.80 and 0.81 µg/mL (Table 2). Based on these results, the selectivity index (SI) was calculated as the ratio between the CC₅₀ and EC₅₀ values to represent the safety for in vitro assays. Both extracts had high SI values (Table 2).

Table 2

Cytotoxicity (CC₅₀), Anti-Zika Virus Profile (EC₅₀), and SI for Fractions From Dictyota Menstrualis.

Compounds	CC50, μg/mL a	EC50, μg/mL b	SI c
F-6	592	2.80 ± 0.10	207.85
FAc-2	482	0.81 ± 0.04	595.06

SI, selectivity index; CC₅₀, 50% cytotoxic concentration;

The mean values ± standard deviation are representative of 3 independent experiments.

^a50% cytotoxic concentration, the concentration that reduces cell viability by 50% compared to untreated controls.

^bConcentration that reduced ZIKV replication by 50% compared to infected controls.

^cSelectivity index was defined as the ratio between CC₅₀ and EC₅₀ and represents the safety for in vitro assays.

To assess whether the compounds with potential inhibitors of ZIKV replication have virucidal activity as a possible mechanism of action, a viral suspension of approximately 10⁶ PFU and the compounds (0.5 µg/mL) were combined and incubated for 2 hours at 37°C. This virus/compound suspension was added to Vero cells and incubated for 3 to 5 days to evaluate whether viral plaque formation was inhibited. As can be seen in Figure 2, FAc-2 showed a high virucidal pattern that achieved a >90% inhibition of viral activity at the 20 µg/mL concentration. Although F-6 did not have a marked virucidal effect, it still produced >30% inhibition, which may be important for the compound’s activity. Virucidal substances inactivate extracellular viral particles by causing damage to the protein layer or by penetrating the virion and destroying the viral genome, which culminates in a reduction of viral infectivity. Studies have shown that a compound’s virucidal activity ³¹ can be an important tool for its use as a protective agent before viral infection and against arboviruses. As there are still no vaccines available for ZIKV, the use of this compound as a preventive measure may be fundamental for controlling the disease and preventing a mass emergence of new cases.

Figure 2

Effect of fractions from Dictyota menstrualis on the infectivity of ZIKV. Vero cells were infected with ZIKV at a multiplicity of infection of 0.1, and the results were evaluated by a plaque assay. Error bars indicate the standard deviation. Experiments were performed in triplicate. 5 µg/mL; 10 µg/mL; 20 µg/mL. ZIKV: Zika virus.

The algal products’ ability to inhibit viral adsorption was assessed by their inhibition of viral plaque formation. After adsorption at 4°C in the presence of 20 µg/mL of the compounds, F-6 inhibited the Vero cell-bound infection by ~70% in a dose-dependent manner, as can be seen in Figure 3. However, FAc-2 did not significantly inhibit adsorption, indicating that the F-6 compound had a further specificity in its ability to inhibit the production of viral plaques. Assays were also performed to study the effect of the compounds on ZIKV adsorption and penetration into the Vero cells. These assays depend on different temperatures in the initial 2 steps, before the viral life cycle. Viral adsorption can occur at both 4 and 37°C, whereas viral penetration only proceeds at 37°C. Therefore, these steps can be investigated in separate experiments. There is no adsorption assay to test whether the virus only binds to cells without penetrating when the exposure to the virus and F-6 was at 4°C. However, plaque formation was considerably reduced, indicating that F-6 either prevented ZIKV from binding to the cells or prevented bound ZIKV from entering the cells. The penetration test confirmed that F-6 had a significant effect after viral binding, with our findings suggesting that F-6 inhibits ZIKV by disrupting its ability to bind to host cell receptors.

Figure 3

Effect of the extract fractions from Dictyota menstrualis on the adsorption of ZIKV. Vero cells were infected with ZIKV at a multiplicity of infection of 1 in the presence or absence of 20 µg/mL of the marine compounds. After adsorption for 1 hour at 4°C, the cells were washed with cold phosphate-buffered saline to remove unabsorbed virus, disrupted by freezing and thawing, and evaluated by the plaque assay. Error bars indicate the standard deviation, and experiments were performed in triplicate. 5 µg/mL; 10 µg/mL; 20 µg/mL. ZIKV: Zika virus.

We evaluated whether the compound might have an additive or synergistic effect when associated with Ribavirin, which has been described as having strong activity against some arboviruses. For this evaluation, we selected the fraction that showed the best inhibition pattern against ZIKV replication (FAc-2). Vero cells were exposed to ZIKV (MOI 0.1) and then treated with the compound and Ribavirin at the concentrations previously found to inhibit viral replication by >90% (10 μg/mL FAc-2; 5 µM Ribavirin) and at suboptimal concentrations (0.25 µg/mL FAc-2; 0.25 µM Ribavirin) that only inhibited viral replication by ~20% (Figure 4). When the suboptimal concentrations of FAc-2 and Ribavirin were combined, however, they showed high synergistic activity, inhibiting viral replication by as much as 90%, a rate greater than the sum of their individual inhibition rates. These results suggest that a combined treatment of FAc-2 and Ribavirin could form the basis for developing novel strategies against ZIKV infection that use low-concentration compounds synergistically to potently inhibit viral replication, which has been widely described for other viruses. ^{32 -35}

Figure 4

Ribavirin synergistically potentiates the antiviral effect of FAc-2 in vitro. Vero cells were infected with Zika virus at a multiplicity of infection of 0.1, incubated for 2 hours, and subsequently treated with different concentrations of the substances, alone and in association, for 18 hours. The medium and cells were then collected and added to new Vero cells to evaluate the reduction in viral plaque formation.

In conclusion, our findings showed that the F-6 crude extract and FAc-2 acetylated crude extract had activity against ZIKV. With regard to the possible mechanism of action of these substances, FAc-2 showed a high virucidal pattern, while F-6 had no marked virucidal effect. However, with respect to the adsorption mechanism, the FAc-2 compound did not significantly inhibit adsorption, while the F-6 compound demonstrated specificity for inhibiting the production of viral plaques. Of interest is that ribavirin synergistically potentiated the antiviral effect of FAc-2. Studies of compounds in association with ribavirin are of great importance because when a synergistic effect is found, it allows the compounds to be used at very low concentrations, significantly reducing their toxicity while maintaining their high antiviral inhibitory activity.

Our results demonstrate the importance of searching the marine environment for antiviral drugs with activity against ZIKV that can significantly reduce the morbidity and mortality of infection. Given the virucidal activity of FAc-2, it may be useful in developing a preventive strategy to significantly reduce the emergence of new cases.

Experimental

Algae and Extraction

Specimens of Dictyota menstrualis (Hoyt), Schnetter, Höring & Weber-Peukert, a common subtidal species, were collected at Enseada do Forno (22°44′00″ S 41°57′25″ W) in the municipality of Armação de Búzios (the northern shore of Rio de Janeiro State, Brazil) during November 2016. The collections were made by snorkeling in shallow water at depths of 2.0 to 5.0 m. Voucher specimens (HUNI 5019) were deposited in the Herbarium of the Universidade Federal do Estado do Rio de Janeiro – UNIRIO (Herbarium Prof. Jorge Pedro Pereira Carauta). The seaweeds were separated from the sediments, epiphytes, and other associated organisms, washed with sea water, and air-dried (approximate temperature 28 to 30°C for 7–10 days) until all water had evaporated.

Two extracts were made from 2 aliquots (~40 g) of the air-dried seaweeds, a crude extract (EDM) and an acetylated crude extract (EDMAc). Initially, an aliquot of 15 g of dry alga was exhaustively extracted with CH₂Cl₂ at room temperature. The biological material was extracted 4 times over 1 month (720 hours) with the solvent under reduced pressure yielding a brownish residue (1.34 g, 9.3% algal dry weight). This EDM was fractionated by flash chromatography over a silica gel (0.004-0.063 mm) column (20 cm × 3 mm) using 400 mL of EtOAc/petroleum ether (2:8). Twenty fractions were collected, which were combined into 8 groups (F-1 to F-8) according to the similarity of the chemical components in their thin-layer chromatography profiles.

The second aliquot (21.6 g) was also exhaustively extracted with CH₂Cl₂ at room temperature and evaporated under reduced pressure; it also yielded a brownish residue (0.9 g, 4.1% algal dry weight). This crude extract was acetylated to increase the amount and stability of the most abundant diterpenes by replacing the secondary hydroxyl groups with acetyl groups. An aliquot of the crude extract (900 mg) was dissolved in pyridine (2 mL) and treated with 5 mL of acetic anhydride (Ac₂O) at room temperature for 48 hours. The reaction mixture was extracted by adding 30 mL Milli-Q H₂O and extracting 3× with pure CHCl₃. To generate the acetylated extract (EDMAc), the organic phase was treated with anhydrous magnesium sulfate and filtered evaporating the solvent.

The acetylated extract was subjected to silica gel chromatography (40 cm × 2.5 mm) eluted with 100% n-hexane to 7:3 EtOAc/n-hexane in aliquots of 200 mL, which produced 80 fractions. The fractionation was monitored by thin-layer chromatography and based on the chromatography profiles, the fractions were combined into 2 groups (FAc-1 and FAc-2). The extract was acetylated to preserve various products that are unstable during the purification process due to the presence of the aldehyde(s). ^{27 -29} A small aliquot (10 mg) from each fraction was dissolved in CDCl₃ to obtain the ¹H NMR (500 MHz) spectrum in a Varian-Unity Plus 500 using trimethylsilyl as the internal reference.

Cytotoxicity Assay

Cell viability was measured by the MTT method. Confluent cultures in 96-well plates were exposed to different concentrations of carrageenan (3 wells/concentration) using incubation conditions equivalent to those used in the antiviral assays. Then 10 µL of Dulbecco’s modified Eagle medium (DMEM) containing MTT (final concentration 0.5 mg/mL) was added to each well. After 2 hours of incubation at 37°C, the supernatant was removed and 200 µL of ethanol was added to each well to solubilize the formazan crystals. After vigorous shaking, the absorbance was measured in a microplate reader at 595 nm. The CC₅₀ was calculated as the concentration of compound necessary to reduce cell viability by 50%.

Antiviral Assay

Antiviral activity was evaluated by 2 methods: a plaque reduction assay and a virus yield inhibition assay. In the plaque reduction test, Vero cell monolayers grown in 24-well plates were infected with ~50 PFU/well of ZIKV serotypes in the absence or presence of serial 2-fold concentrations of the compounds. After adsorption, the residual inoculum was replaced by DMEM containing 1% methylcellulose and the corresponding dose of each compound. Plaques were counted after 6–12 days of incubation at 37°C depending on the virus serotype. The EC₅₀ was calculated as the compound concentration that reduced virus plaque formation by 50%. All determinations were performed twice in duplicate.

In the virus yield inhibition assay, Vero cells were infected with ZIKV at MOIs of 0.01-1 in the presence of different concentrations of the compounds, with 2 wells per concentration. After 48 hours of incubation at 37°C, the cell supernatants were collected and the virus yields were determined by plaque formation in Vero cells. The EC₅₀ values were calculated as specified earlier.

Virucidal Profile

A suspension of ZIKV containing a relative concentration of 200 PFU was mixed with an equivalent volume of the substances at concentrations of 5, 10, or 20 µg/mL and incubated in microtiter plates for 2 hours at 37°C. The preincubated samples were then added to Vero cells in 24-well plates for 2 hours, washed, and incubated under the plate-assay conditions. The virucidal effect was defined by the ability of the compounds to inactivate the particles to not allow infection and to not generate a cytopathic effect, as observed in the virus-only control.

Attachment Assay

The inhibitory effect of the substances on virus adsorption was measured by different experimental approaches. In the infectivity assay for ZIKV adsorption, Vero cells were infected with ZIKV at an MOI of 1 in the presence or absence of 20 µg/mL of the marine compounds. After 1 hour of adsorption at 4°C, the cells were washed with cold phosphate-buffered saline to remove the unabsorbed virus and disrupted by freezing and thawing. The amount of infectious bound virus was then measured by plaque formation.

Synergistic Effect

To evaluate the effect of the algae-derived compounds in association with ribavirin, Vero cells were cultured in 24-well plates until 90% confluence was reached and then infected with ZIKV at an MOI of 0.1. After incubating for 2 hours, they were treated with the previously defined concentrations that completely inhibited infection (>90%) and by suboptimal concentrations (up to 20% less) for 18 hours. Subsequently, the medium and cells were collected and added to new Vero cells and evaluated for viral plaque reduction as described above.