Ayapana Triplinervis Essential Oil and Its Main Component Thymohydroquinone Dimethyl Ether Inhibit Zika Virus at Doses Devoid of Toxicity in Zebraﬁsh

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Article Ayapana triplinervis Essential Oil and Its Main Component Thymohydroquinone Dimethyl Ether Inhibit Zika Virus at Doses Devoid of Toxicity in Zebraﬁsh

Juliano G. Haddad 1, Morgane Picard 1, Sebastien Bénard 2, Claire Desvignes 3, Philippe Desprès 1 , Nicolas Diotel 4,* and Chaker El Kalamouni 1,* 1 Université de la Réunion, INSERM U1187, CNRS UMR 9192, IRD UMR 249, Unité Mixte Processus Infectieux en Milieu Insulaire Tropical, Plateforme Technologique CYROI, 94791 Sainte Clotilde, La Réunion, France; [email protected] (J.G.H.); [email protected] (M.P.); [email protected] (P.D.) 2 Plateforme de recherche CYROI, 2 rue Maxime Rivière, 97490 Sainte Clotilde, La Réunion, France; [email protected] 3 SAS REUNION ECOEX, 2 rue Maxime rivière, 97490 Sainte Clotilde, La Réunion, France; [email protected] 4 Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI), 97490 Saint-Denis, de La Réunion, France * Correspondence: [email protected] (N.D.); [email protected] (C.E.K.); Tel.: +262-262-938822 Academic Editor: Luca Forti Received: 31 August 2019; Accepted: 20 September 2019; Published: 23 September 2019

Abstract: Zika virus (ZIKV) is an emerging mosquito-borne virus of medical concern. ZIKV infection may represent a serious disease, causing neonatal microcephaly and neurological disorders. Nowadays, there is no approved antiviral against ZIKV. Several indigenous or endemic medicinal plants from Mascarene archipelago in Indian Ocean have been found able to inhibit ZIKV infection. The purpose of our study was to determine whether essential oil (EO) from Reunion Island medicinal plant Ayapana triplinervis, whose thymohydroquinone dimethyl ether (THQ) is the main component has the potential to prevent ZIKV infection in human cells. Virological assays were performed on human epithelial A549 cells infected with either GFP reporter ZIKV or epidemic viral strain. Zebrafish assay was employed to evaluate the acute toxicity of THQ in vivo. We showed that both EO and THQ inhibit ZIKV infection in human cells with IC50 values of 38 and 45 µg/mL, respectively. At the noncytotoxic concentrations, EO and THQ reduced virus progeny production by 3-log. Time-of-drug-addition assays revealed that THQ could act as viral entry inhibitor. At the antiviral effective concentration, THQ injection in zebrafish does not lead to any signs of stress and does not impact fish survival, demonstrating the absence of acute toxicity for THQ. From our data, we propose that THQ is a new potent antiviral phytocompound against ZIKV, supporting the potential use of medicinal plants from Reunion Island as a source of natural and safe antiviral substances against medically important mosquito-borne viruses.

Keywords: Ayapana triplinervis; Zika virus; essential oil; antiviral activity; zebraﬁsh; medicinal plant

1. Introduction Zika virus, belonging to the Flavivirus genus of the Flaviviridae family, is an enveloped and single-stranded positive-sense RNA mosquito-borne virus close to dengue virus [1]. ZIKV was ﬁrst isolated in the Zika forest of Uganda in 1947 [2]. The clinical syndromes of ZIKV infection are

Molecules 2019, 24, 3447; doi:10.3390/molecules24193447 www.mdpi.com/journal/molecules Molecules 2019, 24, 3447 2 of 12 similar to those of other arbovirus infections and include, among others, fever, headache, and muscle pain. However, unlike other flavivirus, ZIKV infection has been associated with severe disorders, like Guillain–Barré syndrome and meningoencephalitis in infected adults and congenital defects in affected infants [3–5]. Sexual, vertical, and blood transmissions have also been reported [4,6,7]. Since then, great efforts have been carried out, but still no vaccine or specific antiviral against ZIKV is available [8,9]. The open reading frame of ZIKV genome encodes a single polyprotein, which is post-translationally processed by cellular and viral proteases to yield three structural (Capsid, C; pre-membrane, prM; and Envelope, E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins [10–12]. The ZIKV E-protein mediates the binding of viral particles to the host receptors and the virus enters the target cells via clathrin-mediated endocytosis [12]. We recently reported several indigenous or endemic medicinal plants from Mascarene archipelago which are able to inhibit ZIKV infection through various mechanisms of action [13–15]. Wedemonstrated that Aphloia theiformis and Psiloxilon mauritianum extracts exert a virucidal effect against ZIKV by direct interactions with the virus particles as well as epigallocatechin gallate (EGCG), the main active polyphenol component from green tea [13,14,16,17]. Furthermore, Doratoxylon apetalum extract interfered with ZIKV internalization during viral entry into the host cells, like the flavonoid isoquercitrin (Q3G) [15,18]. As a medicinal plant recently listed in the French Pharmacopeia, Ayapana triplinervis (syn.: Eupatorium ayapana Vent; Eupatorium triplinerve Vahl), belonging to the Asteraceae family, has been traditionally used in folk medicine, among the local people of the Mascarene Islands for its sedative, stimulant, and anti-inflammatory properties [19,20]. Numerous studies demonstrated the antimicrobial, antioxidant, and antifungal activities of A. triplinervis [21–24]. It has been also documented that A. triplinervis essential oil is a rich natural source of thymohydroquinone dimethyl ether, highlighting its antimicrobial activity [25–29]. Several reports have shown the therapeutic roles of THQ and its derivatives as an antimicrobial phytocompounds [29–33]. Herein, we investigated the antiviral activity of A. triplinervis EO and its major compound THQ against ZIKV. We further investigated the possible mechanism of action of THQ for inhibiting ZIKV infection. Finally, we aimed at exploring the innocuity of an acute exposure of an efficient antiviral concentration of THQ in vivo. For this, we took advantage of zebrafish as a relevant model for testing the toxicity of different compounds as previously shown in other studies [34–37]. Indeed, zebrafish is an interesting model sharing a high genomic homology with humans (>70%) as well as evolutionary conserved physiological processes, making it an interesting model for drug development and toxicity analysis [38,39].

2. Results and Discussion

2.1. Noncytotoxic Doses of Ayapana triplinervis EO Inhibit ZIKV Infection in Human Cells Within a project aiming to investigate the potential antiviral activity of medicinal plants from Reunion Island recently registered in the French Pharmacopeia, this study evaluated the anti-ZIKV activity of Ayapana triplinervis essential oil. After extraction of EO from the fresh aerial part of A. triplinervis, we initially performed A. triplinervis EO for cellular cytotoxicity using human A549 cells. Dose-dependent experiments showed that the higher nontoxic concentration of EO (more than 90% of cell viability) was 300 µg/mL (Figure1A). By nonlinear regression analysis, the CC 50 value was calculated as 475 µg/mL (Table1). We reported that ZIKV replicates e fficiently in human lung epithelial A549 cells [40]. Thus, to assess whether EO inhibits ZIKV infection, A549 cells were infected 24 h with the molecular clone of ZIKV expressing the GFP (ZIKVGFP) at the MOI (Multiplicity Of Infection) of 1 in the presence of different concentrations (250, 125, 62,5, 31, 25, 15, 62, 7, 8, 4, and 1 µg/mL) of EO throughout the infection (Figure1B). By flow cytometric analysis, we showed that EO treatment resulted in a severe inhibition of ZIKV infection in A549 cells (Figure1B). The number of GFP-positive cells decreased ~80 % compared to nontreated control cells when 125 µg/mL of EO was added through the experiment (Figure1B). The 50% inhibitory concentration (IC 50) value was evaluated at 38 µg/mL (Table1). Molecules 2019, 24, x FOR PEER REVIEW 3 of 12

Moleculesadded 2019 through, 24, 3447 the experiment (Figure 1B). The 50% inhibitory concentration (IC50) value3 of w 12as evaluated at 38 µg/mL (Table 1).

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FigureFigure 1.1.A. A. triplinervis triplinervisessential essential oil oil (EO) (EO exerts) exerts an antiviralan antiviral activity activity against against Zika Zika virus virus (ZIKV). (ZIKV (A) A549). (A) cellsA549 were cells incubated were incubated with two-fold with two serial-fold dilutions serial dilutions (1000 to (1000 7.8 µ gto/mL) 7.8 ofµg/mL) EO for of 72 EO h. for Cell 72 viability h. Cell wasviability evaluated was evaluated through MTTthrough assay. MTT Results assay. are Results means are SDmeans of five ± SD independent of five independent experiments experiments and are ± GFP expressedand are expressed as relative as valuerelative compared value compared to vehicle. to vehicle. (B) A549 (B cells) A549 were cells infected were infected with ZIKV with ZIKVat MOIGFP at ofMOI 1 in of presence 1 in presence of diff oferent different concentrations concentrations (250, (250, 125, 62,125, 5, 62, 31, 5, 25, 31, 15, 25, 62, 15, 7, 62, 8, 47, and8, 4 1andµg /1mL) µg/mL) of EO. of FlowEO. Flow cytometric cytometric analysis analysis of GFP of GFP fluorescence fluorescence was was performed performed 24 h 24 postinfection. hours postinfection The results. The shownresults are means SD of four independent experiments and are expressed as relative value compared to shown are ±means ± SD of four independent experiments and are expressed as relative value compared untreatedto untreated infected infected cells. cells. Table 1. Cytotoxicity and antiviral activity of EO and THQ. 2.2. Thymohydroquinone Dimethyl Ether Extracted from A. triplinervis is a Potent Inhibitor of ZIKV a b c Compound CC50 (µg/mL) IC (µg/mL) SI To identify the active components present in A. triplinervis50 EO and those responsible for the anti- EO 475 48 38 4.2 12.5 ZIKV activity, the chemical composition has ±been identified ±by GC/MS analysis (Table S1). Thirty- THQ 410 46 45 7.5 9.1 eight volatile organic compounds were identified± , accounting± for 99.18% of the EO composition. The Cytotoxic concentration (CC50) and inhibitory concentration (IC50) were obtained by performing nonlinear regression mainfollowed identified by the compound construction of was the sigmoidal thymoh concentration–responseydroquinone dimethyl curves fromether Figures (THQ),1 and represent2A,B. a Concentrationing 87.06% of b c the totalthat inhibited EO composition cell viability by(Table 50%; S1).Concentration Such a chemical that inhibited composition infection by50%; of A.Selectivity triplinervis index EO (CC is50 /consistentIC50). with a previous report for A. triplinervis harvested in Reunion Island, where the concentration of THQ 2.2.was Thymohydroquinoneestimated from 89.9 Dimethyl to 92.8% Ether [25]. Extracted Thus, we from decided A. triplinervis to investigate is a Potent the Inhibitorantiviral of activity ZIKV of the main compound THQ isolated from A. triplinervis EO after purification by silica gel column To identify the active components present in A. triplinervis EO and those responsible for the chromatography and characterization by GC/MS and NMR (Figure S1). Purified THQ was first tested anti-ZIKV activity, the chemical composition has been identified by GC/MS analysis (Table S1). on A549 cells for cytotoxicity using MTT assay. THQ showed no cytotoxicity at concentrations < 150 Thirty-eight volatile organic compounds were identified, accounting for 99.18% of the EO composition. µg/mL (Figure 2A). The CC50 values was calculated as 410 µg/mL (Table 1). As previously performed The main identified compound was thymohydroquinone dimethyl ether (THQ), representing 87.06% for EO, the antiviral activity of THQ was assessed using ZIKV molecular clone expressing the GFP. of the total EO composition (Table S1). Such a chemical composition of A. triplinervis EO is consistent Flow cytometric analysis showed that THQ severely restricted ZIKV infection in A549 cells, yielding with a previous report for A. triplinervis harvested in Reunion Island, where the concentration of 80% inhibition of ZIKV infection at 125 µg/mL (Figure 2B). The IC50 value was 45 µg/mL (Table 1). THQ was estimated from 89.9 to 92.8% [25]. Thus, we decided to investigate the antiviral activity The selectivity index (SI), which measures the preferential antiviral activity of a drug in a relation to of the main compound THQ isolated from A. triplinervis EO after purification by silica gel column its cytotoxicity [15], was estimated according to the values of CC50 and IC50 (SI = CC50/IC50). The SI chromatography and characterization by GC/MS and NMR (Figure S1). Purified THQ was first tested values were 12.5 and 9.1 for EO and THQ, respectively (Table 1). on A549 cells for cytotoxicity using MTT assay. THQ showed no cytotoxicity at concentrations < 150 To further validate the potential anti-ZIKV activity of THQ, the cotemporary epidemic ZIKV µg/mL (Figure2A). The CC 50 values was calculated as 410 µg/mL (Table1). As previously performed strain (ZIKV PF-25013-18), isolated in French Polynesia during the epidemic in 2013, was used [40]. for EO, the antiviral activity of THQ was assessed using ZIKV molecular clone expressing the GFP. Thus, A549 cells were infected 24h at the MOI of 2 with the clinical strain in presence of increasing Flow cytometric analysis showed that THQ severely restricted ZIKV infection in A549 cells, yielding noncytotoxic concentrations of THQ (Figure 2C). EO was used as positive control. A dose-dependent 80% inhibition of ZIKV infection at 125 µg/mL (Figure2B). The IC 50 value was 45 µg/mL (Table1). effect of THQ on ZIKV growth was observed (Figure 2C). At the noncytotoxic concentration of 125 The selectivity index (SI), which measures the preferential antiviral activity of a drug in a relation to µg/mL, EO and THQ reduced the progeny virus production by 4- and 3-log, respectively (Figure 2C). its cytotoxicity [15], was estimated according to the values of CC50 and IC50 (SI = CC50/IC50). The SI Taken together, these data demonstrated that THQ, the main compound isolated from A. triplinervis values were 12.5 and 9.1 for EO and THQ, respectively (Table1). EO, exerts antiviral activity against ZIKV.

Molecules 2019, 24, 3447 4 of 12 Molecules 2019, 24, x FOR PEER REVIEW 4 of 12

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FigureFigure 2. 2.A. A. triplinervis triplinervis-derived-derived thymohydroquinone thymohydroquinone dimethyl dimethyl ether ether (THQ) (THQ exhibits) exhibits antiviral antiviral activity activity µ againstagainst ZIKV. ZIKV. (A ()A A549) A549 cells cells were were incubated incubated with with two-fold two-fold serial serial dilutions dilutions (1000 (1000 to to 7.8 7.8 gµg/mL)/mL) of of THQ THQ for 72 h. Cell viability was evaluated through MTT assay. Results are means SD of five independent for 72 h. Cell viability was evaluated through MTT assay. Results are means± ± SD of five independent experimentsexperiments and and are are expressed expressed as as relative relative value value compared compared to to vehicle. vehicle. (B ()B A549) A549 cells cells were were infected infected GFP withwith ZIKV ZIKVGFPat at MOIMOI ofof 1 in presence of of different different concentrations concentrations (250, (250, 125, 125, 62, 62, 5, 5,31, 31, 25, 25, 15, 15, 62, 62,7, 8 7,, 4, 8, 4, and 1 µg/mL) of THQ. Flow cytometric analysis of GFP fluorescence was performed 24 hours and 1 µg/mL) of THQ. Flow cytometric analysis of GFP fluorescence was performed 24 hours postinfection. The results shown are means SD of four independent experiments and are expressed as postinfection. The results shown are means± ± SD of four independent experiments and are expressed relative value compared to untreated infected cells. (C) A549 cells were infected with the cotemporary as relative value compared to untreated infected cells. (C) A549 cells were infected with the epidemic ZIKV strain (PF-25013-18) at MOI of 2 and continuously incubated with different non cytotoxic cotemporary epidemic ZIKV strain (PF-25013-18) at MOI of 2 and continuously incubated with concentrations of EO or THQ. ZIKV progeny production was quantified by plaque-forming assay. different non cytotoxic concentrations of EO or THQ. ZIKV progeny production was quantified by Data represent the means SD from four independent experiments. One-way ANOVA and Dunnett’s plaque-forming assay. Data± represent the means  SD from four independent experiments. One-way test were used for statistical analysis (**** p < 0.0001; *** p < 0.001; * p < 0.05). ANOVA and Dunnett’s test were used for statistical analysis (**** p < 0.0001; *** p < 0.001; * p < 0.05). To further validate the potential anti-ZIKV activity of THQ, the cotemporary epidemic ZIKV Table 1. Cytotoxicity and antiviral activity of EO and THQ. strain (ZIKV PF-25013-18), isolated in French Polynesia during the epidemic in 2013, was used [40]. Thus, A549 cells were infectedCompound 24h at theCC MOI50 (µg of/mL) 2 with a theIC50clinical (µg/mL) strain b SI in c presence of increasing noncytotoxic concentrations ofEO THQ (Figure4752C). ±EO 48 was used38 as ± positive4.2 control.12.5 A dose-dependent effect of THQ on ZIKV growthTHQ was observed410 (Figure ± 46 2C). At45 the ± 7.5 noncytotoxic 9.1 concentration of 125

µg/mL,Cytotoxic EO and THQconcentration reduced (CC the50 progeny) and inhibitory virus production concentration by 4- (IC and50) were 3-log, obtained respectively by performing (Figure2 C). Takennonlinear together, regression these data followed demonstrated by the construction that THQ, the of the main sigmoidal compound concentration isolated– fromresponseA. triplinervis curves EO, exertsfrom antiviralFigures 1 activityand 2 (A against and B). ZIKV.a Concentration that inhibited cell viability by 50%; b Concentration that inhibited infection by 50%; c Selectivity index (CC50/IC50). 2.3. Thymohydroquinone Dimethyl Ether Prevents ZIKV Entry in Human Cells 2.3.To Thymohydroquinone characterize the antiviral Dimethyl activity Ether ofPrevents THQ againstZIKV Entry ZIKV, in di Humanfferent experimentalCells approaches were conductedTo characterize (Figure3A). Tothe assess antiviral the eactivityffects on of the THQ viral against entry stage, ZIKV, ZIKV different and THQ experimental were simultaneously approaches co-addedwere conducted to the cells (Figure during 3A). 2 h (Figure To assess3A, the Entry). effects To investigate on the viral whether entry stage, THQ interferes ZIKV and with THQ ZIKV were replication,simultaneously A549 cellsco-added were to first the infected cells durin withg ZIKV 2 h (Figure for 2 h 3 andA, Entry). then treated To investigate with THQ w (Figurehether 3THQA, interferes with ZIKV replication, A549 cells were first infected with ZIKV for 2 h and then treated with THQ (Figure 3A, Replication). To investigate whether EO directly affects the cell-free virions to

Molecules 2019, 24, 3447 5 of 12

Replication).Molecules 2019, To24, investigatex FOR PEER REVIEW whether EO directly affects the cell-free virions to abolish the subsequent5 of 12 infection (virucidal effect), ZIKV particles were pre-incubated with THQ for 2h, and then diluted abolish the subsequent infection (virucidal effect), ZIKV particles were pre-incubated with THQ for 50-fold prior infection (Figure3A, virus-free particles). THQ was also maintained throughout the 2h, and then diluted 50-fold prior infection (Figure 3A, virus-free particles). THQ was also maintained experimental period as positive control condition (Figure3A, throughout). The preincubation of ZIKV throughout the experimental period as positive control condition (Figure 3A, throughout). The with THQ prior to incubation with A549 cells demonstrated that THQ does not exhibit virucidal effect preincubation of ZIKV with THQ prior to incubation with A549 cells demonstrated that THQ does against ZIKV (Figure3B). Also, the treatment of cells with THQ showed no significant e ffect on ZIKV not exhibit virucidal effect against ZIKV (Figure 3B). Also, the treatment of cells with THQ showed replication. The results shown in Figure3B suggest that THQ-mediated inhibition was associated to an no significant effect on ZIKV replication. The results shown in Figure 3B suggest that THQ-mediated inability of ZIKV to initiate a productive infectious cycle. All together, these data suggest that THQ inhibition was associated to an inability of ZIKV to initiate a productive infectious cycle. All together, targetsthese thedata initial suggest stages that ofTHQ ZIKV target infection.s the initial stages of ZIKV infection.

FigureFigure 3. 3. ThymohydroquinoneThymohydroquinone dimethyl dimethyl ether ether interferes interferes with with the theearly early stage stage of ZIKV of ZIKV infection infection.. (A) (ASchematic) Schematic representation representation for for time time-of-drug-addition-of-drug-addition assay assay used used to characterize to characterize anti anti-ZIKV-ZIKV activity activity of ofTHQ THQ (125 (125 µg/mL)µg/mL) in in A549 A549 cells. cells. Yellow Yellow arrows arrows indicate indicate the the presence presence of of THQ THQ.. (B (B) )Results Results of of GFP GFP expressionexpression in in ZIKV-infected ZIKV-infected A549A549 cells, under under different diﬀerent conditions conditions shown shown in panel in panel A, are A, areanalyzed analyzed by bycytometry cytometry assay. assay. The The results results represent represent the the mean mean ± SDSD of of four four ind independentependent experiments experiments and and are are ± expressedexpressed as as relative relative values values compared compared to to vehicle.vehicle. One-wayOne-way ANOVA and Dunnett’s Dunnett’s test test were were used used for for statisticalstatistical analysis analysis (**** (****p p< <0.0001; 0.0001;n.s n.s == not significant). signiﬁcant).

ToTo further further elucidate elucidate the the underlying underlying mechanism mechanism of THQof THQ antiviral antiviral activity, activity we subsequently, we subsequently tested thetested effect the of effect THQ of on THQ ZIKV on entry ZIKV steps. entry Westeps. first We investigated first investigated whether whether THQ THQ was ablewas able to a fftoect affect viral attachment.viral attachment. Thus, binding Thus, binding assays assayswere performed were performed at 4 ◦C at which 4 °C allowswhich virus allow bindings virus binding but prevents but viralprevent entrys viral (Figure entry4A). (Figure Epigallocatechin 4A). Epigallocatechin gallate, EGCG, gallate the, mainEGCG polyphenol, the main polyphen compoundol compound from green tea,from which green is knowntea, which to inhibitis known ZIKV to inhibit binding, ZIKV was binding used as, was positive used controlas positive [14, control16,17]. After[14,16,17 1h] of. After ZIKV attachment1h of ZIKV in attachment the presence in ofthe THQ, presence the temperature of THQ, the wastemperature shifted to was 37 ◦ shiftedC without to 37 THQ °C without to allow THQ ZIKV internalizationto allow ZIKV (Figure internalization4A). Our data(Figure show 4A). that Our the data percentage show that of fluorescence the percentage in ZIKV-infected of fluorescence A549 in cellsZIKV was-infected similar A549 to untreated cells was control similar cells. to untreated However, control EGCG cells. significantly However, EGCG inhibits significantly ZIKV attachment inhibits to theZIKV cell membraneattachment (Figure to the 4cellA). membrane Taken together, (Figure these 4A). results Take demonstraten together, these that THQresults is demonstrate not able to inhibit that ZIKVTHQ binding, is not able suggesting to inhibit thatZIKV a binding postattachment, suggesting step that of thea postattachment infectious virus step cycle of the could infectiou be altered.s virus cycleTo could investigate be altered. whether THQ inhibits ZIKV internalization step, A549 cells were infected with GFP ZIKV To investigateat 4 ◦C for whether 1h without THQ THQ, inhibit thens ZIKV the internalization temperature was step, shifted A549 cells to 37 were◦C toinfecte allowd with virus penetration,ZIKVGFP at in 4 the °C presencefor 1h without of THQ THQ (Figure, then4B). the The temperature natural flavonoid was shifted Q3G, which to 37 is °C known to allow to inhibit virus ZIKVpenetration, internalization, in the presence was used of as THQ positive (Figure control 4B). The [18]. natural Flow cytometric flavonoid Q3G analysis, which showed is known that theto percentageinhibit ZIKV of ZIKV-infected internalization A549, was cells used was as positive severely control reduced [18] in.presence Flow cytometric of THQ, analysis as well asshowed the positive that controlthe percentage Q3G (Figure of ZIKV4B). These-infected results A549 suggest cells was that severely THQ is reduced a potent in inhibitor presence of of ZIKV THQ, internalization as well as the process.positive Thus, control THQ-mediated Q3G (Figure inhibition4B). These of results ZIKV infection suggest thatoccurs THQ early is aftera potent virus inhibitor binding toof the ZIKV cell internalization process. Thus, THQ-mediated inhibition of ZIKV infection occurs early after virus binding to the cell membrane and could be explained by the incapacity of the ZIKV-attached particles to be internalized into the host cell in presence of THQ.

Molecules 2019, 24, 3447 6 of 12 membrane and could be explained by the incapacity of the ZIKV-attached particles to be internalized intoMolecules the host 2019 cell, 24, x in FOR presence PEER REVIEW of THQ. 6 of 12

FigureFigure 4. 4.THQ THQ interferes interferes with with thethe internalizationinternalization step of of ZIKV ZIKV entry. entry. (A (A) A549) A549 cells cells were were infected infected with with GFP ZIKVZIKVGFPat at MOI MOI of of 11 forfor 11 hh at 44°C◦C in in the the presence presence of of THQ THQ (125 (125 µg/mL)µg/mL),, and and then then the the temperature temperature was was shiftedshifted to to 37 37◦C °C in in absence absence of ofTHQ. THQ. EpigallocatechinEpigallocatechin gallate, gallate, EGCG EGCG (1 (10000 µM)µM),, was was used used as aspositive positive GFP control.control. (B (B)) A549 A549 cells cells were were infected infected forfor 1h1h withwith ZIKVGFP atat 4 4°C,◦C, then then the the temperature temperature was was shifted shifted to to 37◦37°CC in in presence presence of of THQ. THQ. Isoquercitrin, Isoquercitrin, Q3GQ3G (100 µM)µM),, was was used used as as positive positive control. control. Flow Flow cytometric cytometric analysisanalysis of of GFP GFP ﬂuorescence fluorescence was was performed performed 2424 h postinfection.postinfection. The The results results shown shown are are means means ± SDSD of of ± fourfour independent independent experiments experiments and and are are expressed expressed as as relative relative value value compared compared to to untreated untreated infected infected cells. One-waycells. One ANOVA-way ANOVA and Dunnett’s and Dunnett’s test were test usedwere forused statistical for statistical analysis analysis (*** p (***< 0.001; p < 0.001; **** p ****< 0.0001; p < n.s0.0001;= not signiﬁcant).n.s = not significant).

2.4.2.4. The The Anti-ZIKV Anti-ZIKV Inhibitor Inhibitor DoseDose ofof ThymohydroquinoneThymohydroquinone Dimethyl Dimethyl Ether Ether Causes Causes no no Toxic Toxic Effect Effect in ina a ZebrafishZebrafish Model Model ToT investigateo investigate the the innocuity innocuity of of the the antiviral antiviral dose dose of of THQ THQin in vivovivo,, we decided decided to to use use zebrafish zebrafish as as a relevanta relevant physiological physiological model. model. Indeed, Indeed, zebrafish zebrafish shares share a highs a high genomic genomic homology homology with with humans humans (>70%) as(>70%) well as as many well as physiological many physiological processes, processes, making making it an interestingit an interesting model model fortoxicity for toxicity test test and and drug developmentdrug development [38,39]. [38,39 We consequently]. We consequently performed performed intraperitoneal intraperitoneal injection injection of THQ of (0.15THQ mg(0.15/g mg/g of body weight)of body for weight) evaluating for evaluating its potential its acutepotential toxicity. acute Thistoxicit dosey. Th (0.15is dose mg (0.15/g of mg/g body of weight) body weight) was chosen was as it correspondschosen as it corresponds to the maximum to the noncytotoxicmaximum noncytotoxic concentration concentration estimated estimatedin vitro. in Any vitr signo. Any of sign suffering of andsuffering behavioral and changesbehavioral were changes carefully were checked carefully every checked day every and fishday survivaland fish wassurvival monitored was monitored for 6 days. Interestingly,for 6 days. Interestingly, no striking behavioral no striking changes behavioral (locomotor changes (locomotor activity, feeding activity, behavior, feeding andbehavior, stress) and have beenstress) detected have been in THQ-treated detected in THQ fish- comparedtreated fish to compared controls. to In controls. addition, In asaddition, shown as by shown the respective by the Kaplan–Meierrespective Kaplan curves,–Meier from curves, the injection from theto day injection 6, THQ-injected to day 6, THQ fish display-injecteda fish 100% display survival a 100% similar tosurvival the control similar (Figure to the5). control Taken (Figure together, 5). theseTaken datatogether, suggest these that data THQ suggest does that not THQ exhibit does acute not exhibit toxicity acute toxicity in vivo in our experimental conditions. in vivo in our experimental conditions.

2.5. Concluding Remarks100 Control (0.4% DMSO)

l THQ (0.150 mg/g of body weight)

Recently, we reportedv that a nonvolatile extract isolated from A. triplinervis in Reunion Island

u n=5

exhibited no antiviralt e50ﬀect against ZIKV [14]. In the present study, we showed that a volatile

c extract from the samer plant and represented by an essential oil was eﬃcacious against ZIKV.

e Whereas the nonvolatileP fraction from A. triplinervis, which is mainly constituted of alkaloids, 0 coumarins, and polyphenols,0 has1 no2 antiviral3 4 eff5 ect6 [23,41]. The terpene derivative-rich A. triplinervis EO was effective against ZIKV.Days We after foundinjection that a major phytocompound in A. triplinervis EO, the thymohydroquinone dimethyl ether, accounts for inhibition of ZIKV in human cells. Interestingly, Figure 5. Acute exposure of THQ does not induce apparent toxicity in fish. Control fish were injected both EOwith and vehicle THQ (0.4% are e ffDMSO)ective againstand treated the ones contemporary were injected epidemic with THQ ZIKV from strain A triplinervis reducing EO. ZIKV After growth injection, fish were observed during 6 days. THQ-treated fish showed normal feeding and locomotor behaviors, and did not display any striking sign of stress. Their survival was not affected by the treatment and was similar to control. n = number of fish per group.

Molecules 2019, 24, x FOR PEER REVIEW 6 of 12

Figure 4. THQ interferes with the internalization step of ZIKV entry. (A) A549 cells were infected with ZIKVGFP at MOI of 1 for 1 h at 4°C in the presence of THQ (125 µg/mL), and then the temperature was shifted to 37 °C in absence of THQ. Epigallocatechin gallate, EGCG (100 µM), was used as positive control. (B) A549 cells were infected for 1h with ZIKVGFP at 4 °C, then the temperature was shifted to 37°C in presence of THQ. Isoquercitrin, Q3G (100 µM), was used as positive control. Flow cytometric analysis of GFP fluorescence was performed 24 h postinfection. The results shown are means ± SD of four independent experiments and are expressed as relative value compared to untreated infected cells. One-way ANOVA and Dunnett’s test were used for statistical analysis (*** p < 0.001; **** p < 0.0001; n.s = not significant).

2.4. The Anti-ZIKV Inhibitor Dose of Thymohydroquinone Dimethyl Ether Causes no Toxic Effect in a Zebrafish Model To investigate the innocuity of the antiviral dose of THQ in vivo, we decided to use zebrafish as a relevant physiological model. Indeed, zebrafish shares a high genomic homology with humans (>70%) as well as many physiological processes, making it an interesting model for toxicity test and drug development [38,39]. We consequently performed intraperitoneal injection of THQ (0.15 mg/g of body weight) for evaluating its potential acute toxicity. This dose (0.15 mg/g of body weight) was chosen as it corresponds to the maximum noncytotoxic concentration estimated in vitro. Any sign of suffering and behavioral changes were carefully checked every day and fish survival was monitored for 6 days. Interestingly, no striking behavioral changes (locomotor activity, feeding behavior, and Molecules 2019, 24, 3447 7 of 12 stress) have been detected in THQ-treated fish compared to controls. In addition, as shown by the respective Kaplan–Meier curves, from the injection to day 6, THQ-injected fish display a 100% survivalup to 3-log. similar To our to the knowledge, control (Figure this is 5 the). Taken ﬁrst time together, that a these noncytotoxic data suggest dose that of EO THQ and do THQes not has exhibit been ademonstratedcute toxicity in eﬃ vivocacious in our against experimental a mosquito-borne conditions. RNA virus of medical concern.

100 Control (0.4% DMSO)

l THQ (0.150 mg/g of body weight)

u n=5

t 50

0 0 1 2 3 4 5 6 Days after injection Figure 5. 5. AcuteAcute exposure exposure of THQ of THQ does does not induce not induce apparent apparent toxicity toxicity in fish in. Control fish. Controlfish were fish injected were withinjected vehicle with (0.4% vehicle DMSO) (0.4% DMSO)and treated and ones treated were ones injected were injected with THQ with from THQ A from triplinervisA triplinervis EO. AfterEO. injection,After injection, fish were fish observed were observed during 6 during days. THQ 6 days.-treated THQ-treated fish showed fish normal showed feeding normal and feeding locomotor and behaviors,locomotor behaviors, and did not and display did not any display striking any striking sign of signstress. of stress.Their survival Their survival was not was affected not affected by the by treatmentthe treatment and andwas wassimilar similar to control to control.. n = number n = number of fish of fishper group per group..

Early viral entry assays revealed that THQ-mediated ZIKV inhibition relates to a blockade in the early stages of virus infection presumably at viral entry level. The antiviral mechanism of THQ is likely close to that of natural flavonoid Q3G as it has been reported elsewhere [18]. Indeed, we hypothesized that THQ-mediated ZIKV inhibition is due to a defect in the endocytosis steps of virus particles. It has recently been reported that clathrin-mediated endocytosis pathway involving Axl/Gas6 as entry factors may play a key role in ZIKV entry into the host-cell [42]. Whether THQ-mediated ZIKV inhibition involves Axl/Gas6 entry factors remains to be investigated. Such studies would also broaden our knowledge on the antiviral mechanisms associated to phytocompounds, such as THQ. In conclusion, our data identified, for the first time, the medicinal plant A. triplinervis as a new source of antiviral phytocompounds targeting the cell entry of viral pathogens [13–15,18,43,44]. We demonstrated that THQ from A. triplinervis EO is a potent inhibitor of ZIKV infection in human cells. It is of great interest to evaluate the toxicity of A. triplinervis major phytocompound THQ in vivo. Consequently, we took advantage of a newly established zebrafish model to test the biosafety of THQ extracted from A. triplinervis [35,37,38]. We noted that inoculation of THQ at the doses showing antiviral effect against ZIKV in vitro caused no obvious stress nor impaired locomotor and feeding behaviors in inoculated zebrafish. The data obtained in a zebrafish experimental model reinforce the great interest that represents THQ as a newly identified antiviral phytocompound, which could be potentially efficacious against different enveloped RNA viruses known for their incidence in terms of public health. The results presented in this study underscore viral entry inhibitors as a promising class of antivirals and add A. triplinervis to the group of medicinal plants and their phytochemicals that interfere with the early stage of ZIKV replication cycle. Whether or not EO and THQ also exert antiviral activity against other medically important flaviviruses such as dengue virus, yellow fever virus, and West Nile virus is a critical issue that remains to be investigated.

3. Materials and Methods

3.1. Cells, Viruses and Reagents Human lung epithelial A549 cells (ATCC, CCL-185, Manassas, VA, USA) and Vero cells (ATCC, CCL-81) were grown in minimum essential medium (MEM: Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS Good: Invitrogen), 2 mmol L 1 · − L-Glutamine, 1 mmol L 1 sodium pyruvate, 100 U mL 1 of penicillin, 0.1 mg mL 1 of streptomycin, · − · − · − and 0.5 µg mL 1 of fungizone (PAN Biotech) under a 5% CO atmosphere at 37 C. The clinical isolate · − 2 ◦ PF-25013-18 of ZIKV (ZIKV-PF13) and the recombinant Zika virus expressing the GFP reporter gene (ZIKVGFP) have been previously described [40,45,46]. The ZIKV progeny production was determined Molecules 2019, 24, 3447 8 of 12 by measuring the quantity of infectious particles released into the supernatant by plaque-forming assay on Vero cells as previously described [13,15]. EGCG and Q3G were purchased from Sigma-Aldrich. The growth culture medium supplemented with 0.4% of DMSO was used as a vehicle control.

3.2. Extraction and Chemical Characterization of Essential Oil The fresh aerial parts of A. triplinervis harvested in the East of Reunion Island were subjected to steam distillation assisted with a turbomixer. After 1h, the EO was collected and dried on Na2SO4 and stored in a dark-sealed vial until analysis. GC analysis was performed on a ThermoFisher gas chromatograph (Trace 1300), equipped with a flame ionization detector. A ZB-5MS capillary column (30 mm, 0.25mm coated with 5% pheny-Arylene and 95% dimethyl polysiloxane, and 0.25 µm film thickness) and helium as the carrier gas (1 mL/min) were used. Percentages of compounds were determined from their peak areas in the GC-FID profiles. GC–MS analysis was performed on the above instrument, coupled with a triple quadrupole mass detector GC–MS/MS (Thermo Scientific TSQ 8000 Evo) with the same column and the same operative conditions used for the analytical GC. The ionization voltage was 70 eV, the ion source temperature was 250 ◦C. Detected compounds were identified based on the following parameters: GC retention index (relative to C8–C23 n-alkanes), values reported in the literature [25,26], matching of mass spectral data with those of the MS libraries (NIST14, Wiley11, FFNSC3 and Adams [47]).

3.3. Purification and Characterization of Thymohydroquinone Dimethyl Ether Thymohydroquinone dimethyl ether was isolated from a sample of A. triplinervis essential oil by flash column chromatography (silica gel). Hexane was used as a mobile phase to afford the desired compound as a colorless oil. Thymohydroquinone dimethyl ether was characterized by 1H NMR spectroscopy and mass spectroscopy [30]. Thymohydroquinone dimethyl ether : Rf: 0.26 (SiO2, 1 Hexane), H NMR (CDCl3, 600 MHz) : 1.21 (d, J = 7.0 Hz, 6H, 2XCH3 (-iPr)), 2.21 (s, 3H, Ar-Me), 3.30 (hept, J = 7.0 Hz, 1H, -CH- (iPr)), 3.79 (s, 3H, -OMe), 3.81 (s, 3H, -OMe), 6.68 (s,1H, Ar-H), 6.73 (s,1H, Ar-H). MS (EI): 194.10, 179.08, 164.07.

3.4. MTT Assay Cell viability was measured using the tetrazolium salt MTT assay as described previously [14,18]. Brieﬂy, A549 cells were treated 72 h at 37 ◦C with EO or THQ within a wide range of concentrations (38–1235 µg/mL). The essential oils and THQ were solubilized in 0.4% DMSO. Following the treatment, 20 µL of 5 mg/ml MTT solution (Sigma) were added to the 96-well plates, and cells were stored at 37 ◦C in the darkness. After 3 h of treatment, supernatant was removed and replaced by 100 µL of DMSO. The MTT medium was removed, and the formazan crystals were solubilized with 50 µL of DMSO. Absorbance was measured at 570 nm with a background subtraction at 690 nm. The CC50 was determined using a nonlinear regression on Graphpad prism software.

3.5. Flow Cytometry Assay For cytometry assay, cells were harvested, fixed with 3.7% PFA in PBS for 20 min, washed twice with PBS, and then subjected to a flow cytometric analysis using Cytoflex (Beckman). Results were analyzed using cytExpert software.

3.6. Virus Inactivation Assay To measure the direct eﬀect of the EO and THQ on viral infectivity (free-virus particles), ZIKVGFP (2 105 PFU) were mixed with EO or THQ at 125 µg/mL and then incubated at 37 C for 2 h. The mixture × ◦ was diluted 50-fold (ﬁnal virus concentration, 1 PFU/cell) in MEM containing 10% FBS to yield a subtherapeutic concentration of EO or THQ, and this mixture was subsequently added to A549 cells monolayer seeded in a 6-well plates. As a comparison, ZIKVGFP was mixed with EO or THQ, diluted Molecules 2019, 24, 3447 9 of 12 immediately to 50-fold (no incubation period), and added to cells for infection. After 2 h of adsorption at 37 ◦C, the diluted inocula were discarded, and cells were washed with PBS twice. Medium overlay was applied and the plates were further incubated at 37 ◦C for 24 h.

3.7. Evaluation of Toxicity In Vivo Three- to six-month-old male adult wild type zebrafish (Danio rerio) were maintained under standard conditions of temperature (28 ◦C), photoperiod (14/10 hr light–dark), and conductivity (400 µS). Zebrafish were fed daily with Gemma Micro ZF 300 (Planktovie). For intraperitoneal injections, fish were deeply anesthetized with 0.02% tricaine (MS-222; REF: A5040, Sigma-Aldrich) and injected with vehicle (PBS—0.4% DMSO), and the main purified compound thymohydroquinone dimethyl ether extracted from Ayapana triplinervis EO (THQ, 0.150 mg/g). This dose was chosen as it corresponds to the maximum noncytotoxic concentration that has been tested in vitro, assuming that 1 gram of zebrafish is closed to 1 mL volume. Animals were carefully checked for stress, locomotor activity, and feeding behavior. All animal experiments were conducted in accordance with the French and European Community Guidelines for the Use of Animals in Research (86/609/EEC and 2010/63/EU) and approved by the local Ethics Committee for animal experimentation of CYROI (APAFIS #2018072310507016_v5). At the end of the procedure, the animals were sacrificed with overdose of tricaine.

3.8. Data Analysis A comparison between the different concentrations was done by a one-way ANOVAtest. All values were expressed as mean SD of at least three independent experiments. All statistical tests were done ± using the software Graph-Pad Prism (version 8.0; GraphPad software, La Jola, CA, USA). Degrees of significance are indicated on the figure as follows; * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001, n.s = not significant.

Supplementary Materials: The following are available online. Table S1: Chemical composition of Ayapana triplinervis essential oil (aerial part) from Reunion Island; Figure S1: Characterization of thymohydroquinone dimethyl ether after purification from A. triplinervis essential oil. Author Contributions: Conceptualization, P.D., N.D., and C.E.K.; Formal analysis, J.G.H., M.P., N.D., and C.E.K.; Funding acquisition, P.D., N.D., and C.E.K.; Investigation, J.G.H., M.P., S.B., C.D., N.D., and C.E.K.; Methodology, J.G.H. and C.E.K.; Supervision, C.E.K.; Validation, J.G.H., P.D., N.D., and C.E.K.; Writing—original draft preparation, C.E.K.; Writing—review and editing, J.G.H., P.D., N.D., and C.E.K. Funding: This research was funded by POE FEDER 2014-20 of the Conseil Régional de La Réunion (ZIKAlert program, N◦ SYNERGIE: RE00001902) and Research Federation BioST (ZIKAYA), Université de La Réunion. J.-G.H. received funding from the ZIKAlert program. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Sample Availability: Samples of A. triplinervis EO are available from the authors.

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